Gene therapy for tuberous sclerosis

Abstract

The present disclosure provides gene therapy compositions and methods for treating tuberous sclerosis. In particular, the present disclosure provides compositions comprising recombinant adeno-associated viruses (rAAVs) that contain AAV capsid proteins and an AAV expression cassette encoding truncated tuberin (cTuberin), as well as methods of using the same.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 210,456, filed June 14, 2021, the entire contents of which are incorporated herein by reference.

[0002] Description of electronically submitted text files The contents of the text file submitted electronically herein: a computer-readable copy of the sequence listing (file name: BGTR_003_01WO_SeqList_ST25.txt, creation date: June 14, 2022, file size approximately 170,338 bytes) is incorporated herein by reference in its entirety. [Background technology]

[0003] Tuberous sclerosis complex (TSC), also called tuberous sclerosis, is a multisystem autosomal dominant genetic disease that can cause noncancerous tumors to grow in the brain and other vital organs, such as kidney, heart, liver, eye, lung, and skin. Such tumors in the brain can cause developmental delay, autism, epilepsy, and hydrocephalus. Life-threatening conditions associated with tuberous sclerosis include renal angiomyolipoma, which can cause internal bleeding, and lymphangioleiomyomatosis (LAM), which can impair breathing.

[0004] Tuberous sclerosis is caused by one or more mutations in the TSC1 and / or TSC2 genes. Tuberous sclerosis resulting from mutations in TSC2 is more severe and more common. TSC1 and TSC2 are tumor growth suppressor genes that code for the proteins hamartin and tuberin, respectively. Tuberin and hamartin form a protein complex that integrates multiple signals to regulate mammalian target of rapamycin (mTOR) signaling, primarily by inhibiting the mTORC1 complex. In addition, tuberin also contains a GTPase activation domain (GAP) domain that downregulates Rheb, an mTORC1 activator.

[0005] Current treatment of tuberous sclerosis involves the administration of rapamycin and its analogues. However, these drugs must be administered continuously and have significant side effects, including impaired brain development and immunosuppression. Administration of rapamycin and its analogues can also result in adverse events due to over-suppression of mTORC1 activity. In addition, some patients do not respond to rapamycin or respond initially and then develop resistance.

[0006] Thus, there is an unmet need for compositions and methods, particularly gene therapy-based compositions and methods, that can be used to treat tuberous sclerosis. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure provides compositions and methods for use in the treatment of tuberous sclerosis complex. In some embodiments, the present disclosure provides a truncated tuberin protein (ctuberin) comprising (i) an N-terminal region capable of binding to hamartin, and (ii) a C-terminal GTPase activating protein (GAP) region, wherein the ctuberin lacks certain amino acid residues of human tuberin (SEQ ID NO:1). In some embodiments, the ctuberin of the present disclosure lacks amino acid residues 451-932 of human tuberin (SEQ ID NO:1). In some embodiments, the ctuberin of the present disclosure lacks amino acid residues 419-932 of human tuberin (SEQ ID NO:1). In some embodiments, the ctuberin of the disclosure comprises (i) an N-terminal region capable of binding hamartin and (ii) a C-terminal GTPase activating protein (GAP) region, wherein the C-terminal region comprises an amino acid sequence having at least 90% identity to SEQ ID NO:7 and lacks amino acid residues 451-932 of SEQ ID NO:1. In some embodiments, the ctuberin of the disclosure comprises (i) an N-terminal region capable of binding hamartin and (ii) a C-terminal GTPase activating protein (GAP) region, wherein the C-terminal region comprises an amino acid sequence having at least 90% identity to one of SEQ ID NOs:10-12, and wherein the ctuberin lacks amino acid residues 451-932 of SEQ ID NO:1.

[0008] In addition to cTuberin proteins, the present disclosure provides nucleic acid molecules encoding any one of the cTuberin proteins disclosed herein, as well as compositions configured to cause expression of any one of the cTuberin proteins disclosed herein in a given cell. For example, the present disclosure provides an adeno-associated virus (AAV) expression cassette comprising, from 5' to 3', a 5'AAV inverted terminal repeat (ITR), any one of the nucleic acid molecules disclosed herein, and a 3'AAV ITR. The present disclosure also provides a recombinant AAV (rAAV) comprising an AAV capsid protein and any one of the nucleic acid molecules or AAV expression cassettes disclosed herein. Furthermore, the present disclosure provides compositions, including pharmaceutical compositions comprising any one of the cTuberin proteins, any one of the nucleic acid molecules, any one of the plasmids, any one of the host cells, or any one of the rAAVs disclosed herein.

[0009] In another aspect, the disclosure provides a method of expressing any one of the cTuberins disclosed herein in a target cell, comprising contacting the target cell with any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the rAAVs disclosed herein, any one of the extracellular vesicles (EVs) disclosed herein, or any one of the compositions disclosed herein, thereby expressing cTuberin in the target cell.

[0010] In a further aspect, the disclosure provides a method of treating tuberous sclerosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the rAAVs disclosed herein, any one of the EVs disclosed herein, or any one of the compositions disclosed herein, thereby treating tuberous sclerosis in the subject. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The use of AAV-based gene therapy to treat tuberous sclerosis, or TSC, caused by mutations in TSC2, has been complicated by the relatively small insertion capacity of AAV vectors (about 4.7 kb) compared to the 5.4 kb cDNA of human tuberin. The present disclosure provides a truncated form of the human tuberin gene (TSC2) that is small enough to be incorporated into an AAV vector and encodes a truncated tuberin (cTuberin). In particular, the present disclosure provides compositions comprising recombinant adeno-associated viruses (rAAVs) that include AAV capsid proteins and an AAV expression cassette encoding cTuberin, as well as methods of using them, including treating tuberous sclerosis.

[0012] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0013] 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 application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this application, representative methods and materials are described herein.

[0014] The terms "a," "an," and "the" as used in this application, including the claims, refer to "one or more." Thus, for example, reference to a "carrier" includes one or more carriers, mixtures of two or more carriers, etc., reference to a "process" includes references to equivalent steps and / or processes known to those skilled in the art, and so forth.

[0015] As used herein, any concentration range, percentage range, ratio range, or integer range, unless otherwise indicated, is to be understood to include any integer value within the stated range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers). The term "about," when immediately preceding a number or numeral, means that the number or numeral ranges from plus or minus 0% to 10%.

[0016] Also, as used herein, "and / or" refers to and covers any and all possible combinations of one or more of the associated listed items, as well as the absence of a combination when construed as an alternative ("or"). The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0017] 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 and the like.

[0018] The term "pharmaceutical acceptable" is used, unless otherwise stated, to characterize a moiety (e.g., a salt, dosage form, or excipient) as being appropriate for use according to sound medical judgment. In general, a pharmaceutical acceptable moiety has one or more benefits that outweigh any adverse effects that the moiety may have. Adverse effects can include, for example, excessive toxicity, irritation, allergic response, and other problems and complications.

[0019] As used herein, "treatment" or "treating", "palliating" and "ameliorating" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to any therapeutically related improvement or effect on one or more diseases, conditions, or symptoms under treatment. The term "treating" in one embodiment includes (1) preventing or delaying the appearance of clinical symptoms of a condition, disorder, or condition developing in a patient who may be affected by or is predisposed to being affected by the condition, disorder, or condition, but who has not yet experienced or exhibited clinical or subclinical symptoms of the condition, disorder, or condition, (2) inhibiting the condition, disorder, or condition (e.g., arresting, reducing, or delaying the onset of disease, or in the case of maintenance treatment, the recurrence of at least one clinical or subclinical symptom thereof), and (3) alleviating the condition (e.g., by causing regression or reducing the severity of the condition, disorder, or condition, or at least one of its clinical or subclinical symptoms). For example, beneficial clinical outcomes include, but are not limited to, delaying or slowing the invasiveness or growth of a tumor or hamartoma, and alleviating symptoms associated with such tumor or hamartoma. For example, in the case of renal angiomyolipoma, tumor size can be monitored by magnetic resonance imaging (MRI), and the shrinkage of cell size resulting from administration of any one of the compositions disclosed herein can be analyzed according to standard procedures, such as those used to monitor the treatment of tuberous sclerosis using rapamycin. Treatment also includes a reduction in mortality or an increase in lifespan of a subject compared to a subject not receiving treatment.

[0020] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to achieve an outcome, for example, to bring about a beneficial or desired result, such as treating tuberous sclerosis or a symptom thereof. The therapeutically effective amount may vary depending on one or more of the subject and disease state being treated, the subject's weight and age, the severity of the disease state, the mode of administration, and the like. The therapeutically effective amount may be an amount sufficient to treat tuberous sclerosis and / or to alleviate, reduce the severity of, eliminate, and / or delay the onset of one or more symptoms of tuberous sclerosis. In some embodiments, the therapeutically effective amount may be an amount sufficient to express tuberin (e.g., tuberin lacking one or more mutations, such as a truncated tuberin provided herein) in the subject.

[0021] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, such as a mammal. A mammal can be, for example, a mouse, a rat, a rabbit, a cat, a dog, a pig, a sheep, a horse, a non-human primate (e.g., a cynomolgus monkey, a chimpanzee), or a human. Also included are subject tissues, cells, or derivatives thereof obtained in vivo or cultured in vitro. In some embodiments, the subject is a human. A human subject can be an adult, a teenager (e.g., 12-18 years old), a child (e.g., 2-14 years old), an infant (e.g., 1 month to 24 months old), or a newborn (up to 1 month old). In some embodiments, an adult is an elderly person, such as about 60 years old or older, such as about 65 years old or older. In some embodiments, the subject is a pregnant woman or a woman who intends to become pregnant. In some embodiments, the subject is under 18 years old.

[0022] An "adeno-associated virus (AAV) expression cassette" is a nucleic acid that is packaged into a recombinant AAV vector and contains sequences encoding one or more transgenes flanked by the 5' inverted terminal repeats (ITRs) and 3' ITRs.

[0023] As used herein, the terms "viral vector," "viral vector," and "gene delivery vector" refer to a viral particle that functions as a nucleic acid delivery vehicle and contains a nucleic acid molecule (e.g., an AAV expression cassette) packaged within the virion. Exemplary viral vectors include adeno-associated viral vectors (AAV).

[0024] As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV (e.g., serotype 1 AAV, also referred to as AAV1), AAV type 2 (e.g., AAV2), AAV type 3 (e.g., AAV3, including types 3A and 3B, AAV3A and AAV3B), AAV type 4 (e.g., AAV4), AAV type 5 (e.g., AAV5), AAV type 6 (e.g., AAV6), AAV type 7 (e.g., AAV7), AAV type 8 (e.g., AAV8), AAV type 9 (e.g., AAV9), AAV type 10 (e.g., AAV10), AAV type 11 (e.g., AAV11), AAV type 12 (e.g., AAV12), AAV type 13 (e.g., AAV rh32.33), AAV type 14 (e.g., AAV rh32.33), AAV type 15 (e.g., AAV rh32.33), AAV type 16 (e.g., AAV rh32.33), AAV type 17 (e.g., AAV rh32.33), AAV type 18 (e.g., AAV rh32.33), AAV type 19 (e.g., AAV rh32.33), AAV type 20 (e.g., AAV rh32.33), AAV type 21 (e.g., AAV rh32.33), AAV type 22 (e.g., AAV rh32.33), AAV type 23 (e.g., AAV rh32.33), AAV type 24 (e.g., AAV rh32.33), AAV type These include rh8 (e.g., AAVrh8), AAV rh10 (e.g., AAVrh10), AAV rh74 (e.g., AAVrh74), AAV hu.68 (e.g., AAVhu.68), avian AAV (e.g., AAAV), bovine AAV (e.g., BAAV), canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAV now known or later discovered.

[0025] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences do not vary across the entire alignment window of elements, e.g., nucleotides or amino acids. The "fractional identity" of an aligned segment of a test sequence and a reference sequence is the number of identical elements shared by the two aligned sequences divided by the total number of elements of the reference sequence segment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. A "percent identity" is 100 times the fractional identity. The degree of identity (homology) between two sequences can be ascertained using computer programs and mathematical algorithms. Percent identity can be calculated using the alignment program Clustal Omega, available at www.ebi.ac.uk / Tools / msa / clustalo, using default parameters. See Sievers et al., "Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega." (2011 October 11) Molecular systems biology 7:539. For purposes of calculating identity to sequences, extensions such as tags are not included.

[0026] As used herein, a nucleic acid sequence (e.g., a coding sequence) and a regulatory sequence are said to be "operably linked" when they are covalently linked in such a manner that expression or transcription of the nucleic acid sequence is under the influence or control of the regulatory sequence. If it is desired that the nucleic acid sequence be translated into a functional protein, if induction of the promoter in the 5' regulatory sequence results in 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 transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein, the two DNA sequences are said to be operably linked.

