Molecular genetic strategies for reactivating juvenile plasticity in target adult neural circuits

Abstract

Transplantation of inhibitory neural progenitor cells reactivates a targeted, time-limited phase of critical plasticity within recipient brain regions. This restoration of youthful plasticity establishes a therapeutic environment leading to circuit reorganization, which has consistently demonstrated improvement in cognitive and functional impairments. Notably, mere overexpression of Calb1 in adult inhibitory neurons via a viral vector is sufficient to reactivate critical plasticity without the need for transplantation. Consequently, methodologies and compositions for enhancing Calb1 expression as a therapeutic means for reactivating plasticity are described herein.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 499,826, filed May 3, 2023, the specification of which is incorporated herein by reference in its entirety.

[0002] Description of Research and Development Funded by the Federal Government This invention was made with government support under Grant Numbers 1F31EY034032 - 01 and R01EY029490 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Field of the Invention The present invention features methodologies and compositions for modulating Calb1 expression as a therapeutic means for re - activating plasticity.

Background Art

[0004] The critical period is a well - defined postnatal developmental window during which neural circuits are particularly receptive to sensory input. This enhanced state of plasticity allows experiences to strongly re - arrange and optimize local wiring for concurrent conditions. Different from the transient changes enabled by adult - type plasticity, the changes that occur during the critical period are strongly fixed and thus control the functional capabilities of the circuit into adulthood. The timing of the critical period is strongly related to the maturation of inhibitory circuits, but it has been difficult to separate mechanisms specific to plasticity from those specific to development. Faithful re - activation of critical - period plasticity is likely mediated by factors expressed during early stages of inhibitory neuron development.

[0005] Inhibitory neural progenitor cells in the brain originate from the basal ganglia primordium, a transient embryonic brain structure in which its subregions give rise to different inhibitory subpopulations with various trajectories: 1) the medial basal ganglia primordium (MGE) which produces parvalbumin (PV) and somatostatin (SOM) inhibitory neurons, 2) the lateral basal ganglia primordium (LGE) which produces striatal PV and SOM inhibitory neurons, and 3) the caudal basal ganglia primordium (CGE) which mostly produces corticovasoactive intestinal polypeptide (VIP) inhibitory neurons. For strong retention of developmental programming, transplanted inhibitory neural progenitor cells mature appropriately and form synergistic connections with host circuits. Unlike LGE and CGE, transplantation of MGE-derived inhibitory neural progenitor cells strongly reactivates critical plasticity within the recipient brain region. Notably, the reactivated critical period occurs not immediately after transplantation, but approximately one month after transplantation, which is the first timing for the maturation of the transplanted inhibitory neurons. This alignment in temporal programming strongly suggests that the transplant reactivation critical period is triggered by factors specific to the transplanted cells.

[0006] Given its remarkable ability to correct dysfunctional circuits, inhibitory neuron transplantation has attracted favorable attention from a wide range of CNS disorders / disorders, including epilepsy, autism, stroke, schizophrenia, Alzheimer's disease, Parkinson's disease, and neuropathic pain. Many of these involve excitation / inhibition imbalances, but pharmacological enhancement of inhibition fails to achieve complete phenotypic mimicry transplantation-mediated functional recovery. Consistent with observations from the juvenile critical period, inhibitory neuron transplantation is likely to rescue the defect by involving mechanisms beyond GABAergic transmission. Despite its broad applications, the underlying cellular and molecular mechanisms of inhibitory neuron transplantation remain unclear. Furthermore, candidate molecules previously thought to be involved in juvenile critical plasticity either do not adequately explain, or are not related to, the transplantation reactivation critical period. [Overview of the Initiative]

[0007] The object of the present invention is to provide compositions and methods that enable the reactivation of plasticity without dependence on the cell vehicle, as specified in the independent claims. Embodiments of the present invention are described in the dependent claims. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.

[0008] In some embodiments, the present invention features a method for increasing neural plasticity (e.g., experience-dependent plasticity) in a target that requires it. The method may include administering a gene therapy drug to a target, which is configured to modulate (e.g., increase) the expression of calbindin (Calb1) in a population of target neurons. In some embodiments, the neural plasticity (e.g., experience-dependent plasticity) of the target is increased by modulating (e.g., increasing) the expression of Calb1. In other embodiments, the neural plasticity (e.g., experience-dependent plasticity) in a population of target neurons is increased by modulating (e.g., increasing) the expression of Calb1. Non-limiting examples of gene therapy drugs include, but are not limited to, viral vectors, exosomes, nanoparticles, small interfering RNAs (siRNAs) and microRNAs (miRNAs), antisense oligonucleotides (ASOs), aptamers, and modified cells. In some embodiments, the population of neurons includes excitatory neurons, inhibitory neurons, or a combination thereof. In some embodiments, the gene therapy drug directly increases the expression of Calb1. In other embodiments, the gene therapy drug indirectly increases the expression of Calb1. In certain embodiments, the method may further include measuring Calb1 expression before administering a gene therapy drug.

[0009] In other embodiments, the present invention features a method for treating a CNS disease or disorder in a subject requiring such treatment. The method may include administering a gene therapy drug to a subject, the gene therapy drug configured to modulate (e.g., increase) the expression of calbindin (Calb1) in a population of nerve cells of the subject. In some embodiments, modulating (e.g., increasing) the expression of Calb1 increases the neuroplasticity (e.g., experience-dependent plasticity) of the subject, thereby promoting the treatment or regression of a CNS disease or disorder. In other embodiments, modulating (e.g., increasing) the expression of Calb1 increases the neuroplasticity (e.g., experience-dependent plasticity) in a population of nerve cells of the subject, thereby promoting the treatment or regression of a CNS disease or disorder. In some embodiments, the CNS disorder or disease may include epilepsy, autism, stroke, schizophrenia, Alzheimer's disease, Parkinson's disease, or neuropathic pain. Non-exclusive examples of gene therapies include, but are not limited to, viral vectors, exosomes, nanoparticles, small interfering RNA (siRNA) and microRNA (miRNA), antisense oligonucleotides (ASOs), aptamers, and modified cells. In some embodiments, the population of nerve cells includes excitatory neurons, inhibitory neurons, or a combination thereof. In some embodiments, the gene therapy directly increases Calb1 expression. In other embodiments, the gene therapy indirectly increases Calb1 expression. In certain embodiments, the method may further include measuring Calb1 expression before administering the gene therapy.

[0010] In certain embodiments, the methods described herein may be carried out within a specific adult neural circuit.

[0011] In some embodiments, the present invention features a method for screening experience-dependent plasticity inducers and agents. The method may include administering (or having administered) one or more factors and agents to a sample and measuring (or having measured) the level expression of calbindin (Calb1). In other embodiments, the method includes obtaining (or having obtained) a sample, administering (or having administered) one or more factors and agents to the sample and measuring (or having measured) the level expression of calbindin (Calb1). In some embodiments, an increase in Calb1 expression indicates that one or more factors and agents induce experience-dependent plasticity. In some embodiments, the sample is a biological sample (e.g., ex vivo tissue, primary culture, iPSC, organoid, etc.).

[0012] In other embodiments, the present invention features a method for screening experience-dependent plasticity inducers and agents. The method may include administering (or having administered) one or more factors and agents to a target or appropriate animal model and measuring (or having measured) the level expression of calbindin (Calb1). In some embodiments, an increase in Calb1 expression indicates that one or more factors and agents induce experience-dependent plasticity. In certain embodiments, the method may further include measuring (or having measured) the level of calbindin expression before administering one or more factors and agents.

[0013] One of the unique and inventive technical features of this invention is the targeting of the specific factor Calb1 to mimic transplantation-induced plasticity. While we do not wish to limit this invention to any theory or mechanism, the technical features of this invention are considered to advantageously provide cell vehicle-independent therapies. None of the currently known prior references or materials possess the unique and inventive technical features of this invention.

[0014] Furthermore, prior references teach that this invention is not directly related to the present invention. For example, the use of embryonic donor tissue makes the direct clinical application of inhibitory neuron transplantation impractical. Moreover, xenotransplantation requires continuous immunosuppression, but existing protocols for iPSC-derived inhibitory neural progenitor cells face limitations in terms of yield and purity. Regardless of their origin, preclinical studies on inhibitory neuron transplantation highlight the susceptibility of immature transplanted cells to active pathological conditions. Therefore, this invention represents a remarkable advance using the specific factor Calb1 to mimic transplantation-induced plasticity without relying on the cell vehicle. As a result, the treatment outlined herein results in improved safety and control.

[0015] Furthermore, technologies aimed at enhancing general plasticity, such as social enrichment, exercise, or exposure to novelty, may only produce temporary effects and are limited by their ability to induce significant change. Additionally, strategies to enhance experience-dependent plasticity in adulthood, such as reducing inhibitory mechanisms or removing natural brakes on juvenile plasticity, do not always result in a seamless transition. For example, reduced inhibition may increase susceptibility to seizures, while eliminating plasticity brakes may hinder memory consolidation.

[0016] Furthermore, the inventive technical features of the present invention contributed to surprising results. For example, the inventors surprisingly found that increasing the expression of a specific factor, namely Calb1, in a subset of neurons resulted in a significant circuit-scale effect. In particular, increased Calb1 expression in adult neurons, especially inhibitory neurons, had a beneficial effect on plasticity and could reproduce transplantation effects even in the absence of additional factors or cells.

[0017] Furthermore, it is noteworthy that, unexpectedly, LGE transplantation does not induce any discernible effect on Calb1 expression levels beyond those observed in non-transplanted adults. A significant increase in Calb1 expression remains clearly associated with MGE transplantation and is consistent with functional analyses performed in previous transplantation studies.

[0018] Any feature or combination of features described herein is included within the scope of the invention, provided that the features included in any such combination are not inconsistent with each other, as is evident from the context, this specification, and the knowledge of those skilled in the art. Additional advantages and aspects of the invention are evident in the following detailed description and claims.

