Treatment of neurological disorders using NHR

Abstract

The present disclosure provides methods of ameliorating or treating a neurological condition or disease in a subject in need thereof by administering to the subject an effective therapeutic amount of a composition comprising (i) recombinant DNA (rDNA) wherein the rDNA comprises a human nuclear hormone receptor (hNHR) gene or fragment thereof; and (ii) a delivery vehicle adapted to deliver the rDNA to nerve cells or tissues to ameliorate or treat the neurological condition or disease.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 423,481, filed on November 7, 2022, the entire content of which is incorporated herein by reference.

[0003] Reference to Electronic Sequence Listing

[0004] The following content of the electronic sequence listing is incorporated herein by reference in its entirety:

[0005] Name of XML file: OCU - 000520PC_SEQ_ID_LIST.xml;

[0006] File size: 13,141 bytes;

[0007] Creation date: November 5, 2023. Technical Field

[0008] The present disclosure relates to methods for ameliorating or treating a neurological disorder or disease in a subject in need thereof by administering to the subject a therapeutically effective amount of a composition comprising (i) recombinant DNA (rDNA), wherein the rDNA comprises a human nuclear hormone receptor (hNHR) gene or a fragment thereof; and (ii) a delivery vehicle adapted to deliver the rDNA to nerve cells or tissues to ameliorate or treat the neurological disorder or disease. Background Art

[0009] Genetic heterogeneity can be observed in many Mendelian monogenic disorders. While environmental influences provide a minor contribution, variations in phenotypic outcomes are generally attributable to allelic heterogeneity or modifier genes, allelic variants different from the mutant gene, which can affect disease onset, progression, and outcome by increasing or decreasing disease severity.

[0010] One of the modified gene therapies being developed by the present applicant involves curing retinal degeneration caused by gene mutations such as NR2E3, RHO, CEP290, PDE6B, etc., for example, by affecting the expression of NR2E3. See U.S. Patent No. 9,855,314, issued on January 2, 2019, and U.S. Patent No. 11,351,225, issued on June 7, 2022, which are incorporated herein by reference in their entireties. Thus, while some modified gene therapies are known, there are many other clinical diseases or disorders for which modified gene therapies are currently unavailable.

[0011] Accordingly, there is a continuing need for modified gene therapies for other clinical diseases or disorders. Summary of the Invention

[0012] It has been found that nuclear hormone receptors (NHRs) play a key role in regulating cellular homeostasis through transcriptional regulation of some downstream genes. Some aspects of the present disclosure provide the use of modified gene therapy to treat various genetic diseases caused by a large number of gene mutations in various genes but leading to the same outcome (phenotype) of a diseased condition. The modified gene alters the phenotype, regardless of the underlying gene mutations causing the disease.

[0013] Without being bound by any theory, it is believed that in some aspects of the present disclosure, the compositions disclosed herein regulate the expression or activity of genes, thereby altering the phenotype of the underlying gene mutations that cause clinical conditions or disorders. In some embodiments, the compositions of the present disclosure reduce the expression or activity of genes that cause neurological conditions or disorders. In other embodiments, the compositions of the present disclosure increase the expression or activity of wild-type (or "normal") genes, thereby ameliorating or treating neurological conditions or disorders.

[0014] A particular aspect of the present disclosure provides a method for ameliorating or treating a neurological condition or disease in a subject in need thereof, the method comprising administering to the subject an effective therapeutically amount of a composition comprising:

[0015] (i) recombinant DNA (rDNA) comprising a human nuclear hormone receptor (hNHR) gene or a fragment thereof; and

[0016] (ii) a delivery vehicle adapted to deliver the rDNA to nerve cells to ameliorate or treat the neurological condition or disease.

[0017] In some embodiments, the delivery vehicle comprises a viral delivery vector. In one particular case, the viral delivery vector includes viral delivery vectors (e.g., capsid proteins) associated with adeno-associated virus (AAV), adenovirus, and lentivirus. In other embodiments, the delivery vehicle comprises adeno-associated virus (AAV). In some other embodiments, the rDNA comprises adeno-associated virus inverted terminal repeats (AAV ITRs). In one particular embodiment, the AAV ITRs comprise AAV2 ITRs. Additionally, in other embodiments, the rDNA further comprises (i) a promoter, (ii) an enhancer, (iii) a polyadenylation portion, or (iv) a combination thereof. In some cases, the polyadenylation portion comprises a simian virus 40 (SV40) polyadenylation (PolyA) region, a bovine growth hormone (bGH) PolyA region, or a combination thereof. In further embodiments, the composition further comprises a cytomegalovirus (CMB) promoter or enhancer, elongation factor 1a (EF1a), chicken β-actin (CBA) promoter, CAG promoter, or a combination thereof.

[0018] In other embodiments, the delivery vehicle comprises a non-viral delivery vehicle, the non-viral delivery vehicle comprising nanoparticles, nanobodies, liposomes, biodegradable polymer complexes, or combinations thereof.

[0019] In some embodiments, the delivery vehicle is adapted to target the brain, using an adsorption ligand such as lectin, cardiolipin, heparin, and cell-penetrating peptides, or using a transporter ligand such as mannose, glutathione, and different amino acids, or using a receptor ligand such as transferrin, OX26 mAb, lactoferrin, apolipoprotein E, polysorbate 80, angiopep-2, candoxin, peptides, RVG29 - rabies virus glycoprotein (29aa peptide), RGD - arginine - glycine - aspartic acid peptide, NGR - asparagine - glycine - arginine peptide. Generally, nanoparticles can be targeted to the brain by modifying their surface with molecules / ligands specifically recognized by receptors or transporters overexpressed in the brain, such as transferrin, lactoferrin, LDL, nAChR, and αvβ3 integrin receptors, or glucose, glutathione, and amino acid transporters.

[0020] In other embodiments, the rDNA comprises a cell- or tissue-specific promoter. Exemplary cell- or tissue-specific promoters useful in the present invention include, but are not limited to, human Syn1, MeCP2, NSE, BM88 promoter - widespread neuronal expression; CaMKII - glutamatergic neuron expression specificity; DLX5 / 6 enhancer - GABAergic neuron specificity; tyrosine hydroxylase - catecholamine neuron specificity; dopamine β-hydroxylase (DBH), PRSx8 (synthetic DBH) - adrenergic and noradrenergic neuron specificity; PCP2 (Purkinje cell protein 2) - Purkinje neuron specificity; FEV, ETS transcription factor (Ple67) - serotonergic neuron specificity; MCH (melanin-concentrating hormone) - dorsal hypothalamus specificity; SLC6A4 (serotonin transporter Ple198), NR2E1 (ple264) - Müller - glial cell specificity; GfABC1D (truncated GFAP), Aldh1A1 - astrocyte specificity; MBP (myelin basic protein), MAG (myelin-associated glycoprotein) - oligodendrocyte specificity; ICAM-2 (intracellular adhesion molecule 2), CLDN5 (claudin 5), Tie-2 (TEK, receptor tyrosine kinase), vWF (von Willebrand factor), FLT1 (vascular endothelial growth factor receptor) - endothelial cell specificity; and combinations thereof.

[0021] In other embodiments, the hNHR gene is selected from NR2E3, NR1C3, NR1D1, RORA, NUPR1, NR2C1, and LXRa. In a particular embodiment, the hNHR gene comprises RORA.

[0022] In further embodiments, the neurological disorder or disease includes intellectual disability, epilepsy, cerebellar ataxia, autism spectrum disorder (ASD), or a combination thereof. In a particular instance, the neurological disorder or disease includes autism spectrum disorder. In yet another instance, the neurological disorder or disease includes intellectual disability. In another instance, the neurological disorder or disease includes epilepsy. In yet another instance, the neurological disorder or disease includes cerebellar ataxia. In yet another instance, the neurological disorder or disease includes Parkinson's disease. In still another instance, the neurological disorder or disease includes Alzheimer's disease.

[0023] In another embodiment, the composition further comprises a cell- or tissue-specific promoter for targeted expression in nerve cells or tissues. Exemplary cell- or tissue-specific promoters useful for the present disclosure include, but are not limited to:

[0024] Human Syn1, MeCP2, NSE, BM88 promoter - widespread neuronal expression;

[0025] CaMKII - glutamatergic neuron expression specificity;

[0026] DLX5 / 6 enhancer - GABAergic neuron specificity;

[0027] Tyrosine hydroxylase - catecholamine neuron specificity;

[0028] Dopamine β-hydroxylase (DBH), PRSx8 (synthetic DBH) - adrenergic and noradrenergic neuron specificity;

[0029] PCP2 (Purkinje cell protein 2) - Purkinje neuron specificity;

[0030] FEV, ETS transcription factor (Ple67) - serotonergic neuron specificity;

[0031] MCH (melanin-concentrating hormone) - dorsal hypothalamus specificity;

[0032] SLC6A4 (serotonin transporter Ple198), NR2E1 (ple264) - Müller glial cell specificity;

[0033] GfABC1D (truncated GFAP), Aldh1A1 - astrocyte specificity;

[0034] MBP (myelin basic protein), MAG (myelin-associated glycoprotein) - oligodendrocyte specific;

[0035] ICAM-2 (intracellular adhesion molecule 2), CLDN5 (claudin 5), Tie-2 (TEK, receptor tyrosine kinase), vWF (von Willebrand factor), FLT1 (vascular endothelial growth factor receptor) - endothelial cell specific; or a combination thereof.

[0036] Another aspect of the present disclosure provides a method for ameliorating or treating a neurological disorder or disease in a subject in need thereof. The method comprises administering to the subject an effective therapeutically amount of a composition comprising (i) a human nuclear hormone receptor (hNHR) gene or a fragment thereof, or its mRNA, or its dbDNA; and (ii) an hNHR delivery vehicle, wherein the hNHR gene or a fragment thereof, or its mRNA, or its dbDNA is selected from NR2E3, NR1C3, NR1D1, RORA, NUPR1, NR2C1, and LXRa, or its mRNA, or its dbDNA.

[0037] In some embodiments, the delivery vehicle comprises a viral delivery vector or a viral capsid protein, wherein the viral delivery vector comprises: a viral delivery vector associated with an adeno-associated virus, a lentivirus, an adenovirus, or an HSV1 viral vector. In other embodiments, the delivery vehicle comprises a nanoparticle or a lipid nanoparticle. Exemplary lipid nanoparticles (LNPs) that can be used in the present disclosure include all LNPs known to those skilled in the art of gene therapy. Some exemplary LNPs are disclosed in U.S. Patent Application Publication No. 2022 / 0184201, which is incorporated herein by reference in its entirety.

[0038] In other embodiments, the nanoparticles comprise liposomes, lipid nanoparticles, polymer nanoparticles, dendrimers, cyclodextrins, silica nanoparticles, polymer complexes, magnetic nanoparticles, gold nanoparticles, quantum dots, or carbon nanotubes.

[0039] In other embodiments, the composition comprises an hNHR gene or a fragment thereof, or its plasmid, or its mRNA, or its dbDNA (doggybone).

[0040] In further embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0041] In one specific embodiment, the composition is administered to the subject more than once.

[0042] In other embodiments, methods of administering the compositions of the present disclosure include intrathecal, intraventricular, intrastriatal, intracerebral, or combinations thereof as local routes of entry into the CNS. Other methods of administration include systemic routes such as intravenous (IV) injection, intramuscular (IM) injection, and the like.

[0043] However, in other embodiments, the method includes administering to a subject from about 10 8 to about 10 14 viral particles.

