Classifications

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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
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  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• • A—HUMAN NECESSITIES
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  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
• • A—HUMAN NECESSITIES
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  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
  • A61K47/6815—Enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
  • C12Y306/01—Hydrolases acting on acid anhydrides (3.6) in phosphorus-containing anhydrides (3.6.1)
  • C12Y306/01009—Nucleotide diphosphatase (3.6.1.9), i.e. nucleotide-pyrophosphatase
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/04—Phosphoric diester hydrolases (3.1.4)
  • C12Y301/04001—Phosphodiesterase I (3.1.4.1)

Definitions

• the disclosure relates to compositions and methods of treating vascular diseases.
• Said ASCII copy, created on July 2, 2021, is named 4427-10502_sequence_ST25.txt and is 343.976 bytes in size.
• Myointimal proliferation or myointimal hyperplasia is a complex pathological process of the vascular system characterized by an abnormal proliferation of smooth muscle cells of the vascular wall. Proliferating smooth muscle cells migrate to the subendothelial area and form the hyperplastic lesion, which can cause stenosis and obstruction of the vascular lumen.
• CAV Cardiac Allograft Vasculopathy
• CAV is often characterized by vascular smooth muscle cell proliferation, accumulation of inflammatory immune cells, and lipid deposition. CAV is a slow progressive disease but complications such as acute graft failure, arrhythmia, infarction, or cardiac death can often manifest without classic symptoms (such as angina) due to graft denervation.
• vasculopathy occurs in, and can severely limit long-term survival of, other solid organ allografts. Because such vasculopathies are difficult to treat, and can affect nearly all vessels of the allograft, they are associated with significant morbidity and mortality for allograft recipients and may require repeat transplantation. Therefore, effective therapies that may prevent or reduce the extent of such vasculopathies in solid organ allografts, such as cardiac allografts, are urgently needed.
• Moyamoya is an occlusive cerebrovascular disorder first reported in 1957 in Japan and is characterized by stenosis of the supraclinoid portion of the internal carotid arteries (ICA) with the formation of an abnormal vascular network at the base of the brain.
• Moyamoya is a general term used to describe two different conditions affecting the intracranial internal carotid artery; moyamoya disease (MMD), a congenital disease causing bilateral arteriopathy which is more prominent among East Asian and Japanese children and adults , and Moyamoya syndrome (MMS), which is idiopathic, and typically seen among Caucasian adults ranging in age from 20 to 40 years.
• MMD moyamoya disease
• MMS Moyamoya syndrome
• MMS myeloma
• MMD myeloma
• autoimmune disorders such as diabetes, lupus or rheumatoid arthritis.
• Treatment options for both MMD and MMS have involved daily aspirin use, lifestyle modifications to maximize cerebral perfusion, and surgical direct or indirect bypass to restore blood flow.
• MMD moyamoya disease
• MMD is prominent amongst the East Asian population presenting in both children and adults with a familial lineage.
• Moyamoya syndrome is prominent amongst Caucasians in the 2 nd /3 rd decades of life, is idiopathic, and usually presents with co-morbidities (autoimmune diseases) Clinical literature often does not distinguish between those with MMD and MMS.
• Chronic hemodialysis is a common treatment for patients suffering from poor kidney function. Such patients often undergo a surgical procedure in which an artificial arterio-venous fistula (AVF) is created usually in their non-dominant arm.
• AVF arterio-venous fistula
• AVF provides a durable vascular access point for the hemodialysis process.
• a common complication with AVF is the occlusion of the AVF or vessels at or adjacent to the location of the AVF.
• Such occlusion can involve, for example, thromboses and intimal hyperplasia, and can result in permanent nerve damage or paralysis of the affected limb, if left untreated (see, e.g., Asif et al. (2006) Clin J Am Soc Nephrol. 1:332-339; Nath et al. (2003) Am J Pathol. 162:2079-90; and Stolic (2013) Med Pric Pract. 22(3):220-228).
• the disclosure relates to a method for reducing and/or preventing allograft vasculopathy in a subject having an allograft, the method comprising: administering to the subject an effective amount of an ENPPl agent or ENPP3 agent to thereby reduce and/or prevent allograft vasculopathy in said subject.
• the disclosure relates to a method for preventing or ameliorating one or more symptoms associated with Moyamoya disease in a subject, the method comprising: administering to the subject an ENPPl agent or ENPP3 agent in an amount sufficient to thereby prevent or ameliorate one or more symptoms associated with Moyamoya disease in the subject.
• the disclosure relates to a method for inhibiting or preventing cerebral vascular occlusion in a subject who is expected to receive or who has received a surgical intervention as a treatment for Moyamoya disease, the method comprising: administering to the subject an ENPPl agent or ENPP3 agent in an amount sufficient to thereby inhibit or prevent cerebral vascular occlusion in the subject.
• the disclosure relates to a method for inhibiting or preventing unwanted vascular smooth muscle cell proliferation in a subject who is expected to receive or who has received a surgical intervention as a treatment for Moyamoya disease, the method comprising: administering to the subject an ENPPl agent or ENPP3 agent in an amount sufficient to thereby inhibit or prevent unwanted vascular smooth muscle cell proliferation in the subject.
• the disclosure also includes a method for inhibiting or slowing progression of Stage I Suzuki grade MMD to Stage II Suzuki grade MMD in a subject, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby inhibit and/or slow progression of Stage I MMD to Stage II MMD in said subject.
• the disclosure also includes a method for inhibiting or slowing progression of Stage I Suzuki grade MMD to Stage III Suzuki grade MMD in a subject, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby inhibit and/or slow progression of Stage I MMD to Stage III MMD in said subject.
• the disclosure relates to method for inhibiting or preventing cerebral vascular occlusion in a subject at risk for developing Moyamoya disease, the method comprising: administering to the subject an ENPP1 agent or ENPP3 agent in an amount sufficient to thereby inhibit or prevent cerebral vascular occlusion in the subject.
• the disclosure relates to a method for inhibiting or preventing unwanted vascular smooth muscle cell proliferation in a subject at risk for developing Moyamoya disease, the method comprising: administering to the subject an ENPP1 agent or ENPP3 agent in an amount sufficient to thereby inhibit or prevent unwanted vascular smooth muscle cell proliferation in the subject.
• the disclosure also relates to a method for treating a subject at risk for developing Moyamoya disease, the method comprising: administering to the subject an ENPP1 agent or ENPP3 agent in an amount sufficient to thereby treat the subject
• the disclosure relates to a method for inhibiting or preventing cerebral vascular occlusion in a subject afflicted with Moyamoya disease, the method comprising: administering to the subject an ENPP1 agent or ENPP3 agent in an amount sufficient to thereby inhibit or prevent cerebral vascular occlusion in the subject.