[0019] The features and advantages of the present invention will become apparent from the following detailed description shown in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0020] [Figure 1A] This figure shows the experimental method used to obtain the data described herein. Donor mice provided embryos from which GABAergic interneurons were isolated, and these neurons were subsequently injected into adult recipients (hosts) having ZsGreen-labeled host interneurons. We have established that donor MGE cells induce a new critical period for experience-dependent plasticity in host animals 35 days after transplantation (Southwell, 2010; Davis, 2015; Zheng, 2021). ZsGreen-labeled host interneurons were sorted and sequenced. Differential expression analysis was performed between host interneurons from MGE-transplanted, LGE-transplanted, and non-transplanted mice during the induced critical period (35 DAT) and post-closure (70 DAT). [Figure 1B]A figure showing the experimental methods used to obtain the data described in this specification. Donor mice provided embryos from which GABAergic interneurons were isolated, and these neurons were then injected into adult recipients (hosts) that had ZsGreen-labeled host interneurons. The inventors established that donor MGE cells induce a new critical period for experience-dependent plasticity in host animals 35 days after transplantation (Southwell, 2010; Davis, 2015; Zheng, 2021). ZsGreen-labeled host interneurons were sorted and sequenced. During the induced critical period (35 DAT), after closure (70 DAT), differential expression analysis was performed among host interneurons from MGE-transplanted, LGE-transplanted, and non-transplanted mice. A schematic diagram detailing the donor tissue sources, the cells obtained from their dissection, and the effects of each transplantation type is shown.

[0021] [Figure 2A] A figure showing verification that the calbindin protein level is elevated and that the elevation is particularly due to MGE transplantation. The transplantation-induced increase in adult calbindin expression is similar to the expression levels seen during the peak of juvenile experience-dependent plasticity. [Figure 2B] A figure showing verification that the calbindin protein level is elevated and that the elevation is particularly due to MGE transplantation. The transplantation-induced increase in adult calbindin expression is similar to the expression levels seen during the peak of juvenile experience-dependent plasticity. Quantification of Calb1 protein and how the expression levels change with age (from p28 to adult non-transplanted) and in transplantation groups (adult non-transplanted compared to MGE recipients and LGE recipients) is shown. [Figure 2C] A figure showing verification that the calbindin protein level is elevated and that the elevation is particularly due to MGE transplantation. The transplantation-induced increase in adult calbindin expression is similar to the expression levels seen during the peak of juvenile experience-dependent plasticity. It shows that the increase in calbindin protein expression is not due to differences in the number of inhibitory neurons between groups.

[0022] [Figure 3A] A schematic diagram of postnatal development that details the transient window of experience-dependent plasticity. Important time points during visual development are marked. [Figure 3B] A schematic diagram of postnatal development that details the transient window of experience-dependent plasticity. Important time points during visual development are marked. It shows an increase and subsequent decrease in calbindin levels that coincide with the peak (increase) of experience-dependent plasticity and the immediate closure (decrease) of its plasticity window during normal development of the mouse visual cortex. This provides a mechanistic rationale for why an induced increase in calbindin levels can recapitulate experience-dependent plasticity. [Figure 3C] A schematic diagram of postnatal development that details the transient window of experience-dependent plasticity. Important time points during visual development are marked. It shows the quantification of Calb1 protein in the cortical layers across age and transplant groups, particularly the increased deep-layer expression of Calb1 after MGE transplantation.

[0023] [Figure 4A] A figure showing the experimental setup of viral injection to overexpress Calb1 in endogenous GABAergic interneurons and imaging to determine changes in circuit plasticity after monocular occlusion. [Figure 4B] A figure showing the experimental setup of viral injection to overexpress Calb1 in endogenous GABAergic interneurons and imaging to determine changes in circuit plasticity after monocular occlusion. It shows successful restricted expression of Calb1 virus in the mouse visual cortex. [Figure 4C] A figure showing the experimental setup of viral injection to overexpress Calb1 in endogenous GABAergic interneurons and imaging to determine changes in circuit plasticity after monocular occlusion. It shows the quantification of a successful increase in Calb1 protein after viral overexpression. [Figure 4D]This figure shows the experimental setup for viral injection to overexpress Calb1 in endogenous GABAergic interneurons and imaging to determine changes in circuit plasticity after monocular detachment. It also shows a physiological map of changes in eye-specific responses before and after monocular detachment between viral recipients and saline-injected controls. [Figure 4E] This figure shows the experimental setup for viral injection to overexpress Calb1 in endogenous GABAergic interneurons and imaging to determine changes in circuit plasticity after monocular detachment. It also shows a physiological map of changes in eye-specific responses before and after monocular detachment between viral recipients and saline-injected controls. [Figure 4F] This figure shows the experimental setup for viral injection to overexpress Calb1 in endogenous GABAergic interneurons and imaging to determine changes in circuit plasticity after monocular deprivation. It shows the induction of experience-dependent plasticity specific to Calb1 overexpression compared to saline-injected controls. The degree of plasticity is similar to the level seen during the peak of experience-dependent plasticity in juvenile neurons. [Figure 4G] This figure shows the experimental setup for viral injection to overexpress Calb1 in endogenous GABAergic interneurons and imaging to determine changes in circuit plasticity after monocular occlusion. It demonstrates that the experience-dependent plasticity induced by Calb1 overexpression is mediated by a decrease in the contra response (a well-established feature of experience-dependent plasticity). [Modes for carrying out the invention]

[0024] Various peptides, solvents, solutions, carriers, and / or components used to prepare compositions used in the methods disclosed herein are disclosed. Various steps, elements, amounts, routes of administration, symptoms, and / or treatments used or observed when carrying out the disclosed methods, as well as the methods themselves, are also disclosed. These and other materials, steps, and / or elements are disclosed herein, and where combinations, subsets, interactions, groups, etc., of these materials are disclosed, specific references to various individual and collective combinations and permutations of these compounds are not expressly disclosed, but it is understood that each is specifically intended and described herein. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit them.

[0025] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the disclosed inventions belong. The singular terms “a,” “an,” and “the” include multiple referents unless the context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The term “includes” means that other elements may be present in addition to the defined elements presented. The use of “includes” indicates inclusion, not limitation. In other words, the term “includes” means “primarily includes, but not necessarily alone.” Furthermore, variations of the word “includes,” such as “comprise” and “comprises,” have the same meaning. In one view, the art described herein relates to the compositions, methods, and their respective components described herein as essential to the invention, but there is still room to include unspecified elements, whether essential or not (“includes”).

[0026] Appropriate methods and materials for carrying out and / or testing embodiments of the embodiments of this disclosure are described below. Such methods and materials are illustrative and not intended to limit the scope. Other methods and materials similar or equivalent to those described herein may be used. For example, prior methods well known in the art relating to this disclosure include, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and supplements up to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th edition, Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, California), “Guide to Protein Purification” in Methods in Enzymology (edited by MP Deutschcer (1990) Academic Press, Inc.)This information is described in various general and more specific references, including PCR Protocols: A Guide to Methods and Applications (Innis et al., 1990, Academic Press, San Diego, California), Culture of Animal Cells: A Manual of Basic Technique, 2nd edition (RI Freshney, 1987, Liss, Inc., New York, New York), Gene Transfer and Expression Protocols, pp. 109–128, edited by E.J. Murray, The Humana Press Inc., Clifton, New Jersey), and Ambion 1998 Catalog (Ambion, Austin, Texas), and these disclosures are incorporated herein by reference in their entirety.

[0027] All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference for all purposes. In the event of any conflict, this specification, including the explanation of terms, shall prevail.

[0028] Methods and materials similar to or equivalent to those described herein may be used to carry out or test the disclosed techniques, but preferred methods and materials are listed below. The materials, methods, and examples are illustrative and not intended to limit the scope of this invention.

[0029] "Subject" refers to an individual and includes, but is not limited to, mammals (e.g., humans, horses, pigs, rabbits, dogs, sheep, goats, non-human primates, cattle, cats, guinea pigs, or rodents), fish, birds, reptiles, or amphibians. This term does not indicate a specific age or sex. Therefore, it is intended to include adult and neonatal subjects, as well as fetuses (whether male or female). "Patient" refers to a subject suffering from a disease or disorder. The term "patient" includes human and veterinary subjects.

[0030] As used herein, the terms “to treat,” “to treat,” or “treatment” refer to both therapeutic actions and preventive or preventive measures aimed at preventing, reducing, slowing (mitigating), inhibiting, or eliminating an undesirable physiological change, symptom, disease, or disorder. For example, a disease could be a CNS disease. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction of the severity of the disease, stabilization (i.e., non-exacerbating) of the disease, delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. “Treatment” may also mean extending survival compared to the expected survival time without treatment. Persons requiring treatment include those who already have a condition or disorder, as well as those who are prone to developing a condition or disorder, or for whom a condition or disorder should be prevented or its onset should be delayed. In some cases, a subject or patient may be identified (e.g., diagnosed) as having a disease or condition prior to administration of the composition of the present invention. Individuals at risk of disease can be identified, for example, by one or a combination of appropriate diagnostic or prognostic assays known in the art.

[0031] As used herein, “clinical improvement” may mean a significant reduction or cessation of the symptoms of the disorder.

[0032] The terms “to manage,” “to control,” and “manage” refer to preventing or delaying the progression, spread, or worsening of a disease or disorder, or one or more of its symptoms. In certain cases, the beneficial effects that a subject may receive from a preventive or therapeutic agent do not result in a cure for the disease or disorder.

[0033] The terms “regression,” “to regress,” and “regression” may refer to a reduction in the size of a tumor or the extent of cancer within the body. In some embodiments, “regression” may refer to a reduction in the severity of the disease and / or a reduction in the size of the tumor. In some embodiments, regression may generally refer to a milder symptom without complete disappearance of the disease. In certain particular cases, the beneficial effects that a subject receives from a preventive or therapeutic agent do not result in a cure for the disease or disorder. In some embodiments, the symptoms of the disease may recur.

[0034] The terms “administering” and “dosing” refer to methods of providing a pharmaceutical preparation, composition, or formulation. The compositions described herein can be administered in several ways, depending on whether topical or systemic treatment is desired and on the area to be treated. Such methods are well known to those skilled in the art and include, but are not limited to, administering the composition orally, intranasally, parenterally (e.g., intravenously and subcutaneously), intramuscularly, intraperitoneally, intrathecally, percutaneously, extracorporeally, or topically.

[0035] As used herein, the term “neuroplasticity” refers to a broad descriptor that summarizes the brain’s receptivity to change its function or connectivity.

[0036] As used herein, the term “experience-dependent plasticity” refers specifically to the brain’s receptiveness to change directed by external cues (e.g., sensory signals / stimuli). Typically, this capacity for change is limited to early postnatal development. The resulting changes become firmly established.

