Patent Application
20240247267
The present disclosure generally relates to the treatment of subjects having a stroke or at risk of developing a stroke, by administering a 1,4-alpha-glucan-branching enzyme (GBE1) inhibitor to the subject, and to methods of identifying subjects having an increased risk of developing a stroke.
Stroke is a medical condition in which poor blood flow to the brain causes cell death. There are two main types of stroke: ischemic, due to lack of blood flow, and hemorrhagic, due to bleeding. Both forms of stroke cause parts of the brain to stop functioning properly. An ischemic stroke is typically caused by blockage of a blood vessel. There are also less common causes of ischemic stroke, such as complications of vascular surgical procedures of the arteries leading to the brain (for example, surgical interventions targeting the carotid artery or aorta). A hemorrhagic stroke is caused by either bleeding directly into the brain (e.g. rupture of a vessel due to high blood pressure) or into the space between the brain's membranes (e.g. rupture of a brain aneurysm). In addition, various techniques, such as T2 FLAIR magnetic resonance imaging (MRI) can be used to detect small vessel ischemic disease, which is a major biomarker of stroke risk.
GBE1 is a 272 kb gene located at 3p12.2. GBE1 protein is 702 amino acids long and is an 80 kDa enzyme that catalyzes the transfer of alpha-1,4-linked glucosyl units from the outer end of a glycogen chain to an alpha-1,6 position on the same or a neighboring glycogen chain. Glycogen is a readily mobilized storage form of glucose, and the extended glycogen polymer is branched by GBE1 to provide glycogen breakdown enzymes, such as glycogen phosphorylase, with many terminal residues for rapid degradation. Branching of the chains is essential to increase the solubility of the glycogen molecule and, consequently, in reducing the osmotic pressure within cells. The highest levels of GBE1 are found in liver and muscle cells and biallelic loss of function mutations (those affecting both copies) of the gene are associated with glycogen storage disease IV also known as Andersen's disease and adult polyglucosan body disease (APBD).
The present disclosure provides methods of treating a subject having a stroke, or at risk of developing a stroke, the methods comprising administering a GBE1 inhibitor to the subject.
The present disclosure also provides methods of treating a subject having a stroke or at risk of developing a stroke by administering a stroke therapeutic agent or stroke therapy, the methods comprising: a) determining or having determined whether the subject has a GBE1 variant nucleic acid molecule, by: obtaining or having obtained a biological sample from the subject; and performing or having performed a sequence analysis on the biological sample to determine if the subject has a genotype comprising a GBE1 variant nucleic acid molecule; and/or b) determining or having determined whether the subject has increased total white matter hyperintensity by imaging the subject; and administering or continuing to administer the stroke therapeutic agent in an amount that is the same as or less than a standard dosage amount or stroke therapy, and/or administering a GBE1 inhibitor to a subject that is GBE1 reference; and administering or continuing to administer the stroke therapeutic agent in an amount that is the same as or less than a standard dosage amount or stroke therapy, and/or a GBE1 inhibitor to a subject that has increased total white matter hyperintensity; wherein the presence of a single copy of the GBE1 variant nucleic acid molecule indicates the subject has a decreased risk of developing a stroke.
The present disclosure also provides methods of identifying a subject having an increased risk of developing a stroke, the methods comprising: determining or having determined the presence or absence of a GBE1 variant nucleic acid molecule in a biological sample obtained from the subject; wherein: when the subject is GBE1 reference, then the subject has an increased risk of developing a stroke; and when the subject is heterozygous for the GBE1 variant nucleic acid molecule, then the subject has a decreased risk of developing a stroke.
The present disclosure also provides combinations of a stroke therapeutic agent and a GBE1 inhibitor for use in the treatment or prevention of a stroke in a subject that is GBE1 reference.
The present disclosure also provides GBE1 inhibitors for use in the treatment or prevention of a stroke in a subject that is GBE1 reference.
Various terms relating to aspects of the present disclosure are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.
Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-expressed basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.
As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
As used herein, the term “about” means that the recited numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. Where a numerical value is used, unless indicated otherwise by the context, the term “about” means the numerical value can vary by +10% and remain within the scope of the disclosed embodiments.
As used herein, the term “comprising” may be replaced with “consisting” or “consisting essentially of” in particular embodiments as desired.
As used herein, the terms “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, “polynucleotide”, or “oligonucleotide” can comprise a polymeric form of nucleotides of any length, can comprise DNA and/or RNA, and can be single-stranded, double-stranded, or multiple stranded. One strand of a nucleic acid also refers to its complement.
As used herein, the term “subject” includes any animal, including mammals. Mammals include, but are not limited to, farm animals (such as, for example, horses, cows, and pigs), companion animals (such as, for example, dogs and cats), laboratory animals (such as, for example, mice, rats, and rabbits), and non-human primates. In some embodiments, the subject is a human. In some embodiments, the human is a patient under the care of a physician.
It has been observed in accordance with the present disclosure that rare GBE1 variant nucleic acid molecules (which variants are heterozygous in a particular subject) associate with a decreased risk of developing a stroke. It is believed that GBE1 variant nucleic acid molecules have not been associated with stroke in humans. Therefore, subjects that are GBE1 reference may be treated with a GBE1 inhibitor such that a stroke is inhibited or prevented, the symptoms thereof are reduced or prevented, and/or development of symptoms is repressed or prevented. It is also believed that such subjects having a stroke may further be treated with one or more stroke therapeutic agents or stroke therapy. In addition, the present disclosure provides methods of leveraging the presence or absence of GBE1 variant nucleic acid molecules in subjects to identify or stratify risk is such subjects of developing a stroke, or to diagnose subjects as having an increased risk of developing a stroke.
For purposes of the present disclosure, any particular subject, such as a human, can be categorized as having one of three GBE1 genotypes: i) GBE1 reference; ii) heterozygous for a GBE1 variant nucleic acid molecule; or iii) homozygous for a GBE1 variant nucleic acid molecule. A subject is GBE1 reference when the subject does not have a copy of a GBE1 variant nucleic acid molecule. A subject is heterozygous for a GBE1 variant nucleic acid molecule when the subject has a single copy of a GBE1 variant nucleic acid molecule. A subject is homozygous for a GBE1 variant nucleic acid molecule when the subject has two copies of a GBE1 variant nucleic acid molecule. Subjects that are homozygous for a GBE1 variant nucleic acid molecule may develop glycogen storage disease IV (i.e., Andersen's disease) and APBD. Treatment of subjects that are heterozygous or homozygous for a GBE1 variant nucleic acid molecule are excluded from the treatment methods described herein.
In any of the embodiments described herein, the GBE1 variant nucleic acid molecule can be any nucleic acid molecule (such as, a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule produced from an mRNA molecule) encoding a GBE1 variant polypeptide having a partial loss-of-function, a complete loss-of-function, a predicted partial loss-of-function, or a predicted complete loss-of-function. A subject who has a GBE1 polypeptide having a partial loss-of-function (or predicted partial loss-of-function) is hypomorphic for GBE1. In some embodiments, the GBE1 variant nucleic acid molecule results in decreased or aberrant expression or activity of GBE1 mRNA or polypeptide. In some embodiments, the GBE1 variant nucleic acid molecule is associated with a reduced in vitro response to GBE1 ligands compared with reference GBE1. In some embodiments, the GBE1 variant nucleic acid molecule is a splice-site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, an in-frame indel variant, or a variant that encodes a truncated GBE1 variant polypeptide. In some embodiments, the GBE1 variant nucleic acid molecule is a missense variant nucleic acid molecule. In some embodiments, the GBE1 variant nucleic acid molecule comprises a single nucleotide polymorphism (SNP). In some embodiments, the GBE1 variant nucleic acid molecule comprises a variation in a coding region. In some embodiments, the GBE1 variant nucleic acid molecule results or is predicted to result in a premature truncation of a GBE1 polypeptide compared to the reference GBE1. In some embodiments, the GBE1 variant nucleic acid molecule is a variant that is predicted to be damaging to the protein function (and hence, in this case, protective to the human) by in vitro prediction algorithms such as Polyphen, SIFT, or similar algorithms. In some embodiments, the GBE1 variant nucleic acid molecule is a variant that causes or is predicted to cause a nonsynonymous amino acid substitution in a GBE1 nucleic acid molecule and whose allele frequency is less than 1/100 alleles in the population from which the subject is selected. In some embodiments, the GBE1 variant nucleic acid molecule is any rare missense variant (allele frequency <0.1%; or 1 in 1,000 alleles), or any splice-site, stop-gain, start-loss, stop-loss, frameshift, or in-frame indel, or other frameshift GBE1 variant.