[0027] As used herein, "codon optimization" refers to modifying a nucleic acid sequence to alter an individual nucleic acid without causing a change in the corresponding encoded amino acid. Sequences modified in this manner are referred to herein as "codon optimized". Methods for performing codon optimization are described in U.S. Patent Nos. 7,561,972, 7,561,973, and 7,888,112, each of which is incorporated by reference in its entirety for all purposes. In some embodiments, the sequence surrounding the translation start site can be converted to a consensus Kozak sequence as described in Kozak et al., Nucleic Acids Res. 15(20):8125-81 48 (1987), which is incorporated by reference in its entirety for all purposes.

[0028] Truncated tuberin (ctuberin) As used herein, truncated tuberin or cTuberin refers to a recombinant tuberin protein having one or more amino acid residues deleted compared to the native tuberin protein sequence. In some embodiments, the native tuberin is human tuberin. In some embodiments, the native tuberin protein sequence has the amino acid sequence of SEQ ID NO: 1, which comprises 1807 amino acid residues. In some embodiments, the cTuberin provided herein lacks at least one amino acid residue of SEQ ID NO: 1. In some embodiments, the cTuberin provided herein lacks at least one amino acid residue of SEQ ID NO: 1 from the region between the N-terminal region and the C-terminal region.

[0029] In some embodiments, the amino acid and nucleic acid sequences of human tuberin are found in NCBI Accession No. NP_000539.2 and GenBank Accession No. X75621.1, respectively. In some embodiments, the amino acid sequences of human tuberin include, but are not limited to, tuberin isoform 4 (NCBI Accession No. NP_001070651.1), tuberin isoform 5 (NCBI Accession No. NP_001107854.1), tuberin isoform 6 (NCBI Accession No. NP_001305756.1), tuberin isoform 7 (NCBI Accession No. NP_001305758.1), tuberin isoform 8 (NCBI Accession No. NP_001305760.1), tuberin isoform 9 (NCBI Accession No. NP_001305770.1), tuberin isoform 10 (NCBI Accession No. NP_001305782.1), tuberin isoform 11 (NCBI Accession No. NP_001305784.1), tuberin isoform 12 (NCBI Accession No. NP_001305786.1), tuberin isoform 13 (NCBI Accession No. NP_001305788.1), tuberin isoform 14 (NCBI Accession No. NP_001305790.1), tuberin isoform 15 (NCBI Accession No. NP_001305792.1), tuberin isoform 16 (NCBI Accession No. NP_001305794.1), tuberin isoform 17 (NCBI Accession No. NP_001305796.1), tuberin isoform 18 (NCBI Accession No. NP_00130 305761.1), tuberin isoform X7 (NCBI accession no. XP_024306181.1), tuberin isoform X8 (NCBI accession no. XP_005255586.2), tuberin isoform X9 (NCBI accession no. XP_016879105.1), tuberin isoform X10 (NCBI accession no. XP_005255588.2), tuberin isoform X11 (NCBI accession no. XP_016879106.1), tuberin isoform X12 (NCBI accession no. XP_016879107.1), and others.

[0030] In some embodiments, ctuberin comprises an N-terminal region capable of binding to hamartin. In some embodiments, ctuberin comprises a C-terminal GTPase activating protein (GAP) region. In some embodiments, the one or more amino acid residues deleted in ctuberin are between the N-terminal region capable of binding to hamartin and the C-terminal GTPase activating protein (GAP) region.

[0031] In some embodiments, cTuberin comprises or consists of an N-terminal region capable of binding to hamartin and a C-terminal GTPase activating protein (GAP) region. In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 75% identity (e.g., at least about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 100%) to SEQ ID NO:4 or 5. In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:4 or 5. In some embodiments, the N-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:4 or 5.

[0032] In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 75% identity (e.g., at least about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) to any one of SEQ ID NOs: 7-12. In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to any one of SEQ ID NOs: 7-12. In some embodiments, the C-terminal region comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 7-12.

[0033] In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO:4, and the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:7. In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:4, and the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:7. In some embodiments, the N-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:4 and the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:7.

[0034] In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:5, and the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:7. In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:5, and the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:7. In some embodiments, the N-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:5 and the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:7.

[0035] In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:4, and the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:8. In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:4, and the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:8. In some embodiments, the N-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:4 and the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:8.

[0036] In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:5, and the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:8. In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:5, and the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:8. In some embodiments, the N-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:5 and the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:8.

[0037] In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:4, and the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:9. In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:4, and the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:9. In some embodiments, the N-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:4 and the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:9.

[0038] In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:5, and the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:9. In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:5, and the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:9. In some embodiments, the N-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:5 and the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:9.

[0039] In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:4, and the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:10. In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:4, and the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:10. In some embodiments, the N-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:4 and the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:10.

[0040] In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:5, and the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:10. In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:5, and the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:10. In some embodiments, the N-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:5 and the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:10.

[0041] In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:4, and the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:11. In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:4, and the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:11. In some embodiments, the N-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:4 and the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:11.

[0042] In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:5, and the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:11. In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:5, and the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:11. In some embodiments, the N-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:5 and the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:11.

[0043] In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:4, and the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:12. In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:4, and the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:12. In some embodiments, the N-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:4 and the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:12.

[0044] In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:5, and the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:12. In some embodiments, the N-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:5, and the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:12. In some embodiments, the N-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:5 and the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:12.

[0045] The disclosure provides ctuberin comprising or consisting of (i) an N-terminal region capable of binding hamartin and (ii) a C-terminal GTPase activating protein (GAP) region, wherein the ctuberin lacks amino acid residues 451 to 932 of SEQ ID NO: 1. The disclosure also provides ctuberin comprising or consisting of (i) an N-terminal region capable of binding hamartin and (ii) a C-terminal GTPase activating protein (GAP) region, wherein the ctuberin lacks amino acid residues 419 to 932 of SEQ ID NO: 1. When amino acid ranges are given, the ranges are inclusive (e.g., ctuberin lacking amino acid residues "419 to 932" or "419 to 932" means that the ctuberin lacks amino acids 419 and 932 of SEQ ID NO: 1, as well as all amino acid residues located therebetween).

[0046] In some embodiments, ctuberin further lacks amino acid residues 947-988 of SEQ ID NO: 1. In some embodiments, ctuberin lacks amino acid residues 451-932 and 947-988 of SEQ ID NO: 1. In some embodiments, ctuberin lacks amino acid residues 419-932 and amino acid residues 947-988 of SEQ ID NO: 1.

[0047] In some embodiments, ctuberin further lacks amino acid residues 1205 to 1271 of SEQ ID NO: 1. In some embodiments, ctuberin lacks amino acid residues 451 to 932 and 1205 to 1271 of SEQ ID NO: 1. In some embodiments, ctuberin lacks amino acid residues 419 to 932 and 1205 to 1271 of SEQ ID NO: 1.

[0048] In some embodiments, cTuberin lacks amino acid residues 451-932, 947-988, and 1205-1271 of SEQ ID NO:1. In some embodiments, cTuberin lacks amino acid residues 419-932, 947-988, and 1205-1271 of SEQ ID NO:1.

[0049] In some embodiments, ctuberin further lacks amino acid residues 1336-1497 of SEQ ID NO:1. In some embodiments, ctuberin lacks amino acid residues 419-932 and 1336-1497 of SEQ ID NO:1. In some embodiments, ctuberin lacks amino acid residues 451-932 and 1336-1497 of SEQ ID NO:1. In some embodiments, ctuberin lacks amino acid residues 419-932, 947-988, 1205-1271, and 1336-1497 of SEQ ID NO:1. In some embodiments, ctuberin lacks amino acid residues 451 ...

[0050] In some embodiments, ctuberin lacks amino acid residues 419-932, 1205-1271, and 1336-1497 of SEQ ID NO:1. In some embodiments, ctuberin lacks amino acid residues 451-932, 1205-1271, and 1336-1497 of SEQ ID NO:1. In some embodiments, ctuberin lacks amino acid residues 419-932, 947-988, and 1336-1497 of SEQ ID NO:1. In some embodiments, ctuberin lacks amino acid residues 451-932, 947-988, and 1336-1497 of SEQ ID NO:1.

[0051] In some embodiments, ctuberin further lacks amino acid residues 933-1109 of human tuberin (SEQ ID NO:1). In some embodiments, ctuberin lacks amino acid residues 451-932 and 933-1109 of human tuberin (SEQ ID NO:1). In some embodiments, ctuberin lacks amino acid residues 419-932 and 933-1109 of human tuberin (SEQ ID NO:1). In some embodiments, ctuberin lacks amino acid residues 419-1109 of SEQ ID NO:1.

[0052] In some embodiments, ctuberin lacks amino acid residues 451-932 of human tuberin (SEQ ID NO:1). In some embodiments, ctuberin lacks amino acid residues 451-1109 of human tuberin (SEQ ID NO:1). In some embodiments, ctuberin lacks amino acid residues 451-1139 of human tuberin (SEQ ID NO:1). In some embodiments, ctuberin lacks amino acid residues 451-1514 of human tuberin (SEQ ID NO:1). In some embodiments, ctuberin lacks amino acid residues 419-932 of human tuberin. In some embodiments, ctuberin lacks amino acid residues 419-1109 of human tuberin (SEQ ID NO:1). In some embodiments, ctuberin lacks amino acid residues 419-1139 of human tuberin (SEQ ID NO:1). In some embodiments, ctuberin lacks amino acid residues 419-1514 of human tuberin (SEQ ID NO:1).

[0053] In some embodiments, ctuberin lacks amino acid residues of one or more exons of human tuberin (SEQ ID NO:1), such as exons 25, 30, and / or 33. In some embodiments, ctuberin lacks amino acid residues of exon 25 (SEQ ID NO:1) of human tuberin. In some embodiments, ctuberin lacks amino acid residues of exon 30 (SEQ ID NO:1) of human tuberin. In some embodiments, ctuberin lacks amino acid residues of exon 33 (SEQ ID NO:1) of human tuberin. In some embodiments, ctuberin lacks amino acid residues of exons 25 and 30 of human tuberin (SEQ ID NO:1). In some embodiments, ctuberin lacks amino acid residues of exons 25, 30, and 33 of human tuberin (SEQ ID NO:1). In some embodiments, ctuberin lacks amino acid residues 451-932 and amino acid residues of exon 25 of human tuberin (SEQ ID NO:1). In some embodiments, ctuberin lacks amino acid residues 451-932 and exons 25 and 30 of human tuberin (SEQ ID NO:1). In some embodiments, ctuberin lacks amino acid residues 451-932 and exons 25, 30, and 33 of human tuberin (SEQ ID NO:1). In some embodiments, ctuberin lacks amino acid residues 419-932 and exon 25 of human tuberin (SEQ ID NO:1). In some embodiments, ctuberin lacks amino acid residues 419-932 and exon 25 of human tuberin (SEQ ID NO:1). In some embodiments, ctuberin lacks amino acid residues 419-932 and exons 25 and 30 of human tuberin (SEQ ID NO:1). In some embodiments, ctuberin lacks amino acid residues 419-932 and exons 25, 30, and 33 of human tuberin (SEQ ID NO:1).

[0054] The present disclosure provides a truncated tuberin (ctuberin) comprising or consisting of (i) an N-terminal region capable of binding to hamartin, and (ii) a C-terminal GTPase-activating protein (GAP) region, wherein the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., at least about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:7, and wherein the ctuberin lacks amino acid residues 419 to 932 or 451 to 932 of human tuberin (SEQ ID NO:1). The present disclosure also provides a truncated tuberin (ctuberin) comprising or consisting of (i) an N-terminal region capable of binding to hamartin and (ii) a C-terminal GTPase activating protein (GAP) region, wherein the C-terminal region comprises or consists of an amino acid sequence having at least 90% identity to SEQ ID NO: 7, and wherein the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO: 1). The present disclosure further provides a truncated tuberin (ctuberin) comprising or consisting of (i) an N-terminal region capable of binding to hamartin and (ii) a C-terminal GTPase activating protein (GAP) region, wherein the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO: 7, and wherein the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO: 1).

[0055] In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:8, and the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO:1). In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:8, and the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO:1). In some embodiments, the C-terminal region comprises or consists of an amino acid sequence of SEQ ID NO:8, and the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO:1).

[0056] In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:9, and the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO:1). In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:9, and the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO:1). In some embodiments, the C-terminal region comprises or consists of an amino acid sequence of SEQ ID NO:9, and the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO:1).

[0057] In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:7, cTuberin lacks amino acid residues 451-932 of human tuberin (SEQ ID NO:1), and the N-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:4. In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:7, the cTuberin lacks amino acid residues 451-932 of human tuberin (SEQ ID NO:1), and the N-terminal region comprises or consists of an amino acid sequence having at least 90% identity to SEQ ID NO:4. In some embodiments, the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:7, the cTuberin lacks amino acid residues 451-932 of human tuberin (SEQ ID NO:1), and the N-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:4.

