CN116670159A

CN116670159A - Compositions and their use for the treatment of Angelman syndrome

Info

Publication number: CN116670159A
Application number: CN202180085876.9A
Authority: CN
Inventors: J·珀西瓦尔; R·施密德; J·M·威尔逊
Original assignee: University of Pennsylvania Penn
Current assignee: University of Pennsylvania Penn
Priority date: 2020-12-01
Filing date: 2021-12-01
Publication date: 2023-08-29

Abstract

The invention provides a rAAV having a vector genome comprising a UBE3A coding sequence. The invention also provides a method for treating one or more symptoms of Angeman's Syndrome (AS) in a patient having defective UBE3A expression in neurons, wherein the method comprises delivering a rAAV having a nucleic acid sequence encoding UBE 3A.

Description

Compositions and their use for the treatment of angermann syndrome

Background

Angermann Syndrome (AS) is a rare genetic disease that affects 500,000 people worldwide. The major symptoms of AS include mental disability, motor dysfunction, ataxia, loss of language, severe epilepsy, and unique behavioral characteristics. Most individuals with AS exhibit loss of function of the maternally inherited UBE3A allele, which encodes the HECT E3 ubiquitin ligase that links ubiquitin to substrates targeting them for degradation. 65-70% of AS cases are caused by class I mutations involving de novo deletions of maternal chromosomes 15q11-q13 [ Angelman H., puppet' child. Report on three cases (A report on three cases), dev Med Child Neurol,1965,7:681-88; birth incidence, genetic discovery and diagnostic age of angermann syndrome in denmark (Angelman syndrome in Denmark. Birth inches, genetic minerals, and age at diagnosis), am J Med Genet A,2013,161A (9): 2197-2203, electronic version 2013, 8, 2; clayton-Smith j, and Laan l., angermann syndrome: summary of clinical and genetic types (Angelman syndrome: a review of the clinical and genetic types), J Med Genet,2003,40 (2): 87-95, electronic edition, month 2, 1 year 2003; khatrin and Man H, autism and angerman syndrome protein Ube3A/E6AP: gene, E3 ligase ubiquitination and neurobiological function (The Autism and Angelman Syndrome Protein Ube A/E6AP: the Gene, E3 Ligase Ubiquination Targets and Neurobiological Functions), front Mol Neurosci,2009,12 (109): 1-12, electronic version 2019, 4, 30).

Three UBE3A isoforms have been described in mice, of which isoforms 2 and 3 have been identified as major UBE3A splice variants in mice. (Valluy, J. Et al., coding independent function of alternative Ube3a transcripts during neuronal development (A coding-independent function of an alternative Ube3a transcript during neuronal development), nat Neurosci,2015,18 (5): 666-673, electronic version 2015, month 13; trezza Ra et al., loss of nuclear UBE3a caused electrophysiological and behavioral deficits in mice and was associated with angerman syndrome (Loss of nuclear UBE3a causes electrophysiological and behavioral deficits in mice and is associated with Angelman syndrome), nat Neurosci,2019,22 (8): 1235-1247, electronic version 2019, month 24; zampeta Fi et al., protected UBE3a subcellular distribution between human and mouse promoted by non-homologous isoforms (Conserved UBE3 version a subcellular distribution between human and mice is facilitated by non-homologous isoforms), hum Mol Genet,2020,29 (18): 3032-3043, electronic 2020, month 2). Loss of hUBE3A isoform 1 occurs in individuals with "mild" angleman syndrome (Sadhwani, A et al, two angleman families with advanced neurodevelopmental carry the highest expressing UBE3A isoform initiation codon variant (Two Angelman families with unusually advanced neurodevelopment carry a start codon variant in the most highly expressed UBE a isosporm), am J Med Genet A,2018,176 (7): 1641-1647, electronic version 2018, 5 months 7), which still expresses nuclear hUBE3A isoform 3 and cytoplasmic hUBE3A isoform 2.

Currently, there is no cure for AS. Current treatments are palliative, focusing on managing medical and developmental problems, including epilepsy. The lack of treatment options underscores the critical unmet need for new therapies for AS.

Disclosure of Invention

Provided herein is a novel adeno-associated virus (AAV) gene replacement therapy based on UBE3A isoform 1, which is useful and well-tolerated. AS assessed in an angermann animal model, the compositions and methods can alleviate motor and behavioral deficits associated with Angermann Syndrome (AS). In one embodiment, a composition includes a recombinant adeno-associated virus (rAAV) stock useful for treating AS, the rAAV comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising: (a) AAV 5' Inverted Terminal Repeats (ITRs); (b) A UBE3A nucleic acid sequence comprising SEQ ID No. 9 or a sequence which is 95% identical to the sequence encoding a UBE3A isoform 1 protein (SEQ ID No. 2), wherein the nucleic acid is operably linked to regulatory elements which regulate the expression of the UBE3A protein in a human cell; (c) a regulatory element that directly expresses UBE3A of (b); and (d) AAV 3' itrs. In certain embodiments, the regulatory element comprises a neuron-specific promoter. In certain embodiments, the neuron-specific promoter is a synapsin promoter. In certain embodiments, the synaptoprotein promoter is a shortened promoter having the nucleic acid sequence of SEQ ID NO. 12. In certain embodiments, the regulatory element comprises a constitutive promoter. In certain embodiments, the regulatory element further comprises one or more enhancers and one or more introns. In certain embodiments, the regulatory sequences further comprise one or more target sequences for miR182 (SEQ ID NO: 20) and/or miR183 (SEQ ID NO: 11), said target sequences being operably linked to the UBE3A nucleic acid sequence. In certain embodiments, the regulatory sequences further comprise one or more target sequences for a miR selected from miR182 and/or miR183, downstream of the UBE3A nucleic acid sequence. In certain embodiments, the regulatory sequence further comprises four target sequences for miR183, which are downstream of the UBE3A nucleic acid sequence. In some embodiments, the regulatory sequence comprises four copies of SEQ ID NO. 11. In certain embodiments, the AAV capsid is an AAVhu68 capsid. In certain embodiments, the AAV capsid is an AAVhu68 capsid resulting from expression of the nucleic acid sequence of SEQ ID NO. 14 or SEQ ID NO. 16. In certain embodiments, the AAV capsid is an AAVrh91 capsid. In certain embodiments, the AAV capsid is an AAVrh91 capsid expressed by the nucleic acid sequence of SEQ ID NO:17 or SEQ ID NO: 19. In certain embodiments, the composition is an aqueous suspension further comprising a physiologically compatible carrier, buffer, adjuvant, and/or diluent.

In certain embodiments, the compositions described herein are useful for treating patients suffering from angermann syndrome. In certain embodiments, the compositions provided herein may be used to treat one or more symptoms of angermann syndrome, optionally wherein the symptoms are selected from one or more of the following: developmental delay, mental disability, severe language disorder, ataxia and/or epilepsy. In certain embodiments, a method is provided for treating one or more symptoms of Angermann Syndrome (AS) in a patient having defective UBE3A expression in neurons by delivering an expression cassette provided herein. In certain embodiments, the expression cassette treats a symptom selected from one or more of the following: developmental delay, mental disability, severe language disorder, ataxia and/or epilepsy. In certain embodiments, the compositions are provided for intrathecal delivery to a patient. In some casesIn embodiments, the patient is injected at least 1x10 ¹⁰ Up to 1x10 ¹³ GC/kg rAAV carrying engineered UBE3A coding sequences.

In certain embodiments, the method provides an improvement in symptoms of angler's disease, including one or more of bradykinesia, mental disability, severe language disorder, ataxia, and/or epilepsy.

Other aspects and advantages of these methods and compositions are further described in the following detailed description.

Drawings

Figures 1A-1C provide schematic diagrams of expression cassettes and vector genomes without miR sequences for modulating dorsal root ganglion expression and toxicity. FIG. 1A provides a schematic representation of the vector genome in which the 5'AAV Inverted Terminal Repeat (ITR) and 3' AAV ITRs flank the hUBE 3A-isoform 1 expression cassette. The expression cassette contains an engineered hUBE 3A-isoform 1 coding sequence (encoding 852 amino acids; SEQ ID NO: 2) under the control of a modified synaptotagmin promoter. The hUBE3A coding sequence includes the amino-terminal zinc finger (AZUL), HECT domain and RCC 1-like domain 2 (HERC 2), the E6 protein binding domain (E6 BD), and the region homologous to the carboxy-terminus of E6-AP (HECT), as well as SV40 polyA of the Ube3A ligase. FIG. 1B provides a schematic representation of the vector genome in which AAV ITRs flank the hUBE 3A-isoform 2 expression cassette. The encoded isoform 2 protein is 875 amino acids in length (SEQ ID NO: 6). FIG. 1C provides a schematic representation of the vector genome in which AAV ITRs flank the hUBE 3A-isoform 3 expression cassette. The encoded isoform 3 protein is 872 amino acids in length (SEQ ID NO: 21).

Fig. 2A and 2B show the results of assessment of vector biodistribution and mRNA expression of miR183 (4 xmiR 183) target sequence (light circle) with 4 copies or raavhu68.ube3a-isoform 1 without 4xmiR183 sequence (dark sequence) in non-human primate (NHP). Fig. 2A shows UBE3A isoform 1 vector biodistribution in NHP as measured in the cerebellum, caudate nucleus, hippocampus, frontal cortex, occipital cortex, medulla, parietal cortex, temporal cortex, thalamus, DRG cervical vertebra, DRG thoracic vertebra, DRG lumbar vertebra, spinal cord cervical vertebra, spinal cord thoracic vertebra, genomic Copy (GC) in spinal cord lumbar vertebra/diploid genome. FIG. 2B shows mRNA expression of UBE3A isoform 1 after treatment with rAAHU68. UBE3A isoform 1 with 4 copies of the miR183 (4 xmiR 183) target sequence (light circles) or without the 4xmiR183 sequence (dark sequences) in the spinal cord and DRG of the NHP. Fig. 2C shows UBE3A isoform 1 as in cerebellum, caudate nucleus, hippocampus, frontal cortex, occipital cortex, medulla, parietal cortex, temporal cortex, thalamus, DRG cervical vertebra, DRG thoracic vertebra, DRG lumbar vertebra, spinal cord cervical vertebra, spinal cord thoracic vertebra, spinal cord lumbar vertebra, brain (negative control).

Figures 3A and 3B show that AAVhu68-UBE 3A-isoform 1±4xmir183 vector did not cause significant Dorsal Root Ganglion (DRG) toxicity in rhesus monkeys (3-4 years old). hUBE3A-1 (group 1) or hUbe3A-1-4xmiR183 (group 2) vector at 3X10 ¹³ GC/animal doses were administered 35 days after the cerebellar medullary pool (ICM) without causing significant AAV-induced dorsal root ganglion toxicity in rhesus monkeys. DRG-related toxicity was observed only in the spinal cord and peripheral nerves of group 1 animals 192285. DRG was not significant in both groups of animals (fig. 3A), only sporadic minimal mononuclear cell infiltration (fig. 3B, circles), and no evidence of neuronal degeneration (3/3 animals, group 1; 3/3 animals, group 2).

FIGS. 4A and 4B show the effect of rAAV hu68. Synaptotagmin-UBE 3A isoform 1 on rhesus peripheral nerves (3-4 years old). Minimal focal axonal disease (focal axonoplath) effects on hindlimb nerves (arrows). Fig. 4A shows animal aavhu68 in peripheral nerves synaptotagmin-UBE 3A isoform 1. Fig. 4B shows that animals 192275 and 192297 exhibited mild multifocal axonal disease (arrow) and mononuclear cell infiltration (oval) exhibited by 192285 in the right median nerve of the forelimb.

FIG. 5 shows treated UBE3A ^m-/p+ Quantification of UBE3A protein positive neurons in neonatal mice. Treatment included passage through the brain chamber (ICV) at 1x10 ¹¹ The GC/animal dose was administered AAV-PHP.B-hSyn-UBE 3A-isoform 1. In cortex, hippocampus, thalamus, hypothalamus and midbrain, 42% -68% are neuronal expressed UBE3A proteins (normalized to UBE3A positive neurons in the corresponding WT tissue).

FIG. 6 shows treated UBE3A ^m-/p+ New born miceQuantification of UBE3A protein positive neurons in (b). Treatment included passage through the brain chamber (ICV) at 1x10 ¹⁰ The GC/animal dose was administered AAV-PHP.B-hSyn-UBE 3A-isoform 1. Between 20% and 50% of the UBE3A proteins expressed by neurons (normalized to UBE3A positive neurons in the corresponding WT tissue) are in the cortex, hippocampus, thalamus, hypothalamus and midbrain.

FIGS. 7A-7I show fluorescence images of the localization of engineered human UBE3A isoform 1 (hUBE 3A-1) transcripts in dorsal root ganglion (cervical, thoracic and dorsal segments) from three treated non-human primates (NHP-1, NHP-2, NHP-3; in a 35 day study). Treatment included 3x10 by the cerebellar medullary pool (ICM) route ¹³ The GC/animal dose was administered AAV-PHP.B-hSyn-UBE 3A-isoform 1. Images of the region of interest were taken at different magnifications and presented at 20x magnification. FIG. 7A shows a fluorescence image of engineered hUBE3A-1 transcript localization in dorsal root ganglion cervical segments from NHP-1. FIG. 7B shows a fluorescence image of engineered hUBE3A-1 transcript localization in dorsal root ganglion cervical segments from NHP-2. FIG. 7C shows a fluorescence image of engineered hUBE3A-1 transcript localization in dorsal root ganglion cervical segments from NHP-3. FIG. 7D shows a fluorescence image of the localization of engineered hUBE3A-1 transcripts in dorsal root ganglion thoracic segments from NHP-1. FIG. 7E shows a fluorescence image of the localization of engineered hUBE3A-1 transcripts in dorsal root ganglion thoracic segments from NHP-2. FIG. 7F shows a fluorescence image of the localization of engineered hUBE3A-1 transcripts in dorsal root ganglion thoracic segments from NHP-3. FIG. 7G shows a fluorescence image of engineered hUBE3A-1 transcript localization in dorsal root ganglion lumbar segment from NHP-1. FIG. 7H shows a fluorescence image of engineered hUBE3A-1 transcript localization in dorsal root ganglion lumbar segment from NHP-2. FIG. 7I shows a fluorescence image of engineered hUBE3A-1 transcript localization in dorsal root ganglion lumbar segment from NHP-3.

FIG. 8 shows UBE3A after injection with AAV-PHP.B-hSyn-hUBE3A-iso1 ICV ^m-/p+ And engineering expression of UBE3A isoform 1 in the brains of wild-type mice.

FIGS. 9A and 9B show the presence of wild-type or AS (UBE 3A) ^m-/p+ ) Mice were 1x10 per animal ¹¹ Dosage ventricles of Genomic Copies (GC)Results of motor coordination behavioural tests performed at 8-10 weeks of age following Intra (ICV) injection of AAV-php.b-synaptotagmin-UBE 3A-isoform 1 or isoform 2 vector. FIG. 9A shows the motor coordination ability of WT and AS mice after treatment with AAV-PHP.B-synaptorin-UBE 3A-isoform 1. FIG. 9B shows the motor coordination ability of WT and AS mice after treatment with AAV-PHP.B-synaptorin-UBE 3A-isoform 2.

FIGS. 10A to 10D show the presence of wild-type or AS (UBE 3A) ^m-/p+ ) Mice were 1x10 per animal ¹¹ Results of nesting performance tests performed at 8-10 weeks of age after injection of AAV-php.b-synaptotagmin-UBE 3A-isoform 1 or isoform 2 vector into the brain room (ICV) at doses of Genomic Copies (GC). FIG. 10A shows nesting scores of WT and AS mice after treatment with AAV-PHP.B-synaptorin-UBE 3A-isoform 1. FIG. 10B shows the unused nesting percentage of WT and AS mice after treatment with AAV-PHP.B-synaptorin-UBE 3A-isoform 1. Figure 10C shows nesting scores of WT and AS mice after treatment with AAV-php.b-synaptorin-UBE 3A-isoform 2. Figure 10D shows the unused nesting percentage of WT and AS mice after treatment with AAV-php.b-synaptorin-UBE 3A-isoform 2.

FIGS. 11A-11D show the presence of wild-type or AS (UBE 3A) ^m-/p+ ) Mice were 1x10 per animal ¹¹ Results of catwalk (stride length and gait improvement) behavior tests performed at 8-10 weeks of age after injection of AAV-php.b-protrusive protein-UBE 3A-isoform 1 vector in the brain room (ICV) at doses of Genomic Copies (GC). FIG. 11A shows the stride length of WT and AS mice right hind limb (RH) after treatment with AAV-PHP.B-synaptoprotein-UBE 3A-isoform 1. FIG. 11B shows the stride length of WT and AS mice left hind Limb (LH) after treatment with AAV-PHP.B-synaptoprotein-UBE 3A-isoform 1. FIG. 11C shows the stride length of WT and AS mice right hind limb (RH) after treatment with AAV-PHP.B-synaptoprotein-UBE 3A-isoform 1. FIG. 11D shows the stride length of WT and AS mice left hind Limb (LH) following treatment with AAV-PHP.B-synopsin-UBE 3A-isoform 1.

FIGS. 12A-2C show the use of AAV-hu68-hSyn-UBE 3A-isoform 1 at 3X10 via the cerebellum bulbar pool (ICM) pathway ¹³ Dorsal root ganglion (dr) in NHP 35 days after GC/animal dose treatmentg) Toxicity study results (plotted as pathology grading scores 0-5). Fig. 12A shows scored pathological grading of the cervical segments of DRGs of NHPs. Fig. 12B shows the scored pathology grading of the thoracic segment of DRG of NHP. Fig. 12C shows the scored pathology grading of the lumbar segment of the DRG of NHP.

FIGS. 13A-13C show the use of AAV-hu68-hSyn-UBE 3A-isoform 1 at 3X10 via the cerebellum bulbar pool (ICM) pathway ¹³ Toxicity study results (plotted as pathogenicity scores 0-5) for spinal cord in NHP 35 days after GC/animal dose treatment. Fig. 13A shows scored pathological grading of cervical segments of NHP. Fig. 13B shows scored pathology grading of spinal cord chest segment of NHP. Fig. 13C shows scored pathological grading of spinal cord lumbar segment of NHP.

FIG. 14 shows the use of AAV-hu68-hSyn-UBE 3A-isoform 1 at 3X10 through the cerebellum bulbar pool (ICM) pathway ¹³ Toxicity study results (plotted as axonal pathology grading scores 0-5) of peripheral nerves in NHP 35 days after GC/animal dose treatment.

FIGS. 15A-15C show the use of AAV-hu68-hSyn-UBE 3A-isoform 1 at 3X10 via the cerebellum bulbar pool (ICM) pathway ¹³ Results of peripheral nerve conduction studies 14 days and 35 days after GC/animal dose treatment. FIG. 15A shows velocity of the left median nerve measured in m/sec. FIG. 15B shows the results of peripheral nerve conduction studies 14 days and 35 days after treatment with AAV-hu68-hSyn-UBE 3A-isoform 1, peak-to-peak (PP) amplitudes were measured in mV. FIG. 15C shows the results of peripheral nerve conduction studies 14 days and 35 days after treatment with AAV-hu68-hSyn-UBE 3A-isoform 1, with Negative Peak (NP) amplitudes measured in mV.

FIGS. 16A and 16B show treated UBE3A ^m-/p+ Quantification of UBE3A isoform 1 or isoform 2 protein positive neurons in neonatal mice, plotted as percentage of positive neurons in cortex, hippocampus, thalamus, hypothalamus and midbrain, normalized to UBE3A positive neurons in WT tissue. Treatment included passage through the brain chamber (ICV) at 1x10 ¹¹ The GC/animal dose was administered AAV-PHP.B-hSyn-UBE 3A-isoform 1 or isoform 2. Figure 16A shows the percentage of UBE3A isoform 1 positive neurons in the cortex, hippocampus, thalamus, hypothalamus and midbrain of mice after treatment. FIG. 16B shows the post-treatmentPercentage of UBE3A isoform 2 positive neurons in cortex, hippocampus, thalamus, hypothalamus and midbrain of mice.

Detailed Description

The expression cassettes provided herein contain an engineered UBE 3A-isoform 1 coding sequence that, when delivered (e.g., by rAAV-mediated gene replacement therapy), expresses UB3A isoform 1 at a level that treats symptoms of angerman syndrome. In certain embodiments, the regulatory elements in the expression cassette comprise up to eight, e.g., four to eight miR183 sequences to regulate Dorsal Root Ganglion (DRG) expression and/or toxicity. In certain embodiments, these DRG targeting sequences are selected for use at high levels of expression and/or when systemic delivery is intended. In other embodiments, these sequences are included when intrathecal delivery is used.

In one embodiment, the expression cassette comprises an engineered UBE3A coding (nucleic acid sequence) operably linked to regulatory elements that regulate expression of the UBE3A protein in the target human cell. In certain embodiments, the UBE3A coding sequence encodes a UBE3A isoform 1 protein that is reproduced in SEQ ID NO. 2. The hUBE3A isoform 1 protein includes multiple domains including the amino-terminal zinc finger (AZUL), HECT domain and RCC 1-like domain 2 (HERC 2), the E6 protein binding domain (E6 BD), and the region of homology to the carboxy-terminus of E6-AP (HECT) of the Ube3A ligase. Suitably, the engineered UBE3A isoform 1 encoding sequence is the nucleic acid sequence of SEQ ID NO. 9 or a sequence encoding a UBE3A isoform 1 protein that is at least 95% identical thereto (SEQ ID NO. 2). In certain embodiments, the sequence is 100% identical to the full length of SEQ ID NO. 9. In other embodiments, the sequence is at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical to SEQ ID NO 9. In certain embodiments, the UBE3A isoform 1 coding sequence is truncated at the 5 'or 3' end, resulting in a carboxy or N-terminal truncation of the UBE3A isoform 1 protein.

