Patent Application
20230212246
The present disclosure relates to the characterization of molecular targets for treating intracranial aneurysms (IA). Intracranial aneurysm (IA) is a cerebrovascular disease that predominantly occurs in the cerebral artery and is characterized by pathologic dilatation of blood vessels. Each intracranial aneurysm (IA) is a weakened area in a cerebral artery wall that leads to abnormal dilatation and rupture causing subarachnoid hemorrhage (SAH), a major cause of hemorrhagic stroke. A rupture of IA induces a subarachnoid hemorrhage (SAH), a type of hemorrhagic stroke that frequently leads to death or severe disability. Due to early age of onset and high mortality, SAH accounts for >25% of years lost for all stroke victims under the age of 65 years. Despite treatment advances, SAH mortality rate is 40% and only half of survivors return to independent life.
There is a critical unmet need for understanding the genetic and molecular basis for IA to improve clinical outcomes through early therapeutic intervention.
This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.
In some aspects, the present disclosure demonstrates that the IA-causing gene THSD1 positively regulates TGFβ signaling. This raises a novel concept that IA disease can be contributed by downregulation of TGFβ signaling, which is in contrast to the previous fibrillin hypothesis in which upregulation of TGFβ signaling plays a pathogenic role in IA development. The present disclosure thus contemplate that overexpression of THSD1 in circle of Willis provides beneficial effects on IA disease by activating endothelial TGFβ signaling. In some aspects, the disclosure proposes metal coils generated by adding TGF-β ligand or other TGF-β activators to coiling materials to improve the outcome of existing surgical treatments on intracranial aneurysm patients. In other aspects, the disclosure contemplates coating metal coils with other proteins that activate latent TGFβ in cell matrix.
Accordingly, disclosed herein are a metal coil conjugated to a TGFβ activator configured for use in an endovascular coiling procedure. In some cases, the TGFβ activator is a TGFβ ligand, such as TGF-β1, TGF-β2, TGF-β3. In other cases the TGFβ activator is a TGFβ signaling activator, such as Plasmin, MMP2, MMP9, Thrombospondin-1. In some instances, a metal in the metal coil is selected from the group consisting of platinum, tungsten, titanium, silver, stainless steel, zirconium, or an alloy thereof. In preferred cases, a metal in the metal coil is platinum. The metal coil ca have a thicknesses ranging from 0.005 mm to 1.0 mm, from 0.010 mm to 1.0 mm, from 0.020 mm to 1.0 mm, from 0.030 mm to 1.0 mm, from 0.040 mm to 1.0 mm, from 0.050 mm to 1.0 mm, from 0.060 mm to 1.0 mm, from 0.070 mm to 1.0 mm, from 0.080 mm to 1.0 mm, from 0.090 mm to 1.0 mm, from 0.1 mm to 1.0 mm, from 0.2 mm to 1.0 mm, from 0.3 mm to 1.0 mm, from 0.4 mm to 1.0 mm, or from 0.5 mm to 1.0 mm. In some instances the metal coil is used to treat a subject at risk of suffering from an aneurysm. In some instances the aneurysm is an intracranial aneurysm. In other instances the aneurysm is an aortic aneurysm. In some instances the subject carries a variant affecting the expression of a Thrombospondin Type 1 Domain Containing 1 (THSD1) gene. Such variants can be in a coding region, in a non-coding region, or in a control sequence of the Thrombospondin Type 1 Domain Containing 1 (THSD1) gene. In some instances, the variant in the THSD1 gene is a single codon substitution in at least one THSD1 allele, specific variants contemplated with the invention have been associated with a phenotype that affects the function of the THSD1 gene and lead to aneurysms such as LSF, R460W, E466G, G600E, P639L, T6531, or S775P. In some instances the subject is a Shuman.