In any of the embodiments described herein, the GBE1 variant genomic nucleic acid molecule may include one or more variations at any of the positions of chromosome 3 (i.e., positions 81489703-81761645) using the nucleotide sequence of the GBE1 reference genomic nucleic acid molecule in the GRCh38/hg38 human genome assembly (see, ENSG00000114480.13, ENST00000429644.7 annotated in the Ensembl database (URL: world wide web at “uswest.ensembl.org/Homo_sapiens/Gene/)) as a reference sequence. The sequences provided in these transcripts for the GBE1 genomic nucleic acid molecule are only exemplary sequences. Other sequences for the GBE1 genomic nucleic acid molecule are also possible.
In any of the embodiments described herein, the GBE1 variant nucleic acid molecule may comprise any one or more of the following genetic variations in the genomic nucleic acid molecule (referring to the chromosome:positions set forth in the GRCh38/hg38 human genome assembly): 3:81648854:A:G (rs192044702), 3:81535193:A:G (rs752625236), 3:81536909A:G (rs539203557), 3:81536926:C:T (rs201029706), 3:81536936:CTT:C, 3:81577973:G:A (rs137852888), 3:81581237:GC:G (rs1703017615), 3:81586127:G:A (rs781198373), 3:81594010:T:A, and 3:81670900:CCCATTTT:C, or an mRNA molecule produced therefrom, or a cDNA molecule produced from the mRNA molecule.
For subjects that are genotyped or determined to be GBE1 reference, such subjects have an increased risk of developing a stroke. These subjects are likely to have increased total white matter hyperintensity. For subjects that are genotyped or determined to be GBE1 reference, such subjects can be treated with a GBE1 inhibitor. For subjects that are genotyped or determined to have one or two GBE1 variant nucleic acid molecules, such subjects may not be treated with a GBE1 inhibitor as they may already be protected from stroke and there may be unwanted side effects from excessive reduction of GBE1 function.
In any of the embodiments described herein, the subject in whom a stroke is prevented by administering a GBE1 inhibitor may be anyone at risk for developing a stroke including, but not limited to, subjects with a genetic predisposition for developing a stroke or subjects undergoing surgical procedures associated with a risk of stroke. Additional risk factors for stroke include, but are not limited to, high blood pressure, high blood cholesterol, tobacco smoking, obesity, diabetes mellitus, heavy alcohol use, a previous transient ischemic attack, a previous stroke, a high distribution of white matter hyperintensities on brain MRI, end-stage kidney disease, congenital heart disease, and atrial fibrillation. A major risk factor for all types of stroke is cerebral small vessel ischemic disease, which is caused by damage to blood vessels from the risk factors for stroke, and can be measured by white matter hyperintensity changes on brain magnetic resonance imaging (MRI), and is known to be a biomarker for stroke. In some embodiments, administering a GBE1 inhibitor to a subject having a stroke may be carried out to prevent development of another occurrence of a future stroke in a subject who has already had a prior stroke. In any of the embodiments described herein, the methods can be used to improve stroke recovery.
In any of the embodiments described herein, the GBE1 predicted loss-of-function polypeptide can be any GBE1 polypeptide having a partial loss-of-function, a complete loss-of-function, a predicted partial loss-of-function, or a predicted complete loss-of-function.
Any one or more (i.e., any combination) of the GBE1 variant nucleic acid molecules described herein can be used within any of the methods described herein to determine whether a subject has an increased or decreased risk of developing a stroke. The combinations of particular variants can form a mask used for statistical analysis of the particular association of GBE1 and an increased or decreased risk of developing a stroke. In some embodiments, the mask used for statistical analysis of the particular association of GBE1 and an increased or decreased risk of developing a stroke can exclude any one or more of these GBE1 variant nucleic acid molecules described herein.
In any of the embodiments described herein, the subject can have a stroke. In any of the embodiments described herein, the subject can be at risk of developing a stroke. In any of the embodiments described herein, the stroke is ischemic stroke or hemorrhagic stroke. In some embodiments, the stroke is ischemic stroke. In some embodiments, the stroke is hemorrhagic stroke.
In any of the embodiments described herein, the methods can be used to treat a complication or co-morbidity of a stroke, or reduce the risk of developing the same.
The present disclosure provides methods of treating a subject having a stroke or at risk of developing a stroke, the methods comprising administering a GBE1 inhibitor to the subject. Although not to limit the present disclosure, treatment may be a primary prevention for small vessel ischemic disease, acute intervention for stroke, secondary prevention after TIA/minor stroke, or periprocedural intervention to reduce ischemic damage from cardiac or other vascular procedures.
In some embodiments, the GBE1 inhibitor comprises an inhibitory nucleic acid molecule. Examples of inhibitory nucleic acid molecules include, but are not limited to, antisense nucleic acid molecules, small interfering RNAs (siRNAs), and short hairpin RNAs (shRNAs). Such inhibitory nucleic acid molecules can be designed to target any region of a GBE1 nucleic acid molecule. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes to a sequence within a GBE1 genomic nucleic acid molecule or mRNA molecule and decreases expression of the GBE1 polypeptide in a cell in the subject. In some embodiments, the GBE1 inhibitor comprises an antisense molecule that hybridizes to a GBE1 genomic nucleic acid molecule or mRNA molecule and decreases expression of the GBE1 polypeptide in a cell in the subject. In some embodiments, the GBE1 inhibitor comprises an siRNA that hybridizes to a GBE1 genomic nucleic acid molecule or mRNA molecule and decreases expression of the GBE1 polypeptide in a cell in the subject. In some embodiments, the GBE1 inhibitor comprises an shRNA that hybridizes to a GBE1 genomic nucleic acid molecule or mRNA molecule and decreases expression of the GBE1 polypeptide in a cell in the subject.
The inhibitory nucleic acid molecules can comprise RNA, DNA, or both RNA and DNA. The inhibitory nucleic acid molecules can also be joined or fused to a heterologous nucleic acid sequence, such as in a vector, or a heterologous label. For example, the inhibitory nucleic acid molecules can be within a vector or as an exogenous donor sequence comprising the inhibitory nucleic acid molecule and a heterologous nucleic acid sequence. The inhibitory nucleic acid molecules can also be joined or fused to a heterologous label. The label can be directly detectable (such as, for example, fluorophore) or indirectly detectable (such as, for example, hapten, enzyme, or fluorophore quencher). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radiolabels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label can also be, for example, a chemiluminescent substance; a metal-containing substance; or an enzyme, where there occurs an enzyme-dependent secondary generation of signal. The term “label” can also refer to a “tag” or hapten that can bind selectively to a conjugated molecule such that the conjugated molecule, when added subsequently along with a substrate, is used to generate a detectable signal. For example, biotin can be used as a tag along with an avidin or streptavidin conjugate of horseradish peroxidate (HRP) to bind to the tag, and examined using a calorimetric substrate (such as, for example, tetramethylbenzidine (TMB)) or a fluorogenic substrate to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3×FLAG, 6×His or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, an epitope tag, or the Fc portion of immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorogenic and chemiluminescent substrates and other labels.
The inhibitory nucleic acid molecules can comprise, for example, nucleotides or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutes. Such nucleotides include a nucleotide that contains a modified base, sugar, or phosphate group, or that incorporates a non-natural moiety in its structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated, and fluorophor-labeled nucleotides.
The inhibitory nucleic acid molecules can also comprise one or more nucleotide analogs or substitutions. A nucleotide analog is a nucleotide which contains a modification to either the base, sugar, or phosphate moieties. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications of A, C, G, and T/U, as well as different purine or pyrimidine bases such as, for example, pseudouridine, uracil-5-yl, hypoxanthin-9-yl(I), and 2-aminoadenin-9-yl. Modified bases include, but are not limited to, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (such as, for example, 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.
Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of the ribose and deoxy ribose as well as synthetic modifications. Sugar modifications include, but are not limited to, the following modifications at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-10alkyl or C2-10alkenyl, and C2-10alkynyl. Exemplary 2′ sugar modifications also include, but are not limited to, —O[(CH2)nO]mCH3, —O(CH2)nOCH3, —O(CH2)nNH2, —O(CH2)nCH3, —O(CH2)n—ONH2, and —O(CH2)nON[(CH2)nCH3)]2, where n and m, independently, are from 1 to about 10. Other modifications at the 2′ position include, but are not limited to, C1-10alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications may also be made at other positions on the sugar, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Modified sugars can also include those that contain modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics, such as cyclobutyl moieties in place of the pentofuranosyl sugar.
Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those that can be modified so that the linkage between two nucleotides contains a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3′-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. These phosphate or modified phosphate linkage between two nucleotides can be through a 3′-5′ linkage or a 2′-5′ linkage, and the linkage can contain inverted polarity such as 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts, and free acid forms are also included. Nucleotide substitutes also include peptide nucleic acids (PNAs).