[0044] In other embodiments, the composition comprises an rAAV vector. In some cases, the composition further comprises rDNA, which includes (i) a promoter, (ii) an enhancer, (iii) a polyadenylation portion, or (iv) combinations thereof. In one specific instance, the polyadenylation portion comprises the simian virus 40 (SV40) polyadenylation (PolyA) region, the bovine growth hormone (bGH) PolyA region, or combinations thereof. In another instance, the vector further comprises a cytomegalovirus (CMB) promoter or enhancer, elongation factor 1a (EF1a), chicken β-actin (CBA) promoter, CAG promoter, a cell- or tissue-specific promoter for targeted expression in nerve cells or tissues, or combinations thereof.

[0045] In further embodiments, the vector is directly delivered to nerve cells or brain tissue of a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Shows the various development and homeostasis of several physiological systems regulated by RORA.

[0047] Figure 2 Shows a specific embodiment of the NHR transgenic expression cassette of the present disclosure. DETAILED DESCRIPTION

[0048] The present disclosure relates to the use of a modified gene called a nuclear hormone receptor (NHR) gene, or a fragment thereof, or its mRNA, or its dbDNA, to treat various clinical conditions or diseases. In particular, NHR transgenes are used herein to ameliorate or treat clinical conditions associated with neurological diseases or disorders. Nuclear receptors are a class of proteins responsible for various activities, including but not limited to sensing steroids, thyroid hormones, cholesterol, and vitamins. It has been shown that these receptors act together with other proteins to regulate the expression of multiple genes, thereby controlling the development, homeostasis, and metabolism of an organism. Thus, these modified genes can be used to correct defects or genetic disorders that manifest as various clinical conditions and / or diseases. It is believed that nuclear receptors bind directly to DNA, thereby regulating the expression of adjacent genes. Thus, these receptors are classified as transcription factors. One of the key properties of nuclear receptors that distinguishes them from other classes of receptors is their direct control of genomic DNA. A variety of NHRs are known to those skilled in the art, including but not limited to NR2E3, NR1C3, NR1D1, RORA (i.e., RORα), N(UPR1, NR2C1, and LXRa.

[0049] The wild-type nucleic acid sequences of human RORA (i.e., RORα) mRNA, NR1D1 mRNA, and variant 1 LxRa mRNA are shown in SEQ ID NOs: 1-3, respectively.

[0050] Human RORAmRNA sequence (CCDS10177):

[0051]

[0052]

[0053] Human Nr1d1 mRNA (Genbank accession number HQ692861.1 (GI: 325495532) (CCDS11361.1)):

[0054]

[0055]

[0056]

[0057] Human nuclear receptor subfamily 1 group H member 3 (NR1H3), transcript variant 1, (CCDS7929.1) mRNA (i.e., human LXRamRNA):

[0058]

[0059]

[0060] CATATGTGGA AGCCCTGCAT GCCTACGTCT CCATCCACCA TCCCCATGAC

[0061] CGACTGATGT TCCCACGGAT GCTAATGAAA CTGGTGAGCC TCCGGACCCT

[0062] GAGCAGCGTC CACTCAGAGC AAGTGTTTGC ACTGCGTCTG CAGGACAAAA

[0063] AGCTCCCACC GCTGCTCTCT GAGATCTGGG ATGTGCACGA ATGA

[0064] (SEQ ID NO:3)

[0065] It should be understood that the scope of the present disclosure also includes allelic variants of SEQ ID NOs: 1-3 known to those skilled in the art. The term "allelic variant" means any one of two or more alternative forms of a gene that occupy the same chromosomal locus. Allelic variations arise naturally through mutation and may result in polymorphisms within a population. Gene mutations can be silent (no change in the encoded polypeptide) or can encode a polypeptide with an altered amino acid sequence. In some embodiments, the allelic variant is a silent mutation variant.

[0066] In addition, the scope of the present disclosure also includes nucleic acids encoding bioactive fragments or variants of Nr1d1, Rora, or LXRa. The bioactive fragment or variant is a "functional equivalent" - a term well understood in the art and further defined in detail herein. Using any method disclosed herein or known in the art to establish the activity of a nuclear hormone receptor, the essential biological activity of the fragment or variant has the following activity relative to the wild-type native polypeptide: about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, and any range derivable therefrom, for example, from about 70% to about 80%, more preferably from about 81% to about 90%; or even more preferably, about 91% to about 99%. As used herein, the term "wild-type" refers to a wild-type that does not result in an undesirable phenotype or a "normal" phenotype that is considered to result in the typical form of a species as it exists in nature.

[0067] When referring to numerical values, the terms "about" and "approximately" are used interchangeably herein and mean within an acceptable error range of a particular value as determined by a person of ordinary skill in the art. Such a determination will depend at least in part on how the value is measured or determined, e.g., the limitations of the measuring system, i.e., the precision required for a particular purpose. For example, in accordance with the practice in the art, the term "about" can mean within 1 or more standard deviations. Alternatively, the term "about" when referring to a number (value) can mean 20%, typically 10%, often 5%, more often 1% of the numerical value. However, generally speaking, unless otherwise specified, in the case of describing a particular value in this application and the claims, the term "about" means within an acceptable error range of the particular value, typically within one standard deviation.

[0068] In the case of these sequences and all other sequences provided herein, a fragment is defined as a portion of a whole that is less than the whole. In addition, the size of a fragment ranges from a single nucleotide or amino acid within a polynucleotide or polypeptide sequence to a nucleotide or amino acid less than the whole polynucleotide or polypeptide sequence. Finally, a fragment is defined as any portion of a complete polynucleotide or polypeptide sequence that lies between the endpoints defined above. For example, fragments of any nuclear hormone receptor gene disclosed herein are about 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 350 nucleotides, 400 nucleotides, 450 nucleotides, 500 nucleotides, 550 nucleotides, 600 nucleotides, 650 nucleotides, 700 nucleotides, 750 nucleotides, 800 nucleotides, 850 nucleotides, 900 nucleotides, 950 nucleotides, 1000 nucleotides, 1100 nucleotides, 1200 nucleotides, 1300 nucleotides, 1400 nucleotides or 1500 nucleotides in length.

[0069] The term "its derivatives" refers to nucleotide sequences having at least about 70%, typically at least about 75%, often at least about 80%, more often at least about 85%, still more often at least about 90%, yet more often at least about 95%, and most often at least about 99% sequence identity or identity with those disclosed in SEQ ID NOs: 1-3.

[0070] The terms "identical" and "percent identity" are used interchangeably herein and in the context of two or more nucleic acids mean that two or more sequences or subsequences are the same or have a specified percentage of identical nucleotides when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm (such as those described below) or by visual inspection.

[0071] Alternatively, in the context of two nucleic acid sequences, the phrase "substantially identical" means two or more sequences or subsequences having at least about 75%, usually at least about 80%, usually at least about 85%, more usually at least about 90%, and most usually at least about 95% or higher nucleotide identity when compared and aligned for maximum correspondence as measured using a sequence comparison algorithm (such as those described below) or by visual inspection. Typically, substantial identity exists over a sequence region of at least about 40 - 60 nucleotides, in other cases over a region of at least 60 - 80 nucleotides, in still other cases over a region of at least 90 - 100 nucleotides, and in yet other cases the sequences are substantially identical over the full length of the sequences being compared (such as the coding region of nucleotides). Some examples of possible modifications include insertion of one or more nucleotides in the sequence, addition of one or more nucleotides at either end of the sequence, or deletion of one or more nucleotides at either end or within the sequence. The degree of identity between two polynucleotides can be readily determined using computer algorithms and methods well known to those of skill in the art.

[0072] For sequence comparison, typically one sequence acts as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters.

[0073] Optimal alignments of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970); by the similarity search method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by the computerized implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection [generally see Current Protocols in Molecular Biology, (edited by Ausubel, F.M. et al.) John Wiley & Sons, Inc., New York (1987 - 1999, including supplements such as supplement 46 (April 1999)]. Sequence comparisons using these programs are typically conducted using the default parameters specific to each program.

[0074] Another example of an algorithm suitable for determining percent sequence identity and percent sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in a database sequence, match or satisfy some positive-valued threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). This initial neighborhood word hit serves as a seed for initiating a search to find longer HSPs that contain them. Then, word hits are extended in both directions along each sequence until the cumulative alignment score can no longer be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for a mismatched residue, always <0). Extension of word hits in each direction is stopped when: the cumulative alignment score drops by the quantity X from its maximum achieved value; the cumulative score becomes zero or less than zero due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. To determine whether a nucleic acid is within the scope of the present disclosure, the default parameters of the BLAST program are suitable. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands as defaults. The TBLASTN program (using protein sequences for nucleotide sequences) uses a word length (W) of 3, an expectation value (E) of 10, and the BLOSUM 62 scoring matrix as defaults. (See Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0075] In addition to calculating the percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which indicates the probability that a match between two nucleotides occurred by chance. For example, if the minimum sum probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.1, typically less than about 0.01, and typically less than about 0.001, the nucleic acid is considered similar to the reference sequence.

[0076] Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. "Substantially hybridize" refers to the complementary hybridization between a probe nucleic acid and a target nucleic acid, and includes minor mismatches that can be accommodated by reducing the stringency of the hybridization medium to achieve the desired detection of the target polynucleotide sequence. The phrases "hybridizing specifically to" or "specifically hybridizing to" mean that when the sequence is present in a complex mixture (e.g., total cellular) DNA or RNA, the molecule binds, duplexes, or hybridizes only to a specific nucleotide sequence under stringent conditions.

[0077] The term "stringent conditions" refers to conditions under which a probe or primer will hybridize to its target sequence but not to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength and pH. In other instances, stringent conditions are selected to be about 20 °C or 25 °C lower than the melting temperature of the sequence, and the probe has exact or nearly exact complementarity to the target. As used herein, the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules dissociates into single strands by half. Methods for calculating the T m m of nucleic acids are well known in the art (see, for example, Berger and Kimmel (1987) Methods in Enzymology, vol. 152: Guide to Molecular Cloning Techniques, San Diego: Academic Press, Inc. and Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vols. 1-3, Cold Spring Harbor Laboratory), both of which are incorporated herein by reference. As shown in standard references, when nucleic acids are in an aqueous solution of 1 M NaCl, a simple estimate of the Tm value can be calculated by the following equation: T m= 81.5 + 0.41(%G+C) (see, e.g., Anderson and Young, “Quantitative Filter Hybridization,” in Nucleic Acid Hybridization (1985)). Other references include more complex calculations that take into account structure as well as sequence features to calculate Tm. The melting temperature of the hybrid (and thus the conditions for stringent hybridization) is affected by various factors such as the length and nature of the probe or primer (DNA, RNA, base composition) and the nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.), as well as the concentration of salts and other components (e.g., presence or absence of formamide, dextran sulfate, polyethylene glycol). The effects of these factors are well known and are discussed in standard references in the art, see, e.g., Sambrook, supra, and Ausubel, supra. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na ions, usually about 0.01 to 1.0 M Na ion concentration (or other salts), pH 7.0 to 8.3, and for short probes or primers (e.g., 10 to 50 nucleotides), the temperature is at least about 30 °C, and for long probes or primers (e.g., greater than 50 nucleotides), the temperature is at least about 60 °C. Stringent conditions can also be achieved by adding destabilizing agents such as formamide.

