Adeno-associated vectors and particles to treat tuberous sclerosis complex caused by TSC1 gene mutations

Abstract

Provided are recombinant adeno-associated virus (rAAV) vectors comprising a transgene to express human TSC1 (hamartin); viral particles comprising said vectors (rAAV particles); methods of their production, methods of their use, including methods for treating Tuberous Sclerosis Complex (TSC) caused by TSC1 gene mutations and symptoms arising therefrom; and kits.

Description

ATTORNEY DOCKET NO.38213.0001P1ADENO-ASSOCIATED VECTORS AND PARTICLES TO TREAT TUBEROUS SCLEROSIS COMPLEX CAUSED BY TSC1 GENE MUTATIONS AND METHODS OF USE AND MANUFACTURESEQUENCE LISTING

[0001] The contents of the electronic sequence listing (38213.0001P1. xml; Size: 218,477 bytes; and Date of Creation: December 19, 2025) 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 TSC1 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), and it affects people of all races and ethnicities equally (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 anti-seizure 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).Attorney Docket No.: 38213.0001P1

[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 more antiepileptic 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, and anti-epileptics including 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 delivers a copy of the TSC1 cDNA that encodes a functional TSC1 protein to the CNS to provide therapeutic benefit to patients suffering from disorders caused by TSC1 gene mutations, such as TSC. The target organ system for this AAV 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.

[0006] 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. In previous studies, survival in two TSC1 knockout mouse models was shown to be improved by delivering a functional TSC1 gene using rAAV9 and rAAVrh8 serotype particles. (Prabhakar et al. 2015, Prabhakar et al. 2019.) However, the rescue of survival was not complete,Attorney Docket No.: 38213.0001P1and the authors of the initial study concluded this was presumably due to a “chimeric state in the brain in which not all the Tscl-null neurons were infected with” the rAAV-TSCl particles. (Prabhakar et al., 2015 at p. 8.) Accordingly, there is a need to develop improved rAAV particles that are capable of more efficient transduction of CNS cells to deliver TSC1 gene therapies.

[0007] The present inventors have developed novel recombinant AAV gene therapy particles designed to cross the BBB and deliver and express therapeutic levels of the TSC1 protein in cells of the CNS.SUMMARY OF INVENTION

[0008] Provided are nucleic acid vector constructs for production of recombinant AAV particles engineered to (i) contain a CNS targeting capsid, and (ii) express a TSCl gene that encodes a functional TSCl (hamartin) protein. The vector constructs are used to generate recombinant AAV (rAAV) particles that are administered for gene therapy in a subject suffering from TSC, by delivering a nucleic acid encoding a functional copy of the TSCl (hamartin) protein to target cells of CNS, thereby providing increased hamartin activity to treat and ameliorate the disease and related disorders. The inventors have developed rAAV particles that deliver the transgene to the relevant tissues of the CNS at therapeutically appropriate levels, such that the transgene is expressed at therapeutic levels in the appropriate tissues of the CNS of the subject.

[0009] Provided are AAV vector constructs wherein AAV inverted terminal repeat (ITR) nucleotide sequences flank an expression cassette that comprises a nucleotide sequence encoding TSCl (hamartin), operably linked to one or more regulatory elements (e.g., Kozak sequences, promoter sequences, enhancer sequences, posttranscriptional regulatory elements, and / or polyadenylation (poly(A)) signal sequences) that promote expression of the nucleotide sequence encoding TSCl (hamartin).

[0010] In embodiments, the TSCl coding sequence encodes a TSCl (hamartin) protein having, comprising or consisting of the amino acid sequence of SEQ ID NO.: 7, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO.: 7. In some embodiments, the TSCl coding sequence comprises a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO.: 8 or a reverse complementary sequence thereof, which encodes a functional TSCl (hamartin) protein, and, in embodiments, comprises the nucleotide sequence of SEQ ID NO.: 8. In some embodiments, the TSCl coding sequence comprises a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO.: 9 orAttorney Docket No.: 38213.0001P1a reverse complementary sequence thereof, which encodes a functional TSC1 (hamartin) protein, and, in embodiments, comprises the nucleotide sequence of SEQ ID NO.: 9. The expression cassette includes in embodiments a promoter sequence operably linked to the nucleotide sequence encoding TSC1 (hamartin), including one of the EFS promoter sequence (SEQ ID NO.: 24), which comprises the core promoter element of the EFla promoter; the chicken beta-actin AGT1 (CBA AGTl) promoter sequence (SEQ ID NO.: 25), the chicken beta-actin AGT2 (CBA AGT2) promoter sequence (SEQ ID NO.: 26), the CMV enhancer / promoter sequence (SEQ ID NO.: 27), the CEG promoter sequence (SEQ ID NO.: 28), the CAG promoter sequence (SEQ ID NO.: 29), or other promoter sequences capable of driving expression of the nucleotide sequence encoding TSC1 (hamartin) in cells of the CNS.

[0011] The expression cassette includes in embodiments a polyadenylation (poly(A)) signal sequence operably linked to the nucleotide sequence encoding TSC1 (hamartin), including one of the human growth hormone polyadenylation signal sequence (SEQ ID NO.: 30 (huGHpA)), variants of the bovine growth hormone polyadenylation signal sequence (SEQ ID NO.: 31 (bGHpA), SEQ ID NO.: 32 (bGH_v3pA)), or other polyadenylation signal sequences that enhance expression of the nucleotide sequence encoding TSC1 (hamartin) in cells of the CNS.

[0012] The expression cassette in embodiments includes additional regulatory sequences, such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) (SEQ ID NO.: 33).

[0013] AAV vector constructs (i.e., recombinant AAV genomes having, comprising, or consisting of the TSC1 transgene, associated regulatory sequences, and ITR sequences) are provided that have nucleotide sequences of SEQ ID NO.: 10 (ITR-EFS-TSCl-bGHv3pA), SEQ ID NO.: 11 (ITR-CMV-TSC1 -bGHpA), SEQ ID NO.: 12 (ITR-EFS-TSCl-huGHpA), SEQ ID NO.: 13 (ITR-CMV-TSC1 -huGHpA), SEQ ID NO.: 14 (ITR-CMV-TSCl-bGH_v3pA), SEQ ID NO.: 15 (ITR-CBA_AGT2-TSCl-bGH_v3pA), or SEQ ID NO.: 16 (ITR-CBA AGT1-TSC1-bGH_v3pA), which encode and express a functional human TSC1 (hamartin) protein, including in CNS cells.

[0014] Expression cassettes (i.e., recombinant AAV genomes having, comprising, or consisting of the TSC1 transgene and associated regulatory sequences, but not including any flanking ITRs sequences) are provided and have nucleotide sequences of SEQ ID NO.: 17 (EFS-TSC1-bGHv3pA), SEQ ID NO.: 18 (CMV-TSC1 -bGHpA), SEQ ID NO.: 19 (EFS-TSC1 -huGHpA ),Attorney Docket No.: 38213.0001P1SEQ ID NO: 20 (CMV-TSCl-huGHpA), SEQ ID NO: 21 (CMV-TSCl-bGH_v3pA), SEQ ID NO.: 22 (CBA_AGT2-TSCl-bGH_v3pA), or SEQ ID NO.: 23 (CBA_AGTl-TSCl-bGH_v3pA), which encode and express a functional human TSC1 (hamartin) protein, including in CNS cells.

[0015] Also provided are plasmids comprising these vector constructs and expression cassettes, said plasmids comprising the following nucleotide sequences (and reverse complements thereof): SEQ ID NO.: 39 (pITR-EFS-TSCl-bGHv3pA), SEQ ID NO.: 40 (pITR-CMV-TSCl-bGHpA), SEQ ID NO.: 41 (pITR-EFS-TSCl-huGHpA), SEQ ID NO.: 42 (pITR-CMV-TSCl-huGHpA) SEQ ID NO.: 43 (pITR- CMV-TSCl-bGH_v3pA), SEQ ID NO.: 44 (p ITR-CBA AGT2-TSC1- bGH_v3pA), and SEQ ID NO.: 45 (pITR-CBA_AGTl-TSCl-bGH_v3pA), which encode and express a functional human TSC1 (hamartin) protein, including in CNS cells.

[0016] Also provided are recombinant AAV particles (hTfRl rAAV particles) comprising capsid sequences containing peptides incorporated into the capsid protein sequence such that they are displayed on the surface of the capsid, wherein the peptides bind the target hTfRl receptor (hTfRl AAV capsids), and further comprising as a transgene a TSC1 gene encoding a functional TSC1 (hamartin) protein operably linked to regulatory elements. Although not to be bound to a particular mechanism of action, rAAV particles comprising capsids incorporating these hTfRl AAV capsid sequences, derived from the AAV9 capsid, are thought to have enhanced ability to cross the blood-brain barrier, likely via hTfRl receptor mediated transcytosis, which allows for efficient delivery throughout the CNS. (Huang et al. 2023.) 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 hTfR1 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.

[0017] In embodiments, the hTfRl AAV particle comprising the TSC1 transgene comprises a capsid polypeptide engineered to include a transferrin receptor (TfRl) binding modification sequence comprising a 7-mer amino acid sequence that is inserted between amino acids 588 andAttorney Docket No.: 38213.0001P1in an analogous position of a capsid polypeptide of another AAV serotype. In embodiments, the 7-mer is selected from YSRIGPN (SEQ ID NO.: 49), YSRNSDN (SEQ ID NO.: 50), LHRLGPN (SEQ ID NO.: 51), LHRLGPD (SEQ ID NO.: 52), LHRAGPD (SEQ ID NO.: 53), YSRIGPD (SEQ ID NO.: 54), LSRIGPD (SEQ ID NO.: 55), LARSGPD (SEQ ID NO.: 56), LHKAGPN (SEQ ID NO: 57), LSRIGPN (SEQ ID NO.: 58), LAKSGPN (SEQ ID NO.: 59), and YARNGPN (SEQ ID NO.: 60). In an example embodiment, the7-mer is YSRIGPN (SEQ ID NO.: 49). Capsids comprising these targeting peptides bind to cells with Tfrl receptors on the surface and transduce such cells better than comparator capsids not having the targeting peptide.

[0018] In embodiments, the hTfRl AAV particle comprises a capsid polypeptide engineered to include a transferrin receptor (TfRl) binding modification sequence comprising a 7-mer amino acid sequence that is 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.: 49), YSRNSDN (SEQ ID NO.: 50), LHRLGPN (SEQ ID NO.: 51), FRSTNGV (SEQ ID NO.: 61), FVSTNGV (SEQ ID NO.: 62), FZ1STNGZ2 (SEQ ID NO.: 63), FRSTNGZ3 (SEQ ID NO.: 64), or VESTNGR (SEQ ID NO.: 65), and wherein the 7-mer is inserted between amino acids 588 and 589 of an AAV9 or AAV9 K449R capsid polypeptide (e.g., SEQ ID NO.: 1 or SEQ ID NO.: 4), 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.: 49), YSRNSDN (SEQ ID NO.: 50), or LHRLGPN (SEQ ID NO.: 51), 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.: 49), 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 atAttorney Docket No.: 38213.0001P1least one amino acid substitution relative to the wild type sequence. In an embodiment, the 7-mer is YSRNSDN (SEQ ID NO.: 50) 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: 51) 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.: 61), FVSTNGV (SEQ ID NO.: 62), FZ1STNGZ2 (SEQ ID NO.: 63), FRSTNGZ3 (SEQ ID NO.: 64), or VESTNGR (SEQ ID NO.: 65) 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.: 61), 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.: 62), 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.: 63) or FRSTNGZ3 (SEQ ID NO.: 64), 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.: 65), 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. Capsids comprising these targeting peptides bind to cells with Tfrl receptors on the surface and transduce such cells better than comparator capsids not having the targeting peptide.

[0019] In some embodiments, the hTfRl AAV particle comprising the TSC1 -encoding recombinant genome comprises a capsid protein having an amino acid sequence of SEQ ID NO.: 2, 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.: 49 (YSRIGPN) between wild-type AAV9 VP1 amino acid residues 588 and 589. In some embodiments, the hTfRl AAV capsid comprising the TSC1 -encoding recombinant genome comprises a capsid protein having an amino acid sequence of SEQ ID NO.: 3, 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.: 49 (YSRIGPN) between wild-type AAV9 VP1 amino acid residues 588 and 589, and further wherein the wild-type AAV9Attorney Docket No.: 38213.0001P1VP1 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 comprising the TSC1 -encoding recombinant genome comprises a capsid protein having an amino acid sequence of SEQ ID NO.: 47, 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.: 61 (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). Capsids comprising these targeting peptides bind to cells with Tfrl receptors on the surface and transduce such cells better than comparator capsids not having the targeting peptide.

[0020] In some embodiments, the hTfRl AAV particle comprising the TSCl-encoding recombinant genome comprises a capsid protein having an amino acid sequence of SEQ ID NO.: 5, which is an engineered variant of the AAV9 K449R VP1 capsid protein having an amino acid sequence of SEQ ID NO.: 4 that includes a 7-amino acid insertion comprising SEQ ID NO.: 49 (YSRIGPN) between AAV9 K449R VP1 amino acid residues 588 and 589. hTfRl AAV particles comprising a VP1 amino acid sequence of SEQ ID NO.: 5 are referred to herein as “AAV hTfRl v.l particles.” In some embodiments, the hTfRl AAV capsid comprising the TSCl-encoding recombinant genome comprises a capsid protein having an amino acid sequence of SEQ ID NO.: 6, which is an engineered variant of the AAV9 K449R VP1 capsid protein having an amino acid sequence of SEQ ID NO.: 4, with a 7-amino acid insertion comprising SEQ ID NO.: 49 (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.: 6 are referred to herein as “AAV hTfRl v.2 particles.” In some embodiments, the hTfRl AAV capsid comprising the TSCl-encoding recombinant genome comprises a capsid protein having an amino acid sequence of SEQ ID NO.: 48, which is an engineered variant of the AAV9 K449R VP1 capsid protein having an amino acid sequence of SEQ ID NO.: 4, with a 7-amino acid insertion comprising SEQ ID NO.: 61 (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 DAttorney Docket No.: 38213.0001P1(Q588D), and the AAV9 K449R VP1 amino acid residue at position 592 is changed from Q to E (Q592E). Capsids comprising these targeting peptides bind to cells with Tfrl receptors on the surface and transduce such cells better than comparator capsids not having the targeting peptide.

[0021] Also provided are recombinant AAV particles (hCA4 rAAV particles) comprising capsid sequences engineered to include peptides incorporated into the capsid protein sequence such that they are displayed on the surface of the capsid, wherein the peptides bind human GPI-linked enzyme Carbonic anhydrase IV (hCA4) (hCA4 rAAV capsids), and further comprising a TSC1 gene encoding a functional TSC1 (hamartin) protein operably linked to regulatory elements. Although not to be bound to a particular mechanism of action, these hCA4 rAAV capsids are thought to have enhanced ability to bind hCA4 and cross the blood-brain barrier, which allows for efficient delivery throughout the CNS. In embodiments, the hCA4 rAAV capsid comprises a modified capsid sequence as set forth in PCT Application No. PCT / US2025 / 023671, published October 16, 2025 as WO / 2025 / 217174, the entirety of which is incorporated herein.

[0022] In embodiments, the hCA4 rAAV capsid comprises a capsid polypeptide 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.: 1 (AAV9 VP1 amino acid sequence) or SEQ ID No.: 4 (AAV9K449R amino acid sequence)), or in an analogous position of a capsid polypeptide of another AAV serotype. In embodiments, the hCA4 amino acid binding modification sequence is a 7-mer amino acid sequence selected from LYDGRSG (SEQ ID NO.: 66), VQRLSVL (SEQ ID NO.: 67), KVSNPVW (SEQ ID NO: 68), RPVQVMA (SEQ ID NO.: 69).

[0023] In embodiments, the hCA4 amino acid binding modification sequence defined by the formula Z1-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 capsid polypeptide (e.g., SEQ ID NO.: 1 or SEQ ID NO.: 4), 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 bindingAttorney Docket No.: 38213.0001P1modification sequence is LYDGRSGY (SEQ ID NO.: 70). 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.: 71). In embodiments of the binding modification formula described above, the n-mer is KVSNPVW, Z1 is S, and Z2 is N, such that the hCA4 amino acid binding modification sequence is SNKVSNPVW (SEQ ID NO.: 72).

[0024] Also provided are recombinant AAV particles (hCD59 rAAV particles) comprising capsid sequences engineered to include peptides incorporated into the capsid protein sequence such that they are displayed on the surface of the capsid, wherein the peptides bind the human CD59 cell surface protein (hCD59) (hCD59 rAAV capsids), and further comprising a TSC1 gene encoding a functional TSC1 (hamartin) protein operably linked to regulatory elements. Although not to be bound to a particular mechanism of action, these hCD59 rAAV particles are thought to have enhanced ability to bind hCD59 and cross the blood-brain barrier, which allows for efficient delivery throughout the CNS. In embodiments, the hCD59 rAAV capsid comprises a modified capsid sequence as set forth in PCT Application No. PCT / US2025 / 023652, published October 16, 2025 as WO / 2025 / 217163, the entirety of which is incorporated herein.

[0025] In embodiments, the hCD59 rAAV capsid comprises a capsid polypeptide engineered to include an hCD59 amino acid binding modification sequence inserted at any position between amino acids 450-461 of an AAV9 or AAV9 K449R capsid polypeptide (e.g., SEQ ID NO.: 1 (AAV9 VP1 amino acid sequence) or SEQ ID No.: 4 (AAV9K449R amino acid sequence), 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.: 73) or GAASLMP (SEQ ID NO.: 74). In other embodiments, the hCD59 rAAV particle comprises a capsid polypeptide engineered such that amino acids 450-461 of an AAV9 or AAV9 K449R capsid polypeptide (e.g., SEQ ID NO.: 1 or SEQ ID No.: 4), 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.: 73) or GAASLMP (SEQ ID NO.: 74).

[0026] Also provided are recombinant AAV particles (ALPL rAAV particle) comprising capsid sequences engineered to include peptides incorporated into the capsid protein sequence such that they are displayed on the surface of the capsid, wherein the peptides bind the human GPI-linkedAttorney Docket No.: 38213.0001P1alkaline phosphatase (ALPL) (ALPL rAAV capsids), and further comprising a TSC1 gene encoding a functional TSC1 (hamartin) protein operably linked to regulatory elements. Although not to be bound to a particular mechanism of action, these ALPL rAAV particles are thought to have enhanced ability to bind human ALPL and cross the blood-brain barrier, which allows for efficient delivery throughout the CNS. In embodiments, the ALPL rAAV capsid comprises a modified capsid sequence as set forth in PCT Application No. PCT / US2023 / 071544, published February 8, 2024 as WO / 2024030976, the entirety of which is incorporated herein.

[0027] Also provided are recombinant AAV particles (rAAV(AE) particles) comprising capsid sequences (rAAV(AE) capsids) engineered to include one or more amino acid substitutions that allow such rAAV(AE) particles to evade immune clearance by neutralizing antibodies, and reduce the incidence of immune response in treated subjects, and further comprising a TSC1 gene encoding a functional TSC1 (hamartin) protein operably linked to regulatory elements. In embodiments, the immune evasion rAAV capsid comprises a modified capsid sequence as set forth in U. S. Provisional Patent Application No. 63 / 720,703, the entirety of which is incorporated herein. The rAAV(AE) particles, in embodiments, further comprise recombinant capsid proteins having the hTfRl or, alternatively, CA4, CD59 or ALPL targeting peptides as described herein and one or more amino acid substitutions as described herein that increase immune evasion (“immune evasion rAAV particle”).

[0028] In embodiments, the immune evasion rAAV particle comprises an engineered AAV capsid polypeptide 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, R550Q, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, 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.

[0029] In embodiments, the immune evasion rAAV particle comprises an engineered AAV capsid protein, 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 capsid protein, wherein the amino acid substitutions further includeAttorney Docket No.: 38213.0001P1from 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 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 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 capsid protein, wherein the additional amino acid substitutions include K462E / Q 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 capsid protein, wherein the additional amino acid substitution includes 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 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.

[0030] In embodiments, the immune evasion rAAV particle comprises an engineered AAV capsid protein, wherein the amino acid substitutions include D327N, N328K, N329D, K332Q, N452D, E500D, A502S, P504T, A510K, R550Q, 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 capsid protein, wherein the amino acid substitutions include D327N, N328K, N329D, K332Q, N452D, E500D, A502S, P504T, A510K, R550Q, D551N, D554N, K557Q and D657N of anAttorney Docket No.: 38213.0001P1AAV9 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 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 capsid protein, wherein the amino acid substitutions include N452K, K462E, E500D, A502S, P504T, A510K, R550Q, D551N, D554N, K557Q, D657N, and N663D. In an embodiment, an engineered AAV 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 evasion rAAV particle comprises an engineered AAV capsid protein, wherein the amino acid substitutions include G455Q / T / K / N, D551N, N552D, K557E / Q, K664E / Q, 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.

[0031] In embodiments, the immune evasion rAAV particle comprises an engineered AAV capsid protein, 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 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 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 capsid protein, wherein an additional amino acid substitution includes D554N 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 theseAttorney Docket No.: 38213.0001P1capsid mutations. In an embodiment, the immune evasion rAAV particle comprises an engineered AAV capsid protein, wherein the additional amino acid substitution includes 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.

[0032] In embodiments, the immune evasion rAAV particle comprises an engineered AAV capsid protein, wherein the amino acid substitutions include G455Q, D551N, N552D, D556K, K557E, K664E, D665N, N668K, and N716D. In embodiments, the immune evasion rAAV particle comprises an engineered AAV capsid protein, wherein the amino acid substitutions include G455T, D551N, N552D, D556K, K557E, K664E, D665N, N668K, and N716D. In embodiments, the immune evasion rAAV particle comprises an engineered AAV capsid protein, wherein the amino acid substitutions include G455K, 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 capsid protein, 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 VP 1 polypeptide of another AAV serotype, and exhibits immune evasion relative to the AAV particle without these capsid mutations.

[0033] Also provided are methods of treating Tuberous Sclerosis Complex (TSC), or increasing TSC1 activity, or reducing hyperactivation of the mTOR pathway, in a subject in need thereof, particularly, in a human subject by administering rAAV particles described herein. Also provided are methods of reducing a disease condition in a subject suffering from TSC by administering 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. Also provided are pharmaceutical gene therapy compositions comprising rAAV particles described herein, including for use in treating TSC, or increasing TSC1 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 ofAttorney Docket No.: 38213.0001P1delivering a nucleic acid encoding a functional copy of the TSC1 (hamartin) protein to target cells of CNS, thereby providing increased hamartin activity to treat and ameliorate disease.

[0034] Also provided are host cells for and methods of producing the recombinant AAV particles as described herein.

[0035] Also provided are methods of increasing TSC1 activity in a subject in need thereof including in cells and tissues of the central nervous system (CNS), by administering rAAV particles described herein.EMBODIMENTS

[0036] Embodiment 1: A recombinant AAV vector construct comprising an expression cassette comprising a polynucleotide encoding a human TSC1 (hamartin) protein comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO.: 7, operably linked to a polynucleotide encoding a promoter that drives expression of TSC1 in cells of the CNS, said expression cassette flanked by AAV inverted terminal repeats (ITRs).

[0037] Embodiment 2: The recombinant AAV vector construct of embodiment 1, wherein the TSC1 (hamartin) protein comprises the amino acid sequence of SEQ ID NO.: 7.

[0038] Embodiment 3: The recombinant AAV vector construct of embodiment 1 or 2, wherein the polynucleotide encoding a promoter comprises any one of the polynucleotide sequences of SEQ ID NO.: 24 (EFS promoter), SEQ ID NO.: 25 (CBA AGTl promoter), SEQ ID NO.: 26 (CBA AGT2 promoter), SEQ ID NO.: 27 (CMV promoter), SEQ ID NO.: 28 (CEG promoter), or SEQ ID NO.: 29 (CAG promoter).

[0039] Embodiment 4: The recombinant AAV vector construct of any one of embodiments 1 through 3, wherein the recombinant AAV vector construct comprises any one of the polynucleotide sequences of SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, or SEQ ID NO.: 16.

[0040] Embodiment 5: The recombinant AAV vector construct of any one of embodiments 1 through 3, wherein the expression cassette comprises any one of the polynucleotide sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, SEQ ID NO.: 19, SEQ ID NO.: 20, SEQ ID NO.: 21, SEQ ID NO.: 22, or SEQ ID NO.: 23.

[0041] Embodiment 6: The recombinant AAV vector construct of any one of embodiments 1 through 3, wherein the recombinant AAV vector construct comprises the polynucleotide sequence of SEQ ID NO.: 10.Attorney Docket No.: 38213.0001P1

[0042] Embodiment 7: The recombinant AAV vector construct of any one of embodiments 1 through 3, wherein the expression cassette comprises the polynucleotide sequence of SEQ ID NO.: 17.

[0043] Embodiment 8: The recombinant AAV vector construct of any one of embodiments 1 through 3, wherein the polynucleotide encoding the TSC1 (hamartin) protein is at least 80% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 8.

[0044] Embodiment 9: The recombinant AAV vector construct of embodiment 8, wherein the polynucleotide encoding a TSC 1 (hamartin) protein is identical to the nucleotide sequence of SEQ ID NO.: 8.

[0045] Embodiment 10: The recombinant AAV vector construct of any one of embodiments 1 through 3, wherein the polynucleotide encoding a TSC1 (hamartin) protein is at least 80% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 9.

[0046] Embodiment 11: The recombinant AAV vector construct of embodiment 10, wherein the polynucleotide encoding a TSC1 (hamartin) protein is identical to SEQ ID NO.: 9.

[0047] Embodiment 12: The recombinant AAV vector construct of any one of embodiments 1 through 11, wherein the ITRs are 5’ AAV2 ITR and 3’ AAV2 ITR, having the polynucleotide sequences of SEQ ID NO.: 34 and SEQ ID NO.: 35, respectively.

[0048] Embodiment 13: The recombinant AAV vector construct of any one of embodiments 1 through 12, wherein the expression cassette further comprises a woodchuck hepatitis virus posttranslational regulatory element (WPRE).

[0049] Embodiment 14: The recombinant AAV vector construct of embodiment 13, wherein the woodchuck hepatitis virus posttranslational regulatory element (WPRE) comprises the polynucleotide sequence of SEQ ID NO.: 33.

