Patent Application
20230330307
This application contains a Sequence Listing which has been submitted electronically as a WIPO Standard ST.26 XML file via Patent Center, created on Jul. 7, 2023, is entitled “4427-10104.xml” and is 161,443 bytes in size. The sequence listing is incorporated herein by reference in its entirety.
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.
Atherosclerosis and neointimal hyperplasia both contribute to cardiovascular disease (CVD), with atherosclerosis resulting in initial native vessel stenosis and neointimal hyperplasia leading to recurrent stenosis after operative intervention. Although stents mitigate the risk of restenosis in selected coronary artery lesions, in-stent restenosis is still a frequent and often intractable clinical problem. Stent placement can directly damage the vessel wall and trigger neointimal hyperplasia that often leads to vessel restenosis, narrowing the lumen despite the stent preventing immediate vessel recoil after angioplasty and later constrictive remodeling. Mechanisms underlying the occurrence and recurrence of neointimal hyperplasia in patients with coronary stents is still not understood.
Neointimal hyperplasia is also the major cause of restenosis after percutaneous coronary interventions such as angioplasty. Neointimal hyperplasia in bypass conduits such as veins and prosthetic grafts greatly limits the long-term success of vascular interventions. Neointimal hyperplasia can affect all forms of vascular grafts, including both venous and prosthetic conduits used in coronary and peripheral arterial bypass, and arteriovenous fistulae (AVF) created for hemodialysis access.
More than 1 million vascular grafts are implanted annually around the world. Up to 50% of these grafts fail within the 1st 18 months following surgery due to the development of neointimal hyperplasia at the anastomosis site. The lack of treatment to prevent this pathology is a major problem and is yet to be addressed effectively. Therefore, there is a need for efficient treatment to prevent and or reduce neointimal hyperplasia in various clinical interventions.
The disclosure is based, at least in part, on the unexpected discovery that administration of soluble ENPP1 or ENPP3 can inhibit the undesirable proliferation of vascular smooth muscle cells in subjects who are not deficient in one or both of ENPP1 protein activity or expression. As set forth in the working examples below, the administration of soluble ENPP1 or ENPP3 inhibited proliferation of vascular smooth muscle cells following a tissue injury in wild type mice not deficient in ENPP1 expression or activity.
Accordingly, in one aspect, the disclosure provides a method for reducing and/or preventing the progression of vascular smooth muscle cell proliferation in a subject having a tissue injury. The method includes administering to the subject a therapeutically effective amount of an ENPP1 or an ENPP3 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation at the site of injury in the subject.
Accordingly, in one aspect, the disclosure provides a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a subject having a tissue injury. The method includes administering to the subject a therapeutically effective amount of an ENPP1 or ENPP3 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation at the site of injury in the subject.
In some embodiments, the subject is not ENPP1 deficient.
In some embodiments of any of the methods described herein, the tissue injury is an injury to any artery or vein. The artery can be, e.g., a coronary artery or carotid artery.
In some embodiments of any of the methods described herein, the tissue injury is a result of stent placement in an artery. In some embodiments of any of the methods described herein, the subject is at risk of developing restenosis. In some embodiments of any of the methods described herein, the subject suffers from restenosis. In some embodiments of any of the methods described herein, the subject suffers from restenosis in an artery.
In yet another aspect, the disclosure features a method for reducing and/or preventing the progression of vascular smooth muscle cell proliferation in a subject who requires surgery. The method comprises: administering to the subject a therapeutically effective amount of an ENPP1 or ENPP3 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation at a surgical site in the subject.
In yet another aspect, the disclosure features a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a subject who requires surgery. The method comprises: administering to the subject a therapeutically effective amount of an ENPP1 or ENPP3 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation at a surgical site in the subject.
In some embodiments, any of the methods described herein can also include detecting the presence of and/or measuring the amount of vascular smooth muscle cell proliferation in the subject, e.g., at the site of an injury or at the site of surgery. In some embodiments, such detecting and/or measuring can occur prior to, during, or following administration of an ENPP1 agent or an ENPP3 agent.
In some embodiments of any of the methods described herein, the ENPP1 agent comprises ENPP1 variants that retain enzymatic activity.
In some embodiments of any of the methods described herein, the ENPP3 agent comprises ENPP3 variants that retain enzymatic activity.
In some embodiments of any of the methods described herein, the agent (e.g., the ENPP1 agent or the ENPP3 agent) is administered prior to the surgery.
In some embodiments of any of the methods described herein, the agent (e.g., the ENPP1 agent or the ENPP3 agent) is administered during surgery.
In some embodiments of any of the methods described herein, the agent (e.g., the ENPP1 agent or the ENPP3 agent) is administered after surgery.
In some embodiments of any of the methods described herein, the agent (e.g., the ENPP1 agent or the ENPP3 agent) is administered prior to, during and/or after surgery.
In some embodiments, any of the methods described herein further comprise performing the surgery.
In some embodiments of any of the methods described herein, the surgery comprises artery bypass grafting.
In some embodiments of any of the methods described herein, the surgery comprises placement of an arterial stent.
In some embodiments of any of the methods described herein, the surgery comprises angioplasty.
In another aspect, the disclosure provides a method of prophylaxis against vascular smooth muscle cell proliferation in a subject who is at risk for non-surgical tissue injury. The method includes administering to the subject a therapeutically effective amount of an ENPP1 or ENPP3 agent to thereby prevent the progression of vascular smooth muscle cell proliferation or reduce the extent of vascular smooth muscle cell proliferation at a site of non-surgical tissue injury in the subject. In some embodiments, the non-surgical tissue injury comprises blunt force trauma. In some embodiments, the subject is at risk of any one of the following: a cardiovascular disorder that is associated with undesirable smooth muscle cell proliferation, atherosclerotic cardiovascular disorder, a myocardial infarction, a stroke, developing coronary artery disease.
In another aspect, the disclosure provides a method of prophylaxis against vascular smooth muscle cell proliferation in a subject who is at risk for non-surgical tissue injury. The method includes administering to the subject a therapeutically effective amount of an ENPP1 or ENPP3 agent to thereby prevent the progression of vascular smooth muscle cell proliferation or reduce the extent of vascular smooth muscle cell proliferation at a site of non-surgical tissue injury in the subject. In some embodiments, the non-surgical tissue injury comprises blunt force trauma. In some embodiments, the subject is at risk of any one of the following: a cardiovascular disorder that is associated with undesirable smooth muscle cell proliferation, atherosclerotic cardiovascular disorder, a myocardial infarction, a stroke, developing coronary artery disease.
In some embodiments of any of the methods described herein, the subject is not ENPP1 Deficient.
In another aspect, the disclosure features a method for treating a subject suffering a myocardial infarction or a stroke. The method comprises administering to the subject a therapeutically effective amount of an ENPP1 or ENPP3 agent to thereby treat the myocardial infarction or stroke.
In another aspect, the disclosure features a method for treating a subject suffering a myocardial infarction or a stroke. The method comprises administering to the subject a therapeutically effective amount of an ENP1 or ENPP3 agent to thereby treat the myocardial infarction or stroke.
In yet another aspect, the disclosure features a method for reducing and/or preventing the progression of vascular smooth muscle cell proliferation in a subject suffering a myocardial infarction or a stroke. The method includes: administering to the subject a therapeutically effective amount of an ENPP1 or ENPP3 agent to thereby reduce and/or prevent the progression of vascular smooth muscle cell proliferation in vasculature associated with the subject's myocardial infarction or stroke.
In yet another aspect, the disclosure features a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a subject suffering a myocardial infarction or a stroke. The method includes: administering to the subject a therapeutically effective amount of an ENPP1 or ENPP3 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation in vasculature associated with the subject's myocardial infarction or stroke.
In some embodiments of any of the methods described herein, the subject is not ENPP1 Deficient.
In some embodiments of any of the methods described herein, the subject is not afflicted with Generalized Arterial Calcification of Infancy (GACI) or Autosomal Recessive Hypophosphatemic Rickets Type 2 (ARHR2).
In some embodiments of any of the methods described herein, the vascular smooth muscle cell proliferation is at the tunica intima of an arterial wall of the subject.
In some embodiments of any of the methods described herein, the tissue injury comprises vascular trauma.
In some embodiments of any of the methods described herein, the surgery comprises coronary intervention, such as scalpel incision or ablation.
In some embodiments of any of the methods described herein, the method includes performing the surgery while simultaneously administering the ENPP1 agent or the ENPP3 agent.
In some embodiments of any of the methods described herein, the method includes administering the ENPP1 agent or the ENPP3 agent prior to surgery or vascular intervention.
In some embodiments of any of the methods described herein, the method includes administering the agent, performing surgery while simultaneously administering the ENPP1 agent or ENPP3 agent, and optionally administering the agent after surgery.
In some embodiments of any of the methods described herein, the method includes administering the ENPP1 agent or ENPP3 agent, performing surgery, and optionally administering the agent after surgery.
In some embodiments of any of the methods described herein, the subject suffers from myocardial ischemia.
In some an embodiments of any of the methods described herein, the ENPP1 agent or ENPP3 agent is administered after treatment for said myocardial infarction and/or said stroke.
In some embodiments of any of the methods described herein, the ENPP1 agent comprises or is an ENPP1 polypeptide.
In some embodiments of any of the methods described herein, the ENPP1 agent comprises or is a nucleic acid encoding an ENPP1 polypeptide.
In some embodiments of any of the methods described herein, the ENPP1 agent comprises or is a viral vector comprising a nucleic acid encoding an ENPP1 polypeptide.
In some embodiments of any of the methods described herein, the ENPP3 agent comprises or is an ENPP3 polypeptide.
In some embodiments of any of the methods described herein, the ENPP3 agent comprises or is a nucleic acid encoding an ENPP3 polypeptide.
In some embodiments of any of the methods described herein, the ENPP3 agent comprises or is a viral vector comprising a nucleic acid encoding an ENPP3 polypeptide.
In some embodiments of any of the methods described herein, the ENPP1 polypeptide comprises the extracellular domain of ENPP1.
In some embodiments of any of the methods described herein, the ENPP1 polypeptide comprises the catalytic domain of ENPP1.
In some embodiments of any of the methods described herein, the ENPP1 polypeptide comprises amino acids 99 to 925 of SEQ ID NO:1.
In some embodiments of any of the methods described herein, the ENPP3 polypeptide comprises amino acids 49 to 875 of SEQ ID NO:7.
In some embodiments of any of the methods described herein, the ENPP1 agent or the ENPP3 agent comprises a heterologous moiety. In some embodiments, the heterologous moiety is a heterologous protein.
In some embodiments of any of the methods described herein, the heterologous moiety increases the half-life of the ENPP1 agent or the ENPP3 agent in a mammal, relative to the half-life of the ENPP1 agent or ENPP3 agent without the heterologous moiety.
In some embodiments of any of the methods described herein, the heterologous moiety is an Fc region of an immunoglobulin molecule, such as an IgG1. In some embodiments, the immunoglobulin is a human immunoglobulin.
In some embodiments of any of the methods described herein, the heterologous moiety is an albumin molecule.
In some embodiments of any of the methods described herein, the heterologous moiety is carboxy-terminal to the ENPP1 polypeptide or ENPP3 polypeptide.
In some embodiments of any of the methods described herein, the ENPP1 agent or the ENPP3 agent comprises a linker.
In some embodiments of any of the methods described herein, the linker separates the ENPP1 polypeptide or ENPP3 polypeptide and the heterologous protein.
In some embodiments of any of the methods described herein, the linker comprises the following amino acid sequence: (GGGGS)n, wherein n is an integer from 1 to 10.
In some embodiments of any of the methods described herein, the heterologous moiety ENPP1 agent or ENPP3 agent is subcutaneously administered to the subject.
In some embodiments of any of the methods described herein, the ENPP1 agent or the ENPP3 agent is intravenously administered to the subject.
In yet another aspect, the disclosure features a coated stent comprising a vascular stent; and a coating on the stent, the coating comprising an ENPP1 agent; and a carrier for said ENPP1 agent, wherein said coating is configured to release said ENPP1 agent from the stent at a rate of 1-10 μg/ml per day.
In some embodiments of any of the stents described herein, the ENPP1 agent in an amount between 1 wt % and 50 wt %, based on a total weight of the coating.
In some embodiments of any of the stents described herein, the ENPP1 agent is selected from a group consisting of: ENPP1, ENPP1-Fc, ENPP1-Albumin, and ENPP1 mRNA.
In some embodiments of any of the stents described herein, the ENPP1 agent comprises ENPP1 variants that retain enzymatic activity.
In some embodiments of any of the stents described herein, the ENPP3 agent comprises ENPP3 variants that retain enzymatic activity.
In some embodiments of any of the stents described herein, the carrier is non-reactive with said ENPP1 agent.
In some embodiments of any of the stents described herein, the carrier comprises a polymeric carrier that is physically bound to said ENPP1 agent.
In some embodiments of any of the stents described herein, the carrier comprises a polymeric carrier that is chemically bound to said ENPP1 agent.
In some embodiments of any of the stents described herein, the carrier comprises a polymeric biodegradable carrier.
In some embodiments of any of the stents described herein, the carrier comprises a nonpolymeric carrier.
In some embodiments of any of the stents described herein, the nonpolymeric carrier is selected from a group consisting of: Vitamin E, Vitamin E acetate, Vitamin E succinate, oleic acid, peanut oil and cottonseed oil.
In some embodiments of any of the methods described herein, the carrier is liquid at body temperature. In some embodiments of any of the methods described herein the carrier is solid at body temperature.
In yet another aspect, the disclosure features a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a subject having a tissue injury, the method comprising: implanting an arterial stent coated with an ENPP1 agent into an artery of the subject proximal to said tissue injury, wherein said implanted stent is configured to release said ENPP1 agent in an amount effective to reduce and/or prevent progression of vascular smooth muscle cell proliferation at a site of injury in the subject, wherein the subject is not ENPP1 deficient, thereby to reduce and/or prevent progression of vascular smooth muscle cell proliferation at said site of injury in said subject.
In some embodiments of any of the methods described herein, the tissue injury comprises stent placement in an artery.
In some embodiments of any of the methods described herein, the tissue injury is due to a prior placement of a non-eluting arterial stent in said artery or due to a prior placement of an eluting arterial stent in said artery which elutes therapeutic agents other than said ENPP1 agent.
In some embodiments of any of the methods described herein, the subject is at risk of developing restenosis.
In yet another aspect, the disclosure features a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a subject who has a condition requiring surgery at a surgical site, the method comprising: implanting an arterial stent coated with an ENPP1 agent into an artery proximal to said surgical site in the subject, wherein said implanted stent is configured to release said ENPP1 agent in an amount effective to reduce and/or prevent progression of vascular smooth muscle cell proliferation, wherein the subject is not ENPP1 deficient, thereby to reduce and/or prevent progression of vascular smooth muscle cell proliferation at said surgical site.
In some embodiments of any of the methods described herein, the agent is administered to the subject prior to, during and/or after surgery.
In some embodiments of any of the methods described herein, further comprises performing the surgery.
In some embodiments of any of the methods described herein, the surgery comprises artery bypass grafting.
In some embodiments of any of the methods described herein, the condition requiring surgery is due to a prior placement of a non-eluting arterial stent in said artery.
In some embodiments of any of the methods described herein, the condition requiring surgery is due to a prior placement of an eluting arterial stent in said artery which elutes therapeutic agents other than said ENPP1 agent.
In some embodiments of any of the methods described herein, the surgery comprises angioplasty.
In yet another aspect, the disclosure features a method for ameliorating a myocardial infarction or a stroke in a subject suffering therefrom, the method comprising: implanting an arterial stent coated with an ENPP1 agent into an artery of said subject, wherein said implanted stent is configured to release said ENPP1 agent in an amount effective to ameliorate a myocardial infarction or stroke, thereby to ameliorating said myocardial infarction or stroke.
In yet another aspect, the disclosure features a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a subject suffering a myocardial infarction or a stroke, the method comprising: implanting an arterial stent coated with an ENPP1 agent into an artery of a subject, wherein said implanted stent is configured to release said ENPP1 agent in an amount effective to reduce and/or prevent progression of vascular smooth muscle cell proliferation in vasculature associated with a myocardial infarction or stroke, thereby to reduce and/or prevent progression of vascular smooth muscle cell proliferation in vasculature of said subject associated with myocardial infarction or stroke.
In some embodiments of any of the methods described herein, the subject is not ENPP1 deficient.
In some embodiments of any of the methods described herein, the ENPP1 agent comprises an ENPP1 polypeptide.
In some embodiments of any of the methods described herein, the ENPP1 agent is a nucleic acid encoding an ENPP1 polypeptide.
In some embodiments of any of the methods described herein, the ENPP1 agent comprises a viral vector comprising a nucleic acid encoding an ENPP1 polypeptide.
In some embodiments of any of the methods described herein, the ENPP1 polypeptide comprises the extracellular domain of ENPP1.
In some embodiments of any of the methods described herein, the ENPP1 polypeptide comprises the catalytic domain of ENPP1.
In some embodiments of any of the methods described herein, the ENPP1 polypeptide comprises amino acids 99 to 925 of SEQ ID NO:1.
In some embodiments of any of the methods described herein, the ENPP1 polypeptide comprises a heterologous protein.
In some embodiments of any of the methods described herein, the heterologous protein increases the circulating half-life of the ENPP1 polypeptide in mammal.
In some embodiments of any of the methods described herein, the heterologous protein is an Fc region of an immunoglobulin molecule.
In some embodiments of any of the methods described herein, the immunoglobulin molecule is an IgG1 molecule.
In some embodiments of any of the methods described herein, the heterologous protein is an albumin molecule.
In some embodiments of any of the methods described herein, the heterologous protein is carboxy-terminal to the ENPP1 polypeptide.
In some embodiments of any of the methods described herein, the ENPP1 agent comprises a linker.
In some embodiments of any of the methods described herein, the linker separates the ENPP1 polypeptide and the heterologous protein.
In some embodiments of any of the methods described herein, the linker comprises the following amino acid sequence: (GGGGS)n, wherein n is an integer from 1 to 10.
In yet another aspect, the disclosure features a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a subject having a tissue injury, the method comprising: implanting an arterial stent coated with an ENPP3 agent into an artery of a subject proximal to said tissue injury, wherein said implanted stent is configured to release said ENPP3 agent in an amount effective to reduce and/or prevent progression of vascular smooth muscle cell proliferation at a site of injury in the subject, thereby to reduce and/or prevent progression of vascular smooth muscle cell proliferation at said site of injury in said subject. In some embodiments of any of the methods described herein, the tissue injury comprises injury to an artery.