[0037] As used herein, the terms “juvenile plasticity” or “juvenile plasticity” (e.g., experience-dependent plasticity) may be used interchangeably and may refer to an increase in the responsiveness of a target neuron to an input signal, similar to the response observed during development (e.g., during early postnatal development).

[0038] Referring here to Figures 1A to 4G, the present invention features compositions and methods that enable the reactivation of transplantation-induced plasticity without depending on the cell vehicle.

[0039] The present invention features a method for increasing the neural plasticity (e.g., experience-dependent plasticity) of a subject (for example, one that requires it). The method may include increasing the expression of calbindin (Calb1) in the target nerve cells (e.g., excitatory neurons, inhibitory neurons, or a combination thereof). In some embodiments, the method includes increasing the expression of Calb1 in a subset of the target nerve cells. In some embodiments, increasing calbindin expression increases the level of neural plasticity of the subject. In other embodiments, increasing calbindin expression increases the level of experience-dependent plasticity of the subject.

[0040] In other embodiments, the method includes increasing the expression of calbindin in a population of neurons of interest (e.g., excitatory or inhibitory neurons). In some embodiments, increasing calbindin expression increases the level of neural plasticity in the neurons of interest. In other embodiments, increasing calbindin expression increases the level of neural plasticity in a population of neurons of interest (e.g., excitatory or inhibitory neurons). In some embodiments, increasing calbindin expression increases the level of experience-dependent plasticity in the neurons of interest. In other embodiments, increasing calbindin expression increases the level of experience-dependent plasticity in a population of neurons of interest (e.g., excitatory or inhibitory neurons).

[0041] In certain embodiments, the assessment of neuroplasticity involves using neurophysiological, behavioral, and histological assays that are well-established within a certain range of the art. One such assay highlighted herein is ocular dominance plasticity, first identified by Hubel and Wiesel. This assay characterizes a specific critical period in early postnatal development in which a transient disparity between the two eyes induces a sustained modification of neurophysiological responses within the visual system, leading to dominance of the dominant eye. In some embodiments, the methods utilized herein may involve evaluating responses from the individual eyes of each subject both before (e.g., adult subject baseline) and after the transient disparity, as described above. A discernible change in response indicates the presence of experience-dependent plasticity (e.g., successful reactivation), while the absence of a significant shift suggests no change in plasticity compared to the adult baseline. However, the present invention may extend beyond the above-described methods for measuring plasticity and may encompass existing domain-specific methodologies, disease-specific methodologies, or other relevant methodologies. Following a similar protocol, baseline measurements can be compared to the effects observed after administration of a composition (e.g., a small molecule or gene therapy drug configured to increase Calb1).

[0042] In some embodiments, the population of nerve cells (e.g., neurons) includes inhibitory neurons or interneurons. In other embodiments, the population of nerve cells (e.g., neurons) includes excitatory neurons, inhibitory neurons, or a combination thereof.

[0043] According to the present invention, increased Calb1 expression can be achieved by a variety of appropriately engineered delivery methods. These methods include, but are not limited to, the use of viral vectors, exosomes, nanoparticles, miRNA / siRNA, ASOs, aptamers, and modified cells. These delivery systems can transport DNA, RNA, factors, or derivatives thereof to directly or indirectly promote the upregulation of Calb1 expression.

[0044] In some embodiments, increased Calb1 expression can be evaluated by a variety of techniques, including RNA / DNA analysis (e.g., Northern blotting, real-time quantitative PCR, sequencing) and protein analysis (e.g., Western blotting, immunohistochemistry). Furthermore, it should be noted that the present invention is not limited to the methods disclosed herein, and any method known in the art can be used.

[0045] The present invention features a method for increasing the neural plasticity (e.g., experience-dependent plasticity) of a subject (e.g., one that requires it). The method may include administering a composition that increases the expression of calbindin (Calb1) (e.g., a composition comprising a small molecule or a gene therapy drug) to a neuronal cell of the subject (e.g., an excitatory neuron, an inhibitory neuron, or a combination thereof). In some embodiments, the method includes administering a composition that increases the expression of Calbindin (e.g., a composition comprising a small molecule or a gene therapy drug) to a subset of the neuronal cells of the subject (e.g., a population of neuronal cells). In some embodiments, increasing the expression of calbindin increases the level of neural plasticity of the subject. In other embodiments, increasing the expression of calbindin increases the level of experience-dependent plasticity of the subject.

[0046] In certain embodiments, the methods described herein may further include evaluating the baseline expression of Calb1 in a target region / cell before administering the composition. Furthermore, an increase in Calb1 expression can be determined by comparing the baseline level of Calb1 expression with its expression after administration of the composition.

[0047] In certain embodiments, the composition directly increases Calb1 expression. In other embodiments, the composition may indirectly promote an increase in Calb1 expression. In some embodiments, the compositions described herein may comprise one or a combination of small molecules, peptides, enzymes, antibodies, oligonucleotides, drugs, and the like.

[0048] In some embodiments, the present invention features a method for increasing neuronal plasticity (e.g., experience-dependent plasticity) in a subject requiring such increase. The method may include administering a composition to a subject, the composition configured to modulate (e.g., increase) the expression of calbindin (Calb1) in a population of neurons of the subject. In some embodiments, increasing the expression of Calb1 increases the neuronal plasticity (e.g., experience-dependent plasticity) of the subject. In other embodiments, increasing the expression of Calb1 increases the neuronal plasticity (e.g., experience-dependent plasticity) in a population of neurons of the subject. In some embodiments, the compositions described herein may include one or a combination of small molecules, peptides, enzymes, antibodies, oligonucleotides, drugs, etc. In some embodiments, the population of neurons includes excitatory neurons, inhibitory neurons, or a combination thereof. In certain embodiments, the method may further include measuring the expression of Calb1 before administering the composition (e.g., including small molecules).

[0049] In other embodiments, the present invention features a method for treating a CNS disease or disorder in a subject requiring such treatment. The method may include administering a composition to a subject, the composition configured to modulate (e.g., increase) the expression of calbindin (Calb1) in a population of nerve cells of the subject. In some embodiments, modulating (e.g., increasing) the expression of Calb1 increases the neuroplasticity (e.g., experience-dependent plasticity) of the subject, thereby promoting the treatment or regression of a CNS disease or disorder. In other embodiments, modulating (e.g., increasing) the expression of Calb1 increases the neuroplasticity (e.g., experience-dependent plasticity) in a population of nerve cells of the subject, thereby promoting the treatment or regression of a CNS disease or disorder. In some embodiments, the CNS disorder or disease may include epilepsy, autism, stroke, schizophrenia, Alzheimer's disease, Parkinson's disease, or neuropathic pain. In some embodiments, the compositions described herein may include one or a combination of small molecules, peptides, enzymes, antibodies, oligonucleotides, drugs, etc. In certain embodiments, the method may further include measuring Calb1 expression before administering the composition.

[0050] The present invention may also feature a method for increasing neural plasticity (e.g., experience-dependent plasticity) in a subject requiring it. In some embodiments, the method comprises administering a gene therapy drug to a subject, wherein the gene therapy drug is configured to increase the expression of calbindin in a population of nerve cells of the subject, thereby increasing the level of experience-dependent plasticity in the subject.

[0051] In certain embodiments, gene therapies directly increase Calb1 expression. For example, a vector incorporating a Calb1 sequence specifically designed to enhance Calb1 expression may be used. In other embodiments, gene therapies may indirectly promote an increase in Calb1 expression. This can be achieved, for example, by a vector containing a regulatory RNA / gene sequence known to regulate Calb1 expression. Such regulation may include inducing cells to increase Calb1 expression or preventing age-related / disease-related declines in Calb1 expression by containing a regulatory RNA / gene sequence known to decrease Calb1 expression. In some embodiments, gene therapies include, but are not limited to, viral vectors, exosomes, nanoparticles, small interfering RNAs (siRNAs) and microRNAs (miRNAs), antisense oligonucleotides (ASOs), aptamers, and modified cells.

[0052] In some embodiments, the method involves administering a viral vector to a target, the viral vector configured to increase the expression of calbindin in a population of target nerve cells (e.g., inhibitory neurons, excitatory neurons, or a combination thereof). In some embodiments, increasing calbindin expression increases the level of experience-dependent plasticity of the target. Non-limiting examples of viral vectors include adeno-associated viruses (AAVs), adenoviruses, and lentiviruses.

[0053] In alternative embodiments, the method comprises administering exosomes (e.g., containing a therapeutic agent) to a target, wherein the exosomes are configured to increase the expression of calbindin in a population of target neurons (e.g., inhibitory neurons, excitatory neurons, or a combination thereof). In some embodiments, increasing calbindin expression increases the level of experience-dependent plasticity of the target. According to the present invention, exosomes represent extracellular vesicles that, upon uptake, can be manipulated to transport therapeutic molecules, such as Calb1 sequences, to surrounding cells.

[0054] In other embodiments, the method comprises administering nanoparticles (e.g., including a therapeutic agent) to a target, wherein the nanoparticles are configured to increase the expression of calbindin in a population of target neurons (e.g., inhibitory neurons, excitatory neurons, or a combination thereof). In some embodiments, increasing calbindin expression increases the level of experience-dependent plasticity of the target. In some embodiments, therapeutic molecules such as Calb1 may be encapsulated within nanoparticles such as lipids or metals to facilitate delivery to target cells.

[0055] In certain embodiments, the methods described herein may target neurons located in diseased / dysregulated areas of the brain in question.

[0056] In some embodiments, the methods described herein can be used to treat CNS disorders / disorders such as epilepsy, autism, stroke, schizophrenia, Alzheimer's disease, Parkinson's disease, and neuropathic pain.

[0057] In certain embodiments, the methods described herein may further include evaluating baseline expression of Calb1 in a target region / cell before administering a gene therapy drug. Furthermore, an increase in Calb1 expression can be determined by comparing the baseline level of Calb1 expression with its expression after administration of a gene therapy drug.

[0058] Methods for enhancing plasticity, such as the experience-dependent plasticity described herein, may benefit a wide range of diseases and dysregulations in brain circuits. For example, induction of plasticity via Calb1 may be applied to a variety of conditions, including but not limited to epilepsy, amblyopia, stroke, schizophrenia-like disorders, Alzheimer's disease, Parkinson's disease, and neuropathic pain. Furthermore, these methods may complement existing neurotherapeutic agents by priming neurons and brain circuits to make them more receptive, thereby potentially enhancing the efficacy of subsequent treatments.

