METHODS AND REAGENTS FOR CHARACTERIZING THSD1 FUNCTION
The disclosure describes global transcriptome profiling in human vascular endothelial cells upon THSD1 knockdown and identification from these studies of specific genes and pathways that are THSD1. The analysis highlights a role for THSD1 in regulating endothelial to mesenchymal transition and other pathways. The analysis also demonstrates that endothelial cell injury is the first event of an intracranial aneurysm. The disclosure provides novel cell lines and reagents used for better understanding the molecular mechanism of THSD1 biology.
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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.
FIELD OF THE INVENTIONThe 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.
SUMMARYThis 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 provided herein are a cell line(s) comprised of an immortalized cell that bears a mutation in a Thrombospondin Type 1 Domain Containing 1 (THSD1) allele. In some instances, the THSD1 allele codes for an isoform of THSD1. The isoform of THSD1 can be encoded by a single codon substitution compared to the wild type allele, such as a single codon substitution that provides a missense mutation compared to the wild type THSD1 protein. The single codon substitution in the THSD1 allele can be, for example, at codon 5, at codon 460, at codon 466, at codon 600, at codon 639, at codon 653, or at codon 775. Such single codon substitution can lead to the following missense mutations, for example, a Leucine for a Phenylalanine (L5F) substitution, an Arginine for a Tryptophan (R460W) substitution, a Glutamic Acid for a Glycine (E466G) substitution, a Glycine for a Glutamic Acid (G600E) substitution, a Proline for a Leucine (P639L) substitution, a Threonine for a Isoleucine (T653I) substitution, a Serine for a Proline (S775P) substitution, or any combination thereof. In some instances the cell line is human and in other instances is murine. In some instances, the cell line is derived from a human endothelial cell, which can be from a brain tissue or from umbilical vein endothelial cells. In some instances, the human endothelial cell is immortalized from a primary cell.
In some aspects provided herein is a chimeric nucleic acid molecule, comprising a nucleic acid sequence of a Thrombospondin Type 1 Domain Containing 1 (THSD1) allele that (a) encodes a THSD1 protein and (b) is modified to comprise a replacement of a sequence encoding the THSD1 protein or portion thereof with a heterologous missense THSD1 protein or a portion thereof, wherein the chimeric nucleic acid molecule encodes a missense THSD1 protein, and optionally, wherein the chimeric nucleic acid sequence further comprises promoter and/or regulatory sequences of the Thsd1 gene. In some instances, the chimeric nucleic acid molecule further comprises a drug selection cassette. Such chimeric nucleic acid molecules may further comprise a 5′ homology arm upstream of the Thrombospondin Type 1 Domain Containing 1 (THSD1) allele; and a 3′ homology arm downstream of the Thrombospondin Type 1 Domain Containing 1 (THSD1) allele. In some instances, the 5′ homology arm and 3′ homology arm undergo homologous recombination with a non-human animal locus of interest, and wherein following homologous recombination with the non-human animal locus of interest, the modified Thsd1 allele replaces the non-human animal Thsd1 allele at the non-human animal Thsd1 locus of interest and is operably linked to an endogenous promoter that drives expression of the non-human animal Thsd1 allele at the non-human animal Thsd1 locus of interest. In some instances the heterologous missense THSD1 protein or a portion thereof comprises (i) a single codon substitution in the THSD1 allele at codon 5; (ii) a single codon substitution in the THSD1 allele at codon 460; (iii) a single codon substitution in the THSD1 allele at codon 466; (iv) a single codon substitution in the THSD1 allele at codon 600; (v) a single codon substitution in the THSD1 allele at codon 639; (vi) a single codon substitution in the THSD1 allele at codon 653; (vii) a single codon substitution in the THSD1 allele at codon 775; or (viii) any combination of (i)-(vii). In some instances, such single codon substitution can lead to the following missense mutations, for example, a Leucine for a Phenylalanine (L5F) substitution, an Arginine for a Tryptophan (R460W) substitution, a Glutamic Acid for a Glycine (E466G) substitution, a Glycine for a Glutamic Acid (G600E) substitution, a Proline for a Leucine (P639L) substitution, a Threonine for a Isoleucine (T653I) substitution, a Serine for a Proline (S775P) substitution, or any combination thereof.