In some embodiments of any of the methods described herein, the tissue injury comprises stent placement in an artery.
In some embodiments of any of the methods described herein, the subject is at risk of developing restenosis.
In yet another aspect, the disclosure features a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a subject who has a condition requiring surgery at a surgical site, the method comprising: implanting an arterial stent coated with an ENPP3 agent into an artery proximal to said surgical site in the subject, wherein said implanted stent is configured to release said ENPP3 agent in an amount effective to reduce and/or prevent progression of vascular smooth muscle cell proliferation, thereby to reduce and/or prevent progression of vascular smooth muscle cell proliferation at said surgical site.
In some embodiments of any of the methods described herein, the agent is administered to the subject prior to, during and/or after surgery.
In some embodiments of any of the methods described herein, further comprises performing the surgery.
In some embodiments of any of the methods described herein, the surgery comprises artery bypass grafting.
In some embodiments of any of the methods described herein, the condition requiring surgery is due to a prior placement of a non-eluting arterial stent in said artery.
In some embodiments of any of the methods described herein, the condition requiring surgery is due to a prior placement of an eluting arterial stent in said artery which elutes therapeutic agents other than said ENPP3 agent.
In some embodiments of any of the methods described herein, the surgery comprises angioplasty.
In yet another aspect, the disclosure features a method for ameliorating a myocardial infarction or a stroke in a subject suffering therefrom, the method comprising: implanting an arterial stent coated with an ENPP3 agent into an artery of said subject, wherein said implanted stent is configured to release said ENPP3 agent in an amount effective to ameliorate a myocardial infarction or stroke, thereby to ameliorating said myocardial infarction or stroke.
In yet another aspect, the disclosure features a method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a subject suffering a myocardial infarction or a stroke, the method comprising: implanting an arterial stent coated with an ENPP3 agent into an artery of a subject, wherein said implanted stent is configured to release said ENPP3 agent in an amount effective to reduce and/or prevent progression of vascular smooth muscle cell proliferation in vasculature associated with a myocardial infarction or stroke, thereby to reduce and/or prevent progression of vascular smooth muscle cell proliferation in vasculature of said subject associated with myocardial infarction or stroke.
In some embodiments of any of the methods described herein, the subject is not ENPP1 deficient.
In some embodiments of any of the methods described herein, the ENPP3 agent comprises an ENPP3 polypeptide.
In some embodiments of any of the methods described herein, the ENPP3 agent is a nucleic acid encoding an ENPP3 polypeptide.
In some embodiments of any of the methods described herein, the ENPP3 agent comprises a viral vector comprising a nucleic acid encoding an ENPP3 polypeptide.
In some embodiments of any of the methods described herein, the ENPP3 polypeptide comprises a heterologous protein.
In some embodiments of any of the methods described herein, the ENPP3 polypeptide comprises the extracellular domain of ENPP3.
In some embodiments of any of the methods described herein, the ENPP3 polypeptide comprises the catalytic domain of ENPP3.
In some embodiments of any of the methods described herein, the ENPP3 polypeptide comprises amino acids 49-875 of SEQ ID NO: 7.
In some embodiments of any of the methods described herein, the ENPP3 polypeptide comprises a heterologous protein.
In some embodiments of any of the methods described herein, the heterologous protein increases the circulating half-life of the ENPP3 polypeptide in mammal.
In some embodiments of any of the methods described herein, the heterologous protein is an Fc region of an immunoglobulin molecule.
In some embodiments of any of the methods described herein, the immunoglobulin molecule is an IgG1 molecule.
In some embodiments of any of the methods described herein, the heterologous protein is an albumin molecule.
In some embodiments of any of the methods described herein, the heterologous protein is carboxy-terminal to the ENPP3 polypeptide.
In some embodiments of any of the methods described herein, the ENPP3 agent comprises a linker
In some embodiments of any of the methods described herein, the linker separates the ENPP3 polypeptide and the heterologous protein.
In some embodiments of any of the methods described herein, the linker comprises the following amino acid sequence: (GGGGS)n, wherein n is an integer from 1 to 10.
In yet another aspect, the disclosure features a coated stent comprising a vascular stent; and a coating on the stent, the coating comprising an ENPP3 agent; and a carrier for said ENPP3 agent, wherein said coating is configured to release said ENPP3 agent from the stent at a rate of 1-10 μg/ml per day.
In some embodiments of any of the methods described herein, the ENPP3 agent is in an amount between 1 wt % and 50 wt %, based on a total weight of the coating.
In some embodiments of any of the methods described herein, the ENPP3 agent is selected from a group consisting of: ENPP3, ENPP3-Fc, ENPP3-Albumin, and ENPP3 mRNA
In some embodiments of any of the methods described herein, the carrier is non-reactive with said ENPP3 agent.
In some embodiments of any of the methods described herein, the carrier comprises a polymeric carrier that is physically bound to said ENPP3 agent.
In some embodiments of any of the methods described herein, the carrier comprises a polymeric carrier that is chemically bound to said ENPP3 agent.
In some embodiments of any of the methods described herein, the carrier comprises a polymeric biodegradable carrier.
In some embodiments of any of the methods described herein, the carrier comprises a nonpolymeric carrier.
In some embodiments of any of the methods described herein, the nonpolymeric carrier is selected from a group consisting of: Vitamin E, Vitamin E acetate, Vitamin E succinate, oleic acid, peanut oil and cottonseed oil.
In some embodiments of any of the methods described herein, the carrier is liquid at body temperature.
In some embodiments of any of the methods described herein, the carrier is solid at body temperature.
Other features and advantages of the disclosure will be apparent from the following detailed description and claims.
The medial area, between the external and internal lamina, remained constant (
Evaluation of the therapeutic effects of ENPP1-Fc was initiated at 7 days post ligation, when neointimal hyperplasia was definitely present. The medial area, between the external and internal lamina, remained constant in all groups of mice (
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described.
For clarity, “NPP1” and “ENPP1” refer to the same protein and are used interchangeably herein. As used herein, the term “ENPP1 protein” or “ENPP1 polypeptide” refers to ectonucleotide pyrophosphatase/phosphodiesterase-1 protein encoded by the ENPP1 gene that is capable of cleaving ATP to generate PPi and also reduces ectopic calcification in soft tissue.
ENPP1 protein is a type II transmembrane glycoprotein and cleaves a variety of substrates, including phosphodiester bonds of nucleotides and nucleotide sugars and pyrophosphate bonds of nucleotides and nucleotide sugars. ENPP1 protein has a transmembrane domain and soluble extracellular domain. The extracellular domain is further subdivided into somatomedin B domain, catalytic domain and the nuclease domain. The sequence and structure of wild-type ENPP1 is described in detail in PCT Application Publication No. WO 2014/126965 to Braddock, et al., which is incorporated herein in its entirety by reference.
ENPP1 polypeptides as used herein encompass 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. as noted below.
ENPP3 polypeptides as used herein encompass polypeptides that exhibit enzymatic activity, mutants of ENPP3 that retain enzymatic activity, fragments of ENPP3 or variants of ENPP3 including deletion variants that exhibit enzymatic activity as noted below.
Some examples of 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/2016/187408—Braddock et al., WO/2017/087936—Braddock et al., and WO2018/027024-Braddock et al., all of which are incorporated by reference in their entireties herein.
“Enzymatically active” with respect to an ENPP1 polypeptide or an ENPP3 polypeptide, or, as used herein, “enzymatic activity” with respect to an ENPP1 polypeptide or an ENPP3 polypeptide, is defined as possessing ATP hydrolytic activity into AMP and PPi and/or AP3a hydrolysis to ADP and AMP. NPP1 and NPP3 readily hydrolyze ATP into AMP and PPi. The steady-state Michaelis-Menten enzymatic constants of NPP1 are determined using ATP as a substrate. NPP1 can be demonstrated to cleave ATP by HPLC analysis of the enzymatic reaction, and the identity of the substrates and products of the reaction are confirmed by using ATP, AMP, and ADP standards. The ATP substrate degrades over time in the presence of NPP1, with the accumulation of the enzymatic product AMP. Using varying concentrations of ATP substrate, the initial rate velocities for NPP1 are derived in the presence of ATP, and the data is fit to a curve to derive the enzymatic rate constants. At physiologic pH, the kinetic rate constants of NPP1 are Km=144 μM and kcat=7.8 s−1.
As used herein the term “plasma pyrophosphate (PPi) levels” refers to the amount of pyrophosphate present in plasma of animals. In certain embodiments, animals include rat, mouse, cat, dog, human, cow and horse. It is necessary to measure PPi in the plasma rather than serum because of release from platelets. There are several ways to measure PPi, one of which is by enzymatic assay using uridine-diphosphoglucose (UDPG) pyrophosphorylase (Lust & Seegmiller, 1976, Clin. Chim. Acta 66:241-249; Cheung & Suhadolnik, 1977, Anal. Biochem. 83:61-63) with modifications.
Typically, plasma PPi levels in healthy human subjects range from about 1 μm to about 3 in some cases between 1-2 μm. A normal level of ENPP1 in plasma refers to the amount of ENPP1 protein required to maintain a normal level of plasma pyrophosphate (PPi) in a healthy subject. A normal level of PPi in healthy humans corresponds to 2-3 μM. Subjects who have a deficiency of ENPP1 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. In patients afflicted with GACI, the PPi levels are found to be less than 1 μm and in some cases are below a detectable level. In patients afflicted with PXE, the PPi levels are below 0.5 μm. (Arterioscler Thromb Vasc Biol. 2014 September; 34(9): 1985-9; Braddock et al., Nat Commun. 2015; 6: 10006.)
As used herein, the term “PPi” refers to pyrophosphate.
As used herein the terms “alteration,” “defect,” “variation” or “mutation” refer 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.
As used herein, the term “ENPP1 precursor protein” refers to ENPP1 polypeptide with its signal peptide sequence at the ENPP1 N-terminus. Upon proteolysis, the signal sequence is cleaved from ENPP1 to provide the ENPP1 protein. Signal peptide sequences useful within the disclosure include, but are not limited to, Albumin signal sequence, Azurocidin signal sequence, ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and/or ENPP5 signal peptide sequence.
As used herein, the term “ENPP3 precursor protein” refers to ENPP3 polypeptide 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, ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and/or ENPP5 signal peptide sequence.
As used herein, the term “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 Azurocidin signal peptide (MTRLTVLALLAGLLASSRA (SE 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. (Optimized signal peptides for the development of high expressing CHO cell lines, Kober et al., Biotechnol Bioeng. 2013 April; 110(4):1164-73)
The term “ENPP1-Fc construct” refers to 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). In certain embodiments, the C-terminus of ENPP1 is fused or conjugated to the N-terminus of the FcR binding domain.
As used herein, the term “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). In certain embodiments, the C-terminus of ENPP1 is fused or conjugated to the N-terminus of the FcR binding domain.
As used herein, the term “Fc” refers to a human IgG (immunoglobulin) Fc domain. Subtypes of IgG such as IgG1, IgG2, IgG3, and IgG4 are contemplated for use as Fc domains. The “Fc region or Fc polypeptide” is the portion of an IgG molecule that correlates to a crystallizable fragment obtained by papain digestion of an IgG molecule. The Fc region comprises the C-terminal half of the two heavy chains of an IgG molecule that are linked by disulfide bonds. It has no antigen binding activity but contains the carbohydrate moiety and the binding sites for complement and Fc receptors, including the FcRn receptor. The Fc fragment contains the entire second constant domain CH2 (residues 231-340 of human IgG1, according to the Kabat numbering system) and the third constant domain CH3 (residues 341-447). The term “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. The term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain contains the CH1, CH2 and CH3 domains of the heavy chain and the CHL domain of the light chain.
As used herein 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 or ENPP3 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 enzymatic activity
The degree of identity between two polypeptides is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)), though other similar algorithms can also be used. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
“Functionally equivalent variants” of ENPP1 or ENPP3 may be obtained by replacing nucleotides within the polynucleotide accounting for codon preference in the host cell that is to be used to produce the ENPP1 or ENPP3 respectively. Such “codon optimization” can be determined via computer algorithms which incorporate codon frequency tables such as “Human high.cod” for codon preference as provided by the University of Wisconsin Package Version 9.0, Genetics Computer Group, Madison, Wis. The variants of ENPP1 or ENPP3 polypeptides are expected to retain at least 50%, 55%, 60%, 70%, 80% or 90% activity of wild type ENPP1 or ENPP3 protein with respect to enzymatic activity.
As used herein, the term “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. In contrast, the term “functionally equivalent” refers to an 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.
As defined herein, the term “subject”, “individual” or “patient” refers to mammal preferably a human.
As defined herein, the term “moiety” refers to a chemical component or biological molecule that can be covalently or non-covalently linked to ENPP1 or ENPP3 protein and has the ability to confer a desired property to the protein to which it is attached. For example, the term moiety can refer to a bone targeting peptide such as polyaspartic acid or polyglutamic acid (of 4-20 consecutive asp or glu residues) or a molecule that extends the half-life of ENPP1 or ENPP3 polypeptide. Some other examples of half-life extending 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).
As defined herein, the phrase “medial area” is the area between lamina elastica externa and lamina elastica interna of an artery.
As defined herein, the phrase “intimal area” and said intimal area is the area between said lamina elastica interna and lumen of an artery.
As defined herein, the phrase “lamina elastica externa” refers to a layer of elastic connective tissue lying immediately outside the smooth muscle of the tunica media of an artery.
As defined herein, the phrase “lamina elastica interna” refers to a layer of elastic tissue that forms the outermost part of the tunica intima of blood vessels.
As defined herein, the phrase “lumen” refers to the interior of a vessel, such as the central space in an artery, vein or capillary through which blood flow occurs.
As defined herein, the phrase “surgery” refers to an invasive medical procedure that involves coronary interventions which result in tissue injury by scalpel incision or radiofrequency ablation or cryoablation or laser ablation.
As defined herein, the phrase “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 coronary interventions such as stenting or angioplasty.
As defined herein, the phrase “deficient for NPP1” or “ENPP1 deficiency” refers to having a loss of function mutation in ENPP1 protein or in a gene encoding the protein that result in a diagnosis of Generalized Arterial Calcination of Infancy. (GACI), or a diagnosis of being at risk of developing or of being afflicted with autosomal recessive hypophosphatemic rickets type 2 (ARHR2).
As defined herein, the phrase “vascular trauma” refers to an injury to a blood vessel—an artery, which carries blood to an extremity, or a vein, which returns blood to the heart. Vascular injuries may also be caused by invasive procedures, such as percutaneous transluminal coronary angioplasty, and vascular bypass surgery.
As defined herein the phrase “accidental trauma” refers to a blood vessel such as artery by a blunt injury that occurs when a blood vessel is crushed or stretched due to exertion of physical force or penetrating injury which occurs when a blood vessel is punctured, torn or severed. Blunt injury occurs during physical alterations such as boxing and penetrating injury occurs due to sharp objects such as knife or bullet wounds. The trauma or injury can be caused by different factors, such as radiation, viral infections, development of immune complexes, and hyperlipidemia.
As defined herein the phrase “restenosis” refers to the 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 becomes 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.
As defined herein the phrase “myointimal proliferation” refers to the proliferation of vascular smooth muscle cells that occurs at the tunica intima of an arterial wall of an individual.
As used herein, 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.
As used herein, the term “prevent” or “prevention” or “reduce” means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been the 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.
As used herein, the phrase “reduce or prevent myointimal or neointimal 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.
As used herein the term “coronary intervention” refers to surgical and non-surgical procedures, such as including balloon angioplasty, angioplasty with stent, rotablation or cutting balloon catherization that are performed to clear blockage and restore blood flow to the blocked blood vessels.
As used herein the term “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 the use of blunt force or sharp objects such as a knife, mechanical injury such fall from elevation, workplace injury due to heavy machinery or vehicular injury such as car accidents.
As used herein the term “site of non-surgical tissue injury” refers to the site at which the tissue injury has occurred which includes but not limited to the brain, spinal cord, coronary arterial vessels, and peripheral arterial vessels
As used herein, the term “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.
As used herein the term “ENPP1 fragment” refers to a fragment or a portion of ENPP1 protein or an active subsequence of the full-length NPP1 having at least an ENPP1 catalytic domain administered in protein form or in the form of a nucleic acid encoding the same.
As used herein, the term “ENPP1 agent” refers to ENPP1 polypeptide or fusion protein or ENPP1 fragment comprising at least catalytic domain capable of producing plasma pyrophosphate (Ppi) by cleavage of adenosine triphosphate (ATP) or a polynucleotide such as cDNA or RNA encoding ENPP1 polypeptide or 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.
As used herein, the term “stent” refers to a tubular support placed inside a blood vessel, canal, or duct to aid healing or relieve an obstruction or prevent narrowing of the passage. Stents generally comprise an expandable mesh coil which is made of metal (ex: stainless steel, Cobalt alloy, Nickel-titanium alloy, manganese alloy, molybdenum alloy, platinum alloy, tungsten alloy) or polymers (ex: Silicone).
As used herein, the term “vascular stent” refers to a tubular support placed inside an artery or vein of a mammal to aid healing or relieve an obstruction or prevent narrowing of the arterial passage.
As used herein, the term “coated stent” or “eluting stent” refers to a stent that is coated with a therapeutic molecule such as protein, chemical compound or nucleic acid that gradually elutes from the stent surface (interior or exterior) at the site of implantation thereby providing therapeutic relief. Therapeutic molecules such as ENPP1 agent or ENPP3 agent can be bonded directly to a metal stent, and some are bonded to a matrix polymer, which acts as a drug reservoir to ensure drug retention during deployment and a uniform distribution on the stent. The types, compositions, and designs of the polymers coated on the stent dictate the eluting kinetic of the sustain time release of the drug over a period of weeks or months following the implantation in situ. The coating materials can be categorized as organic vs inorganic, bioerodable vs nonbioerodable, and synthetic vs naturally occurring substances.
As used herein, the term “coating” refers to composition comprising a polymeric carrier that is used in conjunction with an ENPP1 agent or ENPP3 agent to coat the stents. The coating may be applied in the form a spray or dried film comprising the ENPP1 agent or ENPP3 agent suspended in a polymeric matrix. The polymeric carrier is in an amount sufficient to provide a polymer matrix or support for the ENPP1 agent or ENPP3 agent. The polymer is preferably non-reactive with the ENPP1 agent or ENPP3 agent, i.e., no chemical reaction occurs when the two are mixed.