[0059] In some embodiments, the present invention may feature a method for treating a central nervous system (CNS) disorder or condition. The method may include administering (e.g., by contact) a gene therapy drug that increases the expression of calbindin (Calb1) to target nerve cells (e.g., excitatory neurons, inhibitory neurons, or a combination thereof). In some embodiments, the method includes administering (e.g., by contact) a portion of target nerve cells (e.g., a population of nerve cells) to a specific group of nerve cells. In some embodiments, the CNS disorder or condition may include epilepsy, autism, stroke, schizophrenia, Alzheimer's disease, Parkinson's disease, or neuropathic pain.

[0060] In certain embodiments, the present invention features a gene therapy drug for use in the treatment of CNS diseases or disorders. In certain embodiments, the gene therapy drug increases the expression of Calb1 in nerve cells (e.g., excitatory neurons, inhibitory neurons, or a combination thereof) within a subject. In some embodiments, increasing Calb1 expression in this manner increases the neuroplasticity of the subject, thereby contributing to the treatment or regression of CNS diseases or disorders. Furthermore, in alternative embodiments, upregulation of Calb1 expression increases the experience-dependent plasticity of the subject, further promoting the treatment or regression of CNS diseases or disorders. In some embodiments, the CNS disorders or diseases may include epilepsy, autism, stroke, schizophrenia, Alzheimer's disease, Parkinson's disease, or neuropathic pain.

[0061] In certain embodiments, the methods described herein may be carried out within a specific adult neural circuit.

[0062] The present invention may further include methods for screening experience-dependent plasticity inducers and agents. In certain embodiments, the method includes increasing the expression of calbindin in a target population of neurons of interest. For example, after increasing Calb1 expression, the target cells can be analyzed (e.g., by sequencing) to identify factors or agents that repeatedly and potently co-express. These factors may be co-regulated with Calb1 expression, and regulating their expression may enhance induced plasticity, neuronal function, survival, etc.

[0063] Furthermore, the present invention may encompass methods for screening experience-dependent plasticity inducers and agents. In certain embodiments, the method includes obtaining a sample (e.g., a biological sample such as ex vivo tissue, primary culture, iPSC, or organoid) and administering potential experience-dependent plasticity inducers and agents to the sample. An increase in calbindin expression in the sample serves as an indicator of a factor or agent that can induce experience-dependent plasticity. For example, an in vitro setting allows researchers to screen a comprehensive drug library and determine, among many other metrics, whether a target cell culture shows increased Calb1 expression.

[0064] In other embodiments, the method may involve administering potential experience-dependent plasticity inducers and agents to a subject (e.g., a suitable animal model, e.g., a mouse model). In some embodiments, an increase in calbindin expression indicates a factor or agent that induces experience-dependent plasticity. For example, a personalized treatment or reagent may be administered to a subject, after which the target cells may be evaluated for an increase in Calb1 expression (e.g., as a rapid indicator of successful “rejuvenation”).

[0065] Furthermore, in additional embodiments, the screening method for experience-dependent plasticity inducers and agents may include the use of in silico techniques such as computational modeling.

[0066] The screening methods described herein may include a re-evaluation of the effects of existing FDA-approved or preclinical therapies, devices, etc., on Calb1 expression. Furthermore, the screening process may include testing novel gene candidates involved in experience-dependent plasticity using increased Calb1 expression as an initial indicator. In some embodiments, various combinations of these approaches may also be used.

[0067] In some embodiments, the present invention features a method for increasing neural plasticity in a subject requiring it. The method may include transplanting a population of medial basal ganglia primordia (MGE) cells into the target neural tissue, the transplantation of which increases the expression of calbindin in the population of target neurons, thereby increasing the level of experience-dependent plasticity in the subject.

[0068] In other embodiments, the present invention provides a method for increasing the neural plasticity of the central nervous system of an adult subject requiring such plasticity, comprising transplanting a population of medial basal ganglia primordia (MGE) GABAergic interneurons into the target nerve tissue, wherein the transplantation increases the expression of calbindin in the population of target nerve cells. [Examples]

[0069] Example 1

[0070] The following are non-limiting examples of the present invention. It should be understood that these examples are not intended to limit the present invention in any way. Equivalents or substitutions are within the scope of the present invention.

[0071] Animals: To label host inhibitory neurons, adult mice with green fluorescence inhibitory neurons were generated by mating homozygous VGAT-cre females (VGAT-cre, JAX 028862) with cre-dependent ZsGreen males (Ai6, JAX 007906). The resulting littermates were allowed to reach P120-P180 (4-6 months) before the start of the experiment. To distinguish transplanted cells from endogenous host cells, donor embryos with red fluorescence inhibitory neurons were generated by mating homozygous VGAT-cre females with cre-dependent tdTomato males (Ai14, JAX 007914). Virus injection was performed in hemizygous VGAT-tdTomato recipient mice. Colony genotype was periodically confirmed by PCR (Transnetyx), and successful cre recombination was individually confirmed by fluorescence before each experiment. All transplant recipients were housed individually after head plate placement. All experimental cohorts contained approximately equal numbers of male and female mice.

[0072] Headplate: To ensure stability for downstream intracerebral procedures, custom-printed headplates were fixed to the skulls of adult recipient mice (P120-P180). Prior to surgery, the mice received subcutaneous injections of the analgesic carprofen (0.8 mg / ml; Rimadyl), hydration (Ringer's lactate solution), and eye ointment. Body temperature was maintained throughout the procedure using a feedback-controlled heating pad. After anesthesia with 2% isofluorane, ear bars were attached to temporarily stabilize the head axis. First, topical, injectable lidocaine was administered, followed by surgical exposure of the intact skull, which was reinforced with Vetbond (3M, Vetbond, 1469SB). The V1 adjacent region was further reinforced with layers of dental acrylic (Lang Ortho-Jet Powder and Ortho-Jet Powder Liquid). The headplate was then fixed with a 4-5 mm window centered on V1 using dental acrylic. Finally, the V1 window was sealed with the final layer of Vetbond, and the mice were returned to their heated home cages after awakening. For consistency, all headplates were placed only in the right hemisphere. Mice were given subcutaneous injections of carprofen after placement to reduce pain and inflammation (for up to 3 days).

[0073] Endogenous signal optics imaging (ISOI) for intracranial procedures: To precisely target V1, the central region (bV1) was physiologically determined for individual mice using ISO. After inducing and maintaining a stable plane of light anesthesia with 0.8–1.2% isofluorane, headplate-equipped mice were fixed to the imaging rig. The headplate window was filled with PBS and covered with a 10 mm glass coverslip. PBS was added as needed throughout the session to keep the coverslip level with the top of the headplate. Eye moisture was maintained using periodic application of silicone oil, and body temperature was controlled by a feedback-controlled heating pad.

[0074] Intrinsic signal images were acquired using a custom-designed macroscope (Nikon 135×50mm lens) equipped with a Dalsa 1M30 CCD camera mounted on a headplate. First, images of the surface vascular system were visualized and captured using a green (530nm) light-emitting diode (LED). Then, the camera was refocused to approximately 450–550 μm below the pia mater surface relative to target layer II / III of bV1, and the intrinsic signal was acquired using red (617nm) LED light. Visual stimuli were displayed on an Acer V193 monitor (53×33cm, 60Hz refresh rate, 20cd / m2 average brightness) placed 25cm in front of the mouse, aligned with the mouse's midline and line of sight. The stimuli were generated using the MATLAB® Psychophysics Toolbox extension and consisted of contrast-modulated sweep noise limited to a field of view azimuthal angle of -5° to +15° and a field of view elevation angle of -18° to +36°. Each imaging session consisted of a 5-minute presentation of stimuli to both eyes. A custom Matlab® script using Fourier analysis was used to generate a phase map of the bV1 response. The resulting phase map was then overlaid on vascular images to visualize the bV1 boundary and determine the optimal injection site.

[0075] Inhibitory neuron transplantation for transcriptomics: To prepare donor embryo tissue, VGAT-cre females were housed with cre-dependent tdTomato males for a short period of time, overnight (<20 hours). After successful breeding, pregnant females were euthanized approximately two weeks later by isofluorane and cervical dislocation. E13.5–14.5 VGAT-tdTomato embryos were rapidly dissected in chilled L-15 solution (Gibco, 21083027), and the forebrain was microdissected in a second petri dish of chilled L-15. Prior to MGE and LGE microdissection, the tdTomato signal was confirmed in each forebrain using an epifluorescence scope. The isolated MGE and LGE tissues were then collected in separate tubes of L-15 containing 2% (v / v) DNase I (Roche, 0471672800) and placed on ice.

[0076] Adult VGAT-ZsGreen mice with headplates were anesthetized with 2% isoflurane and fixed to a surgical rig. All recipients received preoperative carprofen, Ringer's solution, and eye ointment. Body temperature was maintained throughout the procedure using a feedback-controlled heating pad, and anesthesia was gradually reduced as needed. The skull beneath the mapped bV1 injection site was thinned using a dental drill (Midwest, 78044) and an FG1 / 4 carbide bur to allow penetration of the injection micropipette.

[0077] The tip of a glass micropipette (Wiretrol 5 μl, Drummond Scientific Company) was stretched to approximately 75 μm and angled. Isolated MGE or LGE was front-loaded into a micropipette prepared immediately before transplantation to dissociate the donor tissue into an injectable cell suspension. The loaded micropipette was then tilted perpendicular to the brain surface curvature of each individual recipient. Injections into the mapped bV1 site were performed at 10 nl / min at approximately 400–600 μm below the surface using a custom-made hydraulic manipulator (Narishige, MO-10). Each recipient received a total of two injections (approximately two isolated MGE / LGE), with 100 nl per injection. After injection, the micropipette was slowly withdrawn to prevent backflow, and the site was resealed with Vetbond. Postoperative mice were returned to heated home cages upon awakening and given subcutaneous injections of carprofen after placement to reduce pain and inflammation (for up to 3 days).

[0078] To maintain consistency, experimental drugs (MGE, LGE, and saline) were injected only into V1 in the right hemisphere. Each transplant cohort contained a balanced number of MGE, LGE, and non-transplant recipients.

