TCF7L2-mediated remyelination in the brain

Abstract

The present application relates to a method of treating a subject having a condition mediated by a deficiency of myelin, the method involving selecting a subject having a condition mediated by a deficiency of myelin and expressing a transcription factor 7-like 2 (TCF7L2) protein in the selected subject under conditions effective to treat the condition. Also disclosed is a method of increasing oligodendrocyte production from glial progenitor cells, the method involving providing a population of glial progenitor cells and expressing a TCF7L2 protein in the provided population of glial progenitor cells under conditions effective to increase oligodendrocyte production compared to oligodendrocyte production in the absence of administration. Also disclosed are, inter alia, genetic constructs suitable for carrying out these methods.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent No. 63 / 274,763, filed November 2, 2021, and U.S. Provisional Patent No. 63 / 378,092, filed October 3, 2022, the contents of which are incorporated herein by reference in their entireties.

[0002] Government Interests This invention was made with Government support under NS110776 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] This application relates to TCF7L2-mediated remyelination in the brain. [Background technology]

[0004] The central nervous system (CNS) is generally organized into "gray matter," which contains the cell bodies and dendritic networks of neurons, and "white matter," which consists of axon bundles wrapped by myelin produced by oligodendrocytes. The myelin sheath has a high lipid fat content, giving it a whitish appearance. Myelin plays a key role in neurotransmission. Damage to oligodendrocytes disrupts the integrity of white matter, resulting in white matter degeneration (demyelination) and loss of neurotransmission within the brain and spinal cord. Inherited or acquired myelin-related disorders affect millions of people and impose a heavy burden on affected individuals and their families. The pathological processes underlying many of these disorders remain poorly understood, and few disease-modifying therapies exist.

[0005] For example, Huntington's disease (HD) is a fatal autosomal dominant progressive neurodegenerative disease caused by the expansion of a CAG triple repeat in the huntingtin (Htt) gene. The polyglutamine expansion occurring in the N-terminal region of the Htt protein leads to the formation of mutant Htt aggregates, which are associated with neurodegeneration that initially occurs in the neostriatum but ultimately affects much of the brain (see de la Monte et al., "Morphometric Demonstration Of Atrophic Changes In The Cerebral Cortex, White Matter, And Neostriatum In Huntington's Disease," Journal of Neuropath.and Exper Neurol 47:516-525 (1988)). Traditionally, HD research has focused on the selective vulnerability of striatal and cortical neurons to the disease process. Recently, however, several studies have also pointed to early white matter loss in HD, and this process has been causally linked to disease progression in HD.In particular, the TRACK HD study identified discrete but progressive white matter atrophy in subclinical HD patients well before the onset of any clinical symptoms (Paulsen et al., “Striatal And White Matter Predictors Of Estimated Diagnosis For Huntington Disease,” Brain Res Bull 82:201-207(2010); Phillips et al., “Deep White Matter In Huntington's Disease,” PloS one 9:e109676(2014); Faria et al., “Linking White Matter And Deep Gray Matter Alterations In Premanifest Huntington Disease,” Neuroimage Clin 11:450-460(2016); Phillips et al., “Major Superficial White Matter Abnormalities in Huntington's Disease,” Front Neurosci 10:197(2016); Bourbon-Teles et al., “Myelin (See “Breakdown in Human Huntington's Disease: Multi-Modal Evidence from Diffusion MRI and Quantitative Magnetization Transfer,” Neuroscience 403:79-92 (2019)). The role of forebrain white matter degeneration in this MRI-defined disease progression, and the extent to which these changes are primary or secondary to neurological dysfunction, remains unclear.

[0006] Previously, it was shown that HD patient-derived glial progenitor cells (hGPCs) generated from human embryonic stem cells (hESCs) displayed profound and systematic transcriptional downregulation of myelination genes in vitro (Osipovitch et al., “Human ESC-Derived Chimeric Mouse Models of Huntington's Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation,” Cell Stem Cell 24:107-122 e107(2019)). Myelin synthesis could be rescued by inducible expression of key myelination drivers SOX10 and MYRF, indicating that the myelin biosynthetic machinery is intact in these cells. Rather, it is the upstream regulators of these latter genes that appear to be defective in HD GPCs, resulting in impaired oligodendrocyte differentiation. HD hGPCs also exhibited impairments in oligodendrocyte maturation and myelination when transplanted into hypomyelinated mouse hosts, suggesting a cell-autonomous nature of their differentiation defect.

[0007] Moreover, in certain neurodegenerative disorders characterized by myelin loss, whether developmental or as a failure to maintain or regenerate myelin, the lack of myelination potential stems from a reduction in TCF7L2-dependent transcription. TCF7L2 is a transcription factor that functions as a signal effector of the Wnt pathway, but it may also be driven through pathways independent of canonical Wnt signaling, and Wnt-dependent transcription can act through intermediates other than TCF7L2. Indeed, it is unclear whether myelination defects occur in adults and in vivo and whether they stem from downregulation of myelinogenic transcription factors such as TCF7L2.

[0008] There is a need for therapies to treat disorders and conditions mediated or characterized by a deficiency in myelin. The present disclosure is directed to overcoming these and other deficiencies in the art. Summary of the Invention

[0009] A first aspect of the present application relates to a method of treating a subject having a condition mediated by a deficiency of myelin.

[0010] In one embodiment, the method includes introducing transcription factor 7-like 2 (TCF7L2) into a subject in need of such treatment and expressing the transcription factor 7-like 2 (TCF7L2) protein in one or more cells of the selected subject. The method may be performed by administering to the subject a genetic construct or expression vector encoding the TCF7L2 protein. Examples of the one or more cells include glial progenitor cells, oligodendrocyte progenitor cells, glial cells, or oligodendrocytes.

[0011] In another embodiment, the method includes administering to a subject in need thereof a host cell comprising a genetic construct or expression vector encoding a TCF7L2 protein. Exemplary host cells include glial progenitor cells, oligodendrocyte progenitor cells, glial cells, or oligodendrocytes.

[0012] Another aspect of the present application relates to a method for increasing oligodendrocyte production from glial progenitor cells, the method comprising expressing TCF7L2 protein in a population of glial progenitor cells and maintaining the population of glial progenitor cells under conditions that allow their development and differentiation. The method can be carried out by administering to the population of glial progenitor cells a genetic construct or expression vector that encodes the TCF7L2 protein.

[0013] The above gene construct may include (i) a nucleic acid molecule encoding TCF7L2 protein, and (ii) a promoter and / or enhancer. The nucleic acid molecule is operably linked to the promoter and / or enhancer and is under the regulatory control of the promoter and / or enhancer. The promoter and / or enhancer may be for a gene selectively or specifically expressed by glial precursor cells. The gene selectively or specifically expressed by glial precursor cells may be one selected from the group consisting of PDGFRA, ZNF488, GPR17, OLIG2, CSPG4, and SOX10.

[0014] The gene construct may be administered in an expression vector, such as a viral vector, a plasmid vector, or a bacterial vector. The viral vector may be one selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and a vaccinia vector.

[0015] In the above method, the genetic construct can be administered in association with a glial progenitor cell-targeting fusogen or glial progenitor cell-selective surface-binding moiety. For example, the genetic construct can be in a particle that includes a progenitor cell-targeting fusogen or glial progenitor cell-selective surface-binding moiety. The particle can be one selected from the group consisting of a virus, a virus-like particle, and a lipid particle. The glial progenitor cell-targeting fusogen or glial progenitor cell-selective surface-binding moiety can be directed to CD140a, NG2 / CSPG4, A2B5 ganglioside, O4 sulfatide, or CD133.

[0016] In the above method, the condition mediated by a deficiency of myelin in the subject may be one selected from the group consisting of childhood leukodystrophies, lysosomal storage diseases, congenital dysmyelination, cerebral palsy, inflammatory demyelination, post-infectious and post-vaccination leukoencephalitis, radiation or chemotherapy induced demyelination, and vascular demyelination. In one example, the subject has a condition involving defects in myelination or remyelination. Examples of conditions include multiple sclerosis, neuromyelitis optica, transverse myelitis, optic neuritis, subcortical stroke, diabetic leukoencephalopathy, hypertensive leukoencephalopathy, age-related white matter disease, white matter dementia, Binswanger's disease, spinal cord injury, radiation or chemotherapy induced demyelination, post-infectious and post-vaccination leukoencephalitis, periventricular leukomalacia, and cerebral palsy. In one embodiment, the condition is a neurodegenerative disease such as Huntington's disease. In another embodiment, the condition is a neuropsychiatric disease such as schizophrenia. In some embodiments, the condition is characterized by downregulation of one or more genes selected from the group consisting of Myrf, Bcas1, Plp1, Mbp, and Mobp. For each of the above methods, the administering can be performed using any suitable means, including intracerebral delivery, intrathecal delivery, intranasal delivery, or via direct injection into the ventricles. The subject can be a mammal, such as a human.

[0017] Another aspect of the present application relates to a genetic construct. The genetic construct comprises a nucleic acid molecule encoding the TCF7L2 protein and a promoter and / or enhancer of a gene selectively or specifically expressed by glial progenitor cells. The nucleic acid molecule is operably linked to and under the regulatory control of the promoter and / or enhancer. The gene selectively or specifically expressed by glial progenitor cells may be selected from the group consisting of PDGFRA, ZNF488, GPR17, OLIG2, CSPG4, and SOX10. The genetic construct may be associated with a glial progenitor cell-targeted fusogen or a glial progenitor cell-selective surface-binding moiety. The glial progenitor cell-targeted fusogen or a glial progenitor cell-selective surface-binding moiety may be directed to CD140a, NG2 / CSPG4, A2B5 ganglioside, O4 sulfatide, or CD133.

[0018] Also provided is an expression vector comprising the above gene construct.The expression vector can be a viral vector, a plasmid vector, or a bacterial vector.Examples of viral vectors include lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and vaccinia vectors.

[0019] Also within the scope of the present disclosure are host cells comprising the genetic constructs or expression vectors described above, or the progeny of the host cells. In some embodiments, the host cells are stem cells or progenitor cells. Examples of stem cells include, but are not limited to, embryonic stem cells, ES-like stem cells, fetal stem cells, adult stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells, and the like. In exemplary embodiments, the cells are mammalian cells. The host cells can be used to express TCF7L2 protein or can be used as therapeutic cells / medicines to treat disorders or conditions described herein.

[0020] This application relates to the discovery that in certain neurodegenerative disorders characterized by myelin loss, whether developmental or as a failure to maintain or regenerate myelin, the lack of myelination stems from a decrease in TCF7L2-dependent transcription. TCF7L2 is a transcription factor that functions as a signal effector of the Wnt pathway, but may also be driven through pathways independent of canonical Wnt signaling, and Wnt-dependent transcription can act through intermediates other than TCF7L2.

[0021] As a result of the above aspects of the present application, it is possible to treat myelin disorders characterized by a differentiation block of resident GPCs, such as Huntington's disease or any neurodegenerative disease with myelin loss, as well as progressive multiple sclerosis and cerebral palsy and selective neuropsychiatric disorders. Since transcription factor networks and predicted signaling pathways defective in GPCs from subjects with childhood-onset schizophrenia, also characterized by significant myelin loss and developmental hypomyelination, similarly show suppression of TCF7L2-dependent transcription, GPC-targeting TCF7L2 may also be expected to rescue hypomyelination in that disorder.

[0022] The data presented here show that HD is associated with a progressive age-related loss of forebrain myelin as well as impaired remyelination after adult demyelination compared to WT mice, as shown in two different transgenic mouse disease models. Collectively, these findings suggest a loss of homeostatic white matter maintenance. This was accentuated by a severe dysregulation of oligodendrocyte lineage-related gene expression predicted to be driven by upstream TCF7L2 signaling. Importantly, forced glial overexpression of TCF7L2 restored functional transcription of key myelination and lipid biosynthesis genes and proved sufficient for in vivo restoration of myelin structure and abundance. Thus, this study provides a novel and effective strategy for the therapeutic rescue of glial dysfunction and thus both synaptic and white matter pathology in HD. [Brief description of the drawings]

[0023] This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0024] [Fig. 1A-1G]Figure 1 shows myelination in presymptomatic and diseased R6 / 2 mice. Figure 1A shows representative transmission electron microscopy micrographs of the corpus callosum of 6- and 12-week WT and R6 / 2 mice. Figures 1B-1C show linear regression analysis of the g-ratio. Figures 1D-1E show the frequency distribution of myelinated axons as a function of their diameter. Figures 1F-1G show the relative number of myelinated axons in the corpus callosum of treated mice as a function of genotype. ns: not significant, unpaired Welch t-test. Scale bar = 0.5 μm. [Figures 2A-2N] Figure 2 shows remyelination after cuprizone treatment in R6 / 2 mice. Figure 2A shows the timeline and experimental design. Figure 2B shows representative transmission electron microscopy micrographs of the corpus callosum during and after cuprizone treatment. Figures 2C-2F show linear regression analysis of the g-ratio. Figures 2G-2J show the frequency distribution of myelinated axons as a function of diameter. Figures 2K-2N show the relative number of myelinated axons in the corpus callosum of treated mice as a function of genotype. ns: not significant, **p<0.01, ***p<0.001 by unpaired Welch t-test. Scale bar = 0.5 μm. [Figure 3A-3E] Figure 3 shows that RNA-Seq of HD GPCs reveals a stall in oligodendrocyte differentiation. Striatal mouse GPCs were isolated from PDGFRa-EGFP bred to either Q175 or R6 / 2 HD mice or their respective littermate controls and transcriptionally analyzed by RNA-Seq. Figure 3A shows principal component analysis of bulk GPC RNA-Seq samples, illustrating the separation of HD glial populations in a time-dependent manner. Figure 3B shows an upset plot of the intersection of all four comparisons, showing gene conservation and independence across models and time points. Figure 3C shows that the average expression of variance-stabilized counts within each HD model was plotted against controls at the same time points to illustrate the increasing severity of DE between early and late time points. Figure 3D shows a heatmap view of the DE genes of interest, indicating which comparison genes are significantly DE. Figure 3E shows the significance of gene ontology enrichment of curated terms within each comparison. [Figure 4A-4C]Network analysis of HD GPC identification of TCF7L2 and myelination enriched modules. Modules were determined via WGCNA and filtered for differentially expressed genes as HD vs. WT GPCs and then analyzed with IPA for GO enrichment. Figure 4A shows that three modules were found to be significantly enriched in terms performing myelination and oligodendrocyte differentiation, most notably black. Figure 4B shows that the black module was also determined to be highly enriched for differentially expressed genes. Figure 4C shows that the gene ontology network representation of the black module generated five neighborhoods with representation of TCF7L2 signaling and myelin related terms. [Diagram 5] Expression analysis of selected genes after overexpression of TCF7L2 in vivo. 10-week-old R6 / 2 mice injected with LV-TetOn-TCF7L2 were divided into two groups: doxycycline-treated control or untreated control. Both control and doxycycline-treated mice were assayed for selected myelination and metabolism genes and expression values ​​were normalized to 18S RNA expression values. Doxycycline-treated LV-TetOn-TCF7L2-injected animals showed increased levels of myelination and lipid biosynthesis genes, while more upstream components of TCF7L2 signaling were minimally affected. *p<0.05, **p<0.01 by Welch's t-test. [Figures 6A-6E]Figure 6 shows that overexpression of TCF7L2 rescued remyelination deficit in R6 / 2 mice. Figure 6A shows the timeline of treatment with cuprizone (CZN) and lentiviral overexpression of TCF7L2 (LV-TCF7L2). Figure 6B shows transmission electron microscopy micrographs of the corpus callosum of cuprizone-treated WT, R6 / 2 and LV-TCF7L2-treated R6 / 2. Figure 6C shows linear regression analysis of g-ratio. Figure 6D shows the percentage of myelinated axons as a function of their diameter and treatment. Figure 6E shows the percentage of remyelinated axons as a function of genotype. ns: not significant, *p<0.05, ****p<0.001 by two-way ANOVA (Figure 6D) or one-way ANOVA with Tukey's multiple test (Figure 6E). Scale bar=0.5 μm. [Figure 7A-7D] Large diameter axonal fibers are preferentially remyelinated in wild type mice compared to R6 / 2. The distribution of remyelinated axonal fibers is shown as a function of their diameter in mice treated with cuprizone for 6-12 weeks. Cohorts of mice were sampled and mice were placed on the cuprizone diet at week 10 (4 weeks after diet initiation, FIG. 7A), at the end of 6 weeks of treatment (FIG. 7B), after 2 weeks of recovery (FIG. 7C), or after 4 weeks of recovery (FIG. 7D). *p>0.05, *p>0.01 by two-way ANOVA. [Figure 8] FIG. 1 shows delayed remyelination of R6 / 2 HD white matter following cuprizone demyelination. Linear regression analysis of the number of remyelinated callosal axons as a function of recovery time comparing cuprizone-treated R6 / 2 and WT mice. *p<0.0001, (F(2,116)=16.15). [Figure 9A-9D]Figure 9 shows isolation of oligodendrocyte precursor cells by FACS from R6 / 2 and zQ175 mice. Striata from 4-8 R6 / 2-PDGFRa-EGFP or zQ175-PDGFRa-EGFP mice were dissociated and GPCs were sorted by FACS based on EGFP expression. Cytometry plots of EGFP+ at early and late time points for R6 / 2 (Figure 9A) and zQ175 (Figure 9B). Relative distribution and total cells collected after cell sorting of GPCs for R6 / 2 (Figure 9C) and zQ175 (Figure 9D) samples. ****p>0.0001; ns: not significant; two-way ANOVA, Tukey's test for multiple comparisons. [Figure 10A-10G] Figure 10 shows that EGFP expression is decreased in the striatum of old mice. EGFP and NG2 immunostaining of the striatum of 12-week-old (Figure 10A) and 1-year-old (Figure 10B) zQ175 mice are color-coded, respectively. Figure 10C shows that EGFP+ cell density was significantly decreased in the striatum of 1-year-old mice (p<0.0001 for age effect, 2-way ANOVA). Stereoscopic counting of Olig2+ cell density in R6 / 2 (Figure 10D) or zQ175 (Figure 10E) mice. Striatal volume of R6 / 2 (Figure 10F) or zQ175 (Figure 10G) mice. *p>0.05, ***p>0.0001, ns: not significant, 2-way ANOVA with Tukey multiple test). Scale (Figures 10A-10B): 50 μm. [Figure 11] Figure 1. Expression of glial precursor and other CNS markers by striatal PDGFRA-EGFP sorted GPCs. FACS-isolated PDGFRA-EGFP striatal cells showed high expression of GPC markers with low expression of off-target CNS cell type markers in all mice analyzed. Counts were batch corrected and normalized via variance stabilizing transformation. [Figure 12]TCF7L2 signaling-related genes that are significantly dysregulated in R6 / 2 and zQ175 mice are shown. TCF7L2 signaling-related genes curated by Ingenuity pathway analysis (IPA) that were found to be significant (FDR<0.01) in both R6 / 2 and zQ175 GPCs are displayed here as gene Z-scores of variance-stabilized transformation counts corrected. The time points and models at which they were differentially expressed are shown in the heatmap on the right. [Figure 13] TCF7L2 isoforms in human GPCs are shown. To identify optimal Tcf7l2 splice isoforms for localized overexpression, as assessed in in vitro generated hGPCs, we refer to previously unpublished isoform data obtained in a more extensive gene expression analysis of human HD and control ESC-derived GPCs (Osipovitch et al., “Human ESC-Derived Chimeric Mouse Models of Huntington's Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation,” Cell Stem Cell 24:107-122 e107 (2019) (incorporated herein by reference in its entirety). Ensemble transcript identifiers are provided for all expressed isoforms. [Figure 14A-14D] Figure 14 shows the transgene expression pattern of LV-TCF7L2-EGFP in the corpus callosum of cuprizone-treated mice. Figure 14A shows that the doxycycline-regulated lentiviral vector enabled DOX expression of TCF7L2. Six-week-old mice were fed cuprizone for 4 weeks before receiving intracallosal injection of LV-TCF7L2-EGFP. Mice were still on the cuprizone diet and were sacrificed one week later. The EGFP reporter was expressed in oligodendrocyte lineage cells (Figure 14B), GPCs (Figure 14C) and astrocytes (Figure 14D) in cuprizone-induced lesions. Scale: 25 μm. [Fig. 15A-15F]Figure 15 shows that HD corpus callosum white matter displays a dysregulated proteome. Corpuses of 12-week-old R6 / 2 and 1-year-old zQ175 (and respective WT littermate controls) mice were dissected and processed for mass spectrometry. Figure 15A shows principal component analysis of HD and WT corpus callosum white matter. Figure 15B shows a Venn diagram of shared peptides. Figures 15C and 15D show volcano plots of differentially expressed peptides (p<0.05). Figure 15E shows heatmaps of selected myelin proteins HD mice and respective WT controls. Figure 15F shows ingenuity pathway analysis of differentially expressed peptides. Significant terms of interest were curated for display (p<0.01). [Figures 16A-16C] Figure 16 shows that striatal GPCs exhibited HD-dependent dysregulation of protein expression consistent with that of gene expression. 12-week-old HD and WT littermate control mice were killed, their striatum dissected and dissociated, and their GPCs were isolated by A2B5-based FACS for mass spectrometry. Figure 16A shows principal component analysis of R6 / 2 and WT striatal GPCs. Figure 16B shows volcano plots of differentially expressed peptides in R6 / 2 vs. WT striatal GPCs (p<0.05). Figure 16C shows scatter plots of log2-fold changes of significantly differentially expressed genes and proteins in both bulk RNA-Seq and mass spectrometry. [Figure 17A-17B] Figure 17 shows that protein levels and mobility of TCF7L2 in HD mouse corpus callosum were not different from WT. Figure 17A shows Western blots of 12 week old corpus callosum for TCF7L2 and B-actin. Figure 17B shows a bar graph of beta-actin normalized TCF7L2 in the corpus callosum of R6 / 2 and littermate WT controls. ns: not significant by unpaired Welch t-test. [Figure 18]Schematic diagram of myelination defects in Huntington's disease mouse models and their rescue by Tcf7l2 expression. Top: R6 / 2 Huntington's disease mice exhibit developmental defects in corpus callosum myelination and slower and more incomplete remyelination after demyelination. Center: Myelination defects of Huntington's disease glial progenitor cells reflect the lack of Tcf7l2 expression in model mice (R6 / 2 or zQ175). Bottom: Tcf7l2 expression rescues myelin gene expression and myelination in R6 / 2 mice in vivo. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] A first aspect of the present application relates to a method of treating a subject having a condition mediated by a deficiency of myelin, the method comprising selecting a subject having a condition mediated by a deficiency of myelin and expressing a transcription factor 7-like 2 (TCF7L2) protein in the selected subject under conditions effective to treat the condition.

[0026] Another aspect of the present application relates to a method for increasing oligodendrocyte production from glial progenitor cells, the method involving providing a population of glial progenitor cells and expressing TCF7L2 protein in the provided population of glial progenitor cells under conditions effective to increase oligodendrocyte production compared to oligodendrocyte production in the absence of administration.

[0027] Another aspect of the present application relates to a genetic construct comprising a nucleic acid molecule encoding a TCF7L2 protein and a promoter and / or enhancer of a gene selectively or specifically expressed by glial progenitor cells, the nucleic acid molecule being under the regulatory control of the promoter and / or enhancer.

[0028] The present application describes a genetic construct comprising a nucleic acid molecule encoding a TCF7L2 protein and a promoter and / or enhancer of a gene selectively expressed by glial progenitor cells, wherein the nucleic acid molecule is under the regulatory control of the promoter and / or enhancer.

[0029] In one embodiment, expressing the transcription factor 7-like 2 (TCF7L2) protein is performed by administering a genetic construct. In some embodiments, the genetic construct comprises a nucleic acid molecule encoding the TCF7L2 protein and a promoter and / or enhancer of a gene selectively or specifically expressed by glial progenitor cells, the nucleic acid molecule being under the regulatory control of the promoter and / or enhancer.

[0030] As used herein, the term "TCF7L2" refers to transcription factor 7-like 2 protein.

[0031] In another embodiment, the gene selectively or specifically expressed by glial progenitor cells is selected from the group consisting of CNP1, GPR17, PDGFRA, ZNF488, OLIG2, CSPG4, and SOX10. In another embodiment, the gene selectivity or gene selectivity specifically expressed by glial cells is selected from the group consisting of CNP1, GPR17, PDGFRA, ZNF488, OLIG2, CSPG4, and SOX10. Listed below are exemplary promoters. Promoter region of CNP1 gene human promoter 2 (P2) (SEQ ID NO: 41) GPR17 (human promoter) (SEQ ID NO: 42) PDGFRA (Genbank: X80389) (SEQ ID NO: 43): TIFF2024542015000002.tif152170CSPG4 (GenBank: DQ241507.1) (SEQ ID NO: 44): TIFF2024542015000003.tif117170

[0032] As used herein, "treating" or "treatment" refers to any indication of successful amelioration of an injury, pathology, or condition, including any objective or subjective parameter, such as alleviation, remission, reduction of symptoms, or making the injury, pathology, or condition more tolerable to the patient, slowing the rate of degeneration or decline, making the end point of degeneration less debilitating, or improving the physical or mental well-being of the subject. The treatment or amelioration of symptoms may be based on objective or subjective parameters, including the results of a physical exam, neurological exam, and / or psychiatric evaluation. "Treating" includes administration of glial progenitor cells to prevent, delay, alleviate, or arrest or inhibit the onset of symptoms or conditions associated with a disease, condition, or disorder.

[0033] "Therapeutic effect" refers to the reduction, elimination, or prevention of a disease, a symptom of a disease, or a side effect of a disease, condition, or disorder in a subject. Treatment can be prophylactic (to prevent or delay the onset or worsening of the disease, condition, or disorder, or to prevent the manifestation of clinical or subclinical symptoms thereof), or therapeutic suppression or alleviation of symptoms after the manifestation of the disease, condition, or disorder.

[0034] As used herein, "subject" refers to any organism that may be treated with the present application. Thus, the term "subject" may include, but is not limited to, any non-human mammal, primate, or human. In one embodiment, the subject is a mammal. In another embodiment, the subject is a mammal, such as a mouse, rat, other rodent, rabbit, dog, cat, pig, sheep, horse, primate, or human. In a further embodiment, the subject is a human.

[0035] 1. Myelin deficiency-mediated conditions and myelin-related disorders One aspect of the present disclosure relates to compositions and methods for treating conditions or myelin-related disorders mediated by myelin deficiency. Such conditions or disorders include any disease or condition associated with demyelination, insufficient myelination and remyelination, or hypomyelination in a subject. Such disorders can be genetic, acquired, or both. Demyelination in the CNS can occur in response to genetic mutations (leukodystrophies), autoimmune diseases (e.g., multiple sclerosis), or trauma (e.g., traumatic brain injury, spinal cord injury, or ischemic stroke). Perturbations in myelin function can play an important role in neurological and psychiatric disorders such as autism spectrum disorder (ASD), Alzheimer's disease, Huntington's disease, multiple system atrophy, Parkinson's disease, fragile X syndrome, schizophrenia, and various leukodystrophies.

[0036] Leukodystrophies are a group of rare, mostly inherited neurological disorders that result from abnormal production, processing, or development of myelin and are the result of gene defects (mutations). Some forms are present at birth, while others may not produce symptoms until the child is a toddler. Some primarily affect adults. Leukodystrophies include Canavan disease, Pelizaeus-Merzbacher disease, hypomyelination with atrophy of the basal ganglia and cerebellum, Krabbe disease (globoid cell leukodystrophy), X-linked adrenoleukodystrophy, metachromatic leukodystrophy, Pelizaeus-Merzbacher-like disease (or hypomyelinating leukodystrophy-2), Niemann-Pick disease type C (NPC), autosomal dominant leukodystrophy with autonomic dysfunction (ADLD), 4H leukodystrophy (Pol III-associated leukodystrophy), Zellweger spectrum disorder (ZSD), childhood ataxia with central nervous system hypomyelination or cache (also called wasting white matter disease or VWMD), Cerebrotendinous These include peripheral demyelinating neuropathy (SOX10-related), central dysmyelinating leukodystrophy (CTX), Alexander disease (AXD), SOX10-related peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome, Hirschsprung disease (PCWH), adult polyglucosan body disease (APBD), hereditary diffuse leukoencephalopathy with axonal spheroid formation (HDLS), Aicardi-Goutiéres syndrome (AGS), and adult Refsum disease.

[0037] Subjects suitable for treatment according to the methods described herein include any human subject having a condition mediated by a deficiency of myelin.

[0038] In another embodiment, the condition mediated by a deficiency in myelin is selected from the group consisting of childhood leukodystrophies, lysosomal storage diseases, congenital dysmyelination, cerebral palsy, inflammatory demyelination, post-infectious and post-vaccination leukoencephalitis, radiation or chemotherapy induced demyelination, and vascular demyelination.

[0039] In a further embodiment, the condition mediated by a deficiency of myelin requires myelination.

[0040] In another embodiment, the condition mediated by myelin deficiency requires remyelination. In some embodiments, the condition requiring remyelination is selected from the group consisting of multiple sclerosis, neuromyelitis optica, transverse osteomyelitis, optic neuritis, subcortical stroke, diabetic leukoencephalopathy, hypertensive leukoencephalopathy, age-related white matter disease, white matter dementia, Binswanger's disease, spinal cord injury, radiation or chemotherapy induced demyelination, post-infectious and post-vaccination leukoencephalitis, periventricular leukomalacia, and cerebral palsy.

[0041] In further embodiments, the condition mediated by a deficiency of myelin is a neurodegenerative disease, hi some embodiments, the neurodegenerative disease is Huntington's disease.

[0042] Huntington's disease is an autosomal dominant neurodegenerative disorder characterized by a relentlessly progressive movement disorder accompanied by devastating psychiatric and cognitive deterioration. Huntington's disease is associated with consistent and severe neostriatal atrophy associated with a marked loss of GABAergic medium spiny neurons, the major output neurons of the striatum. Huntington's disease is characterized by an abnormally long CAG repeat expansion in the first exon of the huntingtin gene. The encoded polyglutamine expansion of the mutant huntingtin protein disrupts its normal function and protein-protein interactions, ultimately resulting in widespread neuropathology that is most rapidly evident in the neostriatum.

[0043] Other neurodegenerative diseases treatable in accordance with the present application include frontotemporal dementia, Alzheimer's disease, Parkinson's disease, multiple system atrophy, and amyotrophic lateral sclerosis.

[0044] In one embodiment, the condition mediated by a deficiency of myelin is a neuropsychiatric disorder, hi some embodiments, the neuropsychiatric disorder is schizophrenia.

[0045] Schizophrenia is a severe mental illness that affects a person's thoughts, emotions, and behavior. Symptoms of schizophrenia generally fall into three categories: 1) psychotic symptoms, which include changes in perception, 2) negative symptoms, which include loss of motivation, apathy, and lack of pleasure, and 3) cognitive symptoms, which include problems with attention, concentration, and memory.

[0046] Other neuropsychiatric disorders that can be treated according to the present application include autism spectrum disorders and bipolar disorders.

[0047] In another embodiment, the gene construct is administered in an expression vector. Suitable expression vectors include viral vectors, plasmid vectors, or bacterial vectors. In another embodiment, the expression vector is a viral vector selected from the group consisting of lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and vaccinia vectors.

[0048] In another embodiment, the genetic construct is administered in association with a glial progenitor cell-targeted fusogen or glial progenitor cell-selective surface binding moiety. The glial progenitor cell-targeted fusogen or glial progenitor cell-selective surface binding moiety may be directed to CD140a, NG2 / CSPG4, A2B5 ganglioside, O4 sulfatide, or CD133.

[0049] As used herein, the term "C140a" also refers to platelet-derived growth factor receptor alpha or PDGFRa or PDGFRα.

[0050] As used herein, the term "NG2 / CSPG4" refers to neuron-glial antigen 2 or chondroitin sulfate proteoglycan 4.

[0051] As used herein, the term "CD133" also refers to prominin-1.

[0052] The glial progenitor cells of the administered preparation can optionally be genetically modified to express a protein of interest other than TCF712. For example, the glial progenitor cells can be modified to express a therapeutic biological molecule, an exogenous targeting moiety, an exogenous marker (e.g., for imaging purposes), etc. The glial progenitor cells of the preparation can optionally be modified to overexpress an endogenous biological molecule, a targeting moiety, and / or a marker.

[0053] As used herein, "glial progenitor cells" refers to cells that have the potential to differentiate into cells of the glial lineage, such as oligodendrocytes and astrocytes. Glial progenitor cells may be astrocyte-biased. Glial progenitor cells may be oligodendrocyte-biased. As used herein, the term "glial cells" refers to a non-neuronal cell population that provides support and nutrients, maintains homeostasis, forms or promotes myelination, and participates in signal transduction in the nervous system. As used herein, "glial cells" encompass fully differentiated cells of the glial lineage, e.g., oligodendrocytes or astrocytes, as well as glial progenitor cells, each of which may be referred to as astroglial cells. In some embodiments, glial progenitor cells are also known as oligodendrocyte precursor cells or NG2 cells.

[0054] The glial progenitor cells of the administered preparation may be astrocyte-biased glial progenitor cells, oligodendrocyte-biased glial progenitor cells, unbiased glial progenitor cells, or a combination thereof. The glial progenitor cells of the administered preparation express one or more markers of a glial cell lineage. For example, in one embodiment, the glial progenitor cells of the administered preparation may express A2B5+. In another embodiment, the glial progenitor cells of the administered preparation are positive for the PDGFαR marker. The PDGFαR marker is optionally a PDGFαR ectodomain, such as CD140a. PDGFαR and CD140a are markers for oligodendrocyte-biased glial progenitor cells. In another embodiment, the glial progenitor cells of the administered preparation are CD44+. CD44 is a marker for astrocyte-biased glial progenitor cells. In another embodiment, the glial progenitor cells of the administered preparation are positive for the CD9 marker. The CD9 marker is optionally a CD9 ectodomain. In one embodiment, the glial progenitor cells of the preparation are A2B5+, CD140a+, and / or CD44+. The above glial progenitor cell surface markers can be used to identify, separate, and / or enrich the preparation for glial progenitor cells prior to administration.

[0055] The glial precursor cell preparation administered is optionally negative for PSA-NCAM marker and / or other neural lineage markers, and / or negative for one or more inflammatory cell markers, for example, negative for CD11 marker, negative for CD32 marker, and / or negative for CD36 marker (a marker for microglia). Optionally, the glial precursor cell preparation is negative for any combination or subset of these additional markers. Thus, for example, the glial precursor cell preparation is negative for any one, two, three, or four of these additional markers.

[0056] Human glial progenitor cells administered in accordance with the present application may be derived from any suitable source of glial cells, such as, but not limited to, human induced pluripotent stem cells (iPSCs), embryonic stem cells, fetal tissue, and / or astrocytes, as described in more detail below.

[0057] iPSCs are pluripotent cells derived from non-pluripotent cells, such as somatic cells.For example, but not limited to, iPSCs can be derived from tissues, peripheral blood, umbilical cord blood, and bone marrow (see, e.g., Cai et al., “Generation of Human Induced Pluripotent Stem Cells from Umbilical Cord Matrix and Amniotic Membrane Mesenchymal Cells,” J. Biol. Chem. 285(15):11227-11234(2010); Giorgetti et al., “Generation of Induced Pluripotent Stem Cells from Human Cord Blood Cells with only Two Factors: Oct4 and Sox2,” Nat. Protocol. 5(4):811-820(2010); Streckfuss-Bomeke et al., “Comparative Study of Human-Induced Pluripotent Stem Cells Derived from Bone Marrow Cells, Hair Keratinocytes, and Skin Fibroblasts,” Eur. Heart. J. doi:10.1093 / eurheartj / ehs203 (July 12, 2012); Hu et al., “Efficient Generation of Transgene-Free Induced Pluripotent Stem Cells from Normal and Neoplastic Bone Marrow and Cord Blood Mononuclear Cells,” Blood doi:10.1182 / blood-2010-07-298331 (Feb. 4, 2011); Sommer et al., “Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood using the STEMCCA Lentiviral Vector,” J. Vis. Exp. 68:e4327 doi:10.3791 / 4327 (2012) (incorporated herein by reference in their entireties).Somatic cells are reprogrammed into an embryonic stem cell-like state using genetic engineering. Exemplary somatic cells suitable for forming iPSCs include fibroblasts (see, e.g., Streckfuss-Bomeke et al., "Comparative Study of Human-Induced Pluripotent Stem Cells Derived from Bone Marrow Cells, Hair Keratinocytes, and Skin Fibroblasts," Eur. Heart J. doi:10.1093 / eurheartj / ehs203 (2012) (incorporated herein by reference in its entirety)), such as dermal fibroblasts obtained by skin samples or biopsies, synoviocytes from synovial tissue, keratinocytes, mature B cells, mature T cells, pancreatic beta cells, melanocytes, hepatocytes, foreskin cells, cheek cells, or lung fibroblasts.

[0058] Methods for producing induced pluripotent stem cells are known in the art and typically involve expressing a combination of reprogramming factors in somatic cells. Suitable reprogramming factors that promote and induce the generation of iPSCs include one or more of Oct4, Klf4, Sox2, c-Myc, Nanog, C / EBPα, Esrrb, Lin28, and Nr5a2. In certain embodiments, at least two reprogramming factors are expressed in somatic cells to successfully reprogram somatic cells. In other embodiments, at least three reprogramming factors are expressed in somatic cells to successfully reprogram somatic cells.

[0059] iPSCs can be derived by methods known in the art, including the use of integrating viral vectors (e.g., lentiviral vectors, inducible lentiviral vectors, and retroviral vectors), excisable vectors (e.g., transposon and floxed lentiviral vectors), and non-integrating vectors (e.g., adenoviral and plasmid vectors) to deliver genes that promote cell reprogramming (see, e.g., Takahashi and Yamanaka, “Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors,” Cell 126:663-676 (2006); Okita.et al., “Generation of Germline-Competent Induced Pluripotent Stem Cells,” Nature 448:313-317 (2007); Nakagawa et al., “Generation of Induced Pluripotent Stem Cells without Myc from Mouse and Human Fibroblasts,” Nat.Biotechnol.26:101-106 (2008), Takahashi et al., Cell 131:1-12 (2007), Meissner et al., “Direct Reprogramming of Genetically Unmodified Fibroblasts into Pluripotent Stem Cells,”Nat.Biotech.25:1177-1181(2007), Yu et al.,“Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells,”Science 318:1917-1920(2007), Park et al., “Reprogramming of Human Somatic Cells to Pluripotency with Defined Factors,” Nature 451:141-146 (2008), and U.S. Patent Application Publication No. 2008 / 0233610, which is incorporated by reference in its entirety. Other methods for generating iPS cells are described in WO2007 / 069666, WO2009 / 006930, WO2009 / 006997, WO2009 / 007852, WO2008 / 118820, U.S. Patent Application Publication No. 2011 / 0200568 to Ikeda et al., U.S. Patent Application Publication No. 2010 / 0156778 to Egusa et al., U.S. Patent Application Publication No. 2012 / 0276070 to Musick, and U.S. Patent Application Publication No. 2012 / 0276636 to Nakagawa, Shi et al., Cell Stem Cell 3(5):568-574 (2008), Kim et al., Nature 454:646-650 (2008), Kim ... 136(3):411-419(2009), Huangfu et al., Nat. Biotechnol. 26:1269-1275(2008), Zhao et al., Cell Stem Cell 3:475-479(2008), Feng et al., Nat. Cell Biol. 11:197-203(2009), and Hanna et al., Cell 133(2):250-264(2008), which are incorporated by reference in their entireties.

[0060] The above-mentioned iPSC generation method can be modified to include small molecules that enhance reprogramming efficiency or even replace reprogramming factors. These small molecules include, but are not limited to, epigenetic modulators such as the DNA methyltransferase inhibitor 5'-azacytidine, the histone deacetylase inhibitor VPA, and the G9a histone methyltransferase inhibitor BIX-01294 in combination with the L-type calcium channel agonist BayK8644. Other small molecule reprogramming factors include those that target signaling pathways such as TGF-β inhibitors and kinase inhibitors (e.g., kenpaullone) (see review by Sommer and Mostoslavsky, "Experimental Approaches for the Generation of Induced Pluripotent Stem Cells," Stem Cell Res. Ther. 1:26 doi:10.1186 / scrt26 (August 10, 2010), which is incorporated herein by reference in its entirety).

[0061] Methods for obtaining highly enriched preparations of glial progenitor cells from iPSCs suitable for the methods described herein are disclosed in WO 2014 / 124087 to Goldman and Wang, and in Wang et al., “Human iPSC-Derived Oligodendrocyte Progenitors Can Myelinate and Rescue a Mouse Model of Congenital Hypomyelination,” Cell Stem Cell 12(2):252-264 (2013), which are incorporated by reference in their entireties.

[0062] In another embodiment, the human glial progenitor cells are derived from embryonic stem cells. Human embryonic stem cells provide a virtually unlimited source of cloned / genetically modified cells potentially useful for tissue replacement therapy. Methods for obtaining highly enriched preparations of glial progenitor cells from embryonic cells suitable for use in the methods of the present disclosure are described in Wang et al., "Human iPSC-derived Oligodendrocyte Progenitor Cells Can Myelinate and Rescue a Mouse Model of Congenital Hypomyelination," Cell Stem Cell 12:252-264 (2013) (incorporated herein by reference in its entirety).

[0063] In another embodiment, the human glial progenitor cells are derived from human fetal tissue. Glial progenitor cells can be extracted directly from fetal brain tissue containing a mixed cell population by using the promoter-specific isolation technique described in US Patent Application Publication Nos. 2004 / 0029269 and 2003 / 0223972 to Goldman, which are incorporated herein by reference in their entireties. The method involves selecting a promoter that functions specifically in glial progenitor cells and introducing a nucleic acid encoding a marker protein under the control of the promoter into the mixed population cells. The mixed cell population can be allowed to express the marker protein, and the cells expressing the marker protein are separated from the cell population, and the separated cells are glial progenitor cells. Human glial progenitor cells can be isolated from the ventricular or subventricular regions of the brain, or from subcortical white matter.

[0064] Glial-specific promoters that can be used to isolate glial precursor cells from mixed cell populations include the CNP promoter (Scherer et al., Neuron 12:1363-75 (1994) (incorporated herein by reference in its entirety)), the NCAM promoter (Holst et al., J. Biol. Chem. 269:22245-52 (1994) (incorporated herein by reference in its entirety)), the myelin basic protein promoter (Wrabetz et al., J. Neurosci. Res. 36:455-71 (1993) (incorporated herein by reference in its entirety)), the JC virus minimal core promoter (Krebs et al., J. Virol. 69:2434-42 (1995) (incorporated herein by reference in its entirety)), the myelin-associated glycoprotein promoter (Laszkiewicz et al., "Structural Characterization of Myelin-associated Glycoprotein Gene Core Promoter," J. Neurosci. Res. 50(6):928-36 (1997) (incorporated herein by reference in its entirety), or protein lipid promoters (Cook et al., "Regulation of Rodent Myelin Proteolipid Protein Gene Expression," Neurosci. Lett. 137(1):56-60 (1992); Wight et al., "Regulation of Murine Myelin Proteolipid Protein Gene Expression," J. Neurosci. Res. 50(6):917-27 (1997); and Cambi et al., Neurochem. Res. 19:1055-60 (1994) (incorporated herein by reference in its entirety). See also U.S. Pat. No. 6,245,564 to Goldman et al., (incorporated herein by reference in its entirety).

[0065] The glial progenitor cell population derived from fetal tissue can be enriched by first removing neurons or neural progenitor cells from a mixed cell population.When separating neuronal progenitor cells from a mixed cell population, they can be removed based on the surface expression of NCAM, PSA-NCAM, or any other surface moiety specific for neurons or neural progenitor cells.Neurons or neural progenitor cells can be separated from the mixed cell population using promoter-based separation techniques. Neuron or neural precursor specific promoters that can be used to separate neural cells from mixed cell populations include the Tα1 tubulin promoter (Gloster et al., J. Neurosci. 14:7319-30 (1994) (incorporated herein by reference in its entirety)), the Hu promoter (Park et al., "Analysis of Upstream Elements in the HuC Promoter Leads to the Establishment of Transgenic Zebrafish with Fluorescent Neurons," Dev. Biol. 227(2):279-93 (2000) (incorporated herein by reference in its entirety)), the ELAV promoter (Yao et al., "Neural Specificity of ELAV Expression: Defining a Drosophila Promoter for Directing Expression to the Nervous System," J. Neurochem. 63(1):41-51 (1994) (incorporated herein by reference in its entirety)), the MAP-1B promoter (Liu et al., Gene 171:307-08 (1996), which are incorporated herein by reference in their entireties, or the GAP-43 promoter. Techniques for introducing nucleic acid molecules of a construct into a plurality of cells and then selecting the cells are described in U.S. Patent No. 6,245,564 to Goldman et al. and U.S. Patent Application Publication No. 2004 / 0029269 to Goldman et al., which are incorporated herein by reference in their entireties.