• the disclosure relates to a method for inhibiting or preventing unwanted vascular smooth muscle cell proliferation in a subject afflicted with Moyamoya disease, the method comprising: administering to the subject an ENPP1 agent or ENPP3 agent in an amount sufficient to thereby inhibit or prevent unwanted cerebral vascular smooth muscle cell proliferation in the subject.
• the disclosure relates to a method for treating a subject afflicted with Moyamoya disease, the method comprising: administering to the subject an ENPP1 agent or ENPP3 agent in an amount sufficient to thereby treat the subject.
• the disclosure relates to a method for treating a subject having Moyamoya disease, the method comprising: administering to the subject an effective amount of an ENPP1 agent or ENPP3 agent to thereby treat said Moyamoya disease in said subject.
• the disclosure relates to a method for treating a subject having Moyamoya syndrome, the method comprising: administering to the subject an effective amount of an ENPP1 agent or ENPP3 agent to thereby treat said Moyamoya syndrome in said subject.
• the disclosure includes a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a cerebral artery of a subject, the method comprising: administering to the subject an effective amount of an ENPP1 agent or ENPP3 agent to thereby reduce and/or prevent progression of said vascular smooth muscle cell proliferation in said cerebral artery of said subject .
• the subject has stage I, stage II or stage III, grade IV Suzuki grade MMD.
• the disclosure also includes a method for inhibiting or slowing progression of Stage I Suzuki grade MMD to Stage II Suzuki grade MMD in a subject, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby inhibit and/or slow progression of Stage I MMD to Stage II MMD in said subject.
• the disclosure features a method for treating a subject having Moyamoya disease, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby treat said peripheral artery disease in said subject.
• the disclosure relates to a method for treating a subject having Moyamoya syndrome, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby treat said Moyamoya syndrome in said subject.
• the disclosure features a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a cerebral artery of a subject having Moyamoya disease, the method comprising: administering to the subject an effective amount of an ENPP1 agent or ENPP3 agent to thereby reduce and/or prevent progression of said vascular smooth muscle cell proliferation in said cerebral artery of said subject.
• the disclosure also relates to a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a cerebral artery of a subject who undergoes surgery on said cerebral artery, the method comprising: administering to the subject an effective amount of an ENPP1 agent or ENPP3 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation in said cerebral artery at a surgical site of said cerebral artery in said subject.
• the disclosure also relates to a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a cerebral artery of a subject who undergoes surgery on said cerebral artery, the method comprising: administering to the subject an effective amount of an ENPP1 agent or ENPP3 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation in said cerebral artery at a surgical site of said cerebral artery in said subject.
• the disclosure features a method for treating a subject having Moyamoya disease, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby treat said peripheral artery disease in said subject.
• the disclosure relates to a method for treating a subject having Moyamoya syndrome, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby treat said Moyamoya syndrome in said subject.
• the disclosure features a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a cerebral artery of a subject having Moyamoya disease, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby reduce and/or prevent progression of said vascular smooth muscle cell proliferation in said cerebral artery of said subject.
• the disclosure also relates to a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a subject’s peripheral vessel at or around the site at which an arterio-venous dialysis shunt has been placed, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation in said peripheral vessel at or around the site the arterio-venous dialysis shunt has been placed.
• the disclosure provides a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a peripheral vessel of a subject who undergoes surgery on said peripheral vessel, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation in said peripheral vessel at a surgical site of said peripheral vessel in said subject, wherein the surgery comprises placement of an arterio-venous dialysis shunt.
• the disclosure relates to a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a peripheral vessel of a subject who requires surgery on said peripheral vessel, wherein the surgery comprises placement of an arterio-venous dialysis shunt, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation in said peripheral vessel at a surgical site of said peripheral vessel in said subject.
• the disclosure also includes a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a peripheral vessel of a subject who undergoes shunt placement in a peripheral vessel, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation in the peripheral vessel.
• the disclosure features a method for reducing and/or preventing stenosis or restenosis in a peripheral vessel of a subject who undergoes shunt placement in the peripheral vessel, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby reduce and/or prevent stenosis or restenosis in the peripheral vessel.
• the disclosure relates to a method for reducing and/or preventing vasculopathy of an allografted vessel in a subject, the method comprising: administering to the subject an effective amount of: (i) an ENPP1 agent or ENPP3 agent and (ii) a complement inhibitor to thereby reduce and/or prevent vasculopathy of the allografted vessel in said subject.
• the vessel is an artery.
• the vessel is a vein.
• the disclosure relates to a method for reducing and/or preventing vasculopathy of an allografted vessel in a subject, the method comprising: administering to the subject an effective amount of an ENPP1 agent or ENPP3 agent to thereby reduce and/or prevent vasculopathy of the allografted vessel in said subject.
• the vessel is an artery. In some embodiments, the vessel is a vein.
• the disclosure relates to a method for reducing and/or preventing vasculopathy of an allografted vessel in a subject, the method comprising: administering to the subject an effective amount of an ENPP1 agent or ENPP3 agent to thereby reduce and/or prevent vasculopathy of the allografted vessel in said subject.
• the vessel is an artery.
• the vessel is a vein.
• the subject has received or is receiving a therapy comprising a complement inhibitor.
• the methods comprise administering to the subject a complement inhibitor.
• the disclosure relates to a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in an allografted vessel in a subject, the method comprising: administering to the subject an effective amount of: (i) an ENPP1 agent or ENPP3 agent and (ii) a complement inhibitor to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation in the allografted vessel in said subject.
• the vessel is an artery. In some embodiments, the vessel is a vein.
• the disclosure relates to a method for reducing and/or preventing allograft vasculopathy (for example, cardiac allograft vasculopathy) in a subject having an allograft, the method comprising: administering to the subject an effective amount of: (i) an ENPP1 agent or ENPP3 agent and (ii) a complement inhibitor to thereby reduce and/or prevent allograft vasculopathy in said subject.
• allograft vasculopathy for example, cardiac allograft vasculopathy
• the disclosure relates to a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in an allografted vessel in a subject, the method comprising: administering to the subject an effective amount of an ENPP1 agent or ENPP3 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation in the allografted vessel in said subject.
• the vessel is an artery. In some embodiments, the vessel is a vein.
• the disclosure relates to a method for reducing and/or preventing allograft vasculopathy (for example, cardiac allograft vasculopathy) in a subject having an allograft and who has received or is receiving a therapy comprising a complement inhibitor, the method comprising: administering to the subject an effective amount of an ENPP1 agent or ENPP3 agent to thereby reduce and/or prevent allograft vasculopathy in said subject.