In some embodiments, the antisense nucleic acid molecules are gapmers, whereby the first one to seven nucleotides at the 5′ and 3′ ends each have 2′-methoxyethyl (2′-MOE) modifications. In some embodiments, the first five nucleotides at the 5′ and 3′ ends each have 2′-MOE modifications. In some embodiments, the first one to seven nucleotides at the 5′ and 3′ ends are RNA nucleotides. In some embodiments, the first five nucleotides at the 5′ and 3′ ends are RNA nucleotides. In some embodiments, each of the backbone linkages between the nucleotides is a phosphorothioate linkage.
In some embodiments, the siRNA molecules have termini modifications. In some embodiments, the 5′ end of the antisense strand is phosphorylated. In some embodiments, 5′-phosphate analogs that cannot be hydrolyzed, such as 5′-(E)-vinyl-phosphonate are used.
In some embodiments, the siRNA molecules have backbone modifications. In some embodiments, the modified phosphodiester groups that link consecutive ribose nucleosides have been shown to enhance the stability and in vivo bioavailability of siRNAs The non-ester groups (—OH, ═O) of the phosphodiester linkage can be replaced with sulfur, boron, or acetate to give phosphorothioate, boranophosphate, and phosphonoacetate linkages. In addition, substituting the phosphodiester group with a phosphotriester can facilitate cellular uptake of siRNAs and retention on serum components by eliminating their negative charge. In some embodiments, the siRNA molecules have sugar modifications. In some embodiments, the sugars are deprotonated (reaction catalyzed by exo- and endonucleases) whereby the 2′-hydroxyl can act as a nucleophile and attack the adjacent phosphorous in the phosphodiester bond. Such alternatives include 2′-O-methyl, 2′-O-methoxyethyl, and 2′-fluoro modifications.
In some embodiments, the siRNA molecules have base modifications. In some embodiments, the bases can be substituted with modified bases such as pseudouridine, 5′-methylcytidine, N6-methyladenosine, inosine, and N7-methylguanosine.
In some embodiments, the siRNA molecules are conjugated to lipids. Lipids can be conjugated to the 5′ or 3′ termini of siRNA to improve their in vivo bioavailability by allowing them to associate with serum lipoproteins. Representative lipids include, but are not limited to, cholesterol and vitamin E, and fatty acids, such as palmitate and tocopherol.
In some embodiments, a representative siRNA has the following formula:
Sense: mN*mN*/i2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN/i2FN/*mN*/32FN/
Antisense: /52FN/*/i2FN/*mN/i2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN/İ2FN/mN/i2FN/mN*N*N
In any of the embodiments described herein, the inhibitory nucleic acid molecules may be administered, for example, as one to two hour i.v. infusions or s.c. injections. In any of the embodiments described herein, the inhibitory nucleic acid molecules may be administered at dose levels that range from about 50 mg to about 900 mg, from about 100 mg to about 800 mg, from about 150 mg to about 700 mg, or from about 175 to about 640 mg (2.5 to 9.14 mg/kg; 92.5 to 338 mg/m2—based on an assumption of a body weight of 70 kg and a conversion of mg/kg to mg/m2 dose levels based on a mg/kg dose multiplier value of 37 for humans).
The present disclosure also provides vectors comprising any one or more of the inhibitory nucleic acid molecules. In some embodiments, the vectors comprise any one or more of the inhibitory nucleic acid molecules and a heterologous nucleic acid. The vectors can be viral or nonviral vectors capable of transporting a nucleic acid molecule. In some embodiments, the vector is a plasmid or cosmid (such as, for example, a circular double-stranded DNA into which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, yeast artificial chromosomes (YACs), Epstein-Barr (EBV)-derived episomes, and other expression vectors known in the art.
The present disclosure also provides compositions comprising any one or more of the inhibitory nucleic acid molecules. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the compositions comprise a carrier and/or excipient. Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-coglycolic-acid) (PLGA) microspheres, liposomes, micelles, inverse micelles, lipid cochleates, and lipid microtubules. A carrier may comprise a buffered salt solution such as PBS, HBSS, etc.
In some embodiments, the GBE1 inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks at a recognition sequence(s) or a DNA-binding protein that binds to a recognition sequence within a GBE1 genomic nucleic acid molecule. The recognition sequence can be located within a coding region of the GBE1 gene, or within regulatory regions that influence the expression of the gene. A recognition sequence of the DNA-binding protein or nuclease agent can be located in an intron, an exon, a promoter, an enhancer, a regulatory region, or any non-protein coding region. The recognition sequence can include or be proximate to the start codon of the GBE1 gene. For example, the recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon. As another example, two or more nuclease agents can be used, each targeting a nuclease recognition sequence including or proximate to the start codon. As another example, two nuclease agents can be used, one targeting a nuclease recognition sequence including or proximate to the start codon, and one targeting a nuclease recognition sequence including or proximate to the stop codon, wherein cleavage by the nuclease agents can result in deletion of the coding region between the two nuclease recognition sequences. Any nuclease agent that induces a nick or double-strand break into a desired recognition sequence can be used in the methods and compositions disclosed herein. Any DNA-binding protein that binds to a desired recognition sequence can be used in the methods and compositions disclosed herein.
Suitable nuclease agents and DNA-binding proteins for use herein include, but are not limited to, zinc finger protein or zinc finger nuclease (ZFN) pair, Transcription Activator-Like Effector (TALE) protein or Transcription Activator-Like Effector Nuclease (TALEN), or Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) systems. The length of the recognition sequence can vary, and includes, for example, recognition sequences that are about 30-36 bp for a zinc finger protein or ZFN pair, about 15-18 bp for each ZFN, about 36 bp for a TALE protein or TALEN, and about 20 bp for a CRISPR/Cas guide RNA.
In some embodiments, CRISPR/Cas systems can be used to modify a GBE1 genomic nucleic acid molecule within a cell. The methods and compositions disclosed herein can employ CRISPR-Cas systems by utilizing CRISPR complexes (comprising a guide RNA (gRNA) complexed with a Cas protein) for site-directed cleavage of GBE1 nucleic acid molecules.
Cas proteins generally comprise at least one RNA recognition or binding domain that can interact with gRNAs. Cas proteins can also comprise nuclease domains (such as, for example, DNase or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Suitable Cas proteins include, for example, a wild type Cas9 protein and a wild type Cpf1 protein (such as, for example, FnCpf1). A Cas protein can have full cleavage activity to create a double-strand break in a GBE1 genomic nucleic acid molecule or it can be a nickase that creates a single-strand break in a GBE1 genomic nucleic acid molecule. Additional examples of Cas proteins include, but are not limited to, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, and homologs or modified versions thereof. In some embodiments, a Cas system, such as Cas12a, can have multiple gRNAs encoded into a single crRNA. Cas proteins can also be operably linked to heterologous polypeptides as fusion proteins. For example, a Cas protein can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. Cas proteins can be provided in any form. For example, a Cas protein can be provided in the form of a protein, such as a Cas protein complexed with a gRNA. Alternately, a Cas protein can be provided in the form of a nucleic acid molecule encoding the Cas protein, such as an RNA or DNA.
In some embodiments, targeted genetic modifications of GBE1 genomic nucleic acid molecules can be generated by contacting a cell with a Cas protein and one or more gRNAs that hybridize to one or more gRNA recognition sequences within a target genomic locus in the GBE1 genomic nucleic acid molecule. The gRNA recognition sequence can include or be proximate to the start codon of a GBE1 genomic nucleic acid molecule or the stop codon of a GBE1 genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located from about 10, from about 20, from about 30, from about 40, from about 50, from about 100, from about 200, from about 300, from about 400, from about 500, or from about 1,000 nucleotides of the start codon or the stop codon.
The gRNA recognition sequences within a target genomic locus in a GBE1 genomic nucleic acid molecule are located near a Protospacer Adjacent Motif (PAM) sequence, which is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease. The canonical PAM is the sequence 5′-NGG-3′ where “N” is any nucleobase followed by two guanine (“G”) nucleobases. gRNAs can transport Cas9 to anywhere in the genome for gene editing, but no editing can occur at any site other than one at which Cas9 recognizes PAM. In addition, 5′-NGA-3′ can be a highly efficient non-canonical PAM for human cells. Generally, the PAM is about 2-6 nucleotides downstream of the DNA sequence targeted by the gRNA. The PAM can flank the gRNA recognition sequence. In some embodiments, the gRNA recognition sequence can be flanked on the 3′ end by the PAM. In some embodiments, the gRNA recognition sequence can be flanked on the 5′ end by the PAM. For example, the cleavage site of Cas proteins can be about 1 to about 10, about 2 to about 5 base pairs, or three base pairs upstream or downstream of the PAM sequence. In some embodiments (such as when Cas9 from S. pyogenes or a closely related Cas9 is used), the PAM sequence of the non-complementary strand can be 5′-NGG-31, where Nis any DNA nucleotide and is immediately 3′ of the gRNA recognition sequence of the non-complementary strand of the target DNA. As such, the PAM sequence of the complementary strand would be 5′-CCN-31, where N is any DNA nucleotide and is immediately 5′ of the gRNA recognition sequence of the complementary strand of the target DNA.