In some aspects the disclosure provides a method for treating a subject at risk of suffering from an aneurysm comprised of inserting into the subject a catheter for delivering a metal coil conjugated to a TGFβ activator configured for use in an endovascular coiling procedure. In some instances, the method further comprises calculating an estimate of an aneurysm volume prior to inserting into the subject the catheter. In some instances, the method further comprises releasing the metal coil conjugated to the TGFβ activator into the aneurysm. In some cases, the TGFβ activator is a TGFβ ligand, such as TGF-β1, TGF-β2, TGF-β3. In other cases the TGFβ activator is a TGFβ signaling activator, such as Plasmin, MMP2, MMP9, Thrombospondin-1. In some instances, a metal in the metal coil is selected from the group consisting of platinum, tungsten, titanium, silver, stainless steel, zirconium, or an alloy thereof. In preferred cases, a metal in the metal coil is platinum. The metal coil ca have a thicknesses ranging from 0.005 mm to 1.0 mm, from 0.010 mm to 1.0 mm, from 0.020 mm to 1.0 mm, from 0.030 mm to 1.0 mm, from 0.040 mm to 1.0 mm, from 0.050 mm to 1.0 mm, from 0.060 mm to 1.0 mm, from 0.070 mm to 1.0 mm, from 0.080 mm to 1.0 mm, from 0.090 mm to 1.0 mm, from 0.1 mm to 1.0 mm, from 0.2 mm to 1.0 mm, from 0.3 mm to 1.0 mm, from 0.4 mm to 1.0 mm, or from 0.5 mm to 1.0 mm. In some instances the metal coil is used to treat a subject at risk of suffering from an aneurysm. In some instances the aneurysm is an intracranial aneurysm. In other instances the aneurysm is an aortic aneurysm. In some instances the subject carries a variant affecting the expression of a Thrombospondin Type 1 Domain Containing 1 (THSD1) gene. Such variants can be in a coding region, in a non-coding region, or in a control sequence of the Thrombospondin Type 1 Domain Containing 1 (THSD1) gene. In some instances, the variant in the THSD1 gene is a single codon substitution in at least one THSD1 allele, specific variants contemplated with the invention have been associated with a phenotype that affects the function of the THSD1 gene and lead to aneurysms such as LSF, R460W, E466G, G600E, P639L, T6531, or S775P. In some instances the subject is a human.
The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:
It should be understood that the drawings are not necessarily to scale, and that like reference numbers refer to like features.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention. The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art.
The rupture of an intracranial aneurysm frequently causes a subarachnoid hemorrhage (SAH), a type of stroke characterized by high morbidity and mortality. Specifically, the present disclosure demonstrates with data from three large IA/SAH families with at least 4 affected individuals where whole exome sequencing has been performed to identify rare variants that segregate with disease. For each family, whole exome sequencing has been performed on at least 15 family members, irrespective of their IA status.
Previously, it has been reported that deleterious Thrombospondin-type 1 domain-containing protein 1 (THSD1) rare variants caused disease in both familial and sporadic cases with supporting evidence from animal models. Of note, whole exome sequencing of large IA families identified (some members of the affected family are shown in the pedigree of
These rare variants were highly enriched in case-control studies in comparison to ethnically matched controls. It was found that Thsd1 loss-of-function leads to brain hemorrhage and premature death in both zebrafish and mice. Further, Thsd1 heterozygous and null mice developed IA and suffered SAH. The study further demonstrated that THSD1 is highly expressed in endothelial cells of the cerebrovasculature, is important for cell adhesion, promotes nascent focal adhesion assembly via Talin interactions, and potentially regulates downstream signaling. For further description of this work see. Z. Xu, D. Kim, et al., NeuroMolecular Medicine (2019) 21:325-343; T. Santiago-Sim, D. Kim, et al., Stroke. 2016; 47:3005-3013. DOI: 10.1161/STROKEAHA.116.014161); Yan-Ning Rui and D. Kim, et al., Cell Physiol Biochem 2017; 43:2200-2211; each of which incorporated by reference in their entireties). However the study did not provide any insights on the THSD1 molecular pathways.
To further study the role of THSD1 in IA/SAH, additional analysis of whole exome sequencing of the IA families described in
The present disclosure considered the differentially expressed genes and characterized autophagy pathways as contributors to IA development and potential targets for therapy. The present disclosure also contemplates that mutations in genes other than THSD1 that affect the TGFβ pathway could render a subject at risk of suffering an IA. The present disclosure characterizes the TGFβ pathway as a novel molecular target for the treatment of subjects at risk of developing an aneurysm.