In some aspects the disclosure provides a method of making a Thrombospondin Type 1 Domain Containing 1 (THSD1) non-human animal comprising modifying an endogenous Thsd1 locus of the non-human animal to encode a heterologous THSD1 protein comprising a missense mutation or a portion thereof. In some instances, the endogenous Thsd1 locus of the non-human animal comprises: (i) a single codon substitution in the THSD1 allele at codon 5; (ii) a single codon substitution in the THSD1 allele at codon 460; (iii) a single codon substitution in the THSD1 allele at codon 466; (iv) a single codon substitution in the THSD1 allele at codon 600; (v) a single codon substitution in the THSD1 allele at codon 639; (vi) a single codon substitution in the THSD1 allele at codon 653; (vii) a single codon substitution in the THSD1 allele at codon 775; or (viii) any combination of (i)-(vii). In some instances, the endogenous Thsd1 locus of the non-human animal comprises: (i) a single codon substitution in the THSD1 allele at codon 5 encoding a Leucine for a Phenylalanine (L5F) substitution; (ii) a single codon substitution in the THSD1 allele at codon 460 encoding an Arginine for a Tryptophan (R460W) substitution; (iii) a single codon substitution in the THSD1 allele at codon 466 encoding a Glutamic Acid for a Glycine (E466G) substitution; (iv) a single codon substitution in the THSD1 allele at codon 600 encoding a Glycine for a Glutamic Acid (G600E) substitution; (v) a single codon substitution in the THSD1 allele at codon 639 encoding a Proline for a Leucine (P639L) substitution; (vi) a single codon substitution in the THSD1 allele at codon 653 encoding a Threonine for a Isoleucine (T653I) substitution; (vii) a single codon substitution in the THSD1 allele at codon 775 encoding a Serine for a Proline (S775P) substitution; (viii) any combination of (i)-(vii). In some instances, the heterologous THSD1 protein comprising the missense mutation or the portion thereof comprise an amino acid sequence of a human THSD1 protein or a portion thereof. In some instances, the non-human animal is a rat or a mouse.
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.
INCORPORATION BY REFERENCEAll 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.
DETAILED DESCRIPTIONSIn 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.
We hypothesized that other high-risk rare genetic variants could cause IA and their identification could provide critical insights into disease pathobiology, however numerous challenges exist in identifying and characterizing genes whose perturbation leads to intracranial aneurysms (IA) and/or vascular endothelial cell dysfunction. Single-family genetic studies are a powerful tool to identify candidate high-risk genetic variants. Yet, these studies are frequently limited by small family pedigrees available for study and usually do not provide a sufficient number of repeats to be statistically significant.
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.
We subsequently used whole exome sequencing and discovered that THSD1 is linked molecularly to endothelial dysfunction mediated through the biological process of endothelial to mesenchymal transition (EndMT).
DefinitionsAll 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 in this specification the “wild-type” or “WT” thrombospondin type 1 domain containing 1 (THSD1) sequence refers to the human sequence identified by NCBI Reference Sequence: NG 047168.1; unless otherwise stated.
As used herein a missense mutation refers to a change in one amino acid in a protein, arising from a point mutation in a single nucleotide. Missense mutation is a type of nonsynonymous substitution in a DNA sequence that is translated into a nonsynonymous substitution in a protein sequence. As used herein, missense mutations do not encompass nonsense mutations—in which a codon is changed to a premature stop codon that results in truncation of the resulting protein.
A used herein, an “immortalized cell line” is a cell line derived from cells that have been artificially manipulated to proliferate indefinitely and can, thus, be cultured over several generations.
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.
A. Cell Lines with Missense Mutations
A variety of cells and non-human animals comprising a modified THSD1 locus (e.g. THSD1 allele) are described herein. A diploid organism has two alleles at each genetic locus. Each pair of alleles represents the genotype of a specific genetic locus. Genotypes are described as homozygous if there are two identical alleles at a particular locus and as heterozygous if the two alleles differ. In many instances, the missense mutations identified and characterized in the disclosure present an autosomal dominant phenotype, and an insertion of one allele having the missense mutation is sufficient to achieve a phenotype associated with an aneurysm in a cell or in an animal.
The cells provided herein can be, for example, any non-human cell comprising an Thsd1 locus or a genomic locus homologous or orthologous to the human CACNG1 locus. The cells can be, e.g., mammalian cells, non-human mammalian cells, and human cells. The term “animal” includes mammals, fishes, and rodents. A mammalian cell can be, for example, a non-human mammalian cell, a rodent cell, a rat cell, or a zebrafish cell. Other non-human mammals include, for example, any non-human animal that carries a homolog of the Thsd1 gene and is suitable for study of phenotypes associated with lack of function of the THSD1 protein. Birds include, for example, chickens, turkeys, ostrich, geese, ducks, and so forth. Domesticated animals and agricultural animals are also included. The term “non-human” excludes humans.