A gRNA is an RNA molecule that binds to a Cas protein and targets the Cas protein to a specific location within a GBE1 genomic nucleic acid molecule. An exemplary gRNA is a gRNA effective to direct a Cas enzyme to bind to or cleave a GBE1 genomic nucleic acid molecule, wherein the gRNA comprises a DNA-targeting segment that hybridizes to a gRNA recognition sequence within the GBE1 genomic nucleic acid molecule. Exemplary gRNAs comprise a DNA-targeting segment that hybridizes to a gRNA recognition sequence present within a GBE1 genomic nucleic acid molecule that includes or is proximate to the start codon or the stop codon. For example, a gRNA can be selected such that it hybridizes to a gRNA recognition sequence that is located from about 5, from about 10, from about 15, from about 20, from about 25, from about 30, from about 35, from about 40, from about 45, from about 50, from about 100, from about 200, from about 300, from about 400, from about 500, or from about 1,000 nucleotides of the start codon or located from about 5, from about 10, from about 15, from about 20, from about 25, from about 30, from about 35, from about 40, from about 45, from about 50, from about 100, from about 200, from about 300, from about 400, from about 500, or from about 1,000 nucleotides of the stop codon. Suitable gRNAs can comprise from about 17 to about 25 nucleotides, from about 17 to about 23 nucleotides, from about 18 to about 22 nucleotides, or from about 19 to about 21 nucleotides. In some embodiments, the gRNAs can comprise 20 nucleotides.
The Cas protein and the gRNA form a complex, and the Cas protein cleaves the GBE1 genomic nucleic acid molecule. The Cas protein can cleave the nucleic acid molecule at a site within or outside of the nucleic acid sequence present in the GBE1 genomic nucleic acid molecule to which the DNA-targeting segment of a gRNA will bind. For example, formation of a CRISPR complex (comprising a gRNA hybridized to a gRNA recognition sequence and complexed with a Cas protein) can result in cleavage of one or both strands in or near (such as, for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the nucleic acid sequence present in the GBE1 genomic nucleic acid molecule to which a DNA-targeting segment of a gRNA will bind.
Such methods can result, for example, in a GBE1 genomic nucleic acid molecule in which a region of the GBE1 genomic nucleic acid molecule is disrupted, the start codon is disrupted, the stop codon is disrupted, or the coding sequence is disrupted or deleted. Optionally, the cell can be further contacted with one or more additional gRNAs that hybridize to additional gRNA recognition sequences within the target genomic locus in the GBE1 genomic nucleic acid molecule. By contacting the cell with one or more additional gRNAs (such as, for example, a second gRNA that hybridizes to a second gRNA recognition sequence), cleavage by the Cas protein can create two or more double-strand breaks or two or more single-strand breaks.
In any of the methods of treatment or prevention described herein, the subject being treated is GBE1 reference (i.e., does not have a GBE1 variant nucleic acid molecule). The GBE1 variant nucleic acid molecule can be any of the GBE1 variant nucleic acid molecules disclosed herein. In some embodiments, the GBE1 variant nucleic acid molecule is a GBE1 variant genomic nucleic acid molecule that comprises the genetic variation 3:81648854:A:G (rs192044702), 3:81535193:A:G (rs752625236), 3:81536909A:G (rs539203557), 3:81536926:C:T (rs201029706), 3:81536936:CTT:C, 3:81577973:G:A (rs137852888), 3:81581237:GC:G (rs1703017615), 3:81586127:G:A (rs781198373), 3:81594010:T:A, or 3:81670900:CCCATTTT:C, or is an mRNA molecule produced therefrom, or is a cDNA molecule produced from the mRNA molecule. In any of the embodiments described herein, the subject being treated has increased risk of stroke, including increased risk as defined by elevated total white matter hyperintensity determined by imaging the subject and/or using other biomarkers.
In some embodiments, the methods of treatment or prevention further comprise detecting the presence or absence of a GBE1 variant nucleic acid molecule in a biological sample from the subject. In some embodiments, the GBE1 variant nucleic acid molecule can be any of the GBE1 variant nucleic acid molecules disclosed herein. In some embodiments, the GBE1 variant nucleic acid molecule is a GBE1 variant genomic nucleic acid molecule that comprises the genetic variation 3:81648854:A:G (rs192044702), 3:81535193:A:G (rs752625236), 3:81536909A:G (rs539203557), 3:81536926:C:T (rs201029706), 3:81536936:CTT:C, 3:81577973:G:A (rs137852888), 3:81581237:GC:G (rs1703017615), 3:81586127:G:A (rs781198373), 3:81594010:T:A, or 3:81670900:CCCATTTT:C, or is an mRNA molecule produced therefrom, or is a cDNA molecule produced from the mRNA molecule. In some embodiments, the methods of treatment or prevention further comprise determining whether a subject has increased total white matter hyperintensity by, for example, imaging techniques such as MRI.
The present disclosure also provides methods of treating a subject with a stroke therapeutic agent or stroke therapy, wherein the subject has a stroke or is at risk of developing a stroke. The methods comprise determining whether the subject has a single copy of a GBE1 variant nucleic acid molecule by obtaining or having obtained a biological sample from the subject, and performing or having performed a sequence analysis on the biological sample to determine if the subject has a genotype comprising the GBE1 variant nucleic acid molecule. The methods can further comprise determining or having determined whether the subject has increased total white matter hyperintensity by imaging the subject. In embodiments where the subject is GBE1 reference, the methods further comprise administering or continuing to administer the stroke therapeutic agent in an amount that is the same as or less than a standard dosage amount or stroke therapy to the subject, and/or administering a GBE1 inhibitor to the subject. In embodiments where the subject has increased total white matter hyperintensity, the methods further comprise administering or continuing to administer the stroke therapeutic agent in an amount that is the same as or less than a standard dosage amount or stroke therapy, and/or a GBE1 inhibitor to the subject. The presence of a single copy of a GBE1 variant nucleic acid molecule indicates the subject has a decreased risk of developing a stroke. In some embodiments, the subject is GBE1 reference. In any of the embodiments described herein, the GBE1 inhibitor is an example of a stroke therapeutic agent. In some embodiments, the GBE1 variant nucleic acid molecule is a GBE1 variant genomic nucleic acid molecule that comprises the genetic variation 3:81648854:A:G (rs192044702), 3:81535193:A:G (rs752625236), 3:81536909A:G (rs539203557), 3:81536926:C:T (rs201029706), 3:81536936:CTT:C, 3:81577973:G:A (rs137852888), 3:81581237:GC:G (rs1703017615), 3:81586127:G:A (rs781198373), 3:81594010:T:A, or 3:81670900:CCCATTTT:C, or is an mRNA molecule produced therefrom, or is a cDNA molecule produced from the mRNA molecule.
For subjects that are genotyped or determined to be either GBE1 reference, such subjects can be administered a GBE1 inhibitor, as described herein. For subjects that are determined to have increased total white matter hyperintensity, such subjects can also be administered a GBE1 inhibitor, as described herein.
Detecting the presence or absence of a GBE1 variant nucleic acid molecule in a biological sample from a subject and/or determining whether a subject has a GBE1 variant nucleic acid molecule can be carried out by any of the methods described herein. In some embodiments, these methods can be carried out in vitro. In some embodiments, these methods can be carried out in situ. In some embodiments, these methods can be carried out in vivo. In any of these embodiments, the nucleic acid molecule can be present within a cell obtained from the subject.
In some embodiments, when the subject is GBE1 reference, the subject is administered a stroke therapeutic agent in an amount that is the same as or less than a standard dosage amount or stroke therapy, and/or a GBE1 inhibitor.
In some embodiments, the treatment or prevention methods comprise detecting the presence or absence of a decrease in the expression of a GBE1 variant mRNA or polypeptide in a biological sample from the subject. In some embodiments, when the subject does not have a decrease in the expression of a GBE1 variant mRNA or polypeptide, the subject is administered a stroke therapeutic agent in an amount that is the same as or less than a standard dosage amount or stroke therapy, and/or a GBE1 inhibitor. In some embodiments, when the subject has a decrease in the expression of a GBE1 variant mRNA or polypeptide, the subject is administered a stroke therapeutic agent in a standard dosage amount or stroke therapy.