[0050] Embodiment 15: A recombinant AAV particle comprising the recombinant AAV vector construct of any one of embodiments 1 through 14 and an AAV capsid that 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.: 1 or SEQ ID NO.: 4, 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.: 49), YSRNSDN (SEQ ID NO.: 50), LHRLGPN (SEQ ID NO.: 51),Attorney Docket No.: 38213.0001P1LHRLGPD (SEQ ID NO.: 52), LHRAGPD (SEQ ID NO.: 53), YSRIGPD (SEQ ID NO.: 54), LSRIGPD (SEQ ID NO.: 55), LARSGPD (SEQ ID NO.: 56), LHKAGPN (SEQ ID NO.: 57), LSRIGPN (SEQ ID NO.: 58), LAKSGPN (SEQ ID NO.: 59), or YARNGPN (SEQ ID NO.: 60), which particle transduces hTfRl receptor-expressing cells or crosses the BBB with greater frequency than an AAV9 capsid particle.

[0051] Embodiment 16: The recombinant AAV particle of embodiment 15, wherein the AAV capsid 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.: 1, and further comprises a 7 amino acid peptide YSRIGPN (SEQ ID NO.: 49) inserted between amino acids 588 and 589.

[0052] Embodiment 17: The recombinant AAV vector of embodiment 16, wherein the AAV capsid comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 2.

[0053] Embodiment 18: The recombinant AAV vector of embodiment 16, wherein the AAV capsid comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 5.

[0054] Embodiment 19: The recombinant AAV particle of embodiment 15, wherein the AAV capsid 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.: 1, and further comprises a 7 amino acid peptide YSRIGPN (SEQ ID NO.: 49) 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.

[0055] Embodiment 20: The recombinant AAV vector of embodiment 19, wherein the AAV capsid comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 3.

[0056] Embodiment 21: The recombinant AAV vector of embodiment 19, wherein the AAV capsid comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 6.

[0057] Embodiment 22: The recombinant AAV particle of embodiment 15, wherein the AAV capsid 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.: 1, and further comprises a 7 amino acid peptide FRSTNGV (SEQ ID NO.: 61) 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.Attorney Docket No.: 38213.0001P1

[0058] Embodiment 23: The recombinant AAV vector of embodiment 22, wherein the AAV capsid comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 47.

[0059] Embodiment 24: The recombinant AAV vector of embodiment 22, wherein the AAV capsid comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 48.

[0060] Embodiment 25: A recombinant AAV particle comprising a recombinant AAV vector construct comprising an expression cassette comprising a polynucleotide encoding a TSC1 (hamartin) protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO.: 7, operably linked to a promoter sequence and a poly(A) signal sequence, flanked by AAV inverted terminal repeats (ITRs) and an AAV capsid that 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.: 1, and further comprises a 7 amino acid peptide YSRIGPN (SEQ ID NO.: 49) inserted between amino acids 588 and 589.

[0061] Embodiment 26: The recombinant AAV particle of embodiment 25 wherein the promoter sequence comprises the polynucleotide sequence of SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27, SEQ ID NO.: 28, or SEQ ID NO.: 29, and the poly(A) signal sequence comprises SEQ ID NO.: 30, SEQ ID NO.: 31, or SEQ ID NO.: 32.

[0062] Embodiment 27: A recombinant AAV particle comprising a recombinant AAV vector construct comprising an expression cassette comprising a polynucleotide encoding a TSC1 (hamartin) protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO.: 7, wherein the polynucleotide is operably linked to a promoter sequence and a poly(A) signal sequence, flanked by AAV inverted terminal repeats (ITRs), and an AAV capsid that 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.: 1, and further comprises a 7 amino acid peptide YSRIGPN (SEQ ID NO.: 49) 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.

[0063] Embodiment 28: The recombinant AAV particle of embodiment 27 wherein the promoter sequence comprises the polynucleotide sequence of SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27, SEQ ID NO.: 28, or SEQ ID NO.: 29, and the poly(A) signal sequence comprises SEQ ID NO.: 30, SEQ ID NO.: 31, or SEQ ID NO.: 32.Attorney Docket No.: 38213.0001P1

[0064] Embodiment 29: A recombinant AAV particle comprising a recombinant AAV vector construct comprising an expression cassette comprising a polynucleotide encoding a TSC1 (hamartin) protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO.: 7, operably linked to a promoter sequence and a poly(A) signal sequence, flanked by AAV inverted terminal repeats (ITRs) and an AAV capsid that 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.: 1, and further comprises a 7 amino acid peptide FRSTNGV (SEQ ID NO.: 61) 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.

[0065] Embodiment 30: The recombinant AAV particle of embodiment 29 wherein the promoter sequence comprises the polynucleotide sequence of SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27, SEQ ID NO.: 28, or SEQ ID NO.: 29, and the poly(A) signal sequence comprises SEQ ID NO.: 30, SEQ ID NO.: 31, or SEQ ID NO.: 32.

[0066] Embodiment 31: A recombinant AAV particle comprising the recombinant AAV vector construct of any one of embodiments 1 through 14 and an AAV capsid that 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.: 1 or SEQ ID NO.: 4, 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.: 66), VQRLSVL (SEQ ID NO.: 67), KVSNPVW (SEQ ID NO.: 68), or RPVQVMA (SEQ ID NO.: 69).

[0067] Embodiment 32: The recombinant AAV particle of embodiment 31 wherein the promoter sequence comprises the polynucleotide sequence of SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27, SEQ ID NO.: 28, or SEQ ID NO.: 29, and the poly(A) signal sequence comprises SEQ ID NO.: 30, SEQ ID NO.: 31, or SEQ ID NO.: 32.

[0068] Embodiment 33: A recombinant AAV particle comprising the recombinant AAV vector construct of any one of embodiments 1 through 14 and an AAV capsid that 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.: 1 or SEQ ID NO.: 4, and further comprises a 7 amino acid peptide LYDGRSG (SEQ ID NO.: 66) inserted between amino acids 588 and 589, and further wherein the amino acid at position 589 is a Y.Attorney Docket No.: 38213.0001P1

[0069] Embodiment 34: The recombinant AAV particle of embodiment 33 wherein the promoter sequence comprises the polynucleotide sequence of SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27, SEQ ID NO.: 28, or SEQ ID NO.: 29, and the poly(A) signal sequence comprises SEQ ID NO.: 30, SEQ ID NO.: 31, or SEQ ID NO.: 32.

[0070] Embodiment 35: A recombinant AAV particle comprising the recombinant AAV vector construct of any one of embodiments 1 through 14 and an AAV capsid that 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.: 1 or SEQ ID NO.: 4, and further comprises a 7 amino acid peptide RPVQVMA (SEQ ID NO.: 69) inserted between amino acids 588 and 589, and further wherein the amino acid at position 589 is an E.

[0071] Embodiment 36: The recombinant AAV particle of embodiment 35 wherein the promoter sequence comprises the polynucleotide sequence of SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27, SEQ ID NO.: 28, or SEQ ID NO.: 29, and the poly(A) signal sequence comprises SEQ ID NO.: 30, SEQ ID NO.: 31, or SEQ ID NO.: 32.

[0072] Embodiment 37: A recombinant AAV particle comprising the recombinant AAV vector construct of any one of embodiments 1 through 14 and an AAV capsid that 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.: 1 or SEQ ID NO.: 4, and further comprises a 7 amino acid peptide KVSNPVW (SEQ ID NO.: 68) 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.

[0073] Embodiment 38: The recombinant AAV particle of embodiment 37 wherein the promoter sequence comprises the polynucleotide sequence of SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27, SEQ ID NO.: 28, or SEQ ID NO.: 29, and the poly(A) signal sequence comprises SEQ ID NO.: 30, SEQ ID NO.: 31, or SEQ ID NO.: 32.

[0074] Embodiment 39: A recombinant AAV particle comprising the recombinant AAV vector construct of any one of embodiments 1 through 14 and an AAV capsid that 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.: 1 or SEQ ID NO.: 4, 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.: 73) or GAASLMP (SEQ ID NO.: 74).Attorney Docket No.: 38213.0001P1

[0075] Embodiment 40: The recombinant AAV particle of embodiment 39 wherein the promoter sequence comprises the polynucleotide sequence of SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27, SEQ ID NO.: 28, or SEQ ID NO.: 29, and the poly(A) signal sequence comprises SEQ ID NO.: 30, SEQ ID NO.: 31, or SEQ ID NO.: 32.

[0076] Embodiment 41: A recombinant AAV particle comprising the recombinant AAV vector construct of any one of embodiments 1 through 14 and an AAV capsid that 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.: 1 or SEQ ID NO.: 4, wherein amino acids 450 and 461 are replaced with a 7 amino acid peptide comprising EFNNGSD (SEQ ID NO.: 73) or GAASLMP (SEQ ID NO.: 74).

[0077] Embodiment 42: The recombinant AAV particle of embodiment 41 wherein the promoter sequence comprises the polynucleotide sequence of SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27, SEQ ID NO.: 28, or SEQ ID NO.: 29, and the poly(A) signal sequence comprises SEQ ID NO.: 30, SEQ ID NO.: 31, or SEQ ID NO.: 32.

[0078] Embodiment 43: A recombinant AAV particle comprising the recombinant AAV vector construct of any one of embodiments 1 through 14 and an AAV capsid that 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.: 1 or SEQ ID NO.: 4, and further comprises an amino acid targeting moiety that binds an ALPL protein.

[0079] Embodiment 44: The recombinant AAV particle of embodiment 43 wherein the promoter sequence comprises the polynucleotide sequence of SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27, SEQ ID NO.: 28, or SEQ ID NO.: 29, and the poly(A) signal sequence comprises SEQ ID NO.: 30, SEQ ID NO.: 31, or SEQ ID NO.: 32.

[0080] Embodiment 45: A recombinant AAV particle comprising the recombinant AAV vector construct of any one of embodiments 1 through 14 and an AAV capsid that 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.: 1 or SEQ ID NO.: 4, 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.: 75).

[0081] Embodiment 46: The recombinant AAV particle of embodiment 45 wherein the promoter sequence comprises the polynucleotide sequence of SEQ ID NO.: 24, SEQ ID NO.: 25,Attorney Docket No.: 38213.0001P1SEQ ID NO.: 26, SEQ ID NO.: 27, SEQ ID NO.: 28, or SEQ ID NO.: 29, and the poly(A) signal sequence comprises SEQ ID NO.: 30, SEQ ID NO.: 31, or SEQ ID NO.: 32.

[0082] Embodiment 47: A recombinant AAV particle comprising the recombinant AAV vector construct of any one of embodiments 1 through 14 and an AAV capsid that 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.: 1 or SEQ ID NO.: 4, 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.: 76).

[0083] Embodiment 48: The recombinant AAV particle of embodiment 47 wherein the promoter sequence comprises the polynucleotide sequence of SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27, SEQ ID NO.: 28, or SEQ ID NO.: 29, and the poly(A) signal sequence comprises SEQ ID NO.: 30, SEQ ID NO.: 31, or SEQ ID NO.: 32.

[0084] Embodiment 49: A recombinant AAV particle comprising the recombinant AAV vector construct of any one of embodiments 1 through 14 and an AAV capsid that comprises a VP1 capsid protein having an amino acid sequence of SEQ ID NO.: 1 or SEQ ID NO.: 4, said VP1 capsid protein further comprising at least three of the following amino acid substitutions: 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, which particle exhibits increased evasion of AAV-neutralizing antibodies relative to the AAV particle without these capsid mutations.

[0085] Embodiment 50: The recombinant AAV particle of embodiment 49 wherein the promoter sequence comprises the polynucleotide sequence of SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27, SEQ ID NO.: 28, or SEQ ID NO.: 29, and the poly(A) signal sequence comprises SEQ ID NO.: 30, SEQ ID NO.: 31, or SEQ ID NO.: 32.

[0086] Embodiment 51: The recombinant AAV particle of any one of embodiments 15 through 48 wherein the amino acid sequence of the VP1 capsid protein further comprises at least three of the following amino acid substitutions: 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,Attorney Docket No.: 38213.0001P1K664Q, K664E, D665N, N668K, and N716D, which particle exhibits increased evasion of AAV-neutralizing antibodies relative to the AAV particle without these capsid mutations.

[0087] Embodiment 52: The recombinant AAV particle of embodiment 51 wherein the promoter sequence comprises the polynucleotide sequence of SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27, SEQ ID NO.: 28, or SEQ ID NO.: 29, and the poly(A) signal sequence comprises SEQ ID NO.: 30, SEQ ID NO.: 31, or SEQ ID NO.: 32.

[0088] Embodiment 53: The recombinant AAV particle of any one of embodiments 49 through 52, wherein said recombinant AAV particle exhibits increased evasion of AAV-neutralizing antibodies relative to a reference AAV particle.

[0089] Embodiment 54: A method of treating a patient having tuberous sclerosis complex (TSC), said method comprising administering to said patient a therapeutically effective amount of a recombinant AAV particle of any one of embodiments 15 through 53.

[0090] Embodiment 55: 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 a recombinant AAV particle of any one of embodiments 15 through 53.

[0091] Embodiment 56: 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 a recombinant AAV particle of any one of embodiments 15 through 53.

[0092] Embodiment 57: 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 a recombinant AAV particle of any one of embodiments 15 through 53.

[0093] Embodiment 58: The method of any one of embodiments 54 through 57 wherein the recombinant AAV particle is administered systemically, intravenously, intracranially, intrathecally, intra-nasally, intra-peritoneally.

[0094] Embodiment 59: The method of embodiment 58 wherein the recombinant AAV particle is administered intravenously.

[0095] Embodiment 60: A pharmaceutical composition for use in treating a patient having tuberous sclerosis Complex (TSC) comprising a recombinant AAV particle of any one of embodiments 15 through 53 and a pharmaceutically acceptable carrier.Attorney Docket No.: 38213.0001P1BRIEF DESCRIPTION OF THE DRAWINGS

[0096] FIG. 1A-1B show (1A) normal exposure and (IB) high exposure images of a Western blot showing expression of TSC1 (hamartin) in TSC1 knockout cell lines 48 hours after transfection with plasmids containing various TSC1 expression cassettes described herein, in comparison to TSC1 knockout cells and wild-type HEK 293T cells that were either untransfected, or transfected with a GFP reporter expression cassette. A band appears at the predicted 130 kD molecular weight for TSC1 (hamartin).

[0097] FIG. 2 is a Western blot showing expression of TSC1 (hamartin) in TSC1 knockout cell lines 48 hours after transduction with recombinant rAAV PHP. B particles encapsidating AAV vector constructs comprising various TSC1 expression cassettes described herein, in comparison to untransduced TSC1 knockout cells and HEK 293T cells, and TSC knockout cells and HEK 293 T cells that were transduced with rAAV PHP. B particles encapsidating AAV vector constructs comprising a GFP reporter expression cassette. A band appears at the predicted 130 kD molecular weight for TSC1 (hamartin).

[0098] FIG. 3A-3C show (3A) the results of in situ hybridization studies to evaluate episome (CMV promoter) distribution in the cortex and hippocampus of TSC 1 CKO mice treated with various doses of rAAV9 or rAAV PHP. B particles encapsidating AAV vector constructs comprising the CMV-hTSCl-Myc-bGHpA expression cassette, in comparison to untreated wildtype mice, untreated TSC1 CKO mice, and TSC1 CKO mice treated with rapamycin; (3B) the results of in situ hybridization studies to evaluate hTSCl transgene RNA distribution in the cortex and hippocampus of TSC1 CKO mice treated with various doses of rAAV9 or rAAV PHP. B particles encapsidating AAV vector constructs comprising the CMV-hTSCl-Myc-bGHpA expression cassette, in comparison to untreated wild-type mice, untreated TSC1 CKO mice, and TSC1 CKO mice treated with rapamycin; (3C) the results of in situ hybridization studies to evaluate episome (CMV promoter) and hTSCl transgene RNA distribution in the liver of TSC1 CKO mice treated with various doses of rAAV9 or rAAV PHP. B particles encapsidating AAV vector constructs comprising the CMV-hTSCl-Myc-bGHpA expression cassette, in comparison to untreated wild-type mice, untreated TSC1 CKO mice, and TSC1 CKO mice treated with rapamycin.

[0099] FIG. 4 shows the results of complete blood counts (reticulocyte percentage and absolute reticulocyte count) in blood taken from hTFRC KI mice receiving various doses of rAAVAttorney Docket No.: 38213.0001P1hTfRlv.1, rAAV9, and rAAV PHP B particles encapsidating AAV vector constructs comprising expression cassettes containing a modified CAG promoter (CEG) driving expression of codon-orthogonal variants of the human glucosylceramidase beta 1 (hGBA) (hGBAco6 or hGBAco7) carrying a C-terminal influenza virus hemagglutinin tag (HA), followed by a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) and human growth hormone polyadenylation signal (huGHpA).

[0100] FIG. 5A-5C show the results of in situ hybridization studies to evaluate hGBAco6-HA or hGBAco7-HA episome distribution in the cortex (5A), thalamus (5B), and cerebellum (5C) of hTFRC KI mice receiving various doses of rAAV hTfRlv.1, rAAV9, and rAAV PHP. B particles encapsidating AAV vector constructs comprising expression cassettes containing a modified CAG promoter (CEG) driving expression of codon-orthogonal variants of the human glucosylceramidase beta 1 (hGBA) (hGBAco6 or hGBAco7) carrying a C-terminal influenza virus hemagglutinin tag (HA), followed by a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) and human growth hormone polyadenylation signal (huGHpA).

[0101] FIG. 6A-6C show the results of in situ hybridization studies to evaluate hGBAco6-HA or hGBAco7-HA episome distribution in cervical (6A), thoracic (6B), and lumbar (6C) sections of spinal cords from hTFRC KI mice receiving various doses of rAAV hTfRlv.1, rAAV9, and rAAV PHP. B particles encapsidating AAV vector constructs comprising expression cassettes containing a modified CAG promoter (CEG) driving expression of codon-orthogonal variants of the human glucosylceramidase beta 1 (hGBA) (hGBAco6 or hGBAco7) carrying a C-terminal influenza virus hemagglutinin tag (HA), followed by a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) and human growth hormone polyadenylation signal (huGHpA).

[0102] FIG. 7 shows the results of in situ hybridization studies to evaluate hGBAco6-HA or hGBAco7-HA episome distribution in the liver of hTFRC KI mice receiving various doses of rAAV hTfRlv.1, rAAV9, and rAAV PHP. B particles encapsidating AAV vector constructs comprising expression cassettes containing a modified CAG promoter (CEG) driving expression of codon-orthogonal variants of the human glucosylceramidase beta 1 (hGBA) (hGBAco6 or hGBAco7) carrying a C-terminal influenza virus hemagglutinin tag (HA), followed by a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) and human growth hormone polyadenylation signal (huGHpA).Attorney Docket No.: 38213.0001P1DETAILED DESCRIPTION DEFINITIONS

[0103] 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.

[0104] 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.

[0105] ‘ ‘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.”Attorney Docket No.: 38213.0001P1

[0106] 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.

[0107] The articles “a” and “an” are used herein to refer to one or to more than one (z.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.

[0108] 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.

[0109] ‘ ‘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.

[0110] 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 line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.

[0111] 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.

[0112] 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 comprisingAttorney Docket No.: 38213.0001P1a 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.

[0113] “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.

[0114] 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.

[0115] 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.

[0116] 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 anAttorney Docket No.: 38213.0001P1otherwise 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.

[0117] 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).

[0118] “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-sense strand 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 whetherAttorney Docket No.: 38213.0001P1one 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.

[0119] 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.).

[0120] 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 N0.: X, and specifically 5’-GGCAT-3’.

[0121] 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’.

[0122] 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 (i.e. miRNA or antisense RNA) or protein i.e. TSC1 (hamartin)).

[0123] 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 particleAttorney Docket No.: 38213.0001P1encoding 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.

[0124] 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.

[0125] 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 when the 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.

[0126] 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.Attorney Docket No.: 38213.0001P1

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] A “target gene” or “gene of interest” (GOI) 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. In embodiments, the target gene is a nucleic acid encoding a functional copy of the TSC1 (hamartin) protein.

[0132] 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 stopAttorney Docket No.: 38213.0001P1codon) or an attenuation in the activity of the target protein, as encoded by and expressed from the genome of a subject.

[0133] In embodiments, the target protein comprises TSC1 (hamartin). TSC1 is the chaperone for tuberin, the protein encoded by 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 “TSC1” or “TSC1 protein” encompasses naturally-occurring versions (i.e. human TSC1) and non-naturally occurring versions of TSC1 (i.e. TSC1 variants with amino-acid additions, deletions, or substitutions within the TSC1 protein sequence which increase or decrease the activity compared to that of naturally occurring TSC1) so long as the protein referred to as TSC1 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 TSC1 has at least, or no more than, 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 TSC1, wherein “corresponding” contemplates and provides support for the naturally-occurring TSC1 protein to which the amino-acid additions, deletions, or substitutions were applied. In embodiments, the TSC1 is a human TSC1 and, in some embodiments, has the amino acid sequence of SEQ ID NO.:7. In alternate embodiments, the TSC1 has an amino acid sequence that has at least 99%, 95%, 90%, 85% or 80% sequence identify to SEQ ID NO.: 7 and has TSC1 activity. In other embodiments, the human TSC1 is encoded by the nucleotide sequence of SEQ ID NO.: 8, or a nucleotide sequence that is at least 99%, 95%, 90%, 85% or 80% identical to SEQ ID NO: 8, and encodes a human TSC1 protein having the amino acid sequence of SEQ ID NO.: 7 or a human TSC1 which has an amino acid sequence that has at least 99%, 95%, 90%, 85% or 80% sequence identify to SEQ ID NO.:1 and has TSC1 activity. In other embodiments, the human TSC1 is encoded by the nucleotide sequence of SEQ ID NO.: 9.

[0134] “ TSC1 activity” refers to the TSC1 protein’s function as a chaperone to stabilize the TSC2 (tuberin) protein, preventing the degradation of TSC2 and thereby enhancing the function of TSC2 to regulate the activity of the mammalian target of rapamycin (mTOR) signaling pathway,Attorney Docket No.: 38213.0001P1as assayed, for example, by measuring the ratio of phosphorylated to unphosphorylated p70S6K protein as described in the Examples set forth herein.

[0135] In some embodiments, a subject or patient treated according to the methods described herein is deficient in TSC1, as encoded by and expressed from the genome of the subject, due for example to an autosomal dominant inheritance of a defective TSC1 gene. In some embodiments, the subject or patient has TSC. In some embodiments, the subject or patient has an attenuated or abolished activity of TSC 1.

[0136] 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 protein, for example, a target protein such as TSC1 (hamartin). Generally, the promoter is operatively linked to the nucleic acid that encodes the target protein in a manner that is capable of promoting 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 sequences (such as a promoter, polyadenylation signal, or other expression sequence) that enhances, modulates, or otherwise facilitates expression of the desired nucleic acid sequence, such as the TSC J gene. Plasmids may be engineered to comprise expression cassettes, as described herein.

[0137] An “AAV vector construct” is a nucleic acid capable of being packaged within a recombinant AAV particle for delivery of a target gene, such as the TSC I gene, to the interior of a cell. AAV vector constructs include both (i) an expression cassette that may include a nucleic acid encoding a protein, such as TSC1 (hamartin), 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 (rAAV particle). ITRs also provide other cis-acting functions for expression of the nucleic acid encoding the target protein 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 acidAttorney Docket No.: 38213.0001P1sequences 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.

[0138] 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.

[0139] 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.

[0140] The nucleotide and amino acid sequences provided herein are set out in Table 1.AAV VECTOR CONSTRUCTS AND EXPRESSION CASSETTES

[0141] In some embodiments, a recombinant adeno-associated virus (rAAV) vector construct is provided, which, at minimum, comprises a TSC1 expression cassette with polynucleotide sequences sufficient for expression of a human TSC1 (hamartin) protein (the “expressing region”), flanked by at least two inverted-terminal repeats (ITRs). In embodiments, the TSC1 expressing region comprises a polynucleotide sequence that encodes TSC1, operably linked to a promoter sequence which promotes CNS expression. In embodiments, the expression cassette includes a polynucleotide sequence that encodes TSC1 that is operably linked to an EFS promoter (SEQ ID. NO.: 24). In other embodiments, the expression cassette includes a polynucleotide sequence that encodes TSC1 (hamartin) that is operably linked to a CBA AGTl promoter (SEQ ID. NO.: 25). In other embodiments, the expression cassette includes a polynucleotide sequence that encodesAttorney Docket No.: 38213.0001P1TSC1 that is operably linked to a CBA AGT2 promoter (SEQ ID. NO.: 26). In other embodiments, the expression cassette includes a polynucleotide sequence that encodes TSC1 (hamartin) that is operably linked to a CMV-derived promoter (SEQ ID. NO.: 27). In other embodiments, the expression cassette includes a polynucleotide sequence that encodes TSC1 that is operably linked to a CEG promoter (SEQ ID. NO.: 28). In other embodiments, the expression cassette includes a polynucleotide sequence that encodes TSC1 that is operably linked to a CAG promoter (SEQ ID. NO.: 29). The TSCJ expression cassette may further include other regulatory sequences such as enhancers, polyadenylation (poly (A)) signal sequences, intron sequences, or WPRE sequences that support the appropriate CNS expression of TSC1.

[0142] In some embodiments, the TSC1 protein comprises an amino acid sequence comprising at least, or no more than, 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.: 7, and exhibits a 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 TSC1 comprises the amino acid sequence of SEQ ID NO.: 7. In some embodiments, the nucleic acid that encodes TSC1 comprises a nucleic acid having at least, or no more than, 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.: 8 or a reverse complementary sequence thereto. In some embodiments, the nucleic acid that encodes TSC1 comprises or consists of SEQ ID NO.: 8 or a reverse complementary sequence thereto. In some embodiments, the nucleic acid that encodes TSC1 comprises or consists of SEQ ID NO.: 9 or a reverse complementary sequence thereto.

[0143] 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, or no more than, 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 9Attorney Docket No.: 38213.0001P1substitutions, additions, deletions, or combinations thereof within, SEQ ID NO.: 34, SEQ ID NO.: 35, or reverse complementary sequences thereto. In some embodiments, the ITR, or at least two ITRs, comprise SEQ ID NO.: 34, SEQ ID NO.:35, 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.: 34 and the ITR at the 3 ’ end of the expression cassette has a nucleotide sequence of SEQ ID NO.: 35 (or the reverse complement of each).

[0144] In embodiments, the polynucleotide sequence that encodes TSC1 (hamartin) is operably linked to a promoter that is capable of driving expression of the TSC1 gene 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 TSC1 (hamartin) is operably linked to a promoter that is capable of driving expression of the TSCJ gene in target cells of the spinal cord, including without limitation cells of cervical, thoracic, and / or lumbar regions of the spinal cord.

