Adeno-associated vectors and particles to treat tuberous sclerosis complex caused by TSC2 gene mutations and methods of use and manufacture

Abstract

Provided herein is a rAAV-mediated TSC2 "split intein" gene therapy that uses two separate rAAV particles, each engineered to deliver a different portion of the human TSC2 coding sequence, to target cells of the CNS such that, when co-expressed, the two portions of the TSC2 protein join via intein-mediated protein splicing to form a full length human TSC2 (tuberin) protein that exhibits TSC2 activity; viral particles (rAAV particles) for use therewith; methods of production, and methods of use, including methods for treating Tuberous Sclerosis Complex (TSC) caused by TSC2 gene mutations and symptoms arising therefrom; and kits.

Description

ATTY DOCKET NO.38213.0002P1ADENO-ASSOCIATED VECTORS AND PARTICLES TO TREAT TUBEROUS SCLEROSIS COMPLEX CAUSED BY TSC2 GENE MUTATIONS AND METHODS OF USE AND MANUFACTURE SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (File name: 38213.0002Pl_l; Size: 549,058 bytes; Created on: January 9, 2026) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] Provided are viral constructs, particles and compositions for use in the treatment of Tuberous Sclerosis Complex (TSC) caused by TSC2 gene mutations.BACKGROUND

[0003] Tuberous Sclerosis Complex (TSC) is a rare neurodevelopmental disorder affecting 1 / 6000 live births (Mowery et al. 2020). There are no differences in prevalence of TSC due to gender or race (Li et al. 2023; Ho et al. 2017). TSC is caused by autosomal dominant, loss-of-function mutations in the TSC1 or TSC2 genes, which encode the proteins for hamartin and tuberin, respectively. Hamartin, the protein encoded by TSC1, is a chaperone for tuberin, the protein encoded by TSC2, which regulates the activity of the mammalian target of rapamycin (mTOR) signaling pathway (Ho et al. 2017). Mutations in the TSC1 or TSC2 genes can lead to hyperactivation of the mTOR pathway, which results in abnormal cell growth, proliferation, metabolism, and epileptogenesis (Zeng et al. 2008). TSC is a leading genetic cause of epilepsy and autism. TSC-associated epilepsy generally begins during the first year of life and is associated with neurodevelopmental and cognitive problems. Management of epilepsy is challenging with existing therapies and medications, and seizures tend to persist in a large proportion of patients despite pharmacological and surgical treatment (Curatolo et al. 2012). Approximately 80 to 90% of individuals with TSC develop epilepsy, and more than 60% of these cases are resistant to antiseizure medications (Chu-Shore et al. 2010). One treatment option for refractory seizures in epilepsy involves invasive surgery; however, this results in only 50-60% of the patients being seizure-free (Curatolo et al. 2012, Moavero et al. 2010, Jansen et al. 2007).

[0004] The vast majority of TSC patients experience seizures, with approximately two thirds of those patients having drug-resistant epilepsy (Chu-Shore et al. 2010), also known as refractory epilepsy, defined as failing to reach epilepsy control after the use of two or moreantiepileptic medications (Engel 2014). Poor seizure control is associated with neurodevelopmental delays, loss of neurocognitive functions and capabilities, loss of learned skills, slower gain in skills, behavioral difficulties, and long-term intellectual disabilities (Tye et al. 2020, Capal et al. 2017). TSC has an extensive body of published data and patient and caregiver testimony, as assembled in the Voice of the Patient report published in 2017 by the TSC Alliance (Ho et al. 2017). As noted in the report, while there are several approved drugs for the management of some symptoms, there is still an unmet need for treatment options, especially with regards to the treatment of drug-resistant epilepsy in TSC patients. For example, mTOR inhibitors including Everolimus; anti-epileptics Vigabatrin, and Epidiolex are approved for TSC-associated epilepsy and partial onset seizures, but many patients remain refractory and report epilepsy has a major impact on daily living (Ho et al. 2017, Chu-Shore et al. 2010). In fact, prophylactic use of either Everolimus or Vigabatrin has failed to demonstrate benefit in pediatric patients as compared to placebo, as detailed by two multicenter double-blind placebo-controlled trials (Kotulska et al.2021, Bebin et al. 2023).

[0005] There is a significant unmet need for improving treatment for TSC-associated disorders. At present, there are no approved cell or gene therapies for TSC. Accordingly, there is a need for a genetic therapy that provides for expression of a functional TSC2 protein in the CNS to provide therapeutic benefit to patients suffering from disorders caused by TSC2 gene mutations, such as TSC.

[0006] Recombinant AAV (rAAV) particles engineered to express therapeutic genes have been used as gene transfer vectors for various gene therapies. (E.g., Simonelli et al., 2009.) However, the use of rAAV to deliver a functional TSC2 protein is problematic because the TSC2 gene is ~5.4 kb, whereas rAAV particles have a maximum transgene capacity of ~5kb. In previous studies, survival in a TSC2 knockout mouse model was shown to be improved by delivering an engineered TSC2 gene that encoded a condensed TSC2 protein using rAAV9 particles. (Cheah et al. 2021). However, the rescue of survival was not complete, and the condensed TSC2 protein used in this study did not exhibit the same in vitro activity as the full-length TSC2 protein. (Id., at 4.) Further, this condensed TSC2 protein excluded amino acids 451 through 1514 of the full-length TSC2 protein, which include HEAT repeat domains that are recognized as contributing to TSC2 dimerization and lysosomal localization, which are known to be required for mTOR regulation. (Yang et al. 2021.) Pathological TSC2 variants associated with TSC have also been identified inthe HEAT repeat domains, further underscoring their importance to TSC2 function. (Ogorek et al., 2020.)

[0007] Another obstacle for rAAV-mediated delivery of TSC2 gene therapy is that the target organ system for this AAV gene therapy approach, the central nervous system (CNS), is predominantly post-mitotic cells and would require sustained transgene expression (Bartel et al.2012, Hammond et al. 2017, Fuentes & Schaffer 2018). As such, genetic therapies intended for treating patients suffering from TSC-related neurological disorders, such as epilepsy, will likely require broad delivery across the CNS. Unfortunately, delivery to the CNS has represented a significant challenge for the field of AAV gene therapy, as it requires crossing the blood-brain barrier (BBB), and naturally occurring AAV capsids have been shown to achieve limited biodistribution and transduction in the CNS.

[0008] Accordingly, there is a need for an improved rAAV TSC2 gene therapy that both (i) transduces a sufficient number of CNS target cells, and (ii) provides for the expression of a full length, functional TSC2 protein to provide a therapeutic benefit to subjects suffering from disorders such as TSC.SUMMARY OF INVENTION

[0009] Provided herein is a rAAV-mediated TSC2 “split intein” gene therapy that uses two separate rAAV particles, each engineered to deliver a different portion of the human TSC2 coding sequence, to target cells of the CNS such that, when expressed, the two portions of the TSC2 protein join via intein-mediated protein splicing to form a full length human TSC2 (tuberin) protein that exhibits TSC2 activity. The first rAAV particle (rAAV-n-TSC2 / n-intein) is engineered to comprise an expression cassette comprising regulatory polynucleotide sequences sufficient to express a polynucleotide sequence (n-TSC2 / n-intein polynucleotide sequence) that encodes an amino (N-terminal) portion of the human TSC2 protein (N-TSC2) that is fused at its C-terminus to the N-terminus of the N-terminal portion of an intein amino acid sequence (N-Intein) (together, N-TSC2 / N-Intein amino acid sequence). The second rAAV particle (rAAV-c-intein / c-TSC2) is engineered to comprise an expression cassette comprising regulatory polynucleotide sequences sufficient to express a polynucleotide sequence (c-intein / c-TSC2 polynucleotide sequence) that encodes the carboxyl (C-terminal) portion of the intein amino acid sequence (C-Intein) that is fused at its C-terminus to the N-terminus of a C-terminal portion of the human TSC2 protein (C-TSC2)(together, C-Intein / C-TSC2 amino acid sequence). Although not to be bound to a particular mechanism of action, it is thought that when these separate n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed at a sufficient level in the same cell, the resulting N-Intein and C-Intein amino acid sequences associate, and are excised from the associated N-TSC2 and C-TSC2 amino acid sequences via intein-mediated protein splicing. During this process, the N-TSC2 and C-TSC2 amino acid sequences are joined together into a full-length TSC2 protein that does not include the excised N-Intein and C-Intein amino acid sequences, and exhibits TSC2 activity. A basic schematic of this “split intein” TSC2 expression strategy is shown in FIG. 1.

[0010] The rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein can utilize various known inteins, so long as (i) both the n-TSC2 / n-intein polynucleotide sequence and the c-intein / c-TSC2 polynucleotide sequence are of suitable length to be included in separate rAAV vector constructs that further comprise regulatory sequences (e.g., promoters, polyadenylation signal sequences, etc.), as well as flanking AAV inverted terminal repeat (ITR) nucleotide sequences, and such rAAV vector constructs are of a suitable length (i.e., less than ~5kb in length) to allow for packaging within an rAAV particle for delivery to target cells, including cells of the CNS, and (ii) when co-expressed in such target cells, the N-TSC2 and C-TSC2 amino acid sequences join together into a full-length protein that has TSC2 protein activity. In embodiments, the n-intein and c-intein polynucleotide sequences are obtained from the DnaE polymerase of Nostoc punctiforme bacteria (for example, Npu DnaE and Cfa DnaE intein sequences), as described, for example, by Shah et al. (2013), Stevens et al. (2017), and in U.S. Patent No. 10,100,080, which are incorporated herein by reference. In other embodiments, the n-intein and c-intein polynucleotide sequences are engineered variants of Npu intein sequences, including for example the NpuGEP and CfaGEP variants described by Stevens et al. (2017), which is incorporated herein by reference. In embodiments, the n-intein polynucleotide sequence has, comprises, or consists of the polynucleotide sequence of SEQ ID NO.: 1, and encodes an N-Intein having the amino acid sequence of SEQ ID NO.: 2, and the c-intein polynucleotide sequence has, comprises, or consists of the polynucleotide sequence of SEQ ID NO.: 3, and encodes a C-Intein having the amino acid sequence of SEQ ID NO.: 4. In other embodiments, the n-intein polynucleotide sequence has, comprises, or consists of the polynucleotide sequence of SEQ ID NO.: 5, and encodes an N-Intein having the amino acid sequence of SEQ ID NO.: 6, and the c-intein polynucleotide sequence has, comprises, or consists of the polynucleotide sequence of SEQ ID NO.: 7, and encodes a C-Intein having the amino acid sequence of SEQ ID NO.: 8.

[0011] In embodiments, the rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein comprises a first polynucleotide sequence (n-TSC2 / n-intein polynucleotide sequence ) encoding an N-terminal portion of the human TSC2 protein fused at its C-terminus to an the N-terminus of an N-Intein polypeptide, the N-Intein polypeptide having, comprising, or consisting of the amino acid sequence of SEQ ID NO.: 2, and a second polynucleotide sequence (c-intein / c-TSC2 polynucleotide sequence) encoding a C-Intein polypeptide having, comprising, or consisting of the amino acid sequence of SEQ ID NO.: 4 fused at its C-terminus to the N-terminus of a C-terminal portion of the human TSC2 protein, whereby when the first and second polynucleotide sequences are coexpressed in the same cell, the N-terminal TSC2 polypeptide and the C-terminal TSC2 polypeptide are joined together (through the intein-mediated protein splicing mechanism) to form a human TSC2 polypeptide (having TSC2 activity) with an amino acid sequence having, comprising, or consisting of either (i) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, the amino acid sequence of, SEQ ID NO.: 10 (human TSC2), or (ii) the amino acid sequence of SEQ ID NO.: 10 (human TSC2) wherein one or more amino acids are replaced with a complementary amino acid, and the resulting polypeptide exhibits TSC2 activity. In embodiments, the first (n-TSC2 / n-intein) polynucleotide sequence and the second (c-intein / c-TSC2) polynucleotide sequence are each of suitable length that, when included in separate rAAV vector constructs that further comprise regulatory sequences (e.g., promoters, polyadenylation signal sequences, etc.), as well as flanking AAV inverted terminal repeat (ITR) nucleotide sequences, such rAAV vector constructs are of a suitable length (i.e., less than ~5kb in length) to allow for packaging within an rAAV particle for delivery to target cells, including cells of the CNS.

[0012] In embodiments, the rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein comprises (i) a first rAAV particle (rAAV-n-TSC2 / n-intein particle) engineered to deliver and express a first polynucleotide sequence encoding an N-terminal portion of the human TSC2 protein fused at its C-terminus to the N-terminus of an N-Intein polypeptide, the N-Intein polypeptide having, comprising, or consisting of the amino acid sequence of SEQ ID NO.: 2, and(ii) a second rAAV particle (rAAV-c-intein / c-TSC2 particle) engineered to deliver and express a second polynucleotide sequence encoding a C-Intein polypeptide having, comprising, or consisting of the amino acid sequence of SEQ ID NO.: 4 fused at its C-terminus to the N-terminus of a C-terminal portion of the human TSC2 protein, whereby when the first and second rAAV particles transduce the same cell, the first and second polynucleotide sequences are coexpressed, and the N-terminal TSC2 polypeptide fragment and the C-terminal TSC2 polypeptide fragment are joined together (through the intein-mediated protein splicing mechanism) to form a polypeptide with an amino acid sequence having, comprising, or consisting of either (i) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, the amino acid sequence of, SEQ ID NO.: 10 (human TSC2), or (ii) the amino acid sequence of SEQ ID NO.: 10 (human TSC2) wherein one or more amino acids are replaced with a complementary amino acid, and the resulting polypeptide exhibits TSC2 activity.

[0013] In embodiments, the rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein comprises a first polynucleotide sequence encoding an N-terminal portion of the human TSC2 protein fused at its C-terminus to the N-terminus of an N-Intein polypeptide, the N-Intein polypeptide having, comprising or consisting of the amino acid sequence of SEQ ID NO.: 6, and a second polynucleotide sequence encoding a C-Intein polypeptide having, comprising or consisting of the amino acid sequence of SEQ ID NO.: 8 fused at its C-terminus to the N-terminus of a C-terminal portion of the human TSC2 protein, whereby when the first and second polynucleotide sequences are coexpressed in the same cell, the N-terminal polypeptide and the C-terminal polypeptide are joined together to form a polypeptide with an amino acid sequence having, comprising, or consisting of either (i) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, the amino acid sequence of, SEQ ID NO.: 10, or (ii) the amino acid sequence of SEQ ID NO.: 10 wherein one or more amino acids are replaced with a complementary amino acid, and the resulting polypeptide exhibits TSC2 activity.

[0014] In embodiments, the rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein comprises (i) a first rAAV particle engineered to deliver and express a firstpolynucleotide sequence encoding an N-terminal portion of the human TSC2 protein fused at its C-terminus to the N-terminus of an N-Intein polypeptide, the N-Intein polypeptide having, comprising or consisting of the amino acid sequence of SEQ ID NO.: 6, and (ii) a second rAAV particle engineered to deliver and express a second polynucleotide sequence encoding a C-Intein polypeptide having, comprising or consisting of the amino acid sequence of SEQ ID NO.: 8 fused at its C-terminus to the N-terminus of a C-terminal portion of the human TSC2 protein, whereby when the first and second rAAV particles transduce the same cell, the first and second polynucleotide sequences are coexpressed, and the N-terminal polypeptide and the C-terminal polypeptide are joined together to form a polypeptide with an amino acid sequence having, comprising, or consisting of either (i) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, the amino acid sequence of, SEQ ID NO.: 10, or (ii) the amino acid sequence of SEQ ID NO. : 10 wherein one or more amino acids are replaced with a complementary amino acid, and the resulting polypeptide exhibits TSC2 activity.

[0015] In embodiments, each of the separate n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences of the first and second rAAV particles described above and herein is operably joined to an upstream (5’) polynucleotide promoter sequence sufficient to drive expression of the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences in target cells, including cells of the CNS, such that when n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed, the N-TSC2 polypeptide and the C-TSC2 polypeptide are joined together to form a full length TSC2 polypeptide having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within the amino acid sequence of SEQ ID NO.: 10. In embodiments, one or both of the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences of the first and second rAAV particles described above and herein are operably joined to an upstream (5’) polynucleotide promoter sequence comprising an EFS promoter (SEQ ID NO.: 67), which includes the core promoter element of the EFla promoter; a CBA AGTl promoter (SEQ ID NO.: 68), which is a variant of the chicken beta-actin promoter; a CBA AGT2 promoter (SEQ ID NO.: 69), which is a variant of the chicken beta-actin promoter; aCMV promoter (SEQ ID NO. : 70), which includes the CMV enhancer / promoter sequences; a CEG promoter (SEQ ID NO.: 71), which is a composite, synthetic promoter that contains CMV enhancer and promoter sequences, and chimeric intron and exon from the chicken P-actin promoter; or a CAG promoter (SEQ ID NO.: 72), which is a composite, synthetic promoter which contains the CMV early enhancer element, the chicken P-actin promoter and the first exon and first intron of the chicken P-actin gene, and the splice acceptor of the rabbit P globin gene (Miyazaki et al. 1989; Niwaet al. 1991), or other promoter sequences capable of driving expression of the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences of the first and second rAAV particles in target cells, including cells of the CNS.

[0016] In embodiments, each of the separate n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences of the first and second rAAV particles described above and herein are operably joined to a downstream (3’) polyadenylation (poly(A) signal sequence that promotes expression of the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences in target cells, including cells of the CNS, such that when n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed, the N-TSC2 polypeptide and the C-TSC2 polypeptide are joined together to form a full length TSC2 polypeptide having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within the amino acid sequence of SEQ ID NO.: 10. In embodiments, one or both of the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences of the first and second rAAV particles described above and herein are operably joined to a downstream (3’) a human growth hormone polyadenylation signal sequence (SEQ ID NO. : 73 (huGHpA)), or to variants of the bovine growth hormone polyadenylation signal sequence (SEQ ID NO.: 74 (bGHpA) and SEQ ID NO.: 75 (bGH_v3pA)), or to other poly(A) signal sequences capable of promoting expression of the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences of the first and second rAAV particles in target cells, including cells of the CNS.

[0017] Also provided herein are (i) n-TSC2 / n-intein expression cassettes comprising (from 5’ to 3’) a promoter sequence that promotes expression in the CNS, including one of the promoter polynucleotide sequences described herein, operably joined to an n-TSC2 / n-intein polynucleotide sequence described herein, operably joined to a polyadenylation signal, including one of thepolyadenylation signal sequences described herein; and (ii) c-intein / c-TSC2 expression cassettes comprising (from 5’ to 3’) a promoter sequence that promotes expression in the CNS, including one of the promoter polynucleotide sequences described herein, operably joined to a c-intein / c-TSC2 polynucleotide sequences described herein, operably joined to a polyadenylation signal, including one of the polyadenylation signal sequences described above and herein.

[0018] Also provided are rAAV vector constructs wherein AAV inverted terminal repeat (1TR) nucleotide sequences flank the n-TSC2 / n-intein expression cassettes described above and herein to allow for the generation of recombinant AAV (rAAV) particles that are administered for gene therapy in a subject suffering from TSC pursuant to the rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein. Also provided are rAAV vector constructs wherein AAV inverted terminal repeat (ITR) nucleotide sequences flank the intein / c-TSC2 expression cassettes described above and herein to allow for the generation of recombinant AAV (rAAV) particles that are administered for gene therapy in a subject suffering from TSC pursuant to the rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein.

[0019] Also provided are plasmids comprising the vector constructs and expression cassettes described above and herein.

[0020] To facilitate this rAAV-mediated TSC2 “split intein” gene therapy, in embodiments either or both of (i) the first rAAV particle (rAAV-n-TSC2 / n-intein particle) and (ii) the second rAAV particle (rAAV-c-intein / c-TSC2 particle) are engineered to include one or more capsid modifications to enhance CNS delivery, evade immune clearance, or both.

[0021] In embodiments, rAAV particles of the present disclosure are engineered to cross the blood-brain barrier (BBB) and transduce cells of the CNS through binding to the hTfRl receptor (hTfRl rAAV particle). In embodiments, these hTfRl rAAV particles comprise a capsid protein that is engineered to bind the hTfRl receptor (hTfRl rAAV capsid protein), and further comprise a recombinant AAV genome containing an expression cassette that encodes either the n-TSC2 / n-intein or the c-intein / c-TSC2 polynucleotide sequences described above, operably linked to regulatory elements. Although not to be bound to a particular mechanism of action, these hTfRl rAAV capsid proteins, derived from the naturally occurring AAV9 capsid, are thought to confer enhanced ability to cross the BBB, likely via human transferrin receptor 1 (hTfRl) receptor mediated transcytosis (RMT), thereby providing for efficient delivery throughout the CNS.

[0022] In embodiments, the rAAV particles of the present disclosure are engineered to bind human GPI-linked enzyme Carbonic anhydrase IV (hCA4) (hCA4 rAAV particle). In embodiments, these hCA4 rAAV particles comprise a capsid protein that is engineered to bind hCA4 (hCA4 rAAV capsid protein), and further comprise a recombinant AAV genome containing an expression cassette that encodes either the n-TSC2 / n-intein or the c-intein / c-TSC2 polynucleotide sequences described above, operably linked to regulatory elements. Although not to be bound to a particular mechanism of action, these hCA4 AAV capsid proteins, derived from the naturally occurring AAV9 capsid, are thought to confer enhanced CNS tropism through hCA4 binding.

[0023] In embodiments, the rAAV particles of the present disclosure are engineered to bind the human CD59 cell surface protein (hCD59) (hCD59 rAAV particle). In embodiments, these 11CD59 rAAV particles comprise a capsid protein that is engineered to bind hCD59 (hCD59 rAAV capsid protein), and further comprise a recombinant AAV genome containing an expression cassette that encodes either the n-TSC2 / n-intein or the c-intein / c-TSC2 polynucleotide sequences described above, operably linked to regulatory elements. Although not to be bound to a particular mechanism of action, these hCD59 rAAV capsid proteins, derived from the naturally occurring AAV9 capsid, are thought to confer enhanced CNS tropism through hCD59 binding.

[0024] In embodiments, the rAAV particles of the present disclosure are engineered to bind human GPI-linked alkaline phosphatase (ALPL) (ALPL rAAV particle). In embodiments, the ALPL rAAV particles comprise a capsid protein that is engineered to bind ALPL (ALPL rAAV capsid protein), and further comprise a recombinant AAV genome containing an expression cassette that encodes either the n-TSC2 / n-intein or the c-intein / c-TSC2 polynucleotide sequences described above, operably linked to regulatory elements. Although not to be bound to a particular mechanism of action, these ALPL rAAV capsid proteins are thought to confer enhanced ability to cross the BBB, likely via ALPL binding, thereby providing for efficient delivery throughout the CNS.

[0025] In embodiments, the rAAV particles of the present disclosure are engineered to include an AAV capsid protein (rAAV(AE) capsid protein) with one or more amino acid substitutions that allow such rAAV particles (rAAV(AE) particles) to evade immune clearance arising from neutralizing antibodies, avoid compliment activation and reduce the incidence ofimmune response in treated subjects, and avoid antibody binding / inhibition that renders the capsids unable to access their target receptor on cells, and further comprise a recombinant AAV genome containing an expression cassette that encodes either the n-TSC2 / n-intein or the c-intein / c-TSC2 polynucleotide sequences described above, operably linked to regulatory elements. In embodiments, the rAAV(AE) capsid protein modifications described above and herein are made in capsid proteins having a targeting peptide, such as a hTfRl, CA4, CD59 or ALPL targeting peptide described above and herein, that increase activity of the rAAV particle incorporating the capsid protein to cross the BBB and / or transduce cells of the CNS, such that the resulting rAAV particles have increased BBB-crossing and CNS cell transduction activity and immune evasion activity, including relative to a reference capsid not having the modifications, such as an AAV9 capsid, and further comprise a recombinant AAV genome containing an expression cassette that encodes either the n-TSC2 / n-intein or the c-intein / c-TSC2 polynucleotide sequences described above, operably linked to regulatory elements.

[0026] Also provided are methods of treating Tuberous Sclerosis Complex (TSC), or increasing TSC2 enzyme activity, or reducing hyperactivation of the mTOR pathway, in a subject in need thereof, particularly, in a human subject. Also included are methods of reducing a disease condition in a subject suffering from TSC by co-administering the first (rAAV-n-TSC2 / n-intein) and second (rAAV-c-intein / c-TSC2) rAAV particles described herein, wherein the disease condition comprises abnormal cell growth and / or proliferation, metabolic disorders, epilepsy, seizure, neurodevelopmental disorders and / or cognitive disorders, particularly in human subjects. Provided are pharmaceutical gene therapy compositions including for use in treating TSC, or increasing TSC2 activity, or reducing hyperactivation of the mTOR pathway in a subject in need thereof and methods of administration, including, but not limited to, intravenous administration or intrathecal administration. The methods provide administration of rAAV particles described herein for gene therapy in a subject suffering from TSC, said rAAV particles being capable of delivering a functional copy of the TSC2 (tuberin) protein to target cells of CNS, thereby providing increased TSC2 (tuberin) activity to treat and ameliorate disease.

[0027] Also provided are host cells for and methods of producing the recombinant AAV particles as described herein.

[0028] Also provided are methods of increasing TSC2 enzyme activity in a subject in need thereof including in cells and tissues of the central nervous system (CNS).EMBODIMENTS

[0029] Embodiment 1. A split-intein system for expressing a protein of interest in a cell, the split-intein system comprising:a) a first rAAV particle comprising a first expression cassette comprising a polynucleotide sequence encoding an N-TSC2 / N-Intein polypeptide, said N-TSC2 / N-Intein polypeptide comprising an N-terminal polypeptide fragment of the human TSC2 protein, fused at its C- terminus to the N-terminus of an N-Intein polypeptide, flanked by ITR sequences, andb) a second rAAV particle comprising a second expression cassette comprising a polynucleotide sequence encoding a C-Intein / C-TSC2 polypeptide, said C-Intein / C-TSC2 polypeptide comprising a C-Intein polypeptide fused at its C-terminus to the N-terminus of a C-terminal polypeptide fragment of the human TSC2 protein, flanked by ITR sequences,wherein the N-terminal polypeptide fragment and C-terminal polypeptide fragment together comprise a full-length human TSC2 protein having an amino acid sequence that is at least 90% identical, at least 95%, at least 99%, or 100% to the amino acid sequence of SEQ ID NO.: 10.

[0030] Embodiment 2. The split-intein system of Embodiment 1, wherein the N-Intein polypeptide comprises or consists of the amino acid sequence of SEQ ID NO.: 2, and the C-Intein polypeptide comprises the amino acid sequence of SEQ ID NO.: 4.

[0031] Embodiment 3. The split-intein system of Embodiment 1, wherein the N-Intein polypeptide comprises the amino acid sequence of SEQ ID NO.: 6, and the C-Intein polypeptide comprises the amino acid sequence of SEQ ID NO.: 8.

[0032] Embodiment 4. The split-intein system of any one of Embodiments 1 through 3, wherein the N-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 12, and the C-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 14.

[0033] Embodiment 5. The split-intein system of any one of Embodiments 1 through 3, wherein the N-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO. : 20, and the C-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 22.

[0034] Embodiment 6. The split-intein system of any one of Embodiments 1 through 3, wherein the N-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO. : 28, and the C-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 30.

[0035] Embodiment 7. The split-intein system of any one of Embodiments 1 through 3, wherein the N-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO. : 36, and the C-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 38.

[0036] Embodiment 8. The split-intein system of any one of Embodiments 1 through 3, wherein the N-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 44, and the C-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 46.

[0037] Embodiment 9. The split-intein system of any one of Embodiments 1 through 3, wherein the N-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 52, and the C-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 54.

[0038] Embodiment 10. The split-intein system of any one of Embodiments 1 through 3, wherein the N-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 60, and the C-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 62.

[0039] Embodiment 11. The split-intein system of any one of Embodiments 1 through 10, wherein the first expression cassette further comprises a first promoter polynucleotide sequence operably joined to the polynucleotide sequence encoding the N-TSC2 / N-Intein polypeptide, and promotes expression of the polynucleotide sequence encoding the N-TSC2 / N-Intein polypeptide in cells of the CNS, and the second expression cassette further comprises a second promoterpolynucleotide sequence operably joined to the polynucleotide sequence encoding the C-Tntein / C-TSC2 polypeptide, and promotes expression of the polynucleotide sequence encoding the C-Intein / C-TSC2 polypeptide in cells of the CNS, and wherein the first promoter polynucleotide sequence can either be the same as or different from the second promoter polynucleotide sequence.

[0040] Embodiment 12. The split-intein system of Embodiment 11, wherein the first promoter polynucleotide sequence comprises any one of the polynucleotide sequences of SEQ ID NO.: 67, SEQ ID NO.: 68, SEQ ID NO.: 69, SEQ ID NO.: 70, SEQ ID NO.: 71, or SEQ ID NO.: 72, and the second promoter polynucleotide sequence comprises any one of the polynucleotide sequences ofSEQ IDNO.: 67, SEQ ID NO.: 68, SEQ ID NO.: 69, SEQ ID NO.: 70, SEQ ID NO.: 71, or SEQ ID NO.: 72, and wherein the first promoter polynucleotide sequence can either be the same as or different from the second promoter polynucleotide sequence.

[0041] Embodiment 13. The split-intein system of any one of Embodiments 11 through 12, wherein the polynucleotide sequence encoding the N-TSC2 / N-Intein polypeptide is operably joined to a first polyadenylation signal sequence, and the polynucleotide sequence encoding the N-TSC2 / N-Intein polypeptide is operably j oined to a second polyadenylation signal sequence, and wherein the first polyadenylation signal sequence can either be the same as or different from the second polyadenylation signal sequence.

[0042] Embodiment 14. The split-intein system of Embodiment 13, wherein the first polyadenylation signal sequence comprises any one of the polynucleotide sequences of SEQ ID NO.: 73, SEQ ID NO.: 74, or SEQ ID NO.: 75, and the second polyadenylation signal sequence comprises any one of the polynucleotide sequences of SEQ ID NO.: 73, SEQ ID NO.: 74, or SEQ ID NO.: 75 , and wherein the first polyadenylation signal sequence can either be the same as or different from the second polyadenylation signal sequence.