Primary cells reach senescence after limited generation and the process of frequently re-establishing fresh cultures from explanted tissues is onerous and often impracticable. Using immortalized primary cells or cell lines provides the ability best understand missense mutations that are not easily captured with the study of knock-down or knock-out cell lines. In notable instances (e.g., sickle cell) the missense mutation has additional properties and changes the function of the protein in a way that cannot be studied or elucidated with the mere review of function of the wild-type protein. Thus, the disclosure provides methods and strategies for studying proteins bearing missense mutations in the THSD1 gene. Primary cells can be immortalized with hTERT-based strategy in a manner that retains characteristics of the differentiated cell types with tissue-specific features, differentiation-specific proteins associated with the cells that the proteins are derived from. Further, technologies such as CRISPR can be used to create knock-in of the identified THSD1 missense mutations, namely LSF, R450X, R460W, E466G, G600E, P639L, T653I, S775P, in established immortalized cell line backgrounds.
Thus, in some aspects, suitable cells provided herein also include primary cells. Primary cells include cells or cultures of cells that have been isolated directly from an organism, organ, or tissue. Primary cells include cells that are neither transformed nor immortal. They include any cell obtained from an organism, organ, or tissue which was not previously passed in tissue culture or has been previously passed in tissue culture but is incapable of being indefinitely passed in tissue culture. Such cells can be rare, for example, endothelial cells (e.g., Human brain microvascular endothelial cells (HBMECs) cells; see Example 1), and can be isolated and immortalized to facilitate the study of a phenotype observed in the family described in Example 1.
Telomerase Reverse Transcriptase (TERT) Expression for Immortalizing Cell Lines
Telomerase is a ribonucleoprotein that can extend the DNA sequence of telomeres, which lets the cells undergo infinite cell divisions through evading the senescence process. This Telomerase Reverse Transcriptase (TERT) expression is generally inactive in most somatic cells, ibut when exogenously expressed, the cells are able to maintain suffcient telomere lengths for avoiding replicative senescence. This approach can be used for immortalizing cell lines with the identified THSD1 missense mutations, namely LSF, R450X, R460W, E466G, G600E, P639L, T653I, S775P.
Viral Gene Based Immortalization
Viral genes can affect the cell cycle by deregulating the biological brakes on the proliferative control of the cells. In some aspects, the disclosure contemplates using viral genes, such as the simian virus 40 (SV40) T-antigen for immortalization of cells; in other instances the disclosure contemplates using viral oncogenes.
The cells can also be any type of undifferentiated or differentiated state. For example, a cell can be a totipotent cell, a pluripotent cell (e.g., a human pluripotent cell or a non-human pluripotent cell such as a mouse embryonic stem (ES) cell or a rat ES cell), or a non-pluripotent cell. Totipotent cells include undifferentiated cells that can give rise to any cell type, and pluripotent cells include undifferentiated cells that possess the ability to develop into more than one differentiated cell types. Such pluripotent and/or totipotent cells can be, for example, ES cells or ES-like cells, such as an induced pluripotent stem (iPS) cells. ES cells include embryo-derived totipotent or pluripotent cells that are capable of contributing to any tissue of the developing embryo upon introduction into an embryo. ES cells can be derived from the inner cell mass of a blastocyst and are capable of differentiating into cells of any of the three vertebrate germ layers (endoderm, ectoderm, and mesoderm).
B. Chimeric Nucleic Acids Encoding Thsd1 Sequences Having Missense MutationsAlso described herein is a chimeric nucleic acid molecule comprising a nucleic acid sequence of a Thrombospondin Type 1 Domain Containing 1 (THSD1) allele that (a) encodes a THSD1 protein and (b) is modified to comprise a replacement of a sequence encoding the THSD1 protein or portion thereof with a heterologous missense THSD1 protein or a portion thereof, wherein the chimeric nucleic acid molecule encodes a missense THSD1 protein, and optionally, wherein the chimeric nucleic acid sequence further comprises promoter and/or regulatory sequences of the Thsd1 gene. In some instances, the chimeric nucleic acid molecule further comprises a drug selection cassette. In some instances, the chimeric nucleic acid molecule further comprises a 5′ homology arm upstream of the Thrombospondin Type 1 Domain Containing 1 (THSD1) allele; and a 3′ homology arm downstream of the Thrombospondin Type 1 Domain Containing 1 (THSD1) allele. In some instances the 5′ homology arm and 3′ homology arm undergo homologous recombination with a non-human animal locus of interest, and wherein following homologous recombination with the non-human animal locus of interest, the modified Thsd1 allele replaces the non-human animal Thsd1 allele at the non-human animal Thsd1 locus of interest and is operably linked to an endogenous promoter that drives expression of the non-human animal Thsd1 allele at the non-human animal Thsd1 locus of interest.