Transforming growth factor β (TGFβ) is an important mediator of a number of cellular processes, including skeletal, ocular, pulmonary, and vascular systems. Aneurysms often result in aortic dissection or rupture of vasculature, which is the leading cause of sudden death in subjects prone to vasculature ruptures and aneurysms. However, there is currently no genotype-specific medical treatment. The common paradigm proposes that increased TGF-β signaling contributes to the complicated pathogenesis of aneurysm formation, particular aortic aneurysm, but a comprehensive understanding of governing molecular mechanisms remained admittedly lacking. See, TGF-β Signaling-Related Genes and Thoracic Aortic Aneurysms and Dissections. Int J Mol Sci. 2018 July; 19(7): 2125.
The present disclosure provides experiments demonstrating that THSD1 positively regulates TGF-β signaling in endothelial cells, thus mechanistically linking a gene associated with aneurysms (THSD1) and the TGF-β pathway.
The methods, compositions, and uses of this disclosure may comprise a treatment method to arrest, reverse, or ameliorate an aneurysm, e.g., an intracranial aneurysm. In some cases, the therapeutic effect is achieved by administrating a therapeutically-effective dose of a TGF-β ligand or other TGF-β signaling activator(s) conjugated to a platinum coil. Endovascular coiling is a procedure performed to block blood flow into an aneurysm. Endovascular coiling is a minimally invasive technique, which means an incision in the skull is not required to treat the brain aneurysm. Rather, a catheter is used to reach the aneurysm in the brain.
During endovascular coiling, a microcatheter is inserted through the initial catheter and passed through the groin up into the artery containing the aneurysm. Typically, when the microcatheter has reached the aneurysm and has been inserted into the aneurysm, an electrical current is used to separate the coil from the catheter. Platinum coils are then released, which seal off the opening of the aneurysm. The coils induce clotting (embolization) of the aneurysm and, in this way, prevent blood from getting into it. The coil is left in place permanently in the aneurysm. Depending on the size of the aneurysm, more than one coil may be needed to completely seal off the aneurysm.
In some aspects, provided herein are TGF-β ligands or other TGF-β signaling activator(s) conjugated to a platinum coil. The coils used in a procedure of the disclosure can be made of soft platinum metal, and they can be shaped like a spring, a rod, or another suitable shape. These coils can be very small and thin, ranging in size from about twice the width of a human hair (largest) to less than one hair's width (smallest). Generally, platinum coils of the disclosure can have thicknesses ranging from 0.005 mm to 1.0 mm, coil widths ranging from 0.01 mm to 1.0 mm.
The metal in the coil can comprise a variety of metals, including, but not limited to platinum, tungsten, titanium, silver, stainless steel, zirconium, or an alloy thereof.
The treatment may comprise treating a subject (e.g. a patient at risk of having an intracranial aneurysm due to the presence of a THSD1 genetic variant or an animal with a similar genetic variant). The disease may be a weakness in a blood vessel in the brain that balloons and fills with blood, for example, a brain aneurysm (also called a cerebral aneurysm or an intracranial aneurysm) is a ballooning arising from a weakened area in the wall of a blood vessel in the brain. The subject may be a human.
Treatment may be provided to the subject before clinical onset of disease. For instance, in specific cases, treatment may be provided upon the identification of a THSD1 variant in a subject, before the onset of a disease. Treatment may be provided to the subject after clinical onset of disease. Treatment may be provided to the subject after 1 day, 1 week, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment may be provided to the subject for more than 1 day, 1 week, 1 month, 6 months, 12 months, 2 years or more after clinical onset of disease. Treatment may be provided to the subject for less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment may also include treating a human in a clinical trial. Because of the genetic aspect of IA, treatment may be provided through the lifetime of a subject that is afflicted with a THSD1 variant that may lead to subarachnoid hemorrhage. In some aspects, treatment will be prescribed to prevent IA in a subject that carries a THSD1 variant associated with IA.
A treatment can comprise performing an endovascular coiling procedure where the platinum coil has TGF-β ligands or other TGF-β signaling activator(s) conjugated to the platinum coil. A treatment can comprise modulating the levels of TGF-β in vivo. A treatment may comprise administering a suitable level of TGF-β ligands or other TGF-β signaling activator(s) for reducing bulge's in the wall of a blood vessel and preventing Subarachnoid hemorrhage.
