Classifications

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  • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
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  • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
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  • C12Y301/04001—Phosphodiesterase I (3.1.4.1)
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  • C12Y306/01—Hydrolases acting on acid anhydrides (3.6) in phosphorus-containing anhydrides (3.6.1)
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• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2207/00—Modified animals
  • A01K2207/30—Animals modified by surgical methods
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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  • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
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  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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Definitions

• PAD Peripheral Artery Disease
• IC intermittent claudication
• PAD is progressive and can lead to necrosis, gangrene, and need for limb amputation. Due to the complexity and multifactorial origins of PAD, as well as the differences in muscular adaptive responses, precise PAD pathophysiological mechanisms are still largely unknown.
• the disclosure is based, at least in part, on the surprising discovery that an ENPP1 polypeptide inhibited the unwanted proliferation of vascular smooth muscle cells that occurs following trauma to peripheral arteries in mammals.
• an ENPP1-Fc fusion protein were assessed with respect to the ability to inhibit stenosis after angioplasty in previously injured and stented peripheral arteries of Yorkshire swine.
• Animals treated with the ENPP1-Fc protein exhibited markedly lower intimal thickening in stented profunda arteries relative to animals treated with a vehicle control, demonstrating that ENPP1 has therapeutic utility in inhibiting and/or preventing intimal thickening and/or proliferation in injured peripheral arteries.
• the disclosure relates to a method for treating a subject having peripheral artery disease, the method comprising: administering to the subject an effective amount of an ENPP1 agent to thereby treat said peripheral artery disease in said subject.
• the disclosure includes a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a peripheral artery of a subject, the method comprising: administering to the subject an effective amount of an ENPP1 agent to thereby reduce and/or prevent progression of said vascular smooth muscle cell proliferation in said peripheral artery of said subject.
• the subject has stage III, stage IV or stage IV, grade III peripheral artery disease.
• the disclosure also includes a method for inhibiting or slowing progression of Stage III peripheral artery disease to Stage IV peripheral artery disease in a subject, the method comprising: administering to the subject an effective amount of an ENPP1 agent to thereby inhibit and/or slow progression of Stage III peripheral artery disease to Stage IV peripheral artery disease in said subject.
• the subject has common femoral artery disease.
• the subject has femoral-popliteal disease.
• the subject has tibial- peroneal disease.
• the disclosure also relates to a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a peripheral artery of a subject who undergoes surgery on said peripheral artery, the method comprising: administering to the subject an effective amount of an ENPP1 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation in said peripheral artery at a surgical site of said peripheral artery in said subject.
• the agent is administered prior to, during and/or after said surgery.
• the surgery comprises placement of a stent.
• the ENPP1 agent comprises an ENPP1 polypeptide.
• the ENPP1 agent comprises ENPP1 variants that retain enzymatic activity. In some embodiments of any of the methods described herein, the ENPP1 agent comprises a nucleic acid encoding an ENPP1 polypeptide. In some embodiments of any of the methods described herein, the ENPP1 agent comprises a viral vector comprising a nucleic acid encoding an ENPP1 polypeptide. In some embodiments of any of the methods described herein, the ENPP1 polypeptide comprises the extracellular domain of ENPP1. In some embodiments of any of the methods described herein, the ENPP1 polypeptide comprises the catalytic domain of ENPP1.
• the ENPP1 polypeptide comprises amino acids 99 to 925 of SEQ ID NO:1. In some embodiments of any of the methods described herein, the ENPP1 polypeptide comprises a heterologous protein. In some embodiments of any of the methods described herein, the heterologous protein increases the circulating half-life of the ENPP1 polypeptide in mammal. In some embodiments of any of the methods described herein, the heterologous protein is an Fc region of an immunoglobulin molecule. In some embodiments of any of the methods described herein, the immunoglobulin molecule is an IgG1 molecule. In some embodiments of any of the methods described herein, the heterologous protein is an albumin molecule.
• the heterologous protein is carboxy-terminal to the ENPP1 polypeptide.
• ENPP1 agent comprises a linker.
• the linker separates the ENPP1 polypeptide and the heterologous protein.
• the linker comprises the following amino acid sequence: (GGGGS) n , wherein n is an integer from 1 to 10.
• the ENPP1 agent is administered to the subject subcutaneously. In some embodiments of any of the methods described herein, the ENPP1 agent is administered to the subject intravenously.
• the subject is a tobacco user, has hypertension, has elevated cholesterol or triglyceride levels, is a diabetic, has renal disease, or is obese.
• the disclosure also includes a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a peripheral artery of a subject who undergoes stent placement in said peripheral artery, the method comprising: administering to the subject an effective amount of an ENPP1 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation in said peripheral artery.
• the disclosure features a method for reducing and/or preventing stenosis or restenosis in a peripheral artery of a subject who undergoes stent placement in said peripheral artery, the method comprising: administering to the subject an effective amount of an ENPP1 agent to thereby reduce and/or prevent stenosis or restenosis in said peripheral artery.
• the agent is administered prior to, during and/or after stent placement.
• the disclosure features a method for treating a subject having peripheral artery disease, the method comprising: administering to the subject an effective amount of an ENPP3 agent to thereby treat said peripheral artery disease in said subject.
• the disclosure features a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a peripheral artery of a subject having peripheral artery disease, the method comprising: administering to the subject an effective amount of an ENPP3 agent to thereby reduce and/or prevent progression of said vascular smooth muscle cell proliferation in said peripheral artery of said subject.
• the subject has stage III, stage IV or stage IV, grade III peripheral artery disease.
• the disclosure features a method for inhibiting or slowing progression of Stage III peripheral artery disease to Stage IV peripheral artery disease in a subject, the method comprising: administering to the subject an effective amount of an ENPP3 agent to thereby inhibit and/or slow progression of Stage III peripheral artery disease to Stage IV peripheral artery disease in said subject.
• the subject has common femoral artery disease.
• the subject has femoral-popliteal disease.
• the subject has tibial- peroneal disease.
• the disclosure features a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a peripheral artery of a subject who requires surgery on said peripheral artery, wherein the subject has peripheral artery disease, the method comprising: administering to the subject an effective amount of an ENPP3 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation in said peripheral artery at a surgical site of said peripheral artery in said subject.
• the agent is administered prior to, during and/or after said surgery.
• the surgery comprises placement of a stent.
• the subject does not have a deficiency of ENPP1.
• the ENPP3 agent comprises an ENPP3 polypeptide. In some embodiments of any of the methods described herein, the ENPP3 agent comprises ENPP3 variants that retain enzymatic activity. In some embodiments of any of the methods described herein, the ENPP3 agent comprises a nucleic acid encoding an ENPP3 polypeptide. In some embodiments of any of the methods described herein, the ENPP3 agent comprises a viral vector comprising a nucleic acid encoding an ENPP3 polypeptide. In some embodiments of any of the methods described herein, the ENPP3 polypeptide comprises the extracellular domain of ENPP3.
• the ENPP3 polypeptide comprises the catalytic domain of ENPP3. In some embodiments of any of the methods described herein, the ENPP3 polypeptide comprises amino acids 49 to 875 of SEQ ID NO:.7 In some embodiments of any of the methods described herein, the ENPP3 polypeptide comprises a heterologous protein. In some embodiments of any of the methods described herein, the heterologous protein increases the circulating half-life of the ENPP3 polypeptide in mammal. In some embodiments of any of the methods described herein, the heterologous protein is an Fc region of an immunoglobulin molecule. In some embodiments of any of the methods described herein, the immunoglobulin molecule is an IgG1 molecule.
• the heterologous protein is an albumin molecule. In some embodiments of any of the methods described herein, the heterologous protein is carboxy-terminal to the ENPP3 polypeptide. In some embodiments of any of the methods described herein, the ENPP3 agent comprises a linker. In some embodiments of any of the methods described herein, the linker separates the ENPP3 polypeptide and the heterologous protein. In some embodiments of any of the methods described herein, the linker comprises the following amino acid sequence: (GGGGS)n, wherein n is an integer from 1 to 10. In some embodiments of any of the methods described herein, the ENPP3 agent is administered to the subject subcutaneously.
• the ENPP3 agent is administered to the subject intravenously.
• the subject is a tobacco user, has hypertension, has elevated cholesterol or triglyceride levels, is a diabetic, has renal disease, or is obese.
• the subject has occlusive peripheral artery disease.
• the disclosure features a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a peripheral artery of a subject who undergoes stent placement in said peripheral artery, the method comprising: administering to the subject an effective amount of an ENPP3 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation in said peripheral artery.
• the disclosure features a method for reducing and/or preventing stenosis or restenosis in a peripheral artery of a subject who undergoes stent placement in said peripheral artery, the method comprising: administering to the subject an effective amount of an ENPP3 agent to thereby reduce and/or prevent stenosis or restenosis in said peripheral artery.
• the ENPP3 agent is administered prior to, during and/or after stent placement.
• the disclosure features a coated stent comprising a vascular stent; and a coating on the stent, the coating comprising an ENPP1 agent; and a carrier for said ENPP1 agent, wherein said coating is configured to release said ENPP1 agent from the stent at a rate of 1-10 ⁇ ug/ml per day.
• the ENPP1 agent in an amount between 1 wt % and 50 wt %, based on a total weight of the coating.
• the ENPP1 agent is selected from a group consisting of: ENPP1, ENPP1-Fc, ENPP1-Albumin, and ENPP1 mRNA.
• the ENPP1 agent comprises ENPP1 variants that retain enzymatic activity.
• the carrier is non-reactive with said ENPP1 agent.
• the carrier comprises a polymeric carrier that is physically bound to said ENPP1 agent.
• the carrier comprises a polymeric carrier that is chemically bound to said ENPP1 agent. In some embodiments of any of the stents described herein, the carrier comprises a polymeric biodegradable carrier. In some embodiments of any of the stents described herein, the carrier comprises a nonpolymeric carrier. In some embodiments of any of the stents described herein, the nonpolymeric carrier is selected from a group consisting of: Vitamin E, Vitamin E acetate, Vitamin E succinate, oleic acid, peanut oil and cottonseed oil. In some embodiments of any of the stents described herein, the carrier is a liquid at body temperature.
• the carrier is a solid at body temperature.
• the disclosure features a coated stent comprising a vascular stent; and a coating on the stent, the coating comprising an ENPP3 agent; and a carrier for said ENPP3 agent, wherein said coating is configured to release said ENPP3 agent from the stent at a rate of 1-10 ⁇ g/ml per day.
• the ENPP3 agent in an amount between 1 wt % and 50 wt %, based on a total weight of the coating.
• the carrier comprises a polymeric carrier that is chemically bound to said ENPP3 agent. In some embodiments of any of the stents described herein, the carrier comprises a polymeric biodegradable carrier. In some embodiments of any of the stents described herein, the carrier comprises a nonpolymeric carrier. In some embodiments of any of the stents described herein, the nonpolymeric carrier is selected from a group consisting of: Vitamin E, Vitamin E acetate, Vitamin E succinate, oleic acid, peanut oil and cottonseed oil. In some embodiments of any of the stents described herein, the carrier is liquid at body temperature.
• the carrier is solid at body temperature.
• the disclosure features a method for treating a subject having peripheral artery disease, the method comprising: implanting an arterial stent coated with an ENPP1 agent into an artery of said subject, wherein said implanted stent is configured to release said ENPP1 agent in an amount effective to thereby treat said peripheral artery disease in said subject.
• the disclosure features a method for inhibiting or slowing progression of Stage III peripheral artery disease to Stage IV peripheral artery disease in a subject, the method comprising: implanting an arterial stent coated with an ENPP1 agent into an artery of said subject, wherein said implanted stent is configured to release said ENPP1 agent in an amount effective to thereby inhibit and/or slow progression of Stage III peripheral artery disease to Stage IV peripheral artery disease in said subject.
• the subject has common femoral artery disease.
• the subject has femoral-popliteal disease.
• the subject has tibial- peroneal disease.
• the condition requiring surgery is due to a prior placement of a non-eluting arterial stent in said artery. In some embodiments of any of the methods described herein, the condition requiring is due to a prior placement of an eluting arterial stent in said artery which elutes therapeutic agents other than said ENPP1 agent. In some embodiments of any of the methods described herein, the subject does not have a deficiency of ENPP1. In some embodiments of any of the methods described herein, the ENPP1 agent comprises an ENPP1 polypeptide. In some embodiments of any of the methods described herein, the ENPP1 agent comprises a nucleic acid encoding an ENPP1 polypeptide.
• the ENPP1 agent comprises a viral vector comprising a nucleic acid encoding an ENPP1 polypeptide.
• the ENPP1 polypeptide comprises the extracellular domain of ENPP1.
• the ENPP1 polypeptide comprises the catalytic domain of ENPP1.
• the ENPP1 polypeptide comprises amino acids 99 to 925 of SEQ ID NO:1.
• the ENPP1 polypeptide comprises a heterologous protein.
• the ENPP1 polypeptide and the heterologous protein comprises the following amino acid sequence: (GGGGS)n, wherein n is an integer from 1 to 10.
• the ENPP1 agent is administered to the subject subcutaneously.
• the ENPP1 agent is administered to the subject intravenously.
• the subject is a tobacco user, has hypertension, has elevated cholesterol or triglyceride levels, is a diabetic, has renal disease, or is obese.
• the disclosure features a method for treating a subject having peripheral artery disease, the method comprising: implanting an arterial stent coated with an ENPP3 agent into an artery of said subject, wherein said implanted stent is configured to release said ENPP3 agent in an amount effective to thereby treat said peripheral artery disease in said subject.
• the disclosure features a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a peripheral artery of a subject having peripheral artery disease, the method comprising: implanting an arterial stent coated with an ENPP3 agent into an artery of said subject, wherein said implanted stent is configured to release said ENPP3 agent in an amount effective to thereby reduce and/or prevent progression of said vascular smooth muscle cell proliferation in said peripheral artery of said subject.
• the subject has stage III, stage IV or stage IV, grade III peripheral artery disease.
• the disclosure features a method for inhibiting or slowing progression of Stage III peripheral artery disease to Stage IV peripheral artery disease in a subject, the method comprising: implanting an arterial stent coated with an ENPP3 agent into an artery of said subject, wherein said implanted stent is configured to release said ENPP1 agent in an amount effective to thereby inhibit and/or slow progression of Stage III peripheral artery disease to Stage IV peripheral artery disease in said subject.
• the subject has common femoral artery disease.
• the subject has femoral-popliteal disease.
• the subject has tibial- peroneal disease.
• the disclosure features a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a peripheral artery of a subject who has a condition requiring surgery on said peripheral artery, wherein the subject has peripheral artery disease, the method comprising: implanting an arterial stent coated with an ENPP3 agent into an artery of said subject, wherein said implanted stent is configured to release said ENPP3 agent in an amount effective to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation in said peripheral artery at a surgical site of said peripheral artery in said subject.
• the agent is administered prior to, during and/or after said surgery.
• the condition requiring surgery is due to a prior placement of a non-eluting arterial stent in said artery. In some embodiments of any of the methods described herein, the condition requiring is due to a prior placement of an eluting arterial stent in said artery which elutes therapeutic agents other than said ENPP3 agent. In some embodiments of any of the methods described herein, the subject does not have a deficiency of ENPP1.
• the ENPP1 agent comprises an ENPP3 polypeptide. In some embodiments of any of the methods described herein, the ENPP3 agent comprises a nucleic acid encoding an ENPP1 polypeptide.
• the ENPP3 agent comprises a viral vector comprising a nucleic acid encoding an ENPP3 polypeptide.
• the ENPP3 polypeptide comprises the extracellular domain of ENPP3.
• the ENPP3 polypeptide comprises the catalytic domain of ENPP3.
• the ENPP3 polypeptide comprises amino acids 49-875 of SEQ ID NO:7.
• the ENPP3 polypeptide comprises a heterologous protein.
• the heterologous protein increases the circulating half-life of the ENPP3 polypeptide in mammal.
• the heterologous protein is an Fc region of an immunoglobulin molecule.
• the immunoglobulin molecule is an IgG1 molecule.
• the heterologous protein is an albumin molecule.
• the heterologous protein is carboxy-terminal to the ENPP3 polypeptide.
• the ENPP3 agent comprises a linker.