[0078] In some embodiments, the composition comprising the recombinant nucleic acid or rDNA is administered by electroporation. Alternatively, the composition is administered by gene delivery based on biodegradable nile red poly(lactide-co-glycolide) (PLGA) nanoparticles, small molecule-based gene delivery, naked DNA delivery, virus-based gene delivery (e.g., adeno-associated virus delivery), or genome editing systems (e.g., CRISPR).

[0079] The nucleic acid sequence encoding the NHR can be obtained using recombinant methods known in the art, e.g., by deriving the gene from a vector known to contain the gene, or by directly isolating it from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be synthesized. The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, PCR-generated linear DNA sequences, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0080] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, carbohydrates, peptides, cationic polymers, and liposomes. An exemplary colloidal system used as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0081] In the case of using non-viral delivery systems, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). On the other hand, nucleic acids can be associated with lipids. Nucleic acids associated with lipids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linking molecules that associate with both liposomes and oligonucleotides, entrapped within liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, combined with lipids, included as a suspension in lipids, included micelles or complexed with micelles, or otherwise associated with lipids. Lipid, lipid / RNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they can exist in bilayer structures, as micelles, or have a "collapsed" structure. They can also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic lipids. For example, lipids include the fat droplets naturally present in the cytoplasm as well as the class of compounds containing long-chain aliphatic hydrocarbons and their derivatives (e.g., fatty acids, alcohols, amines, amino alcohols, and aldehydes).

[0082] Suitable lipids are available from commercial sources. For example, dimyristoyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, N.Y.); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristoyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20 °C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a general term that encompasses a variety of single and multi-layer lipid mediators formed by generating closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure and trap water and dissolved solutes between the lipid bilayers. However, compositions having structures different from the normal vesicular structure in solution are also included. For example, the lipids can assume a micellar structure or exist only as non-uniform aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0083] Optionally, the method further comprises administering a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable" is well recognized in the art and refers to compositions, polymers, and other materials and / or dosage forms that are suitable, within the scope of sound medical judgment, for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. For example, pharmaceutically acceptable materials, compositions, or vehicles, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, are involved in carrying or transporting any supplement or composition or its components from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the supplement and not injurious to the patient. Optionally, the pharmaceutically acceptable carrier is pyrogen-free. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, sunflower seed oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0084] Typically, polynucleotides and / or other biological agents are purified and / or isolated prior to administration. As used herein, an "isolated" or "purified" nucleic acid molecule or polynucleotide is substantially free of other cellular material, or when produced by recombinant techniques is free of culture medium, or when chemically synthesized is free of chemical precursors or other chemicals. A purified compound is at least 60% (dry weight) of the target compound by weight. Typically, the preparation is at least 75% by weight, often at least 90%, and most often at least 99% of the target compound. For example, a purified compound is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99% or 100% (w / w) of the desired compound by weight. Purity is measured by any suitable standard method, such as column chromatography, thin layer chromatography or high performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) does not contain the genes or sequences that flank it in its native state. Purified also defines a degree of sterility that is safe for administration to a human subject, e.g., a reagent that is free of infectious or toxic agents.

[0085] Similarly, "substantially pure" means that the nucleotide has been separated from its naturally associated components. Typically, a nucleotide is substantially pure when it is at least about 60%, about 70%, about 80%, about 90%, about 95% or even about 99% by weight free of nucleotides or nucleic acids, and the naturally occurring organic molecules are associated with it in their natural state.

[0086] The scope of the present disclosure also includes conservatively modified variants of SEQ ID NOs: 1-3. "Conservatively modified variations" of a particular polynucleotide sequence refer to those polynucleotides that encode the same or substantially the same amino acid sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For example, the codons CGU, CGC, CGA, CGG, AGA and AGG all encode the amino acid arginine. Thus, at every position where a codon specifies arginine, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent substitutions" or "silent variations", and they are a type of "conservatively modified variation". Unless otherwise indicated, each polynucleotide sequence encoding a polypeptide described herein also describes every possible silent variation. Thus, silent substitutions are an implicit feature of every nucleic acid sequence encoding an amino acid. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG, which is typically the only codon for methionine) can be modified by standard techniques to produce a functionally identical molecule.

[0087] "Isolated nucleic acid" refers to a nucleic acid that does not contain the genes flanking it in the naturally occurring genome of the organism from which the nucleic acid is derived. The term encompasses, for example: (a) DNA that is part of a naturally occurring genomic DNA molecule but is not flanked by the two nucleic acid sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) nucleic acid incorporated into a vector or the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is different from any naturally occurring vector or genomic DNA; (c) a separate molecule, such as a cDNA, genomic fragment, fragment generated by polymerase chain reaction (PCR), or restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. The isolated nucleic acid molecules according to the invention further include synthetically produced molecules, as well as any nucleic acid that has been chemically altered and / or has a modified backbone. For example, an isolated nucleic acid is a purified cDNA or RNA polynucleotide.

[0088] Although the phrase "nucleic acid molecule" primarily refers to a physical nucleic acid and the phrase "nucleic acid sequence" refers to a linear listing of the nucleotides of a nucleic acid molecule, the two phrases may be used interchangeably.

[0089] The "effective amount" and "effective therapeutic amount" of a formulation or a component of a formulation mean an amount of the formulation or component, alone or in combination, sufficient to provide the desired effect. For example, an "effective amount" means an amount of a compound, alone or in combination, required to reduce or prevent an eye disorder in a mammal. Ultimately, the attending physician or veterinarian determines the appropriate amount and dosage regimen.

[0090] As used herein, the terms "treating" and "treatment" refer to administering an agent or formulation to an individual with clinical symptoms of an adverse condition, disorder, or disease to effect a reduction in the severity and / or frequency of the symptoms, elimination of the symptoms and / or their underlying cause, and / or promotion of the improvement or remediation of the injury.

[0091] The terms "preventing" and "prevention" refer to administering an agent or composition to a clinically asymptomatic individual who is predisposed or susceptible to a particular adverse condition, disorder, or disease, and thus involve preventing the occurrence of the symptoms and / or their underlying cause.

[0092] In some embodiments, the fragments of the present disclosure comprise or consist essentially of specific domains required for or contributing to the functional activity of Nr1d1, Nr2e3, Rora, Nupr1, Nr2c1, or LXRa. For example, nuclear hormone receptors have evolutionarily conserved domains shared by all members of the family, including a highly variable A / B domain, an N-terminal DNA-binding domain, a flexible hinge region, and a C-terminal ligand-binding and dimerization domain.

[0093] Variants encompassed by the invention include nucleic acid or amino acid sequences having the following degrees of sequence identity with Nr1d1, Nr2e3, Rora, Nupr1, Nr2c1, or LXRa: about 50%, about 55%, about 60%, about 65%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, and any ranges derivable therefrom, such as, for example, about 70% to about 80%; typically about 81% to about 90%; and typically about 91% to about 99% identity.

[0094] It should be understood that due to the degeneracy of the genetic code, any variation in the coding sequences of the nucleic acids of the invention that expresses the same sequence of polypeptide is included within the scope of the invention.

[0095] DNA molecules encoding the fragments or variants are produced using any of several known recombinant methods. To produce variants, it is routine to introduce mutations into the coding sequences to produce the desired amino acid sequence variants of the invention. Site-directed mutagenesis is a well-known technique, and protocols and reagents are commercially available (e.g., Zoller, MJ et al., 1982, Nucl Acids Res 10:6487-6500; Adelman, JP et al., 1983, DNA 2:183-93). These mutations include simple deletions or insertions, systematic deletions, insertions or substitutions of base clusters, or single base substitutions.

[0096] In some aspects, the present disclosure includes isolated AAV. As used herein with respect to AAV, the term "isolated" refers to AAV that has been separated from its natural environment (e.g., from a host cell, tissue, or subject) or artificially produced. Isolated AAV can be produced using recombinant methods. Such AAV is referred to herein as "recombinant AAV". Recombinant AAV (rAAV) can have tissue-specific targeting capabilities such that the transgene of the rAAV is specifically delivered to one or more predetermined tissues. The AAV capsid is an important element in determining these tissue-specific targeting capabilities. Thus, an rAAV can be selected that has a capsid suitable for the tissue to be targeted. In some embodiments, the rAAV comprises a capsid protein having an amino acid sequence corresponding to any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh10, AAV11, and variants thereof. Recombinant AAV generally comprises (i.e., contains or encapsulates) the recombinant nucleic acid of the present disclosure. Methods for obtaining recombinant AAV having a desired capsid protein are well known in the art (see, e.g., U.S. Patent Publication No. 2003 / 0138772, which is incorporated herein by reference in its entirety). AAV capsid proteins useful in the rAAV of the present invention include, for example, G. Gao et al., J. Virol., 78(12):6381-6388 (June 2004); G. Gao et al., Proc Natl Acad Sci USA, 100(10):6081-6086 (May 13, 2003); U.S. Patent Application Publication Nos. 2003 / 0138772, 2007 / 0036760, and 2009 / 0197338, and WO 2010 / 138263, all of which relate to AAV capsid proteins and related nucleotide and amino acid sequences and are incorporated herein by reference. Briefly, the methods involve culturing a host cell that contains a nucleic acid sequence encoding an AAV capsid protein or a fragment thereof; a functional rep gene; a recombinant AAV vector consisting of AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to allow packaging of the recombinant AAV vector into the AAV capsid protein.

[0097] Suitable AAVs that can be used in the methods provided herein are disclosed in U.S. Patent Application Publication Nos. 2013 / 0195801, 2012 / 0137379, which are incorporated herein by reference in their entireties.

[0098] Components to be cultured in a host cell for packaging an rAAV vector in an AAV capsid can be provided to the host cell in trans. Alternatively, any one or more of the desired components (e.g., optionally, for example, a recombinant AAV vector, a rep sequence, a cap sequence, and / or helper functions) can be provided by a stable host cell that has been engineered using methods known to those of skill in the art to contain one or more of the desired components. Most suitably, such a stable host cell will contain the desired components under the control of an inducible promoter. However, the desired components can be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein. In another alternative, a selected stable host cell can contain selected components under the control of a constitutive promoter and other selected components under the control of one or more inducible promoters.

[0099] Any suitable NHR genetic element (vector) can be used to deliver the recombinant AAV vector, rep sequence, cap sequence, and helper functions required to produce the rAAV of the present invention to the packaging host cell. The selected genetic element can be delivered by any suitable method, including those described herein. The methods for constructing any embodiment of the present disclosure are known to those of skill in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods for producing rAAV virions are well known, and the choice of a suitable method is not a limitation of the present invention. See, e.g., K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Patent No. 5,478,745.

[0100] In some embodiments, a triple transfection method can be used to generate recombinant AAV, see, e.g., U.S. Patent No. 6,001,650. Generally, recombinant AAV is generated by transfecting a host cell with a recombinant AAV vector (containing the NHR transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. The AAV helper function vector encodes "AAV helper function" sequences (e.g., rep and cap), which act in trans to permit productive AAV replication and encapsidation. In some embodiments, the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (e.g., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for the present disclosure include pHLP19 described in U.S. Patent No. 6,001,650 and the pRep6cap6 vector described in U.S. Patent No. 6,156,303, which are incorporated herein by reference in their entirety. The accessory function vector encodes nucleotide sequences of non-AAV-derived viruses and / or cellular functions (e.g., "accessory functions") on which AAV depends for replication. Accessory functions include those functions required for AAV replication, including but not limited to those involved in AAV gene transcriptional activation, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of the cap expression product, and AAV capsid assembly. The virus-based accessory functions can be derived from any known helper virus, such as adenovirus, herpesvirus (except herpes simplex virus type 1), and vaccinia virus.