[0043] Embodiment 15. The split-intein system of any one of Embodiments 13 through 14 wherein the first promoter polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 67, and the first polyadenylation signal sequence comprises the polynucleotide sequence of SEQ ID NO.: 75.

[0044] Embodiment 16. The split-intein system of any one of Embodiments 13 through 14 wherein the second promoter polynucleotide sequence comprises the polynucleotide sequenceof SEQ ID NO.: 67, and the second polyadenylation signal sequence comprises the polynucleotide sequence of SEQ ID NO.: 75.

[0045] Embodiment 17. The split-intein system of any one of Embodiments 13 through 14 wherein both the first promoter polynucleotide sequence and the second promoter polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 67, and both the first polyadenylation signal sequence and the second polyadenylation signal sequence comprises the polynucleotide sequence of SEQ ID NO.: 75.

[0046] Embodiment 18. The split-intein system of any one of Embodiments 11 through 17, wherein the first expression cassette is flanked by a 5’ AAV inverted terminal repeat (ITR) and a 3’ AAV inverted terminal repeat (ITR), and the second expression cassette is flanked by a 5’ AAV inverted terminal repeat (ITR) and a 3’ AAV inverted terminal repeat (ITR).

[0047] Embodiment 19. The split intein system of Embodiment 18, wherein the 5’ AAV inverted terminal repeat (ITR) comprises the polynucleotide sequence of SEQ ID NO.: 77, and the 3’ AAV inverted terminal repeat comprises the polynucleotide sequence of SEQ ID NO.: 78.

[0048] Embodiment 20. The split-intein system of any one of Embodiments 1 through 2 wherein the first rAAV particle comprises a polynucleotide comprising the polynucleotide sequence of SEQ ID NO.: 93, and the second rAAV particle comprises a polynucleotide comprising the polynucleotide sequence of SEQ ID NO.: 94.

[0049] Embodiment 21. The split-intein system of any one of Embodiments 1 through 2 wherein the first expression cassette comprises the polynucleotide sequence of SEQ ID NO.: 79, and the second expression cassette comprises the polynucleotide sequence of SEQ ID NO.: 80.

[0050] Embodiment 22. The split-intein system of any one of Embodiments 1 through 2 wherein the first rAAV particle comprises a polynucleotide comprising the polynucleotide sequence of SEQ ID NO.: 97, and the second rAAV particle comprises a polynucleotide comprising the polynucleotide sequence of SEQ ID NO.: 98.

[0051] Embodiment 23. The split-intein system of any one of Embodiments 1 through 2 wherein the first expression cassette comprises the polynucleotide sequence of SEQ ID NO.: 81, and the second expression cassette comprises the polynucleotide sequence of SEQ ID NO.: 82.

[0052] Embodiment 24. The split-intein system of any one of Embodiments 1 through 2 wherein the first rAAV particle comprises a polynucleotide comprising the polynucleotide sequence of SEQ ID NO.: 101, and the second rAAV particle comprises a polynucleotide comprising the polynucleotide sequence of SEQ ID NO.: 102.

[0053] Embodiment 25. The split-intein system of any one of Embodiments 1 through 2 wherein the first expression cassette comprises the polynucleotide sequence of SEQ ID NO.: 83, and the second expression cassette comprises the polynucleotide sequence of SEQ ID NO.: 84.

[0054] Embodiment 26. The split-intein system of any one of Embodiments 1 through 2 wherein the first rAAV particle comprises a polynucleotide comprising the polynucleotide sequence of SEQ ID NO.: 105 and the second rAAV particle comprises a polynucleotide comprising the polynucleotide sequence of SEQ ID NO.: 106.

[0055] Embodiment 27. The split-intein system of any one of Embodiments 1 through 2 wherein the first expression cassette comprises the polynucleotide sequence of SEQ ID NO.: 85, and the second expression cassette comprises the polynucleotide sequence of SEQ ID NO.: 86.

[0056] Embodiment 28. The split-intein system of any one of Embodiments 1 through 2 wherein the first rAAV particle comprises a polynucleotide comprising the polynucleotide sequence of SEQ ID NO.: 109, and the second rAAV particle comprises a polynucleotide comprising the polynucleotide sequence of SEQ ID NO. : 110.

[0057] Embodiment 29. The split-intein system of any one of Embodiments 1 through 2 wherein the first expression cassette comprises the polynucleotide sequence of SEQ ID NO.: 87, and the second expression cassette comprises the polynucleotide sequence of SEQ ID NO.: 88.

[0058] Embodiment 30. The split-intein system of any one of Embodiments 1 through 2 wherein the first rAAV particle comprises a polynucleotide comprising the polynucleotide sequence of SEQ ID NO.: 113, and the second rAAV particle comprises a polynucleotide comprising the polynucleotide sequence of SEQ ID NO.: 114.

[0059] Embodiment 31. The split-intein system of any one of Embodiments 1 through 2 wherein the first expression cassette comprises the polynucleotide sequence of SEQ ID NO.: 89, and the second expression cassette comprises the polynucleotide sequence of SEQ ID NO.: 90.

[0060] Embodiment 32. The split-intein system of any one of Embodiments 1 through 2 wherein the first rAAV particle comprises a polynucleotide comprising the polynucleotide sequence of SEQ ID NO.: 117, and the second rAAV particle comprises a polynucleotide comprising the polynucleotide sequence of SEQ ID NO.: 118.

[0061] Embodiment 33. The split-intein system of any one of Embodiments 1 through 2 wherein the first expression cassette comprises the polynucleotide sequence of SEQ ID NO.: 91, and the second expression cassette comprises the polynucleotide sequence of SEQ ID NO.: 92.

[0062] Embodiment 34. The split-intein system of any one of Embodiments 1 through 33, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, and further comprises a 7 amino acid peptide inserted between amino acids 588 and 589, said 7 amino acid peptide comprising YSRIGPN (SEQ ID NO.: 149), YSRNSDN (SEQ ID NO.: 150), LHRLGPN (SEQ ID NO.: 151), LHRLGPD (SEQ ID NO.: 152), LHRAGPD (SEQ ID NO : 153), YSRIGPD (SEQ ID NO.: 154), LSRIGPD (SEQ ID NO.: 155), LARSGPD (SEQ ID NO.: 156), LHKAGPN (SEQ ID NO.: 157), LSRIGPN (SEQ ID NO.: 158), LAKSGPN (SEQ ID NO.: 159), or YARNGPN (SEQ ID NO.: 160), or FRSTNGV (SEQ ID NO.: 61).

[0063] Embodiment 35. The split-intein system of Embodiment 34, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138, and further comprises a 7 amino acid peptide YSRIGPN (SEQ ID NO.: 149) inserted between amino acids 588 and 589.

[0064] Embodiment 36. The split-intein system of Embodiment 35, wherein at least the first rAAV particle or at least the second rAAV particle comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 139.

[0065] Embodiment 37. The split-intein system of Embodiment 35, wherein at least the first rAAV particle or at least the second rAAV particle comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 142.

[0066] Embodiment 38. The split-intein system of Embodiment 34, wherein at least the first rAAV particle or at least the second rAAV particle comprises a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138, and further comprises a 7 amino acid peptide YSRIGPN (SEQ ID NO.: 149) inserted between amino acids 588 and 589, and further wherein the amino acid at position 586 is E, and the amino acid at position 589 is N.

[0067] Embodiment 39. The split-intein system of Embodiment 38, wherein at least the first rAAV particle or at least the second rAAV particle comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 140.

[0068] Embodiment 40. The split-intein system of Embodiment 38, wherein at least the first rAAV particle or at least the second rAAV particle comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 143.

[0069] Embodiment 41. The split-intein system of Embodiment 34, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138, and further comprises a 7 amino acid peptide FRSTNGV (SEQ ID NO.: 161) inserted between amino acids 588 and 589, and further wherein the amino acid at position 588 is D, and the amino acid at position 592 is E.

[0070] Embodiment 42. The split-intein system of Embodiment 41, wherein at least the first rAAV particle or at least the second rAAV particle comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 147.

[0071] Embodiment 43. The split-intein system of Embodiment 41, wherein at least the first rAAV particle or at least the second rAAV particle comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 148.

[0072] Embodiment 44. The split-intein system of any one of Embodiments 1 through 33, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ IDNO.: 138, and further comprises a 7 amino acid peptide YSRIGPN (SEQ ID NO.: 149) inserted between amino acids 588 and 589.

[0073] Embodiment 45. The split-intein system of any one of Embodiments 1 through 33, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138, and further comprises a 7 amino acid peptide YSRIGPN (SEQ ID NO.: 149) inserted between amino acids 588 and 589, and further wherein the amino acid at position 586 is E, and the amino acid at position 589 is N.

[0074] Embodiment 46. The split-intein system of any one of Embodiments 1 through 33, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138, and further comprises a 7 amino acid peptide FRSTNGV (SEQ ID NO.: 161) inserted between amino acids 588 and 589, and further wherein the amino acid at position 588 is D, and the amino acid at position 592 is E.

[0075] Embodiment 47. The split-intein system of any one of Embodiments 1 through 33, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, and further comprises a 7 amino acid peptide inserted between amino acids 588 and 589, said 7 amino acid peptide comprising LYDGRSG (SEQ ID NO.: 166), VQRLSVL (SEQ ID NO.: 167), KVSNPVW (SEQ ID NO.: 168), or RPVQVMA (SEQ ID NO.: 169).

[0076] Embodiment 48. The split-intein system of any one of Embodiments 1 through 33, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, and further comprises a 7 amino acid peptide LYDGRSG (SEQID NO.: 166) inserted between amino acids 588 and 589, and further wherein the amino acid at position 589 is a Y.

[0077] Embodiment 49. The split-intein system of any one of Embodiments 1 through 33, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, and further comprises a 7 amino acid peptide RPVQVMA (SEQ ID NO.: 169) inserted between amino acids 588 and 589, and further wherein the amino acid at position 589 is an E.

[0078] Embodiment 50. The split-intein system of any one of Embodiments 1 through 33, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, and further comprises a 7 amino acid peptide KVSNPVW (SEQ ID NO.: 168) inserted between amino acids 588 and 589, and further wherein the amino acid at position 587 is an S, and the amino acid at position 588 is an N.

[0079] Embodiment 51. The split-intein system of any one of Embodiments 1 through 33, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, and further comprises a 7 amino acid peptide inserted at any position between amino acids 450 and 461, said 7 amino acid peptide comprising EFNNGSD (SEQ ID NO.: 173) or GAASLMP (SEQ ID NO.: 174).

[0080] Embodiment 52. The split-intein system of any one of Embodiments 1 through 33, wherein at least the first rAAV particle or at least the second rAAV particle comprises a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, wherein amino acids 450 and 461 are replaced with a 7 amino acid peptide comprising EFNNGSD (SEQ ID NO.: 173) or GAASLMP (SEQ ID NO.: 174).

[0081] Embodiment 53. The split-intein system of any one of Embodiments 1 through 33, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, and further comprises an amino acid targeting moiety that binds an ALPL protein.

[0082] Embodiment 54. The split-intein system of any one of Embodiments 1 through 33, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, and further comprises a 6 amino acid peptide inserted between amino acids 455 and 456, said 6 amino acid peptide comprising SPHSKA (SEQ ID NO.: 175).

[0083] Embodiment 55. The split-intein system of any one of Embodiments 1 through 33, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, and further comprises a 6 amino acid peptide inserted between amino acids 453 and 454, said 6 amino acid peptide comprising HDSPHK (SEQ ID NO.: 176).

[0084] Embodiment 56. The split-intein system of any one of Embodiments 1 through 33, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, said VP1 capsid protein further comprising one or more of the following amino acid substitutions: D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D.

[0085] Embodiment 57. The split-intein system of any one of Embodiments 34 through 55, wherein at least the first rAAV particle further comprises, or at least the second rAAV particle further comprises, or both the first and second rAAV particles further comprise one or more of thefollowing amino acid substitutions : D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D.

[0086] Embodiment 58. The recombinant AAV particle of any one of Embodiments 34 through 57, wherein at least the first rAAV particle or at least the second rAAV particle exhibits increased evasion of AAV-neutralizing antibodies relative to a reference AAV particle.

[0087] Embodiment 59. A method of treating a patient having tuberous sclerosis complex (TSC), said method comprising administering to said patient a therapeutically effective amount of the first rAAV particle and the second rAAV particle of any one of Embodiments 1 through 58.

[0088] Embodiment 60. A method of restoring the mTOR pathway in a subject in need thereof, said method comprising administering to said patient a therapeutically effective amount of the first rAAV particle and the second rAAV particle of any one of Embodiments 1 through 58.

[0089] Embodiment 61. A method of suppressing the mTOR pathway in a subject in need thereof, said method comprising administering to said patient a therapeutically effective amount of the first rAAV particle and the second rAAV particle of any one of Embodiments 1 through 58.

[0090] Embodiment 62. A method of reducing the amount of phosphorylated p70S6K in target cells of a subject in need thereof, said method comprising administering to said patient a therapeutically effective amount of the first rAAV particle and the second rAAV particle of any one of Embodiments 1 through 58.

[0091] Embodiment 63. The method of any one of Embodiments 59 through 62 wherein the recombinant AAV particle is administered systemically, intravenously, intracranially, intrathecally, intra-nasally, intra-peritoneally.

[0092] Embodiment 64. The method of Embodiment 63 wherein the recombinant AAV particle is administered intravenously.

[0093] Embodiment 65. A pharmaceutical composition for use in treating a patient having tuberous sclerosis Complex (TSC) comprising the first rAAV particle and the second rAAV particle of any one of Embodiments 1 through 58 and a pharmaceutically acceptable carrier.

[0094] Embodiment 66: A plasmid comprising (i) an expression cassette comprising a polynucleotide sequence encoding an N-TSC2 / N-Intein polypeptide, said N-TSC2 / N-Intein polypeptide comprising an N-terminal polypeptide fragment of the human TSC2 protein, fused at its C-terminus to the N-terminus of an N-Intein polypeptide, operably linked to a regulatory sequence that promotes expression in CNS cells, flanked by ITR sequences, or (ii) an expression cassette comprising a polynucleotide sequence encoding a C-Intein / C-TSC2 polypeptide, said C-Intein / C-TSC2 polypeptide comprising a C-Intein polypeptide fused at its C-terminus to the N-terminus of a C-terminal polypeptide fragment of the human TSC2 protein, operably linked to a regulatory sequence that promotes expression in CNS cells, flanked by ITR sequences.

[0095] Embodiment 67. A plasmid comprising the polynucleotide sequence of any one of SEQ IDNOs.: 79-92.

[0096] Embodiment 68. A plasmid comprising the polynucleotide sequence of any one of SEQ IDNOs.: 93, 94, 97, 98, 101, 102, 105, 106, 109, 110, 113, 114, 117, or 118.

[0097] Embodiment 69. A plasmid comprising the polynucleotide sequence of any one of SEQ IDNOs.: 121-134.

[0098] Embodiment 70. A host cell comprising the plasmid of any one of Embodiments 66 through 69.

[0099] Embodiment 71. A method of manufacturing recombinant AAV particles, comprising the following steps in order:a) culturing HEK293 cells;b) transfecting the HEK293 cells with a transfection composition comprising (i) a plasmid comprising the polynucleotide sequence of any one of SEQ ID NOs.: 93, 94, 97, 98, 101, 102, 105, 106, 109, 110, 113, 114, 117, or 118, or a plasmid of embodiment 66, (ii) a plasmid encoding AAV Rep and AAV Cap (pRepCap), and (iii) a plasmid encoding adenoviral helper genes (pHelp);c) culturing the transfected cells under conditions that produce recombinant AAV particles.

[0100] Embodiment 72. The method of Embodiment 71, wherein the plasmid encoding AAV Rep and AAV Cap comprises the polynucleotide sequence of any one of SEQ ID NOs. : 144-146.

[0101] Embodiment 73. The method of Embodiment 71, wherein the recombinant AAV particle is an rAAV hTfRl particle, an rAAV hCA4 particle, an rAAV hCD59 particle, an rAAV ALPL particle, or an rAAV (AE) particle.

[0102] Embodiment 74: A method of manufacturing a pharmaceutical composition, said method comprising:a) manufacturing (i) a first rAAV particle comprising a first expression cassette comprising a first polynucleotide sequence encoding an N-TSC2 / N-Intein polypeptide, said N- TSC2 / N-Intein polypeptide comprising an N-terminal polypeptide fragment of the human TSC2 protein, fused at its C-terminus to the N-terminus of an N-Intein polypeptide, flanked by ITR sequences and (ii) a second rAAV particle comprising a second polynucleotide comprising a second polynucleotide sequence encoding a C-Intein / C-TSC2 polypeptide, said C-Intein / C-TSC2 polypeptide comprising a C-Intein polypeptide fused at its C- terminus to the N-terminus of a C-terminal polypeptide fragment of the human TSC2 protein, flanked by ITR sequences, andb) combining the first rAAV particle and the second rAAV particle.

[0103] Embodiment 75: The method of Embodiment 74, whereina) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 11, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 13, orb) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 19, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 21, orc) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 27, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 29, ord) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 35, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 37, ore) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 43, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 45, orf) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO. : 51, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 53, org) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 59, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 61.

[0104] Embodiment 76: The method of Embodiment 73 or 74, whereina) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 15, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 17, orb) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 23, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 25, orc) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 31, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 33, ord) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 39, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 41, ore) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 47, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 49, orf) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO. : 55, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 57, org) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 63, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 65.

[0105] Embodiment 77: The method of Embodiment 74, whereina) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 79, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 80, orb) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 81, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 82, orc) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 83, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 84, ord) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 85, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 86, ore) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 87, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 88, orf) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO. : 89, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 90, org) the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 91, and the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 92.BRIEF DESCRIPTION OF THE DRAWINGS

[0106] FIG. 1 is a schematic describing the rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein.

[0107] FIG. 2 is a table providing sequence details for the N-TSC2 / N-Npu and C-Npu / C-TSC2 constructs and plasmids used in the transfection experiments that are described in Example 1.

[0108] FIG. 3 is an image of a Western blot showing expression of TSC2 (tuberin) in lysates from TSC2 knockout cell lines after transfection with plasmids containing the N-TSC2 / N-Npu and C-Npu / C-TSC2 constructs that are listed in FIG. 2 and described in Example 1, in comparison to lysates from untransfected TSC2 knockout cells, and TSC2 knockout cells transfected with a control plasmid (pUC19).

[0109] FIG. 4 is a table showing the results of an ELISA to determine levels of unphosphorylated and phosphorylated p70S6 kinase (P70S6K) in lysates from TSC2 knockout cell lines after transfection with plasmids containing the N-TSC2 / N-Npu and C-Npu / C-TSC2 constructs that are listed in FIG. 2 and described in Example 1, in comparison to lysates from untransfected TSC2 knockout cells, TSC2 knockout cells transfected with a control plasmid (pUC19), and TSC2 knockout cells transfected with a plasmid (pTSC2) engineered to express the full-length TSC2 protein.

[0110] FIG. 5A-5B are images of Western blots showing expression of TSC2 (tuberin) in lysates from TSC2 knockout cell lines after transfection with plasmids containing the N-TSC2 / N-Npu and C-Npu / C-TSC2 constructs that are listed in Table 2 and described in Example 2, in comparison to lysates from untransfected TSC2 knockout and HEK 293T cells, and TSC2 knockout cells transfected with a plasmid (pTSC2) engineered to express the full-length TSC2 protein.

[0111] FIG. 6 is a table showing the results of an ELISA to determine levels of unphosphorylated and phosphorylated p70S6 kinase (P70S6K) in lysates from TSC2 knockout cell lines after transfection with plasmids containing the N-TSC2 / N-Npu and C-Npu / C-TSC2 constructs that are described in Example 2 and listed in Table 2, in comparison to lysates from untransfected TSC2 knockout and HEK 293T cells, and lysates from TSC2 knockout cells transfected with a plasmid (pTSC2) engineered to express the full-length TSC2 protein.

[0112] FIG. 7 is an image of a Western blot showing expression of TSC2 (tuberin) in lysates from TSC2 knockout cell lines after transduction with rAAV particles engineered to express the N-TSC2 / N-Npu and C-Npu / C-TSC2 constructs that are listed in Table 3 and describedin Example 3, in comparison to lysates from untransduced HEK 293T cells and untransduced TSC2 knockout cells.

[0113] FIG. 8 is a table showing the results of an ELISA to determine levels of unphosphorylated and phosphorylated p70S6 kinase (P70S6K) in lysates from TSC2 knockout cell lines after transduction with rAAV particles engineered to express the N-TSC2 / N-Npu and C-Npu / C-TSC2 constructs that are listed in Table 3 and described in Example 3, in comparison to lysates from untransduced HEK 293T cells and untransduced TSC2 knockout cells.

[0114] FIG. 9A is an image of a Western blot showing expression of TSC2 (tuberin) in lysates from TSC2 knockout cell lines transduced with rAAV particles engineered to express the N-TSC2 / N-Npu and C-Npu / C-TSC2 constructs and plasmids used in the transfection experiments that are listed in Table 4 and described in Example 4, in comparison to lysates from untransduced TSC2 knockout cells and HEK 293T cells.

[0115] FIG. 9B is an image of a Western blot showing expression of N-TSC2 / N-Npu polypeptide in lysates from TSC2 knockout cell lines transduced with rAAV particles engineered to express the N-TSC2 / N-Npu and C-Npu / C-TSC2 constructs listed in Table 4 and described in Example 4, in comparison to lysates from untransduced TSC2 knockout cells and HEK 293T cells.

[0116] FIG. 10 is an image of a Western blot showing expression of a condensed TSC2 (tuberin) protein (cTSC2) in lysates from TSC2 knockout cell lines after transfection with the pcTSC2 plasmid engineered to express the cTSC2 protein that is described in Example 5 and the pTSC2 plasmid engineered to express the full-length TSC2 protein that is described in Example 5, in comparison to lysates from untransfected HEK 293T cells, HEK 293T cells transfected with a control plasmid (gap) engineered to express a GFP protein, and untransfected TSC2 knockout cells.

[0117] FIG. 11 is an image of a Western blot showing expression of a condensed TSC2 (tuberin) protein (cTSC2) in lysates from TSC2 knockout cell lines after transduction with rAAV particles engineered to express the cTSC2 protein that is described in Example 5, in comparison to lysates from untransduced HEK 293T cells, HEK 293T cells transduced with rAAV particles engineered to express a GFP protein, and untransduced TSC2 knockout cells.

[0118] FIG. 12 is a table showing the results of an ELISA to determine levels of unphosphorylated and phosphorylated p70S6 kinase (P70S6K) in lysates from TSC2 knockout cell lines after (i) transduction with rAAV particles engineered to express the cTSC2 protein as described in Example 5, in comparison to lysates from untransduced HEK 293T and TSC2 knockout cells, and TSC2 knockout cells transfected with a control plasmid (pGFP) engineered to express a GFP protein, or (ii) transfection with the pcTSC2 plasmid engineered to express the cTSC2 protein that is described in Example 5, in comparison to lysates from untransfected HEK 293T and TSC2 knockout cells.DETAILED DESCRIPTION DEFINITIONS

[0119] Recitation of “or” contemplates and supports, “one or more of,” “one or a combination of,” or “and,” as in “and / or.” For example, “A, B, or C” contemplates and supports embodiments with: A alone; B alone; C alone; the combination of A and B; the combination of A and C; the combination of B and C; and the combination of A, B, and C. Further to which, within recitation of “closed” language (e.g. consisting of), as well as within recitation of “open” language (e g. comprising), or the recitation of a list, as in “A, B, or C,” contemplates one or a combination within that list, unless otherwise specified. For example, “consisting of A, B, or C” contemplates and supports embodiments with: A alone; B alone; C alone; the combination of A and B; the combination of A and C; the combination of B and C; and the combination of A, B, and C. Recitation of “and / or” contemplates and supports not only the combination of all within the list (i.e. “A, B, and / or C” contemplates “A, B, and C”), but also “one or more of’ or “one or a combination of.” For example, “A, B, and C” contemplates: A alone; B alone; C alone; the combination of A, B and C; the combination of A and B; the combination of A and C; and the combination of B and C.

[0120] The recitation of a list of alternatives with an “and,” as included, for example, in a discussion of elements “selected from the group consisting of,” contemplates and provides support for combinations within that list, unless otherwise stated. For example, “is selected from the group consisting of A, B, and C” is to be understood to contemplate and support “is selected from the group consisting of A, B, C, and combinations thereof’ and to be coextensive with “is at least one selected from the group consisting of A, B, and C,” such that “group” includes: A alone, B alone,C alone, A and B in combination, A and C in combination, B and C in combination, and A, B, and C in combination.

[0121] “Is at least selected from the group consisting of A, B, and C” is contemplated to include embodiments and supports embodiments wherein what is after “is” is open due to recitation of “at least,” such that it is coextensive with “comprises a member of the group consisting of A, B, and C,” or such that “consisting of’ modifies the meaning of “group” alone and not “is selected from the group.”

[0122] Further, recitation of a component in an embodiment also contemplates and supports exclusion, explicitly, of said component from the embodiment. For example, “comprising A, B, or C” supports embodiments, which comprise A or B, but specifically exclude C.

[0123] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. For example, “comprising an A, a B, or a C” contemplates and supports embodiments comprising two or more A, two or more B, and two or more C.

[0124] Unless defined otherwise, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments pertain. Specific materials and methods are described, but it is understood that any methods and materials similar or equivalent to those described can be used in the practice of embodiments. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. In describing and claiming the present invention, the following terminology will be used.

[0125] ‘About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of .+-.20% or .+-.10%, more preferably .+-.5%, even more preferably .+-.1%, and still more preferably .+-.0.1% from the specified value, as such variations are appropriate to perform the embodiments.

[0126] As used herein, the term “cell line” refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. Spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell linereferred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.

[0127] The terms “express,” “expressing,” or “expression,” as used herein are defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0128] As used herein, the term “host cell” refers to any cell that harbors, or is capable of harboring, a substance of interest. Often a host cell can be a mammalian cell (e.g., a non-human primate, rodent, or human cell). In some aspects, the host cell can be a mammalian cell, a yeast cell, a bacterial cell, an insect cell, a plant cell, or a fungal cell. A host cell can be used as a recipient of an AAV helper construct, an AAV plasmid encoding a recombinant AAV genome comprising a transgene, an accessory function vector, or other transfer DNA associated with the production of recombinant AAV (rAAV) particles. The term includes the progeny of the original cell which has been transfected. Thus, a “host cell” as used herein can refer to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.

[0129] “Identity” as used herein refers to the subunit sequence identity between two polymeric molecules, particularly between two amino acid molecules, such as sequence identity between two polypeptide molecules, or sequence identity between two nucleic acid molecules, such as polynucleotides. When two amino acid sequences have the same residues at the same positions — e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions — e.g., if half (for example, five positions in a polypeptide ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical. In the case of an insertion or deletion, identity is understood to realign those subsequent amino acids that would be identical, and is only considered to be not identical at the insertion or deletion.

[0130] By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0131] As used herein, the terms “intein,” “inteins,” or “intein sequences” refer to polypeptide sequences that self-excise, and ligate the flanking polypeptide sequences (referred to as “extein” sequences) into a new protein through a process known as intein-mediated protein splicing. Both cis- and trans-splicing inteins exist in nature. Cis-splicing inteins exist within a single polypeptide precursor protein, flanked by extein sequences that are joined into a full-length, functional protein via intein-mediated protein splicing. Trans-splicing inteins, or “split inteins,” exist as separate polypeptide sequences that are transcribed and translated from two independent polynucleotides. The trans-splicing requires the co-expression of both “split intein” fragments: a first “split intein” fragment in which an “N-Extein” polypeptide is fused to an “N-Intein” polypeptide, and a second “split intein” fragment in which a “C-Intein” polypeptide is fused to a “C-Extein” polypeptide. Upon co-expression, the two “split intein” fragments associate and catalyze the ligation of N-Extein and C-Extein polypeptides into a functional protein via intein-mediated protein splicing, during which process the “N-Intein” and “C-Intein” sequences are excised. Polynucleotide sequences encoding N-Intein and C-Intein polypeptides may be obtained from the DnaE polymerase of Nostoc punctiforme bacteria (Npu intein sequences), as described, for example, by Shah et al. (2013) and in U.S. Patent No. 10,100,080, which are incorporated herein by reference. Polynucleotide sequences encoding N-Intein and C-Intein polypeptides may also include engineered variants of Npu intein sequences, including for example the NpuGEP and CfaGEP variants described by Stevens et al. (2017). Additional inteins may include, for example: Ssp GyrB inteins, Ssp DnaX inteins, Ter DnaE3 inteins, Ter ThyX inteins, Rma DnaB inteins, and Cne Prp8 inteins, as described in U.S. Pat. No. 8,394,604, which is incorporated herein by reference. FIG. 1 depicts the trans-splicing of two “split-intein” TSC2 polypeptide constructs as described herein (i.e., an N-TSC2 / N-Intein polypeptide and a C-Intein / C-TSC2 polypeptide) to form a full length TSC2 protein.

[0132] By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

[0133] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response, thereby mediating a beneficial therapeutic response in a subject, and preferably in a human subject.

[0134] A “nucleic acid,” as used herein, is interchangeable with “polynucleotide” or “nucleotide sequence.” These terms refer to a discrete sequence that performs a specific function directly or indirectly in a cell. That function includes encoding a sequence of a gene that is transcribed into mRNA and translated into protein, and regulating said transcription (i.e. as a promoter would) and / or translation (i.e. as microRNA would). A nucleic acid inherently has a sequence. Thereby, “a nucleic acid comprising SEQ ID NO.: X” can be used to contemplate and support “a nucleic acid comprising the sequence of SEQ ID NO.: X.” In recombinant molecular biology, discrete nucleic acids can be combined. In some embodiments, a nucleic acid that encodes a protein can be ligated to a promoter or polyadenylation signal (which are nucleic acids), and / or a cis-acting element of a viral vector (i.e. an inverted-terminal repeat (ITR), which is also a nucleic acid). For convenience, a “nucleic acid” might be used to refer to the discrete elements within the larger nucleic acid, which could be referred to as “a polynucleotide,” “an expression region” (i.e. a polynucleotide comprising a promoter and a nucleic acid that encodes a protein), or “a vector” (see definition below).