The present disclosure also provides methods of treating a subject with a stroke therapeutic agent or stroke therapy, wherein the subject has a stroke or is at risk of developing a stroke. The methods comprise determining whether the subject has a decrease in the expression of a GBE1 variant mRNA or polypeptide by obtaining or having obtained a biological sample from the subject, and performing or having performed an assay on the biological sample to determine if the subject a decrease in the expression of a GBE1 variant mRNA or polypeptide. In embodiments where the subject does not have a decrease in the expression of a GBE1 variant mRNA or polypeptide, the methods further comprise administering or continuing to administer the stroke therapeutic agent in an amount that is the same as or less than a standard dosage amount or stroke therapy to the subject, and/or administering a GBE1 inhibitor to the subject. In embodiments where the subject has a decrease in the expression of a GBE1 variant mRNA or polypeptide, the methods further comprise administering or continuing to administer the stroke therapeutic agent in a standard dosage amount or stroke therapy to the subject. The presence of a decrease in the expression of a GBE1 variant mRNA or polypeptide to about 30% to about 80% of its normal level or function indicates the subject has a decreased risk of developing a stroke. In some embodiments, the subject has a decrease in the expression of a GBE1 variant mRNA or polypeptide. In some embodiments, the subject does not have a decrease in the expression of a GBE1 variant mRNA or polypeptide. In any of the embodiments described herein, the GBE1 inhibitor is an example of a stroke therapeutic agent. In some embodiments, the GBE1 variant nucleic acid molecule is a GBE1 variant genomic nucleic acid molecule that comprises the genetic variation 3:81648854:A:G (rs192044702), 3:81535193:A:G (rs752625236), 3:81536909A:G (rs539203557), 3:81536926:C:T (rs201029706), 3:81536936:CTT:C, 3:81577973:G:A (rs137852888), 3:81581237:GC:G (rs1703017615), 3:81586127:G:A (rs781198373), 3:81594010:T:A, or 3:81670900:CCCATTTT:C, or is an mRNA molecule produced therefrom, or is a cDNA molecule produced from the mRNA molecule.
Detecting a decrease in the expression of a GBE1 variant mRNA or polypeptide can be carried out by a variety of known methods. In some embodiments, these methods can be carried out in vitro. In some embodiments, these methods can be carried out in situ. In some embodiments, these methods can be carried out in vivo. In any of these embodiments, the mRNA or polypeptide can be present within a cell obtained from the subject.
In some embodiments, the treatment or prevention methods comprise detecting the presence or absence of a GBE1 variant polypeptide in a biological sample from the subject. In some embodiments, when the subject does not have a GBE1 variant polypeptide, the subject is administered a stroke therapeutic agent in an amount that is the same as or less than a standard dosage amount or stroke therapy, and/or a GBE1 inhibitor. In some embodiments, when the subject has a GBE1 variant polypeptide, the subject is administered a stroke therapeutic agent in standard dosage amount or stroke therapy.
The present disclosure also provides methods of treating a subject with a stroke therapeutic agent or stroke therapy, wherein the subject has a stroke or is at risk of developing a stroke. The methods comprise determining whether the subject has a GBE1 variant polypeptide by obtaining or having obtained a biological sample from the subject and performing or having performed an assay on the biological sample to determine if the subject has a GBE1 variant polypeptide. When the subject does not have a GBE1 variant polypeptide, the subject is administered the stroke therapeutic agent in an amount that is the same as or less than a standard dosage amount or stroke therapy, and/or a GBE1 inhibitor. When the subject has a GBE1 variant polypeptide, the subject is administered the stroke therapeutic agent in a standard dosage amount or stroke therapy. The presence of a GBE1 variant polypeptide at a level or function that is about 30% to about 80% of its normal level or function indicates the subject has a decreased risk of developing a stroke. In some embodiments, the subject has a GBE1 variant polypeptide. In some embodiments, the subject does not have a GBE1 variant polypeptide.
The present disclosure also provides methods of preventing a subject from developing a stroke by administering a stroke therapeutic agent or stroke therapy. In some embodiments, the methods comprise determining whether the subject has a GBE1 variant polypeptide by obtaining or having obtained a biological sample from the subject and performing or having performed an assay on the biological sample to determine if the subject has a GBE1 variant polypeptide. When the subject does not have a GBE1 variant polypeptide, the subject is administered the stroke therapeutic agent in an amount that is the same as or less than a standard dosage amount or stroke therapy, and/or a GBE1 inhibitor. When the subject has a GBE1 variant polypeptide, the subject is administered the stroke therapeutic agent in a standard dosage amount or stroke therapy. The presence of a GBE1 variant polypeptide at a level or function that is about 30% to about 80% of its normal level or function indicates the subject has a decreased risk of developing a stroke. In some embodiments, the subject does not have a GBE1 variant polypeptide.
Detecting the presence or absence of a GBE1 variant polypeptide in a biological sample from a subject and/or determining whether a subject has a GBE1 variant polypeptide can be carried out by any of the methods described herein. In some embodiments, these methods can be carried out in vitro. In some embodiments, these methods can be carried out in situ. In some embodiments, these methods can be carried out in vivo. In any of these embodiments, the polypeptide can be present within a cell obtained from the subject.
In some embodiments, the GBE1 inhibitor is a small molecule. In some embodiments, the small molecule is low molecular weight (<900 daltons) organic compound. In some embodiments, the GBE1 inhibitor is (2R)-2-[(4-methoxyphenyl)methyl]-3,3-dimethylpiperidin-4-one (IMT004); 5-oxo-1-(propan-2-yl)pyrrolidine-3-carboxylic acid (IMT029); 1′H-spiro[piperidine-4,2′-quinazolin]-4′(3′H)-one (IMT009); 1-benzyl-5-oxopyrrolidine-3-carboxylic acid (IMT021); 1-(1H-benzo[d]imidazol-6-yl)-2-methylpropan-2-amine (IMT014); N′-(imino(pyridin-2-yl)methyl)furan-2-carbohydrazide (MB16695); 1-(1H-benzo[d]imidazol-2-yl)ethan-1-amine (IMT012); (3S,5S)-5-(hydroxy(methoxy)methyl) pyrrolidin-3-ol (IMT025); 1H-benzimidazol-2-amine (IMT017); 5-(piperazin-1-yl) [1,2]thiazolo[2,3-c]pyrimidin-8-ium (IMT007); 1-(1H-benzimidazol-2-yl)ethan-1-amine (ZINC05818427); or 1-(1H-benzimidazol-6-yl)-2-methylpropan-2-amine (IMT013); or any combination thereof.
In some embodiments, the GBE1 inhibitor comprises an antibody, or antigen-binding fragment thereof. In some embodiments, the antibody, or antigen-binding fragment thereof, binds specifically to human GBE1. In some embodiments, the antibody is a fully human monoclonal antibody (mAb), or antigen-binding fragment thereof, that specifically binds and neutralizes, inhibits, blocks, abrogates, reduces, or interferes with, at least one activity of GBE1, in particular, human GBE1. In some embodiments, an antibody or fragment thereof can neutralize, inhibit, block, abrogate, reduce, or interfere with, an activity of GBE1 by binding to an epitope of GBE1 that is directly involved in the targeted activity of GBE1. In some embodiments, an antibody or fragment thereof can neutralize, inhibit, block, abrogate, reduce, or interfere with, an activity of GBE1 by binding to an epitope of GBE1 that is not directly involved in the targeted activity of GBE1, but the antibody or fragment binding thereto sterically or conformationally inhibits, blocks, abrogates, reduces, or interferes with, the targeted activity of GBE1. In some embodiments, an antibody or fragment thereof binds to an epitope of GBE1 that is not directly involved in the targeted activity of GBE1 (i.e., a non-blocking antibody), but the antibody or fragment binding thereto results in the enhancement of the clearance of GBE1 from the circulation, compared to the clearance of GBE1 in the absence of the antibody or fragment thereof, thereby indirectly inhibiting, blocking, abrogating, reducing, or interfering with, an activity of GBE1. Clearance of GBE1 from the circulation can be particularly enhanced by combining two or more different non-blocking antibodies that do not compete with one another for specific binding to GBE1. The antibodies can be full-length (for example, an IgG1 or IgG4 antibody) or may comprise only an antigen-binding portion (for example, a Fab, F(ab′)2 or scFv fragment), and may be modified to affect functionality, e.g., to eliminate residual effector functions (Reddy et al., J. Immunol., 2000, 164, 1925-1933).
In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to GBE1 with an equilibrium dissociation constant (KD) of about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, or about 1 nM or less, as measured by surface plasmon resonance assay (for example, BIACORE™). In some embodiments, the antibody exhibits a KD of about 800 pM or less, about 700 pM or less; about 600 pM or less; about 500 pM or less; about 400 pM or less; about 300 pM or less; about 200 pM or less; about 100 pM or less; or about 50 pM or less.
In some embodiments, the anti-GBE1 antibodies have a modified glycosylation pattern. In some applications, modification to remove undesirable glycosylation sites may be useful, or e.g., removal of a fucose moiety to increase antibody dependent cellular cytotoxicity (ADCC) function (see, Shield et al., J. Biol. Chem., 2002, 277, 26733). In other applications, removal of N-glycosylation site may reduce undesirable immune reactions against the therapeutic antibodies or increase affinities of the antibodies. In yet other applications, modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).