In specific instances, the heterologous missense THSD1 protein or a portion thereof comprises (i) a single codon substitution in the THSD1 allele at codon 5; (ii) a single codon substitution in the THSD1 allele at codon 460; (iii) a single codon substitution in the THSD1 allele at codon 466; (iv) a single codon substitution in the THSD1 allele at codon 600; (v) a single codon substitution in the THSD1 allele at codon 639; (vi) a single codon substitution in the THSD1 allele at codon 653; (vii) a single codon substitution in the THSD1 allele at codon 775; or (viii) any combination of (i)-(vii). In more specific instances, the heterologous missense THSD1 protein or a portion thereof comprises (i) a single codon substitution in the THSD1 allele at codon 5 encoding a Leucine for a Phenylalanine (L5F) substitution; (ii) a single codon substitution in the THSD1 allele at codon 460 encoding an Arginine for a Tryptophan (R460W) substitution; (iii) a single codon substitution in the THSD1 allele at codon 466 encoding a Glutamic Acid for a Glycine (E466G) substitution; (iv) a single codon substitution in the THSD1 allele at codon 600 encoding a Glycine for a Glutamic Acid (G600E) substitution; (v) a single codon substitution in the THSD1 allele at codon 639 encoding a Proline for a Leucine (P639L) substitution; (vi) a single codon substitution in the THSD1 allele at codon 653 encoding a Threonine for a Isoleucine (T653I) substitution; (vii) a single codon substitution in the THSD1 allele at codon 775 encoding a Serine for a Proline (S775P) substitution; or (viii) any combination of (i)-(vii).
C. Methods of Making Non-Human Animals Comprising a Heterologous Cacng1 LocusIn some aspects, the disclosure provides a method of making a Thrombospondin Type 1 Domain Containing 1 (THSD1) non-human animal comprising modifying an endogenous Thsd1 locus of the non-human animal to encode a heterologous THSD1 protein comprising a missense mutation or a portion thereof.
Various methods are suitable for making a non-human animal comprising a heterologous locus. Any convenient method or protocol for producing a genetically modified organism is suitable for producing such a genetically modified non-human animal. See, e.g., Cho et al. (2009) Current Protocols in Cell Biology 42:19.11:19.11.1-19.11.22 and Gama Sosa et al. (2010) Brain Struct. Funct. 214(2-3):91-109, each of which is herein incorporated by reference in its entirety for all purposes. For zebra fish methodologies see, e.g., Brief Funct. Genomics. 2016 July; 15(4):322-30. “Genome editing in zebrafish: a practical overview.” See also, Curr Protoc Mol Biol. 2017 Jul. 5; 119:31.9.1-31.9.22. “CRISPR/Cas9-Directed Gene Editing for the Generation of Loss-of-Function Mutants in High-Throughput Zebrafish F0 Screens”. Such genetically modified non-human animals can be generated, for example, through gene knock-in at a targeted Thsd1 locus.
For example, the method of producing a non-human animal comprising a humanized Thsd1 locus can comprise: (1) modifying the genome of a pluripotent cell to comprise the humanized Thsd1 locus; (2) identifying or selecting the genetically modified pluripotent cell comprising the humanized Thsd1 locus; (3) introducing the genetically modified pluripotent cell into a non-human animal host embryo cells in vitro; and (4) implanting and gestating the host embryo cells in a surrogate mother. Optionally, the host embryo comprising modified pluripotent cell (e.g., a non-human ES cell) can be incubated until the blastocyst stage before being implanted into and gestated in the surrogate mother to produce an F0 non-human animal. The surrogate mother can then produce an F0 generation non-human animal comprising the humanized Thsd1 locus.
The methods can further comprise identifying a cell or animal having a modified target genomic locus. Various methods can be used to identify cells and animals having a targeted genetic modification.
The screening step can comprise, for example, a quantitative assay for assessing modification of allele (MOA) of a parental chromosome. For example, the quantitative assay can be carried out via a quantitative PCR, such as a real-time PCR (qPCR). The real-time PCR can utilize a first primer set that recognizes the target locus and a second primer set that recognizes a non-targeted reference locus. The primer set can comprise a fluorescent probe that recognizes the amplified sequence.
Other examples of suitable quantitative assays include fluorescence-mediated in situ hybridization (FISH), comparative genomic hybridization, isothermic DNA amplification, quantitative hybridization to an immobilized probe(s), INVADER® Probes, TAQMAN® Molecular Beacon probes, or ECLIPSE™ probe technology (see, e.g., US 2005/0144655, incorporated herein by reference in its entirety for all purposes).