[0058] In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:7, cTuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO:1), and the N-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:5. In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:7, the cTuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO:1), and the N-terminal region comprises or consists of an amino acid sequence having at least 90% identity to SEQ ID NO:5. In some embodiments, the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:7, the cTuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO:1), and the N-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:5.

[0059] In another aspect, the disclosure provides a truncated tuberin (ctuberin) comprising or consisting of (i) an N-terminal region capable of binding to hamartin and (ii) a C-terminal GTPase activating protein (GAP) region, wherein the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., at least about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to one of SEQ ID NOs: 10-12. The disclosure provides a truncated tuberin (ctuberin) comprising or consisting of (i) an N-terminal region capable of binding to hamartin and (ii) a C-terminal GTPase activating protein (GAP) region, wherein the C-terminal region comprises or consists of an amino acid sequence having at least 90% identity to one of SEQ ID NOs: 10-12. The present disclosure also provides a truncated tuberin (ctuberin) comprising or consisting of (i) an N-terminal region capable of binding to hamartin, and (ii) a C-terminal GTPase-activating protein (GAP) region, wherein the C-terminal region comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 10 to 12.

[0060] In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to one of SEQ ID NOs: 10-12, and the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO: 1). In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to one of SEQ ID NOs: 10-12, and the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO: 1). In some embodiments, the C-terminal region comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 10-12, and cTuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO: 1).

[0061] In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO: 10, and the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO: 1). In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO: 10, and the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO: 1). In some embodiments, the C-terminal region comprises or consists of an amino acid sequence of SEQ ID NO: 10, and the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO: 1).

[0062] In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO: 11, and the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO: 1). In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO: 11, and the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO: 1). In some embodiments, the C-terminal region comprises or consists of an amino acid sequence of SEQ ID NO: 11, and the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO: 1).

[0063] In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO: 12, and the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO: 1). In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO: 12, and the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO: 1). In some embodiments, the C-terminal region comprises or consists of an amino acid sequence of SEQ ID NO: 12, and the ctuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO: 1).

[0064] In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to one of SEQ ID NOs: 10-12, wherein cTuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO: 1), and the N-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO: 5. In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to one of SEQ ID NOs: 10-12, the cTuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO: 1), and the N-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO: 5. In some embodiments, the C-terminal region comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 10-12, the cTuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO: 1), and the N-terminal region comprises or consists of the amino acid sequence of SEQ ID NO: 5.

[0065] In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to one of SEQ ID NOs: 10-12, cTuberin lacks amino acid residues 451-932 of human tuberin (SEQ ID NO: 1), and the N-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO: 4. In some embodiments, the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to one of SEQ ID NOs: 10-12, the cTuberin lacks amino acid residues 451-932 or of human tuberin (SEQ ID NO: 1), and the N-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO: 4. In some embodiments, the C-terminal region comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 10-12, the cTuberin lacks amino acid residues 451-932 or of human tuberin (SEQ ID NO: 1), and the N-terminal region comprises or consists of the amino acid sequence of SEQ ID NO: 4.

[0066] In some embodiments, cTuberin comprises a polypeptide spacer between the N-terminal region and the C-terminal region. In some embodiments, the polypeptide spacer comprises or consists of the sequence of SEQ ID NO:2 (SGGG). In some embodiments, the polypeptide spacer comprises or consists of the sequence of SEQ ID NO:3 (SGGGSGGG SGGGSGGG).

[0067] In some embodiments, cTuberin comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to any one of SEQ ID NOs: 14-19. In some embodiments, cTuberin comprises or consists of an amino acid sequence having at least about 90% identity to any one of SEQ ID NOs: 14-19. In some embodiments, cTuberin comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 14-19.

[0068] c AAV expression cassette encoding tuberin In addition to the amino acid sequences and corresponding cTuberin described herein, the present disclosure further provides a nucleic acid molecule encoding any one of the cTuberin proteins disclosed herein. In some embodiments, the nucleic acid molecule is codon-optimized for expression in a human target cell. In some embodiments, the human target cell is a brain cell, a heart cell, a kidney cell, a skin cell, or a lung cell.

[0069] In some embodiments, the nucleic acid molecule is operably linked to a regulatory control sequence. In some embodiments, the regulatory control sequence comprises a human cytomegalovirus (CMV) promoter, a chicken beta-actin (CBA) promoter, a Rous sarcoma virus (RSV) LTR promoter / enhancer, an SV40 promoter, a dihydrofolate reductase promoter, a phosphoglycerol kinase promoter, a CMV immediate / early gene enhancer / CBA promoter, a synapsin promoter, a CMV-IE promoter / enhancer, a glial fibrillary acidic protein (GFAP) promoter, or a combination thereof. In some embodiments, the regulatory control sequence comprises a CMV immediate / early gene enhancer / CBA promoter, and a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). In some embodiments, the regulatory control sequence comprises a beta-glucuronidase (GUSB) promoter. Further details regarding the GUSB promoter are provided in Shipley et al., Analysis of the 5' Flanking Region of the Human β-Glucuronidase Gene, Genomics 10, 1009-1018 (1991), the entire contents of which are incorporated herein by reference.

[0070] In some embodiments, the nucleic acid molecule comprises or consists of at least about 75% (e.g., at least about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) sequence identity to any one of SEQ ID NOs: 20-26. In some embodiments, the nucleic acid molecule comprises or consists of the sequence of any one of SEQ ID NOs: 20-26. In some embodiments, the nucleic acid molecule comprises or consists of the sequence of SEQ ID NO: 20. In some embodiments, the nucleic acid molecule comprises or consists of the sequence of SEQ ID NO: 21. In some embodiments, the nucleic acid molecule comprises or consists of the sequence of SEQ ID NO: 22. In some embodiments, the nucleic acid molecule comprises or consists of the sequence of SEQ ID NO: 23. In some embodiments, the nucleic acid molecule comprises or consists of the sequence of SEQ ID NO: 24. In some embodiments, the nucleic acid molecule comprises or consists of the sequence of SEQ ID NO: 25. In some embodiments, the nucleic acid molecule comprises or consists of the sequence of SEQ ID NO: 26.

[0071] The present disclosure provides a nucleic acid molecule encoding c-tuberin comprising or consisting of (i) an N-terminal region capable of binding hamartin, and (ii) a C-terminal GTPase activating protein (GAP) region, wherein the c-tuberin lacks at least amino acid residues 451-932 of human tuberin (SEQ ID NO:1), and wherein the nucleic acid molecule is operably linked to a regulatory control sequence comprising, or consisting of, a beta-glucuronidase (GUSB) promoter.

[0072] In some embodiments, the nucleic acid molecule encodes cTuberin lacking amino acid residues 451-1109 of human tuberin (SEQ ID NO:1), the nucleic acid molecule being operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the nucleic acid molecule encodes cTuberin lacking amino acid residues 451-1139 of human tuberin (SEQ ID NO:1), the nucleic acid molecule being operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the nucleic acid molecule encodes cTuberin lacking amino acid residues 451-1514 of human tuberin (SEQ ID NO:1), the nucleic acid molecule being operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the nucleic acid molecule encodes cTuberin lacking amino acid residues 419-932 of human tuberin (SEQ ID NO:1), the nucleic acid molecule being operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the nucleic acid molecule encodes cTuberin lacking amino acid residues 419-1109 of human tuberin (SEQ ID NO:1), the nucleic acid molecule being operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the nucleic acid molecule encodes cTuberin lacking amino acid residues 419-1139 of human tuberin (SEQ ID NO:1), the nucleic acid molecule being operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the nucleic acid molecule encodes ctuberin lacking amino acid residues 419-1514 of human tuberin (SEQ ID NO:1), the nucleic acid molecule being operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the nucleic acid molecule encodes ctuberin lacking amino acid residues of exon 25 of human tuberin (SEQ ID NO:1). In some embodiments, the nucleic acid molecule encodes ctuberin lacking amino acid residues of exon 30 of human tuberin (SEQ ID NO:1).In some embodiments, the nucleic acid molecule encodes ctuberin lacking the amino acid residues of exon 33 of human tuberin (SEQ ID NO:1). In some embodiments, the nucleic acid molecule encodes ctuberin lacking the amino acid residues of exons 25 and 30 of human tuberin (SEQ ID NO:1). In some embodiments, the nucleic acid molecule encodes ctuberin lacking the amino acid residues of exons 25, 30, and 33 of human tuberin (SEQ ID NO:1).

[0073] In some embodiments, the cTuberin lacks amino acid residues 419-932, 451-932, 419-1109, 419-1139, 451-1109, 451-1139, 419-1514, 451-1514, 419-1515, or 451-1515 of human tuberin (SEQ ID NO:1), and the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:6, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the cTuberin lacks amino acid residues 451-1514 or 451-1515 of human tuberin (SEQ ID NO:1), the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:6, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the cTuberin lacks amino acid residues 451-1514 or 451-1515 of human tuberin (SEQ ID NO:1), the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:6, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter.

[0074] In some embodiments, the cTuberin lacks amino acid residues 419-932, 451-932, 419-1109, 419-1139, 451-1109, or 451-1139 of human tuberin (SEQ ID NO:1), the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., at least about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:7, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the cTuberin lacks amino acid residues 419-932, 451-932, 419-1109, 419-1139, 451-1109, or 451-1139 of human tuberin (SEQ ID NO:1), the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:7, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the cTuberin lacks amino acid residues 419-932, 451-932, 419-1109, 419-1139, 451-1109, or 451-1139 of human tuberin (SEQ ID NO:1), the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:7, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter.

[0075] In some embodiments, the cTuberin lacks amino acid residues 419-932, 451-932, 419-1109, or 451-1109 of human tuberin (SEQ ID NO:1), and the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., at least about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:8, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the cTuberin lacks amino acid residues 419-932, 451-932, 419-1109, or 451-1109 of human tuberin (SEQ ID NO:1), the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:8, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the cTuberin lacks amino acid residues 419-932, 451-932, 419-1109, or 451-1109 of human tuberin (SEQ ID NO:1), the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:8, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter.

[0076] In some embodiments, cTuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO:1), the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., at least about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:9, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, cTuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO:1), the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:9, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the cTuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO:1), the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:9, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter.

[0077] In some embodiments, the cTuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO:1), and the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., at least about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to one of SEQ ID NOs:10-12, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the cTuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO:1), the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to one of SEQ ID NOs:10-12, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the cTuberin lacks amino acid residues 419-932 or 451-932 of human tuberin (SEQ ID NO:1), the C-terminal region comprises or consists of the amino acid sequence of one of SEQ ID NOs:10-12, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter.

[0078] In some embodiments, the cTuberin lacks amino acid residues 451-932, 451-1109, 451-1139, 451-1514, 451-1515 of human tuberin (SEQ ID NO:1), and the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., at least about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:4, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the cTuberin lacks amino acid residues 451-932, 451-1109, 451-1139, 451-1514, 451-1515 of human tuberin (SEQ ID NO:1), the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:4, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the cTuberin lacks amino acid residues 451-932, 451-1109, 451-1139, 451-1514, or 451-1515 of human tuberin (SEQ ID NO:1), the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:4, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter.

[0079] In some embodiments, the cTuberin lacks amino acid residues 419-932, 419-1109, 419-1139, 419-1514, or 419-1515 of human tuberin (SEQ ID NO:1), and the C-terminal region comprises or consists of an amino acid sequence having at least about 75% (e.g., at least about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) identity to SEQ ID NO:5, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the cTuberin lacks amino acid residues 419-932, 419-1109, 419-1139, 419-1514, or 419-1515 of human tuberin (SEQ ID NO:1), the C-terminal region comprises or consists of an amino acid sequence having at least about 90% identity to SEQ ID NO:5, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the cTuberin lacks amino acid residues 419-932, 419-1109, 419-1139, 419-1514, or 419-1515 of human tuberin (SEQ ID NO:1), the C-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:5, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter.

[0080] In some embodiments, the cTuberin lacks amino acid residues 451-1514 or 451-1515 of human tuberin (SEQ ID NO:1), the C-terminal region comprises or consists of an amino acid sequence having at least 90% identity to SEQ ID NO:6, the N-terminal region comprises or consists of an amino acid sequence having at least 90% identity to SEQ ID NO:4, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter. In some embodiments, the cTuberin lacks amino acid residues 451-1514 or 451-1515 of human tuberin (SEQ ID NO:1), the C-terminal region comprises or consists of an amino acid sequence of SEQ ID NO:6, the N-terminal region comprises or consists of the amino acid sequence of SEQ ID NO:4, and the nucleic acid molecule is operably linked to a regulatory control sequence comprising or consisting of a beta-glucuronidase (GUSB) promoter.