Further, there are many risk factors for the development of intracranial aneurysms, both inherited and acquired. Females are more prone to aneurysm rupture, with SAH times more common in women. The prevalence of aneurysms is increased in certain genetic diseases; the classic example is autosomal dominant polycystic kidney disease (ADPKD), but other diseases such as Ehlers-Danlos syndrome, neurofibromatosis, al-antitrypsin deficiency also demonstrate a link. In ADPKD, 10% to 15% of patients develop intracranial aneurysms. Marfan's Syndrome was once thought to be linked to intracranial aneurysm formation, but recent evidence suggests that this may not be true. A treatment may comprise identifying a subject that is at risk of developing an aneurysm based on a diagnosis of the subject and subsequently treating the subject with TGF-β ligands or other TGF-β signaling activator(s).
Aneurysms also run in families in the absence of an identified genetic disorder, with a prevalence of 7% to 20% in first or second degree relatives of patients who have suffered a SAH.
All of the functionalities described in connection with one embodiment of the methods, devices or instruments described herein are intended to be applicable to the additional embodiments of the methods, devices and instruments described herein except where expressly stated or where the feature or function is incompatible with the additional embodiments. For example, where a given feature or function is expressly described in connection with one embodiment but not expressly mentioned in connection with an alternative embodiment, it should be understood that the feature or function may be deployed, utilized, or implemented in connection with the alternative embodiment unless the feature or function is incompatible with the alternative embodiment.
Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” refers to one or more cells, and reference to “the system” includes reference to equivalent steps, methods and devices known to those skilled in the art, and so forth. Furthermore, terms such as “first,” “second,” “third,” etc., merely identify one of a number of steps, components, functions, and/or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.
Subjects can be humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. A subject can be of any age. Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, infants.
As used in the specification and claims of this application, the term “administering” includes any method which is effective to result in delivery of an autophagy inhibitor to the subject.
As used in this specification, the term “aneurysm” refers to broad classes of aneurysm, including aneurysms: abdominal aortic, thoracic aortic, and cerebral.
As used in this specification, the term “cerebral aneurysm” or “intracranial aneurysm” (also known as a brain aneurysm) is a weak or thin spot on an artery in the brain that balloons or bulges out and fills with blood. The bulging aneurysm can put pressure on the nerves or brain tissue. It may also burst or rupture, spilling blood into the surrounding tissue (called a hemorrhage). An unruptured aneurysm usually causes no symptoms. A key symptom of a ruptured aneurysm is a sudden, severe headache. Treatments for an unruptured aneurysm include medications to control blood pressure and procedures to prevent a future rupture.
As used in this specification, the term “abdominal aortic” aneurysm (AAA) is a bulge or swelling in the aorta, the main blood vessel that runs from the heart down through the chest and tummy. An AAA can be dangerous if it is not spotted early on. It can get bigger over time and could burst (rupture), causing life-threatening bleeding.
As used in this specification, the term “abdominal aortic” aneurysm (AAA) is a bulge or swelling in the aorta, the main blood vessel that runs from the heart down through the chest and tummy. An AAA can be dangerous if it is not spotted early on. It can get bigger over time and could burst (rupture), causing life-threatening bleeding.
As used in this specification, the term “thoracic aortic” aneurysm is an abnormal widening or ballooning of a portion of an artery due to weakness in the wall of the blood vessel. A thoracic aortic aneurysm occurs in the part of the body's largest artery (the aorta) that passes through the chest.
As used herein the term “coil” can be any type of coil known in the art, such as, for example, a Guglielmi detachable coil (GDC). A metal coil is fabricated with metal. A metal coil can be coated with an absorbable polymeric material to improve long-term anatomic results in the endovascular treatment of intracranial aneurysms. The coil can further be coated to decrease friction to decrease the granulation tissue formation around the coils. In one embodiment, the coating facilitated attachment of TGF-β ligand(s) or TGF-β activator(s) to itself and is used to better deliver therapeutic doses of TGF-β ligand(s) or TGF-β activator(s) to aneurysms.
As used herein, the term TGF-β ligand include TGF-β1, TGF-β2, TGF-β3.
As used herein, the term TGF-β activator include Plasmin, MMP2, MMP9, Thrombospondin-1. These protein or enzymes can cleave the latent form of TGF-β in the extracellular matrix and release active TGF-β from nearby microenvironment.
As used in the specification and claims of this application, the term “at risk” or more specifically a “subject at risk of developing an intracranial aneurysm” is a subject afflicted with a genetic variant, e.g., THSD1 variant, that causes the subarachnoid hemorrhage seen when an aneurysm ruptures.