In certain embodiments, the UBE3A coding sequence encodes a UBE3A isoform 2 protein that is reproduced in SEQ ID NO. 6. The hUBE3A isoform 2 protein includes a number of domains including the amino-terminal zinc finger (AZUL), HECT domain and RCC 1-like domain 2 (HERC 2), the E6 protein binding domain (E6 BD), and the region of homology to the carboxy-terminus of E6-AP (HECT). Suitably, the engineered UBE3A isoform 2 coding sequence is the nucleic acid sequence of SEQ ID NO. 10 or a sequence encoding a UBE3A isoform 2 protein (SEQ ID NO. 6) at least 95% identical thereto. In certain embodiments, the sequence is 100% identical to the full length of SEQ ID NO. 10. In other embodiments, the sequence is at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical to SEQ ID NO. 10. In certain embodiments, the UBE3A isoform 2 coding sequence is truncated at the 5 'or 3' end, resulting in a carboxy or N-terminal truncation of the UBE3A isoform 2 protein. In certain embodiments, the UBE3A coding sequence encodes a UBE3A isoform 3 protein that is reproduced in SEQ ID NO. 21 (UNIPRT ID No: Q05086-3).

In certain embodiments, the expression cassette comprises a UBE3A coding sequence, wherein optionally the UBE3A coding sequence encodes a fusion protein comprising a signal peptide and a functional UBE3A protein. In certain embodiments, the expression cassette comprises a UBE3A coding sequence, wherein optionally the UBE3A coding sequence encodes a fusion protein comprising an uptake peptide fused to a functional UBE3A protein. In certain embodiments, the expression cassette UBE3A coding sequence, wherein optionally the UBE3A coding sequence encodes a fusion protein comprising a UBE3A protein fused to a signal peptide and/or an uptake peptide. In some embodiments, the expression cassette comprises a UBE3A coding sequence, wherein optionally the signal peptide and/or the uptake peptide is located 5 'or 3' of the UBE3A coding sequence to provide a fusion protein comprising the signal peptide and/or the uptake peptide at the N-terminus of the UBE3A protein, a fusion protein comprising the signal peptide and/or the uptake peptide at the C-terminus of the UBE3A protein, or a fusion protein comprising the signal peptide at the N-terminus of the UBE3A protein, or a fusion protein having the signal peptide or the uptake peptide at the C-terminus of the UBE3A protein, or a combination thereof. In certain embodiments, the signal peptide is a Binding Immunoglobulin (BiP) signal peptide. In certain embodiments, the signal peptide is a Gaussia signal peptide. See also U.S. patent No. 9,279,007 B2 ((corresponding to international patent application No. WO2012/071422; binding Immunoglobulin (BiP) signal peptide), US 10,874,750B2 (corresponding to international patent application No. WO2019/213180 A1; binding Immunoglobulin (BiP) signal peptide and Gaussia signal peptide), which are incorporated herein by reference in their entirety.

In certain embodiments, UBE3A isoform 1 expression cassettes are selected for delivery as a single gene replacement therapy. In certain embodiments, the UBE3A isoform 1 expression cassette is selected for delivery as a gene replacement therapy in a regimen involving one or more additional active ingredients (e.g., short-term or long-term enzyme replacement therapy and/or substrate depletion therapy).

In one embodiment, the composition comprises a vector comprising an expression cassette for UBE3A isoform 1. Suitable vectors and vector genomes are described herein.

In other embodiments, a stock of recombinant parvoviral vectors (e.g., recombinant adeno-associated viruses) is provided. The rAAV comprises an AAV capsid and a vector genome packaged therein, the vector genome comprising: (a) AAV 5' Inverted Terminal Repeats (ITRs); (b) A UBE3A nucleic acid sequence comprising SEQ ID No. 9 or a sequence encoding a UBE3A isoform 1 protein (SEQ ID No. 2) which is 95% identical thereto, wherein the nucleic acid is operably linked to regulatory elements which regulate expression of the UBE3A protein in a human cell; (c) a regulatory element that directly expresses UBE3A of (b); and (d) AAV 3' itrs. Desirable AAV capsids include AAVhu68 and AAVrh91, which target desired cells in the Central Nervous System (CNS).

In certain embodiments, a rAAV comprises an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises: (a) AAV 5' Inverted Terminal Repeats (ITRs); (b) optionally a peptide (e.g., a signal peptide or an uptake peptide); (c) A UBE3A nucleic acid sequence comprising SEQ ID No. 9 or a sequence encoding a UBE3A isoform 1 protein (SEQ ID No. 2) which is 95% identical thereto, wherein the nucleic acid sequence is operably linked to regulatory elements which regulate expression of the UBE3A protein in a human cell; (d) optionally a peptide (regulatory peptide and/or uptake peptide); (e) regulatory elements that directly express UBE3A of (b); and (f) AAV 3' itrs. In certain embodiments, the signal peptide is a BiP signal peptide. See also US 9,279,007 B2 (corresponding to international patent application No. WO 2012/071422) and US 10,874,750B2 (corresponding to international patent application No. WO2019/213180 A1), which are incorporated herein by reference in their entirety. In certain embodiments, the regulatory peptide is an IGF peptide. See also WO2021/072372, which is incorporated herein by reference in its entirety. In certain embodiments, the signal peptide is a secretion signal peptide comprising a secretion sequence selected from insulin, GDNF, and IgK. In certain embodiments, the uptake peptide comprises a cellular uptake sequence selected from the group consisting of transmembrane protein, R6W3, HIV TAT, HIV TATk, and pVEC. See also WO 2019/006107, which is incorporated herein by reference in its entirety.

In certain embodiments, UBE3A is optionally a fusion protein comprising a signal peptide and/or an uptake peptide, as described herein. In certain embodiments, the signal peptide and/or the uptake peptide is located at the amino (N) -terminus or the carboxy (C) -terminus. In certain embodiments, the signal peptide is located at the amino (N) -terminus. In certain embodiments, the uptake peptide is located at the N-terminus or the C-terminus.

As used herein, "stock" of rAAV refers to a population of rAAVs. Although its capsid proteins are heterogeneous due to deamidation, rAAV is expected to share the same vector genome as 5 in stock. The stock may include a rAAV having a capsid with, for example, heterogeneous deamidation pattern characteristics of the selected AAV capsid protein and the selected production system. The stock may be produced by a single production system or pooled by multiple runs of the production system. Various production systems may be selected, including but not limited to those described herein.

Unless defined otherwise herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs and with reference to the disclosure, which provides a general guide to many terms used in this application to those of ordinary skill in the art.

As used herein, "disease," "disorder," and "condition" are used interchangeably to indicate an abnormal state in a subject. In one embodiment, the disease is angeman-slower (AS).

As used herein, "patient" or "subject" interchangeably refers to a male or female mammal, including a human, veterinary or farm animal, livestock or companion animal, and animals commonly used in research. In one embodiment, the subjects of these methods and compositions are human patients. In one embodiment, the subject of these methods and compositions is a male or female human.

As used throughout this specification and claims, the terms "comprise," "include," and variations thereof include other components, elements, integers, steps, etc. In contrast, the term "consist of … …" and variations thereof do not include other components, elements, integers, steps, etc.

It should be noted that the term "a/an" means one or more/one or more, for example, "a neuron" should be understood to mean one or more neurons. As such, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

As used herein, unless otherwise indicated, the term "about" means a variability of plus or minus 10% relative to a given reference.

In some cases, the term "e+#" is used to refer to an index. For example, 5E10 is 5x10 ¹⁰ . These terms may be used with one anotherUsed interchangeably.

The nucleic acid sequences described herein may be cloned using conventional molecular biology techniques or generated de novo by DNA synthesis. Nucleic acid encoding aspects of the UBE3A genes described herein are assembled and placed into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., that transfers sequences carried therein to a host cell, e.g., for the production of non-viral delivery systems (e.g., RNA-based systems, naked DNA, etc.) or for the production of viral vectors in packaging host cells and/or for delivery to a host cell of a subject. In one embodiment, the genetic element is a vector. In one embodiment, the genetic element is a plasmid. Methods for preparing such engineered constructs are known to the nucleic acid manipulation skilled person and include genetic engineering, recombinant engineering and synthetic techniques. See, e.g., green and Sambrook, molecular cloning: laboratory Manual (Molecular Cloning: ALaboratory Manual), cold spring harbor laboratory Press (Cold Spring Harbor Press, cold Spring Harbor, NY) of Cold spring harbor, new York City (2012).

Expression cassette

As used herein, an "expression cassette" refers to a nucleic acid molecule that includes a biologically useful nucleic acid sequence (e.g., a gene cDNA, mRNA, etc., encoding a protein, enzyme, or other useful gene product) and a regulatory sequence operably linked thereto that directs or regulates the transcription, translation, and/or expression of the nucleic acid sequence and its gene product. As used herein, "operably linked" sequences include both regulatory sequences that are contiguous or non-contiguous with the nucleic acid sequence and regulatory sequences that function as a trans or cis nucleic acid sequence. Such regulatory sequences typically comprise, for example, one or more of a promoter, enhancer, intron, kozak sequence, polyadenylation sequence, and TATA signal. The expression cassette may contain regulatory sequences upstream (5 ') of the gene sequence, such as one or more of a promoter, enhancer, intron, etc., and one or more of an enhancer, or downstream (3') of the gene sequence, such as the 3 'untranslated region (3' utr) including a polyadenylation site, among other elements. In certain embodiments, the regulatory sequence is operably linked to the nucleic acid sequence of the gene product, wherein the regulatory sequence is separated from the nucleic acid sequence of the gene product by an intervening nucleic acid sequence, i.e., a 5 'untranslated region (5' utr). In certain embodiments, the expression cassette comprises a nucleic acid sequence of one or more gene products. In some embodiments, the expression cassette may be a monocistronic expression cassette or a bicistronic expression cassette. In other embodiments, the term "transgene" refers to one or more DNA sequences from an external source inserted into a target cell. In general, such expression cassettes that can be used to produce viral vectors contain coding sequences for the gene products described herein that flank the packaging signal and other expression control sequences of the viral genome, such as the sequences described herein. In certain embodiments, the vector genome may contain two or more expression cassettes.

In some embodiments, the nucleic acid molecule comprising the coding sequence is a UBE3A coding sequence, and further comprises a promoter, and may also comprise other regulatory sequences therefor. In certain embodiments, the expression cassette used to produce a viral vector (e.g., a viral particle) contains the coding sequence of UBE3A described herein flanking the packaging signal and other expression control sequences of the viral genome, such as the sequences described herein. In some embodiments, the viral vector is an AAV viral vector, wherein the packaging signal is a 5'AAV Inverted Terminal Repeat (ITR) and a 3' AAV ITR. Optionally, the expression cassette (and vector genome) may include one or more dorsal root ganglion (drg) -miRNA target sequences in the UTR, e.g., to reduce drg toxicity and/or axonal lesions. See, e.g., PCT/US2019/67872, filed on 12/20/2019, and now published as WO 2020/132455, U.S. provisional patent application No. 63/023593, filed on 5/12/2020, and U.S. provisional patent application No. 63/038488, filed on 6/12/2020, all of which are entitled "compositions for Drg specific reduction of transgene expression (Compositions for Drg-Specific Reduction of Transgene Expression)", which are incorporated herein in their entirety.

As used herein, the term "operably linked" or "operably linked" refers to both expression control sequences or regulatory elements that are contiguous with a gene of interest and expression control sequences that function in trans or remotely to control the gene of interest.

As described herein, regulatory elements include, but are not limited to: a promoter; an enhancer; a transcription factor; a transcription terminator; efficient RNA processing signals such as splicing and polyadenylation signals (polyA); sequences that stabilize cytoplasmic mRNA, such as woodchuck hepatitis virus (WHP) post-transcriptional regulatory elements (WPRE); sequences that enhance translation efficiency (i.e., kozak consensus sequences).

In one embodiment, the expression cassette comprises regulatory elements that direct expression of sequences encoding one or more elements of a gene replacement system for delivery of UBE 3A. In one embodiment, the regulatory element comprises one or more promoters. In certain embodiments, the expression cassette comprises a constitutive or regulatable promoter. In certain embodiments, the promoter is a tissue-specific (e.g., neuron-specific) promoter. In certain embodiments, suitable promoters may include, but are not limited to, the use of the elongation factor 1alpha (EF 1 alpha) promoter (see, e.g., kim Dw et al, human elongation factor 1alpha promoter as a multifunctional and efficient expression system (Use of the Human elongation factor 1alpha promoter as a versatile and efficient expression system.) (Gene.) (7 month 16 in 1990; 91 (2): 217-23)), the synapsin 1 promoter (see, e.g., kugler S et al, human synapsin 1 promoter providing high neuronal specific long term transgene expression in adult rat brain via adenovirus vectors (Human synopsin 1gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area) gene therapy in 2003, 10 (4): 337-47), shortened synapsin promoters such as provided in the examples herein (see, e.g., SEQ ID NO:12 for coding sequences), the Neuronal Specific Enolase (NSE) promoter (see, e.g., kugler S et al, e.g., 35-16. 35, 35-35 in 35.35, 35.6, 35 to 16, or 13. 13, 35 to 35, 35.16, or 13, respectively, the endocrine cell-35 in Human, in the Human, in accordance with the transduction region (e.g., see, 35-35, 35.g., 35-16, 35, 6, 35, 13), large-scale production of adeno-associated viral vector serotype-9carrying motor neuron survival genes (Large-Scale Production of Adeno-Associated Viral Vector Serotype-9Carrying the Human Survival Motor Neuron Gene,Mol Biotechnol.) month 1 of 2016; 58 (1) 30-6.Doi:10.1007/s 12033-015-9899-5). Other suitable promoters include the CAG promoter, which comprises (C) a Cytomegalovirus (CMV) early enhancer element, (A) a promoter, a first exon and a first intron of the chicken beta-actin gene, and (G) a splice acceptor of the rabbit beta-globin gene. See, e.g., alexopoulou, annika N. Et al BMC cell Biol.9.1 (2008): 2. Although less desirable, other promoters may be used in the vectors described herein, such as viral promoters, constitutive promoters, inducible promoters, regulatable promoters (see, e.g., WO 2011/126808 and WO 2013/04943), or promoters responsive to physiological cues. In certain embodiments, the expression cassette comprises a U6 promoter. In another embodiment, the regulatory element comprises an enhancer. In further embodiments, the enhancer is selected from one or more of the following: APB enhancer, ABPS enhancer, αmic/bik enhancer, TTR enhancer, en34 enhancer, apoE enhancer, CMV enhancer or RSV enhancer. In yet another embodiment, the regulatory element comprises an intron. In further embodiments, the intron is selected from CBA, human beta globin, IVS2, SV40, bGH, alpha globulin, beta globulin, collagen, ovalbumin, or p53. In one embodiment, the regulatory element comprises polyA. In further embodiments, the polyA is synthetic polyA or is derived from bovine growth hormone (bGH), human growth hormone (hGH), SV40 as provided in the examples herein (see, e.g., SEQ ID NO:13 for coding sequences), rabbit β -globin (RGB), or modified RGB (mRGB). In another embodiment, the regulatory element may comprise a WPRE sequence. In yet another embodiment, the regulatory element comprises a Kozak sequence.

In certain embodiments, the expression cassette comprises the nucleic acid sequence of SEQ ID NO. 22 or a sequence at least about 90% identical thereto, which encodes UBE3A comprising the amino acid sequence of SEQ ID NO. 2. In certain embodiments, the expression cassette comprises the nucleic acid sequence of SEQ ID NO. 23 or a sequence at least about 90% identical thereto, which encodes UBE3A comprising the amino acid sequence of SEQ ID NO. 4. In certain embodiments, the expression cassette comprises the nucleic acid sequence of SEQ ID NO. 24 or a sequence at least about 90% identical thereto, which encodes UBE3A comprising the amino acid sequence of SEQ ID NO. 6. In certain embodiments, the expression cassette comprises the nucleic acid sequence of SEQ ID NO. 25 or a sequence at least about 90% identical thereto, which encodes UBE3A comprising the amino acid sequence of SEQ ID NO. 8.

The term "expression" is used herein in its broadest sense and includes the production of RNA, protein or both RNA and protein. With respect to RNA, the term "expression" or "translation" relates in particular to the production of peptides or proteins. Expression may be transient or may be stable.

The expression cassette may be delivered by any suitable delivery system. Suitable non-viral delivery systems are known in the art (see, e.g., ramamoorth and Narvekar. J Clin Diagn Res.2015, 1; 9 (1): GE01-GE06, which is incorporated herein by reference) and can be readily selected by one of skill in the art and can include, for example, naked DNA, naked RNA, dendrimers, PLGA, polymethacrylates, inorganic particles, lipid particles (e.g., lipid nanoparticles or LNP) or chitosan-based formulations.

In one embodiment, the vector is a non-viral plasmid comprising the expression cassette described therein, e.g., "naked DNA," "naked plasmid DNA," RNA, and mRNA; coupled to various compositions and nanoparticles, including, for example, micelles, liposomes, cationic lipid-nucleic acid compositions, polysaccharide compositions and other polymers, lipid and/or cholesterol-based nucleic acid conjugates, and other constructs as described herein. See, for example, X.Su et al, (mol. Pharmaceuticals), 2011,8 (3), pages 774-787; web publication, 2011, 3, 21; WO2013/182683, WO 2010/053572 and WO 2012/170930, all of which are incorporated herein by reference.

Provided herein are compositions comprising nucleic acid sequences encoding one or more elements of a gene replacement system and methods of use thereof for replacing functional UBE 3A.

Optionally, the expression cassette may include a miRNA target sequence in the untranslated region. miRNA target sequences are designed to be specifically recognized by mirnas present in cells in which transgene expression is not desired and/or in which reduced levels of transgene expression are desired. In certain embodiments, the miRNA target sequences are located in the 3'utr, the 5' utr, and/or both the 3 'and 5' utr. In some embodiments, the miRNA target sequence is operably linked to a regulatory sequence in the expression cassette. In certain embodiments, the expression cassette comprises at least two tandem repeats of a DRG-specific miRNA target sequence, wherein the at least two tandem repeats comprise at least one first miRNA target sequence and at least one second miRNA target sequence, which may be the same or different. In certain embodiments, the tandem miRNA target sequences are contiguous or separated by a spacer of 1 to 10 nucleic acids, wherein the spacer is not a miRNA target sequence.

In certain embodiments, the vector genome or expression cassette contains at least one miRNA target sequence that is a miR-183 (or miRNA 183) target sequence. In certain embodiments, the vector genome or expression cassette contains a miR-183 target sequence that includes AGTGAATTCTACCAGTGCCATA (SEQ ID NO: 11), wherein the sequence complementary to the miR-183 seed sequence is underlined. In certain embodiments, the vector genome or expression cassette contains more than one copy (e.g., two or three copies) of a sequence that is 100% complementary to the miR-183 seed sequence. In certain embodiments, the miR-183 target sequence is about 7 nucleotides to about 28 nucleotides in length and comprises at least one region that is at least 100% complementary to the miR-183 seed sequence. In certain embodiments, the miR-183 target sequence contains a sequence that is partially complementary to SEQ ID NO. 11, and thus, when aligned with SEQ ID NO. 11, there are one or more mismatches. In certain embodiments, the miR-183 target sequence comprises a sequence having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches when aligned with SEQ ID NO. 11, wherein the mismatches may be absentContinuous. In certain embodiments, the miR-183 target sequence comprises a region that has 100% complementarity, which region further comprises at least 30% of the length of the miR-183 target sequence. In certain embodiments, the region of 100% complementarity comprises a sequence of 100% complementarity to a miR-183 seed sequence. In certain embodiments, the remainder of the miR-183 target sequence has at least about 80% to about 99% complementarity to miR-183. In certain embodiments, the expression cassette or vector genome comprises a miR-183 target sequence comprising a truncated SEQ ID NO:11, i.e., a sequence lacking at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides at either or both of the 5 'or 3' ends of SEQ ID NO: 11. In certain embodiments, the expression cassette or vector genome comprises a transgene and one miR-183 target sequence. In still other embodiments, the expression cassette or vector genome comprises at least two, three, or four miR-183 target sequences.