The present disclosure also provides compositions comprising a combination of a GBE1 inhibitor and a stroke therapeutic agent. In some embodiments, the stroke therapeutic agents include, but are not limited to, a recombinant tissue plasminogen activator (rtPa). Additional stroke therapies include any therapy used to reduce or manage stroke risk factors such as, for example, warfarin, heparin, dabigatran, novel or direct anticoagulants (apixaban, edoxaban, rivaroxaban, or other factor Xa inhibitors), aspirin, clopidogrel, dipyridamole, cilostazol, and a statin, or any combination thereof. In some embodiments, the stroke therapeutic agent or stroke therapy can be combined with a GBE1 inhibitor. In some embodiments, the treatment therapy for stroke is surgery to open up the arteries to the brain in those with problematic carotid narrowing, mechanical thrombectomy, and hemicraniectomy. These treatment therapies may be delayed or avoided altogether by treatment with a GBE1 inhibitor as described herein.
In any of the embodiments described herein, the amount of the GBE1 inhibitor administered to a subject can be any amount that results in the reduction of the expression or function of GBE1 to about 30% to about 80% of its normal expression or function. The subject can be determined to have a normal expression or function of GBE1 by determining the expression or function of GBE1 prior to initiation of treatment with a GBE1 inhibitor. In some embodiments, the amount of the GBE1 inhibitor administered to a subject can be any amount that results in the reduction of the expression or function of GBE1 to about 35% to about 75% of its normal expression or function. In some embodiments, the amount of the GBE1 inhibitor administered to a subject can be any amount that results in the reduction of the expression or function of GBE1 to about 40% to about 70% of its normal expression or function. In some embodiments, the amount of the GBE1 inhibitor administered to a subject can be any amount that results in the reduction of the expression or function of GBE1 to about 45% to about 65% of its normal expression or function. In some embodiments, the amount of the GBE1 inhibitor administered to a subject can be any amount that results in the reduction of the expression or function of GBE1 to about 50% to about 60% of its normal expression or function. In some embodiments, the amount of the GBE1 inhibitor administered to a subject can be any amount that results in the reduction of the expression or function of GBE1 to about 30%, to about 35%, to about 40%, to about 45%, to about 50%, to about 55%, to about 60%, to about 65%, to about 70%, to about 75%, or to about 80% of its normal expression or function. In some embodiments, the amount of the GBE1 inhibitor administered to a subject can be any amount that results in the reduction of the expression or function of GBE1 to about 50% of its normal expression or function.
Administration of the stroke therapeutic agents and/or GBE1 inhibitors can be repeated, for example, after one day, two days, three days, five days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, eight weeks, two months, or three months. The repeated administration can be at the same dose or at a different dose. The administration can be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more. For example, according to certain dosage regimens a subject can receive therapy for a prolonged period of time such as, for example, 6 months, 1 year, or more.
Administration of the stroke therapeutic agents and/or GBE1 inhibitors can occur by any suitable route including, but not limited to, parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Pharmaceutical compositions for administration are desirably sterile and substantially isotonic and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients, or auxiliaries. The formulation depends on the route of administration chosen. The term “pharmaceutically acceptable” means that the carrier, diluent, excipient, or auxiliary is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.
The terms “treat”, “treating”, and “treatment” and “prevent”, “preventing”, and “prevention” as used herein, refer to eliciting the desired biological response, such as a therapeutic and prophylactic effect, respectively. In some embodiments, a therapeutic effect comprises one or more of a decrease/reduction in stroke, a decrease/reduction in the severity of stroke (such as, for example, a reduction or inhibition of development of a stroke), a decrease/reduction in symptoms and disease-related effects, delaying the onset of symptoms and disease-related effects, reducing the severity of symptoms of disease-related effects, reducing the number of symptoms and disease-related effects, reducing the latency of symptoms and disease-related effects, an amelioration of symptoms and disease-related effects, reducing secondary symptoms, reducing secondary infections, preventing relapse to stroke, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, increasing time to sustained progression, speeding recovery, or increasing efficacy of or decreasing resistance to alternative therapeutics, and/or an increased survival time of the affected host animal, following administration of the agent or composition comprising the agent. A prophylactic effect may comprise a complete or partial avoidance/inhibition or a delay of stroke development/progression (such as, for example, a complete or partial avoidance/inhibition or a delay), and an increased survival time of the affected host animal, following administration of a therapeutic protocol. Treatment of stroke encompasses the treatment of a subject already diagnosed as having any form of stroke at any clinical stage or manifestation, the delay of the onset or evolution or aggravation or deterioration of the symptoms or signs of stroke, and/or preventing and/or reducing the severity of stroke.
In some embodiments, the GBE1 inhibitor and the stroke therapeutic agent are disposed within a pharmaceutical composition. In some embodiments, the GBE1 inhibitor is disposed within a first pharmaceutical composition and the stroke therapeutic agent is disposed within a second pharmaceutical composition. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously. In some embodiments, the first pharmaceutical composition is administered before the second pharmaceutical composition. In some embodiments, the first pharmaceutical composition is administered after the second pharmaceutical composition.
The present disclosure also provides methods of identifying a subject having an increased risk of developing a stroke. In some embodiments, the method comprises determining or having determined in a biological sample obtained from the subject the presence or absence of a single copy of a GBE1 variant nucleic acid molecule (such as a genomic nucleic acid molecule, mRNA molecule, and/or cDNA molecule). When the subject lacks a GBE1 variant nucleic acid molecule (i.e., the subject is genotypically categorized as GBE1 reference), then the subject has an increased risk of developing a stroke. In some embodiments, the GBE1 variant nucleic acid molecule is a GBE1 variant genomic nucleic acid molecule that comprises the genetic variation 3:81648854:A:G (rs192044702), 3:81535193:A:G (rs752625236), 3:81536909A:G (rs539203557), 3:81536926:C:T (rs201029706), 3:81536936:CTT:C, 3:81577973:G:A (rs137852888), 3:81581237:GC:G (rs1703017615), 3:81586127:G:A (rs781198373), 3:81594010:T:A, or 3:81670900:CCCATTTT:C, or is an mRNA molecule produced therefrom, or is a cDNA molecule produced from the mRNA molecule.
Having a single copy of a GBE1 variant nucleic acid molecule is more protective of a subject from developing a stroke than having no copies of a GBE1 variant nucleic acid molecule. Without intending to be limited to any particular theory or mechanism of action, it is believed that a single copy of a GBE1 variant nucleic acid molecule (i.e., heterozygous for a GBE1 variant nucleic acid molecule) is protective of a subject from developing a stroke. A subject that is heterozygous for a GBE1 variant nucleic acid molecule may have reduced signs of cerebral small vessel ischemic disease as assessed by total white matter hyperintensity on imaging, such as by T2 FLAIR MRI scans. Thus, subjects who have increased white matter hyperintensity and a prior stroke or transient ischemic attack are prime candidates for treatment with GBE1 inhibitors.
Determining whether a subject has a GBE1 variant nucleic acid molecule in a biological sample from a subject and/or determining whether a subject has a GBE1 variant nucleic acid molecule can be carried out by any of the methods described herein. In some embodiments, these methods can be carried out in vitro. In some embodiments, these methods can be carried out in situ. In some embodiments, these methods can be carried out in vivo. In any of these embodiments, the nucleic acid molecule can be present within a cell obtained from the subject.
In some embodiments, when a subject is identified as having an increased risk of developing a stroke, the subject is administered a stroke therapeutic agent or stroke therapy, and/or a GBE1 inhibitor, as described herein. For example, when the subject is GBE1 reference, and therefore has an increased risk of developing a stroke, the subject is administered a stroke therapeutic agent in an amount that is the same as or less than a standard dosage amount or stroke therapy, and/or is administered a GBE1 inhibitor. In some embodiments, the subject is GBE1 reference.
The present disclosure also provides methods of detecting the presence or absence of a GBE1 variant nucleic acid molecule (i.e., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule produced from an mRNA molecule) in a biological sample from a subject. It is understood that gene sequences within a population and mRNA molecules encoded by such genes can vary due to polymorphisms such as single-nucleotide polymorphisms.
The biological sample can be derived from any cell, tissue, or biological fluid from the subject. The biological sample may comprise any clinically relevant tissue, such as a bone marrow sample, a tumor biopsy, a fine needle aspirate, or a sample of bodily fluid, such as blood, gingival crevicular fluid, plasma, serum, lymph, ascitic fluid, cystic fluid, cerebrospinal fluid, or urine. In some cases, the sample comprises a buccal swab. The biological sample used in the methods disclosed herein can vary based on the assay format, nature of the detection method, and the tissues, cells, or extracts that are used as the sample. A biological sample can be processed differently depending on the assay being employed. For example, when detecting any GBE1 variant nucleic acid molecule, preliminary processing designed to isolate or enrich the biological sample for the genomic DNA can be employed. A variety of techniques may be used for this purpose. When detecting the level of any GBE1 variant nucleic acid molecule, different techniques can be used enrich the biological sample with mRNA molecules. Various methods to detect the presence or level of an mRNA molecule or the presence of a particular variant genomic DNA locus can be used.