[0079] V1 Isolation, Dissociation, Cell Sorting, and RNA Extraction: At specified experimental time points, mice (35 DAT, 70 DAT, non-transplant littermates) were euthanized with 1.2% tribromoethanol. Post-injection, mice were perfused transcardially for 5 minutes with RNAase inhibitors and transcription inhibitors diluted in chilled L-15 solution. To mitigate cell cycle mismatch, perfusion for each collection cohort was started at the same time and completed within 2 hours. Brains were then dissected, and V1 cells were isolated using stereotactic coordinates centered on mapped bV1 cells (3–4 mm lateral, ±0.5 mm anterior-posterior from the bregma). Isolated V1 tissue was then homogenized into smaller fragments and suspended in additional L-15 / inhibitor solutions in gentleMACS C tubes (Miltenyi Biotec). The tissue clumps were further dissociated using the gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec) and the Adult Brain Dissociation Kit (mouse and rat, Miltenyi Biotec). The resulting cell suspensions were incubated with a microglia antibody (Cd11b-APC) and the cell viability indicator DAPI for 15 minutes.

[0080] Next, the immunolabeled samples were further purified by fluorescence. Following standard FACS screening for cell viability / doublets and fluorescence gating based on control samples, the samples were separated by cell populations (ZsGreen - host inhibitory neurons, APC - microglia). All selected populations were collected in 1 ml of RNA stabilizer Trizol on ice. Finally, RNA was extracted from each sample using an RNA Clean & Concentrator kit and immediately stored at -80°C.

[0081] Immunohistochemistry: Mice were euthanized with 1.2% tribromoethanol (Avertin, 18 ml / g). Post-injection, mice were perfused transcardially with 4% PFA in chilled PBS at 6 ml / min for 5 minutes. The recovered brains were post-fixed overnight and cryoprotected with 30% sucrose. The brains were coronally fragmented into 30 μm free-floating sections using a cryo-gliding microtome (Microm, HM450). To reduce batch effects, samples across experimental / age groups were divided into mixed batches using a custom-designed randomization script. Sections were selected approximately every 200 μm from anterior V1 to posterior V1 to sample V1 representatively. V1 sections were then permeabilized with 0.3% Triton-X in PBS for 1 hour and blocked with 1% normal donkey serum in 0.3% Triton-X / PBS. After incubation with primary antibody overnight at 4°C, sections were washed three times in 0.3% Triton-X / PBS and incubated with secondary antibody at room temperature for 2 hours. Sections were again washed three times in 0.3% Triton-X / PBS, then mounted and covered with coverslips using Fluoroshield (Millipore-Sigma, F6057) containing DAPI.

[0082] To visualize Calb1 protein expression, sections were incubated with donkey anti-rabbit Calb1 primary antibody, followed by donkey anti-rabbit Alexa 647 secondary antibody. To visualize transplanted cells, sections were incubated with donkey anti-RFP (Fisher MA5-15257, 1:250) to amplify the VGAT-tdTomato signal, followed by incubation with donkey anti-mouse 568 secondary antibody. Host cells were visualized without additional amplification, taking into account the intensity of the VGAT-ZsGreen signal.

[0083] Confocal microscopy and image analysis: Stained sections were imaged using a Leica TCS SP8 confocal microscope (20x immersion objective). Image tiling was performed using LAS X image processing software (Leica Microsystem) to obtain z-stacks (20–30 μm) of V1 and adjacent landmarks. To keep the experimental group (i.e., transplanted vs. non-transplanted) blinded, the Calb1 channel was isolated from the exported images and quantified together. The stacks were first z-projected (maximum intensity), and then annotated at the V1 boundary and cortical layer (see Allen Brain Atlas). Background noise was then smoothed by applying minimal Gaussian blur (1 unit). Cells with signal intensity above the background were counted as Calb1+ (cell counter plugin, Fiji).

[0084] To determine co-localization, tdTomato or ZsGreen cells were evaluated and counted for Calb1+ signaling. The percentage of transplanted tdTomato cells expressing Calb1 was calculated by dividing the number of Calb1+ / tdTomato+ cells by the total number of tdTomato+ cells. The percentage of host ZsGreen cells expressing Calb1 was calculated by dividing the number of Calb1+ / ZsGreen+ cells by the total number of ZsGreen+ cells.

[0085] Intracerebral viral injection: To specifically upregulate Calb1 expression in adult inhibitory neurons, P120-180 non-transplanted VGAT-tdTomato mice were injected with a cre-restricted Calb1 overexpression virus (AAV1-CMV-DIO-mCalb1-T2A-eGFP). To visualize the success of AAV integration and expression, an eGFP fluorescent tag was included in the polycistronic construct. After headplate placement and V1 mapping, adult VGAT-tdTomato mice were anesthetized with 2% isoflurane and fixed to a surgical rig. All recipients received preoperative carprofen, Ringer's solution, and eye ointment. Body temperature was maintained throughout the procedure using a feedback-controlled heating pad, and anesthesia was gradually reduced as needed. To penetrate the injection micropipette, the skull beneath the mapped bV1 injection site was thinned using a dental drill (Midwest, 78044) and an FG1 / 4 carbide bur.

[0086] The tip of a glass micropipette (Wiretrol 5 μl, Drummond Scientific Company) was stretched to approximately 75 μm and angled. The virus was front-loaded into the prepared micropipette and tilted perpendicular to the brain surface curvature of each individual recipient. Injections into the mapped bV1 site were performed at 10 nl / min at approximately 400–600 μm below the surface using a custom-made hydraulic manipulator (Narishige, MO-10). Each recipient received a total of two injections, 200 nl per injection. To maintain consistency, the experimental drug (AAV, saline) was injected only into V1 in the right hemisphere. After injection, the micropipette was slowly withdrawn to prevent backflow, and the site was resealed with Vetbond. Postoperative mice were returned to heated home cages upon awakening and given subcutaneous injections of carprofen after placement to reduce pain and inflammation (for up to 3 days). Mice were returned to their normal routine for approximately 3 weeks while stabilizing viral expression.

[0087] Endogenous Signal Optical Imaging (ISOI) for Ocular-Specific Plasticity: To assess whether experimental mice exhibit OD plasticity, ISOI was performed before and after monocular occlusion (MD) to measure changes in the OD index. After inducing and maintaining a stable plane of light anesthesia with 0.8–1.2% isofluorane, headplate-equipped mice were fixed to an imaging rig. The headplate window was filled with PBS and covered with a 10 mm glass coverslip. PBS was added as needed throughout the session to keep the coverslip at the same height as the top of the headplate. Ocular moisture was maintained using periodic application of silicone oil, and body temperature was controlled by a feedback-controlled heating pad.

[0088] Endogenous signal images were acquired using a custom-designed macroscope (Nikon 135×50mm lens) equipped with a Dalsa 1M30 CCD camera mounted on a headplate. First, images of the surface vascular system were visualized and captured using a green (530nm) light-emitting diode (LED). Then, the camera was refocused to approximately 450–550 μm below the pia mater surface relative to target layer II / III of bV1, and the endogenous signal was acquired using red (617nm) LED light. Visual stimuli were displayed on an Acer V193 monitor (53×33cm, 60Hz refresh rate, 20cd / m2 average brightness) placed 25 cm in front of the mouse, aligned with the mouse's midline and line of sight. To capture eye-specific responses, monocular blocks were placed (alternatingly) in front of one eye or the other eye during stimulus presentation and recording.

[0089] The stimuli were generated using the MATLAB® Psychophysics Toolbox extension and consisted of contrast-modulated sweep noise limited to visual field azimuthal angles of -5° to +15° and visual field elevation angles of -18° to +36°. Each recording trial consisted of 5-minute presentations of stimuli at 0° and 180°, with a total of 3-4 recordings per eye. Amplitude and phase maps of the bV1 response were generated using a custom Matlab® script with Fourier analysis. The Fourier maps were then smoothed with a 5x5 Gaussian kernel to calculate the final amplitude map. To account for hemodynamic delay, the phase map was normalized by subtracting the phase of the cortical response at 180° from the phase at 0°. The map of absolute retinal phase is shown with respect to the visual angle relative to the center of the monitor.

[0090] The ODI for each animal was calculated as (CI) / (C+I) (where C is the average response amplitude of the contralateral eye and I is the average response amplitude of the ipsilateral eye, each over 3-4 trials). The OD shift was calculated by subtracting the pre-MD ODI from the post-MD ODI.

[0091] Monocular occlusion: To clarify the presence or absence of underlying circuit plasticity, experimental mice were subjected to a transient alteration of visual experience. Immediately after pre-MD imaging, the eye opposite the injected hemisphere was sutured using Perma-Hand Silk (Ethicon, K809H). Under anesthesia, mice were rapidly transferred from the imaging rig to the surgical rig. 2% isoflurane, preoperative carprofen, and Ringer's solution were administered. Body temperature was maintained throughout the procedure using a feedback-controlled heating pad. Two mattress sutures were applied across the contralateral eyelid (once on each side), with the final knot further secured with a Vetbond dove. Postoperative mice were returned to a heated home cage upon awakening and given subcutaneous injections of carprofen as needed to reduce pain and inflammation (for up to 3 days).

[0092] Eyelid sutures were monitored daily for integrity and carefully removed after 3 days. Mice were returned to their heated home cages for 30 minutes after suture removal to allow the eyes to open fully before post-MD imaging. Mice with sutures that opened too early, cataracts, cloudy eyes, or ptosis were excluded from post-MD imaging.

[0093] Statistics: Experiments were successfully replicated using multiple animals. All quantifications and data analyses were performed blindly and unbiased. To test for significant differences between two experimental groups, either a standard t-test (normal distribution) corrected for Welch or a Mann-Whitney rank test (non-normal distribution) was used. To test for significant differences between three or more groups, either a standard one-way ANOVA followed by a Holm-Sidak multiple comparison test (normal distribution) or a Kruskal-Wallis one-way ANOVA (non-normal distribution) corrected for Dunn's multiple comparison test was used.

[0094] Isolation of host inhibitory neurons from transplanted adult V1: To genetically label host inhibitory neurons in designated transplant recipients, transgenic mice expressing Cre recombinase under the control of the vesicular GABA transporter (VGAT) promoter were crossed with mice possessing Cre-dependent ZsGreen (Figures 1A and 1B). At adulthood (approximately p120), custom printed head plates centered on the right hemisphere V1 were immobilized on the resulting VGAT-ZsGreen littermates (Figures 1A and 1B). To account for anatomical variation between mice, the V1 boundary was physiologically determined pre-transplant via endogenous signal optics imaging (ISOI) (Figures 1A and 1B). This functional imaging method can utilize hemodynamic changes as a readout of neural activity and be combined with visual stimulation to assess the V1 response. Subsequently, the generated bilateral retinotopic maps of V1 were superimposed on images of the surface vascular system to determine the optimal injection site.