An example of a suitable pluripotent cell is an embryonic stem (ES) cell (e.g., a mouse ES cell or a rat ES cell). The modified pluripotent cell can be generated, for example, through recombination by (a) introducing into the cell one or more targeting vectors comprising an insert nucleic acid flanked by 5′ and 3′ homology arms corresponding to 5′ and 3′ target sites, wherein the insert nucleic acid comprises a heterologous Thsd1 locus; and (b) identifying at least one cell comprising in its genome the insert nucleic acid integrated at the target genomic locus. Alternatively, the modified pluripotent cell can be generated by (a) introducing into the cell: (i) a nuclease agent, wherein the nuclease agent induces a nick or double-strand break at a recognition site within the target genomic locus; and (ii) one or more targeting vectors comprising an insert nucleic acid flanked by 5′ and 3′ homology arms corresponding to 5′ and 3′ target sites located in sufficient proximity to the recognition site, wherein the insert nucleic acid comprises the heterologous Thsd1 locus; and (c) identifying at least one cell comprising a modification (e.g., integration of the insert nucleic acid) at the target genomic locus. Any nuclease agent that induces a nick or double-strand break into a desired recognition site can be used. Examples of suitable nucleases include a Transcription Activator-Like Effector Nuclease (TALEN), a zinc-finger nuclease (ZFN), a meganuclease, and Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) systems or components of such systems (e.g., CRISPR/Cas9). See, e.g., US 2013/0309670 and US 2015/0159175, each of which is herein incorporated by reference in its entirety for all purposes.
The donor cell can be introduced into a host embryo at any stage, such as the blastocyst stage or the pre-morula stage (i.e., the 4 cell stage or the 8 cell stage). Progeny that are capable of transmitting the genetic modification though the germline are generated. See, e.g., U.S. Pat. No. 7,294,754, herein incorporated by reference in its entirety for all purposes.
In some instances, the endogenous Thsd1 locus of the non-human animal comprises: (i) a single codon substitution in the THSD1 allele at codon 5; (ii) a single codon substitution in the THSD1 allele at codon 460; (iii) a single codon substitution in the THSD1 allele at codon 466; (iv) a single codon substitution in the THSD1 allele at codon 600; (v) a single codon substitution in the THSD1 allele at codon 639; (vi) a single codon substitution in the THSD1 allele at codon 653; (vii) a single codon substitution in the THSD1 allele at codon 775; or (viii) any combination of (i)-(vii). In specific instances, the endogenous Thsd1 locus of the non-human animal comprises: (i) a single codon substitution in the THSD1 allele at codon 5 encoding a Leucine for a Phenylalanine (L5F) substitution; (ii) a single codon substitution in the THSD1 allele at codon 460 encoding an Arginine for a Tryptophan (R460W) substitution; (iii) a single codon substitution in the THSD1 allele at codon 466 encoding a Glutamic Acid for a Glycine (E466G) substitution; (iv) a single codon substitution in the THSD1 allele at codon 600 encoding a Glycine for a Glutamic Acid (G600E) substitution; (v) a single codon substitution in the THSD1 allele at codon 639 encoding a Proline for a Leucine (P639L) substitution; (vi) a single codon substitution in the THSD1 allele at codon 653 encoding a Threonine for a Isoleucine (T653I) substitution; (vii) a single codon substitution in the THSD1 allele at codon 775 encoding a Serine for a Proline (S775P) substitution; (viii) any combination of (i)-(vii). In specific instances, the non-human animal comprises a heterologous THSD1 protein comprising the missense mutation or the portion thereof comprise an amino acid sequence of a human THSD1 protein or a portion thereof.
The non-human animal can be a rat, a mouse, a zebrafish, or any suitable animal having an ortholog of the human Thsd1 gene.
EMBODIMENTSEMBODIMENT 1. A cell line comprised of an immortalized cell that bears a mutation in a Thrombospondin Type 1 Domain Containing 1 (THSD1) allele.
EMBODIMENT 2. The cell line of embodiment 1, wherein the THSD1 allele codes for an isoform of THSD1.
EMBODIMENT 3. The cell line of embodiment 2, wherein the isoform of THSD1 is encoded by a single codon substitution compared to the wild type allele.
EMBODIMENT 4. The cell line of embodiment 2, wherein the isoform of THSD1 carries a missense mutation compared to the wild type THSD1 protein.
EMBODIMENT 5. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele is at codon 5.
EMBODIMENT 6. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele is at codon 460.
EMBODIMENT 7. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele is at codon 466.
EMBODIMENT 8. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele is at codon 600.
EMBODIMENT 9. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele is at codon 639.
EMBODIMENT 10. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele is at codon 653.
EMBODIMENT 11. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele is at codon 775.
EMBODIMENT 12. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele at codon 5 encoding a Leucine for a Phenylalanine (LSF) substitution.
EMBODIMENT 13. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele at codon 460 encoding an Arginine for a Tryptophan (R460W) substitution.
EMBODIMENT 14. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele at codon 466 encoding a Glutamic Acid for a Glycine (E466G) substitution.