As used herein, the term “solvent” is defined according to its broadest recognized definition and includes any material into which the carrier (polymer) and the ENPP1 agent or ENPP3 agent can dissolve, fully or partially, at room temperature or from 20° C. to 40° C. to form the coating composition. Sterile, double distilled water is a preferred solvent.
As used herein, the term “site of injury” refers to a region in the vasculature where the flow of blood or spinal fluid is constricted due to accumulation of one or more of lipids, cholesterol, calcium, and various types of cells, such as smooth muscle cells and platelets. The site of injury is commonly identified by using Cardiac catheterization. During a cardiac catheterization, a long, narrow tube called a catheter is inserted through a plastic introducer sheath (a short, hollow tube that is inserted into a blood vessel in your arm or leg). The catheter is guided through the blood vessel to the coronary arteries with the aid of an x-ray machine. Contrast material is injected through the catheter and x-ray images (Coronary angiogram) are created as the contrast material moves through the heart's chambers, valves and major vessels. The digital photographs of the contrast material are used to identify the site of the narrowing or blockage in the coronary artery. Additional imaging procedures, called intra-vascular ultrasound (NUS) and fractional flow reserve (FFR), may be performed along with cardiac catheterization in some cases to obtain detailed images of the walls of the blood vessels.
As used herein “site of implant” refers to the region at which the ENPP1 or ENPP3 coated stent is implanted in the vasculature. The coated stents of the invention can be placed at the center of the to the site of tissue injury, immediately adjacent the site of tissue injury or within 200 μm on either side from the center of the site of tissue injury.
As used herein, the term “myocardial infarction” refers to permanent damage to the heart muscle that occurs due to the formation of plaques in the interior walls of the arteries resulting in reduced blood flow to the heart and injuring heart muscles because of lack of oxygen supply. The symptoms of MI include chest pain, which travels from left arm to neck, shortness of breath, sweating, nausea, vomiting, abnormal heart beating, anxiety, fatigue, weakness, stress, depression, and other factors.
As used herein the term “myocardial ischemia” refers to the condition of the heart muscle that is characterized by a decrease in blood supply to the heart tissue which leads to chest pain or angina pectoris, myocardial infarction is the end point of this ischemia that results in the death of heart tissue due to absence of blood supply. Coronary artery disease (CAD) is considered as a common cause of myocardial ischemia.
As used herein the term “blunt force trauma” refers to physical trauma to a body part, either by impact, injury or physical attack or high-velocity impact. Blunt trauma can lead to contusions, abrasions, lacerations, and/or bone fractures. As used herein the term “non-surgical tissue injury” or “penetrating trauma” refers to trauma to a body part which occurs when an object such as a projectile or knife enters a tissue of the body, creating an open wound.
As used herein the term “scalpel incision” refers to incision made in a tissue using a sharp object such as a scalpel during surgical procedure. An incision is a cut made into the tissues of the body to expose the underlying tissue, bone, so that a surgical procedure can be performed.
As used herein the term “ablation” refers to the removal or destruction of a body part or tissue or its function. Ablation may be performed by surgery, hormones, drugs, radiofrequency, heat.
As used herein, 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. The term also includes within its scope amounts effective to enhance normal physiological function.
As used herein, the term “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.
As used here the term “Isolated” means altered or removed from the natural state. For example, 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 a substantially purified form or can exist in a non-native environment such as, for example, a host cell.
As used herein, “substantially purified” refers to being essentially free of other components. For example, 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.
As used herein the term “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.
As used herein, the term “pharmaceutical composition” or “composition” refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient. 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.
As used herein, 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).
As used herein, the term “pathological calcification” refers to the abnormal deposition of calcium salts in soft tissues, secretory and excretory passages of the body causing it to harden. There are two types, dystrophic calcification which occurs in dying and dead tissue and metastatic calcification which elevated extracellular levels of calcium (hypercalcemia), exceeding the homeostatic capacity of cells and tissues. Calcification can involve cells as well as extracellular matrix components such as collagen in basement membranes and elastic fibers in arterial walls. Some examples of tissues prone to calcification include: Gastric mucosa—the inner epithelial lining of the stomach, Kidneys and lungs, Cornea, Systemic arteries and Pulmonary veins.
As used herein, the term “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 being affected, endochondral ossification is ossification that occurs in and replaces cartilage. Intramembranous ossification is the ossification of bone that occurs in and replaces connective tissue. Metaplastic ossification the development of bony substance in normally soft body structures; called also heterotrophic ossification.
As used herein, “reduction of calcification” is observed by using non-invasive methods like X-rays, micro CT and MRI. Reduction of calcification is also inferred by using radio imaging with 99mTc-pyrophosphate (99mPYP) uptake. The presence of calcifications in mice are evaluated via post-mortem by micro-computed tomography (CT) scans and histologic sections taken from the heart, aorta and kidneys with the use of dyes such as Hematoxylin and Eosin (H&E) and Alizarin red by following protocols established by Braddock et al. (Nature Communications volume 6, Article number: 10006 (2015))
A “low level of PPi” refers to a condition in which the subject has less than or equal to 2%-5% of normal levels of plasma pyrophosphate (PPi). Normal levels of Plasma PPi in healthy human subjects is approximately 1.8 to 2.6 μM. (Arthritis and Rheumatism, Vol. 22, No. 8 (August 1979))
As used herein the term “Ectopic calcification” refers to a condition characterized by a pathologic deposition of calcium salts in tissues or bone growth in soft tissues.
As used herein the term “Ectopic calcification of soft tissue” refers to inappropriate biomineralization, typically composed of calcium phosphate, hydroxyapatite, calcium oxalates and ocatcalcium phosphates occurring in soft tissues leading to loss of hardening of soft tissues. “Arterial 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.
As used herein, the term “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.
As used herein, the term “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.
As used herein, the term “Brain calcification” (BC) 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.
The terms “adeno-associated viral vector”, “AAV vector”, “adeno-associated virus”, “AAV virus”, “AAV virion”, “AAV viral particle” and “AAV particle”, as used interchangeably herein, refer to a viral particle composed of at least one AAV capsid protein (preferably by all of the capsid proteins of a particular AAV serotype) and an encapsidated recombinant viral genome. The particle comprises a recombinant viral genome having a heterologous polynucleotide comprising a sequence encoding human ENPP1 or human ENPP3 or a functionally equivalent variant thereof) and a transcriptional regulatory region that at least comprises a promoter flanked by the AAV inverted terminal repeats. The particle is typically referred to as an “AAV vector particle” or “AAV vector”.
As used herein, the term “vector” means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments, the vector is a plasmid, i.e., a circular double stranded DNA loop into which additional DNA segments may be ligated. In some embodiments, the vector is a viral vector, wherein additional nucleotide sequences may be ligated into the viral genome. In some embodiments, 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). In other embodiments, the vectors (e.g., non-episomal mammalian 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. Moreover, certain vectors (expression vectors) are capable of directing the expression of genes to which they are operatively linked.
As used herein, the term “recombinant host cell” (or simply “host cell”), as used herein, 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.
The term “recombinant viral genome”, as used herein, refers to an AAV genome in which at least one extraneous expression cassette polynucleotide is inserted into the naturally occurring AAV genome. The genome of the AAV according to the disclosure typically comprises the cis-acting 5′ and 3′ inverted terminal repeat sequences (ITRs) and an expression cassette.
The term “expression cassette”, as used herein, 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.
The term “transcriptional regulatory region”, as used herein, 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.
The term “promoter”, as used herein, 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.
The term “enhancer”, as used herein, refers to a DNA sequence element to which transcription factors bind to increase gene transcription. Examples of enhancers may be, without limitation, RSV enhancer, CMV enhancer, HCR enhancer, etc. In another embodiment, the enhancer is a liver-specific enhancer, more preferably a hepatic control region enhancer (HCR).
The term “operatively linked”, as used herein, 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). Generally, a promoter operatively linked is contiguous to the sequence of interest. However, an enhancer does not have to be contiguous to the sequence of interest to control its expression. In another embodiment, the promoter and the nucleotide sequence encoding ENPP1 or ENPP3 or a functionally equivalent variant thereof.
The term “effective amount” 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.
The term “Cap protein”, as used herein, refers to a polypeptide having at least one functional activity of a native AAV Cap protein (e.g. VP1, VP2, VP3). Examples of 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. In principle, any Cap protein can be used in the context of the present disclosure.
The term “capsid”, as used herein, refers to the structure in which the viral genome is packaged. A capsid consists of several oligomeric structural subunits made of proteins. For instance, AAV have an icosahedral capsid formed by the interaction of three capsid proteins: VP1, VP2 and VP3.
The term “Rep protein”, as used herein, 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.
The term “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, G B, 1995; Watson R, et al., “Recombinant DNA”, 2nd Ed. Scientific American Books, New York, N.Y., US, 1992; Alberts B, et al., “Molecular Biology of the Cell”, Garland Publishing Inc., New York, N.Y., US, 2008; Innis M, et al., Eds., “PCR Protocols. A Guide to Methods and Applications”, Academic Press Inc., San Diego, Calif., US, 1990; and Schleef M, Ed., “Plasmid for Therapy and Vaccination”, Wiley-VCH Verlag GmbH, Weinheim, Del., 2001).
The term “tissue-specific” promoter is only active in specific types of differentiated cells or tissues. Typically, the downstream gene in a tissue-specific promoter is one which is active to a much higher degree in the tissue(s) for which it is specific than in any other. In this case there may be little or substantially no activity of the promoter in any tissue other than the one(s) for which it is specific.
The term “inducible promoter”, as used herein, refers to a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer. For example, it can be a tetracycline-inducible promoter, a mifepristone (RU-486)-inducible promoter and the like.
The term “constitutive promoter”, as used herein, 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. In another embodiment, the transcriptional regulatory region allows constitutive expression of ENPP1. Examples of 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 β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter (Boshart M, et al., Cell 1985; 41:521-530).
The term “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 EIb polyadenylation signal, the bovine growth hormone polyadenylation signal, the human variant growth hormone polyadenylation signal and the like.
The term “signal peptide”, as used herein, refers to a sequence of amino acid residues (ranging in length from 10-30 residues) bound at the amino terminus of a nascent protein of interest during protein translation. The signal peptide is recognized by the signal recognition particle (SRP) and cleaved by the signal peptidase following transport at the endoplasmic reticulum. (Lodish et al., 2000, Molecular Cell Biology, 4th edition).
As used herein, the term “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.
As used herein, the term “immunosuppression” is a deliberate reduction of the activation or efficacy of the host immune system using immunosuppressant drugs to facilitate immune tolerance towards foreign antigens such as foreign proteins, bone marrow and tissue transplantation. Non limiting examples of immunosuppressant drugs include anti-CD4(GK1.5) antibody, Cyclophosphamide, Azathioprine (Imuran), Mycophenolate mofetil (Cellcept), Cyclosporine (Neoral, Sandimmune, Gengraf), Methotrexate (Rheumatrex), Leflunomide (Arava), Cyclophosphamide (Cytoxan) and Chlorambucil (Leukeran).
Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. 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 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
The present disclosure relates to administration of an ENPP1 or ENPP3 agent, which includes administering sNPP1 and sNPP3 polypeptides and fusion proteins thereof to a subject, and to administration of nucleic acids encoding such polypeptides. Sequences of such polypeptides include the following, without limitation.
MTRLTVLALLAGLLASSRA**A
PSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHI
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGK
MTRLTVLALLAGLLASSRA**A
PSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHI
SYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQ
HKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADK
ESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKE
CCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQE
VCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEP
KNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLS
AILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIK
KQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALARSWSHPQFEK
MTRLTVLALLAGLLASSRA**A
PSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHI
SCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEKRLTRSLCACSDD
MTRLTVLALLAGLLASSRA**A
KQGSCRKKCFDASFRGLENCRCDVACKDRGDCCWDFEDTCVES
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
MTRLTVLALLAGLLASSRA**A
KQGSCRKKCFDASFRGLENCRCDVACKDRGDCCWDFEDTCVES
CSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFL
QHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEAD
KESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNK
ECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQ
EVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEE
PKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYL
SAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQI
KKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALARSWSHPQFE
K
MTRLTVLALLAGLLASSRA**A
KQGSCRKKCFDASFRGLENCRCDVACKDRGDCCWDFEDTCVES
Met Thr Ser Lys Phe Leu Leu Val Ser Phe Ile Leu Ala Ala Leu Ser
Leu Ser Thr Thr Phe Ser Leu Gln**Pro Ser Cys Ala Lys Glu Val Lys
Glu Asp
Met Thr Ser Lys Phe Leu Leu Val Ser Phe Ile Leu Ala Ala Leu Ser
Leu Ser Thr Thr Phe Ser Leu Gln**Pro Ser Cys Ala Lys Glu Val Lys
Glu Asp Gly Gly Ser Gly Gly Ser Met Lys Trp Val Thr Phe Leu Leu
Leu Leu Phe Val Ser Gly Ser Ala Phe Ser Arg Gly Val Phe Arg Arg
Glu Ala His Lys Ser Glu Ile Ala His Arg Tyr Asn Asp Leu Gly Glu
Gln His Phe Lys Gly Leu Val Leu Ile Ala Phe Ser Gln Tyr Leu Gln
Lys Cys Ser Tyr Asp Glu His Ala Lys Leu Val Gln Glu Val Thr Asp
Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Ala Asn Cys Asp Lys
Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Ala Ile Pro Asn Leu
Arg Glu Asn Tyr Gly Glu Leu Ala Asp Cys Cys Thr Lys Gln Glu Pro
Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Ser Leu
Pro Pro Phe Glu Arg Pro Glu Ala Glu Ala Met Cys Thr Ser Phe Lys
Glu Asn Pro Thr Thr Phe Met Gly His Tyr Leu His Glu Val Ala
Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Tyr Tyr Ala
Glu Gln Tyr Asn Glu Ile Leu Thr Gln Cys Cys Ala Glu Ala Asp
Lys Glu Ser Cys Leu Thr Pro Lys Leu Asp Gly Val Lys Glu Lys
Ala Leu Val Ser Ser Val Arg Gln Arg Met Lys Cys Ser Ser Met
Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg
Leu Ser Gln Thr Phe Pro Asn Ala Asp Phe Ala Glu Ile Thr Lys
Leu Ala Thr Asp Leu Thr Lys Val Asn Lys Glu Cys Cys His Gly
Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Glu Leu Ala Lys Tyr
Met Cys Glu Asn Gln Ala Thr Ile Ser Ser Lys Leu Gln Thr Cys
Cys Asp Lys Pro Leu Leu Lys Lys Ala His Cys Leu Ser Glu Val
Glu His Asp Thr Met Pro Ala Asp Leu Pro Ala Ile Ala Ala Asp
Phe Val Glu Asp Gln Glu Val Cys Lys Asn Tyr Ala Glu Ala Lys
Asp Val Phe Leu Gly Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His
Pro Asp Tyr Ser Val Ser Leu Leu Leu Arg Leu Ala Lys Lys Tyr
Glu Ala Thr Leu Glu Lys Cys Cys Ala Glu Ala Asn Pro Pro Ala