[0095] Donor tissue was microdissected from embryos at E13.5–14.5 (Figures 1A and 1B). The dissected donor tissue was then transplanted into adult VGAT-ZsGreen recipients that had received a head plate at least one week prior. Each recipient cohort included 1) a non-transplant age-matched control to account for age-related gene expression, and 2) an LGE transplant control to account for cell transplantation-related gene expression.

[0096] In studies using mouse V1 as a model, visual occlusion is often used to reveal the plasticity underlying local circuits. However, to avoid confounding between gene expression changes that establish the plastic state and changes resulting from the induction of that plasticity, mice were not subjected to such disturbances. After returning to the normal post-transplant routine, the recipient cohort was subsequently euthanized and perfused at two different physiological points in time: a) 35 days after transplantation, during the peak of the transplant reactivation critical period (35 DAT), or b) 70 days after transplantation, after the reactivation period had ended (70 DAT) (Figure). To maintain transcriptome integrity in these in vivo states, all mice were immediately perfused with transcription inhibitors and RNAase inhibitors without fixation.

[0097] V1 was harvested only from the right hemisphere, even in non-transplant mice, to eliminate variations due to hemispheric asymmetry. This was due to the small size of the V1 tissue after dissection (approximately 1 cm). 3 To ensure the quality of downstream processing, V1 samples were pooled together from the same experimental group. Therefore, each reported n represents 2-3 biological replicates. The V1 samples were then dissociated and sorted into separate cell populations by fluorescence-activated cell sorting (FACS). Finally, high-quality RNA samples were isolated from the recovered cells (RIN>8) and prepared into bulk sequencing libraries.

[0098] Calbindin 1 (Calb1): Calbindin 1 is a powerful calcium 2+Known for its binding properties, Calb1 is a mobile cytosolic protein primarily localized to the cell bodies, dendrites, and spines of GABAergic neurons. Compared to other prominent EF hand family members, PV and calretinin, Calb1 is a fast Ca2+ induced 2+ It has been shown to selectively buffer transients. Loss of Calb1 impairs multiple aspects of synaptic plasticity, including proper LTP maintenance, double-stimulus facilitation, and neuronal excitability, which ultimately manifest as dysfunction in learning and memory, motor coordination, and sensory integration.

[0099] Within the central nervous system, Calb1 expression is widely reported to increase during embryogenesis, peak during postnatal development, and then rapidly decline in adulthood. This decline in expression is due to other calcium channels such as PV, which do not decrease with age. 2+ It is different from a binding protein. Importantly, the decrease in Calb1 across brain regions is attributed to programmed downregulation rather than neuronal loss. Therefore, Calb1 appeared to be uniquely positioned at the intersection of both GO prediction and DE comparison for the initial candidate investigation.

[0100] Immunostaining confirms upregulation of Calb1 in MGE transplant recipients: To independently validate RNA-seq results, Calb1 was evaluated by immunostaining of V1 sections from 35 DATs of adult MGE, LGE, and non-transplant mice. In MGE transplant recipients, Calb1 expression levels were significantly higher than in both LGE and non-transplant controls (Figure 2A). LGE transplantation did not significantly alter Calb1 expression levels from non-transplant baseline (Figure 2B). Quantification of VGAT+ cells (transplant and host) across groups confirmed that this increase in post-transplant Calb1 expression was not due to a significantly different number of inhibitory neurons (Figure 2C). Calb1 immunostaining in non-transplant controls showed a clear latitude with enrichment in cortical layers II / III and V, suggesting that MGE transplantation significantly increases Calb1 expression in deeper layers (Figure 2A). Group-wide Calb1 immunostaining is restricted to inhibitory neurons and separates strongly expressed Calb1 cells from weakly expressed ones. Strongly expressed Calb1 cells appear to be mainly localized in deeper layers, while weakly expressed Calb1 cells constitute the majority of the surface population (Figure 2A).

[0101] Postnatal V1 Calb1 expression peaks during the critical period: Calb1 function has been relatively more studied in the hippocampus and cerebellum, but has been little studied in the mouse visual cortex (beyond its application as a neuroanatomical marker). Calb1 levels upregulated by MGE were compared to levels during critical postnatal time points for visual development: p16 (2 days after eye opening, onset of visual experience), p28 (peak of the juvenile critical period), and p35 (closure of the juvenile critical period) (Figure 3A). Interestingly, Calb1 expression has a clear peak at p28 and is already downregulated by p35 (Figure 3B). p35 Calb1 levels do not appear to differ significantly from p100 Calb1 levels. The decrease in Calb1 in adulthood appears to be mainly mediated by loss of expression in deeper cortical layers (Figure 3C). Notably, Calb1 levels upregulated by MGE were not significantly different from p28 expression (Figure 2B), suggesting that MGE transplantation restores adult host Calb1 expression to juvenile levels.

[0102] Upregulation of Calb1 in adult V1 inhibitory neurons restores critical plasticity: To restrict Calb1 overexpression to inhibitory neurons, bilateral V1 was mapped in non-transplanted adult VGAT-cre mice, and the cre-dependent Calb1-overexpressing AAV (AAV1-CMV-DIO-mCalb1-T2A-eGFP) was injected into that site (Figure 4A). Successful upregulation of Calb1 and diffusion of the GFP reporter were established during initial viral optimization and confirmed via post-hoc immunohistochemistry after each experiment (Figure 4B). Three weeks after injection, baseline V1 responses were recorded from each eye using ISOI. Contralateral and ipsilateral responses were used to calculate the Ocular Dominance Index (ODI), a measure of eye-specific response intensity to each other. Immediately after baseline imaging, the eye contralateral to the injected hemisphere was sutured closed to briefly deprive the eyes of visual stimuli. Three days of monocular occlusion (MD) has previously been shown to distinguish juvenile OD plasticity from other adult plasticity mechanisms.

[0103] After 3 days of MD, the eyelid sutures were removed and the V1 response was recorded again from each eye. Post-MD ODI was compared to pre-MD ODI to determine whether the mouse had undergone an OD shift. Adult mice injected with saline did not show OD plasticity after MD (Figure 4F). Surprisingly, adult mice injected with AAV showed a significant shift in OD toward the unoccluded eye, indicating robust reactivation of critical period plasticity (Figure 4F). A more detailed analysis of eye-specific response intensity before and after MD suggests that the OD shift after Calb1 upregulation is primarily mediated by a decrease in response from the occluded eye (Figure 4G). The OD shift in adult mice requires longer occluding and is accompanied by an increase in response from the unoccluded eye, but the loss of response from the occluded eye is more characteristic of OD shifts occurring within the juvenile critical period.

[0104] Example 2

[0105] The following are non-limiting examples of the present invention. It should be understood that these examples are not intended to limit the invention in any way. Equivalents or substitutions are within the scope of the present invention.

[0106] A group of patients with mild to moderate Alzheimer's disease will be administered a series of gene therapies designed to enhance Calb1 expression. The gene therapies will be administered weekly for four weeks. Functional MRI scans will be performed on patients according to the treatment regimen, cognitive function will be assessed using neuropsychological tests, and both patients and the administering physicians will be blinded to whether the patient received treatment or placebo. Post-treatment measurements will be compared to baseline data collected before the start of treatment. This comparison will reveal significant improvements in daily functioning in patients who received gene therapy. No side effects have been reported.

[0107] Example 3

[0108] The following are non-limiting examples of the present invention. It should be understood that these examples are not intended to limit the invention in any way. Equivalents or substitutions are within the scope of the present invention.

[0109] A 28-year-old male diagnosed with epilepsy is prescribed a novel composition containing a small molecule intended to increase the levels of calbindin in a subset of nerve cells. The composition containing the small molecule is administered once daily. After two months of treatment, the patient returns for a follow-up appointment and reports an overall reduction in symptoms. The physician orders confirmatory tests to verify this case-specific improvement. Upon reviewing the test results, the physician confirms the reduction in symptoms and records favorable brain scan findings. No side effects have been reported.

[0110] Example 4

[0111] The following are non-limiting examples of the present invention. It should be understood that these examples are not intended to limit the invention in any way. Equivalents or substitutions are within the scope of the present invention.

[0112] A cohort of patients diagnosed with mild to moderate Parkinson's disease receives a series of gene therapy doses aimed at upregulating the expression of one or more genes selected from a set including Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4. These gene therapies are administered weekly for eight weeks. Cognitive function is then assessed using functional MRI scans and neuropsychological tests, with both patients and administering physicians blinded to treatment assignments. Post-treatment comparison of obtained measurements to baseline data collected before treatment initiation reveals significant improvements in daily functioning in patients who received gene therapy. No side effects have been reported.

[0113] Embodiment

[0114] The following embodiments are for illustrative purposes only and are not limiting.

[0115] Embodiment 1: A method for increasing the neural plasticity of a subject requiring the use thereof, comprising administering a gene therapy drug to the subject, wherein the gene therapy drug is configured to modulate (e.g., increase) the expression of calbindin in a population of nerve cells of the subject, thereby increasing the neural plasticity of the subject.

[0116] Embodiment 2: A method for increasing neural plasticity in a target subject, comprising administering a gene therapy drug to the target, wherein the gene therapy drug is one or a set of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4 in a population of nerve cells of the target. A method configured to regulate (e.g., increase) the expression of a combination of genes, wherein the neural plasticity of a target is increased by regulating (e.g., increasing) the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0117] Embodiment 3: A method for increasing neuronal plasticity in a target, comprising administering a gene therapy drug to the target, wherein the gene therapy drug is one or a pair of genes selected from the group consisting of Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4 in a population of target neurons. A method configured to regulate (e.g., increase) the expression of a combination of genes, wherein the neural plasticity of a target is increased by regulating (e.g., increasing) the expression of one or a combination of genes selected from the group consisting of Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0118] Embodiment 4: The method according to any one of Embodiments 1 to 3, wherein the neural plasticity of a population of target neurons is increased by regulating (e.g., increasing) the expression of calbindin or one or a combination of genes selected from the group consisting of Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0119] Embodiment 5: A method for increasing neuronal plasticity in a subject requiring the use thereof, comprising administering a gene therapy drug to the subject, wherein the gene therapy drug is configured to modulate (e.g., increase) the expression of calbindin in a population of nerve cells of the subject, thereby increasing the neuronal plasticity of the subject.