In certain embodiments, the vector genome or expression cassette contains at least one miRNA target sequence that is a miR-182 target sequence. In certain embodiments, the vector genome or expression cassette contains a miR-182 target sequence that includes AGTGTGAGTTCTACCATTGCCAAA (SEQ ID NO: 20). In certain embodiments, the vector genome or expression cassette contains more than one copy (e.g., two or three copies) of a sequence that is 100% complementary to the miR-182 seed sequence. In certain embodiments, the miR-182 target sequence is about 7 nucleotides to about 28 nucleotides in length and comprises at least one region that is at least 100% complementary to the miR-182 seed sequence. In certain embodiments, the miR-182 target sequence contains a sequence that is partially complementary to SEQ ID NO:20, and thus, when aligned with SEQ ID NO:20, there are one or more mismatches. In certain embodiments, the miR-183 target sequence comprises a sequence having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches when aligned with SEQ ID NO. 20, wherein the mismatches may be discontinuous. In certain embodiments, the miR-182 target sequence comprises a region having 100% complementarity, which region further comprises at least 30% of the length of the miR-182 target sequence. In certain embodiments, the region of 100% complementarity comprises a sequence of 100% complementarity to a miR-182 seed sequence. In certain embodiments, the remainder of the miR-182 target sequence has at least about 80% to about 99% complementarity to miR-182. In certain embodiments, the expression cassette or vector genome comprises a miR-182 target sequence, which comprises a truncated SEQ ID NO:20, i.e., a sequence lacking at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides at either or both of the 5 'or 3' ends of SEQ ID NO: 20. In certain embodiments, the expression cassette or vector genome comprises a transgene and one miR-182 target sequence. In still other embodiments, the expression cassette or vector genome comprises at least two, three, or four miR-182 target sequences.

The term "tandem repeat" as used herein refers to the presence of two or more consecutive miRNA target sequences. These miRNA target sequences may be contiguous, i.e., positioned directly one after the other such that the 3 'end of one target sequence is directly upstream of the 5' end of the next target sequence, without an intermediate sequence, or vice versa. In another embodiment, two or more of the miRNA target sequences are separated by a short spacer sequence. As used herein, a "spacer" is any selected nucleic acid sequence, e.g., a nucleic acid sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length that is positioned between two or more consecutive miRNA target sequences. In certain embodiments, the spacer is 1 to 8 nucleotides in length, 2 to 7 nucleotides in length, 3 to 6 nucleotides in length, four nucleotides in length, 4 to 9 nucleotides, 3 to 7 nucleotides in length, or greater. Suitably, the spacer is a non-coding sequence. In certain embodiments, the spacer may have four (4) nucleotides. In certain embodiments, the spacer is GGAT. In certain embodiments, the spacer is six (6) nucleotides. In certain embodiments, the spacer is CACGTG or GCATGC.

In certain embodiments, the tandem repeat sequence contains two, three, four or more of the same miRNA target sequences. In certain embodiments, the tandem repeat contains at least two different miRNA target sequences, at least three different miRNA target sequences, or at least four different miRNA target sequences, and the like. In certain embodiments, the tandem repeat may contain two or three identical miRNA target sequences different from the fourth miRNA target sequence. In certain embodiments, at least two different sets of tandem repeat sequences may be present in the expression cassette. For example, a 3'UTR may contain a tandem repeat immediately downstream of the transgene, a UTR sequence, and two or more tandem repeat sequences nearer the 3' end of the UTR. In another example, the 5' utr may contain one, two or more miRNA target sequences. In another example, the 3 'may contain a tandem repeat sequence and the 5' utr may contain at least one miRNA target sequence. In certain embodiments, the expression cassette contains two, three, four, or more tandem repeat sequences that begin within about 0 to 20 nucleotides of the stop codon of the transgene. In other embodiments, the expression cassette contains a miRNA tandem repeat sequence of at least 100 to about 4000 nucleotides from the stop codon of the transgene.

See PCT/US19/67872 filed on 12/20 2019 and now published as WO 2020/132455, which is incorporated herein by reference and claims priority from U.S. provisional patent application No. 62/783,956 filed on 21/12/2018, which is incorporated herein by reference. See also U.S. patent application Ser. No. 63/023,593, U.S. patent application Ser. No. 63/038,488, U.S. patent application Ser. No. 63/043,562, U.S. patent application Ser. No. 63/049,299, and U.S. provisional patent application Ser. No. 63/152,042, and International patent application Ser. No. PCT/US21/32003, both filed on even date 12, U.S. patent application Ser. No. 63/079,299, and U.S. provisional patent application Ser. No. 63/152,042, and International patent application Ser. No. PCT/US21/32003, both of which are incorporated herein by reference.

Optionally, the expression cassette may include a UBE3A coding sequence encoding a UBE3A protein, the UBE3A protein being a fusion protein comprising a signal peptide and/or an uptake peptide, as described herein. In certain embodiments, the signal peptide and/or the uptake peptide is located 5 'or 3' of the UBE3A coding sequence.

Vector genomes comprising engineered hUBE 3A-isoform 1 coding sequences are provided herein, for example, in SEQ ID NO:1 (hSyn.hUbe3a-1. GSco.4XmiRNA183.SV40 (with miR183 target sequence)) and SEQ ID NO:3 (hSyn.hUbe3a-1. GSco.SV40, NO miR)).

Vector genomes comprising engineered hUBE 3A-isoform 2 coding sequences are exemplified herein, for example, in SEQ ID NO:5 (hSyn. HUbe3A-2.GSco.4XmiRNA183.SV40 (with miR183 target sequence)) and SEQ ID NO:7 (hSyn. HUbe3A-2.GSco.SV40, NO miR).

It will be appreciated that the compositions in the expression cassettes described herein are intended to apply to the compositions and methods described in this specification.

Carrier body

In certain embodiments, the expression cassette encoding UBE3A is delivered to the neuron by a vector or viral vector, many of which are known and available in the art. In one embodiment, a vector comprising a UBE3A gene as described herein is provided. In one embodiment, a vector comprising an expression cassette as described herein is provided. In one embodiment, the vector is a non-viral vector. In a further embodiment, the non-viral vector is a plasmid. In another embodiment, the vector is a viral vector. Viral vectors include any virus suitable for gene therapy, including but not limited to: bocavirus, adenovirus, adeno-associated virus (AAV), herpes virus, lentivirus, retrovirus, or parvovirus. However, for ease of understanding, adeno-associated viruses are referred to herein as exemplary viral vectors. Thus, in one embodiment, an adeno-associated viral vector is provided comprising a nucleic acid sequence of one or more elements of an expression cassette operably linked to regulatory elements thereof.

As used herein, a "vector" is a biological or chemical moiety comprising a nucleic acid sequence that can be introduced into a suitable target cell to replicate or express the nucleic acid sequence. Examples of vectors include, but are not limited to, recombinant viruses, plasmids, liposomes, polymers, complexes, dendrimers, cell Penetrating Peptide (CPP) conjugates, magnetic particles, or nanoparticles. In one embodiment, the vector is a nucleic acid molecule having an exogenous or heterologous or engineered nucleic acid encoding a functional gene product, which can then be introduced into an appropriate target cell. Such vectors preferably have one or more origins of replication and one or more sites into which recombinant DNA may be inserted. Vectors typically have means by which cells with a vector, e.g., encoding a drug resistance gene, can be selected from cells without a vector. Common vectors include plasmids, viral genomes, and "artificial chromosomes". Conventional methods of vector generation, production, characterization or quantification are available to those skilled in the art.

As used herein, a recombinant viral vector is any suitable viral vector that targets a desired cell. Thus, the recombinant viral vectors described herein preferably target one or more cells and tissues affected by angermann syndrome, including cells of the central nervous system (e.g., brain). These examples provide illustrative recombinant adeno-associated viruses (rAAV). However, other suitable viral vectors may include, for example, recombinant adenoviruses, recombinant parvoviruses such as recombinant bocaviruses, hybrid AAV/bocaviruses, recombinant herpes simplex viruses, recombinant retroviruses, or recombinant lentiviruses. In a preferred embodiment, these recombinant viruses are replication defective.

"replication defective virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, wherein any viral genomic sequence that is also packaged within the viral capsid or envelope is replication defective; that is, it is unable to produce progeny virions, but retains the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome may be engineered to be "gut-free" -contains only genes of interest flanking the signals required to amplify and package the artificial genome), but these genes may be supplied during production. Thus, this is considered to be safe for use in gene therapy because replication and infection by progeny virions does not occur unless the viral enzymes required for replication are present. Such replication-defective viruses may be adeno-associated viruses (AAV), adenoviruses, lentiviruses (integrated or non-integrated), or another suitable viral source.

"plasmid" or "plasmid vector" is generally designated herein by lower case letter p before and/or after the vector name. Plasmids, other cloning and expression vectors, their nature and methods of construction/manipulation thereof, which may be used in accordance with the present invention will be apparent to those skilled in the art. In one embodiment, elements of the vector genome as described herein or the expression cassette as described herein are engineered into suitable genetic elements (vectors) for the production of viral vectors and/or delivery to host cells, e.g., naked DNA, phage, transposons, cosmids, episomes, etc., to transfer sequences carried thereon. The selected vector may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high speed DNA coated aggregates, viral infection, and protoplast fusion. Methods for preparing such constructs are known to the nucleic acid manipulation skilled person and include genetic engineering, recombinant engineering and synthetic techniques. See, e.g., sambrook et al, molecular cloning, A laboratory Manual (Molecular Cloning: ALaboratory Manual), cold Spring Harbor Press, cold Spring Harbor, N.Y..

The term "transgene" or "gene of interest" as used interchangeably herein refers to an exogenous or engineered protein-encoding nucleic acid sequence under the control of a promoter and/or other regulatory elements in an expression cassette, rAAV genome, recombinant plasmid or production plasmid, vector or host cell as described herein.

The term "heterologous" as used in describing a nucleic acid sequence or protein refers to the nucleic acid or protein being derived from a different organism or different species of the same organism than the host cell or subject in which it is expressed. The term "heterologous" when used in reference to a protein or nucleic acid in a plasmid, expression cassette or vector indicates that the protein or nucleic acid is present with another sequence or subsequence that is not in the same relationship to the protein or nucleic acid in question as it is in nature.

As used herein, the term "host cell" may refer to a packaging cell line in which the vector (e.g., recombinant AAV) is produced from a production plasmid. In the alternative, the term "host cell" may refer to any target cell in which expression of the gene products described herein is desired. Thus, "host cell" refers to a prokaryotic or eukaryotic cell (e.g., a human cell or an insect cell) containing exogenous or heterologous DNA introduced into the cell by any means (e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high-speed DNA coated aggregates, viral infection, and protoplast fusion). In certain embodiments herein, the term "host cell" refers to a culture of cells of various mammalian species used for in vitro evaluation of the compositions described herein. In other embodiments herein, the term "host cell" refers to a cell used to produce and package a viral vector or recombinant virus. In further embodiments, the term "host cell" is a neuron, e.g., a neuron of the CNS.

As used herein, the term "target cell" refers to any target cell in which expression of a heterologous nucleic acid sequence or protein is desired. In certain embodiments, the target cell is a neuron of the CNS, in particular a neuron having a mutant or defective maternal UBE3A allele or a neuron lacking UBE3A expression.

As used herein, a "vector genome" refers to a nucleic acid sequence packaged inside a parvoviral (e.g., rAAV) capsid that forms a viral particle. Such nucleic acid sequences comprise AAV Inverted Terminal Repeats (ITRs). In the examples herein, the vector genome contains at least 5 'to 3', AAV 5 'itrs, coding sequences, and AAV 3' itrs. ITRs from AAV2 (AAV other than the capsid source) or other than full-length ITRs may be selected. In certain embodiments, the ITRs are from the same AAV source as the AAV that provides rep function during production or trans-supplementation of AAV. Further, other ITRs, such as self-complementary (scAAV) ITRs, may be used. Further, the vector genome contains regulatory sequences that direct the expression of the gene product. Suitable components of the vector genome are discussed in more detail herein. In one example, a "vector genome" contains at least from 5 'to 3' a vector-specific sequence, a nucleic acid sequence encoding UBE3A operably linked to regulatory control sequences that direct its expression in a target cell, wherein the vector-specific sequence may be a terminal repeat sequence that specifically packages the vector genome into a viral vector capsid or envelope protein. For example, AAV inverted terminal repeats are used for packaging into AAV and certain other parvoviral capsids. Where packaging into a lentiviral vector is desired, lentiviral long terminal repeats may be utilized. Similarly, other terminal repeats (e.g., retroviral long terminal repeats) and the like can be selected.

Vector genomes provided herein encoding UBE3A isoform 1 include, for example, SEQ ID NO 1 (AAV 2-5'ITR-hSyn. HUbe3A-1.GSco.4XmiRNA183.SV40-AAV2-3' ITR), SEQ ID NO 3 (AAV 2-5'ITR-hSyn. HUbe3A-1.GSco.SV40-AAV2-3' ITR).

As used herein, the term "AAV" refers to naturally occurring adeno-associated viruses, adeno-associated viruses obtainable by one of skill in the art and/or obtainable according to the compositions and methods described herein, as well as artificial AAV. Adeno-associated virus (AAV) viral vectors are AAV nuclease (e.g., DNase) resistant particles having an AAV protein capsid, wherein an expression cassette flanked by AAV Inverted Terminal Repeats (ITRs) is packaged for delivery to a target cell. Anti-nuclease recombinant AAV (rAAV) indicates that AAV capsids have been fully assembled and protects these packaged vector genomic sequences from degradation (digestion) during nuclease incubation steps designed to remove contaminating nucleic acids that may be present during production. In many cases, the rAAV described herein is DNase resistant.

In the examples below, the clade F adeno-associated virus is AAVhu68. See WO 2018/160582, which is incorporated herein by reference in its entirety. In other embodiments, the other AAV capsid is selected from a different clade, such as clade A, B, C, D or E, or from an AAV source outside of any of these clades. For example, another suitable capsid is AAVrh91. See published WO2020/223231 at month 11 of 2020, U.S. patent application Ser. No. 63/065,616 at month 8 of 2020 and U.S. patent application Ser. No. 63/109,734 at month 11 of 2020, international patent application Ser. No. PCT/US21/55436, incorporated herein by reference. In certain embodiments, AAV capsids with reduced capsid deamidation may be selected. See, e.g., PCT/US19/19804 and PCT/US18/19861, both filed on 27/2/2019 and incorporated herein by reference in their entirety. See also PCT/US20/030266, filed on 29 at 4/2020, now published as WO2020/223231, and International application No. PCT/US21/45945, filed on 13 at 8/2021, which are incorporated herein by reference.

In other embodiments, the source of the AAV capsid may be any of several tens of naturally occurring and available adeno-associated viruses, as well as engineered or artificial AAV. AAV capsids consist of 60 capsid (cap) protein subunits, VP1, VP2 and VP3, which are symmetrically arranged in icosahedrons, in a ratio of about 1:1:10 to 1:1:20, depending on the AAV chosen. Various AAV can be selected as a source of the AAV viral vector capsids described above. See, for example, U.S. published patent application No. 2007-0036760-A1; U.S. published patent application 2009-0197338-A1; EP 1310571. See also WO 2003/042397 (AAV 7 and other simian AAV), U.S. Pat. No. 7790449 and U.S. Pat. No. 7282199 (AAV 8), WO 2005/033321 and U.S. Pat. No. 7,906,111 (AAV 9), WO 2006/110689 and WO 2003/042397 (AAVrh 10). These documents also describe other AAV that may be selected for AAV production and are incorporated by reference. Among the isolated or engineered and well characterized AAV from human or non-human primate (NHP), human AAV2 is the first AAV developed as a gene transfer vector; it has been widely used for efficient gene transfer experiments in different target tissues and animal models. Unless otherwise specified, the AAV capsids, ITRs, and other selected AAV components described herein can be readily selected from any AAV, including, but not limited to, AAV generally identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, and AAVAnc 80. See, for example, WO 2005/033321, which is incorporated herein by reference. In one embodiment, the AAV capsid is an AAV9 capsid or variant thereof. In certain embodiments, the capsid protein is designated by a number or combination of numbers and letters following the term "AAV" in the rAAV vector name. See also PCT/US19/19804 and PCT/US19/19861, each entitled "Novel Adeno-Associated Virus (AAV) with reduced capsid deamidation" -AAV Vectors and uses thereof (AAV Vectors, AAV Vectors Having Reduced Capsid Deamidation And Uses Therefor) ", and filed on month 27 of 2019, the entire contents of which are incorporated herein by reference.

ITRs or other AAV components can be readily isolated from AAV or engineered using techniques available to those skilled in the art. Such AAV may be isolated, engineered or obtained from an academic, commercial or public source (e.g., american type culture collection (American Type Culture Collection, manassas, VA)) of marassas, virginia. Alternatively, AAV sequences may be engineered by synthesis or other suitable means by reference to published sequences (e.g., published sequences available in the literature or in databases such as GenBank, pubMed). AAV viruses can be engineered by conventional molecular biology techniques, such that the particles can be optimized for cell-specific delivery of nucleic acid sequences, for minimizing immunogenicity, for modulating stability and particle longevity, for efficient degradation, for accurate delivery to the nucleus, and the like.

As used herein, the terms "rAAV" and "recombinant AAV vector" are used interchangeably to mean, but are not limited to, an AAV comprising a capsid protein and a vector genome packaged therein, wherein the vector genome comprises a nucleic acid heterologous to the AAV. rAAV includes "pseudotyped rAAV" in which a viral vector contains a vector genome that contains the inverted terminal repeat of an AAV (e.g., AAV 2) packaged into capsids of different AAV capsid proteins. In one embodiment, the capsid protein is a non-naturally occurring capsid. Such artificial capsids may be produced by any suitable technique using a combination of a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) and a heterologous sequence, which may be obtained from a different selected AAV, a non-contiguous portion of the same AAV, from a non-AAV viral source, or from a non-viral source. The selected genetic elements may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high speed DNA coated aggregates, viral infection, and protoplast fusion. Methods for preparing such constructs are known to the nucleic acid manipulation skilled person and include genetic engineering, recombinant engineering and synthetic techniques. See, e.g., green and Sambrook, molecular cloning: laboratory Manual (Molecular Cloning: ALaboratory Manual), cold spring harbor laboratory Press (Cold Spring Harbor Press, cold Spring Harbor, NY) of Cold spring harbor, new York City (2012).

As used herein, the term "heterogeneous" or any grammatical variation thereof, when used in reference to a vp capsid protein, refers to a population of non-identical elements, e.g., having vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences. SEQ ID NO. 15 provides the encoded amino acid sequence of the AAVhu68 vp1 protein. The term "heterogeneous" used in connection with vp1, vp2 and vp3 proteins (alternatively referred to as isoforms) refers to differences in the amino acid sequences of the vp1, vp2 and vp3 proteins within the capsid. AAV capsids contain a subset of intra-vp 1, intra-vp 2, and intra-vp 3 proteins with modifications from predicted amino acid residues. These sub-populations comprise at least some deamidated asparagine (N or Asn) residues. For example, certain subpopulations comprise at least one, two, three or four highly deamidated asparagine (N) positions of an asparagine-glycine pair, and optionally further comprise other deamidated amino acids, wherein deamidation results in amino acid changes and other optional modifications.

As used herein, unless otherwise indicated, a "sub-population" of vp proteins refers to a group of vp proteins that have at least one defined common property and that consist of at least one group member to less than all members of a reference group. For example, unless otherwise specified, a "sub-population" of vp1 proteins is at least one (1) vp1 protein, and less than all of the vp1 proteins in the assembled AAV capsid. Unless otherwise indicated, a "sub-population" of vp3 proteins may be one (1) vp3 protein that is less than all of the vp3 proteins in the assembled AAV capsid. For example, the vp1 protein may be a sub-population of vp proteins; the vp2 protein may be a separate sub-population of vp proteins, and vp3 is yet another sub-population of vp proteins in the assembled AAV capsid. In another example, vp1, vp2, and vp3 proteins may contain sub-populations with different modifications, e.g., at least one, two, three, or four highly deamidated asparagines, e.g., at an asparagine-glycine pair.

In one aspect, provided herein are AAV vectors comprising an AAV capsid and an expression cassette, wherein the expression cassette comprises a nucleic acid sequence encoding one or more elements of a UBE3A gene and regulatory elements that direct expression of the UBE3A gene elements in a host cell. AAV vectors also include AAV ITR sequences.

ITR is the gene element responsible for genome replication and packaging during vector production and is the only viral cis-element required to produce rAAV. In one embodiment, the ITRs are from an AAV that is different from the AAV supplying the capsid. In preferred embodiments, the ITR sequence from AAV2 or a deleted version thereof (Δitr) can be used for convenience and to speed up regulatory approval. However, ITRs from other AAV sources may be selected. In the case where the source of the ITR is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be referred to as pseudotyped. Typically, the AAV vector genome comprises AAV 5 'ITRs, nucleic acid sequences encoding gene products and any regulatory sequences, as well as AAV 3' ITRs. However, other configurations of these elements may be suitable. In one embodiment, a self-complementary AAV is provided. Shortened versions of the 5' ITR, known as Δitr, have been described in which the D sequence and terminal resolution sites (trs) are deleted. In certain embodiments, the vector genome comprises a 130 base pair shortened AAV2 ITR, wherein the external "a" element is deleted. During amplification of vector DNA using the internal a element as a template, the shortened ITR reverts to 145 base pairs of wild type length. In other embodiments, full length AAV 5 'and 3' itrs are used.