[0135] “Encoding” refers to the inherent property of a nucleic acid to serve as a template, whether directly (i.e. a sense strand) or indirectly (i.e. an antisense, or reverse complementary, strand) for synthesis of peptide, polypeptides, proteins, or other nucleic acids (i.e. rRNA, tRNA, microRNA). A nucleic acid can “encode” whether it is the sense strand, antisense strand, or a double-stranded segment thereof. The sense strand directly encodes the rRNA, tRNA, microRNA, or mRNA, and is sometimes referred to as the “coding” or “plus” strand in the art. The mRNA then serves as the template for translation of a peptide, polypeptide, or protein. The anti-sensestrand is generally considered to be the reverse complementary sequence of the sense strand, and is sometimes called a “non-coding” or “minus” strand in the art (although for present purposes “non-coding” is a misnomer because the non-coding strand still “encodes” the genetic information by perpetuating it during semi-conservative replication by acting as a template for the polymerization of a new, sense strand). Within semi-conservative replication two single strands in double-stranded nucleic acids are separated, and a new strand is polymerized from the information from each of the single-stranded nucleic acids (i.e. single-stranded template), regardless of whether one single- stranded template is the sense strand (e.g. that which is used to transcribe mRNA and thereby, or directly, encode the translate or protein) or the antisense strand. By perpetuating the genetic information, the antisense strand is still encoding the genetic information for, for example, a protein. Accordingly, “a nucleic acid encoding X” includes sense and antisense sequences or strands, whether X is a peptide, a polypeptide, or a protein, or X is a sequence that encodes a rRNA, tRNA, microRNA, antisense RNA, etc.

[0136] Further to which, “nucleic acid encoding X,” includes RNA, DNA, and combinations thereof, since nucleic acids are synthesized from transcription, reverse-transcription, and replication, as naturally occurring processes and man-made processes (recombinant biology, molecular biology, etc.).

[0137] Accordingly, a recited nucleic acid sequence contemplates and supports the complementary version thereof, the reverse complementary version thereof, and double-stranded versions thereof. That is, “a nucleic acid comprising SEQ ID NO.: X” is to be understood, contemplate, and support “a nucleic acid comprising the reverse complementary version of SEQ ID NO.: X” or, using the nomenclature regarding the prime symbol as in “ ‘ “, “a nucleic acid comprising SEQ ID NO.: X’,” unless otherwise specified. For example, “the nucleic acid comprising SEQ ID NO.: X” wherein SEQ ID NO.: X is 5’-ATGCC-3’ contemplates and supports the reverse complementary version of SEQ ID NO.: X, and specifically 5’-GGCAT-3’.

[0138] As noted above, a recited nucleic acid sequence contemplates and supports conversion between RNA and DNA versions thereof. For example, if SEQ ID NO.: X is “5’-ATGCC-3’,” contemplated and supported is 5’-AUGCC-3’, as well as the reverse complementary thereof, 5’-GGCAU-3’.

[0139] With regard to an AAV vector or an AAV particle, the above-noted incorporation of reverse complementary sequences and double-stranded segments into the definition of “a nucleic acid,” and the above-noted use of “encoding” as including sense and antisense strands, is intended to incorporate the means by which the AAV vector can introduce an exogenous nucleic acid sequence that encodes nucleic acid or a protein into the cell. It is further intended to incorporate, in some embodiments, processes whereby said introduction results in the expression of said nucleic acid (e.g., miRNA or antisense RNA) or polypeptide (e.g., the N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptides described herein).

[0140] Take for example, a nucleic acid encoding a protein, and an AAV particle comprising a nucleic acid encoding said protein. When a typical (i.e. naturally occurring) AAV particle encoding one sense or one antisense strand of the nucleic acid that encodes said protein enters the cell, the inverted-terminal repeats (ITRs) prime the synthesis of a reverse complementary sequence to the sense strand or antisense strand of the nucleic acid that encodes said protein. The polymerization thereby forms a segment of double-stranded DNA comprising the sense and antisense strands, regardless of whether the sense version or antisense version was first introduced to the cell. In this regard, the entire nucleic acid including ITRs and sense and antisense nucleic acids encoding a protein can be one single-stranded DNA, which loops upon itself to form a double- stranded segment, wherein the base-pairs the sense and antisense nucleic acids encoding the protein align.

[0141] From this segment of double-stranded DNA, transcription of mRNA and translation of said protein is achieved from said sense strand of DNA, regardless of whether the AAV vector comprised only the sense strand or only the antisense strand when first entering the cell. In this regard, “an AAV vector comprising a nucleic acid encoding protein X” includes, contemplates, and supports embodiments in which the nucleic acid is the sense strand encoding protein X, the antisense strand encoding protein X, a double-stranded nucleic acid encoding protein X, and a single stranded nucleic acid comprising sense and antisense strands wherein the sense and antisense strands form a segment of double-stranded nucleic acid.

[0142] The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence whenthe first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. Thus, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Likewise, a coding sequence is operably linked to a polyadenylation signal sequence if the polyadenylation signal sequence affects the expression of the coding sequence.

[0143] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. In some instances, this sequence may be the core promoter, and in other instances this sequence may also include, or be, an enhancer alone and / or in combination with other regulatory elements which are required for expression of the gene product.

[0144] In certain instances the promoter may comprise enhancer elements, exons, and introns from one or a variety of viruses and animals, and thereby the term “promoter” shall be understood to not be limited to being a non-expressed sequence, nor exclude a non-expressed sequence that is between expressed sequences (i.e. introns), nor be limited to exclude an enhancer alone so long as the combination of sequences used to construct the promoter are capable of initiating the specific transcription of a polynucleotide sequence.

[0145] A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell and without requiring the addition of exogenous factors or the introduction of a different phenotype to the cell. This constitutive promoter can be cell-specific so long as it is produced in the specific, or target, cell under most or all physiological conditions of the cell.

[0146] As used herein, “pharmaceutically acceptable” refers to refers to molecular entities and compositions that do not produce an allergic or similar undesirable reaction when administered to a host.

[0147] As used herein, the term “recombinant cell” refers to a cell into which an exogenous DNA segment (for example, a DNA polynucleotide that leads to the transcription of a biologically active polypeptide, or production of a biologically active nucleic acid, such as an RNA) has been introduced.

[0148] A “target gene” or “gene of interest” (GOT) refers to a nucleic acid encoding a target protein to be expressed within a target cell upon entry of the viral particle or gene delivery vector carrying the target gene into said cell. The target gene includes naturally occurring polymorphisms (i.e. variants) and man-made modifications to the wild-type gene so long as the target protein is still expressed. An example of such man-made modifications includes codon-optimization.

[0149] A “target protein” refers to a man-made or naturally occurring protein of interest to be introduced by vector into a host cell. One some embodiments, the target protein, as encoded in the genome of the host cell, is not functional because of a polymorphism in the gene sequence resulting in some mistranscription, missense, or mistranslation of the gene whereby reduced or no target protein or inoperable target protein is produced (e g. a polymorphism that results in an early stop codon) or an attenuation in the activity of the target protein, as encoded by and expressed from the genome of a subject.

[0150] In embodiments, the target protein comprises TSC2 (tuberin), which regulates the activity of the mammalian target of rapamycin (mTOR) signaling pathway as detected, for example, by measuring a reduction in the amount of phosphorylated p70S6K. It is to be understood and contemplated that “TSC2” or “TSC2 protein” encompasses naturally-occurring versions (i.e. human TSC2) and non-naturally occurring versions of TSC2 (i.e., TSC-2 variants with amino-acid additions, deletions, or substitutions within the TSC2 protein sequence which increase or decrease the activity compared to that of naturally occurring TSC2) so long as the protein referred to as TSC2 has at least the above-noted functional activity of regulating the activity of the mammalian target of rapamycin (mTOR) signaling pathway as detected, for example, by measuring a reduction in the amount of phosphorylated p70S6K. In some embodiments, the non-naturally occurring TSC2 has at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or 200% of the activity of the corresponding naturally-occurring TSC2, wherein “corresponding” contemplates and provides support for the naturally-occurring TSC2 protein to which the amino acid additions, deletions, or substitutions were applied. In embodiments, the TSC2 is a human TSC2 and, in some embodiments, has the amino acid sequence of SEQ ID NO.: 10. In alternate embodiments, the TSC2 has an amino acid sequence that has at least 99%, 95%, 90%, 85% or 80% sequence identify to SEQ ID NO.: 10 and has TSC2 activity.

[0151] In some embodiments, a subject or patient treated according to the methods described herein is deficient in TSC2, as encoded by and expressed from the genome of the subject, due for example to an autosomal dominant inheritance of a defective TSC2 gene. In some embodiments, the subject or patient has TSC. In some embodiments, the subject or patient has an attenuated or abolished activity of TSC2.

[0152] TSC2 activity” refers to regulation or modulation of the activity of the mammalian target of rapamycin (mTOR) signaling pathway, as detected, for example, by measuring a reduction in the amount of phosphorylated p70S6K.

[0153] An “expression cassette” refers to a polynucleotide sequence comprising an expressing region. An expressing (or “expression”) region includes a recombinant polynucleotide comprising a nucleic acid that controls expression (i.e. a promoter) and a nucleic acid that encodes. The nucleic acid that encodes includes a nucleic acid that encodes a polypeptide, including for example the N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptides described herein. Generally, the promoter is operatively linked to the nucleic acid that encodes the target protein in a manner that promotes expression of the protein upon entry of the vector into the host cell. In some embodiments, the promoter can be operably linked by ensuring that there is not codon misalignment. In other embodiments, the expression cassette includes other polynucleotide sequences (e g., promoter, polyadenylation signal, or other expression sequence) that enhance, modulate, or otherwise facilitate expression of the desired nucleic acid sequence, including for example nucleic acid sequences encoding the N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptides described herein. Plasmids may be engineered to comprise expression cassettes, as described herein.

[0154] An “AAV vector construct” is a nucleic acid can be packaged within a recombinant AAV particle for delivery of a target gene or nucleic acid sequence, including for example nucleic acid sequences encoding the N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptides described herein, to the interior of a cell. AAV vector constructs include both (i) an expression cassette that may include a nucleic acid encoding a polypeptide, such as the N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptides described herein, that is operably linked to other sequences (such as a promoter, polyadenylation signal, or other expression sequence), and (ii) inverted-terminal repeat (ITR) sequences that allow the expression cassette to be packaged within an AAV particle (rAAVparticle). In embodiments, the expression cassette is flanked by ITR sequences. ITRs also provide other cis-acting functions for expression of the nucleic acid encoding a target protein or polypeptide in the host cell upon entry of the rAAV particle into the host cell. Such cis-acting functions of ITRs include aiding in concatemer formation for genomic insertion; initiation of second strand formation in the case of a single-stranded (ss) AAV (ssAAV) genome; or initiation of replication and transcription in the case of ssAAV and self-complementary (sc) AAV (scAAV) genomes. In this regard, AAV ITRs can be characterized based on the nucleic acid sequences providing such cis-acting functions that are found in various AAV serotypes. That is, an ITR isolated from an AAV2 serotype can be known as an AAV2 ITR, even though the ITR generally does not contribute to the serotype of an AAV.

[0155] Plasmids may be engineered to comprise AAV vector constructs and / or expression cassettes, as described herein. By way of example, a plasmid can comprise an origin of replication (e.g. ori from cytomegalovirus) which allows for the replication of the target gene within a cell, and such a plasmid is thereby a vector. A viral genetic code may provide a nucleic acid sequence or protein encoded therein that allows for insertion of the gene of interest into the host genome, thereby providing for the replication of the target gene during the replication of, and within, the host cell’s genome.

[0156] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity, and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0157] The nucleotide and amino acid sequences provided herein are set out in Table 1.TSC2 SPLIT INTEIN CONSTRUCTS

[0158] Inteins are polypeptide sequences that self-excise from a given polypeptide sequence, and ligate flanking polypeptide sequences (“exteins”) into a new protein through aprocess known as intein-mediated protein splicing. Both cis- and trans-splicing inteins exist in nature. Cis-splicing inteins exist within a single polypeptide precursor protein, flanked by extein sequences that are joined into a full-length, functional protein via intein-mediated protein splicing. Trans-splicing inteins, or “split inteins” exist as separate polypeptide sequences that are transcribed and translated from two independent genes. The trans-splicing requires the co-expression of both “split intein” fragments: a first “split intein” fragment in which an “N-Extein” polypeptide is fused at its C-terminus to the N-terminus of an “N-Intein” polypeptide, and a second “split intein” fragment in which a “C-Intein” polypeptide is fused at its C-terminus to the N-terminus of a “C-Extein” polypeptide. Upon co-expression, the two “split intein” fragments associate and catalyze the ligation of N-Extein and C-Extein polypeptides into a functional protein via intein-mediated protein splicing, during which process the “N-Intein” and “C-Intein” sequences are excised. Intein-mediated protein splicing does not require assistance from any enzyme or co-factor, but only a proper folded structure of the expressed protein.

[0159] The present inventors have developed an rAAV-mediated TSC2 “split intein” gene therapy that uses two separate rAAV particles, each engineered to deliver a different portion of the human TSC2 coding sequence to target cells of the CNS such that, when expressed, the two portions of the TSC2 protein join via intein-mediated protein splicing to form a full length human TSC2 (or tuberin) protein. The first rAAV particle (rAAV-n-TSC2 / n-intein) is engineered to comprise an expression cassette comprising regulatory polynucleotide sequences sufficient to express a polynucleotide sequence (n-TSC2 / n-intein polynucleotide sequence) that encodes an amino (N-terminal) portion of the human TSC2 protein (N-TSC2) that is fused at its C-terminus to the N-terminus of the N-terminal portion of an intein amino acid sequence (N-Intein) (together, N-TSC2 / N-Intein polypeptide). The second rAAV particle (rAAV-c-intein / c-TSC2) is engineered to comprise an expression cassette comprising regulatory polynucleotide sequences sufficient to express a polynucleotide sequence (c-intein / c-TSC2 polynucleotide sequence) that encodes the carboxyl (C-terminal) portion of the intein amino acid sequence (C-lntein) that is fused at its C-terminus to the N-terminus of a C-terminal portion of the human TSC2 protein (C-TSC2) (together, C-Intein / C-TSC2 polypeptide). Although not to be bound to a particular mechanism of action, it is thought that when these separate n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed at a sufficient level in the same cell, the resulting N-Intein and C-Intein amino acid sequences associate, and are excised from the associated N-TSC2 and C-TSC2 aminoacid sequences via protein splicing. During this process, the N-TSC2 and C-TSC2 amino acid sequences are joined together via intein-mediated protein splicing into a full-length TSC2 protein that does not include the excised N-Intein and C-Intein amino acid sequences. A basic schematic of this “split intein” TSC2 expression strategy is shown in FIG. 1.

[0160] The rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein can utilize various inteins, so long as (i) both the n-TSC2 / n-intein polynucleotide sequence and the c-intein / c-TSC2 polynucleotide sequence are of suitable length to be included in separate rAAV vector constructs that further comprise regulatory sequences (e.g., promoters, polyadenylation signal sequences, etc., particularly that promote expression in the CNS), as well as flanking AAV inverted terminal repeat (ITR) nucleotide sequences, and such rAAV vector constructs are of a suitable length (i.e., less than ~5kb in length) to allow for packaging within an rAAV particle for delivery to target cells, including cells of the CNS, and (ii) when co-expressed in target cells, the N-TSC2 and C-TSC2 amino acid sequences join together into a full-length protein that has TSC2 protein activity. In embodiments, the n-intein and c-intein polynucleotide sequences are obtained from the DnaE polymerase of Nostoc punctiforme bacteria (Npu intein sequences), as described, for example, by Shah et al. (2013). In other embodiments, the n-intein and c-intein polynucleotide sequences are engineered variants of Npu intein sequences, including for example the NpuGEP and CfaGEP variants described by Stevens et al. (2017). In embodiments, the n-intein polynucleotide sequence has, comprises, or consists of the polynucleotide sequence of SEQ ID NO.: 1, and encodes an N-Intein having the amino acid sequence of SEQ ID NO.: 2, and the c-intein polynucleotide sequence has, comprises, or consists of the polynucleotide sequence of SEQ ID NO.: 3, and encodes a C-Intein having the amino acid sequence of SEQ ID NO.: 4. In other embodiments, the n-intein polynucleotide sequence has, comprises, or consists of the polynucleotide sequence of SEQ ID NO.: 5, and encodes an N-Intein having the amino acid sequence of SEQ ID NO.: 6, and the c-intein polynucleotide sequence has, comprises, or consists of the polynucleotide sequence of SEQ ID NO.: 7, and encodes a C-lntein having the amino acid sequence of SEQ ID NO.: 8.

[0161] In embodiments, the rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein comprises a first polynucleotide sequence (n-TSC2 / n-intein polynucleotide sequence) encoding an N-terminal portion of the human TSC2 protein fused at its C-terminus to the N-terminus of an N-Intein polypeptide, the N-Intein polypeptide having, comprising, orconsisting of the amino acid sequence of SEQ ID NO.: 2, and a second polynucleotide sequence (c-intein / c-TSC2 polynucleotide sequence) encoding a C-Intein polypeptide having, comprising, or consisting of the amino acid sequence of SEQ ID NO.: 4 fused at its C-terminus to the N-terminus of a C-terminal portion of the human TSC2 protein, whereby when the first and second polynucleotide sequences are coexpressed in the same cell, the N-terminal polypeptide and the C-terminal polypeptide are joined together (through the intein-mediated protein splicing mechanism) to form a human TSC2 polypeptide (having TSC2 biological activity) with an amino acid sequence having, comprising, or consisting of either (i) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, the amino acid sequence of, SEQ ID NO.: 10 (human TSC2), or (ii) the amino acid sequence of SEQ ID NO.: 10 (human TSC2) wherein one or more amino acids are replaced with a complementary amino acid, and the resulting polypeptide exhibits TSC2 activity. In embodiments, the first (n-TSC2 / n-intein) polynucleotide sequence and the second (c-intein / c-TSC2) polynucleotide sequence are each of suitable length that, when included in separate rAAV vector constructs that further comprise regulatory sequences (e.g., promoters, polyadenylation signal sequences, etc.), as well as flanking AAV inverted terminal repeat (ITR) nucleotide sequences, such rAAV vector constructs are of a suitable length (i.e., less than ~5kb in length) to allow for packaging within an rAAV particle for delivery to target cells, including cells of the CNS.

[0162] In embodiments, the rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein comprises (i) a first rAAV particle engineered to deliver and express a first polynucleotide sequence encoding an N-terminal portion of the human TSC2 protein fused at its C-terminus to the N-terminus of an N-Intein polypeptide, the N-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 2, and (ii) a second rAAV particle engineered to deliver and express a second polynucleotide sequence encoding a C-lntein polypeptide having the amino acid sequence of SEQ ID NO.: 4 fused at its C-terminus to the N-terminus of a C-terminal portion of the human TSC2 protein, whereby when the first and second rAAV particles transduce the same cell, the first and second polynucleotide sequences are coexpressed, and the N-terminal polypeptide and the C-terminal polypeptide are joined together (through the intein-mediated protein splicing mechanism) to form a polypeptide with an amino acid sequence comprising either(i) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, the amino acid sequence of, SEQ ID NO.: 10 (human TSC2), or (ii) the amino acid sequence of SEQ ID NO.: 10 (human TSC2) wherein one or more amino acids are replaced with a complementary amino acid, and the resulting polypeptide exhibits TSC2 activity.[00163J In embodiments, the rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein comprises a first polynucleotide sequence encoding an N-terminal portion of the human TSC2 protein fused at its C-terminus to the N-terminus of an N-Intein polypeptide, the N-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 6, and a second polynucleotide sequence encoding a C-Intein polypeptide having the amino acid sequence of SEQ ID NO. : 8 fused at its C-terminus to the N-terminus of a C-terminal portion of the human TSC2 protein, whereby when the first and second polynucleotide sequences are coexpressed in the same cell, the N-terminal polypeptide and the C-terminal polypeptide are joined together to form a polypeptide with an amino acid sequence comprising either (i) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, the amino acid sequence of, SEQ ID NO.: 10 (human TSC2), or (ii) the amino acid sequence of SEQ ID NO.: 10 (human TSC2) wherein one or more amino acids are replaced with a complementary amino acid, and the resulting polypeptide exhibits TSC2 activity.

[0164] In embodiments, the rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein comprises (i) a first rAAV particle engineered to deliver and express a first polynucleotide sequence encoding an N-terminal portion of the human TSC2 protein fused at its C-terminus to the N-terminus of an N-Intein polypeptide, the N-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 6, and (ii) a second rAAV particle engineered to deliver and express a second polynucleotide sequence encoding a C-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 8 fused at its C-terminus to the N-terminus of a C-terminal portion of the human TSC2 protein, whereby when the first and second rAAV particles transduce the same cell, the first and second polynucleotide sequences are coexpressed, and the N-terminal polypeptide and the C-terminal polypeptide are joined together via intein-mediated protein splicing to form a polypeptide with an amino acid sequence comprising either (i) at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, the amino acid sequence of, SEQ ID NO.: 10 (human TSC2), or (ii) the amino acid sequence of SEQ ID NO.: 10 (human TSC2) wherein one or more amino acids are replaced with a complementary amino acid, and the resulting polypeptide exhibits TSC2 activity.C519 TSC2 “Split Intein” Constructs

[0165] In embodiments, the rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein utilizes “C519 TSC2 split intein” constructs comprising (i) an N-TSC2 / N-Intein polypeptide wherein the N-TSC2 portion of the polypeptide has, comprises, or consists of amino acids 1-518 of the human TSC2 protein, and a (ii) a C-Intein / C-TSC2 polypeptide wherein the C-TSC2 portion of the polypeptide has, comprises, or consists of amino acids 519-1807 of the human TSC2 protein. In embodiments, the “C519 TSC2 split intein” gene therapy comprises: (i) a first rAAV particle engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence encoding an N-TSC2 polypeptide comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, amino acids 1-518 of SEQ ID NO. : 10 (human TSC2) that is fused at its C-terminus to the N-terminus of an N-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 2, and (ii) a second rAAV particle engineered to deliver and express a c-intein / c-TSC2 polynucleotide sequence encoding a C-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 4 that is fused at its C-terminus to the N-terminus of a C-TSC2 polypeptide beginning with a Cysteine amino acid, and further comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, amino acids 520-1807 of SEQ ID NO.: 10 (human TSC2), whereby when the first and second rAAV particles transduce the same cell, the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed, and the N-TSC2 polypeptide and the C-TSC2 polypeptide are joined together via intein-mediated protein splicing to form a full length TSC2 polypeptide comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within the amino acid sequence of SEQ ID NO.: 10 (human TSC2).

[0166] In embodiments, the “C519 TSC2 split intein” gene therapy comprises: (i) a first rAAV particle engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence encoding an N-TSC2 polypeptide comprising amino acids 1-518 of SEQ ID NO.: 10 (human TSC2) that is fused at its C-terminus to the N-terminus of an N-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 2, such that the full-length polypeptide encoded by the n-TSC2 / n-intein polynucleotide sequence comprises the amino acid sequence of SEQ ID NO.: 16; and (ii) a second rAAV particle engineered to deliver and express a c-intein / c-TSC2 polynucleotide sequence encoding a C-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 4 that is fused at its C-terminus to the N-terminus of a C-TSC2 polypeptide comprising amino acids 519-1807 of SEQ ID NO.: 10 (human TSC2), such that the full-length polypeptide encoded by the c-intein / c-TSC2 polynucleotide sequence comprises the amino acid sequence of SEQ ID NO.: 18, whereby when the first and second rAAV particles transduce the same cell, the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed, and the N-TSC2 polypeptide and the C-TSC2 polypeptide are joined together via intein-mediated protein splicing to form a full length TSC2 polypeptide comprising the amino acid sequence of SEQ ID NO.: 10 (human TSC2). In embodiments, the first rAAV particle is engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence comprising the nucleotide sequence of SEQ ID NO.: 15, wherein the n-TSC2 portion of said polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO.: 11 and the n-intein portion of the polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO. 1, and the second rAAV particle is engineered to deliver and express a c-intein / c-TSC2 polynucleotide comprising the nucleotide sequence of SEQ ID NO. : 17, wherein the C-TSC2 portion of said polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO.: 13 and the c-intein portion of the polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO. 3. Exemplary plasmid sequences containing an expression cassette comprising a polynucleotide encoding these “C519 TSC2 split intein” constructs (flanked by an upstream EFS promoter (SEQ ID NO.: 67) and a downstream bGHv3 poly(A) sequence (SEQ ID No.: 75)) are disclosed herein as SEQ ID NO.: 95 (expression cassette encoding a C519 N-TSC2 / N-Intein polypeptide) and SEQ ID NO.: 96 (expression cassette encoding a C519 C-Intein / C-TSC2 polypeptide). The expression cassettes are flanked by AAV ITR sequences.C728 TSC2 “Split Tntein” Constructs

[0167] In embodiments, the rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein utilizes “C728 TSC2 split intein” constructs comprising (i) an N-TSC2 / N-Intein polypeptide wherein the N-TSC2 portion of the polypeptide has, comprises, or consists of amino acids 1-727 of the human TSC2 protein, and a (ii) a C-Intein / C-TSC2 polypeptide wherein the C-TSC2 portion of the polypeptide has, comprises, or consists of amino acids 728-1807 of the human TSC2 protein. In embodiments, the “C728 TSC2 split intein” gene therapy comprises: (i) a first rAAV particle engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence encoding an N-TSC2 polypeptide comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, amino acids 1-727 of SEQ ID NO.: 10 (human TSC2) that is fused at its C-terminus to the N-terminus of an N-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 2, and (ii) a second rAAV particle engineered to deliver and express a c-intein / c-TSC2 polynucleotide sequence encoding a C-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 4 that is fused at its C-terminus to the N-terminus of a C-TSC2 polypeptide beginning with a Cysteine amino acid, and further comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, amino acids 729-1807 of SEQ ID NO.: 10 (human TSC2), whereby when the first and second rAAV particles transduce the same cell, the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed, and the N-TSC2 polypeptide and the C-TSC2 polypeptide are joined together via intein-mediated protein splicing to form a full length TSC2 polypeptide comprising at least 85%, 86%>, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within the amino acid sequence of SEQ ID NO.: 10 (human TSC2).

[0168] In embodiments, the “C728 TSC2 split intein” gene therapy comprises: (i) a first rAAV particle engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence encoding an N-TSC2 polypeptide comprising amino acids 1-727 of SEQ ID NO.: 10 (human TSC2) that is fused at its C-terminus to the N-terminus of an N-Intein polypeptide having theamino acid sequence of SEQ TD NO.: 2, such that the full-length polypeptide encoded by the n-TSC2 / n-intein polynucleotide sequence comprises the amino acid sequence of SEQ ID NO.: 24; and (ii) a second rAAV particle engineered to deliver and express a c-intein / c-TSC2 polynucleotide sequence encoding a C-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 4 that is fused at its C-terminus to the N-terminus of a C-TSC2 polypeptide comprising amino acids 728-1807 of SEQ ID NO.: 10 (human TSC2), such that the full-length polypeptide encoded by the c-intein / c-TSC2 polynucleotide sequence comprises the amino acid sequence of SEQ ID NO.: 26, whereby when the first and second rAAV particles transduce the same cell, the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed, and the N-TSC2 polypeptide and the C-TSC2 polypeptide are joined together via intein-mediated protein splicing to form a full length TSC2 polypeptide comprising the amino acid sequence of SEQ ID NO.: 10 (human TSC2). In embodiments, the first rAAV particle is engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence comprising the nucleotide sequence of SEQ ID NO.: 23, wherein the n-TSC2 portion of said polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO.: 19 and the n-intein portion of the polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO. 1, and the second rAAV particle is engineered to deliver and express a c-intein / c-TSC2 polynucleotide comprising the nucleotide sequence of SEQ ID NO. : 25, wherein the C-TSC2 portion of said polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO.: 21 and the c-intein portion of the polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO. 3. Exemplary plasmid sequences containing an expression cassette comprising a polynucleotide encoding these “C728 TSC2 split intein” constructs (flanked by an upstream EFS promoter (SEQ ID NO.: 67) and a downstream bGHv3 poly(A) sequence (SEQ ID No.: 75)) are disclosed herein as SEQ ID NO.: 99 (expression cassette encoding a C728 N-TSC2 / N-Intein polypeptide) and SEQ ID NO.: 97 (expression cassette encoding a C728 C-Intein / C-TSC2 polypeptide). The expression cassettes are flanked by AAV ITR sequences.C738 TSC2 “Split Intein” Constructs

[0169] In embodiments, the rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein utilizes “C738 TSC2 split intein” constructs comprising (i) an N-TSC2 / N-Intein polypeptide wherein the N-TSC2 portion of the polypeptide has, comprises, or consists of amino acids 1-737 of the human TSC2 protein, and a (ii) a C-Intein / C-TSC2 polypeptide wherein the C-TSC2 portion of the polypeptide has, comprises, or consists of amino acids 738-1807 of the humanTSC2 protein. In embodiments, the “C738 TSC2 split intein” gene therapy comprises: (i) a first rAAV particle engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence encoding an N-TSC2 polypeptide comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, amino acids 1-737 of SEQ ID NO.: 10 (human TSC2) that is fused at its C-terminus to the N-terminus of an N-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 2, and (ii) a second rAAV particle engineered to deliver and express a c-intein / c-TSC2 polynucleotide sequence encoding a C-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 4 that is fused at its C-terminus to the N-terminus of a C-TSC2 polypeptide beginning with a Cysteine amino acid, and further comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, amino acids 739-1807 of SEQ ID NO.: 10 (human TSC2), whereby when the first and second rAAV particles transduce the same cell, the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed, and the N-TSC2 polypeptide and the C-TSC2 polypeptide are joined together via intein-mediated protein splicing to form a full length TSC2 polypeptide comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within the amino acid sequence of SEQ ID NO.: 10 (human TSC2).