[0081] In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 75% (e.g., at least about 99% or more, such as at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or 100%) sequence identity to any one of SEQ ID NOs: 20-26. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 90% sequence identity to any one of SEQ ID NOs: 20-26.

[0082] In some embodiments, the nucleic acid molecule comprises an adeno-associated virus (AAV) expression cassette, the AAV expression cassette comprising, from 5' to 3', a 5' AAV inverted terminal repeat (ITR), any one of the nucleic acid molecules disclosed herein, and a 3' AAV ITR. In some embodiments, the 5' ITR and / or the 3' ITR are derived from AAV2.

[0083] In some embodiments, the AAV expression cassettes disclosed herein contain cis-acting 5' and 3' inverted terminal repeat sequences as further described in BJ Carter, "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155-168 (1990), which is incorporated by reference in its entirety for all purposes. AAV ITR sequences can be obtained from any known AAV, including the currently identified mammalian AAV types disclosed herein.

[0084] In some embodiments, the AAV expression cassette comprises the 5'ITR and / or 3'ITR from AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAV type rh32.33, AAV type rh8, AAV type rh10, AAV type rh74, AAV type hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B. In some embodiments, the AAV expression cassette comprises a 5'ITR from AAV2, a 3'ITR from AAV2, or a combination thereof. In some embodiments, the AAV expression cassette comprises a 5'ITR from AAV2, a 3'ITR from AAV2, or a combination thereof.

[0085] In some embodiments, the 5' AAV ITR sequence comprises or consists of a nucleic acid sequence having at least 80% identity (e.g., at least about 99.5% or more, such as at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%, including all values ​​and subranges therebetween) to the sequence of SEQ ID NO: 27. In some embodiments, the 5' AAV ITR sequence comprises or consists of the sequence of SEQ ID NO: 27.

[0086] In some embodiments, the 3' AAV ITR sequence comprises or consists of a nucleic acid sequence having at least 80% (e.g., about 99.5% or more, such as at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%, including all values ​​and subranges therebetween) identity to the sequence of SEQ ID NO: 28. In some embodiments, the 3' AAV ITR sequence comprises or consists of the nucleic acid sequence of SEQ ID NO: 28.

[0087] In some embodiments, the AAV expression cassettes disclosed herein contain additional expression control elements operably linked to the transgene, including, for example, appropriate transcription initiation, termination, and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals, sequences that stabilize cytoplasmic mRNA, sequences that improve translation efficiency, sequences that improve protein stability, and sequences that improve secretion of the encoded product.

[0088] In some embodiments, the AAV expression cassette disclosed herein comprises an intron. In some embodiments, the intron is located between the promoter / enhancer sequence and the transgene. In some embodiments, the intron is derived from SV-40 and is referred to as the SV-40T intron sequence. In some embodiments, the AAV expression cassette disclosed herein comprises an internal ribosome entry site (IRES). In some embodiments, the AAV expression cassette disclosed herein comprises a nucleic acid encoding a 2A self-cleaving peptide. Exemplary 2A self-cleaving peptides include P2A, E2A, F2A, and T2A. In some embodiments, the AAV expression cassette disclosed herein comprises elements described, for example, in the references cited in Sambrook et al. and pages 3.18 3.26 and 16.17 16.27, each of which is incorporated herein by reference in its entirety for all purposes, as well as in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989.

[0089] In some embodiments, the AAV expression cassette disclosed herein comprises a woodchuck hepatitis virus post-transcriptional element (WPRE). (See, e.g., Wang and Verma, Proc. Natl. Acad. Sci., USA, 96: 3906-3910 (1999)). In some embodiments, the AAV expression cassette disclosed herein comprises a hepatitis B virus post-transcriptional regulatory element (HBVPRE) or an RNA transport element (RTE). In some embodiments, the WPRE or HBVPRE sequence is any of the WPRE or HBVPRE sequences disclosed in U.S. Patent No. 6,136,597 or U.S. Patent No. 6,287,814, both of which are incorporated herein by reference in their entirety.

[0090] In some embodiments, the AAV expression cassettes disclosed herein comprise 5' non-transcribed and 5' non-translated sequences, such as TATA boxes, capping sequences, CAAT sequences, enhancer elements, which are involved in the initiation of transcription and translation, respectively. In some embodiments, the AAV expression cassettes disclosed herein comprise enhancer sequences or upstream activator sequences. In some embodiments, the AAV expression cassettes disclosed herein comprise a 5' leader sequence or signal sequence.

[0091] In some embodiments, the AAV expression cassettes disclosed herein comprise a constitutive promoter, examples of which 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, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter.

[0092] In some embodiments, the AAV expression cassette disclosed herein comprises an inducible promoter. Non-limiting examples of inducible promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system, the ecdysone insect promoter, the tetracycline repression system, the tetracycline induction system, the RU486 induction system, and the rapamycin induction system. Other types of inducible promoters include those that are regulated by a particular physiological state, such as temperature, acute phase, a particular differentiation state of a cell, or a particular cell cycle stage.

[0093] In some embodiments, the AAV expression cassette disclosed herein comprises a native promoter or fragment thereof, or a native expression control element, operably linked to a transgene encoding cTuberin. In some embodiments, the AAV expression cassette disclosed herein comprises a regulatory sequence that confers tissue-specific gene expression capability. In some cases, the tissue-specific regulatory sequence binds to a tissue-specific transcription factor that induces transcription in a tissue-specific manner. Examples of tissue-specific regulatory sequences include, but are not limited to, the following tissue-specific promoters: neuron-specific enolase (NSE) promoter, neurofilament light chain gene promoter, and neuron promoters such as neuron-specific vgf gene promoter.

[0094] In some embodiments, the AAV expression cassette comprises one or more promoters. In some embodiments, the AAV expression cassette comprises a chicken β-actin promoter. In some embodiments, the AAV expression cassette comprises a CB6 promoter. In some embodiments, the CB6 promoter comprises a nucleic acid sequence having at least about 80% identity to the sequence of SEQ ID NO:34 (e.g., at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%, including all values ​​and subranges therebetween). In some embodiments, the CB6 promoter comprises or consists of the nucleic acid sequence of SEQ ID NO:34.

[0095] In some embodiments, the AAV expression cassette comprises a CMV-IE enhancer. In some embodiments, the enhancer is a CMV-IE enhancer. In some embodiments, the CMV-IE enhancer comprises a nucleic acid sequence having at least about 80% identity (e.g., at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, or more, including all values ​​and subranges therebetween) to the sequence of SEQ ID NO: 33. In some embodiments, the CMV-IE enhancer comprises or consists of the nucleic acid sequence of SEQ ID NO: 33.

[0096] In some embodiments, the AAV expression cassette comprises a consensus sequence such as a Kozak sequence (e.g., a DNA sequence that is transcribed into an RNA Kozak sequence). In some embodiments, the AAV expression cassette comprises a Kozak sequence. In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least about 80% identity to the sequence of SEQ ID NO:35 (e.g., at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%, including all values ​​and subranges therebetween). In some embodiments, the Kozak sequence comprises or consists of the nucleic acid sequence of SEQ ID NO:35.

[0097] In some embodiments, the AAV expression cassette disclosed herein comprises one or more binding sites for one or more microRNAs (miRNAs). In some embodiments, the AAV expression cassette comprises a miRNA binding site capable of regulating tissue-specific expression of the cTuberin transgene. In some embodiments, the miRNA binding site capable of regulating tissue-specific expression of the cTuberin transgene is a miR-122 binding site, a miR-133a, or a miR-1 binding site. For example, expression of the cTuberin transgene in the liver can be inhibited by incorporating a binding site for miR-122, such that the mRNA expressed from the transgene binds to and is inhibited by miR-122 in the liver. Expression of the cTuberin transgene in the heart can be inhibited by incorporating a binding site for miR-133a or miR-1, such that the mRNA expressed from the transgene binds to and is inhibited by miR-133a or miR-1 in the heart. In some embodiments, the miRNA target site in the mRNA is in the 5' untranslated region (UTR), 3' UTR, or coding region. Furthermore, the cTuberin transgene may be designed such that multiple miRNAs regulate the mRNA by recognizing the same or multiple sites. The presence of multiple miRNA binding sites may result in the coordinated action of multiple RNA-induced silencing complexes (RISCs) to provide highly efficient inhibition of expression. The target site sequence may comprise at least 5, 10, or more nucleotides, such as 5-100, or 10-60 nucleotides in total. The target site sequence may comprise a sequence of at least 5 nucleotides of the target gene binding site. In some embodiments, the AAV expression cassette comprises a miR-1 binding site, a miR-133a binding site, a miR-122 binding site, or any combination thereof.

[0098] In some embodiments, the AAV expression cassette comprises a polyadenylation (polyA) sequence. The polyA signal may be derived from many suitable species, including, but not limited to, SV-40, human, and bovine. In some embodiments, the polyA sequence is a β-globulin polyA sequence, such as a mammalian β-globulin polyA sequence. In some embodiments, the polyA sequence is a human polyA sequence or a bovine β-globulin polyA sequence. In some embodiments, the AAV expression cassette comprises a rabbit β-globin polyA sequence. In some embodiments, the rabbit β-globin polyA sequence comprises or consists of the nucleic acid sequence of SEQ ID NO:36.

[0099] In some embodiments, the AAV expression cassette comprises, from 5' to 3', (i) a 5' AAV2-based ITR, (ii) a CMV-IE enhancer, (iii) a CB6 promoter, (iv) a transgene encoding any one of the cTuberin proteins disclosed herein, (v) a polyadenylation sequence, and (vi) a 3' AAV2-based ITR. In some embodiments, the AAV expression cassette comprises, from 5' to 3', (i) a 5' AAV2-based ITR comprising the nucleic acid sequence of SEQ ID NO:27, (ii) a CMV-IE enhancer comprising the nucleic acid sequence of SEQ ID NO:33, (iii) a CB6 promoter comprising the nucleic acid sequence of SEQ ID NO:34, (iv) a transgene encoding any one of the cTuberin proteins disclosed herein, (v) a polyadenylation sequence, and (vi) a 3' AAV2-based ITR comprising the nucleic acid sequence of SEQ ID NO:28.

[0100] In some embodiments, the AAV expression cassette comprises, from 5' to 3', (i) a 5' AAV2-based ITR, (ii) a CB6 promoter, (iii) a transgene encoding any one of the cTuberin proteins disclosed herein, (iv) a polyadenylation sequence, and (v) a 3' AAV2-based ITR. In some embodiments, the AAV expression cassette comprises, from 5' to 3', (i) a 5' AAV2-based ITR comprising the nucleic acid sequence of SEQ ID NO:27, (ii) a CB6 promoter comprising the nucleic acid sequence of SEQ ID NO:34, (iii) a transgene encoding any one of the cTuberin proteins disclosed herein, (iv) a polyadenylation sequence, and (v) a 3' AAV2-based ITR comprising the nucleic acid sequence of SEQ ID NO:28.

[0101] In some embodiments, the AAV expression cassette comprises, from 5' to 3', (i) a 5' AAV2-based ITR, (ii) a GUSB promoter, (iii) a transgene encoding any one of the cTuberin proteins disclosed herein, (iv) a polyadenylation sequence, and (v) a 3' AAV2-based ITR. In some embodiments, the AAV expression cassette comprises, from 5' to 3', (i) a 5' AAV2-based ITR comprising the nucleic acid sequence of SEQ ID NO:27, (ii) a GUSB promoter, (iii) a transgene encoding any one of the cTuberin proteins disclosed herein, (iv) a polyadenylation sequence, and (v) a 3' AAV2-based ITR comprising the nucleic acid sequence of SEQ ID NO:28.

[0102] Recombinant adeno-associated virus (rAAV) for treating tuberous sclerosis The present disclosure also provides a plasmid comprising any one of the nucleic acid molecules disclosed herein, and a host cell comprising any one of the nucleic acid molecules or plasmids disclosed herein.

[0103] The present disclosure further provides a method for producing recombinant adeno-associated virus (rAAV).In some embodiments, the method for producing rAAV comprises contacting host cell with any one of the nucleic acid molecules or plasmids disclosed herein.Therefore, the present disclosure further provides the recombinant adeno-associated virus (rAAV) produced by the method for producing rAAV disclosed herein.

[0104] The present disclosure also provides rAAV.In some embodiments, rAAV comprises AAV capsid protein and any one of the nucleic acid molecules or AAV expression cassettes disclosed herein.The present disclosure also provides a composition comprising any one of the cTuberin proteins, any one of the nucleic acid molecules, any one of the plasmids, any one of the host cells, or any one of the rAAVs disclosed herein.