• the methods further comprise administering the complement inhibitor to the subject.
• the disclosure relates to a method for reducing and/or preventing allograft vasculopathy (for example, cardiac allograft vasculopathy) in a subject having an allograft and who has received or is receiving a therapy comprising an ENPP1 agent or ENPP3 agent, the method comprising: administering to the subject an effective amount of a complement inhibitor to thereby reduce and/or prevent allograft vasculopathy in said subject.
• the methods further comprise administering the ENPP1 agent or ENPP3 agent to the subject.
• the disclosure relates to a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in the vasculature of an allograft of a subject having said allograft, the method comprising administering to the subject an effective amount of: (i) an ENPP1 agent or an ENPP3 agent and (ii) a complement inhibitor to thereby reduce and/or prevent progression of said vascular smooth muscle cell proliferation in said vasculature of said allograft of said subject.
• the disclosure relates to a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in the vasculature of an allograft of a subject having said allograft, wherein the subject has received or is receiving a therapy comprising a complement inhibitor, the method comprising administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby reduce and/or prevent progression of said vascular smooth muscle cell proliferation in said vasculature of said allograft of said subject.
• the methods further comprise administering the complement inhibitor to the subject.
• the disclosure relates to a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in the vasculature of an allograft of a subject having said allograft, wherein the subject has received or is receiving a therapy comprising an ENPP1 agent or an ENPP3 agent, the method comprising administering to the subject an effective amount of a complement inhibitor to thereby reduce and/or prevent progression of said vascular smooth muscle cell proliferation in said vasculature of said allograft of said subject.
• the methods further comprise administering the ENPP1 agent or ENPP3 agent to the subject.
• the disclosure also relates to a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a solid organ transplant in a subject having a solid organ transplant and who undergoes surgery on said organ transplant, the method comprising administering to the subject an effective amount of: (i) an ENPP1 agent or an ENPP3 agent and (ii) a complement inhibitor to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation in said solid organ transplant of said subject.
• the disclosure also features a method for delaying or preventing or for prophylaxis against failure of an allografted vessel in a subject having said allografted vessel, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby delay, prevent or provide prophylaxis against failure of the allografted vessel in the subject.
• the subject has received or is receiving a therapy comprising a complement inhibitor.
• the methods comprise administering to the subject a complement inhibitor.
• the disclosure also features a method for delaying or preventing or for prophylaxis against failure of an allografted vessel in a subject having said allografted vessel, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby delay, prevent or provide prophylaxis against failure of the allografted vessel in the subject.
• the subject has received or is receiving a therapy comprising a complement inhibitor.
• the methods comprise administering to the subject a complement inhibitor.
• the disclosure also features a method for delaying solid organ allograft failure in a subject having said solid organ allograft, the method comprising: administering to the subject an effective amount of an: (i) ENPP1 agent or an ENPP3 agent and (ii) a complement inhibitor to thereby delay solid organ allograft failure in the subject.
• the allograft failure can be delayed for at least two months (e.g., at least six months, at least one year, at least two years, at least three years, at least five years, at least seven years, at least 10 years, or even more than 10 years).
• the disclosure also features a method for delaying failure of an allografted vessel in a subject having said allografted vessel, the method comprising: administering to the subject an effective amount of an: (i) ENPP1 agent or an ENPP3 agent and (ii) a complement inhibitor to thereby delay failure of the allografted vessel in the subject.
• the allograft failure can be delayed for at least two months (e.g., at least six months, at least one year, at least two years, at least three years, at least five years, at least seven years, at least 10 years, or even more than 10 years).
• the disclosure also features a method for delaying solid organ allograft failure in a subject having said solid organ allograft, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby delay solid organ allograft failure in the subject.
• the allograft failure can be delayed for at least two months (e.g., at least six months, at least one year, at least two years, at least three years, at least five years, at least seven years, at least 10 years, or even more than 10 years).
• the subject has received or is receiving a therapy comprising a complement inhibitor.
• the methods comprise administering to the subject a complement inhibitor.
• the disclosure relates to a method for reducing and/or preventing stenosis or restenosis in the vasculature of a solid organ allograft of a subject having a solid organ allograft, the method comprising: administering to the subject an effective amount of: (i) an ENPP1 agent or an ENPP3 agent and (ii) a complement inhibitor to thereby reduce and/or prevent stenosis or restenosis in said vasculature of said solid organ allograft.
• the disclosure relates to a method for reducing and/or preventing stenosis or restenosis in the vasculature of a solid organ allograft of a subject having a solid organ allograft, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent and a complement inhibitor to thereby reduce and/or prevent stenosis or restenosis in said vasculature of said solid organ allograft.
• the subject has received or is receiving a therapy comprising a complement inhibitor.
• the methods comprise administering to the subject a complement inhibitor.
• the disclosure also features a method for delaying or preventing or as prophylaxis against solid organ allograft rejection in a subject having said solid organ allograft, the method comprising: administering to the subject an effective amount of an: (i) ENPP1 agent or an ENPP3 agent and (ii) a complement inhibitor to thereby delay or prevent solid organ allograft rejection in the subject.
• the disclosure also features a method for delaying or preventing or as prophylaxis against solid organ allograft rejection in a subject having said solid organ allograft, wherein the subject is receiving or has received a therapy comprising an ENPP1 agent or an ENPP3 agent, the method comprising: administering to the subject an effective amount of a complement inhibitor to thereby delay or prevent solid organ allograft rejection in the subject.
• the method can also include administering to the subject the ENPP1 agent or ENPP3 agent.
• the disclosure also features a method for delaying or preventing or as prophylaxis against rejection of an allografted vessel in a subject having said allografted vessel, the method comprising: administering to the subject an effective amount of an: (i) ENPP1 agent or an ENPP3 agent and (ii) a complement inhibitor to thereby delay or prevent rejection of said vessel in the subject.
• the vessel is an artery. In some embodiments, the vessel is a vein.
• the disclosure also features a method for delaying or preventing or as prophylaxis against rejection of an allografted vessel in a subject having said allografted vessel, wherein the subject is receiving or has received a therapy comprising an ENPP1 agent or an ENPP3 agent, the method comprising: administering to the subject an effective amount of a complement inhibitor to thereby delay or prevent rejection of the allografted vessel in the subject.
• the method can also include administering to the subject the ENPP1 agent or ENPP3 agent.
• the vessel is an artery.
• the vessel is a vein.
• the disclosure relates to a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in the vasculature of an allograft of a subject having said allograft, 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 vasculature of said allograft of said subject.