[0170] In embodiments, the “C738 TSC2 split intein” gene therapy comprises: (i) a first rAAV particle engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence encoding an N-TSC2 polypeptide comprising amino acids 1-737 of SEQ ID NO.: 10 (human TSC2) that is fused at its C-terminus to the N-terminus of an N-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 2, such that the full-length polypeptide encoded by the n-TSC2 / n-intein polynucleotide sequence comprises the amino acid sequence of SEQ ID NO.: 32; and (ii) a second rAAV particle engineered to deliver and express a c-intein / c-TSC2 polynucleotide sequence encoding a C-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 4 that is fused at its C-terminus to the N-terminus of a C-TSC2 polypeptide comprising amino acids 738-1807 of SEQ ID NO.: 10 (human TSC2), such that the full-length polypeptideencoded by the c-intein / c-TSC2 polynucleotide sequence comprises the amino acid sequence of SEQ ID NO.: 34, whereby when the first and second rAAV particles transduce the same cell, the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed, and the N-TSC2 polypeptide and the C-TSC2 polypeptide are joined together via intein-mediated protein splicing to form a full length TSC2 polypeptide comprising the amino acid sequence of SEQ ID NO.: 10 (human TSC2). In embodiments, the first rAAV particle is engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence comprising the nucleotide sequence of SEQ ID NO.: 31, wherein the n-TSC2 portion of said polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO.: 27 and the n-intein portion of the polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO. 1, and the second rAAV particle is engineered to deliver and express a c-intein / c-TSC2 polynucleotide comprising the nucleotide sequence of SEQ ID NO. : 33, wherein the C-TSC2 portion of said polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO.: 29 and the c-intein portion of the polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO. 3. Exemplary plasmid sequences containing an expression cassette comprising a polynucleotide encoding these “C738 TSC2 split intein” constructs (flanked by an upstream EFS promoter (SEQ ID NO.: 67) and a downstream bGHv3 poly(A) sequence (SEQ ID No.: 75)) are disclosed herein as SEQ ID NO.: 103 (expression cassette encoding a C738 N-TSC2 / N-Intein polypeptide) and SEQ ID NO.: 104 (expression cassette encoding a C738 C-Intein / C-TSC2 polypeptide). In embodiments, the expression cassettes are flanked by AAV ITR sequences.C791 TSC2 “Split Intein” Constructs

[0171] In embodiments, the rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein utilizes “C791 TSC2 split intein” constructs comprising (i) an N-TSC2 / N-Intein polypeptide wherein the N-TSC2 portion of the polypeptide has, comprises, or consists of amino acids 1-790 of the human TSC2 protein, and a (ii) a C-Intein / C-TSC2 polypeptide wherein the C-TSC2 portion of the polypeptide has, comprises, or consists of amino acids 791-1807 of the human TSC2 protein. In embodiments, the “C791 TSC2 split intein” gene therapy comprises: (i) a first rAAV particle engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence encoding an N-TSC2 polypeptide comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions,or combinations thereof within, amino acids 1-790 of SEQ ID NO.: 10 (human TSC2) that is fused at its C-terminus to the N-terminus of an N-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 2, and (ii) a second rAAV particle engineered to deliver and express a c-intein / c-TSC2 polynucleotide sequence encoding a C-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 4 that is fused at its C-terminus to the N-terminus of a C-TSC2 polypeptide beginning with a Cysteine amino acid, and further comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, amino acids 792-1807 of SEQ ID NO.: 10 (human TSC2), whereby when the first and second rAAV particles transduce the same cell, the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed, and the N-TSC2 polypeptide and the C-TSC2 polypeptide are joined together via intein-mediated protein splicing to form a full length TSC2 polypeptide comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within the amino acid sequence of SEQ ID NO.: 10 (human TSC2).

[0172] In embodiments, the “C791 TSC2 split intein” gene therapy comprises: (i) a first rAAV particle engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence encoding an N-TSC2 polypeptide comprising amino acids 1-790 of SEQ ID NO.: 10 (human TSC2) that is fused at its C-terminus to the N-terminus of an N-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 2, such that the full-length polypeptide encoded by the n-TSC2 / n-intein polynucleotide sequence comprises the amino acid sequence of SEQ ID NO.: 40; and (ii) a second rAAV particle engineered to deliver and express a c-intein / c-TSC2 polynucleotide sequence encoding a C-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 4 that is fused at its C-terminus to the N-terminus of a C-TSC2 polypeptide comprising amino acids 791-1807 of SEQ ID NO.: 10 (human TSC2), such that the full-length polypeptide encoded by the c-intein / c-TSC2 polynucleotide sequence comprises the amino acid sequence of SEQ ID NO.: 42, whereby when the first and second rAAV particles transduce the same cell, the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed, and the N-TSC2 polypeptide and the C-TSC2 polypeptide are joined together via intein-mediated protein splicing to form a full length TSC2 polypeptide comprising the amino acid sequence of SEQ ID NO.: 10(human TSC2). In embodiments, the first rAAV particle is engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence comprising the nucleotide sequence of SEQ ID NO.: 39, wherein the n-TSC2 portion of said polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO.: 35 and the n-intein portion of the polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO. 1, and the second rAAV particle is engineered to deliver and express a c-intein / c-TSC2 polynucleotide comprising the nucleotide sequence of SEQ ID NO. : 41 , wherein the C-TSC2 portion of said polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO.: 37 and the c-intein portion of the polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO. 3. Exemplary plasmid sequences containing an expression cassette comprising a polynucleotide encoding these “C791 TSC2 split intein” constructs (flanked by an upstream EFS promoter (SEQ ID NO.: 67) and a downstream bGHv3 poly(A) sequence (SEQ ID No.: 75)) are disclosed herein as SEQ ID NO.: 107 (expression cassette encoding a C791 N-TSC2 / N-Intein polypeptide) and SEQ ID NO.: 108 (expression cassette encoding a C791 C-Intein / C-TSC2 polypeptide). In embodiments, the expression cassettes are flanked by AAV ITR sequences.C800 TSC2 “Split Intein” Constructs

[0173] In embodiments, the rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein utilizes “C800 TSC2 split intein” constructs comprising (i) an N-TSC2 / N-Intein polypeptide wherein the N-TSC2 portion of the polypeptide has, comprises, or consists of amino acids 1-799 of the human TSC2 protein, and a (ii) a C-Intein / C-TSC2 polypeptide wherein the C-TSC2 portion of the polypeptide has, comprises, or consists of amino acids 800-1807 of the human TSC2 protein. In embodiments, the “C800 TSC2 split intein” gene therapy comprises: (i) a first rAAV particle engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence encoding an N-TSC2 polypeptide comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, amino acids 1-799 of SEQ ID NO.: 10 (human TSC2) that is fused at its C-terminus to the N-terminus of an N-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 2, and (ii) a second rAAV particle engineered to deliver and express a c-intein / c-TSC2 polynucleotide sequence encoding a C-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 4 that is fused at its C-terminus to the N-terminus of a C-TSC2 polypeptidebeginning with a Cysteine amino acid, and further comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, amino acids 801-1807 of SEQ ID NO.: 10 (human TSC2), whereby when the first and second rAAV particles transduce the same cell, the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed, and the N-TSC2 polypeptide and the C-TSC2 polypeptide are joined together via intein-mediated protein splicing to form a full length TSC2 polypeptide comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within the amino acid sequence of SEQ ID NO.: 10 (human TSC2).

[0174] In embodiments, the “C800 TSC2 split intein” gene therapy comprises: (i) a first rAAV particle engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence encoding an N-TSC2 polypeptide comprising amino acids 1-799 of SEQ ID NO.: 10 (human TSC2) that is fused at its C-terminus to the N-terminus of an N-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 2, such that the full-length polypeptide encoded by the n-TSC2 / n-intein polynucleotide sequence comprises the amino acid sequence of SEQ ID NO.: 48; and (ii) a second rAAV particle engineered to deliver and express a c-intein / c-TSC2 polynucleotide sequence encoding a C-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 4 that is fused at its C-terminus to the N-terminus of a C-TSC2 polypeptide comprising amino acids 800-1807 of SEQ ID NO.: 10 (human TSC2), such that the full-length polypeptide encoded by the c-intein / c-TSC2 polynucleotide sequence comprises the amino acid sequence of SEQ ID NO.: 50, whereby when the first and second rAAV particles transduce the same cell, the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed, and the N-TSC2 polypeptide and the C-TSC2 polypeptide are joined together via intein-mediated protein splicing to form a full length TSC2 polypeptide comprising the amino acid sequence of SEQ ID NO.: 10 (human TSC2). In embodiments, the first rAAV particle is engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence comprising the nucleotide sequence of SEQ ID NO.: 47, wherein the n-TSC2 portion of said polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO.: 43 and the n-intein portion of the polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO. 1, and the second rAAV particle is engineered to deliver andexpress a c-intein / c-TSC2 polynucleotide comprising the nucleotide sequence of SEQ ID NO. : 49, wherein the C-TSC2 portion of said polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO.: 45 and the c-intein portion of the polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO. 3. Exemplary plasmid sequences containing an expression cassette comprising a polynucleotide encoding these “C800 TSC2 split intein” constructs (flanked by an upstream EFS promoter (SEQ ID NO.: 67) and a downstream bGHv3 poly(A) sequence (SEQ ID No.: 75)) are disclosed herein as SEQ ID NO.: Ill (expression cassette encoding a C800 N-TSC2 / N-Intein polypeptide) and SEQ ID NO.: 112 (expression cassette encoding a C800 C-Intein / C-TSC2 polypeptide). In embodiments, the expression cassettes are flanked by AAV ITR sequences.C811 TSC2 “Split Intein” Constructs

[0175] In embodiments, the rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein utilizes “C811 TSC2 split intein” constructs comprising (i) an N-TSC2 / N-Intein polypeptide wherein the N-TSC2 portion of the polypeptide has, comprises, or consists of amino acids 1-810 of the human TSC2 protein, and a (ii) a C-Intein / C-TSC2 polypeptide wherein the C-TSC2 portion of the polypeptide has, comprises, or consists of amino acids 811-1807 of the human TSC2 protein. In embodiments, the “C811 TSC2 split intein” gene therapy comprises: (i) a first rAAV particle engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence encoding an N-TSC2 polypeptide comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, amino acids 1-810 of SEQ ID NO.: 10 (human TSC2) that is fused at its C-terminus to the N-terminus of an N-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 2, and (ii) a second rAAV particle engineered to deliver and express a c-intein / c-TSC2 polynucleotide sequence encoding a C-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 4 that is fused at its C-terminus to the N-terminus of a C-TSC2 polypeptide beginning with a Cysteine amino acid, and further comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, amino acids 812-1807 of SEQ ID NO.: 10 (human TSC2), whereby when the first and second rAAV particles transduce the same cell, the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed, and the N-TSC2 polypeptide and the C-TSC2 polypeptide are joined together via intein-mediated protein splicing to form a full length TSC2 polypeptide comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within the amino acid sequence of SEQ ID NO.: 10.[00176J In embodiments, the “C811 TSC2 split intein” gene therapy comprises: (i) a first rAAV particle engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence encoding an N-TSC2 polypeptide comprising amino acids 1-810 of SEQ ID NO.: 10 (human TSC2) that is fused at its C-terminus to the N-terminus of an N-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 2, such that the full-length polypeptide encoded by the n-TSC2 / n-intein polynucleotide sequence comprises the amino acid sequence of SEQ ID NO.: 56; and (ii) a second rAAV particle engineered to deliver and express a c-intein / c-TSC2 polynucleotide sequence encoding a C-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 4 that is fused at its C-terminus to the N-terminus of a C-TSC2 polypeptide comprising amino acids 811-1807 of SEQ ID NO.: 10 (human TSC2), such that the full-length polypeptide encoded by the c-intein / c-TSC2 polynucleotide sequence comprises the amino acid sequence of SEQ ID NO.: 58, whereby when the first and second rAAV particles transduce the same cell, the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed, and the N-TSC2 polypeptide and the C-TSC2 polypeptide are joined together via intein-mediated protein splicing to form a full length TSC2 polypeptide comprising the amino acid sequence of SEQ ID NO.: 10 (human TSC2). In embodiments, the first rAAV particle is engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence comprising the nucleotide sequence of SEQ ID NO.: 55, wherein the n-TSC2 portion of said polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO.: 51 and the n-intein portion of the polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO. 1, and the second rAAV particle is engineered to deliver and express a c-intein / c-TSC2 polynucleotide comprising the nucleotide sequence of SEQ ID NO. : 57, wherein the C-TSC2 portion of said polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO.: 53 and the c-intein portion of the polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO. 3. Exemplary plasmid sequences containing an expression cassette comprising a polynucleotide encoding these “C811 TSC2 split intein” constructs (flankedby an upstream EFS promoter (SEQ ID NO.: 67) and a downstream bGHv3 poly(A) sequence (SEQ ID No.: 75)) are disclosed herein as SEQ ID NO.: 115 (expression cassette encoding a C811 N-TSC2 / N-Intein polypeptide) and SEQ ID NO.: 116 (expression cassette encoding a C811 C-Intein / C-TSC2 polypeptide). In embodiments, the expression cassettes are flanked by AAV ITR sequences.C1283 TSC2 “Split Intein” Constructs

[0177] In embodiments, the rAAV-mediated TSC2 “split intein” gene therapy strategy disclosed herein utilizes “C1283 TSC2 split intein” constructs comprising (i) an N-TSC2 / N-Intein polypeptide wherein the N-TSC2 portion of the polypeptide has, comprises, or consists of amino acids 1-1282 of the human TSC2 protein, and a (ii) a C-Intein / C-TSC2 polypeptide wherein the C-TSC2 portion of the polypeptide has, comprises, or consists of amino acids 1283-1807 of the human TSC2 protein. In embodiments, the “C1283 TSC2 split intein” gene therapy comprises: (i) a first rAAV particle engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence encoding anN-TSC2 polypeptide comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, amino acids 1-1282 of SEQ ID NO.: 10 (human TSC2) that is fused at its C-terminus to the N-terminus of an N-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 2, and (ii) a second rAAV particle engineered to deliver and express a c-intein / c-TSC2 polynucleotide sequence encoding a C-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 4 that is fused at its C-terminus to the N-terminus of a C-TSC2 polypeptide beginning with a Cysteine amino acid, and further comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, amino acids 1284-1807 of SEQ ID NO. : 10 (human TSC2), whereby when the first and second rAAV particles transduce the same cell, the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed, and the N-TSC2 polypeptide and the C-TSC2 polypeptide are joined together via intein-mediated protein splicing to form a full length TSC2 polypeptide comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions,deletions, or combinations thereof within the amino acid sequence of SEQ ID NO.: 10 (human TSC2).

[0178] In embodiments, the “C1283 TSC2 split intein” gene therapy comprises: (i) a first rAAV particle engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence encoding an N-TSC2 polypeptide comprising amino acids 1-1282 of SEQ ID NO.: 10 (human TSC2) that is fused at its C-terminus to the N-terminus of an N-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 2, such that the full-length polypeptide encoded by the n-TSC2 / n-intein polynucleotide sequence comprises the amino acid sequence of SEQ ID NO.: 64; and (ii) a second rAAV particle engineered to deliver and express a c-intein / c-TSC2 polynucleotide sequence encoding a C-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 4 that is fused at its C-terminus to the N-terminus of a C-TSC2 polypeptide comprising amino acids 1283-1807 of SEQ ID NO.: 10 (human TSC2), such that the full-length polypeptide encoded by the c-intein / c-TSC2 polynucleotide sequence comprises the amino acid sequence of SEQ ID NO.: 66, whereby when the first and second rAAV particles transduce the same cell, the n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed, and the N-TSC2 polypeptide and the C-TSC2 polypeptide are joined together via intein-mediated protein splicing to form a full length TSC2 polypeptide comprising the amino acid sequence of SEQ ID NO.: 10 (human TSC2). In embodiments, the first rAAV particle is engineered to deliver and express an n-TSC2 / n-intein polynucleotide sequence comprising the nucleotide sequence of SEQ ID NO.: 63, wherein the n-TSC2 portion of said polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO.: 59 and the n-intein portion of the polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO. 1, and the second rAAV particle is engineered to deliver and express a c-intein / c-TSC2 polynucleotide comprising the nucleotide sequence of SEQ ID NO. : 65, wherein the C-TSC2 portion of said polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO.: 61 and the c-intein portion of the polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO. 3. Exemplary plasmid sequences containing an expression cassette comprising a polynucleotide encoding these “C1283 TSC2 split intein” constructs (flanked by an upstream EFS promoter (SEQ ID NO.: 67) and a downstream bGHv3 poly(A) sequence (SEQ ID No.: 75)) are disclosed herein as SEQ ID NO.: 119 (expression cassette encoding a C1283 N-TSC2 / N-Intein polypeptide) and SEQ ID NO.: 120 (expression cassetteencoding a Cl 283 C-Intein / C-TSC2 polypeptide). Tn embodiments, the expression cassettes are flanked by AAV ITR sequences.rAAV VECTOR CONSTRUCTS AND EXPRESSION CASSETTES

[0179] In some embodiments, recombinant adeno-associated virus (rAAV) vector constructs are provided, which, at minimum, comprise an expression cassette with nucleotide sequences sufficient for expression of an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein, flanked by at least two inverted-terminal repeats (ITRs). In embodiments, the expression cassette comprises a promoter sequence operably linked to a polynucleotide sequence that encodes an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein. In embodiments, the expression cassette includes a polynucleotide sequence that encodes an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein that is operably linked to an EFS promoter (SEQ ID. NO.: 67). In other embodiments, the polynucleotide sequence that encodes an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein is operably linked to a CBA AGTl promoter (SEQ ID. NO.: 68). In other embodiments, the polynucleotide sequence that encodes an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein is operably linked to a CBA AGT2 promoter (SEQ ID. NO.: 69). In other embodiments, the polynucleotide sequence that encodes an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein is operably linked to a CMV-derived promoter (SEQ ID. NO.: 70). In other embodiments, the polynucleotide sequence that encodes an N-TSC2 / N-lntein or C-lntein / C-TSC2 polypeptide described herein is operably linked to a CEG promoter (SEQ ID. NO.: 71). In other embodiments, the polynucleotide sequence that encodes an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein is operably linked to a C AG promoter (SEQ ID. NO. : 72). The expression cassette may further include other regulatory sequences such as enhancers, polyadenylation (poly (A)) signal sequences, intron sequences, or WPRE sequences.

[0180] In some embodiments, the ITR sequences of the recombinant adeno-associated virus (rAAV) vector construct comprise one or more of AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, or an AAV9 ITR. In some embodiments, the ITR, or at least two ITRs, comprise a nucleic acid having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 77, SEQ ID NO.: 78 or reverse complementary sequences thereto. In some embodiments, the ITR, or at least two ITRs, comprise SEQ ID NO.: 77, SEQ ID NO.: 78, or reverse complementary sequences thereto. In some embodiments, the flanking ITRs are reverse complements of each other such that the ITR at the 5’ end of the expression cassette has a nucleotide sequence of SEQ ID NO.: 77 and the ITR at the 3 ’ end of the expression cassette has a nucleotide sequence of SEQ ID NO. : 78 (or the reverse complement of each).

[0181] In embodiments, the polynucleotide sequence that encodes an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein is operably linked to a promoter that drives expression of the N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide in target cells of the CNS, including without limitation cells of the cortex, thalamus, cerebellum, and / or striatum. In embodiments, the polynucleotide sequence that encodes an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein is operably linked to a promoter that drives expression of the N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide in target cells of the spinal cord, including without limitation cells of cervical, thoracic, and / or lumbar regions of the spinal cord.

[0182] In some embodiments, the expression cassette of the rAAV vector construct comprises a polynucleotide sequence that encodes an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein that is operably linked to a human eukaryotic translation elongation factor 1 (EF 1 a or EF 1 a) promoter. In other embodiments, the promoter is an EFS promoter, which is the core EFla or EFla promoter lacking the EF-1 a intron A sequence. In embodiments, the EFS promoter comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 67, and promotes expression of an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein in appropriate CNS tissues. In some embodiments, the EFS promoter comprises the nucleotide sequence of SEQ ID NO.: 67. Alternately, the EFS promoter is an at least 50, 100, 150, or 200 nucleotide fragment of SEQ ID NO.: 67 (or a reverse complement thereof as appropriate), and has promoter activity that promotes expression of an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein in appropriate CNS tissues.

[0183] In other embodiments, the polynucleotide sequence that encodes an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein is operably linked to a CBA AGTl promoter. The CBA AGT1 promoter is a truncated version of the CAG promoter, which is a composite, synthetic promoter that contains the cytomegalovirus (CMV) early enhancer element, the chicken P-actin promoter and the first exon and first intron of the chicken P-actin gene, and the splice acceptor of the rabbit P globin gene (Miyazaki et al. 1989, Niwa et al. 1991). In embodiments, the CBA AGTl promoter comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 68, and promotes expression of an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein in appropriate CNS tissues. In some embodiments, the CBA AGTl promoter comprises the nucleotide sequence of SEQ ID NO.: 68. Alternately, the CBA AGTl promoter is an at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 nucleotide fragment of SEQ ID NO.: 68 (or a reverse complement thereof as appropriate), and has promoter activity that promotes expression of an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein in appropriate CNS tissues.

[0184] In other embodiments, the polynucleotide sequence that encodes an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein is operably linked to a CBA_AGT2 promoter. The CBA AGT2 promoter is a truncated version of the CAG promoter, and contains cytomegalovirus (CMV) enhancer sequences, the chicken P-actin promoter sequences, and truncated SV40 late 16S intron sequences. In embodiments, the CBA AGT2 promoter comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 69, and promotes expression of an N-TSC2 / N-lntein or C-lntein / C-TSC2 polypeptide described herein in appropriate CNS tissues. In some embodiments, the CBA AGT2 promoter comprises the nucleotide sequence of SEQ ID NO.: 69. Alternately, the CBA_AGT2 promoter is an at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 nucleotide fragment of SEQ ID NO.: 69 (or a reverse complement thereof as appropriate), and haspromoter activity that promotes expression of an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein in appropriate CNS tissues.

[0185] In other embodiments, the polynucleotide sequence that encodes an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein is operably linked to a CMV-derived promoter sequence that contains the cytomegalovirus early enhancer and promoter elements. In embodiments, the CMV-derived promoter comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 70, and promotes expression of an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein in appropriate CNS tissues. In some embodiments, the CMV-derived promoter comprises the nucleotide sequence of SEQ ID NO.: 70. Alternately, the CMV-derived promoter is an at least 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 nucleotide fragment of SEQ ID NO.:27 (or a reverse complement thereof as appropriate), and has promoter activity that promotes expression of an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein in appropriate CNS tissues.

[0186] In other embodiments, the polynucleotide sequence that encodes an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein is operably linked to a CEG promoter sequence. The CEG promoter is a composite, synthetic promoter that contains CMV enhancer and promoter sequences, and chimeric intron and exon from the chicken P-actin promoter. In embodiments, the CEG promoter comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 71, and promotes expression of an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein in appropriate CNS tissues. In some embodiments, the CEG promoter comprises the nucleotide sequence of SEQ ID NO.: 71. Alternately, the CEG promoter is an at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, or 1150 nucleotide fragment of SEQ ID NO.: 71 (or a reverse complement thereof as appropriate), and has promoter activity that promotes expression of an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein in appropriate CNS tissues.

[0187] In some embodiments the polynucleotide sequence that encodes an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein is operably linked to a CAG promoter sequence. A CAG promoter is a composite, synthetic promoter which contains the CMV early enhancer element, the chicken P-actin promoter and the first exon and first intron of the chicken -actin gene, and the splice acceptor of the rabbit P globin gene (Miyazaki et al. 1989, Niwa et al.1991). In embodiments, the CAG promoter comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 72, and promotes expression of an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide in appropriate CNS tissues. In some embodiments, the CAG promoter comprises the nucleotide sequence of SEQ ID NO.: 72. Alternately, the CAGpromoteris an at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, or 1150 nucleotide fragment of SEQ ID NO.: 72 (or a reverse complement thereof as appropriate), and has promoter activity that promotes expression of an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide in appropriate CNS tissues.

[0188] In embodiments, the polynucleotide sequence that encodes an N-TSC2 / N-Intein polypeptide described herein is operably linked to a different promoter than the polynucleotide sequence that encodes a C-Intein / C-TSC2 polypeptide described herein. In embodiments, the polynucleotide sequence that encodes an N-TSC2 / N-Intein polypeptide described herein is operably linked to a stronger promoter than the polynucleotide sequence that encodes a C-Intein / C-TSC2 polypeptide described herein such that, when the N-TSC2 / N-Intein and C-Intein / C-TSC2 polypeptides are co-expressed in appropriate CNS tissues, the N-Intein / NTSC2 polypeptide is expressed at a higher level than the C-Intein / C-TSC2 polypeptide. In embodiments, the polynucleotide sequence that encodes an N-TSC2 / N-Intein polypeptide described herein is operably linked to a weaker promoter than the polynucleotide sequence that encodes a C-Intein / C-TSC2 polypeptide described herein such that, when the N-TSC2 / N-lntein and C-lntein / C-TSC2 polypeptides are co-expressed in appropriate CNS tissues, the N-Intein / NTSC2 polypeptide is expressed at a lower level than the C-Intein / C-TSC2 polypeptide.

[0189] In some embodiments of the rAAV vector construct, the N-TSC2 / N-Intein or C-Intein / C-TSC2) expression cassette is in an anti-sense (e.g. reverse complementary) orientation orin sense orientation. In some embodiments, the rAAV vector construct comprises two or more expression cassettes.

[0190] In some embodiments of the rAAV vector construct, the N-TSC2 / N-Intein or C-Intein / C-TSC2 expression cassette further comprises a nucleic acid that encodes a polyadenylation (poly(A)) signal operably linked to the 3’ end of the polynucleotide sequence that encodes an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide, such that the expressed mRNA has a polyA tail. In some embodiments, the nucleic acid that encodes the poly(A) signal comprises a bGH-polyA-v3 sequence, which is a synthetic poly(A) signal derived from the bovine growth hormone polyadenylation sequence. In some embodiments, the bGH-polyA-v3 sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 75 (or a reverse complement thereof). In some embodiments, the bGH-polyA-v3 sequence comprises the nucleotide sequence of SEQ ID NO.: 75 (or a reverse complement thereof). Alternately, the bGH-polyA-v3 sequence is an at least 50, 75, 100, 125, 150, 175, 200, or 225 nucleotide fragment of SEQ ID NO.: 75 (or a reverse complement thereof), and supports expression of an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide in appropriate CNS tissues.

[0191] In some embodiments, the nucleic acid that encodes the poly(A) signal comprises a bovine growth hormone polyadenylation signal sequence (bGHpA). In some embodiments, the bGHpA sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 74 (or a reverse complement thereof). In some embodiments, the bGHpA sequence comprises the nucleotide sequence of SEQ ID NO.: 74 (or a reverse complement thereof). Alternately, the bGHpA sequence is an at least 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotide fragment of SEQ ID NO.: 74 (or a reverse complement thereof), and supports expression of an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide in appropriate CNS tissues.

[0192] In some embodiments, the nucleic acid that encodes the poly(A) signal comprises a human growth hormone polyadenylation signal (huGHpA). In some embodiments, the huGHpA signal sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 73 (or a reverse complement thereof). In some embodiments, the huGHpA signal sequence comprises the nucleotide sequence of SEQ ID NO.: 73 (or a reverse complement thereof). Alternately, the huGHpA signal sequence is an at least 100, 150, 200, 250, 300, 350, 400, or 450 nucleotide fragment of SEQ ID NO.: 73 (or a reverse complement thereof), and supports expression of an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide in appropriate CNS tissues.

[0193] In some embodiments of the rAAV vector construct, the N-TSC2 / N-Intein or C-Intein / C-TSC2 expression cassette further comprises regulatory elements that may enhance expression of an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide. In some embodiments, the expression cassette further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the WPRE is downstream (3’ of) the polynucleotide sequence encoding an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide, and upstream (5’ of) the poly(A) tail signal. In some embodiments, the WPRE comprises a nucleic acid having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within SEQ ID NO.: 76 (or reverse complement thereof) that enhances expression of an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide. In some embodiments, the WPRE comprises SEQ ID NO.: 76 (or a reverse complement thereof). Alternately, the WPRE is an at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 nucleotide fragment of SEQ ID NO.: 76 (or a reverse complement thereof as appropriate), and enhances expression of an N-TSC2 / N-lntein or C-lntein / C-TSC2 polypeptide in appropriate CNS tissues. In other embodiments, the N-TSC2 / N-Intein or C-Intein / C-TSC2 expression cassette further comprises an intron or a chimeric intron sequence that promotes expression of an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide, and in certain embodiments the intron sequence is inserted between the promoter and the coding sequence.

[0194] In embodiments, the foregoing constructs are flanked by AAV ITR sequences, including AAV2 ITR sequences.

[0195] In some embodiments, the recombinant AAV vector construct is packaged into a recombinant AAV particle, including a particle comprising a modified capsid with enhanced ability to cross the blood brain barrier, including AAV particles incorporating an hTfRl AAV capsid sequence. Provided, thus, are AAV vector constructs that can be incorporated into an AAV particle engineered to cross the blood-brain barrier for expression of an N-TSC2 / N-lntein or C-Intein / C-TSC2 polypeptide in target cells of the CNS. In embodiments, the AAV vector construct may have elements arranged as follows: 5’AAV2ITR-EFS promoter sequence-nucleotide sequence encoding an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide-bGH-polyA-v3 polyadenylation signal sequence-3’ AAV2 ITR. In other embodiments, the AAV vector construct may have elements arranged as follows: EFS promoter sequence- nucleotide sequence encoding an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide-bGH-polyA-v3 polyadenylation signal sequence, flanked by at least two AAV ITRs (5’ and 3’), selected from AAV1 ITRs, AAV2 ITRs, AAV3 ITRs, AAV4 ITRs, AAV5 ITRs, AAV6 ITRs, AAV7 ITRs, AAV8 ITRs, or AAV9 ITRs.