Cys Tyr Gly Thr Val Leu Ala Glu Phe Gln Pro Leu Val Glu Glu
Pro Lys Asn Leu Val Lys Thr Asn Cys Asp Leu Tyr Glu Lys Leu
Gly Glu Tyr Gly Phe Gln Asn Ala Ile Leu Val Arg Tyr Thr Gln
Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu Ala Ala Arg
Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu Pro Glu Asp
Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile Leu Asn
Arg Val Cys Leu Leu His Glu Lys Thr Pro Val Ser Glu His Val
Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys Phe
Ser Ala Leu Thr Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys
Ala Glu Thr Phe Thr Phe His Ser Asp Ile Cys Thr Leu Pro Glu
Lys Glu Lys Gln Ile Lys Lys Gln Thr Ala Leu Ala Glu Leu Val
Lys His Lys Pro Lys Ala Thr Ala Glu Gln Leu Lys Thr Val Met
Asp Asp Phe Ala Gln Phe Leu Asp Thr Cys Cys Lys Ala Ala Asp
Lys Asp Thr Cys Phe Ser Thr Glu Gly Pro Asn Leu Val Thr Arg
Cys Lys Asp Ala Leu Ala Arg Ser Trp Ser His Pro Gln Phe Glu
Lys
Met Thr Ser Lys Phe Leu Leu Val Ser Phe Ile Leu Ala Ala Leu Ser
Leu Ser Thr Thr Phe Ser**Lys Gln Gly Ser Cys Arg Lys Lys Cys Phe
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys
Met Thr Ser Lys Phe Leu Leu Val Ser Phe Ile Leu Ala Ala Leu Ser
Leu Ser Thr Thr Phe Ser**Lys Gln Gly Ser Cys Arg Lys Lys Cys Phe
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Lys Trp
Val Thr Phe Leu Leu Leu Leu Phe Val Ser Gly Ser Ala Phe Ser Arg
Gly Val Phe Arg Arg Glu Ala His Lys Ser Glu Ile Ala His Arg Tyr
Asn Asp Leu Gly Glu Gln His Phe Lys Gly Leu Val Leu Ile Ala Phe
Ser Gln Tyr Leu Gln Lys Cys Ser Tyr Asp Glu His Ala Lys Leu Val
Gln Glu Val Thr Asp Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala
Ala Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys
Ala Ile Pro Asn Leu Arg Glu Asn Tyr Gly Glu Leu Ala Asp Cys Cys
Thr Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp
Asp Asn Pro Ser Leu Pro Pro Phe Glu Arg Pro Glu Ala Glu Ala Met
Cys Thr Ser Phe Lys Glu Asn Pro Thr Thr Phe Met Gly His Tyr
Leu His Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Tyr Tyr Ala Glu Gln Tyr Asn Glu Ile Leu Thr Gln Cys
Cys Ala Glu Ala Asp Lys Glu Ser Cys Leu Thr Pro Lys Leu Asp
Gly Val Lys Glu Lys Ala Leu Val Ser Ser Val Arg Gln Arg Met
Lys Cys Ser Ser Met Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala
Trp Ala Val Ala Arg Leu Ser Gln Thr Phe Pro Asn Ala Asp Phe
Ala Glu Ile Thr Lys Leu Ala Thr Asp Leu Thr Lys Val Asn Lys
Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala
Glu Leu Ala Lys Tyr Met Cys Glu Asn Gln Ala Thr Ile Ser Ser
Lys Leu Gln Thr Cys Cys Asp Lys Pro Leu Leu Lys Lys Ala His
Cys Leu Ser Glu Val Glu His Asp Thr Met Pro Ala Asp Leu Pro
Ala Ile Ala Ala Asp Phe Val Glu Asp Gln Glu Val Cys Lys Asn
Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Thr Phe Leu Tyr Glu
Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser Leu Leu Leu Arg
Leu Ala Lys Lys Tyr Glu Ala Thr Leu Glu Lys Cys Cys Ala Glu
Ala Asn Pro Pro Ala Cys Tyr Gly Thr Val Leu Ala Glu Phe Gln
Pro Leu Val Glu Glu Pro Lys Asn Leu Val Lys Thr Asn Cys Asp
Leu Tyr Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Ile Leu
Val Arg Tyr Thr Gln Lys Ala Pro Gln Val Ser Thr Pro Thr Leu
Val Glu Ala Ala Arg Asn Leu Gly Arg Val Gly Thr Lys Cys Cys
Thr Leu Pro Glu Asp Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu
Ser Ala Ile Leu Asn Arg Val Cys Leu Leu His Glu Lys Thr Pro
Val Ser Glu His Val Thr Lys Cys Cys Ser Gly Ser Leu Val Glu
Arg Arg Pro Cys Phe Ser Ala Leu Thr Val Asp Glu Thr Tyr Val
Pro Lys Glu Phe Lys Ala Glu Thr Phe Thr Phe His Ser Asp Ile
Cys Thr Leu Pro Glu Lys Glu Lys Gln Ile Lys Lys Gln Thr Ala
Leu Ala Glu Leu Val Lys His Lys Pro Lys Ala Thr Ala Glu Gln
Leu Lys Thr Val Met Asp Asp Phe Ala Gln Phe Leu Asp Thr Cys
Cys Lys Ala Ala Asp Lys Asp Thr Cys Phe Ser Thr Glu Gly Pro
Asn Leu Val Thr Arg Cys Lys Asp Ala Leu Ala
Met Thr Ser Lys Phe Leu Leu Val Ser Phe Ile Leu Ala Ala Leu Ser
Leu Ser Thr Thr Phe Ser**Gly Leu Lys Pro Ser Cys Ala Lys Glu Val
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu
Ala Pro Gly Ala Gly Ala**Gly Leu Lys Pro Ser Cys Ala Lys Glu Val
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu
Ala Pro Gly Ala Gly Ala**Pro Ser Cys Ala Lys Glu Val Lys Ser Cys
Asp
Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu
Ala Pro Gly Ala Gly Ala**Pro Ser Cys Ala Lys Glu Val Lys Ser Cys
Asp Leu Ile Asn Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu
Ala Pro Gly Ala Gly Ala**Pro Ser Cys Ala Lys Glu Val Lys Ser Cys
Asp Arg Ser Gly Ser Gly Gly Ser Met Lys Trp Val Thr Phe Leu Leu
Leu Leu Phe Val Ser Gly Ser Ala Phe Ser Arg Gly Val Phe Arg Arg
Glu Ala His Lys Ser Glu Ile Ala His Arg Tyr Asn Asp Leu Gly Glu
Gln His Phe Lys Gly Leu Val Leu Ile Ala Phe Ser Gln Tyr Leu Gln
Lys Cys Ser Tyr Asp Glu His Ala Lys Leu Val Gln Glu Val Thr Asp
Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Ala Asn Cys Asp Lys
Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Ala Ile Pro Asn Leu
Arg Glu Asn Tyr Gly Glu Leu Ala Asp Cys Cys Thr Lys Gln Glu Pro
Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Ser Leu
Pro Pro Phe Glu Arg Pro Glu Ala Glu Ala Met Cys Thr Ser Phe Lys
Glu Asn Pro Thr Thr Phe Met Gly His Tyr Leu His Glu Val Ala
Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Tyr Tyr Ala
Glu Gln Tyr Asn Glu Ile Leu Thr Gln Cys Cys Ala Glu Ala Asp
Lys Glu Ser Cys Leu Thr Pro Lys Leu Asp Gly Val Lys Glu Lys
Ala Leu Val Ser Ser Val Arg Gln Arg Met Lys Cys Ser Ser Met
Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg
Leu Ser Gln Thr Phe Pro Asn Ala Asp Phe Ala Glu Ile Thr Lys
Leu Ala Thr Asp Leu Thr Lys Val Asn Lys Glu Cys Cys His Gly
Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Glu Leu Ala Lys Tyr
Met Cys Glu Asn Gln Ala Thr Ile Ser Ser Lys Leu Gln Thr Cys
Cys Asp Lys Pro Leu Leu Lys Lys Ala His Cys Leu Ser Glu Val
Glu His Asp Thr Met Pro Ala Asp Leu Pro Ala Ile Ala Ala Asp
Phe Val Glu Asp Gln Glu Val Cys Lys Asn Tyr Ala Glu Ala Lys
Asp Val Phe Leu Gly Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His
Pro Asp Tyr Ser Val Ser Leu Leu Leu Arg Leu Ala Lys Lys Tyr
Glu Ala Thr Leu Glu Lys Cys Cys Ala Glu Ala Asn Pro Pro Ala
Cys Tyr Gly Thr Val Leu Ala Glu Phe Gln Pro Leu Val Glu Glu
Pro Lys Asn Leu Val Lys Thr Asn Cys Asp Leu Tyr Glu Lys Leu
Gly Glu Tyr Gly Phe Gln Asn Ala Ile Leu Val Arg Tyr Thr Gln
Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu Ala Ala Arg
Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu Pro Glu Asp
Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile Leu Asn
Arg Val Cys Leu Leu His Glu Lys Thr Pro Val Ser Glu His Val
Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys Phe
Ser Ala Leu Thr Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys
Ala Glu Thr Phe Thr Phe His Ser Asp Ile Cys Thr Leu Pro Glu
Lys Glu Lys Gln Ile Lys Lys Gln Thr Ala Leu Ala Glu Leu Val
Lys His Lys Pro Lys Ala Thr Ala Glu Gln Leu Lys Thr Val Met
Asp Asp Phe Ala Gln Phe Leu Asp Thr Cys Cys Lys Ala Ala Asp
Lys Asp Thr Cys Phe Ser Thr Glu Gly Pro Asn Leu Val Thr Arg
Cys Lys Asp Ala Leu Ala Arg Ser Trp Ser His Pro Gln Phe Glu
Lys
Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu
Ala Pro Gly Ala**Lys Gln Gly Ser Cys Arg Lys Lys Cys Phe Asp Ala
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys
Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu
Ala Pro Gly Ala Gly Leu Lys**Pro Ser Cys Ala Lys Glu Val Lys Ser
Glu Asp Gly Gly Ser Gly Gly Ser Met Lys Trp Val Thr Phe Leu Leu
Leu Leu Phe Val Ser Gly Ser Ala Phe Ser Arg Gly Val Phe Arg Arg
Glu Ala His Lys Ser Glu Ile Ala His Arg Tyr Asn Asp Leu Gly Glu
Gln His Phe Lys Gly Leu Val Leu Ile Ala Phe Ser Gln Tyr Leu Gln
Lys Cys Ser Tyr Asp Glu His Ala Lys Leu Val Gln Glu Val Thr Asp
Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Ala Asn Cys Asp Lys
Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Ala Ile Pro Asn Leu
Arg Glu Asn Tyr Gly Glu Leu Ala Asp Cys Cys Thr Lys Gln Glu Pro
Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Ser Leu
Pro Pro Phe Glu Arg Pro Glu Ala Glu Ala Met Cys Thr Ser Phe Lys
Glu Asn Pro Thr Thr Phe Met Gly His Tyr Leu His Glu Val Ala
Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Tyr Tyr Ala
Glu Gln Tyr Asn Glu Ile Leu Thr Gln Cys Cys Ala Glu Ala Asp
Lys Glu Ser Cys Leu Thr Pro Lys Leu Asp Gly Val Lys Glu Lys
Ala Leu Val Ser Ser Val Arg Gln Arg Met Lys Cys Ser Ser Met
Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg
Leu Ser Gln Thr Phe Pro Asn Ala Asp Phe Ala Glu Ile Thr Lys
Leu Ala Thr Asp Leu Thr Lys Val Asn Lys Glu Cys Cys His Gly
Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Glu Leu Ala Lys Tyr
Met Cys Glu Asn Gln Ala Thr Ile Ser Ser Lys Leu Gln Thr Cys
Cys Asp Lys Pro Leu Leu Lys Lys Ala His Cys Leu Ser Glu Val
Glu His Asp Thr Met Pro Ala Asp Leu Pro Ala Ile Ala Ala Asp
Phe Val Glu Asp Gln Glu Val Cys Lys Asn Tyr Ala Glu Ala Lys
Asp Val Phe Leu Gly Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His
Pro Asp Tyr Ser Val Ser Leu Leu Leu Arg Leu Ala Lys Lys Tyr
Glu Ala Thr Leu Glu Lys Cys Cys Ala Glu Ala Asn Pro Pro Ala
Cys Tyr Gly Thr Val Leu Ala Glu Phe Gln Pro Leu Val Glu Glu
Pro Lys Asn Leu Val Lys Thr Asn Cys Asp Leu Tyr Glu Lys Leu
Gly Glu Tyr Gly Phe Gln Asn Ala Ile Leu Val Arg Tyr Thr Gln
Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu Ala Ala Arg
Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu Pro Glu Asp
Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile Leu Asn
Arg Val Cys Leu Leu His Glu Lys Thr Pro Val Ser Glu His Val
Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys Phe
Ser Ala Leu Thr Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys
Ala Glu Thr Phe Thr Phe His Ser Asp Ile Cys Thr Leu
Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu
Ala Pro Gly Ala**Lys Gln Gly Ser Cys Arg Lys Lys Cys Phe Asp Ala
Phe Leu Leu Leu Leu Phe Val Ser Gly Ser Ala Phe Ser Arg Gly Val
Phe Arg Arg Glu Ala His Lys Ser Glu Ile Ala His Arg Tyr Asn Asp
Leu Gly Glu Gln His Phe Lys Gly Leu Val Leu Ile Ala Phe Ser Gln
Tyr Leu Gln Lys Cys Ser Tyr Asp Glu His Ala Lys Leu Val Gln Glu
Val Thr Asp Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Ala Asn
Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Ala Ile
Pro Asn Leu Arg Glu Asn Tyr Gly Glu Leu Ala Asp Cys Cys Thr Lys
Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn
Pro Ser Leu Pro Pro Phe Glu Arg Pro Glu Ala Glu Ala Met Cys Thr
Ser Phe Lys Glu Asn Pro Thr Thr Phe Met Gly His Tyr Leu His
Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu
Tyr Tyr Ala Glu Gln Tyr Asn Glu Ile Leu Thr Gln Cys Cys Ala
Glu Ala Asp Lys Glu Ser Cys Leu Thr Pro Lys Leu Asp Gly Val
Lys Glu Lys Ala Leu Val Ser Ser Val Arg Gln Arg Met Lys Cys
Ser Ser Met Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala
Val Ala Arg Leu Ser Gln Thr Phe Pro Asn Ala Asp Phe Ala Glu
Ile Thr Lys Leu Ala Thr Asp Leu Thr Lys Val Asn Lys Glu Cys
Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Glu Leu
Ala Lys Tyr Met Cys Glu Asn Gln Ala Thr Ile Ser Ser Lys Leu
Gln Thr Cys Cys Asp Lys Pro Leu Leu Lys Lys Ala His Cys Leu
Ser Glu Val Glu His Asp Thr Met Pro Ala Asp Leu Pro Ala Ile
Ala Ala Asp Phe Val Glu Asp Gln Glu Val Cys Lys Asn Tyr Ala
Glu Ala Lys Asp Val Phe Leu Gly Thr Phe Leu Tyr Glu Tyr Ser
Arg Arg His Pro Asp Tyr Ser Val Ser Leu Leu Leu Arg Leu Ala
Lys Lys Tyr Glu Ala Thr Leu Glu Lys Cys Cys Ala Glu Ala Asn
Pro Pro Ala Cys Tyr Gly Thr Val Leu Ala Glu Phe Gln Pro Leu
Val Glu Glu Pro Lys Asn Leu Val Lys Thr Asn Cys Asp Leu Tyr
Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Ile Leu Val Arg
Tyr Thr Gln Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu
Ala Ala Arg Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu
Pro Glu Asp Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala
Ile Leu Asn Arg Val Cys Leu Leu His Glu Lys Thr Pro Val Ser
Glu His Val Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg
Pro Cys Phe Ser Ala Leu Thr Val Asp Glu Thr Tyr Val Pro Lys
Glu Phe Lys Ala Glu Thr Phe Thr Phe His Ser Asp Ile Cys Thr
Leu Pro Glu Lys Glu Lys Gln Ile Lys Lys Gln Thr Ala Leu Ala
Glu Leu Val Lys His Lys Pro Lys Ala Thr Ala Glu Gln Leu Lys
Thr Val Met Asp Asp Phe Ala Gln Phe Leu Asp Thr Cys Cys Lys
Ala Ala Asp Lys Asp Thr Cys Phe Ser Thr Glu Gly Pro Asn Leu
Val Thr Arg Cys Lys Asp Ala Leu Ala
Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu
Ala Pro Gly Ala**Lys Gln Gly Ser Cys Arg Lys Lys Cys Phe Asp Ala
Gly Ser Ala Phe Ser Arg Gly Val Phe Arg Arg Glu Ala His Lys Ser
Glu Ile Ala His Arg Tyr Asn Asp Leu Gly Glu Gln His Phe Lys Gly
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Glu His Ala Lys Leu Val Gln Glu Val Thr Asp Phe Ala Lys Thr Cys
Val Ala Asp Glu Ser Ala Ala Asn Cys Asp Lys Ser Leu His Thr Leu
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Glu Leu Ala Asp Cys Cys Thr Lys Gln Glu Pro Glu Arg Asn Glu
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Asp Glu His Ala Lys Leu Val Gln Glu Val Thr Asp Phe Ala Lys Thr
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Ala Glu Gln Tyr Asn Glu Ile Leu Thr Gln Cys Cys Ala Glu Ala
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Met Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala
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ct
ccttcctgcgccaaagaagtgaagtcctgcaagggcagatgcttcgagcggaccttcggcaactgtag
ENPP1, or an ENPP1 polypeptide, is prepared as described in US 2015/0359858 A1, which is incorporated herein in its entirety by reference. ENPP1 is a transmembrane protein localized to the cell surface with distinct intramembrane domains. In order to express ENPP1 as a soluble extracellular protein, the transmembrane domain of ENPP1 may be swapped for the transmembrane domain of ENPP2 or a signal peptide sequence such as Azurocidin, which results in the accumulation of soluble, recombinant ENPP1 in the extracellular fluid of the baculovirus cultures. Signal sequences of any other known proteins may be used to target the extracellular domain of ENPP1 for secretion as well, such as but not limited to the signal sequence of the immunoglobulin kappa and lambda light chain proteins. Further, the disclosure should not be construed to be limited to the polypeptides described herein, but also includes polypeptides comprising any enzymatically active truncation of the ENPP1 extracellular domain.
ENPP1 is made soluble by omitting the transmembrane domain. Human ENPP1 (SEQ ID NO:1) was modified to express a soluble, recombinant protein by replacing its transmembrane region (e.g., residues 77-98) with the corresponding subdomain of human ENPP2 (NCBI accession NP 00112433 5, e.g., residues 12-30) or Azurocidin signal sequence (SEQ ID NO: 42).
The modified ENPP1 sequence was cloned into a modified pFastbac FIT vector possessing a TEV protease cleavage site followed by a C-terminus 9-His tag, and cloned and expressed in insect cells, and both proteins were expressed in a baculovirus system as described previously (Albright, et al., 2012, Blood 120:4432-4440; Saunders, et al., 2011, J. Biol. Chem. 18:994-1004; Saunders, et al., 2008, Mol. Cancer Ther. 7:3352-3362), resulting in the accumulation of soluble, recombinant protein in the extracellular fluid.
ENPP3 is poorly exported to the cell surface. Soluble ENPP3 polypeptide is constructed by replacing the signal sequence of ENPP3 with the native signal sequence of other ENPPs or Azurocidin or suitable signal sequences. Several examples of ENPP3 fusion constructs are disclosed in WO 2017/087936. Soluble ENPP3 constructs are prepared by using the signal export signal sequence of other ENPP enzymes, such as but not limited to ENPP7 and/or ENPP5. Soluble ENPP3 constructs are prepared using a signal sequence comprised of a combination of the signal sequences of ENPP1 and ENPP2 (“ENPP1-2-1” or “ENPP121” hereinafter). Signal sequences of any other known proteins may be used to target the extracellular domain of ENPP3 for secretion as well, such as but not limited to the signal sequence of the immunoglobulin kappa and lambda light chain proteins. Further, the disclosure should not be construed to be limited to the constructs described herein, but also includes constructs comprising any enzymatically active truncation of the ENPP3 extracellular domain.
In certain embodiments, the ENPP3 polypeptide is soluble. In some embodiments, the polypeptide of the disclosure includes an ENPP3 polypeptide that lacks the ENPP3 transmembrane domain. In another embodiment, the polypeptide of the disclosure includes an ENPP3 polypeptide wherein the ENPP3 transmembrane domain has been removed and replaced with the transmembrane domain of another polypeptide, such as, by way of non-limiting example, ENPP2, ENPP5 or ENPP7 or Azurocidin signal sequence.
In some embodiments, the polypeptide of the disclosure comprises an IgG Fc domain. In certain embodiments, the polypeptide of the disclosure comprises an albumin domain. In other embodiments, the albumin domain is located at the C terminal region of the ENPP3 polypeptide. In yet other embodiments, the IgG Fc domain is located at the C terminal region of the ENPP3 polypeptide. In yet other embodiments, the presence of IgG Fc domain or albumin domain improves half-life, solubility, reduces immunogenicity and increases the activity of the ENPP3 polypeptide.
In certain embodiments, the polypeptide of the disclosure comprises a signal peptide resulting in the secretion of a precursor of the ENPP3 polypeptide, which undergoes proteolytic processing to yield the ENPP3 polypeptide. In other embodiments, the signal peptide is selected from the group consisting of signal peptides of ENPP2, ENPP5 and ENPP7. In yet other embodiments, the signal peptide is selected from the group consisting of SEQ ID NOs:36-42.
In certain embodiments, the IgG Fc domain or the albumin domain is connected to the C terminal region of the ENPP3 polypeptide by a linker region. In other embodiments, the linker is selected from SEQ ID NOs:43-75, where n is an integer ranging from 1-20.
To produce soluble, recombinant ENPP1 polypeptide for in vitro use, polynucleotide encoding ENPP1 (Human NPP1 (NCBI accession NP 006199)) was fused to the Fc domain of IgG (referred to as “ENPP1-Fc”) and was expressed in stable CHO cell lines. In some embodiments, ENPP1 polynucleotide encoding residues 96 to 925 of NCBI accession NP_006199 were fused to Fc domain to generate ENPP1 polypeptide.