In one embodiment, the regulatory sequences are selected such that the total rAAV vector genome is from about 2.0 to about 5.5 kilobases in size. In one embodiment, the regulatory sequences are selected such that the total rAAV vector genome is from about 2.9 to about 5.5 kilobases in size. In one embodiment, the regulatory sequences are selected such that the total rAAV vector genome is about 2.9kb in size. In one embodiment, it is desirable that the rAAV vector gene be similar to the size of the native AAV genome. Thus, in one embodiment, the regulatory sequences are selected such that the total rAAV vector genome is about 4.7kb in size. In another embodiment, the total rAAV vector genome is less than about 5.2kb in size. The size of the vector genome may be manipulated based on the size of regulatory sequences including promoters, enhancers, introns, poly A, and the like. See Wu et al, mol Ther, month 1 2010, 18 (1): 80-6, which is incorporated herein by reference.

In certain embodiments, provided herein are rAAV for use AS CNS-directed therapy for treating a subject having Angeman Syndrome (AS), wherein the rAAV comprises an AAV capsid and a vector genome packaged therein, the vector genome comprising: (a) AAV 5' Inverted Terminal Repeats (ITRs); (b) A sequence encoding UBE3A operably linked to regulatory elements that direct its expression in a host cell; (c) regulatory elements directing expression; and (d) AAV 3' itrs. In certain embodiments, provided herein are rAAV for use AS CNS-directed therapy for treating a subject having Angeman Syndrome (AS), wherein the rAAV comprises an AAV capsid and a vector genome packaged therein, the vector genome comprising: (a) AAV 5' Inverted Terminal Repeats (ITRs); (b) A sequence encoding UBE3A operably linked to regulatory elements that direct its expression in a host cell; (c) Optionally one or more peptides (e.g., signal peptides and/or uptake peptides); (d) regulatory elements directing expression; and (e) AAV 3' itrs. In one embodiment, the rAAV has a tropism for CNS cells (e.g., a rAAV with AAVhu68 capsid or AAVrh91 capsid), and/or contains a neuron-specific expression control element (e.g., a synaptobrevin promoter). In one aspect, a construct is provided that is a vector (e.g., a plasmid) that can be used to generate a viral vector. In one embodiment, the AAV 5'ITR is an AAV2 ITR and the AAV 3' ITR is an AAV2 ITR. In one embodiment, the rAAV comprises an AAV capsid as described herein. In one embodiment, the rAAV comprises an AAVhu68 capsid. In other embodiments, the rAAV comprises an AAVrh91 capsid. SEQ ID NO. 18 provides the encoded amino acid sequence of the AAVrh91 vp1 protein.

The recombinant adeno-associated viruses (AAV) described herein may be produced using known techniques. See, for example, WO 2003/042397; WO 2005/033321; WO 2006/110689; US 7588772 B2. Such methods involve culturing a host cell containing a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette flanking an AAV Inverted Terminal Repeat (ITR) as described herein; and sufficient helper functions to allow packaging of the expression cassette into AAV capsid proteins. Also provided herein are host cells comprising a nucleic acid sequence encoding an AAV capsid; a functional rep gene; vector genome as described; and sufficient helper functions to allow packaging of the vector genome into AAV capsid proteins. In one embodiment, the host cell is a HEK 293 cell. These methods are described in more detail in WO2017160360A2, which is incorporated herein by reference.

Other methods of producing rAAV that are available to those of skill in the art may be utilized. Suitable methods may include, but are not limited to, baculovirus expression systems or production by yeast. See, e.g., robert m.kotin, large-scale recombinant adeno-associated virus production (Large-scale recombinant adeno-associated virus production) & lt, human molecular genetics & gt, 2011, month 4, 15; 20 R1 is R2-R6. On-line publication at 29, 4, 2011 doi:10.1093/hmg/ddr141; aucin MG et al, using triple infection to generate adeno-associated viral vectors in insect cells: optimization of baculovirus concentration ratio (Production of adeno-associated viral vectors in insect cells using triple infection: optimization of baculovirus concentration ratios.) "biotechnology and bioengineering (Biotechnol bioeng.)" 12/20/2006; 95 1081-92; SAMI s.thakur, production of recombinant adeno-associated viral vectors in yeast (Production of Recombinant Adeno-associated viral vectors in yeast.) submitted to university of florida research institute paper, 2012; kondraov O et al Direct Head-to-Head evaluation of recombinant adeno-associated viral vectors made in human versus insect cells (Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells), "molecular therapy", 8.10.2017. Pii: S1525-0016 (17) 30362-3.Doi:10.1016/J. Ymthe.2017.08.003.[ electronic version prior to printing plate ]; mietzsch M et al, oneBac2.0: sf9 cell line (onebac 2.0: sf9 Cell Lines for Production of AAV1, AAV2, and AAV8 Vectors with Minimal Encapsidation of Foreign DNA.) "human Gene therapy method (Hum Gene ter methods.)" 2017, 2 months, for the production of AAV1, AAV2, and AAV8 vectors that minimize capsid of exogenous DNA; 28 (1) 15-22.Doi:10.1089/hgtb.2016.164.; plum L et al. Production and characterization of novel recombinant adeno-associated virus replication genomes: eukaryotic DNA sources for gene transfer (Production and characterization of novel recombinant adeno-associated virus replicative-form genome: a eukaryotic source of DNA for gene transfer.) "public science library. Synthesis.)" 2013, 8, 1; 8 (8) e69879.doi 10.1371/journal. Fine. 0069879. Print in 2013; galibert L et al, recent progress in large scale production of adeno-associated viral vectors in insect cells towards treatment of neuromuscular diseases (Latest developments in the large-scale production of adeno-associated virus vectors in insect cells toward the treatment of neuromuscular diseases), "invertebrate pathology journal (J inverterbr pathline ]),)" 2011, month 7; 107 journal S80-93.Doi:10.1016/j.jip.2011.05.008; and Kotin RM, large-scale recombinant adeno-associated virus production (Large-scale recombinant adeno-associated virus production.) "human molecular genetics" 2011, 4 months, 15 days; 20 R1R 2-6.Doi:10.1093/Hmg/ddr141 electronic version 2011, 4/29.

Two-step affinity chromatography purification is performed at high salt concentrations followed by purification of the carrier drug product and removal of empty capsids using anion exchange resin chromatography. These methods are described in more detail in WO 2017/160360 entitled "scalable purification method for AAV9 (Scalable Purification Method for AAV 9)" and WO 2017/100674 entitled "scalable purification method for AAV1 (Scalable Purification Method for AAV 1)", which are incorporated herein by reference. Briefly, a method for isolating rAAV particles having packaged genomic sequences from genome-deficient AAV intermediates involves subjecting a suspension comprising recombinant AAV9 or AAV viral particles and AAV capsid protein intermediates to high performance liquid chromatography, wherein the AAV9 viral particles and AAV intermediates bind to a strong anion exchange resin equilibrated at a pH of about 10.2 of rAAV9 and about 9.8 of AAV1 and are subjected to a salt gradient while monitoring the ultraviolet absorbance of the eluate at about 260 and about 280. In this method, AAV complete capsids are collected from eluted fractions when the ratio of a260/a280 reaches an inflection point. In one example, for the affinity chromatography step, the diafiltered product may be applied to an AAV-specific resin effective to capture the selected AAV serotype. Under these ionic conditions, a significant percentage of residual cellular DNA and protein flows through the column, while AAV particles are effectively captured.

Conventional methods for characterizing or quantifying rAAV are available to those skilled in the art. To calculate the content of empty and intact particles, the VP3 band volume of the selected samples (e.g., formulations purified by iodixanol (ioxanol) gradient in the examples herein, where gc# = particle#) was plotted against the loaded GC particles. The resulting linear equation (y=mx+c) is used to calculate the number of particles in the banded volume of the test article peak. The number of particles per 20. Mu.L loaded (pt) was then multiplied by 50 to give particles (pt)/mL. The Pt/mL was divided by GC/mL to give the particle to genome copy ratio (Pt/GC). Pt/mL-GC/mL gave empty Pt/mL. Empty pt/mL divided by pt/mL and x100 gives the percentage of empty particles. Generally, methods for assaying empty capsids and AAV vector particles with packaged genomes are known in the art. See, e.g., grimm et al, (1999) Gene therapy 6:1322-1330; sommer et al, molecular therapy (molecular. Ther.) (2003) 7:122-128. To test denatured capsids, the method comprises subjecting the treated AAV stock to SDS-polyacrylamide gel electrophoresis (consisting of any gel capable of separating three capsid proteins, e.g. a gradient gel containing 3-8% triacetate in buffer), followed by running the gel until sample material is separated and blotting the gel onto a nylon or nitrocellulose membrane (preferably nylon). The anti-AAV capsid antibody is then used as a primary antibody, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody, that binds to denatured capsid proteins (Wobus et al, (2000) 74:9281-9293). A secondary antibody is then used which binds to the primary antibody and comprises a means for detecting binding to the primary antibody, more preferably comprises The anti-IgG antibody of the detection molecule to which it is covalently bound is most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase. A method for detecting binding is used to semi-quantitatively determine binding between a primary antibody and a secondary antibody, preferably a detection method capable of detecting radioisotope emissions, electromagnetic radiation or colorimetric changes, most preferably a chemiluminescent detection kit. For example, for SDS-PAGE, samples can be extracted from the column fractions and heated in SDS-PAGE loading buffer containing a reducing agent (e.g., DTT), and the capsid proteins resolved on a pre-formed gradient polyacrylamide gel (e.g., novex). Silver staining may be performed using SilverXpress (Invitrogen, CA) or other suitable staining methods (i.e., SYPRO ruby or coomassie staining) according to manufacturer's instructions. In one embodiment, the concentration of AAV vector genome (vg) in the column fraction can be measured by quantitative real-time PCR (Q-PCR). The sample is diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After nuclease inactivation, taqMan with specificity for the DNA sequence between the primers is used ^TM The fluorescent probe further dilutes and amplifies the sample. The number of cycles (threshold cycles, ct) required for each sample to reach a defined fluorescence level was measured on a Applied Biosystems Prism 7700 sequence detection system. Plasmid DNA containing the same sequence as that contained in the AAV vector was used to generate a standard curve in the Q-PCR reaction. The values of the cycle threshold (Ct) obtained from the samples were used to determine vector genome titers by normalizing them with respect to the Ct values of the plasmid standard curve. Endpoint determination based on digital PCR may also be used.

In one aspect, an optimized q-PCR method is used that utilizes a broad spectrum of serine proteases, such as proteinase K (e.g., commercially available from Qiagen). More specifically, the optimized qPCR genome titer assay is similar to the standard assay except that after DNase I digestion, the sample is diluted with proteinase K buffer and treated with proteinase K, then heat inactivated. Suitably, the sample is diluted with proteinase K buffer in an amount equal to the sample size. The proteinase K buffer may be concentrated 2-fold or more. Typically, proteinase K treatment is about 0.2mg/mL, but may vary from 0.1mg/mL to about 1 mg/mL. The treatment step is typically conducted at about 55 ℃ for about 15 minutes, but may be conducted at a lower temperature (e.g., about 37 ℃ to about 50 ℃) for a longer period of time (e.g., about 20 minutes to about 30 minutes), or at a higher temperature (e.g., up to about 60 ℃) for a shorter period of time (e.g., about 5 to 10 minutes). Similarly, heat inactivation typically lasts about 15 minutes at about 95 ℃, but the temperature may be reduced (e.g., about 70 ℃ to about 90 ℃) and the time prolonged (e.g., about 20 minutes to about 30 minutes). The sample is then diluted (e.g., 1000-fold) and TaqMan analysis is performed as described in the standard assay.

Additionally or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods for determining single stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, e.g., m.lock et al, method of human Gene therapy (Hum Gene Ther methods), month 4 of 2014; 25 (2) 115-25.Doi:10.1089/hgtb.2013.131. Electronic version 2014, 2 months 14 days.

Methods for determining the ratio between vp1, vp2 and vp3 of capsid proteins are also useful. See, e.g., vamseedhar Rayaprolu et al, comparative analysis of adeno-associated viral capsid stability and kinetics (Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics), "journal of virology", 2013, month 12; 87 (24) 13150-13160; characterization of adenovirus-associated virus-induced polypeptides in Buller RM, rose JA.1978.KB cells (Characterization of adenovirus-associated viruses-induced polypeptides in KB cells.) "J.Virol.25:331-338; and Rose JA, maizel JV, inman JK, shatkin AJ.1971 structural proteins of adeno-associated viruses (Structural proteins of adenovirus-associated viruses.) "J.Virol.8:766-770.

It should be understood that the compositions in the carrier described herein are intended to apply to other compositions and methods described across this specification.

Composition and method for producing the same

An aqueous suspension of AS suitable for administration to treat a subject in need thereof is provided, the suspension comprising an aqueous suspension and a vector comprising an engineered nucleic acid sequence encoding a UBE3A gene operably linked to its regulatory elements AS described herein. In one embodiment, a therapeutically effective amount of the carrier is included in the suspension.

Nucleic acid

In certain embodiments, the pharmaceutical composition comprises an expression cassette comprising a nucleic acid sequence encoding UBE3A isoform 1 and a non-viral delivery system. This may include, for example, naked DNA, naked RNA, inorganic particles, lipid or lipid-like particles, chitosan-based formulations, and other formulations known in the art and described, for example, by Ramamoorth and Narvekar, as referenced above. In other embodiments, the pharmaceutical composition is a suspension comprising an expression cassette comprising the UBE3A gene in a viral vector system. In certain embodiments, the pharmaceutical composition comprises a non-replicating viral vector. Suitable viral vectors may include any suitable delivery vector, such as, for example, a recombinant adenovirus, a recombinant lentivirus, a recombinant bocavirus, a recombinant adeno-associated virus (AAV), or another recombinant parvovirus. In certain embodiments, the viral vector is a recombinant AAV for delivering UBE3A isoform 1 to a patient in need thereof. In certain embodiments, the viral vector is a recombinant AAV for delivering UBE3a isoform 3 to a patient in need thereof.

In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, the composition being, for example, an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition may be transported as a concentrate that is diluted for administration to a subject. In other embodiments, the composition may be lyophilized and reconstituted at the time of administration.

In one embodiment, the suspension further comprises a surfactant, preservative, excipient, and/or buffer dissolved in the aqueous suspension. In one embodiment, the buffer is PBS. Various suitable solutions are known, including those that include one or more of the following: buffered saline, surfactant and physiologically compatible salts or mixtures of salts whose ionic strength is adjusted to correspond to about 100mM sodium chloride (NaCl) to about 250mM sodium chloride, or to equivalent ionic concentrations. Suitable surfactants or combinations of surfactants may be selected from poloxamers, i.e. nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly (propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly (ethylene oxide)), SOLUTOL HS 15 (polyethylene glycol-15 hydroxystearate), LABSOL (polyoxycaprylate glyceride), polyoxy 10 oil ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. The pH may be in the range of 6.5 to 8.5, or 7 to 8.5, or 7.5 to 8. Since the pH of cerebrospinal fluid is about 7.28 to about 7.32, for intrathecal delivery, a pH in this range may be desirable; while for intravenous delivery, a pH of 6.8 to about 7.2 may be desirable. However, the broadest range and other pH within these sub-ranges may be selected for other delivery routes.

Additionally provided is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a carrier comprising a nucleic acid sequence encoding one or more components of UBE3A operably linked to regulatory elements thereof as described herein. 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, gums, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients may also be incorporated into the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar untoward reactions when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like may be used to introduce the compositions of the invention into suitable host cells. Specifically, the UBE3A transgene delivered by the rAAV vector can be formulated for delivery or encapsulation in a lipid particle, liposome, vesicle, nanosphere, nanoparticle, or the like. In one embodiment, a therapeutically effective amount of the carrier is contained in a pharmaceutical composition. The person skilled in the art can easily select a suitable carrier in view of the indication for which the carrier is intended. For example, one suitable carrier includes saline, which may be formulated with a variety of buffer solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil and water. The choice of carrier is not a limitation of the present invention. Other conventional pharmaceutically acceptable carriers, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, methyl parahydroxybenzoate, ethyl vanillin, glycerin, phenol, and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar untoward reactions when administered to a host.

As used herein, the term "dose" or "amount" may refer to the total dose or amount delivered to a subject during treatment or the dose or amount delivered administered in a single unit (or multiple units or divided doses).

The aqueous suspensions or pharmaceutical compositions described herein are designed for delivery to a subject in need thereof by any suitable route or combination of different routes. In one embodiment, the pharmaceutical composition comprises an expression cassette or vector described herein in a formulation buffer suitable for delivery by an Intraventricular (ICV), intrathecal (IT), intracisternal, or Intravenous (IV) route of administration. Alternatively, other routes of administration may be selected (e.g., oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intramuscular, and other parenteral routes).

As used herein, the term "intrathecal delivery" or "intrathecal administration" refers to a route of administration of a drug by injection into a spinal canal, more specifically into the subarachnoid space such that it reaches the cerebrospinal fluid (CSF). Intrathecal delivery may include lumbar puncture, intraventricular (icv), suboccipital/intracisternal, and/or C1-2 puncture. For example, material may be introduced by lumbar puncture to spread throughout the subarachnoid space. In another example, injection into the cisterna magna (in the cisterna; ICM) may be performed. Delivery within the brain pool may increase carrier diffusion and/or reduce toxicity and inflammation caused by administration. See, e.g., christian Hinderer et al, broad gene transfer in the central nervous system of cynomolgus monkeys after delivery of AAV9 into the cerebellum medullary pool (Widespread gene transfer in the central nervous system of cynomolgus macaques following delivery of AAV9 into the cisterna magna), "molecular therapy method clinical development (Mol Ther Methods Clin dev.)) (2014; 1:14051. On-line publication at 12.10.2014. Doi:10.1038/mtm.2014.51. As used herein, the term "intracisternal delivery" or "intracisternal administration" refers to a route of administration of a drug directly into the cerebral ventricle or cerebrospinal fluid of the cerebellar medullary pool (cisterna magna cerebellomedularis), more specifically by subcontestinal puncture or by direct injection into the cerebellar medullary pool (cisterna magna) or by a permanently located tube.

In one aspect, provided herein is a pharmaceutical composition comprising a formulation buffer comprising a carrier as described herein. In certain embodiments, the replication-defective virus composition may be formulated in dosage units such that the replication-defective virus is contained in an amount of about 1.0x10 ⁹ GC to about 1.0x10 ¹⁶ Within the GC range (to treat subjects having an average weight of 70 kg), all integers or fractions within the range are included, and for human patients are preferably 1.0x10 ¹² GC to 1.0x10 ¹⁴ And (3) GC. In one embodiment, the composition is formulated to contain at least 1x10 per dose ⁹ 、2x10 ⁹ 、3x10 ⁹ 、4x10 ⁹ 、5x10 ⁹ 、6x10 ⁹ 、7x10 ⁹ 、8x10 ⁹ Or 9x10 ⁹ GC, contains all integer or fractional amounts within a range. In another embodiment, the composition is formulated to contain at least 1x10 per dose ¹⁰ 、2x10 ¹⁰ 、3x10 ¹⁰ 、4x10 ¹⁰ 、5x10 ¹⁰ 、6x10 ¹⁰ 、7x10 ¹⁰ 、8x10 ¹⁰ Or 9x10 ¹⁰ GC, contains all integer or fractional amounts within a range. In another embodiment, the composition is formulated to contain at least 1x10 per dose ¹¹ 、2x10 ¹¹ 、3x10 ¹¹ 、4x10 ¹¹ 、5x10 ¹¹ 、6x10 ¹¹ 、7x10 ¹¹ 、8x10 ¹¹ Or 9x10 ¹¹ GC, contains all integer or fractional amounts within a range. In another embodiment, the composition is formulated to contain at least 1x10 per dose ¹² 、2x10 ¹² 、3x10 ¹² 、4x10 ¹² 、5x10 ¹² 、6x10 ¹² 、7x10 ¹² 、8x10 ¹² Or 9x10 ¹² GC, contains all integer or fractional amounts within a range. In another embodiment, the composition is formulated to contain at least 1x10 per dose ¹³ 、2x10 ¹³ 、3x10 ¹³ 、4x10 ¹³ 、5x10 ¹³ 、6x10 ¹³ 、7x10 ¹³ 、8x10 ¹³ Or 9x10 ¹³ GC, contains all integer or fractional amounts within a range. In another embodiment, the composition is formulated to contain at least 1x10 per dose ¹⁴ 、2x10 ¹⁴ 、3x10 ¹⁴ 、4x10 ¹⁴ 、5x10 ¹⁴ 、6x10 ¹⁴ 、7x10 ¹⁴ 、8x10 ¹⁴ Or 9x10 ¹⁴ GC, contains all integer or fractional amounts within a range. In another embodiment, the composition is formulated to contain at least 1x10 per dose ¹⁵ 、2x10 ¹⁵ 、3x10 ¹⁵ 、4x10 ¹⁵ 、5x10 ¹⁵ 、6x10 ¹⁵ 、7x10 ¹⁵ 、8x10 ¹⁵ Or 9x10 ¹⁵ GC, contains all integer or fractional amounts within a range. In one embodiment, for human use, the dosage may range from 1x10 per dose ¹⁰ Up to about 1x10 ¹² GC, including all integers or fractional amounts within the range.

In one embodiment, a pharmaceutical composition is provided that includes a formulation buffer comprising a rAAV as described herein. In one embodiment, at about 1x10 ⁹ Genomic Copy (GC)/mL to about 1x10 ¹⁴ GC/mL formulation of rAAV. In a further embodiment, at about 3x10 ⁹ GC/mL to about 3x10 ¹³ GC/mL formulation of rAAV. In yet another embodiment, at about 1x10 ⁹ GC/mL to about 1x10 ¹³ GC/mL formulation of rAAV. In one embodiment, at least about 1x10 ¹¹ GC/mL formulation of rAAV.