[0145] In some embodiments, the TSC1 expression cassette of the AAV vector construct comprises a polynucleotide sequence that encodes TSC1 (hamartin) that is operably linked to a promoter, including a constitutive promoter. In embodiments, the promoter is a human eukaryotic translation elongation factor 1 (EFla or EFla) promoter. In other embodiments, the promoter is an EFS promoter, which is the core EFla or EFla promoter lacking the EF-la intron A sequence. In embodiments, the EFS promoter comprises a nucleotide sequence having at least, or no more than, 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.: 24, and promotes expression of TSC1 in appropriate CNS cells. In some embodiments, the EFS promoter comprises the nucleotide sequence of SEQ ID NO.: 24. Alternately, the EFS promoter is an at least 50, 100, 150, or 200 nucleotide fragment of SEQ ID NO.:24 (or a reverse complement thereof as appropriate), and promotes expression of TSC1 in appropriate CNS cells.

[0146] In other embodiments, the polynucleotide sequence that encodes TSC1 (hamartin) is operably linked to a CBA AGTl promoter. The CBA AGTl 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 [3-actin promoter and the first exon and first intron of the chicken [3-actin gene, and the splice acceptor of the rabbit p globin gene (Miyazaki et al. 1989,Attorney Docket No.: 38213.0001P1Niwa et al. 1991). In embodiments, the CBA AGTl promoter comprises a nucleotide sequence having at least, or no more than, 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.: 25, and promotes expression of TSC1 in appropriate CNS cells. In some embodiments, the CBA AGTl promoter comprises the nucleotide sequence of SEQ ID NO.: 25. Alternately, the CBA_AGT1 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.:25 (or a reverse complement thereof as appropriate), and promotes expression of TSC1 in appropriate CNS cells.

[0147] In other embodiments, the polynucleotide sequence that encodes TSC1 (hamartin) 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, or no more than, 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.: 26, and promotes expression of TSC1 in appropriate CNS cells. In some embodiments, the CBA AGT2 promoter comprises the nucleotide sequence of SEQ ID NO.: 26. 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.:26 (or a reverse complement thereof as appropriate), and promotes expression of TSC1 in appropriate CNS cells.

[0148] In other embodiments, the polynucleotide sequence that encodes TSC1 (hamartin) 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, or no more than, 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.: 27, and promotes expression ofTSCl inappropriate CNS cells. In some embodiments, the CMV-derived promoter comprises the nucleotide sequence of SEQ ID NO.: 27. Alternately, the CMV-derived promoter is an at least 100, 150, 200, 250, 300,Attorney Docket No.: 38213.0001P1350, 400, 450, 500, or 550 nucleotide fragment of SEQ ID NO.:27 (or a reverse complement thereof as appropriate), and promotes expression of TSC1 in appropriate CNS cells.

[0149] In other embodiments, the polynucleotide sequence that encodes TSC1 (hamartin) 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-globin promoter. In embodiments, the CEG promoter comprises a nucleotide sequence having at least, or no more than, 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.: 28, and promotes expression of TSC1 in appropriate CNS cells. In some embodiments, the CEG promoter comprises the nucleotide sequence of SEQ ID NO.: 28. 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.: 28 (or a reverse complement thereof as appropriate), and promotes expression of TSC1 in appropriate CNS cells.

[0150] In some embodiments the promoter is a CAG promoter. 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 P-actin gene, and the splice acceptor of the rabbit globin gene (Miyazaki et al. 1989, Niwa et al. 1991). In embodiments, the CAG promoter comprises a nucleotide sequence having at least, or no more than, 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.: 29, and promotes expression of TSC1 in appropriate CNS cells. In some embodiments, the CAG promoter comprises the nucleotide sequence of SEQ ID NO.: 29. Alternately, the CAG 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.: 29 (or a reverse complement thereof as appropriate), and promotes expression of TSC1 in appropriate CNS cells.

[0151] In other embodiments the polynucleotide sequence that encodes TSC1 (hamartin) is operably linked to a human synapsin I (hSynl) promoter, a rat neuron-specific enolase (rNSE)Attorney Docket No.: 38213.0001P1promoter, or a mouse CaMK2a promoter, or other promoter capable of driving expression of the TSC1 gene in target cells of the CNS.

[0152] In some embodiments of the AAV vector construct, the human TSC1 (hamartin) expression cassette is in an anti-sense (e.g. reverse complementary) orientation or in sense orientation. In some embodiments, the AAV vector construct comprises two or more expression cassettes.

[0153] In some embodiments of the AAV vector construct, the human TSC1 (hamartin) 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 TSC1 (hamartin), such that the expressed TSCl 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, or no more than, 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.: 32 (or a reverse complement thereof), and supports expression of TSCl in appropriate CNS cells. In some embodiments, the bGH-polyA-v3 sequence comprises the nucleotide sequence of SEQ ID NO.: 32 (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.: 32 (or a reverse complement thereof), and supports expression of TSCl in appropriate CNS cells.

[0154] 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, or no more than, 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.: 31 (or a reverse complement thereof), and supports expression of TSCl in appropriate CNS cells. In some embodiments, the bGHpA sequence comprises the nucleotide sequence of SEQ ID NO.: 31 (or a reverse complement thereof). Alternately, the bGHpA sequence is an at least 10, 15, 20, 25, 30,Attorney Docket No.: 38213.0001P135, 40, 45, or 50 nucleotide fragment of SEQ ID NO.: 31 (or a reverse complement thereof), and supports expression of TSC1 in appropriate CNS cells.

[0155] 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, or no more than, 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.: 30 (or a reverse complement thereof), and supports expression of TSC1 in appropriate CNS cells. In some embodiments, the huGHpA signal sequence comprises the nucleotide sequence of SEQ ID NO.: 30 (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.: 30 (or a reverse complement thereof), and supports expression of TSC1 in appropriate CNS cells.

[0156] In some embodiments of the AAV vector construct, the human TSC1 (hamartin) expression cassette further comprises regulatory elements that may enhance TSC1 expression. 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 TSC1 coding sequence and upstream (5’ of) the poly(A) tail signal. In some embodiments, the WPRE comprises a nucleic acid having at least, or no more than, 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.: 33 (or reverse complement thereof) that enhances TSCJ gene expression in appropriate CNS cells. In some embodiments, the WPRE comprises SEQ ID NO.: 33 (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.: 33 (or a reverse complement thereof as appropriate), and enhances TSC1 gene expression in appropriate CNS cells. In other embodiments, the human TSC1 (hamartin) expression cassette further comprises an intron or a chimeric intron sequence that promotes TSC1 expression in appropriate CNS cells, and in certain embodiments the intron sequence is inserted between the promoter and the coding sequence.Attorney Docket No.: 38213.0001P1

[0157] 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, hCA4, hCD59, or ALPL AAV capsid sequence as described herein, and including AAV capsid sequences incorporating modifications that increase immune evasion, as described herein. Provided, thus, are AAV vector constructs that can be incorporated into an AAV particle engineered to cross the blood-brain barrier for gene replacement expression of the TSCl gene in target cells of the CNS. In embodiments, the AAV vector construct may have elements arranged as follows: 5’AAV2 ITR-EFS promoter sequence-TSCl coding sequence-bGH-polyA-v3 polyadenylation signal sequence-3’ AAV2 ITR. In embodiments, such an AAV vector construct has a nucleotide sequence of SEQ ID NO.: 10 (or the reverse complement thereof). In other embodiments, the AAV vector construct may have elements arranged as follows: EFS promoter sequence-TSCl coding sequence-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, in which case in embodiments the AAV vector construct may comprise an expression cassette having the nucleotide sequence of SEQ ID NO.: 17.

[0158] In other embodiments, the AAV vector construct may have elements arranged as follows: 5’AAV2 ITR-CMV promoter sequence-TSCl coding sequence-bovine growth hormone polyadenylation signal sequence (bGHpA)-3’ AAV2 ITR. In embodiments, such an AAV vector construct has a nucleotide sequence of SEQ ID NO.: 11 (or the reverse complement thereof). In other embodiments, the AAV vector construct may have elements arranged as follows: CMV promoter sequence-TSCl coding sequence-bovine growth hormone polyadenylation signal sequence (bGHpA), 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, in which case in embodiments the AAV vector construct may comprise an expression cassette having the nucleotide sequence of SEQ ID NO.: 18.

[0159] In other embodiments, the AAV vector construct may have elements arranged as follows: 5’AAV2 ITR-EFS promoter sequence-TSCl coding sequence-human growth hormone polyadenylation signal sequence (huGHpA)-3’ AAV2 ITR. In embodiments, such an AAV vector construct has a nucleotide sequence of SEQ ID NO.: 12 (or the reverse complement thereof). InAttorney Docket No.: 38213.0001P1other embodiments, the AAV vector construct may have elements arranged as follows: EFS promoter sequence-TSCl coding sequence-human growth hormone polyadenylation signal (huGHpA) polyA 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, in which case in embodiments the AAV vector construct may comprise an expression cassette having the nucleotide sequence of SEQ ID NO.: 19.

[0160] In other embodiments, the AAV vector construct may have elements arranged as follows: 5’AAV2 ITR-CMV promoter sequence-TSCl coding sequence-human growth hormone polyadenylation signal sequence (huGHpA)-3’ AAV2 ITR. In embodiments, such an AAV vector construct has a nucleotide sequence of SEQ ID NO.: 13 (or the reverse complement thereof). In other embodiments, the AAV vector construct may have elements arranged as follows: CMV promoter sequence-TSCl coding sequence-human growth hormone polyadenylation signal (huGHpA) polyA 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, in which case in embodiments the AAV vector construct may comprise an expression cassette having the nucleotide sequence of SEQ ID NO.: 20.

[0161] In other embodiments, the AAV vector construct may have elements arranged as follows: 5’AAV2 ITR-CMV promoter sequence-TSCl coding sequence-bGH-polyA-v3 polyadenylation signal sequence-3’ AAV2 ITR. In embodiments, such an AAV vector construct has a nucleotide sequence of SEQ ID NO.: 14 (or the reverse complement thereof). In other embodiments, the AAV vector construct may have elements arranged as follows: CMV promoter sequence-TSCl coding sequence-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, in which case in embodiments the AAV vector construct may comprise an expression cassette having the nucleotide sequence of SEQ ID NO.: 21.

[0162] In other embodiments, the AAV vector construct may have elements arranged as follows: 5’AAV2 ITR-CBA AGT2 promoter sequence-TSCl coding sequence-bGH-polyA-v3 polyA signal sequence-3’ AAV2 ITR. In embodiments, such an AAV vector construct has a nucleotide sequence of SEQ ID NO.: 15 (or the reverse complement thereof). In other embodiments, the AAV vector construct may have elements arranged as follows: CBA AGT2 promoter sequence-TSClAttorney Docket No.: 38213.0001P1coding sequence-bGH-polyA-v3 polyA 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, in which case in embodiments the AAV vector construct may comprise an expression cassette having the nucleotide sequence of SEQ ID NO.: 22.

[0163] In other embodiments, the AAV vector construct may have elements arranged as follows: 5’AAV2 ITR-CBA AGTl promoter sequence-TSCl coding sequence-bGH-polyA-v3 polyA signal sequence-3’ AAV2 ITR. In embodiments, the AAV vector construct has a nucleotide sequence of SEQ ID NO.: 16 (or the reverse complement thereof). In other embodiments, the AAV vector construct may have elements arranged as follows: CBA AGTl promoter sequence-TSCl coding sequence-bGH-polyA-v3 polyA 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, in which case in embodiments the AAV vector construct may comprise an expression cassette having the nucleotide sequence of SEQ ID NO.: 23.

[0164] In some embodiments, the AAV vector construct comprises a nucleic acid having at least, or no more than, 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.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, SEQ ID NO.: 16, or a reverse complementary sequence thereto, and is an AAV vector construct that expresses TSC1 in appropriate cells, such as cells of the CNS.

[0165] In some embodiments, the expression cassette of the AAV vector construct comprises a nucleic acid having at least, or no more than, 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.: 17, SEQ ID NO.: 18, SEQ ID NO.: 19, SEQ ID NO.: 20, SEQ ID NO.: 21, SEQ ID NO.: 22, or SEQ ID NO.: 23, or a reverse complementary sequence thereto, and is an expression cassette that expresses TSC1 in appropriate cells, such as cells of the CNS.Attorney Docket No.: 38213.0001P1AAV VECTOR PLASMIDS

[0166] In some embodiments, an AAV vector plasmid is provided that is used to prepare a recombinant AAV viral particle having a recombinant genome comprising an AAV vector construct having an expression cassette comprising a nucleotide sequence encoding the TSC1 gene operably linked to regulatory elements that promote expression in appropriate cells, such as 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 hTfRl 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. Exemplary AAV vector plasmid sequences are disclosed herein as SEQ ID NO.: 39, SEQ ID NO.: 40, SEQ ID NO.: 41, SEQ ID NO.: 42, SEQ ID NO.: 43, SEQ ID NO.: 44, SEQ ID NO.: 45, and SEQ ID NO.: 46. The plasmids provided herein include plasmids comprising the AAV vector constructs and expression cassettes described herein.

[0167] The AAV vector plasmids may comprise an expression cassette having a nucleotide sequence of SEQ ID NO.: 17, SEQ ID NO.: 18, SEQ ID NO.: 19, SEQ ID NO: 20, SEQ ID NO.: 21, SEQ ID NO.: 22, or SEQ ID NO.: 23, or a reverse complementary sequence thereto. In certain embodiments, the AAV vector plasmid comprises a nucleic acid having at least, or no more than, 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.: 17, SEQ ID NO.: 18, SEQ ID NO.: 19, SEQ ID NO.: 20, SEQ ID NO.: 21, SEQ ID NO.: 22, or SEQ ID NO.: 23, or a reverse complementary sequence thereto, and is an expression cassette that expresses TSC1 in appropriate cells, such as cells of the CNS.

[0168] AAV vector plasmids may also comprise AAV vector constructs comprising the expression cassettes described above, including but not limited to AAV vector constructs having a nucleotide sequence of SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, or SEQ ID NO.: 16, or a reverse complementary sequence thereto. In certain embodiments, the AAV vector plasmid comprises a nucleic acid having at least, or no more than, 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 IDAttorney Docket No.: 38213.0001P1NO.: 10, SEQ IDNO.: 11, SEQ IDNO.: 12, SEQ IDNO.: 13, SEQ IDNO.: 14, SEQ IDNO.: 15, or SEQ ID NO.: 16, or a reverse complementary sequence thereto, and is an AAV vector construct that expresses TSC1 in appropriate cells, such as cells of the CNS.

[0169] 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 bacterial 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.

[0170] In embodiments, the AAV vector plasmid comprises one of the following sequences: SEQ ID NO.: 39 (pITR-EFS-TSCl-bGHv3pA), SEQ ID NO.: 40 (pITR-CMV-TSCl-bGHpA), SEQ ID NO.: 41 (pITR-EFS-TSCl-huGHpA), SEQ ID NO.: 42 (pITR-CMV-TSCl-huGHpA), SEQ ID NO.: 43 (pITR-CMV-TSCl-bGH_v3pA), SEQ ID NO.: 44 (pITR-CBA_AGT2-TSCl-bGH_v3pA), SEQ ID NO.: 45 (pITR-CBA_AGTl-TSCl-bGH_v3pA), and SEQ ID NO.: 46 (pITR-CMV-TSC 1 -bGHpA).RECOMBINANT AAV PARTICLES

[0171] In some embodiments, the rAAV particle comprises an AAV capsid protein and a recombinant AAV (rAAV) expression cassette that comprises the nucleotide sequence encoding TSC1 operably linked to regulatory elements (rAAV-TSCl particle). In some embodiments, the rAAV expression cassette is a single-stranded DNA. In some embodiments, the rAAV-TSCl particle encapsulates the rAAV expression cassette. In some embodiments, the rAAV-TSCl particle comprises a VP1, a VP2, and a VP3 capsid protein.

[0172] In embodiments, the rAAV-TSCl particle is engineered to cross the blood-brain barrier (BBB) and transduce cells of the CNS through binding to the hTfRl receptor (hTfRl rAAV particle), including to have greater activity to cross the BBB and / or transduce CNS cells than aAttorney Docket No.: 38213.0001P1reference capsid, such as AAV9. In embodiments, the hTfRl rAAV-TSCl particle comprises a capsid protein that is engineered to bind the hTfRl receptor (hTfRl rAAV capsid protein), and further comprises a recombinant AAV genome encoding TSC1, 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.

[0173] In embodiments, the rAAV-TSCl particle is engineered to bind human GPI-linked enzyme Carbonic anhydrase IV (hCA4) (hCA4 rAAV particle). In embodiments, the hCA4-TSCl particle comprises a capsid protein that is engineered to bind hCA4 (hCA4 rAAV capsid protein), and further comprises a recombinant AAV genome encoding TSC1, 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.

[0174] In embodiments, the rAAV-TSCl particle is engineered to bind the human CD59 cell surface protein (hCD59) (hCD59 rAAV particle). In embodiments, the hCD59 rAAV particle comprises a capsid protein that is engineered to bind hCD59 (hCD59 rAAV capsid protein), and further comprises a recombinant AAV genome encoding TSC1, 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.

[0175] In embodiments, the rAAV-TSCl particle is engineered to bind human GPI-linked alkaline phosphatase (ALPL) (ALPL rAAV particle). In embodiments, the ALPL rAAV particle comprises a capsid protein that is engineered to bind ALPL (ALPL rAAV capsid protein), and further comprises a recombinant AAV genome encoding TSC1, 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.

[0176] In embodiments, the rAAV-TSCl particle is 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 by neutralizing antibodies, suchAttorney Docket No.: 38213.0001P1as those directed against AAV, and reduce the incidence of immune response in treated subjects, and comprises a recombinant AAV genome encoding TSC1, operably linked to regulatory elements. The immune evading modifications are, in embodiments, 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.

[0177] In some embodiments, the rAAV-TSCl particle comprises an AAV1 capsid protein, an AAV2 capsid protein, an AAV3 capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV-PHP. B capsid protein, an AAV8-PHP.eB capsid protein, or an AAV-PHP. S capsid protein.hTfRl rAAV particles

[0178] 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 capsid polypeptide (i.e., SEQ ID NO.: 1 or SEQ ID NO.: 4, 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 Tfrl, 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.: 49), YSRNSDN (SEQ ID NO.: 50), LHRLGPN (SEQ ID NO: 51), LHRLGPD (SEQ ID NO.: 52), LHRAGPD (SEQ ID NO.: 53), YSRIGPD (SEQ ID NO.: 54), LSRIGPD (SEQ ID NO.: 55), LARSGPD (SEQ ID NO.: 56), LHKAGPN (SEQ ID NO.: 57), LSRIGPN (SEQ ID NO.: 58), LAKSGPN (SEQ ID NO.: 59), and YARNGPN (SEQ ID NO.: 60). In an example embodiment, then-mer motif is YSRIGPN (SEQ ID NO.: 49).

[0179] In embodiments, the hTfRl rAAV particle comprises an hTfRl rAAV capsid protein including a transferrin receptor (TfRl) binding modification sequence defined by the formula XI -X2-X3-[7-mer]-X4-X5-X6-X7, wherein the 7-mer is one of the amino acid sequences YSRIGPN (SEQ ID NO.: 49), YSRNSDN (SEQ ID NO.: 50), LHRLGPN (SEQ ID NO.: 51), FRSTNGVAttorney Docket No.: 38213.0001P1(SEQ ID NO: 61), FVSTNGV (SEQ ID NO: 62), FZ1STNGZ2 (SEQ ID NO.: 63), FRSTNGZ3 (SEQ ID NO.: 64), or VESTNGR (SEQ ID NO.: 65), and wherein the 7-mer is inserted between amino acids 588 and 589 of an AAV9 or AAV9 K449R capsid polypeptide (e.g., SEQ ID NO.: 1 or SEQ ID NO.: 4), 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 Zi, Z2, and Z3 indicate variant amino acid positions within the 7-mer polypeptide wherein Zi 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.: 49), YSRNSDN (SEQ ID NO.: 50), or LHRLGPN (SEQ ID NO.: 51), 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.: 49), 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.: 50) 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.: 51) 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.: 61), FVSTNGV (SEQ ID NO.: 62), FZ1STNGZ2 (SEQ ID NO.: 63), FRSTNGZ3 (SEQ ID NO.: 64), or VESTNGR (SEQ ID NO.: 65) 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.: 61), 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.: 62), 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 isAttorney Docket No.: 38213.0001P1E; or any combination thereof. In an embodiment, the 7-mer is FZ1STNGZ2 (SEQ ID NO.: 63) or FRSTNGZ3 (SEQ ID NO.: 64), 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.: 65), 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, Zi 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.

[0180] In some embodiments, the hTfR1 AAV particle comprises an hTfRl rAAV capsid protein having an amino acid sequence of SEQ ID NO.: 2, 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.: 49 (YSRIGPN) between wild-type AAV9 VP1 amino acid residues 588 and 589. In some embodiments, the hTfR1 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.: 1), and further includes a 7-amino acid insertion comprising SEQ ID NO.: 49 (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.: 1), and further includes a 7-amino acid insertion comprising SEQ ID NO.: 49 (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 BBB and / or transduction of CNS cells relative to rAAV particles having a capsid protein without the modifications.

[0181] In some embodiments, the hTfRl AAV particle comprises an hTfRl rAAV capsid protein having an amino acid sequence of SEQ ID NO.: 3, 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.: 49 (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 residueAttorney Docket No.: 38213.0001P1at 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.: 1), with a 7-amino acid insertion comprising SEQ ID NO.: 49 (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.: 1), with a 7-amino acid insertion comprising SEQ ID NO.: 49 (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.

[0182] In some embodiments, the hTfRl AAV particle comprises an hTfRl rAAV capsid protein having an amino acid sequence of SEQ ID NO.: 47, 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.: 61 (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 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.: 1), with a 7-amino acid insertion comprising SEQ ID NO.: 61 (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 (SEQAttorney Docket No.: 38213.0001P1ID NO.: 1), with a 7-amino acid insertion comprising SEQ ID NO.: 61 (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-type AAV9 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.

[0183] In some embodiments, the hTfRl AAV particle comprises an hTfRl rAAV capsid protein having an amino acid sequence of SEQ ID NO.: 5, which is an engineered variant of the AAV9 K449R VP1 capsid protein having an amino acid sequence of SEQ ID NO.: 4 that includes a 7-amino acid insertion comprising SEQ ID NO.: 49 (YSRIGPN) between AAV9 K449R VP1 amino acid residues 588 and 589. hTfRl AAV particles comprising a VP1 amino acid sequence of SEQ ID NO.: 5 are referred to herein as “AAV hTfRlv.l 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.: 4), and further includes a 7-amino acid insertion comprising SEQ ID NO.: 49 (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.: 4), and further includes a 7-amino acid insertion comprising SEQ ID NO.: 49 (YSRIGPN) inserted at a position in loop IV and / or loop VIII 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.

[0184] In some embodiments, the hTfRl AAV particle comprises an hTfRl rAAV capsid protein having an amino acid sequence of SEQ ID NO.: 6, which is an engineered variant of the AAV9 K449R VP1 capsid protein having an amino acid sequence of SEQ ID NO.: 4, with a 7-amino acid insertion comprising SEQ ID NO.: 49 (YSRIGPN) between AAV9 K449R VP1 amino acid residues 588 and 589, and further wherein the AAV9 K449R VP1 amino acid residue atAttorney Docket No.: 38213.0001P1position 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.: 6 are referred to herein as “AAV hTfRl v.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.: 4), with a 7-amino acid insertion comprising SEQ ID NO.: 49 (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.: 4), with a 7-amino acid insertion comprising SEQ ID NO.: 49 (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 K449 VP1 amino acid residue at the third upstream amino acid position preceding the 7-amino acid insertion is an E, and the AAV9 K449 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.

[0185] In some embodiments, the hTfRl AAV particle comprises an hTfRl rAAV capsid protein having an amino acid sequence of SEQ ID NO.: 48, which is an engineered variant of the AAV9 K449R VP1 capsid protein having an amino acid sequence of SEQ ID NO.: 4, with a 7-amino acid insertion comprising SEQ ID NO.: 61 (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.: 4), with a 7-amino acid insertion comprising SEQ ID NO.: 61 (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), andAttorney Docket No.: 38213.0001P1the 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 VP 1 amino acid sequence (SEQ ID NO.: 4), with a 7-amino acid insertion comprising SEQ ID NO.: 61 (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 K449 VP1 amino acid residue at the first upstream amino acid position preceding the 7-amino acid insertion is a D, and the AAV9 K449 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

[0186] 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.: 1 or SEQ ID NO.: 4, 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.: 66), VQRLSVL (SEQ ID NO.: 67), KVSNPVW (SEQ ID NO.: 68), RPVQVMA (SEQ ID NO.: 69).

[0187] In embodiments, the hCA4 amino acid binding modification sequence defined by the formula Z1-Z2- [n-mer], [n-mer]-Z3, or Z1-Z2- [n-mer] -Z3, wherein the n-mer is inserted between amino acids 588 and 589 of an AAV9 or AAV9 K449R capsid polypeptide (e.g., SEQ ID NO.: 1 or SEQ ID NO.: 4), 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 bindingAttorney Docket No.: 38213.0001P1modification 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.: 70). 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.: 71). In embodiments of the binding modification formula described above, the n-mer is KVSNPVW, Z1 is S, and Z2 is N, such that the hCA4 amino acid binding modification sequence is SNKVSNPVW (SEQ ID NO.: 72). 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.

[0188] 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.: 1), 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.: 66), VQRLSVL (SEQ ID NO.: 67), KVSNPVW (SEQ ID NO.: 68), RPVQVMA (SEQ ID NO.: 69). 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.: 1), and includes a 7 amino acid peptide LYDGRSG (SEQ ID NO.: 66) 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 wild-type AAV9 VP1 amino acid sequence (SEQ ID NO.: 1), and further includes a 7 amino acid peptide RPVQVMA (SEQ ID NO.: 69) 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.: 1), and further includes a 7 amino acid peptide KVSNPVW (SEQ ID NO.: 68) inserted between amino acids 588 and 589, and the amino acid at position 587 is changed fromAttorney Docket No.: 38213.0001P1A to S (A587S), and the amino acid at position 588 is changed from Q to N (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.

[0189] 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.: 1), 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.: 66), VQRLSVL (SEQ ID NO.: 67), KVSNPVW (SEQ ID NO.: 68), RPVQVMA (SEQ ID NO.: 69). 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.

[0190] 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.: 4), 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.: 66), VQRLSVL (SEQ ID NO.: 67), KVSNPVW (SEQ ID NO.: 68), RPVQVMA (SEQ ID NO.: 69). 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.: 4), and includes a 7 amino acid peptide LYDGRSG (SEQ ID NO.: 66) 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.: 4), and further includes a 7 amino acid peptide RPVQVMA (SEQ ID NO.: 69) 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.: 4),Attorney Docket No.: 38213.0001P1and further includes a 7 amino acid peptide KVSNPVW (SEQ ID NO.: 68) 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 to N (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.