[0101] In some aspects, the present disclosure provides transfected host cells. The term "transfection" is used to refer to the uptake of exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced within the cell membrane. Many transfection techniques are generally known in the art. Such techniques can be used to introduce one or more exogenous nucleic acids (e.g., nucleotide integration vectors and other nucleic acid molecules) into a suitable host cell. A "host cell" refers to any cell that harbors or is capable of harboring a target substance. Generally, the host cell is a mammalian cell, a bacterium, or other suitable cell known to those skilled in the art. The host cell can serve as a recipient for an AAV helper construct, an AAV minigene plasmid, an accessory function vector, or other transfer DNA associated with the production of recombinant AAV. The term includes the progeny of the original cell that has been transfected. Thus, a "host cell" as used herein can refer to a cell that has been transfected with an exogenous DNA sequence. It should be understood that the progeny of a single parental cell may not necessarily be identical in morphology or genomic or total DNA complement to the original parent due to natural, accidental, or deliberate mutations.

[0102] In some aspects, the present disclosure provides isolated cells. As used herein with respect to cells, the term "isolated" refers to cells that have been separated from their natural environment (e.g., from tissue or a subject). As used herein, the term "cell line" refers to a population of cells capable of continuous or extended growth and division in vitro. Typically, a cell line is a clonal population derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in the karyotype during storage or transfer of such clonal populations. Thus, cells derived from the cell line in question may not be identical to the ancestral cells or culture, and the cell line in question includes such variants. As used herein, the term "recombinant cell" refers to a cell that has been introduced with an exogenous DNA segment (e.g., a DNA segment that results in the transcription of a bioactive polypeptide or the production of a bioactive nucleic acid (e.g., RNA)).

[0103] The term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replicating when associated with appropriate control elements and can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. In some embodiments, the vectors that are expected to be useful are those in which the nucleic acid segment to be transcribed is under the transcriptional control of a promoter. A "promoter" is a DNA sequence required to initiate gene-specific transcription that is recognized by the synthetic machinery of the cell or introduced synthetic machinery. The phrases "operably positioned," "under control," or "under transcriptional control" mean that the promoter is in the correct position and orientation relative to the nucleic acid to control the initiation by RNA polymerase and the expression of the gene. The term "expression vector or construct" means any type of genetic construct containing a nucleic acid, wherein part or all of the nucleic acid coding sequence is capable of being transcribed. In some embodiments, expression includes the transcription of nucleic acid, e.g., to produce a bioactive polypeptide product or inhibitory RNA (e.g., shRNA, miRNA) from the transcribed gene.

[0104] The foregoing methods for packaging recombinant vectors in a desired AAV capsid to produce the rAAV of the present disclosure are not meant to be limiting, and other suitable methods will be apparent to those skilled in the art who have read the present disclosure.

[0105] The recombinant nucleic acid of the invention can be a recombinant AAV vector. The recombinant AAV vector can be packaged into a capsid protein and administered to a subject and / or delivered to selected target cells. A "recombinant AAV (rAAV) vector" generally consists of at least a transgene and its regulatory sequences. In some embodiments, the transgene also includes 5'- and 3'-AAV inverted terminal repeats (ITRs). As disclosed elsewhere herein, the transgene can include one or more regions encoding one or more NHRs.

[0106] The AAV sequences of the vector generally contain cis-acting 5' and 3' inverted terminal repeat sequences (see, e.g., B.J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 - 168 (1990)). The length of the ITR sequences ranges from about 100 bp to about 200 bp, typically from about 110 bp to about 175 bp, typically from about 120 bp to about 150 bp, and most typically from about 130 bp to about 140 bp. In some embodiments, substantially the entire sequence encoding the ITR is used in the molecule, although a certain degree of minor modification of these sequences is permitted. The ability to modify these ITR sequences is within the skill in the art. An example of such a molecule employed in the present invention is a “cis-acting” plasmid containing a transgene, wherein the selected transgene sequence and associated regulatory elements are flanked by 5'- and 3'-AAV ITR sequences. The AAV ITR sequences can be obtained from any known AAV, including currently identified mammalian AAV types.

[0107] Thus, the recombinant nucleic acid can contain inverted terminal repeats (ITRs) of an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV.rh10, AAV11, and variants thereof. The recombinant nucleic acid can further include a promoter operably linked to one or more NHRs. The promoter can be a tissue-specific promoter, a constitutive promoter, or an inducible promoter.

[0108] In addition to the major elements identified above for the recombinant AAV vector, the vector can include conventional control elements that are operably linked to the elements of the transgene in a manner that permits its transcription, translation, and / or expression in cells transfected with the vector or infected with the virus generated by the present disclosure. As used herein, “operably linked” sequences include both expression control sequences adjacent to the target gene and expression control sequences that act in trans or at a distance to control the expression of the target gene. Expression control sequences include appropriate transcriptional start, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance protein stability; and, when needed, sequences that enhance the secretion of the encoded product. Many expression control sequences, including natural, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be used.

[0109] As used herein, nucleic acid sequences (e.g., coding sequences) and regulatory sequences are said to be operably linked when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequence. If it is desired to translate a nucleic acid sequence into a functional protein, if the induction of a promoter in a 5'-regulatory sequence results in the transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein, then the two DNA sequences are said to be operably linked. Thus, a promoter region will be operably linked to a nucleic acid sequence if the promoter region is capable of influencing the transcription of the DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide. Similarly, when two or more coding regions are linked in such a way that their transcription from a common promoter results in the expression of two or more proteins that have been translated in-frame, they are operably linked. In some embodiments, the operably linked coding sequences produce a fusion protein. In some embodiments, the operably linked coding sequences produce a functional RNA (e.g., miRNA).

[0110] For nucleic acids encoding proteins, a polyadenylation sequence is typically inserted after the transgene sequence and before the 3'-AAV ITR sequence. The rAAV constructs useful in the present invention may also contain introns, desirably located between the promoter / enhancer sequence and the transgene. One possible intron sequence is derived from SV-40 and is referred to as the SV-40T intron sequence. Any intron can be from the β-actin gene. Another vector element that can be used is an internal ribosome entry site (IRES).

[0111] The exact nature of the regulatory sequences required for gene expression in a host cell can vary between species, tissues, or cell types, but generally should include (as needed) 5'-non-transcribed and 5'-non-translated sequences involved in transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, enhancer elements, etc. In particular, such 5'-non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for the transcriptional control of the operably linked gene. The regulatory sequences may also include enhancer sequences or upstream activator sequences as needed. The vectors of the present invention may optionally include a 5'-leader sequence or signal sequence. The selection and design of an appropriate vector are within the capabilities and judgment of one of ordinary skill in the art.

[0112] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, and the dihydrofolate reductase promoter. Inducible promoters allow for the regulation of gene expression and can be regulated by an exogenously supplied compound, an environmental factor (such as temperature), or the presence of a specific physiological state (such as the acute phase, a specific differentiation state of the cell, or only in replicating cells). Inducible promoters and inducible systems are available from a variety of commercial sources, including, but not limited to, Invitrogen, Clontech, and Ariad. Many other systems have been described and can be readily selected by those skilled in the art. Examples of inducible promoters regulated by an exogenously supplied promoter include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system, the ecdysone insect promoter, the tetracycline repressor system, the tetracycline-inducible system, the RU486-inducible system, and the rapamycin-inducible system. Other types of inducible promoters that may be useful herein are promoters regulated by a specific physiological state (such as temperature, the acute phase, a specific differentiation state of the cell), or only in replicating cells.

[0113] In another embodiment, the native promoter of the transgene or a fragment thereof will be used. In another embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic native expression. In some embodiments, the regulatory sequences confer the ability to express genes in a tissue-specific manner. In some cases, tissue-specific regulatory sequences bind to tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (such as promoters, enhancers, etc.) are well known in the art. In some embodiments, the promoter is the chicken β-actin promoter.

[0114] The compositions disclosed herein are administered in an amount sufficient to transfect cells of the desired tissue and provide a sufficient level of gene transfer and expression without undue adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected tissue (e.g., neurons and other nerve cells or tissues) and subcutaneous, intra-pancreatic, intranasal, parenteral, intravenous, intramuscular, intrathecal, intracerebral, oral, intraperitoneal, by inhalation, or by another route. If desired, routes of administration can be combined. Delivery of certain compositions of the present disclosure to a subject can be, for example, by administration into the bloodstream of the subject. Administration into the bloodstream can be by injection into a vein, artery, or any other blood vessel conduit. Additionally, in certain instances, it may be desirable to deliver the compositions of the present disclosure to the brain tissue, meninges, neuronal cells, glial cells, astrocytes, oligodendrocytes, cerebrospinal fluid (CSF), interstitial space, etc. In some embodiments, the compositions of the present disclosure can be delivered directly to the spinal cord or brain (e.g., prefrontal cortex) using neurosurgical techniques known in the art, such as by stereotactic injection, with a needle, catheter, or related device, by injection into ventricular regions as well as the striatum (e.g., caudate nucleus or putamen of the striatum) and neuromuscular junctions or cerebellar lobules.

[0115] In certain cases, it will be desirable to deliver the compositions of the present disclosure as an appropriately formulated pharmaceutical composition by intrathecal, intracerebral, intravenous, subcutaneous, intra-pancreatic, intranasal, parenteral, intravenous, intramuscular, oral, intraperitoneal, or by inhalation. One of ordinary skill in the art will appreciate that the desired administration of the compositions of the present disclosure can also include ex vivo administration. In some embodiments, ex vivo administration includes (1) isolating the cells or tissue of interest from a subject, (2) contacting the cells or tissue with a sufficient amount of the compositions of the present disclosure to transfect the cells or tissue to provide a sufficient level of gene transfer and expression without undue adverse effects, and (3) transferring the cells or tissue back to the subject. In some embodiments, the cells or tissue can be cultured ex vivo for several days before and / or after transfection.

[0116] Cells or tissue can be isolated from a subject by any suitable method. For example, cells or tissue can be isolated by surgery, biopsy (e.g., biopsy of skin tissue, lung tissue, liver tissue, adipose tissue), or collection of a biological fluid such as blood. In some embodiments, cells are isolated from bone marrow. In some embodiments, cells are isolated from adipose tissue. In some embodiments, cells are isolated from a lipoaspirate. Suitable methods for isolating cells from adipose tissue for ex vivo transfection are known in the art.

[0117] In some embodiments, the isolated cells include stem cells, pluripotent stem cells, neural progenitor cells, adipose-derived stem cells, hepatocytes (e.g., liver cells), hematopoietic stem cells, mesenchymal stem cells, stromal cells, hematopoietic cells, blood cells, fibroblasts, endothelial cells, epithelial cells, or other suitable cells. In some embodiments, the cells to be transfected are induced pluripotent stem cells prepared from cells isolated from a subject.

[0118] When using a viral vector, such as rAAV, it can be delivered to a subject in the form of a composition according to any suitable method known in the art. rAAV that can be suspended in a physiologically compatible carrier (e.g., in a composition) can be administered to a subject, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or non-human primate (e.g., marmoset, macaque). The compositions of the present invention can contain rAAV alone, or rAAV in combination with one or more other viruses (e.g., a second rAAV encoding one or more different transgenes). In some embodiments, the rAAV vector expressing NHR (e.g., RORA) is injected into the CSF of a subject using IT injection in the tail and cisterna magna injection in the head. Given the indication targeted by rAAV, those skilled in the art can readily select a suitable carrier. For example, a suitable carrier includes saline, which can be formulated with a variety of buffer solutions (e.g., phosphate buffer). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. Still others will be obvious to those skilled in the art.