[0066] As an alternative to using promoter-based cell sorting to recover glial progenitor cells from a mixed cell population, immunoisolation procedures can be utilized. In positive immunoisolation techniques, the desired cells (i.e., glial progenitor cells) are isolated based on protein surface markers that are naturally present on progenitor cells. For example, the surface marker A2B5 is an early expression marker for glial progenitor cells (Nunes et al., "Identification and Isolation of Multipotential Neural Progenitor Cells from the Adult Human White Matter," Soc.Neurosci.Abstr. (2001) (incorporated herein by reference in its entirety)). Antibodies specific for A2B5 can be used to separate glial progenitor cells from a mixed population of cell types. Similarly, the surface marker CD44 identifies astrocyte-biased glial progenitor cells (Liu et al., "CD44 Expression Identifies Astrocyte-Restricted Precursor Cells," Dev. Biol. 276:31-46 (2004), incorporated herein by reference in its entirety). Using CD44-conjugated microbead technology, astrocyte-biased glial progenitor cells can be separated from a mixed population of cell types. Oligodendrocyte-biased glial progenitor cells can be separated from a mixed population of cell types based on expression of PDGFαR, PDGFαR ectodomain CD140a, or CD9. Cells expressing markers of non-glial cell types (e.g., neurons, inflammatory cells, etc.) can be removed from preparations of glial cells to further enrich the preparation for the desired glial cell type using immunoisolation techniques. For example, the glial progenitor cell population is preferably negative for the PSA-NCAM marker and / or other markers of neural lineage cells, negative for one or more inflammatory cell markers, e.g., negative for the CD11 marker, negative for the CD32 marker, and / or negative for the CD36 marker, which is a marker for microglia.Exemplary microbead technologies include MACS® Microbeads, MACS® Columns, and MACS® Separators. Additional immunoisolation examples include Wang et al., “Prospective Identification, Direct Isolation, and Expression Profiling of a Telomerase Expressing Subpopulation of Human Neural Stem Cells, Using Sox2 Enhancer-Directed FACS,” J. Neurosci.30:14635-14648 (2010), Keyoung et al., “High-Yield Selection and Extraction of Two Cell Stem Cell 2:553-565 (2008), which is incorporated herein by reference in its entirety.

[0067] The selected preparation of human glial progenitor cells administered according to the methods described herein comprises at least about 80% glial progenitor cells, including, for example, about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% glial progenitor cells. The selected preparation of glial progenitor cells can be relatively free (e.g., containing less than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of other cell types, such as neurons or cells of neuronal lineage, fibrous astrocytes and cells of fibrous astrocyte lineage, and pluripotent stem cells (such as ES cells). Optionally, the exemplary cell population is a substantially pure population of glial progenitor cells.

[0068] 2. Transcription factor 7-like 2 protein Transcription factor 7-like 2, also known as TCF7L2 or TCF4, is a protein that functions as a transcription factor and is encoded by the TCF7L2 gene in humans. The human TCF7L2 gene is located on chromosome 10q25.2-q25.3 and contains 17 exons. As a member of the TCF family, TCF7L2 forms a bipartite transcription factor and can affect several biological pathways, including the Wnt signaling pathway.

[0069] The full-length human TCF7L2 protein contains a β-catenin binding domain, a Groucho binding sequence, an HMG box-DNA binding domain (HGM-DBD), a cysteine ​​clamp (C-clamp) at the N-terminus, and a C-terminus. See, for example, Li et al., Front Cardiovasc Med. 2021 Sep 9;8:701279. doi:10.3389 / fcvm.2021.701279. eCollection 2021. With the help of the HGM-DBD, TCF7L2 can recognize a specific DNA subsequence (5'-xCTTTGATx-3') in the double helix dimple and induce transcription factor activity. The C-clamp contains an alternative DNA binding domain (5'-xTGCCGCx-3') without transcriptional regulatory activity, but the C-clamp is thought to assist the binding of the HGM-DBD to a specific DNA sequence. TCF7L2 exerts dual transcriptional regulatory effects on target genes influenced by the transcriptional coactivator β-catenin or the transcriptional corepressor transducer-like enhancer split (TLE) / Groucho. Upon Wnt signaling stimulation, increased amounts of b-catenin are imported into the nucleus and subsequently assembled into the β-catenin / TCF7L2 complex. Furthermore, β-catenin serves as a scaffold to assist the binding of the β-catenin / TCF7L2 complex to the promoters of target genes, thus enhancing promoter activity. In the absence of Wnt / β-catenin signaling, the corepressive TLE preferentially occupies TCF7L2 by its glutamine-rich (Q) domain and recruits histone methyltransferases or histone deacetylases to silence downstream genes. Taken together, although TCF7L2 contains two DNA-binding domains (HGM-DBD and C-clamp), only the HGM-DBD can activate transcription. Furthermore, TCF7L2 is dually regulated by the transcriptional coactivator β-catenin or the transcriptional corepressor TLE / Groucho. Li et al.,Front Cardiovasc Med.2021 Sep 9;8:701279.doi:10.3389 / fcvm.2021.701279. eCollection 2021.