Suitable volumes for delivering these doses and concentrations can be determined by those skilled in the art. For example, a volume of about 1 μl to 150mL can be selected, with a larger volume being selected for adults. Typically, a suitable volume is about 0.5mL to about 10mL for newborn infants, and about 0.5mL to about 15mL for older infants may be selected. For young children, a volume of about 0.5mL to about 20mL may be selected. For children, a volume of up to about 30mL may be selected. For pre-pubertal adolescents and adolescents, a volume of up to about 50mL may be selected. In still other embodiments, the volume that the patient can receive intrathecal administration is selected to be about 5mL to about 15mL or about 7.5mL to about 10mL. Other suitable volumes and dosages may be determined. Dosages will be adjusted to balance the therapeutic benefit with any side effects, and such dosages may be varied depending on the therapeutic application in which the recombinant vector is employed.

In the case of AAV viral vectors, quantification of genomic copies ("GC") can be used as a measure of the dose contained in an aqueous suspension or pharmaceutical composition. Any method known in the art may be used to determine the number of Genomic Copies (GC) of the replication defective virus composition of the invention. A method for performing AAV GC number titration is as follows: purified AAV vector samples were first treated with DNase to eliminate non-encapsidated AAV genomic DNA or contaminating plasmid DNA from the production process. The DNase resistant particles were then subjected to heat treatment to release the genome from the capsid. The released genome is then quantified by real-time PCR or quantitative PCR using primer/probe sets (typically poly a signals) that target specific regions of the viral genome. The replication-defective virus composition may be formulated in dosage units such that the replication-defective virus is contained in an amount of about 1.0x10 ⁹ GC to about 1.0x10 ¹⁵ Within the scope of GC, and for human patientsPreferably 1.0x10 ¹² GC to 1.0x10 ¹⁴ And (3) GC. Preferably, the concentration of replication defective virus in the formulation is about 1.0x10 ⁹ GC. About 5.0x10 ⁹ GC. About 1.0x10 ¹⁰ GC. About 5.0x10 ¹⁰ GC. About 1.0x10 ¹¹ GC. About 5.0x10 ¹¹ GC. About 1.0x10 ¹² GC. About 5.0x10 ¹² GC. About 1.0x10 ¹³ GC. About 5.0x10 ¹³ GC. About 1.0x10 ¹⁴ GC. About 5.0x10 ¹⁴ GC or about 1.0x10 ¹⁵ And (3) GC. An alternative or additional method for performing AAV GC number titration is by oqPCR or digital droplet PCR (ddPCR), such as, for example, m.lock et al, hum Gene thermethods.2014, month 4; 25 (2) 115-25.Doi:10.1089/hgtb.2013.131. Electronic version 2014, month 2, 14, which is incorporated herein by reference.

It should be understood that the compositions of the pharmaceutical compositions described herein are intended to apply to other compositions, protocols, aspects, embodiments, and methods described across this specification.

Method

In certain embodiments, the expression cassette, nucleic acid or viral or non-viral vector is used in the preparation of a medicament. In certain embodiments, there is provided its use for treating angeman syndrome in a subject in need thereof.

AS used herein, the term "treatment" or "treatment" is defined to encompass the administration of one or more compounds or compositions described herein to a subject for the purpose of ameliorating one or more symptoms of UBE3A deficiency or Angermann Syndrome (AS). Thus, "treatment" may include one or more of the following: reducing the onset or progression of AS, preventing a disease, reducing the severity of a disease symptom, slowing its progression, eliminating a disease symptom, delaying the progression of a disease, or increasing the efficacy of a therapy in a given subject.

The therapy provides UBE3A isoform 1 expression to achieve the desired result, namely treatment of Angermann Syndrome (AS) or one or more symptoms thereof. Such symptoms may include, but are not limited to, one or more of the following: intellectual disability, language disorders, ataxia, epilepsy, epileptic disorders, microcephaly, bradykinesia, and muscular dystrophy with hyperreflexia (see, e.g., buitting K et al, angermann syndrome—rare neurogenetic disease (Angelman syndrome-insight into a rare neurogenetic disorder), nat Rev Neurol,2016,12 (10): 584-593, electronic version, 9, 12 days, which is incorporated herein by reference). As described herein, the desired results may include reducing or eliminating neurophysical complications, including developmental delay, mental disability, severe language impairment, and motor and balance problems.

A "therapeutically effective amount" of a composition provided herein is delivered to a subject to achieve a desired result or to achieve a therapeutic goal. In one embodiment, the therapeutic goal of treating AS is to restore UBE3A isoform 1 expression in a neuron or population of neurons to a functional or non-AS level within the normal range of the patient. In another embodiment, the therapeutic goal of treating AS is to increase UBE3A isoform 1 expression to at least about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 2%, about 1% of normal or non-AS levels, or AS compared to pre-treatment UBE3A expression levels. Patients rescued by delivering UBE3A isoform 1 function to less than 100% activity levels may optionally receive further treatment. In another embodiment, the therapeutic goal of treating AS is to increase the expression of UBE3A isoform 1 in a percentage of target neurons, including about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 2%, or about 1% of neurons in the selected population.

In certain embodiments, provided herein is a method of treating AS by administering to a subject in need thereof an expression cassette, vector, or rAAV that provides UBE3A isoform 1, resulting in expression of functional UBE3A isoform 1 in neurons. In certain embodiments, the method comprises delivering a nucleic acid sequence (the amino acid sequence of SEQ ID NO: 2) that expresses UBE3A isoform 1.

In certain embodiments, provided herein is an enzyme replacement method that results in expression of functional UBE3a isoform 1 in neurons by administering an expression cassette, vector, or rAAV that provides UBE3a isoform 1 to a subject in need thereof. In certain embodiments, the method comprises delivering a nucleic acid sequence (the amino acid sequence of SEQ ID NO: 2) that expresses UBE3a isoform 1. In certain embodiments, provided herein is an enzyme replacement method that results in expression of functional UBE3a isoform 3 in neurons by administering an expression cassette, vector, or rAAV that provides UBE3a isoform 3 to a subject in need thereof. In certain embodiments, the method comprises delivering a nucleic acid sequence (the amino acid sequence of SEQ ID NO: 21) that expresses UBE3a isoform 3

The gene therapies described herein, whether viral or non-viral, can be used in combination with other therapies (secondary therapies), i.e., diagnostic and standard of care for the condition of the subject (patient). AS used herein, the term "secondary therapy" refers to a therapy that can be used in combination with the gene therapies described herein to treat AS. In some embodiments, the gene therapies described herein are administered in combination with one or more secondary therapies for treating AS, such AS administration of an anticonvulsant or dietary restriction (e.g., ketogenesis and hypoglycemia). The secondary therapy may be any therapy that helps to prevent, arrest or ameliorate these AS symptoms. The secondary therapy may be administered prior to, concurrently with, or after administration of the compositions described above. Subjects may be allowed to continue their standard-of-care treatment prior to and concurrently with gene therapy, as determined by their care-givers. In the alternative, the physician may prefer to discontinue the standard of care therapy prior to administration of the gene therapy treatment, and optionally, resume the standard of care therapy as a combination therapy after administration of the gene therapy. In another embodiment, the gene therapies described herein may be combined with genotyping or genetic screening, which are conventional in the art and may include using PCR to identify one or more mutations in the nucleic acid sequence of the UBE3A gene. AS described above, subjects exhibiting symptoms of AS early in life (e.g., 1-3 months) and subjects diagnosed with AS later in life are the intended recipients of the compositions and methods described herein.

"administration" or "route of administration" refers to the delivery of a composition described herein to a subject with or without a pharmaceutical carrier or excipient. The routes of administration may be combined, if desired. In some embodiments, the administration is repeated periodically. Sequential administration may mean a time interval of multiple administrations starting at intervals of days, weeks, months or years. In one embodiment, the compositions described herein are administered one or more times to a subject in need thereof. In one embodiment, administration is at intervals of days, weeks, months or years. In one embodiment, one, two, three or more re-administrations are allowed. Such re-administration may use the same type of carrier or a different carrier. In further embodiments, the vectors described herein can be used alone or in combination with standard of care for patient diagnosis and condition. The nucleic acid molecules and/or vectors described herein can be delivered as a single composition or as multiple compositions. Optionally, two or more different AAV, or multiple viruses may be delivered [ see, e.g., WO 2011/126808 and WO 2013/049493].

In one embodiment, the expression cassettes, vectors, or other compositions described herein for gene therapy are delivered as a single dose per patient. In one embodiment, a therapeutically effective amount of a composition described herein is delivered to a subject. As used herein, "therapeutically effective amount" refers to the amount of an expression cassette or vector or combination thereof.

In one embodiment, the expression cassette is present in the vector genome at about 1x10 per gram of brain mass ⁹ GC to about 1x10 per gram (g) of brain mass ¹³ The delivery of Genome Copies (GCs) in amounts, including all integers or ranges and fractional numbers within the endpoints. In another embodiment, the dose is 1x10 per gram of brain mass ¹⁰ GC to about 1x10 per gram brain mass ¹³ And (3) GC. In particular embodiments, the carrier is administered to the patient at a dose of at least about 1.0x10 ⁹ GC/g, about 1.5x10 ⁹ GC/g, about 2.0x10 ⁹ GC/g, about 2.5x10 ⁹ GC/g, about 3.0x10 ⁹ GC/g, about 3.5x10 ⁹ GC/g, about 4.0x10 ⁹ GC/g, about4.5x10 ⁹ GC/g, about 5.0x10 ⁹ GC/g, about 5.5x10 ⁹ GC/g, about 6.0x10 ⁹ GC/g, about 6.5x10 ⁹ GC/g, about 7.0x10 ⁹ GC/g, about 7.5x10 ⁹ GC/g, about 8.0x10 ⁹ GC/g, about 8.5x10 ⁹ GC/g, about 9.0x10 ⁹ GC/g, about 9.5x10 ⁹ GC/g, about 1.0x10 ¹⁰ GC/g, about 1.5x10 ¹⁰ GC/g, about 2.0x10 ¹⁰ GC/g, about 2.5x10 ¹⁰ GC/g, about 3.0x10 ¹⁰ GC/g, about 3.5x10 ¹⁰ GC/g, about 4.0x10 ¹⁰ GC/g, about 4.5x10 ¹⁰ GC/g, about 5.0x10 ¹⁰ GC/g, about 5.5x10 ¹⁰ GC/g, about 6.0x10 ¹⁰ GC/g, about 6.5x10 ¹⁰ GC/g, about 7.0x10 ¹⁰ GC/g, about 7.5x10 ¹⁰ GC/g, about 8.0x10 ¹⁰ GC/g, about 8.5x10 ¹⁰ GC/g, about 9.0x10 ¹⁰ GC/g, about 9.5x10 ¹⁰ GC/g, about 1.0x10 ¹¹ GC/g, about 1.5x10 ¹¹ GC/g, about 2.0x10 ¹¹ GC/g, about 2.5x10 ¹¹ GC/g, about 3.0x10 ¹¹ GC/g, about 3.5x10 ¹¹ GC/g, about 4.0x10 ¹¹ GC/g, about 4.5x10 ¹¹ GC/g, about 5.0x10 ¹¹ GC/g, about 5.5x10 ¹¹ GC/g, about 6.0x10 ¹¹ GC/g, about 6.5x10 ¹¹ GC/g, about 7.0x10 ¹¹ GC/g, about 7.5x10 ¹¹ GC/g, about 8.0x10 ¹¹ GC/g, about 8.5x10 ¹¹ GC/g, about 9.0x10 ¹¹ GC/g, about 9.5x10 ¹¹ GC/g, about 1.0x10 ¹² GC/g, about 1.5x10 ¹² GC/g, about 2.0x10 ¹² GC/g, about 2.5x10 ¹² GC/g, about 3.0x10 ¹² GC/g, about 3.5x10 ¹² GC/g, about 4.0x10 ¹² GC/g, about 4.5x10 ¹² GC/g, about 5.0x10 ¹² GC/g, about 5.5x10 ¹² GC/g, about 6.0x10 ¹² GC/g, about 6.5x10 ¹² GC/g, about 7.0x10 ¹² GC/g, about 7.5x10 ¹² GC/g, about 8.0x10 ¹² GC/g, about 8.5x10 ¹² GC/g, about 9.0x10 ¹² GC/g, about 9.5x10 ¹² GC/g, about 1.0x10 ¹³ GC/g, about 1.5x10 ¹³ GC/g, about 2.0x10 ¹³ GC/g, about 2.5x10 ¹³ GC/g, about 3.0x10 ¹³ GC/g, about 3.5x10 ¹³ GC/g, about4.0x10 ¹³ GC/g, about 4.5x10 ¹³ GC/g, about 5.0x10 ¹³ GC/g, about 5.5x10 ¹³ GC/g, about 6.0x10 ¹³ GC/g, about 6.5x10 ¹³ GC/g, about 7.0x10 ¹³ GC/g, about 7.5x10 ¹³ GC/g, about 8.0x10 ¹³ GC/g, about 8.5x10 ¹³ GC/g, about 9.0x10 ¹³ GC/g, about 9.5x10 ¹³ GC/g, or about 1.0x10 ¹⁴ GC/g brain mass.

In certain embodiments, the regimen may involve additional treatments, including compositions comprising a gene editing system. See, for example, U.S. patent application Ser. Nos. 63/016,712, filed on 28 th month 4 of 2020, and U.S. patent application Ser. No. 63/118,299, filed on 25 th 11 of 2020, titled "composition for treating Angelmann syndrome and use thereof (Compositions and uses for Treatment of Angelman Syndrome)". Such treatment may be performed prior to treatment with the gene replacement therapies described herein, and may utilize a vector having a capsid different from that used for the initial treatment. Other combinations of AAV capsids may be selected by those skilled in the art.

In certain embodiments, the treatment regimen may involve co-expression of UBE3A isoform 1 with UBE3A isoform 3. In certain embodiments, the treatment may involve a combination therapy of aav. Hbbe 3A-isoform 1 and hbbe 3A enzyme replacement therapy (e.g., with an isoform 3 and/or isoform 1 enzyme). In certain embodiments, the treatment regimen may involve combination therapy with an aav.hube3a-isoform 1 gene therapeutic vector and an immunomodulatory regimen. Such immunomodulation regimens may include, for example, but are not limited to, immunosuppressants such as glucocorticoids, steroids, antimetabolites, T-cell inhibitors, macrolides (e.g., rapamycin or rapamycin analogs), and cytostatic agents, including alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies, or agents active against immunoaffinity. The immunosuppressant may comprise nitrogen mustard (nitrogen mustard), nitrosourea (nitrosourcea), platinum compounds, methotrexate (methotrextrate), azathioprine (azathioprine), mercaptopurine (merapatopurin), fluorouracil (fluorouracil), dactinomycin (dactinomycin), anthracycline (anthracycline), mitomycin C (mitomycin C), bleomycin (bleomycin), mithramycin (mithramycin), IL-2 receptor (CD 25) or CD3 directed antibodies, anti-IL-2 antibodies, cyclosporine (cycloporin), tacrolimus (tacrolimus), IFN- β, IFN- γ, opioids or TNF- α binding agents. In certain embodiments, immunosuppressive therapy may be initiated prior to administration of gene therapy. Such therapies may involve co-administration of two or more drugs (e.g., prednisone, mycophenolate Mofetil (MMF), and/or sirolimus (i.e., rapamycin)) within the same day. One or more of these drugs may be continued to be used at the same dose or at an adjusted dose after administration of the gene therapy. Such therapies may last for about 1 week, about 15 days, about 30 days, about 45 days, about 60 days, or longer, as desired. Still other combination therapies may include, for example, anti-IgG enzymes that have been described as useful for eliminating anti-AAV antibodies (and thus may allow administration to a patient to test for antibody levels of a selected AAV capsid above a threshold level) and/or delivering, for example, anti-FcRN antibodies described in U.S. provisional patent application No. 63/040,381 entitled "composition and method for treating a gene therapy patient (Compositions and Methods for Treatment of Gene Therapy Patients)" filed on month 6 of 2020, and/or a) a steroid or combination of steroids and/or (b) an IgG lyase; (c) an Fc-IgE binding inhibitor; (d) an Fc-IgM binding inhibitor; (e) an Fc-IgA binding inhibitor; and/or (f) one or more of gamma interferon.

Generally, the method comprises administering to a mammalian subject in need thereof a pharmaceutically effective amount of a composition comprising a recombinant adeno-associated virus (AAV) carrying a nucleic acid sequence encoding one or more elements of a UBE3A gene substitution (expression) system under the control of regulatory sequences, and a pharmaceutically acceptable carrier. In one embodiment, such methods are designed to treat, delay or stop progression of AS in a mammalian subject.

In one embodiment, a method of treating AS is provided by administering to a subject in need thereof a vector, rAAV, aqueous suspension or medicament AS described hereinA composition. In one embodiment, the rAAV is at about 1x10 ¹⁰ Up to about 1x10 ¹⁵ Genomic Copy (GC)/kg body weight delivery. In certain embodiments, the subject is a human. In one embodiment, the rAAV is administered multiple times. In further embodiments, the rAAV is administered at intervals of days, weeks, months, or years.

Examples

The invention will now be described with reference to the following examples. These examples are for illustrative purposes only and the invention should not be construed as limited to these examples but rather should be construed to encompass any and all variations that become apparent as a result of the teachings provided herein.

Angerman Syndrome (AS) is a rare neurological disorder affecting approximately one hundred thousand people worldwide. AS is characterized by mental and physical disability, seizures and sleep disorders, and intestinal function. Many of these defects are caused by loss of the maternal inherited ubiquitin protein ligase E3A (UBE 3A) allele. Currently, treatment options for AS are limited. AAV-based gene replacement therapies represent a promising strategy to restore UBE3A isoform expression and reduce AS severity. However, the UBE3A gene encodes three isoforms, and it is currently unclear which UBE3A isoform is the most effective candidate. We compared AAV vectors in AS (UBE 3A) ^m-/p+ ) The efficacy of delivering engineered human UBE3A isoforms in a mouse model addresses this uncertainty. Western blotting and immunohistochemical analysis showed that intraventricular injection of AAV-UBE3A human isoforms 1 and 2 vectors into neonatal control and AS mice resulted in robust protein expression. The isochrom 1 surrogate significantly improved gait, nesting and motor coordination in a dose dependent manner in AS mice. Unlike isotype 1, isotype 2 further compromises the nesting and motor coordination ability of AS mice. Toxicology studies in non-human primates showed that injection of high doses of AAV-UBE3A isoform 1 vector into the cerebellar medullary pool had no significant side effects. Taken together, these data indicate that AAV-UBE3A isoform 1 vectors are most effective in therapy and are also promising in safety. These preclinical findings represent an important step in the development of AS gene replacement therapies.

Example 1-engineering of hUBE3A isoform 1 and engineered hUBE3A isoform 2 coding sequences under the direction of a modified human synaptoprotein promoter

For the first set of studies in mice, we designed a series of transgenes containing engineered human UBE3A isoform 1 (SEQ ID NO: 9) or UBE3A isoform 2 (SEQ ID NO: 10) that had a modified human synaptoprotein (hSyn) promoter (SEQ ID NO: 12). SV40 polyadenylation sequence (SEQ ID NO: 13) is included to promote transcript stability. On both sides of the promoter-transgene-SV 40 construct are Inverted Terminal Repeats (ITRs). The plasmid was packaged into php.b, an AAV9 variant, which robustly transduced neurons and glial cells of the C57BL6/J mouse central nervous system.

The coding sequence for the modified human synapsin promoter is provided in SEQ ID NO. 12.

The resulting vector genome was reproduced in SEQ ID NO. 3 (hSyn. HUbe3a-1. GSco.SV40) and SEQ ID NO. 7 (isotype 2hSyn. HUbe3a-2. GSco.SV40).

Aavphp.b capsids (US 9,585,971) are trans plasmids generated in packaging host cells using triple transfection techniques in trans plasmids comprising AAV2rep coding sequences and php.b VP1 coding sequences, co-transfected with cis plasmids containing the vector genome and expressing essential adenovirus helper functions not provided by packaging host cells.

EXAMPLE 2 rAAV mediated delivery of hUBE3A to mice and NHPs

Expression test of hUBE3A isoform 1 and isoform 2 in adult mouse brain

We injected (intraventricular-ICV) AAV-php.b-synaptotagmin-UBE 3A isoform 1 and AAV-php.b-synaptotagmin-UBE 3A-isoform 2 into the brains of wild-type adult mice by western blotting and quantified transgene expression, confirming the highest expressing vector (fig. 8).

The method comprises the following steps: 5x10 ¹¹ GC/mice were injected with retroorbital (IV) into adult wild-type WT (UBE 3A) ^m+/p+ ) And UBE3AKO/null (UBE 3A) ^m-/p+ ) In litters. Brains were harvested 14 days after injection for western blotting. The cDNA was then subcloned into the kitIn plasmids with intact ITRs to obtain optimal expression.