Alternately the ENPP1 polypeptide can also be expressed from HEK293 cells, Baculovirus insect cell system or CHO cells or Yeast Pichia expression system using suitable vectors. The ENPP1 polypeptide can be produced in either adherent or suspension cells. Preferably the ENPP1 polypeptide is expressed in CHO cells. To establish stable cell lines the nucleic acid sequence encoding ENPP1 constructs are cloned into an appropriate vector for large scale protein production.
ENPP3 is produced by establishing stable transfections in either CHO or HEK293 mammalian cells. ENPP3 polynucleotide encoding ENPP3 (Human NPP3 (UniProtKB/Swiss-Prot: O14638.2) was fused to the Fc domain of IgG (referred to as “ENPP3-Fc”) and was expressed in stable CHO cell lines. In some embodiments, ENPP3 polynucleotide encoding residues 49-875 of UniProtKB/Swiss-Prot: O14638.2 was fused to Fc domain to generate ENPP3 polypeptide. The ENPP3 polypeptide can be produced in either adherent or suspension cells. To establish stable cell lines the nucleic acid sequence encoding NPP3 fusion polypeptides of the disclosure into an appropriate vector for large scale protein production. There are a variety of these vectors available from commercial sources and any of those can be used. ENPP3 polypeptides are produced following the protocols established in WO 2017/087936, the contents of which are hereby incorporated by reference in their entirety. ENPP1 polypeptides are produced following the protocols established in Albright, et al, 2015, Nat Commun. 6:10006, the contents of which are hereby incorporated by reference in their entirety.
A suitable plasmid containing the desired polypeptide constructs of ENPP1 or ENPP3 can be stably transfected into expression plasmid using established techniques such as electroporation or lipofectamine, and the cells can be grown under antibiotic selection to enhance for stably transfected cells. Clones of single, stably transfected cells are then established and screened for high expressing clones of the desired fusion protein. Screening of the single cell clones for ENPP1 or ENPP3 polypeptide expression can be accomplished in a high-throughput manner in 96 well plates using the synthetic enzymatic substrate pNP-TMP as previously described (Saunders, et al, 2008, Mol. Cancer Therap. 7(10):3352-62; Albright, et al, 2015, Nat Commun. 6:10006).
Upon identification of high expressing clones for ENPP3 or ENPP1 polypeptides through screening, protein production can be accomplished in shaking flasks or bio-reactors previously described for ENPP1 (Albright, et al, 2015, Nat Commun. 6:10006). Purification of ENPP3 or ENPP1 polypeptides can be accomplished using a combination of standard purification techniques known in the art. These techniques are well known in the art and are selected from techniques such as column chromatography, ultracentrifugation, filtration, and precipitation. Column chromatographic purification is accomplished using affinity chromatography such as protein-A and protein-G resins, metal affinity resins such as nickel or copper, hydrophobic exchange chromatography, and reverse-phase high-pressure chromatography (HPLC) using C8-C14 resins. Ion exchange may also be employed, such as anion and cation exchange chromatography using commercially available resins such as Q-sepharose (anion exchange) and SP-sepharose (cation exchange), blue sepharose resin and blue-sephadex resin, and hydroxyapatite resins. Size exclusion chromatography using commercially available S-75 and S200 Superdex resins can also be employed, as known in the art. Buffers used to solubilize the protein and provide the selection media for the above described chromatographic steps, are standard biological buffers known to practitioners of the art and science of protein chemistry.
Some examples of buffers that are used in preparation include citrate, phosphate, acetate, tris(hydroxymemyl)aminomethane, saline buffers, glycine-HCL buffers, Cacodylate buffers, and sodium barbital buffers, which are well known in art. Using a single technique, or a series of techniques in combination, and the appropriate buffer systems purified ENPP3 and the crude starting material side by side on a Coomasie stained polyacrylamide gel after a single purification step. The ENPP3 protein can then be additionally purified using additional techniques and/or chromatographic steps as described above, to reach substantially higher purity such as −99% purity adjusted to the appropriate pH, one can purify the ENPP1 or ENPP3 polypeptides described to greater than 99% purity from crude material.
Following purification, ENPP1-Fc or ENPP3-Fc was dialyzed into PBS supplemented with Zn2+ and Mg2+(PBSplus) concentrated to between 5 and 7 mg/ml, and frozen at −80° C. in aliquots of 200-500 μl. Aliquots were thawed immediately prior to use and the specific activity of the solution was adjusted to 31.25 au/ml (or about 0.7 mg/ml depending on the preparation) by dilution in PBSplus.
In another embodiment, the hsNPP1 or hsNPP3 is administered in one or more doses containing about 1.0 mg/kg to about 5.0 mg/kg NPP1 or about 1.0 mg/kg to about 5.0 mg/kg NPP3 respectively. In another embodiment, the hsNPP1 or hsNPP3 is administered in one or more doses containing about 1.0 mg/kg to about 10.0 mg/kg NPP1 or about 1.0 mg/kg to about 10.0 mg/kg NPP3.
The time period between doses of the hsNPP1 or hsNPP3 is at least 2 days and can be longer, for example at least 3 days, at least 1 week, 2 weeks or 1 month. In one embodiment, the administration is weekly, bi-weekly, or monthly.
The recombinant hsNPP1 or hsNPP3 can be administered in any suitable way, such as intravenously, subcutaneously, or intraperitoneally.
The recombinant hsNPP1 or hsNPP3 can be administered in combination with one or more additional therapeutic agents. Exemplary therapeutic agents include, but are not limited to Bisphosphonate, Statins, Fibrates, Niacin, Aspirin, Clopidogrel, and warfarin.
In some embodiments, the recombinant hsNPP1 or hsNPP3 and additional therapeutic agents are administered separately and are administered concurrently or sequentially. In some embodiments, the recombinant hsNPP1 or hsNPP3 is administered prior to the administration of the additional therapeutic agent. In some embodiments, the recombinant hsNPP1 or hsNPP3 is administered after the administration of the additional therapeutic agent. In other embodiments, the recombinant hsNPP1 or hsNPP3 and additional therapeutic agents are administered together.
Viral Vectors for In Vivo Expression of ENPP1 and ENPP3
The nucleic acids encoding the polypeptide(s) useful within the disclosure may be used in gene therapy protocols for the treatment of the diseases or disorders contemplated herein. The improved construct encoding the polypeptide(s) can be inserted into the appropriate gene therapy vector and administered to a patient to treat or prevent the diseases or disorder of interest.
Vectors, such as viral vectors, have been used in the prior art to introduce genes into a wide variety of different target cells. Typically, the vectors are exposed to the target cells so that transformation can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired polypeptide (e.g., a receptor). The transfected nucleic acid may be permanently incorporated into the genome of each of the targeted cells, providing long lasting effect, or alternatively, the treatment may have to be repeated periodically. In certain embodiments, the (viral) vector transfects liver cells in vivo with genetic material encoding the polypeptide(s) of the disclosure.
A variety of vectors, both viral vectors and plasmid vectors are known in the art (see for example U.S. Pat. No. 5,252,479 and WO 93/07282). In particular, a number of viruses have been used as gene transfer vectors, including papovaviruses, such as SV40, vaccinia virus, herpes viruses including HSV and EBV, and retroviruses. Many gene therapy protocols in the prior art have employed disabled murine retroviruses. Several recently issued patents are directed to methods and compositions for performing gene therapy (see for example U.S. Pat. Nos. 6,168,916; 6,135,976; 5,965,541 and 6,129,705). Each of the foregoing patents is incorporated by reference in its entirety herein. Hence, genetic material such as a polynucleotide comprising an NPP1 or an NPP3 sequence can be introduced to a mammal in order to treat VSMC proliferation.
Certain modified viruses are often used as vectors to carry a coding sequence because after administration to a mammal, a virus infects a cell and expresses the encoded protein. Modified viruses useful according to the disclosure are derived from viruses which include, for example: parvovirus, picornavirus, pseudorabies virus, hepatitis virus A, B or C, papillomavirus, papovavirus (such as polyoma and SV40) or herpes virus (such as Epstein-Barr Virus, Varicella Zoster Virus, Cytomegalovirus, Herpes Zoster and Herpes Simplex Virus types 1 and 2), an RNA virus or a retrovirus, such as the Moloney murine leukemia virus or a lentivirus (i.e. derived from Human Immunodeficiency Virus, Feline Immunodeficiency Virus, equine infectious anemia virus, etc.). Among DNA viruses useful according to the disclosure are: Adeno-associated viruses adenoviruses, Alphaviruses, and Lentiviruses.
A viral vector is generally administered by injection, most often intravenously (by IV) directly into the body, or directly into a specific tissue, where it is taken up by individual cells. Alternately, a viral vector may be administered by contacting the viral vector ex vivo with a sample of the patient's cells, thereby allowing the viral vector to infect the cells, and cells containing the vector are then returned to the patient. Once the viral vector is delivered, the coding sequence expressed and results in a functioning protein. Generally, the infection and transduction of cells by viral vectors occur by a series of sequential events as follows: interaction of the viral capsid with receptors on the surface of the target cell, internalization by endocytosis, intracellular trafficking through the endocytic/proteasomal compartment, endosomal escape, nuclear import, virion uncoating, and viral DNA double-strand conversion that leads to the transcription and expression of the recombinant coding sequence interest. (Colella et al., Mol Ther Methods Clin Dev. 2017 Dec. 1; 8:87-104.).
Adeno-Associated Viral Vectors According to the Disclosure
AAV refers to viruses belonging to the genus Dependovirus of the Parvoviridae family. The AAV genome is approximately 4.7 kilobases long and is composed of linear single-stranded deoxyribonucleic acid (ssDNA) which may be either positive- or negative-sensed. The genome comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap. The rep frame is made of four overlapping genes encoding non-structural replication (Rep) proteins required for the AAV life cycle. The cap frame contains overlapping nucleotide sequences of structural VP capsid proteins: VP1, VP2 and VP3, which interact together to form a capsid of icosahedral symmetry.
The terminal 145 nucleotides are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex. Following wild type AAV infection in mammalian cells, the rep genes (i.e. Rep78 and Rep52) are expressed from the P5 promoter and the P19 promoter, respectively, and both Rep proteins have a function in the replication of the viral genome. A splicing event in the rep ORF results in the expression of actually four Rep proteins (i.e. Rep78, Rep68, Rep52 and Rep40). However, it has been shown that the unspliced mRNA, encoding Rep78 and Rep52 proteins, in mammalian cells are sufficient for AAV vector production. Also in insect cells the Rep78 and Rep52 proteins suffice for AAV vector production.
AAV is a helper-dependent virus, that is, it requires co-infection with a helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) in order to form functionally complete AAV virions. In the absence of co-infection with a helper virus, AAV establishes a latent state in which the viral genome inserts into a host cell chromosome or exists in an episomal form, but infectious virions are not produced. Subsequent infection by a helper virus “rescues” the integrated genome, allowing it to be replicated and packaged into viral capsids, thereby reconstituting the infectious virion. While AAV can infect cells from different species, the helper virus must be of the same species as the host cell. Thus, for example, human AAV replicates in canine cells that have been co-infected with a canine adenovirus.
To produce infectious recombinant AAV (rAAV) containing a heterologous nucleic acid sequence, a suitable host cell line can be transfected with an AAV vector containing the heterologous nucleic acid sequence, but lacking the AAV helper function genes, rep and cap. The AAV-helper function genes can then be provided on a separate vector. Also, only the helper virus genes necessary for AAV production (i.e., the accessory function genes) can be provided on a vector, rather than providing a replication-competent helper virus (such as adenovirus, herpesvirus, or vaccinia).
Collectively, the AAV helper function genes (i.e., rep and cap) and accessory function genes can be provided on one or more vectors. Helper and accessory function gene products can then be expressed in the host cell where they will act in trans on rAAV vectors containing the heterologous nucleic acid sequence. The rAAV vector containing the heterologous nucleic acid sequence will then be replicated and packaged as though it were a wild-type (wt) AAV genome, forming a recombinant virion. When a patient's cells are infected with the resulting rAAV virions, the heterologous nucleic acid sequence enters and is expressed in the patient's cells.
Because the patient's cells lack the rep and cap genes, as well as the accessory function genes, the rAAV cannot further replicate and package their genomes. Moreover, without a source of 5 rep and cap genes, wtAAV cannot be formed in the patient's cells.
The AAV vector typically lacks rep and cap frames. Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products (i.e. AAV Rep and Cap proteins), and wherein the host cell has been transfected with a vector which encodes and expresses a protein from the adenovirus open reading frame E4orf6.
Delivery of a protein of interest to the cells of a mammal is accomplished by first generating an AAV vector comprising DNA encoding the protein of interest and then administering the vector to the mammal. Thus, the disclosure should be construed to include AAV vectors comprising DNA encoding the polypeptide(s) of interest. Once armed with the present disclosure, the generation of AAV vectors comprising DNA encoding this/these polypeptide(s)s will be apparent to the skilled artisan.
In one embodiment, the disclosure relates to an adeno-associated viral (AAV) expression vector comprising a sequence encoding mammal ENPP1 or mammal ENPP3, and upon administration to a mammal the vector expresses an ENPP1 or ENPP3 precursor in a cell, the precursor including an Azurocidin signal peptide fused at its carboxy terminus to the amino terminus of ENPP1 or ENPP3. The ENPP1 or ENPP3 precursor may include a stabilizing domain, such as an IgG Fc region or human albumin. Upon secretion of the precursor from the cell, the signal peptide is cleaved off and enzymatically active soluble mammal ENPP1 or ENPP3 is provided extracellularly.
An AAV expression vector may include an expression cassette comprising a transcriptional regulatory region operatively linked to a nucleotide sequence comprising a transcriptional regulatory region operatively linked to a recombinant nucleic acid sequence encoding a polypeptide comprising a Azurocidin signal peptide sequence and an ectonucleotide pyrophosphatase/phosphodiesterase (ENPP1) polypeptide sequence.
In some embodiments, the expression cassette comprises a promoter and enhancer, the Kozak sequence GCCACCATGG, a nucleotide sequence encoding mammal NPP1 protein or a nucleotide sequence encoding mammal NPP3 protein, other suitable regulatory elements and a polyadenylation signal.
In some embodiments, the AAV recombinant genome of the AAV vector according to the disclosure lacks the rep open reading frame and/or the cap open reading frame.
The AAV vector according to the disclosure comprises a capsid from any serotype. In general, the AAV serotypes have genomic sequences of significant homology at the amino acid and the nucleic acid levels, provide an identical set of genetic functions, and replicate and assemble through practically identical mechanisms. In particular, the AAV of the present disclosure may belong to the serotype 1 of AAV (AAV1), AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV.
Examples of the sequences of the genome of the different AAV serotypes may be found in the literature or in public databases such as GenBank. For example, GenBank accession numbers NC_001401.2 (AAV2), NC_001829.1 (AAV4), NC_006152.1 (AAV5), AF028704.1 (AAV6), NC_006260.1 (AAV7), NC_006261.1 (AAV8), AX753250.1 (AAV9) and AX753362.1 (AAV10).
In some embodiments, the adeno-associated viral vector according to the disclosure comprises a capsid derived from a serotype selected from the group consisting of the AAV2, AAV5, AAV7, AAV8, AAV9, AAV10 and AAVrh10 serotypes. In another embodiment, the serotype of the AAV is AAV8. If the viral vector comprises sequences encoding the capsid proteins, these may be modified so as to comprise an exogenous sequence to direct the AAV to a particular cell type or types, or to increase the efficiency of the delivery of the targeted vector to a cell, or to facilitate purification or detection of the AAV, or to reduce the host response.
In certain embodiments, the rAAV vector of the disclosure comprises several essential DNA elements. In certain embodiments, these DNA elements include at least two copies of an AAV ITR sequence, a promoter/enhancer element, a transcription termination signal, any necessary 5′ or 3′ untranslated regions which flank DNA encoding the protein of interest or a biologically active fragment thereof. The rAAV vector of the disclosure may also include a portion of an intron of the protein on interest. Also, optionally, the rAAV vector of the disclosure comprises DNA encoding a mutated polypeptide of interest.
In certain embodiments, the vector comprises a promoter/regulatory sequence that comprises a promiscuous promoter which is capable of driving the expression of a heterologous gene to high levels in many different cell types. Such promoters include but are not limited to the cytomegalovirus (CMV) immediate early promoter/enhancer sequences, the Rous sarcoma virus promoter/enhancer sequences and the like. In certain embodiments, the promoter/regulatory sequence in the rAAV vector of the disclosure is the CMV immediate early promoter/enhancer. However, the promoter sequence used to drive expression of the heterologous gene may also be an inducible promoter, for example, but not limited to, a steroid inducible promoter, or maybe a tissue specific promoter, such as, but not limited to, the skeletal a-actin promoter which is muscle tissue specific and the muscle creatine kinase promoter/enhancer, and the like.
In certain embodiments, the rAAV vector of the disclosure comprises a transcription termination signal. While any transcription termination signal may be included in the vector of the disclosure, in certain embodiments, the transcription termination signal is the SV40 transcription termination signal.
In certain embodiments, the rAAV vector of the disclosure comprises isolated DNA 5 encoding the polypeptide of interest, or a biologically active fragment of the polypeptide of interest. The disclosure should be construed to include any mammalian sequence of the polypeptide of interest, which is either known or unknown. Thus, the disclosure should be construed to include genes from mammals other than humans, which polypeptide functions in a substantially similar manner to the human polypeptide. Preferably, the nucleotide sequence comprising the gene encoding the polypeptide of interest is about 50% homologous, more preferably about 70% homologous, even more preferably about 80% homologous and most preferably about 90% homologous to the gene encoding the polypeptide of interest.
Further, the disclosure should be construed to include naturally occurring variants or recombinantly derived mutants of wild type protein sequences, which variants or mutants render the polypeptide encoded thereby either as therapeutically effective as full-length polypeptide, or even more therapeutically effective than full-length polypeptide in the gene therapy methods of the disclosure.
The disclosure should also be construed to include DNA encoding variants which retain the polypeptide's biological activity. Such variants include proteins or polypeptides which have been or may be modified using recombinant DNA technology, such that the protein or polypeptide possesses additional properties which enhance its suitability for use in the methods described herein, for example, but not limited to, variants conferring enhanced stability on the protein in plasma and enhanced specific activity of the protein. Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function.