• the disclosure relates to a method for reducing and/or preventing stenosis or restenosis in the vasculature of a solid organ allograft of a subject having a solid organ allograft , the method comprising: administering to the subject an effective amount of an ENPP1 agent or ENPP3 agent to thereby reduce and/or prevent stenosis or restenosis in said solid organ allograft
• the disclosure relates to a method for prolonging the survival of a solid organ allograft in a subject having a solid organ allograft, the method comprising administering to said subject an ENPP1 agent or ENPP3 agent in an amount sufficient to thereby prolong survival of said solid organ allograft in said subject
• the disclosure relates to a method for inhibiting or preventing vasculopathy in a solid organ allograft of a subject having a solid organ allograft, the method comprising administering to said subject an ENPP1 agent or ENPP3 agent in an amount sufficient to inhibit or prevent vasculopathy in the solid organ allograft.
• the disclosure relates to a method for inhibiting or preventing vasculopathy of an allografted blood vessel in a subject having a blood vessel allograft, the method comprising administering to a subject an ENPP1 agent or ENPP3 agent in an amount sufficient to prevent or inhibit vasculopathy of said allografted vessel.
• the disclosure relates to a method for inhibiting or preventing vascular smooth muscle cell proliferation in an allografted blood vessel in a subject having a blood vessel allograft, the method comprising administering to said subject an ENPP1 agent or ENPP3 agent in an amount sufficient to prevent or inhibit vascular smooth muscle cell proliferation in said allografted vessel
• the disclosure relates to a method for prolonging the survival of an allografted blood vessel in a subject having a blood vessel allograft, the method comprising administering to said subject an ENPP1 agent or ENPP3 agent in an amount sufficient to thereby prolong survival of said allografted blood vessel.
• the disclosure also relates to a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a solid organ transplant in a subject having a solid organ transplant and who undergoes surgery on said organ transplant, 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 solid organ transplant of said subject.
• the disclosure also features a method for preventing or for prophylaxis against solid organ allograft failure in a subject having said solid organ allograft, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby prevent or provide prophylaxis against solid organ allograft failure in the subject.
• the disclosure also features a method for delaying solid organ allograft failure in a subject having said solid organ allograft, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby delay solid organ allograft failure in the subject.
• the allograft failure can be delayed for at least two months (e.g., at least six months, at least one year, at least two years, at least three years, at least five years, at least seven years, at least 10 years, or even more than 10 years).
• the disclosure relates to a method for reducing and/or preventing vasculopathy of an allografted vessel in a subject, the method comprising: administering to the subject an effective amount of: (i) an ENPP1 agent or ENPP3 agent and (ii) a complement inhibitor to thereby reduce and/or prevent vasculopathy of the allografted vessel in said subject.
• the vessel is an artery. In some embodiments, the vessel is a vein.
• the disclosure relates to a method for reducing and/or preventing vasculopathy of an allografted vessel in a subject, the method comprising: administering to the subject an effective amount of an ENPP1 agent or ENPP3 agent to thereby reduce and/or prevent vasculopathy of the allografted vessel in said subject.
• the vessel is an artery. In some embodiments, the vessel is a vein.
• the disclosure relates to a method for reducing and/or preventing vasculopathy of an allografted vessel in a subject, the method comprising: administering to the subject an effective amount of an ENPP1 agent or ENPP3 agent to thereby reduce and/or prevent vasculopathy of the allografted vessel in said subject.
• the vessel is an artery.
• the vessel is a vein.
• the subject has received or is receiving a therapy comprising a complement inhibitor.
• the methods comprise administering to the subject a complement inhibitor.
• the disclosure relates to a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in an allografted vessel in a subject, the method comprising: administering to the subject an effective amount of an ENPP1 agent or ENPP3 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation in the allografted vessel in said subject.
• the vessel is an artery.
• the vessel is a vein.
• the subject has received or is receiving a therapy comprising a complement inhibitor.
• the methods comprise administering to the subject a complement inhibitor.
• the disclosure relates to a method for reducing and/or preventing allograft vasculopathy (for example, cardiac allograft vasculopathy) in a subject having an allograft, the method comprising: administering to the subject an effective amount of: (i) an ENPP1 agent or ENPP3 agent and (ii) a complement inhibitor to thereby reduce and/or prevent allograft vasculopathy in said subject.
• allograft vasculopathy for example, cardiac allograft vasculopathy
• the disclosure relates to a method for reducing and/or preventing allograft vasculopathy (for example, cardiac allograft vasculopathy) in a subject having an allograft and who has received or is receiving a therapy comprising a complement inhibitor, the method comprising: administering to the subject an effective amount of an ENPP1 agent or ENPP3 agent to thereby reduce and/or prevent allograft vasculopathy in said subject.
• the methods further comprise administering the complement inhibitor to the subject.
• the agent is administered prior to, during and/or after said surgery. In some embodiments of any of the methods described herein, the agent is administered prior to, during and/or after shunt placement.
• surgery and/or shunt placement further comprises introduction into the subject of a dialysis catheter.
• any of the methods described herein can comprise administering to the subject one or more of an anticoagulant, an antibiotic, and an antihypertensive.
• any of the methods described herein can comprise monitoring the subject for an occlusion of the shunt, such as a thrombosis.
• any of the methods described herein further include administering to the patient one or more immunosuppressants.
• the ENPP1 agent comprises ENPP1 variants that retain enzymatic activity.
• the ENPP3 agent comprises ENPP3 variants that retain enzymatic activity.
• the subject is one who is receiving or who has received one or more of an anticoagulant, an antibiotic, and an antihypertensive.
• the subject has received and/or is receiving an immunosuppressive therapy in conjunction with the solid organ allograft transplantation, such as one or more immunosuppressants.
• the subject has received and/or is receiving in conjunction with the solid organ allograft transplantation one or more of a statin drug, a vasodialator, an anticoagulant (e.g., aspirin), and an immunosuppressant.
• a statin drug e.g., aspirin
• a vasodialator e.g., aspirin
• an anticoagulant e.g., aspirin
• an immunosuppressant e.g., aspirin
• any of the methods described herein further include administering to the patient one or more of a statin drug, a vasodialator, an anticoagulant (e.g., aspirin), and an immunosuppressant. In some embodiments, any of the methods described herein further include performing revascularization surgery on the solid organ allograft.
• a statin drug e.g., aspirin
• an anticoagulant e.g., aspirin
• immunosuppressant e.g., aspirin
• any of the methods described herein further include performing revascularization surgery on the solid organ allograft.
• the subject is expected to undergo, has undergone, or is undergoing revascularization surgery on the solid organ allograft.