[0191] 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.: 4), 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.: 66), VQRLSVL (SEQ ID NO.: 67), KVSNPVW (SEQ ID NO.: 68), RPVQVMA (SEQ ID NO.: 69). 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

[0192] 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 capsid polypeptide (i.e.., SEQ ID NO.: 1 or SEQ ID NO.: 4, 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.: 73) or GAASLMP (SEQ ID NO.: 74). 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 capsid polypeptide (e.g., SEQ ID NO.: 1 or SEQ ID No.: 4), 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.: 73) or GAASLMP (SEQ ID NO.: 74). rAAV particlesAttorney Docket No.: 38213.0001P1incorporating 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.

[0193] 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.: 1), 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.: 73) or GAASLMP (SEQ ID NO.: 74). 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.: 1), 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.: 73) or GAASLMP (SEQ ID NO.: 74). 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.: 1), 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.: 73) or GAASLMP (SEQ ID NO.: 74). 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.

[0194] 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.: 4), 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.: 73) or GAASLMP (SEQ ID NO.: 74). 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.: 4), wherein K449R AAV9 VP1 amino acid residues 450-461 are removed andAttorney Docket No.: 38213.0001P1replaced with a 7-mer amino acid sequence selected from EFNNGSD (SEQ ID NO.: 73) or GAASLMP (SEQ ID NO.: 74). 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.: 4), 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.: 73) or GAASLMP (SEQ ID NO.: 74). 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

[0195] In embodiments, the ALPL rAAV particle comprises the capsid of an AAV9 or AAV9 K449R capsid polypeptide (i.e., SEQ ID NO.: 1 or SEQ ID NO.: 4, 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.

[0196] 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.

[0197] 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.: 1), and further includes a 6-mer amino acid insertion between wild-type AAV9 VP1 amino acid residuesAttorney Docket No.: 38213.0001P1455 and 456, with said 6-mer amino acid sequence consisting of SPHSKA (SEQ ID NO.: 75). 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.: 1), 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.: 76). 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.

[0198] 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.: 4), 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.: 75). 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.: 4), 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.: 76). 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.rAAV (AE) particles

[0199] 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 VP1 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, R550Q, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 or AAV9 K449R capsid polypeptide (e.g., SEQ ID NO.: 1 or SEQ ID NO.: 4), or in an analogous position of a VP1 capsid protein of another AAVAttorney Docket No.: 38213.0001P1serotype, and exhibits evasion of anti-AAV antibodies and immune response relative to the AAV particle without these capsid mutations.

[0200] 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 ALPL capsid 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.

[0201] In embodiments, the immune evasion rAAV particle comprises an engineered AAV VP 1 capsid protein, including an AAV9, AAV9 K449, hTfRl, 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 capsidAttorney Docket No.: 38213.0001P1protein, wherein the additional amino acid substitutions include N328K and N329D 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 K462E / Q 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 capsid protein, wherein the additional amino acid substitution includes 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 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.

[0202] 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, R550Q, 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, R550Q, 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 capsidAttorney Docket No.: 38213.0001P1mutations. 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 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 G455Q / T / K / N, D551N, N552D, K557E / Q, K664E / Q, D665N, and N668K.

[0203] 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 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.

[0204] 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 G455Q, D551N,Attorney Docket No.: 38213.0001P1N552D, 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 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 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.

[0205] 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 kDa and about 62 kDa. 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.Attorney Docket No.: 38213.0001P1

[0206] 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.

[0207] 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 construct any 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.)

[0208] 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 hTfRlv.l 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.: 36, SEQ ID NO.: 37, or SEQ ID NO.: 38. 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).Attorney Docket No.: 38213.0001P1

[0209] 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, AAVDNA 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

[0210] 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) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, and Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier. 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.

[0211] 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 capsid comprises a VP1 protein that has an amino acid sequence of SEQ ID NO.: 2 or SEQ ID NO.: 5 (i.e., AAV hTfRlv.l particles), or SEQ ID NO.: 3 or SEQ ID NO.: 6 (i.e., AAV hTfRlv.2 particles), as described herein. In some embodiments, the hTfR.1 AAV packaging plasmid comprises the nucleotide sequence of SEQ ID NO.: 36, SEQ ID NO.: 37, or SEQ ID NO.: 38. In some embodiments, the cap is AAV9 cap.Attorney Docket No.: 38213.0001P1

[0212] In some embodiments, the AAV vector plasmid comprises the AAV vector construct having a nucleotide sequence of SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, or SEQ ID NO.: 16, or a reverse complementary sequence thereto. In other embodiments, the AAV vector plasmid comprises an expression cassette having a nucleotide sequence of SEQ ID NO.: 17, SEQ ID NO.: 18, SEQ ID NO.: 19, SEQ ID NO.: 20, SEQ ID NO.: 21, SEQ ID NO.: 22, or SEQ ID NO.: 23, or a reverse complementary sequence thereto.

[0213] 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.: 36, SEQ ID NO.: 37, or SEQ ID NO.: 38. In some embodiments, the method comprises transfecting a cell with an AAV vector construct plasmid comprising the AAV vector construct of SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, or SEQ ID NO.: 16, 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 of SEQ ID NO.: 17, SEQ ID NO.: 18, SEQ ID NO.: 19, SEQ ID NO.: 20, SEQ ID NO.: 21, SEQ ID NO.: 22, or SEQ ID NO.: 23, or a reverse complementary sequence thereto.

[0214] In some embodiments, 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 embodiments, RNA from the transfected cells can provide a template for RT-PCR amplification of cDNA and theAttorney Docket No.: 38213.0001P1detection 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 embodiments, 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, epi somes, 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.

[0215] 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.

[0216] 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.Attorney Docket No.: 38213.0001P1METHODS OF TREATMENT

[0217] Methods of treating TSC by administering rAAV-mediated TSC1 gene therapy are provided. In embodiments and as described herein, the TSC1 gene therapy utilizes rAAV particles comprising capsids engineered to cross the blood-brain barrier (BBB) and transduce cells of 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 embodiments, the rAAV particles (for example, the AAVhTfRlv.1 or AAVhTfRlv.2 particles described herein) comprise capsids engineered to bind the hTfRl receptor, and further comprise an AAV vector construct encoding TSC1, operably linked to regulatory elements. In other embodiments, the TSC1 gene therapy utilizes rAAV particles comprising AAV9, AAV9 K449, hCA4, hCD59, or ALPL VP1 capsid protein, or rAAV(AE) capsid protein, as described herein.

[0218] In embodiments, upon transduction, the AAV vector construct will be released from encapsulation from the capsid protein. In some embodiments, the AAV vector construct 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 AAV vector construct. In some embodiments, the cell will express the nucleic acid encoding the protein. In some embodiments, the cell will express the protein. In some embodiments, the cell will express the nucleic acid encoding TSC1. In some embodiments, the cell, including a CNS cell, will express the TSC1 protein. In some embodiments, TSC1 will undergo its enzymatic activity in the target cell (i.e. by suppressing the function of mTOR by inhibiting the Ras homologue enriched in brain (Rheb) protein).

[0219] In embodiments, a method for treating TSC or related disorders is provided. In some embodiments, a method of regulating mTOR signaling activity in a subject, including in the CNS of the 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, neurodevelopmental 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 administering to the subject a therapeutically effective amount of one or more of the AAV particles described herein. InAttorney Docket No.: 38213.0001P1embodiments, the method comprises administering to the subject a therapeutically effective amount of a recombinant hTfRl AAV particle described herein that provides for expression of TSC1 in transduced cells as gene therapy for TSC1. In some embodiments, a method of reducing p70S6K levels by administration of a therapeutically effective amount of one or more of the recombinant hTfRl AAV particles described herein as gene therapy for a subject in need thereof is provided.

[0220] In some embodiments, a subject treated with gene therapy by administering to the subject a therapeutically effective amount of a recombinant hTfRl AAV particle of the present disclosure (for example, the rAAVhTfRlv.l or rAAVhTfRlv.2 particles described herein) engineered to express TSC1 according to the present methods has TSC. In some embodiments, a treated subject does not express TSC1 protein or expresses undetectable levels of TSC1 protein. In some embodiments, a treated subject expresses a modified or mutant form of TSC1. In some embodiments, a treated subject suffers from autosomal dominant TSC due to modifications or mutations in TSC1. In some embodiments, a treated subject suffers from TSC caused by TSC1 haploinsufficiency. In some embodiments, a treated subject lacks TSC1 activity or has undetectable levels of TSC1 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 TSC 1 protein expression compared to a subject that does not have TSC. In some embodiments, a treated subject has reduced levels of TSC 1 activity compared to a subject that does not have TSC.

[0221] In embodiments, a subject treated with gene therapy by administering to the subject a therapeutically effective amount of a recombinant hTfRl AAV particle of the present disclosure (for example, the rAAVhTfRlv.l or rAAVhTfRlv.2 particles described herein) engineered to express TSC1 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 is refractory to or does not tolerate, due to adverse effects, a drug approved by the FDA for treatment of TSC due to side effects of the drug treatment. In embodiments, treatmentAttorney Docket No.: 38213.0001P1with a drug approved by the FDA for treatment of TSC is contraindicated or otherwise not appropriate for administration to a treated subject.

[0222] In embodiments, a subject treated with gene therapy by administering to the subject a therapeutically effective amount of a recombinant hTfRl AAV particle of the present disclosure (for example, the rAAVhTfR.lv.1 or rAAVhTfRlv.2 particles described herein) engineered to express TSC1 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 is refractory to or does not tolerate, due to adverse effects, 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.

[0223] In some embodiments, a method of treating dysregulated mTOR signaling activity in a subject in need thereof with gene therapy by administering to the subject a therapeutically effective amount of a recombinant hTfRl AAV particle of the present disclosure (for example, the rAAVhTfR.lv.1 or rAAVhTfR.lv.2 particles described herein) engineered to express TSC1 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 TSC1 (or TSC2) 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 reducing MTOR-dependent phosphorylation of p70S6K in a subject in need is provided.

[0224] In embodiments, gene therapy by administering to the subject a therapeutically effective amount of a recombinant hTfRl AAV particle of the present disclosure (for example, theAttorney Docket No.: 38213.0001P1rAAVhTfR.lv.1 or rAAVhTfR.lv.2 particles described herein) engineered to express TSC1 according to the present methods leads to increased expression of TSC1 in transduced cells. In some embodiments, TSC1 protein expression is increased in cells or tissues of the CNS as compared to the TSC1 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, TSC1 protein expression is increased in brain tissues. In some embodiments, TSC1 protein expression is increased in cortex and / or cerebral tissues. In some embodiments, TSC1 protein expression is increased in neuronal cells and / or glial cells. In some embodiments, TSC1 protein expression is increased in Purkinje neurons. In some embodiments, TSC1 protein expression is increased in spinal cord tissues. In all cases, the increase is relative to the TSC1 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 TSC1 expression makes a therapeutic difference to the patient by ameliorating one or more symptoms of TSC.

[0225] In embodiments, gene therapy by administering to the subject a therapeutically effective amount of a recombinant hTfR.1 AAV particle of the present disclosure (for example, the rAAVhTfR.lv.1 or rAAVhTIRlv.2 particles described herein) engineered to express TSC1 according to the present methods leads to increased TSC2 enzyme activity via increased TSC1 activity, which leads to TSC 1 -mediated TSC2 stabilization in transduced cells. In some embodiments, TSC1 activity is increased in cells or tissues of the CNS. In some embodiments, TSC1 activity is increased in brain tissues. In some embodiments, TSC1 activity is increased in cortex and / or cerebral tissues. In some embodiments, TSC1 activity is increased in neuronal cells and / or glial cells. In some embodiments, TSC1 activity is increased in Purkinje neurons. In some embodiments, TSC1 activity is increased in spinal cord tissues. In all cases, the increase is relative to the TSC1 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, TSC1 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 TSC1 protein deficiency. In some embodiments, TSC1 activity isAttorney Docket No.: 38213.0001P1increased 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 TSC1 protein deficiency.

[0226] In embodiments, gene therapy by administering a therapeutically effective amount of a recombinant hTfRl AAV particle of the present disclosure (for example, the rAAVhTfR.lv.1 or rAAVhTfR.lv.2 particles described herein) engineered to express TSC1 to a subject in need thereof according to the present methods 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 patient / caregiver seizure diaries, electroencephalogram (EEG) monitoring, or magnetic resonance imaging (MRI).

[0227] In some embodiments, a method of alleviating the severity of neurodevelopmental and / or cognitive disorders (for example, autism spectrum disorders, intellectual disability, attention deficit-hyperactivity) through gene therapy comprising administering to the subject a therapeutically effective amount of a recombinant hTfR.1 AAV particle of the present disclosure (for example, the rAAVhTfR.lv.1 or rAAVhTfR.lv.2 particles described herein) engineered to express TSC1 according to the present methods is provided in which the reduction in severity of a neurodevelopmental and / or cognitive disorder (for example, autism spectrum disorders, intellectual disability, attention deficit-hyperactivity) is 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 an assisted mainstreamAttorney Docket No.: 38213.0001P1schooling 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.)

[0228] In embodiments, administering to the subject a therapeutically effective amount of a recombinant hTfRl AAV particle of the present disclosure (for example, the rAAVhTfR.lv.1 or rAAVhTfR.lv.2 particles described herein) engineered to express TSC1 for gene therapy leads to a reduction of abnormal cell growth and / or proliferation in a subject suffering from TSC. Examples of proliferative disorders associated with TSC1 include Renal Angiomyolipoma and Lymphangioleiomyomatosis (LAM). Reduction of abnormal cell growth and / or proliferation may be assessed according to methods known in the art.

[0229] In some embodiments, a recombinant hTfRl AAV particle of the present disclosure (for example, the rAAVhTfRlv.1 or rAAVhTfRlv.2 particles described herein) engineered to express TSC1 for gene therapy is administered prior to the onset of puberty. In some embodiments, a recombinant hTfRl AAV particle of the present disclosure is administered to a young pediatric subject. 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.

[0230] In some embodiments, the TSC1 gene therapy methods described herein further comprise 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.

[0231] In some embodiments, the treated subject has a TSC1 gene that comprises a mutation of at least one of 328-2 A^G, 747delTA, 903C^T, 1250+1 G^A, 1331insA, 1719C^T, 1801delAG, 2105delAAAG, 2122delAC, 2323delAGTT, 2324dupGTTACTC, 2365delG, R786X, 2632dell4bp, 2871insT, 2887insA, or 2895delAG. In some embodiments, the subject has a TSC1 protein that comprises a mutation of at least one of: S91X, W103X, S334X, R509X, A726E, W750X, or S836X (Jones et al. 1997, Jones et al. (1999). In some embodiments, the treatedAttorney Docket No.: 38213.0001P1subject has a TSC1 gene that comprises a mutation as described in scientific publications such as Martin et al. (2017), Nellist et al. (2009), or Sanack et al. (2005).

[0232] In some embodiments, the subject has a TSC1 -related mutation that is identified on the Leiden Open Variation Database (https: / / databases.lovd.nl / shared / genes / TSCl). In some embodiments, the subject has a TSC1 and / or TSC1 mutation that has not yet been identified, described, or cataloged.

[0233] In some embodiments, administration of or treatment with a recombinant hTfRl AAV particle of the present disclosure (for example, the rAAVhTfR.lv.1 or rAAVhTfR.lv.2 particles described herein) engineered to express TSC1 for gene therapy 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, intra-ci sterna magna (ICM) administration, or administration by inhalation. In some embodiments, the TSC1 gene therapy comprises contacting the AAV particle, 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.

[0234] In some embodiments, the administration, treating, contacting, or effective amount comprises at least, or no more than, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, or 1015viral genomes (vg) / mL, or total viral particles of a recombinant hTfR.1 AAV particle of the present disclosure (for example, the rAAVhTfR.lv.1 or rAAVhTfR.lv.2 particles described herein) engineered to express TSC1 for gene therapy. In some embodiments, the administration, treating, contacting, or effective amount comprises at least, or no more than, 109, 1010, 1011, 1012, 1013, 1014, or 1015viral genomes (vg) / kg of the treated subject’ s body weight.

[0235] 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 followingAttorney Docket No.: 38213.0001P1is 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 repaired target polynucleotide in one or more cells, tissues, or organs isolated from the subject. In some embodiments, success of treatment is determined by detecting the presence of polypeptide encoded by the repaired target polynucleotide in one or more cells, tissues, or organs isolated from the subject.

[0236] In some embodiments, a recombinant hTfRl AAV particle of the present disclosure (for example, the rAAVhTfR.lv.1 or rAAVhTfR.lv.2 particles described herein) engineered to express TSC1 can be administered for gene therapy according to the present methods in a composition at a concentration of at least, or no more than, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% weight by weight, weight by volume.

[0237] In some embodiments, the composition 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.

[0238] In embodiments, a recombinant AAV particle of the present disclosure (for example, the rAAVhTfR.lv.1 or rAAVhTfR.lv.2 particles described herein) engineered to express TSC1 for gene therapy according to the present methods is administered in a pharmaceutical composition comprising 200 mM NaCl, 20 mM Tris, 1 mM MgC12, 0.005% Pluronic F-68, pH 8. In embodiments, a recombinant AAV particle of the present disclosure is administered in aAttorney Docket No.: 38213.0001P1pharmaceutical composition comprising 20 mM Tris (pH 8.0), 1 mM magnesium chloride (MgC12), 200 mM sodium chloride (NaCl) and 0.005% poloxamer 188. In embodiments, a recombinant AAV particle of the present disclosure is administered in a pharmaceutical composition comprising 180 mM sodium chloride, 10 mM sodium phosphate, and 0.001% Poloxamer 188 (pH 7.3). In embodiments, a recombinant AAV particle of the present disclosure is administered in a pharmaceutical composition comprising mannitol (20 mg / mL), poloxamer 188 (2.0 mg / mL), sodium chloride (8.2 mg / mL), sodium phosphate monobasic dihydrate (0.23 mg / mL), sodium phosphate dibasic dodecahydrate (3.05 mg / mL) and Water for Injection, USP (pH 6.9-7.8). In embodiments, a recombinant AAV particle of the present disclosure is administered in a pharmaceutical composition comprising potassium chloride (3 mM), sodium chloride (337 mM), potassium dihydrogen phosphate (2 mM), disodium hydrogen phosphate (8 mM), and poloxamer 188 (0.001%). In embodiments, a recombinant AAV particle of the present disclosure is administered in a pharmaceutical composition comprising sucrose (50 mg / mL), polysorbate-20 (0.22 mg / mL), potassium chloride (0.2 mg / mL), potassium phosphate (0.2 mg / mL), sodium chloride (8 mg / mL), and sodium phosphate (1.2 mg / mL). In embodiments, a recombinant AAV particle of the present disclosure is administered in a pharmaceutical composition comprising other appropriate pharmaceutically acceptable buffers or excipients. The formulation may be frozen until ready for use and then thawed and administered. In some embodiments, the compositions disclosed herein can comprise an rAAV particle alone, or in combination with one or more other viruses (e.g., a second rAAV particle comprising polynucleotide sequences encoding 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 rAAV particles, each having one or more different transgenes.

[0239] Recombinant AAV particles (including hTfRl AAV particles such as the rAAVhTfRlv. l or rAAVhTfRlv.2 particles described herein) can be administered in sufficient amounts to transfect the cells of a desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse effects. In some embodiments, acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., intracranial injection for delivery to the CNS), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parental routes of administration. In some embodiments, the route of administration can be byAttorney Docket No.: 38213.0001P1intracerebroventricular injection. In some embodiments, the route of administration can be by intravenous injection. Routes of administration may be combined, if desired.

[0240] The dose of rAAV particles (including hTfRl AAV particles such as the rAAVhTfRlv.1 or rAAVhTfRlv.2 particles described herein) required to achieve a “therapeutic effect,” e.g., the units of dose in genome copies / per kilogram of body weight (GC / kg) or vector genomes (vg) per kilogram of body weight (kg), the units of dose in genome copies per brain volume, and units of dose in genome copies per CSF volume, will vary based on several factors including, but not limited to: the route of rAAV particle administration, the level of TSC1 expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product.

[0241] In some embodiments, an effective amount of an rAAV particle (including hTfRl AAV particles such as the rAAVhTfRlv.1 or rAAVhTfRlv.2 particles described herein) can be an amount sufficient to target a desired tissue. The effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among animal and tissue. For example, an effective amount of the rAAV particle can be in the range from about 1 ml to about 100 ml of solution containing from about 106to 1016genome copies e.g., from 1 x 106to 1 x 1016, inclusive). In methods disclosed herein, the therapeutically effective dose is between 6 X 1013vg / kg to 6 X 1014vg / kg, including 7 X 1013vg / kg, 8 X 1013vg / kg, 9 X 1013vg / kg, 1 X 1014vg / kg, 2 X 1014vg / kg, 3 X 1014vg / kg, 4 X 1014vg / kg, or 5 X 1014vg / kg (or alternatively, genome copies per brain volume, CSF volume or other measurement appropriate for ICV or ICM delivery). In some embodiments, a dosage between about 1011to 1012per kg or appropriate measurement rAAV genome copies can be appropriate. In some embodiments, a dosage of between about 1011to 1013per kg or appropriate measurement rAAV genome copies can be appropriate. In some embodiments, a dosage of between about 1011to 1014per kg or appropriate measurement rAAV genome copies can be appropriate. In some embodiments, a dosage of between about 1011to 1013per kg or appropriate measurement rAAV genome copies can be appropriate. In some embodiments, a dosage of about 1 x 1014vector genome (vg) copies per kg or appropriate measurement can be appropriate. In some embodiments, the dosage can vary or be reduced when specifically targeting one or more brain region(s). In some embodiments, a dosage between about 107to 108rAAV genome copies per kg or appropriate measurement can be appropriate. In some embodiments, a dosage of between about 108to 109Attorney Docket No.: 38213.0001P1rAAV genome copies per kg or appropriate measurement can be appropriate. In some embodiments, a dosage of between about 109to 1010rAAV genome copies per kg or appropriate measurement can be appropriate. In some embodiments, a dosage of between about IO10to TO11rAAV genome copies per kg or other appropriate measurement can be appropriate.

[0242] In some aspects, a potential side-effect for administering an AAV particle (including hTfRl AAV particles such as the rAAVhTfR.lv.1 or rAAVhTfR.lv.2 particles described herein) to a subject can be an immune response in the subject to the AAV particle, including inflammation, and, and may depend on the route of administration, particularly when the administration of an AAV particle is systemic. In some embodiments, a subject can be immunosuppressed prior to administration of one or more rAAVs as described herein.

[0243] As used herein, “immunosuppressed” or “immunosuppression” refers to a decrease in the activation or efficacy of an immune response in a subject. Immunosuppression can be induced in a subject using one or more (e.g., multiple, such as 2, 3, 4, 5, or more) agents, including, but not limited to, rituximab, methylprednisolone, prednisolone, sirolimus, immunoglobulin injection, prednisone, methotrexate, an interleukin-6 inhibitor, an anti-interleukin-6 antibody, an interleukin-6 receptor inhibitor, an anti-interleukin-6 receptor antibody, and any combination thereof.

[0244] In some embodiments, methods disclosed herein can further comprise the step of inducing immunosuppression (e.g., administering one or more immunosuppressive agents) in a subject prior to the subject being administered an rAAV particle (including hTfR.1 AAV particles such as the rAAVhTfR.lv.1 or rAAVhTfR.lv.2 particles described herein). In some embodiments, a subject can be immunosuppressed (e.g., immunosuppression is induced in the subject) between about 30 days and about 0 days (e.g., any time between 30 days until administration of the rAAV particle, inclusive) prior to administration of the rAAV particle to the subject. In some embodiments, the subject can be pretreated with immune suppression agent (e.g., rituximab, sirolimus, and / or prednisone) for at least 7 days. In some embodiments, immunosuppression of a subject maintained during and / or after administration of a rAAV particle or pharmaceutical composition. In some embodiments, a subject can be immunosuppressed (e.g., administered one or more immunosuppressants) for between 1 day and 1 year after administration of the rAAV particle or pharmaceutical composition. In some embodiments, rAAV particle compositions can be formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV particle concentrations are present (e.g., ~1013GC / ml or more). Methods for reducingAttorney Docket No.: 38213.0001P1aggregation of rAAVs include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright et al 2005.)

[0245] In some embodiments, these formulations can contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and can be conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of active compound in each pharmaceutical composition can be prepared in such a way that a suitable dosage can be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological halflife, route of administration, product shelf life, as well as other pharmacological considerations can be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens can be desirable.

[0246] In some aspects, it will be desirable to deliver the rAAV particles in suitably formulated pharmaceutical compositions as disclosed herein either intracranially, subcutaneously, intrapancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally, or orally, intraperitoneally, intracerebroventricularly, by subpial injection, or by inhalation. In some aspects, the administration modalities as described in U. S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 can be used to deliver rAAVs. In some embodiments, a preferred mode of administration can be by intracerebroventricular or intrathecal injection.

[0247] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form can be sterile and fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, forAttorney Docket No.: 38213.0001P1example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars or sodium chloride can be included. Prolonged absorption of the injectable compositions can be achieved by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0248] For administration of an injectable aqueous solution, for example, the solution can be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These aqueous solutions can be suitable for intravenous, intracranial, intramuscular, subcutaneous, intracerebroventricular, and intraperitoneal administration. In this connection, a sterile aqueous medium can be employed. Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0249] Sterile injectable solutions can be prepared by incorporating the rAAV particle (including hTfRl AAV particles such as the rAAVhTfR.lv.1 or rAAVhTfR.lv.2 particles described herein) in the required amount in the appropriate solvent with various of the other ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions can be prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation can be vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. Supplementary active ingredients can also be incorporated into the compositions.

[0250] In some aspects, the methods can include administering one or more additional therapeutic agents to a subject who has been administered an rAAV or pharmaceutical composition as described herein.

[0251] In some aspects, administering the rAAV particles (including hTfRl AAV particles such as the rAAVhTfRlv.l or rAAVhTfRlv.2 particles described herein) for TSC1 gene therapy to a subject promotes expression of TSC1 by 10-fold compared to a control (untreated) subject. In some aspects, administering the rAAV particles described herein to a subject promotes expressionAttorney Docket No.: 38213.0001P1of TSC1 by 5-fold to 100-fold compared to control (e.g., 5-fold to 10-fold, 10-fold to 15-fold, 10-fold to 20-fold, 15-fold to 25-fold, 20-fold to 30-fold, 25-fold to 35-fold, 30-fold to 40-fold, 35-fold to 45-fold, 40-fold to 60-fold, 50-fold to 75-fold, 60-fold to 80-fold, 75-fold to 100-fold compared to a control (untreated) subject). In some aspects, administering the rAAV particles described herein to a subject promotes expression of TSC1 in a subject (e.g., promotes expression of TSC1 in the CNS of a subject) by between a 5% and 200% increase (e.g., 5-50%, 25-75%, 50-100%, 75-125%, 100-200%, or 100-150% etc.) compared to a control (untreated) subject.