The disclosure is not limited to the specific rAAV vector exemplified in the experimental examples; rather, the disclosure should be construed to include any suitable AAV vector, including, but not limited to, vectors based on AAV-1, AAV-3, AAV-4 and AAV-6, and the like. Also included in the disclosure is a method of treating a mammal having a disease or disorder in an amount effective to provide a therapeutic effect.
The method comprises administering to the mammal an rAAV vector encoding the polypeptide of interest. Preferably, the mammal is a human. Typically, the number of viral vector genomes/mammal which are administered in a single injection ranges from about 1×108 to about 5×1016. Preferably, the number of viral vector genomes/mammal which are administered in a single injection is from about 1×1010 to about 1×1015; more preferably, the number of viral vector genomes/mammal which are administered in a single injection is from about 5×1010 to about 5×1015; and, most preferably, the number of viral vector genomes which are administered to the mammal in a single injection is from about 5×1010 to about 5×1014.
When the method of the disclosure comprises multiple site simultaneous injections, or several multiple site injections comprising injections into different sites over a period of several hours (for example, from about less than one hour to about two or three hours) the total number of viral vector genomes administered may be identical, or a fraction thereof or a multiple thereof, 15 to that recited in the single site injection method.
For administration of the rAAV vector of the disclosure in a single site injection, in certain embodiments a composition comprising the virus is injected directly into an organ of the subject (such as, but not limited to, the liver of the subject).
For administration to the mammal, the rAAV vector may be suspended in a pharmaceutically acceptable carrier, for example, HEPES buffered saline at a pH of about 7.8. Other useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey). The rAAV vector of the disclosure may also be provided in the form of a kit, the kit comprising, for example, a freeze-dried preparation of vector in a dried salts formulation, sterile water for suspension of the vector/salts composition and instructions for suspension of the vector and administration of the same to the mammal.
The published application, US 2017/0290926—Smith et al., the contents of which are incorporated by reference in their entirety herein, describe in detail the process by which AAV vectors are generated, delivered and administered.
The present disclosure provides compositions and methods for the production and delivery of recombinant double-stranded RNA molecules (dsRNA that encode ENPP1 or ENPP3 polypeptides described herein. The double stranded RNA particle (dsRP) can contain a dsRNA molecule enclosed in a capsid or coat protein. The dsRNA molecule can be a viral genome or portion of a genome, which can be derived from a wild-type viral genome. The RNA molecule can encode an RNA-dependent RNA polymerase (RDRP) and a polyprotein that forms at least part of a capsid or coat protein. The RNA molecule can also contain an RNA sub-sequence that encodes an ENPP1 or ENPP3 polypeptides that are translated by the cellular components of a host cell. When the dsRP is transfected into a host cell the sub-sequence can be translated by the cellular machinery of the host cell to produce the ENPP1 or ENPP3 polypeptides.
In another aspect the disclosure provides a method of producing a protein product in a host cell. The method includes transfecting a host cell with a dsRP having a recombinant double-stranded RNA molecule (dsRNA) and a capsid or coat protein. The RNA molecule can encode an RNA-dependent RNA polymerase and a polyprotein that forms at least part of the capsid or coat protein, and the dsRP can be able to replicate in the host cell. The RNA molecule has at least one RNA sub-sequence that encodes ENPP1 or ENPP3 polypeptides that is translated by cellular components of the host cell.
In another aspect the disclosure provides an RNA molecule translatable by a host cell. The RNA molecule can be any RNA molecule that encodes the ENPP1 or ENPP3 polypeptides described herein. In one embodiment the RNA molecule encodes an RNA-dependent RNA polymerase and a polyprotein that forms at least part of a capsid or coat protein of a dsRP and, optionally, can have at least one sub-sequence of RNA that encodes an additional protein product.
Production of dsRP
A dsRP of the disclosure can also be produced by presenting to a host cell a plasmid or other DNA molecule encoding a dsRP of the disclosure or encoding the genes of the dsRP. The plasmid or DNA molecule containing nucleotide sequences encoding desired protein such as ENPP1 or ENPP3 polypeptide is then transfected into the host cell and the host cell begins producing the dsRP of the disclosure. The dsRP can also be produced in the host cell by presenting to the host cell an RNA molecule encoding the genes of the dsRP. The RNA molecule can be (+)-strand RNA.
Once the dsRP of the disclosure has been presented to the host cell (or a plasmid encoding the genes of the dsRP of the disclosure, or an RNA molecule encoding the genes of the dsRP), the dsRP will be produced within the host cell using the cellular components of the host cell. The dsRP of the disclosure is therefore self-sustaining within the host cell and is propagated within the host cell. The host cell can be any suitable host cell such as, for example, a eukaryotic cell, a mammalian cell, a fungal cell, a bacterial cell, an insect cell, or a yeast cell. The host cell can propagate a recombinant dsRP after a recombinant dsRNA molecule of the disclosure or a DNA molecule encoding a dsRP of the disclosure is presented to and taken up by the host cell.
Methods of Producing a dsRNA Virus or dsRP
The disclosure also provides methods of producing a dsRP of the disclosure. A double-stranded or single-stranded RNA or DNA molecule can be presented to a host cell. The amplification of the dsRNA molecules in the host cell utilizes the natural production and assembly processes already present in many types of host cells (e.g., yeast). The disclosure can thus be applied by presenting to a host cell a single-stranded or double-stranded RNA or DNA molecule of the disclosure, which is taken up by the host cell and is utilized to produce the recombinant dsRP and protein or peptide encoded by the RNA sub-sequence using the host cell's cellular components. The disclosure can also be applied by providing to the host cell a linear or circular DNA molecule (e.g., a plasmid or vector) containing one or more sequences coding for an RNA-dependent RNA polymerase, a polyprotein that forms at least part of the capsid or coat protein of the dsRP, and a sub-sequence encoding the protein of interest such as ENPP1 or ENPP3 polypeptides as disclosed herein.
The presentation of a dsRNA or ssRNA molecule of the disclosure can be performed in any suitable way such as, for example, by presenting an RNA molecule of the disclosure directly to the host cell as “naked” or unmodified single-stranded or double-stranded RNA. The RNA molecule can be transfected (or transformed) into a yeast, bacterial, or mammalian host cell by any suitable method, for example by electroporation, exposure of the host cell to calcium phosphate, or by the production of liposomes that fuse with the cell membrane and deposit the viral sequence inside. It can also be performed by a specific mechanism of direct introduction of dsRNA from killer viruses or heterologous dsRNA into the host cell. This step can be optimized using a reporter system, such as red fluorescent protein (RFP), or by targeting a specific constitutive gene transcript within the host cell genome. This can be done by using a target with an obvious phenotype or by monitoring by quantitative reverse transcriptase PCR (RT-PCR).
In some embodiments a DNA molecule (e.g., a plasmid or other vector) that encodes an RNA molecule of the disclosure is introduced into the host cell. The DNA molecule can contain a sequence coding for the RNA molecule of a dsRP of the disclosure. The DNA molecule can code for an entire genome of the dsRP, or a portion thereof. The DNA molecule can further code for the at least one sub-sequence of RNA that produces the additional (heterologous) protein product. The DNA sequence can also code for gag protein or gag-pol protein, and as well as any necessary or desirable promoters or other sequences supporting the expression and purpose of the molecule. The DNA molecule can be a linear DNA, a circular DNA, a plasmid, a yeast artificial chromosome, or may take another form convenient for the specific application.
In one embodiment the DNA molecule can further comprise T7 ends for producing concatamers and hairpin structures, thus allowing for propagation of the virus or dsRP sequence in the host cell. The DNA molecule can be transfected or transformed into the host cell and then, using the host cellular machinery, transcribed and thus provide the dsRNA molecule having the at least one sub-sequence of RNA to the host cell. The host cell can then produce the encoded desired ENPP1 or ENPP3 polypeptide. The dsRNA can be packaged in the same manner that a wild-type virus would be, using the host cell's metabolic processes and machinery. The ENPP1 or ENPP3 polypeptide is also produced using the host cell's metabolic processes and cellular components.
The patent, U.S. Ser. No. 10/266,834 by Brown et al., the contents of which are incorporated by reference in their entirety herein, describes in detail the process by which dsRNA particles that encode polypeptides are generated, delivered and administered.
ENPP1 Coated Stents and ENPP3 Coated Stents
Stents are typically elongated structures used to keep open lumens (e.g., openings in the body) found in various parts of the body so that the parts of the body containing those lumens may function properly. Stents are often used in the treatment of atherosclerosis, a disease of the vascular system in which arteries become partially, and sometimes completely, occluded with substances that may include lipids, cholesterol, calcium, and various types of cells, such as smooth muscle cells and platelets.
Stents located within any lumen in the body may not always prevent partial or complete restenosis. In particular, stents do not always prevent the re-narrowing of an artery following Percutaneous transluminal angioplasty (PTA). In some cases, the introduction and presence of the stent itself in the artery or vein can create regions of trauma or tissue injury such as, e.g., tears in the inner lining of the artery, called the endothelium requiring further surgeries post stent placement.
It is believed that such trauma or tissue injury can trigger migration of vascular smooth muscle cells, which are usually separated from the arterial lumen by the endothelium, into the arterial lumen, where they proliferate to create a mass of cells that may, in a matter of days or weeks, occlude the artery. Such re-occlusion, which is sometimes seen after PTA, is an example of restenosis. Coating a stent with therapeutic agent such as ENPP1 agent or ENPP3 agent is expected to prevent and/or reduce vascular smooth muscle cell proliferation which in return reduces the occurrence of or treats restenosis.
In some embodiments, the patient is need of surgery and/or has tissue injury due to the presence of a prior implanted non-eluting stent.
In some embodiments, the patient is need of surgery and/or has tissue injury due to the presence of a prior implanted eluting stent that elutes therapeutic agents other than ENPP1 agent or ENPP3 agent.
In some embodiments, the prior stent that had caused the tissue injury is removed and replaced with ENPP1 agent coated stent.
In some embodiments, the prior stent that had caused the tissue injury is removed and replaced with ENPP3 agent coated stent.
In some embodiments, the prior stent that had caused the tissue injury is not removed and the ENPP1 agent coated stent is implanted adjacent to the prior stent.
In some embodiments, the prior stent that had caused the tissue injury is not removed and the ENPP3 agent coated stent is implanted adjacent to the prior stent.
ENPP1 or ENPP3 coated stents are typically hollow, cylindrical structures made from struts or interconnected filaments. Stents are usually implanted at their site of use in the body by attaching them in a compressed state to a catheter that is directed through the body to the site of stent use. Vascular stents are frequently used in blood vessels to open the vessel and provide improved blood flow. The stent can be expanded to a size which enables it to keep the lumen open by supporting the walls of the lumen once it is positioned at the desired site. Vascular stents can be collapsed to reduce their diameter so that the stent can be guided through a patient's arteries or veins to reach the site of deployment. Stents are typically either coupled to the outside of the balloon for expansion by the expanding balloon or are self-expanding upon removal of a restraint such as a wire or sleeve maintaining the stent in its collapsed state.
Vascular stents are often made of metal to provide the strength necessary to support the occluded arterial walls. Two of the preferred metals are Nitinol alloys of nickel and titanium, and stainless steel. Other materials that can be used in fabricating stents are ceramics, polymers, and plastics. The polymer may be a polymer having no functional groups. Alternatively, the polymer may be one having functional groups, but none that are reactive with the ENPP1 agent or ENPP3 agent. The polymer may include a biodegradable polymer. For example, the polymer may include a polymer selected from the group consisting of polyhydroxy acids, polyanhydrides, polyphosphazenes, polyalkylene oxalates, biodegradable polyamides, polyorthoesters, polyphosphoesters, polyorthocarbonates, and blends or copolymers thereof. The polymer may also include a biostable polymer, alone or in combination with a biodegradable polymer. For example, the polymer may include a polymer selected from the group consisting of polyurethanes, silicones, polyacrylates, polyesters, polyalkylene oxides, polyalcohols, polyolefins, polyvinyl chlorides, cellulose and its derivatives, fluorinated polymers, biostable polyamides, and blends or copolymers thereof.
The effect of different stent designs on the drug distribution pattern has been scrutinized in experimental studies and also tested in clinical trials (Hwang C W, Wu D, Edelman ER. 2001. Physiological transport forces govern drug distribution for stent-based delivery. Circulation, 104: 600-5; & Takebayashi H, Mintz G S, Cartier S G, et al. 2004. Nonuniform strut distribution correlates with more neointimal hyperplasia after Sirolimus-eluting stent implantation. Circulation, 110:3430-4). Although a large number of stent designs have been developed to date, only the multicellular design is currently most commonly used; they can be categorized into “closed cell” and “open cell” configurations (Rogers C D K. 2002. Drug-eluting stents: role of stent design, delivery vehicle, and drug selection. Rev Cardiovasc Med, 3(Suppl 5): S10-15.). A closed cell stent has a uniform cell expansion and constant cell spacing when deployed in a curved vascular segment, which gives more uniform drug distribution (Rogers 2002). An open cell stent has a greater variation in the surface coverage between the inner and outer curvatures in the curved segment but gives better conformability to curved surface at the expense of less uniform drug distribution (Rogers 2002). The majority of current stents use a closed cell design. The optimal stent design for drug delivery would have a large stent surface area, a small cell gap, and minimal strut deformation after deployment while maintaining conformability, radial support, and flexibility to reach the complex coronary lesions. Several examples of the different geometrical stent structures are described in Paisal et al. (Muhammad Sufyan Amir Paisal et al 2017 IOP Conf. Ser.: Mater. Sci. Eng. 165 012003)
ENPP1 coated stents or ENPP3 coated stents are prepared by applying a coating composition comprising an effective amount of ENPP1 agent or ENPP3 agent respectively. The coating composition preferably includes an amount of the ENPP1 agent or ENPP3 agent that is sufficient to be therapeutically effective for inhibiting regrowth of plaque or inhibiting restenosis or preventing vascular smooth cell proliferation.
In one embodiment, the coating composition comprises from about 1 wt % to about 50 wt % ENPP1 polypeptide, based on the total weight of the coating composition. In another embodiment, the coating composition comprises from about 5 wt % to about 30 wt % ENPP1 polypeptide. In yet another embodiment, the coating composition comprises from about 10 wt % to about 20 wt % ENPP1 polypeptide.
In one embodiment, the coating composition comprises from about 1 wt % to about 50 wt % ENPP3 polypeptide, based on the total weight of the coating composition. In another embodiment, the coating composition comprises from about 5 wt % to about 30 wt % ENPP3 polypeptide. In yet another embodiment, the coating composition comprises from about 10 wt % to about 20 wt % ENPP3 polypeptide.
In one embodiment, the coating composition comprises from about 1 μg/ml to about 10 mg/ml of ENPP1 polypeptide. In another embodiment, the coating composition comprises from about 100 μg/ml to 5 mg/ml ENPP1 polypeptide. In yet another embodiment, the coating composition comprises from about 500 μg/ml to about 2 mg/ml ENPP1 polypeptide.
In a related embodiment, the ENPP1 polypeptide of the coating composition is ENPP1-Fc.
In a related embodiment, the ENPP1 polypeptide of the coating composition is ENPP1-Albumin.
In one embodiment, the coating composition comprises from about 1 μg/ml to about 10 mg/ml of ENPP3 polypeptide. In another embodiment, the coating composition comprises from about 100 μg/ml to 5 mg/ml ENPP3 polypeptide. In yet another embodiment, the coating composition comprises from about 500 μg/ml to about 2 mg/ml ENPP3 polypeptide.
In a related embodiment, the ENPP3 polypeptide of the coating composition is ENPP3-Fc.
In a related embodiment, the ENPP3 polypeptide of the coating composition is ENPP3-Albumin.
In one embodiment, the coating composition comprises from about 1 ng/μ1 to about 1000 μg/μ1 of ENPP1 mRNA. In another embodiment, the coating composition comprises from about 100 ng/μ1 to 10 μg/μ1 ENPP1 mRNA. In yet another embodiment, the coating composition comprises from about 50 ng/μ1 to about 5 μg/μl ENPP1 mRNA.
In one embodiment, the coating composition comprises from about 1 ng/μ1 to about 1000 μg/μ1 of ENPP1-Fc mRNA. In another embodiment, the coating composition comprises from about 100 ng/μ1 to 10 μg/μ1 ENPP1-Fc mRNA. In yet another embodiment, the coating composition comprises from about 50 ng/μl to about 5 μg/μl ENPP1-Fc mRNA.
In one embodiment, the coating composition comprises from about 1 ng/μ1 to about 1000 μg/μ1 of ENPP1-Albumin mRNA. In another embodiment, the coating composition comprises from about 100 ng/μ1 to 10 μg/μ1 ENPP1-Albumin mRNA. In yet another embodiment, the coating composition comprises from about 50 ng/μ1 to about 5 μg/μl ENPP1-Albumin mRNA.
In one embodiment, the coating composition comprises from about 1 ng/μ1 to about 1000 μg/μ1 of ENPP3 mRNA. In another embodiment, the coating composition comprises from about 100 ng/μ1 to 5 μg/μ1 ENPP3 mRNA. In yet another embodiment, the coating composition comprises from about 500 ng/μ1 to about 2 μg/μl ENPP3 mRNA.
In one embodiment, the coating composition comprises from about 1 ng/μ1 to about 1000 μg/μ1 of ENPP3-Fc mRNA. In another embodiment, the coating composition comprises from about 100 ng/μ1 to 5 μg/μ1 ENPP3-Fc mRNA. In yet another embodiment, the coating composition comprises from about 500 ng/μl to about 2 μg/μl ENPP3-Fc mRNA.
In one embodiment, the coating composition comprises from about 1 ng/μ1 to about 1000 μg/μ1 of ENPP3-Albumin mRNA. In another embodiment, the coating composition comprises from about 100 ng/μ1 to 5 μg/μ1 ENPP3-Albumin mRNA. In yet another embodiment, the coating composition comprises from about 500 ng/μl to about 2 μg/μl ENPP3-Albumin mRNA.
Stents may be coated with a substance, such as a biodegradable or biostable polymer, to improve the biocompatibility of the stent, making it less likely to cause an allergic or other immunological response in a patient. A coating substance may also add to the strength of the stent. Some known coating substances include organic acids, their derivatives, and synthetic polymers that are either biodegradable or biostable. Biostable coating substances do not degrade in the body, biodegradable coating substances can degrade in the body.