[0120] Embodiment 6: A method for increasing neural plasticity in a target subject, comprising administering a gene therapy drug to the target, wherein the gene therapy drug contains one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4 in a population of target neurons. A method configured to regulate (e.g., increase) the expression of a combination of genes, wherein the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4 is regulated (e.g., increased), thereby increasing the neuronal plasticity of the target.

[0121] Embodiment 7: A method for increasing neural plasticity in a target that requires it, comprising administering a gene therapy drug to the target, wherein the gene therapy drug contains one or a combination of genes selected from the group consisting of Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4 in a population of target neurons. A method for increasing the neuronal plasticity of a target neuron by regulating (e.g., increasing) the expression of one or a combination of genes selected from the group consisting of Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0122] Embodiment 8: The method according to any one of Embodiments 1 to 7, wherein the experience-dependent plasticity of a population of neurons of interest is regulated (increased) by regulating (e.g., increasing) the expression of calbindin or one or a combination of genes selected from the group consisting of Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0123] Embodiment 9: A method for increasing the neural plasticity of a subject requiring it, comprising administering a gene therapy drug to the subject, wherein the gene therapy drug is configured to modulate (e.g., increase) the expression of calbindin in a population of nerve cells of the subject, thereby increasing the experience-dependent plasticity of the subject.

[0124] Embodiment 10: The method according to any one of Embodiments 1 to 9, wherein the gene therapy modulates (e.g., increases) the expression of one or a combination of genes selected from the group consisting of Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0125] Embodiment 11: A method for increasing neural plasticity in a subject requiring it, comprising administering a gene therapy drug to the subject, wherein the gene therapy drug expresses one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4. A method for increasing the experience-dependent plasticity of a subject by regulating (e.g., increasing) the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0126] Embodiment 12: The method according to any one of Embodiments 1 to 11, wherein the population of nerve cells includes excitatory neurons, inhibitory neurons, or a combination thereof.

[0127] Embodiment 13: The method according to any one of Embodiments 1 to 12, wherein the therapeutic agent comprises a viral vector, exosomes, nanoparticles, small interfering RNA (siRNA) and microRNA (miRNA), antisense oligonucleotide (ASO), aptamer, modified cells, etc.

[0128] Embodiment 14: The method according to any one of Embodiments 1 to 13, wherein the gene therapy directly modulates (increases) the expression of one or a combination of genes selected from the group consisting of calbindin or Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0129] Embodiment 15: The method according to any one of Embodiments 1 to 13, wherein the gene therapy drug indirectly modulates (increases) the expression of one or a combination of genes selected from the group consisting of calbindin, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0130] Embodiment 16: A method for increasing the neural plasticity of a subject requiring such action, comprising administering a viral vector to the subject, wherein the viral vector is configured to modulate (increase) the expression of calbindin (Calb1) in a population of nerve cells of the subject, thereby increasing the level of experience-dependent plasticity of the subject.

[0131] Embodiment 17: The method according to Embodiment 16, wherein the viral vector directly increases the expression of calbindin.

[0132] Embodiment 18: The method according to Embodiment 16, wherein the viral vector indirectly increases the expression of calbindin.

[0133] Embodiment 19: The method according to any one of Embodiments 16 to 18, wherein the viral vector further modulates the expression of one or a combination of genes selected from the group consisting of Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0134] Embodiment 20: A method for increasing neural plasticity in a target requiring the same, comprising administering a viral vector to the target, wherein the viral vector contains one gene selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4 in a population of target neurons. A method comprising a mechanism configured to regulate (increase) the expression of a combination of genes, wherein the level of experience-dependent plasticity of a subject is increased by regulating (increasing) the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0135] Embodiment 21: The method according to Embodiment 20, wherein the viral vector directly modulates (increases) the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0136] Embodiment 22: The method according to Embodiment 20, wherein the viral vector indirectly regulates (increases) the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0137] Embodiment 23: The method according to any one of Embodiments 16 to 22, wherein the population of nerve cells includes excitatory neurons, inhibitory neurons, or a combination thereof.

[0138] Embodiment 24: The method according to any one of Embodiments 16 to 23, further comprising measuring the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4 in a population of nerve cells before administration of a viral vector.

[0139] Embodiment 25: A method for increasing the neural plasticity of a subject requiring such action, comprising administering exosomes to the subject, wherein the exosomes are configured to modulate (increase) the expression of calbindin (Calb1) in a population of nerve cells of the subject, thereby increasing the level of experience-dependent plasticity of the subject.

[0140] Embodiment 26: The method according to Embodiment 25, wherein the exosome directly regulates (e.g., increases) the expression of Calb1.

[0141] Embodiment 27: The method according to Embodiment 25, wherein the exosome indirectly regulates (e.g., increases) the expression of Calb1.

[0142] Embodiment 28: The method according to any one of Embodiments 25 to 27, wherein the exosome further modulates (e.g., increases) the expression of one or a combination of genes selected from the group consisting of Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0143] Embodiment 29: A method for increasing neural plasticity in a target that requires the same, comprising administering exosomes to the target, wherein the exosomes contain one of the genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4 in a population of target neurons or A method configured to regulate (increase) the expression of a combination of genes, wherein the level of experience-dependent plasticity of a subject is increased by regulating (increasing) the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0144] Embodiment 30: The method according to Embodiment 29, wherein the exosome directly regulates (e.g., increases) the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0145] Embodiment 31: The method according to Embodiment 29, wherein the exosome indirectly regulates (e.g., increases) the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0146] Embodiment 32: The method according to any one of Embodiments 25 to 31, wherein the population of nerve cells includes excitatory neurons, inhibitory neurons, or a combination thereof.

[0147] Embodiment 33: The method according to any one of Embodiments 25 to 32, further comprising measuring the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4 in a population of nerve cells before administration of exosomes.

[0148] Embodiment 34: A method for increasing the neural plasticity of a subject requiring the same, comprising administering nanoparticles to the subject, wherein the nanoparticles are configured to modulate (increase) the expression of calbindin (Calb1) in a population of nerve cells of the subject, thereby increasing the level of experience-dependent plasticity of the subject.

[0149] Embodiment 35: The method according to Embodiment 34, wherein nanoparticles directly regulate (increase) the expression of Calb1.

[0150] Embodiment 36: The method according to Embodiment 34, wherein nanoparticles indirectly regulate (increase) the expression of Calb1.

[0151] Embodiment 37: The method according to any one of Embodiments 34 to 36, wherein the nanoparticles further regulate the expression of one or a combination of genes selected from the group consisting of Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0152] Embodiment 38: A method for increasing neural plasticity in a target, comprising administering nanoparticles to the target, wherein the nanoparticles contain one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4 in a population of neurons of the target. A method configured to regulate (increase) the expression of a combination of genes, wherein the level of experience-dependent plasticity of a subject is increased by regulating (increasing) the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0153] Embodiment 39: The method according to Embodiment 38, wherein nanoparticles directly regulate (increase) the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0154] Embodiment 40: The method according to Embodiment 38, wherein nanoparticles indirectly regulate (increase) the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0155] Embodiment 41: The method according to any one of Embodiments 34 to 40, wherein the population of nerve cells includes excitatory neurons, inhibitory neurons, or a combination thereof.

[0156] Embodiment 42: The method according to any one of Embodiments 34 to 41, further comprising measuring the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4 in a population of nerve cells before administration of nanoparticles.

[0157] Embodiment 43: A method for increasing the neural plasticity of an object requiring the same, comprising increasing the expression of calbindin in a population of nerve cells of the object, wherein the neural plasticity of the object is increased by increasing the expression of calbindin.

[0158] Embodiment 44: A method for increasing the neural plasticity of a subject requiring it, comprising increasing the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4 in a population of neurons of the subject, thereby increasing the neural plasticity of the subject.

[0159] Embodiment 45: A method for increasing the neural plasticity of a subject requiring the same, comprising increasing the expression of one or a combination of genes selected from the group consisting of Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4 in a population of neurons of the subject, wherein the neural plasticity of the subject is increased by increasing the expression of one or a combination of genes selected from the group consisting of Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0160] Embodiment 46: The method according to any one of Embodiments 43 to 45, wherein the neural plasticity of a population of target neurons is increased by increasing the expression of calbindin.

[0161] Embodiment 47: A method for increasing the neural plasticity of a target population, comprising increasing the expression of calbindin in a population of target neurons, wherein the neural plasticity of the target population is increased by increasing the expression of calbindin.

[0162] Embodiment 48: A method for increasing the neural plasticity of a population of neurons in which it is required, comprising increasing the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4 in a population of neurons in which it is required, thereby increasing the neural plasticity of a population of neurons in which it is required.

[0163] Embodiment 49: A method for increasing the neural plasticity of a population of neurons in which it is required, comprising increasing the expression of one or a combination of genes selected from the group consisting of Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4 in a population of neurons in which it is required, thereby increasing the neural plasticity of a population of neurons in which it is required.

[0164] Embodiment 50: The method according to any one of Embodiments 43 to 49, wherein the experience-dependent plasticity of a population of target neurons is increased by increasing the expression of calbindin.

[0165] Embodiment 51: A method for increasing the neural plasticity of an object requiring it, comprising increasing the expression of calbindin in a population of neurons of the object, wherein the level of experience-dependent plasticity of the object is increased by increasing the expression of calbindin.

[0166] Embodiment 52: A method for increasing the neural plasticity of an object requiring it, comprising increasing the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4 in a population of neurons of the object, wherein the level of experience-dependent plasticity of the object is increased by increasing the expression of one or a combination of genes selected from the group consisting of Calb1, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0167] Embodiment 53: A method for increasing the neural plasticity of an object requiring the same, comprising increasing the expression of one or a combination of genes selected from the group consisting of Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4 in a population of neurons of the object, wherein the level of experience-dependent plasticity of the object is increased by increasing the expression of one or a combination of genes selected from the group consisting of Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0168] Embodiment 54: The method according to any one of Embodiments 43 to 53, wherein the population of nerve cells includes excitatory neurons, inhibitory neurons, or a combination thereof.

[0169] Embodiment 55: The method according to any one of Embodiments 43 to 54, comprising administering one or more gene therapies to a population of target neurons, thereby increasing the expression of calbindin, or one or a combination of genes selected from the group consisting of Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0170] Embodiment 56: The method according to any one of Embodiments 43 to 55, wherein one or more gene therapy drugs include a viral vector, exosomes, nanoparticles, small interfering RNA (siRNA) and microRNA (miRNA), antisense oligonucleotide (ASO), aptamer, modified cells, etc.