• the linker separates the ENPP3 polypeptide and the heterologous protein.
• the linker comprises the following amino acid sequence: (GGGGS)n, wherein n is an integer from 1 to 10.
• the ENPP3 agent is administered to the subject subcutaneously.
• the ENPP3 agent is administered to the subject intravenously.
• the subject is a tobacco user, has hypertension, has elevated cholesterol or triglyceride levels, is a diabetic, has renal disease, or is obese.
• Figure 1 is a series of photographs of representative profunda artery images captured by angiography at day 14 and day 42 post stent implantation.
• the two control images illustrate a narrowing of the profunda due to intimal proliferation at day 42 relative to the morphology of the vessel at day 14.
• the upper and lower boundary of the stent within the vessel is identified in each photograph by rectangles.
• Figure 2 is a series of photographs of representative profunda artery images captured by Optical Coherence Tomography (OCT) at day 14 and day 42 post stent implantation.
• the two control images illustrate a pronounced intimal thickening within the profunda at day 42 relative to the morphology of the vessel at day 14.
• OCT Optical Coherence Tomography
• Figure 3 is a bar graph depicting the percent area of stenosis at day 14 and day 42 in the profunda of pigs treated with ENPP1-Fc (Treatment) or given vehicle control (Control), as measured by OCT.
• Figure 4A is a cross-section of an artery experiencing restenosis in the presence of an uncoated stent.
• the endothelium 12 normally serves as a solid barrier between the layer of smooth muscle cells 14 and the arterial lumen 20. Small tears 16 in the endothelium 12 can expose smooth muscle cells 14, which can then migrate into the arterial lumen 20 and hyper proliferate into a mass 18 which can partially or completely occlude the lumen 20 even though an uncoated stent 21 is placed, during a procedure 60 such as angioplasty, in the artery 10 to keep the arterial lumen 20 open.
• Fig.4B is a cross-section of an artery 10 containing a coated stent 22.
• the stent has a coating 24 containing a carrier and a bioactive compound such as ENPP1 agent 65 that inhibits and or prevents restenosis.
• a stent having this coating 24 the tears 16 shown in FIG. 4A in the endothelium 12 may be reduced or eliminated. Additionally, the mass 18 created by a proliferation of smooth muscle cells 14, as shown in FIG.4A, is eliminated or substantially reduced.
• DETAILED DESCRIPTION Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure, the preferred methods and materials are described.
• ENPP1 protein or “ENPP1 polypeptide” refers to ectonucleotide pyrophosphatase/phosphodiesterase-1 protein encoded by the ENPP1 gene that is capable of cleaving ATP to generate PPi and also reduces ectopic calcification in soft tissue.
• ENPP1 protein is a type II transmembrane glycoprotein and cleaves a variety of substrates, including phosphodiester bonds of nucleotides and nucleotide sugars and pyrophosphate bonds of nucleotides and nucleotide sugars.
• ENPP1 protein has a transmembrane domain and soluble extracellular domain. The extracellular domain is further subdivided into somatomedin B domain, catalytic domain and the nuclease domain.
• the sequence and structure of wild-type ENPP1 is described in detail in PCT Application Publication No. WO 2014/126965 to Braddock, et al., which is incorporated herein in its entirety by reference.
• ENPP1 polypeptides as used herein encompasses polypeptides that exhibit ENPP1 enzymatic activity, mutants of ENPP1 that retain ENPP1 enzymatic activity, fragments of ENPP1 or variants of ENPP1 including deletion variants that exhibit ENPP1 enzymatic activity.
• ENPP1 enzymatic activity refers to the ability of the ENPP1 polypeptide to cleave Adenosine Triphosphate (ATP) into plasma pyrophosphate (PPi), as noted below.
• ENPP3 polypeptides as used herein encompasses polypeptides that exhibit ATP cleavage enzymatic activity, mutants of ENPP3 that retain ATP cleavage enzymatic activity, fragments of ENPP3 or variants of ENPP3 including deletion variants that exhibit ATP cleavage enzymatic activity.
• ATP cleavage enzymatic activity refers to the ability of the ENPP3 polypeptide to cleave Adenosine Triphosphate (ATP ) into plasma pyrophosphate (PPi), as noted below.
• ENPP1 and ENPP3 polypeptides, mutants, or mutant fragments thereof have been previously disclosed in International PCT Application Publications No. WO/2014/126965- Braddock et al., WO/2017/187408-Braddock et al., WO/2017/087936- Braddock et al., and WO2018/027024-Braddock et al., all of which are incorporated by reference in their entireties herein.
• Enzymatically active with respect to an ENPP1 polypeptide or an ENPP3 polypeptide is defined as possessing ATP hydrolytic activity into AMP and PPi and/or AP3a hydrolysis to ADP and AMP.
• NPP1 and NPP3 readily hydrolyze ATP into AMP and PPi.
• the steady-state Michaelis-Menten enzymatic constants of NPP1 are determined using ATP as a substrate.
• NPP1 can be demonstrated to cleave ATP by HPLC analysis of the enzymatic reaction, and the identity of the substrates and products of the reaction are confirmed by using ATP, AMP, and ADP standards.
• the ATP substrate degrades over time in the presence of NPP1, with the accumulation of the enzymatic product AMP.
• the initial rate velocities for NPP1 are derived in the presence of ATP, and the data is fit to a curve to derive the enzymatic rate constants.
• PPi levels refers to the amount of pyrophosphate present in plasma of animals.
• animals include rat, mouse, cat, dog, human, cow and horse.
• UDPG uridine-diphosphoglucose
• plasma PPi levels in healthy human subjects range from about 1 ⁇ m to about 3 ⁇ M, in some cases between 1-2 ⁇ m.
• Subjects who have defective ENPP1 expression tend to exhibit low ppi levels which range from at least 10% below normal levels, at least 20% below normal levels, at least 30% below normal levels, at least 40% below normal levels, at least 50% below normal levels, at least 60% below normal levels, at least 70% below normal levels, at least 80% below normal levels and combinations thereof.
• GCI Generalized Arterial Calcification of Infancy
• PPi Pseudoxanthoma Elasticum
• alteration refers to a mutation in a gene in a cell that affects the function, activity, expression (transcription or translation) or conformation of the polypeptide it encodes, including missense and nonsense mutations, insertions, deletions, frameshifts and premature terminations.
• ENPP1 precursor protein refers to ENPP1 with its signal peptide sequence at the ENPP1 N-terminus. Upon proteolysis, the signal sequence is cleaved from ENPP1 to provide the ENPP1 protein.
• Signal peptide sequences useful within the disclosure include, but are not limited to, Albumin signal sequence, Azurocidin signal sequence, ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and/or ENPP5 signal peptide sequence.
• ENPP3 precursor protein refers to ENPP3 with its signal peptide sequence at the ENPP3 N-terminus.
• the signal sequence is cleaved from ENPP3 to provide the ENPP3 protein.
• Signal peptide sequences useful within the disclosure include, but are not limited to, Albumin signal peptide sequence, Azurocidin signal peptide sequence, ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and/or ENPP5 signal peptide sequence.
• Azurocidin signal peptide sequence refers to the signal peptide derived from human azurocidin.
• Azurocidin also known as cationic antimicrobial protein CAP37 or heparin-binding protein (HBP), is a protein that in humans is encoded by the AZU1 gene.
• the nucleotide sequence encoding Azurocin signal peptide MTRLTVLALLAGLLASSRA (SEQ ID NO: 42) is fused with the nucleotide sequence of NPP1 or NPP3 gene which when encoded generates ENPP1 precursor protein or ENPP3 precursor protein.
• ENPP1-Fc construct refers to ENPP1 (e.g., the extracellular domain of ENPP1) recombinantly fused and/or chemically conjugated (including both covalent and non-covalent conjugations) to an FcR binding domain of an IgG molecule (preferably, a human IgG).
• IgG molecule preferably, a human IgG
• the C-terminus of ENPP1 is fused or conjugated to the N-terminus of the FcR binding domain.
• ENPP3-Fc construct refers to ENPP3 recombinantly fused and/or chemically conjugated (including both covalent and non-covalent conjugations) to an FcR binding domain of an IgG molecule (preferably, a human IgG).
• the C-terminus of ENPP1 is fused or conjugated to the N-terminus of the FcR binding domain.
• Fc refers to a human IgG (immunoglobulin) Fc domain. Subtypes of IgG such as IgG1, IgG2, IgG3, and IgG4 are contemplated for use as Fc domains.
• the “Fc region or Fc polypeptide” is the portion of an IgG molecule that correlates to a crystallizable fragment obtained by papain digestion of an IgG molecule.
• the Fc region comprises the C-terminal half of the two heavy chains of an IgG molecule that are linked by disulfide bonds. It has no antigen binding activity but contains the carbohydrate moiety and the binding sites for complement and Fc receptors, including the FcRn receptor.
• the Fc fragment contains the entire second constant domain CH2 (residues 231-340 of human IgG1, according to the Kabat numbering system) and the third constant domain CH3 (residues 341- 447).
• IgG hinge-Fc region or “hinge-Fc fragment” refers to a region of an IgG molecule consisting of the Fc region (residues 231 -447) and a hinge region (residues 216- 230) extending from the N-terminus of the Fc region.
• constant domain refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site.
• the constant domain contains the CH1, CH2 and CH3 domains of the heavy chain and the CHL domain of the light chain.
• the term “functional equivalent variant”, as used herein, relates to a polypeptide substantially homologous to the sequences of ENPP1 or ENPP3 (defined above) and that preserves the enzymatic and biological activities of ENPP1 or ENPP3, respectively.
• Methods for determining whether a variant preserves the biological activity of the native ENPP1 or ENPP3 are widely known to the skilled person and include any of the assays used in the experimental part of said application.
• functionally equivalent variants of ENPP1 or ENPP3 delivered by viral vectors is encompassed by the present disclosure.
• the functionally equivalent variants of ENPP1 or ENPP3 are polypeptides substantially homologous to the native ENPP1 or ENPP3 respectively.
• substantially homologous relates to a protein sequence when said protein sequence has a degree of identity with respect to the ENPP1 or ENPP3 sequences described above of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% respectively and still retaining at least 50%, 55%, 60%, 70%, 80% or 90% activity of wild type ENPP1 or ENPP3 protein with respect to ATP cleavage.
• the degree of identity between two polypeptides is determined using computer algorithms and methods that are widely known for the persons skilled in the art.
• the identity between two amino acid sequences is preferably determined by using the BLASTP algorithm (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md.20894, Altschul, S., et al., J. Mol. Biol.215: 403-410 (1990)), though other similar algorithms can also be used.
• BLASTP BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md.20894, Altschul, S., et al., J. Mol. Biol.215: 403-410 (1990)
• BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity.
• Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
• “Functionally equivalent variants” of ENPP1 or ENPP3 may be obtained by replacing nucleotides within the polynucleotide accounting for codon preference in the host cell that is to be used to produce the ENPP1 or ENPP3 respectively.
• Such “codon optimization” can be determined via computer algorithms which incorporate codon frequency tables such as “Human high.cod” for codon preference as provided by the University of Wisconsin Package Version 9.0, Genetics Computer Group, Madison, Wis.
• the variants of ENPP1 or ENPP3 polypeptides are expected to retain at least 50%, 55%, 60%, 70%, 80% or 90% activity of wild type ENPP1 or ENPP3 protein with respect to ATP cleavage.
• wild-type refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the human NPP1 or NPP3 genes.
• functionally equivalent refers to a NPP1 or NPP3 gene or gene product that displays modifications in sequence and/or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product.
• Naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics (including altered nucleic acid sequences) when compared to the wild-type gene or gene product.
• the term “moiety” refers to a chemical component or biological molecule that can be covalently or non-covalently linked to ENPP1 or ENPP3 polypeptide and has the ability to confer a desired property to the protein to which it is attached.
• the term moiety can refer to a bone targeting peptide such as polyaspartic acid or polyglutamic acid (of 4-20 consecutive asp or glu residues) or a molecule that extends the half-life of ENPP1 or ENPP3 polypeptide.
• moieties include Fc, albumin, transferrin, polyethylene glycol (PEG), homo-amino acid polymer (HAP), proline- alanine-serine polymer (PAS), elastin-like peptide (ELP), and gelatin-like protein (GLK).
• the term “subject”, “individual” or “patient” refers to mammal preferably a human who does not possess a loss of function mutation in the NPP1 gene, such as those loss of function mutations that result in pathological calcification and pathological ossification diseases such as Generalized Arterial Calcification of Infancy (GACI) , Autosomal Recessive Hypophosphatemic Rickets Type 2 (ARHR2), Infantile idiopathic arterial calcification (IIAC), Ossification of the Posterior Longitudinal Ligament (OPLL), hypophosphatemic rickets, osteoarthritis, calcification of atherosclerotic plaques, hereditary and non-hereditary forms of osteoarthritis, ankylosing spondylitis, hardening of the arteries occurring with aging, calciphylaxis resulting from end stage renal disease and progeria.
• GCI Generalized Arterial Calcification of Infancy
• ARHR2 Autosom
• NPP1 in serum refers to the amount of NPP1 required to maintain a normal level of plasma pyrophosphate (PPi) in a healthy subject.
• a normal level of PPi corresponds to 2-3 ⁇ M.
• the phrase “medial area” is the area between lamina elastica externa and lamina elastica interna of an artery.
• the phrase “intimal area” and said intimal area is the area between said lamina elastica interna and lumen of an artery.
• lamina elastica externa refers to a layer of elastic connective tissue lying immediately outside the smooth muscle of the tunica media of an artery.
• the phrase “lamina elastica interna” refers to a layer of elastic tissue that forms the outermost part of the tunica intima of blood vessels.
• the phrase “lumen” refers to the interior of a vessel, such as the central space in an artery, vein or capillary through which blood flow occurs.
• the phrase “surgery” refers to an invasive medical procedure that involves vascular interventions which result in tissue injury by scapel incision or radiofrequency ablation or cryoablation or laser ablation.
• tissue injury refers to proliferation or onset of proliferation and migration of vascular smooth muscle eventually resulting in the thickening of arterial walls and decreased arterial lumen space resulting restenosis after percutaneous vascular interventions such as stenting or angioplasty.
• the phrase “deficient for NPP1” or “ENPP1 deficiency” refers to a reduction in an amount of NPP1 protein or in NPP1 activity relative to a normal serum level of NPP1 protein or normal activity of NPP1, wherein such a reduction results in a disease or disorder of pathological calcification and/or pathological ossification.
• pathological diseases include but are not limited to GACI and ARHR2.
• ENPP1 deficiency does not refer to small reductions in an amount of NPP1 protein and/or NPP1 activity that do not result in a disease or disorder of pathological calcification and/or pathological ossification.
• vascular trauma refers to injury to a blood vessel—an artery, which carries blood to an extremity or an organ, or a vein, which returns blood to the heart.
• vascular injuries may also be caused by invasive procedures, such as vascular bypass surgery.
• the phrase “accidental trauma” refers to a blood vessel such as artery by a blunt injury which occurs when a blood vessel is crushed or stretched due to exertion of physical force or penetrating injury which occurs when a blood vessel is punctured, torn or severed. Blunt injury occurs during physical alterations such as boxing and penetrating injury occurs due to sharp objects such as knife or bullet wounds. The trauma or injury can be caused by different factors, such as radiation, viral infections, development of immune complexes, and hyperlipidemia.
• restenosis refers to recurrence of stenosis. Stenosis refers to the narrowing of a blood vessel, leading to restricted blood flow.
• Restenosis usually pertains to an artery or other large blood vessel that has become narrowed, received treatment to clear the blockage and subsequently become re-narrowed. Restenosis is commonly detected by using one or more of ultrasound, X-ray computed tomography (CT), nuclear imaging, optical imaging or contrast enhanced image or immunohistochemical detection.
• CT computed tomography
• myointimal proliferation refers to the proliferation of vascular smooth muscle cells that occurs at the tunica intima of an arterial wall of an individual.
• ENPP1 fragment refers to a fragment or a portion of ENPP1 protein or an active subsequence of the full-length NPP1 having at least an ENPP1 catalytic domain administered in protein form or in the form of a nucleic acid encoding the same.