[0070] Human or mouse TCF7L2 has multiple splice variants or isoforms that show different expression patterns or play different roles during development (Helgason et al., Nature Genetics 39:218-225(2007)). Below are some of the human TCF7L2 variants / isoforms. All of these human splice variants / isoforms can be used in the expression cassettes, genetic constructs, vectors, compositions, or methods disclosed herein. Below are listed some exemplary Tcf7l2 human isoforms, related nucleic acid sequences, and related amino acid sequences. [Table 1] >NM_001146274.2 Homo sapiens transcription factor 7-like 2 (TCF7L2), transcript variant 1, (SEQ ID NO: 45) GTCAATAATCTCCGCTCCCAGACTACTCCGTTCCTCCGGATTTCGATCCCCCTTTTTCTATCTGTCAATC AGCGCCGCCTTTGAACTGAAAAGCTCTCAGTCTAACTTCAACTCACTCAAATCCGAGCGGCACGAGCACC TCCTGTATCTTCGGCTTCCCCCCCTTTGCTCTTTATATCTGACTTCTTGTTGTTGTTGGTGTTTTTTT TTTTTTTACCCCCCTTTTTTATTTATTATTTTTTTGCACATTGATCGGATCCTTGGGAACGAGAGAAAAAA AGAAACCCAAACTCACGCGTGCAGAAGATCTCCCCCCCCTTCCCCTCCCCTCCTCCCTCTTTTCCCCTCC CCAGGAGAAAAAGACCCCCAAGCAGAAAAAAGTTCACCTTGGACTCGTCTTTTTCTTGCAATATTTTTTG GGGGGGCAAAACTTTTTGGGGGTGATTTTTTTTGGCTTTTCTTCCTCCTTCATTTTCTTCCAAAATTGC TGCTGGTGGGTGAAAAAAAAATGCGCAGCTGAACGGCGGTGGAGGGGATCTAGGCGCCAACGACGA ACTGATTTCCTTCCAAAGACGAGGGCGAACAGGAGAGAAGAGCTCCGAAAACTCCTCGGCAGAGAGGGAT TTAGCTGATGTCAAATCGTCTCTAGTCAATGAATCAGAAACGAATCAAAAACAGCTCCTCCGATTCCGAGG CGGAAAGACGGCCTCCGCCTCGCTCCGTCCGAAAGTTTCCGAGAAATCCCGGAAAGTTTGGAAGAAGCGGC CAAGAGGCAAGATGGAGGGCTCTTTAAGGGGCCACCGTATCCCGGCTACCCCTTCATCATGATCCCCGAC CTGACGAGCCCCTACCTCCCCAACGGATCGCTCTCGCCCACCGCCCGAACCCTCCATTTTCAGTCCGGCA GCACACATTACTCTGCGTACAAAACGATTGAACACCAGATTGCAGTTCAGTATCTCCAGATGAAATGGCC ACTGCTTGATGTCCAGGCAGGAGCCTCCAGAGTAGACAGCCCTCAAGGATGCCCGGTCCCCATCACCG GCACACATTGTCTCTAACAAAGTGCCAGTGGTGCAGCACCCTCACCATGTCCACCCCCTCACGCCTCTTA TCACGTACAGCAATGAACACTTCACGCCGGGAAACCCACCTCCACACTTACCAGCCGACGTAGACCCCAA AAACGGAATCCCACGGCCTCCGCACCCTCCAGATATATCCCCGATTACCCACTATCGCCTGGCACCGTA GGACAAATCCCCCATCCGCTAGGATGGTTAGTACCACAGCAAGGTCAACCAGTGTACCCAATCACGACAG GAGGATTCAGACACCCCTACCCCACAGCTCTGACCGTCAATGCTTCCATGTCCAGGTTCCCTCCCCATAT GGTCCCACCACATCATACGCTACACACGACGGGCATTCCGCATCCGGCCATAGTCACACCAACAGTCAAA CAGGAATCGTCCCAGAGTGATGTCGGCTCACTCCATAGTTCAAAGCATCAGGACTCCAAAAAGGAAGAAG AAAAGAAGAAGCCCCACATAAAGAAACCTCTTAATGCATTCATGTTGTATATGAAGGAAATGAGAGCAAA GGTCGTAGCTGAGTGCACGTTGAAAGAAAGCGCGGCCATCAACCAGATCCTTGGGCGGAGGTGGCATGCA CTGTCCAGAGAAGAGCAAGCGAAATACTACGAGCTGGCCCGGAAGGAGCGACAGCTTCATATGCAACTGT ACCCCGGCTGGTCCGCGCGGGATAACTATGGAAAGAAGAAGAAGAGGAAAAGGGACAAGCAGCCGGGAGA GACCAATGATGCAAATACTCCAAAGAAGTGTCGGGCACTGTTCGGGCTTGACCGACAGACTTTATGGTGC AAACCGTGCAGGAGAAAAAAAAAGTGCGTTCGCTACATACAAGGTGAAGGCAGCTGCCTCAGCCCACCCT CTTCAGATGGAAGCTTACTAGATTCGCCTCCCCCCTCCCCGAACCTGCTAGGCTCCCCTCCCCGAGACGC CAAGTCACAGACTGAGCAGACCCAGCCTCTGTCGCTGTCCCTGAAGCCCGACCCCCTGGCCCACCTGTCC ATGATGCCTCCGCCACCCGCCCTCCTGCTCGCTGAGGCCACCCACAAGGCCTCGCCCCTCTGTCCCAACG GGGCCCTGGACCTGCCCCCAGCCGCTTTGCAGCCTGCCGCCCCCTCCTCATCAATTGCACAGCCGTCGAC TTCTTCCTTACATTCCCACAGCTCCCTGGCCGGGACCCAGCCCCAGCGCCTGTCGCTCGTCACCAAGTCT TTAGATAGCTTTAGCGTCGTGAACCCCGCTGCTTTGTTTATGGTTTTGTTTCACTTTTCTTTAATTTGCC CCCCACCCCCACCTTGAAAGGTTTTGTTTTGTACTCTCTTAATTTTGTGCCATGTGGCTACATTAGTTGA TGTTTATCGAGTTCATTGGTCAATATTTGACCCATTCTTATTTCAATTTCTCCTTTTAAAATATGTAGATG AGAGAAGAACCTCATGATTCTACCAAAATTTTTATCAACAGCTGTTTAAAGTCTTTGTAGCGTTTAAAAA ATATATATATATACATAACTGTTATGTAGTTCGGATAGCTTAGTTTTAAAAGACTGATTAAAAAAAAAA AGAAAAAAAAAGCAATTTTGAAGCAGCCCTCCAGAAGGAGTTGGTTCTGTATTATTGTATTAAATACGA GCTTGCGAACCAATCATTTACATCTGGTTTTAAACCGTAAGGGCACCATGAATGCAGTGCCGTTACTT TTTTTTTTTTTCTCTGTGAAACAACTCTTTATTGTGATGTTACTTGTTATTGTTTAAAATGTACAGAAAC AAAGGGTAAAATGGTTAATATACCTTGTTCCATGGTGTTGTTCTTTTGGGGGGAGGGGACGCTACTCA ACACTTAATAGAATCACAACGCTGTTGGGCCAGTAGTAGTATTTATTGCTTTAGAGATTGCTTGTCGTACCTG TATGTCGTCCCTTTTTAAATATGTTTTCCTTTTTCTTGAAACTGTATAAAGTTTTTTTCCCCCTTAGCAT AAGCATCTTATATATAACAACTCATTTGTACAAGGTTTTTAAGTTTATATATAAAATGTGTATATATATT TTGTTTCCCCTTTTTGACTTTTTTTTTTCTGTATGAAACCCAGATGTCACCAAATGGACATTAATAGTT GCATTAAGGATCAGTAGCTAGCATTAACAAAAGTTGCTTTAAAAGCCATTATGTAAAACAAGACTTGAAAATGA GTGAGGGAATTTTAGCGACACTGTCTGAGCAGCAGTGGGAACCATCTTCGTTTTCCCCTTTGAACTCCCAG TGGGATGCCCTACCCTGCGCCCTTAGGACCCGGACTGACCGTGTACAAAACTTTTACGTGCCAAAATTCTC AGTGAATTTAGCTTTCTCCCCTCTTTTTGATGCTGTAATTTTTGTTCATCATGTTTTGCTGTGATGTTACA TAGGTAGATTTGTATGTAGTTTTAATGTCACCTATAACAAAATGGTGTTTGGTAGCAGATTGTCCAGAAAG CATTTTAAATGAAGAGGTATAAACCCTTAAGGGCCAAAATTCTGTATATTAGATTACTCTTAAACGAAAA ACCAGCTGCCGCTTTTATGTACACATATTACATACGAGTAGGCAGCAGACTTTAAAAATAAAAAACCT AGGCATGTTGATGTTGCAAAATGCTGTATAAAGCTGAAACCTGTTCATTCAGTGCCATTGTAGTTGACAT GAAGCGATTGTAAAACTGTCTCCGATTTTTCTCTGGTTTATTAAAATGCTAACTATAACATTTTTTGTGA ATACTTTGAATGTTTCCTAACAGTTGTGATGTTACTGTTCCGTTTTATGCTCTTATTCCAAGTTCATTTT TAATGGTTTGGAAGCCATTTTTGTAATGAATAAATGTTCATGCTGTACAGTATCTGTAGCATGCCGTTCT GGATTAATAAAAGCAACTTAGTATGTGCAGATAAA Protein sequence (SEQ ID NO:46): MPQLNGGGGDDLGANDELISFKDEGEQEEKSSENSSAERDLADVKSSLVNESETNQNSSSDSEAERRPPPRSESFRDKSRESLEEAAKRQDGGLFKGPPYPGYPFIMIPDLTSPYLPNGSLSPTARTLHFQSGSTHYSAYKTIEHQIAVQYLQMKWPLLDVQAGSLQSRQALKDARSPSPAHIVSNKVPVVQHPHHVHPLTPLITYSNEHFTPGNPPPPHLPADVDPKTGIPRPPHPPDISPYYPLSPGTVGQIPHPLGWLVPQQGQPVYPITTGG FRHPYPTALTVNASMSRFPPHMVPPHHTLHTTGIPHPPAIVTPTVKQESSQSDVGSLHSSKHQDSKKEEEKKKPHIKKPLNAFMLYMKEMRAKVVAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSAR DNYGKKKKRKRDKQPGETNDANTPKKCRALFGLDRQTLWCKPCRRKKKCVRYIQGEGSCLSPPSSDGSLLDSPPPSPNLLGSPPRDAKSQTEQTQPLSLSLKPDPLAHLSMMPPPPALLLAEATHKASALCPNGALDLPPAALQPA APSSSIAQPSTSSLHSHSSLAGTQPQPLSLVTKSLE >NM_030756.5 Homo sapiens transcription factor 7-like 2 (TCF7L2), transcript variant 2 (SEQ ID NO: 47); GTCAATAATCTCCGCTCCCAGACTACTCCGTTCCTCCGGATTTCGATCCCCCTTTTTCTATCGTCAATC AGCGCCGCCTTTGAACTGAAAGCTCTCAGTCTAACTTCAACTCACTCAAATCCGAGCGGCACGAGCACC TCCTGTATCTTCGGCTTCCCCCCCCCTTTGCTCTTTATATCTGACTTCTTGTTGTTGTTGGTGTTTTTTT TTTTTTTACCCCCCTTTTTTTATTATTTTTTTGCACATTGATCGGATCCTTGGGAACGAGAGAAAAA AGAAACCCAAACTCACGCGTGCAGAAGATCTCCCCCCCCTTCCCCTCCCCTCCTCCCTCTTTTCCCTCC CCAGGAGAAAAAGACCCCCAAGCAGAAAAAAGTTCACCTTGGACTCGTCTTTTTCTTGCAATATTTTTTG GGGGGGCAAAACTTTTTGGGGGTGATTTTTTTTGGCTTTTCTTCCTCCTTCATTTTCTTCCAAAATTGC TGCTGGTGGGTGAAAAAAAAATGCGCAGCTGAACGGCGGTGGAGGGGATCTAGGCGCCAACGACGA ACTGATTTCCTTCCAAAGACGAGGGCGAACAGGAGAGAAGAGCTCCGAAAACTCCTCGGCAGAGAGGGAT TTAGCTGATGTCAAATCGTCTCTAGTCAATGAATCAGAAACGAATCAAAAACAGCTCCTCCGATTCCGAGG CGGAAAGACGGCCTCCGCCTCGCTCCGTCCGAAAGTTTCCGAGAAATCCCGGAAAGTTTGGAAGAAGCGGC CAAGAGGCAAGATGGAGGGCTCTTTAAGGGGCCACCGTATCCCGGCTACCCCTTCATCATGATCCCCGAC CTGACGAGCCCCTACCTCCCCAACGGATCGCTCTCGCCCACCGCCCGAACCTATCTCCAGATGAAATGGC CACTGCTTGATGTCCAGGCAGGGAGCCTCCAGAGTAGACAAGCCCTCAAGGATGCCCGGTCCCCATCACC GGCACACATTGTCTCTAACAAAGTGCCAGTGGTGCAGCACCCTCACCATGTCCACCCCCTCACGCCTCTT ATCACGTACAGCAATGAACACTTCACGCCGGGAAACCCACCTCCACACTTACCAGCCGACGTAGACCCCA AAACAGGAATCCCACGGCCTCCGCACCCTCCAGATATATCCCCGTATTACCCACTATCGCCTGGCACCGT AGGACAAATCCCCCATCCGCTAGGATGGTTAGTACCACAGCAAGGTCAACCAGTGTACCCAATCACGACA GGAGGATTCAGACACCCCTACCCCACAGCTCTGACCGTCAATGCTTCCATGTCCAGGTTCCCTCCCCATA TGGTCCCACCACATCATACGCTACACACGACGGGCATTCCGCATCCGGCCATAGTCACACCAACAGTCAA ACAGGAATCGTCCCAGAGTGATGTCGGCTCACTCCATAGTTCAAAGCATCAGGACTCCAAAAAGGAAGAA GAAAAGAAGAAGCCCCACATAAAGAAACCTCTTAATGCATTCATGTTGTATATGAAGGAAATGAGAGCAA AGGTCGTAGCTGAGTGCACGTTGAAAGAAAGCGCGGCCATCAACCAGATCCTTGGGCGGAGGTGGCATGC ACTGTCCAGAGAAGAGCAAGCGAAATACTACGAGCTGGCCCGGAAGGAGCGACAGCTTCATATGCAACTG TACCCCGGCTGGTCCGCGCGGGATAACTATGGAAAGAAGAAGAAGAGGAAAAGGGACAAGCAGCCGGGAG AGACCAATGAACACAGCGAATGTTTCCTAAATCCTTGCCTTTCACTTCCTCCGATTACAGACCTGAGCGC TCCTAAGAAATGCCGAGCGCGCTTTGGCCTTGATCAACAGAATAACTGGTGCGGCCCTTGCAGGAGAAAA AAAAAGTGCGTTCGCTACATACAAGGTGAAGGCAGCTGCCTCAGCCCACCCTCTTCAGATGGAAGCTTAC TAGATTCGCCTCCCCCCTCCCCGAACCTGCTAGGCTCCCCTCCCCGAGACGCCAAGTCACAGACTGAGCA GACCCAGCCTCTGTCGCTGTCCCTGAAGCCCGACCCCCTGGCCCACCTGTCCATGATGCCTCCGCCACCC GCCCTCCTGCTCGCTGAGGCCACCCACAAGGCCTCCGCCCTCTGTCCCAACGGGGCCCTGGACCTGCCCC CAGCCGCTTTGCAGCCTGCCGCCCCCTCCTCATCAATTGCACAGCCGTCGACTTCTTCCTTACATTCCCA CAGCTCCCTGGCCGGGACCCAGCCCCAGCCGCTGTCGCTCGTCACCAAGTCTTTAGAATAGCTTTAGCGT CGTGAACCCCGCTGCTTTGTTTATGGTTTTGTTTCACTTTTCTTAATTTGCCCCCCACCCCCACCTTGAA AGGTTTTGTTTTGTACTCTCTTAATTTTGTGCCATGTGGCTACATTAGTTGATGTTTATCGAGTTCATTG GTCAATATTTGACCCATTCTTATTTCAATTTCTCCTTTTAAATATGTAGATGAGAGAAGAACCTCATGAT TCTACCAAAATTTTTATCAACAGCTGTTTAAAGTCTTTGTAGCGTTTAAAAAATATATATATATACATAA CTGTTATGTAGTTCGGATAGCTTAGTTTTAAAAGACTGATTAAAAAACAAAAAGAAAAAAAAAGCAATTT TGAAGCAGCCCTCCAGAAGGAGTTGGTTCTGTATTATTTGTATTAAATACGAGCTTGCGAACCAATCATT TTACATCTGGTTTTTAAACCGTAAGGGCACCATGAATGCAGTGCCGTTACTTTTTTTTTTTTTTTCTGTG TGAAACAACTCTTATTGTGATGTTACTTGTTATTGTTTAAATGTACAGAAACAAAGGGTAAAAATGTGTT AATATACCTTGTTCCATGGTGTTGTTCTTTTGGGGGGAGGGGACGCTACTCAACACTTAATAGAATCACA ACGCTGTTGGGCCAGTAGTATTTATTGCTTTAGAGATTGCTTGTCGTACCTGTATGTCGTCCCTTTTTAA ATATGTTTTCCTTTTTCTTGAAACTGTATAAAGTTTTTTTCCCCCTTAGCATAAGCATCTTATATATAAC AACTCATTTGTACAAGGTTTTTAAGTTTATATATAAAATGTGTATATATATTTTTGTTTCCCCTTTTTGA CTTTTTTTTTCTGTATGAAACCCAGATGTCACCAAATGGACATTAATAGTTGCATTAAGGATCAGTAGC ATTAACAAAAGTGCTTTAAAAGCCATTATGTAAAACAAGACTTGAAAATGAGTGAGGGAATTTTAGCGA CACTGTCTGAGCAGCAGTGGGAACCATCTTCGTTTCCCCTTTGAACTCCCAGTGGGATGCCCTACCCTGC GCCCTTAGGACCCGGACTGACCGTGTACAAAACTTTACGTGCCAAAATTCTCAGTGAATTTAGCTTTCTC CCTCTTTTTGATGCTGTAATTTTTGTTCATCATGTTTTGCTGTGATGTTACATAGGTAGATTTGTATGTA GTTTTAATGTCACCTATAACAAAATGTGTTTGGTAGCAGATTGTCCAGAAAGCATTTTAAATGAAGAGGT ATAAACCCTTAAGGGCCAAAATTCTGTATATTAGATTACTCTTAAACGAAAAACCAGCTGCCGCTTTTAT GTACACATATTACATACGAGTAGGCAGCAGACTTTAAAAATAAAAAAAACCTAGGCATGTTGATGTTGCA AAATGCTGTATAAAGCTGAAACCTGTTCATTCAGTGCCATTGTAGTTGACATGAAGCGATTGTAAAACTG TCTCCGATTTTTCTCTGGTTTATTAAAATGCTAACTATAACATTTTTTGTGAATACTTTGAATGTTTCCT AACAGTTGTGATGTTACTGTTCCGTTTTATGCTCTTATTCCAAGTTCATTTTTAATGGTTTGGAAGCCAT TTTTGTAATGAATAAATGTTCATGCTGTACAGTATCTGTAGCATGCCGTTCTGGATTAATAAAAGCAACT TAGTATGTGCAGATAAA Protein sequence (SEQ ID NO:48): TIFF2024542015000005.tif87170>NM_001146283.2 Homo sapiens transcription factor 7-like 2 (TCF7L2), transcript variant 3 (SEQ ID NO: 49); GTCAATAATCTCCGCTCCCAGACTACTCCGTTCCTCCGGATTTCGATCCCCCTTTTTCTATCTGTCAATC AGCGCCGCCTTTGAACTGAAAGCTCTCAGTCTAACTTCAACTCACTCAAATCCGAGCGGCACGAGCACC TCCTGTATCTTCGGCTTCCCCCCCCCTTTGCTCTTTATATCTGACTTCTTGTTGTTGTTGGTGTTTTTTT TTTTTTTACCCCCCTTTTTTTATTATTTTTTTGCACATTGATCGGATCCTTGGGAACGAGAGAAAAA AGAAACCCAAACTCACGCGTGCAGAAGATCTCCCCCCCCTTCCCCTCCCCTCCTCCCTCTTTTCCCTCC CCAGGAGAAAAAGACCCCCAAGCAGAAAAAAGTTCACCTTGGACTCGTCTTTTTCTTGCAATATTTTTTG GGGGGGCAAAACTTTTTGGGGGTGATTTTTTTTGGCTTTTCTTCCTCCTTCATTTTCTTCCAAAATTGC TGCTGGTGGGTGAAAAAAAAATGCGCAGCTGAACGGCGGTGGAGGGGATCTAGGCGCCAACGACGA ACTGATTTCCTTCCAAAGACGAGGGCGAACAGGAGAGAAGAGCTCCGAAAACTCCTCGGCAGAGAGGGAT TTAGCTGATGTCAAATCGTCTCTAGTCAATGAATCAGAAACGAATCAAAAACAGCTCCTCCGATTCCGAGG CGGAAAGACGGCCTCCGCCTCGCTCCGTCCGAAAGTTTCCGAGAAATCCCGGAAAGTTTGGAAGAAGCGGC CAAGAGGCAAGATGGAGGGCTCTTTAAGGGGCCACCGTATCCCGGCTACCCCTTCATCATGATCCCCGAC CTGACGAGCCCCTACCTCCCCAACGGATCGCTTCCGCCCACCGCCCGAACCTATCTCCAGATGAAATGGC CACTGCTTGATGTCCAGGCAGGGAGCCTCCAGAGTAGACAAGCCCTCAAGGATGCCCGGTCCCCATCACC GGCACACATTGTCAGCCCCCTCCCTTGCTGCACTCAGGGACATGACTGTCAGCACTTCTACCCCCCCTCA GACTTCACTGTCAGCACTCAAGTCTTCAGGGACATGAAAAGGAGCCACTCCTTACAAAAAGTTGGGGAGC CCTGGTGTATTGAGTCTAACAAAGTGCCAGTGGTGCAGCACCCTCACCATGTCCACCCCCTCACGCCTCT TATCACGTACAGCAATGAACACTTCACGCCGGGAAACCCACCTCCACACTTACCAGCCGACGTAGACCCC AAAACAGGAATCCCACGGCCTCCGCACCCTCCAGATATATCCCCGTATTACCCACTATCGCCTGGCACCG TAGGACAAATCCCCCATCCGCTAGGATGGTTAGTACCACAGCAAGGTCAACCAGTGTACCCAATCACGAC AGGAGGATTCAGACACCCCTACCCCACAGCTCTGACCGTCAATGCTTCCATGTCCAGGTTCCCTCCCCAT ATGGTCCCACCACATCATACGCTACACACGACGGGCATTCCGCATCCGGCCATAGTCACACCAACAGTCA AACAGGAATCGTCCCAGAGTGATGTCGGCTCACTCCATAGTTCAAAGCATCAGGACTCCAAAAAGGAAGA AGAAAAGAAGAAGCCCCACATAAAGAAACCTCTTAATGCATTCATGTTGTATATGAAGGAAATGAGAGCA AAGGTCGTAGCTGAGTGCACGTTGAAAGAAAGCGCGGCCATCAACCAGATCCTTGGGCGGAGGTGGCATG CACTGTCCAGAGAAGAGCAAGCGAAATACTACGAGCTGGCCCGGAAGGAGCGACAGCTTCATATGCAACT GTACCCCGGCTGGTCCGCGCGGGATAACTATGGAAAGAAGAAGAAGAGGAAAAGGGACAAGCAGCCGGGA GAGACCAATGGAGAAAAAAAAAGTGCGTTCGCTACATACAAGGTGAAGGCAGCTGCCTCAGCCCACCCTC TTCAGATGGAAGCTTACTAGATTCGCCTCCCCCCTCCCCGAACCTGCTAGGCTCCCCTCCCCGAGACGCC AAGTCACAGACTGAGCAGACCCAGCCTCTGTCGCTGTCCCTGAAGCCCGACCCCCTGGCCCACCTGTCCA TGATGCCTCCGCCACCCGCCCTCCTGCTCGCTGAGGCCACCCACAAGGCCTCCGCCCTCTGTCCCAACGG GGCCCTGGACCTGCCCCCAGCCGCTTTGCAGCCTGCCGCCCCCTCCTCATCAATTGCACAGCCGTCGACT TCTTCCTTACATTCCCACAGCTCCCTGGCCGGGACCCAGCCCCAGCCGCTGTCGCTCGTCACCAAGTCTT TAGAATAGCTTTAGCGTCGTGAACCCCGCTGCTTTGTTTATGGTTTTGTTTCACTTTTCTTAATTTGCCC CCCACCCCCACCTTGAAAGGTTTTGTTTTGTACTCTCTTAATTTTGTGCCATGTGGCTACATTAGTTGAT GTTTATCGAGTTCATTGGTCAATATTTGACCCATTCTTATTTCAATTTCTCCTTTTAAATATGTAGATGA GAGAAGAACCTCATGATTCTACCAAAATTTTTATCAACAGCTGTTTAAAGTCTTTGTAGCGTTTAAAAAA TATATATATATACATAACTGTTATGTAGTTCGGATAGCTTAGTTTTAAAGACTGATTAAAAAACAAAA GAAAAAAAAGCAATTTTGAAGCAGCCCTCCAGAAGGAGTTGGTTCGTATTATTGTATTAAATACGAG CTTGCGAACCAATCATTTACATCTGGTTTTAAACCGTAAGGGCACCATGAATGCAGTGCCGTTACTTT TTTTTTTTTTTCTGTGTGAAACAACTCTTTATTGTGATGTTACTTGTTATTGTTTAAATGTACAGAAACA AAGGGTAAAATGTGTTAATATACCTTGTTCCATGGTGTTGTTCTTTTGGGGGGAGGGGACGCTACTCAA CACTTAATAGAATCACAACGCTGTTGGGCCAGTAGTATTATTGCTTTAGAGATTGCTTGTCGTACCTGT ATGTCGTCCCTTTTTAAATATGTTTTCCTTTTTCTTGAAACTGTATAAAGTTTTTTCCCCCTTAGCATA AGCATCTTATATATAACAACTCATTTGTACAAGGTTTTTAAGTTTATATATAAAATGTGTATATATATTT TTGTTTCCCCTTTTTGACTTTTTTTTTCTGTTATGAAACCCCAGATGTCACCAAATGGACATTAATAGTTG CATTAAGGATCAGTAGCATTAACAAAGTTGCTTTAAAAGCCATTATGTAAAACAAGACTTGAAAATGAG TGAGGGAATTTTAGCGACACTGTCTGAGCAGCAGTGGGAACCATCTTCGTTTCCCCTTTGAACTCCCAGT GGGATGCCCTACCCTGCGCCCTTAGGACCCGGACTGACCGTGTACAAAACTTTACGTGCCAAAAATTCTCA GTGAATTTAGCTTTCTCCCTCTTTTTGATGCTGTAATTTTTGTTCATCATGTTTTGCTGTGATGTTACAT AGGTAGATTTGTATGTAGTTTTAATGTCACCTATAACAAAATGTGTTTGGTAGCAGATTGTCCAGAAAGC ATTTTAAATGAAGAGGTATAAACCCTTAAGGGCCAAAATTCTGTATATTAGATTACTCTTAAACGAAAAA CCAGCTGCCGCTTTTATGTACACATATTACATACGAGTAGGCAGCAGACTTTAAAAATAAAAAACCTA GGCATGTTGATGTTGCAAAATGCTGTATAAAGCTGAAACCTGTTCATTCAGTGCCATTGTAGTTGACATG AAGCGATTGTAAAACTGTCTCCGATTTTTCTCTGGTTTATTAAAATGCTAACTATAACATTTTTTGTGAA TACTTTGAATGTTTCCTAACAGTTGTGATGTTACTGTTCCGTTTTATGCTCTTATTCCAAGTTCATTTTT AATGGTTTGGAAGCCATTTTTGTAATGAATAAATGTTCATGCTGTACAGTATCTGTAGCATGCCGTTCTG GATTAATAAGCAACTTAGTATGTGCAGATAAA Protein sequence (SEQ ID NO:50): MPQLNGGGGDDLGANDELISFKDEGEQEEKSSENSSAERDLADVKSSLVNESETNQNSSSDSEAERRPPPRSESFRDKSRESLEEAAKRQDGGLFKGPPYPGYPFIMIPDLTSPYLPNGSLSPTARTYLQMKWPLLDVQAGSLQSRQALKDARSPSPAHIVSPLPCCTQGHDCQHFYPPSDFTVSTQVFRDMKRSHSLQKVGEPWCIESNKVPVVQHPHHVHPLTPLITYSNEHFT PGNPPPHLPADVDPKTGIPRPHPHPPDISPYYPLSPGTVGQIPHPLGWLVPQQGQPVYPITTGGFRHPYPTALTVNASMSRFPPHMVPPHHTLHTTGIPHPPAIVTPTVKQESSQSDVGSLHSSKHQD SKKEEEKKKPHIKKPLNAFMLYMKEMRAKVVAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKKRKRDKQPGETNGEKKSAFATYKVKAAASAHPLQMEAY >NM_001146284.2 Homo sapiens transcription factor 7-like 2 (TCF7L2), transcript variant 4 (SEQ ID NO: 51); GTCAATAATCTCCGCTCCCAGACTACTCCGTTCCTCCGGATTTCGATCCCCCTTTTTCTATCTGTCAATC AGCGCCGCCTTTGAACTGAAAAGCTCTCAGTCTAACTTCAACTCACTCAAATCCGAGCGGCACGAGCACC TCCTGTATCTTCGGCTTCCCCCCCTTTGCTCTTTATATCTGACTTCTTGTTGTTGTTGGTGTTTTTTT TTTTTTTACCCCCCTTTTTTATTTATTATTTTTTTGCACATTGATCGGATCCTTGGGAACGAGAGAAAAAA AGAAACCCAAACTCACGCGTGCAGAAGATCTCCCCCCCCTTCCCCTCCCCTCCTCCCTCTTTTCCCCTCC CCAGGAGAAAAAGACCCCCAAGCAGAAAAAAGTTCACCTTGGACTCGTCTTTTTCTTGCAATATTTTTTG GGGGGGCAAAACTTTTTGGGGGTGATTTTTTTTGGCTTTTCTTCCTCCTTCATTTTCTTCCAAAATTGC TGCTGGTGGGTGAAAAAAAAATGCGCAGCTGAACGGCGGTGGAGGGGATCTAGGCGCCAACGACGA ACTGATTTCCTTCCAAAGACGAGGGCGAACAGGAGAGAAGAGCTCCGAAAACTCCTCGGCAGAGAGGGAT TTAGCTGATGTCAAATCGTCTCTAGTCAATGAATCAGAAACGAATCAAAAACAGCTCCTCCGATTCCGAGG CGGAAAGACGGCCTCCGCCTCGCTCCGTCCGAAAGTTTCCGAGAAATCCCGGAAAGTTTGGAAGAAGCGGC CAAGAGGCAAGATGGAGGGCTCTTTAAGGGGCCACCGTATCCCGGCTACCCCTTCATCATGATCCCCGAC CTGACGAGCCCCTACCTCCCCAACGGATCGCTTCCGCCCACCGCCCGAACCTATCTCCAGATGAAATGGC CACTGCTTGATGTCCAGGCAGGAGCCTCCAGAGTAGACAGCCCTCAAGGATGCCCGGTCCCCATCACC GGCACACATTGTCTCTAACAAAGTGCCAGTGGTGCAGCACCCTCACCATGTCCACCCCCTCCACGCCTCTT ATCACGTACAGCAATGAACACTTCACGCCGGGAAACCCACCTCCACACTTACCAGCCGACGTAGACCCCA AAACAGGAATCCCACGGCCTCCGCACCCTCCAGATATATCCCGTATTACCCACTATCGCCTGGCACCGTT AGGACAAATCCCCCATCCGCTAGGATGGCAAGGTCAACCAGTGTACCCAATCACGACAGGAGGATTCAGA CACCCCTACCCCACAGCTCTGACCGTCAATGCTTCCATGTCCAGGTTCCCTCCCCATATGGTCCCACCAC ATCATACGCTACACACGACGGGCATTCCGCATCCGGCCATAGTCACACCAACAGTCAAACAGGAATCGTC CCAGAGTGATGTCGGCTCACTCCATAGTTCAAAGCATCAGGACTCCAAAAAGGAAGAAGAAAAGAAGAAG CCCCACATAAAGAAACCTCTTAATGCATTCATGTTGTATATGAAGGAAATGAGAGCAAAGGTCGTAGCTG AGTGCACGTTGAAAGAAAGCGCGGCCATCAACCAGATCCTTGGGCGGAGGTGGCATGCACTGTCCAGAGA AGAGCAAGCGAAATACTACGAGCTGGCCCGGAAGGAGCGACAGCTTCATATGCAACTGTACCCCGGCTGG TCCGCGCGGGATAACTATGGAAAGAAGAAGAAGAGGAAAAGGGACAAGCAGCCGGGAGAGACCAATGAAC ACAGCGAATGTTTCCTAAATCCTTGCCTTTCACTTCCTCCGATTACAGGAGAAAAAAAAAGTGCGTTCGC TACATACAAGGTGAAGGCAGCTGCCTCAGCCCACCCTCTTCAGATGGAAGCTTACTAGATTCGCCTCCCC CCTCCCCGAACCTGCTAGGCTCCCCTCCCCGAGACGCCAAGTCACAGACTGAGCAGACCCAGCCTCTGTC GCTGTCCCTGAAGCCCGACCCCCTGGCCCACCTGTCCATGATGCCTCCGCCACCCGCCCTCCTGCTCGCT GAGGCCACCCACAAGGCCTCCGCCCTCTGTCCCAACGGGGCCCTGGACCTGCCCCCAGCCGCTTTGCAGC CTGCCGCCCCCTCCTCATCAATTGCACAGCCGTCGACTTCTTCCTTACATTCCCACAGCTCCCTGGCCGG GACCCAGCCCCAGCCGCTGTCGCTCGTCACCAAGTCTTTAGAATAGCTTTAGCGTCGTGAACCCCGCTGC TTTGTTTATGGTTTTGTTTCACTTTTCTTAATTTGCCCCCCACCCCCACCTTGAAAGGTTTTGTTTTGTA CTCTCTTAATTTTGTGCCATGTGGCTACATTAGTTGATGTTTATCGAGTTCATTGGTCAATATTTGACCC ATTCTTATTTCAATTTCTCCTTTTAAATATGTAGATGAGAGAAGAACCTCATGATTCTACCAAAATTTTT ATCAACAGCTGTTTAAAGTCTTTGTAGCGTTTAAAAAATATATATATATACATAACTGTTATGTAGTTCG GATAGCTTAGTTTTAAAAGACTGATTAAAAAACAAAAAGAAAAAAAAAGCAATTTTGAAGCAGCCCTCCA GAAGGAGTTGGTTCTGTATTATTTGTATTAAATACGAGCTTGCGAACCAATCATTTTACATCTGGTTTTT AAACCGTAAGGGCACCATGAATGCAGTGCCGTTACTTTTTTTTTTTTTTTCTGTGTGAAACAACTCTTAT TGTGATGTTACTTGTTATTGTTTAAATGTACAGAAACAAAGGGTAAAAATGTGTTAATATACCTTGTTCC ATGGTGTTGTTCTTTTGGGGGGAGGGGACGCTACTCAACACTTAATAGAATCACAACGCTGTTGGGCCAG TAGTATTTATTGCTTTAGAGATTGCTTGTCGTACCTGTATGTCGTCCCTTTTTAAATATGTTTTCCTTTT TCTTGAAACTGTATAAAGTTTTTTTCCCCCTTAGCATAAGCATCTTATATATAACAACTCATTTGTACAA GGTTTTTAAGTTTATATATAAAATGTGTATATATATTTTTGTTTCCCCTTTTTTGACTTTTTTTTCTGT ATGAAACCCAGATGTCACCAAATGGACATTAATAGTTGCATTAAGGATCAGTAGCATTAACAAGTTGC TTTAAAAGCCATTATGTAAAACAAGACTTGAAAATGAGTGAGGGAATTTTAGCGACACTGTCTGAGCAGC AGTGGGAACCATCTTCGTTTCCCCTTTGAACTCCCAGTGGGATGCCCTACCCTGCGCCCTTAGGACCCGG ACTGACCGTGTACAAAACTTTACGTGCCAAAATTCTCAGTGAATTTAGCTTTCTCCCTCTTTTTGATGCT GTAATTTTTGTTCATCATGTTTTGCTGTGATGTTACATAGGTAGATTTGTATGTAGTTTTAATGTCACCT ATAACAAAATGTGTTTGGTAGCAGATTGTCCAGAAAGCATTTTAAATGAAGAGGTATAAACCCTTAAGGG CCAAAATTCTGTATATTAGATTACTCTTAAACGAAACCAGCTGCCGCTTTTATGTACACATATTACAT ACGAGTAGGCAGCAGACTTTAAAAATAAAAAACCTAGGCATGTTGATGTTGCAAAATGCTGTATAAAG CTGAAACCTGTTCATTCAGTGCCATTGTAGTTGACATGAAGCGATTGTAAAACTGTCTCCGATTTTTCTC TGGTTTATTAAAATGCTAACTATAACATTTTTTGTGAATACTTTGAATGTTTCCTAACAGTTGTGATGTT ACTGTTCCGTTTTATGCTCTTATTCCAAGTTCATTTTTAATGGTTTGGAAGCCATTTTTGTAATGAATAA ATGTTCATGCTGTACAGTATCTGTAGCATGCCGTTCTGGATTAATAAAAGCAACTTAGTATGTGCAGATA AA Protein sequence (SEQ ID NO:52): MPQLNGGGGDDLGANDELISFKDEGEQEEKSSENSSAERDLADVKSSLVNESETNQNSSSDSEAERRPPPRSESFRDKSRESLEEAAKRQDGGLFKGPPYPGYPFIMIPDLTSPYLPNGSLSPTARTYLQMKWPLLDVQAGSLQSRQALKDARSPSPAHIVSNKVPVVQHPHHVHPLTPLITYSNEHFTPGNPPHLPADVDPKTGIPRPPHPPDISPY YPLSPGTVGQIPPHPLGWQGQPVYPITTGGFRHPYPTALTVNASMSRFPPHMVPPHHTLHTTGIPHPPAIVTPTVKQESSQSDVGSLHSSKHQDSKKEEEKKKPHIKKPLNAFMLYMKEM RAKVVAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKKRKRDKQPGETNEHSECFLNPCLSLPPITGEKKSAFATYKVKAAASAHPLQMEAY >NM_001146285.2 Homo sapiens transcription factor 7-like 2 (TCF7L2), transcript variant 5 (SEQ ID NO:53); GTCAATAATCTCCGCTCCCAGACTACTCCGTTCCTCCGGATTTCGATCCCCCTTTTTCTATCTGTCAATC AGCGCCGCCTTTGAACTGAAAAGCTCTCAGTCTAACTTCAACTCACTCAAATCCGAGCGGCACGAGCACC TCCTGTATCTTCGGCTTCCCCCCCCCTTTGCTCTTTATATCTGACTTCTTGTTGTTGTTGGTGTTTTTTT TTTTTTTACCCCCCTTTTTTTATTATTTTTTTGCACATTGATCGGATCCTTGGGAACGAGAGAAAAA AGAAACCCAAACTCACGCGTGCAGAAGATCTCCCCCCCCTTCCCCTCCCCTCCTCCCTCTTTTCCCTCC CCAGGAGAAAAAGACCCCCAAGCAGAAAAAAGTTCACCTTGGACTCGTCTTTTTCTTGCAATATTTTTTG GGGGGGCAAAACTTTTTGGGGGTGATTTTTTTTGGCTTTTCTTCCTCCTTCATTTTCTTCCAAAATTGC TGCTGGTGGGTGAAAAAAAAATGCGCAGCTGAACGGCGGTGGAGGGGATCTAGGCGCCAACGACGA ACTGATTTCCTTCCAAAGACGAGGGCGAACAGGAGAGAAGAGCTCCGAAAACTCCTCGGCAGAGAGGGAT TTAGCTGATGTCAAATCGTCTCTAGTCAATGAATCAGAAACGAATCAAAAACAGCTCCTCCGATTCCGAGG CGGAAAGACGGCCTCCGCCTCGCTCCGTCCGAAAGTTTCCGAGAAATCCCGGAAAGTTTGGAAGAAGCGGC CAAGAGGCAAGATGGAGGGCTCTTTAAGGGGCCACCGTATCCCGGCTACCCCTTCATCATGATCCCCGAC CTGACGAGCCCCTACCTCCCCAACGGATCGCTTCCGCCCACCGCCCGAACCTATCTCCAGATGAAATGGC CACTGCTTGATGTCCAGGCAGGAGCCTCCAGAGTAGACAGCCCTCAAGGATGCCCGGTCCCCATCACC GGCACACATTGTCTCTAACAAAGTGCCAGTGGTGCAGCACCCTCACCATGTCCACCCCCTCACGCCTCTT ATCACGTACAGCAATGAACACTTCACGCCGGGAAACCCACCTCCACACTTACCAGCCGACGTAGACCCCA AAACAGGAATCCCACGGCCTCCGCACCCTCCAGATATATCCCCGTATTACCCACTATCGCCTGGCACCGT AGGACAAATCCCCCATCCGCTAGGATGGTTAGTACCACAGCAAGGTCAACCAGTGTACCCAATCACGACA GGAGGATTCAGACACCCCTACCCCACAGCTCTGACCGTCAATGCTTCCATGTCCAGGTTCCCTCCCCATA TGGTCCCACCACATCATACGCTACACACGACGGGCATTCCGCATCCGGCCATAGTCACACCAACAGTCAA ACAGGAATCGTCCCAGAGTGATGTCGGCTCACTCCATAGTTCAAAGCATCAGGACTCCAAAAAGGAAGAA GAAAAGAAGAAGCCCCACATAAAGAAACCTCTTAATGCATTCATGTTGTATATGAAGGAAATGAGAGCAA AGGTCGTAGCTGAGTGCACGTTGAAAGAAAGCGCGGCCATCAACCAGATCCTTGGGCGGAGGTGGCATGC ACTGTCCAGAGAAGAGCAAGCGAAATACTACGAGCTGGCCCGGAAGGAGCGACAGCTTCATATGCAACTG TACCCCGGCTGGTCCGCGCGGGATAACTATGGAAAGAAGAAGAAGAGGAAAAGGGACAAGCAGCCGGGAG AGACCAATGACCTGAGCGCTCCTAAGAAATGCCGAGCGCGCTTTGGCCTTGATCAACAGAATAACTGGTG CGGCCCTTGCAGGAGAAAAAAAAAGTGCGTTCGCTACATACAAGGTGAAGGCAGCTGCCTCAGCCCACCC TCTTCAGATGGAAGCTTACTAGATTCGCCTCCCCCCTCCCCGAACCTGCTAGGCTCCCCTCCCCGAGACG CCAAGTCACAGACTGAGCAGACCCAGCCTCTGTCGCTGTCCCTGAAGCCCGACCCCCTGGCCCACCTGTC CATGATGCCTCCGCCACCCGCCCTCCTGCTCGCTGAGGCCACCCACAAGGCCTCCGCCCTCTGTCCCAAC GGGGCCCTGGACCTGCCCCCAGCCGCTTTGCAGCCTGCCGCCCCCTCCTCATCAATTGCACAGCCGTCGA CGGCCCTTGCAGGAGAAAAAAAAAGTGCGTTCGCTACATACAAGGTGAAGGCAGCTGCCTCAGCCCACCC TTTAGAATAGCTTTAGCGTCGTGAACCCCGCTGCTTTGTTTATGGTTTTGTTTCACTTTTCTTAATTTGC CCCCCACCCCCACCTTGAAAGGTTTTGTTTTGTACTCTCTTAATTTTGTGCCATGTGGCTACATTAGTTG ATGTTTATCGAGTTCATTGGTCAATATTTGACCCATTCTTATTTCAATTTCTCCTTTTAAATATGTAGAT GAGAGAAGAACCTCATGATTCTACCAAAATTTTTATCAACAGCTGTTTAAAGTCTTTGTAGCGTTTAAAA AATATATATATATACATAACTGTTATGTAGTTCGGATAGCTTAGTTTTAAAAGACTGATTAAAAAACAAA AAGAAAAAAAAAGCAATTTTGAAGCAGCCCTCCAGAAGGAGTTGGTTCTGTATTATTTGTATTAAATACG AGCTTCGAACCAATCATTTTACATCTGGTTTTTAAACCGTAAGGGCACCATGAATGCAGTGCCGTTACT TTTTTTTTTTTTTTCTGTGTGAAACAACTCTTATTGTGATGTTACTTGTTATTGTTTAAATGTACAGAAA CAAAGGGTAAAAATGTGTTAATATACCTTGTTCCATGGTGTTGTTCTTTTGGGGGGAGGGGACGCTACTC AACACTTAATAGAATCACAACGCTGTTGGGCCAGTAGTAGTATTTATTGCTTTAGAGATTGCTTGTCGTACCT GTATGTCGTCCCTTTTTAAATATGTTTTCCTTTTTCTTGAAACTGTATAAAGTTTTTTCCCCCTTAGCA TAAGCATCTTATATATAACAACTCATTTGTACAAGGTTTTTAAGTTTATATATAAAATGTGTATATATAT TTTTGTTTCCCCTTTTTGACTTTTTTTTTTCTGTATGAAACCCAGATGTCACCAAATGGACATTAATAGT TGCATTAAGGATCAGTAGCATTAACAAAAGTTGCTTTAAAAGCCATTATGTAAAACAAGACTTGAAAATG AGTGAGGGAATTTTAGCGACACTGTCTGAGCAGCAGTGGGAACCATCTTCGTTTCCCCTTTGAACTCCCA GTGGGATGCCCTACCCTGCGCCCTTAGGACCCGGACTGACCGTGTACAAAACTTTTACGTGCCAAAATTCT CAGTGAATTTAGCTTTCTCCCTCTTTTTGATGCTGTAATTTTTGTTCATCATGTTTTGCTGTGATGTTAC ATAGGTAGATTTGTATGTAGTTTTAATGTCACCTATAACAAAATGGTGTTTGGTAGCAGATTGTCCAGAAA GCATTTTAAATGAAGAGGTATAAACCCTTAAGGGCCAAAATTCTGTATATTAGATTACTCTTAAACGAAA AACCAGCTGCCGCTTTTATGTACACATATTACATACGAGTAGGCAGCAGACTTTAAAAATAAAAAAAACC TAGGCATGTTGATGTTGCAAAATGCTGTATAAAGCTGAAACCTGTTCATTCAGTGCCATTGTAGTTGACA TGAAGCGATTGTAAAACTGTCTCCGATTTTTCTCTGGTTTATTAAAATGCTAACTATAACATTTTTTGTG AATACTTTGAATGTTTCCTAACAGTTGTGATGTTACTGTTCCGTTTTATGCTCTTATTCCAAGTTCATTT TTAATGGTTTGGAAGCCATTTTTGTAATGAATAAATGTTCATGCTGTACAGTATCTGTAGCATGCCGTTC TGGATTAATAAGCAACTTAGTATGTGCAGATAAA Protein sequence (SEQ ID NO:54): MPQLNGGGGDDLGANDELISFKDEGEQEEKSSENSSAERDLADVKSSLVNESETNQNSSSDSEAERRPPPRSESFRDKSRESLEEAAKRQDGGLFKGPPYPGYPFIMIPDLTSPYLPNGSLSPTARTYLQMKWPLLDVQAGSLQSRQALKDARSPSPAHIVSNKVPVVQHPHHVHPLTPLITYSNEHFTPGNPPPPHLPADVDPKTGIPRPPHPPDISPYYPLSPGTVGQIPHPLGWLVPQQGQPVYPITTGGFRHPYPTALTVNASMSRFPPHMV PPHHTLHTTGIPHPAIVTPTVKQESSQSDVGSLHSSKHQDSKKEEEKKKPHIKKPLNAFMLYMKEMRAKVVAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKKRKRDKQPGETNDLSA PKKCRARFGLDQQNNWCGPCRRKKKCVRYIQGEGSCLSPPSSDGSLLDSPPPSPNLLGSPPRDAKSQTEQTQPLSLSLKPDPLAHLSMMPPPPALLLAEATHKASALCPNGALDLPPAALQPAAPSSSIAQPSTSSLHSHSSLAGT QPQPLSLVTKSLE >NM_001146286.2 Homo sapiens transcription factor 7-like 2 (TCF7L2), transcript variant 6 (SEQ ID NO: 55); GTCAATAATCTCCGCTCCCAGACTACTCCGTTCCTCCGGATTTCGATCCCCCTTTTTCTATCTGTCAATC AGCGCCGCCTTTGAACTGAAAAGCTCTCAGTCTAACTTCAACTCACTCAAATCCGAGCGGCACGAGCACC TCCTGTATCTTCGGCTTCCCCCCCTTTGCTCTTTATATCTGACTTCTTGTTGTTGTTGGTGTTTTTTT TTTTTTTACCCCCCTTTTTTTATTATTTTTTTGCACATTGATCGGATCCTTGGGAACGAGAGAAAAA AGAAACCCAAACTCACGCGTGCAGAAGATCTCCCCCCCCTTCCCCTCCCCTCCTCCCTCTTTTCCCTCC CCAGGAGAAAAAGACCCCCAAGCAGAAAAAAGTTCACCTTGGACTCGTCTTTTTCTTGCAATATTTTTTG GGGGGGCAAAACTTTTTGGGGGTGATTTTTTTTGGCTTTTCTTCCTCCTTCATTTTCTTCCAAAATTGC TGCTGGTGGGTGAAAAAAAAATGCGCAGCTGAACGGCGGTGGAGGGGATCTAGGCGCCAACGACGA ACTGATTTCCTTCCAAAGACGAGGGCGAACAGGAGAGAAGAGCTCCGAAAACTCCTCGGCAGAGAGGGAT TTAGCTGATGTCAAATCGTCTCTAGTCAATGAATCAGAAACGAATCAAAAACAGCTCCTCCGATTCCGAGG CGGAAAGACGGCCTCCGCCTCGCTCCGTCCGAAAGTTTCCGAGAAATCCCGGAAAGTTTGGAAGAAGCGGC CAAGAGGCAAGATGGAGGGCTCTTTAAGGGGCCACCGTATCCCGGCTACCCCTTCATCATGATCCCCGAC CTGACGAGCCCCTACCTCCCCAACGGATCGCTTCCGCCCACCGCCCGAACCTATCTCCAGATGAAATGGC CACTGCTTGATGTCCAGGCAGGAGCCTCCAGAGTAGACAGCCCTCAAGGATGCCCGGTCCCCATCACC GGCACACATTGTCTCTAACAAAGTGCCAGTGGTGCAGCACCCTCACCATGTCCACCCCCTCCACGCCTCTT ATCACGTACAGCAATGAACACTTCACGCCGGGAAACCCACCTCCACACTTACCAGCCGACGTAGACCCCA AAACAGGAATCCCACGGCCTCCGCACCCTCCAGATATATCCCCGTATTACCCACTATCGCCTGGCACCGT AGGACAAATCCCCCATCCGCTAGGATGGTTAGTACCACAGCAAGGTCAACCAGTGTACCCAATCACGACA GGAGGATTCAGACACCCCTACCCCACAGCTCTGACCGTCAATGCTTCCATGTCCAGGTTCCCTCCCCATA TGGTCCCACCACATCATACGCTACACACGACGGGCATTCCGCATCCGGCCATAGTCACACCAACAGTCAA ACAGGAATCGTCCCAGAGTGATGTCGGCTCACTCCATAGTTCAAAGCATCAGGACTCCAAAAAGGAAGAA GAAAAGAAGAAGCCCCACATAAAGAAACCTCTTAATGCATTCATGTTGTATATGAAGGAAATGAGAGCAA AGGTCGTAGCTGAGTGCACGTTGAAAGAAAGCGCGGCCATCAACCAGATCCTTGGGCGGAGGTGGCATGC ACTGTCCAGAGAAGAGCAAGCGAAATACTACGAGCTGGCCCGGAAGGAGCGACAGCTTCATATGCAACTG TACCCCGGCTGGTCCGCGCGGGATAACTATGGAAAGAAGAAGAAGAGGAAAAGGGACAAGCAGCCGGGAG AGACCAATGGAGAAAAAAAAAGTGCGTTCGCTACATACAAGGTGAAGGCAGCTGCCTCAGCCCACCCTCT TCAGATGGAAGCTTACTAGATTCGCCTCCCCCCTCCCCGAACCTGCTAGGCTCCCCTCCCCGAGACGCCA AGTCACAGACTGAGCAGACCCAGCCTCTGTCGCTGTCCCTGAAGCCCGACCCCCTGGCCCACCTGTCCAT GATGCCTCCGCCACCCGCCCTCCTGCTCGCTGAGGCCACCCACAAGGCCTCCGCCCTCTGTCCCAACGGG GCCCTGGACCTGCCCCCAGCCGCTTTGCAGCCTGCCGCCCCCTCCTCATCAATTGCACAGCCGTCGACTT CTTCCTTACATTCCCACAGCTCCCTGGCCGGGACCCAGCCCCAGCCGCTGTCGCTCGTCACCAAGTCTTT AGAATAGCTTTAGCGTCGTGAACCCCGCTGCTTTGTTTATGGTTTTGTTTCACTTTTCTTAATTTGCCCC CCACCCCCACCTTGAAAGGTTTTGTTTTGTACTCTCTTAATTTTGTGCCATGTGGCTACATTAGTTGATG TTTATCGAGTTCATTGGTCAATATTTGACCCATTCTTATTTCAATTTCTCCTTTTAAATATGTAGATGAG AGAAGAACCTCATGATTCTACCAAAATTTTTATCAACAGCTGTTTAAAGTCTTTGTAGCGTTTAAAAAAT ATATATATATACATAACTGTTATGTAGTTCGGATAGCTTAGTTTTAAAAGACTGATTAAAAAACAAAAAG AAAAAAAAAGCAATTTTGAAGCAGCCCTCCAGAAGGAGTTGGTTCTGTATTATTTGTATTAAATACGAGC TTGCGAACCAATCATTTTACATCTGGTTTTTAAACCGTAAGGGCACCATGAATGCAGTGCCGTTACTTTT TTTTTTTTTTTCTGTGTGAAACAACTCTTATTGTGATGTTACTTGTTATTGTTTAAATGTACAGAAACAA AGGGTAAATTGTGTTAATATACCTTGTTCCATGGTGTTGTTCTTTTGGGGGGAGGGGACGCTACTCAAC ACTTAATAGAATCACAACGCTGTTGGGCCAGTAGTATTTATTGCTTTAGAGATTGCTTGTCGTACCTGTA TGTCGTCCCTTTTTAAATATGTTTTCCTTTTTCTTGAAACTGTATAAAGTTTTTTTCCCCCTTAGCATAA GCATCTTATATATAACAACTCATTTGTACAAGGTTTTTAAGTTTATATATAAAATGTGTATATATATTTT TGTTTCCCCCTTTTTGACTTTTTTTTTTCTGTATGAAACCCAGATGTCACCAAATGGACATTAATAGTTGC ATTAAGGATCAGTAGCATTAACAAAAGTTGCTTTAAAAGCCATTATGTAAAACAAGACTTGAAAATGAGT GAGGGAATTTTAGCGACACTGTCTGAGCAGCAGTGGGAACCATCTTCGTTTCCCCTTTGAACTCCCAGTG GGATGCCCTACCCTTGCGCCCTTAGGACCCGGACTGACCGTGTACAAAACTTTTACGTGCCAAAATTCTCAG TGAATTTAGCTTTCTCCCTCTTTTTGATGCTGTAATTTTTGTTCATCATGTTTTGCTGTGATGTTACATA GGTAGATTTGTATGTAGTTTTAATGTCACCTATAACAAAATGTGTTTGGTAGCAGATTGTCCAGAAAGCA TTTTAAATGAAGAGGTATAAACCCTTAAGGGCCAAAATTCTGTATATTAGATTACTCTTAAACGAAAAAC CAGCTGCCGCTTTTATGTACACATATTACATACGAGTAGGCAGCAGACTTTAAAAATAAAAAACCTAG GCATGTTGATGTTGCAAAATGCTGTATAAAGCTGAAACCTGTTCATTCAGTGCCATTGTAGTTGACATGA AGCGATTGTAAAACTGTCTCCGATTTTTCTCTGGTTTATTAAAATGCTAACTATAACATTTTTTGTGAAT ACTTTGAATGTTTCCTAACAGTTGTGATGTTACTGTTCCGTTTTATGCTCTTATTCCAAGTTCATTTTTA ATGGTTTGGAAGCCATTTTTGTAATGAATAAATGTTCATGCTGTACAGTATCTGTAGCATGCCGTTCTGG ATTAATAAAAGCAACTTAGTATGTGCAGATAAA Protein sequence (SEQ ID NO:56): MPQLNGGGGDDLGANDELISFKDEGEQEEKSSENSSAERDLADVKSSLVNESETNQNSSSDSEAERRPPPRSESFRDKSRESLEEAAKRQDGGLFKGPPYPGYPFIMIPDLTSPYLPNGSLSPTARTILTYLQMKWPLLDVQAGSLQSRQALKDARSPSPAHIVSNKVPVVQHPHHVHPLTPLITYSNEHFTPGNPPHLPADVDPKTGIPRPPHP PDISPYYPLSPGTVGQIPPHPLGWLVPQQGQPVYPITTGGFRHPYPTALTVNASMSRFPPHMVPPHHTLHTTGIPHPPAIVTPTVKQESSQSDVGSLHSSKHQDSKKEEEKKKPHI KKPLNAFMLYMKEMRAKVVAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKKRKRDKQPGETNGEKKSAFATYKVKAAAASAHPLQMEAY >NM_001198525.2 Homo sapiens transcription factor 7-like 2 (TCF7L2), transcript variant 7 (SEQ ID NO:57); GTCAATAATCTCCGCTCCCAGACTACTCCGTTCCTCCGGATTTCGATCCCCCTTTTTCTATCTGTCAATC AGCGCCGCCTTTGAACTGAAAGCTCTCAGTCTAACTTCAACTCACTCAAATCCGAGCGGCACGAGCACC TCCTGTATCTTCGGCTTCCCCCCCCCTTTGCTCTTTATATCTGACTTCTTGTTGTTGTTGGTGTTTTTTT TTTTTTTACCCCCCTTTTTTTATTATTTTTTTGCACATTGATCGGATCCTTGGGAACGAGAGAAAAA AGAAACCCAAACTCACGCGTGCAGAAGATCTCCCCCCCCTTCCCCTCCCCTCCTCCCTCTTTTCCCTCC CCAGGAGAAAAAGACCCCCAAGCAGAAAAAAGTTCACCTTGGACTCGTCTTTTTCTTGCAATATTTTTTG GGGGGGCAAAACTTTTTGGGGGTGATTTTTTTTGGCTTTTCTTCCTCCTTCATTTTCTTCCAAAATTGC TGCTGGTGGGTGAAAAAAAAATGCGCAGCTGAACGGCGGTGGAGGGGATCTAGGCGCCAACGACGA ACTGATTTCCTTCCAAAGACGAGGGCGAACAGGAGAGAAGAGCTCCGAAAACTCCTCGGCAGAGAGGGAT TTAGCTGATGTCAAATCGTCTCTAGTCAATGAATCAGAAACGAATCAAAAACAGCTCCTCCGATTCCGAGG CGGAAAGACGGCCTCCGCCTCGCTCCGTCCGAAAGTTTCCGAGAAATCCCGGAAAGTTTGGAAGAAGCGGC CAAGAGGCAAGATGGAGGGCTCTTTAAGGGGCCACCGTATCCCGGCTACCCCTTCATCATGATCCCCGAC CTGACGAGCCCCTACCTCCCCAACGGATCGCTTCCGCCCACCGCCCGAACCTATCTCCAGATGAAATGGC CACTGCTTGATGTCCAGGCAGGGAGCCTCCAGAGTAGACAAGCCCTCAAGGATGCCCGGTCCCCATCACC GGCACACATTGTCTCTAACAAAGTGCCAGTGGTGCAGCACCCTCACCATGTCCACCCCCTCACGCCTCTT ATCACGTACAGCAATGAACACTTCACGCCGGGAAACCCACCTCCACACTTACCAGCCGACGTAGACCCCA AAACAGGAATCCCACGGCCTCCGCACCCTCCAGATATATCCCCGTATTACCCACTATCGCCTGGCACCGT AGGACAAATCCCCCATCCGCTAGGATGGTTAGTACCACAGCAAGGTCAACCAGTGTACCCAATCACGACA GGAGGATTCAGACACCCCTACCCCACAGCTCTGACCGTCAATGCTTCCATGTCCAGCTTTCTGTCTTCTA GGTTCCCTCCCCATATGGTCCCACCACATCATACGCTACACACGACGGGCATTCCGCATCCGGCCATAGT CACACCAACAGTCAAACAGGAATCGTCCCAGAGTGATGTCGGCTCACTCCATAGTTCAAAGCATCAGGAC TCCAAAAAGGAAGAAGAAAAGAAGAAGCCCCACATAAAGAAACCTCTTAATGCATTCATGTTGTATATGA AGGAAATGAGAGCAAAGGTCGTAGCTGAGTGCACGTTGAAAGAAAGCGCGGCCATCAACCAGATCCTTGG GCGGAGGTGGCATGCACTGTCCAGAGAAGAGCAAGCGAAATACTACGAGCTGGCCCGGAAGGAGCGACAG CTTCATATGCAACTGTACCCCGGCTGGTCCGCGCGGGATAACTATGGAAAGAAGAAGAAGAGGAAAAGGG ACAAGCAGCCGGGAGAGACCAATGAACACAGCGAATGTTTCCTAAATCCTTGCCTTTCACTTCCTCCGAT TACAGACCTGAGCGCTCCTAAGAAATGCCGAGCGCGCTTTGGCCTTGATCAACAGAATAACTGGTGCGGC CCTTGCAGATGCAAATACTCCAAAGAAGTGTCGGGCACTGTTCGGGCTTGACCGACAGACTTTATGGTGC AAACCGTGCAGGAGAAAAAAAAAGTGCGTTCGCTACATACAAGGTGAAGGCAGCTGCCTCAGCCCACCCT CTTCAGATGGAAGCTTACTAGATTCGCCTCCCCCCTCCCCGAACCTGCTAGGCTCCCCTCCCCGAGACGC CAAGTCACAGACTGAGCAGACCCAGCCTCTGTCGCTGTCCCTGAAGCCCGACCCCCTGGCCCACCTGTCC ATGATGCCTCCGCCACCCGCCCTCCTGCTCGCTGAGGCCACCCACAAGGCCTCCGCCCTCTGTCCCAACG GGGCCCTGGACCTGCCCCCAGCCGCTTTGCAGCCTGCCGCCCCCTCCTCATCAATTGCACAGCCGTCGAC TTCTTCCTTACATTCCCACAGCTCCCTGGCCGGGACCCAGCCCCAGCCGCTGTCGCTCGTCACCAAGTCT TTAGAATAGCTTTAGCGTCGTGAACCCCGCTGCTTTGTTTATGGTTTTGTTTCACTTTTCTTAATTTGCC CCCCACCCCCACCTTGAAAGGTTTTGTTTTGTACTCTCTTAATTTTGTGCCATGTGGCTACATTAGTTGA TGTTTATCGAGTTCATTGGTCAATATTTGACCCATTCTTATTTCAATTTCTCCTTTTAAATATGTAGATG AGAGAAGAACCTCATGATTCTACCAAAATTTTTATCAACAGCTGTTTAAAGTCTTTGTAGCGTTTAAAAA ATATATATATATACATAACTGTTATGTAGTTCGGATAGCTTAGTTTTAAAAGACTGATTAAAAAAAAAA AGAAAAAAAAAGCAATTTTGAAGCAGCCCTCCAGAAGGAGTTGGTTCTGTATTATTGTATTAAATACGA GCTTGCGAACCAATCATTTACATCTGGTTTTAAACCGTAAGGGCACCATGAATGCAGTGCCGTTACTT TTTTTTTTTTTCTCTGTGAAACAACTCTTTATTGTGATGTTACTTGTTATTGTTTAAAATGTACAGAAAC AAAGGGTAAAATGGTTAATATACCTTGTTCCATGGTGTTGTTCTTTTGGGGGGAGGGGACGCTACTCA ACACTTAATAGAATCACAACGCTGTTGGGCCAGTAGTATTATTGCTTTAGAGATTGCTTGTCGTACCTG TATGTCGTCCCTTTTTAAATATGTTTCCTTTTCTTGAAACTGTATAAAGTTTTTTCCCCCTTAGCAT AAGCATCTTATATATAACAACTCATTTGTACAAGGTTTTTAAGTTTATATATAAAATGTGTATATATATT TTTGTTTCCCCTTTTTGACTTTTTTTTTCTCTGAATGAAACCCAGATGTCACCAAATGGACATTAATAGTT GCATTAAGGATCAGTAGCATTAACAAAAGTTGCTTTAAAAGCCATTATGTAAAACAAGACTTGAAAATGA GTGAGGGAATTTTAGCGACACTGTCTGAGCAGCAGTGGGAACCATCTTCGTTTCCCCTTTGAACTCCCAG TGGGATGCCCTACCCTGCGCCCTTAGGACCCGGACTGACCGTGTACAAAACTTTTACGTGCCAAAATTCTC AGTGAATTTAGCTTTCTCCCCTCTTTTTGATGCTGTAATTTTTGTTCATCATGTTTTGCTGTGATGTTACA TAGGTAGATTTGTATGTAGTTTTAATGTCACCTATAACAAAATGGTGTTTGGTAGCAGATTGTCCAGAAAG CATTTTAAATGAAGAGGTATAAACCCTTAAGGGCCAAAATTCTGTATATTAGATTACTCTTAAACGAAAA ACCAGCTGCCGCTTTTATGTACACATATTACATACGAGTAGGCAGCAGACTTTAAAAATAAAAAACCT AGGCATGTTGATGTTGCAAAATGCTGTATAAAGCTGAAACCTGTTCATTCAGTGCCATTGTAGTTGACAT GAAGCGATTGTAAAACTGTCTCCGATTTTTCTCTGGTTTATTAAAATGCTAACTATAACATTTTTTGTGA ATACTTTGAATGTTTCCTAACAGTTGTGATGTTACTGTTCCGTTTTATGCTCTTATTCCAAGTTCATTTT TAATGGTTTGGAAGCCATTTTTGTAATGAATAAATGTTCATGCTGTACAGTATCTGTAGCATGCCGTTCT GGATTAATAAGCAACTTAGTATGTGCAGATAAA Protein sequence (SEQ ID NO:58): MPQLNGGGGDDLGANDELISFKDEGEQEEKSSENSSAERDLADVKSSLVNESETNQNSSSDSEAERRPPPRSESFRDKSRESLEEAAKRQDGGLFKGPPYPGYPFIMIPDLTSPYLPNGSLSPTARTILTYLQMKWPLLDVQAGSLQSRQALKDARSPSPAHIVSNKVPVVQHPHHVHPLTPLITYSNEHFTPGNPPHLPADVDPKTGIPRPPHPPDISPYYPLSPGTVGQI PHPLGWLVPQQGQPVYPITTGGFRHPYPTALTVNASMSSFLSSRFPPHMVPPHHTLHTTGIPHPAIVTPTVKQESSQSDVGSLHSSKHQDSKKEEEKKKPHIKKPLNAFMLYMKEMRAKVVAE CTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKKRKRDKQPGETNEHSECFLNPCLSLPPITDLSAPKKCRARFGLDQQNNWCGPCRCKYSKEVSGTVRA >NM_001198526.2 Homo sapiens transcription factor 7-like 2 (TCF7L2), transcript variant 8 (SEQ ID NO:59); GTCAATAATCTCCGCTCCCAGACTACTCCGTTCCTCCGGATTTCGATCCCCCTTTTTCTATCTGTCAATC AGCGCCGCCTTTGAACTGAAAAGCTCTCAGTCTAACTTCAACTCACTCAAATCCGAGCGGCACGAGCACC TCCTGTATCTTCGGCTTCCCCCCCTTTGCTCTTTATATCTGACTTCTTGTTGTTGTTGGTGTTTTTTT TTTTTTTACCCCCCTTTTTTATTTATTATTTTTTTGCACATTGATCGGATCCTTGGGAACGAGAGAAAAAA AGAAACCCAAACTCACGCGTGCAGAAGATCTCCCCCCCCTTCCCCTCCCCTCCTCCCTCTTTTCCCCTCC CCAGGAGAAAAAGACCCCCAAGCAGAAAAAAGTTCACCTTGGACTCGTCTTTTTCTTGCAATATTTTTTG GGGGGGCAAAACTTTTTGGGGGTGATTTTTTTTGGCTTTTCTTCCTCCTTCATTTTCTTCCAAAATTGC TGCTGGTGGGTGAAAAAAAAATGCGCAGCTGAACGGCGGTGGAGGGGATCTAGGCGCCAACGACGA ACTGATTTCCTTCCAAAGACGAGGGCGAACAGGAGAGAAGAGCTCCGAAAACTCCTCGGCAGAGAGGGAT TTAGCTGATGTCAAATCGTCTCTAGTCAATGAATCAGAAACGAATCAAAAACAGCTCCTCCGATTCCGAGG CGGAAAGACGGCCTCCGCCTCGCTCCGTCCGAAAGTTTCCGAGAAATCCCGGAAAGTTTGGAAGAAGCGGC CAAGAGGCAAGATGGAGGGCTCTTTAAGGGGCCACCGTATCCCGGCTACCCCTTCATCATGATCCCCGAC CTGACGAGCCCCTACCTCCCCAACGGATCGCTTCCGCCCACCGCCCGAACCTATCTCCAGATGAAATGGC CACTGCTTGATGTCCAGGCAGGAGCCTCCAGAGTAGACAGCCCTCAAGGATGCCCGGTCCCCATCACC GGCACACATTGTCTCTAACAAAGTGCCAGTGGTGCAGCACCCTCACCATGTCCACCCCCTCCACGCCTCTT ATCACGTACAGCAATGAACACTTCACGCCGGGAAACCCACCTCCACACTTACCAGCCGACGTAGACCCCA AAACAGGAATCCCACGGCCTCCGCACCCTCCAGATATATCCCGTATTACCCACTATCGCCTGGCACCGTT AGGACAAATCCCCCATCCGCTAGGATGGTTAGTACCACAGCAAGGTCAACCAGTGTACCCAATCACGACA GGAGGATTCAGACACCCCTACCCCACAGCTCTGACCGTCAATGCTTCCATGTCCAGGTTCCCTCCCCATA TGGTCCCACCACATCATACGCTACACACGACGGGCATTCCGCATCCGGCCATAGTCACACCAACAGTCAA ACAGGAATCGTCCCAGAGTGATGTCGGCTCACTCCATAGTTCAAAGCATCAGGACTCCAAAAAGGAAGAA GAAAAGAAGAAGCCCCACATAAAGAAACCTCTTAATGCATTCATGTTGTATATGAAGGAAATGAGAGCAA AGGTCGTAGCTGAGTGCACGTTGAAAGAAAGCGCGGCCATCAACCAGATCCTTGGGCGGAGGTGGCATGC ACTGTCCAGAGAAGAGCAAGCGAAATACTACGAGCTGGCCCGGAAGGAGCGACAGCTTCATATGCAACTG TACCCCGGCTGGTCCGCGCGGGATAACTATGGAAAGAAGAAGAAGAGGAAAAGGGACAAGCAGCCGGGAG AGACCAATGATGCAAATACTCCAAAGAAGTGTCGGGCACTGTTCGGGCTTGACCGACAGACTTTATGGTG CAAACCGTGCAGGAGAAAAAAAAAGTGCGTTCGCTACATACAAGGTGAAGGCAGCTGCCTCAGCCCACCC TCTTCAGATGGAAGCTTACTAGATTCGCCTCCCCCCTCCCCGAACCTGCTAGGCTCCCCTCCCCGAGACG CCAAGTCACAGACTGAGCAGACCCAGCCTCTGTCGCTGTCCCTGAAGCCCGACCCCCTGGCCCACCTGTC CATGATGCCTCCGCCACCCGCCCTCCTGCTCGCTGAGGCCACCCACAAGGCCTCCGCCCTCTGTCCCAAC GGGGCCCTGGACCTGCCCCCAGCCGCTTTGCAGCCTGCCGCCCCCTCCTCATCAATTGCACAGCCGTCGA CTTCTTCCTTACATTCCCACAGCTCCCTGGCCGGGACCCAGCCCCAGCCGCTGTCGCTCGTCACCAAGTC TTTAGAATAGCTTTAGCGTCGTGAACCCCGCTGCTTTGTTTATGGTTTTGTTTCACTTTTCTTAATTTGC CCCCCACCCCCACCTTGAAAGGTTTTGTTTTGTACTCTCTTAATTTTGTGCCATGTGGCTACATTAGTTG ATGTTTATCGAGTTCATTGGTCAATATTTGACCCATTCTTATTTCAATTTCTCCTTTTAAATATGTAGAT GAGAGAAGAACCTCATGATTCTACCAAAATTTTTATCAACAGCTGTTTAAAGTCTTTGTAGCGTTTAAAA AATATATATATATACATAACTGTTATGTAGTTCGGATAGCTTAGTTTTAAAAGACTGATTAAAAAACAAA AAGAAAAAAAAAGCAATTTTGAAGCAGCCCTCCAGAAGGAGTTGGTTCTGTATTATTTGTATTAAATACG AGCTTGCGAACCAATCATTTTACATCTGGTTTTTAAACCGTAAGGGCACCATGAATGCAGTGCCGTTACT TTTTTTTTTTTTTTCTGTGTGAAACAACTCTTATTGTGATGTTACTTGTTATTGTTTAAATGTACAGAAA CAAAGGGTAAAAATGTGTTAATATACCTTGTTCCATGGTGTTGTTCTTTTGGGGGGAGGGGACGCTACTC AACACTTAATAGAATCACAACGCTGTTGGGCCAGTAGTAGTATTTATTGCTTTAGAGATTGCTTGTCGTACCT GTATGTCGTCCCTTTTTAAATATGTTTTCCTTTTTCTTGAAACTGTATAAAGTTTTTTCCCCCTTAGCA TAAGCATCTTATATATAACAACTCATTTGTACAAGGTTTTTAAGTTTATATATAAAATGTGTATATATAT TTTTGTTTCCCCTTTTTGACTTTTTTTTTTCTGTATGAAACCCAGATGTCACCAAATGGACATTAATAGT TGCATTAAGGATCAGTAGCATTAACAAAAGTTGCTTTAAAAGCCATTATGTAAAACAAGACTTGAAAATG AGTGAGGGAATTTTAGCGACACTGTCTGAGCAGCAGTGGGAACCATCTTCGTTTCCCCTTTGAACTCCCA GTGGGATGCCCTACCCTGCGCCCTTAGGACCCGGACTGACCGTGTACAAAACTTTTACGTGCCAAAATTCT CAGTGAATTTAGCTTTCTCCCTCTTTTTGATGCTGTAATTTTTGTTCATCATGTTTTGCTGTGATGTTAC ATAGGTAGATTTGTATGTAGTTTTAATGTCACCTATAACAAAATGGTGTTTGGTAGCAGATTGTCCAGAAA GCATTTTAAATGAAGAGGTATAAACCCTTAAGGGCCAAAATTCTGTATATTAGATTACTCTTAAACGAAA AACCAGCTGCCGCTTTTATGTACACATATTACATACGAGTAGGCAGCAGACTTTAAAAATAAAAAAAACC TAGGCATGTTGATGTTGCAAAATGCTGTATAAAGCTGAAACCTGTTCATTCAGTGCCATTGTAGTTGACA TGAAGCGATTGTAAAACTGTCTCCGATTTTTCTCTGGTTTATTAAAATGCTAACTATAACATTTTTTGTG AATACTTTGAATGTTTCCTAACAGTTGTGATGTTACTGTTCCGTTTTATGCTCTTATTCCAAGTTCATTT TTAATGGTTTGGAAGCCATTTTTGTAATGAATAAATGTTCATGCTGTACAGTATCTGTAGCATGCCGTTC TGGATTAATAAAAGCAACTTAGTATGTGCAGATAAA Protein sequence (SEQ ID NO:60): MPQLNGGGGDDLGANDELISFKDEGEQEEKSSENSSAERDLADVKSSLVNESETNQNSSSDSEAERRPPPRSESFRDKSRESLEEAAKRQDGGLFKGPPYPGYPFIMIPDLTSPYLPNGSLSPTARTILTYLQMKWPLLDVQAGSLQSRQALKDARSPSPAHIVSNKVPVVQHPHHVHPLTPLITYSNEHFTPGNPPPPHLPADVDPKTGIPRPPHPPDISPYYPLSPGTVGQIPHPLGWLVPQQGQPVYPITTGGFRHPYPTALTVNASMSRFPPHMVPPHHTL HTTGIPPAIVTPTVKQESSQSDVGSLHSSKHQDSKKEEEKKKPHIKKPLNAFMLYMKEMRAKVVAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKKRKRDKQPGETNDANTPKKCRALFGL DRQTLWCKPCRKKKCVRYIQGEGSCLSPPSSDGSLLDSPPPSPNLLGSPPRDAKSQTEQTQPLSLSLKPDPLAHLSMMPPPPALLLAEATHKASALCPNGALDLPPAALQPAAPSSSIAQPSTSSLHSHSSLAGTQPQPLSLVTKSLE >NM_001198527.2 Homo sapiens transcription factor 7-like 2 (TCF7L2), transcript variant 9 (SEQ ID NO: 61); GTCAATAATCTCCGCTCCCAGACTACTCCGTTCCTCCGGATTTCGATCCCCCTTTTTCTATCGTCAATC AGCGCCGCCTTTGAACTGAAAGCTCTCAGTCTAACTTCAACTCACTCAAATCCGAGCGGCACGAGCACC TCCTGTATCTTCGGCTTCCCCCCCCCTTTGCTCTTTATATCTGACTTCTTGTTGTTGTTGGTGTTTTTTT TTTTTTTACCCCCCTTTTTTTATTATTTTTTTGCACATTGATCGGATCCTTGGGAACGAGAGAAAAA AGAAACCCAAACTCACGCGTGCAGAAGATCTCCCCCCCCTTCCCCTCCCCTCCTCCCTCTTTTCCCTCC CCAGGAGAAAAAGACCCCCAAGCAGAAAAAAGTTCACCTTGGACTCGTCTTTTTCTTGCAATATTTTTTG GGGGGGCAAAACTTTTTGGGGGTGATTTTTTTTGGCTTTTCTTCCTCCTTCATTTTCTTCCAAAATTGC TGCTGGTGGGTGAAAAAAAAATGCGCAGCTGAACGGCGGTGGAGGGGATCTAGGCGCCAACGACGA ACTGATTTCCTTCCAAAGACGAGGGCGAACAGGAGAGAAGAGCTCCGAAAACTCCTCGGCAGAGAGGGAT TTAGCTGATGTCAAATCGTCTCTAGTCAATGAATCAGAAACGAATCAAAAACAGCTCCTCCGATTCCGAGG CGGAAAGACGGCCTCCGCCTCGCTCCGTCCGAAAGTTTCCGAGAAATCCCGGAAAGTTTGGAAGAAGCGGC CAAGAGGCAAGATGGAGGGCTCTTTAAGGGGCCACCGTATCCCGGCTACCCCTTCATCATGATCCCCGAC CTGACGAGCCCCTACCTCCCCAACGGATCGCTCTCGCCCACCGCCCGAACCTATCTCCAGATGAAATGGC CACTGCTTGATGTCCAGGCAGGGAGCCTCCAGAGTAGACAAGCCCTCAAGGATGCCCGGTCCCCATCACC GGCACACATTGTCTCTAACAAAGTGCCAGTGGTGCAGCACCCTCACCATGTCCACCCCCTCACGCCTCTT ATCACGTACAGCAATGAACACTTCACGCCGGGAAACCCACCTCCACACTTACCAGCCGACGTAGACCCCA AAACAGGAATCCCACGGCCTCCGCACCCTCCAGATATATCCCCGTATTACCCACTATCGCCTGGCACCGT AGGACAAATCCCCCATCCGCTAGGATGGCAAGGTCAACCAGTGTACCCAATCACGACAGGAGGATTCAGA CACCCCTACCCCACAGCTCTGACCGTCAATGCTTCCATGTCCAGGTTCCCTCCCCATATGGTCCCACCAC