[0252] As used herein, the term “treating” refers to the application or administration of a composition (e.g., an isolated nucleic acid or rAAV particle as described herein, including hTfRl AAV particles such as the rAAVhTfR.lv.1 or rAAVhTfR.lv.2 particles) for TSC1 to a subject who has TSC or a TSC-related disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward a disease.

[0253] Alleviating TSC or a TSC-related disorder includes delaying the development or progression of the disease, or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, “delaying” the development of a disease means to defer, hinder, slow, retard, stabilize, and / or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and / or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

[0254] Administration of an rAAV particle described herein (including hTfR.1 AAV particles such as the rAAVhTfR.lv.1 or rAAVhTfR.lv.2 particles described herein) for TSC1 gene therapy to a human subject suffering from TSC will, within 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, 50 weeks or 1 year after the administration, result in reduction in one or more biomarkers or symptoms of TSC.

[0255] “Development” or “progression” of a disease means initial manifestations and / or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers toAttorney Docket No.: 38213.0001P1progression that can be undetectable. As used herein the terms development or progression refer to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a disease can be associated with low levels of TSC1 expression (e.g., TSC1 deficiency).

[0256] In some aspects, a subject has or is suspected of having a disease or disorder associated with TSC1. In some aspects, a subject having a disease or disorder associated with TSC1 comprises a subject with at least one TSC1 allele having a loss-of-function mutation. In some aspects, aTSCl allele having a loss-of-function mutation comprises a deletion, a frameshift mutation, a splice site mutation, a missense mutation, a truncation mutation or a nonsense mutation. A subject may have two TSC1 alleles having the same loss-of-function mutations (homozygous state) or two TSC1 alleles having different loss-of-function mutations (compound heterozygous state).

[0257] In some aspects, the rAAV particles disclosed herein can be administered 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. Pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., to the central nervous system), by oral, inhalation (including intranasal and intratracheal delivery), intraocular, intracerebroventricular, intravenous, intramuscular, subcutaneous, intradermal, and other parental routes of administration. Routes of administration can be combined, if desired.KITS

[0258] Disclosed herein are kits comprising any of the agents described herein. In some aspects, any of the agents disclosed herein can be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic or research applications. A kit can include one or more containers housing the components of the disclosure and instructions for use. Specifically, such kits may include one or more agents described herein, along with instructions describing the intended application and the proper use of these agents. In some aspects, the agents in a kit can be in a pharmaceutical formulation and dosage suitable for a particular application and for a method of administration of the agents. Kits for research purposes can contain the components in appropriate concentrations or quantities for running various experiments.

[0259] Also disclosed herein are kits for producing rAAV particles engineered to express TSC1, as described herein. In some aspects, the kit can comprise a container housing an isolated nucleic acid encoding a TSC1 protein or a portion thereof. In some aspects, the kits can further compriseAttorney Docket No.: 38213.0001P1instructions for producing the rAAV particle. In some aspects, the kit further comprises at least one container housing a recombinant AAV vector, wherein the recombinant AAV vector comprises a transgene (i.e., TSC1).

[0260] In some aspects, the kits can comprise a container housing a recombinant AAV particle as described herein. In some aspects, the kits can further comprises a container housing a pharmaceutically acceptable carrier. For example, a kit can comprise one container housing a rAAV particle and a second container housing a buffer suitable for injection of the rAAV particle into a subject. In some aspects, the container can be a syringe.

[0261] In some aspects, the kits can be designed to facilitate use of the methods described herein by researchers and can take many forms. Each of the compositions of the kit, where applicable, may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder). In some aspects, some of the compositions can be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit. As used herein, “instructions” can define a component of instruction and / or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions can be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), internet, and / or web-based communications, etc. The written instructions can be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use or sale for animal administration.

[0262] The kits disclosed herein can also contain any one or more of the components described herein in one or more containers. In some embodiments, the kits can include instructions for mixing one or more components of the kit and / or isolating and mixing a sample and applying to a subject. The kits can include a container housing agents described herein. The agents can be in the form of a liquid, gel or solid (powder). The agents can be prepared sterilely, packaged in syringe and shipped refrigerated. Alternatively, it can be housed in a vial or other container for storage. A second container can have other agents prepared sterilely. Alternatively, the kits can include the active agents premixed and shipped in a syringe, vial, tube, or other container. The kits can have one or more or all of the components required to administer the agents to an animal, such as aAttorney Docket No.: 38213.0001P1syringe, topical application devices, or iv needle tubing and bag, particularly in the case of the kits for producing specific somatic animal models.

[0263] The kits disclosed can have a variety of forms, such as a blister pouch, a shrink wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box or a bag. The kits can be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped. The kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kits can also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration etc.