[0196] In embodiments, recombinant AAV vector constructs are designed to express “C519 TSC2 splitintein” constructs, whereby a first AAV vector construct is engineered toexpress an N-TSC2 / N-Intein polypeptide, said first AAV vector construct having, comprising, or consisting of a nucleic acid having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 93, and a second AAV vector construct is engineered to express a C-Intein / C-TSC2 polypeptide, said second AAV vector construct having, comprising, or consisting of a nucleic acid having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 94. In embodiments, the recombinant AAV vector constructs designed to express “C519 TSC2 split intein” constructs comprise a first AAV vector construct engineered to express an N-TSC2 / N-Intein polypeptide, said first AAV vector construct having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO.: 93, and a second AAV vector construct engineered to express a C-Intein / C-TSC2 polypeptide, said second AAV vector construct having,comprising, or consisting of the nucleic acid sequence of SEQ ID NO.: 94. In each of these vector constructs, the nucleotide sequences encoding the N-TSC2 / N-Intein and C-Intein / C-TSC2 polypeptide are operably linked to an upstream (5’) EFS promoter sequence, and a downstream (3’) bGH-polyA-v3 polyadenylation signal sequence, with flanking 5’ and 3’ AAV ITRs to allow the vector constructs to be separately packaged as recombinant AAV particles.

[0197] In embodiments, recombinant AAV vector constructs are designed to express “C728 TSC2 split intein” constructs, whereby a first AAV vector construct is engineered to express an N-TSC2 / N-Intein polypeptide, said first AAV vector construct comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 97, and a second AAV vector construct is engineered to express a C-Intein / C-TSC2 polypeptide, said second AAV vector construct comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 98. In embodiments, the recombinant AAV vector constructs designed to express “C728 TSC2 split intein” constructs comprise a first AAV vector construct engineered to express an N-TSC2 / N-Intein polypeptide, said first AAV vector construct having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO.: 97, and a second AAV vector construct engineered to express a C-Intein / C-TSC2 polypeptide, said second AAV vector construct having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO.: 98. In each of these vector constructs, the nucleotide sequences encoding the N-TSC2 / N-Intein and C-Intein / C-TSC2 polypeptide are operably linked to an upstream (5’) EFS promoter sequence, and a downstream (3’) bGH-polyA-v3 polyadenylation signal sequence, with flanking 5’ and 3’ AAV ITRs to allow the vector constructs to be separately packaged as recombinant AAV particles.

[0198] In embodiments, recombinant AAV vector constructs are designed to express “C738 TSC2 split intein” constructs, whereby a first AAV vector construct is engineered to express an N-TSC2 / N-Intein polypeptide, said first AAV vector construct comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,or 99.9% identity to, or 1 , 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 101, and a second AAV vector construct is engineered to express a C-Intein / C-TSC2 polypeptide, said second AAV vector construct comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 102. In embodiments, the recombinant AAV vector constructs designed to express “C738 TSC2 split intein” constructs comprise a first AAV vector construct engineered to express an N-TSC2 / N-Intein polypeptide, said first AAV vector construct having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO.: 101, and a second AAV vector construct engineered to express a C-Intein / C-TSC2 polypeptide, said second AAV vector construct having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO.: 102. In each of these vector constructs, the nucleotide sequences encoding the N-TSC2 / N-Intein and C-Intein / C-TSC2 polypeptide are operably linked to an upstream (5’) EFS promoter sequence, and a downstream (3’) bGH-polyA-v3 polyadenylation signal sequence, with flanking 5’ and 3’ AAV ITRs to allow the vector constructs to be separately packaged as recombinant AAV particles.

[0199] In embodiments, recombinant AAV vector constructs are designed to express “C791 TSC2 split intein” constructs, whereby a first AAV vector construct is engineered to express an N-TSC2 / N-Intein polypeptide, said first AAV vector construct comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 105, and a second AAV vector construct is engineered to express a C-Intein / C-TSC2 polypeptide, said second AAV vector construct comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 106. In embodiments, the recombinant AAV vector constructs designed to express “C791 TSC2 split intein” constructs comprise a first AAV vector construct engineered to express an N-TSC2 / N-Intein polypeptide, said first AAV vector construct having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO.: 105, and a second AAVvector construct engineered to express a C-Intein / C-TSC2 polypeptide, said second AAV vector construct having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO.: 106. In each of these vector constructs, the nucleotide sequences encoding the N-TSC2 / N-Intein and C-Intein / C-TSC2 polypeptide are operably linked to an upstream (5’) EFS promoter sequence, and a downstream (3’) bGH-polyA-v3 polyadenylation signal sequence, with flanking 5’ and 3’ AAV ITRs to allow the vector constructs to be separately packaged as recombinant AAV particles.[00200J In embodiments, recombinant AAV vector constructs are designed to express “C800 TSC2 split intein” constructs, whereby a first AAV vector construct is engineered to express an N-TSC2 / N-Intein polypeptide, said first AAV vector construct comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 109, and a second AAV vector construct is engineered to express a C-Intein / C-TSC2 polypeptide, said second AAV vector construct comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 110. In embodiments, the recombinant AAV vector constructs designed to express “C800 TSC2 split intein” constructs comprise a first AAV vector construct engineered to express an N-TSC2 / N-Intein polypeptide, said first AAV vector construct having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO.: 109, and a second AAV vector construct engineered to express a C-Intein / C-TSC2 polypeptide, said second AAV vector construct having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO.: 110. In each of these vector constructs, the nucleotide sequences encoding the N-TSC2 / N-Intein and C-Intein / C-TSC2 polypeptide are operably linked to an upstream (5’) EFS promoter sequence, and a downstream (3’) bGH-polyA-v3 polyadenylation signal sequence, with flanking 5’ and 3’ AAV ITRs to allow the vector constructs to be separately packaged as recombinant AAV particles.

[0201] In embodiments, recombinant AAV vector constructs are designed to express “C811 TSC2 split intein” constructs, whereby a first AAV vector construct is engineered to express an N-TSC2 / N-Intein polypeptide, said first AAV vector construct comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 113, and a second AAV vector construct is engineered to express a C-Intein / C-TSC2 polypeptide, said second AAV vector construct comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 114. In embodiments, the recombinant AAV vector constructs designed to express “C811 TSC2 split intein” constructs comprise a first AAV vector construct engineered to express an N-TSC2 / N-Intein polypeptide, said first AAV vector construct having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO.: 113, and a second AAV vector construct engineered to express a C-Intein / C-TSC2 polypeptide, said second AAV vector construct having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO.: 114. In each of these vector constructs, the nucleotide sequences encoding the N-TSC2 / N-Intein and C-Intein / C-TSC2 polypeptide are operably linked to an upstream (5’) EFS promoter sequence, and a downstream (3’) bGH-polyA-v3 polyadenylation signal sequence, with flanking 5’ and 3’ AAV ITRs to allow the vector constructs to be separately packaged as recombinant AAV particles.

[0202] In embodiments, recombinant AAV vector constructs are designed to express “C1283 TSC2 split intein” constructs, whereby a first AAV vector construct is engineered to express an N-TSC2 / N-Intein polypeptide, said first AAV vector construct comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 117, and a second AAV vector construct is engineered to express a C-Intein / C-TSC2 polypeptide, said second AAV vector construct comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 118. In embodiments, the recombinant AAV vector constructs designed to express “C1283 TSC2 split intein” constructs comprise a first AAV vector construct engineered to express an N-TSC2 / N-Intein polypeptide, said first AAV vector constructhaving, comprising, or consisting of the nucleic acid sequence of SEQ ID NO.: 117, and a second AAV vector construct engineered to express a C-Intein / C-TSC2 polypeptide, said second AAV vector construct having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO.: 118. In each of these vector constructs, the nucleotide sequences encoding the N-TSC2 / N-Intein and C-Intein / C-TSC2 polypeptide are operably linked to an upstream (5’) EFS promoter sequence, and a downstream (3’) bGH-polyA-v3 polyadenylation signal sequence, with flanking 5’ and 3’ AAV ITRs to allow the vector constructs to be separately packaged as recombinant AAV particles.

[0203] In embodiments, expression cassettes are designed to express “C519 TSC2 split intein” constructs, whereby a first expression cassette is engineered to express anN-TSC2 / N-Intein polypeptide, said first expression cassette comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 79, and a second expression cassette is engineered to express a C-Intein / C-TSC2 polypeptide, said second expression cassette comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 80. In embodiments, the expression cassettes designed to express “C519 TSC2 split intein” constructs comprise a first expression cassette engineered to express an N-TSC2 / N-Intein polypeptide, said first expression cassette having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO. : 79, and a second expression cassette engineered to express a C-Intein / C-TSC2 polypeptide, said second expression cassette having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO. : 80. These expression cassettes may be flanked by at least two AAV ITRs (5’ and 3’), selected from AAV1 ITRs, AAV2 ITRs, AAV3 ITRs, AAV4 ITRs, AAV5 ITRs, AAV6 ITRs, AAV7 ITRs, AAV8 ITRs, or AAV9 ITRs to create AAV vector constructs suitable for the production of rAAV particles expressing the “C519 TSC2 split intein” constructs. In each of these expression cassettes, the nucleotide sequences encoding the N-TSC2 / N-Intein and C-Intein / C-TSC2 polypeptide are operably linked to an upstream (5’) EFS promoter sequence, and a downstream (3’) bGH-polyA-v3 polyadenylation signal sequence. Such expression cassettes mayalso be cloned into plasmids or other recombinant viral particles for expression in target cells of interest, including cells of the CNS.

[0204] In embodiments, expression cassettes are designed to express “C728 TSC2 split intein” constructs, whereby a first expression cassette is engineered to express anN-TSC2 / N-Intein polypeptide, said first expression cassette comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 81, and a second expression cassette is engineered to express a C-Intein / C-TSC2 polypeptide, said second expression cassette comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 82. In embodiments, the expression cassettes designed to express “C728 TSC2 split intein” constructs comprise a first expression cassette engineered to deliver an N-TSC2 / N-Intein polypeptide, said first expression cassette having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO. : 81 , and a second expression cassette engineered to express a C-Intein / C-TSC2 polypeptide, said second expression cassette having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO. : 82. These expression cassettes may be flanked by at least two AAV ITRs (5’ and 3’), selected from AAV1 ITRs, AAV2 ITRs, AAV3 ITRs, AAV4 ITRs, AAV5 ITRs, AAV6 ITRs, AAV7 ITRs, AAV8 ITRs, or AAV9 ITRs to create AAV vector constructs suitable for the production of rAAV particles expressing the “C728 TSC2 split intein” constructs. In each of these expression cassettes, the nucleotide sequences encoding the N-TSC2 / N-Intein and C-Intein / C-TSC2 polypeptide are operably linked to an upstream (5’) EFS promoter sequence, and a downstream (3’) bGH-polyA-v3 polyadenylation signal sequence. Such expression cassettes may also be cloned into plasmids or other recombinant viral particles for expression in target cells of interest, including cells of the CNS.

[0205] In embodiments, expression cassettes are designed to express “C738 TSC2 split intein” constructs, whereby a first expression cassette is engineered to express anN-TSC2 / N-Intein polypeptide, said first expression cassette comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 83, and a second expression cassette is engineered to express a C-Intein / C-TSC2 polypeptide, said second expression cassette comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 84. In embodiments, the expression cassettes designed to express “C738 TSC2 split intein” constructs comprise a first expression cassette engineered to express an N-TSC2 / N-Intein polypeptide, said first expression cassette having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO. : 83, and a second expression cassette engineered to express a C-Intein / C-TSC2 polypeptide, said second expression cassette having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO. : 84. These expression cassettes may be flanked by at least two AAV ITRs (5’ and 3’), selected from AAV1 ITRs, AAV2 ITRs, AAV3 ITRs, AAV4 ITRs, AAV5 ITRs, AAV6 ITRs, AAV7 ITRs, AAV8 ITRs, or AAV9 ITRs to create AAV vector constructs suitable for the production of rAAV particles expressing the “C738 TSC2 split intein” constructs. In each of these expression cassettes, the nucleotide sequences encoding the N-TSC2 / N-Intein and C-Intein / C-TSC2 polypeptide are operably linked to an upstream (5’) EFS promoter sequence, and a downstream (3’) bGH-polyA-v3 polyadenylation signal sequence. Such expression cassettes may also be cloned into plasmids or other recombinant viral particles for expression in target cells of interest, including cells of the CNS.

[0206] In embodiments, expression cassettes are designed to express “C791 TSC2 split intein” constructs, whereby a first expression cassette is engineered to express anN-TSC2 / N-Intein polypeptide, said first expression cassette comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 85, and a second expression cassette is engineered to express a C-Intein / C-TSC2 polypeptide, said second expression cassette comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to,or 1 , 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 86. In embodiments, the expression cassettes designed to express “C791 TSC2 split intein” constructs comprise a first expression cassette engineered to express an N-TSC2 / N-Intein polypeptide, said first expression cassette having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO. : 85, and a second expression cassette engineered to express a C-Intein / C-TSC2 polypeptide, said second expression cassette having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO. : 86. These expression cassettes may be flanked by at least two AAV ITRs (5’ and 3’), selected from AAV1 ITRs, AAV2 ITRs, AAV3 ITRs, AAV4 ITRs, AAV5 ITRs, AAV6 ITRs, AAV7 ITRs, AAV8 ITRs, or AAV9 ITRs to create AAV vector constructs suitable for the production of rAAV particles expressing the “C791 TSC2 split intein” constructs. In each of these expression cassettes, the nucleotide sequences encoding the N-TSC2 / N-Intein and C-Intein / C-TSC2 polypeptide are operably linked to an upstream (5’) EFS promoter sequence, and a downstream (3’) bGH-polyA-v3 polyadenylation signal sequence. Such expression cassettes may also be cloned into plasmids or other recombinant viral particles for expression in target cells of interest, including cells of the CNS.

[0207] In embodiments, expression cassettes are designed to express “C800 TSC2 split intein” constructs, whereby a first expression cassette is engineered to express anN-TSC2 / N-Intein polypeptide, said first expression cassette comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 87, and a second expression cassette is engineered to express a C-Intein / C-TSC2 polypeptide, said second expression cassette comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 88. In embodiments, the expression cassettes designed to express “C800 TSC2 split intein” constructs comprise a first expression cassette engineered to express an N-TSC2 / N-Intein polypeptide, said first expression cassette having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO. : 87, and a second expression cassette engineered to express a C-Intein / C-TSC2 polypeptide, said second expression cassette having, comprising, or consisting of the nucleicacid sequence of SEQ ID NO.: 88. These expression cassettes may be flanked by at least two AAV ITRs (5’ and 3’), selected from AAV1 ITRs, AAV2 ITRs, AAV3 ITRs, AAV4 ITRs, AAV5 ITRs, AAV6 ITRs, AAV7 ITRs, AAV8 ITRs, or AAV9 ITRs to create AAV vector constructs suitable for the production of rAAV particles expressing the “C800 TSC2 split intein” constructs. In each of these expression cassettes, the nucleotide sequences encoding the N-TSC2 / N-Intein and C-Intein / C-TSC2 polypeptide are operably linked to an upstream (5’) EFS promoter sequence, and a downstream (3’) bGH-polyA-v3 polyadenylation signal sequence. Such expression cassettes may also be cloned into plasmids or other recombinant viral particles for expression in target cells of interest, including cells of the CNS.

[0208] In embodiments, expression cassettes are designed to express “C811 TSC2 split intein” constructs, whereby a first expression cassette is engineered to express anN-TSC2 / N-Intein polypeptide, said first expression cassette comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 89, and a second expression cassette is engineered to express a C-Intein / C-TSC2 polypeptide, said second expression cassette comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 90. In embodiments, the expression cassettes designed to express “C811 TSC2 split intein” constructs comprise a first expression cassette engineered to express an N-TSC2 / N-Intein polypeptide, said first expression cassette having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO. : 89, and a second expression cassette engineered to express a C-Intein / C-TSC2 polypeptide, said second expression cassette having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO.: 90. These expression cassettes may be flanked by at least two AAV ITRs (5’ and 3’), selected from AAV1 ITRs, AAV2 ITRs, AAV3 ITRs, AAV4 ITRs, AAV5 ITRs, AAV6 ITRs, AAV7 ITRs, AAV8 ITRs, or AAV9 ITRs to create AAV vector constructs suitable for the production of rAAV particles expressing the “C811 TSC2 split intein” constructs. In each of these expression cassettes, the nucleotide sequences encoding the N-TSC2 / N-Intein and C-Intein / C-TSC2 polypeptide are operably linked to an upstream (5’) EFS promoter sequence, and adownstream (3’) bGH-polyA-v3 polyadenylation signal sequence. Such expression cassettes may also be cloned into plasmids or other recombinant viral particles for expression in target cells of interest, including cells of the CNS.

[0209] In embodiments, expression cassettes are designed to express “C1283 TSC2 split intein” constructs, whereby a first expression cassette is engineered to express anN-TSC2 / N-Intein polypeptide, said first expression cassette comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 91, and a second expression cassette is engineered to express a C-Intein / C-TSC2 polypeptide, said second expression cassette comprising a nucleic acid having, comprising, or consisting of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 92. In embodiments, the expression cassettes designed to express “C1283 TSC2 split intein” constructs comprise a first expression cassette engineered to express an N-TSC2 / N-Intein polypeptide, said first expression cassette having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO. : 91, and a second expression cassette engineered to express a C-Intein / C-TSC2 polypeptide, said second expression cassette having, comprising, or consisting of the nucleic acid sequence of SEQ ID NO. : 92. These expression cassettes may be flanked by at least two AAV ITRs (5’ and 3’), selected from AAV1 ITRs, AAV2 ITRs, AAV3 ITRs, AAV4 ITRs, AAV5 ITRs, AAV6 ITRs, AAV7 ITRs, AAV8 ITRs, or AAV9 ITRs to create AAV vector constructs suitable for the production of rAAV particles expressing the “C1283 TSC2 split intein” constructs. In each of these expression cassettes, the nucleotide sequences encoding the N-TSC2 / N-Intein and C-Intein / C-TSC2 polypeptide are operably linked to an upstream (5’) EFS promoter sequence, and a downstream (3’) bGH-polyA-v3 polyadenylation signal sequence. Such expression cassettes may also be cloned into plasmids or other recombinant viral particles for expression in target cells of interest, including cells of the CNS.AAV VECTOR PLASMIDS

[0210] In some embodiments, AAV vector plasmids are provided that are used to prepare recombinant AAV viral particles having a recombinant genome comprising an AAV vector construct having an expression cassette comprising a nucleotide sequence encoding an N-TSC2 / N-Intein or C-Intein / C-TSC2 polypeptide described herein, operably linked to regulatory elements that promote expression in appropriate tissues and cells, such as tissues and cells of the CNS, flanked by ITRs that allow the recombinant genome to be packaged as an rAAV particle, including an rAAV particle comprising an hTfR.1 AAV capsid. The plasmids provided herein generally have an origin of replication and selectable markers to permit replication of the plasmid and use in host cells for generating the recombinant AAV viral particles described herein.

[0211] Exemplary AAV vector plasmid sequences comprising an AAV vector construct having an expression cassette comprising a nucleotide sequence encoding “C519 TSC2 split intein” constructs are disclosed herein as SEQ ID NO.: 121 (encoding a C519 N-TSC2 / N-Intein polypeptide) and SEQ ID NO.: 122 (encoding a C519 C-Intein / C-TSC2 polypeptide).

[0212] Exemplary AAV vector plasmid sequences comprising an AAV vector construct having an expression cassette comprising a nucleotide sequence encoding “C728 TSC2 split intein” constructs are disclosed herein as SEQ ID NO.: 123 (encoding a C728 N-TSC2 / N-Intein polypeptide) and SEQ ID NO.: 124 (encoding a C728 C-Intein / C-TSC2 polypeptide).

[0213] Exemplary AAV vector plasmid sequences comprising an AAV vector construct having an expression cassette comprising a nucleotide sequence encoding “C738 TSC2 split intein” constructs are disclosed herein as SEQ ID NO.: 125 (encoding a C738 N-TSC2 / N-Intein polypeptide) and SEQ ID NO.: 126 (encoding a C738 C-Intein / C-TSC2 polypeptide).

[0214] Exemplary AAV vector plasmid sequences comprising an AAV vector construct having an expression cassette comprising a nucleotide sequence encoding “C791 TSC2 split intein” constructs are disclosed herein as SEQ ID NO.: 127 (encoding a C791 N-TSC2 / N-Intein polypeptide) and SEQ ID NO.: 128 (encoding a C791 C-Intein / C-TSC2 polypeptide).

[0215] Exemplary AAV vector plasmid sequences comprising an AAV vector construct having an expression cassette comprising a nucleotide sequence encoding “C800 TSC2 splitintein” constructs are disclosed herein as SEQ ID NO.: 129 (encoding a C800 N-TSC2 / N-Intein polypeptide) and SEQ ID NO.: 130 (encoding a C800 C-Intein / C-TSC2 polypeptide).

[0216] Exemplary AAV vector plasmid sequences comprising an AAV vector construct having an expression cassette comprising a nucleotide sequence encoding “C811 TSC2 split intein” constructs are disclosed herein as SEQ ID NO.: 131 (encoding a C811 N-TSC2 / N-Intein polypeptide) and SEQ ID NO.: 132 (encoding a C811 C-Intein / C-TSC2 polypeptide).

[0217] Exemplary AAV vector plasmid sequences comprising an AAV vector construct having an expression cassette comprising a nucleotide sequence encoding “Cl 283 TSC2 split intein” constructs are disclosed herein as SEQ ID NO.: 133 (encoding a C1283 N-TSC2 / N-Intein polypeptide) and SEQ ID NO.: 134 (encoding a C1283 C-Intein / C-TSC2 polypeptide).

[0218] In embodiments, the AAV vector plasmid further comprises a bacterial expressing region. In some embodiments, the bacterial expressing region comprises a bacterial promoter and a nucleic acid that encodes a bacterial selecting region. In some embodiments, the nucleic acid that encodes the bacterial selecting region is operably linked to the bacterial promoter. In some embodiments, the nucleic acid that encodes the bacterial selecting region comprises a nucleic acid that encodes an antibiotic resistance gene or protein. In some embodiments, the antibiotic resistance gene or protein comprises an ampicillin resistance gene (AmpR) or a kanamycin resistance gene sequence (KanR). In some embodiments the bacteria promoter comprises AmpR promoter or a KanR promoter. In some embodiments, the AAV vector plasmid further comprises an origin of replication. In some embodiments, the origin of replication comprises a CMV origin of replication (ori). In some embodiments, the AAV vector plasmid further comprises a eukaryotic expressing region.RECOMBINANT AAV PARTICLES

[0219] To facilitate this rAAV-mediated TSC2 “split intein” gene therapy, in embodiments either or both of (i) the first rAAV particle (rAAV-n-TSC2 / n-intein particle) and (ii) the second rAAV particle (rAAV-c-intein / c-TSC2 particle) are engineered to include one or more capsid modifications to enhance CNS delivery, including hTfRl rAAV particles, hCA4 rAAV particles, hCD59 rAAV particles, ALPL rAAV particles, and rAAV(AE) particles, as described below.hTfRl rAAV particles

[0220] In embodiments, rAAV particles of the present disclosure are engineered to cross the blood-brain barrier (BBB) and transduce cells of the CNS through binding to the hTfRl receptor (hTfRl rAAV particle). In embodiments, these hTfRl rAAV particles comprise a capsid protein that is engineered to bind the hTfRl receptor (hTfRl rAAV capsid protein), and further comprise a recombinant AAV genome containing an expression cassette that encodes an n-TSC2 / n-intein or c-intein / c-TSC2 polynucleotide sequence (as described herein), operably linked to regulatory elements. Although not to be bound to a particular mechanism of action, these hTfRl rAAV capsid proteins, derived from the naturally occurring AAV9 capsid, are thought to confer enhanced ability to cross the BBB, likely via human transferrin receptor 1 (hTfRl) receptor mediated transcytosis (RMT), thereby providing for efficient delivery throughout the CNS. In embodiments, the hTfRl AAV particle comprises a modified capsid sequence as set forth in PCT Application No. PCT / US2023 / 070285, published January 18, 2024 as WO 2024 / 016003, the entirety of which is incorporated herein. In other embodiments, the hTfRl AAV particle comprises a modified capsid sequence as set forth in PCT Application No. PCT / US2025 / 12207, published July 24, 2025 as WO 2025 / 155923, the entirety of which is incorporated herein, including disclosures of AAV particles engineered to incorporate hTfRl targeting sequences that facilitate enhanced CNS transduction in the brains of mice that were engineered to express human TFRC, relative to a comparator AAV particle, such as an AAV9 particle.

[0221] In embodiments, the hTfRl rAAV particle comprises an hTfRl rAAV capsid protein including a transferrin receptor (TfRl) binding modification sequence comprising a 7-mer amino acid sequence inserted between amino acids 588 and 589 of an AAV9 or AAV9 K449R VP1 capsid polypeptide (i.e., SEQ ID NO.: 138 or SEQ ID NO.: 141, respectively), or in an analogous position of a capsid polypeptide of another AAV serotype wherein the rAAV particle incorporating the modified capsid protein exhibits increased binding to Tfirl, crossing the BBB and / or transduction of CNS cells relative to an rAAV particle having a capsid protein without the modifications. In embodiments, the 7-mer is selected from YSRIGPN (SEQ ID NO.: 149), YSRNSDN (SEQ ID NO.: 150), LHRLGPN (SEQ ID NO.: 151), LHRLGPD (SEQ ID NO.: 152), LHRAGPD (SEQ ID NO.: 153), YSRIGPD (SEQ ID NO.: 154), LSRIGPD (SEQ ID NO.: 155), LARSGPD (SEQ ID NO.: 156), LHKAGPN (SEQ ID NO.: 157), LSRIGPN (SEQ ID NO : 158),LAKSGPN (SEQ ID NO : 159), YARNGPN (SEQ ID NO : 160) and FRSTNGV (SEQ ID NO.: 161). In an example embodiment, the 7-mer motif is YSRIGPN (SEQ ID NO.: 149).

[0222] In embodiments, the hTfRl rAAV particle comprises an hTfRl rAAV capsid protein including a transferrin receptor (TfRl) binding modification sequence defined by the formula Xl-X2-X3-[7-mer]-X4-X5-X6-X7, wherein the 7-mer is one of the amino acid sequences YSRIGPN (SEQ ID NO.: 149), YSRNSDN (SEQ ID NO.: 150), LHRLGPN (SEQ ID NO.: 151), FRSTNGV (SEQ ID NO.: 161), FVSTNGV (SEQ ID NO.: 162), FZ1STNGZ2 (SEQ ID NO.: 163), FRSTNGZ3 (SEQ ID NO.: 164), or VESTNGR (SEQ ID NO.: 165), and wherein the 7-mer is inserted between amino acids 588 and 589 of an AAV9 or AAV9 K449R VP1 capsid polypeptide (e.g., SEQ ID NO.: 138 or SEQ ID NO.: 141), or in an analogous position of a capsid polypeptide of another AAV serotype, and wherein XI, X2, X3, X4, X5, X6, X7 indicate an amino acid substitution at one or more of these positions in the capsid polypeptide flanking the inserted 7-mer (i.e., residues 586, 587, 588, 589, 590, 591 and 592 of AAV9 or corresponding positions of any other AAV serotypes), and Zl, Z2, and Z3 indicate variant amino acid positions within the 7-mer polypeptide wherein Zl is selected from A, D, H, N, Q, and S; Z2 is K or R; and Z3 is selected from L, M, and R. In an embodiment, the 7-mer includes one of YSRIGPN (SEQ ID NO.: 149), YSRNSDN (SEQ ID NO.: 150), or LHRLGPN (SEQ ID NO.: 151), and wherein XI is A, G, E, L, N, Q, S, W, or M; X2 includes A, F, I, L, M, N, Q, P, T, V, or Y; X3 includes Q; X4 includes any amino acid; X5 includes D, F, G, I, L, M, Q, P, S, T, or V; X6 includes A, C, F, G, H, I, P, S, T, V, W, or Y; X7 includes D, E, Q, or T; or any combination thereof where there is at least one amino acid substitution relative to the wild type sequence. In an embodiment, the 7-mer includes the amino acid sequence YSRIGPN (SEQ ID NO.: 149), and wherein XI is A, G, E, L, N, Q, S, W, or M; X2 is A, F, I, L, M, N, Q, P, T, V, or Y; X3 is Q; X4 is A, E, F, H, I, L, M, N, P, Q, V, Y, D, or G; X5 is D, F, G, I, L, M, Q, P, S, T, or V; X6 is A, C, F, G, H, I, P, S, T, V, W, or Y; X7 is D, E, Q, or T; or any combination thereof where there is at least one amino acid substitution relative to the wild type sequence. In an embodiment, the 7-mer is YSRNSDN (SEQ ID NO.: 150) and XI is S, or W; X2 is V, I, or F; X3 is Q; X4 is any amino acid; X5 is Q or T; X6 is A; X7 is Q; or any combination thereof. In an embodiment, the 7-mer is LHRLGPN (SEQ ID NO.: 151) and XI is S, A, L, or M; X2 is A or P; X3 is Q; X4 is A, E, F, H, I, L, M, N, P, Q, V, or Y; X5 is Q; X6 is A, P, S, or T; X7 is D, E, Q, or T; or any combination thereof. In an embodiment, the 7-mer is FRSTNGV (SEQ ID NO.: 161), FVSTNGV (SEQ ID NO.: 162), FZ1STNGZ2 (SEQ IDNO.: 163), FRSTNGZ3 (SEQ ID NO.: 164), or VESTNGR (SEQ ID NO.: 165) and wherein XI is S; X2 is A, S, M, or D; X3 is F, H, I, L, M, N, Q, R, Y, D, or E; X4 is A, S, or M; X5 is Q or P; X6 is A, F, H, Q, or S; X7 is A, D, E, F, Q, S, or T; or any combination thereof. In an embodiment, the 7-mer is FRSTNGV (SEQ ID NO.: 161), and XI is S; X2 is A or S; X3 is D; X4 is A, or S; X5 is Q or P; X6 is A, F, H, Q, or S; X7 is D, E, Q, or T; or any combination thereof. In an embodiment, the 7-mer is FVSTNGV (SEQ ID NO.: 162), and XI is S; X2 is A, S, or M; X3 is Q, E, or D; X4 is A, or M; X5 is Q or P; X6 is A; X7 is E; or any combination thereof. In an embodiment, the 7-mer is FZ1STNGZ2 (SEQ ID NO : 163) or FRSTNGZ3 (SEQ ID NO.: 164), and XI is S; X2 is A or S; X3 is Q or D; X4 is A; X5 is Q; X6 is A; X7 is E; or any combination thereof. In an embodiment, the 7-mer is VESTNGR (SEQ ID NO.: 165), and XI is S; X2 is S or D; X3 is F, H, I, L, M, N, Q, R, or Y; X4 is A; X5 is Q or P; X6 is A; X7 is A, D, E, F, Q, S, or T; or any combination thereof. In an embodiment, Z1 is selected from A, D, H, N, Q, and S; Z2 is K or R; and Z3 is selected from L, M, and R. . rAAV particles incorporating such modified capsid proteins exhibit increased binding to hTfRl, crossing the BBB and / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.