ATCATACGCTACACACGACGGGCATTCCGCATCCGGCCATAGTCACACCAACAGTCAAACAGGAATCGTC CCAGAGTGATGTCGGCTCACTCCATAGTTCAAAGCATCAGGACTCCAAAAAGGAAGAAGAAAAGAAGAAG CCCCACATAAAGAAACCTCTTAATGCATTCATGTTGTATATGAAGGAAATGAGAGCAAAGGTCGTAGCTG AGTGCACGTTGAAAGAAAGCGCGGCCATCAACCAGATCCTTGGGCGGAGGTGGCATGCACTGTCCAGAGA AGAGCAAGCGAAATACTACGAGCTGGCCCGGAAGGAGCGACAGCTTCATATGCAACTGTACCCCGGCTGG TCCGCGCGGGATAACTATGGAAAGAAGAAGAAGAGGAAAAGGGACAAGCAGCCGGGAGAGACCAATGAAC ACAGCGAATGTTTCCTAAATCCTTGCCTTTCACTTCCTCCGATTACAGACCTGAGCGCTCCTAAGAAATG CCGAGCGCGCTTTGGCCTTGATCAACAGAATAACTGGTGCGGCCCTTGCAGTCTTTGAATTTGGAATATT ACAATGGAGAAAAAAAAAGTGCGTTCGCTACATACAAGGTGAAGGCAGCTGCCTCAGCCCACCCTCTTCA GATGGAAGCTTACTAGATTCGCCTCCCCCCTCCCCGAACCTGCTAGGCTCCCCTCCCCGAGACGCCAAGT CACAGACTGAGCAGACCCAGCCTCTGTCGCTGTCCCTGAAGCCCGACCCCCTGGCCCACCTGTCCATGAT GCCTCCGCCACCCGCCCTCCTGCTCGCTGAGGCCACCCACAAGGCCTCCGCCCTCTGTCCCAACGGGGCC CTGGACCTGCCCCCAGCCGCTTTGCAGCCTGCCGCCCCCTCCTCATCAATTGCACAGCCGTCGACTTCTT CCTTACATTCCCACAGCTCCCTGGCCGGGACCCAGCCCCAGCCGCTGTCGCTCGTCACCAAGTCTTTAGA ATAGCTTTAGCGTCGTGAACCCCGCTGCTTTGTTTATGGTTTTGTTTCACTTTTCTTAATTTGCCCCCCA CCCCCACCTTGAAAGGTTTTGTTTTGTACTCTCTTAATTTTGTGCCATGTGGCTACATTAGTTGATGTTT ATCGAGTTCATTGGTCAATATTTGACCCATTCTTATTTCAATTTCTCCTTTTAAATATGTAGATGAGAGA AGAACCTCATGATTCTACCAAAATTTTTATCAACAGCTGTTTAAAGTCTTTGTAGCGTTTAAAAAATATA TATATATACATAACTGTTATGTAGTTCGGATAGCTTAGTTTTAAAAGACTGATTAAAAAACAAAAAGAAA AAAAAAGCAATTTTGAAGCAGCCCTCCAGAAGGAGTTGGTTCTGTATTATTTGTATTAAATACGAGCTTG CGAACCAATCATTTTACATCTGGTTTTTAAACCGTAAGGGCACCATGAATGCAGTGCCGTTACTTTTTTT TTTTTTTTCTGTGTGAAACAACTCTTATTGTGATGTTACTTGTTATTGTTTAAATGTACAGAAACAAAGG GTAAAAATGTGTTAATATACCTTGTTCCATGGTGTTGTTCTTTTGGGGGGAGGGGACGCTACTCAACACT TAATAGAATCACAACGCTGTTGGGCCAGTAGTATTTATTGCTTTAGAGATTGCTTGTCGTACCTGTATGT CGTCCCTTTTTAAATATGTTTTCCTTTTTCTTGAAACTGTATAAAGTTTTTTTCCCCCTTAGCATAAGCA TCTTATATATAACAACTCATTTGTACAAGGTTTTTAAGTTTATATATAAAATGTGTATATATATTTTTGT TTCCCCTTTTTGACTTTTTTTTTTCTGTATGAAACCCAGATGTCACCAAATGGACATTAATAGTTGCATT AAGGATCAGTAGCATTAACAAAAGTTGCTTTAAAAGCCATTATGTAAAACAAGACTTGAAAATGAGTGAG GGAATTTTAGCGACACTGTCTGAGCAGCAGTGGGAACCATCTTCGTTTCCCCTTTGAACTCCCAGTGGGA TGCCCTACCCTGCGCCCTTAGGACCCGGACTGACCGTGTACAAAACTTTTACGTGCCAAAATTCTCAGTGA ATTTAGCTTTCTCCCTCTTTTTGATGCTGTAATTTTTGTTCATCATGTTTTGCTGTGATGTTACATAGGT AGATTTGTATGTAGTTTTAATGTCACCTATAACAAAATGTGTTTGGTAGCAGATTGTCCAGAAGAAGCATTT TAAATGAAGAGGTATAAACCCTTAAGGGCCAAAATTCTGTATATTAGATTACTCTTAAACGAAAAACCAG CTGCCGCTTTTATGTACACATATTACATACGAGTAGGCAGCAGACTTTAAAAATAAAAAAAACCTAGGCA TGTTGATGTTGCAAAATGCTGTATAAAGCTGAAACCTGTTCATTCAGTGCCATTGTAGTTGACATGAAGC GATTGTAAAACTGTCTCCGATTTTTCTCTGGTTTATTAAAATGCTAACTATAACATTTTTTGTGAATACT TTGAATGTTTCCTAACAGTTGTGATGTTACTGTTCCGTTTTATGCTCTTATTCCAAGTTCATTTTTAATG GTTTGGAAGCCATTTTTGTAATGAATAAATGTTCATGCTGTACAGTATCTGTAGCATGCCGTTCTGGATT AATAAGCAACTTAGTATGTGCAGATAAA Protein sequence (SEQ ID NO:62): MPQLNGGGGDDLGANDELISFKDEGEQEEKSSENSSAERDLADVKSSLVNESETNQNSSSDSEAERRPPPRSESFRDKSRESLEEAAKRQDGGLFKGPPYPGYPFIMIPDLTSPYLPNGSLSPTARTYLQMKWPLLDVQAGSLQSRQALKDARSPSPAHIVSNKVPVVQHPHHVHPLTPLITYSNEHFTPGNPPHLPADVDPKTGIPRPPHPPDISPY YPLSPGTVGQIPPHPLGWQGQPVYPITTGGFRHPYPTALTVNASMSRFPPHMVPPHHTLHTTGIPHPPAIVTPTVKQESSQSDVGSLHSSKHQDSKKEEEKKKPHIKKPLNAFMLYMKEM RAKVVAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKKRKRDKQPGETNEHSECFLNPCLSLPPITDLSAPKKCRARFGLDQQNNWCGPCSL >NM_001198528.2 Homo sapiens transcription factor 7-like 2 (TCF7L2), transcript variant 10 (SEQ ID NO: 63); GTCAATAATCTCCGCTCCCAGACTACTCCGTTCCTCCGGATTTCGATCCCCCTTTTTCTATCTGTCAATC AGCGCCGCCTTTGAACTGAAAAGCTCTCAGTCTAACTTCAACTCACTCAAATCCGAGCGGCACGAGCACC TCCTGTATCTTCGGCTTCCCCCCCTTTGCTCTTTATATCTGACTTCTTGTTGTTGTTGGTGTTTTTTT TTTTTTTACCCCCCTTTTTTATTTATTATTTTTTTGCACATTGATCGGATCCTTGGGAACGAGAGAAAAAA AGAAACCCAAACTCACGCGTGCAGAAGATCTCCCCCCCCTTCCCCTCCCCTCCTCCCTCTTTTCCCCTCC CCAGGAGAAAAAGACCCCCAAGCAGAAAAAAGTTCACCTTGGACTCGTCTTTTTCTTGCAATATTTTTTG GGGGGGCAAAACTTTTTGGGGGTGATTTTTTTTGGCTTTTCTTCCTCCTTCATTTTCTTCCAAAATTGC TGCTGGTGGGTGAAAAAAAAATGCGCAGCTGAACGGCGGTGGAGGGGATCTAGGCGCCAACGACGA ACTGATTTCCTTCCAAAGACGAGGGCGAACAGGAGAGAAGAGCTCCGAAAACTCCTCGGCAGAGAGGGAT TTAGCTGATGTCAAATCGTCTCTAGTCAATGAATCAGAAACGAATCAAAAACAGCTCCTCCGATTCCGAGG CGGAAAGACGGCCTCCGCCTCGCTCCGTCCGAAAGTTTCCGAGAAATCCCGGAAAGTTTGGAAGAAGCGGC CAAGAGGCAAGATGGAGGGCTCTTTAAGGGGCCACCGTATCCCGGCTACCCCTTCATCATGATCCCCGAC CTGACGAGCCCCTACCTCCCCAACGGATCGCTTCCGCCCACCGCCCGAACCTATCTCCAGATGAAATGGC CACTGCTTGATGTCCAGGCAGGAGCCTCCAGAGTAGACAGCCCTCAAGGATGCCCGGTCCCCATCACC GGCACACATTGTCTCTAACAAAGTGCCAGTGGTGCAGCACCCTCACCATGTCCACCCCCTCCACGCCTCTT ATCACGTACAGCAATGAACACTTCACGCCGGGAAACCCACCTCCACACTTACCAGCCGACGTAGACCCCA AAACAGGAATCCCACGGCCTCCGCACCCTCCAGATATATCCCGTATTACCCACTATCGCCTGGCACCGTT AGGACAAATCCCCCATCCGCTAGGATGGTTAGTACCACAGCAAGGTCAACCAGTGTACCCAATCACGACA GGAGGATTCAGACACCCCTACCCCACAGCTCTGACCGTCAATGCTTCCATGTCCAGGTTCCCTCCCCATA TGGTCCCACCACATCATACGCTACACACGACGGGCATTCCGCATCCGGCCATAGTCACACCAACAGTCAA ACAGGAATCGTCCCAGAGTGATGTCGGCTCACTCCATAGTTCAAAGCATCAGGACTCCAAAAAGGAAGAA GAAAAGAAGAAGCCCCACATAAAGAAACCTCTTAATGCATTCATGTTGTATATGAAGGAAATGAGAGCAA AGGTCGTAGCTGAGTGCACGTTGAAAGAAAGCGCGGCCATCAACCAGATCCTTGGGCGGAGGTGGCATGC ACTGTCCAGAGAAGAGCAAGCGAAATACTACGAGCTGGCCCGGAAGGAGCGACAGCTTCATATGCAACTG TACCCCGGCTGGTCCGCGCGGGATAACTATGGAAAGAAGAAGAAGAGGAAAAGGGACAAGCAGCCGGGAG AGACCAATGAACACAGCGAATGTTTCCTAAATCCTTGCCTTTCACTTCCTCCGATTACAGACCTGAGCGC TCCTAAGAAATGCCGAGCGCGCTTTGGCCTTGATCAACAGAATAACTGGTGCGGCCCTTGCAGTCTTTGA ATTTGGAATATTACAATGGAGAAAAAAAAAGTGCGTTCGCTACATACAAGGTGAAGGCAGCTGCCTCAGC CCACCCTCTTCAGATGGAAGCTTACTAGATTCGCCTCCCCCCTCCCCGAACCTGCTAGGCTCCCCTCCCC GAGACGCCAAGTCACAGACTGAGCAGACCCAGCCTCTGTCGCTGTCCCTGAAGCCCGACCCCCTGGCCCA CCTGTCCATGATGCCTCCGCCACCCGCCCTCCTGCTCGCTGAGGCCACCCACAAGGCCTCCGCCCTCTGT CCCAACGGGGCCCTGGACCTGCCCCCAGCCGCTTTGCAGCCTGCCGCCCCCTCCTCATCAATTGCACAGC CGTCGACTTCTTCCTTACATTCCCACAGCTCCCTGGCCGGGACCCAGCCCCAGCCGCTGTCGCTCGTCAC CAAGTCTTTAGAATAGCTTTAGCGTCGTGAACCCCGCTGCTTTGTTTATGGTTTTGTTTCACTTTTCTTA ATTTGCCCCCCACCCCCACCTTGAAAGGTTTTGTTTTGTACTCTCTTAATTTTGTGCCATGTGGCTACAT TAGTTGATGTTTATCGAGTTCATTGGTCAATATTTGACCCATTCTTATTTCAATTTCTCCTTTTAAATAT GTAGATGAGAGAAGAACCTCATGATTCTACCAAAATTTTTATCAACAGCTGTTTAAAGTCTTTGTAGCGT TTAAAAAATATATATATATACATAACTGTTATGTAGTTCGGATAGCTTAGTTTTAAAAGACTGATTAAAA AACAAAAAGAAAAAAAAAGCAATTTTGAAGCAGCCCTCCAGAAGGAGTTGGTTCTGTATTATTTGTATTA AATACGAGCTTGCGAACCAATCATTTTACATCTGGTTTTTAAACCGTAAGGGCACCATGAATGCAGTGCC GTTACTTTTTTTTTTTTTTTCTGTGTGAAACAACTCTTATTGTGATGTTACTTGTTATTGTTTAAATGTA CAGAAACAAAGGGTAAATTGTGTTAATATACCTTGTTCCATGGTGTTGTTCTTTTGGGGGGAGGGGACG CTACTCAACACTTAATAGAATCACAACGCTGTTGGGCCAGTAGTATTTATTGCTTTAGAGATTGCTTGTC GTACCTGTATGTCGTCCCTTTTTAAATATGTTTTCCTTTTTCTTGAAACTGTATAAAGTTTTTTCCCCC TTAGCATAAGCATCTTATATATAACAACTCATTTGTACAAGGTTTTTAAGTTTATATATAAAATGTGTAT ATATATTTTTGTTTCCCCTTTTTGACTTTTTTTTTTCTGTATGAAACCCAGATGTCACCAAATGGACATT AATAGTTGCATTAAGGATCAGTAGCATTAACAAAAGTTGCTTTAAAAGCCATTATGTAAAACAAGACTTG AAATGAGTGAGGGAATTTTAGCGACACTGTCTGAGCAGCAGTGGGAACCATCTTCGTTTCCCCTTTGAA CTCCCCAGTGGGATGCCCTACCCTGCGCCCTTAGGACCCGGACTGACCGTGTACAAAACTTTACGTGCCAA AATTCTCAGTGAATTTAGCTTTCTCCCTCTTTTTGATGCTGTAATTTTTGTTCATCATGTTTTGCTGTGA TGTTACATAGGTAGATTTGTATGTAGTTTTAATGTCACCTATAACAAAATGTGTTTGGTAGCAGATTGTC CAGAAAGCATTTTAAATGAAGAGGTATAAACCCTTAAGGGCCAAAATTCTGTATATTAGATTACTCTTAA ACGAAAAACCAGCTGCCGCTTTTATGTACACATATTACATACGAGTAGGCAGCAGACTTTAAAAATAAAA AAAACCTAGGCATGTTGATGTTGCAAAATGCTGTATAAAGCTGAAACCTGTTCATTCAGTGCCATTGTAG TTGACATGAAGCGATTGTAAAACTGTCTCCGATTTTTCTCTGGTTTATTAAAATGCTAACTATAACATTT TTTGTGAATACTTTGAATGTTTCCTAACAGTTGTGATGTTACTGTTCCGTTTTATGCTCTTATTCCAAGT TCATTTTTAATGGTTTGGAAGCCATTTTTGTAATGAATAAATGTTCATGCTGTACAGTATCTGTAGCATG CCGTTCTGGATTAATAAAAGCAACTTAGTATGTGCAGATAAA Protein sequence (SEQ ID NO:64): MPQLNGGGGDDLGANDELISFKDEGEQEEKSSENSSAERDLADVKSSLVNESETNQNSSSDSEAERRPPPRSESFRDKSRESLEEAAKRQDGGLFKGPPYPGYPFIMIPDLTSPYLPNGSLSPTARTILTYLQMKWPLLDVQAGSLQSRQALKDARSPSPAHIVSNKVPVVQHPHHVHPLTPLITYSNEHFTPGNPPHLPADVDPKTGIPRPPHPPDISPYYP LSPGTVGQIPHPLGWLVPQQGQPVYPITTGGFRHPYPTALTVNASMSRFPPHMVPPHHTLHTTGIPHPPAIVTPTVKQESSQSDVGSLHSSKHQDSKKEEEKKKPHIKKPLNAFMLYMKE MRAKVVAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKKRKRDKQPGETNEHSECFLNPCLSLPPITDLSAPKKCRARFGLDQQNNWCGPCSL >NM_001198529.2 Homo sapiens transcription factor 7-like 2 (TCF7L2), transcript variant 11 (SEQ ID NO: 65); GTCAATAATCTCCGCTCCCAGACTACTCCGTTCCTCCGGATTTCGATCCCCCTTTTTCTATCGTCAATC AGCGCCGCCTTTGAACTGAAAGCTCTCAGTCTAACTTCAACTCACTCAAATCCGAGCGGCACGAGCACC TCCTGTATCTTCGGCTTCCCCCCCCCTTTGCTCTTTATATCTGACTTCTTGTTGTTGTTGGTGTTTTTTT TTTTTTTACCCCCCTTTTTTTATTATTTTTTTGCACATTGATCGGATCCTTGGGAACGAGAGAAAAA AGAAACCCAAACTCACGCGTGCAGAAGATCTCCCCCCCCTTCCCCTCCCCTCCTCCCTCTTTTCCCTCC CCAGGAGAAAAAGACCCCCAAGCAGAAAAAAGTTCACCTTGGACTCGTCTTTTTCTTGCAATATTTTTTG GGGGGGCAAAACTTTTTGGGGGTGATTTTTTTTGGCTTTTCTTCCTCCTTCATTTTCTTCCAAAATTGC TGCTGGTGGGTGAAAAAAAAATGCGCAGCTGAACGGCGGTGGAGGGGATCTAGGCGCCAACGACGA ACTGATTTCCTTCCAAAGACGAGGGCGAACAGGAGAGAAGAGCTCCGAAAACTCCTCGGCAGAGAGGGAT TTAGCTGATGTCAAATCGTCTCTAGTCAATGAATCAGAAACGAATCAAAAACAGCTCCTCCGATTCCGAGG CGGAAAGACGGCCTCCGCCTCGCTCCGTCCGAAAGTTTCCGAGAAATCCCGGAAAGTTTGGAAGAAGCGGC CAAGAGGCAAGATGGAGGGCTCTTTAAGGGGCCACCGTATCCCGGCTACCCCTTCATCATGATCCCCGAC CTGACGAGCCCCTACCTCCCCAACGGATCGCTCTCGCCCACCGCCCGAACCTATCTCCAGATGAAATGGC CACTGCTTGATGTCCAGGCAGGGAGCCTCCAGAGTAGACAAGCCCTCAAGGATGCCCGGTCCCCATCACC GGCACACATTGTCTCTAACAAAGTGCCAGTGGTGCAGCACCCTCACCATGTCCACCCCCTCACGCCTCTT ATCACGTACAGCAATGAACACTTCACGCCGGGAAACCCACCTCCACACTTACCAGCCGACGTAGACCCCA AAACAGGAATCCCACGGCCTCCGCACCCTCCAGATATATCCCCGTATTACCCACTATCGCCTGGCACCGT AGGACAAATCCCCCATCCGCTAGGATGGTTAGTACCACAGCAAGGTCAACCAGTGTACCCAATCACGACA GGAGGATTCAGACACCCCTACCCCACAGCTCTGACCGTCAATGCTTCCATGTCCAGGTTCCCTCCCCATA TGGTCCCACCACATCATACGCTACACACGACGGGCATTCCGCATCCGGCCATAGTCACACCAACAGTCAA ACAGGAATCGTCCCAGAGTGATGTCGGCTCACTCCATAGTTCAAAGCATCAGGACTCCAAAAAGGAAGAA GAAAAGAAGAAGCCCCACATAAAGAAACCTCTTAATGCATTCATGTTGTATATGAAGGAAATGAGAGCAA AGGTCGTAGCTGAGTGCACGTTGAAAGAAAGCGCGGCCATCAACCAGATCCTTGGGCGGAGGTGGCATGC ACTGTCCAGAGAAGAGCAAGCGAAATACTACGAGCTGGCCCGGAAGGAGCGACAGCTTCATATGCAACTG TACCCCGGCTGGTCCGCGCGGGATAACTATGGAAAGAAGAAGAAGAGGAAAAGGGACAAGCAGCCGGGAG AGACCAATGAACACAGCGAATGTTTCCTAAATCCTTGCCTTTCACTTCCTCCGATTACAGGAGAAAAAAA AAGTGCGTTCGCTACATACAAGGTGAAGGCAGCTGCCTCAGCCCACCCTCTTCAGATGGAAGCTTACTAG ATTCGCCTCCCCCCTCCCCGAACCTGCTAGGCTCCCCTCCCCGAGACGCCAAGTCACAGACTGAGCAGAC CCAGCCTCTGTCGCTGTCCCTGAAGCCCGACCCCCTGGCCCACCTGTCCATGATGCCTCCGCCACCCGCC CTCCTGCTCGCTGAGGCCACCCACAAGGCCTCCGCCCTCTGTCCCAACGGGGCCCTGGACCTGCCCCCAG CCGCTTTGCAGCCTGCCGCCCCCTCCTCATCAATTGCACAGCCGTCGACTTCTTCCTTACATTCCCACAG CTCCCTGGCCGGGACCCAGCCCCAGCCGCTGTCGCTCGTCACCAAGTCTTTAGAATAGCTTTAGCGTCGT GAACCCCGCTGCTTTGTTTATGGTTTTGTTTCACTTTTCTTAATTTGCCCCCCACCCCCACCTTGAAAGG TTTTGTTTTGTACTCTCTTAATTTTGTGCCATGTGGCTACATTAGTTGATGTTTATCGAGTTCATTGGTC AATATTTGACCCATTCTTATTTCAATTTCTCCTTTTAAATATGTAGATGAGAGAAGAACCTCATGATTCT ACCAAAATTTTTATCAACAGCTGTTTAAAGTCTTTGTAGCGTTTAAAAAATATATATATATACATAACTG TTATGTAGTTCGGATAGCTTAGTTTTAAAAGACTGATTAAAAAACAAAAAGAAAAAAAAAGCAATTTTGA AGCAGCCCTCCAGAAGGAGTTGGTTCTGTATTATTTGTATTAAATACGAGCTTGCGAACCAATCATTTTA CATCTGGTTTTTAAACCGTAAGGGCACCATGAATGCAGTGCCGTTACTTTTTTTTTTTTTTTCTGTGTGA AACAACTCTTATTGTGATGTTACTTGTTATTGTTTAAATGTACAGAAACAAAGGGTAAAAATGTGTTAAT ATACCTTGTTCCATGGTGTTGTTCTTTTGGGGGGAGGGGACGCTACTCAACACTTAATAGAATCACAACG CTGTTGGGCCAGTAGTATTTATTGCTTTAGAGATTGCTTGTCGTACCTGTATGTCGTCCCTTTTTAAATA TGTTTTCCTTTTTCTTGAAACTGTATAAAGTTTTTTTCCCCCTTAGCATAAGCATCTTATATATAACAAC TCATTTGTACAAGGTTTTTAAGTTTATATATAAAATGTGTATATATATTTTTGTTTCCCCTTTTTGACTT TTTTTTTTCTGTATGAAACCCAGATGTCACCAAATGGACATTAATAGTTGCATTAAGGATCAGTAGCATT AACAAAAGTTGCTTTAAAAGCCATTATGTAAAACAAGACTTGAAAATGAGTGAGGGAATTTTAGCGACAC TGTCTGAGCAGCAGTGGGAACCATCTTCGTTTCCCCTTTGAACTCCCAGTGGGATGCCCTACCCTGCGCC CTTAGGACCCGGACTGACCGTGTACAAAACTTTACGTGCCAAAATTCTCAGTGAATTTAGCTTTCTCCCT CTTTTGATGCTTGTAATTTTTGTTCATCATGTTTTGCTGTGATGTTACATAGGTAGATTTGTATGTAGTT TTAATGTCACCTATAACAAAATGTGTTTGGTAGCAGATTGTCCAGAAAGCATTTTAAATGAAGAGGTATA AACCCTTAAGGGCCAAAATTCTGTATATTAGATTACTCTTAAACGAAAAACCAGCTGCCGCTTTTATGTA CACATATTACATACGAGTAGGCAGCAGACTTTAAAAATAAAAAAAACCTAGGCATGTTGATGTTGCAAAA TGCTGTATAAAGCTGAAACCTGTTCATTCAGTGCCATTGTAGTTGACATGAAGCGATTGTAAAACTGTCT CCGATTTTTCTCTGGTTTATTAAAATGCTAACTATAACATTTTTTGTGAATACTTTGAATGTTTCCTAAC AGTTGTGATGTTACTGTTCCGTTTTATGCTCTTATTCCAAGTTCATTTTTAATGGTTTGGAAGCCATTTT TGTAATGAATAAATGTTCATGCTGTACAGTATCTGTAGCATGCCGTTCTGGATTAATAAAAGCAACTTAG TATGTGCAGATAAA Protein sequence (SEQ ID NO:66): MPQLNGGGGDDLGANDELISFKDEGEQEEKSSENSSAERDLADVKSSLVNESETNQNSSSDSEAERRPPPRSESFRDKSRESLEEAAKRQDGGLFKGPPYPGYPFIMIPDLTSPYLPNGSLSPTARTILTYLQMKWPLLDVQAGSLQSRQALKDARSPSPAHIVSNKVPVVQHPHHVHPLTPLITYSNEHFTPGNPPHLPADVDPKTGIPRPPHPPDISPYYP LSPGTVGQIPHPLGWLVPQQGQPVYPITTGGFRHPYPTALTVNASMSRFPPHMVPPHHTLHTTGIPHPPAIVTPTVKQESSQSDVGSLHSSKHQDSKKEEEKKKPHIKKPLNAFMLYMKE MRAKVVAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKKRKRDKQPGETNEHSECFLNPCLSLPPITGEKKSAFATYKVKAAAASAHPLQMEAY >NM_001198530.2 Homo sapiens transcription factor 7-like 2 (TCF7L2), transcript variant 12 (SEQ ID NO: 67); GTCAATAATCTCCGCTCCCAGACTACTCCGTTCCTCCGGATTTCGATCCCCCTTTTTCTATCTGTCAATC AGCGCCGCCTTTGAACTGAAAAGCTCTCAGTCTAACTTCAACTCACTCAAATCCGAGCGGCACGAGCACC TCCTGTATCTTCGGCTTCCCCCCCTTTGCTCTTTATATCTGACTTCTTGTTGTTGTTGGTGTTTTTTT TTTTTTTACCCCCCTTTTTTATTTATTATTTTTTTGCACATTGATCGGATCCTTGGGAACGAGAGAAAAAA AGAAACCCAAACTCACGCGTGCAGAAGATCTCCCCCCCCTTCCCCTCCCCTCCTCCCTCTTTTCCCCTCC CCAGGAGAAAAAGACCCCCAAGCAGAAAAAAGTTCACCTTGGACTCGTCTTTTTCTTGCAATATTTTTTG GGGGGGCAAAACTTTTTGGGGGTGATTTTTTTTGGCTTTTCTTCCTCCTTCATTTTCTTCCAAAATTGC TGCTGGTGGGTGAAAAAAAAATGCGCAGCTGAACGGCGGTGGAGGGGATCTAGGCGCCAACGACGA ACTGATTTCCTTCCAAAGACGAGGGCGAACAGGAGAGAAGAGCTCCGAAAACTCCTCGGCAGAGAGGGAT TTAGCTGATGTCAAATCGTCTCTAGTCAATGAATCAGAAACGAATCAAAAACAGCTCCTCCGATTCCGAGG CGGAAAGACGGCCTCCGCCTCGCTCCGTCCGAAAGTTTCCGAGAAATCCCGGAAAGTTTGGAAGAAGCGGC CAAGAGGCAAGATGGAGGGCTCTTTAAGGGGCCACCGTATCCCGGCTACCCCTTCATCATGATCCCCGAC CTGACGAGCCCCTACCTCCCCAACGGATCGCTCTCGCCCACCGCCCGAACCTCTAACAAAGTGCCAGTGG TGCAGCACCCTCACCATGTCCACCCCCTCCAGCCCTCTTATCACGTACAGCAATGAACACTTCACGCCGGG AAACCCACCTCACACTTACCAGCCGACGTAGACCCCAAAACAGGAATCCCACGGCCTCCGCACCCTCCA GATATATCCCCGATTACCCACTATCGCCTGGCACCGTAGGACAAATCCCCCATCCGCTAGGATGGTTAG TACCACAGCAAGGTCAACCAGTGTACCCAATCACGACAGGAGGATTCAGACACCCCTACCCCACAGCTCT GACCGTCAATGCTTCCATGTCCAGGTTCCCTCCCCATATGGTCCCACCACATCATACGCTACACACGACG GGCATTCCGCATCCGGCCATAGTCACACCAACAGTCAAACAGGAATCGTCCCAGAGTGATGTCGGCTCAC TCCATAGTTCAAAGCATCAGGACTCCAAAAAGGAAGAAGAAAAGAAGAAGCCCCACATAAAGAAACCTCT TAATGCATTCATGTTGTATATGAAGGAAATGAGAGCAAAGGTCGTAGCTGAGTGCACGTTGAAAGAAAGC GCGGCCATCAACCAGATCCTTGGGCGGAGGTGGCATGCACTGTCCAGAGAAGAGCAAGCGAAATACTACG AGCTGGCCCGGAAGGAGCGACAGCTTCATATGCAACTGTACCCCGGCTGGTCCGCGCGGGATAACTATGG AAAGAAGAAGAAGAGGAAAAGGGACAAGCAGCCGGGAGAGACCAATGGAGAAAAAAAAAGTGCGTTCGCT ACATACAAGGTGAAGGCAGCTGCCTCAGCCCACCCTCTTCAGATGGAAGCTTACTAGATTCGCCTCCCCC CTCCCCGAACCTGCTAGGCTCCCCTCCCCGAGACGCCAAGTCACAGACTGAGCAGACCCAGCCTCTGTCG CTGTCCCTGAAGCCCGACCCCCTGGCCCACCTGTCCATGATGCCTCCGCCACCCGCCCTCCTGCTCGCTG AGGCCACCCACAAGGCCTCCGCCCTCTGTCCCAACGGGGCCCTGGACCTGCCCCCAGCCGCTTTGCAGCC TGCCGCCCCCTCCTCATCAATTGCACAGCCGTCGACTTCTTCCTTACATTCCCACAGCTCCCTGGCCGGG ACCCAGCCCCAGCCGCTGTCGCTCGTCACCAAGTCTTTAGAATAGCTTTAGCGTCGTGAACCCCGCTGCT TTGTTTATGGTTTTGTTTCACTTTTCTTAATTTGCCCCCCACCCCCACCTTGAAAGGTTTTGTTTTGTAC TCTCTTAATTTGTGCCATTGGCTACATTAGTTGATGTTTATCGAGTTCATTGGTCAATATTTGACCCA TTCTTATTTCAATTTCTCCTTTTAAAATATGTAGATGAGAGAAAACCTCATGATTCTACCAAAATTTTTA TCAACAGCTGTTTAAAGTCTTTGTAGCGTTAAAAAAATATATATATATACATAACTGTTATGTAGTTCGG ATAGCTTAGTTTTAAAGACTGATTAAAAAACAAAAGAAAAAAAAGCAATTTTGAAGCAGCCCTCCAG AAGGAGTTGGTTCTGTATTATTTGTATTAAATACGAGCTTGCGAACCAATCATTTTACATCTGGTTTTTA AACCGTAAGGGCACCATGAATGCAGTGCCGTTACTTTTTTTTTTTTTTCTGTGTGGAAACAACTCTTATT GTGATGTTACTTGTTATTGTTTTAAATGTACAGAACAAAGGGTAAAAATGTGTTAAATATCTTGTTCCA TGGTGTTGTTCTTTTGGGGGGAGGGGACGCTACTCAACACTTAATAGAATCACAACGCTGTTGGGCCAGT AGTATTATTGCTTTAGAGATTGCTTGTCGTACCTGTATGTCGTCCCTTTTAAATATGTTTTCCTTTTTT CTTGAAACTGTATAAAGTTTTTTTCCCCCTTAGCATAAGCATCTTATATATAACAACTCATTTGTACAAG GTTTTTAAGTTTATATATAAAATGTGTATATATATTTTTGTTTCCCCTTTTTTGACTTTTTTTTCTGTA TGAAACCCAGATGTCACCAAATGGACATTAATAGTTGCATTAAGGATCAGTAGCATTAACAAAAGTTGCT TTAAAAGCCATTATGTAAAACAAGACTTGAAAATGAGTGAGGGAATTTTAGCGACACTGTCTGAGCAGCA GTGGGAACCATCTTCGTTTCCCCTTTGAACTCCCAGTGGGATGCCCTACCCTGCGCCCTTAGGACCCGGA CTGACCGTGTACAAAACTTTACGTGCCAAAATTCTCAGTGAATTTAGCTTTCTCCCTCTTTTTGATGCTG TATTTTTGTTCATCATGTTTTGCTGTGATGTTACATAGGTAGATTTGTATGTAGTTTTAATGTCACCTA TAACAAAATGTGTTTGGTAGCAGATTGTCCAGAAAGCATTTTAAATGAAGAGGTATAAACCCTTAAGGGC CAAAATTCTGTATATTAGATTACTCTTAAACGAAACCAGCTGCCGCTTTTATGTACACATATTACATA CGAGTAGGCAGCAGACTTTAAAAATAAAAAACCTAGGCATGTTGATGTTGCAAAATGCTGTATAAAGC TGAAACCTGTTCATTCAGTGCCATTGTAGTTGACATGAAGCGATTGTAAAACTGTCTCCGATTTTTCTCT GGTTTATTAAAATGCTAACTATAACATTTTTTGTGAATACTTTGAATGTTTCCTAACAGTTGTGATGTTA CTGTTCCGTTTTATGCTCTTATTCCAAGTTCATTTTTAATGGTTTGGAAGCCATTTTTGTAATGAATAAA TGTTCATGCTGTACAGTATCTGTAGCATGCCGTTCTGGATTAATAAAAGCAACTTAGTATGTGCAGATAA A Protein sequence (SEQ ID NO:68): MPQLNGGGGDDLGANDELISFKDEGEQEEKSSENSSAERDLADVKSSLVNESETNQNSSSDSEAERRPPPRSESFRDKSRESLEEAAKRQDGGLFKGPPYPGYPFIMIPDLTSPYLPNGSLSPTARTSNKVPVVQHPHHVHPLTPLITYSNEHFTPGNPPPPHLPADVDPKTGIPRPPHPPDISPYYPLSPGTVGQIPHPLGWLVPQQGQPVYPITTGGFRHPYPTALTVNASMSRFPPHMVPPHHTLHTTGIPHPAIVTPTVKQESSQSDVGSLHSSKHQDSKKEEEKKKPHIKKPLNAFMLYMKEMRAKVVAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKKRKRDKQPGETNGEKKSAFATYKVKAAASAHPLQMEA >NM_001198531.2 Homo sapiens transcription factor 7-like 2 (TCF7L2), transcript variant 13 (SEQ ID NO: 69); GTCAATAATCTCCGCTCCCAGACTACTCCGTTCCTCCGGATTTCGATCCCCCTTTTTCTATCTGTCAATC AGCGCCGCCTTTGAACTGAAAAGCTCTCAGTCTAACTTCAACTCACTCAAATCCGAGCGGCACGAGCACC TCCTGTATCTTCGGCTTCCCCCCCTTTGCTCTTTATATCTGACTTCTTGTTGTTGTTGGTGTTTTTTT TTTTTTTACCCCCCTTTTTTATTTATTATTTTTTTGCACATTGATCGGATCCTTGGGAACGAGAGAAAAAA AGAAACCCAAACTCACGCGTGCAGAAGATCTCCCCCCCCTTCCCCTCCCCTCCTCCCTCTTTTCCCTCC CCAGGAGAAAAAGACCCCCAAGCAGAAAAAAGTTCACCTTGGACTCGTCTTTTTCTTGCAATATTTTTTG GGGGGGCAAAACTTTTTGGGGGTGATTTTTTTTGGCTTTTCTTCCTCCTTCATTTTCTTCCAAAATTGC TGCTGGTGGGTGAAAAAAAAATGCGCAGCTGAACGGCGGTGGAGGGGATCTAGGCGCCAACGACGA ACTGATTTCCTTCCAAAGACGAGGGCGAACAGGAGAGAAGAGCTCCGAAAACTCCTCGGCAGAGAGGGAT TTAGCTGATGTCAAATCGTCTCTAGTCAATGAATCAGAAACGAATCAAAAACAGCTCCTCCGATTCCGAGG CGGAAAGACGGCCTCCGCCTCGCTCCGTCCGAAAGTTTCCGAGAAATCCCGGAAAGTTTGGAAGAAGCGGC CAAGAGGCAAGATGGAGGGCTCTTTAAGGGGCCACCGTATCCCGGCTACCCCTTCATCATGATCCCCGAC CTGACGAGCCCCTACCTCCCCAACGGATCGCTCTCGCCCACCGCCCGAACCCTCCATTTTCAGTCCGGCA GCACACATTACTCTGCGTACAAAACGATTGAACACCAGATTGCAGTTCAGTATCTCCAGATGAAATGGCC ACTGCTTGATGTCCAGGCAGGAGCCTCCAGAGTAGACAGCCCTCAAGGATGCCCGGTCCCCATCACCG GCACACATTGTCTCTAACAAAGTGCCAGTGGTGCAGCACCCTCACCATGTCCACCCCCTCACGCCTCTTA TCACGTACAGCAATGAACACTTCACGCCGGGAAACCCACCTCCACACTTACCAGCCGACGTAGACCCCAA AACAGGAATCCCACGGCCTCCGCACCCTCCAGATATATCCCCGTATTACCCACTATCGCCTGGCACCGTA GGACAAATCCCCCATCCGCTAGGATGGTTAGTACCACAGCAAGGTCAACCAGTGTACCCAATCACGACAG GAGGATTCAGACACCCCTACCCCACAGCTCTGACCGTCAATGCTTCCATGTCCAGGTTCCCTCCCCATAT GGTCCCACCACATCATACGCTACACACGACGGGCATTCCGCATCCGGCCATAGTCACACCAACAGTCAAA CAGGAATCGTCCCAGAGTGATGTCGGCTCACTCCATAGTTCAAAGCATCAGGACTCCAAAAAGGAAGAAG AAAAGAAGAAGCCCCACATAAAGAAACCTCTTAATGCATTCATGTTGTATATGAAGGAAATGAGAGCAAA GGTCGTAGCTGAGTGCACGTTGAAAGAAAGCGCGGCCATCAACCAGATCCTTGGGCGGAGGTGGCATGCA CTGTCCAGAGAAGAGCAAGCGAAATACTACGAGCTGGCCCGGAAGGAGCGACAGCTTCATATGCAACTGT ACCCCGGCTGGTCCGCGCGGGATAACTATGGAAAGAAGAAGAAGAGGAAAAGGGACAAGCAGCCGGGAGA GACCAATGGAGAAAAAAAAAGTGCGTTCGCTACATACAAGGTGAAGGCAGCTGCCTCAGCCCACCCTCTT CAGATGGAAGCTTACTAGATTCGCCTCCCCCCTCCCCGAACCTGCTAGGCTCCCCTCCCCGAGACGCCAA GTCACAGACTGAGCAGACCCAGCCTCTGTCGCTGTCCCTGAAGCCCGACCCCCTGGCCCACCTGTCCATG ATGCCTCCGCCACCGCCCTCCTGCTCGCTGAGGCCACCCACAAGGCCTCCGCCCTCTGTCCCAACGGGG CCCTGGACCTGCCCCCAGCCGCTTTGCAGCCTGCCGCCCCCTCCTCATCAATTGCACAGCCGTCGACTTC TTCCTTACATTCCCACAGCTCCTGGCCGGGACCCAGCCCCAGCGCTGTCGCTCGTCCACCAAGTCTTTA GAATAGCTTTAGCGTCGTGAACCCCGCTGCTTTGTTTATGGTTTTGTTTCACTTTTCTTTAATTTGCCCCC CACCCCCACCTTGAAAGGTTTTGTTTTTGTACTCTCTTAATTTTGTGCCATGTGGCTACATTAGTTGATGT TTATCGAGTTCATTGGTCAATATTTGACCCATTCTTATTTCAATTTCTCCTTTTAAATATGTAGATGAGA GAAGAACCTCATGATTCTACCAAAATTTTTATCAACAGCTGTTTAAAGTCTTTGTAGCGTTTAAAAAATA TATATATACATAACTGTTATGTAGTTCGGATAGCTTAGTTTAAAAGACTGATTAAAAAAAAAAAAGA AAAAAAAAGCAATTTTGAAGCAGCCCTCCAGAAGGAGTTGGTTCTGTATTATTGTATTAAATACGAGCT TGCGAACCAATCATTTACATCTGGTTTTAAACCGTAAGGGCACCATGAATGCAGTGCCGTTACTTTTT TTTTTTTTTCTGTGTGAAACAACTCTTTATTGTGATGTTACTTGTTATTGTTTAAAATGTACAGAAAAAAA GGGTAAAAATGTGTTAATATACCTTGTTCCATGGTGTTGTTCTTTTGGGGGGAGGGGACGCTACTCAACA CTTATAGAATCACAACGCTGTTGGGCCAGTAGTAGTATTTATTGCTTTAGAGATTGCTTGTCGTACCTGTAT GTCGTCCCTTTTTAAATATGTTTTCCTTTTTCTTGAAACTGTATAAAGTTTTTTTCCCCCTTAGCATAAG CATCTTATATATAACAACTCATTTGTACAAGGTTTTTAAGTTTATATATAAAATGTGTATATATATTTTT GTTTCCCCTTTTTGACTTTTTTTTTTCTGTATGAACCCAGATGTCACCAAATGGACATTAATAGTTGCA TTAAGGATCAGTAGCATTAACAAAAGTTGCTTTAAAAGCCATTATGTAAAACAAGACTTGAAAATGAGTG AGGGAATTTTAGCGACACTGTCTGAGCAGCAGTGGGAACCATCTTCGTTTCCCCTTTGAACTCCCAGTGG GATGCCCTACCCTGCGCCCTTAGGACCCGGACTGACCGTGTACAAAACTTTTACGTGCCAAAATTCTCAGT GATTTAGCTTTCTCCCTCTTTTTGATGCTGTAATTTTTGTTCATCATGTTTTGCTGTGATGTTACATAG GTAGATTTGTATGTAGTTTTAATGTCACCTATAACAAAATGTGTTTGGTAGCAGATTGTCCAGAAAGCAT TTTAAATGAAGAGGTATAAACCCTTAAGGGCCAAAATTCTGTATATTAGATTACTCTTAAACGAAAAACC AGCTGCCGCTTTTATGTACACATATTACATACGAGTAGGCAGCAGACTTTAAAAATAAAAAAAACCTAGG CATGTTGATGTTGCAAAATGCTGTATAAAGCTGAAACCTGTTCATTCAGTGCCATTGTAGTTGACATGAA GCGATTGTAAAACTGTCTCCGATTTTTCTCTGGTTTATTAAAATGCTAACTATAACATTTTTTGTGAATA CTTTGAATGTTTCCTAACAGTTGTGATGTTACTGTTCCGTTTTATGCTCTTATTCCAAGTTCATTTTTAA TGGTTTGGAAGCCATTTTTGTAATGAATAAATGTTCATGCTGTACAGTATCTGTAGCATGCCGTTCTGGA TTAATAAAAGCAACTTAGTATGTGCAGATAAA Protein sequence (SEQ ID NO: 70): MPQLNGGGGDDLGANDELISFKDEGEQEEKSSENSSAERDLADVKSSLVNESETNQNSSSDSEAERRPPPRSESFRDKSRESLEEAAKRQDGGLFKGPPYPGYPFIMIPDLTSPYLPNGSLSPTARTLHFQSGSTHYSAYKTIEHQIAVQYLQMKWPLLDVQAGSLQSRQALKDARSPSPAHIVSNKVPVVQHPHHVHPLTPLITYSNEHFTPGNPPPHLPADV DPKTGIPRPHPHPPDISPYYPLSPGTVGQIPPHPLGWLVPQQGQPVYPITTGGFRHPYPTALTVNASMSRFPPHMVPPHHTLHTTGIPPHPAIVTPTVKQESSQSDVGSLHSSKHQDSKKEEE KKKPHIKKPLNAFMLYMKEMRAKVVAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKKRKRDKQPGETNGEKKSAFATYKVKAAAASAHPLQMEAY >NM_001349870.2 Homo sapiens transcription factor 7-like 2 (TCF7L2), transcript variant 14 (SEQ ID NO: 71); AGATAAAGTGCCTGCCTCCTGCACTCGGTTGATCATTTTCCCTCATGGCTGACCGTAATTTCTTTGCTAC TCTTAGCGGCAGCTCTGTCCCTGCTGCCCAAGGTAACGGGTGAGGGAGGGGACTTTGCTGAGGTGCCCTG AGCTGGCTGGGGAACATGGACCTTCCCCCTTCCTTCACGGATTCTGTAGTGGCAAAGATTAGAAAGTAGA AGGAGTTTTTAAAATTTTTTTTAAAAAATTAAATCTGCAACCAGCTGAACTCTAGATTATTAGTGGACTT TTTTGTTTTTTATTTTTAAGATTTTTTTAGGGATGTGTGTACAGGGGGAAGGATTAACAAGATAATTCTA GGCAATATGAGCGACAACTTTGAGCATTTTGAGCCTACTCGGCCAGGAATCTGGAGCCATTTCGATCTTT TTAGATGCTTTTTTTCAGTTCTTTAAGTGAAAGGTTTAAAGGGAGGGGAAGCTGGGAGGAAAAAGAAAAG TGAGAAAAGCAGAAAGGGAGGAGGAAGAAAAACTGGCCCAGTCCCATCTCGAAGGTACAGAGAAAGTTTT TTTAGAGGTGGAGAGAGGGAAAACCAAGGCTGTTATGTACCCTAGGGACACAATTTTAGTGGGAATGGAA GATTTGAGTGGTAAGGGAGGCGCAGTGGCCTCCGGGGTCTCTCTGTTCCAGCAGTTGGGCGGTTGGCACC TCAATTTGAATTGCTGGTCTAACAAAGTGCCAGTGGTGCAGCACCCTCACCATGTCCACCCCCTCACGCC TCTTATCACGTACAGCAATGAACACTTCACGCCGGGAAACCCACCTCCACACTTACCAGCCGACGTAGAC CCCAAAACAGGAATCCCACGGCCTCCGCACCCTCCAGATATATCCCCGTATTACCCACTATCGCCTGGCA CCGTAGGACAAATCCCCCATCCGCTAGGATGGTTAGTACCACAGCAAGGTCAACCAGTGTACCCAATCAC GACAGGAGGATTCAGACACCCCTACCCCACAGCTCTGACCGTCAATGCTTCCATGTCCAGGTTCCCTCCC CATATGGTCCCACCACATCATACGCTACACACGACGGGCATTCCGCATCCGGCCATAGTCACACCAACAG TCAAACAGGAATCGTCCCAGAGTGATGTCGGCTCACTCCATAGTTCAAAGCATCAGGACTCCAAAAAGGA AGAAGAAAAGAAGAAGCCCCACATAAAGAAACCTCTTAATGCATTCATGTTGTATATGAAGGAAATGAGA GCAAAGGTCGTAGCTGAGTGCACGTTGAAAGAAAGCGCGGCCATCAACCAGATCCTTGGGCGGAGGTGGC ATGCACTGTCCAGAGAAGAGCAAGCGAAATACTACGAGCTGGCCCGGAAGGAGCGACAGCTTCATATGCA ACTGTACCCCGGCTGGTCCGCGCGGGATAACTATGGAAAGAAGAAGAAGAGGAAAAGGGACAAGCAGCCG GGAGAGACCAATGAACACAGCGAATGTTTCCTAAATCCTTGCCTTTCACTTCCTCCGATTACAGACCTGA GCGCTCCTAAGAAATGCCGAGCGCGCTTTGGCCTTGATCAACAGAATAACTGGTGCGGCCCTTGCAGTCT TTGAATTTGGAATATTACAATGGAGAAAAAAAAAGTGCGTTCGCTACATACAAGGTGAAGGCAGCTGCCT CAGCCCACCCTCTTCAGATGGAAGCTTACTAGATTCGCCTCCCCCCTCCCCGAACCTGCTAGGCTCCCCT CCCCGAGACGCCAAGTCACAGACTGAGCAGACCCAGCCTCTGTCGCTGTCCCTGAAGCCCGACCCCCTGG CCCACCTGTCCATGATGCCTCCGCCACCCGCCCTCCTGCTCGCTGAGGCCACCCACAAGGCCTCCGCCCT CTGTCCCAACGGGGCCCTGGACCTGCCCCCAGCCGCTTTGCAGCCTGCCGCCCCCTCCTCATCAATTGCA CAGCCGTCGACTTCTTCCTTACATTCCCACAGCTCCCTGGCCGGGACCCAGCCCCAGCCGCTGTCGCTCG TCACCAAGTCTTTAGAATAGCTTTAGCGTCGTGAACCCCGCTGCTTTGTTTATGGTTTTGTTTCACTTTT CTTAATTTGCCCCCCACCCCCACCTTGAAAGGTTTTGTTTTGTACTCTCTTAATTTTGTGCCATGTGGCT ACATTAGTTGATGTTTATCGAGTTCATTGGTCAATATTTGACCCATTCTTATTTCAATTTCTCCTTTTAA ATATGTAGATGAGAGAAGAACCTCATGATTCTACCAAAATTTTTATCAACAGCTGTTTAAAGTCTTTGTA GCGTTTAAAAAATATATATATATACATAACTGTTATGTAGTTCGGATAGCTTAGTTTTAAAAGACTGATT AAAAAACAAAAAGAAAAAAAAAGCAATTTTGAAGCAGCCCTCCAGAAGGAGTTGGTTCTGTATTATTTGT ATTAAATACGAGCTTGCGAACCAATCATTTTACATCTGGTTTTTAAACCGTAAGGGCACCATGAATGCAG TGCCGTTACTTTTTTTTTTTTTTCTGTGTGAAACAACTCTTATTGTGATGTTACTTGTTATTGTTTAAA TGTACAGAAACAAAGGGTAAAAATGTGTTAATATACCTTGTTCCATGGTGTTGTTCTTTTGGGGGGAGGG GACGCTACTCAACACTTAATAGAATCACAACGCTGTTGGGCCAGTAGTATTTATTGCTTTAGAGATTGCT TGTCGTACCTGTATGTCGTCCCTTTTTAAATATGTTTTCCTTTTTCTTGAAACTGTATAAAGTTTTTTC CCCCTTAGCATAAGCATCTTATATATAACAACTCATTTGTACAAGGTTTTTAAGTTTATATATAAAAATGT GTATATATATTTTTGTTTCCCCTTTTTTGACTTTTTTTTTTCTGTATGAAACCCAGATGTCACCAAATGGA CATTAATAGTTGCATTAAGGATCAGTAGCATTAACAAAAGTTGCTTTAAAAGCCATTATGTAAAACAAGA CTTGAAAATGAGTGAGGGAATTTTAGCGACACTGTCTGAGCAGCAGTGGGAACCATCTTCGTTTCCCCTT TGAACTCCCAGTGGGATGCCCTACCCTGCGCCCTTAGGACCCGGACTGACCGTGTACAAAACTTTACGTG CCAAAATTCTCAGTGAATTTAGCTTTCTCCCTCTTTTTGATGCTGTAATTTTTGTTCATCATGTTTTGCT GTGATGTTACATAGGTAGATTTGTATGTAGTTTTAATGTCACCTATAACAAAATGTGTTTGGTAGCAGAT TGTCCAGAAAGCATTTTAAATGAAGAGGTATAAACCCTTAAGGGCCAAAATTCTGTATATTAGATTACTC TTAAACGAAAAACCAGCTGCCGCTTTTATGTACACATATTACATACGAGTAGGCAGCAGACTTTAAAAAT AAAAAAAACCTAGGCATGTTGATGTTGCAAAATGCTGTATAAAGCTGAAACCTGTTCATTCAGTGCCATT GTAGTTGACATGAAGCGATTGTAAAACTGTCTCCGATTTTTCTCTGGTTTATTAAAATGCTAACTATAAC ATTTTTTGTGAATACTTTGAATGTTTCCTAACAGTTGTGATGTTACTGTTCCGTTTTATGCTCTTATTCC AAGTTCATTTTTAATGGTTTGGAAGCCATTTTTGTAATGAATAAATGTTCATGCTGTACAGTATCTGTAG CATGCCGTTCTGGATTAATAAAAGCAACTTAGTATGTGCAGATAAA Protein sequence (SEQ ID NO:72): MYPRDTILVGMEDLSGKGGAVASGVSLFQQLGGWHLNLNCWSNKVPVVQHPHHVHPLTPLITYSNEHFTPGNPPPHLPADVDPKTGIPRPPHPPDISPYYPLSPGTVGQIPHPLGWLVPQQGQPVYPITTGGFRHPYPTALTVNASMSRFPPHMVPPHHTLHTTGIPHPAIVTPTVKQESSQSDVGSLHSSKHQDSKKEEEKKKPHIKKPLNAFMLYMKEMRAKVVAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKKRKRDKQPGETNEHSECFLNPCLSLPPITDLSAPKKCRARFGLDQQNNWCGPCS >NM_001349871.1 Homo sapiens transcription factor 7-like 2 (TCF7L2), transcript variant 15 (SEQ ID NO: 73); ATTGTGCTCTTCCCTATCAAGCGAGATAATTCTGTGAATGGAACTGTGCGTGAGCATCTTGTCTGGCGGC GCTGCTGGTGTGTGCTGCTCCCCTGTGCCGCGGGTGCGCGGCGGCGGCGCGGGCTGCAGGGCGGGTGCTG CCCTCCAGTGGAGCCCGCGGCCGGCGCGGGCCCTGGGCAGCATCTGGGCGCAGGCAGCATGCCCGCGAGC CGGCAGCGGGAAGGACAGCGCCGCAACCCTCTCTAGATGTCTAACAAAGTGCCAGTGGTGCAGCACCCTC ACCATGTCCACCCCCTCACGCCTCTTATCACGTACAGCAATGAACACTTCACGCCGGGAAACCCACCTCC ACACTTACCAGCCGACGTAGACCCCAAAACAGGAATCCCACGGCCTCCGCACCCTCCAGATATATCCCCG TATTACCCACTATCGCCTGGCACCGTAGGACAAATCCCCCATCCGCTAGGATGGTTAGTACCACAGCAAG GTCAACCAGTGTACCCAATCACGACAGGAGGATTCAGACACCCCTACCCCACAGCTCTGACCGTCAATGC TTCCATGTCCAGGTTCCCTCCCCATATGGTCCCACCACATCATACGCTACACACGACGGGCATTCCGCAT CCGGCCATAGTCACACCAACAGTCAAACAGGAATCGTCCCAGAGTGATGTCGGCTCACTCCATAGTTCAA AGCATCAGGACTCCAAAAAGGAAGAAGAAAAGAAGAAGCCCCACATAAAGAAACCTCTTAATGCATTCAT GTTGTATATGAAGGAAATGAGAGCAAAGGTCGTAGCTGAGTGCACGTTGAAAGAAAGCGCGGCCATCAAC CAGATCCTTGGGCGGAGGTGGCATGCACTGTCCAGAGAAGAGCAAGCGAAATACTACGAGCTGGCCCGGA AGGAGCGACAGCTTCATATGCAACTGTACCCCGGCTGGTCCGCGCGGGATAACTATGGAAAGAAGAAGAA GAGGAAAAGGGACAAGCAGCCGGGAGAGACCAATGAACACAGCGAATGTTTCCTAAATCCTTGCCTTTCA CTTCCTCCGATTACAGACCTGAGCGCTCCTAAGAAATGCCGAGCGCGCTTTGGCCTTGATCAACAGAATA ACTGGTGCGGCCCTTGCAGTCTTTGAATTTGGAATATTACAATGGAGAAAAAAAAAGTGCGTTCGCTACA TACAAGGTGAAGGCAGCTGCCTCAGCCCACCCTCTTCAGATGGAAGCTTACTAGATTCGCCTCCCCCCTC CCCGAACCTGCTAGGCTCCCCTCCCCGAGACGCCAAGTCACAGACTGAGCAGACCCAGCCTCTGTCGCTG TCCCTGAAGCCCGACCCCCTGGCCCACCTGTCCATGATGCCTCCGCCACCCGCCCTCCTGCTCGCTGAGG CCACCCACAAGGCCTCCGCCCTCTGTCCCAACGGGGCCCTGGACCTGCCCCCAGCCGCTTTGCAGCCTGC CGCCCCCTCCTCATCAATTGCACAGCCGTCGACTTCTTCCTTACATTCCCACAGCTCCCTGGCCGGGACC CAGCCCCAGCCGCTGTCGCTCGTCACCAAGTCTTTAGAATAGCTTTAGCGTCGTGAACCCCGCTGCTTTG TTTATGGTTTTGTTTCACTTTTCTTAATTTGCCCCCCACCCCCACCTTGAAAGGTTTTGTTTTGTACTCT CTTAATTTTGTGCCATGTGGCTACATTAGTTGATGTTTATCGAGTTCATTGGTCAATATTTGACCCATTC TTATTTCAATTTCTCCTTTTAAATATGTAGATGAGAGAAGAACCTCATGATTCTACCAAAATTTTTATCA ACAGCTGTTTAAAGTCTTTGTAGCGTTTAAAAAATATATATATATACATAACTGTTATGTAGTTCGGATA GCTTAGTTTTAAGACTGATTAAAAAACAAAGAAGAAAAGAAGGCAATTTTGAAGCAGCCCTCCAGAAG GAGTTGGTTCTGTATTATTTGTATTAAATACGAGCTTGCGAACCAATCATTTTACATCTGGTTTTTAAAC CGTAAGGGCACCATGAATGCAGTGCCGTTACTTTTTTTTTTTTTTCTGTGTGAAACAACTCTTATTGTG ATGTTACTTGTTATTGTTTAAATGTACAGAAACAAAGGGTAAAAATGTGTTAATATACCTTGTTCCATGG TGTTGTTCTTTTGGGGGGAGGGGACGCTACTCAACACTTAATAGAATCACAACGCTGTTGGGCCAGTAGT ATTTATTGCTTTAGAGATTGCTTGTCGTACCTGTATGTCGTCCCTTTTTAAATATGTTTTCCTTTTTCTT GAAACTGTATAAAGTTTTTTTCCCCCTTAGCATAAGCATCTTATATATAACAACTCATTTGTACAAGGTT TTTAAGTTTATATATAAAATGTGTATATATATTTTTGTTTCCCCTTTTTTGACTTTTTTTTCTGTATGA AACCCGATGTCACCAAATGGACATTAATAGTTGCATTAAGGATCAGTAGCATTAACAAAAGTTGCTTTA AAAGCCATTATGTAAAACAAGACTTGAAAATGAGTGAGGGAATTTTAGCGACACTGTCTGAGCAGCAGTG GGAACCATCTTCGTTTCCCCTTTGAACTCCCAGTGGGATGCCCTACCCTGCGCCCTTAGGACCCGGACTG ACCGTGTACAAAACTTTACGTGCCAAAATTCTCAGTGAATTTAGCTTTCTCCCTCTTTTTGATGCTGTAA TTTTTGTTCATCATGTTTTGCTGTGATGTTACATAGGTAGATTTGTATGTAGTTTTAATGTCACCTATAA CAAAATGTGTTTGGTAGCAGATTGTCCAGAAAGCATTTTAAATGAAGAGGTATAAACCCTTAAGGGCCAA AATTCTGTATATTAGATTACTCTTAAACGAAACCAGCTGCCGCTTTTATGTACACATATTACATACGA GTAGGCAGCAGACTTTAAAAATAAAAAACCTAGGCATGTTGATGTTGCAAAATGCTGTATAAAGCTGA AACCTGTTCATTCAGTGCCATTGTAGTTGACATGAAGCGATTGTAAAACTGTCTCCGATTTTTCTCTGGT TTATTAAAATGCTAACTATAACATTTTTTGTGAATACTTTGAATGTTTCCTAACAGTTGTGATGTTACTG TTCCGTTTTATGCTCTTATTCCAAGTTCATTTTTAATGGTTTGGAAGCCATTTTTGTAATGAATAAATGT TCATGCTGTACAGTATCTGTAGCATGCCGTTCTGGATCATTAAAAGCAACTTAGTATGTGCAGATAAA Protein sequence (SEQ ID NO:74): MSNKVPVVQHPHHVHPLTPLITYSNEHFTPGNPPPHLPADVDPKTGIPRPPHPPDISPYYPLSPGTVGQIPHPLGWLVPQQGQPVYPITTGGFRHPYPTALTVNASMSRFPPHMVPPHHTLHTTGIPHPAIVTPTVKQESSQSDVGSLHSSKHQDSKKEEEKKKPHIKKPLNAFMLYMKEMRAKVVAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKKRKRDKQPGETNEHSECFLNPCLSLPPITDLSAPKKCRARFGLDQQNNWCGPCSL >NM_001363501.2 Homo sapiens transcription factor 7-like 2 (TCF7L2), transcript variant 16 (SEQ ID NO: 75); GTCAATAATCTCCGCTCCCAGACTACTCCGTTCCTCCGGATTTCGATCCCCCTTTTTCTATCTGTCAATC AGCGCCGCCTTTGAACTGAAAAGCTCTCAGTCTAACTTCAACTCACTCAAATCCGAGCGGCACGAGCACC TCCTGTATCTTCGGCTTCCCCCCCTTTGCTCTTTATATCTGACTTCTTGTTGTTGTTGGTGTTTTTTT TTTTTTTACCCCCCTTTTTTATTTATTATTTTTTTGCACATTGATCGGATCCTTGGGAACGAGAGAAAAAA AGAAACCCAAACTCACGCGTGCAGAAGATCTCCCCCCCCTTCCCCTCCCCTCCTCCCTCTTTTCCCCTCC CCAGGAGAAAAAGACCCCCAAGCAGAAAAAAGTTCACCTTGGACTCGTCTTTTTCTTGCAATATTTTTTG GGGGGGCAAAACTTTTTGGGGGTGATTTTTTTTGGCTTTTCTTCCTCCTTCATTTTTCTTCCAAAATTGC TGCTGGTGGGTGAAAAAAAAATGCGCAGCTGAACGGCGGTGGAGGGGATCTAGGCGCCAACGACGA ACTGATTTCCTTCCAAAGACGAGGGCGAACAGGAGAGAAGAGCTCCGAAAACTCCTCGGCAGAGAGGGAT TTAGCTGATGTCAAATCGTCTCTAGTCAATGAATCAGAAACGAATCAAAAACAGCTCCTCCGATTCCGAGG CGGAAAGACGGCCTCCGCCTCGCTCCGTCCGAAAGTTTCCGAGAAATCCCGGAAAGTTTGGAAGAAGCGGC CAAGAGGCAAGATGGAGGGCTCTTTAAGGGGCCACCGTATCCCGGCTACCCCTTCATCATGATCCCCGAC CTGACGAGCCCCTACCTCCCCAACGGATCGCTCTCGCCCACCGCCCGAACCCTCCATTTTCAGTCCGGCA GCACACATTACTCTGCGTACAAAACGATTGAACACCAGATTGCAGTTCAGTATCTCCAGATGAAATGGCC ACTGCTTGATGTCCAGGCAGGAGCCTCCAGAGTAGACAGCCCTCAAGGATGCCCGGTCCCCATCACCG GCACACATTGTCTCTAACAAAGTGCCAGTGGTGCAGCACCCTCACCATGTCCACCCCCTCACGCCTCTTA TCACGTACAGCAATGAACACTTCACGCCGGGAAACCCACCTCCACACTTACCAGCCGACGTAGACCCCAA AAACGGAATCCCACGGCCTCCGCACCCTCCAGATATATCCCCGATTACCCACTATCGCCTGGCACCGTA GGACAAATCCCCCATCCGCTAGGATGGTTAGTACCACAGCAAGGTCAACCAGTGTACCCAATCACGACAG GAGGATTCAGACACCCCTACCCCACAGCTCTGACCGTCAATGCTTCCATGTCCAGGTTCCCTCCCCATAT GGTCCCACCACATCATACGCTACACACGACGGGCATTCCGCATCCGGCCATAGTCACACCAACAGTCAAA CAGGAATCGTCCCAGAGTGATGTCGGCTCACTCCATAGTTCAAAGCATCAGGACTCCAAAAAGGAAGAAG AAAAGAAGAAGCCCCACATAAAGAAACCTCTTAATGCATTCATGTTGTATATGAAGGAAATGAGAGCAAA GGTCGTAGCTGAGTGCACGTTGAAAGAAAGCGCGGCCATCAACCAGATCCTTGGGCGGAGGTGGCATGCA CTGTCCAGAGAAGAGCAAGCGAAATACTACGAGCTGGCCCGGAAGGAGCGACAGCTTCATATGCAACTGT ACCCCGGCTGGTCCGCGCGGGATAACTATGGAAAGAAGAAGAAGAGGAAAAGGGACAAGCAGCCGGGAGA GACCAATGAACACAGCGAATGTTTCCTAAATCCTTGCCTTTCACTTCCTCCGATTACAGGAGAAAAAAAA AGTGCGTTCGCTACATACAAGGTGAAGGCAGCTGCCTCAGCCCACCCTCTTCAGATGGAAGCTTACTAGA TTCGCCTCCCCCCTCCCCGAACCTGCTAGGCTCCCCTCCCCGAGACGCCAAGTCACAGACTGAGCAGACC CAGCCTCTGTCGCTGTCCCTGAAGCCCGACCCCCTGGCCCACCTGTCCATGATGCCTCCGCCACCCGCCC TCCTGCTCGCTGAGGCCACCCACAAGGCCTCCGCCCTCTGTCCCAACGGGGCCCTGGACCTGCCCCCAGC CGCTTTGCAGCCTGCCGCCCCCTCCTCATCAATTGCACAGCCGTCGACTTCTTCCTTACATTCCCACAGC TCCCTGGCCGGGACCCAGCCCCAGCCGCTGTCGCTCGTCACCAAGTCTTTAGAATAGCTTTAGCGTCGTG AACCCCGCTGCTTTGTTTATGGTTTTGTTTCACTTTTCTTAATTTGCCCCCCACCCCCACCTTGAAAGGT TTTGTTTTGTACTCTCTTAATTTTGTGCCATGTGGCTACATTAGTTGATGTTTATCGAGTTCATTGGTCA ATATTTGACCCATTCTTATTTCAATTTCTCCTTTTAAATATGTAGATGAGAGAAGAACCTCATGATTCTA CCAAAATTTTTATCAACAGCTGTTTAAAGTCTTTGTAGCGTTTAAAAAATATATATATATACATAACTGT TATGTAGTTCGGATAGCTTAGTTTTAAAAGACTGATTAAAAAACAAAAAGAAAAAAAAAGCAATTTTGAA GCAGCCCTCCAGAAGGAGTTGGTTCTGTATTATTTGTATTAAATACGAGCTTGCGAACCAATCATTTTAC ATCTGGTTTTTAAACCGTAAGGGCACCATGAATGCAGTGCCGTTACTTTTTTTTTTTTTTTCTGTGTGAA ACAACTCTTATTGTGATGTTACTTGTTATTGTTTAAATGTACAGAAACAAAGGGTAAAAATGTGTTAATA TACCTTGTTCCATGGTGTTGTTCTTTTGGGGGGAGGGGACGCTACTCAACACTTAATAGAATCACAACGC TGTTGGGCCAGTAGTATTTATTGCTTTAGAGATTGCTTGTCGTACCTGTATGTCGTCCCTTTTTAAATAT GTTTTCCTTTTTCTTGAAACTGTATAAAGTTTTTTTCCCCCTTAGCATAAGCATCTTATATATAACAACT CATTTGTACAAGGTTTTTAAGTTTATATATAAAATGTGTATATATATTTTTGTTTCCCCTTTTTGACTTT TTTTTTTCTGTATGAAACCCAGATGTCACCAAATGGACATTAATAGTTGCATTAAGGATCAGTAGCATTA ACAAAAGTTGCTTTAAAAGCCATTATGTAAAACAAGACTTGAAAATGAGTGAGGGAATTTTAGCGACACT GTCTGAGCAGCAGTGGGAACCATCTTCGTTTCCCCTTTGAACTCCCAGTGGGATGCCCTACCCTGCGCCC TTAGGACCCGGACTGACCGTGTACAAAACTTTACGTGCCAAAATTCTCAGTGAATTTAGCTTTCTCCCTC TTTTTGATGCTGTAATTTTTGTTCATCATGTTTTGCTGTGATGTTACATAGGTAGATTTGTATGTAGTTT TAATGTCACCTATAACAAAATGTGTTTGGTAGCAGATTGTCCAGAAAGCATTTTAAATGAAGAGGTATAA ACCCTTAAGGGCCAAAATTCTGTATATTAGATTACTCTTAAACGAAAAACCAGCTGCCGCTTTTATGTAC ACATATTACATACGAGTAGGCAGCAGACTTTAAAAATAAAAAAAACCTAGGCATGTTGATGTTGCAAAAT GCTGTATAAAGCTGAAACCTGTTCATTCAGTGCCATTGTAGTTGACATGAAGCGATTGTAAAACTGTCTC CGATTTTTCTCTGGTTTATTAAAATGCTAACTATAACATTTTTTGTGAATACTTTGAATGTTTCCTAACA GTTGTGATGTTACTGTTCCGTTTTATGCTCTTATTCCAAGTTCATTTTTAATGGTTTGGAAGCCATTTTT GTAATGAATAAATGTTCATGCTGTACAGTATCTGTAGCATGCCGTTCTGGATTAATAAAAGCAACTTAGT ATGTGCAGATAAA Protein sequence (SEQ ID NO:76): MPQLNGGGGDDLGANDELISFKDEGEQEEKSSENSSAERDLADVKSSLVNESETNQNSSSDSEAERRPPPRSESFRDKSRESLEEAAKRQDGGLFKGPPYPGYPFIMIPDLTSPYLPNGSLSPTARTLHFQSGSTHYSAYKTIEHQIAVQYLQMKWPLLDVQAGSLQSRQALKDARSPSPAHIVSNKVPVVQHPHHVHPLTPLITYSNEHFTPGNPPHLPADVDPKTGIPRP PHPPDISPYYPLSPGTVGQIPPHPLGWLVPQQGQPVYPITTGGFRHPYPTALTVNASMSRFPPHMVPPHHTLHTTGIPPHPAIVTPTVKQESSQSDVGSLHSSKHQDSKKEEEKKKPHIKKPLNAF MLYMKEMRAKVVAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKKRKRDKQPGETNEHSECFLNPCLSLPPITGEKKSAFATYKVKAAAASAHPLQMEAY >NM_001367943.1 Homo sapiens transcription factor 7-like 2 (TCF7L2), transcript variant 17, mRNA ((SEQ ID NO: 77)) GTCAATAATCTCCGCTCCCAGACTACTCCGTTCCTCCGGATTTCGATCCCCCTTTTTCTATCTGTCAATC AGCGCCGCCTTTGAACTGAAAAGCTCTCAGTCTAACTTCAACTCACTCAAATCCGAGCGGCACGAGCACC TCCTGTATCTTCGGCTTCCCCCCCCCTTTGCTCTTTATATCTGACTTCTTGTTGTTGTTGGTGTTTTTTT TTTTTTTACCCCCCTTTTTTTATTATTTTTTTGCACATTGATCGGATCCTTGGGAACGAGAGAAAAA AGAAACCCAAACTCACGCGTGCAGAAGATCTCCCCCCCCTTCCCCTCCCCTCCTCCCTCTTTTCCCTCC CCAGGAGAAAAAGACCCCCAAGCAGAAAAAAGTTCACCTTGGACTCGTCTTTTTCTTGCAATATTTTTTG GGGGGGCAAAACTTTTTGGGGGTGATTTTTTTTGGCTTTTCTTCCTCCTTCATTTTCTTCCAAAATTGC TGCTGGTGGGTGAAAAAAAAATGCGCAGCTGAACGGCGGTGGAGGGGATCTAGGCGCCAACGACGA ACTGATTTCCTTCCAAAGACGAGGGCGAACAGGAGAGAAGAGCTCCGAAAACTCCTCGGCAGAGAGGGAT TTAGCTGATGTCAAATCGTCTCTAGTCAATGAATCAGAAACGAATCAAAAACAGCTCCTCCGATTCCGAGG CGGAAAGACGGCCTCCGCCTCGCTCCGTCCGAAAGTTTCCGAGAAATCCCGGAAAGTTTGGAAGAAGCGGC CAAGAGGCAAGATGGAGGGCTCTTTAAGGGGCCACCGTATCCCGGCTACCCCTTCATCATGATCCCCGAC CTGACGAGCCCCTACCTCCCCAACGGATCGCTCTCGCCCACCGCCCGAACCCTCCATTTTCAGTCCGGCA GCACACATTACTCTGCGTACAAAACGATTGAACACCAGATTGCAGTTCAGTATCTCCAGATGAAATGGCC ACTGCTTGATGTCCAGGCAGGGAGCCTCCAGAGTAGACAAGCCCTCAAGGATGCCCGGTCCCCATCACCG GCACACATTGTCTCTAACAAAGTGCCAGTGGTGCAGCACCCTCACCATGTCCACCCCCTCACGCCTCTTA TCACGTACAGCAATGAACACTTCACGCCGGGAAACCCACCTCCACACTTACCAGCCGACGTAGACCCCAA AACAGGAATCCCACGGCCTCCGCACCCTCCAGATATATCCCCGTATTACCCACTATCGCCTGGCACCGTA GGACAAATCCCCCATCCGCTAGGATGGTTAGTACCACAGCAAGGTCAACCAGTGTACCCAATCACGACAG GAGGATTCAGACACCCCTACCCCACAGCTCTGACCGTCAATGCTTCCATGTCCAGGTTCCCTCCCCATAT GGTCCCACCACATCATACGCTACACACGACGGGCATTCCGCATCCGGCCATAGTCACACCAACAGTCAAA CAGGAATCGTCCCAGAGTGATGTCGGCTCACTCCATAGTTCAAAGCATCAGGACTCCAAAAAGGAAGAAG AAAAGAAGAAGCCCCACATAAAGAAACCTCTTAATGCATTCATGTTGTATATGAAGGAAATGAGAGCAAA GGTCGTAGCTGAGTGCACGTTGAAAGAAAGCGCGGCCATCAACCAGATCCTTGGGCGGAGGTGGCATGCA CTGTCCAGAGAAGAGCAAGCGAAATACTACGAGCTGGCCCGGAAGGAGCGACAGCTTCATATGCAACTGT ACCCCGGCTGGTCCGCGCGGGATAACTATGGAAAGAAGAAGAAGAGGAAAAGGGACAAGCAGCCGGGAGA GACCAATGAACACAGCGAATGTTTCCTAAATCCTTGCCTTTCACTTCCTCCGATTACAGACCTGAGCGCT CCTAAGAAATGCCGAGCGCGCTTTGGCCTTGATCAACAGAATAACTGGTGCGGCCCTTGCAGGAGAAAAA AAAAGTGCGTTCGCTACATACAAGGTGAAGGCAGCTGCCTCAGCCCACCCTCTTCAGATGGAAGCTTACT AGATTCGCCTCCCCCCTCCCCGAACCTGCTAGGCTCCCCTCCCCGAGACGCCAAGTCACAGACTGAGCAG ACCCAGCCTCTGTCGCTGTCCCTGAAGCCCGACCCCCTGGCCCACCTGTCCATGATGCCTCCGCCACCCG CCCTCCTGCTCGCTGAGGCCACCCACAAGGCCTCCGCCCTCTGTCCCAACGGGGCCCTGGACCTGCCCCC AGCCGCTTTGCAGCCTGCCGCCCCCTCCTCATCAATTGCACAGCCGTCGACTTCTTCCTTACATTCCCAC AGCTCCCTGGCCGGGACCCAGCCCCAGCCGCTGTCGCTCGTCACCAAGTCTTTAGAATAGCTTTAGCGTC GTGAACCCCGCTGCTTTGTTTATGGTTTTGTTTCACTTTTCTTAATTTGCCCCCCACCCCCACCTTGAAA GGTTTTGTTTTGTACTCTCTTAATTTTGTGCCATGTGGCTACATTAGTTGATGTTTATCGAGTTCATTGG TCAATATTTGACCCATTCTTATTTCAATTTCTCCTTTTAAATATGTAGATGAGAGAAGAACCTCATGATT CTACCAAAATTTTTATCAACAGCTGTTTAAAGTCTTTGTAGCGTTTAAAAAATATATATATATACATAAC TGTTATTGTAGTTCGGATAGCTTAGTTTTAAGACTGATTAAAAAACAAAGAGAAAAAAGCAATTTT GAAGCAGCCCTCCAGAAGGAGTTGGTTCTGTATTATTTGTATTAAATACGAGCTTGCGAACCAATCATTT TACATCTGGTTTTTAAACCGTAAGGGCACCATGAATGCAGTGCCGTTACTTTTTTTTTTTTTTTCTGTGT GAAACAACTCTTATTGTGATGTTACTTGTTATTGTTTAAATGTACAGAAACAAAGGGTAAAAATGTGTTA ATATACCTTGTTCCATGGTGTTGTTCTTTTGGGGGGAGGGGACGCTACTCAACACTTAATAGAATCACAA CCGCTGTTGGGCCAGTAGTATTTATTGCTTTAGAGATTGCTTGTCGTACCTGTATGTCGTCCCTTTTTAAA TATGTTTTCCTTTTTCTTGAAACTGTATAAAGTTTTTTTCCCCCTTAGCATAAGCATCTTATATATAACA ACTCATTTGTACAAGGTTTTTAAGTTTATATATAAAATGTGTATATATATTTTTGTTTCCCCTTTTTGAC TTTTTTTTTTCTGTATGAAACCCAGATGTCACCAAATGGACATTAATAGTTGCATTAAGGATCAGTAGCA TTAACAAAAGTTGCTTTAAAAGCCATTATGTAAAACAAGACTTGAAAATGAGTGAGGGAATTTTAGCGAC ACTGTCTGAGCAGCAGTGGGAACCATCTTCGTTTCCCCTTTGAACTCCCAGTGGGATGCCCTACCCTGCG CCCTTAGGACCCGGACTGACCGTGTACAAAACTTTTACGTGCCAAAATTCTCAGTGAATTTAGCTTTCTCC CTCTTTTTGATGCTGTAATTTTTGTTCATCATGTTTTGCTGTGATGTTACATAGGTAGATTTGTATGTAG TTTTAATGTCACCTATAACAAAATGTGTTTGGTAGCAGATTGTCCAGAAAGCATTTTAAATGAAGAGGTA TAAACCCTTAAGGGCCAAAATTCTGTATATTAGATTACTCTTAAACGAAAAACCAGCTGCCGCTTTTATG TACACATATTACATACGAGTAGGCAGCAGACTTTAAAAATAAAAAAAACCTAGGCATGTTGATGTTGCAA AATGCTGTATAAAGCTGAAACCTGTTCATTCAGTGCCATTGTAGTTGACATGAAGCGATTGTAAAACTGT CTCCGATTTTTCTCTGGTTTATTAAAATGCTAACTATAACATTTTTTGTGAATACTTTGAATGTTTCCTA ACAGTTGTGATGTTACTGTTCCGTTTTATGCTCTTATTCCAAGTTCATTTTTAATGGTTTGGAAGCCATT TTTGTAATGAATAAATGTTCATGCTGTACAGTATCTGTAGCATGCCGTTCTGGATTAATAAAAGCAACTT AGTAGTGCAGATAAA Protein sequence (SEQ ID NO:78): MPQLNGGGGDDLGANDELISFKDEGEQEEKSSENSSAERDLADVKSSLVNESETNQNSSSDSEAERRPPPRSESFRDKSRESLEEAAKRQDGGLFKGPPYPGYPFIMIPDLTSPYLPNGSLSPTARTLHFQSGSTHYSAYKTIEHQIAVQYLQMKWPLLDVQAGSLQSRQALKDARSPSPAHIVSNKVPVVQHPHHVHPLTPLITYSNEHFTPGNPPPPHLPADVDPKTGIPRPPHPPDISPYYPLSPGTVGQIPHPLGWLVPQQGQPVYPITTGG FRHPYPTALTVNASMSRFPPHMVPPHHTLHTTGIPHPPAIVTPTVKQESSQSDVGSLHSSKHQDSKKEEEKKKPHIKKPLNAFMLYMKEMRAKVVAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSAR DNYGKKKKRKRDKQPGETNEHSECFLNPCLSLPPITDLSAPKKCRARFGLDQQNNWCGPCRRKKKCVRYIQGEGSCLSPPSSDGSLLDSPPPSPNLLGSPPRDAKSQTEQTQPLSLSLKPDPLAHLSMMPPPPALLLAEATHKASA LCPNGALDLPPAALQPAAPSSSIAQPSTSSLHSHSSLAGTQPQPLSLVTKSLE