In some embodiments, detecting a GBE1 variant nucleic acid molecule in a subject comprises performing a sequence analysis on a biological sample obtained from the subject to determine whether a GBE1 genomic nucleic acid molecule in the biological sample, and/or a GBE1 mRNA molecule in the biological sample, and/or a GBE1 cDNA molecule produced from an mRNA molecule in the biological sample, is present in the sample. In some embodiments, the methods detect the GBE1 variant genomic nucleic acid molecule that comprises the genetic variation 3:81648854:A:G (rs192044702), 3:81535193:A:G (rs752625236), 3:81536909A:G (rs539203557), 3:81536926:C:T (rs201029706), 3:81536936:CTT:C, 3:81577973:G:A (rs137852888), 3:81581237:GC:G (rs1703017615), 3:81586127:G:A (rs781198373), 3:81594010:T:A, or 3:81670900:CCCATTTT:C, or an mRNA molecule produced therefrom, or a cDNA molecule produced from the mRNA molecule.
In some embodiments, the methods of detecting the presence or absence of a GBE1 variant nucleic acid molecule (such as, for example, a genomic nucleic acid molecule, an mRNA molecule, and/or a cDNA molecule produced from an mRNA molecule) in a subject comprise performing an assay on a biological sample obtained from the subject. The assay determines whether a nucleic acid molecule in the biological sample comprises a particular nucleotide sequence.
In some embodiments, the biological sample comprises a cell or cell lysate. Such methods can further comprise, for example, obtaining a biological sample from the subject comprising a GBE1 genomic nucleic acid molecule or mRNA molecule, and if mRNA, optionally reverse transcribing the mRNA into cDNA. Such assays can comprise, for example determining the identity of these positions of the particular GBE1 nucleic acid molecule. In some embodiments, the method is an in vitro method.
In some embodiments, the determining step, detecting step, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of the GBE1 genomic nucleic acid molecule, the GBE1 mRNA molecule, or the GBE1 cDNA molecule in the biological sample that comprises a genetic variation compared to the corresponding GBE1 reference molecule. In some embodiments, the sequenced portion comprises one or more variations that cause a loss-of-function (partial or complete) or are predicted to cause a loss-of-function (partial or complete).
In some embodiments, the assay comprises sequencing the entire nucleic acid molecule. In some embodiments, only a GBE1 genomic nucleic acid molecule is analyzed. In some embodiments, only a GBE1 mRNA is analyzed. In some embodiments, only a GBE1 cDNA obtained from the GBE1 mRNA is analyzed.
Alteration-specific polymerase chain reaction techniques can be used to detect mutations such as SNPs in a nucleic acid sequence. Alteration-specific primers can be used because the DNA polymerase will not extend when a mismatch with the template is present.
In some embodiments, the nucleic acid molecule in the sample is mRNA and the mRNA is reverse-transcribed into a cDNA prior to the amplifying step. In some embodiments, the nucleic acid molecule is present within a cell obtained from the subject.
In some embodiments, the assay comprises contacting the biological sample with a primer or probe, such as an alteration-specific primer or alteration-specific probe, that specifically hybridizes to a GBE1 variant genomic sequence, variant mRNA sequence, or variant cDNA sequence and not the corresponding GBE1 reference sequence under stringent conditions and determining whether hybridization has occurred.
In some embodiments, the determining step, detecting step, or sequence analysis comprises: a) amplifying at least a portion of the GBE1 nucleic acid molecule that encodes the GBE1 polypeptide; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe; and d) detecting the detectable label.
In some embodiments, the assay comprises RNA sequencing (RNA-Seq). In some embodiments, the assays also comprise reverse transcribing mRNA into cDNA, such as by the reverse transcriptase polymerase chain reaction (RT-PCR).
In some embodiments, the methods utilize probes and primers of sufficient nucleotide length to bind to the target nucleotide sequence and specifically detect and/or identify a polynucleotide comprising a GBE1 variant genomic nucleic acid molecule, variant mRNA molecule, or variant cDNA molecule. The hybridization conditions or reaction conditions can be determined by the operator to achieve this result. The nucleotide length may be any length that is sufficient for use in a detection method of choice, including any assay described or exemplified herein. Such probes and primers can hybridize specifically to a target nucleotide sequence under high stringency hybridization conditions. Probes and primers may have complete nucleotide sequence identity of contiguous nucleotides within the target nucleotide sequence, although probes differing from the target nucleotide sequence and that retain the ability to specifically detect and/or identify a target nucleotide sequence may be designed by conventional methods. Probes and primers can have about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity or complementarity with the nucleotide sequence of the target nucleic acid molecule.
Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods involve nucleic acid hybridization methods other than sequencing, including using labeled primers or probes directed against purified DNA, amplified DNA, and fixed cell preparations (fluorescence in situ hybridization (FISH)). In some methods, a target nucleic acid molecule may be amplified prior to or simultaneous with detection. Illustrative examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic SDA (tSDA).
In hybridization techniques, stringent conditions can be employed such that a probe or primer will specifically hybridize to its target. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence to a detectably greater degree than to other non-target sequences, such as, at least 2-fold, at least 3-fold, at least 4-fold, or more over background, including over 10-fold over background. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater degree than to other nucleotide sequences by at least 2-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater degree than to other nucleotide sequences by at least 3-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater degree than to other nucleotide sequences by at least 4-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater degree than to other nucleotide sequences by over 10-fold over background. Stringent conditions are sequence-dependent and will be different in different circumstances.
Appropriate stringency conditions which promote DNA hybridization, for example, 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2×SSC at 50° C., are known or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Typically, stringent conditions for hybridization and detection will be those in which the salt concentration is less than about 1.5 M Na+ ion, typically about 0.01 to 1.0 M Na+ ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (such as, for example, 10 to 50 nucleotides) and at least about 60° C. for longer probes (such as, for example, greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.
In some embodiments, such isolated nucleic acid molecules comprise or consist of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, or at least about 5000 nucleotides. In some embodiments, such isolated nucleic acid molecules comprise or consist of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, or at least about 25 nucleotides. In some embodiments, the isolated nucleic acid molecules comprise or consist of at least about 18 nucleotides. In some embodiments, the isolated nucleic acid molecules comprise or consists of at least about 15 nucleotides. In some embodiments, the isolated nucleic acid molecules consist of or comprise from about 10 to about 35, from about 10 to about 30, from about 10 to about 25, from about 12 to about 30, from about 12 to about 28, from about 12 to about 24, from about 15 to about 30, from about 15 to about 25, from about 18 to about 30, from about 18 to about 25, from about 18 to about 24, or from about 18 to about 22 nucleotides. In some embodiments, the isolated nucleic acid molecules consist of or comprise from about 18 to about 30 nucleotides. In some embodiments, the isolated nucleic acid molecules comprise or consist of at least about 15 nucleotides to at least about 35 nucleotides.
In some embodiments, such isolated nucleic acid molecules hybridize to GBE1 variant nucleic acid molecules (such as genomic nucleic acid molecules, mRNA molecules, and/or cDNA molecules) under stringent conditions. Such nucleic acid molecules can be used, for example, as probes, primers, alteration-specific probes, or alteration-specific primers as described or exemplified herein, and include, without limitation primers, probes, antisense RNAs, shRNAs, and siRNAs, each of which is described in more detail elsewhere herein and can be used in any of the methods described herein.
In some embodiments, the isolated nucleic acid molecules hybridize to at least about 15 contiguous nucleotides of a nucleic acid molecule that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to GBE1 variant nucleic acid molecules. In some embodiments, the isolated nucleic acid molecules consist of or comprise from about 15 to about 100 nucleotides, or from about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecules consist of or comprise from about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecules consist of or comprise from about 15 to about 35 nucleotides.
In some embodiments, the alteration-specific probes and alteration-specific primers comprise DNA. In some embodiments, the alteration-specific probes and alteration-specific primers comprise RNA.
In some embodiments, the probes and primers described herein (including alteration-specific probes and alteration-specific primers) have a nucleotide sequence that specifically hybridizes to any of the nucleic acid molecules disclosed herein, or the complement thereof. In some embodiments, the probes and primers specifically hybridize to any of the nucleic acid molecules disclosed herein under stringent conditions.