• the revascularization surgery comprises angioplasty, a bypass graft, and/or a stent placement.
• the agent is administered prior to, during and/or after said surgery.
• the surgery comprises balloon angioplasty and/or placement of a stent.
• the methods described herein further comprise performing the surgery.
• the ENPP1 agent comprises an ENPP1 polypeptide.
• 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 heterologous protein increases the circulating half-life of the ENPP1 polypeptide in mammal.
• the heterologous protein is an Fc region of an immunoglobulin molecule.
• the immunoglobulin molecule is an IgGl molecule.
• 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.
• 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 subject has stage I, stage II or stage III, Suzuki grade MMD.
• the disclosure features a method for inhibiting or slowing progression of Stage I Suzuki grade MMD peripheral artery disease to Stage III Suzuki grade MMD 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 I Suzuki grade MMD to Stage III Suzuki grade MMD in said subject.
• the disclosure features a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a cerebral artery of a subject who requires surgery on said cerebral artery, wherein the subject has Moyamoya disease, the method comprising: administering to the subject an effective amount of an ENPP1 agent or an ENPP3 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation in said cerebral artery at a surgical site of said cerebral artery in said subject.
• the cerebral artery is one or more of an external carotid artery (EC A), an internal carotid artery (ICA), a middle cerebral artery (MCA) and an anterior cerebral artery (ACA).
• EC A external carotid artery
• ICA internal carotid artery
• MCA middle cerebral artery
• ACA anterior cerebral artery
• the ENPP3 agent is administered prior to, during and/or after stent placement.
• the solid organ allograft is a cardiac allograft.
• the solid organ allograft is a lung allograft, a liver allograft, or a kidney allograft.
• the complement inhibitor is a complement component C5 inhibitor, such as an anti-C5 antibody, e.g., eculizumab or ravulizumab-cwvz.
• the complement inhibitor is an inhibitor of complement component Cl (including Cls and Clq), C2, C3, C4, C5, C6, C7, C8, and/or C9, such as an antibody that binds to and inhibits the function of any one of such complement components.
• complement component Cl including Cls and Clq
• C2, C3, C4, C5, C6, C7, C8, and/or C9 such as an antibody that binds to and inhibits the function of any one of such complement components.
• the complement inhibitor is compstatin or an analog thereof.
• the complement inhibitor is a C5a inhibitor, a C5aR inhibitor, a C3 inhibitor, a Factor D inhibitor, a Factor B inhibitor, a C4 inhibitor, a Clq inhibitor, a Cls inhibitor, or any combination thereof.
• the complement inhibitor is a lectin pathway inhibitor, such as an anti-MASP2 antibody (e.g., OMS721).
• the disclosure relates to a method for reducing and/or preventing stenosis or restenosis in the vasculature of a solid organ allograft of a subject having a solid organ allograft, 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 vasculature of said solid organ allograft.
• the disclosure relates to a method for reducing and/or preventing vasculopathy of an allograft in a subject having allograft vasculopathy, the method comprising administering to the subject an effective amount of an ENPP3 agent to thereby treat said allograft vasculopathy in said subject.
• the disclosure relates to a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in the vasculature of an allograft of a subject having said allograft, 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 vasculature of said allograft of said subject.
• the disclosure also relates to a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a solid organ transplant in a subject having a solid organ transplant and who undergoes surgery on said organ transplant, 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 solid organ transplant of said subject.
• the agent is administered prior to, during and/or after said surgery.
• the surgery comprises balloon angioplasty and/ or placement of a stent.
• the subject does not have a deficiency of ENPP1.
• the ENPP3 agent comprises an ENPP3 polypeptide.
• the ENPP3 agent comprises a nucleic acid encoding an ENPP3 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 to 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 IgGl 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.
• the subject has cerebral arterial occlusions.
• the disclosure relates to a method for reducing and/or preventing stenosis or restenosis in the vasculature of a solid organ allograft of a subject having a solid organ allograft , 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 solid organ allograft.
• Figure 1 shows the schematic diagram of prophylactic treatment regimen of control and experimental mice prior and after transplant.
• the experimental mice are treated 7 days prior to aortic transplantation with ENPPl-Fc at an exemplary dosage of lOmg/kg weight by subcutaneous injection every day.
• the control cohorts are injected with vehicle containing tris buffered saline, at pH 7.4. All mice are then dissected at 28 days after transplantation and the mice are approximately 10 weeks of age.
• Figure 2 shows a schematic diagram of heart transplant in mouse. It also shows morphometrical measurements of 5 pm sections of the transplanted aorta. The medial area, the intimal area and the intima/media ratio (EM ratio) of each section are calculated.
• Figure 3 shows a schematic version of Porcine model of heterotopic heart transplantation.
• 3 (A) shows the donor heart is harvested after cardiac standstill achieved by using cold cardioplegic solution (Plegisol).
• 3(B) shows that the graft is maintained in the ice- saline slurry and prepared for implantation by creating an atrial septal defect and defunction the mitral valve to minimize left ventricular atrophy and intracavity thrombus formation.
• 3(C) shows the recipient’s inferior vena cava (IVC) and the infrarenal aorta were isolated.
• 3(D) shows the graft heart is implanted by anastomosing the donor pulmonary artery to the recipient’s IVC and the donor ascending aorta to the abdominal aorta of the recipient. Graft function was monitored by using (E) electrocardiography (ECG) and (F) echocardiography (UCG). Arrows indicate electrical spikes attributed to heterotopic cardiac allograft. ( Hsu et al, Transplantation. 2018 Dec; 102(12): 2002-2011.)
• Figure 4 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 5 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.
• OCT Optical Coherence Tomography
• 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
• in animals treated with ENPPl-Fc little visible intimal thickening was observed between day 14 and day 42. The extent of stenosis is highlighted in the day 42 photographs.
• Figure 6 is a bar graph depicting the percent area of stenosis at day 14 and day 42 in the profunda of pigs treated with ENPPl-Fc (Treatment) or given vehicle control (Control), as measured by OCT.
• Figure 7 shows the schematic diagram of prophylactic treatment regimen of control and experimental mice prior and after brain surgery to induce MMD.
• the experimental mice are treated 7 days prior to surgery with ENPPl-Fc at an exemplary dosage of lOmg/kg weight by subcutaneous injection every day.
• the control cohorts are injected with vehicle containing tris buffered saline, at pH 7.4. All mice are then dissected at 28 days after transplantation and the mice are approximately 10 weeks of age.
• FIG. 8 shows the process of creating MMD model by Internal Carotid Artery Stenosis.