[0105] In some embodiments, the rAAV comprises an AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAV rh32.33, AAV rh8, AAV rh10, AAV rh74, AAV hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, or AAV PHP.B capsid protein.

[0106] In some embodiments, the rAAV comprises AAV9 capsid protein, AAV8 capsid protein, and / or AAVrhlO capsid protein. In some embodiments, the rAAV comprises AAV9 capsid protein. In some embodiments, the rAAV comprises AAV8 capsid protein. In some embodiments, the rAAV comprises AAVrhlO capsid protein. In some embodiments, the rAAV is a pseudotyped AAV, comprising AAV capsid protein of one serotype and AAV ITRs from a different serotype. In some embodiments, the rAAV comprises a chimeric AAV capsid, or a humanized AAV capsid. In some embodiments, the rAAV is a self-complementary AAV (scAAV). In some embodiments, the rAAV is a single-chain AAV.

[0107] In some embodiments, preparing rAAV particles involves culturing a host cell that contains a recombinant AAV vector comprised of a nucleic acid sequence encoding an AAV capsid protein or fragment thereof, a functional rep gene, an AAV expression cassette encoding any one of the AAV inverted terminal repeats (ITRs) and the cTuberin protein disclosed herein, and sufficient helper functions to allow packaging of the recombinant AAV vector into the AAV capsid protein. In some embodiments, the components that are cultured in the host cell to package the rAAV vector into the AAV capsid are provided to the host cell in trans. In some embodiments, any one or more of the necessary components (e.g., recombinant AAV vector, rep sequences, cap sequences, and / or helper functions) are provided by a stable host cell that has been engineered to contain one or more of the necessary components.

[0108] In some embodiments, the stable host cell will contain the required component(s) under the control of an inducible or constitutive promoter. In some embodiments, the selected stable host cell contains the selected component(s) under the control of a constitutive promoter and the other selected component(s) under the control of one or more inducible promoters. For example, stable host cells can be generated that are derived from 293 cells (containing E1 helper functions under the control of a constitutive promoter) but contain the rep and / or cap proteins under the control of an inducible promoter. The recombinant AAV vectors, rep sequences, cap sequences, and helper functions required to produce the rAAV disclosed herein can be delivered to the packaging host cell using any suitable genetic elements (e.g., vectors). Further details regarding methods for preparing rAAV particles are provided in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY; K. Fisher et al., J. Virol., 70:520-532 (1993); and U.S. Pat. No. 5,478,745, the contents of each of which are incorporated herein in their entirety for all purposes.

[0109] In some embodiments, recombinant AAV is produced using the triple transfection method described in U.S. Patent No. 6,001,650, the entire contents of which are incorporated herein for all purposes. In some embodiments, recombinant AAV is produced by transfecting host cells with a recombinant AAV vector (including an AAV expression cassette encoding cTuberin) packaged within an AAV particle, an AAV helper function vector, and an accessory function vector. The AAV helper function vector encodes "AAV helper function" sequences (i.e., rep and cap) that function in trans for productive AAV replication and encapsidation. Non-limiting examples of AAV helper function vectors include the pHLP19 and pRep6cap6 vectors described in U.S. Patent Nos. 6,001,650 and 6,156,303, respectively, the entire contents of each of which are incorporated herein for all purposes. The accessory function vector encodes nucleotide sequences for non-AAV derived viral and / or cellular functions (i.e., "accessory functions") on which AAV depends for replication. Accessory functions include functions required for AAV replication, including, but not limited to, moieties involved in activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of the cap expression product, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus.

[0110] In some embodiments, recombinant AAV is produced using baculovirus vector.Baculovirus vector is used to produce recombinant AAV in insect cells (e.g., Spodoptera frugiperda (Sf9) cells).More details regarding the production of AAV encoding cTuberin can be found in US Patent Publication No. 2020 / 0079824, the entire contents of which are incorporated herein by reference for all purposes.

[0111] Pharmaceutical Compositions The present disclosure further provides a pharmaceutical composition comprising (a) any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the host cells disclosed herein, or any one of the rAAVs disclosed herein; and (b) a pharma- ceutically acceptable carrier.

[0112] In some embodiments, the compositions disclosed herein include at least one pharma- ceutically acceptable carrier, excipient, and / or vehicle, such as a solvent, buffer, solution, dispersion medium, coating, antibacterial agent, antifungal agent, isotonic agent, and absorption delaying agent. In some embodiments, the pharma- ceutically acceptable carrier, excipient, and / or vehicle includes saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, or combinations thereof. In some embodiments, the pharma- ceutically acceptable carrier, excipient, and / or vehicle includes phosphate buffered saline, sterile saline, lactose, sucrose, calcium phosphate, dextran, agar, pectin, peanut oil, sesame oil, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), or suitable mixtures thereof. In some embodiments, the compositions disclosed herein further comprise an emulsifier or wetting agent, or a pH buffering agent. Such species may be present in small amounts (e.g., less than 10% by weight of the composition, such as less than 5% by weight of the composition, less than 1% by weight of the composition, less than 2% by weight of the composition, or even less).

[0113] In some embodiments, the compositions disclosed herein further comprise one or more other pharmaceutical ingredients, such as one or more preservatives or chemical stabilizers. Examples of preservatives and chemical stabilizers include, but are not limited to, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, and albumin. In some embodiments, the compositions disclosed herein may further comprise antibacterial and / or antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal; isotonicity agents, such as sugars and sodium chloride; and / or agents that delay absorption, such as aluminum monostearate and gelatin. In some embodiments, the compositions disclosed herein comprise a surfactant, such as pluronic F68 (also known as Poloxamer 188, LUTROL F68).

[0114] In some embodiments, the compositions disclosed herein contain particularly high rAAV concentrations (e.g., about 10 13 The composition is formulated to reduce aggregation of AAV particles when rAAV is present in the presence of 100 ng / ml or more. Methods for reducing aggregation of rAAV include the addition of detergents, pH adjustment, and salt concentration adjustment, as further described in Wright, et al., Molecular Therapy (2005) 12, 171-178, the entire contents of which are incorporated herein by reference for all purposes.

[0115] In some embodiments, the pharmaceutical composition is in the form of an injectable solution or dispersion, such as an aqueous solution or dispersion.In some embodiments, the pharmaceutical composition is a sterile powder for the extemporaneous preparation of a sterile injectable solution or dispersion.Dispersions can be prepared in water, glycerol, liquid polyethylene glycol, oil, or any combination thereof.Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. can be used to introduce the pharmaceutical compositions disclosed herein.

[0116] The present disclosure also provides an extracellular vesicle (EV) comprising any one of the cTuberin proteins disclosed herein or any of the nucleic acid molecules disclosed herein. In some embodiments, a pharmaceutical composition comprises an extracellular vesicle (EV) comprising any one of the cTuberin proteins disclosed herein or any of the nucleic acid molecules disclosed herein. Extracellular vesicles, including but not limited to exosomes, microvesicles, microparticles, circulating microvesicles, detached microvesicles, nanovesicles, nanoparticles, apoptotic bodies, and membrane vesicles, are fragments of plasma membrane, e.g., in the range of 20 nanometers (nm) to 10 micrometers (μm), and are detached from nearly all cell types. In some embodiments, EVs are isolated and purified using filtration, differential centrifugation, ultracentrifugation, flotation of vesicles in gradients (sucrose, OptiPrep™), and immunoaffinity capture utilizing antibodies against membrane proteins.Further details are provided in Simpson RJ, Mathivanan S (2012) Extracellular Microvesicles: The Need for Internationally Recognised Nomenclature and Stringent Purification Criteria. J Proteomics; van der Pol et al., Classification, functions, and clinical relevance of extracellular vesicles, Pharmacol Rev. 2012 July; 64(3): 676-705; Raposo and Stoorvogel, Extracellular vesicles: exosomes, microvesicles, and friends, J Cell Biol. 201 3 Feb. 18; 200(4): 373-83; and Witwer et al., Standardization of sample collection, isolation and analysis methods in extracellular vesicle research, J Extracell Vesicles. 2013 May 27; 2, the contents of each of which are incorporated herein by reference in their entirety for all purposes.

[0117] How to Treat Tuberous Sclerosis The present disclosure also provides a method of expressing any one of the cTuberins disclosed herein in a target cell, comprising contacting the target cell with any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the rAAVs disclosed herein, any one of the EVs disclosed herein, or any one of the compositions disclosed herein, thereby expressing cTuberin in the target cell.

[0118] In addition, the present disclosure provides a method of inhibiting mTORC1 and / or Rheb activity, comprising contacting a target cell with any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the rAAVs disclosed herein, any one of the EVs disclosed herein, or any one of the compositions disclosed herein, thereby inhibiting mTORC1 and / or Rheb activity in the target cell.

[0119] In some embodiments, the target cell is a brain cell, a heart cell, a kidney cell, a skin cell, a lung cell, or any combination thereof. In some embodiments, the contacting step is performed in vitro, ex vivo, or in vivo. In some embodiments, the contacting step is performed in vivo in a subject in need of treatment. In some embodiments, the contacting step comprises administering a therapeutically effective amount of a nucleic acid molecule, a plasmid, a rAAV, or a composition to the subject.

[0120] The present disclosure also provides a method of treating tuberous sclerosis or a symptom thereof in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the rAAVs disclosed herein, any one of the EVs disclosed herein, or any one of the compositions disclosed herein, thereby treating tuberous sclerosis in the subject. In a related aspect, the present disclosure provides a method of alleviating, reducing the severity, eliminating, and / or delaying the onset of one or more symptoms of tuberous sclerosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the rAAVs disclosed herein, or any one of the compositions disclosed herein, thereby alleviating, reducing the severity, eliminating, and / or delaying the onset of one or more symptoms of tuberous sclerosis in the subject.

[0121] The present disclosure further provides a method of treating renal angiomyolipoma or a symptom thereof in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the rAAVs disclosed herein, any one of the EVs disclosed herein, or any one of the compositions disclosed herein, thereby treating renal angiomyolipoma in the subject. In some embodiments, treating renal angiomyolipoma results in involution, shrinkage, elimination, or slowing of the growth of renal angiomyolipoma.

[0122] Additionally, the present disclosure provides a method of treating lymphangioleiomyomatosis (LAM) or a symptom thereof in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the rAAVs disclosed herein, any one of the EVs disclosed herein, or any one of the compositions disclosed herein, thereby treating LAM in the subject. In a related aspect, the present disclosure provides a method of alleviating, reducing the severity, eliminating, and / or delaying the onset of one or more symptoms of lymphangioleiomyomatosis (LAM) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the rAAVs disclosed herein, or any one of the compositions disclosed herein, thereby alleviating, reducing the severity, eliminating, and / or delaying the onset of one or more symptoms of LAM in the subject.

[0123] The present disclosure also provides a method of treating brain dysfunction in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the rAAVs disclosed herein, any one of the EVs disclosed herein, or any one of the compositions disclosed herein, thereby treating the brain dysfunction in the subject. In a related aspect, the present disclosure provides a method of alleviating, reducing the severity, eliminating, and / or delaying the onset of one or more symptoms of brain dysfunction in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the rAAVs disclosed herein, or any one of the compositions disclosed herein, thereby alleviating, reducing the severity, eliminating, and / or delaying the onset of one or more symptoms of brain dysfunction in the subject.

[0124] In some embodiments, tuberous sclerosis is associated with, correlated with, or caused by a decrease in the activity of tuberin. In some embodiments, the subject is afflicted with tuberous sclerosis. In some embodiments, the subject is at risk of developing at least one symptom of tuberous sclerosis.

[0125] In some embodiments, the subject has been diagnosed with tuberous sclerosis. In some embodiments, the subject is diagnosed with tuberous sclerosis based on clinical criteria such as the presence of seizures; developmental delay; white spots on the skin (vitiligo); identification of cardiac tumor rhabdomyoma; identification of tumors in the brain, heart, liver, or kidneys; examination of the skin for a wide variety of skin features; fingernails and toenails for nail fibroma; teeth and gums for dental cavities and / or gingival fibroma; eyes for retinal lesions; facial angiofibroma; and / or the presence of vitiligo. In some embodiments, the subject is a human subject. In some embodiments, the subject is under the age of 18, such as 12-18 years old, 8-12 years old, 6-12 years old, 2-18 years old, 0-2 years old, or any range therein. In some embodiments, the subject is a newborn or infant. In some embodiments, the infant is diagnosed with tuberous sclerosis based on clinical criteria such as the presence of cardiac rhabdomyoma at birth, or infantile spasms within the first 6 months of life.