• ENPP1 agent refers to ENPP1 polypeptide or fusion protein or ENPP1 fragment comprising at least catalytic domain capable of producing plasma pyrophosphate (Ppi) by cleavage of adenosine triphosphate (ATP) or a polynucleotide such as cDNA or RNA encoding ENPP1 fusion protein or ENPP1 fragment comprising at least catalytic domain capable of producing PPi by enzymatic cleavage of ATP or a vector such as a viral vector containing a polynucleotide encoding the same.
• ablation refers to the removal or destruction of a body part or tissue or its function.
• Ablation may be performed by surgery, hormones, drugs, radiofrequency, heat.
• the phrase “reduce or prevent myointimal proliferation” refers to the ability of soluble NPP1 upon administration to reduce the level of proliferation vascular smooth muscle cells at the site of tissue injury thereby reducing the thickening of arterial walls and prevent the occurrence of or reduce the level of restenosis of the artery.
• non-surgical tissue injury refers to injuries sustained to a tissue or blood vessel during a traumatic event including but not limited to physical altercations involving use of blunt force or sharp objects such as knife, mechanical injury such fall from elevation, workplace injury due to heavy machinery or vehicular injury such as car accidents.
• the term “stent” refers to a tubular support placed inside a blood vessel, canal, or duct to aid healing or relieve an obstruction or prevent narrowing of the passage.
• Stents generally comprise an expandable mesh coil which is made of metal (ex: stainless steel, Cobalt alloy, Nickel-titanium alloy, manganese alloy, molybdenum alloy, platinum alloy, tungsten alloy) or polymers (ex: Silicone).
• metal ex: stainless steel, Cobalt alloy, Nickel-titanium alloy, manganese alloy, molybdenum alloy, platinum alloy, tungsten alloy
• polymers ex: Silicone
• the term “vascular stent” refers to a tubular support placed inside an artery or vein of a mammal to aid healing or relieve an obstruction or prevent narrowing of the arterial passage.
• the term “coated stent” or “eluting stent” refers to a stent that is coated with a therapeutic molecule such as protein, chemical compound or nucleic acid that gradually elutes from the stent surface (interior or exterior) at the site of implantation thereby providing therapeutic relief.
• Therapeutic molecules such as ENPP1 agent or ENPP3 agent can be bonded directly to a metal stent, and some are bonded to a matrix polymer, which acts as a drug reservoir to ensure drug retention during deployment and a uniform distribution on the stent.
• the types, compositions, and designs of the polymers coated on the stent dictate the eluting kinetic of the sustain time release of the drug over a period of weeks or months following the implantation in situ.
• the coating materials can be categorized as organic vs inorganic, bioerodable vs nonbioerodable, and synthetic vs naturally occurring substances.
• the term “coating” refers to composition comprising a polymeric carrier that is used in conjunction with an ENPP1 agent or ENPP3 agent to coat the stents.
• the coating may be applied in the form a spray or dried film comprising the ENPP1 agent or ENPP3 agent suspended in a polymeric matrix.
• the polymeric carrier is in an amount sufficient to provide a polymer matrix or support for the ENPP1 agent or ENPP3 agent.
• the polymer is preferably non-reactive with the ENPP1 agent or ENPP3 agent, i.e., no chemical reaction occurs when the two are mixed.
• solvent is defined according to its broadest recognized definition and includes any material into which the carrier (polymer) and the ENPP1 agent or ENPP3 agent can dissolve, fully or partially, at room temperature or from 20° C. to 40° C to form the coating composition. Sterile, double distilled water is a preferred solvent.
• the term “site of injury” refers to a region in the vasculature where the flow of blood or spinal fluid is constricted due to accumulation of one or more of lipids, cholesterol, calcium, and various types of cells, such as smooth muscle cells and platelets.
• the site of injury is commonly identified by using Cardiac catheterization.
• a cardiac catheterization a long, narrow tube called a catheter is inserted through a plastic introducer sheath (a short, hollow tube that is inserted into a blood vessel in your arm or leg). The catheter is guided through the blood vessel to the coronary arteries with the aid of an x-ray machine.
• Contrast material is injected through the catheter and x-ray images (Coronary angiogram) are created as the contrast material moves through the heart’s chambers, valves and major vessels.
• the digital photographs of the contrast material are used to identify the site of the narrowing or blockage in the coronary artery. Additional imaging procedures, called intra-vascular ultrasound (IVUS) and fractional flow reserve (FFR), may be performed along with cardiac catheterization in some cases to obtain detailed images of the walls of the blood vessels.
• IVUS intra-vascular ultrasound
• FFR fractional flow reserve
• site of implant refers to the region at which the ENPP1 or ENPP3 coated stent is implanted in the vasculature.
• the coated stents of the invention can be placed at the center of the to the site of tissue injury, immediately adjacent the site of tissue injury or within 200 ⁇ m on either side from the center of the site of tissue injury.
• myocardial infarction refers to a permanent damage to the heart muscle that occurs due to the formation of plaques in the interior walls of the arteries resulting in reduced blood flow to the heart and injuring heart muscles because of lack of oxygen supply.
• the symptoms of MI include chest pain, which travels from left arm to neck, shortness of breath, sweating, nausea, vomiting, abnormal heart beating, anxiety, fatigue, weakness, stress, depression, and other factors.
• Blunt force trauma refers to a physical trauma to a body part, either by impact, injury or physical attack or high-velocity impact. Blunt trauma can lead to contusions, abrasions, lacerations, and/or bone fractures.
• non-surgical tissue injury or “penetrating trauma” refers to trauma to a body part which occurs when an object such as a projectile or knife enters a tissue of the body, creating an open wound.
• scapel incision refers to incision made in a tissue using a sharp object such as a scapel during surgical procedure.
• Peripheral artery disease refers to the narrowing of the peripheral arteries serving the legs, stomach, arms and head. (“Peripheral” in this case means away from the heart, in the outer regions of the body.) PAD most commonly affects arteries in the legs. PAD generally occurs due to atherosclerosis, a buildup of cholesterol and fatty deposits (plaque) which narrows or blocks blood flow to the arteries leading to the arms, legs and feet. The supply of oxygen to cells is also limited due to the plaque buildup in the artery walls.
• PAD Planar Artery Disease
• asymptomatic, claudication, critical limb ischemia, and acute limb ischemia There are at least two major classification criterion that are routine used in art to classify the level of severity of PAD, Fontaine Classification system and Rutherford Classification system (Overview of Classification Systems in Peripheral Artery Disease, Rulon L.
• Claudication refers to fatigue, discomfort, or pain in the lower extremities, typically the calves, which is reproducibly brought on by exercise and relieved by rest.
• Critical Limb Ischemia refers to a condition wherein the patient experiences chronic ischemic rest pain, nocturnal recumbent pain, or ischemic skin lesions that may include ulcers or gangrene.
• Acute Limb Ischemia refers to patients with a sudden decrease in limb perfusion causing an immediate threat to limb viability.
• the term “Fontaine Classification System” refers to classification system developed by Fontaine et al.(Fontaine R, Kim M, Kieny R. Surgical treatment of peripheral circulation disorders [in German]. Helv Chir Acta 1954;21(5–6):499–533). This classification system grades the clinical presentation of patients to four stages. The system is solely based on clinical symptoms, without other diagnostic tests and is as shown in table below. l
• the term “Rutherford Classification System” refers to classification system developed by Rutherford et al. (Rutherford RB, Flanigan DP, Gupta SK, et al. Suggested standards for reports dealing with lower extremity ischemia.
• the Rutherford system classifies PAD into acute and chronic limb ischemia, emphasizing that each presentation requires different treatment algorithms.
• the Rutherford classification also associates patient clinical symptoms with objective findings, including Doppler, arterial brachial indices (ABI), and pulse volume recordings.
• Rutherford’s chronic limb ischemia classification most resembles Fontaine’s classification, with the addition of objective noninvasive data such as treadmill test. Treadmill protocols are well described in other publications. (H ⁇ yer C, Sandermann J, Petersen LJ. The toe-brachial index in the diagnosis of peripheral arterial disease. J Vasc Surg 2013;58(1): 231–238).
• Treadmill exercise testing with and without preexercise and postexercise ABIs helps differentiate claudication from pseudoclaudication in patients with exertional leg symptoms. Patients who cannot perform treadmill testing can undergo similar stress testing using plantar flexion or thigh blood pressure cuff compression to cause reactive hyperemia. Rutherford’s classification for chronic limb ischemia is shown in table below. Abbreviations: AP, ankle pressure; PVR, pulse volume recording; TM, transmetatarsal; TP, toe pressure.
• toe pressure refers to the measurement of the blood pressure in the toe compared to the blood pressure in the arm
• arm pressure refers to the measurement of the blood pressure in the lower leg compared to the blood pressure in the arm
• pulse volume recording refers to a noninvasive vascular test in which blood pressure cuffs and a hand-held ultrasound device (such as a Doppler or transducer) are used to obtain information about arterial blood flow in the arms and legs.
• stage III peripheral artery disease refers to the PAD disease stage as indicated by the Fontaine classification system
• stage IV peripheral artery disease refers to a PAD disease stage as classified by the Fontaine classification system
• stage IV, grade III peripheral artery disease refers to a PAD disease stage as classified by the Rutherford classification system
• common femoral artery disease refers to a disease state wherein occlusion due to PAD occurs in the femoral artery of the patient.
• femoral-popliteal disease refers to a disease state wherein patients have obstructive occlusions due to PAD in the femoropopliteal artery.
• tibial-peroneal disease refers to a disease state wherein patients have obstructive occlusions due to PAD in a segment of the artery, referred to as tibial-peroneal trunk, below the knee, distal to the origin of the anterior tibial artery off the popliteal artery, and proximal to the branch point of the posterior tibial artery and the fibular artery.
• the term “subject who requires surgery” refers to a patient who is not ENPP1 deficient and has arterial occlusion in the peripheral arteries such as femoral, femoropopliteal or tibial-peroneal arteries.
• site of surgery refers to the region of the artery upon which a tissue injury has occurred either due to vascular trauma or accidental trauma.
• angioplasty refers to a medical procedure that opens up a blocked or narrowed artery around the heart.
• a “low level of PPi” refers to a condition in which the subject has at least 0.1% - 0.99% less than 2%-5% of normal levels of plasma pyrophosphate (PPi). Normal levels of Plasma PPi in healthy human subjects are in the range of 1.8 to 2.6 ⁇ M.
• treatment is defined as the application or administration of soluble NPP1 (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a disease or disorder, a symptom of a disease or disorder or the potential to develop a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the potential to develop the disease or disorder.
• the term “prevent” or “prevention” or “reduce” means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease.
• the term "effective amount” refers to an amount of an agent (e.g., NPPl fusion or NPP3 fusion polypeptides) which, as compared to a corresponding subject who has not received such an amount, sufficient to provide improvement of a condition, disorder, disease, or to provide a decrease in progression or advancement of a condition, disorder, or disease.
• polypeptide refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds.
• isolated means altered or removed from the natural state.
• nucleic acid or a polypeptide naturally present in a living animal is not “isolated,” but the same nucleic acid or polypeptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
• An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
• substantially purified refers to being essentially free of other components.
• a substantially purified polypeptide is a polypeptide that has been separated from other components with which it is normally associated in its naturally occurring state. Non-limiting embodiments include 95% purity, 99% purity, 99.5% purity, 99.9% purity and 100% purity.
• oligonucleotide or “polynucleotide” is a nucleic acid ranging from at least 2, in certain embodiments at least 8, 15 or 25 nucleotides in length, but may be up to 50, 100, 1000, or 5000 nucleotides long or a compound that specifically hybridizes to a polynucleotide.
• pharmaceutical composition or “composition” refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient.
• the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained; for example, phosphate- buffered saline (PBS)
• PBS phosphate- buffered saline
• pathological calcification refers to the abnormal deposition of calcium salts in blood vessels, soft tissues, secretory and excretory passages of the body causing it to harden.
• Calcification can involve cells as well as extracellular matrix components such as collagen in basement membranes and elastic fibers in arterial walls.
• tissues prone to calcification include: Gastric mucosa – the inner epithelial lining of the stomach, Kidneys and lungs, Cornea, heart valves, Systemic arteries and Pulmonary veins.
• pathological ossification refers to a pathological condition in which bone arises in tissues not in the osseous system and in connective tissues usually not manifesting osteogenic properties.
• Ossification is classified into three types depending on the nature of the tissue or organ being affected, endochondral ossification is ossification that occurs in and replaces cartilage. Intramembranous ossification is ossification of bone that occurs in and replaces connective tissue. Metaplastic ossification the development of bony substance in normally soft body structures; called also heterotrophic ossification. As used herein, “reduction of calcification” is observed by using non-invasive methods like X-rays, micro CT and MRI. Reduction of calcification is also inferred by using radio imaging with 99mTc-pyrophosphate (99mPYP) uptake.
• 99mTc-pyrophosphate 99mPYP
• mice The presence of calcifications in mice are evaluated via post-mortem by micro-computed tomography (CT) scans and histologic sections taken from the heart, aorta and kidneys with the use of dyes such as Hematoxylin and Eosin (H&E) and Alizarin red by following protocols established by Braddock et al. (Nature Communications volume 6, Article number: 10006 (2015))
• CT micro-computed tomography
• H&E Hematoxylin and Eosin
• Alizarin red Alizarin red
• Ectopic calcification of soft tissue refers to inappropriate biomineralization, typically composed of calcium phosphate, hydroxyapatite, calcium oxalates and ocatacalcium phosphates occurring in soft tissues leading to loss of hardening of soft tissues.
• Articleerial calcification refers to ectopic calcification that occurs in arteries and heart valves leading to hardening and or narrowing of arteries. Calcification in arteries is correlated with atherosclerotic plaque burden and increased risk of myocardial infarction, increased ischemic episodes in peripheral vascular disease, and increased risk of dissection following angioplasty.
• Vascular calcification refers to ectopic calcification that occurs in veins that reduces the elasticity of the veins and restricts blood flow which can then lead to increase in blood pressure and coronary defects.
• Vascular calcification refers to the pathological deposition of mineral in the vascular system. It has a variety of forms, including intimal calcification and medial calcification, but can also be found in the valves of the heart. Vascular calcification is associated with atherosclerosis, diabetes, certain heredity conditions, and kidney disease, especially CKD. Patients with vascular calcification are at higher risk for adverse cardiovascular events. Vascular calcification affects a wide variety of patients.
• Idiopathic infantile arterial calcification is a rare form of vascular calcification where the arteries of neonates calcify.
• Brain calcification refers to a nonspecific neuropathology wherein deposition of calcium and other mineral in blood vessel walls and tissue parenchyma occurs leading to neuronal death and gliosis.
• Brain calcification is” often associated with various chronic and acute brain disorders including Down’s syndrome, Lewy body disease, Alzheimer’s disease, Parkinson’s disease, vascular dementia, brain tumors, and various endocrinologic conditions
• Calcification of heart tissue refers to accumulation of deposits of calcium (possibly including other minerals) in tissues of the heart, such as aorta tissue and coronary tissue.
• AAV vector refers to a viral particle composed of at least one AAV capsid protein (preferably by all of the capsid proteins of a particular AAV serotype) and an encapsidated recombinant viral genome.
• the particle comprises a recombinant viral genome having a heterologous polynucleotide comprising a sequence encoding human ENPP1 or human ENPP3 or a functionally equivalent variant thereof,) and a transcriptional regulatory region that at least comprises a promoter flanked by the AAV inverted terminal repeats.
• the particle is typically referred to as an “AAV vector particle” or “AAV vector”.
• the term “vector” means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
• the vector is a plasmid, i.e., a circular double stranded DNA loop into which additional DNA segments may be ligated.
• the vector is a viral vector, wherein additional nucleotide sequences may be ligated into the viral genome.
• the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
• the vectors e.g., non-episomal mammalian vectors
• the vectors is integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
• certain vectors are capable of directing the expression of genes to which they are operatively linked.
• the term "recombinant host cell” or simply "host cell”
• host cell means a cell into which an exogenous nucleic acid and/or recombinant vector has been introduced. It should be understood that "recombinant host cell” and “host cell” mean not only the particular subject cell but also the progeny of such a cell.
• progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
• the term “recombinant viral genome”, as used herein, refers to an AAV genome in which at least one extraneous expression cassette polynucleotide is inserted into the naturally occurring AAV genome.
• the genome of the AAV according to the disclosure typically comprises the cis-acting 5′ and 3′ inverted terminal repeat sequences (ITRs) and an expression cassette.
• expression cassette refers to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements, which permit transcription of a particular nucleic acid in a target cell.
• the expression cassette of the recombinant viral genome of the AAV vector according to the disclosure comprises a transcriptional regulatory region operatively linked to a nucleotide sequence encoding ENPP1 or ENPP3 or a functionally equivalent variant thereof.
• transcriptional regulatory region refers to a nucleic acid fragment capable of regulating the expression of one or more genes.
• the transcriptional regulatory region according to the disclosure includes a promoter and, optionally, an enhancer.
• promoter refers to a nucleic acid fragment that functions to control the transcription of one or more polynucleotides, located upstream the polynucleotide sequence(s), and which is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites, and any other DNA sequences including, but not limited to, transcription factor binding sites, repressor, and activator protein binding sites, and any other sequences of nucleotides known in the art to act directly or indirectly to regulate the amount of transcription from the promoter. Any kind of promoters may be used in the disclosure including inducible promoters, constitutive promoters and tissue-specific promoters.
• enhancer refers to a DNA sequence element to which transcription factors bind to increase gene transcription.
• enhancers may be, without limitation, RSV enhancer, CMV enhancer, HCR enhancer, etc.
• the enhancer is a liver-specific enhancer, more preferably a hepatic control region enhancer (HCR).
• operatively linked refers to the functional relation and location of a promoter sequence with respect to a polynucleotide of interest (e.g. a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence).
• a promoter operatively linked is contiguous to the sequence of interest.
• an enhancer does not have to be contiguous to the sequence of interest to control its expression.
• the promoter and the nucleotide sequence encoding ENPP1 or ENPP3 or a functionally equivalent variant thereof refers to a nontoxic but sufficient amount of a viral vector encoding ENPP1 or ENPP3 to provide the desired biological result. That result may be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
• Cap protein refers to a polypeptide having at least one functional activity of a native AAV Cap protein (e.g. VP1, VP2, VP3).
• Cap proteins examples include the ability to induce formation of a capsid, facilitate accumulation of single-stranded DNA, facilitate AAV DNA packaging into capsids (i.e. encapsidation), bind to cellular receptors, and facilitate entry of the virion into host cells.
• any Cap protein can be used in the context of the present disclosure.
• a capsid consists of several oligomeric structural subunits made of proteins. For instance, AAV have an icosahedral capsid formed by the interaction of three capsid proteins: VP1, VP2 and VP3.
• Rep protein refers to a polypeptide having at least one functional activity of a native AAV Rep protein (e.g. Rep 40, 52, 68, 78).
• a “functional activity” of a Rep protein is any activity associated with the physiological function of the protein, including facilitating replication of DNA through recognition, binding and nicking of the AAV origin of DNA replication as well as DNA helicase activity.
• adeno-associated virus ITRs or “AAV ITRs”, as used herein, refers to the inverted terminal repeats present at both ends of the DNA strand of the genome of an adeno- associated virus. The ITR sequences are required for efficient multiplication of the AAV genome.
• tissue-specific promoter is only active in specific types of differentiated cells or tissues. Typically, the downstream gene in a tissue-specific promoter is one which is active to a much higher degree in the tissue(s) for which it is specific than in any other. In this case there may be little or substantially no activity of the promoter in any tissue other than the one(s) for which it is specific.
• inducible promoter refers to a promoter that is physiologically or developmentally regulated, e.g.
• a chemical inducer for example, it can be a tetracycline-inducible promoter, a mifepristone (RU-486)-inducible promoter and the like.
• a chemical inducer for example, it can be a tetracycline-inducible promoter, a mifepristone (RU-486)-inducible promoter and the like.
• the transcriptional regulatory region allows constitutive expression of ENPP1.
• constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the ⁇ -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter (Boshart M, et al., Cell 1985; 41:521-530).
• RSV Rous sarcoma virus
• CMV cytomegalovirus
• SV40 promoter the dihydrofolate reductase promoter
• ⁇ -actin promoter the ⁇ -actin promoter
• PGK phosphoglycerol kinase
• polyadenylation signal relates to a nucleic acid sequence that mediates the attachment of a polyadenine stretch to the 3′ terminus of the mRNA.
• Suitable polyadenylation signals include, without limitation, the SV40 early polyadenylation signal, the SV40 late polyadenylation signal, the HSV thymidine kinase polyadenylation signal, the protamine gene polyadenylation signal, the adenovirus 5 EIb polyadenylation signal, the bovine growth hormone polyadenylation signal, the human variant growth hormone polyadenylation signal and the like.
• signal peptide refers to a sequence of amino acid residues (ranging in length from 10-30 residues) bound at the amino terminus of a nascent protein of interest during protein translation.
• the signal peptide is recognized by the signal recognition particle (SRP) and cleaved by the signal peptidase following transport at the endoplasmic reticulum. (Lodish et al., 2000, Molecular Cell Biology, 4th edition).
• immune response or “immune reaction” refers to the host's immune system to antigen in an invading (infecting) pathogenic organism, or to introduction or expression of foreign protein.
• the immune response is generally humoral and local; antibodies produced by B cells combine with antigen in an antigen-antibody complex to inactivate or neutralize antigen. Immune response is often observed when human proteins are injected into mouse model systems. Generally, the mouse model system is made immune tolerant by injecting immune suppressors prior to the introduction of a foreign antigen to ensure better viability.
• immunesuppression is a deliberate reduction of the activation or efficacy of the host immune system using immunesuppresant drugs to facilitate immune tolerance towards foreign antigens such as foreign proteins, organ transplants, bone marrow and tissue transplantation.
• Non limiting examples of immunosuppressant drugs include anti-CD4(GK1.5) antibody, Cyclophosphamide, Azathioprine (Imuran), Mycophenolate mofetil (Cellcept), Cyclosporine (Neoral, Sandimmune, Gengraf), Methotrexate (Rheumatrex), Leflunomide (Arava), Cyclophosphamide (Cytoxan) and Chlorambucil (Leukeran). Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure.
• a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range.
• description of a range such as from 1to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1to 4, from 1to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
• the present disclosure relates to administration of an ENPP1 or ENPP3 agent to treat PAD, which includes administering sNPP1 and sNPP3 polypeptides and fusion proteins thereof to a subject, and to administration of nucleic acids encoding such polypeptides. Sequences of such polypeptides include the following, without limitation.
• ENPP1 Cloning and Expression of ENPP1 and ENPP3 fusion polypeptides ENPP1, or an ENPP1 polypeptide, is prepared as described in US 2015/0359858 Al, which is incorporated herein in its entirety by reference.
• ENPP1 is a transmembrane protein localized to the cell surface with distinct intramembrane domains.
• the transmembrane domain of ENPP1 may be swapped for the transmembrane domain of ENPP2 or a signal peptide sequence such as Azurocidin, which results in the accumulation of soluble, recombinant ENPP1 in the extracellular fluid of the baculovirus cultures.
• ENPP1 is made soluble by omitting the transmembrane domain.
• Human ENPP1 (SEQ ID NO:1) was modified to express a soluble, recombinant protein by replacing its transmembrane region (e.g., residues 77-98) with the corresponding subdomain of human ENPP2 (NCBI accession NP 001124335, e.g., residues 12-30) or Azurocidin signal sequence (SEQ ID 42).
• the modified ENPP1 sequence was cloned into a modified pFastbac FIT vector possessing a TEV protease cleavage site followed by a C-terminus 9-F lIS tag, and cloned and expressed in insect cells, and both proteins were expressed in a baculovirus system as described previously (Albright, et al., 2012, Blood 120:4432-4440; Saunders, et al., 2011, J. Biol. Chem.18:994-1004; Saunders, et al., 2008, Mol. Cancer Ther.7:3352-3362), resulting in the accumulation of soluble, recombinant protein in the extracellular fluid.
• ENPP3 is poorly exported to the cell surface.
• Soluble ENPP3 polypeptide is constructed by replacing the signal sequence of ENPP3 with the native signal sequence of other ENPPs or Azurocidin or suitable signal sequences.
• ENPP3 fusion constructs are disclosed in WO 2017/087936.
• Soluble ENPP3 constructs are prepared by using the signal export signal sequence of other ENPP enzymes, such as but not limited to ENPP7 and/or ENPP5.
• Soluble ENPP3 constructs are prepared using a signal sequence comprised of a combination of the signal sequences of ENPP1 and ENPP2 ("ENPP1-2-1" or "ENPP121" hereinafter).
• ENPP3 extracellular domain of ENPP3 for secretion as well, such as but not limited to the signal sequence of the immunoglobulin kappa and lambda light chain proteins.
• the disclosure should not be construed to be limited to the constructs described herein, but also includes constructs comprising any enzymatically active truncation of the ENPP3 extracellular domain.
• the ENPP3 polypeptide is soluble.
• the polypeptide of the disclosure includes an ENPP3 polypeptide that lacks the ENPP3 transmembrane domain.
• the polypeptide of the disclosure includes an ENPP3 polypeptide wherein the ENPP3 transmembrane domain has been removed and replaced with the transmembrane domain of another polypeptide, such as, by way of non- limiting example, ENPP2, ENPPS or ENPP7 or Azurocidin signal sequence.
• the polypeptide of the disclosure comprises an IgG Fc domain.
• the polypeptide of the disclosure comprises an albumin domain.
• the albumin domain is located at the C terminal region of the ENPP3 polypeptide.
• the IgG Fc domain is located at the C terminal region of the ENPP3 polypeptide.
• the presence of IgG Fc domain or albumin domain improves half-life, solubility, reduces immunogenicity and increases the activity of the ENPP3 polypeptide.
• the polypeptide of the disclosure comprises a signal peptide resulting in the secretion of a precursor of the ENPP3 polypeptide, which undergoes proteolytic processing to yield the ENPP3 polypeptide.
• the signal peptide is selected from the group consisting of signal peptides of ENPP2, ENPP5 and ENPP7.
• the signal peptide is selected from the group consisting of SEQ ID NOs: 36-42.
• the IgG Fc domain or the albumin domain is connected to the C terminal region of the ENPP3 polypeptide by a linker region.
• the linker is selected from SEQ ID NOs: 43-75, where n is an integer ranging from 1-20.
• ENPP1 polynucleotide encoding residues 96 to 925 of NCBI accession NP_006199 were fused to Fc domain to generate ENPP1 polypeptide.
• the ENPP1 polypeptide can also be expressed from HEK293 cells, Baculovirus insect cell system or CHO cells or Yeast Pichia expression system using suitable vectors.
• the ENPP1 polypeptide can be produced in either adherent or suspension cells.
• the ENPP1 polypeptide is expressed in CHO cells.
• the nucleic acid sequence encoding ENPP1 constructs are cloned into an appropriate vector for large scale protein production.
• ENPP3 is produced by establishing stable transfections in either CHO or HEK293 mammalian cells.
• ENPP3 polynucleotide encoding ENPP3 (Human NPP3 (UniProtKB/Swiss-Prot: O14638.2) was fused to the Fc domain of IgG (referred to as "ENPP3-Fc") and was expressed in stable CHO cell lines.
• ENPP3 polynucleotide encoding residues 49-875 of UniProtKB/Swiss-Prot: O14638.2 was fused to Fc domain to generate ENPP3 polypeptide.
• the ENPP3 polypeptide can be produced in either adherent or suspension cells.
• NPP3 fusion polypeptides of the disclosure into an appropriate vector for large scale protein production.
• these vectors available from commercial sources and any of those can be used.
• ENPP3 polypeptides are produced following the protocols established in WO 2017/087936, the contents of which are hereby incorporated by reference in their entirety.
• ENPP1 polypeptides are produced following the protocols established in Albright, et al, 2015, Nat Commun.6:10006, the contents of which are hereby incorporated by reference in their entirety.
• a suitable plasmid containing the desired polypeptide constructs of ENPP1 or ENPP3 can be stably transfected into expression plasmid using established techniques such as electroporation or lipofectamine, and the cells can be grown under antibiotic selection to enhance for stably transfected cells. Clones of single, stably transfected cells are then established and screened for high expressing clones of the desired fusion protein. Screening of the single cell clones for ENPP1 or ENPP3 polypeptide expression can be accomplished in a high-throughput manner in 96 well plates using the synthetic enzymatic substrate pNP-TMP as previously described (Saunders, et al, 2008, Mol.
• ENPP3 or ENPP1 polypeptides can be accomplished in shaking flasks or bio-reactors previously described for ENPP1 (Albright, et al, 2015, Nat Commun.6:10006).
• Purification of ENPP3 or ENPP1 polypeptides can be accomplished using a combination of standard purification techniques known in the art. These techniques are well known in art and are selected from techniques such as column chromatograph, ultracentrifugation, filtration, and precipitation.
• chromatographic purification is accomplished using affinity chromatography such as protein-A and protein-G resins, metal affinity resins such as nickel or copper, hydrophobic exchange chromatography, and reverse-phase high-pressure chromatography (HPLC) using C8-C14 resins.
• Ion exchange may also be employed, such as anion and cation exchange chromatography using commercially available resins such as Q- sepharose (anion exchange) and SP-sepharose (cation exchange), blue sepharose resin and blue-sephadex resin, and hydroxyapatite resins.
• Size exclusion chromatography using commercially available S-75 and S200 Superdex resins can also be employed, as known in the art.
• Buffers used to solubilize the protein and provide the selection media for the above described chromatographic steps are standard biological buffers known to practitioners of the art and science of protein chemistry. Some examples of buffers that are used in preparation include citrate, phosphate, acetate, tris(hydroxymemyl)aminomethane, saline buffers, glycine-HCL buffers, Cacodylate buffers, and sodium barbital buffers, which are well known in art. Using a single techniques, or a series of techniques in combination, and the appropriate buffer systems purified ENPP3 and the crude starting material side by side on a Coomasie stained polyacrylamide gel after a single purification step.
• ENPP3 protein can then be additionally purified using additional techniques and/or chromatographic steps as described above, to reach substantially higher purity such as ⁇ 99% purity adjusted to the appropriate pH, one can purify the ENPP1 or ENPP3 polypeptides described to greater than 99% purity from crude material.
• ENPP1-Fc or ENPP3-Fc was dialyzed into PBS supplemented with Zn2+ and Mg2+ (PBSplus) concentrated to between 5 and 7 mg/ml, and frozen at -80 °C in aliquots of 200-500 ⁇ l.
• the hsNPP1 or hsNPP3 is administered in one or more doses containing about 1.0 mg/kg to about 5.0 mg/kg NPP1 or about 1.0 mg/kg to about 5.0 mg/kg NPP3 respectively.
• the hsNPP1 or hsNPP3 is administered in one or more doses containing about 1.0 mg/kg to about 10.0 mg/kg NPP1 or about 1.0 mg/kg to about 10.0 mg/kg NPP3.
• the time period between doses of the hsNPP1 or hsNPP3 is at least 2 days and can be longer, for example at least 3 days, at least 1 week, 2 weeks or 1 month. In one embodiment, the administration is weekly, bi-weekly, or monthly.
• the recombinant hsNPP1 or hsNPP3 can be administered in any suitable way, such as intravenously, subcutaneously, or intraperitoneally.
• the recombinant hsNPP1 or hsNPP3 can be administered in combination with one or more additional therapeutic agents.
• Exemplary therapeutic agents include, but are not limited to Bisphosphonate, Statins, Fibrates, Niacin, Aspirin, Clopidogrel, and warfarin.
• the recombinant hsNPP1 or hsNPP3 and additional therapeutic agent are administered separately and are administered concurrently or sequentially. In some embodiments, the recombinant hsNPP1 or hsNPP3 is administered prior to administration of the additional therapeutic agent. In some embodiments, the recombinant hsNPP1 or hsNPP3 is administered after administration of the additional therapeutic agent. In other embodiments, the recombinant hsNPP1 or hsNPP3 and additional therapeutic agent are administered together.
• nucleic Acid Administration and Therapy Viral Vectors for in vivo expression of ENPP1 and ENPP3 The nucleic acids encoding the polypeptide(s) useful within the disclosure may be used in gene therapy protocols for the treatment of the diseases or disorders contemplated herein.