[0223] In some embodiments, the hTfRl AAV particle comprises an hTfRl rAAV capsid protein having an amino acid sequence of SEQ ID NO. : 139, which is an engineered variant of the wild-type AAV9 VP1 capsid protein having an amino acid sequence of SEQ ID NO.: 1 that includes a 7-amino acid insertion comprising SEQ ID NO.: 149 (YSRIGPN) between wild-type AAV9 VP1 amino acid residues 588 and 589. In some embodiments, the hTfRl AAV particle comprises a VP1 amino acid sequence that is an engineered variant of the wild-type AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the wild-type AAV9 VP1 amino acid sequence (SEQ ID NO.: 138), and further includes a 7-amino acid insertion comprising SEQ ID NO.: 149 (YSRIGPN) between wild-type AAV9 VP1 amino acid residues 588 and 589. In some embodiments, the hTfRl AAV particle comprises a VP1 amino acid sequence that is an engineered variant of the wild-type AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the wild-type AAV9 VP1 amino acid sequence (SEQ ID NO.: 138), and further includes a 7-amino acid insertion comprising SEQ ID NO.: 149 (YSRIGPN) inserted at a position in loop IV and / or loop VIII that will be displayed on the VP1 capsid surface. rAAV particles incorporating such modified capsid proteins exhibit increased binding to hTfRl, crossing the BBBand / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.

[0224] In some embodiments, the hTfRl AAV particle comprises an hTfRl rAAV capsid protein having an amino acid sequence of SEQ ID NO. : 140, which is an engineered variant of the wild-type AAV9 VP1 capsid protein having an amino acid sequence of SEQ ID NO.: 1, with a 7-amino acid insertion comprising SEQ ID NO.: 149 (YSRIGPN) between wild-type AAV9 VP1 amino acid residues 588 and 589, and further wherein the wild-type AAV9 VP1 amino acid residue at position 586 is changed from S to E (S586E), and the wild-type AAV9 VP1 amino acid residue at position 589 is changed from A to N (A589N). In some embodiments, the hTfRl AAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the wild-type AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the wild-type AAV9 VP1 amino acid sequence (SEQ ID NO.: 138), with a 7-amino acid insertion comprising SEQ ID NO.: 149 (YSRIGPN) between wild-type AAV9 VP1 amino acid residues 588 and 589, and further wherein the wild-type AAV9 VP1 amino acid residue at position 586 is changed from S to E (S586E), and the wild-type AAV9 VP1 amino acid residue at position 589 is changed from A to N (A589N). In some embodiments, the hTfRl AAV capsid protein has a VP1 amino acid sequence which is 99%, 98%, 95%, 90% or 85% identical to the wild-type AAV9 VP1 amino acid sequence (SEQ ID NO.: 138), with a 7-amino acid insertion comprising SEQ ID NO.: 149 (YSRIGPN), inserted at a position in loop IV and / or loop VIII that will be displayed on the VP1 capsid surface, and further wherein the AAV9 VP1 amino acid residue at the third upstream amino acid position preceding the 7-amino acid insertion is an E, and the AAV9 VP1 amino acid residue at the first downstream amino acid position succeeding the 7-amino acid insertion is an N. rAAV particles incorporating such modified capsid proteins exhibit increased binding to hTfRl, crossing the BBB and / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.

[0225] In some embodiments, the hTfRl AAV particle comprises an hTfRl rAAV capsid protein having an amino acid sequence of SEQ ID NO. : 147, which is an engineered variant of the wild-type AAV9 VP1 capsid protein having an amino acid sequence of SEQ ID NO.: 138, with a 7-amino acid insertion comprising SEQ ID NO.: 161 (FRSTNGV) between wild-type AAV9 VP 1 amino acid residues 588 and 589, and further wherein the wild-type AAV9 VP1 amino acid residue at position 588 is changed from Q to D (Q588D), and the wild-type AAV9 VP1 amino acid residueat position 592 is changed from Q to E (Q592E). Tn some embodiments, the hTfRl AAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the wild-type AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the wild-type AAV9 VP1 amino acid sequence (SEQ ID NO.: 138), with a 7-amino acid insertion comprising SEQ ID NO.: 161 (FRSTNGV) between wild-type AAV9 VP1 amino acid residues 588 and 589, and further wherein the wild-type AAV9 VP1 amino acid residue at position 588 is changed from Q to D (Q588D), and the wild-type AAV9 VP1 amino acid residue at position 592 is changed from Q to E (Q592E). In some embodiments, the hTfRl AAV capsid protein has a VP1 amino acid sequence which is 99%, 98%, 95%, 90% or 85% identical to the wild-type AAV9 VP1 amino acid sequence (SEQ ID NO.: 138), with a 7-amino acid insertion comprising SEQ ID NO.: 161 (FRSTNGV), inserted at a position in loop IV and / or loop VIII that will be displayed on the VP1 capsid surface, and further wherein the wild-type AAV9 VP1 amino acid residue at the first upstream amino acid position preceding the 7-amino acid insertion is a D, and the wild-typeAAV9 VP1 amino acid residue at the fourth downstream amino acid position succeeding the 7-amino acid insertion is an E. rAAV particles incorporating such modified capsid proteins exhibit increased binding to hTfRl, crossing the BBB and / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.

[0226] In some embodiments, the hTfRl AAV particle comprises an hTfRl rAAV capsid protein having an amino acid sequence of SEQ ID NO. : 142, which is an engineered variant of the AAV9 K449R VP1 capsid protein having an amino acid sequence of SEQ ID NO.: 141 that includes a 7-amino acid insertion comprising SEQ ID NO.: 149 (YSRIGPN) between AAV9 K449R VP1 amino acid residues 588 and 589. hTfRl AAV particles comprising a VP1 amino acid sequence of SEQ ID NO.: 142 are referred to herein as “AAV hTfRlv.1 particles.” In some embodiments, the hTfRl AAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the AAV9 K449R VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the AAV9 K449R VP1 amino acid sequence (SEQ ID NO.: 141), and further includes a 7-amino acid insertion comprising SEQ ID NO.: 149 (YSRIGPN) between AAV9 K449R VP1 amino acid residues 588 and 589. In some embodiments, the hTfRl AAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the AAV9 K449R VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the AAV9 K449R VP1 amino acid sequence (SEQ ID NO.: 141), and further includes a 7-amino acid insertion comprising SEQ ID NO.: 149 (YSRIGPN) insertedat a position in loop TV and / or loop VITI that will be displayed on the AAV9 K449R VP1 capsid surface. rAAV particles incorporating such modified capsid proteins exhibit increased binding to hTfRl, crossing the BBB and / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.

[0227] In some embodiments, the hTfRl AAV particle comprises an hTfRl rAAV capsid protein having an amino acid sequence of SEQ ID NO. : 143, which is an engineered variant of the AAV9 K449R VP1 capsid protein having an amino acid sequence of SEQ ID NO.: 141, with a 7-amino acid insertion comprising SEQ ID NO.: 149 (YSRIGPN) between AAV9 K449R VP1 amino acid residues 588 and 589, and further wherein the AAV9 K449R VP1 amino acid residue at position 586 is changed from S to E (S586E), and the AAV9 K449R VP1 amino acid residue at position 589 is changed from A to N (A589N). hTfRl AAV particles comprising a VP1 amino acid sequence of SEQ ID NO.: 143 are referred to herein as “AAV hTfRlv.2 particles.” In some embodiments, the hTfRl AAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the AAV9 K449R VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the AAV9 K449R VP1 amino acid sequence (SEQ ID NO.: 141), with a 7-amino acid insertion comprising SEQ ID NO.: 149 (YSRIGPN) between AAV9 K449R VP1 amino acid residues 588 and 589, and further wherein the AAV9 K449R VP1 amino acid residue at position 586 is changed from S to E (S586E), and the AAV9 K449R VP1 amino acid residue at position 589 is changed from A to N (A589N). In some embodiments, the hTfRl AAV capsid protein has a VP1 amino acid sequence which is 99%, 98%, 95%, 90% or 85% identical to the AAV9 K449R VP1 amino acid sequence (SEQ ID NO.: 141), with a 7-amino acid insertion comprising SEQ ID NO.: 149 (YSRIGPN), inserted at a position in loop IV and / or loop VIII that will be displayed on the AAV9 K449R VP1 capsid surface, and further wherein the AAV9 K449R VP1 amino acid residue at the third upstream amino acid position preceding the 7-amino acid insertion is an E, and the AAV9 K449R VP1 amino acid residue at the first downstream amino acid position succeeding the 7-amino acid insertion is an N. rAAV particles incorporating such modified capsid proteins exhibit increased binding to hTfRl, crossing the BBB and / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.

[0228] In some embodiments, the hTfRl AAV particle comprises an hTfRl rAAV capsid protein having an amino acid sequence of SEQ ID NO. : 148, which is an engineered variant of the AAV9 K449R VP1 capsid protein having an amino acid sequence of SEQ ID NO.: 141, with a 7-amino acid insertion comprising SEQ ID NO.: 161 (FRSTNGV) between AAV9 K449R VP1 amino acid residues 588 and 589, and further wherein the AAV9 K449R VP1 amino acid residue at position 588 is changed from Q to D (Q588D), and the AAV9 K449R VP1 amino acid residue at position 592 is changed from Q to E (Q592E). In some embodiments, the hTfRl AAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the AAV9 K449R VP1 capsid protein that is 99%, 98%, 95%, 90%> or 85% identical to the AAV9 K449R VP1 amino acid sequence (SEQ ID NO.: 141), with a 7-amino acid insertion comprising SEQ ID NO.: 161 (FRSTNGV) between AAV9 K449R VP1 amino acid residues 588 and 589, and further wherein the AAV9 K449R VP1 amino acid residue at position 588 is changed from Q to D (Q588D), and the AAV9 K449R VP1 amino acid residue at position 592 is changed from Q to E (Q592E). In some embodiments, the hTfRl AAV capsid protein has a VP1 amino acid sequence which is 99%, 98%, 95%, 90% or 85% identical to the AAV9 K449R VP1 amino acid sequence (SEQ ID NO.: 141), with a 7-amino acid insertion comprising SEQ ID NO.: 161 (FRSTNGV), inserted at a position in loop IV and / or loop VIII that will be displayed on the AAV9 K449R VP1 capsid surface, and further wherein the AAV9 K449R VP1 amino acid residue at the first upstream amino acid position preceding the 7-amino acid insertion is a D, and the AAV9 K449R VP1 amino acid residue at the fourth downstream amino acid position succeeding the 7-amino acid insertion is an E. rAAV particles incorporating such modified capsid proteins exhibit increased binding to hTfRl, crossing the BBB and / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.hCA4 rAAV particles

[0229] In embodiments, the rAAV particles of the present disclosure are engineered to bind human GPI-linked enzyme Carbonic anhydrase IV (hCA4) (hCA4 rAAV particle). In embodiments, these hCA4 rAAV particles comprise a capsid protein that is engineered to bind hCA4 (hCA4 rAAV capsid protein), and further comprise a recombinant AAV genome containing an expression cassette that encodes an n-TSC2 / n-intein or c-intein / c-TSC2 polynucleotide sequence (as described herein), operably linked to regulatory elements. Although not to be bound to a particular mechanism of action, these hCA4 AAV capsid proteins, derived from the naturally occurring AAV9 capsid, are thought to confer enhanced CNS tropism through hCA4 binding.

[0230] In embodiments, the hCA4 rAAV particle comprises an hCA4 rAAV capsid protein engineered to include an hCA4 amino acid binding modification sequence inserted between amino acids 588 and 589 of an AAV9 or AAV9 K449R capsid polypeptide (i.e., SEQ ID NO.: 138 or SEQ ID NO.: 141, respectively), or in an analogous position of a capsid polypeptide of another AAV serotype. In embodiments, the hCA4 amino acid binding modification sequence is an n-mer that is selected from SEQ ID NOs.: 1-7 and 64-4320 from PCT Application No. PCT / US2025 / 023671, published October 16, 2025 as WO 2025 / 217174, the entirety of which is incorporated herein. An example embodiment selects the n-mer from any amino acid sequence in Table A from U.S. Provisional Application Number 63 / 631,410, the entirety of which is incorporated herein. In embodiments, the hCA4 amino acid binding modification sequence is a 7-mer amino acid sequence selected from LYDGRSG (SEQ ID NO.: 166), VQRLSVL (SEQ ID NO.: 167), KVSNPVW (SEQ ID NO.: 168), RPVQVMA (SEQ ID NO.: 169).

[0231] In embodiments, the hCA4 amino acid binding modification sequence defined by the formula Zl-Z2-[n-mer], [n-mer]-Z3, or Zl-Z2-[n-mer]-Z3, wherein the n-mer is inserted between amino acids 588 and 589 of an AAV9 or AAV9 K449R VP1 capsid polypeptide (e.g., SEQ ID NO.: 138 or SEQ ID NO.: 141), or in an analogous position of a capsid polypeptide of another AAV serotype, and wherein Zl, Z2, and Z3 indicate an amino acid modification at one or more amino acid positions in the AAV capsid polypeptide flanking the inserted n-mer. In embodiments of the binding modification formula described above, Zl is S. In embodiments of the binding modification formula described above, Z2 is N. In embodiments of the binding modification formula described above, Z3 is Y or E. In embodiments of the binding modification formula described above, the n-mer is LYDGRSG, and Z3 is Y, such that the hCA4 amino acid binding modification sequence is LYDGRSGY (SEQ ID NO.: 170). In embodiments of the binding modification formula described above, the n-mer is RPVQVMA, and Z3 is E, such that the hCA4 amino acid binding modification sequence is RPVQVMAE (SEQ ID NO.: 171). In embodiments of the binding modification formula described above, the n-mer is KVSNPVW, Zl is S, and Z2 is N, such that the hCA4 amino acid binding modification sequence is SNKVSNPVW (SEQ ID NO. : 172). rAAV particles incorporating such modified capsid proteins exhibit increased binding to hCA4, crossing the BBB and / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.

[0232] In some embodiments, the hCA4 rAAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the wild-type AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the wild-type AAV9 VP1 amino acid sequence (SEQ ID NO.: 138), and further includes a 7-mer amino acid insertion between wild-type AAV9 VP1 amino acid residues 588 and 589, with said 7-mer amino acid sequence selected from LYDGRSG (SEQ ID NO.: 166), VQRLSVL (SEQ ID NO.: 167), KVSNPVW (SEQ ID NO.: 168), RPVQVMA (SEQ ID NO.: 169). In embodiments, the hCA4 rAAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the wild-type AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the wild-type AAV9 VP1 amino acid sequence (SEQ ID NO.: 138), and includes a 7 amino acid peptide LYDGRSG (SEQ ID NO.: 166) inserted between amino acids 588 and 589, and the amino acid at position 589 is changed from A to Y (A589Y) In embodiments, the hCA4 rAAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the wild-type AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the wildtype AAV9 VP1 amino acid sequence (SEQ ID NO.: 138), and further includes a 7 amino acid peptide RPVQVMA (SEQ ID NO.: 169) inserted between amino acids 588 and 589, and the amino acid at position 589 is changed from A to E (A589E). In embodiments, the hCA4 rAAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the wild-type AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the wild-type AAV9 VP1 amino acid sequence (SEQ ID NO.: 138), and further includes a 7 amino acid peptide KVSNPVW (SEQ ID NO.: 168) inserted between amino acids 588 and 589, and the amino acid at position 587 is changed from A to S (A587S), and the amino acid at position 588 is changed from Q toN (Q588N). rAAV particles incorporating such modified capsid proteins exhibit increased binding to hCA4, crossing the BBB and / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.

[0233] In some embodiments, the hCA4 rAAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the wild-type AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the wild-type AAV9 VP1 amino acid sequence (SEQ ID NO.: 138), and further includes a 7-mer amino acid insertion at a position in loop IV and / or loop VIII that will be displayed on the VP1 capsid surface, with said 7-mer amino acid sequence selected from LYDGRSG (SEQ ID NO.: 166), VQRLSVL (SEQ ID NO.: 167), KVSNPVW (SEQ ID NO.: 168), RPVQVMA (SEQ ID NO.: 169). rAAV particles incorporating such modified capsidproteins exhibit increased binding to hCA4, crossing the BBB and / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.

[0234] In some embodiments, the hCA4 rAAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the K449R AAV9 VP1 capsid protein that is 99%, 98%, 95%>, 90% or 85% identical to the K449R AAV9 VP1 amino acid sequence (SEQ ID NO.: 141), and further includes a 7-mer amino acid insertion between K449R AAV9 VP1 amino acid residues 588 and 589, with said 7-mer amino acid sequence selected from LYDGRSG (SEQ ID NO.: 166), VQRLSVL (SEQ ID NO.: 167), KVSNPVW (SEQ ID NO.: 168), RPVQVMA (SEQ ID NO.: 169). In embodiments, the hCA4 rAAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the K449R AAV9 VP1 capsid protein that is 99%o, 98%, 95%, 90% or 85%o identical to the K449R AAV9 VP1 amino acid sequence (SEQ ID NO.: 141), and includes a 7 amino acid peptide LYDGRSG (SEQ ID NO.: 166) inserted between amino acids 588 and 589, and the amino acid at position 589 is changed from A to Y (A589Y) In embodiments, the hCA4 rAAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the K449R AAV9 VP1 capsid protein that is 99%, 98%, 95%>, 90%> or 85%> identical to the K449R AAV9 VP1 amino acid sequence (SEQ ID NO.: 141), and further includes a 7 amino acid peptide RPVQVMA (SEQ ID NO.: 169) inserted between amino acids 588 and 589, and the amino acid at position 589 is changed from A to E (A589E). In embodiments, the hCA4 rAAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the K449R AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the K449R AAV9 VP1 amino acid sequence (SEQ ID NO.: 141), and further includes a 7 amino acid peptide KVSNPVW (SEQ ID NO.: 168) inserted between amino acids 588 and 589, and the amino acid at position 587 is changed from A to S (A587S), and the amino acid at position 588 is changed from Q toN (Q588N). rAAV particles incorporating such modified capsid proteins exhibit increased binding to hCA4, crossing the BBB and / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.

[0235] In some embodiments, the hCA4 rAAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the K449R AAV9 VP1 capsid protein that is 99%, 98%, 95%o, 90% or 85% identical to the K449R AAV9 VP1 amino acid sequence (SEQ ID NO.: 141), and further includes a 7-mer amino acid insertion at a position in loop IV and / or loop VIII that will be displayed on the VP1 capsid surface, with said 7-mer amino acid sequence selected fromLYDGRSG (SEQ ID NO . : 166), VQRLS VL (SEQ ID NO . : 167), KVSNPVW (SEQ ID NO : 168), RPVQVMA (SEQ ID NO.: 169). rAAV particles incorporating such modified capsid proteins exhibit increased binding to hCA4, crossing the BBB and / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.hCD59 rAAV particles

[0236] In embodiments, the rAAV particles of the present disclosure are engineered to bind the human CD59 cell surface protein (hCD59) (hCD59 rAAV particle). In embodiments, these hCD59 rAAV particles comprise a capsid protein that is engineered to bind hCD59 (hCD59 rAAV capsid protein), and further comprise a recombinant AAV genome containing an expression cassette that encodes an n-TSC2 / n-intein or c-intein / c-TSC2 polynucleotide sequence (as described herein), operably linked to regulatory elements. Although not to be bound to a particular mechanism of action, these hCD59 rAAV capsid proteins, derived from the naturally occurring AAV9 capsid, are thought to confer enhanced CNS tropism through hCD59 binding.

[0237] In embodiments, the hCD59 rAAV particle comprises an hCD59 rAAV capsid protein engineered to include an hCD59 amino acid binding modification sequence inserted at any position between amino acid residues 450-461 of an AAV9 or AAV9 K449R VP1 capsid polypeptide (i.e., SEQ ID NO.: 138 or SEQ ID NO.: 141, respectively), or in an analogous position of a capsid polypeptide of another AAV serotype. In embodiments, the hCD59 amino acid binding modification sequence is a 7-mer amino acid sequence selected from EFNNGSD (SEQ ID NO.: 173) or GAASLMP (SEQ ID NO.: 174). In embodiments, the hCD59 amino acid binding modification sequence is selected from SEQ ID NO: 89-5983 from PCT Application No. PCT / US2025 / 023652, published October 16, 2025 as WO 2025 / 217163, the entirety of which is incorporated here. In embodiments, the hCD59 amino acid binding modification sequence is selected from any amino acid sequences in Table A from US Provisional Application Number 63 / 631,415, the entirety of which is incorporated here. In other embodiments, the hCD59 rAAV capsid protein is engineered such that amino acid residues 450-461 of an AAV9 or AAV9 K449R VP1 capsid polypeptide (e.g., SEQ ID NO.: 138 or SEQ ID NO.: 141), or the analogous amino acids of a capsid polypeptide of another AAV serotype, are removed and replaced with an hCD59 amino acid binding modification sequence selected from EFNNGSD (SEQ ID NO.: 173) or GAASLMP (SEQ ID NO.: 174). rAAV particles incorporating such modified capsid proteinsexhibit increased binding to hCD59, crossing the BBB and / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.

[0238] In some embodiments, the hCD59 rAAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the wild-type AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the wild-type AAV9 VP1 amino acid sequence (SEQ ID NO.: 138), and further includes a 7-mer amino acid insertion at any position between wild-type AAV9 VP1 amino acid residues 450-461, with said 7-mer amino acid sequence selected from EFNNGSD (SEQ ID NO.: 173) or GAASLMP (SEQ ID NO.: 174). In some embodiments, the hCD59 rAAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the wild-type AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the wild-type AAV9 VP1 amino acid sequence (SEQ ID NO.: 138), wherein wild-type AAV9 VP1 amino acid residues 450-461 are removed and replaced with a 7-mer amino acid sequence selected from EFNNGSD (SEQ ID NO.: 173) or GAASLMP (SEQ ID NO.: 174). In some embodiments, the hCD59 rAAV protein has a VP1 amino acid sequence that is an engineered variant of the wild-type AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the wild-type AAV9 VP1 amino acid sequence (SEQ ID NO.: 138), and further includes a 7-mer amino acid insertion at a position in loop IV and / or loop VIII that will be displayed on the VP1 capsid surface, with said 7-mer amino acid sequence selected from EFNNGSD (SEQ ID NO.: 173) or GAASLMP (SEQ ID NO.: 174). rAAV particles incorporating such modified capsid proteins exhibit increased binding to hCD59, crossing the BBB and / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.

[0239] In some embodiments, the hCD59 rAAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the K449R AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the K449R AAV9 VP1 amino acid sequence (SEQ ID NO.: 141), and further includes a 7-mer amino acid insertion at any position between K449R AAV9 VP1 amino acid residues 450-461, with said 7-mer amino acid sequence selected from EFNNGSD (SEQ ID NO.: 173) or GAASLMP (SEQ ID NO.: 174). In some embodiments, the hCD59 rAAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the K449R AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the K449R AAV9 VP1 amino acid sequence (SEQ ID NO.: 141), wherein K449R AAV9 VP1 amino acid residues 450-461 are removed and replaced with a 7-mer amino acid sequence selected from EFNNGSD (SEQ ID NO.:173) or GAASLMP (SEQ ID NO.: 174). In some embodiments, the hCD59 rAAV protein has a VP1 amino acid sequence that is an engineered variant of the K449R AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the K449R AAV9 VP1 amino acid sequence (SEQ ID NO.: 141), and further includes a 7-mer amino acid insertion at a position in loop IV and / or loop VIII that will be displayed on the VP1 capsid surface, with said 7-mer amino acid sequence selected from EFNNGSD (SEQ ID NO.: 173) or GAASLMP (SEQ ID NO.: 174). rAAV particles incorporating such modified capsid proteins exhibit increased binding to hCD59, crossing the BBB and / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.ALPL rAAV particles

[0240] In embodiments, the rAAV particles of the present disclosure are engineered to bind human GPI-linked alkaline phosphatase (ALPL) (ALPL rAAV particle). In embodiments, the ALPL rAAV particles comprise a capsid protein that is engineered to bind ALPL (ALPL rAAV capsid protein), and further comprise a recombinant AAV genome containing an expression cassette that encodes an n-TSC2 / n-intein or c-intein / c-TSC2 polynucleotide sequence (as described herein), operably linked to regulatory elements. Although not to be bound to a particular mechanism of action, these ALPL rAAV capsid proteins are thought to confer enhanced ability to cross the BBB, likely via ALPL binding, thereby providing for efficient delivery throughout the CNS.

[0241] In embodiments, the ALPL rAAV particle comprises the capsid of an AAV9 or AAV9 K449R capsid polypeptide (i.e., SEQ ID NO.: 138 or SEQ ID NO.: 141, respectively) engineered to include an ALPL amino acid binding modification sequence. In an embodiment, the ALPL amino acid binding modification sequence binds to an ALPL polypeptide. Design considerations for ALPL amino acid binding modification sequences can be found in International Patent Application Publication Number WO 2024030976, the entirety of which is incorporated here.

[0242] In embodiments, the ALPL amino acid binding modification sequence is selected from SEQ ID NO.: 200-940, 1800-2241, 2242-2886, or 2887-3076 from International Patent Application Publication Number WO 2024030976. In embodiments, the ALPL amino acid binding modification sequence is selected from any amino acid sequences in Table 1, 2A, 2B, 2C, or 13-19 from International Patent Application Publication Number WO 2024030976. In embodiments, the ALPL rAAV particle comprises a capsid having an amino acid sequence selected from any of the amino acid sequences in Table 4 from International Patent Application Publication Number WO 2024030976. . rAAV particles incorporating such modified capsid proteins exhibit increased binding to ALPL, crossing the BBB and / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.[00243J In some embodiments, the ALPL rAAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the wild-type AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the wild-type AAV9 VP1 amino acid sequence (SEQ ID NO.: 138), and further includes a 6-mer amino acid insertion between wild-type AAV9 VP1 amino acid residues 455 and 456, with said 6-mer amino acid sequence consisting of SPHSKA (SEQ ID NO.: 175). In some embodiments, the ALPL rAAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the wild-type AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the wild-type AAV9 VP1 amino acid sequence (SEQ ID NO. : 138), and further includes a 6-mer amino acid insertion between wild-type AAV9 VP1 amino acid residues 453 and 454, with said 6-mer amino acid sequence consisting of HDSPHK (SEQ ID NO.: 176). rAAV particles incorporating such modified capsid proteins exhibit increased binding to ALPL, crossing the BBB and / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.

[0244] In some embodiments, the ALPL rAAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the K449R AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the K449R AAV9 VP1 amino acid sequence (SEQ ID NO.: 141), and further includes a 6-mer amino acid insertion between wild-type AAV9 VP1 amino acid residues 455 and 456, with said 6-mer amino acid sequence consisting of SPHSKA (SEQ ID NO.: 175). In some embodiments, the ALPL rAAV capsid protein has a VP1 amino acid sequence that is an engineered variant of the K449R AAV9 VP1 capsid protein that is 99%, 98%, 95%, 90% or 85% identical to the K449R AAV9 VP1 amino acid sequence (SEQ ID NO.: 141), and further includes a 6-mer amino acid insertion between wild-type AAV9 VP1 amino acid residues 453 and 454, with said 6-mer amino acid sequence consisting of HDSPHK (SEQ ID NO.: 176). rAAV particles incorporating such modified capsid proteins exhibit increased binding to ALPL, crossingthe BBB and / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.rAAV (AE) particles

[0245] In embodiments, the rAAV particles of the present disclosure are engineered to include an AAV capsid protein (rAAV(AE) capsid protein) with one or more amino acid substitutions that allow such rAAV particles (rAAV(AE) particles) to evade immune clearance arising from neutralizing antibodies, avoid compliment activation and immune response and reduce the incidence of immune response in treated subjects, and to avoid antibody binding / inhibition that renders the capsids unable to access their target receptor on cells, and further comprise a recombinant AAV genome containing an expression cassette that encodes an n-TSC2 / n-intein or the c-intein / c-TSC2 polynucleotide sequences (as described herein), operably linked to regulatory elements.

[0246] Design considerations for rAAV(AE) particles can be found in U.S. Provisional Patent Application No. 63 / 720,703, the entirety of which is incorporated here. In embodiments, the rAAV(AE) particle comprises an engineered AAV VP 1 capsid protein comprising one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 or AAV9 K449R capsid polypeptide (i.e.., SEQ ID NO.: 138 or SEQ ID NO.: 141, respectively), or in an analogous position of a VP1 capsid protein of another AAV serotype, and exhibits evasion of anti-AAV antibodies and immune response relative to the AAV particle without these capsid mutations.

[0247] In embodiments, the rAAV(AE) particle comprises an AAV9, hTfRl, hCA4, hCD59, or ALPL capsid protein (as described above) that is further engineered to include one or more VP1 capsid protein amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R550Q, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D, and exhibits immune evasion relative to the AAV particle without these capsid mutations (“immune evasion rAAV particle”). In embodiments, the immune evasion rAAV particle comprises an hTfRl, hCA4, hCD59, or ALPLcapsid protein (as described above) that is further engineered to include the amino acid sequence modifications of one or more of the AE1.5, AE1.9, AE1.11, AE1.15, AE1.18, AE2.1, AE2.2, AE2.15, and AE2.18 rAAV(AE) capsid variants described in U.S. Provisional Patent Application No. 63 / 720,703, and exhibits immune evasion relative to the AAV particle without these capsid mutations.