[0126] In some embodiments, tuberous sclerosis is associated with, correlated with, or caused by a mutation in the TSC2 gene. In some embodiments, the subject has a mutation in the TSC2 gene. In some embodiments, the subject has a mutation in one or both alleles of TSC2 in at least one cell in the body. In some embodiments, the mutation is an inherited germline mutation. In some embodiments, the mutation is a somatic mutation. In some embodiments, the mutation in one allele of TSC2 is an inherited mutation and the mutation in the second allele of TSC2 is a somatic mutation. In some embodiments, the subject at risk of developing at least one symptom of tuberous sclerosis is a subject with an inherited germline mutation in one allele of TSC2. In some embodiments, the subject at risk of developing at least one symptom of tuberous sclerosis is a subject in which one or both parents are carriers of one or more mutant tuberin gene alleles.

[0127] In some embodiments, the subject has a mutation in both alleles of TSC2 in at least one cell in the body. In some embodiments, the subject has a mutation in both alleles of TSC2 in at least one cell of the brain, heart, kidney, skin, lung, and / or other organ. In some embodiments, the subject is homozygous or compound heterozygous for a mutation in TSC2.

[0128] In some embodiments, the subject has a mutation in the TSC1 gene. In some embodiments, the subject has a mutation in the TSC1 gene and a mutation in the TSC2 gene.

[0129] In some embodiments, a subject is diagnosed as having or at risk of developing tuberous sclerosis by testing for the presence of any one or more of the mutations in TSC2 described herein in a biological sample from the subject. In some embodiments, the mutation in the TSC2 gene can be any amino acid modification, such as, for example, an amino acid insertion, deletion, splice site mutation, and / or an amino acid substitution. Details regarding mutations in the TSC2 gene are further described in Reyna-Fabian, ME, Sci Rep 10, 6589 (2020); Gilbert JR, et al., Neurogenetics. 1998 Aug; 1(4): 267-72; Avgeris, S., Sci Rep 7, 16697 (2017), and Rosset C, et al., Genet Mol Biol. 2017; 40(1): 69-79, the contents of each of which are incorporated herein in their entirety for all purposes. In some embodiments, the subject has a mutation in exon 33, exon 37, and / or exon 38 of the TSC2 gene. In some embodiments, the subject has a mutation in exon 33 of the TSC2 gene.

[0130] In some embodiments, administration of a therapeutically effective amount of a therapeutic provided herein (e.g., a therapeutically effective amount of any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the rAAVs disclosed herein, or any one of the compositions disclosed herein) reduces the severity of any one of the symptoms of tuberous sclerosis disclosed herein. In some embodiments, administration of a therapeutically effective amount of a therapeutic provided herein delays the onset of any one of the symptoms of tuberous sclerosis disclosed herein. In some embodiments, administration of a therapeutically effective amount of a therapeutic provided herein eliminates any one of the symptoms of tuberous sclerosis disclosed herein. In some embodiments, administration of a therapeutically effective amount of a therapeutic provided herein alleviates any one of the symptoms of tuberous sclerosis disclosed herein. In some embodiments, symptoms of tuberous sclerosis are the presence of any one or more of the following: tumors or hamartomas in the brain, heart, liver, kidneys, eyes, or skin; subependymal growths or nodules; subependymal giant cell astrocytoma; cortical nodules; brain dysfunction; seizures; developmental delay; vitiligo; cardiac tumors rhabdomyomas; nail fibromas; dental cavities and / or gingival fibromas; retinal lesions; facial angiofibromas; renal angiomyolipomas; lymphangioleiomyomatosis (LAM); internal bleeding; autism; epilepsy; hydrocephalus; and / or the presence of vitiligo. In some embodiments, the symptoms of tuberous sclerosis are any one of those described in Randle SC., Pediatr Ann. 2017 Apr 1;46(4):e166-e171, Uysal SP, Turk J Med Sci. 2020 Nov 3;50(SI-2):1665-1676, and Henske EP, et al. Nat Rev Dis Primers. 2016 May 26;2:16035, the contents of each of which are incorporated by reference in their entirety for all purposes.

[0131] In some embodiments, administration is associated with, correlates with, or results in a reduction in the size of a tumor or hamartoma in the subject. In some embodiments, administration is associated with, correlates with, or results in a reduction in the size of a tumor or hamartoma in the subject by at least about 5% (e.g., at least about 95% or more, such as at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or 100%), compared to a control subject with tuberous sclerosis who has not been administered a composition disclosed herein.

[0132] In some embodiments, administration is associated with, correlates with, or results in a reduction in the number of tumors or hamartomas in the subject. In some embodiments, administration is associated with, correlates with, or results in a reduction in the number of tumors or hamartomas in the subject by at least about 5% (e.g., at least about 95% or more, such as at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or 100%), compared to a control subject with tuberous sclerosis who has not been administered a composition disclosed herein.

[0133] Without being bound by theory, it is believed that administration of rapamycin to subjects with tuberous sclerosis is associated with side effects resulting from over-inhibition of mTORC1, whereas expression of cTuberin using the compositions and methods disclosed herein may not cause over-inhibition of mTORC1. This is because subjects with tuberous sclerosis associated with mutations in TSC2 may have normal levels of hamartin, which would ensure normal inhibition of mTORC1. Furthermore, rapamycin and its analogs can only inhibit mTORC1, whereas expression of cTuberin using the disclosed compositions produces mTORC1-independent Rheb-dependent pathological effects in addition to inhibition of mTORC1. Thus, the compositions and methods disclosed herein may be more effective than rapamycin-based therapy for tuberous sclerosis.

[0134] In some embodiments, the method includes administering a therapeutically effective amount of rAAV, wherein the therapeutically effective amount is about 10 5 Genome copies ~10 20 Genome copies, e.g., about 10 6 Genome copies / kg, approximately 10 7 Genome copies / kg, approximately 10 8 Genome copies / kg, approximately 10 9 Genome copies / kg, approximately 10 10 Genome copies / kg, approximately 10 11 Genome copies / kg, approximately 10 12 Genome copies / kg, approximately 10 13 Genome copies / kg, approximately 10 14 Genome copies / kg, approximately 10 15 Genome copies / kg, approximately 10 16 Genome copies / kg, approximately 10 17 Genome copies / kg, approximately 10 18 genome copies / kg, or approximately 10 19 In some embodiments, the method includes administering a therapeutically effective amount of rAAV, the therapeutically effective amount being in the range of 10 genome copies / kg, including all values ​​and subranges therebetween. 10 Genome copies ~1014 In some embodiments, the method includes administering a therapeutically effective amount of rAAV, the therapeutically effective amount being in the range of 10 genome copies per kilogram. 9 Genome copies ~10 15 in the range of genome copies.

[0135] In some embodiments, the therapeutically effective amount is about 10 5 ~10 20 Genome copies, e.g., about 10 per subject 6 Genome copies, approximately 10 per subject 7 Genome copies, approximately 10 per subject 8 Genome copies, approximately 10 per subject 9 Genome copies, approximately 10 per subject 10 Genome copies, approximately 10 per subject 11 Genome copies, approximately 10 per subject 12 Genome copies, approximately 10 per subject 13 Genome copies, approximately 10 per subject 14 Genome copies, approximately 10 per subject 15 Genome copies, approximately 10 per subject 16 Genome copies, approximately 10 per subject 17 Genome copies, approximately 10 per subject 18 genome copies, or approximately 10 per subject 19 In some embodiments, the therapeutically effective amount is about 10 9 ~10 16 in the range of genome copies.

[0136] In some embodiments, a therapeutically effective amount is from about 1 microliter (μl) to about 100 mL of solution, e.g., about 10 μl, about 50 μl, about 100 μl, about 125 μl, about 150 μl, about 175 μl, about 200 μl, about 250 μl, about 300 μl, about 350 μl, about 400 μl, about 450 μl, about 500 μl, about 550 μl, about 600 μl, about The rAAV may be administered in a volume of about 650 μl, about 700 μl, about 750 μl, about 800 μl, about 850 μl, about 900 μl, about 950 μl, about 1 milliliter (mL), about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 60 mL, about 70 mL, about 80 mL, about 90 mL, or about 100 mL, including all values ​​and subranges therebetween. The volume used may depend on the dose of rAAV and the route of administration. For example, for intrathecal or intracerebral administration, volumes ranging from about 1 μl to about 10 μl, or from about 10 μl to about 100 μl may be used. For intravenous administration, volumes ranging from about 10 μl to about 100 μl, or from about 100 μl to 1 mL, or from about 1 mL to about 10 mL, or more, may be used.

[0137] In some embodiments, more than one administration (e.g., 2, 3, 4 or more administrations) may be used to achieve a desired level of gene expression over variously spaced periods, e.g., daily, weekly, monthly, yearly, etc.

[0138] In some embodiments, administration is by injection into the central nervous system. Other modes of administration that may be used include dermal, oral, rectal, transmucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or intraovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular (including administration to skeletal muscle, diaphragm muscle, and / or cardiac muscle), intradermal, intrapleural, intracerebral, and intraarticular), topical (e.g., to both skin and mucosal surfaces, including airway surfaces and transdermal administration), intralymphatic, and the like, and direct injection of a tissue or organ (e.g., to the liver, skeletal muscle, cardiac muscle, diaphragm muscle, or brain). In some embodiments, administration is by intraventricular injection, or intracranial injection. In some embodiments, the cTuberin, nucleic acid molecule, plasmid, composition or rAAV is administered intravascularly, intrarenal artery or vein, intrapulmonary, intracisternal, intracerebral, intrathecal, intravenous, intraventricular, intraventricular, intraperitoneal, or intradermal.

[0139] In some embodiments, the method comprises administering a therapeutically effective amount of cTuberin, a nucleic acid molecule, a plasmid, a composition, or a rAAV to a subject having renal angiomyolipoma, for example, by intravascular injection into a renal artery or vein. In some embodiments, the therapeutically effective amount of cTuberin, a nucleic acid molecule, a plasmid, a composition, or a rAAV targets renal angiomyolipoma. In some embodiments, the method comprises administering a therapeutically effective amount of cTuberin, a nucleic acid molecule, a plasmid, a composition, or a rAAV to a subject having lymphangioleiomyomatosis (LAM) by intravascular injection. In some embodiments, the therapeutically effective amount of cTuberin, a nucleic acid molecule, a plasmid, a composition, or a rAAV targets LAM. In some embodiments, the subject has brain dysfunction. In some embodiments, the cTuberin, a nucleic acid molecule, a plasmid, a composition, or a rAAV is provided into the subarachnoid space.

[0140] In some embodiments, the cTuberin, nucleic acid molecule, plasmid, composition, or rAAV is provided (e.g., administered) to brain cells, heart cells, kidney cells, skin cells, or lung cells. In some embodiments, the nucleic acid molecule, plasmid, host cell, rAAV, or composition is administered to the bloodstream of the subject. Administration to the bloodstream may be by injection into a vein, artery, or any other vascular conduit. In some embodiments, the nucleic acid molecule, plasmid, cell, rAAV, or composition is administered intravascularly. In some embodiments, the nucleic acid molecule, plasmid, host cell, rAAV, or composition is administered intravenously.

[0141] In some embodiments, the nucleic acid molecule, plasmid, host cell, rAAV, or composition is delivered to brain tissue, meninges, neurons, glial cells, astrocytes, oligodendrocytes, cerebrospinal fluid (CSF), interstitial spaces, etc. In some embodiments, the recombinant AAV can be delivered directly to the spinal cord or brain using neurosurgical techniques such as stereotactic injection with needles, catheters, or related devices, by injection into the ventricular region, as well as into the striatum (e.g., the caudate or putamen of the striatum), and neuromuscular junctions, or cerebellar lobules.

[0142] In some embodiments, administration may include administering a nucleic acid molecule, plasmid, host cell, rAAV, or composition by two or more routes at the same or different times.

[0143] In some embodiments, the methods disclosed herein include administering another secondary therapy to the subject. In some embodiments, the secondary therapy includes administration of an anti-seizure drug. Non-limiting examples of anti-seizure drugs include carbamazepine, phenytoin, valproic acid, oxcarbazepine, lamotrigine, gabapentin, topiramate, and phenobarbital, and zonisamide. In some embodiments, the secondary therapy includes administration of rapamycin or an analog thereof. The secondary therapy may be administered to the subject sequentially or simultaneously.

[0144] The present disclosure also provides kits that include one or more agents (e.g., any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the rAAVs disclosed herein, or any one of the compositions disclosed herein). In some embodiments, the kit is a pharmaceutical, diagnostic, or research kit for use in pharmaceutical, diagnostic, or research applications. The kit may include one or more containers that contain the agents disclosed herein and instructions for use. In certain embodiments, the agents in the kit are pharmaceutical formulations and dosages suitable for a particular application and method of administration of the agents. In some embodiments, the container is a syringe, a vial, a tube, a topical application device, an IV needle tubing and bag, or another container.