• the improved construct encoding the polypeptide(s) can be inserted into the appropriate gene therapy vector and administered to a patient to treat or prevent the diseases or disorder of interest.
• Vectors such as viral vectors, have been used in the prior art to introduce genes into a wide variety of different target cells. Typically, the vectors are exposed to the target cells so that transformation can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired polypeptide (e.g., a receptor).
• the transfected nucleic acid may be permanently incorporated into the genome of each of the targeted cells, providing long lasting effect, or alternatively the treatment may have to be repeated periodically.
• the (viral) vector transfects liver cells in vivo with genetic material encoding the polypeptide(s) of the disclosure.
• vectors both viral vectors and plasmid vectors are known in the art (see for example U.S. Patent No.5,252,479 and WO 93/07282).
• viruses have been used as gene transfer vectors, including papovaviruses, such as SV40, vaccinia virus, herpes viruses including HSV and EBV, and retroviruses.
• Modified viruses useful according to the disclosure are derived from viruses which include, for example: parvovirus, picornavirus, pseudorabies virus, hepatitis virus A, B or C, papillomavirus, papovavirus (such as polyoma and SV40) or herpes virus (such as Epstein- Barr Virus, Varicella Zoster Virus, Cytomegalovirus, Herpes Zoster and Herpes Simplex Virus types 1 and 2), an RNA virus or a retrovirus, such as the Moloney murine leukemia virus or a lentivirus (i.e. derived from Human Immunodeficiency Virus, Feline Immunodeficiency Virus, equine infectious anemia virus, etc.).
• viruses which include, for example: parvovirus, picornavirus, pseudorabies virus, hepatitis virus A, B or C, papillomavirus, papovavirus (such as polyoma and SV40) or herpes virus (such as
• a viral vector is generally administered by injection, most often intravenously (by IV) directly into the body, or directly into a specific tissue, where it is taken up by individual cells.
• a viral vector may be administered by contacting the viral vector ex vivo with a sample of the patient's cells, thereby allowing the viral vector to infect the cells, and cells containing the vector are then returned to the patient. Once the viral vector is delivered, the coding sequence expressed and results in a functioning protein.
• Adeno-Associated Viral Vectors refers to viruses belonging to the genus Dependovirus of the Parvoviridae family.
• the AAV genome is approximately 4.7 kilobases long and is composed of linear single-stranded deoxyribonucleic acid (ssDNA) which may be either positive- or negative- sensed.
• the genome comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap.
• the rep frame is made of four overlapping genes encoding non-structural replication (Rep) proteins required for the AAV life cycle.
• the cap frame contains overlapping nucleotide sequences of structural VP capsid proteins: VP1, VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry.
• the terminal 145 nucleotides are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex.
• the rep genes i.e. Rep78 and Rep52
• both Rep proteins have a function in the replication of the viral genome.
• a splicing event in the rep ORF results in the expression of actually four Rep proteins (i.e. Rep78, Rep68, Rep52 and Rep40).
• AAV is a helper-dependent virus, that is, it requires co-infection with a helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) in order to form functionally complete AAV virions.
• helper virus e.g., adenovirus, herpesvirus, or vaccinia virus
• AAV establishes a latent state in which the viral genome inserts into a host cell chromosome or exists in an episomal form, but infectious virions are not produced.
• helper virus Subsequent infection by a helper virus "rescues" the integrated genome, allowing it to be replicated and packaged into viral capsids, thereby reconstituting the infectious virion.
• AAV can infect cells from different species, the helper virus must be of the same species as the host cell.
• human AAV replicates in canine cells that have been co-infected with a canine adenovirus.
• rAAV infectious recombinant AAV
• a suitable host cell line can be transfected with an AAV vector containing the heterologous nucleic acid sequence, but lacking the AAV helper function genes, rep and cap.
• the AAV-helper function genes can then be provided on a separate vector. Also, only the helper virus genes necessary for AAV production (i.e., the accessory function genes) can be provided on a vector, rather than providing a replication-competent helper virus (such as adenovirus, herpesvirus, or vaccinia). Collectively, the AAV helper function genes (i.e., rep and cap) and accessory function genes can be provided on one or more vectors. Helper and accessory function gene products can then be expressed in the host cell where they will act in trans on rAAV vectors containing the heterologous nucleic acid sequence.
• the rAAV vector containing the heterologous nucleic acid sequence will then be replicated and packaged as though it were a wild-type (wt) AAV genome, forming a recombinant virion.
• wt wild-type
• the heterologous nucleic acid sequence enters and is expressed in the patient's cells. Because the patient's cells lack the rep and cap genes, as well as the accessory function genes, the rAAV cannot further replicate and package their genomes. Moreover, without a source of 5 rep and cap genes, wtAAV cannot be formed in the patient's cells.
• the AAV vector typically lacks rep and cap frames.
• Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products (i.e. AAV Rep and Cap proteins), and wherein the host cell has been transfected with a vector which encodes and expresses a protein from the adenovirus open reading frame E4orf6.
• Delivery of a protein of interest to the cells of a mammal is accomplished by first generating an AAV vector comprising DNA encoding the protein of interest and then administering the vector to the mammal.
• the disclosure should be construed to include AAV vectors comprising DNA encoding the polypeptide(s) of interest.
• the disclosure relates to an adeno-associated viral (AAV) expression vector comprising a sequence encoding mammal ENPP1 or mammal ENPP3, and upon administration to a mammal the vector expresses an ENPP1 or ENPP3 precursor in a cell, the precursor including an Azurocidin signal peptide fused at its carboxy terminus to the amino terminus of ENPP1 or ENPP3.
• the ENPP1 or ENPP3 precursor may include a stabilizing domain, such as an IgG Fc region or human albumin.
• An AAV expression vector may include an expression cassette comprising a transcriptional regulatory region operatively linked to a nucleotide sequence comprising a transcriptional regulatory region operatively linked to a recombinant nucleic acid sequence encoding a polypeptide comprising an Azurocidin signal peptide sequence and an ectonucleotide pyrophosphatase/phosphodiesterase (ENPP1) polypeptide sequence.
• the expression cassette comprises a promoter and enhancer, the Kozak sequence GCCACCATGG, a nucleotide sequence encoding mammal NPP1 protein or a nucleotide sequence encoding mammal NPP3 protein, other suitable regulatory elements and a polyadenylation signal.
• the AAV recombinant genome of the AAV vector according to the disclosure lacks the rep open reading frame and/or the cap open reading frame.
• the AAV vector according to the disclosure comprises a capsid from any serotype.
• the AAV serotypes have genomic sequences of significant homology at the amino acid and the nucleic acid levels, provide an identical set of genetic functions, and replicate and assemble through practically identical mechanisms.
• the AAV of the present disclosure may belong to the serotype 1 of AAV (AAV1), AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV.
• AAV1 AAV1
• AAV2 AAV3
• AAV4 AAV5
• AAV6, AAV7, AAV8, AAV9 AAV10
• AAVrh10 AAV11
• avian AAV bovine AAV
• GenBank GenBank
• the adeno-associated viral vector according to the disclosure comprises a capsid derived from a serotype selected from the group consisting of the AAV2, AAV5, AAV7, AAV8, AAV9, AAV10 and AAVrh10 serotypes.
• the serotype of the AAV is AAV8.
• the viral vector comprises sequences encoding the capsid proteins, these may be modified so as to comprise an exogenous sequence to direct the AAV to a particular cell type or types, or to increase the efficiency of delivery of the targeted vector to a cell, or to facilitate purification or detection of the AAV, or to reduce the host response.
• the rAAV vector of the disclosure comprises several essential DNA elements. In certain embodiments, these DNA elements include at least two copies of an AAV ITR sequence, a promoter/enhancer element, a transcription termination signal, any necessary 5' or 3' untranslated regions which flank DNA encoding the protein of interest or a biologically active fragment thereof.
• the rAAV vector of the disclosure may also include a portion of an intron of the protein on interest.
• the rAAV vector of the disclosure comprises DNA encoding a mutated polypeptide of interest.
• the vector comprises a promoter/regulatory sequence that comprises a promiscuous promoter which is capable of driving expression of a heterologous gene to high levels in many different cell types.
• promoters include but are not limited to the cytomegalovirus (CMV) immediate early promoter/enhancer sequences, the Rous sarcoma virus promoter/enhancer sequences and the like.
• the promoter/regulatory sequence in the rAAV vector of the disclosure is the CMV immediate early promoter/enhancer.
• the promoter sequence used to drive expression of the heterologous gene may also be an inducible promoter, for example, but not limited to, a steroid inducible promoter, or may be a tissue specific promoter, such as, but not limited to, the skeletal a-actin promoter which is muscle tissue specific and the muscle creatine kinase promoter/enhancer, and the like.
• the rAAV vector of the disclosure comprises a transcription termination signal. While any transcription termination signal may be included in the vector of the disclosure, in certain embodiments, the transcription termination signal is the SV40 transcription termination signal.
• the rAAV vector of the disclosure comprises isolated DNA 5 encoding the polypeptide of interest, or a biologically active fragment of the polypeptide of interest.
• the disclosure should be construed to include any mammalian sequence of the polypeptide of interest, which is either known or unknown.
• the disclosure should be construed to include genes from mammals other than humans, which polypeptide functions in a substantially similar manner to the human polypeptide.
• the nucleotide sequence comprising the gene encoding the polypeptide of interest is about 50% homologous, more preferably about 70% homologous, even more preferably about 80% homologous and most preferably about 90% homologous to the gene encoding the polypeptide of interest.
• the disclosure should be construed to include naturally occurring variants or recombinantly derived mutants of wild type protein sequences, which variants or mutants render the polypeptide encoded thereby either as therapeutically effective as full-length polypeptide, or even more therapeutically effective than full-length polypeptide in the gene therapy methods of the disclosure.
• the disclosure should also be construed to include DNA encoding variants which retain the polypeptide's biological activity.
• variants include proteins or polypeptides which have been or may be modified using recombinant DNA technology, such that the protein or polypeptide possesses additional properties which enhance its suitability for use in the methods described herein, for example, but not limited to, variants conferring enhanced stability on the protein in plasma and enhanced specific activity of the protein.
• Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function.
• the disclosure is not limited to the specific rAAV vector exemplified in the experimental examples; rather, the disclosure should be construed to include any suitable AAV vector, including, but not limited to, vectors based on AAV-1, AAV-3, AAV-4 and AAV-6, and the like. Also included in the disclosure is a method of treating a mammal having a disease or disorder in an amount effective to provide a therapeutic effect.
• the method comprises administering to the mammal an rAAV vector encoding the polypeptide of interest.
• the mammal is a human.
• the number of viral vector genomes/mammal which are administered in a single injection ranges from about 1x108 to about 5 x1016.
• the number of viral vector genomes/mammal which are administered in a single injection is from about lx10 10 to about lx10 15 ; more preferably, the number of viral vector genomes/mammal which are administered in a single injection is from about 5 x 10 10 to about 5 x10 15 ; and, most preferably, the number of viral vector genomes which are administered to the mammal in a single injection is from about 5x 10 10 to about 5 x 10 14 .
• the total number of viral vector genomes administered may be identical, or a fraction thereof or a multiple thereof, 15 to that recited in the single site injection method.
• a composition comprising the virus is injected directly into an organ of the subject (such as, but not limited to, the liver of the subject).
• the rAAV vector may be suspended in a pharmaceutically acceptable carrier, for example, HEPES buffered saline at a pH of about 7.8.
• compositions include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).
• the rAAV vector of the disclosure may also be provided in the form of a kit, the kit comprising, for example, a freeze-dried preparation of vector in a dried salts formulation, sterile water for suspension of the vector/salts composition and instructions for suspension of the vector and administration of the same to the mammal
• kit comprising, for example, a freeze-dried preparation of vector in a dried salts formulation, sterile water for suspension of the vector/salts composition and instructions for suspension of the vector and administration of the same to the mammal
• RNA based in vivo expression of ENPP1 and ENPP3 polypeptides The present disclosure provides compositions and methods for the production and delivery of recombinant double-stranded RNA molecules (dsRNA that encode ENPP1 or ENPP3 polypeptides described herein.
• the double stranded RNA particle (dsRP) can contain a dsRNA molecule enclosed in a capsid or coat protein.
• the dsRNA molecule can be a viral genome or portion of a genome, which can be derived from a wild-type viral genome.
• the RNA molecule can encode an RNA-dependent RNA polymerase (RDRP) and a polyprotein that forms at least part of a capsid or coat protein.
• RDRP RNA-dependent RNA polymerase
• the RNA molecule can also contain an RNA sub-sequence that encodes an ENPP1 or ENPP3 polypeptides that are translated by the cellular components of a host cell.
• the dsRP is transfected into a host cell the sub- sequence can be translated by the cellular machinery of the host cell to produce the ENPP1 or ENPP3 polypeptides.
• the disclosure provides a method of producing a protein product in a host cell. The method includes transfecting a host cell with a dsRP having a recombinant double-stranded RNA molecule (dsRNA) and a capsid or coat protein.
• dsRNA recombinant double-stranded RNA molecule
• the RNA molecule can encode an RNA-dependent RNA polymerase and a polyprotein that forms at least part of the capsid or coat protein, and the dsRP can be able to replicate in the host cell.
• the RNA molecule has at least one RNA sub-sequence that encodes ENPP1 or ENPP3 polypeptides that is translated by cellular components of the host cell.
• the disclosure provides an RNA molecule translatable by a host cell.
• the RNA molecule can be any RNA molecule that encodes the ENPP1 or ENPP3 polypeptides described herein.
• RNA molecule encodes an RNA- dependent RNA polymerase and a polyprotein that forms at least part of a capsid or coat protein of a dsRP and, optionally, can have at least one sub-sequence of RNA that encodes an additional protein product.
• Production of dsRP A dsRP of the disclosure can also be produced by presenting to a host cell a plasmid or other DNA molecule encoding a dsRP of the disclosure or encoding the genes of the dsRP.
• the plasmid or DNA molecule containing nucleotide sequences encoding desired protein such as ENPP1 or ENPP3 polypeptide is then transfected into the host cell and the host cell begins producing the dsRP of the disclosure.
• the dsRP can also be produced in the host cell by presenting to the host cell an RNA molecule encoding the genes of the dsRP.
• the RNA molecule can be (+)-strand RNA.
• the dsRP of the disclosure is therefore self-sustaining within the host cell and is propagated within the host cell.
• the host cell can be any suitable host cell such as, for example, a eukaryotic cell, a mammalian cell, a fungal cell, a bacterial cell, an insect cell, or a yeast cell.
• the host cell can propagate a recombinant dsRP after a recombinant dsRNA molecule of the disclosure or a DNA molecule encoding a dsRP of the disclosure is presented to and taken up by the host cell.
• Methods of Producing a dsRNA Virus or dsRP The disclosure also provides methods of producing a dsRP of the disclosure.
• a double-stranded or single-stranded RNA or DNA molecule can be presented to a host cell.
• the amplification of the dsRNA molecules in the host cell utilizes the natural production and assembly processes already present in many types of host cells (e.g., yeast).
• the disclosure can thus be applied by presenting to a host cell a single-stranded or double-stranded RNA or DNA molecule of the disclosure, which is taken up by the host cell and is utilized to produce the recombinant dsRP and protein or peptide encoded by the RNA sub-sequence using the host cell's cellular components.
• the disclosure can also be applied by providing to the host cell a linear or circular DNA molecule (e.g., a plasmid or vector) containing one or more sequences coding for an RNA-dependent RNA polymerase, a polyprotein that forms at least part of the capsid or coat protein of the dsRP, and a sub-sequence encoding the protein of interest such as ENPP1 or ENPP3 polypeptides as disclosed herein.
• the presentation of a dsRNA or ssRNA molecule of the disclosure can be performed in any suitable way such as, for example, by presenting an RNA molecule of the disclosure directly to the host cell as “naked” or unmodified single-stranded or double-stranded RNA.