[0248] In embodiments, the immune evasion rAAV particle comprises an engineered AAV VP1 capsid protein, including an AAV9, AAV9 K449, hTfR.1, hCA4, hCD59, or ALPL capsid protein (as described above), wherein the amino acid substitutions include N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations. In embodiments, the immune evasion rAAV particle comprises an engineered AAV VP1 capsid protein, wherein the amino acid substitutions further include from 1 to 12 amino acid substitutions chosen from D327N, N328K, N329D, K332Q, K462E / Q, R533Q, D657N, D657N, N663D, K664Q, N665N, and N668K of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations. In embodiments, the immune evasion rAAV particle comprises an engineered AAV VP1 capsid protein, wherein the amino acid substitutions further include D327N, K332Q, and D657N of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations. In embodiments, the immune evasion rAAV particle comprises an engineered AAV VP1 capsid protein, wherein the additional amino acid substitutions include N328K and N329D of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations. In embodiments, the immune evasion rAAV particle comprises an engineered AAV VP1 capsid protein, wherein the additional amino acid substitutions include K462E / Q and N668K of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations. In embodiments, the immune evasion rAAV particle comprises an engineered AAV capsid protein, wherein the additional amino acid substitution includes N668K of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype, and exhibits immuneevasion relative to the AAV particle without these capsid mutations. Tn embodiments, the immune evasion rAAV particle comprises an engineered AAV capsid protein, wherein the additional amino acid substitution includes N663D of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations.

[0249] In embodiments, the immune evasion rAAV particle comprises an engineered AAV capsid protein, including an AAV9, AAV9 K449, hTfRl, hCA4, hCD59, or ALPL VP1 capsid protein (as described above), wherein the amino acid substitutions include D327N, N328K, N329D, K332Q, N452D, E500D, A502S, P504T, A510K, R55OQ, D551N, D554N, K557Q, D657N, N663D, and D665N of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations. In embodiments, the immune evasion rAAV particle comprises an engineered AAV VP1 capsid protein, wherein the amino acid substitutions include D327N, N328K, N329D, K332Q, N452D, E500D, A502S, P504T, A510K, R55OQ, D551N, D554N, K557Q and D657N of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations. In embodiments, the immune evasion rAAV particle comprises an engineered AAV VP1 capsid protein, wherein the amino acid substitutions include D327N, K332Q, N452D, E500D, A502S, P504T, A510K, R550Q, D551N, D554N, K557Q, D657N, N663D, and N668K of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations. In embodiments, the immune evasion rAAV particle comprises an engineered AAV VP1 capsid protein, wherein the amino acid substitutions include N452K, K462E, E500D, A502S, P504T, A510K, R550Q, D551N, D554N, K557Q, D657N, and N663D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations. In embodiments, the immune evasion rAAV particle comprises an engineered AAV VP1 capsid protein, wherein the amino acid substitutions include N452K, K462Q, E500D, A502S, P504T, A510K, R533Q, R550Q, D551K, D554N, K557Q, K664Q, and N668K of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations. In embodiments, the immune evasionrAAV particle comprises an engineered AAV VP1 capsid protein, wherein the amino acid substitutions include G455Q / T / K / N, D551N, N552D, K557E / Q, K664E / Q, D665N, and N668K.

[0250] In embodiments, the immune evasion rAAV particle comprises an engineered AAV capsid protein, including an AAV9, AAV9 K449, hTfR.1, hCA4, hCD59, or ALPL VP1 capsid protein (as described above), wherein amino acid substitutions further include from 1 to 5 amino acid substitutions selected from D554N, D556K / N, N663D, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations. In embodiments, the immune evasion rAAV particle comprises an engineered AAV VP1 capsid polypeptide, wherein an additional amino acid substitution includes D556K / N of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations. In embodiments, the immune evasion rAAV particle comprises an engineered AAV VP1 capsid polypeptide, wherein an additional amino acid substitution includes D554N. In embodiments, the immune evasion rAAV particle comprises an engineered AAV capsid polypeptide, wherein the additional amino acid substitution includes N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations.

[0251] In embodiments, the immune evasion rAAV particle comprises an engineered AAV capsid protein, including an AAV9, AAV9 K449, hTfR.1, hCA4, hCD59, or ALPL VP1 capsid protein (as described above), wherein the amino acid substitutions include G455Q, D551N, N552D, D556K, K557E, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations. In embodiments, the immune evasion rAAV particle comprises an engineered AAV VP1 capsid polypeptide, wherein the amino acid substitutions include G455T, D551N, N552D, D556K, K557E, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations. In embodiments, the immune evasion rAAV particle comprises an engineered AAV VP1 capsid polypeptide, wherein the amino acid substitutions include G455K, D551N, N552D, D556K, K557E, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or ananalogous position of a VP1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations. In embodiments, the immune evasion rAAV particle comprises an engineered AAV VP1 capsid polypeptide, wherein the amino acid substitutions include G455N, D551N, N552D, D554N, D556N, K557E, N663D, K664Q, D665N, and N668K of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations.

[0252] Methods for obtaining recombinant AAVs having a desired capsid protein can be obtained from, for example U.S. Patent Application Publication Number 2003 / 0138772, the entirety of which is incorporated herein. Typically, the methods involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein or fragment thereof; a functional rep gene; sufficient helper genes to permit packaging of the recombinant AAV vector construct into the AAV capsid proteins; and a recombinant AAV vector plasmid comprising the AAV vector construct. Typically, capsid proteins are structural proteins encoded by the cap gene of an AAV. In some aspects, wherein the capsid protein comprises VP1, VP2, and VP3, said VP1, VP2, and VP3 are transcribed from a single cap gene via alternative splicing. In some aspects, the molecular weights of VP1, VP2 and VP3 are respectively about 87 kDa, about 72 EDa and about 62 EDa. In some aspects, upon translation, capsid proteins form a spherical 60-mer protein shell around the viral genome. In some aspects, capsid proteins protect a viral genome, deliver a genome and / or interact with a host cell. In some aspects, capsid proteins deliver the viral genome to a host in a tissue specific manner.

[0253] In some aspects, components to be cultured in the host cell to package a recombinant AAV vector in an AAV capsid can be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and / or helper functions) can be provided by a stable host cell which has been engineered to contain one or more of the required components.

[0254] The recombinant AAV vector, rep sequences, cap sequences, and helper functions or genes useful for producing the rAAV described herein can be delivered to the packaging host cell using any appropriate genetic element (e.g., a plasmid). The selected genetic element can be delivered by any suitable method, including those described herein. The methods used to constructany of compositions disclosed herein are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. (See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.) Similarly, methods of generating rAAV particles are well known and the selection of a suitable method is not a limitation on the present disclosure. (See, e.g., K. Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.)[00255J In some aspects, recombinant AAVs can be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650). Typically, the recombinant AAVs can be produced by transfecting a host cell with a plasmid comprising a recombinant AAV vector construct (comprising a transgene and expression sequences flanked by ITRs) to be packaged into AAV particles, a packaging plasmid comprising AAV helper function sequences, and a plasmid comprising accessory function sequences. An AAV packaging plasmid encodes the “AAV helper function” sequences (i.e., rep and cap), which function in trans for productive AAV replication and encapsidation with the cap gene encoding the capsid proteins of desired serotype, for example, encoding hTfRl AAV capsid proteins such as AAV hTfR.lv.1 and AAV hTfR.lv.2, which, when incorporated into a capsid, are thought to have enhanced ability to cross the BBB, likely via human transferrin receptor 1 (hTfRl) receptor mediated transcytosis (RMT), thereby providing for efficient delivery throughout the CNS. In some embodiments, the hTfRl AAV packaging plasmid comprises the sequence of SEQ ID NO.: 144, SEQ ID NO.: 145, or SEQ ID NO.: 146. In some aspects, the AAV packaging plasmid can support efficient rAAV particle production without generating any detectable wild-type AAV particles (i.e., AAV particles containing functional rep and cap genes).

[0256] The accessory function plasmid encodes nucleotide sequences for non-AAV derived viral and / or cellular functions upon which AAV is dependent for replication (i.e., “accessory functions”). The accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, 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.Cells

[0257] Disclosed herein are transfected host cells. The term “transfection” is used to refer to the uptake of foreign DNA by a cell, and a cell has been “transfected” when exogenous DNA has been introduced through the cell membrane. Examples of methods of transfection include Graham et al. 1973, Chu et al. 1981, Sambrook et al. (1989), and Davis et al. (1986). Such techniques can be used to introduce one or more exogenous nucleic acids, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells.

[0258] In one aspect, a cell is provided. In some embodiments, the cell comprises an AAV packaging plasmid and an AAV vector construct plasmid. In some embodiments, the AAV packaging plasmid comprises rep and cap. In some embodiments, the cap encodes a VP1, a VP2, and a VP3. In some embodiments, the rep encodes rep78, rep68, rep 52, and rep 40. In certain embodiments, the packaging plasmid comprises a nucleotide sequence that encodes an hTfRl rAAV capsid protein that targets the hTfRl receptor (hTfRl AAV packaging plasmid). These hTfRl AAV capsid proteins, derived from the naturally occurring AAV9 capsid, use human transferrin receptor 1 (hTfRl) to cross the blood-brain barrier (BBB) via receptor mediated transcytosis (RMT), which allows for efficient delivery throughout the CNS. In some embodiments, the hTfRl AAV packaging plasmid comprises a nucleotide sequence that encodes an AAV capsid comprising a VP1 protein that has an amino acid sequence of SEQ ID NO.: 139 or SEQ ID NO.: 142 (i.e., AAV hTfRl v.l particles), or SEQ ID NO.: 140 or SEQ ID NO.: 143 (i.e., AAV hTfRlv.2 particles), as described herein. In some embodiments, the AAV packaging plasmid comprises a nucleotide sequence that encodes an AAV capsid comprising a VP1 protein that has an amino acid sequence ofSEQ IDNO.: 147 or SEQ ID NO.: 148. In some embodiments, the hTfRl AAV packaging plasmid comprises the nucleotide sequence of SEQ ID NO.: 144, SEQ ID NO.: 145, or SEQ ID NO.: 146. In some embodiments, the cap is AAV9 cap.

[0259] In some embodiments, the AAV vector plasmid comprises an AAV vector construct having, comprising, or consisting of the nucleotide sequence of SEQ ID NO.: 121 (encoding a C519N-TSC2 / N-Intein polypeptide), SEQ ID NO.: 122 (encoding a C519 C-Intein / C-TSC2 polypeptide), SEQ ID NO.: 123 (encoding a C728 N-TSC2 / N-Intein polypeptide), SEQ ID NO.: 124 (encoding a C728 C-Intein / C-TSC2 polypeptide), SEQ ID NO.: 125 (encoding a C738 N-TSC2 / N-Intein polypeptide), SEQ ID NO.: 126 (encoding a C738 C-Intein / C-TSC2polypeptide), SEQ TD NO.: 127 (encoding a C791 N-TSC2 / N-Intein polypeptide), SEQ ID NO.: 128 (encoding a C791 C-Intein / C-TSC2 polypeptide), SEQ ID NO.: 129 (encoding a C800 N-TSC2 / N-Intein polypeptide), SEQ ID NO.: 130 (encoding a C800 C-Intein / C-TSC2 polypeptide), SEQ ID NO.: 131 (encoding a C811 N-TSC2 / N-Intein polypeptide), SEQ ID NO.: 132 (encoding a C811 C-Intein / C-TSC2 polypeptide), SEQ ID NO.: 134 (encoding a C1283 N-TSC2 / N-Intein polypeptide), or SEQ ID NO.: 133 (encoding a C1283 C-Intein / C-TSC2 polypeptide), or a reverse complementary sequence thereto. In embodiments, the AAV vector plasmid comprises an expression cassette having a nucleotide sequence of SEQ ID NO.: 79, SEQ ID NO.: 80, SEQ ID NO.: 81, SEQ ID NO.: 82, SEQ ID NO.: 83, SEQ ID NO.: 84, SEQ ID NO.: 85, SEQ ID NO.: 86, SEQ ID NO.: 87, SEQ ID NO.: 88, SEQ ID NO.: 89, SEQ ID NO.: 90, SEQ ID NO.: 91, or SEQ ID NO.: 92, or a reverse complementary sequence thereto.

[0260] In another aspect, a method of producing the AAV particle is provided. In some embodiments, the method comprises transfecting a cell with a packaging plasmid and at least one of a vector plasmid or the AAV vector construct. In some embodiments, the method comprises transfecting a cell with an AAV vector plasmid or AAV vector construct comprising the recombinant AAV vector or the recombinant scAAV vector. In some embodiments, the method comprises transfecting a cell with a packaging plasmid comprising cap and rep. In some embodiments, the cap encodes the VP1, the VP2, and the VP3. In some embodiments, the rep encodes rep78, rep68, rep 52, and rep 40. In certain embodiments, the method comprises transfecting a cell with a packaging plasmid comprising an hTfRl rAAV capsid sequence that targets the hTfRl receptor (hTfRl AAV packaging plasmid). In some embodiments, the method comprises transfecting a cell with an hTfRl AAV packaging plasmid comprising the sequence of SEQ ID NO.: 144, SEQ ID NO.: 145, or SEQ ID NO.: 146.

[0261] In some embodiments, the method comprises transfecting a cell with an AAV vector construct plasmid comprising the AAV vector construct polynucleotide of SEQ ID NO.: 93, SEQ ID NO.: 94, SEQ ID NO.: 97, SEQ ID NO : 98, SEQ ID NO.: 101, SEQ ID NO.: 102, SEQ ID NO.: 105, SEQ ID NO.: 106, SEQ ID NO.: 109, SEQ ID NO.: 110, SEQ ID NO.: 113, SEQ ID NO.: 114, SEQ ID NO.: 117, or SEQ ID NO.: 118, or a reverse complementary sequence thereto. In other embodiments, the method comprises transfecting a cell with an AAV vector construct plasmid comprising the expression cassette polynucleotide of SEQ ID NO.: 79, SEQ ID NO.: 80, SEQ ID NO.: 81, SEQ ID NO.: 82, SEQ ID NO.: 83, SEQ ID NO.: 84, SEQ ID NO.: 85,SEQ ID NO.: 86, SEQ ID NO : 87, SEQ TD NO.: 88, SEQ IDNO.: 89, SEQ IDNO.: 90, SEQ ID NO.: 91, or SEQ ID NO.: 92, , or a reverse complementary sequence thereto.

[0262] In some aspects, the method disclosed herein can involve transfecting cells with total cellular DNAs isolated from the tissues that potentially harbor proviral AAV genomes at very low abundance and supplementing with helper virus function (e.g., adenovirus) to trigger and / or boost AAV rep and cap gene transcription in the transfected cell. In some aspects, RNA from the transfected cells can provide a template for RT-PCR amplification of cDNA and the detection of novel AAVs. In cases where cells are transfected with total cellular DNAs isolated from the tissues that potentially harbor proviral AAV genomes, it is often desirable to supplement the cells with factors that promote AAV gene transcription. For example, the cells can also be infected with a helper virus, such as an Adenovirus or a Herpes Virus. In some aspects, the helper functions can be provided by an adenovirus. The adenovirus can be a wild-type adenovirus, and can be of human or non-human origin, for example, non-human primate (NHP) origin. Similarly, adenoviruses known to infect non-human animals (e.g., chimpanzees, mouse) can also be employed in the methods of the disclosure (See, e.g., U.S. Pat. No. 6,083,716). In addition to wild-type adenoviruses, recombinant viruses or non-viral vectors (e.g., plasmids, episomes, etc.) carrying the necessary helper functions can be utilized. Such recombinant viruses are known in the art and may be prepared according to published techniques. See, e.g., U.S. Pat. No. 5,871,982 and U.S. Pat. No. 6,251,677, which describe a hybrid Ad / AAV virus. A variety of adenovirus strains are available from the American Type Culture Collection, Manassas, Va., or available by request from a variety of commercial and institutional sources. Further, the sequences of many such strains are available from a variety of databases including, e.g., PubMed and GenBank.

[0263] Cells can also be transfected with a vector (e.g., helper vector) which provides helper functions to the AAV. The vector providing helper functions can provide adenovirus functions, including, e.g., Ela, Elb, E2a, E4ORF6. The sequences of adenovirus gene providing these functions can be obtained from any known adenovirus serotype, such as serotypes 2, 3, 4, 7, 12 and 40, and further including any of the presently identified human types known in the art. Thus, in some aspects, the methods involve transfecting the cell with a vector expressing one or more genes necessary for AAV replication, AAV gene transcription, and / or AAV packaging.

[0264] In some aspects, an isolated capsid gene can be used to construct and package recombinant AAV vectors, using methods well known in the art, to determine functional characteristics associated with the novel capsid protein encoded by the gene. For example, isolated capsid genes can be used to construct and package recombinant AAV (rAAV) vectors comprising a reporter gene (e.g., B-Galactosidase, GFP, Luciferase, etc.). The rAAV vector can then be delivered to an animal (e.g., mouse) and the tissue targeting properties of the isolated capsid gene can be determined by examining the expression of the reporter gene in various tissues (e.g., heart, liver, kidneys) of the animal. Other methods for characterizing isolated capsid genes are disclosed herein and still others are well known in the art.METHODS OF TREATMENT

[0265] Methods of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein are provided. In embodiments, the method of treating TSC2 involves co-administration of two separate rAAV particles to a subject in need, wherein each rAAV particle is engineered to deliver a different portion of the human TSC2 coding sequence to target cells of the CNS such that, when expressed, the two portions of the TSC2 protein join via intein-mediated protein splicing to form a full length human TSC2 (tuberin) protein that exhibits TSC2 activity. The first rAAV particle (rAAV-n-TSC2 / n-intein) is engineered to comprise an expression cassette comprising regulatory polynucleotide sequences sufficient to express a polynucleotide sequence (n-TSC2 / n-intein polynucleotide sequence) that encodes an amino (N-terminal) fragment of the TSC2 protein (N-TSC2) that is fused at its C-terminus to the N-terminus of the N-terminal portion of an intein amino acid sequence (together, N-TSC2 / N-Intein amino acid sequence). The second rAAV particle (rAAV-c-intein / c-TSC2) is engineered to comprise an expression cassette comprising regulatory polynucleotide sequences sufficient to express a polynucleotide sequence (c-intein / c-TSC2 polynucleotide sequence) that encodes the carboxyl (C-terminal) portion of the intein amino acid sequence (C-Intein) that is fused at its C-terminus to the N-terminus of a C-terminal fragment of the TSC2 protein (together, C-Intein / C-TSC2 amino acid sequence). Although not to be bound to a particular mechanism of action, it is thought that when these separate n-TSC2 / n-intein and c-intein / c-TSC2 polynucleotide sequences are coexpressed at a sufficient level in the same cell, the resulting N-Intein and C-Intein amino acid sequences associate, and are excised from the associated N-TSC2 and C-TSC2 amino acid sequences via intein-mediated protein splicing. During this process, the N-TSC2 and C-TSC2 amino acid sequences are joinedtogether into a full-length TSC2 protein that does not include the excised N-Intein and C-Intein amino acid sequences, and exhibits TSC2 activity. A schematic of this rAAV-mediated TSC2 “split intein” gene therapy strategy is shown in FIG. 1.

[0266] In embodiments, the method of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein comprises co-administration to a subject in need of both (i) a first rAAV particle engineered to deliver and express a first polynucleotide sequence encoding an N-terminal portion of the human TSC2 protein fused at its C-terminus to the N-terminus of an N-Intein polypeptide, the N-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 2, and (ii) a second rAAV particle engineered to deliver and express a second polynucleotide sequence encoding a C-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 4 fused at its C-terminus to the N-terminus of a C-terminal portion of the human TSC2 protein, whereby when the first and second rAAV particles transduce the same cell, the first and second polynucleotide sequences are coexpressed, and the N-terminal polypeptide and the C-terminal polypeptide are joined together (through the intein-mediated protein splicing mechanism) to form a polypeptide having, comprising, or consisting of an amino acid sequence that is either (i) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, the amino acid sequence of, SEQ ID NO.: 10, or (ii) the amino acid sequence of SEQ ID NO.: 10 wherein one or more amino acids are replaced with a complementary amino acid, and the resulting polypeptide exhibits TSC2 activity.

[0267] In embodiments, the method of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein comprises co-administration to a subject in need of both (i) a first rAAV particle engineered to deliver and express a first polynucleotide sequence encoding an N-terminal portion of the human TSC2 protein fused at its C-terminus to the N-terminus of an N-Intein polypeptide, the N-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 6, and (ii) a second rAAV particle engineered to deliver and express a second polynucleotide sequence encoding a C-Intein polypeptide having the amino acid sequence of SEQ ID NO.: 8 fused at its C-terminus to the N-terminus of a C-terminal portion of the human TSC2 protein, whereby when the first and second rAAV particles transduce the same cell, the first and second polynucleotide sequences are coexpressed, and the N-terminal polypeptide and the C-terminal polypeptide are joined together via intein-mediated protein splicing to form a polypeptide having, comprising, or consisting of an amino acid sequence that is either (i) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%., 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within, the amino acid sequence of, SEQ ID NO.: 10, or (ii) the amino acid sequence of SEQ ID NO.: 10 wherein one or more amino acids are replaced with a complementary amino acid, and the resulting polypeptide exhibits TSC2 activity.

[0268] In embodiments, the method of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein comprises co-administration to a subject in need of both a first rAAV particle engineered to deliver and express the C519 N-TSC2 / N-Intein polypeptide described herein, and a second rAAV particle engineered to deliver and express the C519 C-Intein / C-TSC2 polypeptide described herein, whereupon the resulting N-Intein and C-Intein amino acid sequences associate, and are excised from the associated N-TSC2 and C-TSC2 amino acid sequences via intein-mediated protein splicing, and the N-TSC2 and C-TSC2 amino acid sequences are joined together via intein-mediated protein splicing into a full-length TSC2 protein that does not include the excised N-Intein and C-Intein amino acid sequences, and exhibits TSC2 activity.

[0269] In embodiments, the method of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein comprises co-administration to a subject in need of both a first rAAV particle engineered to deliver and express the C728 N-TSC2 / N-Intein polypeptide described herein, and a second rAAV particle engineered to deliver and express the C728 C-Intein / C-TSC2 polypeptide described herein, whereupon the resulting N-Intein and C-Intein amino acid sequences associate, and are excised from the associated N-TSC2 and C-TSC2 amino acid sequences via intein-mediated protein splicing, and the N-TSC2 and C-TSC2 amino acid sequences are joined together via intein-mediated protein splicing into a full-length TSC2 protein that does not include the excised N-Intein and C-Intein amino acid sequences, and exhibits TSC2 activity.

[0270] In embodiments, the method of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein comprises co-administration to a subject in needof both a first rAAV particle engineered to deliver and express the C738 N-TSC2 / N-Intein polypeptide described herein, and a second rAAV particle engineered to deliver and express the C738 C-Intein / C-TSC2 polypeptide described herein, whereupon the resulting N-Intein and C-Intein amino acid sequences associate, and are excised from the associated N-TSC2 and C-TSC2 amino acid sequences via intein-mediated protein splicing, and the N-TSC2 and C-TSC2 amino acid sequences are joined together via intein-mediated protein splicing into a full-length TSC2 protein that does not include the excised N-Intein and C-Intein amino acid sequences, and exhibits TSC2 activity.

[0271] In embodiments, the method of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein comprises co-administration to a subject in need of both a first rAAV particle engineered to deliver and express the C791 N-TSC2 / N-Intein polypeptide described herein, and a second rAAV particle engineered to deliver and express the C791 C-Intein / C-TSC2 polypeptide described herein, whereupon the resulting N-Intein and C-Intein amino acid sequences associate, and are excised from the associated N-TSC2 and C-TSC2 amino acid sequences via intein-mediated protein splicing, and the N-TSC2 and C-TSC2 amino acid sequences are joined together via intein-mediated protein splicing into a full-length TSC2 protein that does not include the excised N-Intein and C-Intein amino acid sequences, and exhibits TSC2 activity.

[0272] In embodiments, the method of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein comprises co-administration to a subject in need of both a first rAAV particle engineered to deliver and express the C800 N-TSC2 / N-Intein polypeptide described herein, and a second rAAV particle engineered to deliver and express the C800 C-Intein / C-TSC2 polypeptide described herein, whereupon the resulting N-Intein and C-Intein amino acid sequences associate, and are excised from the associated N-TSC2 and C-TSC2 amino acid sequences via intein-mediated protein splicing, and the N-TSC2 and C-TSC2 amino acid sequences are joined together via intein-mediated protein splicing into a full-length TSC2 protein that does not include the excised N-Intein and C-Intein amino acid sequences, and exhibits TSC2 activity.

[0273] In embodiments, the method of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein comprises co-administration to a subject in needof both a first rAAV particle engineered to deliver and express the C811 N-TSC2 / N-Intein polypeptide described herein, and a second rAAV particle engineered to deliver and express the C811 C-Intein / C-TSC2 polypeptide described herein, whereupon the resulting N-Intein and C-Intein amino acid sequences associate, and are excised from the associated N-TSC2 and C-TSC2 amino acid sequences via intein-mediated protein splicing, and the N-TSC2 and C-TSC2 amino acid sequences are joined together via intein-mediated protein splicing into a full-length TSC2 protein that does not include the excised N-Intein and C-Intein amino acid sequences, and exhibits TSC2 activity.

[0274] In embodiments, the method of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein comprises co-administration to a subject in need of both a first rAAV particle engineered to deliver and express the C1283 N-TSC2 / N-Intein polypeptide described herein, and a second rAAV particle engineered to deliver and express the C1283 C-Intein / C-TSC2 polypeptide described herein, whereupon the resulting N-Intein and C-Intein amino acid sequences associate, and are excised from the associated N-TSC2 and C-TSC2 amino acid sequences via intein-mediated protein splicing, and the N-TSC2 and C-TSC2 amino acid sequences are joined together via intein-mediated protein splicing into a full-length TSC2 protein that does not include the excised N-Intein and C-Intein amino acid sequences, and exhibits TSC2 activity.

[0275] In embodiments, the method of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein comprises co-administration to a subject in need of both a first rAAV particle engineered to express an N-TSC2 / N-Intein polypeptide described herein, and a second rAAV particle engineered to express a C-Intein / C-TSC2 polypeptide, wherein the first rAAV particle is administered in a greater amount than the second rAAV particle, or alternatively wherein the second rAAV particle is administered in a greater amount than the first rAAV particle. In embodiments, the method of treating TSC involves co-administration of a first rAAV particle engineered to express the N-TSC2 / N-Intein polypeptide described herein, and a second rAAV particle engineered to express the C-Intein / C-TSC2 polypeptide at a first rAAV particle: second rAAV particle ratio of about 10:1, about 9:1, about8:l, about7:l, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10.

[0276] In embodiments, the method of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein utilizes rAAV particles comprising capsids engineered to cross the blood-brain barrier (BBB) and transduce cells of the CNS through binding to the hTfRl receptor (hTfRl rAAV particle). In embodiments, these hTfRl rAAV particles comprise a capsid protein that is engineered to bind the hTfRl receptor (hTfRl rAAV capsid protein), and further comprise a recombinant AAV genome containing an expression cassette that comprises n-TSC2 / n-intein or c-intein / c-TSC2 polynucleotide sequences described herein, operably linked to regulatory elements. Although not to be bound to a particular mechanism of action, these hTfRl rAAV capsid proteins, derived from the naturally occurring AAV9 capsid, are thought to confer enhanced ability to cross the BBB, likely via human transferrin receptor 1 (hTfRl) receptor mediated transcytosis (RMT), thereby providing for efficient delivery and expression of N-TSC2 / N-Intein and c-Intein / c-TSC2 polypeptides described herein to cells of the CNS, whereupon the resulting N-Intein and C-Intein amino acid sequences associate, and are excised from the associated N-TSC2 and C-TSC2 amino acid sequences via intein-mediated protein splicing, and the N-TSC2 and C-TSC2 amino acid sequences are joined together into a full-length TSC2 protein that does not include the excised N-Intein and C-Intein amino acid sequences, and exhibits TSC2 activity.

[0277] In embodiments, the method of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein utilizes rAAV particles comprising capsids engineered to bind human GPI-linked enzyme Carbonic anhydrase IV (hCA4) (hCA4 rAAV particle). In embodiments, these hCA4 rAAV particles comprise a capsid protein that is engineered to bind hCA4 (hCA4 rAAV capsid protein), and further comprise a recombinant AAV genome containing an expression cassette that comprises n-TSC2 / n-intein or c-intein / c-TSC2 polynucleotide sequences described herein, operably linked to regulatory elements. Although not to be bound to a particular mechanism of action, these hCA4 AAV capsid proteins, derived from the naturally occurring AAV9 capsid, are thought to confer enhanced CNS tropism through hCA4 binding, thereby providing for efficient delivery and expression of N-TSC2 / N-Intein and c-Intein / c-TSC2 polypeptides described herein to cells of the CNS, whereupon the resulting N-Intein and C-Intein amino acid sequences associate, and are excised from the associated N-TSC2 and C-TSC2 amino acid sequences via intein-mediated protein splicing, and the N-TSC2 and C-TSC2amino acid sequences are joined together into a full-length TSC2 protein that does not include the excised N-Intein and C-Intein amino acid sequences, and exhibits TSC2 activity.

[0278] In embodiments, the method of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein utilizes rAAV particles engineered to bind the human CD59 cell surface protein (hCD59) (hCD59 rAAV particle). In embodiments, these hCD59 rAAV particles comprise a capsid protein that is engineered to bind hCD59 (hCD59 rAAV capsid protein), and further comprise a recombinant AAV genome containing an expression cassette that comprises n-TSC2 / n-intein or c-intein / c-TSC2 polynucleotide sequences described herein, operably linked to regulatory elements. Although not to be bound to a particular mechanism of action, these hCD59 rAAV capsid proteins, derived from the naturally occurring AAV9 capsid, are thought to confer enhanced CNS tropism through hCD59 binding, thereby providing for efficient delivery and expression of N-TSC2 / N-Intein and c-Intein / c-TSC2 polypeptides described herein to cells of the CNS, whereupon the resulting N-Intein and C-Intein amino acid sequences associate, and are excised from the associated N-TSC2 and C-TSC2 amino acid sequences via intein-mediated protein splicing, and the N-TSC2 and C-TSC2 amino acid sequences are joined together into a full-length TSC2 protein that does not include the excised N-Intein and C-Intein amino acid sequences, and exhibits TSC2 activity.