B. Determination of AAV-UBE3A isoform vectors most effective in therapy

We directly compared the ability of the highest expressing AAV-php.b-synaptoprotein-UBE 3A-isoform 1 (isoform 1) to rescue motor and behavioral deficits in the AS mouse model with AAV-php.b-synaptoprotein-UBE 3A-isoform 2 (isoform 2) vectors. Nascent wild type or AS (UBE 3A) ^m-/p+ ) Mice were 1x10 per animal ¹¹ Dosage of genomic copies in brain room (ICV) either isoform 1 or isoform 2 vectors are injected. Behavioral testing (8-10 weeks of age), test sequence: (1) walk to show, (2) sport activity, (3) bull stick, (4) nest. Two months later, AS mice injected with isotype 1 instead of isotype 2 showed statistically significant improvements in gait (stride length figures 11A-11D), nesting ability (figures 10A and 10B), and motor coordination ability (figures 9A and 9B). In fact, expression of isoform 2 aggravates the defect in nesting ability of AS mice (fig. 10C and 10D). 1x10 per animal ¹⁰ Lower doses of genomic copies injected with isotype 1 were less effective, but did increase nesting ability in AS mice. We also observed that overexpression of either isotype 1 or 2 in wild-type mice adversely affected several behavioral areas, indicating the importance of tightly controlling UBE3A isotype expression. In summary, head-to-head testing indicated that nuclear UBE3A isoform 1 vectors have greater therapeutic capacity than cytoplasmic isoform 2 vectors in reducing behavioral defects in AS mice. By and from wild-type UBE3A ^m-/p+ And treated UBE3A ^m-/p+ Neuronal markers NeuN and nuclei co-stained in the sagittal brain portion of mice (i.e. cortex, hippocampus, thalamus, hypothalamus, midbrain), immunofluorescence images confirmed the expression and localization of engineered human UBE3A isoform 1 protein. The fractions (percentages) of UBE3A isoform 1 (fig. 5 and 6) and UBE3A isoform 2 (fig. 7) protein positive neurons in different brain regions were determined and quantified using the visioparm software (fig. 5 and 6).

At 1x10 ¹⁰ At GC dose, AAV-PHP.B-hSyn-UBE 3A-isoform 1 dose achieved 50% expression of wild-type endogenous UBE3A isoforms without significant impact on locomotor activity, locomotor coordination ability, or nestingThe behavioral deficit in this respect, and improved some gait abnormalities. FIG. 5 shows treated UBE3A ^m-/p+ Quantification of UBE3A protein positive neurons in mice (statistical analysis: mean ± SD, unpaired student t test). Treatment included passage through the brain chamber (ICV) at 1x10 ¹⁰ The GC/animal dose was administered AAV-PHP.B-hSyn-UBE 3A-isoform 1.

At 1x10 ¹¹ At higher doses of GC, AAV-php.b-hSyn-UBE 3A-isoform 1 administration achieved-100% expression of wild-type endogenous UBE3A isoforms, significantly improving nesting ability (fig. 10A). UBE3A treated according to the method of Deacon RMJ,2006, to evaluate the nesting ability of mice, nat Protoc 1 (3): 1117-9 ^m-/p+ The mice were nest using significantly more nesting material (fig. 10B). UBE 3A-isoform 1 doses had no effect on hypoactivity but were normalized to previously published UBE3A ^mp+ Abnormal gait in mice (stride length assessment as described in Heck et al, analysis of brain function in Ube3a deficient mice revealed genotype-specific behavior, 2008, hum Mol genetics, 17 (14): 2181-2189; electronic edition 2008, 4 months 15). UBE3A was significantly reduced by administration of UBE 3A-isoform 1 (fig. 9A) but not by administration of isoform 2 (fig. 9B) ^m-/p+ Motor coordination ability of mice is deficient. FIG. 6 shows treated UBE3A ^m-/p+ Quantification of UBE3A protein-positive neurons in mice (statistical analysis: unpaired t-test). Treatment included passage through the brain chamber (ICV) at 1x10 ¹¹ The GC/animal dose was administered AAV-PHP.B-hSyn-UBE 3A-isoform 1. The quantification of protein expression from fig. 5 is summarized in table 1 below.

Table 1.

Brain part	UBE 3A-1-percent positive neurons
		Cortex layer	～68％
Hippocampus japonicus	～63％
		Thalamus and thalamus	～58％
Hypothalamus (hypothalamus)	～42％
		Midbrain	～47％

Therapeutic effects of UBE 3A-isoform substitution: isotype 1: (i) normalizing stride length (clinically relevant gait deficit), (ii) improving motor coordination ability, (iii) improving nesting behavior, (iv) defective to UBE3 (UBE 3A) ^m-/p+ ) The hypokinesia of the mice had no effect. Isotype 2: (i) Impairment of nesting behaviour and (ii) no effect on hypokinesia (as in type 1) or motor coordination.

Additionally, as noted above, the data indicate that robust UBE3A isoform 1 protein expression can be seen in most neurons in several brain regions. UBE3A isoform 1 protein expression showed dose-dependency in 1X10 ¹¹ GC/animal and 1X10 ¹⁰ The percentage of UBE3A positive neurons was higher in GC/animal treated animals.

Preclinical studies in AS mice indicate that AAV-php.b-synaptoprotein-UBE 3A-isoform 1 is well expressed in AS mice and reduces motor and behavioral deficits. We next investigated the therapeutic potential of isotype 1 vector by assessing its safety in rhesus monkeys at high doses, as the immune system and brain structure of rhesus monkeys are similar to humans. First, we generated an AAV-hu 68-synaptorin-UBE 3A-isoform 1 vector. AAVhu68 is also an internally developed AAV9 variant, which performs well in non-human primates. We use 3x10 ¹³ GC/animal high dose AAVhu68-hSyn-UBE 3A-isoform 1 vector was injected into the large pool cerebrospinal fluid of three rhesus monkeys (NHP-1, NHP-2, NHP-3). After 35 days, macaques were removed and assessed for transgene expression, immune response, and adverse effects. None of the animals showed clinically significant symptoms or neurological problems in the cage side evaluation. Blood chemistry tests indicate that the treated macaque has normal blood coagulation function and liver and kidney function. We observed low levels of isoform 1 vector in several brain regions, including medulla and parietal and temporal cortex, and higher levels of isoform 1 vector in the dorsal root ganglion of the spine (fig. 2A). Furthermore, we detected low expression of human UBE 3A-isoform 1mRNA in several brain regions including the dorsal root ganglion of the spinal column (fig. 2B and 2C). Fluorescence images of dorsal root ganglion (cervical, thoracic and dorsal segments) of treated NHPs confirm localization of engineered human UBE3A isoform 1 transcripts. Use of RNAscope probes specific for engineered human sequences to determine UBE3A isoform 1 transcript localization (fig. 7A-7I) and counterstain with DAPI (cell nucleus). Images of the region of interest were taken at different magnifications and presented at 20x magnification. Fig. 7A-7I show fluorescence images of the localization of engineered human UBE3A isoform 1 (hbbe 3A-1) transcripts in dorsal root ganglion (neck, chest and dorsal segment) from three treated non-human primates (NHP; in a 35 day study). Treatment included 3x10 by the cerebellar medullary pool (ICM) route ¹³ The GC/animal dose was administered AAV-hu68-hSyn-UBE 3A-isoform 1. The image of the region of interest is presented at 20x magnification. FIG. 7A shows a fluorescence image of engineered hUBE3A-1 transcript localization in dorsal root ganglion cervical segments from NHP-1. FIG. 7B shows a fluorescence image of engineered hUBE3A-1 transcript localization in dorsal root ganglion cervical segments from NHP-2. FIG. 7C shows a fluorescence image of engineered hUBE3A-1 transcript localization in dorsal root ganglion cervical segments from NHP-3. FIG. 7D shows a fluorescence image of the localization of engineered hUBE3A-1 transcripts in dorsal root ganglion thoracic segments from NHP-1. FIG. 7E shows a fluorescence image of the localization of engineered hUBE3A-1 transcripts in dorsal root ganglion thoracic segments from NHP-2. FIG. 7F shows engineered hUBE3A-1 transduce in dorsal root ganglion thoracic segments from NHP-3Fluorescent image of transcript localization. FIG. 7G shows a fluorescence image of engineered hUBE3A-1 transcript localization in dorsal root ganglion lumbar segment from NHP-1. FIG. 7H shows a fluorescence image of engineered hUBE3A-1 transcript localization in dorsal root ganglion lumbar segment from NHP-2. FIG. 7I shows a fluorescence image of engineered hUBE3A-1 transcript localization in dorsal root ganglion lumbar segment from NHP-3.

The ELISPOT analysis showed minimal immune response, indicating a modest immune response to the hu68 capsid as expected. Finally, dorsal Root Ganglion (DRG) toxicity is a platfonn problem for non-human primate and potentially human central nervous system targeting and high doses of AAV. However, we observed only slight DRG (fig. 12 to 12C) and neuronal toxicity in the spinal cord (fig. 13A to 13C) and peripheral nerves (fig. 13A to 13C) of one animal. Taken together, these findings indicate that high dose ICM delivery of AAVhu68-hSyn-UBE 3A-isoform 1 vector is well tolerated in non-human primates. Additionally, it was concluded from this dataset that robust UBE3A isoform 1 transcript localization could be detected in all DRG fragments, consistent with the pPCR analysis. Despite robust transgenic DRG expression, there was no significant AAV toxicity in the treated NHPs.

EXAMPLE 3 NHP test point study to investigate safety and expression of hUBE3A isoform 1+ -miR 183 target sequence in rhesus monkeys

Because of concerns about non-human primate and human high dose AAV-induced DRG toxicity, we simultaneously employed microrna strategies to down-regulate isoform 1 protein expression in DRGs, thereby alleviating DRG toxicity. Inclusion of the miR183 target site in the transgenic 3' utr (UBE 3A-isoform 1 in this example) selectively promotes RISC complex-mediated degradation of isoform 1 transgenic mRNA in DRG sensory neurons while retaining the therapeutic transgene in the target neurons in the brain. We inserted four copies of the miR183 binding site between the 3' end of the highest expressing engineered isoform 1 or isoform 2 and the SV40 polyadenylation site and packaged them into AAVhu68 capsids. SEQ ID NO. 11 provides a sequence that copies the miRNA183 (or miR 183) target sequence. In this way, we produced AAV-hu 68-synaptorin-UBE 3A-isoform 1-4xmiR183.AAVhu68 capsids are produced in packaging host cells using triple transfection techniques in a trans plasmid comprising the AAV2 rep coding sequence and the hu68 VP1 coding sequence of SEQ ID No. 14, co-transfected with a cis plasmid containing the vector genome and a trans plasmid expressing essential adenovirus helper functions not provided by the packaging host cells.

At 1x10 ¹¹ Intravenous injection of the 4xmiR183 cassette at the dose of GC/animal did not significantly affect UBE 3A-isoform 1 or isoform 2 protein expression in adult wild-type or AS mice.

We next compared the safety and expression profile of rAAV hu 68-synaptorin-UBE 3A-isoform 1-4xmiR183 vector in rhesus monkeys with the rAAV hu 68-synaptorin-UBE 3A-isoform 1-miR183 vector previously analyzed. AAV-hu 68-synaptoprotein-UBE 3A-isoform 1-4xmiR183 was expressed as 3X10 ¹³ GC/animals were injected into the cerebellum pool of three macaques and after 35 days the macaques were removed for analysis. During the study, all animals were in clinically insignificant state. We observed low levels of isoform 1-miR183 vector in some brain regions, with higher levels of medulla in dorsal root ganglion and spinal cord as expected. Furthermore, we detected low expression of human isoform 1-miR183 mRNA in the dorsal root ganglion and spinal cord, which is generally comparable to isoform 1. Like isoform 1, isoform 1-miR183 is well tolerated in non-human primates, is less immunogenic, and has no effect on blood coagulation, liver and kidney function. Similarly, we did not observe significant DRG or neuronal toxicity in macaques treated with isoform 1-miR 183. See fig. 3A-B. The effect of rAAV hu68 on the peripheral nerve of synaptotagmin-UBE 3A-isoform 1 is shown in FIGS. 4A-4B.

Vector biodistribution and mRNA expression were assessed and are shown in figures 2A-2C.

Taken together, these findings indicate that high dose ICM delivery of AAVhu68-hSyn-UBE 3A-isoform 1-miR183 vectors is well tolerated in non-human primates. Such a vector, while not useful for non-human primates, may help to reduce DRG and neuronal toxicity if it is present in human clinical trials.

(sequence Listing free text)

The following information is provided for a sequence containing free text under the numeric identifier <223 >.

All documents cited in this specification are incorporated herein by reference. The sequence listing filed herewith under the name "21-9579pct_seqlisting_st25" and sequences and text therein are incorporated by reference. U.S. provisional application No. 63/119,860, filed on 1 of 12 months in 2020, and U.S. provisional application No. 63/179,807, filed on 26 of 4 months in 2021, are incorporated herein by reference. Although the invention has been described with reference to specific embodiments, it will be understood that modifications may be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Sequence listing

<110> assignee of university of pennsylvania

<120> composition and use thereof for the treatment of angermann syndrome

<130> UPN-21-9579.PCT

<150> US 63/119,860

<151> 2020-12-01

<150> US 63/179,807

<151> 2021-04-26

<160> 25

<170> patent version 3.5

<210> 1

<211> 3804

<212> DNA

<213> artificial sequence

<220>

<223> vector genome hSyn. HUbe3a-1.GSco.4XmiRNA183.SV40

<220>

<221> repeat_region

<222> (1)..(130)

<223> ITR

<220>

<221> promoter

<222> (213)..(678)

<223> human synaptoprotein

<220>

<221> misc_feature

<222> (690)..(695)

<223> Kozak

<220>

<221> CDS

<222> (696)..(3257)

<223> hUBE3a-1

<220>

<221> misc_feature

<222> (779)..(779)

<223> mutation

<220>

<221> misc_feature

<222> (824)..(824)

<223> mutation

<220>

<221> misc_feature

<222> (1028)..(1028)

<223> mutation

<220>

<221> misc_feature

<222> (1128)..(1128)

<223> mutation

<220>

<221> misc_feature

<222> (1181)..(1181)

<223> mutation

<220>

<221> misc_feature

<222> (1217)..(1217)

<223> mutation

<220>

<221> misc_feature

<222> (1283)..(1283)

<223> mutant SD/AS

<220>

<221> misc_feature

<222> (1715)..(1715)

<223> mutation

<220>

<221> misc_feature

<222> (1721)..(1721)

<223> mutation

<220>

<221> misc_feature

<222> (1766)..(1766)

<223> mutation

<220>

<221> misc_feature

<222> (1805)..(1805)

<223> mutation

<220>

<221> misc_feature

<222> (1814)..(1814)

<223> mutation

<220>

<221> misc_feature

<222> (1991)..(1991)

<223> mutation

<220>

<221> misc_feature

<222> (2063)..(2063)

<223> mutation

<220>

<221> misc_feature

<222> (2111)..(2111)

<223> mutation

<220>

<221> misc_feature

<222> (2294)..(2294)

<223> mutation

<220>

<221> misc_feature

<222> (2439)..(2440)

<223> mutant SD/AS

<220>

<221> misc_feature

<222> (2723)..(2723)

<223> mutation

<220>

<221> misc_feature

<222> (2957)..(2957)

<223> mutation

<220>

<221> misc_feature

<222> (3023)..(3023)

<223> mutation

<220>

<221> misc_feature

<222> (3056)..(3056)

<223> mutant SD/AS

<220>

<221> misc_feature

<222> (3152)..(3152)

<223> mutation

<220>

<221> misc_feature

<222> (3161)..(3161)

<223> mutation

<220>

<221> misc_feature

<222> (3230)..(3230)

<223> mutation

<220>

<221> misc_feature

<222> (3264)..(3285)

<223> miRNA183

<220>

<221> misc_feature

<222> (3318)..(3311)

<223> miRNA183

<220>

<221> misc_feature

<222> (3318)..(3339)

<223> miRNA183

<220>

<221> misc_feature

<222> (3346)..(3367)

<223> miRNA183

<220>

<221> polyA_Signal

<222> (3379)..(3610)

<223> SV40 polyA

<220>

<221> repeat_region

<222> (3675)..(3804)

<223> ITR

<400> 1

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcc tctagaacta tagctagcat gcctgcagag ggccctgcgt atgagtgcaa 240