[0119] Optionally, in addition to rAAV and the carrier, the compositions of the present invention can also contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0120] The dose of rAAV virions required to achieve the desired effect or “therapeutic effect”, e.g., the dose unit in vector genomes per kilogram body weight (vg / kg), will vary based on several factors, including but not limited to: the rAAV administration route, the level of gene or RNA expression required to achieve the therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. Those skilled in the art can readily determine the rAAV virion dose range to treat a subject suffering from a specific disease or disorder based on the above factors and other factors well known in the art. The effective amount of rAAV is typically in the range of about 10 μL to about 100 mL of solution, with each subject containing about 10 9from 0 to 10 16 genomic copies. Other volumes of solution can be used. The volume used will generally depend especially on the size of the subject, the dose of rAAV, and the route of administration. For example, for intravenous administration, volumes in the range of 10 μL to 100 μL, 100 μL to 1 mL, 1 mL to 10 mL, or more can be used. In some cases, a dose of about 10 10 to 10 12 rAAV genomic copies per subject is suitable. In some embodiments, rAAV is administered at a dose of 10 10 、10 11 、10 12 、10 13 、10 14 or 10 15 genomic copies per subject. In some embodiments, rAAV is administered at a dose of 10 10 、10 11 、10 12 、10 13 or 10 14 genomic copies / kg.

[0121] The formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, and developing suitable dosing and treatment regimens for using the specific compositions described herein in various treatment regimens is also well known to those skilled in the art. Generally, these formulations can contain at least about 0.1% of the active ingredient or more, although the percentage of the active ingredient can of course vary and can conveniently be between about 1% or 2% and about 70% or 80% or more of the total weight or volume of the formulation. Of course, the amount of the active ingredient in each therapeutically useful composition can be prepared such that a suitable dose will be obtained in any given unit dose of the compound. Those skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and thus, a variety of dosing and treatment regimens may be desirable.

[0122] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The dispersions can also be prepared in glycerol, liquid polyethylene glycols and their mixtures, and in oils. Under ordinary conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms. In many cases, the forms are sterile and fluid to such an extent that easy injection is possible. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycols, etc.), suitable mixtures thereof and / or vegetable oils. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by the use of agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0123] For the administration of injectable aqueous solutions, for example, if necessary, the solution can be appropriately buffered and first made isotonic with sufficient saline or glucose to the liquid diluent. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, the sterile aqueous media that can be used are known to those skilled in the art. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous perfusion fluid or injected at the proposed infusion site. Some variation in dosage will necessarily occur depending on the condition of the host. In any case, the person responsible for administration will determine the appropriate dosage for the individual host.

[0124] Sterile injectable solutions are prepared by incorporating the required amount of the active composition of the present disclosure into a suitable solvent (as required) having the various other ingredients enumerated herein, and then filtering to sterilize. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional required ingredients from its previously sterile-filtered solution.

[0125] The compositions disclosed herein may also be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. Upon formulation, the solution will be administered in a manner compatible with the dosage formulation and in an amount therapeutically effective. The formulations are readily administered in a variety of dosage forms such as injectable solutions, drug release capsules, etc.

[0126] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.

[0127] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. can be used to introduce the compositions of the present invention into suitable host cells. In particular, the transgenes delivered by rAAV vectors can be formulated for encapsulation and delivery in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, etc. Such formulations are preferably pharmaceutically acceptable formulations for introducing the nucleic acids or rAAV constructs disclosed herein. The formation and use of liposomes are generally known to those skilled in the art. Recently, liposomes with improved serum stability and circulating half-life have been developed (U.S. Patent No. 5,741,516). In addition, various methods of liposomes and liposome-like formulations as potential drug carriers have been described (U.S. Patents Nos. 5,567,434, 5,552,157, 5,565,213, 5,738,868, and 5,795,587).

[0128] Liposomes have been successfully used in many cell types that are generally resistant to transfection by other methods. In addition, liposomes are not subject to the DNA length limitations typical of virus-based delivery systems. Liposomes have been effectively used to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors, and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials have been completed to examine the effectiveness of liposome-mediated drug delivery. Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also known as multilamellar vesicles (MLV)). MLV generally have a diameter of 25 nm to 4 μm. Sonication of MLV results in the formation of diameters between 200 and Small unilamellar vesicles (SUVs) within the range that contain an aqueous solution in their core. Alternatively, a nanocapsule formulation of rAAV can be used. Nanocapsules can generally capture substances in a stable and reproducible manner. Such ultrafine particles (sized at about 0.1 μm) should be designed using polymers that can degrade in vivo. Biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements are considered for use.

[0129] In addition to the above delivery methods, the following techniques are also considered as alternative methods for delivering the rAAV composition to a host. Sonophoresis (such as ultrasound) has been used and described in U.S. Patent No. 5,656,016 as a means for enhancing the rate and efficacy of drug penetration into and through the circulatory system. Other expected alternative drug delivery methods are intraosseous injection (U.S. Patent No. 5,779,708), microchip devices (U.S. Patent No. 5,797,898), transdermal matrices (U.S. Patents Nos. 5,770,219 and 5,783,208), and feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0130] Therapeutic methods

[0131] This disclosure is based on the discovery of a gene therapy method, in which a modified gene is directly or indirectly administered to neurons or nerve cells to treat or prevent various neurological disorders or diseases. Exemplary neurological disorders or diseases that can be treated using the methods of this disclosure include, but are not limited to, intellectual developmental disorders, epilepsy, cerebellar ataxia, autism spectrum disorder (ASD), Parkinson's disease, Alzheimer's disease, neurodegeneration of unknown etiology, or combinations thereof.

[0132] In one embodiment, the disclosure is further characterized by an expression vector, which includes a vector containing a recombinant polynucleotide, the recombinant polynucleotide containing an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), RNAs, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating the recombinant polynucleotide. The expression vectors of this disclosure can also include one or more regulatory elements, such as heterologous promoters. A specific recombinant polynucleotide containing an expression control sequence is shown in Figure 2In this recombinant DNA or polynucleotide, the transgenic hRORA is under the transcriptional control of the cytomegalovirus (CMV) enhancer and contains the chicken β-actin promoter (CBA) promoter, the Kozak sequence at the transcription start site, and the SV40 polyadenylation sequence. It can be seen that the NHR (e.g., hRORA) expression cassette is inserted between two AAV2 ITRs. In particular, Figure 2 The recombinant polynucleotide containing hRORA as shown contains the nucleotide sequence of SEQ ID NO: 4.

[0133] cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc

[0134] ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag

[0135] cgagcgagcg cgcagagagg gagtggccaa ctccatcact

[0136] aggggttcct gcggcctaag gcaattgaga tctcgacatt

[0137] gattattgac tagttattaa tagtaatcaa ttacggggtc

[0138] attagttcat agcccatata tggagttccg cgttacataa

[0139] cttacggtaa atggcccgcc tggctgaccg cccaacgacc

[0140] cccgcccatt gacgtcaata atgacgtatg ttcccatagt

[0141] aacgccaata gggactttcc attgacgtca atgggtggag

[0142] tatttacggt aaactgccca cttggcagta catcaagtgt

[0143] atcatatgcc aagtacgccc cctattgacg tcaatgacgg

[0144] taaatggccc gcctggcatt atgcccagta catgacctta

[0145] tgggactttc ctacttggca gtacatctac gtattagtca

[0146] tcgctattac catggtcgag gtgagcccca cgttctgctt

[0147] cactctcccc atctcccccc cctccccacc cccaattttg

[0148] tatttattta ttttttaatt attttgtgca gcgatggggg

[0149] cggggggggg gggggggcgc gcgccaggcg gggcggggcg

[0150] gggcgagggg cggggcgggg cgaggcggag aggtgcggcg

[0151] gcagccaatc agagcggcgc gctccgaaag tttcctttta

[0152] tggcgaggcg gcggcggcgg cggccctata aaaagcgaag

[0153] cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc

[0154] gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc

[0155] tctgactgac cgcgttactc ccacaggtga gcgggcggga

[0156] cggcccttct cctccgggct gtaattagcg cttggtttaa

[0157] tgacggcttg tttcttttct gtggctgcgt gaaagccttg

[0158] aggggctccg ggagggccct ttgtgcgggg ggagcggctc

[0159] ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg

[0160] tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc

[0161] gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg

[0162] gagcgcggcc gggggcggtg ccccgcggtg cggggggggc

[0163]

[0164]

[0165] agtgagcgag cgagcgcgca gctgcctgca

[0166] gg(SEQ ID NO:4)

[0167] A variety of known nucleic acid vectors can be used in these methods, such as recombinant viruses, such as recombinant adeno-associated virus (rAAV), recombinant adenovirus, recombinant retrovirus, recombinant poxvirus, and other viruses known in the art, as well as plasmids, cosmids, and phages. Many publications known in the art discuss the use of a variety of such vectors for gene delivery. See, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, latest edition; Kay, M.A. et al., 2001, Nat. Med., 7:33-40; and Walther W et al., 2000, Drugs 60:249-71).

[0168] The compositions of the present disclosure further comprise recombinant DNA, such as a recombinant human nuclear hormone receptor (hNHR) gene or a fragment thereof, or its mRNA, or its dbDNA. Regulatory elements can be found endogenously upstream or downstream of the gene, or they can be exogenous regulatory elements that do not regulate genes in nature and are introduced by recombinant DNA techniques known in the art. The regulatory elements can be operably linked to the genes of the present disclosure or fragments thereof, or genes encoding the proteins of the present disclosure or fragments thereof. Methods for assembling recombinant vectors are well known. See, for example, WO 00 / 15822 and other references cited therein, all of which are incorporated herein by reference. After delivering the vector to a subject, such as to a subject's nerve cells or brain tissue, the nucleic acid is optionally integrated into the genome of the cell.

[0169] The compositions of the present disclosure or the rDNA can also include appropriate sequences operably linked to the coding sequence or ORF to facilitate the expression of the nuclear hormone receptor of the present disclosure in the targeted host cell. "Operably linked" sequences include expression control sequences adjacent to the coding sequence, such as promoters, and expression control sequences that act in trans or distally to control the expression of the polypeptide product.

[0170] Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance the stability of nucleic acids or proteins; and, when needed, sequences that enhance protein processing and / or secretion. Many different expression control sequences, including natural and non-natural, constitutive, inducible, and / or tissue-specific, are known in the art and can be used herein. This depends on the type of expression desired.

[0171] Expression control sequences in eukaryotic cells generally include promoters, enhancers (e.g., enhancers derived from immunoglobulin genes, SV40, CMV, etc.), and polyadenylation sequences (which can include splice donor and acceptor sites). The polyadenylation sequence is generally inserted 3' of the coding sequence and 5' of the 3' ITR sequence. PolyA from bovine growth hormone is an example of a suitable sequence.

[0172] The promoter can be selected from several constitutive or inducible promoters that can drive the expression of the selected transgene in the context of nerve cells or brain tissue. Generally, the promoter used is "cell-specific", meaning it is selected to direct the expression of the selected transgene in a particular nerve cell type or brain tissue.

[0173] The rAAV used in the present disclosure can be constructed and produced using the materials and methods described herein as well as those well-known in the art. The methods for generating the constructs of the present disclosure are conventional and include genetic engineering, recombinant engineering, and synthetic techniques readily understood by those of ordinary skill in the art.