• the RNA molecule can be transfected (or transformed) into a yeast, bacterial, or mammalian host cell by any suitable method, for example by electroporation, exposure of the host cell to calcium phosphate, or by the production of liposomes that fuse with the cell membrane and deposit the viral sequence inside. It can also be performed by a specific mechanism of direct introduction of dsRNA from killer viruses or heterologous dsRNA into the host cell. This step can be optimized using a reporter system, such as red fluorescent protein (RFP), or by targeting a specific constitutive gene transcript within the host cell genome. This can be done by using a target with an obvious phenotype or by monitoring by quantitative reverse transcriptase PCR (RT-PCR).
• RT-PCR quantitative reverse transcriptase PCR
• a DNA molecule that encodes an RNA molecule of the disclosure is introduced into the host cell.
• the DNA molecule can contain a sequence coding for the RNA molecule of a dsRP of the disclosure.
• the DNA molecule can code for an entire genome of the dsRP, or a portion thereof.
• the DNA molecule can further code for the at least one sub-sequence of RNA that produces the additional (heterologous) protein product.
• the DNA sequence can also code for gag protein or gag-pol protein, and as well as any necessary or desirable promoters or other sequences supporting the expression and purpose of the molecule.
• the DNA molecule can be a linear DNA, a circular DNA, a plasmid, a yeast artificial chromosome, or may take another form convenient for the specific application.
• the DNA molecule can further comprise T7 ends for producing concatamers and hairpin structures, thus allowing for propagation of the virus or dsRP sequence in the host cell.
• the DNA molecule can be transfected or transformed into the host cell and then, using the host cellular machinery, transcribed and thus provide the dsRNA molecule having the at least one sub-sequence of RNA to the host cell.
• the host cell can then produce the encoded desired ENPP1 or ENPP3 polypeptide.
• the dsRNA can be packaged in the same manner that a wild-type virus would be, using the host cell's metabolic processes and machinery.
• the ENPP1 or ENPP3 polypeptide is also produced using the host cell's metabolic processes and cellular components.
• ENPP1 Coated Stents and ENPP3 Coated stents Stents are typically elongated structures used to keep open lumens (e.g., openings in the body) found in various parts of the body so that the parts of the body containing those lumens may function properly.
• Stents are often used in the treatment of atherosclerosis, a disease of the vascular system in which arteries become partially, and sometimes completely, occluded with substances that may include lipids, cholesterol, calcium, and various types of cells, such as smooth muscle cells and platelets. Stents located within any lumen in the body may not always prevent partial or complete restenosis. In particular, stents do not always prevent the re-narrowing of an artery following Percutaneous transluminal angioplasty (PTA). In some cases, the introduction and presence of the stent itself in the artery or vein can create regions of trauma or tissue injury such as, e.g., tears in the inner lining of the artery, called the endothelium requiring further surgeries post stent placement.
• PTA Percutaneous transluminal angioplasty
• vascular smooth muscle cells which are usually separated from the arterial lumen by the endothelium, into the arterial lumen, where they proliferate to create a mass of cells that may, in a matter of days or weeks, occlude the artery.
• vascular smooth muscle cells which are usually separated from the arterial lumen by the endothelium, into the arterial lumen, where they proliferate to create a mass of cells that may, in a matter of days or weeks, occlude the artery.
• Such re-occlusion which is sometimes seen after PTA, is an example of restenosis.
• Coating a stent with therapeutic agent such as ENPP1 agent or ENPP3 agent is expected to prevent and/or reduce vascular smooth muscle cell proliferation which in return reduces the occurrence of or treats restenosis.
• the patient is need of surgery and/or has tissue injury due to the presence of a prior implanted non-eluting stent.
• the patient is need of surgery and/or has tissue injury due to the presence of a prior implanted eluting stent that elutes therapeutic agents other than ENPP1 agent or ENPP3 agent.
• the prior stent that had caused the tissue injury is removed and replaced with ENPP1 agent coated stent.
• the prior stent that had caused the tissue injury is removed and replaced with ENPP3 agent coated stent.
• the prior stent that had caused the tissue injury is not removed and the ENPP1 agent coated stent is implanted adjacent to the prior stent.
• the prior stent that had caused the tissue injury is not removed and the ENPP3 agent coated stent is implanted adjacent to the prior stent.
• ENPP1 or ENPP3 coated stents are typically hollow, cylindrical structures made from struts or interconnected filaments. Stents are usually implanted at their site of use in the body by attaching them in a compressed state to a catheter that is directed through the body to the site of stent use. Vascular stents are frequently used in blood vessels to open the vessel and provide improved blood flow. The stent can be expanded to a size which enables it to keep the lumen open by supporting the walls of the lumen once it is positioned at the desired site.
• Vascular stents can be collapsed to reduce their diameter so that the stent can be guided through a patient’s arteries or veins to reach the site of deployment.
• Stents are typically either coupled to the outside of the balloon for expansion by the expanding balloon or are self- expanding upon removal of a restraint such as a wire or sleeve maintaining the stent in its collapsed state.
• Vascular stents are often made of metal to provide the strength necessary to support the occluded arterial walls. Two of the preferred metals are Nitinol alloys of nickel and titanium, and stainless steel. Other materials that can be used in fabricating stents are ceramics, polymers, and plastics. The polymer may be a polymer having no functional groups.
• the polymer may be one having functional groups, but none that are reactive with the ENPP1 agent or ENPP3 agent.
• the polymer may include a biodegradable polymer.
• the polymer may include a polymer selected from the group consisting of polyhydroxy acids, polyanhydrides, polyphosphazenes, polyalkylene oxalates, biodegradable polyamides, polyorthoesters, polyphosphoesters, polyorthocarbonates, and blends or copolymers thereof.
• the polymer may also include a biostable polymer, alone or in combination with a biodegradable polymer.
• the polymer may include a polymer selected from the group consisting of polyurethanes, silicones, polyacrylates, polyesters, polyalkylene oxides, polyalcohols, polyolefins, polyvinyl chlorides, cellulose and its derivatives, fluorinated polymers, biostable polyamides, and blends or copolymers thereof.
• Nonuniform strut distribution correlates with more neointimal hyperplasia after Sirolimus- eluting stent implantation. Circulation, 110:3430–4).
• stent designs have been developed to date, only the multicellular design is currently most commonly used; they can be categorized into “closed cell” and “open cell” configurations (Rogers CDK.2002. Drug-eluting stents: role of stent design, delivery vehicle, and drug selection. Rev Cardiovasc Med, 3(Suppl 5): S10–15.).
• a closed cell stent has a uniform cell expansion and constant cell spacing when deployed in a curved vascular segment, which gives more uniform drug distribution (Rogers 2002).
• An open cell stent has a greater variation in the surface coverage between the inner and outer curvatures in the curved segment but gives better conformability to curved surface at the expense of less uniform drug distribution (Rogers 2002).
• the majority of current stents use a closed cell design.
• the optimal stent design for drug delivery would have a large stent surface area, a small cell gap, and minimal strut deformation after deployment while maintaining conformability, radial support, and flexibility to reach the complex coronary lesions.
• ENPP1 coated stents or ENPP3 coated stents are prepared by applying a coating composition comprising an effective amount of ENPP1 agent or ENPP3 agent respectively.
• the coating composition preferably includes an amount of the ENPP1 agent or ENPP3 agent that is sufficient to be therapeutically effective for inhibiting regrowth of plaque or inhibiting restenosis or preventing vascular smooth cell proliferation.
• the coating composition comprises from about 1 wt % to about 50 wt % ENPP1 polypeptide, based on the total weight of the coating composition. In another embodiment, the coating composition comprises from about 5 wt % to about 30 wt % ENPP1 polypeptide.
• the coating composition comprises from about 10 wt % to about 20 wt % ENPP1 polypeptide. In one embodiment, the coating composition comprises from about 1 wt % to about 50 wt % ENPP3 polypeptide, based on the total weight of the coating composition. In another embodiment, the coating composition comprises from about 5 wt % to about 30 wt % ENPP3 polypeptide. In yet another embodiment, the coating composition comprises from about 10 wt % to about 20 wt % ENPP3 polypeptide. In one embodiment, the coating composition comprises from about 1 ⁇ g/ml to about 10 mg/ml of ENPP1 polypeptide.
• the coating composition comprises from about 100 ⁇ g/ml to 5mg/ml ENPP1 polypeptide. In yet another embodiment, the coating composition comprises from about 500 ⁇ g/ml to about 2 mg/ml ENPP1 polypeptide. In a related embodiment, the ENPP1 polypeptide of the coating composition is ENPP1-Fc. In a related embodiment, the ENPP1 polypeptide of the coating composition is ENPP1-Albumin. In one embodiment, the coating composition comprises from about 1 ⁇ g/ml to about 10 mg/ml of ENPP3 polypeptide. In another embodiment, the coating composition comprises from about 100 ⁇ g/ml to 5mg/ml ENPP3 polypeptide.
• the coating composition comprises from about 500 ⁇ g/ml to about 2 mg/ml ENPP3 polypeptide.
• the ENPP3 polypeptide of the coating composition is ENPP3-Fc.
• the ENPP3 polypeptide of the coating composition is ENPP3-Albumin.
• the coating composition comprises from about 1ng/ ⁇ l to about 1000 ⁇ g/ ⁇ l of ENPP1 mRNA.
• the coating composition comprises from about 100 ng/ ⁇ l to 10 ⁇ g/ ⁇ l ENPP1 mRNA.
• the coating composition comprises from about 50 ng/ ⁇ l to about 5 ⁇ g/ ⁇ l ENPP1 mRNA.
• the coating composition comprises from about 1ng/ ⁇ l to about 1000 ⁇ g/ ⁇ l of ENPP1-Fc mRNA. In another embodiment, the coating composition comprises from about 100 ng/ ⁇ l to 10 ⁇ g/ ⁇ l ENPP1 -Fc mRNA. In yet another embodiment, the coating composition comprises from about 50 ng/ ⁇ l to about 5 ⁇ g/ ⁇ l ENPP1-Fc mRNA. In one embodiment, the coating composition comprises from about 1ng/ ⁇ l to about 1000 ⁇ g/ ⁇ l of ENPP1-Albumin mRNA. In another embodiment, the coating composition comprises from about 100 ng/ ⁇ l to 10 ⁇ g/ ⁇ l ENPP1-Albumin mRNA.
• the coating composition comprises from about 50 ng/ ⁇ l to about 5 ⁇ g/ ⁇ l ENPP1-Albumin mRNA. In one embodiment, the coating composition comprises from about 1ng/ ⁇ l to about 1000 ⁇ g/ ⁇ l of ENPP3 mRNA. In another embodiment, the coating composition comprises from about 100 ng/ ⁇ l to 5 ⁇ g/ ⁇ l ENPP3 mRNA. In yet another embodiment, the coating composition comprises from about 500 ng/ ⁇ l to about 2 ⁇ g/ ⁇ l ENPP3 mRNA. In one embodiment, the coating composition comprises from about 1ng/ ⁇ l to about 1000 ⁇ g/ ⁇ l of ENPP3-Fc mRNA.
• the coating composition comprises from about 100 ng/ ⁇ l to 5 ⁇ g/ ⁇ l ENPP3-Fc mRNA. In yet another embodiment, the coating composition comprises from about 500 ng/ ⁇ l to about 2 ⁇ g/ ⁇ l ENPP3-Fc mRNA. In one embodiment, the coating composition comprises from about 1ng/ ⁇ l to about 1000 ⁇ g/ ⁇ l of ENPP3-Albumin mRNA. In another embodiment, the coating composition comprises from about 100 ng/ ⁇ l to 5 ⁇ g/ ⁇ l ENPP3-Albumin mRNA. In yet another embodiment, the coating composition comprises from about 500 ng/ ⁇ l to about 2 ⁇ g/ ⁇ l ENPP3-Albumin mRNA.
• Stents may be coated with a substance, such as a biodegradable or biostable polymer, to improve the biocompatibility of the stent, making it less likely to cause an allergic or other immunological response in a patient.
• a coating substance may also add to the strength of the stent.
• Some known coating substances include organic acids, their derivatives, and synthetic polymers that are either biodegradable or biostable. Biostable coating substances do not degrade in the body, biodegradable coating substances can degrade in the body.
• the coating composition comprises an effective amount of carrier which helps in the coating process to ensure that the therapeutic molecules such as ENPP1 agent or ENPP3 agent adhere to the stent surface and also facilitate in eluting the therapeutic agent into the body at the site of stent placement.
• the carrier could be a liquid carrier or a solid carrier.
• the coating composition may alternatively comprise more than one solid compound in a solid carrier.
• the coating composition may further comprise both a liquid carrier and a solid carrier.
• the coating composition may also comprise more than one type of nonpolymeric or polymeric compound in the carrier and may further comprise both a polymeric material and a nonpolymeric material in a solid or liquid carrier.
• two or more types of biodegradable compounds may be blended together to obtain a liquid carrier for use in the coating composition.
• the biodegradable compounds can be liquids before they are mixed together, e.g., forming a homogeneous solution, mixture, or suspension.
• biodegradable compounds may be solids before they are mixed with other liquid biodegradable compounds.
• the solid biodegradable compounds preferably dissolve when they are mixed with the liquid biodegradable compounds, resulting in a liquid carrier composition containing the different biodegradable compounds.
• the biodegradable carrier component of the coating composition is a solid, which dissolves when mixed with the biologically active component and any other components included in the coating composition.
• the carrier could be a polymeric carrier. Some polymeric carriers are synthetic polymers.
• Examples of synthetic polymers that serve as reservoir matrices include but not limited to poly-n-butyl methacrylate, polyethylene-vinyl acetate, poly (lactide-co- ⁇ - caprolactone) copolymer, Fibrin, cellulose, Phosphorylcholine.
• Some eluting stent comprise porous 300 ⁇ m ceramic layer containing therapeutic molecule -loaded nanocavities.
• the carriers in the coating composition may be either biodegradable or biostable.
• Biodegradable polymers are often used in synthetic biodegradable sutures. These polymers include polyhydroxy acids.
• Polyhydroxy acids suitable for use in the present invention include poly-L-lactic acids, poly- DL-lactic acids, polyglycolic acids, polylactides including homopolymers and copolymers of lactide (including lactides made from all stereo isomers of lactic acids, such as D-,L-lactic acid and meso lactic acid), polylactones, polycaprolactones, polyglycolides, polyparadioxanone, poly 1,4-dioxepan- 2-one, poly 1,5-dioxepan-2-one, poly 6,6-dimethyl-1, 4-dioxan-2-one, polyhydroxyvalerate, polyhydroxybuterate, polytrimethylene carbonate polymers, and blends of the foregoing.
• Polylactones suitable for use in the present invention include polycaprolactones such as poly(e-caprolactone), polyvalerolactones such as poly(d-valerolactone), and polybutyrolactones such as poly(butyrolactone).
• Other biodegradable polymers that can be used are polyanhydrides, polyphosphazenes, biodegradable polyamides such as synthetic polypeptides such as polylysine and polyaspartic acid, polyalkylene oxalates, polyorthoesters, polyphosphoesters, and polyorthocarbonates. Copolymers and blends of any of the listed polymers may be used. Polymer names that are identical except for the presence or absence of brackets represent the same polymers.
• Biostable polymers suitable for use in the present invention include, but are not limited to polyurethanes, silicones such as polyalkyl siloxanes such as polydimethyl siloxane and polybutyl methacrylate, polyesters such as poly(ethylene terephthalate), polyalkylene oxides such as polyethylene oxide or polyethylene glycol, polyalcohols such as polyvinyl alcohols and polyethylene glycols, polyolefins such as poly- 5 ethylene, polypropylene, poly(ethylene-propylene) rubber and natural rubber, polyvinyl chloride, cellulose and modified cellulose derivatives such as rayon, rayon-triacetate, cellulose acetate, cellulose acetate butyrate, cellophane, cellulose nitrate, cellulose propionate, cellulose ethers such as carboxymethyl cellulose and hydroxyalkyl celluloses, fluorinated polymers such as polytetrafluoroethylene (Teflon), and bio stable polyamides such as Nylon 66
• the coating composition further comprises an effective amount of a non-polymeric carrier.
• the non-polymeric carrier can include one or more of fatty acid, biocompatible oil, or wax.
• non-polymeric biodegradable carriers include liquid oleic acid, vitamin E, peanut oil, and cottonseed oil, which are liquids that are both hydrophobic and biocompatible.