• 8A shows orientation of the mouse during the surgical procedure. Head (teeth), forepaws and tail are restrained, and incision is made in the midline of the neck (red dashed line).
• White box indicates region of images that follow.
• 8B) shows opening of the cervical region exposing the trachea, sternocleidomastoid (SCM) muscle and posterior belly of the digastric (PBD) muscle.
• 8C shows suture (SI -2) placement retracting the SCM and PBD to expose the common, internal and external carotid (CCA, ICA, ECA) arteries.
• SI -2 suture
• 8D shows Identification of the occipital artery (OA), vagus nerve (VN) and ICA.
• 8E) shows suture ligation of the OA and dashed line showing cut to better expose the ICA.
• 8F shows cut OA with ICA exposed and isolated using 6 ⁇ 0 suture.
• 8G shows micro-coil placement on ICA deep to ECA (as seen in H).
• Figure 9 is a diagram of hemodialysis blood flow from a subject’s arm, which contains a dialysis shunt, into a tube, past a pressure monitor, a blood pump, and a heparin pump, which prevents clotting. Blood flows past another pressure monitor before entering the dialyzer, or filter. Filtered blood continues past a venous pressure monitor, an air trap and air detector, and an air detector clamp, and returns to the subject’s arm.
• FIG 10 is a view of an implantable shunt 2 positioned in the upper right chest area 100 of a subject.
• the implantable dialysis shunt 2 may also be implanted into other areas of the body, so long as it is implanted in reasonable proximity to a medium sized artery, typically between 6 and 8 mm, for use with the implantable dialysis shunt 2.
• the implantable dialysis shunt preferably comprises an arterial port 4 and a venous port 6 connected to each other in a single structure.
• the ports 4, 6 may be separate structures which may include features to permit their attachment to each other.
• An arterial graft 12 generally extends through the arterial port 4 while a venous graft 18 extends from the venous port 6.
• the arterial graft 12 is preferably connected at each of its ends to the sidewall of an artery 26 while the end of the venous graft 18 is connected to a vein 34.
• the arterial graft 12 may be connected to the artery 26 by a pair of end-to-end anasomoses.
• the venous graft 18 may take the form of a venous catheter which is inserted into the vein 34 such that it may enter the central venous system.
• Dialysis may be conducted by tapping the arterial port 4 with an arterial catheter 102 and the venous port with a venous catheter 104. Each of the arterial and venous catheters 102, 104 are connected to a dialysis machine.
• 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 ah, 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 ah, WO/2017/087936- Braddock et al., and W02018/027024-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 NPPl are determined using ATP as a substrate.
• NPPl 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 NPPl, with the accumulation of the enzymatic product AMP.
• the initial rate velocities for NPPl are derived in the presence of ATP, and the data is fit to a curve to derive the enzymatic rate constants.
• ENPP1 precursor protein refers to ENPPl with its signal peptide sequence at the ENPPl N-terminus. Upon proteolysis, the signal sequence is cleaved from ENPPl to provide the ENPPl protein.
• Signal peptide sequences useful within the disclosure include, but are not limited to, Albumin signal sequence, Azurocidin signal sequence, ENPPl 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. Upon proteolysis, 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, ENPPl 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.
• ENPPl-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).
• the C-terminus of ENPPl 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).
• IgG molecule preferably, a human IgG
• the C-terminus of ENPPl is fused or conjugated to the N-terminus of the FcR binding domain.
• the term “Ac” refers to a human IgG (immunoglobulin) Fc domain. Subtypes of IgG such as IgGl, IgG2, IgG3, and IgG4 are contemplated for use as Fc domains.
• the “Ac 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 IgGl, according to the Rabat 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 CHI, 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.
• Particularly, functionally equivalent variants of ENPP1 or ENPP3 delivered by viral vectors is encompassed by the present disclosure.
• the functionally equivalent variants of ENPP1 orENPP3 are polypeptides substantially homologous to the native ENPP1 or ENPP3 respectively.
• the expression “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 ah, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et ah, J. Mol. Biol. 215: 403-410 (1990)), though other similar algorithms can also be used.
• 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 ENPPl or ENPP3 protein with respect to ATP cleavage.
• ENPPl fragment refers to a fragment or a portion of ENPPl protein or an active subsequence of the full-length NPP1 having at least an ENPPl catalytic domain administered in protein form or in the form of a nucleic acid encoding the same.
• ⁇ NRR1 age/iG 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.
• Ppi plasma pyrophosphate
• ATP adenosine triphosphate
• 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 polynucleo
• 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.
• “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of + 20% or + 10%, more preferably + 5%, even more preferably + 1%, and still more preferably + 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
• the term “ moiety ” refers to a chemical component or biological molecule that can be covalently or non-covalently linked to ENPPl 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 ENPPl 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).
• PEG polyethylene glycol
• HAP homo-amino acid polymer
• PAS proline- alanine-serine polymer
• ELP elastin-like peptide
• GLK gelatin-like protein
• the term “ subject ⁇ “ individuar or “ patienf” 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
• Such a patient will have a normal level of NPP1 in serum which 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 mM.
• plasma pyrophosphate (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 lpm to about 3 pM, in some cases between 1-2 pm.
• 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 refers to inorganic pyrophosphate.
• a “tow level ofPPf 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 mM. +/- 0.1 mM (Arthritis and Rheumatism, Vol. 22, No. 8 (August 1979))
• 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.
• MI myocardial infarction
• 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.
• MMD myelogenous leukemia
• moyamoya syndrome refers to a steno-occlusive disease of the cerebral arteries, involving smooth muscle cell proliferation with intima hyperplasia causing arterial stenosis and occlusion around the circle of Willis. It involves development of new blood vessels resembling a “puff of smoke” (“moyamoya”) in the subcortical region. MMD occurs in children and adults with two peaks- at around age 5- 10 and a second peak between the third and fifth decade of life.
• MMD cases are carriers of RNF213 and or R4810K mutations.
• Treatment options for both MMD and MMS involve daily aspirin use, lifestyle modifications to maximize cerebral perfusion, and surgical direct or indirect bypass to restore blood flow.
• Diagnostic criteria for definitive MMD were revised to include patients with both bilateral and unilateral presentation of terminal carotid artery stenosis (ICA) with an abnormal vascular network at the base of the brain.
• ICA terminal carotid artery stenosis
• Suzuki system of grading the patient population has been used for MMD.
• Definitive diagnosis of MMD requires catheter angiography in unilateral cases, whereas bilateral cases can be promptly diagnosed by either catheter angiography or magnetic resonance imaging/angiography (MRI/MRA).