[0174] Briefly, in order to package an rAAV construct into an rAAV virion, the sequences necessary for expressing AAV rep and AAV cap or functional fragments thereof, as well as the helper genes necessary for AAV production, must be present in the host cell. See, for example, U.S. Patent Publication 2007 / 0015238, which describes the production of pseudotyped rAAV virion vectors encoding AAV Rep and Cap proteins of different serotypes and AdV transcripts providing helper functions. For example, the AAV rep and cap sequences can be introduced into the host cell in any known manner, including but not limited to transfection, electroporation, liposome delivery, membrane fusion, gene gun delivery of DNA-coated pellets, viral infection, and protoplast fusion.

[0175] In another embodiment, the nucleic acids of the present disclosure can be delivered by nanoparticles. Nanoparticles are lipid-based colloidal particles, for example, with a diameter less than 100 nm. Nanoparticles intended for drug and gene delivery can be characterized by various parameters, including particle size, size distribution, morphology, ζ potential, drug loading, injectability and injectability, in vitro drug release, and stability. Depending on the intended use, the formulation of the nanoparticles varies with the lipid composition, the ratio of nucleic acid to lipid, and the formulation method. Nanoparticle assembly methods are known in the art and are described, for example, in Kompella et al., "Nanoparticles for Drug and Gene Delivery in Treating Diseases of the Eye"; Methods in Pharmacology and Toxicology, 2014, pp. 291-316, which is incorporated herein by reference in its entirety.

[0176] Genome editing systems can also be used to deliver the nucleic acids of the present disclosure. Examples of such genome editing systems include but are not limited to the CRISPR / Cas system, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). In such systems, the nucleic acids of the present disclosure can be readily incorporated into the host cell genome and expressed. In some embodiments, the mutant form of the pathogenic gene (i.e., RORα) can be "edited", or selectively excised, and replaced with any of the nucleic acids described herein. Expression is regulated by endogenous or exogenous regulatory elements, and the expression of these nucleic acids ameliorates or alleviates the symptoms of the clinical condition or disease.

[0177] The methods and compositions described herein relate to the restoration or normalization of phenotypes. As used herein, "restoration" or "normalization" refers to increasing or decreasing the expression level or activity of a defective gene in a subject to a level similar to that of a subject not suffering from a neurological disorder or disease (i.e., a subject not exhibiting the neurological disorder or disease disclosed herein). Restoration or normalization of neuronal or brain tissue activity can be measured or determined by various tests well known to those skilled in the art.

[0178] As described in detail below, delivery of the NHR gene to neuronal cells or brain tissue effectively ameliorates clinical, morphological, and functional deficits associated with various genetic defects that cause the observed phenotypic neurological disorders or diseases disclosed herein.

[0179] For many Mendelian monogenic disorders, genetic heterogeneity has been observed. While environmental influences may provide a minor contribution, variation in phenotypic outcomes is often attributable to allelic heterogeneity or modifier genes. Modifier genes are allelic variants distinct from the mutant gene that can alter neurological disease or disorder outcomes by increasing or decreasing disease severity and affecting disease onset and progression. Identification of modifier genes has significant implications for prediction of disease progression and development of new treatment strategies.

[0180] The data provided herein illustrate the use of modifier genes for treating various neurological disorders or diseases. In some embodiments, neuronal or brain tissue integrity and function are rescued by gene therapy methods through delivery of a modifier gene rather than replacement of the disease-causing gene. The methods described herein identify gene modifiers that inhibit neurological disorders or diseases caused by several different genes converging on specific nodes or pathways within a signaling network. Since genes act in networks rather than individually, the impact of any gene delivery is on the network as a whole rather than just a single gene. These studies suggest that viable treatment options with broad impact arise from genetically modified genes capable of modulating disease states by affecting entire gene networks that regulate specific biological processes rather than individual genes.

[0181] Administration of a gene modifier ameliorates or treats clinical, morphological, and / or functional deficits associated with a primary gene mutation. In a specific embodiment of the present disclosure, retinoic acid-related orphan receptor alpha (RORα), also known as NR1F1 (nuclear receptor subfamily 1, group F, member 1) is used in the modifier gene therapy of the present disclosure. RORα plays important roles in lipid metabolism, oxidative stress response, and regulation of anti-inflammatory responses, etc.

[0182] RORα belongs to the NR1 subfamily of nuclear hormone receptors and binds to DNA as a monomer or as a homodimer at ROR response elements (ROREs), which contain the core motif 5'-AGGTCA-3' preceded by a short A-T rich sequence, upstream of several genes to enhance their expression. RORα is one of the key regulators of embryonic development, cell differentiation, immunity, circadian rhythm, and lipid, steroid, xenobiotic, and glucose metabolism. Although RORα has intrinsic transcriptional activity, some natural ligands such as oxysterols that act as agonists (25-hydroxy cholesterol) or inverse agonists (7-oxysterols) enhance or inhibit RORα transcriptional activity, respectively. RORα regulates the transcription of several genes by recruiting different combinations of cofactors to the regulatory regions, depending on the tissue, time, and promoter context. Some of the genes regulated by RORα include: (i) the circadian expression of several CLOCK genes, including CLOCK, ARNTL / BMAL1, NPAS2, and CRY1; (ii) cerebellar development, such as the sonic hedgehog (SHH) gene and other genes involved in calcium-mediated signal transduction; (iii) photoreceptor development, such as OPN1SW, OPN1SM, and ARR3; (iv) skeletal muscle development with MYOD1; (v) lipid metabolism, such as apolipoproteins APOA1, APOA5, APOC3, and PPARγ, and the genes CYP7B1 and SULT2A1 encoding phase I and II proteins involved in lipid, steroid, and xenobiotic metabolism in the liver; (vi) hepatic glucose metabolism through CRY1 regulating G6 (PC1 and PCK1); (vii) adipocyte differentiation, such as CEBPB and PPARγ; (viii) the lineage specification of naive CD4 T helper cells into Th17 cells; (ix) hypoxia signaling such as HIF-1; and (x) anti-inflammatory effects, such as i-κB, which inhibits pro-inflammatory NF-κB signaling. See Figure 1 。

[0183] In some embodiments, the compositions of the present disclosure are administered locally to nerve cells or neurons or brain tissue. In another embodiment, the compositions are administered intranasally.

[0184] The composition is administered at a concentration of 0.001 μg to 100 μg, such as 0.01 μg, 0.1 μg, 0.5 μg, 1.0 μg, 1.5 μg, 2.0 μg, 5.0 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg or 100 μg. The composition is administered in a volume of 0.01 μL to 10 μL, such as 0.1 μL, 0.25 μL, 0.5 μL, 1 μL, 1.5 μL, 2 μL, 2.5 μL, 3 μL, 3.5 μL, 4 μL, 4.5 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL or 10 μL. The composition is administered once a day, once a week, once a month, every 3 months, every 6 months or every 12 months. The composition is administered for a duration of 1 day, 1 week, 1 month, 3 months, 6 months, 1 year, 2 years or 5 years. Alternatively, the composition is administered only once.

[0185] The composition comprising nucleic acid can also be administered by electroporation. Alternatively, the composition can be administered by biodegradable nile red poly(lactide-co-glycolide) (PLGA) nanoparticle-based gene delivery, small molecule-based gene delivery, naked DNA delivery, virus-based gene delivery (such as adeno-associated virus delivery) or genome editing systems (such as CRISPR). Figure 2 A specific embodiment of the RORA transgenic expression cassette that can be used to ameliorate or treat a neurological disorder or disease in a subject is shown. In a particular embodiment, the expression cassette is provided in an rAAV vector. In a further embodiment, the rAAV vector is an AAV serotype 5-based (AAV5) vector, AAV2 vector, AAV8 vector or AAV9 vector. In a particular embodiment, the rAAV vector is an AAV5 vector. In another embodiment, the rAAV vector is an AAV8 vector.

[0186] Other features and advantages of the invention will become apparent from the following description of its preferred embodiments and the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All foreign patents and patent applications cited herein are incorporated herein by reference. The GenBank and NCBI submissions indicated by the accession numbers cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods and examples are illustrative only and not limiting.

[0187] Examples

[0188] The present disclosure relates to the use of modified genes known as nuclear hormone receptor (NHR) genes to treat various clinical conditions or diseases. Nuclear receptors are a class of proteins responsible for various activities, including but not limited to sensing steroids, thyroid hormones, cholesterol, and vitamins. It has been shown that these receptors act together with other proteins to regulate the expression of multiple genes, thereby controlling the development, homeostasis, and metabolism of an organism. Thus, these modified genes can be used to correct defects or genetic disorders manifested as various clinical conditions and / or diseases. It is believed that nuclear receptors bind directly to DNA, thereby regulating the expression of adjacent genes. Thus, these receptors are classified as transcription factors. One of the key properties of nuclear receptors that distinguishes them from other classes of receptors is their direct control of genomic DNA. A variety of NHRs are known to those skilled in the art, including but not limited to RORA (i.e., RORα), NR1D1, and LXRa.

[0189] Some aspects of the present disclosure provide methods for treating and / or preventing various clinical conditions and disorders using compositions that modify or restore the signal transduction pathways and / or functions of various genes. For example, the present disclosure provides methods for treating clinical diseases or conditions associated with neurological disorders. Exemplary neurological disorders that can be treated using the methods of the present disclosure include but are not limited to intellectual developmental disorder, epilepsy, cerebellar ataxia, autism spectrum disorder (ASD), Parkinson's disease, Alzheimer's disease, neurodegeneration of unknown etiology, or combinations thereof.

[0190] Administration of the gene modifier improves clinical, morphological, and functional defects associated with primary gene mutations. In a specific embodiment of the present disclosure, retinoic acid-related orphan receptor α (RORα), also known as nuclear receptor subfamily 1 group F1 (NR1F1), is used in the modified gene therapy of the present disclosure. RORα plays an important role in lipid metabolism, oxidative stress response, and regulates anti-inflammatory responses, etc.

[0191] RORα belongs to the NR1 subfamily of nuclear hormone receptors and binds to DNA as a monomer or as a homodimer at ROR response elements (ROREs), which contain the core motif 5'-AGGTCA-3' preceded by a short A-T rich sequence, upstream of several genes to enhance their expression. RORα is one of the key regulators of embryonic development, cell differentiation, immunity, circadian rhythm, and lipid, steroid, xenobiotic, and glucose metabolism. Although RORα has intrinsic transcriptional activity, some natural ligands such as oxysterols that act as agonists (25-hydroxy cholesterol) or inverse agonists (7-oxysterols) enhance or inhibit RORα transcriptional activity, respectively. RORα regulates the transcription of several genes by recruiting different combinations of cofactors to the regulatory regions, depending on the tissue, time, and promoter context. Some of the genes regulated by RORα include: (i) the circadian expression of several CLOCK genes, including CLOCK, ARNTL / BMAL1, NPAS2, and CRY1; (ii) cerebellar development, such as the sonic hedgehog (SHH) gene and other genes involved in calcium-mediated signal transduction; (iii) photoreceptor development, such as OPN1SW, OPN1SM, and ARR3; (iv) skeletal muscle development with MYOD1; (v) lipid metabolism, such as apolipoproteins APOA1, APOA5, APOC3, and PPARγ, and genes CYP7B1 and SULT2A1 encoding phase I and II proteins involved in lipid, steroid, and xenobiotic metabolism in the liver; (vi) hepatic glucose metabolism through the regulation of G6PC1 and PCK1 by CRY1; (vii) adipocyte differentiation, such as CEBPB and PPARγ; (viii) the lineage specialization of naive CD4 T helper cells into Th17 cells; (ix) hypoxia signaling, such as HIF-1; and (x) anti-inflammatory effects, such as i-κB, which inhibits pro-inflammatory NF-κB signaling. See Figure 1 。

[0192] RORα stagerer (RORα sg / RORα sg ) mouse model : The first homozygous staggerer mouse was observed in 1955 and was identified by its staggering gait, mild tremors, hypotonus, and hypoplastic cerebellar cortex with reduced granule and Purkinje cells. Twenty-five years later, the mutation was genetically mapped to a 160-kilobase interval on mouse chromosome 9 that contains the RORα gene. The mutation removes the exon-encoding portion of the RORα ligand-binding domain, resulting in a truncated protein. Since then, it has been shown in RORα sg / RORα sg Phenotypes involving multiple physiological systems were observed in mice. Neurological phenotypes included abnormal gait, posture, coordination, and motor learning, accompanied by ataxia, hypoactivity, tremors, and associated defects in the morphology of the cerebellum, cerebrum, and olfactory bulb. Staggerer mice exhibited abnormal metabolic phenotypes such as elevated levels of thyroid-stimulating hormone and norepinephrine, depletion of neuronal aspartate, taurine, and gamma-aminobutyric acid (GABA) levels, and reduced circulating cholesterol leading to abnormal lipid homeostasis.