• the nonpolymeric or polymeric carrier can be a liquid at room and body temperature.
• the nonpolymeric or polymeric carrier can be a solid at room and body temperature, or a solid at room temperature and a liquid at body temperature.
• the polymer solution can be formed into a film and the film then applied to the stent. Any of a variety of conventional methods of forming films can be used.
• the polymer, ENPP1 agent or ENPP3 agent and solvent are preferably mixed into solution and then poured onto a smooth, flat surface such that a coating film is formed after the solution is dried to remove the solvent.
• the film can then be cut to fit the stent on which it is to be used.
• the film may then be mounted, such as by wrapping, on the outer surface of a stent.
• the coated stent is prepared by spraying the stent with the liquid carrier comprising the therapeutic agent such as ENPP1 agent or ENPP3 agent resulting in a coating of uniform thickness on the struts of the stent.
• the stent may be dip coated or immersed in the coating solution comprising carrier and therapeutic agent, such that the solution completely coats the struts of the stent.
• the stent may be painted with the coating solution comprising carrier and therapeutic agent, such as with a paint brush. In each of these coating applications, the entirety of both the outer and inner surfaces of the stent are preferably coated, although only portions of either or both surfaces may be coated in some embodiments.
• the coating composition comprises a bioactive component and a biodegradable carrier component.
• the coating composition comprises from 0.1% to 100% by weight of a biologically active component and from 1% to 99% by weight of a biodegradable carrier component. More preferably, the coating composition comprises from 0.1% to 50% by weight of a biologically active component and from 50% to 99.9% by weight of a biodegradable carrier component.
• the coating composition can be prepared in a number of ways including by simply mixing the bioactive component and the carrier component together to form a mixture, e.g., a solution or suspension. Alternatively, the bioactive component and the carrier component together are mixed in a suitable solvent, the coating is applied to the stent, and the solvent is removed.
• the coating composition is applied to the stent in its expanded state.
• examples of other medical devices that can be coated in accordance with aspects of the inventions disclosed herein include catheters, heart valves, pacemaker leads, annuloplasty rings and other medical implants.
• coated angioplasty balloons and other coated medical devices can also comprise one of the coating compositions disclosed herein.
• stents are preferred.
• the coating composition may be applied to the stent (or other medical device) by any number of ways, e.g, by spraying the coating composition onto the stent, by immersing the stent in the coating composition, or by painting the stent with the coating composition.
• a stent is coated in its expanded (i.e., enlarged diameter) form so that a sufficient amount of the coating composition will be applied to coat the entire surface of the expanded stent.
• the excess coating composition on the surface of the stent may be removed, such as by brushing off the excess coating composition with a paint brush.
• both the outer and inner surfaces of the stent are coated.
• compositions described herein preferably remain on a stent, partially or in substantial part, after the stent has been introduced to the body, for at least several days , for several weeks and more preferably for several months thereby slowly releasing the therapeutic agents such as ENPP1 agent or ENPP3 agent into the blood stream.
• Pharmaceutical Compositions and Formulations The disclosure provides pharmaceutical compositions comprising a polypeptide of the disclosure within the methods described herein. Such a pharmaceutical composition is in a form suitable for administration to a subject, or the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these.
• the various components of the pharmaceutical composition may be present in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
• the pharmaceutical compositions useful for practicing the method of the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day.
• the pharmaceutical compositions useful for practicing the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 500 mg/kg/day.
• the relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the disclosure will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
• the composition may comprise between about 0.1% and about 100% (w/w) active ingredient.
• Pharmaceutical compositions that are useful in the methods of the disclosure may be suitably developed for inhalational, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, intravenous or another route of administration.
• Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically- based formulations.
• the route(s) of administration is readily apparent to the skilled artisan and depends upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.
• the formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
• a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
• the amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one- third of such a dosage.
• the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
• the regimen of administration may affect what constitutes an effective amount. For example, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection.
• the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
• administration of the compound of the disclosure to a subject elevates the subject's plasma PPi to a level that is close to normal, where a normal level of PPi in mammals is 1-3 ⁇ M.
• “Close to normal” refers to 0 to 1.2 ⁇ M or 0-40% below or above normal, 30 nM to 0.9 ⁇ M or 1-30% 15 below or above normal, 0 to 0.6 ⁇ M or 0-20% below or above normal, or 0 to 0.3 ⁇ M or 0-10% below or above normal.
• compositions of the present disclosure may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder in the patient.
• An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response.
• Dosage is determined based on the biological activity of the therapeutic compound which in turn depends on the half-life and the area under the plasma time of the therapeutic compound curve.
• the polypeptide according to the disclosure is administered at an appropriate time interval of every 2 days, or every 4 days, or every week or every month so as to achieve a continuous level of plasma PPi that is either close to the normal (1-3 ⁇ M) level or above (30-50% higher than) normal levels of PPi.
• Therapeutic dosage of the polypeptides of the disclosure may also be determined based on half-life or the rate at which the therapeutic polypeptide is cleared out of the body.
• the polypeptide according to the disclosure is administered at appropriate time intervals of either every 2 days, or every 4 days, every week or every month so as to achieve a constant level of enzymatic activity of ENPP1 or ENPP3 polypeptides. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
• a non- limiting example of an effective dose range for a therapeutic compound of the disclosure is from about 0.01 and 50 mg/kg of body weight/per day. In certain embodiments, the effective dose range for a therapeutic compound of the disclosure is from about 50 ng to 500 ng/kg, preferably 100 ng to 300 ng/kg of bodyweight.
• the compound can be administered to a patient as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non- limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days.
• a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
• the frequency of the dose is readily apparent to the skilled artisan and depends upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, and the type and age of the patient.
• Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
• a medical doctor, e.g., physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
• the physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
• the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more.
• the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks.
• the frequency of administration of the various combination compositions of the disclosure varies from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors.
• the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physical taking all other factors about the patient into account.
• the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.
• Routes of Administration of any of the compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
• inhalational e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchi
• compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like.
• the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.
• Parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue.
• Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
• parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.
• Example 1 Efficacy of ENPP1-Fc Fusion Protein in In-Stent Restenosis Model
• the efficacy of an ENPP1-Fc fusion protein was evaluated in large animal model of peripheral vascular injury – specifically, in-stent restenosis lesions in the peripheral vasculature of domestic (Yorkshire) swine.
• Therapeutic effects of the ENPP1-Fc fusion protein were assessed with respect to the ability to inhibit stenosis after angioplasty in previously injured and stented peripheral arteries of England swine.
• peripheral arterial sites were created for induction of neointimal response in each animal; one site was selected in each of four arteries (bilateral profunda and superficial femoral arteries). All target sites were injured on Day 0 to create the in-stent restenosis model, 10 days prior to the first dose of ENPP1-Fc or a vehicle only control, and 14 days before repeat injury.
• the four peripheral artery sites were mapped using quantitative vascular angiography (QVA) in order to select the treatment site and correctly sized balloon and stent.
• QVA quantitative vascular angiography
• the injury was created by overstretch of the artery with a standard angioplasty balloon catheter at a target 130% overstretch; three inflations were performed. Immediately following injury, a bare metal stent was deployed.
• Peripheral stents were self-expandable, targeting approximately a 120% overstretch.
• ENPP1-Fc treatment occurred systemically starting on Day 10 and was dosed every 4 days subcutaneously until termination.
• all vessels were assessed by angiography and Optical Coherence Tomography (OCT).
• OCT Optical Coherence Tomography
• the previously injured and stented artery sites were subjected to a re-injury event consisting of overstretch of the artery with a single inflation of a standard angioplasty balloon catheter at the same pressure/diameter as the original pre-stent injury was done (130% of the baseline reference diameter).
• final post-procedural angiography and OCT were also recorded for select peripheral sites.
• arteries underwent repeat imaging with angiography and endovascular imaging (OCT).
• OCT endovascular imaging
• the treated peripheral segments were explanted and stored in 10% neutral buffered formalin.
• angiography revealed a pronounced narrowing of the profunda at day 42 relative to the morphology of the vessel at day 14 in animals given the vehicle control.
• OCT endovascular imaging
• Example 2 Efficacy of ENPP3-Fc Fusion Protein in In-Stent Restenosis Model The efficacy of an ENPP3-Fc fusion protein is evaluated in large animal model of peripheral vascular injury – specifically, in-stent restenosis lesions in the peripheral vasculature of domestic (Yorkshire) swine.
• Therapeutic effects of the ENPP3-Fc fusion protein are assessed with respect to the ability to inhibit stenosis after angioplasty in previously injured and stented peripheral arteries of Yorkshire swine.
• Four peripheral arterial sites are created for induction of neointimal response in each animal as described in Example 1. All target sites are injured on Day 0 to create the in-stent restenosis model, 10 days prior to the first dose of ENPP3-Fc or a vehicle only control, and 14 days before repeat injury.
• the four peripheral artery sites are mapped using quantitative vascular angiography (QVA) in order to select the treatment site and correctly sized balloon and stent.
• QVA quantitative vascular angiography
• the injury is created by overstretch of the artery with a standard angioplasty balloon catheter at a target 130% overstretch; three inflations are performed. Immediately following injury, a bare metal stent is deployed. Peripheral stents are self-expandable, targeting approximately a 120% overstretch.
• ENPP3-Fc treatment is initiated systemically starting on Day 10 and is dosed every 4 days subcutaneously until termination. On Day 14, all vessels are assessed by angiography and Optical Coherence Tomography (OCT).
• a re-injury event consisting of overstretch of the artery with a single inflation of a standard angioplasty balloon catheter at the same pressure/diameter as the original pre-stent injury (130% of the baseline reference diameter).
• final post-procedural angiography and OCT are recorded for select peripheral sites.
• arteries underwent repeat imaging with angiography and endovascular imaging (OCT). The treated peripheral segments are explanted and stored in 10% neutral buffered formalin.
• ENPP3-Fc would exhibit lower % area of stenosis as compared to the vehicle control group. It is expected that ENPP3 polypeptides would be useful for, among other things, inhibiting the intimal thickening associated with injury of and/or to peripheral arteries.
• ENPP1 polypeptides ENPP1 or ENPP1-Fc or ENPP1- Albumin
• ENPP1 polypeptides ENPP1 or ENPP1-Fc or ENPP1-Ablumin
• This therapy is based on the delivery of ENPP1 mRNA (or ENPP1-Fc mRNA or ENPP1-Albumin mRNA) to the endothelial cells, which then in turn express the ENPP1 protein at the site of the stent implant after mRNA translation.
• the purified mRNA is dephosphorylated using the Antarctic Phosphatase Kit (New England Biolabs) and once again purified with the RNeasy Kit (Qiagen). The same procedure is repeated to generate enhanced green fluorescent protein (eGFP) mRNA using eGFP DNA.
• eGFP enhanced green fluorescent protein
• ENPP1 mRNA transfected HEK293 cells are incubated with 20 ⁇ M ATP (möLab, Langenfeld, Germany) or PBS as control for 10 min at 37°C on a shaking platform (Polymax 1040, Heidolph, Schwabach, Germany).
• the ATP substrate degrades over time in the presence of ENPP1, with the accumulation of the enzymatic product AMP.
• the initial rate velocities for ENPP1 are derived in the presence of ATP, and the data is fit to a curve to derive the enzymatic rate constants.
• the generated ENPP1 mRNA is first coated on thermanox plastic slides.
• the stent coating is thus simulated using thermanox plastic slides (Nunc, Thermo scientific, USA).
• 100.000 HEK293 cells per well are seeded on a 12-well plate.
• 2 ⁇ l Lipofectamin as well as 10 ⁇ g ENPP1 mRNA are mixed with 50 ⁇ l Opti-MEM and incubated at room temperature for 20 min.
• PLGA polylactic-co-glycolic-acid
• ethyl acetate final concentration 200 ⁇ g/ml
• 200 ⁇ l of the PLGA solution are mixed with the transfection complexes.
• the thermanox slides are coated with the solution in a step-by-step approach at room temperature.
• eGFP mRNA and sterilized water are used as controls.
• the HEK293 cells are supplied with a new medium before the dried slides are plated face down onto the cells.
• the cells are incubated with the slides at 37°C and 5% CO2 for 24 hrs, 48 hrs and 72 hrs and then analyzed using a FACScan cytometer.
• the expression of ENPP1of HEK293 cells is measured using flow cytometry.
• the ENPP1 coated thermonox slide exposed cells and control cells are stained with anti-ENPP1- fluorescein isothiocyanate (FITC) antibody.
• FITC anti-ENPP1- fluorescein isothiocyanate
• Flow cytometric analysis of the HEK293 cells after incubation with the ENPP1mRNA/PLGA covered thermanox slides are expected to show that the ENPP1 mRNA is released from the PLGA coating, whereby increase in ENPP1 expression is expected to be detectable after 24 hours, 48 hours and 72 hours post exposure to slides.
• 0.5-1 ⁇ g of the ENPP1 mRNA is expected to be sufficient to induce increase of the ENPP1 protein expression in HEK cells exposed to ENPP1 mRNA coated thermonox slides even after 24 hours of exposure.
• the ENPP1 expressed at the site of the stent implant is expected to prevent intimal proliferation and reduce platelet occlusion in peripheral arteries.
• EXAMPLE 4 Preparation and implantation of ENPP1 coated stent for the treatment of Atherosclerotic Peripheral Blood Vessels
• a juvenile pig animal model is used for implanting the ENPP1-coated stent to determine the efficacy of an ENPP1 coated stent to inhibit neointima formation, restenosis and inflammation.
• An ENPP1 agent coated stent is prepared and then implanted in peripheral artery.
• Four peripheral arterial sites are created for induction of neointimal response in each animal; one site was selected in each of four arteries (bilateral profunda and superficial femoral arteries) as described in Example 1.
• ENPP1 coated stent Any stent is amenable to be coated with ENPP1 agent.
• Common examples of commercial sources that sell stents for use include Abbot, Boston Scientific, Medtronic, Alvimedica, Lepu Medical Technology, Cordis, Balton or Biotronik.
• Peripheral arterial stents are shorter in length (12-18 mm) with a diameter range from 5-8 mm are commonly used for placement in iliac and femoral arteries. Henry et al. describes in detail the different types of stent lengths and diameters available for peripheral arteries (Henry et al., Tex Heart Inst J.
• a plain stent such as a bare metal stent can be converted to ENPP1 coated eluting stent by placing a polymeric film comprising ENPP1 mRNA inside the stent or by spraying a polymeric or nonpolymeric solution comprising ENPP1 mRNA or ENPP1polypeptide on to the stent surface.
• ENPP1 polymeric film Some examples of ENPP1 polymeric film are shown below, the ENPP1 polymeric film can be placed inside stents to create ENPP1 coated eluting stents.
• nonpolymeric carrier such as Vitamin E, Vitamin E acetate, Vitamin E succinate, oleic acid, peanut oil and cottonseed oil
• ENPP1 agent coating composition A) – 10 mg PCL (poly caprolactone) polymer and 100 ⁇ g ENPP1 mRNA (or ENPP1-Fc mRNA or ENPP1-Albumin mRNA) are dissolved in sterile double distilled water at room temperature. The solution is poured onto a glass plate and the solvent is allowed to evaporate for 12-24 hours.
• the ENPP1-loaded PCL film is removed from the glass plate and is cut to 1.5 cm by 1.5 cm size which is then further trimmed to fit the size of the stent.
• the ENPP1 mRNA (or ENPP1-Fc mRNA or ENPP1-Albumin mRNA) comprising polymeric film is then mounted on the stainless stent.
• the same process can be repeated for preparing a stent coated with a vector expressing ENPP1 polypeptide (ENPP1 or ENPP1-Fc or ENPP1-Albumin) by using 50 ⁇ g of vector DNA.
• ENPP1 agent coating composition (B) – 10 mg EVA (ethylene-vinyl acetate) polymer and 100 ⁇ g ENPP1 mRNA (or ENPP1-Fc mRNA or ENPP1-Albumin mRNA) are dissolved in sterile double distilled water at room temperature. The solution is poured onto a glass plate and the solvent is allowed to evaporate for 12-24 hours. After almost complete removal of the solvent, the ENPP1-mRNA (or ENPP1-Fc mRNA or ENPP1-Albumin mRNA) loaded EVA film is removed from the glass plate and was cut to 1.5 cm by 1.5 cm size.