• MRI/MRA magnetic resonance imaging/angiography
• Cerebral vascular occlusion refers to the temporary or permanent blockage of blood vessels in the brain. Restrictions in blood flow may occur from vessel narrowing (stenosis), clot formation (thrombosis), blockage (embolism) or blood vessel rupture (hemorrhage). Lack of sufficient blood flow (ischemia) affects brain tissue and may cause a stroke.
• Suzuki classification System refers to classification system developed by Suzuki et al. (Suzuki J, Takaku A. Cerebrovascular "moyamoya” disease. Disease showing abnormal net-like vessels in base of brain. Arch Neurol. 1969; 20(3):288 ⁇ 99.). This classification system grades the clinical presentation of patients to four stages. The vast majority of patients will progress through some or all of the Suzuki stages, although progression may occur at different rates, and appears to occur more rapidly in children than in adolescents or adults. The system is solely based on conventional angiography and is as shown in table below.
• ICA internal carotid artery
• ECA external carotid artery
• ACA anterior Cerebral Artery
• MCA medial cerebral artery
• the MCA arises from the internal carotid and continues into the lateral sulcus where it then branches and projects to many parts of the lateral cerebral cortex. It also supplies blood to the anterior temporal lobes and the insular cortices.
• conventional angiography refers to Angiography or arteriography is a medical imaging technique used to visualize the inside, or lumen, of blood vessels and organs of the body, with particular interest in the arteries, veins, and the heart chambers. This is traditionally done by injecting a radio-opaque contrast agent into the blood vessel and imaging using X-ray based techniques such as fluoroscopy.
• catheter angiography refers to a medical procedure wherein a catheter, x-ray imaging guidance and an injection of contrast material to examine blood vessels in key areas of the body such as brain or heart for abnormalities such as aneurysms and disease such as atherosclerosis (plaque).
• MRA magnetic resonance angiography
• 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.
• 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.
• stenosis slows and reduces blood flow through an AY fistula, causing problems with the quality of dialysis treatment, prolonged bleeding after puncture, or pain in the fistula. Stenosis can also lead to a blocked or clotted access.
• scapel incision refers to incision made in a tissue using a sharp object such as a scapel during surgical procedure.
• An incision is a cut made into the tissues of the body to expose the underlying tissue, bone, or organ so that a surgical procedure can be performed.
• 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.
• arterio-venous shunt or “A V shunt” or simply “shunt” refers to an implanted device which includes a tube to which an artery and vein is attached.
• a shunt connects the arterial and venous cannulas and provides a larger than normal volume of blood flow for effective hemodialysis.
• a shunt can be located in any part of the body, and is most often located in an arm, a leg or the chest area below the right collarbone.
• coated shunt refers to shunts that are capable of slowly eluting therapeutic compounds or polypeptides such as ENPP1 or ENPP3 to reduce the amount of vascular smooth muscle cell proliferation at the site of surgery, typically performed to remove blockage of the arteries.
• hemodialysis refers to a treatment that is required to compensate for abnormal kidney function, in which wastes and water are filtered out of blood and the filtered cleaner blood is returned to the body. Hemodialysis helps control blood pressure and balance important minerals, such as potassium, sodium, and calcium, in a subject’s blood.
• distala refers to an abnormal or surgically made passage between a hollow or tubular organ and the body surface, or between two hollow or tubular organs.
• tissue refers to a tubular support placed inside a blood vessel, canal, or duct to aid healing or relieve an obstruction.
• the term “vessel” refers to a tubular structure carrying blood through the tissues and organs; a vein, artery, or capillary.
• the term “complement inhibitor” refer to a molecule (e.g., a protein (such as an antibody), a small molecule, or a peptide) that prevents or reduces activation and/or propagation of the complement cascade that results in the formation of C3a or signaling through the C3a receptor, C5a or signaling through the C5a receptor, or formation of terminal complement.
• Complement inhibitors are well known in the art and described in, e.g., Zipfel et al. (2019) Front Immunol 10:2166. See also, e.g., U.S. Patent No. 5,679,345, the disclosure of which is incorporated by reference in its entirety.
• 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.
• medial area is the area between lamina elastica externa and lamina elastica interna of an artery.
• 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.
• lamina elastica interna refers to a layer of elastic tissue that forms the outermost part of the tunica intima of blood vessels.
• lumen refers to the interior of a vessel, such as the central space in an artery, vein or capillary through which blood flow occurs.
• vasculopathy refers to disease of the vasculature.
• Vasculature refers to the arrangement of blood vessels in the body or in an organ, such as a solid organ transplant, or in a body part.
• a “ blood vesseF refers to one or more of an artery, arteriole, capillary and vein in the body of a subject or of a solid organ allograft of a subject.
• vascularlitis refers to inflammation of veins, arteries, capillaries, or lymph vessels.
• a “vascularized graft ” refers to a graft after the recipient vasculature has been connected with the vessels in the graft.
• cardiac allograft vasculopathy CA Vf refers to a vascular complication of allograft or solid organ transplantation such as heart wherein the blood vessels supplying the transplanted heart gradually narrow and restrict its blood flow, subsequently leading to impairment of the heart muscle or sudden death. Diagnosis of CAV is by regular follow-up and monitoring of the transplanted organ such as heart for early signs of disease.
• invasive diagnostics including coronary angiography and intravascular ultrasound
• non-invasive investigations including dobutamine stress echocardiography, positron emission tomography, computed tomographic angiography (CT angiography) and the levels of a variety of biomarkers such as C-reactive protein, serum brain natriuretic peptide, troponin and serum microRNA 628-5p.
• allograft refers to the transplant of an organ or tissue from a donor to a recipient of the same species. Allografts account for many human organ and tissue transplants, including those from cadaveric, living related, and living unrelated donors.
• a “solid organ allograft” refers to an allograft of a solid organ.
• a “solid organ” is an internal organ that has a firm tissue consistency and is neither hollow (such as the organs of the gastrointestinal tract) nor liquid (such as blood).
• a solid organ includes but is not limited to kidney, liver, cornea, intestines, heart, lung and pancreas.
• transplant rejection refers to a condition wherein the transplanted organ or tissue is rejected by the recipient's immune system, which destroys the allograft and results in long-term loss of function in transplanted organs via fibrosis of the transplanted tissue blood vessels.
• the phrase “ prolonging the survival of an allograft ’ refers to the prevention of rejection of a transplanted donor organ or tissue by the recipient immune system and to improve the lifespan of the transplanted organ. Survival of an allograft may be prolonged by at least 12 months, 18 months, 2 years, 3 years, 4 years, 5 years, 8 years, 10 years or longer relative to allograft survival absent treatment.