EMBODIMENT 15. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele at codon 600 encoding a Glycine for a Glutamic Acid (G600E) substitution.
EMBODIMENT 16. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele at codon 639 encoding a Proline for a Leucine (P639L) substitution.
EMBODIMENT 17. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele at codon 653 encoding a Threonine for a Isoleucine (T653I) substitution.
EMBODIMENT 18. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele at codon 775 encoding a Serine for a Proline (S775P) substitution.
EMBODIMENT 19. The cell line of any of embodiments 2-18 wherein the cell line is human.
EMBODIMENT 20. The cell line of embodiment 1, wherein the cell line is derived from a human endothelial cell.
EMBODIMENT 21. The cell line of embodiment 20, wherein the human endothelial cell is derived from a brain tissue.
EMBODIMENT 22. The cell line of embodiment 20, wherein the human endothelial cell is derived from umbilical vein endothelial cells.
EMBODIMENT 23. The cell line of embodiment 20, wherein the human endothelial cell is immortalized from a primary cell.
EMBODIMENT 24. A chimeric nucleic acid molecule, comprising a nucleic acid sequence of a Thrombospondin Type 1 Domain Containing 1 (THSD1) allele that (a) encodes a THSD1 protein and (b) is modified to comprise a replacement of a sequence encoding the THSD1 protein or portion thereof with a heterologous missense THSD1 protein or a portion thereof, wherein the chimeric nucleic acid molecule encodes a missense THSD1 protein, and optionally, wherein the chimeric nucleic acid sequence further comprises promoter and/or regulatory sequences of the Thsd1 gene.
EMBODIMENT 25. The chimeric nucleic acid molecule of embodiment Error! Reference source not found., wherein the chimeric nucleic acid molecule further comprises a drug selection cassette.
EMBODIMENT 26. The chimeric nucleic acid molecule of any one of embodiments Error! Reference source not found.—Error! Reference source not found., further comprising: (i) a 5′ homology arm upstream of the Thrombospondin Type 1 Domain Containing 1 (THSD1) allele; and (ii) a 3′ homology arm downstream of the Thrombospondin Type 1 Domain Containing 1 (THSD1) allele.
EMBODIMENT 27. The chimeric nucleic acid molecule of embodiment Error! Reference source not found., wherein the 5′ homology arm and 3′ homology arm undergo homologous recombination with a non-human animal locus of interest, and wherein following homologous recombination with the non-human animal locus of interest, the modified Thsd1 allele replaces the non-human animal Thsd1 allele at the non-human animal Thsd1 locus of interest and is operably linked to an endogenous promoter that drives expression of the non-human animal Thsd1 allele at the non-human animal Thsd1 locus of interest.
EMBODIMENT 28. The chimeric nucleic acid molecule of any one of embodiments Error! Reference source not found.-Error! Reference source not found., wherein the heterologous missense THSD1 protein or a portion thereof comprises (i) a single codon substitution in the THSD1 allele at codon 5; (ii) a single codon substitution in the THSD1 allele at codon 460; (iii) a single codon substitution in the THSD1 allele at codon 466; (iv) a single codon substitution in the THSD1 allele at codon 600; (v) a single codon substitution in the THSD1 allele at codon 639; (vi) a single codon substitution in the THSD1 allele at codon 653; (vii) a single codon substitution in the THSD1 allele at codon 775; or (viii) any combination of (i)-(vii).
EMBODIMENT 29. The chimeric nucleic acid molecule of any one of embodiments Error! Reference source not found., wherein the heterologous missense THSD1 protein or a portion thereof comprises (i) a single codon substitution in the THSD1 allele at codon 5 encoding a Leucine for a Phenylalanine (L5F) substitution; (ii) a single codon substitution in the THSD1 allele at codon 460 encoding an Arginine for a Tryptophan (R460W) substitution; (iii) a single codon substitution in the THSD1 allele at codon 466 encoding a Glutamic Acid for a Glycine (E466G) substitution; (iv) a single codon substitution in the THSD1 allele at codon 600 encoding a Glycine for a Glutamic Acid (G600E) substitution; (v) a single codon substitution in the THSD1 allele at codon 639 encoding a Proline for a Leucine (P639L) substitution; (vi) a single codon substitution in the THSD1 allele at codon 653 encoding a Threonine for a Isoleucine (T653I) substitution; (vii) a single codon substitution in the THSD1 allele at codon 775 encoding a Serine for a Proline (S775P) substitution; (viii) any combination of (i)-(vii).
EMBODIMENT 30. A method of making a Thrombospondin Type 1 Domain Containing 1 (THSD1) non-human animal comprising modifying an endogenous Thsd1 locus of the non-human animal to encode a heterologous THSD1 protein comprising a missense mutation or a portion thereof.