[0264] The instructions included within the kit can involve methods for detecting a latent AAV in a cell. In addition, kits of the disclosure can include instructions, a negative and / or positive control, containers, diluents and buffers for the sample, sample preparation tubes and a printed or electronic table of reference AAV sequence for sequence comparisons.Table 1 SequencesName SEQ ID SequenceNO.:wt AAV9 1 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGP GNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSF VP1 aminoGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQacid PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADG sequence VGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYF GYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNG VKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDG SQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLI DQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQ NNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGR DNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGM VWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPT AFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAV NTEGVYSEPRPIGTRYLTRNL AAV 2 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGP GNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFhTfR.lv.1GGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ VP1 amino PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADG acid VGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFsequence GYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:(wt AAV VKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDG SQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIbkgd)DQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQ NNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGR DNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQYSRIGPNAQAQTGWVQN QGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTP VPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKS NNVE FAVNTEGVYS E PRP I GTRYLTRNL AAV 3 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGP hTfRlv.2 GNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSF GGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ VP1 amino PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADG acid VGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYF sequence GYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNG (wt AAV VKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDG SQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIbkgd) DQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQ NNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGR DNVDADKVMITNEEEIKTTNPVATESYGQVATNHQEAQYSRIGPNNQAQTGWVQN QGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTP VPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKS NNVE FAVNTEGVYS E PRP I GTRYLTRNL AAV9 4 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGP GNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFK449RVP1GGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQamino acid PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADG sequence VGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYF GYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNG VKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDG SQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLI DQYLYYLSRTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQ NNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGR DNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGM VWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPT AFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAV NTEGVYSEPRPIGTRYLTRNL AAV 5 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGP GNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFhTfRlv.lGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ VP1 amino PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADG acid VGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYF sequence GYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNG (K449R VKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDG SQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLI AAV bkgd) DQYLYYLSRTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQ NNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGR DNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQYSRIGPNAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:VPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKS NNVE FAVNTEGVYS E PRP I GTRYLTRNL AAV 6 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGP GNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFhTfRlv.2GGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ VP1 amino PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADG acid VGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYF sequence GYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNG (K449R VKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDG SQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLI AAV bkgd) DQYLYYLSRTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQ NNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGR DNVDADKVMITNEEEIKTTNPVATESYGQVATNHQEAQYSRIGPNNQAQTGWVQN QGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTP VPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKS NNVE FAVNTEGVYS E PRP I GTRYLTRNLhuman 7 MAQQANVGELLAMLDSPMLGVRDDVTAVFKENLNSDRGPMLVNTLVDYYLETSSQ PALHILTTLQEPHDKHLLDRINEYVGKAATRLSILSLLGHVIRLQPSWKHKLSQA TSC1PLLPSLLKCLKMDTDVVVLTTGVLVLITMLPMIPQSGKQHLLDFFDIFGRLSSWC(hamartin) LKKPGHVAEVYLVHLHASVYALFHRLYGMYPCNFVSFLRSHYSMKENLETFEEVV amino acid KPMMEHVRIHPELVTGSKDHELDPRRWKRLETHDVVIECAKISLDPTEASYEDGY sequence SVSHQISARFPHRSADVTTSPYADTQNSYGCATSTPYSTSRLMLLNMPGQLPQTL SSPSTRLITEPPQATLWSPSMVCGMTTPPTSPGNVPPDLSHPYSKVFGTTAGGKG TPLGTPATSPPPAPLCHSDDYVHISLPQATVTPPRKEERMDSARPCLHRQHHLLN DRGSEEPPGSKGSVTLSDLPGFLGDLASEEDSIEKDKEEAAISRELSEITTAEAE PVVPRGGFDSPFYRDSLPGSQRKTHSAASSSQGASVNPEPLHSSLDKLGPDTPKQ AFTPIDLPCGSADESPAGDRECQTSLETSIFTPSPCKIPPPTRVGFGSGQPPPYD HLFEVALPKTAHHFVIRKTEELLKKAKGNTEEDGVPSTSPMEVLDRLIQQGADAH SKELNKLPLPSKSVDWTHFGGSPPSDEIRTLRDQLLLLHNQLLYERFKRQQHALR NRRLLRKVIKAAALEEHNAAMKDQLKLQEKDIQMWKVSLQKEQARYNQLQEQRDT MVTKLHSQIRQLQHDREEFYNQSQELQTKLEDCRNMIAELRIELKKANNKVCHTE LLLSQVSQKLSNSESVQQQMEFLNRQLLVLGEVNELYLEQLQNKHSDTTKEVEMM KAAYRKELEKNRSHVLQQTQRLDTSQKRILELESHLAKKDHLLLEQKKYLEDVKL QARGQLQAAESRYEAQKRITQVFELEILDLYGRLEKDGLLKKLEEEKAEAAEAAE ERLDCCNDGCSDSMVGHNEEASGHNGETKTPRPSSARGSSGSRGGGGSSSSSSEL STPEKPPHQRAGPFSSRWETTMGEASASIPTTVGSLPSSKSFLGMKARELFRNKS ESQCDEDGMTSSLSESLKTELGKDLGVEAKIPLNLDGPHPSPPTPDSVGQLHIMD YNETHHEHShuman 8 atggcccaacaagcaaatgtcggggagcttcttgccatgctggactcccccatgc tgggtgtgcgggacgacgtgacagctgtctttaaagagaacctcaattctgaccg TSC1tggccctatgcttgtaaacaccttggtggattattacctggaaaccagctctcag (hamartin) ccggcattgcacatcctgaccaccttgcaagagccacatgacaagcacctcttgg nucleotide acaggattaacgaatatgtgggcaaagccgccactcgtttatccatcctctcgtt sequence actgggtcatgtcataagactgcagccatcttggaagcataagctctctcaagca cctcttttgccttctttactaaaatgtctcaagatggacactgacgtcgttgtcc tcacaacaggcgtcttggtgttgataaccatgctaccaatgattccacagtctgg gaaacagcatcttcttgatttctttgacatttttggccgtctgtcatcatggtgc ctgaagaaaccaggccacgtggcggaagtctatctcgtccatctccatgccagtg tgtacgcactctttcatcgcctttatggaatgtacccttgcaacttcgtctcctttttgcgttctcattacagtatgaaagaaaacctggagacttttgaagaagtggtcAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:aagccaatgatggagcatgtgcgaattcatccggaattagtgactggatccaagg accatgaactggaccctcgaaggtggaagagattagaaactcatgatgttgtgat cgagtgtgccaaaatctctctggatcccacagaagcctcatatgaagatggctat tctgtgtctcaccaaat ct cagcccgctttcctcatcgttcagccgatgt caeca ccagcccttatgctgacacacagaatagctatgggtgtgctacttctacccctta ctccacgtctcggctgatgttgttaaatatgccagggcagctacctcagactctg agttccccatcgacacggctgataactgaaccaccacaagctactctttggagcc catctatggtttgtggtatgaccactcctccaacttctcctggaaatgtcccacc tgatctgtcacacccttacagtaaagtctttggtacaactgcaggtggaaaagga actcctctgggaaccccagcaacctctcctcctccagccccactctgtcattcgg atgactacgtgcacatttcactcccccaggccacagtcacaccccccaggaagga agagagaatggattctgcaagaccatgtctacacagacaacaccatcttctgaat gacagaggatcagaagagccacctggcagcaaaggttctgtcactctaagtgatc ttccagggtttttaggtgatctggcctctgaagaagatagtattgaaaaagataa agaagaagctgcaatatctagagaactttctgagatcaccacagcagaggcagag cctgtggttcctcgaggaggctttgactctcccttttaccgagacagtctcccag gttctcagcggaagacccactcggcagcctccagttctcagggcgccagcgtgaa ccctgagcctttacactcctccctggacaagcttgggcctgacacaccaaagcaa gcctttactcccatagacctgccctgcggcagtgctgatgaaagccctgcgggag acagggaatgccagacttctttggagaccagtatcttcactcccagtccttgtaa aattccacctccgacgagagtgggctttggaagcgggcagcctcccccgtatgat catctttttgaggtggcattgccaaagacagcccatcattttgtcatcaggaaga ctgaggagctgttaaagaaagcaaaaggaaacacagaggaagatggtgtgccctc t acct ccccaatggaagt get ggacagactgatacagcagggagcagacgcgcac agcaaggagctgaacaagttgcctttacccagcaagtctgtcgactggacccact ttggaggctctcctccttcagatgagatccgcaccctccgagaccagttgctttt actgcacaaccagttactctatgagcgttttaagaggcagcagcatgccctccgg aacaggcggctcctccgcaaggtgatcaaagcagcagctctggaggaacataatg ctgccatgaaagatcagttgaagttacaagagaaggacatccagatgtggaaggt tagtctgcagaaagaacaagctagatacaatcagctccaggagcagcgtgacact atggtaaccaagctccacagccagatcagacagctgcagcatgaccgagaggaat tctacaaccagagccaggaattacagacgaagctggaggactgcaggaacatgat tgcggagctgcggatagaactgaagaaggccaacaacaaggtgtgtcacactgag ctgctgctcagtcaggtttcccaaaagctctcaaacagtgagtcggtccagcagc agatggagttcttgaacaggcagctgttggttcttggggaggtcaacgagctcta tttggaacaactgcagaacaagcactcagataccacaaaggaagtagaaatgatg aaagccgcctatcggaaagagctagaaaaaaacagaagccatgttctccagcaga ctcagaggcttgatacctcccaaaaacggattttggaactggaatctcacctggc caagaaagaccaccttcttttggaacagaagaaatatctagaggatgtcaaactc caggcaagaggacagctgcaggccgcagagagcaggtatgaggctcagaaaagga taacccaggtgtttgaattggagatcttagatttatatggcaggttggagaaaga tggcctcctgaaaaaacttgaagaagaaaaagcagaagcagctgaagcagcagaa gaaaggcttgactgttgtaatgacgggtgctcagattccatggtagggcacaatg aagaggcatctggccacaacggtgagaccaagacccccaggcccagcagcgcccg gggcagtagtggaagcagaggtggtggaggcagcagcagcagcagcagcgagctt tctaccccagagaaacccccacaccagagggcaggcccattcagcagtcggtggg agacgactatgggagaagcgtctgccagcatccccaccactgtgggctcacttcc cagttcaaaaagcttcctgggtatgaaggctcgagagttatttcgtaataagagc gagagccagtgtgatgaggacggcatgaccagtagcctttctgagagcctaaaga cagaactgggcaaagacttgggtgtggaagccaagattcccctgaacctagatgg ccctcacccgtctcccccgaccccggacagtgttggacagctacatatcatggac tacaatgagactcatcatgaacacagctaaAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:codon- 9 atggcccagcaggcgaatgtgggtgagctccttgcaatgctggacagtcccatgt tgggcgtccgagacgacgttactgccgtgttcaaagaaaaccttaatagtgacag optimizedgggaccaatgctcgttaatactcttgtcgactattacctggagacatcaagccag human cctgccttgcatatcttgacgacgttgcaggaaccgcacgacaagcatcttctgg TSC1 atcgaataaacgagtatgtgggcaaagcagcaactcgcctctcaattctctccct (hamartin) cttgggacacgtcatacgactccagccatcctggaagcataagctgagtcaggcc nucleotide cccttgctgcctagccttctgaagtgccttaagatggacacggatgtggtcgttc tcactaccggagtgttggttcttatcacaatgctgcctatgattcctcagtctgg sequence aaagcagcacctgctcgatttctttgacatatttggacgcctttcttcttggtgt ttgaaaaaaccgggacatgtagctgaagtctacttggtgcacctccatgcttctg tgtacgcattgtttcataggctttatggtatgtacccctgtaatttcgttagttt tctccgatctcactacagtatgaaagaaaatctggaaacgtttgaagaggtcgtc aaaccgatgatggagcacgtaaggatacaccccgagttggtaactggtagtaagg atcacgaacttgaccctcggagatggaaaaggttggagactcacgacgttgtaat cgaatgtgctaagataagccttgacccaaccgaggctagctatgaggatggctat agcgttagccatcagataagtgccagattcccacatcggagcgcagatgtgacga catcaccgtatgctgacacgcagaactcttacggttgtgccacctctactcctta cagtacatctcggttgatgcttcttaatatgcccgggcagctcccacagacactt tccagcccgagtacacgcctcataaccgagccaccacaggcaacactctggtcac caagcatggtttgtggaatgactacgccgccgacaagcccagggaatgtcccccc cgatctgagtcacccgtattctaaggtcttcgggacaacggccggtgggaagggg act ccattgggt act ccggct acct cacctccgccggctcctctctgccactctg atgattacgttcacatcagtctgccccaagcgacagtgacccctccccgcaaaga agaaaggatggattcagctagaccttgcctgcaccgacaacatcaccttctcaac gatcggggaagcgaagaaccgcccggaagcaaagggtccgttacgttgtctgact tgcccggattccttggtgacctggcatcagaggaagattctatagaaaaagataa ggaagaagctgccattagccgagaactttcagaaatcaccacagccgaggctgaa ccagttgttccacggggcggcttcgattcccctttttatagagattctttgccag gaagccaaaggaagacccattctgcggctagcagcagtcagggagcaagtgtcaa ccctgagccccttcattcctcactcgacaagcttggacctgatacaccaaagcag gcttttactccgatcgacttgccgtgtggaagtgctgatgagtctcccgccggag acagggagtgccagacttcactcgaaacctcaattttcacgccctcaccatgtaa aattcccccgcccactcgggtggggtttggctcaggtcagccacccccttacgat cacctgtttgaggttgctttgcccaagaccgcccaccacttcgttatccgaaaga cagaggaactcctcaaaaaagctaagggcaacactgaggaagacggtgtgccaag tacctctcctatggaggtcctggatcgcttgatccagcagggtgcggatgcccat agtaaagaactcaacaagttgccgttgccgtctaagtctgtcgattggactcatt tcgggggaagcccccctagcgatgaaataagaacgctccgagaccaactgcttct tttgcacaaccaactcttgtacgagcgcttcaagaggcaacagcacgcactccgg aatcgccggctcttgaggaaagtaataaaggcagccgctctggaggagcacaatg cggcgatgaaggaccagttgaagcttcaggagaaagacatccaaatgtggaaggt ctccctccagaaagaacaagcgcggtataatcagctgcaagaacaacgagacact atggttacaaaactgcactctcaaataaggcaactccagcatgacagagaagagt tttacaaccagtcccaagaattgcaaaccaaacttgaagactgccgcaacatgat cgctgagttgaggatcgagctcaaaaaggcaaacaacaaagtgtgtcacacagag cttctcctcagtcaggtttcccagaagctttccaactccgaatctgtgcagcaac agatggagttccttaatcgccaactcctggtgcttggtgaggtaaacgagctgta tttggaacaacttcagaacaaacatagcgatacgactaaagaagtagaaatgatg aaggcagcataccggaaggagcttgagaaaaacagaagtcacgtcctccaacaaa cacaaagactcgacacatctcagaaacggattctggagctggagagtcatttggc gaaaaaggatcatctgttgcttgagcagaaaaagtacctggaagatgttaagctc caggctagggggcagctccaagctgctgaaagcagatatgaagcacaaaagcggattacgcaagttttcgagttggaaatcctggacctgtacggaagattggaaaaggaAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:tggtctcctcaaaaaactggaggaggaaaaagcggaagcagcggaggccgccgag gagaggctcgactgctgcaatgacggctgctcagactccatggttggtcacaacg aggaggctagcggtcataatggagagacaaaaacaccgaggccgagctccgcgag gggttccagtggctctaggggtggaggtggcagcagctcttcttccagcgaactg tccactcctgagaaaccccctcaccaacgggcaggccccttctcatctaggtggg agaccacgatgggtgaagcgagcgcctccattccaactaccgtaggcagcctccc ctcttccaaaagtttcctcggaatgaaagcccgcgagctgttcaggaataaatcc gagtcacaatgtgatgaggacggtatgacctctagcctgtccgagtctttgaaaa cagagctcgggaaggatctgggcgtcgaagctaagatccctctgaatttggatgg accgcacccgtcaccacccacacctgatagtgtcggccagttgcatattatggat tataacgaaactcatcatgagcacagcITR-EFS- 10 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcga cctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaa TSC1- ctccatcactaggggttcctctcgagggctccggtgcccgtcagtgggcagagcg bGHv3 cacatcgcccacagtccccgagaagttggggggaggggtcggcaattgatccggt gcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctcc gcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaa cgttctttttcgcaacgggtttgccgccagaacacagggccaccatggcccagca ggcgaatgtgggtgagctccttgcaatgctggacagtcccatgttgggcgtccga gacgacgttactgccgtgttcaaagaaaaccttaatagtgacaggggaccaatgc tcgttaatactcttgtcgactattacctggagacatcaagccagcctgccttgca tatcttgacgacgttgcaggaaccgcacgacaagcatcttctggatcgaataaac gagtatgtgggcaaagcagcaactcgcctctcaattctctccctcttgggacacg tcatacgactccagccatcctggaagcataagctgagtcaggcccccttgctgcc tagccttctgaagtgccttaagatggacacggatgtggtcgttctcactaccgga gtgttggttcttatcacaatgctgcctatgattcctcagtctggaaagcagcacc tgctcgatttctttgacatatttggacgcctttcttcttggtgtttgaaaaaacc gggacatgtagctgaagtctacttggtgcacctccatgcttctgtgtacgcattg tttcataggctttatggtatgtacccctgtaatttcgttagttttctccgatctc actacagtatgaaagaaaatctggaaacgtttgaagaggtcgtcaaaccgatgat ggagcacgtaaggatacaccccgagttggtaactggtagtaaggatcacgaactt gaccctcggagatggaaaaggttggagactcacgacgttgtaatcgaatgtgcta agataagccttgacccaaccgaggctagctatgaggatggctatagcgttagcca tcagataagtgccagattcccacatcggagcgcagatgtgacgacatcaccgtat gctgacacgcagaactcttacggttgtgccacctctactccttacagtacatctc ggttgatgcttcttaatatgcccgggcagctcccacagacactttccagcccgag tacacgcctcataaccgagccaccacaggcaacactctggtcaccaagcatggtt tgtggaatgactacgccgccgacaagcccagggaatgtcccccccgatctgagtc acccgtattctaaggtcttcgggacaacggccggtgggaaggggactccattggg tactccggctacctcacctccgccggctcctctctgccactctgatgattacgtt cacatcagtctgccccaagcgacagtgacccctccccgcaaagaagaaaggatgg attcagctagaccttgcctgcaccgacaacatcaccttctcaacgatcggggaag cgaagaaccgcccggaagcaaagggtccgttacgttgtctgacttgcccggattc cttggtgacctggcatcagaggaagattctatagaaaaagataaggaagaagctg ccattagccgagaactttcagaaatcaccacagccgaggctgaaccagttgttcc acggggcggcttcgattcccctttttatagagattctttgccaggaagccaaagg aagacccattctgcggctagcagcagtcagggagcaagtgtcaaccctgagcccc ttcattcctcactcgacaagcttggacctgatacaccaaagcaggcttttactcc gatcgacttgccgtgtggaagtgctgatgagtctcccgccggagacagggagtgc cagacttcactcgaaacctcaattttcacgccctcaccatgtaaaattcccccgc ccactcgggtggggtttggctcaggtcagccacccccttacgatcacctgtttgaggttgctttgcccaagaccgcccaccacttcgttatccgaaagacagaggaactcAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:ctcaaaaaagctaagggcaacactgaggaagacggtgtgccaagtacctctccta tggaggtcctggatcgcttgatccagcagggtgcggatgcccatagtaaagaact caacaagttgccgttgccgtctaagtctgtcgattggactcatttcgggggaagc ccccctagcgatgaaataagaacgctccgagaccaactgcttcttttgcacaacc aactcttgtacgagcgcttcaagaggcaacagcacgcactccggaatcgccggct cttgaggaaagtaataaaggcagccgctctggaggagcacaatgcggcgatgaag gaccagttgaagcttcaggagaaagacatccaaatgtggaaggtctccctccaga aagaacaagcgcggtataatcagctgcaagaacaacgagacactatggttacaaa actgcactctcaaataaggcaactccagcatgacagagaagagttttacaaccag tcccaagaattgcaaaccaaacttgaagactgccgcaacatgatcgctgagttga ggatcgagctcaaaaaggcaaacaacaaagtgtgtcacacagagcttctcctcag tcaggtttcccagaagctttccaactccgaatctgtgcagcaacagatggagttc cttaatcgccaactcctggtgcttggtgaggtaaacgagctgtatttggaacaac ttcagaacaaacatagcgatacgactaaagaagtagaaatgatgaaggcagcata ccggaaggagcttgagaaaaacagaagtcacgtcctccaacaaacacaaagactc gacacatctcagaaacggattctggagctggagagtcatttggcgaaaaaggatc atctgttgcttgagcagaaaaagtacctggaagatgttaagctccaggctagggg gcagctccaagctgctgaaagcagatatgaagcacaaaagcggattacgcaagtt ttcgagttggaaatcctggacctgtacggaagattggaaaaggatggtctcctca aaaaactggaggaggaaaaagcggaagcagcggaggccgccgaggagaggctcga ctgctgcaatgacggctgctcagactccatggttggtcacaacgaggaggctagc ggtcataatggagagacaaaaacaccgaggccgagctccgcgaggggttccagtg gctctaggggtggaggtggcagcagctcttcttccagcgaactgtccactcctga gaaaccccctcaccaacgggcaggccccttctcatctaggtgggagaccacgatg ggtgaagcgagcgcctccattccaactaccgtaggcagcctcccctcttccaaaa gtttcctcggaatgaaagcccgcgagctgttcaggaataaatccgagtcacaatg tgatgaggacggtatgacctctagcctgtccgagtctttgaaaacagagctcggg aaggatctgggcgtcgaagctaagatccctctgaatttggatggaccgcacccgt caccacccacacctgatagtgtcggccagttgcatattatggattataacgaaac tcatcatgagcacagctgatactgtgccttctagttgccagccatctgttgtttg cccctcccccttgccttccttgaccctggaaggtgccactcccactgtcctttcc taataaaatgaggaaattgcatcacattgtctgagtaggtgtcattctattctgg ggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggca tgctggggatgcagtgggctctatgggcggccgcaggaacccctagtgatggagt tggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggt cgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcag ITR-CMV- 11 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcga TSCl-bGH cctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaa ctccatcactaggggttcctctcgaggattaatagtaatcaattacggggtcatt agttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccg cctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttc ccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacg gtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccct attgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgacct tatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccat ggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacgg ggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaa atcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatggg cggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgt cagatccgctagcgccaccatggcccagcaggcgaatgtgggtgagctccttgca atgctggacagtcccatgttgggcgtccgagacgacgttactgccgtgttcaaagaaaaccttaatagtgacaggggaccaatgctcgttaatactcttgtcgactattaAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:cctggagacatcaagccagcctgccttgcatatcttgacgacgttgcaggaaccg cacgacaagcatcttctggatcgaataaacgagtatgtgggcaaagcagcaactc gcctctcaattctctccctcttgggacacgtcatacgactccagccatcctggaa gcataagctgagtcaggcccccttgctgcctagccttctgaagtgccttaagatg gacacggatgtggtcgttctcactaccggagtgttggttcttatcacaatgctgc ctatgattcctcagtctggaaagcagcacctgctcgatttctttgacatatttgg acgcctttcttcttggtgtttgaaaaaaccgggacatgtagctgaagtctacttg gtgcacctccatgcttctgtgtacgcattgtttcataggctttatggtatgtacc cctgtaatttcgttagttttctccgatctcactacagtatgaaagaaaatctgga aacgtttgaagaggtcgtcaaaccgatgatggagcacgtaaggatacaccccgag ttggtaactggtagtaaggatcacgaacttgaccctcggagatggaaaaggttgg agactcacgacgttgtaatcgaatgtgctaagataagccttgacccaaccgaggc tagctatgaggatggct at agcgtt agecat cagataagtgccagattcccacat cggagcgcagatgtgacgacatcaccgtatgctgacacgcagaactcttacggtt gtgccacctctactccttacagtacatctcggttgatgcttcttaatatgcccgg gcagctcccacagacactttccagcccgagtacacgcctcataaccgagccacca caggcaacactctggtcaccaagcatggtttgtggaatgactacgccgccgacaa gcccagggaatgtcccccccgatctgagtcacccgtattctaaggtcttcgggac aacggccggtgggaaggggactccattgggtactccggctacctcacctccgccg gctcctctctgccactctgatgattacgttcacatcagtctgccccaagcgacag tgacccctccccgcaaagaagaaaggatggattcagctagaccttgcctgcaccg acaacatcaccttctcaacgatcggggaagcgaagaaccgcccggaagcaaaggg tccgttacgttgtctgacttgcccggattccttggtgacctggcatcagaggaag attctatagaaaaagataaggaagaagctgccattagccgagaactttcagaaat caccacagccgaggctgaaccagttgttccacggggcggcttcgattcccctttt tatagagattctttgccaggaagccaaaggaagacccattctgcggctagcagca gtcagggagcaagtgtcaaccctgagccccttcattcctcactcgacaagcttgg acctgatacaccaaagcaggcttttactccgatcgacttgccgtgtggaagtgct gatgagtctcccgccggagacagggagtgccagacttcactcgaaacctcaattt tcacgccct caeca tgtaaaattcccccgcccactcgggtggggtttggctcagg tcagccacccccttacgatcacctgtttgaggttgctttgcccaagaccgcccac cacttcgttatccgaaagacagaggaactcctcaaaaaagctaagggcaacactg aggaagacggtgtgccaagtacctctcctatggaggtcctggatcgcttgatcca gcagggtgcggatgcccatagtaaagaactcaacaagttgccgttgccgtctaag tctgtcgattggactcatttcgggggaagcccccctagcgatgaaataagaacgc tccgagaccaactgcttcttttgcacaaccaactcttgtacgagcgcttcaagag gcaacagcacgcactccggaatcgccggctcttgaggaaagtaataaaggcagcc gctctggaggagcacaatgcggcgatgaaggaccagttgaagcttcaggagaaag acatccaaatgtggaaggtctccctccagaaagaacaagcgcggtataatcagct gcaagaacaacgagacactatggttacaaaactgcactctcaaataaggcaactc cagcatgacagagaagagttttacaaccagtcccaagaattgcaaaccaaacttg aagactgccgcaacatgatcgctgagttgaggatcgagctcaaaaaggcaaacaa caaagtgtgtcacacagagcttctcctcagtcaggtttcccagaagctttccaac tccgaatctgtgcagcaacagatggagttccttaatcgccaactcctggtgcttg gtgaggtaaacgagctgtatttggaacaacttcagaacaaacatagcgatacgac taaagaagtagaaatgatgaaggcagcataccggaaggagcttgagaaaaacaga agtcacgtcctccaacaaacacaaagactcgacacatctcagaaacggattctgg agctggagagtcatttggcgaaaaaggatcatctgttgcttgagcagaaaaagta cctggaagatgttaagctccaggctagggggcagctccaagctgctgaaagcaga tatgaagcacaaaagcggattacgcaagttttcgagttggaaatcctggacctgt acggaagattggaaaaggatggtctcctcaaaaaactggaggaggaaaaagcgga agcagcggaggccgccgaggagaggctcgactgctgcaatgacggctgctcagactccatggttggtcacaacgaggaggctagcggtcataatggagagacaaaaacacAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:cgaggccgagctccgcgaggggttccagtggctctaggggtggaggtggcagcag ctcttcttccagcgaactgtccactcctgagaaaccccctcaccaacgggcaggc cccttctcatctaggtgggagaccacgatgggtgaagcgagcgcctccattccaa ctaccgtaggcagcctcccctcttccaaaagtttcctcggaatgaaagcccgcga gctgttcaggaataaatccgagtcacaatgtgatgaggacggtatgacctctagc ctgtccgagtctttgaaaacagagctcgggaaggatctgggcgtcgaagctaaga tccctctgaatttggatggaccgcacccgtcaccacccacacctgatagtgtcgg ccagttgcatattatggattataacgaaactcatcatgagcacagcgagcagaag ctgatcagcgaggaggacctgtgaggggatccgtcgactagagctcgctgatcag cctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgcc ttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaa attgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggc aggacagcaagggggaggattgggaagacaatagcaggcatgctggggagagatc tgagggcggccgcaggaacccctagtgatggagttggccactccctctctgcgcg ctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgc ccgggcggcctcagtgagcgagcgagcgcgcagITR-EFS- 12 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcga cctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaa TSC1- ctccatcactaggggttcctctcgagggctccggtgcccgtcagtgggcagagcg huGHpA cacatcgcccacagtccccgagaagttggggggaggggtcggcaattgatccggt gcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctcc gcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaa cgttctttttcgcaacgggtttgccgccagaacacagggccaccatggcccagca ggcgaatgtgggtgagctccttgcaatgctggacagtcccatgttgggcgtccga gacgacgttactgccgtgttcaaagaaaaccttaatagtgacaggggaccaatgc tcgttaatactcttgtcgactattacctggagacatcaagccagcctgccttgca tatcttgacgacgttgcaggaaccgcacgacaagcatcttctggatcgaataaac gagtatgtgggcaaagcagcaactcgcctctcaattctctccctcttgggacacg tcatacgactccagccatcctggaagcataagctgagtcaggcccccttgctgcc tagccttctgaagtgccttaagatggacacggatgtggtcgttctcactaccgga gtgttggttcttatcacaatgctgcctatgattcctcagtctggaaagcagcacc tgctcgatttctttgacatatttggacgcctttcttcttggtgtttgaaaaaacc gggacatgtagctgaagtctacttggtgcacctccatgcttctgtgtacgcattg tttcataggctttatggtatgtacccctgtaatttcgttagttttctccgatctc actacagtatgaaagaaaatctggaaacgtttgaagaggtcgtcaaaccgatgat ggagcacgtaaggatacaccccgagttggtaactggtagtaaggatcacgaactt gaccctcggagatggaaaaggttggagactcacgacgttgtaatcgaatgtgcta agataagccttgacccaaccgaggctagctatgaggatggctatagcgttagcca tcagataagtgccagattcccacatcggagcgcagatgtgacgacatcaccgtat gctgacacgcagaactcttacggttgtgccacctctactccttacagtacatctc ggttgatgcttcttaatatgcccgggcagctcccacagacactttccagcccgag tacacgcctcataaccgagccaccacaggcaacactctggtcaccaagcatggtt tgtggaatgactacgccgccgacaagcccagggaatgtcccccccgatctgagtc acccgtattctaaggtcttcgggacaacggccggtgggaaggggactccattggg tactccggctacctcacctccgccggctcctctctgccactctgatgattacgtt cacatcagtctgccccaagcgacagtgacccctccccgcaaagaagaaaggatgg attcagctagaccttgcctgcaccgacaacatcaccttctcaacgatcggggaag cgaagaaccgcccggaagcaaagggtccgttacgttgtctgacttgcccggattc cttggtgacctggcatcagaggaagattctatagaaaaagataaggaagaagctg ccattagccgagaactttcagaaatcaccacagccgaggctgaaccagttgttcc acggggcggcttcgattcccctttttatagagattctttgccaggaagccaaaggaagacccattctgcggctagcagcagtcagggagcaagtgtcaaccctgagccccAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:ttcattcctcactcgacaagcttggacctgatacaccaaagcaggcttttactcc gatcgacttgccgtgtggaagtgctgatgagtctcccgccggagacagggagtgc cagacttcactcgaaacctcaattttcacgccctcaccatgtaaaattcccccgc ccactcgggtggggtttggctcaggtcagccacccccttacgatcacctgtttga ggttgctttgcccaagaccgcccaccacttcgttatccgaaagacagaggaactc ctcaaaaaagctaagggcaacactgaggaagacggtgtgccaagtacctctccta tggaggtcctggatcgcttgatccagcagggtgcggatgcccatagtaaagaact caacaagttgccgttgccgtctaagtctgtcgattggactcatttcgggggaagc ccccctagcgatgaaataagaacgctccgagaccaactgcttcttttgcacaacc aactcttgtacgagcgcttcaagaggcaacagcacgcactccggaatcgccggct cttgaggaaagtaataaaggcagccgctctggaggagcacaatgcggcgatgaag gaccagttgaagcttcaggagaaagacatccaaatgtggaaggtctccctccaga aagaacaagcgcggtataatcagctgcaagaacaacgagacactatggttacaaa actgcactctcaaataaggcaactccagcatgacagagaagagttttacaaccag tcccaagaattgcaaaccaaacttgaagactgccgcaacatgatcgctgagttga ggatcgagctcaaaaaggcaaacaacaaagtgtgtcacacagagcttctcctcag tcaggtttcccagaagctttccaactccgaatctgtgcagcaacagatggagttc cttaatcgccaactcctggtgcttggtgaggtaaacgagctgtatttggaacaac ttcagaacaaacatagcgatacgactaaagaagtagaaatgatgaaggcagcata ccggaaggagcttgagaaaaacagaagtcacgtcctccaacaaacacaaagactc gacacatctcagaaacggattctggagctggagagtcatttggcgaaaaaggatc atctgttgcttgagcagaaaaagtacctggaagatgttaagctccaggctagggg gcagctccaagctgctgaaagcagatatgaagcacaaaagcggattacgcaagtt ttcgagttggaaatcctggacctgtacggaagattggaaaaggatggtctcctca aaaaactggaggaggaaaaagcggaagcagcggaggccgccgaggagaggctcga ctgctgcaatgacggctgctcagactccatggttggtcacaacgaggaggctagc ggtcataatggagagacaaaaacaccgaggccgagctccgcgaggggttccagtg gctctaggggtggaggtggcagcagctcttcttccagcgaactgtccactcctga gaaaccccctcaccaacgggcaggccccttctcatctaggtgggagaccacgatg ggtgaagcgagcgcctccattccaactaccgtaggcagcctcccctcttccaaaa gtttcctcggaatgaaagcccgcgagctgttcaggaataaatccgagtcacaatg tgatgaggacggtatgacctctagcctgtccgagtctttgaaaacagagctcggg aaggatctgggcgtcgaagctaagatccctctgaatttggatggaccgcacccgt caccacccacacctgatagtgtcggccagttgcatattatggattataacgaaac tcatcatgagcacagctgagggtggcatccctgtgacccctccccagtgcctctc ctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaa gttgcatcattttgtctgactaggtgtccttctataatattatggggtggagggg ggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtc tattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccg cctcctgggttcaagcgattctcctgcctcagcctcccgagttgttgggattcca ggcatgcatgaccaggctcagctaatttttgtttttttggtagagacggggtttc accatattggccaggctggtctccaactcctaatctcaggtgatctacccacctt ggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgtcct tgcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcg ctcgctcactgaggccgcccgggctttgcccgggcggcctcagtgagcgagcgag cgcgcagITR-CMV- 13 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcga cctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaa TSC1- ctccatcactaggggttcctctcgagtagttattaatagtaatcaattacggggt huGHpA cattagttcatagcccatatatggagttccgcgttacataacttacggtaaatgg cccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtattAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:tacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgcc ccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatg accttatgggactttcctacttggcagtacatctacgtattagtcatcgctatta ccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactc acggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcac caaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaa tgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaa ccgtcagatcgccaccatggcccagcaggcgaatgtgggtgagctccttgcaatg ctggacagtcccatgttgggcgtccgagacgacgttactgccgtgttcaaagaaa accttaatagtgacaggggaccaatgctcgttaatactcttgtcgactattacct ggagacatcaagccagcctgccttgcatatcttgacgacgttgcaggaaccgcac gacaagcatcttctggatcgaataaacgagtatgtgggcaaagcagcaactcgcc tctcaattctctccctcttgggacacgtcatacgactccagccatcctggaagca taagctgagtcaggcccccttgctgcctagccttctgaagtgccttaagatggac acggatgtggtcgttctcactaccggagtgttggttcttatcacaatgctgccta tgattcctcagtctggaaagcagcacctgctcgatttctttgacatatttggacg cctttcttcttggtgtttgaaaaaaccgggacatgtagctgaagtctacttggtg cacctccatgcttctgtgtacgcattgtttcataggctttatggtatgtacccct gtaatttcgttagttttctccgatctcactacagtatgaaagaaaatctggaaac gtttgaagaggtcgtcaaaccgatgatggagcacgtaaggatacaccccgagttg gtaactggtagtaaggatcacgaacttgaccctcggagatggaaaaggttggaga ctcacgacgttgtaatcgaatgtgctaagataagccttgacccaaccgaggctag ct atgaggatggct at agcgtt agecat cagataagtgccagattcccacatcgg agcgcagatgtgacgacatcaccgtatgctgacacgcagaactcttacggttgtg ccacctctactccttacagtacatctcggttgatgcttcttaatatgcccgggca gctcccacagacactttccagcccgagtacacgcctcataaccgagccaccacag gcaacactctggtcaccaagcatggtttgtggaatgactacgccgccgacaagcc cagggaatgtcccccccgatctgagtcacccgtattctaaggtcttcgggacaac ggccggtgggaaggggactccattgggtactccggctacctcacctccgccggct cctctctgccactctgatgattacgttcacatcagtctgccccaagcgacagtga cccctccccgcaaagaagaaaggatggattcagctagaccttgcctgcaccgaca acatcaccttctcaacgatcggggaagcgaagaaccgcccggaagcaaagggtcc gttacgttgtctgacttgcccggattccttggtgacctggcatcagaggaagatt ctatagaaaaagataaggaagaagctgccattagccgagaactttcagaaatcac cacagccgaggctgaaccagttgttccacggggcggcttcgattcccctttttat agagattctttgccaggaagccaaaggaagacccattctgcggctagcagcagtc agggagcaagtgtcaaccctgagccccttcattcctcactcgacaagcttggacc tgatacaccaaagcaggcttttactccgatcgacttgccgtgtggaagtgctgat gagtctcccgccggagacagggagtgccagacttcactcgaaacctcaattttca cgccct caeca tgtaaaattcccccgcccactcgggtggggtttggctcaggtca gccacccccttacgatcacctgtttgaggttgctttgcccaagaccgcccaccac ttcgttatccgaaagacagaggaactcctcaaaaaagctaagggcaacactgagg aagacggtgtgccaagtacctctcctatggaggtcctggatcgcttgatccagca gggtgcggatgcccatagtaaagaactcaacaagttgccgttgccgtctaagtct gtcgattggactcatttcgggggaagcccccctagcgatgaaataagaacgctcc gagaccaactgcttcttttgcacaaccaactcttgtacgagcgcttcaagaggca acagcacgcactccggaatcgccggctcttgaggaaagtaataaaggcagccgct ctggaggagcacaatgcggcgatgaaggaccagttgaagcttcaggagaaagaca tccaaatgtggaaggtctccctccagaaagaacaagcgcggtataatcagctgca agaacaacgagacactatggttacaaaactgcactctcaaataaggcaactccag catgacagagaagagttttacaaccagtcccaagaattgcaaaccaaacttgaag actgccgcaacatgatcgctgagttgaggatcgagctcaaaaaggcaaacaacaaagtgtgtcacacagagcttctcctcagtcaggtttcccagaagctttccaactccAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:gaatctgtgcagcaacagatggagttccttaatcgccaactcctggtgcttggtg aggtaaacgagctgtatttggaacaacttcagaacaaacatagcgatacgactaa agaagtagaaatgatgaaggcagcataccggaaggagcttgagaaaaacagaagt cacgtcctccaacaaacacaaagactcgacacatctcagaaacggattctggagc tggagagt cat ttggcgaaaaaggatcatctgttgcttgagcagaaaaagt acct ggaagatgttaagctccaggctagggggcagctccaagctgctgaaagcagatat gaagcacaaaagcggattacgcaagttttcgagttggaaatcctggacctgtacg gaagattggaaaaggatggtctcctcaaaaaactggaggaggaaaaagcggaagc agcggaggccgccgaggagaggctcgactgctgcaatgacggctgctcagactcc atggttggtcacaacgaggaggctagcggtcataatggagagacaaaaacaccga ggccgagctccgcgaggggttccagtggctctaggggtggaggtggcagcagctc ttcttccagcgaactgtccactcctgagaaaccccctcaccaacgggcaggcccc ttctcatctaggtgggagaccacgatgggtgaagcgagcgcctccattccaacta ccgtaggcagcctcccctcttccaaaagtttcctcggaatgaaagcccgcgagct gttcaggaataaatccgagtcacaatgtgatgaggacggtatgacctctagcctg tccgagtctttgaaaacagagctcgggaaggatctgggcgtcgaagctaagatcc ctctgaatttggatggaccgcacccgtcaccacccacacctgatagtgtcggcca gttgcatattatggattataacgaaactcatcatgagcacagctgagggtggcat ccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgc ccaccagccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtc cttctataatattatggggtggaggggggtggtatggagcaaggggcaagttggg aagacaacctgtagggcctgcggggtctattgggaaccaagctggagtgcagtgg cacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctgcc tcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaattt ttgtttttttggtagagacggggtttcaccatattggccaggctggtctccaact cctaatctcaggtgatctacccaccttggcctcccaaattgctgggattacaggc gtgaaccactgctcccttccctgtccttgcggccgcaggaacccctagtgatgga gttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaag gtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgca gITR-CMV- 14 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcga cctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaa TSC1- ctccatcactaggggttcctctcgagtagttattaatagtaatcaattacggggt bGHpA_v3 cattagttcatagcccatatatggagttccgcgttacataacttacggtaaatgg cccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtat gttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatt tacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgcc ccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatg accttatgggactttcctacttggcagtacatctacgtattagtcatcgctatta ccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactc acggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcac caaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaa tgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaa ccgtcagatcgccaccatggcccagcaggcgaatgtgggtgagctccttgcaatg ctggacagtcccatgttgggcgtccgagacgacgttactgccgtgttcaaagaaa accttaatagtgacaggggaccaatgctcgttaatactcttgtcgactattacct ggagacatcaagccagcctgccttgcatatcttgacgacgttgcaggaaccgcac gacaagcatcttctggatcgaataaacgagtatgtgggcaaagcagcaactcgcc tctcaattctctccctcttgggacacgtcatacgactccagccatcctggaagca taagctgagtcaggcccccttgctgcctagccttctgaagtgccttaagatggac acggatgtggtcgttctcactaccggagtgttggttcttatcacaatgctgcctatgattcctcagtctggaaagcagcacctgctcgatttctttgacatatttggacgAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:cctttcttcttggtgtttgaaaaaaccgggacatgtagctgaagtctacttggtg cacctccatgcttctgtgtacgcattgtttcataggctttatggtatgtacccct gtaatttcgttagttttctccgatctcactacagtatgaaagaaaatctggaaac gtttgaagaggtcgtcaaaccgatgatggagcacgtaaggatacaccccgagttg gtaactggtagtaaggatcacgaacttgaccctcggagatggaaaaggttggaga ctcacgacgttgtaatcgaatgtgctaagataagccttgacccaaccgaggctag ct atgaggatggct at agcgtt agecat cagataagtgccagattcccacatcgg agcgcagatgtgacgacatcaccgtatgctgacacgcagaactcttacggttgtg ccacctctactccttacagtacatctcggttgatgcttcttaatatgcccgggca gctcccacagacactttccagcccgagtacacgcctcataaccgagccaccacag gcaacactctggtcaccaagcatggtttgtggaatgactacgccgccgacaagcc cagggaatgtcccccccgatctgagtcacccgtattctaaggtcttcgggacaac ggccggtgggaaggggactccattgggtactccggctacctcacctccgccggct cctctctgccactctgatgattacgttcacatcagtctgccccaagcgacagtga cccctccccgcaaagaagaaaggatggattcagctagaccttgcctgcaccgaca acatcaccttctcaacgatcggggaagcgaagaaccgcccggaagcaaagggtcc gttacgttgtctgacttgcccggattccttggtgacctggcatcagaggaagatt ctatagaaaaagataaggaagaagctgccattagccgagaactttcagaaatcac cacagccgaggctgaaccagttgttccacggggcggcttcgattcccctttttat agagattctttgccaggaagccaaaggaagacccattctgcggctagcagcagtc agggagcaagtgtcaaccctgagccccttcattcctcactcgacaagcttggacc tgatacaccaaagcaggcttttactccgatcgacttgccgtgtggaagtgctgat gagtctcccgccggagacagggagtgccagacttcactcgaaacctcaattttca cgccct caeca tgtaaaattcccccgcccactcgggtggggtttggctcaggtca gccacccccttacgatcacctgtttgaggttgctttgcccaagaccgcccaccac ttcgttatccgaaagacagaggaactcctcaaaaaagctaagggcaacactgagg aagacggtgtgccaagtacctctcctatggaggtcctggatcgcttgatccagca gggtgcggatgcccatagtaaagaactcaacaagttgccgttgccgtctaagtct gtcgattggactcatttcgggggaagcccccctagcgatgaaataagaacgctcc gagaccaactgcttcttttgcacaaccaactcttgtacgagcgcttcaagaggca