gtgggtttta ggaccaggat gaggcggggt gggggtgcct acctgacgac cgaccccgac 300

ccactggaca agcacccaac ccccattccc caaattgcgc atcccctatc agagaggggg 360

aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc gcggacagtg 420

ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg cgcgctgacg 480

tcactcgccg gtcccccgca aactcccctt cccggccacc ttggtcgcgt ccgcgccgcc 540

gccggcccag ccggaccgca ccacgcgagg cgcgagatag gggggcacgg gcgcgaccat 600

ctgcgctgcg gcgccggcga ctcagcgctg cctcagtctg cggtgggcag cggaggagtc 660

gtgtcgtgcc tgagagcgca gtcgaattcg ccacc atg aag agg gca gca gca 713

Met Lys Arg Ala Ala Ala

1 5

aag cac ctg atc gag aga tac tat cac cag ctg acc gag gga tgc gga 761

Lys His Leu Ile Glu Arg Tyr Tyr His Gln Leu Thr Glu Gly Cys Gly

10 15 20

aac gag gca tgt aca aac gag ttc tgc gcc tcc tgt ccc acc ttt ctg 809

Asn Glu Ala Cys Thr Asn Glu Phe Cys Ala Ser Cys Pro Thr Phe Leu

25 30 35

agg atg gat aac aac gcc gcc gcc atc aag gcc ctg gag ctg tac aag 857

Arg Met Asp Asn Asn Ala Ala Ala Ile Lys Ala Leu Glu Leu Tyr Lys

40 45 50

atc aac gcc aag ctg tgc gac ccc cac cct agc aag aag ggc gcc agc 905

Ile Asn Ala Lys Leu Cys Asp Pro His Pro Ser Lys Lys Gly Ala Ser

55 60 65 70

tcc gcc tat ctg gag aac tcc aag ggc gcc cct aac aat agc tgt tcc 953

Ser Ala Tyr Leu Glu Asn Ser Lys Gly Ala Pro Asn Asn Ser Cys Ser

75 80 85

gag atc aag atg aat aag aag ggc gcc cgg atc gat ttc aag gac gtg 1001

Glu Ile Lys Met Asn Lys Lys Gly Ala Arg Ile Asp Phe Lys Asp Val

90 95 100

acc tac ctg aca gag gag aag gtg tac gag atc ctg gag ctg tgc cgg 1049

Thr Tyr Leu Thr Glu Glu Lys Val Tyr Glu Ile Leu Glu Leu Cys Arg

105 110 115

gag aga gag gat tac agc cca ctg atc aga gtg atc ggc aga gtg ttc 1097

Glu Arg Glu Asp Tyr Ser Pro Leu Ile Arg Val Ile Gly Arg Val Phe

120 125 130

tct agc gcc gag gcc ctg gtg cag tcc ttt aga aag gtg aag cag cac 1145

Ser Ser Ala Glu Ala Leu Val Gln Ser Phe Arg Lys Val Lys Gln His

135 140 145 150

aca aag gag gag ctg aag tct ctg cag gcc aag gac gag gac aag gac 1193

Thr Lys Glu Glu Leu Lys Ser Leu Gln Ala Lys Asp Glu Asp Lys Asp

155 160 165

gag gac gag aag gag aag gca gcc tgt tct gcc gca gca atg gag gag 1241

Glu Asp Glu Lys Glu Lys Ala Ala Cys Ser Ala Ala Ala Met Glu Glu

170 175 180

gac agc gag gca tcc tct agc cgg atc ggc gat tcc tct caa ggc gac 1289

Asp Ser Glu Ala Ser Ser Ser Arg Ile Gly Asp Ser Ser Gln Gly Asp

185 190 195

aac aat ctg cag aag ctg ggc ccc gac gac gtg agc gtg gat atc gac 1337

Asn Asn Leu Gln Lys Leu Gly Pro Asp Asp Val Ser Val Asp Ile Asp

200 205 210

gcc atc cgg aga gtg tac aca aga ctg ctg agc aac gag aag atc gag 1385

Ala Ile Arg Arg Val Tyr Thr Arg Leu Leu Ser Asn Glu Lys Ile Glu

215 220 225 230

acc gcc ttc ctg aac gcc ctg gtg tat ctg agc cct aat gtg gag tgc 1433

Thr Ala Phe Leu Asn Ala Leu Val Tyr Leu Ser Pro Asn Val Glu Cys

235 240 245

gat ctg acc tac cac aac gtg tac tcc cgg gac cca aac tac ctg aat 1481

Asp Leu Thr Tyr His Asn Val Tyr Ser Arg Asp Pro Asn Tyr Leu Asn

250 255 260

ctg ttc atc atc gtg atg gag aac aga aat ctg cac tcc ccc gag tat 1529

Leu Phe Ile Ile Val Met Glu Asn Arg Asn Leu His Ser Pro Glu Tyr

265 270 275

ctg gag atg gcc ctg cct ctg ttt tgt aag gcc atg tcc aag ctg cct 1577

Leu Glu Met Ala Leu Pro Leu Phe Cys Lys Ala Met Ser Lys Leu Pro

280 285 290

ctg gca gca cag ggc aag ctg atc agg ctg tgg tct aag tac aac gcc 1625

Leu Ala Ala Gln Gly Lys Leu Ile Arg Leu Trp Ser Lys Tyr Asn Ala

295 300 305 310

gat cag atc agg cgc atg atg gag acc ttc cag cag ctg atc aca tac 1673

Asp Gln Ile Arg Arg Met Met Glu Thr Phe Gln Gln Leu Ile Thr Tyr

315 320 325

aaa gtg atc tct aac gag ttt aat agc cgc aac ctg gtg aac gac gac 1721

Lys Val Ile Ser Asn Glu Phe Asn Ser Arg Asn Leu Val Asn Asp Asp

330 335 340

gac gcc atc gtg gcc gcc tct aag tgc ctg aag atg gtg tac tac gcc 1769

Asp Ala Ile Val Ala Ala Ser Lys Cys Leu Lys Met Val Tyr Tyr Ala

345 350 355

aac gtg gtg ggc ggc gag gtg gac aca aac cac aac gag gag gac gac 1817

Asn Val Val Gly Gly Glu Val Asp Thr Asn His Asn Glu Glu Asp Asp

360 365 370

gag gag cca atc ccc gag agc tcc gag ctg acc ctg cag gag ctg ctg 1865

Glu Glu Pro Ile Pro Glu Ser Ser Glu Leu Thr Leu Gln Glu Leu Leu

375 380 385 390

gga gag gag cgg aga aat aag aag gga cca agg gtg gat cct ctg gag 1913

Gly Glu Glu Arg Arg Asn Lys Lys Gly Pro Arg Val Asp Pro Leu Glu

395 400 405

acc gag ctg ggc gtg aag aca ctg gac tgc aga aag cct ctg atc cca 1961

Thr Glu Leu Gly Val Lys Thr Leu Asp Cys Arg Lys Pro Leu Ile Pro

410 415 420

ttc gag gag ttt atc aac gag ccc ctg aac gag gtg ctg gag atg gat 2009

Phe Glu Glu Phe Ile Asn Glu Pro Leu Asn Glu Val Leu Glu Met Asp

425 430 435

aag gac tac acc ttc ttt aag gtg gag aca gag aac aag ttc agc ttt 2057

Lys Asp Tyr Thr Phe Phe Lys Val Glu Thr Glu Asn Lys Phe Ser Phe

440 445 450

atg acc tgt cct ttc atc ctg aac gcc gtg acc aag aat ctg ggc ctg 2105

Met Thr Cys Pro Phe Ile Leu Asn Ala Val Thr Lys Asn Leu Gly Leu

455 460 465 470

tac tac gat aac agg atc cgc atg tac tcc gag agg cgc atc acc gtg 2153

Tyr Tyr Asp Asn Arg Ile Arg Met Tyr Ser Glu Arg Arg Ile Thr Val

475 480 485

ctg tat tct ctg gtg cag ggc cag cag ctg aat cct tac ctg agg ctg 2201

Leu Tyr Ser Leu Val Gln Gly Gln Gln Leu Asn Pro Tyr Leu Arg Leu

490 495 500

aag gtg cgg aga gac cac atc atc gat gac gcc ctg gtg cgc ctg gag 2249

Lys Val Arg Arg Asp His Ile Ile Asp Asp Ala Leu Val Arg Leu Glu

505 510 515

atg atc gcc atg gag aat cca gcc gat ctg aag aag cag ctg tac gtg 2297

Met Ile Ala Met Glu Asn Pro Ala Asp Leu Lys Lys Gln Leu Tyr Val

520 525 530

gag ttt gag gga gag cag gga gtg gac gag gga ggc gtg tct aag gag 2345

Glu Phe Glu Gly Glu Gln Gly Val Asp Glu Gly Gly Val Ser Lys Glu

535 540 545 550

ttc ttt cag ctg gtg gtg gag gag atc ttc aac ccc gat atc ggc atg 2393

Phe Phe Gln Leu Val Val Glu Glu Ile Phe Asn Pro Asp Ile Gly Met

555 560 565

ttt acc tac gac gag agc aca aag ctg ttc tgg ttt aat cct tct tcc 2441

Phe Thr Tyr Asp Glu Ser Thr Lys Leu Phe Trp Phe Asn Pro Ser Ser

570 575 580

ttc gag acc gag ggc cag ttt aca ctg atc ggc atc gtg ctg ggc ctg 2489

Phe Glu Thr Glu Gly Gln Phe Thr Leu Ile Gly Ile Val Leu Gly Leu

585 590 595

gcc atc tac aac aat tgt atc ctg gac gtg cac ttc cca atg gtg gtg 2537

Ala Ile Tyr Asn Asn Cys Ile Leu Asp Val His Phe Pro Met Val Val

600 605 610

tat agg aag ctg atg ggc aag aag ggc acc ttt cgc gat ctg ggc gac 2585

Tyr Arg Lys Leu Met Gly Lys Lys Gly Thr Phe Arg Asp Leu Gly Asp

615 620 625 630

tcc cac ccc gtg ctg tac cag tct ctg aag gat ctg ctg gag tat gag 2633

Ser His Pro Val Leu Tyr Gln Ser Leu Lys Asp Leu Leu Glu Tyr Glu

635 640 645

ggc aac gtg gag gat gac atg atg atc acc ttc cag atc tcc cag aca 2681

Gly Asn Val Glu Asp Asp Met Met Ile Thr Phe Gln Ile Ser Gln Thr

650 655 660

gac ctg ttt ggc aac cca atg atg tac gat ctg aag gag aac ggc gac 2729

Asp Leu Phe Gly Asn Pro Met Met Tyr Asp Leu Lys Glu Asn Gly Asp

665 670 675

aag atc ccc atc aca aac gag aat aga aag gag ttc gtg aac ctg tac 2777

Lys Ile Pro Ile Thr Asn Glu Asn Arg Lys Glu Phe Val Asn Leu Tyr

680 685 690

agc gat tat atc ctg aat aag tcc gtg gag aag cag ttc aag gcc ttt 2825

Ser Asp Tyr Ile Leu Asn Lys Ser Val Glu Lys Gln Phe Lys Ala Phe

695 700 705 710

agg cgc ggc ttc cac atg gtg acc aac gag agc cct ctg aag tat ctg 2873

Arg Arg Gly Phe His Met Val Thr Asn Glu Ser Pro Leu Lys Tyr Leu

715 720 725

ttt agg cca gag gag atc gag ctg ctg atc tgc ggc tcc cgc aat ctg 2921

Phe Arg Pro Glu Glu Ile Glu Leu Leu Ile Cys Gly Ser Arg Asn Leu

730 735 740

gac ttt cag gcc ctg gag gag acc aca gag tac gac ggc ggc tat acc 2969

Asp Phe Gln Ala Leu Glu Glu Thr Thr Glu Tyr Asp Gly Gly Tyr Thr

745 750 755

agg gac tct gtg ctg atc cgc gag ttc tgg gag atc gtg cac agc ttt 3017

Arg Asp Ser Val Leu Ile Arg Glu Phe Trp Glu Ile Val His Ser Phe

760 765 770

aca gac gag cag aag cgg ctg ttc ctg cag ttt acc acc ggc acc gac 3065

Thr Asp Glu Gln Lys Arg Leu Phe Leu Gln Phe Thr Thr Gly Thr Asp

775 780 785 790

aga gca cca gtg gga gga ctg ggc aag ctg aag atg atc atc gcc aag 3113

Arg Ala Pro Val Gly Gly Leu Gly Lys Leu Lys Met Ile Ile Ala Lys

795 800 805

aac ggc cca gac aca gag agg ctg ccc acc agc cac acc tgt ttc aac 3161

Asn Gly Pro Asp Thr Glu Arg Leu Pro Thr Ser His Thr Cys Phe Asn

810 815 820

gtg ctg ctg ctg ccc gag tac tcc tct aag gag aag ctg aag gag cgc 3209

Val Leu Leu Leu Pro Glu Tyr Ser Ser Lys Glu Lys Leu Lys Glu Arg

825 830 835

ctg ctg aag gcc atc acc tac gcc aag ggc ttt ggc atg ctg tga tga 3257

Leu Leu Lys Ala Ile Thr Tyr Ala Lys Gly Phe Gly Met Leu

840 845 850

ggtaccagtg aattctacca gtgccatagg atagtgaatt ctaccagtgc catacacgtg 3317

agtgaattct accagtgcca tagcatgcag tgaattctac cagtgccata ggcggccgct 3377

tcgagcagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg 3437

aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag 3497

ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga 3557

gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga 3617

tcttcctaga gcatggctac gtagataagt agcatggcgg gttaatcatt aactacaagg 3677

aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg 3737

ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag 3797

cgcgcag 3804

<210> 2

<211> 852

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 2

Met Lys Arg Ala Ala Ala Lys His Leu Ile Glu Arg Tyr Tyr His Gln

1 5 10 15

Leu Thr Glu Gly Cys Gly Asn Glu Ala Cys Thr Asn Glu Phe Cys Ala

20 25 30

Ser Cys Pro Thr Phe Leu Arg Met Asp Asn Asn Ala Ala Ala Ile Lys

35 40 45

Ala Leu Glu Leu Tyr Lys Ile Asn Ala Lys Leu Cys Asp Pro His Pro

50 55 60

Ser Lys Lys Gly Ala Ser Ser Ala Tyr Leu Glu Asn Ser Lys Gly Ala

65 70 75 80

Pro Asn Asn Ser Cys Ser Glu Ile Lys Met Asn Lys Lys Gly Ala Arg

85 90 95

Ile Asp Phe Lys Asp Val Thr Tyr Leu Thr Glu Glu Lys Val Tyr Glu

100 105 110

Ile Leu Glu Leu Cys Arg Glu Arg Glu Asp Tyr Ser Pro Leu Ile Arg

115 120 125

Val Ile Gly Arg Val Phe Ser Ser Ala Glu Ala Leu Val Gln Ser Phe

130 135 140

Arg Lys Val Lys Gln His Thr Lys Glu Glu Leu Lys Ser Leu Gln Ala

145 150 155 160

Lys Asp Glu Asp Lys Asp Glu Asp Glu Lys Glu Lys Ala Ala Cys Ser

165 170 175

Ala Ala Ala Met Glu Glu Asp Ser Glu Ala Ser Ser Ser Arg Ile Gly

180 185 190

Asp Ser Ser Gln Gly Asp Asn Asn Leu Gln Lys Leu Gly Pro Asp Asp

195 200 205

Val Ser Val Asp Ile Asp Ala Ile Arg Arg Val Tyr Thr Arg Leu Leu

210 215 220

Ser Asn Glu Lys Ile Glu Thr Ala Phe Leu Asn Ala Leu Val Tyr Leu

225 230 235 240

Ser Pro Asn Val Glu Cys Asp Leu Thr Tyr His Asn Val Tyr Ser Arg

245 250 255

Asp Pro Asn Tyr Leu Asn Leu Phe Ile Ile Val Met Glu Asn Arg Asn

260 265 270

Leu His Ser Pro Glu Tyr Leu Glu Met Ala Leu Pro Leu Phe Cys Lys

275 280 285

Ala Met Ser Lys Leu Pro Leu Ala Ala Gln Gly Lys Leu Ile Arg Leu

290 295 300

Trp Ser Lys Tyr Asn Ala Asp Gln Ile Arg Arg Met Met Glu Thr Phe

305 310 315 320

Gln Gln Leu Ile Thr Tyr Lys Val Ile Ser Asn Glu Phe Asn Ser Arg

325 330 335

Asn Leu Val Asn Asp Asp Asp Ala Ile Val Ala Ala Ser Lys Cys Leu

340 345 350

Lys Met Val Tyr Tyr Ala Asn Val Val Gly Gly Glu Val Asp Thr Asn

355 360 365

His Asn Glu Glu Asp Asp Glu Glu Pro Ile Pro Glu Ser Ser Glu Leu

370 375 380

Thr Leu Gln Glu Leu Leu Gly Glu Glu Arg Arg Asn Lys Lys Gly Pro

385 390 395 400

Arg Val Asp Pro Leu Glu Thr Glu Leu Gly Val Lys Thr Leu Asp Cys

405 410 415

Arg Lys Pro Leu Ile Pro Phe Glu Glu Phe Ile Asn Glu Pro Leu Asn

420 425 430

Glu Val Leu Glu Met Asp Lys Asp Tyr Thr Phe Phe Lys Val Glu Thr

435 440 445

Glu Asn Lys Phe Ser Phe Met Thr Cys Pro Phe Ile Leu Asn Ala Val

450 455 460

Thr Lys Asn Leu Gly Leu Tyr Tyr Asp Asn Arg Ile Arg Met Tyr Ser

465 470 475 480

Glu Arg Arg Ile Thr Val Leu Tyr Ser Leu Val Gln Gly Gln Gln Leu

485 490 495

Asn Pro Tyr Leu Arg Leu Lys Val Arg Arg Asp His Ile Ile Asp Asp

500 505 510

Ala Leu Val Arg Leu Glu Met Ile Ala Met Glu Asn Pro Ala Asp Leu

515 520 525

Lys Lys Gln Leu Tyr Val Glu Phe Glu Gly Glu Gln Gly Val Asp Glu

530 535 540

Gly Gly Val Ser Lys Glu Phe Phe Gln Leu Val Val Glu Glu Ile Phe

545 550 555 560

Asn Pro Asp Ile Gly Met Phe Thr Tyr Asp Glu Ser Thr Lys Leu Phe

565 570 575

Trp Phe Asn Pro Ser Ser Phe Glu Thr Glu Gly Gln Phe Thr Leu Ile

580 585 590

Gly Ile Val Leu Gly Leu Ala Ile Tyr Asn Asn Cys Ile Leu Asp Val

595 600 605

His Phe Pro Met Val Val Tyr Arg Lys Leu Met Gly Lys Lys Gly Thr

610 615 620

Phe Arg Asp Leu Gly Asp Ser His Pro Val Leu Tyr Gln Ser Leu Lys

625 630 635 640

Asp Leu Leu Glu Tyr Glu Gly Asn Val Glu Asp Asp Met Met Ile Thr

645 650 655

Phe Gln Ile Ser Gln Thr Asp Leu Phe Gly Asn Pro Met Met Tyr Asp

660 665 670

Leu Lys Glu Asn Gly Asp Lys Ile Pro Ile Thr Asn Glu Asn Arg Lys

675 680 685

Glu Phe Val Asn Leu Tyr Ser Asp Tyr Ile Leu Asn Lys Ser Val Glu

690 695 700

Lys Gln Phe Lys Ala Phe Arg Arg Gly Phe His Met Val Thr Asn Glu

705 710 715 720

Ser Pro Leu Lys Tyr Leu Phe Arg Pro Glu Glu Ile Glu Leu Leu Ile

725 730 735

Cys Gly Ser Arg Asn Leu Asp Phe Gln Ala Leu Glu Glu Thr Thr Glu

740 745 750

Tyr Asp Gly Gly Tyr Thr Arg Asp Ser Val Leu Ile Arg Glu Phe Trp

755 760 765

Glu Ile Val His Ser Phe Thr Asp Glu Gln Lys Arg Leu Phe Leu Gln

770 775 780

Phe Thr Thr Gly Thr Asp Arg Ala Pro Val Gly Gly Leu Gly Lys Leu

785 790 795 800

Lys Met Ile Ile Ala Lys Asn Gly Pro Asp Thr Glu Arg Leu Pro Thr

805 810 815

Ser His Thr Cys Phe Asn Val Leu Leu Leu Pro Glu Tyr Ser Ser Lys

820 825 830

Glu Lys Leu Lys Glu Arg Leu Leu Lys Ala Ile Thr Tyr Ala Lys Gly

835 840 845

Phe Gly Met Leu

850

<210> 3

<211> 3700

<212> DNA

<213> artificial sequence

<220>

<223> vector genome hSyn.hUbe3a-1.GSco.SV40

<220>

<221> repeat_region

<222> (1)..(130)

<223> ITR

<220>

<221> promoter

<222> (213)..(678)

<223> human synapsin promoter

<220>

<221> misc_feature

<222> (690)..(695)

<223> Kozak

<220>

<221> CDS

<222> (696)..(3257)

<223> hUBE3a-1

<220>

<221> misc_feature

<222> (779)..(779)

<223> mutation

<220>

<221> misc_feature

<222> (824)..(824)

<223> mutation

<220>

<221> misc_feature

<222> (1028)..(1028)

<223> mutation

<220>

<221> misc_feature

<222> (1181)..(1181)

<223> mutation

<220>

<221> misc_feature

<222> (1193)..(1193)

<223> mutation

<220>

<221> misc_feature

<222> (1217)..(1217)

<223> mutation

<220>

<221> misc_feature

<222> (1283)..(1283)

<223> mutant SD/AS

<220>

<221> misc_feature

<222> (1715)..(1715)

<223> mutation

<220>

<221> misc_feature

<222> (1721)..(1721)

<223> mutation

<220>

<221> misc_feature

<222> (1766)..(1766)

<223> mutation

<220>

<221> misc_feature

<222> (1805)..(1805)

<223> mutation

<220>

<221> misc_feature

<222> (1814)..(1814)

<223> mutation

<220>

<221> misc_feature

<222> (1991)..(1991)

<223> mutation

<220>

<221> misc_feature

<222> (2063)..(2063)

<223> mutation

<220>

<221> misc_feature

<222> (2111)..(2111)

<223> mutation

<220>

<221> misc_feature

<222> (2294)..(2294)

<223> mutation

<220>

<221> misc_feature

<222> (2439)..(2440)

<223> mutant SD/AS

<220>

<221> misc_feature

<222> (2723)..(2723)

<223> mutation

<220>

<221> misc_feature

<222> (2957)..(2957)

<223> mutation

<220>

<221> misc_feature

<222> (3023)..(3023)

<223> mutation

<220>

<221> misc_feature

<222> (3056)..(3056)

<223> mutant SD/AS

<220>

<221> misc_feature

<222> (3152)..(3152)

<223> mutation

<220>

<221> misc_feature

<222> (3161)..(3161)

<223> mutation

<220>

<221> misc_feature

<222> (3230)..(3230)

<223> mutation

<220>

<221> polyA_Signal

<222> (3275)..(3700)

<223> SV40 polyA

<220>

<221> repeat_region

<222> (3571)..(3700)

<223> ITR

<400> 3

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcc tctagaacta tagctagcat gcctgcagag ggccctgcgt atgagtgcaa 240