[0071] In one example, the following human TCF7L2-210 sequence is used, which is identical to the sequence of variant 2 above, except for the underlined / bolded residues: TIFF2024542015000006.tif87170

[0072] Listed below are several exemplary Tcf7l2 mouse isoforms, associated nucleic acid sequence information, and associated amino acid sequence information. All of these variants / isoforms may be used in the expression cassettes, genetic constructs, vectors, compositions, or methods disclosed herein. [Table 2] Tcf7l2 isoform 6 [Mus musculus] (protein) (SEQ ID NO: 80) MPQLNGGGGGDDLGANDELISFKDEGEQEEKSSENSSAERDLADVKSSLVNESETNQNSSSDSEAERRPPPRSESFRDKSRESLEEAAKRQDGGLFKGPPYPGYPFIMIPD LTSPYLPNGSLSPARTYLQMKWPLLDVQAGSLQSRQALKDARSPSPAHIVSNKVPVVQHPHHVHPLTPLITYSNEHFTPGNPPPHLPADVDPKTGIPRPHPPDISPYYP LSPGTVGQIPHPLGWLVPQQGQPVYPITTGGFRHPYPTALTVNASMSRFPPHMVPPHHTLHTTGIPHPPAIVTPTVKQESSQSDVGSLHSSKHQDSKKEEEKKKPHIKKPL NAFMLYMKEMRAKVVAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKKRKRDKQPGETNGEKKSAFATYKVKAAAASAHPLQMEAY Tcf7l2 isoform 6 [Mus musculus] (nucleic acid) (SEQ ID NO: 81) CTCGCCCTGTCAAATAATCTCCGCTCCCAGACTACTCCGTTCCTCCGGATTTCGATCCCCCTTTTTCTATC TGTCAATCAGCGCCGCCTTTGAACTGAAAAGCTCAGTCTAACTTCAACTCACTCAAATCCGAGCGGCACG AGATCCTTCGGTGTCTTCGGCTTCCCCCCCCTTTGCTCTTATATCTGACTTCTTCTTGTTGTTGTTGTTG TTTTTTTTACCCCCTTTTTATTTATTATTATTTTTTTTTTTTGCACATTGATCGGATCCTTGGGAACGA GAGAAAAAAGAAAACCCAAACTCACGCGTGCAGAAGATCTCCCCCCTTCCCCTCCCCTCCTCCCTCTTTT CCCCTCCCCAGGAGAGAAAGACCCCAAAGCAGGAAAAAAAAAGTTAACCTTGGACTCGTCTTTTTCTTG CAGTATTTTTTTGGGGGGGACTCGCAAAACTTTTTTTTTTTTTTTTTTTTTTTTTGCTTTTTTCCTC CTTCATTTTTCTTCCAAAATTGCTGCTGCTGGTGGGTGAAAAAATGCGCAGCTGAACGGCGGTGGAGGGAG ATGACCTAGGCGCTAACGACGAGCTGATCTCCTTTCAAAGACGAAGGCGAGCAGGAGGAGAAGAACTCGGA AAACTCCTCGGCGGAAAGGGATTTAGCCGATGTCAAGTCCTCGCTGGTCAATGAATCAGAGACGAATCAA AACAGCTCCTCCGATTCCGAGGCGGAAAGACGGCCTCCGCCTCGCTCCGAAAGTTTCCGAGATAAATCCC GGGAAAGTTTGGAAGAAGCGGCCAAGAGGCAAGATGGAGGGCTCTTTAAGGGGGCCACCGTATCCCGGCTA CCCCTTCATCATGATCCCCCGACCTGACGAGCCCCTACCTCCCCAACGGATCGCTCTCGCCCACCGCCGA ACCTATCTTCAGAATGAAATGGCCACTGCTTGATGTCCAAGCAGGAAGCCTCCAGAGCAGACAAACCCTCA AGGATGCTCGTTCGCCGTCGCCAGCACACATCGTTTCGAACAAAGTACCGGTGGTGCAACACCCCCCACCA TGTCCACCCACTCACGCCTCTCATCACGTACAGCAATGAACACTTCACCCCGGGAAATCCACCTCCGCAC TTACCAGCTGACGTAGACCCCAAAACAGGAATCCCAAGGCCTCCGCACCCTCCAGATATCTCTCCATATT ACCCGCTGTCGCCCGGCACCGTAGGACAAATCCCCCATCCGCTAGGATGGTTAGTACCACAGCAAGGTCA GCCTGTGTACCCAATCACGACAGGAGGATTCAGACACCCCTACCCCACAGCGCTGACAGTCAACGCATCT ATGTCTAGGTTCCCTCCCCATATGGTCCCTCCCCATCACACTCTGCACACGACCGGCATCCCTCACCCGG CCATCGTCACACCGACAGTCAAGCAGGAATCCTCCCAGAGTGACGTCGGCTCACTCCACAGCTCAAAGCA TCAGGACTCCAAAAAGGAAGAAGAGAAGAAGAAGCCCCACATAAAGAAGCCCCTTAATGCATTCATGTTG TATATGAAAGAGATGAGAGCGAAGGTGGTGGCCGAATGCACATTGAAAGAGAGTGCAGCCATCAACCAGA TTCTCGGGCGCAGGTGGCACGCCCTGTCCAGGGAAGAACAGGCAAAATACTACGAGCTGGCCCGGAAGGA ACGACAGCTTCACATGCAGCTGTACCCTGGCTGGTCTGCACGGGATAACTATGGGAAGAAGAAGAAGAGA AAAAGAGACAAGCAGCCGGGGGAAACCAACGAACACAGCGAATGTTTCCTAAATCCTTGCCTTTCGCTTC CTCCGATCACAGACCTGAGCGCTCCTAAGAAATGCCGAGCGCGCTTTGGCCTTGATCAACAGAATAACTG GTGCGGCCCCTGCAGGAGAAAAAAAAAGTGCGTTCGCTACATACAAGGTGAAGGCAGCTGCCTCAGCCCA CCCTCTTCAGATGGAAGCTTACTAGACTCGCCTCCCCCCTCACCGCATCTGCTAGGCTCCCCTCCCCAAG ACGCCAAGTCACAGACTGAGCAGACCCAGCCGCTCTCGCTGTCCCTGAAGCCTGATCCTCTGGCCCACCT GTCCATGATGCCTCCGCCACCCGCGCTCCTGTTGGCCGAAGCTGCCCACGGCAAGGCCTCTGCCCTCTGT CCCAATGGGGCTCTGGACCTGCCACCTGCCGCTCTGCAGCCGTCCATGGTCCCTTCCTCATCGCTCGCAC AACCATCAACTTCTTCCTTACATTCCCACAACTCGCTGGCTGGAACGCAACCCCAGCCTCTGTCTCTGGT GACCAAGTCTTTAGAATAGCTCCGCCTCCTCGTCCTCCATGCCCCTGCTTGATTGAAGTGTTTCCTTCTG GTTCTTGGTTCGCCCTTCCCCGCTTGGAAAGCTTTTGTTTTGTACTCTTTAATTTTGTGCCGCCGTGGCT ACATGAGTTAATGTTTATGGAGTTCATTGGTCAATATTTGACCCATTCTTATTTCAATTTCTCCTTTTAA ATATGTAGATGAGAAAAACCTCATGATTCTACCAAAATTTTTATCAACAGCTGTTTAAAGTCTTTGTAGC GTTTAAAAAATATATTATATATACATAACTGTTATGTAGTTCGGATAGCTTAGTTTTAAAAGACTGATTT AAAAAACAAAAAGAAAAAAAAGAGAGAGAAGCAATTTTGAAGCACCCTCCAGAAGGAGTTGGTTCTGTGT TATTTGTATTAAATACGAGCTTGCGAACCAATCACTTTACACCTTGGTATTTAAACCACGAGGGCACAAG GAATGCAGTGCCGTTTCTTTCTTTCTTTTTTTTTCTTTTTTTTTTTTTTCCTCCCAGTGTGAAACAACTTTT ACCTTGATGTTACTTGTTATTGTTTAAATGTACAGAAACAAAGGGTTAAAATGTGTTAATATACCTTGTT CCATGGTGTTGTTCTTTTGGGGGGGAGGGGACACCACTCAACAATGAATGGAATCAACACTGTTGGACCA GTAGTATTTATTGCTTTAGAGGTTGCTTGCTGTACCTGTATGTCCGTCCAATTTTAAATATGTTTTCCCT TTTTTTGAAACTGTGTAAAGTCCCCCCCAAACCTTAGCGTAAGCATCTTATATATAACAACTCATTTGTA CAAGGTTTTTAAGTTTATATATAAAATGTGTATATATTTTTTTGTTTCCTTTTTTTTTTTTTTTTGGCGT ACGTTCGTTCGTTCGTTTGTTTGTTTCTGTATAAACCCAGATGCCACCAAATGGACATTGACATTCGCA TTAAGGATCAGTAGCATTAACAAAACTTGCTTTAAGCCATTCTGTAAAACAAGACTTGAACATTAGCG AGAGGGGTCTGAGCAGCCGTCTGAGCAGCCGTGGGAACTGCCCCGTTTCCCCCTTGACCTCCTAGTCAACT GCCCTGTGCTCTTAGAACATCGGACTGACCGTGTGCAAAACTTTTATGTGCCAAAATTCTCAGTGACTTTA GCTTTCTCCCTCCTTTTTGATGCTGTACTTTCTGTTCGCCATGTTTTGCTGTGATGTTACATAGATAGAT TTGTATGTAGTTTTTAATGTCACCTATAACAAAATGTGTTTGGTAGCAGACTGTCCAGAAAGCATTTTAAA TGAAGAGGTCTAAACCCTTAAGGGCCAAAAAAAATCCTGTATATTAGATTACTCTTAAATGAAAAAGAAA AAAAGAAAAAAAAAAACCCCAGCTGCCGCTTTTATGTATGCATATTACATACAAGTAGGTAGTGACCTTT AAAAACAAGAAAACCTAGGCATGCCGATGTTGTAAAATGCTGTATAAAGCTGAGACCTGTCCTTCAGTG CCATCGTAGTTGACATGAAGCGATTGTAAAACTGTCTCCGGTTTTCTCTGGTTTATTAAAATGCTAACTA TAACATTTTCTTTTCTTTTTTTTTTTTTGTGAATACTTTGAATGTCTCCCTAACAGTTGTGGTGTTGCTG TTCTGTTCGATGCTTATTCCAAGTTCATTTTTTTGAATGGTTTTGAAGCCATTTTGTAATGAATTAAATG TCCATGCTGTACAGTACCTGTAGCATGCCTGCCGTTCTGGATTAATAAAAGCAACTTAGTATGTGCA

[0073] TCF7L2 has a highly conserved protein domain that is conserved in several species, including human, mouse, rat, chicken, fish and fruit fly.For example, human TCF7L2 has 90.5% homology with mouse transcript.Therefore, TCF7L2 of non-human species can also be used in the expression cassette, gene construct, vector, composition or method disclosed herein.