[0145] In some embodiments, the kit contains a nucleic acid molecule encoding cTuberin (e.g., in an rAAV as described herein) or a first pharmaceutical composition comprising cTuberin, and a second pharmaceutical composition comprising one or more drugs used in the treatment of tuberous sclerosis, such as rapamycin and its analogs. In some embodiments, the kit contains instructions for administering the two compositions sequentially or simultaneously, or for mixing the two pharmaceutical compositions prior to administration. ********

[0146] The headings used herein are merely for organizational purposes and should not be construed as limiting the subject matter described. All documents or portions of documents cited herein, including but not limited to patents, patent applications, articles, books, and papers, are expressly incorporated herein by reference in their entirety and for all purposes. If one or more of the incorporated documents or portions of documents defines a term and it contradicts the definition of that term in this application, the definition set forth in this application shall prevail. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein should not be construed as an acknowledgment or any form of implication that they constitute valid prior art or form part of the general general knowledge in any country in the world.

[0147] It is specifically contemplated that the various features described herein can be used in any combination, unless the context dictates otherwise. EXAMPLES

[0148] Example 1: Generation of new truncated tuberin (cTuberin) constructs By cloning different nucleic acid constructs into suitable expression plasmids, various cTuberin proteins containing deletions of specific amino acid residues are generated. The constructs and amino acid deletions are listed in Table 1 below. [Table 1-1] [Table 1-2]

[0149] An AAV vector is generated, comprising an AAV expression cassette comprising a nucleic acid sequence encoding each of the cTuberins listed in Table 1. The AAV expression cassette comprises a CB6 promoter with or without a CMV-IE upstream response element (URE) or a GUSB promoter operably linked to a nucleic acid sequence encoding each of the variants as shown in Table 1. The AAV vector is packaged into an AAV particle using a triple transfection method. In some cases, the AAV is a type 1 AAV (e.g., AAV1) or a type 9 AAV (e.g., AAV9). In some cases, the vector comprises one or more ITR elements, such as one or more AAV2 ITR elements.

[0150] AAV vectors such as those described above can be prepared by any useful method. In some embodiments, AAV vectors are prepared using transfected (e.g., triple-transfected) cell cultures (e.g., HEK293 suspension cultures) to produce the desired product. Subsequent processing can include one or more lysis, ion exchange chromatography, filtration (including ultrafiltration), affinity chromatography, and / or dilution steps. The sequence of the vector can be confirmed using various sequencing methods. The titer (e.g., gc / mL) can be determined using PCR amplification or other methods.

[0151] Example 2: Expression of cTuberin in cell culture Cells (e.g., COS-7 cells) are transfected with a vector plasmid containing the nucleic acid sequence corresponding to cTuberin as described in Example 1. After 24 hours, expression of cTuberin is detected by immunoblotting (e.g., Western blotting) with an anti-Tuberin / TSC2 antibody.

[0152] To test the activity of cTuberin, cells (e.g., COS-7 cells) are transfected with various AAV constructs, including, for example, GFP, pAAV-CBA-cTSC2, TSC2-FLAG, pAAV-CBA-cTSC2+TSC1-FLAG, TSC1-FLAG+TSC2-FLAG, and TSC1-FLAG vectors. The expression levels of phosphorylated S6 (pS6), S6, and GAPDH are detected by immunoblotting (e.g., Western blotting). Although pS6 kinase levels are normally elevated in the absence of tuberin activity, cells transfected with the plasmids provided herein may show low pS6 levels, which would indicate reduced pS6 kinase activity.

[0153] Example 3: Expression of cTuberin in vivo The efficacy of the vectors of the present disclosure was confirmed by using Tcs2 as described by Onda et al. (Onda et al., J. Clin. Invest. 104(6):687-695, 1999). c / c Further evaluation will be performed using mice lacking tuberin, such as floxed mice. c / c The allele is converted to a null allele and the lacZ allele expresses β-galactosidase. Such mice have been observed to be healthy and have normal life spans.

[0154] Intracerebroventricular (ICV) and / or retroorbital (RO) injections are performed using the selected vectors described in Example 1. ICV injections are performed early in the mouse life cycle, such as postnatal day (PND) 0, 1, 2, 3, 4, or 5. RO injections are performed later in the mouse life cycle, such as between PND 7-35, such as PND 21. Injected titers are 1 x 10 per milliliter. 11 Genome copies (gc / mL) ~ 1 x 10 14 The survival time will be monitored. For pS6, the brains of tuberin-deficient and treated mice will be studied using hematoxylin and eosin (H&E) staining and / or immunohistochemistry analysis.

[0155] One study will evaluate the effect of a cTuberin vector, such as that described in Example 1, on survival. c / c Mice are injected ICV on PND0 or PND3 with a Cre-carrying vector such as AAV1-CBA-Cre, or RO on P21 with a c-tuberin vector as described in Example 1, or not injected. At PND3, the cerebrospinal fluid (CSF) barrier may be somewhat less permeable than at P0, so less depletion of tuberin in the brain may be observed. Survival time is monitored. The survival time of mice injected with the c-tuberin vector was expected to be longer than that of mice injected with the Cre vector. The survival time of mice injected with the c-tuberin vector may be comparable to that of mice not injected (e.g., more than 175 days).

[0156] In another study, the efficacy of the cTuberin vector described in Example 1 is evaluated. All mice are injected ICV on PND0 or PND3 with a Cre-carrying vector, such as AAV1-CBA-Cre. A first group of mice is then RO injected on PND21 with the cTuberin vector described in Example 1, and a second group of mice is not injected again. The survival time of mice injected with the cTuberin vector is expected to be longer than that of mice not injected with the cTuberin vector. The brains of tuberin-deficient and cTuberin-treated mice are studied for pS6 after sacrifice (e.g., on PND27) using H&E staining or IHC. In mice lacking tuberin, ependymal cell proliferation, enlargement of pyramidal cells in the hippocampus, subependymal nodes, and multiple subependymal nodes, as well as proliferation, can be observed. In cTuberin-treated mice, the brain tissue may appear more similar to that of normal uninjected (e.g., control) brains.

[0157] Another study evaluates the efficacy of a combination of cTuberin vectors, such as those described in Example 1, with anticonvulsants. TSC2 mutations are associated with early onset and high frequency of seizures. Thus, a combination of cTuberin vectors with anticonvulsants may be effective in treating subjects with TSC2. All mice are ICV injected with a Cre-carrying vector, such as AAV1-CBA-Cre, at P0 or P3. A first group of mice is then RO injected with a cTuberin vector, as described in Example 1, from PND3 onwards, and a second group of mice is not injected again. A group of mice injected with the cTuberin vector is treated with an anticonvulsant, such as vigabatrin (e.g., 200 mg / kg), and another group of mice is not treated with the anticonvulsant. The survival time of mice injected with a cTuberin vector and treated with an anticonvulsant is expected to be longer than that of mice not injected with a cTuberin vector and not treated with a drug, mice not injected with a cTuberin vector and treated with a drug, and mice injected with a cTuberin vector and not treated with an anticonvulsant.

[0158] In further studies, the efficacy of cTuberin vectors such as those described in Example 1 is tested in vivo against lymphangioleiomyomatosis (LAM) tumors subcutaneously injected into NOD-SCID II2R gamma (NSG) mice. Fluc-expressing TSC2-null immortalized angiomyolipoma cells are suspended in serum medium, mixed with matrigel, and implanted subcutaneously on the backs of NSG mice. After several weeks (e.g., 4 weeks), mice are intraperitoneally injected with the Fluc substrate D-luciferin (LUCNA-1G) and the signal is detected with a Spectral and anesthesia system. Tumor volume is monitored via bioluminescence, e.g., at 1, 4, 6, 9, and 14 weeks. Tumors are injected or not injected with cTuberin vectors, e.g., at 4 and 9 weeks. By 14 weeks, tumors injected with cTuberin vectors are expected to stop increasing in size, while tumors not injected may continue to expand in volume.

[0159] Additional experimental details and related work can be found, for example, in International Patent Publication No. PCT / US2018 / 033247, filed May 17, 2018, which is incorporated by reference in its entirety. Numbered embodiments Embodiment 1: A truncated tuberin (c-tuberin) comprising (i) an N-terminal region capable of binding to hamartin, and (ii) a C-terminal GTPase-activating protein (GAP) region, said c-tuberin lacking amino acid residues 419 to 932 of SEQ ID NO: 1. Embodiment 2. The cTuberin according to embodiment 1, wherein said cTuberin further lacks amino acid residues 947 to 988 of SEQ ID NO:1. Embodiment 3. A cTuberin according to embodiment 1 or 2, wherein said cTuberin further lacks amino acid residues 1205 to 1271 of SEQ ID NO:1. Embodiment 4. cTuberin according to any one of embodiments 1 to 3, wherein said cTuberin further lacks amino acid residues 1336 to 1497 of SEQ ID NO:1. Embodiment 5. A cTuberin according to embodiment 1, wherein the C-terminal region comprises an amino acid sequence having at least 90% identity to one of SEQ ID NOs: 10 to 12. Embodiment 6. The cTuberin according to embodiment 1, wherein said cTuberin further lacks amino acid residues 933 to 1109 of SEQ ID NO:1. Embodiment 7. A cTuberin according to embodiment 6, wherein the C-terminal domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:8. Embodiment 8. A cTuberin according to any one of embodiments 1 to 7, wherein the N-terminal domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:5. Embodiment 9. A truncated tuberin (c-tuberin) comprising (i) an N-terminal region capable of binding to hamartin, and (ii) a C-terminal GTPase-activating protein (GAP) region, wherein said C-terminal region comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 7, and wherein said c-tuberin lacks amino acid residues 451 to 932 of SEQ ID NO: 1. Embodiment 10. The cTuberin of embodiment 9, wherein said cTuberin lacks amino acid residues 419 to 932 of SEQ ID NO:1. Embodiment 11. A cTuberin according to embodiment 9 or 10, wherein said C-terminal region comprises an amino acid sequence having at least 90% identity to SEQ ID NO:8. Embodiment 12. A cTuberin according to embodiment 9 or 10, wherein said C-terminal region comprises an amino acid sequence having at least 90% identity to SEQ ID NO:9. Embodiment 13. A cTuberin according to embodiment 9, wherein said N-terminal region comprises an amino acid sequence having at least 90% identity to SEQ ID NO:4. Embodiment 14. A cTuberin according to any one of embodiments 9 to 12, wherein the N-terminal region comprises an amino acid sequence having at least 90% identity to SEQ ID NO:5. Embodiment 15. The cTuberin of embodiment 9, wherein said cTuberin comprises an amino acid sequence having at least 90% identity to SEQ ID NO:14. Embodiment 16. The cTuberin of embodiment 9, wherein said cTuberin comprises an amino acid sequence having at least 90% identity to SEQ ID NO:15. Embodiment 17. The cTuberin of embodiment 9, wherein said cTuberin comprises an amino acid sequence having at least 90% identity to SEQ ID NO:16. Embodiment 18. A truncated tuberin (c-tuberin) comprising (i) an N-terminal region capable of binding to hamartin, and (ii) a C-terminal GTPase-activating protein (GAP) region, wherein said C-terminal region comprises an amino acid sequence having at least 90% identity to one of SEQ ID NOs: 10 to 12, and wherein said c-tuberin lacks amino acid residues 451 to 932 of SEQ ID NO: 1. Embodiment 19. The cTuberin of embodiment 18, wherein said cTuberin lacks amino acid residues 419 to 932 of SEQ ID NO:1. Embodiment 20. A cTuberin according to embodiment 18 or 19, wherein said C-terminal region comprises an amino acid sequence having at least 90% identity to SEQ ID NO:10. Embodiment 21. A cTuberin according to embodiment 18 or 19, wherein said C-terminal region comprises an amino acid sequence having at least 90% identity to SEQ ID NO:11. Embodiment 22. A cTuberin according to embodiment 18 or 19, wherein said C-terminal region comprises an amino acid sequence having at least 90% identity to SEQ ID NO:12. Embodiment 23. A cTuberin according to embodiment 18, wherein said cTuberin comprises an amino acid sequence having at least 90% identity to SEQ ID NO:17. Embodiment 24. The cTuberin of embodiment 18, wherein said cTuberin comprises an amino acid sequence having at least 90% identity to SEQ ID NO:18. Embodiment 25. A cTuberin according to embodiment 18, wherein said cTuberin comprises an amino acid sequence having at least 90% identity to SEQ ID NO:19. Embodiment 26. A cTuberin according to any one of embodiments 18 to 25, wherein the N-terminal region comprises an amino acid sequence having at least 90% identity to SEQ ID NO:5. Embodiment 27. A cTuberin according to any one of embodiments 1 to 26, wherein said cTuberin comprises a spacer sequence between said N-terminal region and said C-terminal region. Embodiment 28. A cTuberin according to embodiment 27, wherein the spacer sequence comprises the sequence of SEQ ID NO:2. Embodiment 29. A cTuberin according to embodiment 28, wherein the spacer sequence comprises the sequence of SEQ ID NO:3. Embodiment 30. A nucleic acid molecule encoding cTuberin according to any one of embodiments 1 to 29.