• heart allograft refers to a solid organ transplant involving a donor heart transplanted into a recipient or grafting of one or more donor arteries or veins into a recipient’s heart. Graft rejection in heart allografts is commonly diagnosed by performing Endomyocardial biopsy.
• kidney allograft refers to a solid organ transplant involving a donor kidney transplanted into a recipient or grafting of one or more donor arteries or veins into a recipient’s kidney. Graft rejection in kidney allografts is commonly diagnosed by monitoring Urine protein levels such total protein-to-creatinine ratio, albumin- to-creatinine ratio, serum creatinine level and glomerular filtration rate.
• liver allograft refers to a solid organ transplant involving a donor liver transplanted into a recipient or grafting of one or more donor arteries or veins into a recipient’s liver. Graft rejection in liver allografts is diagnosed by monitoring Transaminase, bilirubin, and alkaline phosphatase levels.
• lung allograft refers to refers to a solid organ transplant involving a donor lung transplanted into a recipient or grafting of one or more donor arteries or veins into a recipient’s lung. Graft rejection in lung allografts is diagnosed by bronchoscopy with transbronchial biopsies and pulmonary function testing.
• allografted vesseT or Allografted vasculature refers to the grafting of one or more donor blood vessels such as artery, vein, capillary and/or arteriole into the recipient.
• allografted artery refers to the grafting of one or more donor arteries into the recipient.
• allografted vein refers to the grafting of one or more donor veins into the recipient.
• endomyocardial biopsy refers to a procedure that percutaneously obtains small amounts of myocardial tissue for diagnostic, therapeutic, and research purposes. It is primarily used to (1) follow the transplanted heart for myocardial rejection; (2) diagnose specific inflammatory, infiltrative, or familial myocardial disorders; and (3) sample unknown myocardial masses.
• transbronchial lung biopsy refers to a biopsy from the lung obtained by endoscopically-guided forceps, which is useful in evaluating lesions in the transplant distributed along bronchovascular bundles and in the central lung zones.
• 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.
• 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.
• 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.
• the term “treatment ’ or “ treating ’ 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.
• Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
• 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., NPP1 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.
• An effective amount also may result in treating, healing, preventing or ameliorating a condition, disease, or disorder.
• 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.
• a 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.
• 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.
• Multiple techniques of administering a compound exist in the art including, but not limited to, subcutaneous, intravenous, oral, aerosol, inhalational, rectal, vaginal, transdermal, intranasal, buccal, sublingual, parenteral, intrathecal, intragastrical, ophthalmic, pulmonary, and topical administration.
• 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.
• dystrophic calcification which occurs in dying and dead tissue
• metastatic calcification which elevated extracellular levels of calcium (hypercalcemia)
• 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.
• ectopic calcification refers to a condition characterized by a pathologic deposition of calcium salts in tissues or bone growth in soft tissues.
• 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.
• venous 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.
• adeno-associated viral 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 “44 F vector particle ” or “44 F 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.
• recombinant 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. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such 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.
• recombinant viral genome 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.
• effective amounC 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).
• functional activities of Cap proteins 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.
• capsid ’ refers to the structure in which the viral genome is packaged.
• a capsid consists of several oligomeric structural subunits made of proteins.
• 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. Another property of these sequences is their ability to form a hairpin. This characteristic contributes to its self-priming which allows the primase-independent synthesis of the second DNA strand. Procedures for modifying these ITR sequences are known in the art ( Brown T, “Gene Cloning”, Chapman & Hall, London, GB, 1995; Watson R, et al, “Recombinant DNA ” , 2 nd Ed.
• tissue-specific ” promoter is only active in specific types of differentiated cells or tissues.
• 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. by the application of a chemical inducer.
• a chemical inducer e.g., it can be a tetracycline-inducible promoter, a mifepristone (RU-486)-inducible promoter and the like.
• constitutive promoter refers to a promoter whose activity is maintained at a relatively constant level in all cells of an organism, or during most developmental stages, with little or no regard to cell environmental conditions.
• 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 b-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter (Boshart M, etal, Cell 1985; 41:521-530 ).
• RSV Rous sarcoma virus
• CMV cytomegalovirus
• SV40 promoter the dihydrofolate reductase promoter
• the b-actin promoter the phosphoglycerol kinase (PGK) promoter
• PGK phosphoglycerol kinase
• polyadenylation signal as used herein, 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 Elb 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 ah, 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.
• immunosuppressant drugs include anti -CD4(GK 1.5) 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. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from lto 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from lto 4, from lto 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 ENPPl or ENPP3 agent to treat PAD, which includes administering sNPPl 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. Sequences
• Gly lie lie asp Asn Lys Met Tyr Asp Pro Lys Met Asn Ala Ser
• Gly Gin Pro Leu Trp lie Thr Ala Thr Lys Gin Gly Val Lys Ala Gly 260 265 270
• Ala Lys Tyr Asp Pro Lys Ala lie lie Ala Asn Leu Thr Cys Lys Lys 405 410 415
• Trp Asp Thr Leu Met Pro Asn lie Asn Lys Leu Lys Thr Cys Gly lie 130 135 140
• Gly Ser Glu Val Ala lie Asn
• Gly Ser Phe Pro Ser lie Tyr Met Pro 225 230 235 240
• Glu Asn lie Glu Val Tyr Asn Leu Met Cys Asp Leu Leu Arg lie Gin 465 470 475 480
• Thr Leu Lys Lys Tyr Lys lie Ala Cys lie Val Leu Leu Ala Leu Leu 20 25 30 Val lie Met Ser Leu Gly Leu Gly Leu Gly Leu Gly Leu Gly Leu Arg Lys Leu 35 40 45
• Trp Asp Thr Leu Met Pro Asn lie Asn Lys Leu Lys Thr Cys Gly lie 180 185 190
• Gly Ser Glu Val Ala lie Asn
• Gly Ser Phe Pro Ser lie Tyr Met Pro 275 280 285
• Glu Asn lie Glu Val Tyr Asn Leu Met Cys Asp Leu Leu Arg lie Gin 515 520 525
• Trp Asn Glu Ala Val Pro lie Trp Val Thr Asn Gin Leu Gin Glu
• Lys lie Asp Asp Leu lie Gly Asp Leu Val Gin Arg Leu Lys Met
• Glu Glu Arg lie Leu Ala Val Leu Glu Trp Leu Gin Leu Pro Ser
• Glu Glu Arg lie Leu Ala Val Leu Glu Trp Leu Gin Leu Pro Ser
• 565 570 575 lie Tyr His Met Thr Val Pro Tyr Gly Arg Pro Arg He Leu Leu

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