[0279] In embodiments, the method of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein utilizes rAAV particles engineered to bind human GPI-linked alkaline phosphatase (ALPL) (ALPL rAAV particle). In embodiments, the ALPL rAAV particles comprise a capsid protein that is engineered to bind ALPL (ALPL rAAV capsid protein), and further comprise a recombinant AAV genome containing an expression cassette that comprises n-TSC2 / n-intein or c-intein / c-TSC2 polynucleotide sequences described herein, operably linked to regulatory elements. Although not to be bound to a particular mechanism of action, these ALPL rAAV capsid proteins are thought to confer enhanced ability to cross the BBB, likely via ALPL binding, thereby providing for efficient delivery throughout the CNS, thereby providing for efficient delivery and expression of N-TSC2 / N-Intein and c-Intein / c-TSC2 polypeptides described herein to cells of the CNS, whereupon the resulting N-Intein and C-Intein amino acid sequences associate, and are excised from the associated N-TSC2 and C-TSC2 amino acid sequences via intein-mediated protein splicing, and the N-TSC2 and C-TSC2 amino acidsequences are joined together into a full-length TSC2 protein that does not include the excised N-Intein and C-Intein amino acid sequences, and exhibits TSC2 activity.

[0280] In embodiments, the method of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein utilizes rAAV particles engineered to include an AAV capsid protein (rAAV(AE) capsid protein) with one or more amino acid substitutions that allow such rAAV particles (rAAV(AE) particles) to evade immune clearance arising from neutralizing antibodies, avoid compliment activation and immune response and reduce the incidence of immune response in treated subjects, and to avoid antibody binding / inhibition that renders the capsids unable to access their target receptor on cells, and further comprise a recombinant AAV genome containing an expression cassette that comprises n-TSC2 / n-intein or c-intein / c-TSC2 polynucleotide sequences described herein, operably linked to regulatory elements. Although not to be bound to a particular mechanism of action, these rAAV(AE) capsid proteins are thought to confer enhanced CNS transduction by evading immune clearance, thereby providing for efficient delivery and expression of N-TSC2 / N-Intein and c-Intein / c-TSC2 polypeptides described herein to cells of the CNS, whereupon the resulting N-Intein and C-Intein amino acid sequences associate, and are excised from the associated N-TSC2 and C-TSC2 amino acid sequences via intein-mediated protein splicing, and the N-TSC2 and C-TSC2 amino acid sequences are joined together into a full-length TSC2 protein that does not include the excised N-Intein and C-Intein amino acid sequences, and exhibits TSC2 activity.

[0281] In embodiments, the method of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein utilizes rAAV particles comprising 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 AAV-PHP.B capsid protein, an AAV-PHP.eB capsid protein, or an AAV-PHP.S capsid protein.

[0282] In embodiments, upon co-transduction of both a first rAAV particle comprising a first vector construct engineered to express an N-TSC2 / N-Intein polypeptide as described herein, and a second rAAV particle comprising a second vector construct engineered to express a C-Intein / C-TSC2 polypeptide as described herein, the AAV vector constructs will be released from encapsulation by the capsid protein. In some embodiments, the first and second AAV vectorconstructs will be released into the cytosol or nucleus of the cell. In some embodiments, the cell’s transcription machinery will bind to the promoter of the expression cassette of the first and second AAV vector constructs. In some embodiments, the cell will express the nucleic acid encoding the N-TSC2 / N-Intein and C-Intein / C-TSC2 polypeptides. In some embodiments, the cell will express both the N-TSC2 / N-Intein polypeptide and the C-Intein / C-TSC2 polypeptide. In some embodiments, the co-expressed N-TSC2 / N-Intein and C-Intein / C-TSC2 polypeptides associate, and the N-Intein and C-Intein amino acid sequences are excised from the associated N-TSC2 and C-TSC2 amino acid sequences via intein-mediated protein splicing, such that the N-TSC2 and C-TSC2 amino acid sequences are joined together into a full-length TSC2 protein that does not include the excised N-Intein and C-Intein amino acid sequences, and exhibits TSC2 activity by regulating the function of mTOR by inhibiting the Ras homologue enriched in brain (Rheb) protein.

[0283] In embodiments, a method for treating TSC or related disorders by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein is provided. In some embodiments, a method of regulating mTOR signaling activity in a subject is provided. In some embodiments, a method of reducing a disease condition in a subject that suffers from TSC is provided. In some embodiments, said disease condition comprises abnormal cell growth and / or proliferation, metabolic disorders, epilepsy, seizure, neurodevel opmental disorders and / or cognitive disorders (for example, autism spectrum disorders, intellectual disability, attention deficit-hyperactivity), and sleep disorders, particularly in human subjects. In some embodiments, the method comprises co-admini st ering to the subject a therapeutically effective amount of both a first rAAV particle comprising a first vector construct engineered to express an N-TSC2 / N-Intein polypeptide as described herein, and a second rAAV particle comprising a second vector construct engineered to express a C-Intein / C-TSC2 polypeptide as described herein to provide for expression of TSC2 in transduced cells as gene therapy for TSC. In some embodiments, a method of reducing p70S6K levels by co-administration of both a first rAAV particle comprising a first vector construct engineered to express an N-TSC2 / N-Intein polypeptide as described herein, and a second rAAV particle comprising a second vector construct engineered to express a C-Intein / C-TSC2 polypeptide as described herein as gene therapy for a subject in need thereof is provided.

[0284] In some embodiments, a subject treated with gene therapy by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein according to the presentmethods has TSC. In some embodiments, a treated subject does not express TSC2 protein or expresses undetectable levels of TSC2 protein. In some embodiments, a treated subject expresses a modified or mutant form of TSC2. In some embodiments, a treated subject suffers from autosomal dominant TSC due to modifications or mutations in TSC2. In some embodiments, a treated subject suffers from TSC caused by TSC2 haploinsufficiency. In some embodiments, a treated subject lacks TSC2 enzymatic activity or has undetectable levels of TSC2 enzymatic activity. In some embodiments, a treated subject has dysregulated mTOR signaling activity compared to a subject that does not have TSC. In some embodiments, a treated subject has lower levels of TSC2 protein expression compared to a subject that does not have TSC. In some embodiments, a treated subject has reduced levels of TSC2 enzymatic activity compared to a subject that does not have TSC.

[0285] In some embodiments, a subject treated with gene therapy by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein according to the present methods has previously been treated with, or is undergoing concomitant treatment with, a drug approved by the FDA for treatment of TSC, such as Afinitor® (everolimus), Rapamune® (sirolimus), Hyftor® (sirolimus), Sabril® (vigabatrin), Vigadrone® (vigabatrin), or Epidiolex® (cannabidiol). In embodiments, a treated subject is unresponsive or refractive to treatment with a drug approved by the FDA for treatment of TSC. In embodiments, a treated subject was forced to abandon treatment with a drug approved by the FDA for treatment of TSC due to side effects of the drug treatment. In embodiments, treatment with a drug approved by the FDA for treatment of TSC is contraindicated or otherwise not appropriate for administration to a treated subject.

[0286] In some embodiments, a subject treated with gene therapy by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein according to the present methods has previously been treated with, or is undergoing concomitant treatment with, a drug approved by the FDA for treatment of seizures or epilepsy. In embodiments, a treated subject is unresponsive or refractive to treatment with a drug approved by the FDA for treatment of seizures or epilepsy. In embodiments, a treated subject was forced to abandon treatment with a drug approved by the FDA for treatment of seizures or epilepsy due to side effects of the drug treatment. In embodiments, treatment with a drug approved by the FDA for treatment of seizures or epilepsy is contraindicated or otherwise not appropriate for administration to a treated subject. In embodiments, the treated subject suffers from refractory epilepsy.

[0287] In some embodiments, a method of treating dysregulated mTOR signaling activity in a subject in need thereof by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein according to the present methods is provided. Ribosomal protein S6 kinase beta-1 (S6K1), also known as p70S6 kinase (p70S6K) is a protein kinase that is downstream of and selectively phosphorylated by mTOR in order to regulate its role in ribosome biogenesis through the phosphorylation of ribosomal protein S6, which results in ribosome recruitment and protein translation. The TSC1 / TSC2 complex suppresses the function of mTOR by inhibiting the Ras homologue enriched in brain (Rheb) protein; in its active state, Rheb stimulates mTOR, so TSCl / TSC2-mediated inhibition of Rheb results in the inhibition of mTOR. Loss of TSC2 activity thus results in the activation of mTOR and, consequently, in mTOR-dependent increased phosphorylation of p70S6K (Wu et al. 2007). Accordingly, in embodiments, a method of treating dysregulated mTOR signaling activity by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein, resulting in expression of TSC2 as described herein, and thereby reducing mTOR-dependent phosphorylation of p70S6K in a subject in need, is provided.

[0288] In some embodiments, administering the rAAV-mediated TSC2 “split intein” gene therapy described herein to a subject in need according to the present methods leads to increased expression of TSC2 in transduced cells. In some embodiments, TSC2 protein expression is increased in cells or tissues of the CNS as compared to the TSC2 protein expression levels either prior to the administration, or relative to the levels in a similarly situated subject as determined in a natural history study. In some embodiments, TSC2 protein expression is increased in brain tissues. In some embodiments, TSC2 protein expression is increased in cortex and / or cerebral tissues. In some embodiments, TSC2 protein expression is increased in neuronal cells and / or glial cells. In some embodiments, TSC2 protein expression is increased in Purkinje neurons. In some embodiments, TSC2 protein expression is increased in spinal cord tissues. In all cases, the increase is relative to the TSC2 protein expression levels either prior to the administration, or relative to the levels in a similarly situated subject as determined in a natural history study, and wherein the increased TSC2 expression makes a therapeutic difference to the patient by ameliorating one or more symptoms of TSC.

[0289] In some embodiments, administering the rAAV-mediated TSC2 “split intein” gene therapy described herein to a subject in need according to the present methods leads to increased TSC2 enzyme activity in transduced cells. In some embodiments, TSC2 enzyme activity isincreased in cells or tissues of the CNS. In some embodiments, TSC2 enzyme activity is increased in brain tissues. In some embodiments, TSC2 enzyme activity is increased in cortex and / or cerebral tissues. In some embodiments, TSC2 enzyme activity is increased in neuronal cells and / or glial cells. In some embodiments, TSC2 enzyme activity is increased in Purkinje neurons. In some embodiments, TSC2 enzyme activity is increased in spinal cord tissues. In all cases, the increase is relative to the TSC2 enzyme activity either prior to the gene therapy administration or relative to the levels in a similarly situated subject as determined in a natural history study and wherein the reduction that makes a therapeutic difference to the patient by ameliorating one or more symptoms of the disease. In some embodiments, TSC2 enzyme activity is increased in cells or tissues of the CNS by about 1000%, about 1500%, about 2000%, about 2500%, about 3000%, about 3500%, about 4000%, about 4500%, about 5000%, about 5500%, about 6000%, about 6500%, about 7000%, or about 7500% compared to a subject without a TSC2 enzyme deficiency. In some embodiments, TSC enzyme activity is increased in brain by about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, or about 550% compared to a subject without a TSC2 enzyme deficiency.

[0290] In some embodiments, administering the rAAV-mediated TSC2 “split intein” gene therapy described herein to a subject in need leads to a reduction of the incidence and / or severity of epilepsy or seizures relative to a similarly situated subject that has not been administered such treatment. The incidence and / or severity of epilepsy may be assessed according to methods known in the art. For example, a reduction of the incidence and / or severity of epilepsy or seizure in treated subjects may be characterized according to the International League Against Epilepsy (ILAE) classification model (Sarmast et al. 2020). Treated subjects may also be monitored to determine the incidence of focal seizures and / or epileptic spasms over a given time period, as these symptoms are known to be elevated in patients suffering from TSC (Nabbout et al. 2018). Likewise, subjects treated in the first year of life may be monitored to determine the incidence of infantile spasms known to be exhibited by patients suffering from TSC during the first year of life. The incidence and severity of TSC-related epilepsy and / or seizures may also be monitored using electroencephalogram (EEG) monitoring, or magnetic resonance imaging (MRI).

[0291] In some embodiments, administering the rAAV-mediated TSC2 “split intein” gene therapy described herein to a subject in need leads to a reduction in severity of a neurodevelopmental and / or cognitive disorder (for example, autism spectrum disorders,Illintellectual disability, attention deficit-hyperactivity) relative to a similarly situated subject that has not been administered such treatment. Severity of a neurodevelopmental and / or cognitive disorder (for example, autism spectrum disorders, intellectual disability, attention deficithyperactivity) may be assessed according to methods known in the art. For example, TSC-related intellectual disability may be determined to be present in a subject when formal developmental assessment reveals a developmental quotient of less than 70, when unassisted mainstream schooling is impossible due to intellectual disability (as opposed to behavioral problems), or when an adult requires institutionalization or supervision within the community (Jones et al. 1999.)

[0292] In some embodiments, administering the rAAV-mediated TSC2 “split intein” gene therapy described herein to a subject in need according to the present methods leads to a reduction of abnormal cell growth and / or proliferation in a subject suffering from TSC. Examples of proliferative disorders associated with TSC include Renal Angiomyolipoma and Lymphangioleiomyomatosis (LAM). Reduction of abnormal cell growth and / or proliferation may be assessed according to methods known in the art.

[0293] In some embodiments, the rAAV-mediated TSC2 “split intein” gene therapy described herein is administered to a subject in need according to the present methods prior to the onset of puberty. In some embodiments, the rAAV-mediated TSC2 “split intein” gene therapy described herein is administered to a young pediatric subject in need. In some embodiments, a “young pediatric subject” is about 4 years old, about 3.5 years old, about 3 years old, about 2.5 years old, about 2 years old, about 1.5 years old, about 1 year old or about 6 months old. In some embodiments, the AAV particle is administered to a pediatric subject about 5 years old, about 6 years old, about 7 years old, about 8 years old, about 9 years old, about 10 years old, about 11 years old, about 12 years old, about 13 years old, about 14 years old, about 15 years old, about 16 years old or about 17 years old. In some embodiments, a recombinant hTfRl AAV particle of the present disclosure is delivered to an adult subject that is about 17 years old or older.

[0294] In some embodiments, administration of the rAAV-mediated TSC2 “split intein” gene therapy described herein according to the present methods further comprises the step of monitoring the treated subject’s levels of phosphorylated p70S6K. Methods of measuring levels of phosphorylated p70S6K are known in the art and include immunohistochemistry, enzymatic assays, mass spectrometry (GC / MS and LC / MS), etc.

[0295] In some embodiments, the treated subject has a TSC2 gene that comprises a mutation of at least one of the mutations cataloged for TSC2 in the Leiden Open Variation Database. (Jones et al. 1997, Jones et al. 1999.) In some embodiments, the subject has a TSC2 protein that comprises a mutation of at least one of the mutations cataloged for TSC2 in the Leiden Open Variation Database. In some embodiments, the subject has a TSC2-related mutation that is identified on the Leiden Open Variation Database. In some embodiments, the subject has a TSC2 and / or TSC2 mutation that has not yet been identified, described, or cataloged.

[0296] In some embodiments, administration of the rAAV-mediated TSC2 “split intein” gene therapy described herein according to the present methods comprises: intravenous administration, intra-arterial, intramuscular administration, intracardiac administration, intrathecal administration, subventricular administration, epidural administration, intracerebral administration, intracerebroventricular administration, sub-retinal administration, intravitreal administration, intraarticular administration, intraocular administration, intraperitoneal administration, intrauterine administration, intradermal administration, subcutaneous administration, transdermal administration, transmucosal administration, or administration by inhalation. In some embodiments, administration of the rAAV-mediated TSC2 “split intein” gene therapy described herein according to the present methods comprises contacting both a first rAAV particle engineered to express an N-TSC2 / N-Intein polypeptide as described herein, and a second rAAV particle engineered to express a C-Intein / C-TSC2 polypeptide as described herein, or an effective amount thereof, with at least one cell or tissue of the CNS, the brain, the cerebellum, the brainstem, the basal ganglia, the hypothalamus, the preoptic area, a hippocampus, a striatum, a cortex, a motor cortex, a prefrontal cortex, a somatosensory cortex, a temporal cortex, a visual cortex, an occipital lobe, a temporal lobe, a parietal lobe, or a frontal lobe of the subject.

[0297] In some embodiments, administering, treating, contacting, or providing an effective amount comprises at least 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, or IO15vector genomes (vg), vg / mL, or total viral particles of a first rAAV particle engineered to express an N-TSC2 / N-Intein polypeptide as described herein, and at least 104, 103, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, or 1015vector genomes (vg), vg / mL, or total viral particles of a second rAAV particle engineered to express a C-Intein / C-TSC2 polypeptide as described herein.

[0298] In some embodiments, the administering or treating can comprise a single treatment. In other embodiments, a treatment may be followed by one or more subsequent treatments. In some embodiments, successful treatment and / or repair is determined when one or more of the following is detected: alleviation or amelioration of one or more of symptoms of the treated subject’s disease, disorder, or condition, diminishment of extent of the subject’s disease, disorder, or condition, stabilized (i.e., not worsening) state of a disease, disorder, or condition, delay or slowing of the progression of the disease, disorder, or condition, and amelioration or palliation of the disease, disorder, or condition. In some embodiments, success of treatment is determined by detecting the presence of functional TSC2 in one or more cells, tissues, or organs isolated from the subject.

[0299] In some embodiments, a composition comprising rAAV particles engineered to deliver the rAAV-mediated TSC2 “split intein” gene therapy described herein optionally comprises a suitable carrier. Suitable carriers can be selected for the indication for which the rAAV is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Examples of other suitable carriers include but are not limited to sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. Optionally, the compositions disclosed herein can also include, in addition to the rAAV particle and carrier(s), other pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0300] In embodiments, the method of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein comprises co-administration to a subject in need of both a first rAAV particle engineered to express an N-TSC2 / N-Intein polypeptide as described herein, and a second rAAV particle engineered to express a C-Intein / C-TSC2 polypeptide as described herein, in a pharmaceutical composition comprising in combination with one or more other viruses (e.g., a third rAAV particle encoding having one or more different transgenes). In some aspects, a composition can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different additional rAAV particles, each having one or more different transgenes.

[0301] In embodiments, the method of treating TSC by administering the rAAV-mediated TSC2 “split intein” gene therapy described herein comprises co-administration to a subject in need of a pharmaceutical composition comprising both a first rAAV particle engineered to express an N-TSC2 / N-Intein polypeptide as described herein, and a second rAAV particle engineered to express a C-Intein / C-TSC2 polypeptide as described herein in sufficient amounts to transduce the cells of a desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse effects. In some aspects, acceptabl...

Claims

CLAIMSWe claim:

1. A split-intein system for expressing a protein of interest in a cell, the split-intein system comprising:a) a first rAAV particle comprising a first expression cassette comprising a polynucleotide sequence encoding an N-TSC2 / N-Intein polypeptide, said N-TSC2 / N-Intein polypeptide comprising an N-terminal polypeptide fragment of the human TSC2 protein, fused at its C- terminus to the N-terminus of an N-Intein polypeptide, operably linked to a first promoter nucleotide sequence and flanked by AAV ITR sequences, andb) a second rAAV particle comprising a second expression cassette comprising a polynucleotide sequence encoding a C-Intein / C-TSC2 polypeptide, said C-Intein / C-TSC2 polypeptide comprising a C-Intein polypeptide fused at its C-terminus to the N-terminus of a C-terminal polypeptide fragment of the human TSC2 protein, operably linked to a second promoter nucleotide sequence and flanked by AAV ITR sequences,wherein the N-terminal polypeptide fragment and C-terminal polypeptide fragment together comprise a full-length human TSC2 protein having an amino acid sequence that is at least 90% identical, at least 95%, at least 99%, or 100% to the amino acid sequence of SEQ ID NO.: 10.

2. The split-intein system of Claim 1, wherein the N-Intein polypeptide comprises the amino acid sequence of SEQ ID NO.: 2, and the C-Intein polypeptide comprises the amino acid sequence of SEQ ID NO. : 4.

3. The split-intein system of Claim 1, wherein the C-Intein polypeptide comprises the amino acid sequence of SEQ ID NO.: 6, and the N-Intein polypeptide comprises the amino acid sequence of SEQ ID NO.: 8.

4. The split-intein system of Claim 1, wherein the N-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 12, and the C-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 14.

5. The split-intein system of Claim 1, wherein the N-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 28, and the C-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 30.

6. The split-intein system of Claim 1, wherein the N-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 36, and the C-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 38.

7. The split-intein system of Claim 1, wherein the N-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 44, and the C-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 46.

8. The split-intein system of Claim 1, wherein the N-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 52, and the C-terminal polypeptide fragment comprises the amino acid sequence of SEQ ID NO.: 54.

9. The split-intein system of any one of Claims 1-8, wherein the first promoter polynucleotide sequence and the second promoter polynucleotide sequence both promote expression in the CNS and are the same as different from the second promoter polynucleotide sequence.

10. The split-intein system of Claim 9, wherein the polynucleotide sequence encoding the N-TSC2 / N-Intein polypeptide is operably joined to a first polyadenylation signal sequence, and the polynucleotide sequence encoding the C-Intein / C-TSC2 polypeptide is operably joined to a second polyadenylation signal sequence, and wherein the first polyadenylation signal sequence can either be the same as or different from the second polyadenylation signal sequence.

11. The split-intein system of Claim 10, wherein the first promoter polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 67, and the first polyadenylation signal sequence comprises the polynucleotide sequence of SEQ ID NO.: 75.

12. The split-intein system of Claim 10, wherein the second promoter polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 67, and the second polyadenylation signal sequence comprises the polynucleotide sequence of SEQ ID NO.: 75.

13. The split-intein system of Claim 10, wherein both the first promoter polynucleotide sequence and the second promoter polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO.: 67, and both the first polyadenylation signal sequence and the second polyadenylation signal sequence comprise the polynucleotide sequence of SEQ ID NO.: 75.

14. The split-intein system of Claim 13, wherein the first expression cassette is flanked by a 5’ AAV inverted terminal repeat (ITR) and a 3’ AAV inverted terminal repeat (ITR), and the second expression cassette is flanked by a 5’ AAV inverted terminal repeat (ITR) and a 3’ AAV inverted terminal repeat (ITR).

15. The split intein system of Claim 14, wherein the 5’ AAV inverted terminal repeat (ITR) comprises the polynucleotide sequence of SEQ ID NO.: 77, and the 3’ AAV inverted terminal repeat comprises the polynucleotide sequence of SEQ ID NO.: 78.

16. The split-intein system of Claim 1, wherein the first rAAV particle comprises a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO.: 93, and the second rAAV particle comprises a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO.: 94.

17. The split-intein system of Claim 1, wherein the first rAAV particle comprises a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO.: 97, and the second rAAV particle comprises a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO.: 98.

18. The split-intein system of Claim 1, wherein the first rAAV particle comprises a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO.: 101, and the second rAAV particle comprises a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO.: 102.

19. The split-intein system of Claim 1, wherein the first rAAV particle comprises a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO.: 105 and the second rAAV particle comprises a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO.: 106.

20. The split-intein system of Claim 1, wherein the first rAAV particle comprises a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO.: 109, and the second rAAV particle comprises a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO.: 110.

21. The split-intein system of Claim 1, wherein the first rAAV particle comprises a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO.: 113, and the second rAAV particle comprises a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO.: 11422. The split-intein system of Claim 1, wherein the first rAAV particle comprises a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO.: 117, and the second rAAV particle comprises a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO.: 118.

23. The split-intein system of any one of Claims 1-8 or 16-22, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, and further comprises a 7 amino acid peptide inserted between amino acids 588 and 589, said 7 amino acid peptide comprising YSRIGPN (SEQ ID NO.: 149), YSRNSDN (SEQ ID NO.: 150), LHRLGPN (SEQ ID NO.: 151), LHRLGPD (SEQ ID NO.: 152), LHRAGPD (SEQ ID NO.: 153), YSRIGPD (SEQ ID NO.: 154), LSRIGPD (SEQ ID NO.: 155), LARSGPD (SEQ ID NO.: 156), LHKAGPN (SEQ ID NO.: 157), LSRIGPN (SEQ ID NO.: 158), LAKSGPN (SEQ ID NO.: 159), YARNGPN (SEQ ID NO.: 160), or FRSTNGV (SEQ ID NO.: 161).

24. The split-intein system of Claim 23, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138, and further comprises a 7 amino acid peptide YSRIGPN (SEQ ID NO.: 149) inserted between amino acids 588 and 589.

25. The split-intein system of Claim 24, wherein at least the first rAAV particle or at least the second rAAV particle comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 139.

26. The split-intein system of Claim 25, wherein at least the first rAAV particle or at least the second rAAV particle comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 142.

27. The split-intein system of Claim 23, wherein at least the first rAAV particle or at least the second rAAV particle comprises a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO. : 138, and further comprises a 7 amino acid peptide YSRIGPN (SEQ ID NO.: 149) inserted between amino acids 588 and 589, and further wherein the amino acid at position 586 is E, and the amino acid at position 589 is N.

28. The split-intein system of Claim 27, wherein at least the first rAAV particle or at least the second rAAV particle comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 140.

29. The split-intein system of Claim 27, wherein at least the first rAAV particle or at least the second rAAV particle comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 143.

30. The split-intein system of Claim 23, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138, and further comprises a 7 amino acid peptide FRSTNGV (SEQ ID NO.: 161) inserted between amino acids 588 and 589, and further wherein the amino acid at position 588 is D, and the amino acid at position 592 isE.

31. The split-intein system of Claim 30, wherein at least the first rAAV particle or at least the second rAAV particle comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 147.

32. The split-intein system of Claim 30, wherein at least the first rAAV particle or at least the second rAAV particle comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 148.

33. The split-intein system of any one of Claims 1-8 or 16-22, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, and further comprises a 7 amino acid peptide inserted between amino acids 588 and 589, said 7 amino acid peptide comprising LYDGRSG (SEQ ID NO.: 166), VQRLSVL (SEQ ID NO.: 167), KVSNPVW (SEQ ID NO.: 168), or RPVQVMA (SEQ ID NO.: 169).

34. The split-intein system of any one of Claims 1-8 or 16-22, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, and further comprises a 7 amino acid peptide LYDGRSG (SEQ ID NO.: 166) inserted between amino acids 588 and 589, and further wherein the amino acid at position 589 is a Y.

35. The split-intein system of any one of Claims 1-8 or 16-22, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, and further comprises a 7 amino acid peptide RPVQVMA (SEQ ID NO.: 169) inserted between amino acids 588 and 589, and further wherein the amino acid at position 589 is an E.

36. The split-intein system of any one of Claims 1-8 or 16-22, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, and further comprises a 7 amino acid peptide KVSNPVW (SEQ ID NO.: 168)inserted between amino acids 588 and 589, and further wherein the amino acid at position 587 is an S, and the amino acid at position 588 is an N.

37. The split-intein system of any one of Claims 1-8 or 16-22, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO. : 141, and further comprises a 7 amino acid peptide inserted at any position between amino acids 450 and 461, said 7 amino acid peptide comprising EFNNGSD (SEQ ID NO.: 173) or GAASLMP (SEQ ID NO.: 174).

38. The split-intein system of any one of Claims 1-8 or 16-22, wherein at least the first rAAV particle or at least the second rAAV particle comprises a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, wherein amino acids 450 and 461 are replaced with a 7 amino acid peptide comprising EFNNGSD (SEQ ID NO.: 173) or GAASLMP (SEQ ID NO.: 174).

39. The split-intein system of any one of Claims 1-8 or 16-22, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, and further comprises an amino acid targeting moiety that binds an ALPL protein.

40. The split-intein system of any one of Claims 1-8 or 16-22, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, and further comprises a 6 amino acid peptide inserted between amino acids 455 and 456, said 6 amino acid peptide comprising SPHSKA (SEQ ID NO.: 175).

41. The split-intein system of any one of Claims 1-8 or 16-22, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is atleast 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, and further comprises a 6 amino acid peptide inserted between amino acids 453 and 454, said 6 amino acid peptide comprising HDSPHK (SEQ ID NO.: 176).

42. The split-intein system of any one of Claims 1-8 or 16-22, wherein at least the first rAAV particle comprises, or at least the second rAAV particle comprises, or both the first and second rAAV particles comprise a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 138 or SEQ ID NO.: 141, said VP1 capsid protein further comprising one or more of the following amino acid substitutions: D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, andN716D.

43. The recombinant AAV particle of Claim 42, wherein at least the first rAAV particle or at least the second rAAV particle exhibits increased evasion of AAV-neutralizing antibodies relative to a reference AAV particle.

44. A method of treating a patient having tuberous sclerosis complex (TSC), said method comprising administering to said patient a therapeutically effective amount of the first rAAV particle and the second rAAV particle of any one of Claims 1-8 or 16-22.

45. A method of restoring the mTOR pathway in a subject in need thereof, said method comprising administering to said patient a therapeutically effective amount of the first rAAV particle and the second rAAV particle of any one of Claims 1-8 or 16-22.

46. A method of suppressing the mTOR pathway in a subject in need thereof, said method comprising administering to said patient a therapeutically effective amount of the first rAAV particle and the second rAAV particle of any one of Claims 1-8 or 16-22.

47. A method of reducing the amount of phosphorylated p70S6K in target cells of a subject in need thereof, said method comprising administering to said patient a therapeutically effective amount of the first rAAV particle and the second rAAV particle of any one of Claims 1-8 or 16-22.

48. A pharmaceutical composition for use in treating a patient having tuberous sclerosis Complex (TSC) comprising the first rAAV particle and the second rAAV particle of any one of Claims 1-8 or 16-22 and a pharmaceutically acceptable carrier.

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