[0074] The terms "TCF7L2" and "transcription factor 7-like 2" also encompass functional fragments or derivatives that substantially retain the transcription factor activity of TCF7L2 as described herein. Typically, a functional fragment or derivative retains at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of its transcription factor activity. It is also contemplated that a TCF7L2 protein may contain conservative amino acid substitutions that do not substantially alter its activity. Suitable conservative substitutions of amino acids are known to those of skill in the art and may generally be made without altering the biological activity of the resulting molecule. Those of skill in the art will generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. Conservative and non-conservative amino acid substitutions are described herein.

[0075] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the activity of TCF7L2. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art. A conservative modification or functional equivalent of a peptide, polypeptide, or protein disclosed herein refers to a polypeptide derivative of the peptide, polypeptide, or protein, such as a protein having one or more substitutions, point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof. Substantially retains activity relative to the parent peptide, polypeptide, or protein (such as those disclosed herein). Generally, a conservative modification or functional equivalent is at least 60% identical (e.g., any number between 60% and 100%, e.g., 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the parent (e.g., one of the human or non-human TCF7L2 sequences disclosed herein).

[0076] Amino acid substitutions can be made by selecting substitutions whose effects on maintaining (a) the structure of the peptide backbone in the area of ​​substitution, (b) the charge or hydrophobicity of the molecule at the target position, or (c) the bulk of the side chain are not different from those of the demyelination. For example, naturally occurring residues can be divided into groups based on side chain properties: (1) hydrophobic amino acids (norleucine, methionine, alanine, valine, leucine, and isoleucine), (2) neutral hydrophilic amino acids (cysteine, serine, threonine, asparagine, and glutamine), (3) acidic amino acids (aspartic acid and glutamic acid), (4) basic amino acids (histidine, lysine, and arginine), (5) amino acids that affect chain orientation (glycine and proline), and (6) aromatic amino acids (tryptophan, tyrosine, and phenylalanine). Substitutions made within these groups can be considered conservative substitutions. Examples of substitutions include, but are not limited to, alanine to valine, arginine to lysine, asparagine to glutamine, aspartic acid to glutamic acid, cysteine ​​to serine, glutamine to asparagine, glutamic acid to aspartic acid, glycine to proline, histidine to arginine, isoleucine to leucine, leucine to isoleucine, lysine to arginine, methionine to leucine, phenylalanine to leucine, proline to glycine, serine to threonine, threonine to serine, tryptophan to tyrosine, tyrosine to phenylalanine, and / or valine to leucine. Exemplary substitutions are shown in the following table. Amino acid substitutions may be introduced into human TCF7L2 and the products may be screened for retention of human TCF7L2 biological activity. [Table 3]

[0077] 3. Gene constructs, expression cassettes and expression vectors The present disclosure also provides a genetic construct, such as an expression cassette, comprising or consisting of a nucleic acid encoding a transcription factor 7-like 2 protein. Such a nucleic acid may not inherently comprise a promoter, but the expression cassette may further comprise a promoter or enhancer. In that case, the nucleic acid is operably linked to and under the regulatory control of the promoter and / or enhancer of a gene selectively or specifically expressed by glial progenitor cells. Thus, an expression cassette according to the present invention may comprise, in the 5' to 3' direction, a promoter, a coding sequence, and optionally a terminator or other elements. The expression cassette allows for easy transfer of a nucleic acid sequence of interest into an organism, preferably a cell, preferably a diseased cell.

[0078] The expression cassette of the present disclosure can be preferably included in a vector. Thus, the vector of the present disclosure allows for the transformation of cells with a nucleic acid sequence of interest. Correspondingly, the present disclosure provides a host cell comprising an expression cassette according to the present disclosure or a recombinant nucleic acid according to the present disclosure. The recombinant nucleic acid can also comprise a promoter or enhancer to allow the expression of the nucleic acid sequence of interest.

[0079] Exogenous genetic material (e.g., a nucleic acid, an expression cassette, or an expression vector encoding one or more therapeutic agents) can be introduced into a target cell of interest in vivo by gene transfer methods such as transfection or transduction to provide a genetically modified cell. A variety of expression vectors (i.e., vehicles for facilitating the delivery of exogenous genetic material to a target cell) are known to those skilled in the art. As used herein, "exogenous genetic material" refers to natural or synthetic nucleic acids or oligonucleotides that are not naturally found in a cell, or that, when naturally found in a cell, are not transcribed or expressed at biologically significant levels by the cell. Thus, "exogenous genetic material" includes, for example, non-naturally occurring nucleic acids that can be transcribed into RNA.

[0080] As used herein, "transfection of a cell" refers to the acquisition of new genetic material by a cell by incorporating added nucleic acid (DNA, RNA, or hybrids thereof) without the use of a viral delivery vehicle. Thus, transfection refers to the introduction of nucleic acid into a cell using physical or chemical methods. Several transfection techniques are known to those skilled in the art, including calcium phosphate nucleic acid co-precipitation, strontium phosphate nucleic acid co-precipitation, DEAE-dextran, electroporation, cationic liposome-mediated transfection, and tungsten particle-facilitated microparticle bombardment. In contrast, "transduction of a cell" refers to the process of transferring nucleic acid into a cell using a DNA or RNA virus. RNA viruses (e.g., retroviruses) for the transfer of nucleic acid into cells can be used as transducing chimeric viruses. The exogenous genetic material contained in the virus can be integrated into the genome of the transduced cell. A cell transduced with a chimeric DNA virus (e.g., an adenovirus carrying DNA encoding a therapeutic agent) may not have the exogenous genetic material integrated into its genome, but may be capable of expressing exogenous genetic material carried extrachromosomally within the cell.

[0081] Typically, exogenous genetic material may include a heterologous gene (encoding a therapeutic RNA or protein) along with a promoter to control the transcription of the new gene. A promoter characteristically has a specific nucleotide sequence required to initiate transcription. Optionally, the exogenous genetic material further includes additional sequences (i.e., enhancers) required to obtain the desired gene transcription activity. The exogenous genetic material may be introduced into the cell genome immediately downstream of the promoter such that the promoter and the coding sequence are operably linked to allow transcription of the coding sequence. Viral expression vectors may include an exogenous promoter element to control the transcription of the inserted exogenous gene. Examples of such exogenous promoters include constitutive promoters, inducible promoters, and tissue or cell type specific promoters.

[0082] Naturally occurring constitutive promoters control the expression of essential cellular functions. As a result, genes under the control of a constitutive promoter are expressed under all conditions of cell growth. Exemplary constitutive promoters include the promoters of the following genes that code for specific constitutive or "housekeeping" functions: hypoxanthine phosphoribosyltransferase, dihydrofolate reductase, adenosine deaminase, phosphoglycerol kinase, pyruvate kinase, phosphoglycerol mutase, actin promoter, ubiquitin, elongation factor-1, and other constitutive promoters known to those skilled in the art. In addition, many viral promoters function constitutively in eukaryotic cells. These include, among others, the early and late promoters of SV40, the long terminal repeats (LTRs) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Thus, any of the above constitutive promoters can be used to control the transcription of a heterologous gene insert.

[0083] Genes under the control of inducible promoters are expressed only in the presence of, or are largely controlled by, an inducing agent (e.g., transcription under the control of the metallothionein promoter is greatly increased in the presence of certain metal ions). Inducible promoters contain responsive elements (REs) that stimulate transcription when their inducers bind. For example, there are REs for serum factors, steroid hormones, retinoic acid, and cyclic AMP. Promoters containing specific REs can be selected to obtain an inducible response, and in some cases, the REs themselves can be attached to different promoters, thereby conferring inducibility to the recombinant gene. Thus, by selecting the appropriate promoters (constitutive vs. inducible promoters, strong vs. weak promoters), it is possible to control both the presence and expression levels of therapeutic agents in genetically modified cells. When a gene encoding a therapeutic agent is under the control of an inducible promoter, delivery of the therapeutic agent in situ can be triggered by exposing the genetically modified cells in situ to conditions to allow transcription of the therapeutic agent, for example, by injection of a specific inducer of the inducible promoter that controls transcription of the agent. For example, in situ expression by genetically modified cells of a therapeutic agent encoded by a gene under the control of a metallothionein promoter is enhanced by contacting the genetically modified cells in situ with a solution containing the appropriate (i.e., inducing) metal ion.

[0084] Thus, the amount of therapeutic agent delivered in situ is regulated by controlling factors such as: (1) the nature of the promoter used to direct transcription of the inserted gene (i.e., whether the promoter is constitutive or inducible, strong or weak), (2) the number of copies of the exogenous gene inserted into the cells, (3) the number of transduced / transfected cells administered (e.g., implanted) to the patient, (4) the size of the implant (e.g., graft or encapsulated expression system), (5) the number of implants, (6) the length of time the transduced / transfected cells or implants are left in place, and (7) the rate of production of the therapeutic agent by the genetically modified cells. Selection and optimization of these factors for delivery of a therapeutically effective dose of a particular therapeutic agent is deemed to be within the scope of one of ordinary skill in the art without undue experimentation, taking into account the factors disclosed above and the clinical profile of the patient.

[0085] In addition to at least one promoter and at least one heterologous nucleic acid encoding a therapeutic agent, the expression vector may contain a selection gene, such as a neomycin resistance gene or a fluorescent protein gene, to facilitate the selection of cells transfected or transduced with the expression vector. Alternatively, cells are transfected with two or more expression vectors, at least one vector containing a gene encoding a therapeutic agent and the other vector containing a selection gene. Selection of a suitable promoter, enhancer, selection gene, and / or signal sequence is deemed to be within the scope of one of ordinary skill in the art without undue experimentation.

[0086] The coding sequence of the present disclosure can be inserted into any type of target cell or host cell. In the context of an expression vector, the vector can be easily introduced into a host cell, such as a mammalian, bacterial, yeast, or insect cell, by any method in the art. For example, the expression vector can be transferred into the host cell by physical, chemical, or biological means.

[0087] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0088] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0089] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0090] As disclosed herein, the above-mentioned transcription factor 7-like 2 protein can be used to treat a disorder in a subject. In some embodiments, the polynucleotide encoding the protein can be inserted into or encoded by a vector, such as a plasmid or a viral vector. Preferably, the polynucleotide is inserted into or encoded by a viral vector. A variety of virus-derived vectors can be used for transfection and integration into the mammalian cell genome. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses (AAV), herpes viruses, and lentiviruses. In some embodiments, the protein can be encoded by a retroviral vector, such as a lentiviral vector (see, for example, U.S. Patent Nos. 5,399,346, 5,124,263, 4,650,764, and 4,980,289, the contents of each of which are incorporated herein by reference in their entirety). In some specific embodiments, the viral vector is an AAV vector.

[0091] Lentiviral Vectors Lentiviruses, such as HIV, are "slow viruses." Vectors derived from lentiviruses can be expressed in host cells for long periods of time, for example, via ex vivo transducing stem or progenitor cells, after several doses in patients. For most diseases and disorders, including genetic diseases, cancer, and neurological diseases, long-term expression is essential for successful treatment. With regard to the safety of lentiviral vectors, several strategies are currently known in the art to eliminate the replication capacity of lentiviral vectors. See, for example, US2021 / 0401868 and 2021 / 0403517, each of which is incorporated herein by reference in its entirety. For example, deletion of promoter and enhancer elements from the U3 region of the long terminal repeat (LTR) would result in no LTR-directed transcription. The resulting vector is called "self-inactivating" (SIN).

[0092] Lentiviral vectors are particularly suitable for achieving long-term gene transfer, since they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as CNS cells. They also have the additional advantage of being less immunogenic. In general, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction nuclease sites, and one or more selection markers (e.g., WO01 / 96584 and WO01 / 29058, and U.S. Patent No. 6,326,193). Several vector promoter sequences are available for the expression of the transgene. One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is EF1a. However, other constitutive promoter sequences can also be used, including, but not limited to, Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters (such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter). Inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0093] The present disclosure provides recombinant lentiviruses capable of infecting dividing and non-dividing cells, such as oligodendrocytes or oligodendrocyte precursor cells. The viruses are useful for transferring and expressing nucleic acid sequences in vivo and ex vivo. The lentiviral vectors of the present disclosure can be lentiviral transfer plasmids or infectious lentiviral particles. Constructs such as lentiviral vectors, helper constructs, envelope constructs, etc. for use in lentiviral delivery systems are described, for example, in US2021 / 0401868 and 2021 / 0403517, each of which is incorporated herein by reference in its entirety.

[0094] Adenovirus Adenovirus is a eukaryotic DNA virus that can be modified to efficiently deliver nucleic acid to various cell types in vivo, and is widely used in gene therapy protocols, including those for targeting genes to neuronal and glial cells.For nucleic acid therapy, various replication-defective adenoviruses and minimal adenovirus vectors have been described (see, for example, PCT Patent Publication Nos. 1994 / 26914, 1995 / 02697, 1994 / 28152, 1994 / 12649, 1995 / 02697 and 1996 / 22378, each of which is incorporated by reference in its entirety).Such adenovirus vectors can also be used to deliver therapeutic molecules of the present disclosure to cells.

[0095] Adeno-associated virus Adeno-associated viruses are widely used gene therapy vectors due to their clinical safety record, non-pathogenicity, ability to infect non-dividing cells (such as neurons), and ability to provide long-term gene expression after a single administration. Currently, many human and non-human primate AAV serotypes have been identified. AAV vectors have demonstrated safety in hundreds of clinical trials worldwide, and clinical efficacy has been shown in trials for hemophilia B, spinal muscular atrophy, alpha-1 antitrypsin, and Leber congenital amaurosis.

[0096] Due to their safety, non-pathogenicity, and ability to infect neurons, AAVs such as AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, and AAV9 are commonly used gene therapy vectors for CNS applications. However, after direct CNS injection, these serotypes exhibit dominant neuronal tropism and expression in oligodendrocytes is low, especially when gene expression is driven by a constitutive promoter, limiting their potential use in treating white matter diseases. AAV1 / 2, AAV2, and AAV8 have been shown to transduce oligodendrocytes. The reliance on cell-specific promoters for expression specificity allows for non-selective cellular uptake and the possibility of leaky transgene expression via cryptic promoter activity in non-oligodendrocyte lineage cells.

[0097] The approach described herein to alleviate these problems involves using AAV serotypes with high tropism for oligodendrocytes or glial progenitor cells, e.g., glial progenitor cells such as oligodendrocyte progenitors. Recently, using DNA shuffling and directed evolution, a chimeric AAV capsid, AAV / Olig001, with strong selectivity for oligodendrocytes has been described (Powell et al., 2016, Gene Ther 23:807-814). AAV / Olig001 was subsequently shown to transduce neonatal oligodendrocytes in a mouse model of Canavan disease (Francis et al., 2021. Mol Ther Methods Clin Dev 20:520-534). Other approaches, such as random mutagenesis and peptide library insertion, can be used to generate capsid libraries that can be screened for tropism and selectivity for oligodendrocytes or glial progenitor cells.

[0098] As mentioned above, the term "adeno-associated virus" and / or "AAV" refers to a parvovirus with a linear single-stranded DNA genome and variants thereof. The term encompasses all subtypes and both naturally occurring and recombinant forms, unless otherwise required. Parvoviruses, including AAV, are useful as gene therapy vectors because they can enter cells and introduce nucleic acids (e.g., transgenes) into the nucleus. In some embodiments, the introduced nucleic acid (e.g., rAAV vector genome) forms circular concatemers that persist as episomes in the nucleus of the transduced cell. In some embodiments, the transgene is inserted into a specific site within the host cell genome. Site-specific integration is believed to be more likely to result in a predictable long-term expression profile, as opposed to random integration. The insertion site of AAV into the human genome is referred to as AAVS1. Once introduced into a cell, the RNA or polypeptide encoded by the nucleic acid can be expressed by the cell. Because AAV is not associated with any pathogenic disease in humans, nucleic acids delivered by AAV can be used to express therapeutic RNAs or polypeptides for the treatment of diseases, disorders, and / or conditions in human subjects.

[0099] Multiple serotypes of AAV exist in nature, and at least 15 wild-type serotypes have been identified in humans to date (i.e., AAV1-AAV15). Naturally occurring and variant serotypes are distinguished from other AAV serotypes by possessing serologically distinct protein capsids. Examples include AAV1, AAV2, AAV, AAV3 (including AAV3A and AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV12, AAVrhlO, AAVrh74 (see WO2016 / 210170), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, as well as recombinantly produced variants (such as capsid variants with insertions, deletions, and substitutions), such as the variants designated AAV2i8, NP4, NP22, NP66, DJ, DJ / 8, DJ / 9, LK3, RHM4-1, among others. For example, "primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, and "bovine AAV" refers to AAV that infects bovine mammals.

[0100] Serotype uniqueness is determined based on the lack of cross-reactivity between antibodies against one AAV compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences and antigenic determinants (e.g., due to differences in VP1, VP2, and / or VP3 sequences of AAV serotypes). However, some naturally occurring AAV or artificial AAV variants (e.g., recombinant AAV) may not show serological differences with any of the currently known serotypes. These viruses can then be considered as subgroups of the corresponding type, or more simply, as variant AAV. Thus, as used herein, the term "serotype" refers to both serologically distinct viruses, as well as viruses that are not serologically distinct but may be within a subgroup or variant of a given serotype.

[0101] A comprehensive list and alignment of the amino acid sequences of the capsids of known AAV serotypes is provided by Marsic et al. (2014) Molecular Therapy 22(11):1900-1909. The genomic sequences of various serotypes of AAV, as well as the sequences of the native ITRs, rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Nos. NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2), AF043303 (AAV2), NC_001729 (AAV3), NC_001863 (AAV3B), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), NC_001862 (AAV6), AF513851 (AAV7), AF513852 (AAV8), and NC_006261 (AAV8), the disclosures of which are incorporated herein by reference. For example, Srivistava et al. (1983) J. Virology 45:555, Chiorini et al. (1998) J. Virology 71:6823, Chiorini et al. (1999) J. Virology 73:1309, Bantel-Schaal et al. (1999) J. Virology 73:939, Xiao et al. al. (1999) J. Virology 73:3994, Muramatsu et al. (1996) Virology 221:208, Shade et al. (1986) J. Virol. 58:921, Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854, Morris et al. (2004) Virology 33:375-383, International Patent Publication Nos. 00 / 28061, 99 / 61601, 98 / 11244, 2013 / 063379, 2014 / 194132, 2015 / 121501, and U.S. Pat. Nos. 6,156,303 and 7,906,111.

[0102] As discussed herein, "recombinant adeno-associated virus" or "rAAV" is distinguished from wild-type AAV by replacing all or part of the endogenous viral genome with a non-native sequence. The incorporation of a non-native sequence into the virus defines the viral vector as a "recombinant" vector, and thus an "rAAV vector." The rAAV vector may contain a heterologous polynucleotide encoding a desired RNA or protein or polypeptide (e.g., an RNA molecule disclosed herein). The recombinant vector sequence may be encapsidated or packaged into an AAV capsid and is referred to as a "rAAV vector," "rAAV vector particle," "rAAV viral particle," or simply "rAAV."

[0103] The present disclosure provides rAAV vectors that include polynucleotide sequences that are not of AAV origin (e.g., polynucleotides that are heterologous to AAV). The heterologous polynucleotide may be flanked by at least one, and possibly two, AAV terminal repeat sequences (e.g., inverted terminal repeats). The heterologous polynucleotide flanked by ITRs, also referred to herein as the "vector genome", typically encodes a gene of interest, such as an RNA or a polypeptide of interest, or a target for therapeutic treatment. Delivery or administration of the rAAV vector to a subject (e.g., a patient) provides the encoded RNA / protein / peptide to the subject. Thus, the rAAV vector can be used to transcribe / deliver heterologous polynucleotides for expression, for example, to treat various diseases, disorders, and conditions.

[0104] rAAV vector genomes generally retain 145 bases of ITRs in cis to the heterologous nucleic acid sequences that replace the viral rep and cap genes. Such ITRs are useful for producing recombinant AAV vectors, although non-AAV terminal repeats, including modified AAV ITRs and partially or completely synthetic sequences, can also serve this purpose. The ITRs form hairpin structures and function as primers for host cell-mediated synthesis of complementary DNA strands, for example, after infection. The ITRs also play a role in viral packaging, integration, and the like. The ITRs are the only AAV viral elements required in cis for AAV genome replication and packaging into rAAV vectors. The rAAV vector genomes contain two ITRs, which are typically present at the 5' and 3' ends of the vector genome, that optionally contain heterologous sequences (e.g., a transgene encoding a gene of interest, or a nucleic acid sequence of interest, including but not limited to antisense and siRNA, and CRISPR molecules, among others). The 5' and 3' ITRs may both contain the same sequence, or each may contain a different sequence. The AAV ITRs can be derived from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, or any other AAV.

[0105] The rAAV vectors of the present disclosure may comprise ITRs from an AAV serotype (e.g., wild-type AAV2, fragments or variants thereof) that is different from the serotype of the capsid (e.g., AAV8, Olig001). Such rAAV vectors that comprise at least one ITR from one serotype but a capsid from a different serotype may be referred to as hybrid viral vectors (see U.S. Patent No. 7,172,893). The AAV ITRs may comprise the entire wild-type ITR sequence or may be variants, fragments or modifications thereof, but retain functionality.

[0106] In some embodiments, the rAAV vector genome is linear, single-stranded, and flanked by AAV ITRs. Prior to transcription and translation of the heterologous gene, the free 3'-OH of one of the self-priming ITRs must be used by a DNA polymerase (e.g., a DNA polymerase in the transduced cell) to convert the approximately 4700 nucleotide single-stranded DNA genome into a double-stranded form to initiate second strand synthesis. In some embodiments, the full-length single-stranded vector genome (i.e., sense and antisense) anneals to generate a full-length double-stranded vector genome. This can occur when multiple rAAV vectors carrying genomes of opposite polarity (i.e., sense or antisense) transduce the same cell at the same time. Regardless of how they are produced, once the double-stranded vector genome is formed, the cell can transcribe and translate the double-stranded DNA and express the heterologous gene.

[0107] The efficiency of transgene expression from rAAV vectors can be hindered by the need to convert single-stranded rAAV genomes (ssAAV) to double-stranded DNA prior to expression. This step can be avoided by using self-complementary AAV genomes (scAAV) that can package inverted repeat genomes that can fold into double-stranded DNA without the need for DNA synthesis or base pairing between multiple vector genomes. See, for example, U.S. Patent No. 8,784,799; McCarty, (2008) Molec. Therapy 16(10):1648-1656; and McCarty et al., (2001) Gene Therapy 8:1248-1254; McCarty et al., (2003) Gene Therapy 10:2112-2118.

[0108] The viral capsid of the rAAV vector may be a wild-type AAV or a variant AAV, such as AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAVrh74 (see WO2016 / 210170), AAV12, AAV2i8, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, RHM4-1 (SEQ ID NO: 5 in WO2015 / 013313), RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, RHM15-6, AAV The AAV vector may be derived from hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9,45, AAV2i8, AAV29G, AAV2,8G9, AVV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAV avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, snake AAV, caprine AAV, shrimp AAV, ovine AAV and variants thereof (see, e.g., Fields et al., VIROLOGY, volume 2, chapter 69(4 th ed., Lippincott-Raven Publishers). The capsid may be derived from several AAV serotypes disclosed in U.S. Pat. No. 7,906,111, Gao et al. (2004) J. Virol. 78:6381, Morris et al. (2004) Virol. 33:375; WO2013 / 063379; WO2014 / 194132, including the true type AAV (AAV-TT) variant disclosed in WO2015 / 121501, and RHM4-1, RHM15-1 through RHM15-6, and variants thereof, disclosed in WO2015 / 013313. The full complement of AAV cap proteins includes VP1, VP2, and VP3. An ORF comprising a nucleotide sequence encoding an AAV VP capsid protein may contain less than the full complement AAV Cap protein, or the full complement of AAV cap proteins may be provided.

[0109] In some embodiments, an rAAV vector that comprises capsid proteins encoded by nucleotide sequences from two or more AAV serotypes (e.g., wildtype AAV serotypes, variant AAV serotypes) is referred to as a "chimeric vector" or "chimeric capsid" (see U.S. Pat. No. 6,491,907, the disclosure of which is incorporated herein by reference in its entirety). In some embodiments, the chimeric capsid protein is encoded by nucleic acid sequences from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more AAV serotypes. In some embodiments, the recombinant AAV vector comprises capsid sequences derived from, for example, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh74, AAVrh10, AAV2i8, or variants thereof, resulting in a chimeric capsid protein comprising a combination of amino acids from any of the aforementioned AAV serotypes (see Rabinowitz et al. (2002) J. Virology 76(2):791-801). Alternatively, the chimeric capsid may comprise a VP1 from one serotype, a VP2 from a different serotype, a VP3 from yet a different serotype, and a mixture of combinations thereof. For example, the chimeric viral capsid may comprise an AAV1 cap protein or subunit, and at least one AAV2 cap protein or subunit. The chimeric capsid can include, for example, an AAV capsid having one or more B19cap subunits, e.g., the AAV cap protein or subunit can be replaced by a B19 cap protein or subunit. For example, in one embodiment, the VP3 subunit of the AAV capsid can be replaced by the VP2 subunit of B19. In some embodiments, the chimeric capsid is an Olig001 capsid as described in WO2021 / 221995 and WO2014 / 052789 (incorporated herein by reference).

[0110] In some embodiments, the chimeric vector is engineered to exhibit altered tropism or tropism for a particular tissue or cell type. The term "tropism" refers to preferential entry of the virus into a particular cell (e.g., oligodendrocyte) or tissue type, and / or preferential interaction with the cell surface that facilitates entry into a particular cell or tissue type. AAV tropism is generally determined by specific interactions between different viral capsid proteins and their cognate cellular receptors (Lykken et al. (2018) J. Neurodev. Disord. 10:16). Preferably, once the virus or viral vector enters the cell, sequences (e.g., heterologous sequences such as transgenes) carried by the vector genome (e.g., rAAV vector genome) are expressed.

[0111] "Tropical profile" refers to a pattern of transduction of one or more target cells in various tissues and / or organs. For example, a chimeric AAV capsid may have a tropism profile characterized by efficient transduction of oligodendrocytes or oligodendrocyte precursor cells with only low transduction of neurons, astrocytes and other CNS cells. See WO2014 / 052789, incorporated herein by reference. Such chimeric capsids may be considered "oligodendrocyte or oligodendrocyte precursor cell specific" that exhibits tropism for oligodendrocytes or oligodendrocyte precursor cells, and are referred to herein as "oligotropic" when they preferentially transduce oligodendrocytes or oligodendrocyte precursor cells over neurons, astrocytes and other CNS cell types when administered directly to the CNS. In some embodiments, at least about 80% of the cells transduced with the oligodendrocyte or oligodendrocyte precursor cell specific capsid are oligodendrocytes or oligodendrocyte precursor cells, e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the transduced cells are oligodendrocytes or oligodendrocyte precursor cells.

[0112] In some embodiments, the rAAV vectors are useful for treating or preventing "disorders associated with oligodendrocyte dysfunction." As used herein, the term "associated with oligodendrocyte dysfunction" refers to a disease, disorder, or condition in which oligodendrocytes are damaged, lost, or function inappropriately compared to otherwise identical normal oligodendrocytes. This term includes diseases, disorders, and conditions in which oligodendrocytes are directly affected, as well as diseases, disorders, or conditions in which oligodendrocytes malfunction secondary to damage to other cells. In some embodiments, the disorder associated with oligodendrocyte dysfunction is demyelination.

[0113] 4. Use and Treatment Methods Gene therapy The nucleic acids, gene constructs, expression cassettes, and expression vectors described herein can be used for gene therapy treatment and / or prevention of diseases, disorders, or conditions. In particular, it can be used to treat or prevent diseases, disorders, or conditions associated with oligodendrocyte or myelin deficiency or dysfunction by increasing the expression of transcription factor 7-like 2 protein, as well as any other condition and / or disease in which increasing the expression of the protein can provide therapeutic benefit or improvement, such as diseases, disorders, or conditions mediated by or associated with a decrease in the level or function of the protein, as compared to the level or function of the protein in otherwise healthy individuals.

[0114] As used herein, myelin disorders, myelin diseases, myelin-related disorders, myelin-related diseases, myelin disorders, disorders mediated by myelin deficiency, and myelin diseases are used interchangeably. These include any disease, condition (e.g., those resulting from traumatic spinal cord injury and cerebral infarction) or disorder in a subject associated with demyelination, insufficient myelination and remyelination, or hypomyelination. Such disorders may be inherited, acquired, or both. It may result from myelination-related disorders or demyelination due to various neurotoxic insults. As used herein, "demyelination" refers to the act of demyelination, or loss of the myelin sheath that insulates nerves, and is a feature of several neurodegenerative autoimmune diseases, including multiple sclerosis, transverse osteomyelitis, chronic inflammatory demyelinating polyneuropathy, and Guillain-Barre syndrome. Leukodystrophies are caused by inherited enzyme deficiencies that cause abnormal formation, destruction, and / or abnormal turnover of myelin sheaths within the CNS white matter. Both acquired and inherited myelin disorders have poor prognosis leading to severe disability. Thus, some embodiments of the present disclosure may include a method of treating a neurodegenerative autoimmune disease in a subject. Remyelination of neurons requires oligodendrocytes. As used herein, the term "remyelination" refers to the regeneration of the myelin sheath of nerves by replacing or restoring the function of myelin-producing cells.

[0115] Myelin-related diseases or disorders that may be treated or ameliorated by the methods of the invention include diseases, disorders or injuries associated with hypomyelination or demyelination in brain cells, e.g., CNS neurons, of a subject. Such diseases include, but are not limited to, diseases and disorders in which the myelin sheath surrounding neurons is absent, incomplete, not properly formed, or deteriorated. Such diseases include, but are not limited to, multiple sclerosis (MS), neuromyelitis optica (NMO), progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelinolysis (CPM), adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease (PMD), Wallerian degeneration, optic neuritis, transverse osteomyelitis, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, post-radiation injury, neurological complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, acute disseminated encephalitis, Guillian-Barre syndrome, Marie-Charcot-Tooth disease, and Bell's palsy.

[0116] Myelin-related diseases or disorders that may be treated or ameliorated by the methods of the present invention include diseases or disorders characterized by myelin deficiency. Insufficient myelination in the central nervous system has been implicated in a wide range of neurological disorders. Among these are forms of cerebral palsy in which congenital deficit in forebrain myelination in children with periventricular leukomalacia contributes to neurological morbidity (Goldman et al., 2008). Goldman, SA, Schanz, S., and Windrem, MS (2008). Stem cell-based strategies for treating pediatric disorders of myelin. Hum Mol Genet. 17, R76-83. At the other end of the age spectrum, myelin loss and ineffective repair may contribute to aging-related cognitive decline (Kohama et al., 2011) Kohama, SG, Rosene, DL, and Sherman, LS (2011) Age (Dordr). Age-related changes in human and non-human primate white matter: from myelination disturbances to cognitive decline. It is therefore contemplated that effective compositions and methods that enhance myelination and / or remyelination could have substantial therapeutic benefit in halting disease progression and restoring function in a wide range of myelin-related disorders.

[0117] In some embodiments, the compositions of the present invention may be administered to subjects who do not have and / or are not suspected of having a myelin-related disorder to enhance or promote myelin-dependent processes. In some embodiments, the compositions described herein may be administered to subjects to promote myelination of CNS neurons to enhance cognition, which is known to be a myelin-dependent process in cognitively healthy subjects. In certain embodiments, the compositions described herein may be administered in combination with a cognitive enhancing (nootropic) agent. Exemplary agents include any drug, supplement, or other substance that improves cognitive function in healthy individuals, particularly executive function, memory, creativity, or motivation. Non-limiting examples include racetams (e.g., piracetam, oxiracetam, and aniracetam), dietary supplements (e.g., bacopa monnieri, panax ginseng, ginko biloba, and GABA), stimulants (e.g., amphetamine medications, methylphenidate, eugeroics, xanthines, and nicotine), L-theanine, tolcapone, levodopa, atomoxetine, and desipramine.

[0118] The total dosage of a therapeutic agent (e.g., a protein, a polynucleotide encoding a protein, or a vector such as an rAAV vector or cell) will be a therapeutically effective amount depending on several factors, including the subject's overall health, the subject's disease state, the severity of the condition, the observation of improvement, and the selected formulation and route of administration of the agent. Determining a therapeutically effective amount is within the capabilities of one of ordinary skill in the art. The exact formulation, route of administration, and dosage can be selected by the individual physician, taking into account the subject's condition.

[0119] In certain embodiments, the cells or nucleotide compositions described herein increase the amount of one or more myelin proteins (e.g., MBP, MAG, MOG, MOBP, PLP1, GPR37, ASPA, CNP, MYRF, BCAS1, PLP1, UGT8, TF, LPAR1, and FA2H) by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 100%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 8 The compound may be administered in an amount effective to enhance myelin production in the CNS of a subject by increasing myelin production by 0%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000%.

[0120] In other embodiments, the cell or nucleotide compositions may be administered in an amount effective to promote survival of CNS neurons in a subject by increasing the number of surviving neurons by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% compared to the number of surviving CNS neurons or neurons in an untreated CNS neuron or subject.

[0121] Another strategy for treating a subject suffering from a myelin-related disorder is to administer a therapeutically effective amount of a cell or nucleotide composition described herein together with a therapeutically effective amount of an oligodendrocyte differentiation and / or proliferation inducer and / or an anti-neurodegenerative disease agent. Examples of anti-neurodegenerative disease agents include L-dopa, cholinesterase inhibitors, anticholinergics, dopamine agonists, steroids, and immunomodulators including interferons, monoclonal antibodies, and glatiramer acetate. Thus, in a further aspect of the present disclosure, the compositions described herein can be administered as part of a combination therapy with adjunctive therapy for treating neurodegenerative and myelin-related disorders.

[0122] The phrase "combination therapy" encompasses administration of the oligodendrocyte precursor differentiation-inducing compositions described herein and therapeutic agents as part of a specific treatment regimen intended to provide beneficial effects from the synergistic action of these therapeutic agents. When administered in combination, the oligodendrocyte precursor differentiation-inducing compounds and therapeutic agents can be formulated as separate compositions. The administration of these combined therapeutic agents is typically carried out over a defined period of time (usually minutes, hours, days, or weeks, depending on the combination selected).

[0123] The genetic nucleic acids, genetic constructs, expression cassettes, and expression vectors of the present application may be administered by intracerebral delivery, intrathecal delivery, intranasal delivery, or via direct injection into the ventricles of the brain.

[0124] The genetic construct of the present application can also be administered directly to the airways in the form of an aerosol.For use as an aerosol, the compound of the present application in solution or suspension can be packaged in a pressurized aerosol container with a suitable propellant, for example, a hydrocarbon propellant such as propane, butane, or isobutane with conventional adjuvants.The material of the present application can also be administered in a non-pressurized form, such as a nebulizer or atomizer.

[0125] In one embodiment, the one or more genetic constructs activate transcription of one or more of the genes described herein via CRISPR-Cas9 guided nucleases (Gimenez et al., "CRISPR-on System for the Activation of the Endogenous human INS gene," Gene Therapy 23:543-547 (2016); Wiedenheft et al., "RNA-Guided Genetic Silencing Systems in Bacteria and Archaea," Nature 482:331-338 (2012); Zhang et al., "Multiplex Genome Engineering Using CRISPR / Cas Systems," Science 339(6121):819-23 (2013); and Gaj et al., "ZFN, TALEN, and CRISPR / Cas-based Methods for Genome Engineering," Cell 20:111-115 (2013)). 31(7):397-405 (2013), which is incorporated herein by reference in its entirety. CRISPR-Cas9 is a genetic technology that allows sequence-specific control of gene expression in prokaryotic and eukaryotic cells by inducible nuclease double-stranded DNA breaks. It is based on the CRISPR (clustered regularly interspaced palindromic repeats) pathway derived from the bacterial immune system.

[0126] In the above-described embodiments, one or more gene constructs can be packaged into a suitable delivery vehicle or carrier for delivery to a subject. Suitable delivery vehicles include, but are not limited to, viruses, virus-like particles, bacteria, bacteriophages, biodegradable microspheres, microparticles, nanoparticles, exosomes, liposomes, collagen minipellets, and cochleates. These and other biological gene delivery vehicles are well known to those skilled in the art (see, for example, Seow and Wood, "Biological Gene Delivery Vehicles: Beyond Viral Vectors," Mol. Therapy 17(5):767-777 (2009), which is incorporated herein by reference in its entirety).

[0127] In one embodiment, the genetic construct is packaged into a therapeutic expression vector to facilitate delivery. Suitable expression vectors are well known in the art and include, but are not limited to, viral vectors such as adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, or herpes viral vectors.

[0128] The viral vector or other suitable expression vector comprises a sequence encoding the genetic construct of the present application and any suitable promoter and / or enhancer for expressing the genetic construct. Suitable promoters include, for example, but are not limited to, U6 or HI RNA pol III promoter sequences and cytomegalovirus promoters. Selection of other suitable promoters is within the skill of the art. The expression vector may also comprise an inducible or regulatable promoter for expression of the inhibitory nucleic acid molecule in a tissue or cell specific manner.

[0129] Gene therapy vectors carrying therapeutic gene constructs or nucleic acid molecules are administered to a subject, for example, by intravenous injection, local administration (U.S. Patent No. 5,328,470 to Nabel et al., incorporated herein by reference in its entirety), or by stereotactic injection (see, e.g., Chen et al., "Gene Therapy for Brain Tumors: Regression of Experimental Gliomas by Adenovirus Mediated Gene Transfer In vivo," Proc. Nat'l. Acad. Sci. USA 91:3054-3057 (1994), incorporated herein by reference in its entirety). Pharmaceutical preparations of therapeutic vectors can include the therapeutic vector in an acceptable diluent or can comprise a slow release matrix in which the therapeutic delivery vehicle is embedded. Alternatively, where the complete therapeutic delivery vector can be produced intact from recombinant cells, e.g., in the case of retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the therapeutic delivery system. Gene therapy vectors typically utilize constitutive regulatory elements that are responsive to endogenous transcription factors.

[0130] Another suitable approach for delivery of the genetic constructs of the present disclosure involves the use of liposomal or nanoparticle delivery vehicles.

[0131] In another embodiment of the present application, the delivery vehicle is a nanoparticle. A variety of nanoparticle delivery vehicles are known in the art and are suitable for delivery of the genetic constructs of the present application (see, for example, van Vlerken et al., "Multi-functional Polymeric Nanoparticles for Tumour-Targeted Drug Delivery," Expert Opin. Drug Deliv. 3(2):205-216 (2006), which is incorporated herein by reference in its entirety).Suitable nanoparticles include poly(beta amino esters) (Sawicki et al., “Nanoparticle Delivery of Suicide DNA for Epithelial Ovarian Cancer Cell Therapy,” Adv. Exp. Med. Biol. 622:209-219 (2008) (hereby incorporated by reference in its entirety)), polyethyleneimine-alt-poly(ethylene glycol) copolymers (Park et al., “Degradable Polyethylenimine-alt-Poly(ethylene glycol) Copolymers As Novel Gene Carriers,” J. Control Release 105(3):367-80 (2005) and Park et al., “Intratumoral Administration of Anti-KITENIN shRNA-Loaded PEI-alt-PEG Nanoparticles Suppressed Colon Carcinoma Established Subcutaneously in Mice,” J Nanosci. Nanotechnology 10(1):111-115 (2005)). 10(5):3280-3(2010) (incorporated herein by reference in its entirety), poly(d,l-lactide-coglycolide) (Chan et al., "Antisense Oligonucleotides: From Design to Therapeutic Application," Clin. Exp. Pharm. Physiol. 33:533-540(2006) (incorporated herein by reference in its entirety)), and liposome-entrapped siRNA nanoparticles (Kenny et al., "Novel Multifunctional Nanoparticle Mediates siRNA Tumor Delivery, Visualization and Therapeutic Tumor Reduction In vivo," J. Control Release 149(2):111-116(2011) (incorporated herein by reference in its entirety)).Other nanoparticle vehicles suitable for use in the present application include the microcapsule nanotube devices disclosed in U.S. Patent Publication No. 2010 / 0215724 to Prakash et al., which is incorporated by reference in its entirety.

[0132] In another embodiment, the genetic construct is contained in a liposomal delivery vehicle. The term "liposome" refers to a vesicle consisting of a spherical bilayer or amphiphilic lipids arranged in a bilayer. Liposomes are unilamellar or multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage of being able to fuse with the cell wall. Non-cationic liposomes cannot fuse efficiently with the cell wall, but are taken up by macrophages in vivo.

[0133] Some advantages of liposomes include their biocompatibility and biodegradability, the incorporation of a wide range of water- and lipid-soluble drugs, and the protection they provide of encapsulated molecules from metabolism and degradation. Important considerations in the preparation of liposomal formulations are the lipid surface charge, vesicle size, and the aqueous volume of the liposomes.

[0134] Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because liposome membranes are structurally similar to biological membranes, when liposomes are applied to tissues, they begin to merge with cell membranes, and as the merger of liposomes with cells progresses, the contents of the liposomes are discharged into the cells where the active agent can act.

[0135] Methods for preparing liposomes include those disclosed in Bangham et al., "Diffusion of Univalent Ions Across the Lamellae of Swollen Phospholipids," J. Mol. Biol. 13:238-52 (1965), U.S. Patent No. 5,653,996 to Hsu, U.S. Patent No. 5,643,599 to Lee et al., U.S. Patent No. 5,885,613 to Holland et al., U.S. Patent No. 5,631,237 to Dzau et al., and U.S. Patent No. 5,059,421 to Loughrey et al., which are incorporated by reference in their entireties.

[0136] As disclosed herein, in another embodiment, the genetic construct, expression cassette, or expression vector may be administered in association with a glial progenitor cell-targeted fusogen or glial progenitor cell-selective surface-binding moiety, e.g., the genetic construct, expression cassette, or expression vector may be present in or associated with a fusosome.

[0137] As used herein, a "fusogen" refers to an agent or molecule that creates an interaction between two membrane-bound lumens. In embodiments, the fusogen facilitates fusion of the membranes. In other embodiments, the fusogen creates a connection, e.g., a pore, between two lumens (e.g., the lumen of a liposome and the cytoplasm of a target cell, or the lumen of a viral vector and the cytoplasm of a target cell). In some embodiments, the fusogen comprises a protein or a complex of two or more proteins having a targeting domain or binding moiety. In some examples, the targeting domain or binding moiety specifically targets or binds to a molecule on a glial progenitor cell or a glial progenitor cell. Examples of molecules include, but are not limited to, CD140a, NG2 / CSPG4, A2B5 ganglioside, O4 sulfatide, or CD133. The targeting domain or binding moiety can be a receptor ligand, a peptide / polypeptide, an antibody, or an antigen-binding portion thereof that specifically binds to a molecule or marker on a glial progenitor cell or a glial progenitor cell. Non-limiting examples of human and non-human fusogens are described, for example, in US2021 / 0198698 and US2021 / 0137839, which are incorporated by reference in their entireties.

[0138] As used herein, "fusosome" refers to a bilayer of amphipathic lipids that surrounds a lumen or cavity and a fusogen that interacts with the amphipathic lipid bilayer. In some embodiments, fusosomes contain nucleic acid. In some embodiments, fusosomes are membrane-encapsulated preparations. In some embodiments, fusosomes are derived from a source cell.

[0139] Fusosomes can take a variety of forms. For example, in some embodiments, the fusosomes described herein are derived from a source cell. The fusosomes may be or include, for example, extracellular vesicles, microvesicles, nanovesicles, exosomes, microparticles, or any combination thereof. In some embodiments, the fusosomes are naturally released from the source cell, and in some embodiments, the source cell is treated to enhance the formation of fusosomes. In some embodiments, the fusosomes are about 10-10,000 nm in diameter, e.g., about 30-100 nm in diameter. In some embodiments, the fusosomes include one or more synthetic lipids.

[0140] In some embodiments, the fusosome is or comprises a virus, e.g., a retrovirus, e.g., a lentivirus. For example, in some embodiments, the fusosome bilayer of amphipathic lipids is or comprises a viral envelope. The viral envelope can comprise a fusogen, e.g., a fusogen endogenous to the virus, or a pseudotyped fusogen. In some embodiments, the lumen or cavity of the fusosome comprises a viral nucleic acid, e.g., a retroviral nucleic acid, e.g., a lentiviral nucleic acid. The viral nucleic acid can be a viral genome. In some embodiments, the fusosome further comprises one or more viral nonstructural proteins, e.g., in its cavity or lumen.

[0141] Fusosomes can have a variety of structures or properties that facilitate delivery of a payload to a target cell. For example, in some embodiments, the fusosome and the source cell together contain sufficient nucleic acid to create a particle that can fuse with a target cell. In embodiments, these nucleic acids encode proteins that have one or more (e.g., all) of the following activities: gag polyprotein activity, polymerase activity, integrase activity, protease activity, and fusogenic activity.