[0193] Intellectual disability with or without epilepsy or cerebellar ataxia (IDDECA) : The RORα gene in humans is located on the long (q) arm of chromosome 15, and microdeletions at 15q22.2 have been reported to overlap RORα in individuals with IDDECA. A multi-center study identified three copy number variant deletions, one disruptive duplication, and nine de novo point mutations (three truncating, one classic splice site, and five missense mutations) involving the RORα gene in 16 individuals (13 families) with variable neurodevelopmental delay and IDDECA. See Table 1.

[0194] Table 1. Genotypes and clinical outcomes of RORA mutations in human participants. See Guissart et al., Am J Hum Genet, 2018, 102, pp. 744 - 759.

[0195]

[0196] Guissart et al. used a zebrafish model to reproduce the neuroanatomical findings found in 16 affected humans and RORα sg mice. While most mutations showed loss of function or haploinsufficiency, two missense mutations at the DNA-binding domain showed dominant toxic effects. IDDECA presents (A) cognitive and motor phenotypes characterized by moderate to severe intellectual disability (ID) with ataxia, severe cerebellar vermis hypoplasia, and generalized epilepsy, or (B) cognitive and behavioral phenotypes with autism spectrum disorder (ASD), mild ID, normal cognition, and often associated with epilepsy. Rescue of cerebellar pathology caused by RORα truncated splice variants (TSVs) in the zebrafish model using wild-type human RORα mRNAId.

[0197] Mutations in individuals 6, 7, 8, and 13 (showing ASD) disrupt the RORα ligand-binding domain, consistent with reports that RORα is a candidate for ASD. Analysis indicates that the RORα protein is recruited to the promoter regions of approximately 2,544 genes across the human genome, particularly genes controlling neuronal differentiation, adhesion, survival, synaptogenesis, synaptic transmission, plasticity, axonogenesis, cortical and cerebellar development, cognition, memory, and spatial learning. Independent ChIP-qPCR analysis confirmed the binding of the RORα protein to the promoter regions of selected ASD-related genes: A2BP1, CYP19A1, ITPR1, NLGN1, and NTRK2. It has been shown that the expression levels of these ASD genes are reduced in human neuronal cells and prefrontal cortex tissues repressed for RORα in individuals with ASD. Additionally, two RORα polymorphisms (rs11639084 and rs4774388) have been shown to be associated with ASD risk. Treatment with the synthetic RORα agonist SR1078 reduced repetitive behaviors in an autism BTBR mouse model, indicating that RORa upregulation by the compositions disclosed herein is a viable gene therapy approach for intellectual developmental disorders with or without epilepsy or cerebellar ataxia (IDDECA) and ASD.

[0198] The use of the RORα gene therapy as disclosed herein is applicable for treating individuals showing ASD or individuals at risk of developing ASD.

[0199] Autism spectrum disorder : Wild-type human RORA can rescue cerebellar defects caused by RORA-TSV in zebrafish, while the R462Q mutant cannot. Some studies have linked nuclear receptor defects to autism in humans. RORα polymorphisms (rs11639084 and rs4774388) are associated with ASD risk. Global methylation profiling reveals that RORα protein levels are significantly reduced in the brains of individuals with ASD due to epigenetic alterations at the RORa gene. Multiple genes associated with ASD are direct RORα targets, and reduced RORa expression leads to reduced expression of these genes that cause ASD. The lack of Purkinje cells has been identified as a neuroanatomical abnormality in the brains of individuals with ASD. RORα has been shown to be crucial in the development of Purkinje cells. Thus, increased RORα expression is a treatment for ASD. Individuals with ASD show significant disruption of their circadian rhythm cycle, and RORa plays a role in the regulation of the circadian rhythm. Environmental and metabolic factors of ASD have also been reported to affect RORa expression levels. Gender differences in the expression of RORα and its target genes in the brain have been investigated as potential contributors to gender bias in autism. Additionally, relative to WT mice, RORα sg mice show behaviors associated with autism, including abnormal spatial learning, reduced exploration, limited maze patrol, and increased perseverative behavior.

[0200] Reduction of RORα protein in autistic brains: Twin studies in which one sibling has autism and the other does not have autism have revealed increased CpG island methylation at the upstream RORα promoter locus in twins with ASD. As a result, lymphoblastoid cell lines (LCLs) from twins with ASD show reduced expression of the RORα protein. Figures 4 and 5. Autopsies of age-matched case-control individuals also show reduced expression of the RORα protein in the prefrontal cortex and cerebellum of autistic individuals. These findings are important because studies on sg RORα mice have shown that the RORα protein is involved in several processes associated with autism, including Purkinje cell differentiation, cerebellar development, brain lipid homeostasis, protection against oxidative stress and inflammation, and the circadian rhythm.

[0201] RORα in Purkinje cells and cerebellar development : Purkinje cells express RORα very early in development, and this continues into adulthood. In sg RORα mice, most Purkinje cells die within the first month of life. The surviving Purkinje cells fail to mature and develop into spiny branches. RORα is required for transient dendritic retraction during early Purkinje cell development (necessary to establish a mature dendritic tree). RORα deficiency in adult mice also produces defects in Purkinje cells, such as premature dendritic atrophy and death, higher levels of FoxP2, an immature "capuchon" stage of climbing fibers from brainstem olivary neurons, and multiple innervation of Purkinje cells by climbing fibers as opposed to more mature single innervation. Thus, RORα is a terminal differentiation gene that defines the functional properties of mature Purkinje cells from development to maintenance throughout their life. Analysis of the genetic program in developing Purkinje cells daily during prenatal development in mice reveals that RORα binds to promoter sites and controls the expression of Shh, Slc1a6, Itpr1, Pcp4, and Pcp2. These RORα target genes provide a mitogenic drive and are also necessary for the reciprocal signaling between Purkinje, granule, and molecular cells during cerebellar development. RORα sg studies in mice have suggested a possible role of RORα in cell proliferation, neuronal differentiation, and the expression of mature neuronal markers (Ki67, DCX, and NeuN, respectively) in the dentate gyrus. The dentate gyrus is the first region where all sensory modalities converge to form a unique representation that binds different sensory stimuli together, thus playing a role in learning and memory. Liver X receptor (LXR), a nuclear receptor closely related to RORα, has been associated with abnormal dentate gyrus development and autism spectrum disorder. RORα sgExogenous RORα expression in mice (Figure 5) partially restored normal Purkinje cell counts and neuronal architecture in the cerebellum, indicating that the compositions disclosed herein can be used to treat ASD.

[0202] RORα in brain lipid metabolism : Polyunsaturated fatty acids (PUFAs), such as ω-6 and ω-3 fatty acids, have been shown to play a role in early brain development. The major PUFA species, arachidonic acid (n-6) and docosahexaenoic acid (DHA; n-3), are essential for neuronal growth, synaptogenesis, neuronal survival, and neurotransmitter regulation. Although abnormal neural lipid metabolism in individuals with ASD has not been extensively studied, abnormal lipid metabolism has been reported as one of the plasma biomarkers, and PUFA intervention in animal models can mitigate autistic-like cognitive and social behaviors. RORα regulates lipoprotein homeostasis and RORα sg mice exhibit abnormal lipid metabolism (reduced serum cholesterol and triglycerides) due to decreased expression of ApoA1 and ApoC3, respectively. RORα can regulate adipogenesis and mitochondrial fatty acid oxidation by inhibiting the expression of peroxisome proliferator-activated receptor-γ (PPARγ), its coactivator PGC1, and lipin1. Recent reports have shown that RORα deficiency delays the accumulation of all fatty acids during the critical period of brain development. However, the deficiency of the ω-3 PUFA species - DHA persists in adult RORα sg mice and cannot be rescued by dietary DHA supplementation. Similarly, a meta-analysis of case-control cohorts found that although reported dietary intakes did not differ from those of the control group, blood DHA levels were selectively reduced in children with ASD (≤12 years old). Although DHA supplementation reversed some impairments in ASD mouse models (BTBR and serotonin transporter knockout), human trials reporting increased blood DHA after dietary supplementation failed to observe improvement in social behavior in children with ASD. This provides additional evidence that RORα deficiency may affect the efficacy of dietary DHA supplementation by slowing DHA incorporation into brain phospholipids and / or accelerating DHA loss from brain phospholipids. Thus, in humans, upregulating RORa by gene therapy is a viable option for individuals with ASD (Figure 5) in combination with supplements and other medical options for symptom management.

[0203] RORα protects neurons against oxidative stress : Oxidative stress is a common feature in autism cases and may be further exacerbated by the presence of genetically susceptible alleles. Limited antioxidant capacity, high energy demand, and high levels of iron and PUFAs in the brain increase its vulnerability to oxidative stress. Postmortem studies of brain tissue from individuals with ASD showed elevated levels of oxidative damage and reduced antioxidant capacity compared to age-matched control subjects.

[0204] Individuals with ASD show higher levels of lipid hydroperoxides (from fatty acid oxidation); malondialdehyde (from lipid peroxidation); 8-hydroxy-2'-deoxyguanosine (from oxidative DNA damage); protein carbonyls (from protein oxidation); 3-nitrotyrosine (from protein nitration); and carboxyethylpyrrole (lipid-derived oxidized protein modification). Overexpression of human RORα1 in cultured mouse cortical neurons increases the expression of the antioxidant proteins glutathione peroxidase 1 (Gpx1) and peroxiredoxin 6 (Prx6), reduces the level of reactive oxygen species (ROS) (Figure 5), and protects neurons from apoptosis induced by oxidative stressors such as β-amyloid peptide, c2-ceramide, and H 2 O 2 . Another study reported that maternal diabetes in mice induces oxidative stress in the brains of their offspring and leads to autistic-like behavior (ALB). Oxidation in the mouse offspring (both stress and ALB) is accompanied by downregulation of RORα and its target genes CYP19A1 (aromatase) and Sod2 (superoxide dismutase). Postnatal overexpression of RORα in the offspring rescued ALB and neuronal oxidative stress, while sh-RNA knockdown had the opposite effect and worsened ALB.