• EVA ethylene-vinyl acetate
• ENPP1 mRNA (or ENPP1-Fc mRNA or ENPP1-Albumin mRNA) comprising polymeric film is then mounted on the stainless stent.
• the same process can be repeated for preparing a stent coated with a vector expressing ENPP1 polypeptide (ENPP1 or ENPP1-Fc or ENPP1-Albumin) by using 50 ⁇ g of vector DNA.
• ENPP1 comprising spray solutions are shown below, the spray solutions can be applied onto stents to create ENPP1 coated eluting stents.
• nonpolymeric carrier such as Vitamin E, Vitamin E acetate, Vitamin E succinate, oleic acid, peanut oil and cottonseed oil can be added to the spray solution improve the stability of ENPP1 agent.
• ENPP1 agent coating composition C)- 10 mg PCL (poly caprolactone) polymer and 100 ⁇ g ENPP1 mRNA is dissolved in sterile double distilled water at room temperature.100 ⁇ l polymeric PCL solution comprising the ENPP1 mRNA (or ENPP1-Fc mRNA or ENPP1-Albumin mRNA) is sprayed onto a stent (6 mmx20 mm) using a semi-automated nebulizer apparatus.
• the nebulizer spray system provides means of rotating and traversing the length of the stent at a controlled rate.
• the traversing component of the apparatus contained a glass nebulizer system that applies nebulized polycaprolactone solution to the stent at a rate of 3 ml per minute. Once applied, the polymer coating is "reflowed" by application of 60° C heated air for approximately 5 seconds. The process of reflowing the polymer provides better adherence to the stent surface. The same process can be repeated for preparing a stent coated with a vector expressing ENPP1 polypeptide (ENPP1 or ENPP1-Fc or ENPP1-Albumin) by using 50 ⁇ g of vector DNA.
• ENPP1 polypeptide ENPP1 or ENPP1-Fc or ENPP1-Albumin
• ENPP1 agent coating composition (D)-A 1% solution of uncured two-part silicone rubber is dissolved in trichloroethylene and then sprayed on to the stent using a nebulizer spray system as described above in (C).
• the coated stent is dried at room temperature for 15 minutes to allow the trichloroethylene to evaporate.
• the coated stent comprising silicone is heated in a vacuum oven for a period of four hours in order to crosslink the silicone coating.
• the coated stents are removed from the oven and allowed to cool for a period of 1 hour.100 ⁇ g ENPP1 mRNA is dissolved in sterile double distilled water at room temperature.
• a volume of 100 ⁇ 1 of ENPP1 comprising spray solution is applied to the silicone coating of each stent in dropwise fashion.
• the crosslinked silicone absorbs the solution, where the solvent subsequently evaporates at room temperature, leaving behind the ENPP1 mRNA (or ENPP1-Fc mRNA or ENPP1-Albumin mRNA) entrapped within the silicone.
• ENPP1 mRNA or ENPP1-Fc mRNA or ENPP1-Albumin mRNA
• ENPP1-Albumin mRNA ENPP1-Albumin
• ENPP1 agent coating composition E-10 mg PCL (poly caprolactone) polymer and ENPP1 polypeptide (any one of ENPP1 or ENPP1-Fc or ENPP1-albumin) is dissolved in sterile double distilled water at room temperature to reach an ENPP1 polypeptide concentration of 10 mg/ml.
• 100 ⁇ l polymeric PCL solution comprising the ENPP1 polypeptide (10 mg/ml) is sprayed onto a stent as described in (C)
• ENPP1 agent coating composition F)- The coated stent comprising silicone are prepared as discussed in (d). The coated stents are removed from the oven and allowed to cool for a period of 1 hour.
• ENPP1 polypeptide (ENPP1 or ENPP1-Fc or ENPP1-Albumin) is dissolved in a sterile double distilled water at room temperature to reach an ENPP1 polypeptide concentration of 10 mg/ml.
• a volume of 100 ⁇ l of ENPP1 comprising spray solution (10 mg/ml) is applied to the silicone coating of each stent in dropwise fashion.
• the crosslinked silicone absorbs the solution, where the solvent subsequently evaporates at room temperature, leaving behind the ENPP1 mRNA entrapped within the silicone.
• Animal Model Thirty 4-to-5-month-old juvenile pigs with the weight of 25–35 kg are procured from commercial sources.
• Thirty stainless steel vents are obtained from one or more commercial sources such as Abbot, Boston Scientific, Medtronic, Alvimedica, Lepu Medical Technology, Cordis, Balton or Biotronik. Thirty stainless steel stents thus obtained are coated with ENPP1 mRNA following the protocol shown above for coating. Thirty bare metal stents (BMSs) are obtained from Abbott to be used as control set. The ENPP1 coated stent is then sterilized using ethylene oxide, compressed, and mounted on a balloon angioplasty catheter. It is then deployed at a site in an artery using standard balloon angioplasty techniques. Same is done for the control set using bare metal stents. All target sites are injured on Day 0 to create the in-stent restenosis model.
• BMSs bare metal stents
• the four peripheral artery sites are mapped using quantitative vascular angiography (QVA) as described in Example.
• QVA quantitative vascular angiography
• the injury is created as described in Example 1.
• the stents are randomly assigned and placed in the peripheral (bilateral profunda and superficial femoral) arteries (one stent per artery) of 30 pigs, one coated stent per pig. The pigs are then maintained on 100 mg aspirin per day. On Day 14, all vessels are assessed by angiography and Optical Coherence Tomography (OCT).
• OCT Optical Coherence Tomography
• a re-injury event consisting of overstretch of the artery with a single inflation of a standard angioplasty balloon catheter at the same pressure/diameter as the original pre-stent injury was done (130% of the baseline reference diameter).
• final post-procedural angiography and OCT are recorded for select peripheral sites.
• arteries underwent repeat imaging with angiography and endovascular imaging (OCT). The treated peripheral segments are explanted and stored in 10% neutral buffered formalin.
• Lumen area, Stent area, Neointimal thickness, Neointimal area and % of Stenosis is calculated for pigs with ENPP1 coated stents and pigs with bare metal stents. It is expected that the profunda arteries of animals treated with ENPP1-Fc coated stents would have a higher lumen area at compared to the vehicle control group treated with bare metal stents. The stent area is expected to be similar between both groups. Neointimal thickness and neointimal area are expected to be reduced in animals treated with ENPP1-Fc coated stents relative to the vehicle control animals with bare metal stents.
• EXAMPLE 5 Preparation and implantation of ENPP3 coated stent for the treatment of Atherosclerotic Peripheral Blood Vessels
• a juvenile pig animal model is used for implanting the ENPP3-coated stent to determine the efficacy of an ENPP3 coated stent to inhibit neointima formation, restenosis and inflammation.
• An ENPP3 agent coated stent is prepared and then implanted in peripheral artery.
• Four peripheral arterial sites are created for induction of neointimal response in each animal; one site was selected in each of four arteries (bilateral profunda and superficial femoral arteries) as described in Example 1.
• Preparation of ENPP3 coated stent Any stent is amenable to be coated with ENPP3 agent.
• Common examples of commercial sources that sell stents for use include Abbot, Boston Scientific, Medtronic, Alvimedica, Lepu Medical Technology, Cordis, Balton or Biotronik.
• Peripheral arterial stents are shorter in length (12-18 mm) with a diameter range from 5-8 mm are commonly used for placement in iliac and femoral arteries.
• a plain stent such as a bare metal stent can be converted to ENPP3 coated eluting stent by placing a polymeric film comprising ENPP3 mRNA inside the stent or by spraying a polymeric or nonpolymeric solution comprising ENPP3 mRNA or ENPP3polypeptide on to the stent surface.
• ENPP3 polymeric film Some examples of ENPP3 polymeric film are shown below, the ENPP3 polymeric film can be placed inside stents to create ENPP3 coated eluting stents.
• nonpolymeric carrier such as Vitamin E, Vitamin E acetate, Vitamin E succinate, oleic acid, peanut oil and cottonseed oil
• nonpolymeric carrier such as Vitamin E, Vitamin E acetate, Vitamin E succinate, oleic acid, peanut oil and cottonseed oil
• ENPP3 agent coating composition A) – 10 mg PCL (poly caprolactone) polymer and 100 ⁇ g ENPP1 mRNA (or ENPP3-Fc mRNA or ENPP3-Albumin mRNA) are dissolved in sterile double distilled water at room temperature. The solution is poured onto a glass plate and the solvent is allowed to evaporate for 12-24 hours.
• the ENPP3-loaded PCL film is removed from the glass plate and is cut to 1.5 cm by 1.5 cm size which is then further trimmed to fit the size of the stent.
• the ENPP3 mRNA (or ENPP3-Fc mRNA or ENPP3-Albumin mRNA) comprising polymeric film is then mounted on the stainless stent.
• the same process can be repeated for preparing a stent coated with a vector expressing ENPP1 polypeptide (ENPP3 or ENPP3-Fc or ENPP3-Albumin) by using 50 ⁇ g of vector DNA.
• ENPP3 agent coating composition B) – 10 mg EVA (ethylene-vinyl acetate) polymer and 100 ⁇ g ENPP3 mRNA (or ENPP3-Fc mRNA or ENPP3-Albumin mRNA) are dissolved in sterile double distilled water at room temperature. The solution is poured onto a glass plate and the solvent is allowed to evaporate for 12-24 hours. After almost complete removal of the solvent, the ENPP3-mRNA (or ENPP3-Fc mRNA or ENPP3-Albumin mRNA) loaded EVA film is removed from the glass plate and was cut to 1.5 cm by 1.5 cm size.
• EVA ethylene-vinyl acetate
• ENPP3 mRNA (or ENPP3-Fc mRNA or ENPP3-Albumin mRNA) comprising polymeric film is then mounted on the stainless stent.
• the same process can be repeated for preparing a stent coated with a vector expressing ENPP3 polypeptide (ENPP3 or ENPP3-Fc or ENPP3-Albumin) by using 50 ⁇ g of vector DNA.
• ENPP3 comprising spray solutions are shown below, the spray solutions can be applied onto stents to create ENPP3 coated eluting stents.
• nonpolymeric carrier such as Vitamin E, Vitamin E acetate, Vitamin E succinate, oleic acid, peanut oil and cottonseed oil can be added to the spray solution improve the stability of ENPP3 agent.
• ENPP3 agent coating composition C)- 10 mg PCL (poly caprolactone) polymer and 100 ⁇ g ENPP3 mRNA is dissolved in sterile double distilled water at room temperature.100 ⁇ l polymeric PCL solution comprising the ENPP3 mRNA (or ENPP3-Fc mRNA or ENPP3-Albumin mRNA) is sprayed onto a stent (6 mmx20 mm) using a semi-automated nebulizer apparatus.
• PCL poly caprolactone
• the nebulizer spray system provides means of rotating and traversing the length of the stent at a controlled rate.
• the traversing component of the apparatus contained a glass nebulizer system that applies nebulized polycaprolactone solution to the stent at a rate of 3 ml per minute. Once applied, the polymer coating is "reflowed" by application of 60° C heated air for approximately 5 seconds. The process of reflowing the polymer provides better adherence to the stent surface. The same process can be repeated for preparing a stent coated with a vector expressing ENPP3 polypeptide (ENPP3 or ENPP3-Fc or ENPP3-Albumin) by using 50 ⁇ g of vector DNA.
• ENPP3 polypeptide ENPP3 or ENPP3-Fc or ENPP3-Albumin
• ENPP3 agent coating composition (D)-A 1% solution of uncured two-part silicone rubber is dissolved in trichloroethylene and then sprayed on to the stent using a nebulizer spray system as described above in (C).
• the coated stent is dried at room temperature for 15 minutes to allow the trichloroethylene to evaporate.
• the coated stent comprising silicone is heated in a vacuum oven for a period of four hours in order to crosslink the silicone coating.
• the coated stents are removed from the oven and allowed to cool for a period of 1 hour.100 ⁇ g ENPP3 mRNA is dissolved in sterile double distilled water at room temperature.
• a volume of 100 ⁇ 1 of ENPP3 comprising spray solution is applied to the silicone coating of each stent in dropwise fashion.
• the crosslinked silicone absorbs the solution, where the solvent subsequently evaporates at room temperature, leaving behind the ENPP3 mRNA (or ENPP3-Fc mRNA or ENPP3-Albumin mRNA) entrapped within the silicone.
• ENPP3 mRNA or ENPP3-Fc mRNA or ENPP3-Albumin mRNA
• ENPP3-Albumin mRNA ENPP3-Albumin
• ENPP3 agent coating composition E-10 mg PCL (poly caprolactone) polymer and ENPP3 polypeptide (any one of ENPP3 or ENPP3-Fc or ENPP3-albumin) is dissolved in sterile double distilled water at room temperature to reach an ENPP3 polypeptide concentration of 10 mg/ml.
• 100 ⁇ l polymeric PCL solution comprising the ENPP3 polypeptide (10 mg/ml) is sprayed onto a stent as described in (C)
• the coated stent comprising silicone are prepared as discussed in (d). The coated stents are removed from the oven and allowed to cool for a period of 1 hour.
• ENPP3 polypeptide (ENPP3 or ENPP3-Fc or ENPP3-Albumin) is dissolved in a sterile double distilled water at room temperature to reach an ENPP3 polypeptide concentration of 10 mg/ml.
• a volume of 100 ⁇ l of ENPP3 comprising spray solution (10 mg/ml) is applied to the silicone coating of each stent in dropwise fashion.
• the crosslinked silicone absorbs the solution, where the solvent subsequently evaporates at room temperature, leaving behind the ENPP3 mRNA entrapped within the silicone.
• Animal Model Thirty 4-to-5-month-old juvenile pigs with the weight of 25–35 kg are procured from commercial sources.
• Thirty stainless steel vents are obtained from one or more commercial sources such as Abbot, Boston Scientific, Medtronic, Alvimedica, Lepu Medical Technology, Cordis, Balton or Biotronik. Thirty stainless steel stents thus obtained are coated with ENPP1 mRNA following the protocol shown above for coating. Thirty bare metal stents (BMSs) are obtained from Abbott to be used as control set. The ENPP3 coated stent is then sterilized using ethylene oxide, compressed, and mounted on a balloon angioplasty catheter. It is then deployed at a site in an artery using standard balloon angioplasty techniques. Same is done for the control set using bare metal stents. All target sites are injured on Day 0 to create the in-stent restenosis model.
• BMSs bare metal stents
• the four peripheral artery sites are mapped using quantitative vascular angiography (QVA) as described in Example.
• QVA quantitative vascular angiography
• the injury is created as described in Example 1.
• the stents are randomly assigned and placed in the peripheral (bilateral profunda and superficial femoral) arteries (one stent per artery) of 30 pigs, one coated stent per pig. The pigs are then maintained on 100 mg aspirin per day. On Day 14, all vessels are assessed by angiography and Optical Coherence Tomography (OCT).
• OCT Optical Coherence Tomography
• a re-injury event consisting of overstretch of the artery with a single inflation of a standard angioplasty balloon catheter at the same pressure/diameter as the original pre-stent injury was done (130% of the baseline reference diameter).
• final post-procedural angiography and OCT are recorded for select peripheral sites.
• arteries underwent repeat imaging with angiography and endovascular imaging (OCT). The treated peripheral segments are explanted and stored in 10% neutral buffered formalin.
• Lumen area, Stent area, Neointimal thickness, Neointimal area and % of Stenosis is calculated for pigs with ENPP3 coated stents and pigs with bare metal stents. It is expected that the profunda arteries of animals treated with ENPP3-Fc coated stents would have a higher lumen area at compared to the vehicle control group treated with bare metal stents. The stent area is expected to be similar between both groups. Neointimal thickness and neointimal area are expected to be reduced in animals treated with ENPP3-Fc coated stents relative to the vehicle control animals with bare metal stents.
• animals treated with ENPP3-Fc are expected to have a markedly lower % area of stenosis as compared to the vehicle control group.
• in situ administration of ENPP3 agent by using ENPP3 coated stents is expected to prevent and effectively treat myointimal proliferation and/or restenosis at the site of injury in peripheral arteries.

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