[0142] cell therapy Also within the scope of the present disclosure are host cells, including the genetic constructs, cassettes, or expression vectors described above, or progeny cells of the host cells. The host cells can be stem cells or progenitor cells. Examples of stem cells include embryonic stem cells, ES-like stem cells, fetal stem cells, adult stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells, and the like. In some embodiments, the host cells are glial progenitor cells, e.g., oligodendrocyte progenitor cells. The host cells or their progeny can be used as therapeutic cells or drugs to treat the disorders or conditions described herein.

[0143] Suitable methods for introducing cells (such as the host cells described above or their progeny) into the striatum, forebrain, brainstem, and / or cerebellum of a subject are well known to those of skill in the art and include, but are not limited to, injection, deposition, and transplantation as described herein.

[0144] In one embodiment, glial progenitor cells are bilaterally transplanted into multiple sites in a subject as described in U.S. Patent No. 7,524,491 to Goldman; Windrem et al., "Neonatal Chimerization With Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2:553-565 (2008); Han et al., "Forebrain Engraftment by Human Glial Progenitor Cells Enhances Synaptic Plasticity and Learning Adult Mice," Cell Stem Cell 12:342-353 (2013); and Wang et al., "Human iPSCs-Derived Oligodendrocyte Progenitor Cells Can Myelinate and Rescue a Mouse Model of Congenital Hypomyelination," Cell Stem Cell 12:252-264 (2013). Methods for grafting neural tissue and cells into a host brain are described by Neural Grafting in the Mammalian CNS, Ch. 3-8 (Bjorklund and Stenevi eds., Elsevier, Amsterdam 1985), U.S. Patent No. 5,082,670 to Gage et al., and U.S. Patent No. 6,497,872 to Weiss et al., which are incorporated by reference in their entireties. Typical procedures include intraparenchymal, intracallosal, intrathecal, intracerebral, intranasal, and intravenous transplants, as well as direct injection into the ventricles.

[0145] Intraparenchymal transplantation is achieved by injection or deposition of tissue in the host brain so that it will attach to the brain parenchyma upon transplantation. The two main procedures for intraparenchymal transplantation are: 1) injecting donor cells into the host brain parenchyma, or 2) preparing a cavity by surgical means to expose the host brain parenchyma, and then depositing the graft into the cavity (Neural Grafting in the Mammalian CNS, Ch.3 (Bjorklund and Stenevi eds., Elsevier, Amsterdam 1985) (incorporated herein by reference in its entirety). Both methods provide substantial bonding between the donor cells and the host brain tissue upon transplantation, and both promote anatomical integration between the graft and the host brain tissue. This is important if the donor cells are required to become an integral part of the host brain and survive for the life of the host.

[0146] Glial progenitor cells can also be delivered into the corpus callosum, as described in US Patent Application Publication No. 2003 / 0223972 to Goldman, the entirety of which is incorporated herein by reference.Glial progenitor cells can also be delivered directly to the forebrain subcortex, specifically to the anterior and posterior anlage of the corpus callosum.Glial progenitor cells can also be delivered to the cerebellar white matter to gain access to the main cerebellum and brainstem.Glial progenitor cells can also be delivered to the spinal cord.

[0147] Alternatively, cells can be placed in the ventricles, for example, the brain ventricles. Transplantation of cells in the ventricles can be achieved by injection of donor cells, or by growing cells in a matrix such as 30% collagen to form a solid tissue plug, which can then be implanted in the ventricles to prevent dislocation of transplanted cells. In the case of subdural implantation, cells can be injected around the surface of the brain after making a slit in the dura.

[0148] Suitable techniques for glial cell delivery are described above. In one embodiment, the preparation of glial precursor cells is administered to one or more of the brain, brain stem, spinal cord, or a combination thereof.

[0149] Delivery of cells to a subject can include either single-step or multi-step injections directly into the nervous system. Adult and fetal oligodendrocyte progenitor cells are widely distributed within the brain of transplant recipients, but for widespread disorders, multiple injection sites can be performed to optimize treatment. Injections are optionally directed to regions of the central nervous system, such as white matter bundles (e.g., anterior and posterior anlage), such as the corpus callosum, dorsal columns, cerebellar peduncle, cerebral peduncle, etc. Such injections can be performed unilaterally or bilaterally, using precise stereotactic methods, such as stereotactic surgery, optionally with accompanying imaging methods (e.g., high-resolution MRI imaging). Those skilled in the art will recognize that brain regions vary by species, but will also recognize equivalent brain regions across mammalian species.

[0150] The cell transplant is optionally injected as dissociated cells, but may also be provided by local placement of non-dissociated cells. In either case, the cell transplant optionally includes an acceptable solution. Such acceptable solutions include solutions that avoid undesirable biological activity and contamination. Include an appropriate amount of a pharma- ceutically acceptable salt to make the formulation isotonic. Examples of pharma- ceutically acceptable solutions include, but are not limited to, saline, Ringer's solution, dextrose solution, and culture medium. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5.

[0151] Injection of the dissociated cell transplant can be a streaming injection across the inlet, outlet, or both inlet and outlet pathways of an injection device (e.g., a cannula, needle, or tube). Automation can be used to provide uniform inlet and outlet rates and injection rates and volumes.

[0152] The number of glial progenitor cells administered to a subject may range from approximately 10 to approximately 10 cells per administration (e.g., injection site), depending on the size and species of the recipient and the volume of tissue requiring cell replacement. 2 ~10 8 A single administration (e.g., injection) dose can range from 10 3 ~10 5cells, 10 4 ~10 7 Cells and 10 5 ~10 8 The amount of cells can range from 10 to 200 mg / kg, or any total amount for the transplant recipient patient.

[0153] Pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.In all cases, the form must be sterile and must be fluid to the extent that easy injectability exists.It must be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0154] Since the CNS is an immunologically privileged site, the administered cells, including xenogeneic ones, can survive, and optionally, immunosuppressants or typical regimens of immunosuppressants are not used in the treatment method. Optionally, however, immunosuppressants can also be administered to the subject. Immunosuppressants and their administration regimens are known to those skilled in the art and include agents such as azathioprine, azathioprine sodium, cyclosporine, daltroban, gusperimus trihydrochloride, sirolimus, and tacrolimus. The dosage range and duration of the regimen can vary with the disorder being treated, the degree of rejection, the activity of the specific immunosuppressant used, the age, weight, overall health, sex, and diet of the subject, the time of administration, the route of administration, the excretion rate of the specific immunosuppressant used, the duration and frequency of treatment, and the drugs used in combination. Those skilled in the art can determine the tolerated dose and duration of immunosuppression. The dosing regimen can be adjusted by the individual physician in the event of any contraindications or changes in the subject's condition.

[0155] 5. Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions or medicaments for preventing or treating inherited or acquired disorders of myelin. In some embodiments, the pharmaceutical compositions include one or more of the protein molecules, polynucleotides, expression cassettes, expression vectors (e.g., viral vector genomes, expression vectors, rAAV vectors), and host cells.

[0156] The pharmaceutical composition further comprises a pharma- ceutically acceptable carrier, adjuvant, diluent, excipient and / or other agent. A pharma- ceutically acceptable carrier, adjuvant, diluent, excipient or other agent is not biologically or otherwise undesirable, e.g., the material can be administered to a subject without causing undesirable biological effects that outweigh the beneficial biological effects of the material. Any suitable pharma- cetically acceptable carrier or excipient can be used in the preparation of a pharmaceutical composition according to the present invention (see, e.g., Remington The Science and Practice of Pharmacy, Adeboye Adejare (Editor) Academic Press, November 2020).

[0157] Pharmaceutical compositions are typically sterile, pyrogen-free, and stable under the conditions of manufacture and storage. Pharmaceutical compositions may be formulated as solutions (e.g., water, saline, dextrose solution, buffer solutions, or other pharma- ceutical sterile fluids), microemulsions, liposomes, or other ordered structures compatible with high product (e.g., viral vector particles, microparticles, or nanoparticles) concentrations.

[0158] In some embodiments, pharmaceutical compositions comprising the proteins, polynucleotides, expression cassettes, expression vectors, vector genomes, host cells, or rAAV vectors of the present disclosure are formulated in water or buffered saline. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. In some embodiments, it may be preferable to include isotonicity agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged adsorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. In some embodiments, the nucleic acids, vectors, and / or host cells of the present disclosure may be administered in compositions containing slow release polymers or other carriers that protect the product against rapid release, including controlled release formulations, for example, implants and microencapsulated delivery systems.

[0159] In some embodiments, the pharmaceutical compositions of the present disclosure are parenteral pharmaceutical compositions, including compositions suitable for intravenous, intraarterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intraarticular, intraparenchymal (IP), intrathecal (IT), intracerebroventricular (ICV) and / or intracisternal (ICM) administration. In some embodiments, the pharmaceutical compositions of the present disclosure are formulated for administration by ICV injection. In some embodiments, the vector (e.g., a viral vector such as AAV) may be formulated in 350 mM NaCl and 5% D-sorbitol in PBS.

[0160] 6. Method of Administration The above molecules, or polynucleotides, or vectors (e.g., vector genomes, rAAV vectors) can be administered to a subject (e.g., a patient) or target cells to treat the subject. The administration of the vector to a human subject or an animal in need thereof can be performed by any means known in the art for administering vectors. Examples of target cells include cells of the CNS, preferably oligodendrocytes or their precursor cells.

[0161] The vectors can be administered in addition to and as an adjunct to standard care treatments. That is, the vectors can be co-administered with another agent, compound, drug, treatment or treatment regimen at the same time, contemporaneously, or at a predetermined dosage interval, as determined by one of skill in the art using routine methods. The uses disclosed herein include administering the rAAV vectors of the present disclosure in addition to and / or in accordance with a dosing schedule in addition to and / or in conjunction with standard treatments for diseases known in the art.

[0162] In some embodiments, the combination composition comprises one or more immunosuppressants. In some embodiments, the combination composition comprises a rAAV vector comprising a transgene (e.g., a polynucleotide encoding an RNA molecule disclosed herein) and one or more immunosuppressants. In some embodiments, the method comprises administering or delivering to a subject a rAAV vector comprising a transgene, and administering to the subject an immunosuppressant prophylactically prior to administration of the vector or after administration of the vector (i.e., before or after symptoms of a response to the vector and / or the protein provided thereby become evident).

[0163] In one embodiment, the vectors (e.g., rAAV vectors) of the present disclosure are administered systemically. Exemplary methods of systemic administration include, but are not limited to, intravenous (e.g., portal vein), intraarterial (e.g., femoral artery, hepatic artery), intravascular, subcutaneous, intradermal, intraperitoneal, transmucosal, intrapulmonary, intralymphatic, and intramuscular administration, and the like, as well as direct tissue or organ injection. Those skilled in the art will appreciate that systemic administration can deliver nucleic acids to all tissues. In some embodiments, direct tissue or organ administration includes administration to areas directly affected by oligodendrocyte deficiency (e.g., the brain and / or central nervous system). In some embodiments, the vectors of the present disclosure and pharmaceutical compositions thereof are administered to the brain parenchyma (i.e., by intraparenchymal administration), to the spinal canal or subarachnoid space to reach the cerebrospinal fluid (CSF) (i.e., by intrathecal administration), to the ventricles of the brain (i.e., by intraventricular administration), and / or to the cisterna magna of the brain (i.e., by intracisternal administration).

[0164] Thus, in some embodiments, vectors of the present disclosure are administered by direct injection into the brain (e.g., into the parenchyma, ventricles, cisterna magna, etc.) and / or into the CSF (e.g., into the spinal canal or subarachnoid space) to treat myelin disorders. Target cells of vectors of the present disclosure include cells located in the cortex, subcortical white matter of the corpus callosum, striatum, and / or cerebellum. In some embodiments, target cells of vectors of the present disclosure are oligodendrocytes or their precursor cells. Additional routes of administration may also include local application of vectors under direct visualization, e.g., superficial cortical application, or other stereotactic application.

[0165] In some embodiments, the vector of the present disclosure is administered by at least two routes. For example, the vector is administered systemically and also directly to the brain. When administered via at least two routes, the administration of the vector can be, but need not be, simultaneous or contemporaneous. Instead, administration via different routes can be performed separately with a time interval between each administration.

[0166] The above-mentioned proteins, or polynucleotides encoding the proteins, or vector genomes, or vectors comprising the polynucleotides (e.g., rAAV vectors) can be used for ex vivo transduction of cells or for direct administration to a subject (e.g., direct administration to the CNS of a patient with a disease). In some embodiments, the transduced cells (e.g., host cells) are administered to a subject to treat or prevent a disease, disorder, or condition (e.g., cell therapy of a disease). For example, a rAAV vector comprising a therapeutic nucleic acid (e.g., encoding a protein) can be administered to oligodendrocytes or their precursor cells, preferably in a biologically effective amount.

[0167] The dosage of the vector depends, for example, on the mode of administration, the disease or condition being treated, the stage and / or aggressiveness of the disease, the condition of the individual subject (age, sex, weight, etc.), the particular viral vector, the stability of the expressed protein, the host immune response to the vector, and / or the gene being delivered. In general, the dose should be at least 1×10 per kg of subject body weight to achieve a therapeutic effect. 8 or more, e.g., 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 or more in the range of vector genomes (vg).

[0168] In some embodiments, a polynucleotide encoding a protein described herein may be administered as a component of a DNA molecule (e.g., a recombinant nucleic acid) having appropriate regulatory elements (e.g., a promoter) for expression in a target cell (e.g., an oligodendrocyte). The polynucleotide may be administered as a component of a plasmid or viral vector, such as a rAAV vector. The rAAV vector may be administered in vivo to a patient in need of treatment by delivering the vector directly (e.g., directly to the CNS). The rAAV vector may also be administered ex vivo to a patient by in vitro administration of the vector to cells from a donor patient in need of treatment, followed by reintroduction of the transduced cells into the donor (e.g., cell therapy).

[0169] 7. Kit The present disclosure provides a kit that includes packaging material and one or more components. The kit typically includes a label or package insert that includes a description of the components or an in vitro, in vivo, or ex vivo use of the components therein. The kit may include a collection of such components, such as the above-mentioned polynucleotides, nucleic acids, expression cassettes, expression vectors (e.g., viral vector genomes, expression vectors, rAAV vectors), and host cells or their progeny, and optionally a second active agent, such as a compound, therapeutic agent, drug, or composition.

[0170] A kit refers to a physical structure that contains one or more components of the kit. The packaging material can maintain the components sterile and can be made of materials commonly used for such purposes (e.g., paper, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0171] The label or insert may include identity of one or more components therein, dose, mechanism of action, clinical pharmacology of the active ingredients including pharmacokinetics and pharmacodynamics. The label or insert may include information identifying manufacture, lot number, place and date of manufacture, expiration date. The label or insert may include information regarding the disease for which the kit components may be used (e.g., inherited or acquired disorders of myelin such as HD). The label or insert may include instructions for the clinician or subject for using one or more of the kit components in a method, use, or treatment protocol or regimen. The instructions may include dosage, frequency of duration, and instructions for practicing any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0172] The label or package insert may include information regarding potential side effects, complications or reactions, for example, warnings to the subject or clinician regarding situations in which it is not appropriate to use a particular composition.

[0173] 8. Definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which this invention belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. When used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well unless the context clearly dictates otherwise. The following terms have the following meanings:

[0174] As used herein, the term "about" or "approximately" refers to a measurable value such as amount, homology or length of biological activity of a polynucleotide or polypeptide sequence, dosage, time, temperature, and is meant to encompass a variation of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 1%, 0.5%, or even 0.1%, unless otherwise stated, clear from the context, or where such number exceeds 100% of the possible value.

[0175] As used herein, the term "homologous" or "homology" refers to two or more reference entities (e.g., nucleic acid or polypeptide sequences) that share at least partial identity over a given region or portion. For example, if an amino acid position in two peptides is occupied by the same amino acid, the peptides are homologous at that position. In particular, a homologous peptide retains an activity or function associated with the unmodified or reference peptide, and a modified peptide generally has an amino acid sequence that is "substantially homologous" to the amino acid sequence of the unmodified sequence. When referring to a polypeptide, nucleic acid, or fragment thereof, "substantial homology" or "substantial similarity" means that there is sequence identity in at least about 70%-99% of the sequence when optimally aligned with another polypeptide, nucleic acid (or its complementary strand) or fragment thereof, with appropriate insertions or deletions. The degree of homology (identity) between two sequences can be ascertained using computer programs or mathematical algorithms known in the art. Such algorithms that calculate percent sequence homology (or identity) generally account for sequence gaps and mismatches over the comparison region or area.

[0176] A nucleic acid or polynucleotide refers to a DNA molecule (e.g., cDNA or genomic DNA), an RNA molecule (e.g., mRNA), or a DNA or RNA analog. A DNA or RNA analog can be synthesized from nucleotide analogs. A nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.

[0177] An isolated or recombinant nucleic acid refers to a nucleic acid whose structure is not identical to that of any naturally occurring nucleic acid or to that of any fragment of naturally occurring genomic nucleic acid. Thus, the term encompasses, for example, (a) a DNA having the sequence of a portion of a naturally occurring genomic DNA molecule, but not flanked by both sequences that flank that portion of the molecule in the genome of the organism in which it occurs in nature, (b) a nucleic acid that is incorporated into a prokaryotic or eukaryotic vector or genomic DNA in such a way that the resulting molecule is not identical to any naturally occurring vector or genomic DNA, (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment, and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene that codes for a fusion protein. The nucleic acid described above can be used to express the proteins of the present disclosure. For this purpose, the nucleic acid can be operably linked to suitable regulatory sequences to generate an expression vector.

[0178] A "recombinant nucleic acid" is a combination of nucleic acid sequences that are joined together using recombinant techniques and procedures used to join nucleic acid sequences together.

[0179] The terms "heterologous" DNA molecule and "heterologous" nucleic acid, as used herein, refer to a molecule that is derived from a source foreign to a particular host cell, or that, if derived from the same source, has been modified from its original form, respectively. Thus, a heterologous gene in a host cell includes a gene that is endogenous to a particular host cell, but has been modified, for example, by the use of shuffling or recombination. When used to describe two nucleic acid segments, these terms mean that the two nucleic acid segments are not derived from the same gene, or, if they form the same gene, one or both of them are modified from their original form. These terms also include non-naturally occurring multiple copies of a naturally occurring DNA molecule. Thus, the terms refer to a nucleic acid segment that is foreign or heterologous to the cell, or that is homologous to the cell, but is in a location within the host cell nucleic acid where the element is not normally found. The exogenous DNA segment is expressed, resulting in an exogenous RNA or polypeptide. A "homologous DNA molecule" is a DNA molecule that is naturally associated with the host cell into which it is introduced.

[0180] "Regulatory sequences" include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and / or inducible sequences. The design of an expression vector may depend on factors such as the choice of the host cell to be transformed, the level of expression of protein or RNA desired. The expression vector can be introduced into a host cell to produce an RNA or polypeptide of interest. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one that initiates RNA at high frequency.

[0181] A "promoter" is a nucleotide sequence that initiates and regulates transcription of a polynucleotide. Promoters can include inducible promoters (expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. The term "promoter" or "regulatory element" is intended to include full-length promoter regions as well as functional (e.g., transcription or translation controlling) segments of these regions.

[0182] "Operably linked" refers to an arrangement of elements such that the components so described are configured to perform their normal functions. Thus, a given promoter operably linked to a nucleic acid sequence can affect the expression of that sequence if the appropriate enzymes are present. The promoter need not be contiguous with the sequence, so long as it functions to induce expression of the sequence. Thus, for example, there can be an intervening untranslated but transcribed sequence between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence. Thus, the term "operably linked" is intended to encompass any spacing or orientation of the promoter element and DNA sequence of interest that allows initiation of transcription of the DNA sequence of interest upon recognition of the promoter element by the transcription complex.

[0183] As used herein, the term "gene construct" or "nucleic acid construct" refers to a non-naturally occurring nucleic acid molecule resulting from the use of recombinant DNA technology (e.g., recombinant nucleic acid). A gene or nucleic acid construct is a nucleic acid molecule, either single-stranded or double-stranded, that has been modified to contain segments of nucleic acid sequences that are joined and arranged in a manner not found in nature. A nucleic acid construct can be a "cassette" or "vector" (e.g., a plasmid, a rAAV vector genome, an expression vector, etc.), i.e., a nucleic acid molecule designed to deliver exogenously formed DNA to a host cell.

[0184] As used herein, "expression cassette" refers to a nucleic acid sequence capable of directing the expression of a particular nucleotide sequence in a suitable host cell, which may include a promoter operably linked to the nucleotide sequence of interest, which may be operably linked to a termination signal. It may also include sequences necessary for proper translation of the nucleotide sequence. The coding region usually encodes an RNA or protein of interest. The expression cassette containing the nucleotide sequence of interest may be chimeric. The expression cassette may also be naturally occurring but obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or a regulatable promoter, which initiates transcription only when the host cell is exposed to some specific stimulus. In the case of a multicellular organism, the promoter may also be specific to a particular tissue or organ or developmental stage.

[0185] A vector refers to a nucleic acid molecule that can transport another nucleic acid to which it is linked. A vector may or may not be capable of autonomous replication, or may or may not be capable of integrating into a host DNA. Examples of vectors include plasmids, cosmids, or viral vectors. A vector contains a nucleic acid in a form suitable for expressing a nucleic acid of interest in a host cell. Preferably, a vector contains one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed.

[0186] As used herein, the terms "overexpressing," "overexpress," "overexpressed," or "overexpression," when referring to the production of a nucleic acid or protein in a host cell, mean that the nucleic acid or protein is produced in an amount that is greater than the amount that is produced in its naturally occurring environment. The term is intended to encompass the overexpression of endogenous, as well as exogenous or heterologous nucleic acids and proteins. Thus, these terms and the like are intended to encompass increasing the expression of a nucleic acid or protein in a cell to a level that is greater than the level that the cell naturally contains. In certain embodiments, the expression level or amount of a nucleic acid or protein in a cell is increased by at least 5%, 10%, 20% 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% compared to the level or amount naturally contained in the cell.

[0187] In the context of mutant or diseased cells, terms such as "overexpressing," "overexpress," "overexpressed," and "overexpression" are intended to encompass increasing expression of a nucleic acid or protein to levels greater than those contained in a mutant, diseased, wild-type, or non-diseased cell. In certain embodiments, the expression level or amount of a nucleic acid or protein in a mutant or diseased cell is increased by at least 5%, 10%, 20% 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% compared to the level or amount contained in a mutant, diseased, wild type, or non-diseased cell.

[0188] As used herein, the term "prevent" or "prevention" refers to a delay in the onset and / or a reduction in the frequency and / or severity of one or more signs or symptoms of a particular disease, disorder or condition. In some embodiments, prevention is assessed on a population basis such that an agent is considered to "prevent" a particular disease, disorder or condition if a statistically significant reduction in the onset, frequency and / or intensity of one or more signs or symptoms of the disease, disorder or condition is observed in a population susceptible to the disease, disorder or condition. Prevention may be considered complete if the onset of the disease, disorder or condition is delayed for a predetermined period of time.

[0189] As used herein, the term "therapeutically effective amount" refers to an amount that produces the desired therapeutic effect for which it is administered. In some embodiments, the term refers to an amount sufficient to treat a disease, disorder, or condition when administered according to a therapeutic dosing regimen to a population suffering from or susceptible to the disease, disorder, or condition. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity and / or delays the onset of one or more symptoms of a disease, disorder, and / or condition. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not actually require that successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be an amount that, when administered to a patient in need of such treatment, provides a specific desired pharmacological response in a significant number of subjects.

[0190] As used herein, the term "stem cell" refers to a cell that has the ability to both replace itself and differentiate into more specialized cells. Their self-renewal ability generally persists for the life of an organism. Pluripotent stem cells can give rise to all of the body's various cell types. Multipotent stem cells can give rise to a limited subset of cell types. For example, hematopoietic stem cells can give rise to the various types of cells found in the blood, but cannot give rise to other types of cells. Multipotent stem cells can also be referred to as somatic stem cells, tissue stem cells, lineage-specific stem cells, and adult stem cells. The non-stem cell progeny of multipotent stem cells are progenitor cells (also referred to as restricted progenitor cells). Progenitor cells give rise to fully differentiated cells, but give rise to a more restricted set of cell types than stem cells. Progenitor cells also have a relatively limited ability to self-renew, and as they divide and differentiate, they eventually wear out and are replaced by new progenitor cells derived from their upstream multipotent stem cells.

[0191] As used herein, "therapeutic cells" refers to a cell population that ameliorates a patient's condition, disease, and / or injury. Therapeutic cells can be autologous (i.e., derived from the patient), allogeneic (i.e., derived from an individual of the same species but different from the patient), or xenogeneic (i.e., derived from a species but different from the patient). Therapeutic cells can be homogeneous (i.e., composed of a single cell type) or heterogeneous (i.e., composed of multiple cell types). The term "therapeutic cells" includes both therapeutic active cells and progenitor cells that can differentiate into therapeutic active cells. EXAMPLES

[0192] The following examples are intended to illustrate the practice of embodiments of the present disclosure, but are not intended to limit its scope in any way.

[0193] Example Overview The examples presented here will help to explore whether HD is associated with a failure of homeostatic myelin maintenance and adult remyelination and to link impaired myelination of HD hGPCs to white matter degeneration in HD patients. Using the R6 / 2 mouse model of HD, we compared the ultrastructure of adult R6 / 2 and wild-type corpus callosum white matter and subsequently their response to cuprizone-induced demyelination. While age-related and progressive hypomyelination was observed in untreated R6 / 2 mice, cuprizone-treated R6 / 2 mice were further demyelinated by cuprizone treatment (Figure 18, top panel). RNA sequencing of both corpus callosum white matter and isolated GPCs from R6 / 2 and zQ175 mice (the latter a long-lived model of late-onset HD) then revealed a systematic downregulation of genes related to oligodendrocyte differentiation and myelination in both models compared to controls. Gene co-expression and network analysis predicted suppressed Tcf7l2 signaling as the primary driver of this expression pattern. Proteomic analysis of corpus callosum and striatal white matter from both R6 / 2 and zQ175 mice then confirmed that TCF7L2-regulated myelin proteins were downregulated compared to wild-type controls, and the relative suppression of myelin protein expression increased with age in both HD models (Figure 18, middle panel). TCF7L2 was overexpressed by lentivirus in the R6 / 2 striatum, and found to be sufficient to rescue oligodendrocyte gene expression, accompanied by upregulation of many metabolic regulatory and myelination genes required for both astrocyte and oligodendrocyte differentiation. Proteomic analysis of corpus callosum and striatal white matter from both R6 / 2 and zQ175 mice then confirmed that TCF7L2-regulated myelin proteins were downregulated compared to wild-type controls, and the relative suppression of myelin protein expression increased with age in both HD models. Based on this, we assessed the ability of lentiviral Tcf7l2 expression to regulate remyelination in cuprizone-treated R6 / 2 mice. These studies demonstrated that TCF7L2 overexpression was sufficient to restore the normal time course and efficacy of remyelination (Figure 18, bottom panel).Collectively, these data suggest that impaired expression of Tcf7l2-dependent genes may be an underlying cause of impaired glial differentiation and white matter damage in HD, and that inducible overexpression of Tcf7l2 may be a viable strategy for alleviating white matter damage in HD.

[0194] Materials and Methods of the Examples Animals. All experiments were approved by the Institutional Animal Care and Use Committee of the University of Rochester. + Wild-type females receiving ovarian transplants from (120 CAG) donor mice were purchased from Jackson Laboratories (Bar Harbor, ME). zQ175 (190Q) breeders were obtained from Charles River through the CHDI foundation. These mice were bred to PDGFRa-EGFP mice and genotyped after weaning, and double heterozygous mice were further analyzed to determine their CAG repeat number by PCR using primers encoding products spanning the repeat region, as previously described (see Benraiss et al., “Sustained Mobilization Of Endogenous Neural Progenitors Delays Disease Progression In A Transgenic Model Of Huntington's Disease,” Cell Reports 36:109308, (2013) (incorporated herein by reference in its entirety). As indicated, all experiments included 4–8 mice / group. For remyelination studies, mice were fed 0.2% (w / w) cuprizone ad libitum in the diet (Bio-serv) for 6 weeks. All samples contained equal numbers of males and females.

[0195] Transmission electron microscopy and image processing. Mice were perfused with 2.0% paraformaldehyde / 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) containing 0.2 M sucrose. After 24 h of primary fixation, tissues were sectioned coronally, rinsed in the same buffer, and postfixed for 90 min in cacodylate buffered 1.5% osmium tetroxide mixed with an equal volume of 1.5% potassium ferrocyanide. Sections were rinsed twice in distilled water, dehydrated in a graded series of ethanol to 100%, transferred to propylene oxide, infiltrated with EPON / Araldite resin, embedded, and polymerized at 60° C. for 48 h. Epoxy-embedded tissue blocks were cut at 1 micron on glass slides with a glass knife and stained with toluidine blue on a hot plate to specifically identify defined regions of the corpus callosum that were cross-sectionally myelinated. Thin sections (70 nm) of the target area were cut using a diamond knife in an ultramicrotome and collected on carbon-coated nickel grids. Grids were stained with aqueous uranyl acetate and lead citrate and examined using a Hitachi 7650 TEM equipped with an attached Gatan 11 megapixel Erlangshen digital camera and digital micrograph software. For g-ratio (axon diameter / fiber diameter ratio), 20 randomly acquired images of each sample were processed using Image J software. Images were converted to binary images using the Image J plugin G-ratio calculator (Cellular Imaging Facility, Univ. of Lausanne) and all axons in any given micrograph were measured for G-ratio. Due to the irregular shape of the elements, the approximate diameter was calculated as the average of the two diameters measured for each axon (see Madsen et al., “Mitochondrial DNA Double-Strand Breaks in Oligodendrocytes Cause Demyelination, Axonal Injury, and CNS Inflammation,” J Neurosci 37:10185-10199 (2017), which is incorporated by reference in its entirety).

[0196] Dissection of striatal tissue. Mice were euthanized with carbon dioxide, perfused transcardially with sterile Hank's Balanced Salt Solution (HBSS), and the brains were removed. The brains were immersed in ice-cold sterile HBSS for approximately 5 minutes to facilitate microdissection. Under a dissecting microscope, the subventricular region was removed and discarded, and the striatum from each mouse was dissected and placed in sterile HBSS on ice. Striata from all mice of a litter were pooled together according to genotype. Striatal tissue was transferred to a Petri dish containing sterile HBSS, cut into small pieces using a sterile disposable scalpel, transferred to a sterile tube, and then incubated in papain / DNase dissociation solution at 37°C for 50 minutes. Minimal essential medium containing 5% serum plus 0.5% BSA (MEM-BSA) was then added to inactivate the papain. The tissue was triturated by repeated pipetting to achieve a single cell suspension. The cells were then pelleted and resuspended in MEM-BSA and layered first onto a 90% Percoll gradient followed by a second 30% Percoll gradient, and the solution was centrifuged, the myelin sheath and debris were removed from the tube, and the cell pellet was resuspended in MEM containing 20 U / ml DNase.

[0197] Flow cytometry and cell sorting. Flow cytometry of PDGFRa-EGFP glial precursor cells was performed on freshly dissociated striatal cells from wild-type, R6 / 2 or Q175 mice. For FACS sorting, mouse striata were pooled together (n=3-4 / group for R6 / 2 and 12-week zQ175 and WT controls, 4-8 / group for 1-year-old zQ175 and WT). All samples were cultured at 1-1.5 × 10 6Cells were resuspended to a concentration of 1000 cells / ml and then passed through a 35 μm tube-top cell strainer prior to flow cytometry. DAPI was added at 1 μg / ml. Flow cytometry analysis and FACS were performed on a BD FACSAria IIIU (Becton Dickinson, San Jose, CA). Cells were analyzed by forward and side scatter for EGFP fluorescence through a 530±30 nm bandpass filter and DAPI fluorescence through a 450±50 nm bandpass. Non-fluorescent cells were used to set background fluorescence and a 0.5% false positive rate was allowed. EGFP isolated by FACS + and EGFP - Striatal cells were pelleted, frozen on dry ice, and stored at -80°C until time of RNA extraction.

[0198] RNA preparation, amplification, and labeling. RNA was extracted from pelleted / frozen cells using the Qiagen RNeasy Plus Mini kit. RNA concentration was determined using a Nanodrop. A portion of the RNA was then used for bioanalysis to confirm RNA integrity. EGFP + RNA isolated from cells was used to generate sequencing libraries using the TruSeq RNA v2 kit and sequenced on an Illumina HiSeq2500 platform for approximately 45 million 2×125 bp reads per sample.

[0199] RNA-Seq analysis of FACS-isolated GPCs. Reads were demultiplexed and cleaned using Trimmomatic (Bolger et al., "Trimmomatic: A Flexible Trimmer For Illumina Sequence Data," Bioinformatics 30:2114-2120 (2014) (incorporated herein by reference in its entirety)). Reads were aligned to mouse genome GRCm38.p6 and mapped to Ensembl reference 92 via STAR 2.5.2b (Dobin et al., “STAR: Ultrafast Universal RNA-Seq Aligner,” Bioinformatics 29:15-21 (2013), incorporated herein by reference in its entirety). quantMode was set to TranscriptomeSAM. Gene abundance and expected counts were then calculated using RSEM 1.3.0 (Li and Dewey, “RSEM: Accurate Transcript Quantification From RNA-Seq Data With Or Without A Reference Genome,” BMC Bioinformatics 12:323 (2011), incorporated herein by reference in its entirety). Expected counts were imported into R via tximport for differential expression analysis (Soneson et al., “Differential Analyses For RNA-Seq: Transcript-Level Estimates Improve Gene-Level Inferences,” F1000Research 4:1521 (2015); R Core Team, “R: A Language And Environment For Statistical Computing,” R Foundation for Statistical Computing Vienna, Austria (2017) (incorporated by reference in its entirety).Following regression of strain effects from generalized linear models, differential expression between control and HD model Glia was performed at each time point using DESeq2 (Risso et al., “GC-Content Normalization For RNA-Seq Data,” BMC Bioinformatics 12:480 (2011); Love et al., “Moderated Estimation Of Fold Change And Dispersion For RNA-Seq Data With Deseq2,” Genome biology 15:550 (2014) (incorporated herein by reference in its entirety)). Genes with adjusted p-values ​​less than 0.01 were considered significant. Only moderately expressed genes were retained for functional analysis (mean transcripts per million (TPM) >1 in either group) because they were likely to be biologically significant. Differentially expressed genes between both groups were analyzed with Ingenuity Pathway Analysis (IPA) (QIAGEN) for functional enrichment.

[0200] For functional analysis and inference of gene interactions across all time points and models of HD, gene ontology networks were constructed according to weighted gene expression correlation network analysis (WGCNA) (Langfelder and Horvath, “WGCNA: An R Package For Weighted Correlation Network Analysis.” BMC Bioinformatics 9:559 (2008), incorporated herein by reference in its entirety). Five modules were discovered from WGCNA. These modules were then filtered for significant differentially expressed genes and fed into IPA. Three modules were enriched for terms related to glial maturation and myelination, with the black module being the most prominent. Significant terms were filtered by biological relevance and used to construct a functional IPA term network in which terms and gene nodes were connected via undirected edges. Network visualization was performed in Cytoscape (Shannon, "Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks," Genome Res 13:2498-2504 (2003) (incorporated herein by reference in its entirety)) and neighborhood determination was performed in Gephi (Bastian et al., "Gephi: An Open Source Software for Exploring and Manipulating Networks," Proc. Third Int'l ICWSM Conference 3(1):361-362 (2009) (incorporated herein by reference in its entirety). Nodes were clustered within their respective neighborhoods and slightly rearranged aesthetically. Gene expression data are available via GEO under the accession number GSE181370.

[0201] Western immunoblotting. Twelve-week-old R6 / 2 mice and their wild-type littermate controls (n=3 each) were perfused with HBSS, their corpus callosum were homogenized, and proteins were extracted in radioimmunoprecipitation assay buffer (RIPA buffer, Sigma) in the presence of protease and phosphatase inhibitors (Halt™ protease and phosphatase inhibitor Cocktail Thermo Scientific). 10 μg of protein was loaded onto an electrophoresis gel (4-12% SDS-PAGE gel, NuPAGE™ 4-12% Bis-Tris gel, Invitrogen) and transferred onto an Immobilon membrane (Immobilon-FL Transfer Membrane PVDF, Millipore). After transfer, blots were stained with Ponceau (Sigma incubated in blocking buffer (SuperBlock™ Blocking Buffer in TBS, Thermo Scientific)) followed by staining with TCF7L2 antibody (Cell Signaling Technologies, Clone C48H11) or β-actin antibody (Cell Signaling Technologies). Membranes were then treated with HRP-conjugated goat anti-rabbit secondary antibody (Cell Signaling Technologies) followed by SuperSignal™ West Pico+Chemiluminescent Substrate and imaged on a ChemiDoc Imaging System (BioRad). Band intensity was quantified using Image J and data was normalized to the expression of the housekeeping protein β-actin.

[0202] Quantitative Mass Spectrometry (MS) Callosal tissue was collected as described above (n=4) and samples were processed as previously described (Hutti et al., “Global Analysis of Protein Degradation in Prion Infected Cells,” Sci Rep 10:10800, 2020, incorporated herein by reference in its entirety). Briefly, whole corpus callosum or A2B5 FACS immunosorted striatal neuroglial progenitor cells were pelleted and washed with 50 μL of 5% SDS, 100 mM, sonicated, and then briefly centrifuged to remove debris. Samples (15 μg from each sample) were then reduced, alkylated, and digested overnight with trypsin using S-Traps (Protifi). Samples were then frozen, dried in a Speed ​​Vac (Labconco), and then resuspended in 0.1% trifluoroacetic acid prior to analysis. The peptide samples were then placed on a homemade 30 cm C18 column containing 1.8 μm beads (Sepax) and then loaded onto a high-performance liquid chromatograph Easy nLC-1200 HPLC (Thermo Fisher) connected to a mass spectrometer (Fusion Lumos Tribrid, Thermo Fisher). Raw data analysis was performed by the SEQUEST search engine within the Proteome Discoverer software platform, version 2.4 (Thermo Fisher), using the SwissProt mus musculus database. Search parameters included a maximum of two missed cleavages by trypsin, a tolerance of 10 ppm for MS1 masses, and a tolerance of 0.6 Da for MS2 masses, while oxidation of methionine was set as a variable modification and carbamidomethyl was set as a fixed modification. Finally, relative protein abundance between samples was performed using the Minora node and Percolator for false discovery rate, excluding peptides with q values ​​above 0.01. The raw data are available in the Proteomics Identification Database (https: / / www.ebi.ac.uk / pride / ).

[0203] FACS Isolation of Striatal GPCs Striata were processed as above until debris was removed through a Percoll gradient. Cells were then blocked for 5 min and stained with APC-conjugated A2B5 for 20 min. As previously reported (Gard and Pfeiffer, “Two Proliferative Stages of the Oligodendrocyte Lineage (A2B5+O4- and O4+GalC-) Under Different Mitogenic Control,” Neuron 5:615-625 (1990); Roy et al., “Identification, Isolation, and Promoter-defined Separation of Mitotic Oligodendrocyte Progenitor Cells from the Adult Human Subcortical White Matter,” J Neurosci (2011) 23:1311-1315 (2011)). 19:9986-9995 (1999) (incorporated herein by reference in its entirety), PDGFRa expression was found to decrease with age in these models, but A2B5 expression was maintained and remains selectively expressed by GPCs in white matter, so A2B5 was used to extract striatal GPCs. Cells were washed and then FACS-isolated for A2B5 positivity before pelleting, drying, and flash-frozen. These pellets were then submitted for quantitative mass spectrometry.

[0204] Assessment of human TCF7L2 isoform distribution. Raw Fastq files from the public HD hGPC dataset (Osipovitch et al., “Human ESC-Derived Chimeric Mouse Models of Huntington's Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation,” Cell Stem Cell 24:107-122 e107 (2019) (incorporated herein by reference in its entirety)) were obtained from GSE105041 and aligned to GRCh38 using ensemble 95 gene annotations via STAR2.5.4b in two-pass mode across all samples and quantified with RSEM 1.3.1. Isoform abundances were imported into R via Tximport (1.8.0).

[0205] Viral construction and injection. Human TCF7L2 (NM_030756.5) was cloned into pTANK-TRE-CAG-rtTA3G-WPRE under the control of a tetracycline-inducible promoter (Osipovitch et al., “Human ESC-Derived Chimeric Mouse Models of Huntington's Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation,” Cell Stem Cell 24:107-122 e107 (2019) (incorporated herein by reference in its entirety)). Viral particles pseudotyped with vesicular stomatitis virus G glycoprotein were produced and transfected into 293HEK cells (3.8 10 9The virus was titrated at limiting dilutions on the 10-week-old R6 / 2 mice at the following coordinates from the bregma: anterior / posterior +1.1 mm, medial / lateral ±1.5 mm, dorsal / ventral -2.3 mm from the dura. Immediately after injection, a cohort of mice received doxycycline (introduced in their water) to allow expression of TCF7L2, whereas other mice did not receive doxycycline and thus served as matched controls. At 12 weeks of age, all mice were sacrificed and their striata were dissected and processed for RNA extraction.

[0206] Histology. Animals were killed using sodium pentobarbital, perfused transcardially with saline, then administered 4% paraformaldehyde, and their brains were processed for immunocytochemistry as previously described (Benraiss et al., “Cell-Intrinsic Glial Pathology Is Conserved Across Human and Murine Models Of Huntington's Disease,” Cell Reports 36:109308 (2016) (incorporated herein by reference in its entirety). Sagittal sections (20 μm) spanning the bone marrow were processed for immunostaining with anti-EGFP (chicken anti-EGFP, Rockland) in combination with anti-Olig2 (goat anti-olig2, R&D Systems), anti-Ng2 (rabbit anti-NG2, Millipore), or anti-GFAP (mouse monoclonal anti-human GFAP, Covance Research).

[0207] QPCR. RNA was isolated using TRIZOL via the RNeasy Mini kit (Qiagen, Germany) according to the manufacturer's instructions and quantified on a Nanodrop spectrophotometer. First-strand cDNA was synthesized using TaqMan Reverse Transcription Reagent (Applied Biosystems, USA). Real-time PCR samples were prepared in triplicate with 5 ng of RNA in FastStart Universal SybrGreen Mastermix (Roche Diagnostics, Germany) and amplified on a CFX Connect Real-Time System Thermocycler (Bio-Rad, USA). Primer sequences are listed in Table 1. Each PCR was followed by melting curve analysis to confirm reaction specificity. Results were normalized to 18S gene expression within samples. Fold changes were calculated using the ΔΔCt method (Pfaffl, “A New Mathematical Model for Relative Quantification in Real-time RT-PCR,” Nucleic Acids Research 29:e45 (2001), which is incorporated herein by reference in its entirety). [Table 4]

[0208] Statistical analysis. Data were analyzed using GraphPad Prism V8 (GraphPad Software Inc., La Jolla, CA, USA). Data were analyzed using GraphPad Prism 8.0 (GraphPad, San Diego, CA). Unpaired t-tests were used to compare two groups, whereas two-way ANOVA (followed by Tukey's post-hoc comparison test) was used to compare four or more groups. Frequency distribution plots of the number of myelinated axons as a function of diameter were analyzed using nonlinear regression with a Lorentzian distribution curve fitting model. Quantitative results are presented as mean ± SEM, and statistical significance was accepted at p < 0.05.

[0209] Example 1 - Ultrastructural imaging analysis reveals progressive hypomyelination in HD mouse models Human ESC-derived mHTT-expressing GPCs show delayed oligodendrocyte differentiation after neonatal transplantation into hypomyelinated shiverer mice (Osipovitch et al., “Human ESC-Derived Chimeric Mouse Models of Huntington's Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation,” Cell Stem Cell 24:107-122 e107 (2019) (incorporated herein by reference in its entirety)). This delayed differentiation is due to a cell-autonomous mHTT-dependent block in terminal glial differentiation from bipotential hGPCs. This finding suggested that homeostatic maintenance of mature myelin, as well as remyelination of lesions following adult demyelination, may be impaired in vivo, as each of these processes requires unhindered oligodendrocyte differentiation from the parenchymal GPC pool. We next assessed myelin maintenance and repair to determine whether it was disrupted in vivo in the R6 / 2 mouse model of juvenile HD. These mice harbor the first exon of mutant human huntingtin (mHTT) with a 120CAG repeat expansion. Mice show symptoms as early as 8 weeks, then rapidly deteriorate and die early at 16-17 weeks.