[0205] RORα protects neurons against neuroinflammation : A prominent feature common to individuals with ASD is persistent neuroinflammation across a wide age range, with elevated levels of cytokines and chemokines (such as IL-6, TGF1, TNF, CCL2, and CCL17) in the cerebellum and other regions of the brain. The transcriptome tissue pattern in the brains of individuals with ASD shows abnormalities in gene co-expression networks associated with immune activation. Disrupted monocyte / macrophage function has been reported in ASD under resting conditions, with reduced production of the regulatory (anti-inflammatory) cytokines TGFβ1 and IL-10, and elevated antibody levels against cerebellar proteins, all of which are associated with a worsening behavioral phenotype. Astrocytes are multifunctional macroglial cells that provide structural and metabolic support to neurons, absorb neurotransmitters, regulate ion concentration and synaptic transmission, maintain the blood-brain barrier, act as chemical sensors, promote myelination, axon regeneration, and drive molecular oscillations in the circadian clock. The role of astrocytes in neuroinflammation has been documented. As effectors of innate immunity in the brain, astrocytes are believed to be mainly activated by the NF-κB signaling pathway and produce high levels of IL-6 through a RORα-dependent mechanism. Although it has been reported that astrocytes from RORα sg mice have lower resting IL-6 levels than WT mice, after stimulation with the pro-inflammatory cytokines IL-1 and TNF, the IL-6 levels in RORαsg astrocytes are significantly higher, indicating a pro-neuroinflammatory drive in the absence of RORα (Figure 5).

[0206] RORα protects nerves in in vitro models of neuroinflammation in Parkinson's disease and Alzheimer's disease Yuan: To determine whether RORa plays a role in neuronal death, Boukhtuche et al. (2006) overexpressed the human RORa1 (hRORa1) isoform in neurons by transient transfection of a plasmid encoding hRORa1 (pSG5-hRORa1) or by infecting cultures with a lentivirus-derived vector encoding hRORa1 (LentihRORa1). The hRORa1-overexpressing neurons were then exposed to three different apoptotic stimuli, β-amyloid (Alzheimer's disease model), c2-ceramide (Parkinson's disease / Alzheimer's disease model), and H 2 O 2 (oxidative stress model), and their survival rates were evaluated. Overexpression of hRORa1 not only protected cortical neurons from apoptosis but also significantly improved the survival rate after exposure to apoptotic stimuli. A recent prospective study showed an association between sleep duration and Parkinson's disease in carriers of the RORA genotype rs2028122 (Shao et al., 2022). Another study (Li et al., 2022) showed that RORA was downregulated in a Parkinson's disease model and that melatonin improved the disease by upregulating RORA expression. Another study on nucleoside diphosphate kinase A (a neuroprotective agent in Parkinson's disease) revealed its mechanism of action through RORA (Anantha et al., 2021). Network analysis studies showed that a significant cross-section of genes differentially expressed in the hippocampus of an Alzheimer's disease mouse model was associated with RORA (Acquaah-Mensah et al., 2015 / Darshini et al., 2019). Another study determined that RORA regulates microglia that mediate the development of Alzheimer's disease (Jian et al., 2021).

[0207] hRORA transgenic : Generate a transgenic hRORA under the transcriptional control of the cytomegalovirus (CMV) enhancer and containing the chicken β-actin promoter (CBA) promoter, Kozak sequence at the transcription start site, and SV40 polyadenylation sequence. The generated hRORA transgene contains the sequences (DNA sequence lengths and element names) of the following different gene elements: 1-130 (130bp) left AAV2 ITR; 155-534 (380bp) CMV enhancer (CAG); 536-813 (278bp) chicken β-actin promoter (CAG); 814-1830 (1017bp) chimeric intron (CAG); 1908-1917 (10bp) Kozak sequence; 1914-3485 (1572bp) hRORA; 3646-3767 (122bp) SV40 polyA signal sequence; and 3812-3952 (141bp) right AAV2 ITR.

[0208] The transgenic hRORA is administered to nerve cells, tissues, and organs to determine the therapeutic efficacy for various neurological disorders and diseases.

[0209] The foregoing discussion of the invention has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to one or more forms disclosed herein. Although the description of the invention has included a description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g., within the skill and knowledge of those skilled in the art after understanding the disclosure. It is intended to obtain rights to include alternative embodiments to the extent permitted, including alternative, interchangeable, and / or equivalent structures, functions, ranges, or steps, whether or not such alternative, interchangeable, and / or equivalent structures, functions, ranges, or steps are disclosed herein, and not to publicly dedicate any patentable subject matter. All references cited herein are incorporated by reference in their entirety.

Claims

1. A method for ameliorating or treating a neurological disorder or disease in a subject in need of treatment, the method comprising administering to the subject in need of such treatment an effective therapeutically amount of a therapeutic agent, the therapeutic agent comprising: (a) recombinant DNA (rDNA), recombinant RNA, or a combination thereof, of a human nuclear hormone receptor (hNHR) gene or a fragment thereof; and (b) a delivery vehicle adapted to deliver the hNHR gene or a fragment thereof to a nerve cell, tissue, organ, or combination thereof to ameliorate or treat the neurological disorder or disease.

2. The method according to claim 1, wherein the delivery vehicle comprises a viral vector.

3. The method according to claim 2, wherein the viral vector comprises a viral vector associated with adeno-associated virus (AAV), adenovirus, and lentivirus.

4. The method according to claim 1, wherein the rDNA further comprises (i) a promoter, (ii) an enhancer, (iii) a polyadenylation moiety, or (iv) a combination thereof.

5. The method according to claim 4, wherein the polyadenylation moiety comprises a simian virus 40 (SV40) polyadenylation (PolyA) region, a bovine growth hormone (bGH) PolyA region, or a combination thereof.

6. The method according to claim 1, wherein the rDNA further comprises a cytomegalovirus (CMV) promoter or enhancer, elongation factor 1a (EF1a), chicken β-actin (CBA) promoter, CAG promoter, or a combination thereof.

7. The method according to claim 1, wherein the delivery vehicle is adapted to target brain cells.

8. The method according to claim 7, wherein the delivery vehicle comprises: (i) a peptide ligand; (ii) different amino acids as transporter ligands: or (iii) a receptor ligand, an arginine-glycine-aspartic acid (RGD) peptide, or an asparagine-glycine-arginine (NGR) peptide.

9. The method according to claim 1, wherein the delivery vehicle further comprises a cell- or tissue-specific promoter, and wherein the cell- or tissue-specific promoter comprises: human Syn1, MeCP2, NSE, or BM88 promoter; CaMKII; DLX5 / 6 enhancer; tyrosine hydroxylase; dopamine β-hydroxylase (DBH) or PRSx8 (synthetic DBH); PCP2 (Purkinje cell protein 2); FEV, ETS transcription factor (Ple67); MCH (melanin-concentrating hormone); SLC6A4 (serotonin transporter Ple198) or NR2E1 (ple264); GfABC1D (truncated GFAP) or Aldh1A1; MBP (myelin basic protein) or MAG (myelin-associated glycoprotein); ICAM-2 (intracellular adhesion molecule 2), CLDN5 (claudin 5), Tie-2 (TEK, receptor tyrosine kinase), vWF (von Willebrand factor), or FLT1 (vascular endothelial growth factor receptor); or a combination thereof.

10. The method according to claim 1, wherein the delivery vehicle comprises a non-viral delivery agent, and the non-viral delivery agent comprises nanoparticles, nanobodies, liposomes, biodegradable polymer complexes, or combinations thereof.

11. The method according to claim 10, wherein the nanoparticles comprise liposomes, lipid nanoparticles, polymer nanoparticles, dendrimers, cyclodextrins, silica nanoparticles, polymer complexes, magnetic nanoparticles, gold nanoparticles, quantum dots, carbon nanotubes, or combinations thereof.

12. The method according to claim 1, wherein the hNHR gene or a fragment thereof is expressed by a plasmid, RNA, or dbDNA (doggybone).

13. The method according to claim 1, wherein the hNHR gene is selected from NR1D1, RORA, and LXRa.

14. The method according to claim 13, wherein the hNHR gene comprises RORA.

15. The method according to claim 1, wherein the neurological disorder or disease comprises intellectual disability, epilepsy, cerebellar ataxia, autism spectrum disorder (ASD), Parkinson's disease, Alzheimer's disease, neurodegeneration of unknown etiology, or combinations thereof.

16. The method according to claim 15, wherein the neurological disorder or disease comprises autism spectrum disorder.

17. The method according to claim 15, wherein the neurological disorder or disease comprises intellectual disability.

18. The method according to claim 15, wherein the neurological disorder or disease comprises epilepsy.

19. The method according to claim 15, wherein the neurological disorder or disease comprises cerebellar ataxia.

20. The method according to claim 15, wherein the neurological disorder or disease comprises Parkinson's disease.

21. The method according to claim 15, wherein the neurological disorder or disease comprises Alzheimer's disease.

22. The method according to claim 15, wherein the neurological disorder or disease comprises neurodegeneration of unknown etiology.

23. A method for ameliorating or treating a neurological disorder or disease in a subject in need thereof, the method comprising administering to the subject an effective therapeutically amount of a composition comprising a human nuclear hormone receptor (hNHR) gene or a fragment thereof and an hNHR delivery vehicle, wherein the hNHR gene or a fragment thereof is selected from NR1D1, RORA, and LXRa.

24. The method according to claim 23, wherein the delivery vehicle comprises: (i) a viral delivery vehicle associated with an adeno-associated virus (rAAV), lentivirus, adenovirus, or HSV1 viral vector; or (ii) a non-viral delivery vehicle comprising nanoparticles, nanobodies, liposomes, biodegradable polymer complexes, or combinations thereof.

25. The method according to claim 23, wherein the composition further comprises a pharmaceutically acceptable carrier.

26. The method according to claim 23, wherein the composition is administered to the subject more than once.

27. The method according to claim 23, wherein about 10 8 to about 10 14 particles of the hNHR delivery vehicle are administered to the subject.

28. A recombinant adeno-associated virus (AAV) gene therapy particle comprising an AAV capsid protein and a nucleic acid sequence encoding an hRORA protein or a bioactive portion thereof operably linked to a promoter, and first and second AAV inverted terminal repeat (ITR) sequences flanking the sequence encoding the hRORA protein or portion thereof.

29. The recombinant AAV gene therapy particle according to claim 28, wherein the nucleic acid sequence further comprises a cytomegalovirus enhancer; a chicken β-actin promoter; a chimeric intron; and a simian virus 40 polyadenylation region.

30. The recombinant AAV gene therapy particle according to claim 28, wherein the AAV capsid protein comprises a capsid protein from an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVRh10, AAV11, and variants thereof.

31. The recombinant AAV vector according to claim 28, wherein the nucleic acid sequence comprises a sequence having at least 95% identity to the nucleic acid sequence of SEQ ID NO:

4.

32. A method for ameliorating or treating a neurological disorder or disease in a subject in need thereof, the method comprising administering to a subject in need of such treatment an effective therapeutically amount of a composition comprising: Recombinant DNA (rDNA) for ameliorating or treating a neurological disorder or disease, wherein the rDNA comprises a nucleic acid sequence having at least 95% identity to the nucleic acid sequence of SEQ ID NO: 4; and A delivery vehicle adapted to deliver the recombinant DNA to a nerve cell, tissue, organ, or combination thereof of the subject.

33. The method according to claim 32, wherein the delivery vehicle comprises a viral vector.

34. The method according to claim 33, wherein the viral vector comprises an adeno-associated virus (AAV) capsid protein.

35. The method according to claim 34, wherein the AAV capsid protein comprises a capsid protein from an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVRh10, AAV11, and variants thereof.

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