The term DNA “control sequences” refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites, nuclear localization sequences, enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell.
Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
EMBODIMENT 1. A metal coil conjugated to a TGFβ activator configured for use in an endovascular coiling procedure.
EMBODIMENT 2. The metal coil of embodiment Error! Reference source not found., wherein the TGFβ activator is a TGFβ ligand.
EMBODIMENT 3. The metal coil of embodiment Error! Reference source not found., wherein the TGFβ ligand is selected from the group consisting of TGF-β1, TGF-β2, TGF-β3.
EMBODIMENT 4. The metal coil of embodiment Error! Reference source not found., wherein the TGFβ activator is a TGFβ signaling activator.
EMBODIMENT 5. The metal coil of embodiment Error! Reference source not found., wherein the TGFβ signaling activator is selected from the group consisting of Plasmin, MMP2, MMP9, Thrombospondin-1.
EMBODIMENT 6. The metal coil of embodiment Error! Reference source not found., wherein a metal in the metal coil is selected from the group consisting of platinum, tungsten, titanium, silver, stainless steel, zirconium, or an alloy thereof.
EMBODIMENT 7. The metal coil of embodiment Error! Reference source not found., wherein the metal in the metal coil is platinum.
EMBODIMENT 8. The metal coil of embodiment Error! Reference source not found., wherein the metal coil has a thicknesses ranging from 0.005 mm to 1.0 mm.
EMBODIMENT 9. The metal coil of embodiment Error! Reference source not found., wherein the metal coil has a width ranging from 0.01 mm to 1.0 mm.
EMBODIMENT 10. The metal coil of embodiment Error! Reference source not found., wherein the metal coil is used to treat a subject at risk of suffering from an aneurysm.
EMBODIMENT 11. The metal coil of embodiment Error! Reference source not found., wherein the aneurysm is an intracranial aneurysm.
EMBODIMENT 12. The metal coil of embodiment Error! Reference source not found., wherein the aneurysm is an aortic aneurysm.
EMBODIMENT 13. The metal coil of embodiment Error! Reference source not found., wherein the subject carries a variant affecting the expression of a Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.
EMBODIMENT 14. The metal coil of embodiment Error! Reference source not found., wherein the variant is in a coding region of the Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.
EMBODIMENT 15. The metal coil of embodiment Error! Reference source not found., wherein the variant is in a control sequence of a non-coding region of the Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.
EMBODIMENT 16. The metal coil of embodiment Error! Reference source not found., wherein the variant in the THSD1 gene is a single codon substitution in at least one THSD1 allele.
EMBODIMENT 17. The metal coil of embodiment Error! Reference source not found., wherein the single codon substitution is LSF, R460W, E466G, G600E, P639L, T6531, or S775P.
EMBODIMENT 18. The metal coil of embodiment Error! Reference source not found., wherein the subject is a human.
EMBODIMENT 19. A method for treating a subject at risk of suffering from an aneurysm comprised of inserting into the subject a catheter for delivering a metal coil conjugated to a TGFβ activator configured for use in an endovascular coiling procedure.
EMBODIMENT 20. The method of embodiment 19, further comprising calculating an estimate of an aneurysm volume prior to inserting into the subject the catheter.
EMBODIMENT 21. The method of embodiment Error! Reference source not found., further comprising releasing the metal coil conjugated to the TGFβ activator into the aneurysm.
EMBODIMENT 22. The method of embodiment Error! Reference source not found., wherein the TGFβ activator is a TGFβ ligand.
EMBODIMENT 23. The method of embodiment 22, wherein the TGFβ ligand is selected from the group consisting of TGF-β1, TGF-β2, TGF-β3.
EMBODIMENT 24. The method of embodiment Error! Reference source not found., method of claim Error! Reference source not found., wherein the TGFβ activator is a TGFβ signaling activator.
EMBODIMENT 25. The method of embodiment 24, wherein the TGFβ signaling activator is selected from the group consisting of Plasmin, MMP2, MMP9, Thrombospondin-1.
EMBODIMENT 26. The method of embodiment Error! Reference source not found., wherein a metal in the metal coil is selected from the group consisting of platinum, tungsten, titanium, silver, stainless steel, zirconium, or an alloy thereof.
EMBODIMENT 27. The method of embodiment 26, wherein the metal in the metal coil is platinum.