[0171] Embodiment 57: The method according to Embodiment 55 or Embodiment 56, wherein the gene therapy directly increases the expression of one or a combination of genes selected from the group consisting of calbindin, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0172] Embodiment 58: The method according to Embodiment 55 or Embodiment 56, wherein the gene therapy drug indirectly increases the expression of calbindin or one or a combination of genes selected from the group consisting of Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0173] Embodiment 59: A method for screening experience-dependent plasticity inducers and agents, comprising: a) obtaining or having obtained a sample; b) administering one or more factors and agents to the sample; and c) measuring the level expression of calbindin, wherein an increase in calbindin expression indicates that one or more factors and agents induce experience-dependent plasticity.

[0174] Embodiment 60: The method according to Embodiment 59, wherein the sample is a biological sample.

[0175] Embodiment 61: The method according to Embodiment 59 or Embodiment 60, wherein the sample includes ex vivo tissue, primary culture, iPSC, organoid, etc.

[0176] Embodiment 62: A method for screening experience-dependent plasticity inducers and agents, comprising: a) administering one or more factors and agents to a target; and b) measuring the level expression of calbindin, wherein an increase in calbindin expression indicates that one or more factors and agents induce experience-dependent plasticity.

[0177] Embodiment 63: The method according to Embodiment 62, wherein the subject is an animal model (e.g., a suitable animal model).

[0178] Embodiment 64: The method according to any one of Embodiments 59 to 63, further comprising measuring the level of calbindin expression before administering one or more factors and agents.

[0179] Embodiment 65: A method for increasing neural plasticity in an object requiring such plasticity, comprising transplanting a population of medial basal ganglia primordia (MGE) cells into the target neural tissue, wherein the transplantation increases the expression of calbindin in the population of target neurons, thereby increasing the level of experience-dependent plasticity in the object.

[0180] Embodiment 66: A method for increasing neural plasticity in an object requiring it, comprising increasing the expression of at least one of the following genes: calbindin, Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4, wherein the gene is overexpressed in at least one of cortical layers IV, V, or VI.

[0181] Embodiment 67: A method for increasing the neural plasticity of the central nervous system of an adult subject requiring it, comprising transplanting a population of medial basal ganglia primordia (MGE) GABAergic interneurons into the target nerve tissue, wherein the transplantation increases the expression of calbindin in the target neuronal population, and the following genes: Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nf A method for increasing experience-dependent circuit plasticity in adult subjects by altering the expression of at least one of kb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4, and increasing the expression of calbindin and at least one of Grin2b, Itpka, Neurod6, Panx2, Msi1, Cck, Nrgn, Caln1, Thy1, Marcksl1, Ncs1, Ctbp2, Git2, Nfkb1, Tgfbr1, Tgfbr2, Tgfb1, and Dlg4.

[0182] As used herein, the term “approximately” refers to ±10% of the referenced number.

[0183] While preferred embodiments of the present invention have been described, it will be readily apparent to those skilled in the art that modifications can be made that do not exceed the scope of the appended claims. Therefore, the scope of the present invention should be limited only by the scope of the appended claims. In some embodiments, the figures presented in this patent application are drawn to scale, including angles, dimensional ratios, etc. In some embodiments, the figures are representative only, and the scope of the claims is not limited by the dimensions in the figures. In some embodiments, the description of the invention described herein using the phrase "comprising" includes embodiments that may be described as "consisting essentially of" or "consisting of," and thus the written description requirement for claiming one or more embodiments of the present invention using the phrase "consisting essentially of" or "consisting of" is satisfied.

Claims

1. A method for increasing the neuroplasticity of a subject requiring increased neuroplasticity, comprising administering a gene therapy drug to the subject, wherein the gene therapy drug is configured to regulate the expression of calbindin (Calb1) in a population of nerve cells of the subject, and thereby increasing the neuroplasticity of the subject by regulating the expression of Calb1.

2. A method for increasing neuronal plasticity in a subject requiring increased neuronal plasticity, comprising administering a gene therapy drug to the subject, wherein the gene therapy drug is configured to regulate the expression of calbindin (Calb1) in a population of nerve cells of the subject, and by regulating the expression of Calb1, the neuronal plasticity of the subject is increased.

3. A method for increasing the neuroplasticity of a subject requiring increased neuroplasticity, comprising administering a gene therapy drug to the subject, wherein the gene therapy drug is configured to regulate the expression of calbindin (Calb1) in a population of neurons of the subject, and by regulating the expression of Calb1, the experience-dependent plasticity of the subject is increased.

4. The method according to any one of claims 1 to 3, wherein the gene therapy drug increases the expression of Calb1.

5. The method according to any one of claims 1 to 4, wherein the population of nerve cells includes excitatory neurons, inhibitory neurons, or a combination thereof.

6. The method according to any one of claims 1 to 5, wherein the therapeutic agent comprises a viral vector, exosomes, nanoparticles, small interfering RNA (siRNA) and microRNA (miRNA), antisense oligonucleotide (ASO), aptamer, modified cells, etc.

7. The method according to any one of claims 1 to 6, wherein the gene therapy drug directly increases the expression of Calb1.

8. The method according to any one of claims 1 to 7, wherein the gene therapy drug indirectly increases the expression of Calb1.

9. The method according to any one of claims 1 to 8, further comprising measuring the expression of Calb1 before administering the gene therapy drug.

10. A method for increasing the neuroplasticity of a subject requiring increased neuroplasticity, comprising administering a viral vector to the subject, wherein the viral vector is configured to regulate the expression of calbindin (Calb1) in a population of neurons of the subject, and the level of experience-dependent plasticity of the subject is increased by regulating the expression of Calb1.

11. The method according to claim 10, wherein the viral vector increases the expression of Calb1.

12. The method according to claim 10 or 11, wherein the viral vector directly increases the expression of Calb1.

13. The method according to claim 10 or claim 11, wherein the viral vector indirectly increases the expression of Calb1.

14. The method according to any one of claims 10 to 13, wherein the population of nerve cells includes excitatory neurons, inhibitory neurons, or a combination thereof.

15. The method according to any one of claims 10 to 14, further comprising measuring the expression of Calb1 before administering the viral vector.

16. A method for increasing the neuronal plasticity of a subject requiring increased neuronal plasticity, comprising administering exosomes to the subject, wherein the exosomes are configured to regulate the expression of calbindin (Calb1) in a population of neurons of the subject, and the level of experience-dependent plasticity of the subject is increased by regulating the expression of Calb1.

17. The method according to claim 16, wherein the exosome increases the expression of Calb1.

18. The method according to claim 16 or claim 17, wherein the exosome directly increases the expression of Calb1.

19. The method according to claim 16 or claim 17, wherein the exosome indirectly increases the expression of Calb1.

20. The method according to any one of claims 16 to 19, wherein the population of nerve cells includes excitatory neurons, inhibitory neurons, or a combination thereof.

21. The method according to any one of claims 16 to 20, further comprising measuring the expression of Calb1 before administering the exosomes.

22. A method for increasing the neuroplasticity of a subject requiring increased neuroplasticity, comprising administering nanoparticles to the subject, wherein the nanoparticles are configured to modulate the expression of calbindin (Calb1) in a population of neurons of the subject, and by modulating the expression of Calb1, the level of experience-dependent plasticity of the subject is increased.

23. The method according to claim 22, wherein the nanoparticles increase the expression of Calb1.

24. The method according to claim 22 or claim 23, wherein the nanoparticles directly increase the expression of Calb1.

25. The method according to claim 22 or claim 23, wherein the nanoparticles indirectly increase the expression of Calb1.

26. The method according to any one of claims 22 to 25, wherein the population of nerve cells includes excitatory neurons, inhibitory neurons, or a combination thereof.

27. The method according to any one of claims 22 to 26, further comprising measuring the expression of Calb1 before administering the exosomes.

28. A method for increasing the experience-dependent plasticity of an object requiring increased experience-dependent plasticity, comprising increasing the expression of calbindin (Calb1) in a population of nerve cells of the object, wherein increasing the expression of Calb1 increases the neural plasticity of the object.

29. A method for increasing the experience-dependent plasticity of an object requiring increased experience-dependent plasticity, comprising increasing the expression of calbindin (Calb1) in a population of nerve cells of the object, wherein increasing the expression of Calb1 increases the level of experience-dependent plasticity of the object.

30. The method according to claim 28 or 29, wherein the population of nerve cells includes excitatory neurons, inhibitory neurons, or a combination thereof.

31. The method according to any one of claims 28 to 30, wherein increasing the expression of Calb1 comprises administering one or more gene therapy drugs to the population of target nerve cells.

32. The method according to any one of claims 28 to 31, wherein the one or more gene therapy drugs include a viral vector, an exosome, nanoparticles, small interfering RNA (siRNA) and microRNA (miRNA), an antisense oligonucleotide (ASO), an aptamer, a modified cell, and the like.

33. The method according to claim 31 or claim 32, wherein the gene therapy drug directly increases the expression of Calb1.

34. The method according to claim 31 or claim 32, wherein the gene therapy drug indirectly increases the expression of Calb1.

35. A method for screening experience-dependent plasticity inducers and agents, a) Administering one or more factors and agents to the sample; b) Measuring the level expression of calbindin and Includes, A method demonstrating that an increase in calbindin expression indicates that one or more of the factors and agents induce experience-dependent plasticity.

36. The method according to claim 35, further comprising obtaining or having obtained a sample.

37. A method for screening experience-dependent plasticity inducers and agents, a) Obtaining a sample, or having obtained one; b) Administering one or more factors and agents to the sample; c) Measuring the level expression of calbindin and Includes, A method demonstrating that an increase in calbindin expression indicates that one or more of the factors and agents induce experience-dependent plasticity.

38. The method according to any one of claims 35 to 37, wherein the sample is a biological sample.

39. The method according to any one of claims 35 to 38, wherein the sample includes ex vivo tissue, primary culture, iPSC, organoid, etc.

40. A method for screening experience-dependent plasticity inducers and agents, a) To administer one or more factors and agents as the target; b) Measuring the level expression of calbindin and Includes, A method demonstrating that an increase in calbindin expression indicates that one or more of the factors and agents induce experience-dependent plasticity.

41. The method according to claim 40, wherein the subject is an animal model.

42. The method according to any one of claims 35 to 41, further comprising measuring the level of calbindin expression before administering the one or more factors and agents.

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