EMBODIMENT 31. The non-human animal of embodiment Error! Reference source not found., wherein the endogenous Thsd1 locus of the non-human animal comprises: (i) a single codon substitution in the THSD1 allele at codon 5; (ii) a single codon substitution in the THSD1 allele at codon 460; (iii) a single codon substitution in the THSD1 allele at codon 466; (iv) a single codon substitution in the THSD1 allele at codon 600; (v) a single codon substitution in the THSD1 allele at codon 639; (vi) a single codon substitution in the THSD1 allele at codon 653; (vii) a single codon substitution in the THSD1 allele at codon 775; or (viii) any combination of (i)-(vii).
EMBODIMENT 25. The non-human animal of claim Error! Reference source not found., wherein the endogenous Thsd1 locus of the non-human animal comprises: (i) a single codon substitution in the THSD1 allele at codon 5 encoding a Leucine for a Phenylalanine (L5F) substitution; (ii) a single codon substitution in the THSD1 allele at codon 460 encoding an Arginine for a Tryptophan (R460W) substitution; (iii) a single codon substitution in the THSD1 allele at codon 466 encoding a Glutamic Acid for a Glycine (E466G) substitution; (iv) a single codon substitution in the THSD1 allele at codon 600 encoding a Glycine for a Glutamic Acid (G600E) substitution; (v) a single codon substitution in the THSD1 allele at codon 639 encoding a Proline for a Leucine (P639L) substitution; (vi) a single codon substitution in the THSD1 allele at codon 653 encoding a Threonine for a Isoleucine (T653I) substitution; (vii) a single codon substitution in the THSD1 allele at codon 775 encoding a Serine for a Proline (S775P) substitution; (viii) any combination of (i)-(vii).
EMBODIMENT 26. The non-human animal of any one of embodiments Error! Reference source not found.-Error! Reference source not found., wherein the heterologous THSD1 protein comprising the missense mutation or the portion thereof comprise an amino acid sequence of a human THSD1 protein or a portion thereof.
EMBODIMENT 27. The non-human animal of any one of embodiments Error! Reference source not found.-Error! Reference source not found., wherein the non-human animal is a rat or a mouse.
EXAMPLESThe 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.
Example 1: Identification of Novel THSD1 Variants in Human SamplesSingle-family genetic studies are a powerful tool to identify candidate high-risk genetic variants.
For each family, whole exome sequencing has been performed on at least 15 family members, irrespective of their IA status. The detailed analysis identified a THSD1 nonsense mutation that segregated in all 9 affected and was absent in 13 unaffected family members. The following additional THSD1 missense variants were discovered in 507 unrelated patients/probands where each perturbed THSD1 cell adhesion activity. They are LSF, R460W, E466G, G600E, P639L, T653I, or S775P.
Example 2: Molecular Dissection of THSD1 Function in Vascular Endothelial Cells from Global TranscriptomicsGenetic 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.
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 role for THSD1 in regulating endothelial to mesenchymal transition.
Example 3: Endothelial Dysfunction Mediated by THSD1 Through the Biological Process of Endothelial to Mesenchymal Transition (EndMT)Regulation of Endothelial to Mesenchymal Transition as a Target for Treating IA
Endothelial to mesenchymal transition (EndMT) is a biological process whereby an endothelial cell undergoes a series of molecular events that lead to a change in phenotype toward a mesenchymal cell like phenotype. During this process, endothelial cells adopt a mesenchymal phenotype displaying typical mesenchymal cell morphology and functions, including the promotion of inflammatory response. In addition, endothelial cells lose the expression of endothelial cell-specific proteins and initiate the expression of mesenchymal cell-specific genes and the production.
We applied global transcriptomics approaches to identify genes disrupted in THSD1 knockdown studies and obtained RNAseq data for two THSD1 knock-down endothelial cell lines. Both cell lines support the evidence that THSD1 mediates EndMT function (THSD1 Knock-down induces the activations of EMT—loss of endothelial normal function, but increased inflammatory cell), see
A variety of chimeric nucleic acid molecules encoding a nucleic acid sequence of a Thrombospondin Type 1 Domain Containing 1 (THSD1) allele that (a) encodes a THSD1 protein modified to comprise a replacement of a sequence encoding the THSD1 protein or portion thereof with a heterologous missense THSD1 protein or a portion thereof were engineered. First, two synonymous mutations (DM) were introduced into a WT THSD1 sequence to make an siRNA-resistant form of THSD1.