acagcacgcactccggaatcgccggctcttgaggaaagtaataaaggcagccgct ctggaggagcacaatgcggcgatgaaggaccagttgaagcttcaggagaaagaca tccaaatgtggaaggtctccctccagaaagaacaagcgcggtataatcagctgca agaacaacgagacactatggttacaaaactgcactctcaaataaggcaactccag catgacagagaagagttttacaaccagtcccaagaattgcaaaccaaacttgaag actgccgcaacatgatcgctgagttgaggatcgagctcaaaaaggcaaacaacaa agtgtgtcacacagagcttctcctcagtcaggtttcccagaagctttccaactcc gaatctgtgcagcaacagatggagttccttaatcgccaactcctggtgcttggtg aggtaaacgagctgtatttggaacaacttcagaacaaacatagcgatacgactaa agaagtagaaatgatgaaggcagcataccggaaggagcttgagaaaaacagaagt cacgtcctccaacaaacacaaagactcgacacatctcagaaacggattctggagc tggagagt cat ttggcgaaaaaggatcatctgttgcttgagcagaaaaagt acct ggaagatgttaagctccaggctagggggcagctccaagctgctgaaagcagatat gaagcacaaaagcggattacgcaagttttcgagttggaaatcctggacctgtacg gaagattggaaaaggatggtctcctcaaaaaactggaggaggaaaaagcggaagc agcggaggccgccgaggagaggctcgactgctgcaatgacggctgctcagactcc atggttggtcacaacgaggaggctagcggtcataatggagagacaaaaacaccga ggccgagctccgcgaggggttccagtggctctaggggtggaggtggcagcagctc ttcttccagcgaactgtccactcctgagaaaccccctcaccaacgggcaggcccc ttctcatctaggtgggagaccacgatgggtgaagcgagcgcctccattccaacta ccgtaggcagcctcccctcttccaaaagtttcctcggaatgaaagcccgcgagct gttcaggaataaatccgagtcacaatgtgatgaggacggtatgacctctagcctgtccgagtctttgaaaacagagctcgggaaggatctgggcgtcgaagctaagatccAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:ctctgaatttggatggaccgcacccgtcaccacccacacctgatagtgtcggcca gttgcatattatggattataacgaaactcatcatgagcacagctgatactgtgcc ttctagttgccagccatctgttgtttgcccctcccccttgccttccttgaccctg gaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcacatt gtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaaggg ggaggattgggaagacaatagcaggcatgctggggatgcagtgggctctatgggc ggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctc gctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcg gcctcagtgagcgagcgagcgcgcagITR- 15 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcga CBA AGT cctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaa ctccatcactaggggttcctctcgagcgttacataacttacggtaaatggcccgc 2-TSC1- ctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcc bGH_v3 catagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacgg taaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgcccccta ttgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgacctt atgggactttcctacttggcagtacatcacgttctgcttcactctccccatctcc cccccctccccacccccaattttgtatttatttattttttaattattttgtgcag cgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcga ggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcg ctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagc gaagcgcgcggcgggcgggagaactgaaaaaccagaaagttaactggtaagttta gtctttttgtcttttatttcaggtccggtggtgcaaatcaaagaactgctcctca gtggatgttgcctttacttctaggcctgtacggaagtgttacttctgctctaaaa gctgccaccatggcccagcaggcgaatgtgggtgagctccttgcaatgctggaca gtcccatgttgggcgtccgagacgacgttactgccgtgttcaaagaaaaccttaa tagtgacaggggaccaatgctcgttaatactcttgtcgactattacctggagaca tcaagccagcctgccttgcatatcttgacgacgttgcaggaaccgcacgacaagc atcttctggatcgaataaacgagtatgtgggcaaagcagcaactcgcctctcaat tctctccctcttgggacacgtcatacgactccagccatcctggaagcataagctg agtcaggcccccttgctgcctagccttctgaagtgccttaagatggacacggatg tggtcgttctcactaccggagtgttggttcttatcacaatgctgcctatgattcc tcagtctggaaagcagcacctgctcgatttctttgacatatttggacgcctttct tcttggtgtttgaaaaaaccgggacatgtagctgaagtctacttggtgcacctcc atgcttctgtgtacgcattgtttcataggctttatggtatgtacccctgtaattt cgttagttttctccgatctcactacagtatgaaagaaaatctggaaacgtttgaa gaggtcgtcaaaccgatgatggagcacgtaaggatacaccccgagttggtaactg gtagtaaggatcacgaacttgaccctcggagatggaaaaggttggagactcacga cgttgtaatcgaatgtgctaagataagccttgacccaaccgaggctagctatgag gatggctatagcgtt agecat cagataagtgccagattcccacatcggagcgcag atgtgacgacatcaccgtatgctgacacgcagaactcttacggttgtgccacctc tactccttacagtacatctcggttgatgcttcttaatatgcccgggcagctccca cagacactttccagcccgagtacacgcctcataaccgagccaccacaggcaacac tctggtcaccaagcatggtttgtggaatgactacgccgccgacaagcccagggaa tgtcccccccgatctgagtcacccgtattctaaggtcttcgggacaacggccggt gggaaggggactccattgggtactccggctacctcacctccgccggctcctctct gccactctgatgattacgttcacatcagtctgccccaagcgacagtgacccctcc ccgcaaagaagaaaggatggattcagctagaccttgcctgcaccgacaacatcac cttctcaacgatcggggaagcgaagaaccgcccggaagcaaagggtccgttacgt tgtctgacttgcccggattccttggtgacctggcatcagaggaagattctataga aaaagataaggaagaagctgccattagccgagaactttcagaaatcaccacagccgaggctgaaccagttgttccacggggcggcttcgattcccctttttatagagattAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:ctttgccaggaagccaaaggaagacccattctgcggctagcagcagtcagggagc aagtgtcaaccctgagccccttcattcctcactcgacaagcttggacctgataca ccaaagcaggcttttactccgatcgacttgccgtgtggaagtgctgatgagtctc ccgccggagacagggagtgccagacttcactcgaaacctcaattttcacgccctc accatgtaaaattcccccgcccactcgggtggggtttggctcaggtcagccaccc ccttacgatcacctgtttgaggttgctttgcccaagaccgcccaccacttcgtta tccgaaagacagaggaactcctcaaaaaagctaagggcaacactgaggaagacgg tgtgccaagtacctctcctatggaggtcctggatcgcttgatccagcagggtgcg gatgcccatagtaaagaactcaacaagttgccgttgccgtctaagtctgtcgatt ggactcatttcgggggaagcccccctagcgatgaaataagaacgctccgagacca actgcttcttttgcacaaccaactcttgtacgagcgcttcaagaggcaacagcac gcactccggaatcgccggctcttgaggaaagtaataaaggcagccgctctggagg agcacaatgcggcgatgaaggaccagttgaagcttcaggagaaagacatccaaat gtggaaggtctccctccagaaagaacaagcgcggtataatcagctgcaagaacaa cgagacactatggttacaaaactgcactctcaaataaggcaactccagcatgaca gagaagagttttacaaccagtcccaagaattgcaaaccaaacttgaagactgccg caacatgatcgctgagttgaggatcgagctcaaaaaggcaaacaacaaagtgtgt cacacagagcttctcctcagtcaggtttcccagaagctttccaactccgaatctg tgcagcaacagatggagttccttaatcgccaactcctggtgcttggtgaggtaaa cgagctgtatttggaacaacttcagaacaaacatagcgatacgactaaagaagta gaaatgatgaaggcagcataccggaaggagcttgagaaaaacagaagtcacgtcc tccaacaaacacaaagactcgacacatctcagaaacggattctggagctggagag tcatttggcgaaaaaggatcatctgttgcttgagcagaaaaagtacctggaagat gttaagctccaggctagggggcagctccaagctgctgaaagcagatatgaagcac aaaagcggattacgcaagttttcgagttggaaatcctggacctgtacggaagatt ggaaaaggatggtctcctcaaaaaactggaggaggaaaaagcggaagcagcggag gccgccgaggagaggctcgactgctgcaatgacggctgctcagactccatggttg gtcacaacgaggaggctagcggtcataatggagagacaaaaacaccgaggccgag ctccgcgaggggttccagtggctctaggggtggaggtggcagcagctcttcttcc agcgaactgtccactcctgagaaaccccctcaccaacgggcaggccccttctcat ctaggtgggagaccacgatgggtgaagcgagcgcctccattccaactaccgtagg cagcctcccctcttccaaaagtttcctcggaatgaaagcccgcgagctgttcagg aataaatccgagtcacaatgtgatgaggacggtatgacctctagcctgtccgagt ctttgaaaacagagctcgggaaggatctgggcgtcgaagctaagatccctctgaa tttggatggaccgcacccgtcaccacccacacctgatagtgtcggccagttgcat attatggattataacgaaactcatcatgagcacagctgatactgtgccttctagt tgccagccatctgttgtttgcccctcccccttgccttccttgaccctggaaggtg ccactcccactgtcctttcctaataaaatgaggaaattgcatcacattgtctgag taggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggat tgggaagacaatagcaggcatgctggggatgcagtgggctctatgggcggccgca ggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcact gaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcag tgagcgagcgagcgcgcagITR- 16 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcga CBA AGT cctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaa ctccatcactaggggttcctctcgagcgttacataacttacggtaaatggcccgc 1-TSC1- ctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcc bGH_v3 catagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacgg taaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgcccccta ttgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgacctt atgggactttcctacttggcagtacatctatcgaggccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattaAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:ttttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcgg ggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatca gagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccc tataaaaagcgaagcgcgcggcgggcgggagcgggatcagccaccgcggtggcgg cctagagtcgacgagaactgaaaaaccagaaagttaactggtaagtttagtcttt ttgtcttttatttcaggtcccggatccggtggtggtgcaaatcaaagaactgctc ctcagtggatgttgcctttacttctaggcctgtacggaagtgttacttctgctct aaaagctgccaccatggcccagcaggcgaatgtgggtgagctccttgcaatgctg gacagtcccatgttgggcgtccgagacgacgttactgccgtgttcaaagaaaacc ttaatagtgacaggggaccaatgctcgttaatactcttgtcgactattacctgga gacatcaagccagcctgccttgcatatcttgacgacgttgcaggaaccgcacgac aagcatcttctggatcgaataaacgagtatgtgggcaaagcagcaactcgcctct caattctctccctcttgggacacgtcatacgactccagccatcctggaagcataa gctgagtcaggcccccttgctgcctagccttctgaagtgccttaagatggacacg gatgtggtcgttctcactaccggagtgttggttcttatcacaatgctgcctatga ttcctcagtctggaaagcagcacctgctcgatttctttgacatatttggacgcct ttcttcttggtgtttgaaaaaaccgggacatgtagctgaagtctacttggtgcac ctccatgcttctgtgtacgcattgtttcataggctttatggtatgtacccctgta atttcgttagttttctccgatctcactacagtatgaaagaaaatctggaaacgtt tgaagaggtcgtcaaaccgatgatggagcacgtaaggatacaccccgagttggta actggtagtaaggatcacgaacttgaccctcggagatggaaaaggttggagactc acgacgttgtaatcgaatgtgctaagataagccttgacccaaccgaggctagcta tgaggatggct at agcgtt agecat cagataagtgccagattcccacatcggagc gcagatgtgacgacatcaccgtatgctgacacgcagaactcttacggttgtgcca cctctactccttacagtacatctcggttgatgcttcttaatatgcccgggcagct cccacagacactttccagcccgagtacacgcctcataaccgagccaccacaggca acactctggtcaccaagcatggtttgtggaatgactacgccgccgacaagcccag ggaatgtcccccccgatctgagtcacccgtattctaaggtcttcgggacaacggc cggtgggaaggggactccattgggtactccggctacctcacctccgccggctcct ctctgccactctgatgattacgttcacatcagtctgccccaagcgacagtgaccc ctccccgcaaagaagaaaggatggattcagctagaccttgcctgcaccgacaaca tcaccttctcaacgatcggggaagcgaagaaccgcccggaagcaaagggtccgtt acgttgtctgacttgcccggattccttggtgacctggcatcagaggaagattcta tagaaaaagataaggaagaagctgccattagccgagaactttcagaaatcaccac agccgaggctgaaccagttgttccacggggcggcttcgattcccctttttataga gattctttgccaggaagccaaaggaagacccattctgcggctagcagcagtcagg gagcaagtgtcaaccctgagccccttcattcctcactcgacaagcttggacctga tacaccaaagcaggcttttactccgatcgacttgccgtgtggaagtgctgatgag tctcccgccggagacagggagtgccagacttcactcgaaacctcaattttcacgc cct caeca tgtaaaattcccccgcccactcgggtggggtttggctcaggtcagcc acccccttacgatcacctgtttgaggttgctttgcccaagaccgcccaccacttc gttatccgaaagacagaggaactcctcaaaaaagctaagggcaacactgaggaag acggtgtgccaagtacctctcctatggaggtcctggatcgcttgatccagcaggg tgcggatgcccatagtaaagaactcaacaagttgccgttgccgtctaagtctgtc gattggactcatttcgggggaagcccccctagcgatgaaataagaacgctccgag accaactgcttcttttgcacaaccaactcttgtacgagcgcttcaagaggcaaca gcacgcactccggaatcgccggctcttgaggaaagtaataaaggcagccgctctg gaggagcacaatgcggcgatgaaggaccagttgaagcttcaggagaaagacatcc aaatgtggaaggtctccctccagaaagaacaagcgcggtataatcagctgcaaga acaacgagacactatggttacaaaactgcactctcaaataaggcaactccagcat gacagagaagagttttacaaccagtcccaagaattgcaaaccaaacttgaagact gccgcaacatgatcgctgagttgaggat egage tcaaaaaggcaaacaacaaagtgtgtcacacagagcttctcctcagtcaggtttcccagaagctttccaactccgaaAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:tctgtgcagcaacagatggagttccttaatcgccaactcctggtgcttggtgagg taaacgagctgtatttggaacaacttcagaacaaacatagcgatacgactaaaga agtagaaatgatgaaggcagcataccggaaggagcttgagaaaaacagaagtcac gtcctccaacaaacacaaagactcgacacatctcagaaacggattctggagctgg agagtcatttggcgaaaaaggatcatctgttgcttgagcagaaaaagtacctgga agatgttaagctccaggctagggggcagctccaagctgctgaaagcagatatgaa gcacaaaagcggattacgcaagttttcgagttggaaatcctggacctgtacggaa gattggaaaaggatggtctcctcaaaaaactggaggaggaaaaagcggaagcagc ggaggccgccgaggagaggctcgactgctgcaatgacggctgctcagactccatg gttggtcacaacgaggaggctagcggtcataatggagagacaaaaacaccgaggc cgagctccgcgaggggttccagtggctctaggggtggaggtggcagcagctcttc ttccagcgaactgtccactcctgagaaaccccctcaccaacgggcaggccccttc tcatctaggtgggagaccacgatgggtgaagcgagcgcctccattccaactaccg taggcagcctcccctcttccaaaagtttcctcggaatgaaagcccgcgagctgtt caggaataaatccgagtcacaatgtgatgaggacggtatgacctctagcctgtcc gagtctttgaaaacagagctcgggaaggatctgggcgtcgaagctaagatccctc tgaatttggatggaccgcacccgtcaccacccacacctgatagtgtcggccagtt gcatattatggattataacgaaactcatcatgagcacagctgatactgtgccttc tagttgccagccatctgttgtttgcccctcccccttgccttccttgaccctggaa ggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcacattgtc tgagtaggtgtcattctattctggggggtggggtggggcaggacagcaaggggga ggattgggaagacaatagcaggcatgctggggatgcagtgggctctatgggcggc cgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgct cactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcc tcagtgagcgagcgagcgcgcagEFS-TSC1- 17 ggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagt bGHv3 tggggggaggggtcggcaattgatccggtgcctagagaaggtggcgcggggtaaa ctgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaa ccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccg ccagaacacagggccaccatggcccagcaggcgaatgtgggtgagctccttgcaa tgctggacagtcccatgttgggcgtccgagacgacgttactgccgtgttcaaaga aaaccttaatagtgacaggggaccaatgctcgttaatactcttgtcgactattac ctggagacatcaagccagcctgccttgcatatcttgacgacgttgcaggaaccgc acgacaagcatcttctggatcgaataaacgagtatgtgggcaaagcagcaactcg cctctcaattctctccctcttgggacacgtcatacgactccagccatcctggaag cataagctgagtcaggcccccttgctgcctagccttctgaagtgccttaagatgg acacggatgtggtcgttctcactaccggagtgttggttcttatcacaatgctgcc tatgattcctcagtctggaaagcagcacctgctcgatttctttgacatatttgga cgcctttcttcttggtgtttgaaaaaaccgggacatgtagctgaagtctacttgg tgcacctccatgcttctgtgtacgcattgtttcataggctttatggtatgtaccc ctgtaatttcgttagttttctccgatctcactacagtatgaaagaaaatctggaa acgtttgaagaggtcgtcaaaccgatgatggagcacgtaaggatacaccccgagt tggtaactggtagtaaggatcacgaacttgaccctcggagatggaaaaggttgga gactcacgacgttgtaatcgaatgtgctaagataagccttgacccaaccgaggct agctatgaggatggct at agcgtt agecat cagataagtgccagattcccacatc ggagcgcagatgtgacgacatcaccgtatgctgacacgcagaactcttacggttg tgccacctctactccttacagtacatctcggttgatgcttcttaatatgcccggg cagctcccacagacactttccagcccgagtacacgcctcataaccgagccaccac aggcaacactctggtcaccaagcatggtttgtggaatgactacgccgccgacaag cccagggaatgtcccccccgatctgagtcacccgtattctaaggtcttcgggaca acggccggtgggaaggggactccattgggtactccggctacctcacctccgccggctcctctctgccactctgatgattacgttcacatcagtctgccccaagcgacagtAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:gacccctccccgcaaagaagaaaggatggattcagctagaccttgcctgcaccga caacatcaccttctcaacgatcggggaagcgaagaaccgcccggaagcaaagggt ccgttacgttgtctgacttgcccggattccttggtgacctggcatcagaggaaga ttctatagaaaaagataaggaagaagctgccattagccgagaactttcagaaatc accacagccgaggctgaaccagttgttccacggggcggcttcgattccccttttt atagagattctttgccaggaagccaaaggaagacccattctgcggctagcagcag tcagggagcaagtgtcaaccctgagccccttcattcctcactcgacaagcttgga cctgatacaccaaagcaggcttttactccgatcgacttgccgtgtggaagtgctg atgagtctcccgccggagacagggagtgccagacttcactcgaaacctcaatttt cacgccct caeca tgtaaaattcccccgcccactcgggtggggtttggctcaggt cagccacccccttacgatcacctgtttgaggttgctttgcccaagaccgcccacc acttcgttatccgaaagacagaggaactcctcaaaaaagctaagggcaacactga ggaagacggtgtgccaagtacctctcctatggaggtcctggatcgcttgatccag cagggtgcggatgcccatagtaaagaactcaacaagttgccgttgccgtctaagt ctgtcgattggactcatttcgggggaagcccccctagcgatgaaataagaacgct ccgagaccaactgcttcttttgcacaaccaactcttgtacgagcgcttcaagagg caacagcacgcactccggaatcgccggctcttgaggaaagtaataaaggcagccg ctctggaggagcacaatgcggcgatgaaggaccagttgaagcttcaggagaaaga catccaaatgtggaaggtctccctccagaaagaacaagcgcggtataatcagctg caagaacaacgagacactatggttacaaaactgcactctcaaataaggcaactcc agcatgacagagaagagttttacaaccagtcccaagaattgcaaaccaaacttga agactgccgcaacatgatcgctgagttgaggat egage tcaaaaaggcaaacaac aaagtgtgtcacacagagcttctcctcagtcaggtttcccagaagctttccaact ccgaatctgtgcagcaacagatggagttccttaatcgccaactcctggtgcttgg tgaggtaaacgagctgtatttggaacaacttcagaacaaacatagcgatacgact aaagaagtagaaatgatgaaggcagcataccggaaggagcttgagaaaaacagaa gtcacgtcctccaacaaacacaaagactcgacacatctcagaaacggattctgga gctggagagtcatttggcgaaaaaggatcatctgttgcttgagcagaaaaagtac ctggaagatgttaagctccaggctagggggcagctccaagctgctgaaagcagat atgaagcacaaaagcggattacgcaagttttcgagttggaaatcctggacctgta cggaagattggaaaaggatggtctcctcaaaaaactggaggaggaaaaagcggaa gcagcggaggccgccgaggagaggctcgactgctgcaatgacggctgctcagact ccatggttggtcacaacgaggaggctagcggtcataatggagagacaaaaacacc gaggccgagctccgcgaggggttccagtggctctaggggtggaggtggcagcagc tcttcttccagcgaactgtccactcctgagaaaccccctcaccaacgggcaggcc ccttctcatctaggtgggagaccacgatgggtgaagcgagcgcctccattccaac taccgtaggcagcctcccctcttccaaaagtttcctcggaatgaaagcccgcgag ctgttcaggaataaatccgagtcacaatgtgatgaggacggtatgacctctagcc tgtccgagtctttgaaaacagagctcgggaaggatctgggcgtcgaagctaagat ccctctgaatttggatggaccgcacccgtcaccacccacacctgatagtgtcggc cagttgcatattatggattataacgaaactcatcatgagcacagctgatactgtg ccttctagttgccagccatctgttgtttgcccctcccccttgccttccttgaccc tggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcaca ttgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaag ggggaggattgggaagacaatagcaggcatgctggggatgcagtgggctctatgg CMV- 18 attaatagtaatcaattacggggtcattagttcatagcccatatatggagttccg cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgc TSCl-bGHccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttcc attgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatca agtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggccc gcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaaAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:tgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgac gtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgta acaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtcta tataagcagagctcgtttagtgaaccgtcagatccgctagcgccaccatggccca gcaggcgaatgtgggtgagctccttgcaatgctggacagtcccatgttgggcgtc cgagacgacgttactgccgtgttcaaagaaaaccttaatagtgacaggggaccaa tgctcgttaatactcttgtcgactattacctggagacatcaagccagcctgcctt gcatatcttgacgacgttgcaggaaccgcacgacaagcatcttctggatcgaata aacgagtatgtgggcaaagcagcaactcgcctctcaattctctccctcttgggac acgtcatacgactccagccatcctggaagcataagctgagtcaggcccccttgct gcctagccttctgaagtgccttaagatggacacggatgtggtcgttctcactacc ggagtgttggttcttatcacaatgctgcctatgattcctcagtctggaaagcagc acctgctcgatttctttgacatatttggacgcctttcttcttggtgtttgaaaaa accgggacatgtagctgaagtctacttggtgcacctccatgcttctgtgtacgca ttgtttcataggctttatggtatgtacccctgtaatttcgttagttttctccgat ctcactacagtatgaaagaaaatctggaaacgtttgaagaggtcgtcaaaccgat gatggagcacgtaaggatacaccccgagttggtaactggtagtaaggatcacgaa cttgaccctcggagatggaaaaggttggagactcacgacgttgtaatcgaatgtg ctaagataagccttgacccaaccgaggctagctatgaggatggctatagcgttag ccatcagataagtgccagattcccacatcggagcgcagatgtgacgacatcaccg tatgctgacacgcagaactcttacggttgtgccacctctactccttacagtacat ctcggttgatgcttcttaatatgcccgggcagctcccacagacactttccagccc gagtacacgcctcataaccgagccaccacaggcaacactctggtcaccaagcatg gtttgtggaatgactacgccgccgacaagcccagggaatgtcccccccgatctga gtcacccgtattctaaggtcttcgggacaacggccggtgggaaggggactccatt gggtactccggctacctcacctccgccggctcctctctgccactctgatgattac gttcacatcagtctgccccaagcgacagtgacccctccccgcaaagaagaaagga tggattcagctagaccttgcctgcaccgacaacatcaccttctcaacgatcgggg aagcgaagaaccgcccggaagcaaagggtccgttacgttgtctgacttgcccgga ttccttggtgacctggcatcagaggaagattctatagaaaaagataaggaagaag ctgccattagccgagaactttcagaaatcaccacagccgaggctgaaccagttgt tccacggggcggcttcgattcccctttttatagagattctttgccaggaagccaa aggaagacccattctgcggctagcagcagtcagggagcaagtgtcaaccctgagc cccttcattcctcactcgacaagcttggacctgatacaccaaagcaggcttttac tccgatcgacttgccgtgtggaagtgctgatgagtctcccgccggagacagggag tgccagacttcactcgaaacctcaattttcacgccctcaccatgtaaaattcccc cgcccactcgggtggggtttggctcaggtcagccacccccttacgatcacctgtt tgaggttgctttgcccaagaccgcccaccacttcgttatccgaaagacagaggaa ctcctcaaaaaagctaagggcaacactgaggaagacggtgtgccaagtacctctc ctatggaggtcctggatcgcttgatccagcagggtgcggatgcccatagtaaaga actcaacaagttgccgttgccgtctaagtctgtcgattggactcatttcggggga agcccccctagcgatgaaataagaacgctccgagaccaactgcttcttttgcaca accaactcttgtacgagcgcttcaagaggcaacagcacgcactccggaatcgccg gctcttgaggaaagtaataaaggcagccgctctggaggagcacaatgcggcgatg aaggaccagttgaagcttcaggagaaagacatccaaatgtggaaggtctccctcc agaaagaacaagcgcggtataatcagctgcaagaacaacgagacactatggttac aaaactgcactctcaaataaggcaactccagcatgacagagaagagttttacaac cagtcccaagaattgcaaaccaaacttgaagactgccgcaacatgatcgctgagt tgaggatcgagctcaaaaaggcaaacaacaaagtgtgtcacacagagcttctcct cagtcaggtttcccagaagctttccaactccgaatctgtgcagcaacagatggag ttccttaatcgccaactcctggtgcttggtgaggtaaacgagctgtatttggaac aacttcagaacaaacatagcgatacgactaaagaagtagaaatgatgaaggcagcataccggaaggagcttgagaaaaacagaagtcacgtcctccaacaaacacaaagaAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:ctcgacacatctcagaaacggattctggagctggagagtcatttggcgaaaaagg atcatctgttgcttgagcagaaaaagtacctggaagatgttaagctccaggctag ggggcagctccaagctgctgaaagcagatatgaagcacaaaagcggattacgcaa gttttcgagttggaaatcctggacctgtacggaagattggaaaaggatggtctcc tcaaaaaactggaggaggaaaaagcggaagcagcggaggccgccgaggagaggct cgactgctgcaatgacggctgctcagactccatggttggtcacaacgaggaggct agcggtcataatggagagacaaaaacaccgaggccgagctccgcgaggggttcca gtggctctaggggtggaggtggcagcagctcttcttccagcgaactgtccactcc tgagaaaccccctcaccaacgggcaggccccttctcatctaggtgggagaccacg atgggtgaagcgagcgcctccattccaactaccgtaggcagcctcccctcttcca aaagtttcctcggaatgaaagcccgcgagctgttcaggaataaatccgagtcaca atgtgatgaggacggtatgacctctagcctgtccgagtctttgaaaacagagctc gggaaggatctgggcgtcgaagctaagatccctctgaatttggatggaccgcacc cgtcaccacccacacctgatagtgtcggccagttgcatattatggattataacga aactcatcatgagcacagcgagcagaagctgatcagcgaggaggacctgtgaggg gatccgtcgactagagctcgctgatcagcctcgactgtgccttctagttgccagc catctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcc cactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgt cattctattctggggggtggggtggggcaggacagcaagggggaggattgggaag acaatagcaggcatgctggggagagatctgaggEFS-TSC1- 19 ggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagt huGHpA tggggggaggggtcggcaattgatccggtgcctagagaaggtggcgcggggtaaa ctgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaa ccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccg ccagaacacagggccaccatggcccagcaggcgaatgtgggtgagctccttgcaa tgctggacagtcccatgttgggcgtccgagacgacgttactgccgtgttcaaaga aaaccttaatagtgacaggggaccaatgctcgttaatactcttgtcgactattac ctggagacatcaagccagcctgccttgcatatcttgacgacgttgcaggaaccgc acgacaagcatcttctggatcgaataaacgagtatgtgggcaaagcagcaactcg cctctcaattctctccctcttgggacacgtcatacgactccagccatcctggaag cataagctgagtcaggcccccttgctgcctagccttctgaagtgccttaagatgg acacggatgtggtcgttctcactaccggagtgttggttcttatcacaatgctgcc tatgattcctcagtctggaaagcagcacctgctcgatttctttgacatatttgga cgcctttcttcttggtgtttgaaaaaaccgggacatgtagctgaagtctacttgg tgcacctccatgcttctgtgtacgcattgtttcataggctttatggtatgtaccc ctgtaatttcgttagttttctccgatctcactacagtatgaaagaaaatctggaa acgtttgaagaggtcgtcaaaccgatgatggagcacgtaaggatacaccccgagt tggtaactggtagtaaggatcacgaacttgaccctcggagatggaaaaggttgga gactcacgacgttgtaatcgaatgtgctaagataagccttgacccaaccgaggct agctatgaggatggct at agcgtt agecat cagataagtgccagattcccacatc ggagcgcagatgtgacgacatcaccgtatgctgacacgcagaactcttacggttg tgccacctctactccttacagtacatctcggttgatgcttcttaatatgcccggg cagctcccacagacactttccagcccgagtacacgcctcataaccgagccaccac aggcaacactctggtcaccaagcatggtttgtggaatgactacgccgccgacaag cccagggaatgtcccccccgatctgagtcacccgtattctaaggtcttcgggaca acggccggtgggaaggggactccattgggtactccggctacctcacctccgccgg ctcctctctgccactctgatgattacgttcacatcagtctgccccaagcgacagt gacccctccccgcaaagaagaaaggatggattcagctagaccttgcctgcaccga caacatcaccttctcaacgatcggggaagcgaagaaccgcccggaagcaaagggt ccgttacgttgtctgacttgcccggattccttggtgacctggcatcagaggaaga ttctatagaaaaagataaggaagaagctgccattagccgagaactttcagaaatcaccacagccgaggctgaaccagttgttccacggggcggcttcgattcccctttttAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:atagagattctttgccaggaagccaaaggaagacccattctgcggctagcagcag tcagggagcaagtgtcaaccctgagccccttcattcctcactcgacaagcttgga cctgatacaccaaagcaggcttttactccgatcgacttgccgtgtggaagtgctg atgagtctcccgccggagacagggagtgccagacttcactcgaaacctcaatttt cacgccct caeca tgtaaaattcccccgcccactcgggtggggtttggctcaggt cagccacccccttacgatcacctgtttgaggttgctttgcccaagaccgcccacc acttcgttatccgaaagacagaggaactcctcaaaaaagctaagggcaacactga ggaagacggtgtgccaagtacctctcctatggaggtcctggatcgcttgatccag cagggtgcggatgcccatagtaaagaactcaacaagttgccgttgccgtctaagt ctgtcgattggactcatttcgggggaagcccccctagcgatgaaataagaacgct ccgagaccaactgcttcttttgcacaaccaactcttgtacgagcgcttcaagagg caacagcacgcactccggaatcgccggctcttgaggaaagtaataaaggcagccg ctctggaggagcacaatgcggcgatgaaggaccagttgaagcttcaggagaaaga catccaaatgtggaaggtctccctccagaaagaacaagcgcggtataatcagctg caagaacaacgagacactatggttacaaaactgcactctcaaataaggcaactcc agcatgacagagaagagttttacaaccagtcccaagaattgcaaaccaaacttga agactgccgcaacatgatcgctgagttgaggat egage tcaaaaaggcaaacaac aaagtgtgtcacacagagcttctcctcagtcaggtttcccagaagctttccaact ccgaatctgtgcagcaacagatggagttccttaatcgccaactcctggtgcttgg tgaggtaaacgagctgtatttggaacaacttcagaacaaacatagcgatacgact aaagaagtagaaatgatgaaggcagcataccggaaggagcttgagaaaaacagaa gtcacgtcctccaacaaacacaaagactcgacacatctcagaaacggattctgga gctggagagtcatttggcgaaaaaggatcatctgttgcttgagcagaaaaagtac ctggaagatgttaagctccaggctagggggcagctccaagctgctgaaagcagat atgaagcacaaaagcggattacgcaagttttcgagttggaaatcctggacctgta cggaagattggaaaaggatggtctcctcaaaaaactggaggaggaaaaagcggaa gcagcggaggccgccgaggagaggctcgactgctgcaatgacggctgctcagact ccatggttggtcacaacgaggaggctagcggtcataatggagagacaaaaacacc gaggccgagctccgcgaggggttccagtggctctaggggtggaggtggcagcagc tcttcttccagcgaactgtccactcctgagaaaccccctcaccaacgggcaggcc ccttctcatctaggtgggagaccacgatgggtgaagcgagcgcctccattccaac taccgtaggcagcctcccctcttccaaaagtttcctcggaatgaaagcccgcgag ctgttcaggaataaatccgagtcacaatgtgatgaggacggtatgacctctagcc tgtccgagtctttgaaaacagagctcgggaaggatctgggcgtcgaagctaagat ccctctgaatttggatggaccgcacccgtcaccacccacacctgatagtgtcggc cagttgcatattatggattataacgaaactcatcatgagcacagctgagggtggc atccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagt gcccaccagccttgtcctaataaaattaagttgcatcattttgtctgactaggtg tccttctataatattatggggtggaggggggtggtatggagcaaggggcaagttg ggaagacaacctgtagggcctgcggggtctattgggaaccaagctggagtgcagt ggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctg cctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaat ttttgtttttttggtagagacggggtttcaccatattggccaggctggtctccaa ctcctaatctcaggtgatctacccaccttggcctcccaaattgctgggattacag gcgtgaaccactgctcccttccctgtccttCMV- 20 cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgc ccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttcc TSC1- attgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatca huGHpA agtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggccc gcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtaca tctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:gtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgta acaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtcta tataagcagagctcgtttagtgaaccgtcagatcgccaccatggcccagcaggcg aatgtgggtgagctccttgcaatgctggacagtcccatgttgggcgtccgagacg acgttactgccgtgttcaaagaaaaccttaatagtgacaggggaccaatgctcgt taatactcttgtcgactattacctggagacatcaagccagcctgccttgcatatc ttgacgacgttgcaggaaccgcacgacaagcatcttctggatcgaataaacgagt atgtgggcaaagcagcaactcgcctctcaattctctccctcttgggacacgtcat acgactccagccatcctggaagcataagctgagtcaggcccccttgctgcctagc cttctgaagtgccttaagatggacacggatgtggtcgttctcactaccggagtgt tggttcttatcacaatgctgcctatgattcctcagtctggaaagcagcacctgct cgatttctttgacatatttggacgcctttcttcttggtgtttgaaaaaaccggga catgtagctgaagtctacttggtgcacctccatgcttctgtgtacgcattgtttc ataggctttatggtatgtacccctgtaatttcgttagttttctccgatctcacta cagtatgaaagaaaatctggaaacgtttgaagaggtcgtcaaaccgatgatggag cacgtaaggatacaccccgagttggtaactggtagtaaggatcacgaacttgacc ctcggagatggaaaaggttggagactcacgacgttgtaatcgaatgtgctaagat aagccttgacccaaccgaggctagctatgaggatggctatagcgttagccatcag ataagtgccagattcccacatcggagcgcagatgtgacgacatcaccgtatgctg acacgcagaactcttacggttgtgccacctctactccttacagtacatctcggtt gatgcttcttaatatgcccgggcagctcccacagacactttccagcccgagtaca cgcctcataaccgagccaccacaggcaacactctggtcaccaagcatggtttgtg gaatgactacgccgccgacaagcccagggaatgtcccccccgatctgagtcaccc gtattctaaggtcttcgggacaacggccggtgggaaggggactccattgggtact ccggctacctcacctccgccggctcctctctgccactctgatgattacgttcaca tcagtctgccccaagcgacagtgacccctccccgcaaagaagaaaggatggattc agctagaccttgcctgcaccgacaacatcaccttctcaacgatcggggaagcgaa gaaccgcccggaagcaaagggtccgttacgttgtctgacttgcccggattccttg gtgacctggcatcagaggaagattctatagaaaaagataaggaagaagctgccat tagccgagaactttcagaaatcaccacagccgaggctgaaccagttgttccacgg ggcggcttcgattcccctttttatagagattctttgccaggaagccaaaggaaga cccattctgcggctagcagcagtcagggagcaagtgtcaaccctgagccccttca ttcctcactcgacaagcttggacctgatacaccaaagcaggcttttactccgatc gacttgccgtgtggaagtgctgatgagtctcccgccggagacagggagtgccaga cttcactcgaaacctcaattttcacgccctcaccatgtaaaattcccccgcccac tcgggtggggtttggctcaggtcagccacccccttacgatcacctgtttgaggtt gctttgcccaagaccgcccaccacttcgttatccgaaagacagaggaactcctca aaaaagctaagggcaacactgaggaagacggtgtgccaagtacctctcctatgga ggtcctggatcgcttgatccagcagggtgcggatgcccatagtaaagaactcaac aagttgccgttgccgtctaagtctgtcgattggactcatttcgggggaagccccc ctagcgatgaaataagaacgctccgagaccaactgcttcttttgcacaaccaact cttgtacgagcgcttcaagaggcaacagcacgcactccggaatcgccggctcttg aggaaagtaataaaggcagccgctctggaggagcacaatgcggcgatgaaggacc agttgaagcttcaggagaaagacatccaaatgtggaaggtctccctccagaaaga acaagcgcggtataatcagctgcaagaacaacgagacactatggttacaaaactg cactctcaaataaggcaactccagcatgacagagaagagttttacaaccagtccc aagaattgcaaaccaaacttgaagactgccgcaacatgatcgctgagttgaggat cgagctcaaaaaggcaaacaacaaagtgtgtcacacagagcttctcctcagtcag gtttcccagaagctttccaactccgaatctgtgcagcaacagatggagttcctta atcgccaactcctggtgcttggtgaggtaaacgagctgtatttggaacaacttca gaacaaacatagcgatacgactaaagaagtagaaatgatgaaggcagcataccgg aaggagcttgagaaaaacagaagtcacgtcctccaacaaacacaaagactcgacacatctcagaaacggattctggagctggagagtcatttggcgaaaaaggatcatctAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:gttgcttgagcagaaaaagtacctggaagatgttaagctccaggctagggggcag ctccaagctgctgaaagcagatatgaagcacaaaagcggattacgcaagttttcg agttggaaatcctggacctgtacggaagattggaaaaggatggtctcctcaaaaa actggaggaggaaaaagcggaagcagcggaggccgccgaggagaggctcgactgc tgcaatgacggctgctcagactccatggttggtcacaacgaggaggctagcggtc ataatggagagacaaaaacaccgaggccgagctccgcgaggggttccagtggctc taggggtggaggtggcagcagctcttcttccagcgaactgtccactcctgagaaa ccccctcaccaacgggcaggccccttctcatctaggtgggagaccacgatgggtg aagcgagcgcctccattccaactaccgtaggcagcctcccctcttccaaaagttt cctcggaatgaaagcccgcgagctgttcaggaataaatccgagtcacaatgtgat gaggacggtatgacctctagcctgtccgagtctttgaaaacagagctcgggaagg atctgggcgtcgaagctaagatccctctgaatttggatggaccgcacccgtcacc acccacacctgatagtgtcggccagttgcatattatggattataacgaaactcat catgagcacagctgagggtggcatccctgtgacccctccccagtgcctctcctgg ccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttg catcattttgtctgactaggtgtccttctataatattatggggtggaggggggtg gtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtctatt gggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctc ctgggttcaagcgattctcctgcctcagcctcccgagttgttgggattccaggca tgcatgaccaggctcagctaatttttgtttttttggtagagacggggtttcacca tattggccaggctggtctccaactcctaatctcaggtgatctacccaccttggcc tcccaaattgctgggattacaggcgtgaaccactgctcccttccctgtcctt CMV- 21 tagttattaatagtaatcaattacggggtcattagttcatagcccatatatggag ttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacc TSC1- cccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggac bGHpA v3 tttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagta catcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaat ggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggca gtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtac atcaatgggcgtggatagcggtttgactcacggggatttccaagtctccacccca ttgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatg tcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggag gtctatataagcagagctcgtttagtgaaccgtcagatcgccaccatggcccagc aggcgaatgtgggtgagctccttgcaatgctggacagtcccatgttgggcgtccg agacgacgttactgccgtgttcaaagaaaaccttaatagtgacaggggaccaatg ctcgttaatactcttgtcgactattacctggagacatcaagccagcctgccttgc atatcttgacgacgttgcaggaaccgcacgacaagcatcttctggatcgaataaa cgagtatgtgggcaaagcagcaactcgcctctcaattctctccctcttgggacac gtcatacgactccagccatcctggaagcataagctgagtcaggcccccttgctgc ctagccttctgaagtgccttaagatggacacggatgtggtcgttctcactaccgg agtgttggttcttatcacaatgctgcctatgattcctcagtctggaaagcagcac ctgctcgatttctttgacatatttggacgcctttcttcttggtgtttgaaaaaac cgggacatgtagctgaagtctacttggtgcacctccatgcttctgtgtacgcatt gtttcataggctttatggtatgtacccctgtaatttcgttagttttctccgatct cactacagtatgaaagaaaatctggaaacgtttgaagaggtcgtcaaaccgatga tggagcacgtaaggatacaccccgagttggtaactggtagtaaggatcacgaact tgaccctcggagatggaaaaggttggagactcacgacgttgtaatcgaatgtgct aagataagccttgacccaaccgaggctagctatgaggatggctatagcgttagcc atcagataagtgccagattcccacatcggagcgcagatgtgacgacatcaccgta tgctgacacgcagaactcttacggttgtgccacctctactccttacagtacatct cggttgatgcttcttaatatgcccgggcagctcccacagacactttccagcccgagtacacgcctcataaccgagccaccacaggcaacactctggtcaccaagcatggtAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:ttgtggaatgactacgccgccgacaagcccagggaatgtcccccccgatctgagt cacccgtattctaaggtcttcgggacaacggccggtgggaaggggactccattgg gt act ccggct acct cacctccgccggctcctctctgccactctgatgattacgt tcacatcagtctgccccaagcgacagtgacccctccccgcaaagaagaaaggatg gattcagctagaccttgcctgcaccgacaacatcaccttctcaacgatcggggaa gcgaagaaccgcccggaagcaaagggtccgttacgttgtctgacttgcccggatt ccttggtgacctggcatcagaggaagattctatagaaaaagataaggaagaagct gccattagccgagaactttcagaaatcaccacagccgaggctgaaccagttgttc cacggggcggcttcgattcccctttttatagagattctttgccaggaagccaaag gaagacccattctgcggctagcagcagtcagggagcaagtgtcaaccctgagccc cttcattcctcactcgacaagcttggacctgatacaccaaagcaggcttttactc cgatcgacttgccgtgtggaagtgctgatgagtctcccgccggagacagggagtg ccagacttcactcgaaacctcaattttcacgccctcaccatgtaaaattcccccg cccactcgggtggggtttggctcaggtcagccacccccttacgatcacctgtttg aggttgctttgcccaagaccgcccaccacttcgttatccgaaagacagaggaact cctcaaaaaagctaagggcaacactgaggaagacggtgtgccaagtacctctcct atggaggtcctggatcgcttgatccagcagggtgcggatgcccatagtaaagaac tcaacaagttgccgttgccgtctaagtctgtcgattggactcatttcgggggaag cccccctagcgatgaaataagaacgctccgagaccaactgcttcttttgcacaac caactcttgtacgagcgcttcaagaggcaacagcacgcactccggaatcgccggc tcttgaggaaagtaataaaggcagccgctctggaggagcacaatgcggcgatgaa ggaccagttgaagcttcaggagaaagacatccaaatgtggaaggtctccctccag aaagaacaagcgcggtataatcagctgcaagaacaacgagacactatggttacaa aactgcactctcaaataaggcaactccagcatgacagagaagagttttacaacca gtcccaagaattgcaaaccaaacttgaagactgccgcaacatgatcgctgagttg aggatcgagctcaaaaaggcaaacaacaaagtgtgtcacacagagcttctcctca gtcaggtttcccagaagctttccaactccgaatctgtgcagcaacagatggagtt ccttaatcgccaactcctggtgcttggtgaggtaaacgagctgtatttggaacaa cttcagaacaaacatagcgatacgactaaagaagtagaaatgatgaaggcagcat accggaaggagcttgagaaaaacagaagtcacgtcctccaacaaacacaaagact cgacacatctcagaaacggattctggagctggagagtcatttggcgaaaaaggat catctgttgcttgagcagaaaaagtacctggaagatgttaagctccaggctaggg ggcagctccaagctgctgaaagcagatatgaagcacaaaagcggattacgcaagt tttcgagttggaaatcctggacctgtacggaagattggaaaaggatggtctcctc aaaaaactggaggaggaaaaagcggaagcagcggaggccgccgaggagaggctcg actgctgcaatgacggctgctcagactccatggttggtcacaacgaggaggctag cggtcataatggagagacaaaaacaccgaggccgagctccgcgaggggttccagt ggctctaggggtggaggtggcagcagctcttcttccagcgaactgtccactcctg agaaaccccctcaccaacgggcaggccccttctcatctaggtgggagaccacgat gggtgaagcgagcgcctccattccaactaccgtaggcagcctcccctcttccaaa agtttcctcggaatgaaagcccgcgagctgttcaggaataaatccgagtcacaat gtgatgaggacggtatgacctctagcctgtccgagtctttgaaaacagagctcgg gaaggatctgggcgtcgaagctaagatccctctgaatttggatggaccgcacccg tcaccacccacacctgatagtgtcggccagttgcatattatggattataacgaaa ctcatcatgagcacagctgatactgtgccttctagttgccagccatctgttgttt gcccctcccccttgccttccttgaccctggaaggtgccactcccactgtcctttc ctaataaaatgaggaaattgcatcacattgtctgagtaggtgtcattctattctg gggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggc atgctggggatgcagtgggctctatggAttorney Docket No.: 38213.0001P1Name SEQ ID SequenceNO.:CBA AGT 22 cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgc ccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttcc 2-TSC1- attgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatca bGH_v3 agtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggccc gcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtaca tcacgttctgcttcactctccccatctcccccccctccccacccccaattttgta tttatttattttttaattattttgtgcagcgatgggggcgggggggggggggggg cgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagag gtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgag gcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagaactg aaaaaccagaaagttaactggtaagtttagtctttttgtcttttatttcaggtcc ggtggtgcaaatcaaagaactgctcctcagtggatgttgcctttacttctaggcc tgtacggaagtgttacttctgctctaaaagctgccaccatggcccagcaggcgaa tgtgggtgagctccttgcaatgctggacagtcccatgttgggcgtccgagacgac gttactgccgtgttcaaagaaaaccttaatagtgacaggggaccaatgctcgtta atactcttgtcgactattacctggagacatcaagccagcctgccttgcatatctt gacgacgttgcaggaaccgcacgacaagcatcttctggatcgaataaacgagtat gtgggcaaagcagcaactcgcctctcaattctctccctcttgggacacgtcatac gactccagccatcctggaagcataagctgagtcaggcccccttgctgcctagcct tctgaagtgccttaagatggacacggatgtggtcgttctcactaccggagtgttg gttcttatcacaatgctgcctatgattcctcagtctggaaagcagcacctgctcg atttctttgacatatttggacgcctttcttcttggtgtttgaaaaaaccgggaca t gt age tgaagtctacttggtgcacctccatgcttctgtgtacgcattgtt teat aggctttatggtatgtacccctgtaatttcgttagttttctccgatctcactaca gtatgaaagaaaatctggaaacgtttgaagaggtcgtcaaaccgatgatggagca cgtaaggatacaccccgagttggtaactggtagtaaggatcacgaacttgaccct cggagatggaaaaggttggagactcacgacgttgtaatcgaatgtgctaagataa gccttgacccaaccgaggctagctatgaggatggctatagcgtt agecat cagat aagtgccagattcccacatcggagcgcagatgtgacgacatcaccgtatgctgac acgcagaactcttacggttgtgccacctctactccttacagtacatctcggttga tgcttcttaatatgcccgggcagctcccacagacactttccagcccgagtacacg cctcataaccgagccaccacaggcaacactctggtcaccaagcatggtttgtgga atgactacgccgccgacaagcccagggaatgtcccccccgatctgagtcacccgt attctaaggtcttcgggacaacggccggtgggaaggggactccattgggtactcc ggctacctca...