In some embodiments, the primers, including alteration-specific primers, can be used in second generation sequencing or high throughput sequencing. In some instances, the primers, including alteration-specific primers, can be modified. In particular, the primers can comprise various modifications that are used at different steps of, for example, Massive Parallel Signature Sequencing (MPSS), Polony sequencing, and 454 Pyrosequencing. Modified primers can be used at several steps of the process, including biotinylated primers in the cloning step and fluorescently labeled primers used at the bead loading step and detection step. Polony sequencing is generally performed using a paired-end tags library wherein each molecule of DNA template is about 135 bp in length. Biotinylated primers are used at the bead loading step and emulsion PCR. Fluorescently labeled degenerate nonamer oligonucleotides are used at the detection step. An adaptor can contain a 5′-biotin tag for immobilization of the DNA library onto streptavidin-coated beads.
The probes and primers described herein can be used to detect a nucleotide variation within any of the GBE1 variant nucleic acid molecules disclosed herein. The primers described herein can be used to amplify any GBE1 variant nucleic acid molecule, or a fragment thereof.
In the context of the disclosure “specifically hybridizes” means that the probe or primer (such as, for example, the alteration-specific probe or alteration-specific primer) does not hybridize to a nucleic acid sequence encoding a GBE1 reference genomic nucleic acid molecule, a GBE1 reference mRNA molecule, and/or a GBE1 reference cDNA molecule.
In some embodiments, the probes (such as, for example, an alteration-specific probe) comprise a label. In some embodiments, the label is a fluorescent label, a radiolabel, or biotin.
The present disclosure also provides supports comprising a substrate to which any one or more of the probes disclosed herein is attached. Solid supports are solid-state substrates or supports with which molecules, such as any of the probes disclosed herein, can be associated. A form of solid support is an array. Another form of solid support is an array detector. An array detector is a solid support to which multiple different probes have been coupled in an array, grid, or other organized pattern. A form for a solid-state substrate is a microtiter dish, such as a standard 96-well type. In some embodiments, a multiwell glass slide can be employed that normally contains one array per well.
The genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be from any organism. For example, the genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be human or an ortholog from another organism, such as a non-human mammal, a rodent, a mouse, or a rat. It is understood that gene sequences within a population can vary due to polymorphisms such as single-nucleotide polymorphisms.
Also provided herein are functional polynucleotides that can interact with the disclosed nucleic acid molecules. Examples of functional polynucleotides include, but are not limited to, antisense molecules, aptamers, ribozymes, triplex forming molecules, and external guide sequences. The functional polynucleotides can act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional polynucleotides can possess a de novo activity independent of any other molecules.
The isolated nucleic acid molecules disclosed herein can comprise RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules can also be linked or fused to a heterologous nucleic acid sequence, such as in a vector, or a heterologous label. For example, the isolated nucleic acid molecules disclosed herein can be within a vector or as an exogenous donor sequence comprising the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecules can also be linked or fused to a heterologous label. The label can be directly detectable (such as, for example, fluorophore) or indirectly detectable (such as, for example, hapten, enzyme, or fluorophore quencher). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radiolabels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label can also be, for example, a chemiluminescent substance; a metal-containing substance; or an enzyme, where there occurs an enzyme-dependent secondary generation of signal. The term “label” can also refer to a “tag” or hapten that can bind selectively to a conjugated molecule such that the conjugated molecule, when added subsequently along with a substrate, is used to generate a detectable signal. For example, biotin can be used as a tag along with an avidin or streptavidin conjugate of horseradish peroxidate (HRP) to bind to the tag, and examined using a calorimetric substrate (such as, for example, tetramethylbenzidine (TMB)) or a fluorogenic substrate to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3×FLAG, 6×his or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, an epitope tag, or the Fc portion of immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorogenic and chemiluminescent substrates and other labels.
Percent identity (or percent complementarity) between particular stretches of nucleotide sequences within nucleic acid molecules or amino acid sequences within polypeptides can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Herein, if reference is made to percent sequence identity, the higher percentages of sequence identity are preferred over the lower ones.
The present disclosure also provides the combination of a stroke therapeutic agent and a GBE1 inhibitor for use in the treatment or prevention of a stroke in a subject that is GBE1 reference. The stroke therapeutic agents can be any of the stroke therapeutic agents described herein. The GBE1 inhibitors can be any of the GBE1 inhibitors described herein.
The present disclosure also provides the combination of a stroke therapeutic agent and a GBE1 inhibitor for use in the preparation of a medicament for treating or preventing a stroke in a subject that is GBE1 reference. The stroke therapeutic agents can be any of the stroke therapeutic agents described herein. The GBE1 inhibitors can be any of the GBE1 inhibitors described herein.
In some embodiments, the GBE1 inhibitor and the stroke therapeutic agent are disposed within a pharmaceutical composition. In some embodiments, the GBE1 inhibitor is disposed within a first pharmaceutical composition and the stroke therapeutic agent is disposed within a second pharmaceutical composition. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously. In some embodiments, the first pharmaceutical composition is administered before the second pharmaceutical composition. In some embodiments, the first pharmaceutical composition is administered after the second pharmaceutical composition.
The present disclosure also provides GBE1 inhibitors for use in the treatment or prevention of a stroke in a subject that is GBE1 reference and/or in a subject that has increased total white matter hyperintensity. The GBE1 inhibitors can be any of the GBE1 inhibitors described herein.
The present disclosure also provides GBE1 inhibitors for use in the preparation of a medicament for treating or preventing a stroke in a subject that is GBE1 reference and/or in a subject that has increased total white matter hyperintensity. The GBE1 inhibitors can be any of the GBE1 inhibitors described herein.
All patent documents, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present disclosure can be used in combination with any other feature, step, element, embodiment, or aspect unless specifically indicated otherwise. Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.
The following examples are provided to describe the embodiments in greater detail. They are intended to illustrate, not to limit, the claimed embodiments. The following examples provide those of ordinary skill in the art with a disclosure and description of how the compounds, compositions, articles, devices and/or methods described herein are made and evaluated and are intended to be purely exemplary and are not intended to limit the scope of any claims. Efforts have been made to ensure accuracy with respect to numbers (such as, for example, amounts, temperature, etc.), but some errors and deviations may be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.
An exome wide association analysis was performed to discover genetic variants affecting total white matter hyperintensity volumes on brain MRI and the chance of having a clinical diagnosis of stroke. DNA was extracted from whole blood and/or saliva samples from participants from biobanks and electronic health record cohorts recruited by collaborators of the Regeneron Genetics Center. DNA was sheared and hybridized to an exome capture kit, which removes most DNA that is not protein coding. DNA libraries were prepared for sequencing on a standard Illumina DNA sequencer. Sequence data was mapped to the reference genome and variant sequences were identified for all participants. These variant sequences were utilized in standard genome-wide statistical analyses to identify associations with phenotypes. Phenotypes included total white matter hyperintensity, which was quantified by an automated algorithm that detects areas of T2 FLAIR white matter hyperintense signal on brain MRI scans and made publicly available by the UK Biobank. The other phenotype was diagnosis of stroke, including any stroke or ischemic stroke, as defined by a combination of international classification of diseases (ICD) 10 codes. Table 1 reports the association between an aggregate of rare variants (<1%) predicted to cause loss-of-function (as described above) in the GBE1 transcript or protein. Table 2 reports further associations for other functional categories, including missense mutation or synonymous mutation (no predicted function change). Table 3 reports the association between individual variants in GBE1 and the risk for ischemic stroke and the amount of total white matter hyperintensity.
An ExWAS study was performed and showed a protective effect of GBE1 loss-of-function variants for stroke (see, Table 1).
Abbreviations: minor allele frequency, MAF, reference counts, Ref, heterozygous counts, Het, homozygous counts, Alt.
A GBE1 burden mask study was performed and showed a protective effect of GBE1 loss-of-function variants for stroke (see, Table 2).
The top rare variant from exome sequencing was 3:81648854:A:G (p=1.7E−10; effect=−0.67; MAF=0.0012; carriers=88).
Another study was performed and showed that GBE1 pLoF mask and main protective variant carriers have reduced total white matter hyperintensity (TWMH) (see,
Another study for ischemic stroke vs TWMH in the UKB cohort across variants was performed (see, Table 3).
Another study for GBE1 pLoF mask and top variants with stroke subtyping in cohorts was performed (see,
Another study was performed and showed that GBE1 function was via reduced gene dosage (see,
Table 4 shows a relationship between total white matter hyperintensity and stroke. Genetic correlation analysis using linkage disequilibrium score regression demonstrated total white matter hyperintensity (TWMH) and stroke are genetically correlated. Table 4 describes a genetic correlation between each trait (left column) and TWMH (2nd and 3rd columns). TWMH is strongly correlated to the risk of any cerebrovascular disease and 10 ischemic stroke. The correlation between TWMH and other stroke risk factors is minimal, suggesting TWMH independently contributes to stroke risk.
Various modifications of the described subject matter, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, and the like) cited in the present application is incorporated herein by reference in its entirety and for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 63440935 | Jan 2023 | US |