The coating composition comprises an effective amount of carrier which helps in the coating process to ensure that the therapeutic molecules such as ENPP1 agent or ENPP3 agent adhere to the stent surface and also facilitate in eluting the therapeutic agent into the body at the site of stent placement. The carrier could be a liquid carrier or a solid carrier. The coating composition may alternatively comprise more than one solid compound in a solid carrier. The coating composition may further comprise both a liquid carrier and a solid carrier. In a still further aspect, the coating composition may also comprise more than one type of nonpolymeric or polymeric compound in the carrier and may further comprise both a polymeric material and a nonpolymeric material in a solid or liquid carrier.
In another embodiment, two or more types of biodegradable compounds (polymers or non-polymers) may be blended together to obtain a liquid carrier for use in the coating composition. The biodegradable compounds can be liquids before they are mixed together, e.g., forming a homogeneous solution, mixture, or suspension. Alternatively, some of the biodegradable compounds may be solids before they are mixed with other liquid biodegradable compounds. The solid biodegradable compounds preferably dissolve when they are mixed with the liquid biodegradable compounds, resulting in a liquid carrier composition containing the different biodegradable compounds.
In another embodiment, the biodegradable carrier component of the coating composition is a solid, which dissolves when mixed with the biologically active component and any other components included in the coating composition.
The carrier could be a polymeric carrier. Some polymeric carriers are synthetic polymers. Examples of synthetic polymers that serve as reservoir matrices include but not limited to poly-n-butyl methacrylate, polyethylene-vinyl acetate, poly (lactide-co-Σ-caprolactone) copolymer, Fibrin, cellulose, Phosphorylcholine. Some eluting stent comprise porous 300 μm ceramic layer containing therapeutic molecule-loaded nanocavities. Examples of drug eluting stents, stent structures and stent designs can be found in Drug-Eluting Stent: A Review and Update, Vasc Health Risk Manag. 2005 December; 1(4): 263-276 and Modern Stents: Where Are We Going?, Rambam Maimonides Med J. 2020 April; 11(2): e0017.
The carriers in the coating composition may be either biodegradable or biostable. Biodegradable polymers are often used in synthetic biodegradable sutures. These polymers include polyhydroxy acids. Polyhydroxy acids suitable for use in the present invention include poly-L-lactic acids, poly-DL-lactic acids, polyglycolic acids, polylactides including homopolymers and copolymers of lactide (including lactides made from all stereo isomers of lactic acids, such as D-,L-lactic acid and meso lactic acid), polylactones, polycaprolactones, polyglycolides, polyparadioxanone, poly 1,4-dioxepan-2-one, poly 1,5-dioxepan-2-one, poly 6,6-dimethyl-1, 4-dioxan-2-one, polyhydroxyvalerate, polyhydroxybuterate, polytrimethylene carbonate polymers, and blends of the foregoing.
Polylactones suitable for use in the present invention include polycaprolactones such as poly(e-caprolactone), polyvalerolactones such as poly(d-valerolactone), and polybutyrolactones such as poly(butyrolactone). Other biodegradable polymers that can be used are polyanhydrides, polyphosphazenes, biodegradable polyamides such as synthetic polypeptides such as polylysine and polyaspartic acid, polyalkylene oxalates, polyorthoesters, polyphosphoesters, and polyorthocarbonates. Copolymers and blends of any of the listed polymers may be used. Polymer names that are identical except for the presence or absence of brackets represent the same polymers.
Biostable polymers suitable for use in the present invention include, but are not limited to polyurethanes, silicones such as polyalkyl siloxanes such as polydimethyl siloxane and polybutyl methacrylate, polyesters such as poly(ethylene terephthalate), polyalkylene oxides such as polyethylene oxide or polyethylene glycol, polyalcohols such as polyvinyl alcohols and polyethylene glycols, polyolefins such as poly-5 ethylene, polypropylene, poly(ethylene-propylene) rubber and natural rubber, polyvinyl chloride, cellulose and modified cellulose derivatives such as rayon, rayon-triacetate, cellulose acetate, cellulose acetate butyrate, cellophane, cellulose nitrate, cellulose propionate, cellulose ethers such as carboxymethyl cellulose and hydroxyalkyl celluloses, fluorinated polymers such as polytetrafluoroethylene (Teflon), and bio stable polyamides such as Nylon 66 and polycaprolactam. Fixed animal tissues such as glutaraldehyde fixed bovine pericardium can also be used. Polyesters and polyamides can be either biodegradable or biostable. Ester and amide bonds are susceptible to hydrolysis, which can contribute to biodegradation.
In some cases, the coating composition further comprises an effective amount of a non-polymeric carrier. The non-polymeric carrier can include one or more of fatty acid, biocompatible oil, or wax. Examples of non-polymeric biodegradable carriers include liquid oleic acid, vitamin E, peanut oil, and cottonseed oil, which are liquids that are both hydrophobic and biocompatible. In some cases, the nonpolymeric or polymeric carrier, can be a liquid at room and body temperature. In some cases, the nonpolymeric or polymeric carrier can be a solid at room and body temperature, or a solid at room temperature and a liquid at body temperature.
In another embodiment, the polymer solution can be formed into a film and the film then applied to the stent. Any of a variety of conventional methods of forming films can be used. For example, the polymer, ENPP1 agent or ENPP3 agent and solvent are preferably mixed into solution and then poured onto a smooth, flat surface such that a coating film is formed after the solution is dried to remove the solvent. The film can then be cut to fit the stent on which it is to be used. The film may then be mounted, such as by wrapping, on the outer surface of a stent.
In another embodiment, the coated stent is prepared by spraying the stent with the liquid carrier comprising the therapeutic agent such as ENPP1 agent or ENPP3 agent resulting in a coating of uniform thickness on the struts of the stent. In another embodiment, the stent may be dip coated or immersed in the coating solution comprising carrier and therapeutic agent, such that the solution completely coats the struts of the stent. Alternatively, the stent may be painted with the coating solution comprising carrier and therapeutic agent, such as with a paint brush. In each of these coating applications, the entirety of both the outer and inner surfaces of the stent are preferably coated, although only portions of either or both surfaces may be coated in some embodiments.
As discussed above, the coating composition comprises a bioactive component and a biodegradable carrier component. Preferably, the coating composition comprises from 0.1% to 100% by weight of a biologically active component and from 1% to 99% by weight of a biodegradable carrier component. More preferably, the coating composition comprises from 0.1% to 50% by weight of a biologically active component and from 50% to 99.9% by weight of a biodegradable carrier component. The coating composition can be prepared in a number of ways including by simply mixing the bioactive component and the carrier component together to form a mixture, e.g., a solution or suspension. Alternatively, the bioactive component and the carrier component together are mixed in a suitable solvent, the coating is applied to the stent, and the solvent is removed. Preferably the coating composition is applied to the stent in its expanded state.
In addition to stents, examples of other medical devices that can be coated in accordance with aspects of the inventions disclosed herein include catheters, heart valves, pacemaker leads, annuloplasty rings and other medical implants. In other specific embodiments, coated angioplasty balloons and other coated medical devices can also comprise one of the coating compositions disclosed herein. However, stents are preferred. The coating composition may be applied to the stent (or other medical device) by any number of ways, e.g, by spraying the coating composition onto the stent, by immersing the stent in the coating composition, or by painting the stent with the coating composition. Preferably, a stent is coated in its expanded (i.e., enlarged diameter) form so that a sufficient amount of the coating composition will be applied to coat the entire surface of the expanded stent. When the stent is immersed in the coating composition, the excess coating composition on the surface of the stent may be removed, such as by brushing off the excess coating composition with a paint brush. In each of these coating applications, preferably both the outer and inner surfaces of the stent are coated.
The coating compositions described herein preferably remain on a stent, partially or in substantial part, after the stent has been introduced to the body, for at least several days, for several weeks and more preferably for several months thereby slowly releasing the therapeutic agents such as ENPP1 agent or ENPP3 agent into the blood stream.
The disclosure provides pharmaceutical compositions comprising a polypeptide of the disclosure within the methods described herein. Such a pharmaceutical composition is in a form suitable for administration to a subject, or the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The various components of the pharmaceutical composition may be present in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
In an embodiment, the pharmaceutical compositions useful for practicing the method of the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In other embodiments, the pharmaceutical compositions useful for practicing the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 500 mg/kg/day.
The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the disclosure will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between about 0.1% and about 100% (w/w) active ingredient.
Pharmaceutical compositions that are useful in the methods of the disclosure may be suitably developed for inhalational, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, intravenous or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations. The route(s) of administration is readily apparent to the skilled artisan and depends upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.
The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
As used herein, a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. The unit dosage form may be for a single daily dose or one of the multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
The regimen of administration may affect what constitutes an effective amount. For example, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation. In certain embodiments, administration of the compound of the disclosure to a subject elevates the subject's plasma PPi to a level that is close to normal, where a normal level of PPi in mammals is 1-3 μM. “Close to normal” refers to 0 to 1.2 μM or 0-40% below or above normal, 30 nM to 0.9 μM or 1-30% 15 below or above normal, 0 to 0.6 μM or 0-20% below or above normal, or 0 to 0.3 μM or 0-10% below or above normal.
Administration of the compositions of the present disclosure to a patient, such as a mammal, such as a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. Dosage is determined based on the biological activity of the therapeutic compound which in turn depends on the half-life and the area under the plasma time of the therapeutic compound curve. The polypeptide according to the disclosure is administered at an appropriate time interval of every 2 days, or every 4 days, or every week or every month so as to achieve a continuous level of plasma PPi that is either close to the normal (1-3 μM) level or above (30-50% higher than) normal levels of PPi. Therapeutic dosage of the polypeptides of the disclosure may also be determined based on half-life or the rate at which the therapeutic polypeptide is cleared out of the body. The polypeptide according to the disclosure is administered at appropriate time intervals of either every 2 days, or every 4 days, every week or every month so as to achieve a constant level of enzymatic activity of ENPP1 or ENPP3 polypeptides.
For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the disclosure is from about 0.01 and 50 mg/kg of body weight/per day. In certain embodiments, the effective dose range for a therapeutic compound of the disclosure is from about 50 ng to 500 ng/kg, preferably 100 ng to 300 ng/kg of bodyweight. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
The compound can be administered to a patient as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose is readily apparent to the skilled artisan and depends upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, and the type and age of the patient. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
A medical doctor, e.g., physician, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. The frequency of administration of the various combination compositions of the disclosure varies from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physical taking all other factors about the patient into account.
In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.
Routes of administration of any of the compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. The formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.
“Parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.
The present disclosure is further exemplified by the following examples. The examples are for illustrative purpose only and are not intended, nor should they be construed as limiting the disclosure in any manner.
Mice
The tip-toe walking (ttw/ttw) mice and WT mice were used in the following experiments. ttw/ttw mice were bred onto a C57BL/6J background for more than ten generations, and ttw/ttw mice and wild-type (WT) littermate control (male and female) animals were generated through heterozygous mating.
Plasma Collection
Whole blood from ttw/ttw mice and WT mice (by cardiac puncture), was collected in syringes containing trisodium ethylenediaminetetraacetic acid (EDTA) and maintained on ice until the separation of plasma and erythrocytes by centrifugation (1000×g, 4° C., 20 min) was performed. The plasma was then depleted of platelets by filtration (2200×g, 4° C., 20 min) through a 300,000-kDa mass cutoff filter and stored at −20° C. until further processing.
It is known that damage to a blood vessel induces an inflammatory response and endothelial activation, resulting in smooth muscle cell proliferation and narrowing of the lumen of the vessel. (Exp Mol Med. 2018 Oct. 29; 50(10):1-12). Carotid artery ligation in WT mice and ttw/ttw mice was performed to create a model of mechanical injury and was then used to study the effect of ENPP1-Fc on smooth muscle cell proliferation at the site of injury. Thus, the main aim of the experiment was to determine the therapeutic effect of ENPP1-Fc on myointimal hyperplasia in WT mice and homozygous ttw/ttw mice.
ttw/ttw and wildtype (WT) littermate control (male and female) animals were generated by heterozygous mating. The pups were weaned at 3-4 weeks of age and then maintained on normal chow diet. Animals were blindly numbered during weaning, independent on genotype. ENPP1 genotyping was then performed by the polymerase chain reaction analysis of tail DNA by following the protocols described in Okawa et al. (Okawa A, Nakamura I, Goto S, Moriya H, Nakamura Y, Ikegawa S. Mutation in Npps in a mouse model of ossification of the posterior longitudinal ligament of the spine. Nature genetics. 1998; 19(3):271-3).
Left carotid artery ligation surgery may be performed on young mice, for example 6-8 week old mice. Left carotid artery ligation surgery was performed in a 7 week-old WT (n=5) and ttw/ttw mice (n=5). Mice were anesthetized by isoflurane inhalation (Forene®, Abbott GmbH & Co. KG, Wiesbaden), at an initial concentration of 1 l/min oxygen to 3 vol % isoflurane, maintaining a concentration of 0.6 l/min oxygen to 1-1.5 vol % isoflurane. Carprofen was used for analgesia (5 mg/kg bodyweight through a subcutaneous injection; Rimadyl®, Pfizer, Berlin, Germany). Left carotid arteries were exposed through a small midline incision in the neck and ligated with a 5-0 nylon silk suture approximately 2 mm proximal from the carotid bifurcation. All animals recovered well from the procedure and showed no signs of a stroke.
Seven days after carotid artery ligation, ENPP1-Fc or vehicle is administered to a model mouse, for example, the ttw/ttw mouse. At 7 days after carotid ligation, intimal hyperplasia in ttw/ttw mice is present in vessels, but the I/M ratio is lower at 7 days compared to 14 days post-ligated ttw/ttw mice (p<0.001 for intimal area and I/M ratio,
To determine whether ENPP1-Fc has a therapeutic effect if administered after the carotid ligation, 7 week-old WT and ttw/ttw mice were subjected to carotid ligation and allowed to recover. Both mice were then treated with either vehicle (Tris buffered saline, pH 7.4/Control cohort) or ENPP1-Fc (Experimental cohort) at 10 mg/kg bodyweight by subcutaneous injection every other day. ENPP1-Fc treatment (10 mg/kg bodyweight subcutaneously injected every other day) was initiated 7 days after carotid ligation and continued for 7 days until the carotid arteries were harvested at 14 days post ligation. Carotid arteries were fixed with 4% paraformaldehyde in PBS for morphological analyses.
Serial sections (sections of 5 μm each) were collected. For morphometrical measurements of the ligated carotid arteries, sections immediately proximal of the ligation site were taken. By using every fifth section, a total of 12 sections (every 25 μm) per animal were analyzed proximal from the ligation site, spanning a distance of approximately 250 μm. Morphometric analyses were performed by using Elastica van Gieson stain (Roth, Karlsruhe, Germany). (See
Statistical analyses were performed using Student's t test (unpaired two-sample testing for means). Comparisons of multiple groups used one-way ANOVA, followed by the Bonferroni's post hoc test, performed with GraphPad Prism software version 7. Probability values of p<0.05 were considered significant.
ENPP1 deficiency resulted in neointimal lesion formation after carotid ligation injury in ttw/ttw mice and hence ttw/ttw mice had higher levels of VSMC proliferation when compared with the WT mice. Representative stained sections from either 100 or 200 μm caudal from the ligation in ttw/ttw-mice and WT mice showed that the carotid ligation caused intimal hyperplasia, resulting in the narrowing of the lumen, with more severe narrowing closer to the ligature (100 μm) and less severe occlusion further away (200 μm) (See
In ttw/ttw mice the degree of intimal hyperplasia was increased, as the lumen at 200 μm caudal from the ligation was almost completely occluded. Quantitative analyses of sequential sections of ligated common carotid arteries showed that ttw/ttw mice had significantly increased neointimal proliferation compared to WT mice after ligation-induced vascular remodeling for 14 days (See
The results demonstrated that subcutaneous administration of recombinant ENPP1-Fc fusion protein treats intimal hyperplasia in mice models of vascular injury in both ENPP1 deficient (ttw/ttw) and ENPP1-non deficient (WT) mice. This surprising finding suggests that ENPP1 has therapeutic potential for treating intimal hyperplasia in patients who suffer from VSMC proliferation due to surgical tissue injury, myocardial infarction, stroke, and even non-surgical tissue injury.
The main aim of the experiment is to determine the prophylactic effect of ENPP1-Fc on intimal hyperplasia in WT mice and homozygous ttw/ttw mice. The scheme of prophylactic treatment using ENPP1-Fc is shown in
In this preventive approach, both mice (WT & ttw/ttw mice) were treated for 7 days prior to carotid ligation, and treatment was continued for 14 days post-surgery or carotid ligation. Left carotid artery ligation surgery was performed in a 7 week-old WT and ttw/ttw mice following the procedures outlined in Example I. Mice were then euthanized using CO2 inhalation 14 days after carotid ligation following the same protocols as in Example I.
To determine the preventive effect of ENPP1 on intimal hyperplasia, both mice (WT & ttw/ttw mice) were treated with either vehicle (Control cohort) or ENPP1-Fc (Experimental cohort) for 7 days prior to carotid ligation, and treatment was continued for 14 days post-surgery.
14 days after surgery, both WT- and ttw/ttw-mice treated with ENPP1-Fc showed greatly reduced medial area (
For further investigation of apoptosis in carotids from WT- and ttw/ttw-mice, a sub cohort which were treated with vehicle alone was allowed to stay ligated for 21 days and TUNEL staining was preformed using in situ cell death detection kit (TMR red, Roche Diagnotics GmbH, Penzberg, Germany) following the manufacturer's instructions. For negative control, staining was performed without TUNEL enzyme; for positive control, sample DNA was degraded by DNAse I grade I for 10 min at room temperature.
The WT mice treated with ENPP1-Fc showed greatly reduced intimal hyperplasia compared to WT mice treated with vehicle. Likewise, the ttw/ttw mice treated with ENPP1-Fc showed greatly reduced intimal hyperplasia compared to ttw/ttw mice treated with vehicle. Histological Elastica van Gieson staining of 14 days ligated mice showed much less intimal hyperplasia in ENPP1-Fc treated WT- and ttw/ttw-mice than those treated with vehicle, ENPP1-Fc treated ttw/ttw-mice approaching the degree seen in ENPP1-Fc treated WT animals (See
WT- and ttw/ttw-mice ligated for 21 days and preventively treated with ENPP1-Fc for 28 days also showed a greatly reduced medial area (
Interestingly, the carotids of vehicle treated ttw/ttw-mice ligated for 21 days had a smaller intimal area than those of vehicle treated WT-mice (
The results of quantitative analyses of the neointimal and medial areas, as well as the I/M ratio of ligated common carotid arteries obtained in vehicle-treated WT mice showed to be similar to those of WT mice without treatment. Likewise, the neointimal and medial areas, as well as the I/M ratio of ligated common carotid arteries obtained in vehicle-treated ttw/ttw mice showed to be similar to ttw/ttw mice without treatment.