Claims

Attorney Docket No.: 38213.0001P1CLAIMSWe claim:

1. A recombinant AAV vector construct comprising an expression cassette comprising a polynucleotide encoding a human TSC1 (hamartin) protein comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO.: 7, operably linked to a polynucleotide encoding an EFS promoter that drives expression of TSC1 in cells of the CNS, said expression cassette flanked by AAV inverted terminal repeats (ITRs).

2. The recombinant AAV vector construct of claim 1, wherein the TSC1 (hamartin) protein comprises the amino acid sequence of SEQ ID NO.: 7.

3. The recombinant AAV vector construct of claim 1, wherein the polynucleotide encoding an EFS promoter comprises the polynucleotide sequence of SEQ ID NO.: 24.

4. The recombinant AAV vector construct of claim 1 further comprising a poly(A) signal sequence comprising the polynucleotide sequences of SEQ ID NO.: 32 or SEQ ID NO.: 30.

5. The recombinant AAV vector construct of claim 3, wherein the recombinant AAV vector constmct comprises any one of the polynucleotide sequences of SEQ ID NO.: 10 or SEQ ID NO.: 12.

6. The recombinant AAV vector constmct of claim 3, wherein the expression cassette comprises any one of the polynucleotide sequences of SEQ ID NO.: 17 or SEQ ID NO.: 19.

7. The recombinant AAV vector constmct of claim 5, wherein the recombinant AAV vector constmct comprises SEQ ID NO.: 10.

8. The recombinant AAV vector constmct of claim 6, wherein the expression cassette comprises SEQ ID NO.: 17.Attorney Docket No.: 38213.0001P19. The recombinant AAV vector construct of claim 1, wherein the polynucleotide encoding the TSC1 (hamartin) protein is at least 80% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 8.

10. The recombinant AAV vector construct of claim 9, wherein the polynucleotide encoding a TSC1 (hamartin) protein is identical to the nucleotide sequence of SEQ ID NO.: 8.

11. The recombinant AAV vector construct of claim 1, wherein the polynucleotide encoding a TSC1 (hamartin) protein is at least 80% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO.: 9.

12. The recombinant AAV vector construct of claim 11, wherein the polynucleotide encoding a TSC1 (hamartin) protein is identical to the polynucleotide sequence of SEQ ID NO.: 9.

13. The recombinant AAV vector construct of claim 1, wherein the ITRs are 5’ AAV2 ITR and 3’ AAV2 ITR, having the polynucleotide sequences of SEQ ID NO.: 34 and SEQ ID NO.: 35, respectively.

14. The recombinant AAV vector construct of claim 1, wherein the expression cassette further comprises a woodchuck hepatitis virus posttranslational regulatory element (WPRE).

15. The recombinant AAV vector construct of claim 14, wherein the woodchuck hepatitis virus posttranslational regulatory element (WPRE) is identical to SEQ ID NO.: 33.

16. A recombinant AAV particle comprising the recombinant AAV vector construct of claim 1, wherein the AAV capsid 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.: 1 or SEQ ID NO.: 4, 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.: 49), YSRNSDN (SEQ ID NO.: 50), LHRLGPN (SEQ ID NO.: 51), LHRLGPD (SEQ ID NO.: 52), LHRAGPD (SEQ ID NO.: 53), YSRIGPD (SEQ ID NO.: 54), LSRIGPD (SEQ ID NO.: 55), LARSGPD (SEQ ID NO.: 56), LHKAGPN (SEQ ID NO.: 57), LSRIGPN (SEQ ID NO.: 58),Attorney Docket No.: 38213.0001P1LAKSGPN (SEQ ID NO.: 59), or YARNGPN (SEQ ID NO.: 60), or FRSTNGV (SEQ ID NO.: 61), wherein a capsid incorporating the VP1 capsid protein has increased binding to cells expressing hTfRl relative to a reference capsid not having the 7 amino acid peptide.

17. The recombinant AAV particle of claim 16, wherein the AAV capsid 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.: 1, and further comprises a 7 amino acid peptide YSRIGPN (SEQ ID NO.: 49) inserted between amino acids 588 and 589.

18. The recombinant AAV vector of claim 16, wherein the AAV capsid comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 2.

19. The recombinant AAV vector of claim 16, wherein the AAV capsid comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 5.

20. The recombinant AAV particle of claim 16, wherein the AAV capsid 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.: 1, and further comprises a 7 amino acid peptide YSRIGPN (SEQ ID NO.: 49) 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.

21. The recombinant AAV vector of claim 20, wherein the AAV capsid comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 3.

22. The recombinant AAV vector of claim 20, wherein the AAV capsid comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 6.

23. The recombinant AAV particle of claim 16, wherein the AAV capsid 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.: 1, and further comprises a 7 amino acid peptide FRSTNGV (SEQ ID NO.: 61) 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.

24. The recombinant AAV vector of claim 23, wherein the AAV capsid comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 47.Attorney Docket No.: 38213.0001P125. The recombinant AAV vector of claim 23, wherein the AAV capsid comprises a VP1 capsid protein having an amino acid sequence that is identical to SEQ ID NO.: 48.

26. A recombinant AAV particle comprising a recombinant AAV vector construct comprising an expression cassette comprising a polynucleotide encoding a TSC1 (hamartin) protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO.: 7, operably linked to a polynucleotide encoding a promoter that drives expression of TSC1 in cells of the CNS, wherein the capsid of the recombinant AAV 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.: 1, and further comprises a 7 amino acid peptide YSRIGPN (SEQ ID NO.: 49) inserted between amino acids 588 and 589, wherein a capsid incorporating the VP1 capsid protein has increased binding to cells expressing hTfRl relative to a reference capsid not having the 7 amino acid peptide.

27. A recombinant AAV particle comprising a recombinant AAV vector construct comprising an expression cassette comprising a polynucleotide encoding a TSC1 (hamartin) protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO.: 7, operably linked to a polynucleotide encoding a promoter that drives expression of TSC1 in cells of the CNS, wherein the capsid of the recombinant AAV 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.: 1, and further comprises a 7 amino acid peptide YSRIGPN (SEQ ID NO.: 49) 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, wherein a capsid incorporating the VP1 capsid protein has increased binding to cells expressing hTfRl relative to a reference capsid not having the 7 amino acid peptide.

28. A recombinant AAV particle comprising a recombinant AAV vector construct comprising an expression cassette comprising a polynucleotide encoding a TSC1 (hamartin) protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO.: 7, operably linked to a polynucleotide encoding a promoter that drives expression of TSC1 in cells of the CNS, wherein the capsid of the recombinant AAV particle comprises a VP1 capsid protein having an amino acid sequence that is at least 90% identical, at least 95% identical, or at leastAttorney Docket No.: 38213.0001P199% identical to SEQ ID NO.: 1, and further comprises a 7 amino acid peptide FRSTNGV (SEQ ID NO.: 61) 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, wherein a capsid incorporating the VP1 capsid protein has increased binding to cells expressing hTfRl relative to a reference capsid not having the 7 amino acid peptide.

29. A method of treating a patient having tuberous sclerosis complex (TSC), said method comprising administering to said patient a therapeutically effective amount of a recombinant AAV particle of any one of claims 16 through 28.

30. The method of claim 29, wherein the recombinant AAV particle is administered systemically, intravenously, intracranially, intrathecally, intra-nasally, or intra-peritoneally.

31. The method of claim 30, wherein the recombinant AAV particle is administered intravenously.

32. 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 a recombinant AAV particle of any one of claims 16 through 28.

33. The method of claim 32, wherein the recombinant AAV particle is administered systemically, intravenously, intracranially, intrathecally, intra-nasally, or intra-peritoneally.

34. The method of claim 33, wherein the recombinant AAV particle is administered intravenously.

35. 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 a recombinant AAV particle of any one of claims 16 through 28.

36. The method of claim 35, wherein the recombinant AAV particle is administered systemically, intravenously, intracranially, intrathecally, intra-nasally, or intra-peritoneally.

37. The method of claim 36, wherein the recombinant AAV particle is administered intravenously.Attorney Docket No.: 38213.0001P138. 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 a recombinant AAV particle of any one of claims 16 through 28.

39. The method of claim 38, wherein the recombinant AAV particle is administered systemically, intravenously, intracranially, intrathecally, intra-nasally, or intra-peritoneally.

40. The method of claim 39, wherein the recombinant AAV particle is administered intravenously.

41. A pharmaceutical composition for use in treating a patient having tuberous sclerosis Complex (TSC) comprising a recombinant AAV particle of any one of claims 16 through 28 and a pharmaceutically acceptable carrier.

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