[0210] To assess myelination in the R6 / 2 brain, we first examined the ultrastructure of corpus callosum myelin in both R6 / 2 mice and their wild-type controls at two separate time points: pre-symptomatic (6 weeks) and diseased (12 weeks, n=4 mice for all groups). Analysis of electron microscopy (EM) images of the corpus callosum was performed using the ImageJ plugin G-ratio calculator to measure the G-ratio of the inner to outer diameter of the myelin sheath (Goebbels et al., “Elevated Phosphatidylinositol 3,4,5-Trisphosphate In Glia Triggers Cell-Autonomous Membrane Wrapping And Myelination,” J Neurosci 30:8953-8964 (2010) (incorporated herein by reference in its entirety). The percentage of myelinated fibers within each study field was also scored (Figure 1), as was the size distribution of myelinated axons as a function of genotype (Figures 7A-7D). At 6 weeks, the g-ratio of myelinated axons in R6 / 2 mice was not significantly different from that of wild-type mice (F[1,1219] = 2.18 by linear regression, p = 0.14, Figures 1A and 1B). In contrast, by 12 weeks, the average g-ratio of myelinated R6 / 2 callosal axons was significantly higher than that of WT controls, suggesting that R6 / 2 axons have fewer and / or thinner myelin wraps per axon than WT mice (F[1,799] = 5.09, p = 0.024, Figures 1A and 1C). Lorentzian nonlinear regression analysis of the frequency of myelinated fibers as a function of diameter (Motulsky & Brown, “Detecting Outliers When Fitting Data With Nonlinear Regression-A New Method Based on Robust Nonlinear Regression and The False Discovery Rate,” BMC Bioinformatics 7:123 (2006), which is incorporated by reference in its entirety, revealed a shift toward smaller axons in R6 / 2 compared to WT mice.This difference was already significant at 6 weeks of age and further widened by 12 weeks (6 weeks: F[3,66] = 35.8, p < 0.0021, 12 weeks: F[3,38] = 8.83, p = 0.0001; Figures 1D and 1E, see also Figures 7A-7D). However, the proportion of myelinated fibers did not differ between R6 / 2 and WT mice at either time point (6 weeks: p = 0.58, 12 weeks: p = 0.09, by t-test) (Figures 1F and 1G). These data indicate that, compared to WT mice, R6 / 2HD mice acquire a progressive age-related loss of callosal myelin, reflected by a tonic decline in both the caliber and myelin thickness of their callosal axons.

[0211] Example 2 - Remyelination is significantly delayed in HD model R6 / 2 mice The cellular pathology that leads to developmental hypomyelination in HD mice may manifest as impaired remyelination. The copper chelator cuprizone, a well-established oral demyelinating toxin, was administered to both R6 / 2 and WT mice starting at 6 weeks of age for up to 6 weeks (Stidworthy et al., “Quantifying The Early Stages of Remyelination Following Cuprizone-Induced Demyelination,” Brain Pathol 13:329-339 (2003) (incorporated herein by reference in its entirety)). Mice were sacrificed at one of four time points (n=4 mice / genotype / time point): either 4 weeks after diet initiation, 10 weeks of age; 12 weeks, immediately after the end of the cuprizone diet; or 2 or 4 weeks later, either 14 or 16 weeks (FIG. 2A). All brains were processed for transmission electron microscopy and imaged as previously described, with quantitative assessment of axon diameter, g-ratio, and myelinated fiber frequency. At 4 weeks after starting the cuprizone diet, the corpus callosum of both R6 / 2 and WT mice showed fewer myelinated axons that differed in g-ratio (Fig. 2C, F(1,315) = 6.35, p = 0.012, by linear regression), but not in their distribution as a function of diameter (Fig. 2G, F(3,34) = 0.26, p = 0.50, by nonlinear regression), nor in their percentage of total myelinated axons (Fig. 2K, p = 0.63, by t-test). Among cuprizone-treated mice, remyelination was relatively deficient and delayed in time in R6 / 2 mice compared to WT controls at all studied time points. Indeed, by 12 weeks of age, the efficacy of remyelination was significantly different in R6 / 2 compared to WT mice (Figures 2D-2N). Remyelinated callosal axons in cuprizone-treated R6 / 2HD mice had higher g-ratios—indicating thinner myelin—than WT littermates both immediately after cessation of the cuprizone diet at 12 weeks, and 2 and 4 weeks after their recovery (12 weeks: F(1,812) = 2.412, p = 0.016; 14 weeks: F(1,1603) = 77.80, p < 0.0001; 16 weeks: F(1,1398) = 10.69, p < 0.001, by linear regression; Figures 2D-2F).Nonlinear regression analysis of the frequency of myelinated fibers as a function of diameter revealed that after cuprizone treatment, fibers with larger diameters were less likely to be myelinated in HD mice than in WT mice (12 weeks: F(3,24) = 3.79, p = 0.017 by nonlinear regression; 14 weeks: F(3,20) = 45.86, p < 0.0001; 16 weeks: F(3,24) = 14.66, p < 0.0001) (Figures 2H-2J and 7).

[0212] The percentage of remyelinated axons did not differ between WT and R6 / 2 mice at the earlier 10 and 12 week time points when mice were still receiving cuprizone (10 weeks: p = 0.62; 12 weeks: p = 0.29 by t-test). In contrast, the percentage of remyelinated axons was significantly higher in WT than R6 / 2 mice 2 and 4 weeks after cuprizone cessation, further indicating that WT mice recover and remyelinate significantly more quickly (14 weeks: p < 0.001; 16 weeks: p < 0.01 by t-test; Figures 2L-2N). Regression analysis of the number of remyelinated axons as a function of recovery time showed that with recovery, the percentage of myelinated fibers in R6 / 2 was consistently and significantly lower compared to WT littermates (F(2,116)=16.15, p<0.0001, by linear regression; FIG. 8). Thus, remyelination was significantly delayed and ultimately defective in cuprizone-demelinated R6 / 2 mice compared to WT controls.

[0213] Example 3 - Proteomic analysis of HD mouse white matter reveals reduced myelin protein expression To complement the ultrastructural analysis with a biochemical assessment of the relative myelination of HD and WT brains, mass spectrometry of colonic white matter was performed to more definitively establish and characterize hypomyelination in HD mouse brains. Two different HD mouse models were used to ensure that any disease-related correlations to hypomyelination were not model-specific. In particular, in addition to R6 / 2, zQ175 mice were used, which express a full-length mutant HTT with approximately 190 CAG repeats. zQ175 mice develop milder symptoms than R6 / 2 mice and have a normal life span, with most motor and behavioral symptoms beginning at 1 year of age. Thus, these mice reflect a later-onset form of HD (Menalled et al., “Comprehensive Behavioral and Molecular Characterization of a New Knock-in Mouse Model of Huntington's Disease:zQ175,” PLoS one,7:e49838 (2012); Carty et al., “Characterization of HTT Inclusion Size, Location, and Timing in the zQ175 Mouse Model of Huntington's Disease:an in vivo High-Content Imaging Study,” PloS one 10:e0123527 (2015) (both of which are incorporated by reference in their entireties).

[0214] Using these two different transgenics, the corpus callosum of 12-week-old R6 / 2 and 12-month-old zQ175 mice, in addition to their WT littermate controls, was sampled to identify conserved differences between HD and WT mice in their white matter proteomes (n=4 mice / group). Principal component analysis (PCA) of these samples revealed a sharp separation of diseased R6 / 2 corpus callosum white matter from WT, and a smaller but clear separation of zQ175 white matter from WT control white matter (Figure 15A). Differential expression analysis between these proteomes revealed 2,443 unique dysregulated peptides (1,076 downregulated and 1,367 upregulated; FDR<0.05) in R6 / 2 corpus callosum white matter, and 722 unique dysregulated peptides (304 downregulated, 418 upregulated) in zQ175 white matter (Figures 15B, 15C, and 15D). Of these differentially expressed cohorts, 416 peptides were found to be dysregulated in both HD models (Figure 15B). Numerous myelin proteins were differentially downregulated in R6 / 2 corpus callosum white matter, including Mbp, Mag, Mog, Mobp, Plp1, Gpr37, Aspa, and Cnp (Figures 15C and 15E). Downregulation of myelin proteins was also evident in 12-month-old zQ175 corpus callosum white matter, but not to the same dramatic extent as seen in R6 / 2 mice, and among those downregulated were Ugt8, Tf, Aspa, Lpar1, and Fa2h (Figures 15D and 15E).

[0215] Ingenuity pathway analysis of differentially expressed proteins in R6 / 2 corpus callosum white matter revealed predicted activation of HTT, PTEN, CREB1, and AMPK signaling, consistent with signatures consistent with both neurodegeneration and neurodegenerative motor disorders (Figure 15F). Concurrently, TCF7L2, BDNF, S1P, neuregulin, and ceramide signaling were all predicted to be suppressed in R6 / 2 white matter. Functional suppression of lipid synthesis and metabolism, metal ion transport, and RNA translation was also predicted in R6 / 2 mice. Similarly, 1-year-old zQ175 corpus callosum white matter showed milder functional dysregulation than R6 / 2, but AMPK signaling and neurodegenerative motor disorders were also predicted to be activated in zQ175, whereas TCF7L2 and neuregulin signaling, lipid synthesis and metabolism, and metal iron transport were all predicted to be suppressed (Figure 15F). Collectively, these proteomic data provide a biochemical correlate of the findings that HD is associated with dysmyelination and has a largely common molecular signature across HD genotypes.

[0216] Example 4 - GPCs derived from different HD models shared a common transcription defect We defined the transcriptional basis of the apparent defects in R6 / 2 and zQ175 HD mice in both myelin maintenance and reparative myelination. Previously, we identified human GPCs derived from HD-derived pluripotent stem cells in vitro as exhibiting oligodendrocyte impairment, reflected by downregulation of a critical set of glial transcription factors, including OLIG2, NKX2.2, SOX10, and MYRF (Osipovitch et al., “Human ESC-Derived Chimeric Mouse Models of Huntington's Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation,” Cell Stem Cell 24:107-122 e107 (2019) (incorporated herein by reference in its entirety)). To understand whether the defective developmental myelination and failed remyelination in R6 / 2 mice is transcriptionally altered, we used high-throughput RNA sequencing to examine the transcriptional profile of CD140a / PDGFRA-defined GPCs, which comprise the major source of oligodendrocytes in both mouse and human CNS (Sim et al., “Complementary Patterns Of Gene Expression By Human Oligodendrocyte Progenitors And Their Environment Predict Determinants Of Progenitor Maintenance And Differentiation,” Ann Neurol 59:763-779 (2011) (incorporated herein by reference in its entirety)). In addition, to ensure that any differential gene expression shown was not model-specific, a second mouse model was used, the zQ175 mouse, which expresses a full-length mutant HTT with 190 CAG repeats.Unlike R6 / 2 mice, zQ175 mice develop milder symptoms from 1 year of age and have a normal life span, thus representing a later-onset form of HD (Menalled et al., "Comprehensive Behavioral And Molecular Characterization Of A New Knock-In Mouse Model Of Huntington's Disease:zQ175," PloS one 7:e49838 (2012); Carty et al., "Characterization of HTT Inclusion Size, Location, and Timing in the zQ175 Mouse Model Of Huntington's Disease:an in vivo High-Content Imaging Study," PloS one 10:e0123527 (2015) (incorporated herein by reference in their entireties)).

[0217] To reliably identify and isolate GPCs from zQ175 mice, each mouse line was bred to PDGFRA-EGFP reporter mice (Hamilton et al., “Evolutionary Divergence of Platelet-Derived Growth Factor Alpha Receptor Signaling Mechanisms,” Mol Cell Biol 23:4013-4025 (2003) (incorporated herein by reference in its entirety)) to obtain proliferative GPC reporters for each HD line. Both presymptomatic (6 weeks for R6 / 2, 12 weeks for zQ175) and symptomatic (12 weeks for R6 / 2, 1 year for zQ175) mice were analyzed. GPCs were acutely isolated via FACS from the striatum of HD transgenic mice and their littermate controls at each time point, and 3–8 mice were pooled for each sample, depending on the age of the group ( FIG. 9 ). In that regard, 1-year-old mice (both zQ175 as well as their WT littermate controls) expressed significantly less EGFP than younger mice. +zQ175 had 100% EGFP counts (age effect: p<0.0001, F(1,20)=53.9 by two-way ANOVA; FIG. 9). This age-related decline in EGFP counts was consistent with the 100% EGFP counts in the striatum of zQ175 at both time points. + This was confirmed by cell stereological estimation (age effect: p<0.0001, F(1,23)=155.0; Figure 10). However, there was no difference in Olig2 stereological counts between 12-week-old and 1-year-old mice in either WT or zQ175 mice (genotype effect: p=0.91, F(1,22)=0.01; age effect: p=0.29, F(1,22)=1.16; Figure 10). Thus, EGFP + The decline in cells was not due to a loss of GPCs, but rather due to age-associated downregulation of PDGFRa expression in aged mice. Indeed, 12-week-old R6 / 2 mice had Olig. + We show that increased cell density, i.e., likely results from preserved numbers in the setting of reduced striatal volume in diseased mice, again suggesting that the decline in PDGFRA-driven EGFP expression in 1-year-old mice was a reflection of age rather than disease (Figure 10).

[0218] After batch correction, principal component analysis of these RNA-Seq samples revealed tight clustering of control and diseased mice that was more pronounced at later time points (Figure 3A). All groups showed transcriptional signatures consistent with the GPC phenotype (Figure 11). In R6 / 2, 598 genes were differentially expressed (FDR-adjusted p<0.01) at week 6, while 2988 genes were dysregulated at week 12. In contrast, zQ175 showed milder transcriptional dysregulation, with only 13 genes dysregulated at week 12 and 1066 genes dysregulated at year 1. Many dysregulated genes were shared between both R6 / 2 and zQ175 at both presymptomatic and diseased stages (Figure 3B). Scatter plots of normalized and variance-stabilized numbers of trait transcripts showed greater expression fold changes in R6 / 2 than zQ175 mice (Figure 3C).

[0219] Among the differentially expressed genes, several myelination genes were downregulated in both R6 / 2 and zQ175. These included Myrf, Bcas1, Plp1, Mbp, and Mobp (Figure 3D). Expression of genes prominent in early precursors and astrocytes was found to be enriched in both disease models, including Vim, Bmp4, S100b, Id3, Clu, and Lingo1. Functional analysis by IPA of the differentially expressed gene sets of R6 / 2 and zQ175 showed significant enrichment of terms involved in myelination, including myelination, lipid synthesis, and oligodendrocyte differentiation (Figure 3E). Upstream signaling related to myelination was also inhibited in both models, including SOX10-dependent transcription and TCF7L2-dependent transcription, each of which was significantly suppressed in HD GPCs.

[0220] Correlation of transcriptional and proteomic data was then performed by assaying 12-week-old striatal R6 / 2 and WT littermate control GPCs via mass spectrometry. This model and time point was chosen because its differential gene expression predicted the greatest degree of relative myelination. Striatal GPCs were dissected, dissociated, and isolated by sorting on A2B5, which targets ganglioside epitopes enriched on GPCs (n=3) (Gard and Pfeiffer, “Two Proliferative Stages of the Oligodendrocyte Lineage (A2B5+O4- and O4+GalC-) under Different Mitogenic Control,” Neuron 5:615-625 (1990); Roy et al., “Identification, Isolation, and Promoter-defined Separation of Mitotic Oligodendrocyte Progenitor Cells from the Adult Human Subcortical White Matter,” J Neurosci 19:9986-9995 (1999) (incorporated herein by reference in its entirety)). Unlike PDGFRa, A2B5 was not cleaved by papain, which was used for the enzymatic dissociation of tissue in this study, and was therefore ideal for acute dissociation and isolation of GPCs. PCA of the detected peptides revealed a tight clustering of R6 / 2 striatal GPCs and their separation from WT GPCs (Figure 16A). Differential expression analysis revealed 212 proteins that were dysregulated in R6 / 2 striatal GPCs compared to WT GPCs (Figure 16B). Examination of the intersection of differentially expressed proteins and genes revealed that 69.6% of genes and transcripts displayed consistent directionality (Figure 16C). Among these, R6 / 2 striatal GPCs notably showed sharply reduced levels of myelin and oligodendrocyte-enriched proteins Mobp, Fasn, and Ndrg1, consistent with disease-associated suppression of the myelination program in HD GPCs.

[0221] Example 5 - Network analysis revealed mHTT length-dependent defects in myelination To understand whether the common patterns of differential gene expression by R6 / 2 and zQ175GPC might identify potential upstream regulators of their common cellular pathology, the data were analyzed across all conditions using weighted gene expression correlation network analysis (WGCNA; (Langfelder and Horvath, “WGCNA: an R Package for Weighted Correlation Network Analysis,” BMC Bioinformatics 9:559 (2008) (incorporated herein by reference in its entirety)). This analysis yielded five distinct modules, termed black, turquoise, blue, magenta, and green (FIG. 4A). Ingenuity Pathway Analysis (IPA) of differentially expressed genes within each module showed a selective advantage of myelin-related terms within the black module (FIG. 4B). Applicants therefore proceeded to further investigate the black module through the construction of a functional IPA network.

[0222] Modularity analysis within black modules (Bastian et al., “Gephi: An Open Source Software for Exploring and Manipulating Networks,” Proc. Third Int'l ICWSM Conference 3(1):361-362 (2009), which is incorporated herein by reference in its entirety, generated five neighborhoods of closely related genes and IPA terms. "Neighborhood 1" had several myelination-related genes and terms, including Cnp, Mog, Mbp, Bcas1, Mobp, and Plp1 (FIG. 4C). "Neighborhood 2" included TCF7L2 signaling and related downstream targets, including the cholesterol biosynthesis pathway, enriched by differential expression of Cyp51a1, Hmgcs1, Idi1, and Dhcr7. "Neighborhood 3" isolated terms related to the autophagic and lysosomal pathways, while "Neighborhood 4" contained terms dealing with morphology and cytoskeletal remodeling. Finally, "Neighborhood 5" terms consisted of terms referring to glycolipid metabolism and peripheral myelination and demyelination.

[0223] Thus, the black module was heavily weighted in genes and associated terms related to oligodendrocyte differentiation and subsequent myelination. Among these, the most significantly dysregulated signaling pathway from the IPA analysis was TCF7L2 (Figure 3E), whose activation was predicted to be strongly suppressed in the HD model (Activation Z-score: 6 weeks: R6 / 2 = -7.99, 12 weeks: R6 / 2 = -11.15, 1 year: zQ175 = -6.57). This predicted suppression was due to the dysregulation of 38 TCF7L2 signaling pathway genes (Figure 12). These include transcripts involved in myelination and oligodendrocyte differentiation, cell cycle progression, and lipid and phospholipid synthesis (Figure 12). Collectively, these data suggest a causal relationship between TCF7L2 signaling and defective myelination in HD GPCs and further suggest that TCF7L2-dependent transcription may be defective in mHTT-expressing GPCs, thus implicating a rate-limiting determinant of myelination and maintenance in HD. However, neither Tcf7l2 gene expression nor protein levels or isoforms themselves appeared to be dysregulated in the model (Figures 17A and 17B), suggesting the involvement of additional, as yet unidentified partners or interactors in determining the effective transcriptional tone of Tcf7l2.

[0224] Example 6 - Rescue of key myelination genes by overexpression of TCF7L2 in R6 / 2 mice in vivo The possibility that mHTT-dependent reduction in TCF7L2 signaling may be causative in the myelination defects of HD was assessed by determining whether overexpression of TCF7L2 was sufficient to rescue myelination gene expression in GPCs from R6 / 2 mice. Because TCF7L2 has multiple splice variants that play different roles during development (Helgason et al., “Refining the Impact of TCF7L2 Gene Variants On Type 2 Diabetes And Adaptive Evolution,” Nature Genetics 39:218-225 (2007); Young et al., “Developmentally Regulated Tcf7l2 Splice Variants Mediate Transcriptional Repressor Functions During Eye Formation,” Elife 8 (2019) (incorporated herein by reference in their entirety)). TCF7L2 isoform expression was investigated in publicly available HD and control hESC-derived GPC RNA-Seq datasets (Osipovitch et al., “Human ESC-Derived Chimeric Mouse Models of Huntington's Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation,” Cell Stem Cell 24:107-122 e107 (2019) (incorporated herein by reference in its entirety)). The TCF7L2-210 isoform was identified as the most significantly enriched GPC isoform (Figure 13). Due to its high therapeutic relevance, the human transcript was used, and analysis in Blastn revealed 90.5% homology with the mouse transcript. The coding sequence was then inserted into the Tet-On lentiviral system to drive forced expression in vivo (Figure 14A). The resulting lentiviral TCF7L2 was then transfected into 10-week-old R6 / 2 mice (3.8 × 10 6cfu / 1 μl) were injected intrastriatally. Mice were then equally divided into two groups, one treated with doxycycline and the other untreated control group (n=5 mice per group). Two weeks after lentiviral TCF7L2 injection, RT-QPCR was performed on dissected striatal tissue to evaluate treatment-related changes in the expression of predicted TCF7L2-dependent targets. In particular, the expression of myelination genes such as Myrf, Mag, Plp1, Mbp and Trf was evaluated. Lipid biosynthesis genes Srebf1, Srebf2, and Hmgcr were monitored, recognizing that TCF7L2 has a role in regulating myelogenesis and lipid metabolism.

[0225] The expression levels of these TCF7L2 targets were significantly increased, whereas other TCF7L2 targets, such as Ctnnb1 (Hammond et al., “The Wnt Effector Transcription Factor 7-like 2 Positively Regulates Oligodendrocyte Differentiation In A Manner Independent Of Wnt / Beta-Catenin Signaling,” J Neurosci 35:5007-5022(2015) (incorporated herein by reference in its entirety)), Dkk1, Lrp6 (Su et al., “Effects Of The Extracellular Matrix On Myelin Development And Regeneration In The Central Nervous System,” Tissue Cell 69:101444(2021) (incorporated herein by reference in its entirety)), Fzd8, Kaiso / Zbtb33 (Zhao et al., “Dual Regulatory Switch Through Interactions Of Tcf7l2 / Tcf4 With Stage-Specific Partners Propels Oligodendroglial Maturation,” Nature communications 7:10883(2016) (incorporated herein by reference in its entirety)), Stk11 (Nguyen-Tu et al., “Transcription Factor-7-Like 2 (TCF7L2) Gene Acts Downstream Of The Lkb1 / Stk11 Kinase to Control mTOR Signaling, Beta Cell Growth, and Insulin Secretion,” J Biol Chem 293:14178-14189(2018) (incorporated herein by reference in its entirety)), and Tle1 (Dugas et al.The expression levels of other WNT signaling-related genes that are not targets of TCF7L2 (Functional Genomic Analysis of Oligodendrocyte Differentiation, J Neurosci 26:10967-10983 (2006) (incorporated herein by reference in its entirety)) were significantly increased (Figure 5). These data indicate that overexpression of TCF7L2 was sufficient to rescue the expression of key myelin biosynthesis and metabolism genes that are otherwise downregulated in R6 / 2HD mice.

[0226] Example 7 - TCF7L2 rescues remyelination failure in R6 / 2 mice in vivo. To determine whether lentiviral overexpression of TCF7L2 (LV-TCF7L2) was sufficient to rescue the remyelination failure of R6 / 2 mice, a cohort of wild-type mice (n=3) was subjected to cuprizone demyelination beginning at 6 weeks of age. Then, at 10 weeks of age, mice were stereotactically injected with LV-TCF7L2 (FIG. 14) into the corpus callosum just above the fornix, the region most rapidly and completely demyelinated by cuprizone (Schmidt et al., “Regional Heterogeneity of Cuprizone-Induced Demyelination: Topographical Aspects of The Midline of the Corpus Callosum,” J Mol Neurosci 49:80-88 (2013) (incorporated herein by reference in its entirety)). Immediately after viral injection, mice were orally administered doxycycline to activate the expression of TCF7L2. Mice were then killed 2 weeks later at 12 weeks of age, and their brains were cryosectioned and immunolabeled. In these WT control mice, LV-TCF7L2-transduced corpus callosum cells, easily identified by the EGFP reporter, were predominantly OLIG2 + and NG2 +The LV-TCF7L2 vector was found to be composed of oligodendrocyte lineage cells expressing GFAP, some of which expressed astroglial GFAP (Figure 14), thus establishing the ability of the LV-TCF7L2 vector to efficiently transduce and express in normal corpus callosum GPCs and their progeny.

[0227] Next, to evaluate whether forced expression of TCF7L2 would then enhance oligodendrocyte differentiation and improve myelination in HD mice, a group of R6 / 2 mice and their wild-type littermate controls were fed cuprizone starting at 6 weeks of age (R6 / 2, n=8; wild-type, n=4) (Figure 6A). Age-matched groups of R6 / 2 controls were fed a normal diet (n=4). Of note, it has been shown that doxycycline itself does not affect myelination (Osipovitch et al., “Human ESC-Derived Chimeric Mouse Models of Huntington's Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation,” Cell Stem Cell 24:107-122 e107 (2019) (incorporated herein by reference in its entirety)). R6 / 2 mice were then given an intracallosal injection of LV-TCF7L2-EGFP at 10 weeks of age. At 12 weeks of age, all mice were placed back on a normal diet and all were sacrificed 2 weeks later (Figure 6A).

[0228] Electron microscopy image analysis of remyelinated callosal axons, performed as described above (Figures 1 and 2), showed that myelin sheaths from cuprizone-exposed and LV-TCF7L2-treated R6 / 2 mice had significantly lower g-ratios than those from untreated R6 / 2 mice. Importantly, remyelinated axons from LV-TCF7L2-treated R6 / 2 mice were substantially normalized, with g-ratios similar to those of wild-type mice (WT vs. R6 / 2, p<0.0001; WT vs. R6 / 2+LV-TCF7L2, p=0.34; R6 / 2 vs. R6 / 2+LV-TCF7L2, p=0.0001; all by linear regression) (Figures 6B-6C). Analysis of the distribution of remyelinated fibers as a function of diameter showed that myelinated axons in R6 / 2 mice treated with LV-TCF7L2 had larger axon diameters and the distribution was similar to that of wild-type mice (treatment effect: F[4,27]=6.05; p=0.001 by two-way ANOVA) (Figure 6D). Moreover, LV-TCF7L2 also restored the relative distribution of myelinated axon sizes in wild-type mice (F[2.57]=22.07; p<0.0001, one-way ANOVA; Figure 6E). Taken together, this data indicates that forced expression of TCF7L2 is sufficient to rescue the myelination defect in R6 / 2 mice. Study of the Example

[0229] Huntington's disease (HD) is characterized by defective oligodendrocyte differentiation and white matter disease. Here, applicants investigated the role of GPC dysfunction in adult myelin maintenance in HD. Progressive age-related myelin loss was first noted in both R6 / 2 and zQ175 HD mice compared to wild-type controls. Then, as adults, R6 / 2 mice showed a significant delay in remyelination after cuprizone demyelination. RNA sequencing and proteomic analysis of corpus callosum white matter and GPC isolated from both R6 / 2 and zQ175 mice revealed a systematic downregulation of genes related to oligodendrocyte differentiation and myelination compared to controls. Gene co-expression and network analysis predicted suppressed TCF7L2 signaling as the primary driver of this expression pattern. Overexpression of TCF7L2 in vivo was found to be sufficient to restore both myelin gene expression and normal myelination in demyelinated R6 / 2 mice. These data demonstrate a causal link between impaired TCF7L2-dependent transcription and the poor development and maintenance of myelin in HD and provide a mechanism for its therapeutic repair.

[0230] Huntington's disease (HD) has been considered primarily a neurological disease due to its associated significant loss of striatal and cortical neurons. However, a growing body of research suggests that glial and white matter pathology is not only present at early stages but also plays a contributing role in the pathogenesis of HD (Poudel et al., "Longitudinal Change in White Matter Microstructure In Huntington's Disease: The IMAGE-HD Study," Neurobiol Dis 74:406-412 (2015); McColgan et al., "Brain Regions Showing White Matter Loss in Huntington's Disease Are Enriched for Synaptic and Metabolic Genes," Biol Psychiatry 83:456-465 (2018) (incorporated herein by reference in its entirety)). Applicants have previously shown that HD-derived human glial progenitor cells and their derived astrocytes exhibit abnormal patterns of gene expression in an mHtt-dependent manner (Osipovitch et al., “Human ESC-Derived Chimeric Mouse Models of Huntington's Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation,” Cell Stem Cell 24:107-122 e107 (2019) (incorporated herein by reference in its entirety)). When transplanted into hypomyelinating shiverer mouse hosts, these cells exhibited delayed, and ultimately defective, myelination, which could be rescued by MYRF-SOX10 expression in vivo. Retention of the oligodendrocyte differentiation defect by these HD hGPCs after transplantation into shiverers suggested the cell-intrinsic nature of their maturation defect.This concept was confirmed in a mouse model of HD by targeted depletion of mutant Htt in resident GPCs, which rescued their myelination capacity and partially reversed the motor defects of HD mice (Ferrari Bardile et al., “Intrinsic Mutant HTT-Mediated Defects in Oligodendroglia Cause Myelination Deficits and Behavioral Abnormalities in Huntington Disease,” PNAS 116:9622-9627 (2019) (incorporated herein by reference in its entirety)).

[0231] However, it is unclear whether such myelination defects occur both in the adult and in vivo and whether they result from downregulation of myelination transcription factors. To this end, Applicants have investigated myelination in R6 / 2 HD mice and shown that the g-ratio, a reliable predictor of functional and structural axonal myelination, is significantly higher in 12-week-old R6 / 2 mice compared to healthy WT controls, indicating a thin and / or missing myelin sheath (Hildebrand and Hahn, “Relation Between Myelin Sheath Thickness and Axon Size in Spinal Cord White Matter of Some Vertebrate Species,” J Neurol Sci 38:421-434 (1978); Chomiak and Hu, “What is the Optimal Value of the g-ratio for Myelinated Fibers in the Rat CNS? A Theoretical Approach,” PloS one 4:e7754 (2009) (incorporated herein by reference in its entirety). This suggests that maintenance of myelin sheath thickness and integrity is a continuous process that is affected by mutant Htt in adults as the disease progresses.

[0232] Remyelination was also investigated by feeding adult mice a cuprizone-containing diet (Blakemore, "Demyelination of the Superior Cerebellar Peduncle in the Mouse Induced by Cuprizone," Journal of the Neurological Sciences 20:63-72 (1973); Stidworthy et al., "Quantifying the Early Stages of Remyelination Following Cuprizone-Induced Demyelination," Brain Pathol 13:329-339 (2003) (incorporated herein by reference in their entireties)). Once the mice had recovered from 6 weeks of dietary cuprizone treatment, R6 / 2 mice showed a significant and progressive delay in remyelination, as quantified by both a higher mean g-ratio and lower myeloid fiber density, compared to WT controls. Interestingly, a similar process of delayed remyelination in response to demyelination was observed in YAC128 mice, another full-length mutant HTT model of HD (Teo et al., “Impaired Remyelination in a Mouse Model of Huntington Disease,” Mol Neurobiol 56:6873-6882 (2019) (incorporated herein by reference in its entirety)).

[0233] To better define the mechanistic basis of this observation, gene expression of GPCs was profiled in two models of HD, R6 / 2 and zQ175 mice, which are models of juvenile and adult onset HD, respectively. GPCs from both models were isolated from both presymptomatic and disease manifest mice, thereby improving the identification and isolation of those pathways involved in the progressive deficits of remyelination in these animals. Indeed, gene expression analysis showed that HD GPCs exhibited striking changes in gene expression in diseased mice - significantly more dysregulated in R6 / 2 than zQ175, likely due to the more severe phenotype of the former. Taken together, these data in three very different models (R6 / 2, zQ175 and YAC128) all suggested a cell-intrinsic defect in oligodendrocyte maturation and myelination that is maintained in vivo.

[0234] Subsequent pathway analysis revealed that TCF7L2 signaling was the most dysregulated pathway in HD and GPC across models and predicted that this pathway was strongly suppressed. Tcf7l2 is a member of the TCF / LEF family, a key downstream effector of Wnt / β-catenin signaling in Wnt activation (Arce et al., "Diversity of LEF / TCF Action in Development and Disease," Oncogene 25:7492-7504 (2006) (incorporated herein by reference in its entirety)). Tcf7l2 regulates myelination as well as cholesterol biosynthesis genes (Saher et al., “High Cholesterol Level Is Essential For Myelin Membrane Growth,” Nat Neurosci 8:468-475(2005); Fancy et al., “Dysregulation of the Wnt Pathway Inhibits Timely Myelination and Remyelination in The Mammalian CNS,” Genes Dev 23:1571-1585(2009); Zhao et al., “Dual Regulatory Switch Through Interactions of Tcf7l2 / Tcf4 with Stage-Specific Partners Propels Oligodendroglial Maturation,” Nature Communications 7:10883(2016) (incorporated herein by reference in their entireties)), both of which are disrupted in HD (Valenza et al., “Cholesterol Defect is Marked Across Multiple Rodent Models of Huntington's Disease,” Nature Communications 7:10883(2016) (incorporated herein by reference in their entireties)). Disease and is Manifest in Astrocytes,”J Neurosci 30:10844-10850(2010), Benraiss et al., “Cell-intrinsic Glial Pathology is Conserved Across Human and Murine Models Of Huntington's Disease,” Cell Reports 36:109308 (2021) (incorporated by reference in its entirety)), suggesting the importance of TCF7Ll2-dependent transcription in mHTT-expressing glial and oligodendrocyte precursor cells (Huang et al., “Mutant Huntingtin Downregulates Myelin Regulatory Factor-Mediated Myelin Gene Expression and Affects Mature Oligodendrocytes,” Neuron 85:1212-1226 (2015); Zhao et al., “Dual Regulatory Switch Through Interactions of Tcf7l2 / Tcf4 with Stage-Specific Partners Propels Oligodendroglial Maturation,” Nature Communications 7:10883 (2016); Osipovitch et al., “Human ESC-Derived Chimeric Mouse Models of Huntington's Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation,” Cell Stem Cell 24:107-122 e107(2019) (incorporated by reference in its entirety). TCF7L2 controls oligodendrocyte differentiation and remyelination through multiple mechanisms, and its expression is tightly regulated during oligodendrocyte development (Fu et al., “Tcf7l2 is Tightly Controlled During Myelin Formation,” Cell Mol Neurobiol 32:345-352(2012); Zhao et al., “Dual Regulatory Switch Through Interactions of Tcf7l2 / Tcf4 with Stage-Specific Partners Propels Oligodendroglial Maturation,” Nature communications 7:10883(2016); Weng et al., “Transcription Factor 7 like 2 Promotes Oligodendrocyte Differentiation and Remyelination,” Mol Med Rep 16:1864-1870(2017) (incorporated by reference in their entireties). Previous studies have demonstrated that TCF7L2 suppresses the bone morphogenetic protein signaling pathway, which has been shown to inhibit oligodendrocyte differentiation while promoting astrocyte differentiation (Mabie et al., “Bone Morphogenetic Proteins Induce Astroglial Differentiation of Oligodendroglial-Astroglial Progenitor Cells,” J Neurosci 17:4112-4120(1997); Sim et al., “Bone Morphogenetic Proteins Induce Astroglial Differentiation of Oligodendroglial Progenitor Cells,” J Neurosci 17:4112-4120(2006); Morell et al., “Inducible Expression of Noggin Selectively Expands Neural Progenitors in the Adult SVZ,” Stem Cell Res 14:79-94(2015); Zhang et al., “The Wnt Effector TCF7l2 Promotes Oligodendroglial Differentiation by Repressing Autocrine BMP 4-Mediated Signaling,” J Neurosci 41:1650-1664 (2021) (incorporated by reference in its entirety). TCF7L2 also functions as an effector of Wnt signaling, a key pathway for oligodendrogenesis (Fancy et al., “Dysregulation of the Wnt Pathway Inhibits Timely Myelination and Remyelination in The Mammalian CNS,” Genes & Development 23:1571-1585 (2009b) (incorporated by reference in its entirety). Importantly, at the onset of oligodendrocyte differentiation, TCF7L2 interacts with the transcriptional corepressor Kaiso / Zbtb33 to block β-catenin signaling, reinforcing oligodendrocyte fate, which then promotes further oligodendrocyte maturation via interaction with Sox10 (Zhao et al., “Dual Regulatory Switch Through Interactions of Tcf7l2 / Tcf4 with Stage-Specific Partners Propels Oligodendroglial Maturation,” Nature Communications 7:10883 (2016) (incorporated herein by reference in its entirety)). However, beyond its cell-autonomous effects on oligodendrocyte differentiation, Tcf7l2 signaling may also participate in driving oligodendrocyte fate through paracrine mechanisms. As mentioned above, the effects of Tcf2 may include not only relieving the differentiation block of mHtt-expressing GPCs, but also rescuing astrocyte cholesterol synthesis and lipogenesis, which may then support oligodendrocyte myelination. .

[0235] Interestingly, TCF7L2 gene expression itself was not significantly downregulated in either R6 / 2 or zQ175 mice, so it seems unlikely that mHtt acts to suppress its transcription. Rather, the data herein suggest that other checkpoints in the Wnt-regulated pathway downstream of TCF7L2, which are important for the induction of oligo- and myelination, may be pathologically rate-limited in HD but may be compensated for by overexpression of TCF7L2. Thus, additional modulators of Tcf7l2-dependent transcription may be causally involved in the suppression of downstream Tcf7l2 signaling in HD white matter, such that Tcf7l2 overexpression remains sufficient to overcome that suppression and rescue myelination. Indeed, several partners have been identified for Tcf7l2-dependent transcriptional activation (see Zhao et al., “Dual Regulatory Switch Through Interactions of Tcf7l2 / Tcf4 with Stage-Specific Partners Propels Oligodendroglial Maturation,” Nature Communications 7:10883 (2016), incorporated herein by reference in its entirety), whose relative expression levels may regulate Tcf7l2-dependent gene expression whose levels and activities have yet to be examined in HD. Regardless of the identity of these agonistic transcriptional modulators, the data strongly argue that overexpression of TCF7L2 is necessary and sufficient for the correct expression of both myelination and lipid biosynthesis genes in HD GPCs and can rescue the remyelination failure of cuprizone-treated R6 / 2 mice.

[0236] Together, these data indicate that HD is associated with a progressive age-related loss of forebrain myelin as well as impaired remyelination after adult demyelination compared to WT mice, as shown in two different transgenic mouse models of the disease. Collectively, these findings suggest a loss of homeostatic white matter maintenance. This was accentuated by the severe dysregulation of oligodendrocyte lineage-related gene expression predicted to be driven by upstream TCF7L2 signaling. Importantly, forced glial overexpression of TCF7L2 restored functional transcription of key myelination and lipid biosynthesis genes and proved sufficient for in vivo restoration of myelin structure and abundance. Thus, the present study may provide a novel and effective strategy for the therapeutic rescue of glial dysfunction and thus both synaptic and white matter pathology in HD.

[0237] It will be appreciated that variations of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems or applications, and various presently unforeseen or unanticipated substitutions, modifications, variations, or improvements there...

Claims

1. 1. A host cell comprising a genetic construct or expression vector encoding transcription factor 7-like 2 (TCF7L2) or TCF7L2 protein for use in a method of treating a subject having a condition mediated by a lack of myelin, said method comprising: introducing said TCF7L2 into said subject in need of treatment; expressing a transcription factor 7-like 2 protein in one or more cells of the subject, or administering the host cell to the subject.

2. Expressing transcription factor 7-like 2 protein in a glial progenitor cell population; maintaining said population of glial progenitor cells under conditions that allow their development and differentiation, The expressing comprises administering to the glial progenitor cell population a genetic construct comprising: a nucleic acid molecule encoding the transcription factor 7-like 2 protein; a promoter and / or enhancer of a gene selectively or specifically expressed by glial progenitor cells; the nucleic acid molecule is operably linked to and under the regulatory control of the promoter and / or enhancer; The method is carried out by administering a genetic construct.

3. wherein the expressing comprises administering to the subject or glial progenitor cell population a genetic construct comprising: a nucleic acid molecule encoding the transcription factor 7-like 2 protein; a promoter and / or enhancer of a gene selectively or specifically expressed by glial progenitor cells; the nucleic acid molecule is operably linked to and under the regulatory control of the promoter and / or enhancer; administered by administering a genetic construct, The TCF7L2 or host cell of claim 1.

4. The TCF7L2 or host cell of claim 3, wherein the gene selectively or specifically expressed by glial progenitor cells is selected from the group consisting of PDGFRA, ZNF488, GPR17, OLIG2, CSPG4, and SOX10.

5. the genetic construct is administered in an expression vector; the expression vector is a viral vector, a plasmid vector, or a bacterial vector; and The viral vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and a vaccinia vector. The TCF7L2 or host cell of claim 4.

6. The TCF7L2 or host cell of claim 5, wherein the genetic construct is administered in association with a glial progenitor cell-targeting fusogen or a glial progenitor cell-selective surface-binding moiety.

7. The TCF7L2 or host cell of claim 6, wherein the genetic construct is within a particle comprising the glial progenitor cell-targeted fusogen or the glial progenitor cell-selective surface-binding moiety.

8. The TCF7L2 or host cell of claim 7 , wherein the particle is one selected from the group consisting of a virus, a virus-like particle, and a lipid particle.

9. The TCF7L2 or host cell of any one of claims 6 to 8, wherein the glial progenitor cell-targeting fusogen or glial progenitor cell-selective surface-binding moiety is directed against CD140a, NG2 / CSPG4, A2B5 ganglioside, O4 sulfatide or CD133.

10. 9. The TCF7L2 or host cell of any one of claims 1 and 3 to 8, wherein the condition is selected from the group consisting of childhood leukodystrophy, lysosomal storage diseases, congenital dysmyelination, cerebral palsy, inflammatory demyelination, post-infectious and post-vaccination leukoencephalitis, radiation- or chemotherapy-induced demyelination, and vascular demyelination.

11. the subject has a condition involving defective remyelination, and 9. The TCF7L2 or host cell of any one of claims 1 and 3 to 8, wherein the condition is selected from the group consisting of multiple sclerosis, neuromyelitis optica, transverse osteomyelitis, optic neuritis, subcortical stroke, diabetic leukoencephalopathy, hypertensive leukoencephalopathy, age-related white matter disease, leukodementia, Binswanger's disease, spinal cord injury, radiation or chemotherapy-induced demyelination, post-infectious and post-vaccination leukoencephalitis, periventricular leukomalacia, and cerebral palsy.

12. The TCF7L2 or host cell of any one of claims 1 and 3 to 8, wherein the condition is a neurodegenerative disease.

13. 13. The TCF7L2 or host cell of claim 12, wherein the condition is Huntington's disease.

14. The TCF7L2 or host cell of any one of claims 1 and 3 to 8, wherein the condition is a neuropsychiatric disorder.

15. 15. The TCF7L2 or host cell of claim 14, wherein the condition is schizophrenia.

16. The TCF7L2 or host cell of any one of claims 3 to 8, wherein said administering is performed using intracerebral delivery, intrathecal delivery, intranasal delivery, or via direct injection into the ventricles of the brain.

17. The TCF7L2 or host cell according to any one of claims 1 and 3 to 8, wherein the subject is a mammal.

18. The TCF7L2 or host cell of claim 17, wherein the subject is a human.

19. 9. The TCF7L2 or host cell of any one of claims 1 and 3 to 8, wherein the condition is characterized by downregulation of one or more genes selected from the group consisting of Myrf, Bcas1, Plp1, Mbp, and Mobp.

20. A genetic construct comprising: a nucleic acid molecule encoding a transcription factor 7-like 2 protein; a promoter and / or enhancer of a gene selectively or specifically expressed by glial progenitor cells, wherein said nucleic acid molecule is operably linked to and under the regulatory control of said promoter and / or enhancer; A gene construct, wherein the gene selectively or specifically expressed by glial progenitor cells is selected from the group consisting of PDGFRA, ZNF488, GPR17, OLIG2, CSPG4, and SOX10.

21. 21. An expression vector comprising the genetic construct of claim 20, wherein the expression vector is a viral vector, a plasmid vector, or a bacterial vector.

22. 22. The expression vector of claim 21, wherein the viral vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and a vaccinia vector.

23. The genetic construct of claim 20 , wherein the genetic construct is associated with a glial progenitor cell-targeting fusogen or a glial progenitor cell-selective surface-binding moiety.

24. The genetic construct of claim 23, wherein the glial progenitor cell-targeting fusogen or glial progenitor cell-selective surface-binding moiety is directed against CD140a, NG2 / CSPG4, A2B5 ganglioside, O4 sulfatide, or CD133.

25. A host cell, or a descendant of said host cell, comprising a genetic construct or an expression vector according to any one of claims 20 to 24.

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