EMBODIMENT 28. The method of embodiment Error! Reference source not found., wherein the metal coil has a thicknesses ranging from 0.005 mm to 1.0 mm.
EMBODIMENT 29. The method of embodiment Error! Reference source not found., wherein the metal coil has a width ranging from 0.01 mm to 1.0 mm.
EMBODIMENT 30. The method of embodiment Error! Reference source not found., wherein the metal coil is used to treat a subject at risk of suffering from an aneurysm.
EMBODIMENT 31. The method of embodiment Error! Reference source not found., wherein the aneurysm is an intracranial aneurysm.
EMBODIMENT 32. The method of embodiment Error! Reference source not found., wherein the aneurysm is an aortic aneurysm.
EMBODIMENT 33. The method of embodiment Error! Reference source not found., wherein the subject carries a variant affecting the expression of a Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.
EMBODIMENT 34. The method of embodiment 33, wherein the variant is in a coding region of the Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.
EMBODIMENT 35. The method of embodiment 33, wherein the variant is in a control sequence of a non-coding region of the Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.
EMBODIMENT 36. The method of embodiment 33, wherein the variant in the THSD1 gene is a single codon substitution in at least one THSD1 allele.
EMBODIMENT 37. The method of embodiment 36, wherein the single codon substitution is LSF, R460W, E466G, G600E, P639L, T6531, or S775P.
EMBODIMENT 38. The method of embodiment Error! Reference source not found., wherein the subject is a human.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.
The practice of some molecular techniques described herein may employ, unless otherwise indicated, techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, and genetic engineering technology, which are within the skill of those who practice in the art. Such techniques and descriptions can be found in standard laboratory manuals such as Westerfield, M. (2000). The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). 4th ed., Univ. of Oregon Press, Eugene.; all of which are herein incorporated in their entirety by reference for all purposes.
Genetic factors play a significant role in IA pathogenesis as illustrated by family studies and several IA predisposing syndromes. 7%-20% of all patients have a known family history and a family history is the strongest risk factor for disease. Excluding syndromes that account for less than 1% of all IA cases, candidate IA genes have been primarily identified by genome-wide association studies and more recently, by whole exome sequencing in affected families. Yet, little is known about the genetic causes of IA providing minimum insight for the understanding and development of therapeutic targets that could treat the disease.
Single-family genetic studies are a powerful tool to identify candidate high-risk genetic variants.
Deleterious Thrombospondin-type 1 domain-containing protein 1 (THSD1) rare variants cause disease in both familial and sporadic cases with supporting evidence from animal models. THSD1 is predominantly expressed in vascular endothelial cells. The work identified deleterious variants in thrombospondin-type 1 domain-containing protein 1 (THSD1) that can cause IA and SAH. Initial characterization of Thsd1 in two vertebrate models including zebrafish and mice lead to the discovery that THSD1 mediated cerebral hemorrhage is located in subarachnoid space in mice. For further description of this work see. Z. Xu, D. Kim, et al., NeuroMolecular Medicine (2019) 21:325-343; T. Santiago-Sim, D. Kim, et al., Stroke. 2016; 47:3005-3013. DOI: 10.1161/STROKEAHA.116.014161); Yan-Ning Rui and D. Kim, et al., Cell Physiol Biochem 2017; 43:2200-2211; each of which incorporated by reference).
However, the mechanism of action utilized by the discovered THSD1 variants to drive disease remained elusive. Further, there was little information describing genes and pathways regulated by THSD1 using global transcriptomics that could be used to inform the mechanism of action of THSD1. Thus, on its own the identification of THSD1 in the context of AI was not sufficient to inform a therapeutic strategy.
The present disclosure contemplated that THSD1 regulated genes may contribute to IA pathogenesis and that modulating their function may be beneficial as an IA treatment or in other diseases with aberrant THSD1 expression. The present disclosure provides results from global transcriptome profiling in human vascular endothelial cells upon THSD1 knockdown that identifies THSD1-regulated specific genes and pathways that are critical for mediating its function, providing potential targets for therapeutic intervention in IA.
The instant disclosure provides RNAseq experiments in two THSD1 knock-down endothelial cell lines. The RNAseq results from both cell lines support the evidence that THSD1 regulates multiple signaling pathways: Integrin, Src, PI3/AKT/mTor, and Rho signaling that are functionally linked to Focal Adhesion Kinase (FAK) signaling. A few of these pathways were selected for further analysis and characterization.