The intimal area of WT mice receiving subcutaneous ENPP1-Fc was significantly reduced compared to vehicle-treated WT mice, whereas the medial area, between the external and internal lamina, remained constant. The I/M ratio showed show a statistically significant decrease in ENPP1-Fc treated WT mice compared to vehicle-treated WT mice (See
Furthermore, the preventive treatment of carotid ligated ttw/ttw-mice led to more decreased intimal areas and I/M ratios compared to therapeutic treatment (See
The main aim of the experiment is to determine the therapeutic effect of ENPP3-Fc on intimal hyperplasia in WT mice and homozygous ttw/ttw mice. ENPP3-Fc is prepared using previously established protocols described elsewhere. Left carotid artery ligation surgery is performed in a 6 week-old WT and ttw/ttw mice following protocols described in Example 1.
To determine whether ENPP3-Fc could have a therapeutic effect if administered after the carotid ligation, 6 week-old WT and ttw/ttw mice are subjected to carotid ligation and allowed to recover. Both mice are then treated with either vehicle (Tris buffered saline, pH 7.4/Control cohort) or ENPP3-Fc (Experimental cohort) at 10 mg/kg bodyweight by subcutaneous injection every other day. ENPP3-Fc treatment (10 mg/kg bodyweight subcutaneously injected every other day) is initiated 7 days after carotid ligation and continued for 7 days until the carotid arteries are harvested at 14 days post ligation. Carotid arteries are fixed with 4% paraformaldehyde in PBS for morphological analyses.
Serial sections (sections of 5 μm each) are collected and analyzed following the protocols described in Example 1. Statistical analyses are performed as described in Example I. ENPP1 deficiency resulted in neointimal lesion formation after carotid ligation injury in ttw/ttw mice and hence ttw/ttw mice had higher levels of VSMC proliferation when compared with the WT mice as seen in Example I.
In ttw/ttw mice the degree of intimal hyperplasia increased, as the lumen at 200 μm caudal from the ligation was nearly occluded. Quantitative analyses of sequential sections of ligated common carotid arteries shows that ttw/ttw mice had significantly increased neointimal proliferation compared to WT mice after ligation-induced vascular remodeling for 14 days.
It is expected that VSMC proliferation will decrease in ttw/ttw mice upon administration of ENPP3-Fc since these mutant mice are deficient in ENPP1 protein. It is expected that the VSMC proliferation in WT mice will be reduced upon ENPP3-Fc administration. Such results will evidence that ENPP3-Fc protein has a therapeutic effect by decreasing VSMC proliferation in blood vasculature caused by mechanical injury.
The results are expected to demonstrate that subcutaneous administration of recombinant ENPP3-Fc fusion protein can treat intimal hyperplasia in mice models of vascular injury in both ENPP1 deficient (ttw/ttw) and ENPP1 non-deficient (WT) mice. Thus, ENPP3-Fc may serve as a therapeutic for treating intimal hyperplasia in patients who suffer from VSMC proliferation caused due to surgical tissue injury, myocardial infarction, stroke, and even non-surgical tissue injury.
The main aim of the experiment is to determine the prophylactic effect of ENPP3-Fc on intimal hyperplasia in WT mice and homozygous ttw/ttw mice. The scheme of prophylactic treatment using ENPP3-Fc is similar to the schematic shown in
In this preventive approach, both mice (WT & ttw/ttw mice) are treated for 7 days prior to carotid ligation, and treatment is continued for 14 days post-surgery or carotid ligation. Left carotid artery ligation surgery is performed in a 6 week-old WT and ttw/ttw mice following the procedures outlined in Example I. Mice are then euthanized using CO2 inhalation 14 days after carotid ligation following the same protocols as in Example I.
To determine the preventive effect of ENPP3 on intimal hyperplasia, both mice (WT & ttw/ttw mice) are treated with either vehicle (Control cohort) or ENPP3-Fc (Experimental cohort) for 7 days prior to carotid ligation, and treatment continued for 14 days post-surgery. The WT mice treated with ENPP3-Fc are expected to show greatly reduced intimal hyperplasia in comparison to WT mice treated with vehicle. Likewise, the ttw/ttw mice treated with ENPP3-Fc are expected to show greatly reduced intimal hyperplasia compared to ttw/ttw mice treated with vehicle.
The results of quantitative analyses of the neointimal and medial areas, as well as the I/M ratio of ligated common carotid arteries obtained in vehicle-treated WT mice are expected to be similar to those of WT mice without treatment. Likewise, the neointimal and medial areas, as well as the I/M ratio of ligated common carotid arteries obtained in vehicle-treated ttw/ttw mice are expected to be similar to those of ttw/ttw mice without treatment.
The intimal area of WT mice receiving subcutaneous ENPP3-Fc is expected to be significantly reduced compared to vehicle-treated WT mice, whereas the medial area, between the external and internal lamina, is expected to be constant. The I/M ratio is expected to show a statistically significant decrease in ENPP3-Fc treated WT mice compared to vehicle-treated WT mice indicating that the prophylactic treatment of ENPP3-Fc prior to carotid ligation will have a protective effect by lowering the level of VSMC proliferation. Thus, ENPP3-Fc administration is expected to prevent and effectively treat myointimal proliferation and stenosis in carotid ligated WT mice in addition to carotid ligated ttw/ttw mice. The experiment is expected to demonstrate that administration of ENPP3 prior to and after carotid ligation protects against intimal hyperplasia even in WT mice.
Atherosclerosis is the most common inflammatory disease of arterial vessels, which can lead to life-threatening myocardial infarction or ischemic stroke. The main aim of the experiment is to determine the ability of ENPP1 or ENPP1-Fc eluting stents to inhibit neointima formation and inflammation thereby reducing thrombosis and/or vessel occlusion which increases the risk of hemorrhagic complications post cardiac surgery.
Without being bound to any one theory, it is expected that inducing the overexpression of ENPP1 or ENPP1-Fc at the site of the implanted stent would result in one or more (i) a decrease in platelet activation, (ii) a reduction in restenosis and inflammatory responses, and (iii) a decrease in VSMC proliferation, following stent implantation. This therapy is based on the delivery of ENPP1 mRNA (or ENPP1-Fc mRNA or ENPP1-Albumin mRNA) to the endothelial cells, which then in turn express the ENPP1 protein at the site of the stent implant after mRNA translation.
Production of ENPP1 mRNA
pcDNA 3.3 plasmid (Eurofins Genomics GmbH, Ebersberg, Germany) containing ENPP1 DNA templates is amplified using the HotStar HiFidelity Polymerase Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The PCR product (PCR cycler: Eppendorf, Wesseling, Germany) is purified with the Qiaquick PCR Purification Kit (Qiagen). In vitro transcribed mRNA is generated with the MEGAscript1 T7 Kit (Ambion, Glasgow, Scotland) according to the manufacturer's instructions.
To modify the mRNA a 3″-0-Mem7 G(5′)ppp(5′)G RNA Cap Structure Analog (New England Biolabs, Frankfurt, Germany) is added to the reaction as well as pseudouridine-5′-triphosphate and 5-methylcytidine-5′-triphosphate (TriLink Biotech, San Diego, CA, USA), which are substituted for UTP and CTP, respectively. For RNase inhibition 1 μl of RNase inhibitor (Thermo Scientific, Waltham) is added per reaction. The in vitro transcribed mRNA is then purified with the RNeasy Kit (Qiagen). The purified mRNA is dephosphorilized using the Antarctic Phosphatase Kit (New England Biolabs) and once again purified with the RNeasy Kit (Qiagen). The same procedure is repeated to generate enhanced green fluorescent protein (eGFP) mRNA using eGFP DNA. (Avci-Adali M, Behring A, Keller T, Krajewski S, Schlensak C, Wendel H P (2014), Optimized conditions for successful transfection of human endothelial cells with in vitro synthesized and modified mRNA for induction of protein expression. J Biol Eng 8: 8).
The functionality of the generated ENPP1 mRNA is validated by measuring free phosphate after hydrolysis of ATP by transfected HEK293 cells. ENPP1 mRNA transfected HEK293 cells are incubated with 20 μM ATP (möLab, Langenfeld, Germany) or PBS as control for 10 min at 37° C. on a shaking platform (Polymax 1040, Heidolph, Schwabach, Germany). The ATP substrate degrades over time in the presence of ENPP1, with the accumulation of the enzymatic product AMP. Using varying concentrations of ATP substrate, the initial rate velocities for ENPP1 are derived in the presence of ATP, and the data is fit to a curve to derive the enzymatic rate constants.
In order to develop a bioactive stent coating, which allows local delivery of ENPP1mRNA and transfection of endothelial cells in vivo, the generated ENPP1 mRNA is first coated on thermanox plastic slides. The stent coating is thus simulated using thermanox plastic slides (Nunc, Thermo scientific, USA). First, 100.000 HEK293 cells per well are seeded on a 12-well plate.
After 24 hours, 2 μl Lipofectamin as well as 10 μg ENPP1 mRNA are mixed with 50 μl Opti-MEM and incubated at room temperature for 20 min. Meanwhile, 10 μl from a polylactic-co-glycolic-acid (PLGA) (Evoniks, Darmstadt) stock solution (20 mg/ml)) is diluted in 990 μl ethyl acetate (final concentration 200 μg/ml). Then 200 μl of the PLGA solution are mixed with the transfection complexes.
The thermanox slides are coated with the solution in a step-by-step approach at room temperature. eGFP mRNA and sterilized water are used as controls. The HEK293 cells are supplied with a new medium before the dried slides are plated face down onto the cells. The cells are incubated with the slides at 37° C. and 5% CO2 for 24 hrs, 48 hrs and 72 hrs and then analyzed using a FACScan cytometer.
The expression of ENPP1 of HEK293 cells was measured using flow cytometry. The ENPP1 coated thermonox slide exposed cells and control cells are stained with anti-ENPP1-fluorescein isothiocyanate (FITC) antibody. Flow cytometric analysis of the HEK293 cells after incubation with the ENPP1mRNA/PLGA covered thermanox slides are expected to show that the ENPP1 mRNA is released from the PLGA coating, whereby increase in ENPP1 expression is expected to be detectable after 24 hours, 48 hours and 72 hours post exposure to slides.
Compared to control HEK293 cells, (which were exposed thermonox slides coated with Lipofectamine alone) 0.5-1 μg of the ENPP1 mRNA is expected to be sufficient to induce increase of the ENPP1 protein expression in HEK cells exposed to ENPP1 mRNA coated thermonox slides even after 24 hours of exposure.
Without being bound to any one theory, it is proposed herein that the ENPP1 expressed at the site of the stent implant is expected to prevent intimal proliferation and reduce platelet occlusion thereby the risk of hemorrhagic complications post cardiac surgery as seen from the results of Examples 1 and 2.
An ENPP1 agent coated stent is prepared and then implanted in a coronary artery. In this example, a juvenile pig animal model is used for implanting the ENPP1-coated stent to determine the efficacy of an ENPP1 coated stent to inhibit neointima formation, restenosis and inflammation.
Preparation of ENPP1 Coated Stent
Any stent is amenable to be coated with ENPP1 agent. Common examples of commercial sources that sell stents for use include Abbot, Boston Scientific, Medtronic, Alvimedica, Lepu Medical Technology, Cordis, Balton or Biotronik.
For example, a plain stent such as a bare metal stent can be converted to ENPP1 coated eluting stent by placing a polymeric film comprising ENPP1 mRNA inside the stent or by spraying a polymeric or nonpolymeric solution comprising ENPP1 mRNA or ENPP1 polypeptide on to the stent surface.
Some examples of ENPP1 polymeric film are shown below, the ENPP1 polymeric film can be placed inside stents to create ENPP1 coated eluting stents. Optionally nonpolymeric carrier such as Vitamin E, Vitamin E acetate, Vitamin E succinate, oleic acid, peanut oil and cottonseed oil can be added to the solution improve the stability of ENPP1 agent in the polymeric film
Some examples of ENPP1 comprising spray solutions are shown below, the spray solutions can be applied onto stents to create ENPP1 coated eluting stents. Optionally nonpolymeric carrier such as Vitamin E, Vitamin E acetate, Vitamin E succinate, oleic acid, peanut oil and cottonseed oil can be added to the spray solution improve the stability of ENPP1 agent.
Animal Model
Thirty 4-to-5-month-old juvenile pigs with the weight of 25-35 kg are procured from commercial sources. Thirty stainless steel vents are obtained from one or more commercial sources such as Abbot, Boston Scientific, Medtronic, Alvimedica, Lepu Medical Technology, Cordis, Balton or Biotronik. Thirty stainless steel stents thus obtained are coated with ENPP1 mRNA following the protocol shown above for coating. Thirty bare metal stents (BMSs) are obtained from Abbott to be used as control set. The ENPP1 coated stent is then sterilized using ethylene oxide, compressed, and mounted on a balloon angioplasty catheter. It is then deployed at a site in an artery using standard balloon angioplasty techniques.
The stents are randomly assigned and placed in the left anterior descending, circumflex, or right coronary arteries (one stent per artery) of 30 pigs, one coated stent per pig. The pigs are then maintained on 75 mg clopidogrel and 100 mg aspirin per day and sacrificed after 7 days and 14 days, respectively.
Seven or 14 days after stent implantation, the animals are euthanized using intravenous injection of pentobarbital euthanasia solution (100 mg/kg), and the stented coronary arteries were harvested. The arteries are sectioned into 3 to 5 mm segments from the proximal, middle, and distal part of the stents, fixed in 4% formalin for 48 h, and embedded in paraffin. The sections are subjected to histology and morphometrical measurements to determine intimal, medial area and I/M ratios following the protocols described in Example 1. The intimal area of arterial sections obtained from pigs receiving ENPP1 coated stents is expected to be significantly reduced compared to arterial sections from pigs having non-eluting stainless-steel bare mesh stent. The I/M ratio is expected to show a statistically significant decrease in the arterial sections of pigs with ENPP1 coated stents compared to pigs with non-eluting stainless-steel stents. Thus, in situ administration of ENPP1 agent by using ENPP1 coated stents is expected to prevent and effectively treat myointimal proliferation and/or restenosis at the site of injury.
An ENPP3 agent coated stent is prepared and then implanted in a coronary artery. In this example, a juvenile pig animal model is used for implanting the ENPP3-coated stent to determine the efficacy of an ENPP3 coated stent to inhibit neointima formation, restenosis and inflammation.
Preparation of ENPP3 Coated Stent
Any stent is amenable to be coated with ENPP3 agent. Common examples of commercial sources that sell stents for use include Abbot, Boston Scientific, Medtronic, Alvimedica, Lepu Medical Technology, Cordis, Balton or Biotronik.
For example, a plain stent such as a bare metal stent can be converted to ENPP3 coated stent by placing a polymeric film comprising ENPP3 mRNA inside the stent or by spraying a polymeric or nonpolymeric solution comprising ENPP3 mRNA or ENPP3 polypeptide on to the stent surface.
Some examples of ENPP3 polymeric film are shown below, the ENPP3 polymeric film can be placed inside stents to create ENPP3 coated eluting stents. Optionally nonpolymeric carrier such as Vitamin E, Vitamin E acetate, Vitamin E succinate, oleic acid, peanut oil and cottonseed oil can be added to the solution improve the stability of ENPP3 agent in the polymeric film
Some examples of ENPP3 comprising spray solutions are shown below, the spray solutions can be applied onto stents to create ENPP3 coated eluting stents. Optionally nonpolymeric carrier such as Vitamin E, Vitamin E acetate, Vitamin E succinate, oleic acid, peanut oil and cottonseed oil can be added to the spray solution improve the stability of ENPP3 agent.
Animal Model
Thirty 4-to-5-month-old juvenile pigs with the weight of 25-35 kg are procured from commercial sources as described in Example 5. Thirty stainless steel vents are obtained from commercial sources. Thirty stainless steel stents thus obtained are coated with ENPP3 mRNA following the protocol shown above for coating. Thirty bare metal stents (BMSs) are obtained from Abbott to be used as control set. The ENPP3 coated stent is then sterilized using ethylene oxide, compressed, and mounted on a balloon angioplasty catheter. It is then deployed at a site in an artery using standard balloon angioplasty techniques.
The stents are randomly assigned and placed in the left anterior descending, circumflex, or right coronary arteries (one stent per artery) of 30 pigs, one coated stent per pig. The pigs are then maintained on 75 mg clopidogrel and 100 mg aspirin per day and sacrificed after 7 days and 14 days, respectively. Seven or 14 days after stent implantation, the animals are euthanized using intravenous injection of pentobarbital euthanasia solution (100 mg/kg), and the stented coronary arteries were harvested. The arteries are sectioned into 3 to 5 mm segments from the proximal, middle, and distal part of the stents, fixed in 4% formalin for 48 h, and embedded in paraffin.
The sections are subjected to histology and morphometrical measurements to determine intimal, medial area and I/M ratios following the protocols described in Example 1. The intimal area of arterial sections obtained from pigs receiving ENPP3 coated stents is expected to be significantly reduced compared to arterial sections from pigs having non-eluting stainless-steel bare mesh stent. The I/M ratio is expected to show a statistically significant decrease in the arterial sections of pigs with ENPP3 eluting stents compared to pigs with non-eluting stainless-steel stents. Thus, in situ administration of ENPP3 agent by using ENPP3 coated eluting stents is expected to prevent and effectively treat myointimal proliferation and/or restenosis at the site of injury.
The disclosure of each and every U.S. and foreign patent and pending patent application and publication referred to herein is specifically incorporated herein by reference in its entirety, as are the contents of Sequence Listing and Figures.
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims. Any combination of the embodiments disclosed in the any plurality of the dependent claims or Examples is contemplated to be within the scope of the disclosure.
From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions, including the use of different signal sequences to express functional variants of ENPP1 or ENPP3 or combinations thereof in different viral vectors having different promoters or enhancers or different cell types known in art to treat any diseases characterized by the presence of pathological calcification or ossification are within the scope according to the disclosure. Other embodiments according to the disclosure are within the following claims.
Recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or sub combination) of listed elements. Recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Other embodiments are within the following claims.
This application is a continuation of International Patent Application No. PCT/US2021/034576, filed May 27, 2021, which claims priority to U.S. Application No. 63/030,870 filed on May 27, 2020, the content of each is herein incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63030870 | May 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2021/034576 | May 2021 | US |
| Child | 18058712 | US |