Next, each individual rare variant associated with pathogenesis was made by PCR mutagenesis and subcloned into pLVX-TetOne vector with a C-terminal tagging of FLAG. pLVX-TetOne vector is a lentiviral vector and can be induced by addition of doxycycline. The constructs created for the experiment included chimeric nucleic acid molecules wherein the heterologous missense THSD1 protein or a portion thereof comprised (i) a single codon substitution in the THSD1 allele at codon 5 encoding a Leucine for a Phenylalanine (L5F) substitution; (ii) a single codon substitution in the THSD1 allele at codon 460 encoding an Arginine for a Tryptophan (R460W) substitution; (iii) a single codon substitution in the THSD1 allele at codon 466 encoding a Glutamic Acid for a Glycine (E466G) substitution; (iv) a single codon substitution in the THSD1 allele at codon 600 encoding a Glycine for a Glutamic Acid (G600E) substitution; (v) a single codon substitution in the THSD1 allele at codon 639 encoding a Proline for a Leucine (P639L) substitution; (vi) a single codon substitution in the THSD1 allele at codon 653 encoding a Threonine for a Isoleucine (T653I) substitution; (vii) a single codon substitution in the THSD1 allele at codon 775 encoding a Serine for a Proline (S775P) substitution; (viii) any combination of (i)-(vii).
Table 5 illustrates exemplary nucleic acids constructs made:
Next, the expression of THSD1 rare variants in human brain endothelial cells. A pLVX-TetOne backbone was used to generate lentiviruses together with co-plasmids psPAX2 (2 ug) and pMD2.G (2 ug). Human brain endothelial cells (D3) were infected by lentiviruses and the cells stably expressing each variant were selected against puromycin (8 ug/ml) for 3 days. Each stable cell line was then induced by doxycycline/DOX (10 ng/ml) for 2 days and whole cell lysates were collected for examining protein expression by Western blot against FLAG tag. See
Identified Conserved Sequences Suitable for Assessing Cross Species Efficacies of Treatments:
A protein alignment between human and zebrafish THSD1 was performed. See
Mutations R449X, R459W and T6651 were found to be conserved between human and zebrafish. Subsequently, the nucleic acid constructs encoding these sequences were introduced into zebrafish thsd1 by PCR mutagenesis. Specifically, the thsd1 variant or the WT sequence was subcloned into pCS2+ zebrafish vector for expression. These constructs were used as tools to study cerebrovascular integrity mediated by thsd1 WT and rare variants in the zebrafish animal model selected to model a human condition.
In-Vivo Zebrafish Model
The experiment generated non-human animals carrying conserved human mutations for the study of therapies in-vivo.
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.
Claims
1. A cell line comprised of an immortalized cell that bears a mutation in a Thrombospondin Type 1 Domain Containing 1 (THSD1) allele.
2. The cell line of claim 1, wherein the THSD1 allele codes for an isoform of THSD1.
3. The cell line of claim 2, wherein the isoform of THSD1 is encoded by a single codon substitution compared to the wild type allele.
4. The cell line of claim 2, wherein the isoform of THSD1 carries a missense mutation compared to the wild type THSD1 protein.
5. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele is at codon 5.
6. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele is at codon 460.
7. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele is at codon 466.
8. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele is at codon 600.
9. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele is at codon 639.
10. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele is at codon 653.
11. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele is at codon 775.
12. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele at codon 5 encoding a Leucine for a Phenylalanine (L51-) substitution.
13. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele at codon 460 encoding an Arginine for a Tryptophan (R460W) substitution.
14. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele at codon 466 encoding a Glutamic Acid for a Glycine (E466G) substitution.
15. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele at codon 600 encoding a Glycine for a Glutamic Acid (G600E) substitution.
16. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele at codon 639 encoding a Proline for a Leucine (P639L) substitution.
17. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele at codon 653 encoding a Threonine for a Isoleucine (T653I) substitution.
18. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele at codon 775 encoding a Serine for a Proline (S775P) substitution.
19. The cell line of claim 1 wherein the cell line is human.
20. The cell line of claim 1, wherein the cell line is derived from a human endothelial cell.
21. The cell line of claim 20, wherein the human endothelial cell is derived from a brain tissue.
22. The cell line of claim 20, wherein the human endothelial cell is derived from umbilical vein endothelial cells.
23. The cell line of claim 20, wherein the human endothelial cell is immortalized from a primary cell.
Type: Application
Filed: May 25, 2022
Publication Date: Jul 6, 2023
Applicant: THE BOARD OF REGENTS OF THE UNIVERSITY OF TEXAS SYSTEM (Austin, TX)
Inventors: Dong H. Kim (Houston, TX), Yuanqing Yan (Houston, TX), Yanning Rui (Houston, TX), Zhen Xu (Houston, TX), John Hagan (Houston, TX)
Application Number: 17/824,612