Materials and Methods
Cell Culture
HEK293T cells were maintained in DMEM medium (Corning, 10-013-CV) containing 10% fetal bovine serum (Invitrogen, 10082147), 100 IU penicillin, and 100 μg/ml streptomycin. Transfections of small interfering RNAs and plasmid DNA were performed using lipofectamine 2000 (Life Technologies, 11668027) according to the manufacturer's instructions. Alternatively, for cells such as endothelial cells that are hard to transfect, we will utilize lentiviral system to generate stable cell lines.
Knock-down Experiments
Knockdown experiments in human vascular endothelial cells were performed using two distinct cell lines [HUVECs and Human brain microvascular endothelial cells(HBMECs)] using four siRNAs (two control siRNAs and two THSD1-specific siRNAs) to minimize erroneous findings due to off-target effects.
Transcriptome Profiling
Bioinformatic analyses of the global transcriptome were performed on rRNA-depleted RNA samples by RNA-Seq. Table 1 illustrates results of the analysis. As shown on Table 1, THSD1 regulates multiple signaling pathways: Integrin, Src, PI3/AKT/mTor, and Rho signaling that are functionally linked to Focal Adhesion Kinase (FAK) signaling) as well as TGFβ signaling.
We identified a number of genes that are affected by the lack of THSD1 in the knock-down cell lines and are likely regulated by THSD1. A subset of these genes likely contributes to disease pathobiology and may be targets for therapeutic intervention. Table 2. Describes genes differentially expressed in THSD1 knockdowns.
Table 3. lists genes differentially expressed in THSD1 knockdown HUVECs.
Table 4 lists genes differentially expressed in THSD1 knockdown HUVECs
Bioinformatic analyses highlighted a potential role for the TGFβ Signaling pathway, and other pathways, in the pathology of IA. We evaluated the potential link to the TGFβ signaling pathway below.
THSD1 is required for TGFβ signaling in endothelial cells
Intracranial aneurysm (IA) is a weakened area in the wall of cerebral artery that leads to a bulging in a brain blood vessel. See, e.g.,
TGFβ is a polypeptide that plays diverse roles in cell proliferation and differentiation, apoptosis, and extracellular matrix formation. TGFβ transduces its signals via types I and II receptors, encoded by TGFBR1 and TGFBR2. The ligand-bound type-II receptor phosphorylates the glycine/serine-rich domain of the type-I receptor, which activates signal transduction. The gene that is responsible for most cases of Marfan syndrome which increases the risk for IA, the FBN1 gene encoding fibrillin-1, is required for effective TGFβ activation. The expression of genes normally stimulated by TGFβ, such as collagen and connective tissue growth factor, was upregulated in tissue of Loeys-Dietz syndrome patients who are more predisposed to IA disease. Furthermore, aneurysms that develop in an accepted mouse model of MFS (fbn1C139G/+) are associated with increased TGFβ signaling and can be prevented by TGFβ antagonists.
The present disclosure considered that the IA-causing gene THSD1 positively regulates TGFβ signaling. This raises a novel concept that IA disease can be contributed to by downregulation of TGFβ signaling, which is in contrast to the existing fibrillin hypothesis in which upregulation of TGFβ signaling plays a pathogenic role in IA development.
Materials and Methods
Cell Culture
HEK293T cells were maintained in DMEM medium (Corning, 10-013-CV) containing 10% fetal bovine serum (Invitrogen, 10082147), 100 IU penicillin, and 100 μg/ml streptomycin. Transfections of small interfering RNAs and plasmid DNA were performed using lipofectamine 2000 (Life Technologies, 11668027) according to the manufacturer's instructions. Alternatively, for cells such as endothelial cells that are hard to transfect, we will utilize lentiviral system to generate stable cell lines.
Western Bot
Cells were lysed in 1% Triton lysis buffer and sonicated briefly before centrifuged at 18506 g for 30 min at 4° C. Total cell lysates were added by 2X SDS sample buffer and then subjected to discontinuous SDS-PAGE analysis. Proteins were transferred to nitro-cellulose membranes using a Bio-Rad (Hercules, Calif.) mini transfer apparatus followed by blocking with 5% nonfat milk. Primary antibodies and secondary antibodies were used usually at 1:1000 and 1:10000 dilutions respectively before using an Odyssey system to detect the fluorescence signal.