[0041] Embodiment 31. The nucleic acid molecule of embodiment 30, wherein the nucleic acid molecule is codon-optimized for expression in a human target cell. Embodiment 32. The nucleic acid molecule of embodiment 31, wherein the human target cell is a brain cell, a heart cell, a kidney cell, a skin cell, or a lung cell. Embodiment 33. The nucleic acid molecule according to any one of embodiments 30 to 32, wherein the nucleic acid molecule is operably linked to a regulatory control sequence. Embodiment 34 The nucleic acid molecule of embodiment 33, wherein the regulatory control sequence comprises a human cytomegalovirus (CMV) promoter, a chicken beta-actin (CBA) promoter, a Rous sarcoma virus (RSV) LTR promoter / enhancer, an SV40 promoter, a dihydrofolate reductase promoter, a phosphoglycerol kinase promoter, a CMV immediate / early gene enhancer / CBA promoter, a synapsin promoter, or a glial fibrillary acidic protein (GFAP) promoter. Embodiment 35. The nucleic acid molecule of embodiment 33, wherein the regulatory control sequence comprises a human cytomegalovirus (CMV) immediate / early gene enhancer / chicken beta-actin (CBA) promoter, and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0036] Embodiment 36. The nucleic acid molecule of embodiment 33, wherein the regulatory control sequence comprises a beta-glucuronidase (GUSB) promoter. Embodiment 37. The nucleic acid molecule according to any one of embodiments 30 to 36, wherein said nucleic acid molecule has at least 90% sequence identity to any one of SEQ ID NOs: 21 to 26. Embodiment 38. A nucleic acid molecule encoding c-tuberin comprising (i) an N-terminal region capable of binding to hamartin, and (ii) a C-terminal GTPase activating protein (GAP) region, said c-tuberin lacking amino acid residues 451 to 932 of SEQ ID NO:1, said nucleic acid molecule being operably linked to a regulatory control sequence comprising a beta-glucuronidase (GUSB) promoter. Embodiment 39. The nucleic acid molecule of embodiment 38, wherein the cTuberin lacks amino acid residues 451 to 1515 of SEQ ID NO:1.

[0039] Embodiment 40. The nucleic acid molecule of embodiment 38 or 39, wherein the C-terminal region comprises an amino acid sequence having at least 90% identity to SEQ ID NO:6. Embodiment 41. The nucleic acid molecule according to any one of embodiments 38 to 40, wherein the N-terminal region comprises an amino acid sequence having at least 90% identity to SEQ ID NO:4.

[0046] Embodiment 42. A nucleic acid molecule comprising an adeno-associated virus (AAV) expression cassette, the AAV expression cassette comprising, from 5' to 3': i) a 5' AAV inverted terminal repeat (ITR); ii) a nucleic acid molecule according to any one of embodiments 25 to 35; and iii) 3' AAV ITR. Embodiment 43. The nucleic acid molecule of embodiment 42, wherein the 5'ITR and / or the 3'ITR are derived from AAV2.

[0081] Embodiment 44. The nucleic acid molecule of embodiment 42 or 43, wherein the 5' AAV ITR sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:27. Embodiment 45. The nucleic acid molecule of any one of embodiments 42 to 44, wherein the 3′ AAV ITR sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:28. Embodiment 46. The nucleic acid molecule of any one of embodiments 42 to 45, wherein the AAV expression cassette further comprises a polyadenylation sequence. Embodiment 47. The nucleic acid molecule of any one of embodiments 42 to 46, wherein the AAV expression cassette further comprises a Kozak sequence. Embodiment 48. A plasmid comprising the nucleic acid molecule according to any one of embodiments 30 to 47. Embodiment 49. A host cell comprising a nucleic acid molecule according to any one of embodiments 30 to 47, or a plasmid according to embodiment 48. Embodiment 50. A composition comprising a nucleic acid molecule according to any one of embodiments 30 to 47, a plasmid according to embodiment 48, or a host cell according to embodiment 49. Embodiment 51. A method for producing a recombinant adeno-associated virus (rAAV), comprising contacting a host cell with a nucleic acid molecule described in any one of embodiments 30 to 47 or a plasmid described in embodiment 48.

[0081] Embodiment 52. A recombinant adeno-associated virus (rAAV) produced by the method of embodiment 51. Embodiment 53. A recombinant adeno-associated virus (rAAV) comprising an AAV capsid protein and a nucleic acid molecule described in any one of embodiments 30 to 47. Embodiment 54. The rAAV of embodiment 52 or 53, wherein the rAAV comprises an AAV1 capsid protein, an AAV2 capsid protein, an AAV3 capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV10 capsid protein, an AAVrhlO capsid protein, an AAV11 capsid protein, and / or an AAV12 capsid protein. Embodiment 55. A method for expressing cTuberin in a target cell, comprising contacting the target cell with a nucleic acid molecule described in any one of embodiments 30 to 47, a plasmid described in embodiment 48, a composition described in embodiment 50, or an rAAV described in any one of embodiments 52 to 54, thereby expressing cTuberin in the target cell.

[0046] Embodiment 56. The method of embodiment 55, wherein the contacting step is carried out in vitro, ex vivo, or in vivo. Embodiment 57. The method of embodiment 56, wherein the contacting step is performed in vivo in a subject in need of treatment.

[0046] Embodiment 58. The method of embodiment 57, wherein the contacting step comprises administering a therapeutically effective amount of the nucleic acid molecule, the plasmid, the composition, or the rAAV to the subject. Embodiment 59: A method for treating a subject having tuberous sclerosis complex (TSC), comprising administering to the subject a therapeutically effective amount of cTuberin described in any one of embodiments 1-29, a nucleic acid molecule described in any one of embodiments 30-47, one or more extracellular vesicles (EVs) comprising a nucleic acid molecule described in any one of embodiments 30-47, a plasmid described in embodiment 48, a composition described in embodiment 50, or a rAAV described in any one of embodiments 52-54, thereby treating TSC in the subject. Embodiment 60. A method of treating a subject having renal cancer, comprising administering to the subject a therapeutically effective amount of cTuberin described in any one of embodiments 1 to 29, a nucleic acid molecule described in any one of embodiments 30 to 47, one or more extracellular vesicles (EVs) comprising a nucleic acid molecule described in any one of embodiments 30 to 47, a plasmid described in embodiment 48, a composition described in embodiment 50, or an rAAV described in any one of embodiments 52 to 54, thereby treating renal cancer in the subject. Embodiment 61. The method of any one of embodiments 57 to 60, wherein the cTuberin, the nucleic acid molecule, the plasmid, the composition, or the rAAV is administered intravascularly, intrarenal artery or vein, intrapulmonary, intracisternomatous, intracerebral, intrathecal, intravenous, intraventricular, intralateral ventricular, intraperitoneal, or dermal. Embodiment 62. The method of any one of embodiments 57-61, wherein the subject has renal angiomyolipoma. Embodiment 63. The method of embodiment 62, wherein the cTuberin, the nucleic acid molecule, the plasmid, the composition, or the rAAV targets the renal angiomyolipoma. Embodiment 64. The method of any one of embodiments 57-63, wherein the subject has lymphangioleiomyomatosis (LAM). Embodiment 65. The method of embodiment 64, wherein the cTuberin, the nucleic acid molecule, the plasmid, the composition, or the rAAV targets the LAM. Embodiment 66. The method of any one of embodiments 57-65, wherein the subject has brain dysfunction. Embodiment 67. The method of embodiment 66, wherein the cTuberin, the nucleic acid molecule, the plasmid, the composition, or the rAAV is provided to the subarachnoid space. Embodiment 68. The method of any one of embodiments 57 to 67, wherein the cTuberin, the nucleic acid molecule, the plasmid, the composition, or the rAAV is administered to a brain cell, a heart cell, a kidney cell, a skin cell, or a lung cell. Embodiment 69. The method of any one of embodiments 57-68, wherein the subject is administered rapamycin. Embodiment 70. The method of any one of embodiments 57 to 69, wherein the subject is a human. Embodiment 71. The method of any one of embodiments 57-70, wherein the subject is under 18 years of age. Embodiment 72. The method of embodiment 71, wherein the subject is an infant. Embodiment 73. The method of any one of embodiments 57-72, wherein the subject has been diagnosed with tuberous sclerosis complex. Embodiment 74. The method of any one of embodiments 57-73, wherein the subject has a mutation in the TSC2 gene.

[0073] Embodiment 75. The method of embodiment 74, wherein the subject has a mutation in exon 33, exon 37, and / or exon 38 of the TSC2 gene. Embodiment 76. The method of any one of embodiments 57-75, wherein the subject has one or more of the following: cortical nodules, subependymal nodules, and subependymal giant cell astrocytomas.

Claims

1. A shortened tuberine (c-tuberine) comprising (i) an N-terminal region capable of binding to hamartin, and (ii) a C-terminal GTPase activating protein (GAP) region, wherein the c-tuberine lacks amino acid residues 419-932 of SEQ ID NO:

1.

2. The c-tuberine according to claim 1, wherein the c-tuberine further lacks (a) amino acid residues 947-988 of SEQ ID NO: 1, (b) amino acid residues 1205-1271 of SEQ ID NO: 1, (c) amino acid residues 1336-1497 of SEQ ID NO: 1, or (d) amino acid residues 933-1109 of SEQ ID NO:

1.

3. The c-tuberine according to claim 1, wherein the C-terminal region comprises an amino acid sequence having at least 90% identity with one of sequence numbers 8 or 10 to 12.

4. The c-tuberine according to claim 1, wherein the N-terminal domain comprises an amino acid sequence having at least 90% identity with SEQ ID NO:

5.

5. A shortened tuberine (c-tuberine) comprising (i) an N-terminal region capable of binding to hamartin, and (ii) a C-terminal GTPase activating protein (GAP) region, wherein the C-terminal region comprises an amino acid sequence having at least 90% identity with one of SEQ ID NOs: 7 or SEQ ID NOs: 10-12, and the c-tuberine lacks amino acid residues 451-932 of SEQ ID NO:

1.

6. The c-tuberine according to claim 5, wherein the c-tuberine lacks amino acid residues 419 to 932 of SEQ ID NO:

1.

7. The c-tuberine according to claim 5, wherein the C-terminal region comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 8 or 9.

8. The c-tuberine according to claim 5, wherein the N-terminal region comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 4 or 5.

9. The c-tuberine according to claim 5, wherein the c-tuberine comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 14 to 19.

10. The c-tuberine according to claim 5, wherein the c-tuberine includes a spacer array between the N-terminal region and the C-terminal region.

11. The c-tuberine according to claim 10, wherein the spacer array includes the array of sequence number 2 or 3.

12. A nucleic acid molecule encoding c-tuberine as described in Claim 1.

13. The nucleic acid molecule according to claim 12, wherein the nucleic acid molecule has at least 90% sequence identity with any one of sequence numbers 21 to 26.

14. A nucleic acid molecule encoding c-tuberine comprising (i) an N-terminal region capable of binding to hamartin, and (ii) a C-terminal GTPase activating protein (GAP) region, wherein the c-tuberine lacks amino acid residues 451-932 of SEQ ID NO: 1, and the nucleic acid molecule is operably linked to a regulatory sequence comprising a beta-glucuronidase (GUSB) promoter.

15. A nucleic acid molecule comprising an adeno-associated virus (AAV) expression cassette, wherein the AAV expression cassette is configured such that it is divided from 5' to 3'. 5'AAV inverted terminal repeat (ITR) and, The nucleic acid molecule according to claim 12, The nucleic acid molecule comprising 3'AAV ITR.

16. A plasmid comprising the nucleic acid molecule described in Claim 12.

17. A host cell comprising the nucleic acid molecule described in Claim 12, or a plasmid containing the same.

18. A composition comprising the nucleic acid molecule described in claim 12, a plasmid containing the same, or a host cell containing the plasmid containing the same.

19. A method for producing recombinant adeno-associated virus (rAAV), comprising contacting a host cell with the nucleic acid molecule described in claim 12 or a plasmid containing the same.

20. Recombinant adeno-associated virus (rAAV) produced by the method described in Claim 19.

21. Recombinant adeno-associated virus (rAAV) comprising an AAV capsid protein and the nucleic acid molecule described in claim 12.

22. A composition for use in a method for expressing c-tuberine in target cells, wherein the composition comprises the nucleic acid molecule described in claim 12, and the method comprises contacting the target cells with the nucleic acid molecule.

23. A composition for treating a subject having tuberous sclerosis complex (TSC), comprising the rAAV described in claim 21.

24. A composition for treating a subject having renal cancer, comprising the rAAV described in Claim 21.