TGFβ Signaling
Gain-of-function of THSD1 promotes TGFβ signaling
To further investigate a potential role for the TGFβ Signaling pathway in the pathology of aneurysms, specifically IA, we treated HEK293T cells with TGFβ1 in different time points. As shown in
Loss-of-function of THSD1 inhibits TGFβ signaling
To further investigate a potential role for the TGFβ Signaling pathway in the pathology of aneurysms, specifically IA, we also tested TGFβ treatment in different endothelial cells including HBMEC and HUVEC. HBMEC is a primary brain endothelial cells and HUVEC is primary umbilical vein endothelial cells. As shown in
These data suggest that THSD1 is required for TGFβ signaling in endothelial cells.
To evaluate the specificity of the requirement for THSD1 for TGFβ signaling in endothelial cells we also tested Activin treatment. As shown in
In-vivo mammalian model
To interrogate the consequence of Thsd1 loss in mammals, we used the Thsd1 knockout mouse that contains a knockin of a fluorescent Venus reporter. Thsd1Venus/+ and Thsd1Venus/Venus mice survived to weaning age in expected Mendelian ratios. However, brain magnetic resonance imaging revealed mild to severe dilatation of cerebral ventricles, consistent with hydrocephalus in a subset of mutant mice as young as 8 weeks (not shown here, previously reported). Mecanistically, the knock-in cassette containing a Venus reporter and a Neo resistant gene with poly-A sequence was inserted right after the first start codon of Thsd1, the splicing between exon 2 and exon 3 of thsd1 was interrupted, leading to the early termination of thsd1 transcription and translation.
Materials and Methods
MicroFil® injection in mice
MICROFIL® compounds will fill and opacify microvascular and other spaces of non-surviving animals and postmortem tissue under physiological injection pressure. The continuous, closed vascular system tends itself to flow through injection or perfusion techniques. Following injection, MICROFIL® compounds cure to form a three dimensional cast of the vasculature.
Prepare MicroFil® (Flow Tech, Inc. Carver, Mass.) casting solution according to the manufacturer's instruction: Mix 5 ml of MV diluent with 4 ml of filtered MV-112 compound (yellow). Add 450 μl (5%) of catalyst (MV curing agent). Use 10 ml syringe to inject MicroFil® mixture into the left ventricle after the blood was flushed out by saline. Inject MicroFil® mixture slowly into the left ventricle at approximately 3 ml/min.
Thsd1Venus/+ and Thsd1Venus/Venus mice survived to weaning age in expected Mendelian ratios. However, brain magnetic resonance imaging revealed mild to severe dilatation of cerebral ventricles, consistent with hydrocephalus in a subset of mutant mice as young as 8 weeks (not shown here, previously reported).
A microcatheter for delivering the metal coil conjugated to the TGFβ ligand or the TGFβ signaling activator is navigated to the location of aneurysm and partially inserted into the aneurysm cavity. The metal coil can be substantially similar to the metal coil prototype conjugated to a TGFβ activator illustrated in
While this invention is satisfied by embodiments in many different forms, as described in detail in connection with preferred embodiments of the invention, it is understood that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated and described herein. Numerous variations may be made by persons skilled in the art without departure from the spirit of the invention. The scope of the invention will be measured by the appended claims and their equivalents. The abstract and the title are not to be construed as limiting the scope of the present invention, as their purpose is to enable the appropriate authorities, as well as the general public, to quickly determine the general nature of the invention. In the claims that follow, unless the term “means” is used, none of the features or elements recited therein should be construed as means-plus-function limitations pursuant to 35 U.S.C. § 112, 6.
The present application claims priority to U.S. Provisional Application Ser. No. 63/296,817, filed Jan. 5, 2022; U.S. Provisional Application Ser. No. 63/296,820, filed Jan. 5, 2022; U.S. Provisional Application Ser. No. 63/296,821, filed Jan. 5, 2022; and U.S. Provisional Application Ser. No. 63/296,825, filed Jan. 5, 2022, the contents of each being hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63296817 | Jan 2022 | US | |
| 63296820 | Jan 2022 | US | |
| 63296821 | Jan 2022 | US | |
| 63296825 | Jan 2022 | US |