gtgggtttta ggaccaggat gaggcggggt gggggtgcct acctgacgac cgaccccgac 300

ccactggaca agcacccaac ccccattccc caaattgcgc atcccctatc agagaggggg 360

aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc gcggacagtg 420

ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg cgcgctgacg 480

tcactcgccg gtcccccgca aactcccctt cccggccacc ttggtcgcgt ccgcgccgcc 540

gccggcccag ccggaccgca ccacgcgagg cgcgagatag gggggcacgg gcgcgaccat 600

ctgcgctgcg gcgccggcga ctcagcgctg cctcagtctg cggtgggcag cggaggagtc 660

gtgtcgtgcc tgagagcgca gtcgaattcg ccacc atg aag agg gca gca gca 713

Met Lys Arg Ala Ala Ala

1 5

aag cac ctg atc gag aga tac tat cac cag ctg acc gag gga tgc gga 761

Lys His Leu Ile Glu Arg Tyr Tyr His Gln Leu Thr Glu Gly Cys Gly

10 15 20

aac gag gca tgt aca aac gag ttc tgc gcc tcc tgt ccc acc ttt ctg 809

Asn Glu Ala Cys Thr Asn Glu Phe Cys Ala Ser Cys Pro Thr Phe Leu

25 30 35

agg atg gat aac aac gcc gcc gcc atc aag gcc ctg gag ctg tac aag 857

Arg Met Asp Asn Asn Ala Ala Ala Ile Lys Ala Leu Glu Leu Tyr Lys

40 45 50

atc aac gcc aag ctg tgc gac ccc cac cct agc aag aag ggc gcc agc 905

Ile Asn Ala Lys Leu Cys Asp Pro His Pro Ser Lys Lys Gly Ala Ser

55 60 65 70

tcc gcc tat ctg gag aac tcc aag ggc gcc cct aac aat agc tgt tcc 953

Ser Ala Tyr Leu Glu Asn Ser Lys Gly Ala Pro Asn Asn Ser Cys Ser

75 80 85

gag atc aag atg aat aag aag ggc gcc cgg atc gat ttc aag gac gtg 1001

Glu Ile Lys Met Asn Lys Lys Gly Ala Arg Ile Asp Phe Lys Asp Val

90 95 100

acc tac ctg aca gag gag aag gtg tac gag atc ctg gag ctg tgc cgg 1049

Thr Tyr Leu Thr Glu Glu Lys Val Tyr Glu Ile Leu Glu Leu Cys Arg

105 110 115

gag aga gag gat tac agc cca ctg atc aga gtg atc ggc aga gtg ttc 1097

Glu Arg Glu Asp Tyr Ser Pro Leu Ile Arg Val Ile Gly Arg Val Phe

120 125 130

tct agc gcc gag gcc ctg gtg cag tcc ttt aga aag gtg aag cag cac 1145

Ser Ser Ala Glu Ala Leu Val Gln Ser Phe Arg Lys Val Lys Gln His

135 140 145 150

aca aag gag gag ctg aag tct ctg cag gcc aag gac gag gac aag gac 1193

Thr Lys Glu Glu Leu Lys Ser Leu Gln Ala Lys Asp Glu Asp Lys Asp

155 160 165

gag gac gag aag gag aag gca gcc tgt tct gcc gca gca atg gag gag 1241

Glu Asp Glu Lys Glu Lys Ala Ala Cys Ser Ala Ala Ala Met Glu Glu

170 175 180

gac agc gag gca tcc tct agc cgg atc ggc gat tcc tct caa ggc gac 1289

Asp Ser Glu Ala Ser Ser Ser Arg Ile Gly Asp Ser Ser Gln Gly Asp

185 190 195

aac aat ctg cag aag ctg ggc ccc gac gac gtg agc gtg gat atc gac 1337

Asn Asn Leu Gln Lys Leu Gly Pro Asp Asp Val Ser Val Asp Ile Asp

200 205 210

gcc atc cgg aga gtg tac aca aga ctg ctg agc aac gag aag atc gag 1385

Ala Ile Arg Arg Val Tyr Thr Arg Leu Leu Ser Asn Glu Lys Ile Glu

215 220 225 230

acc gcc ttc ctg aac gcc ctg gtg tat ctg agc cct aat gtg gag tgc 1433

Thr Ala Phe Leu Asn Ala Leu Val Tyr Leu Ser Pro Asn Val Glu Cys

235 240 245

gat ctg acc tac cac aac gtg tac tcc cgg gac cca aac tac ctg aat 1481

Asp Leu Thr Tyr His Asn Val Tyr Ser Arg Asp Pro Asn Tyr Leu Asn

250 255 260

ctg ttc atc atc gtg atg gag aac aga aat ctg cac tcc ccc gag tat 1529

Leu Phe Ile Ile Val Met Glu Asn Arg Asn Leu His Ser Pro Glu Tyr

265 270 275

ctg gag atg gcc ctg cct ctg ttt tgt aag gcc atg tcc aag ctg cct 1577

Leu Glu Met Ala Leu Pro Leu Phe Cys Lys Ala Met Ser Lys Leu Pro

280 285 290

ctg gca gca cag ggc aag ctg atc agg ctg tgg tct aag tac aac gcc 1625

Leu Ala Ala Gln Gly Lys Leu Ile Arg Leu Trp Ser Lys Tyr Asn Ala

295 300 305 310

gat cag atc agg cgc atg atg gag acc ttc cag cag ctg atc aca tac 1673

Asp Gln Ile Arg Arg Met Met Glu Thr Phe Gln Gln Leu Ile Thr Tyr

315 320 325

aaa gtg atc tct aac gag ttt aat agc cgc aac ctg gtg aac gac gac 1721

Lys Val Ile Ser Asn Glu Phe Asn Ser Arg Asn Leu Val Asn Asp Asp

330 335 340

gac gcc atc gtg gcc gcc tct aag tgc ctg aag atg gtg tac tac gcc 1769

Asp Ala Ile Val Ala Ala Ser Lys Cys Leu Lys Met Val Tyr Tyr Ala

345 350 355

aac gtg gtg ggc ggc gag gtg gac aca aac cac aac gag gag gac gac 1817

Asn Val Val Gly Gly Glu Val Asp Thr Asn His Asn Glu Glu Asp Asp

360 365 370

gag gag cca atc ccc gag agc tcc gag ctg acc ctg cag gag ctg ctg 1865

Glu Glu Pro Ile Pro Glu Ser Ser Glu Leu Thr Leu Gln Glu Leu Leu

375 380 385 390

gga gag gag cgg aga aat aag aag gga cca agg gtg gat cct ctg gag 1913

Gly Glu Glu Arg Arg Asn Lys Lys Gly Pro Arg Val Asp Pro Leu Glu

395 400 405

acc gag ctg ggc gtg aag aca ctg gac tgc aga aag cct ctg atc cca 1961

Thr Glu Leu Gly Val Lys Thr Leu Asp Cys Arg Lys Pro Leu Ile Pro

410 415 420

ttc gag gag ttt atc aac gag ccc ctg aac gag gtg ctg gag atg gat 2009

Phe Glu Glu Phe Ile Asn Glu Pro Leu Asn Glu Val Leu Glu Met Asp

425 430 435

aag gac tac acc ttc ttt aag gtg gag aca gag aac aag ttc agc ttt 2057

Lys Asp Tyr Thr Phe Phe Lys Val Glu Thr Glu Asn Lys Phe Ser Phe

440 445 450

atg acc tgt cct ttc atc ctg aac gcc gtg acc aag aat ctg ggc ctg 2105

Met Thr Cys Pro Phe Ile Leu Asn Ala Val Thr Lys Asn Leu Gly Leu

455 460 465 470

tac tac gat aac agg atc cgc atg tac tcc gag agg cgc atc acc gtg 2153

Tyr Tyr Asp Asn Arg Ile Arg Met Tyr Ser Glu Arg Arg Ile Thr Val

475 480 485

ctg tat tct ctg gtg cag ggc cag cag ctg aat cct tac ctg agg ctg 2201

Leu Tyr Ser Leu Val Gln Gly Gln Gln Leu Asn Pro Tyr Leu Arg Leu

490 495 500

aag gtg cgg aga gac cac atc atc gat gac gcc ctg gtg cgc ctg gag 2249

Lys Val Arg Arg Asp His Ile Ile Asp Asp Ala Leu Val Arg Leu Glu

505 510 515

atg atc gcc atg gag aat cca gcc gat ctg aag aag cag ctg tac gtg 2297

Met Ile Ala Met Glu Asn Pro Ala Asp Leu Lys Lys Gln Leu Tyr Val

520 525 530

gag ttt gag gga gag cag gga gtg gac gag gga ggc gtg tct aag gag 2345

Glu Phe Glu Gly Glu Gln Gly Val Asp Glu Gly Gly Val Ser Lys Glu

535 540 545 550

ttc ttt cag ctg gtg gtg gag gag atc ttc aac ccc gat atc ggc atg 2393

Phe Phe Gln Leu Val Val Glu Glu Ile Phe Asn Pro Asp Ile Gly Met

555 560 565

ttt acc tac gac gag agc aca aag ctg ttc tgg ttt aat cct tct tcc 2441

Phe Thr Tyr Asp Glu Ser Thr Lys Leu Phe Trp Phe Asn Pro Ser Ser

570 575 580

ttc gag acc gag ggc cag ttt aca ctg atc ggc atc gtg ctg ggc ctg 2489

Phe Glu Thr Glu Gly Gln Phe Thr Leu Ile Gly Ile Val Leu Gly Leu

585 590 595

gcc atc tac aac aat tgt atc ctg gac gtg cac ttc cca atg gtg gtg 2537

Ala Ile Tyr Asn Asn Cys Ile Leu Asp Val His Phe Pro Met Val Val

600 605 610

tat agg aag ctg atg ggc aag aag ggc acc ttt cgc gat ctg ggc gac 2585

Tyr Arg Lys Leu Met Gly Lys Lys Gly Thr Phe Arg Asp Leu Gly Asp

615 620 625 630

tcc cac ccc gtg ctg tac cag tct ctg aag gat ctg ctg gag tat gag 2633

Ser His Pro Val Leu Tyr Gln Ser Leu Lys Asp Leu Leu Glu Tyr Glu

635 640 645

ggc aac gtg gag gat gac atg atg atc acc ttc cag atc tcc cag aca 2681

Gly Asn Val Glu Asp Asp Met Met Ile Thr Phe Gln Ile Ser Gln Thr

650 655 660

gac ctg ttt ggc aac cca atg atg tac gat ctg aag gag aac ggc gac 2729

Asp Leu Phe Gly Asn Pro Met Met Tyr Asp Leu Lys Glu Asn Gly Asp

665 670 675

aag atc ccc atc aca aac gag aat aga aag gag ttc gtg aac ctg tac 2777

Lys Ile Pro Ile Thr Asn Glu Asn Arg Lys Glu Phe Val Asn Leu Tyr

680 685 690

agc gat tat atc ctg aat aag tcc gtg gag aag cag ttc aag gcc ttt 2825

Ser Asp Tyr Ile Leu Asn Lys Ser Val Glu Lys Gln Phe Lys Ala Phe

695 700 705 710

agg cgc ggc ttc cac atg gtg acc aac gag agc cct ctg aag tat ctg 2873

Arg Arg Gly Phe His Met Val Thr Asn Glu Ser Pro Leu Lys Tyr Leu

715 720 725

ttt agg cca gag gag atc gag ctg ctg atc tgc ggc tcc cgc aat ctg 2921

Phe Arg Pro Glu Glu Ile Glu Leu Leu Ile Cys Gly Ser Arg Asn Leu

730 735 740

gac ttt cag gcc ctg gag gag acc aca gag tac gac ggc ggc tat acc 2969

Asp Phe Gln Ala Leu Glu Glu Thr Thr Glu Tyr Asp Gly Gly Tyr Thr

745 750 755

agg gac tct gtg ctg atc cgc gag ttc tgg gag atc gtg cac agc ttt 3017

Arg Asp Ser Val Leu Ile Arg Glu Phe Trp Glu Ile Val His Ser Phe

760 765 770

aca gac gag cag aag cgg ctg ttc ctg cag ttt acc acc ggc acc gac 3065

Thr Asp Glu Gln Lys Arg Leu Phe Leu Gln Phe Thr Thr Gly Thr Asp

775 780 785 790

aga gca cca gtg gga gga ctg ggc aag ctg aag atg atc atc gcc aag 3113

Arg Ala Pro Val Gly Gly Leu Gly Lys Leu Lys Met Ile Ile Ala Lys

795 800 805

aac ggc cca gac aca gag agg ctg ccc acc agc cac acc tgt ttc aac 3161

Asn Gly Pro Asp Thr Glu Arg Leu Pro Thr Ser His Thr Cys Phe Asn

810 815 820

gtg ctg ctg ctg ccc gag tac tcc tct aag gag aag ctg aag gag cgc 3209

Val Leu Leu Leu Pro Glu Tyr Ser Ser Lys Glu Lys Leu Lys Glu Arg

825 830 835

ctg ctg aag gcc atc acc tac gcc aag ggc ttt ggc atg ctg tga tga 3257

Leu Leu Lys Ala Ile Thr Tyr Ala Lys Gly Phe Gly Met Leu

840 845 850

ggtaccggcg gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca 3317

caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat 3377

ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt 3437

ttcaggttca gggggagatg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg 3497

gtaaaatcga taaggatctt cctagagcat ggctacgtag ataagtagca tggcgggtta 3557

atcattaact acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc 3617

tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc 3677

tcagtgagcg agcgagcgcg cag 3700

<210> 4

<211> 852

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 4

Met Lys Arg Ala Ala Ala Lys His Leu Ile Glu Arg Tyr Tyr His Gln

1 5 10 15

Leu Thr Glu Gly Cys Gly Asn Glu Ala Cys Thr Asn Glu Phe Cys Ala

20 25 30

Ser Cys Pro Thr Phe Leu Arg Met Asp Asn Asn Ala Ala Ala Ile Lys

35 40 45

Ala Leu Glu Leu Tyr Lys Ile Asn Ala Lys Leu Cys Asp Pro His Pro

50 55 60

Ser Lys Lys Gly Ala Ser Ser Ala Tyr Leu Glu Asn Ser Lys Gly Ala

65 70 75 80

Pro Asn Asn Ser Cys Ser Glu Ile Lys Met Asn Lys Lys Gly Ala Arg

85 90 95

Ile Asp Phe Lys Asp Val Thr Tyr Leu Thr Glu Glu Lys Val Tyr Glu

100 105 110

Ile Leu Glu Leu Cys Arg Glu Arg Glu Asp Tyr Ser Pro Leu Ile Arg

115 120 125

Val Ile Gly Arg Val Phe Ser Ser Ala Glu Ala Leu Val Gln Ser Phe

130 135 140

Arg Lys Val Lys Gln His Thr Lys Glu Glu Leu Lys Ser Leu Gln Ala

145 150 155 160

Lys Asp Glu Asp Lys Asp Glu Asp Glu Lys Glu Lys Ala Ala Cys Ser

165 170 175

Ala Ala Ala Met Glu Glu Asp Ser Glu Ala Ser Ser Ser Arg Ile Gly

180 185 190

Asp Ser Ser Gln Gly Asp Asn Asn Leu Gln Lys Leu Gly Pro Asp Asp

195 200 205

Val Ser Val Asp Ile Asp Ala Ile Arg Arg Val Tyr Thr Arg Leu Leu

210 215 220

Ser Asn Glu Lys Ile Glu Thr Ala Phe Leu Asn Ala Leu Val Tyr Leu

225 230 235 240

Ser Pro Asn Val Glu Cys Asp Leu Thr Tyr His Asn Val Tyr Ser Arg

245 250 255

Asp Pro Asn Tyr Leu Asn Leu Phe Ile Ile Val Met Glu Asn Arg Asn

260 265 270

Leu His Ser Pro Glu Tyr Leu Glu Met Ala Leu Pro Leu Phe Cys Lys

275 280 285

Ala Met Ser Lys Leu Pro Leu Ala Ala Gln Gly Lys Leu Ile Arg Leu

290 295 300

Trp Ser Lys Tyr Asn Ala Asp Gln Ile Arg Arg Met Met Glu Thr Phe

305 310 315 320

Gln Gln Leu Ile Thr Tyr Lys Val Ile Ser Asn Glu Phe Asn Ser Arg

325 330 335

Asn Leu Val Asn Asp Asp Asp Ala Ile Val Ala Ala Ser Lys Cys Leu

340 345 350

Lys Met Val Tyr Tyr Ala Asn Val Val Gly Gly Glu Val Asp Thr Asn

355 360 365

His Asn Glu Glu Asp Asp Glu Glu Pro Ile Pro Glu Ser Ser Glu Leu

370 375 380

Thr Leu Gln Glu Leu Leu Gly Glu Glu Arg Arg Asn Lys Lys Gly Pro

385 390 395 400

Arg Val Asp Pro Leu Glu Thr Glu Leu Gly Val Lys Thr Leu Asp Cys

405 410 415

Arg Lys Pro Leu Ile Pro Phe Glu Glu Phe Ile Asn Glu Pro Leu Asn

420 425 430

Glu Val Leu Glu Met Asp Lys Asp Tyr Thr Phe Phe Lys Val Glu Thr

435 440 445

Glu Asn Lys Phe Ser Phe Met Thr Cys Pro Phe Ile Leu Asn Ala Val

450 455 460

Thr Lys Asn Leu Gly Leu Tyr Tyr Asp Asn Arg Ile Arg Met Tyr Ser

465 470 475 480

Glu Arg Arg Ile Thr Val Leu Tyr Ser Leu Val Gln Gly Gln Gln Leu

485 490 495

Asn Pro Tyr Leu Arg Leu Lys Val Arg Arg Asp His Ile Ile Asp Asp

500 505 510

Ala Leu Val Arg Leu Glu Met Ile Ala Met Glu Asn Pro Ala Asp Leu

515 520 525

Lys Lys Gln Leu Tyr Val Glu Phe Glu Gly Glu Gln Gly Val Asp Glu

530 535 540

Gly Gly Val Ser Lys Glu Phe Phe Gln Leu Val Val Glu Glu Ile Phe

545 550 555 560

Asn Pro Asp Ile Gly Met Phe Thr Tyr Asp Glu Ser Thr Lys Leu Phe

565 570 575

Trp Phe Asn Pro Ser Ser Phe Glu Thr Glu Gly Gln Phe Thr Leu Ile

580 585 590

Gly Ile Val Leu Gly Leu Ala Ile Tyr Asn Asn Cys Ile Leu Asp Val

595 600 605

His Phe Pro Met Val Val Tyr Arg Lys Leu Met Gly Lys Lys Gly Thr

610 615 620

Phe Arg Asp Leu Gly Asp Ser His Pro Val Leu Tyr Gln Ser Leu Lys

625 630 635 640

Asp Leu Leu Glu Tyr Glu Gly Asn Val Glu Asp Asp Met Met Ile Thr

645 650 655

Phe Gln Ile Ser Gln Thr Asp Leu Phe Gly Asn Pro Met Met Tyr Asp

660 665 670

Leu Lys Glu Asn Gly Asp Lys Ile Pro Ile Thr Asn Glu Asn Arg Lys

675 680 685

Glu Phe Val Asn Leu Tyr Ser Asp Tyr Ile Leu Asn Lys Ser Val Glu

690 695 700

Lys Gln Phe Lys Ala Phe Arg Arg Gly Phe His Met Val Thr Asn Glu

705 710 715 720

Ser Pro Leu Lys Tyr Leu Phe Arg Pro Glu Glu Ile Glu Leu Leu Ile

725 730 735

Cys Gly Ser Arg Asn Leu Asp Phe Gln Ala Leu Glu Glu Thr Thr Glu

740 745 750

Tyr Asp Gly Gly Tyr Thr Arg Asp Ser Val Leu Ile Arg Glu Phe Trp

755 760 765

Glu Ile Val His Ser Phe Thr Asp Glu Gln Lys Arg Leu Phe Leu Gln

770 775 780

Phe Thr Thr Gly Thr Asp Arg Ala Pro Val Gly Gly Leu Gly Lys Leu

785 790 795 800

Lys Met Ile Ile Ala Lys Asn Gly Pro Asp Thr Glu Arg Leu Pro Thr

805 810 815

Ser His Thr Cys Phe Asn Val Leu Leu Leu Pro Glu Tyr Ser Ser Lys

820 825 830

Glu Lys Leu Lys Glu Arg Leu Leu Lys Ala Ile Thr Tyr Ala Lys Gly

835 840 845

Phe Gly Met Leu

850

<210> 5

<211> 3873

<212> DNA

<213> artificial sequence

<220>

<223> vector genome hSyn. HUbe3a-2.GSco.4XmiRNA183.SV40

<220>

<221> repeat_region

<222> (1)..(130)

<223> ITR

<220>

<221> promoter

<222> (213)..(678)

<223> human synapsin promoter

<220>

<221> misc_feature

<222> (690)..(695)

<223> Kozak

<220>

<221> CDS

<222> (696)..(3326)

<223> hUBE3a-2

<220>

<221> misc_feature

<222> (848)..(848)

<223> mutation

<220>

<221> misc_feature

<222> (893)..(893)

<223> mutation

<220>

<221> misc_feature

<222> (1097)..(1097)

<223> mutation

<220>

<221> misc_feature

<222> (1250)..(1250)

<223> mutation

<220>

<221> misc_feature

<222> (1262)..(1262)

<223> mutation

<220>

<221> misc_feature

<222> (1286)..(1286)

<223> mutation

<220>

<221> misc_feature

<222> (1352)..(1352)

<223> mutant SD/AS

<220>

<221> misc_feature

<222> (1784)..(1784)

<223> mutation

<220>

<221> misc_feature

<222> (1790)..(1790)

<223> mutation

<220>

<221> misc_feature

<222> (1835)..(1835)

<223> mutation

<220>

<221> misc_feature

<222> (1874)..(1874)

<223> mutation

<220>

<221> misc_feature

<222> (1883)..(1883)

<223> mutation

<220>

<221> misc_feature

<222> (2060)..(2060)

<223> mutation

<220>

<221> misc_feature

<222> (2132)..(2132)

<223> mutation

<220>

<221> misc_feature

<222> (2180)..(2180)

<223> mutation

<220>

<221> misc_feature

<222> (2363)..(2363)

<223> mutation

<220>

<221> misc_feature

<222> (2508)..(2508)

<223> mutant SD/AS

<220>

<221> misc_feature

<222> (2792)..(2792)

<223> mutation

<220>

<221> misc_feature

<222> (3026)..(3026)

<223> mutation

<220>

<221> misc_feature

<222> (3092)..(3092)

<223> mutation

<220>

<221> misc_feature

<222> (3125)..(3125)

<223> mutant SD/AS

<220>

<221> misc_feature

<222> (3221)..(3221)

<223> mutation

<220>

<221> misc_feature

<222> (3230)..(3230)

<223> mutation

<220>

<221> misc_feature

<222> (3299)..(3299)

<223> mutation

<220>

<221> misc_feature

<222> (3333)..(3354)

<223> miRNA183

<220>

<221> misc_feature

<222> (3359)..(3380)

<223> miRNA183

<220>

<221> misc_feature

<222> (3387)..(3408)

<223> miRNA183

<220>

<221> misc_feature

<222> (3415)..(3436)

<223> miRNA183

<220>

<221> polyA_Signal

<222> (3448)..(3679)

<223> SV40 polyA

<220>

<221> repeat_region

<222> (3744)..(3873)

<223> ITR

<400> 5

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcc tctagaacta tagctagcat gcctgcagag ggccctgcgt atgagtgcaa 240

gtgggtttta ggaccaggat gaggcggggt gggggtgcct acctgacgac cgaccccgac 300

ccactggaca agcacccaac ccccattccc caaattgcgc atcccctatc agagaggggg 360

aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc gcggacagtg 420

ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg cgcgctgacg 480

tcactcgccg gtcccccgca aactcccctt cccggccacc ttggtcgcgt ccgcgccgcc 540

gccggcccag ccggaccgca ccacgcgagg cgcgagatag gggggcacgg gcgcgaccat 600

ctgcgctgcg gcgccggcga ctcagcgctg cctcagtctg cggtgggcag cggaggagtc 660

gtgtcgtgcc tgagagcgca gtcgaattcg ccacc atg gag aag ctg cac cag 713

Met Glu Lys Leu His Gln

1 5

tgc tac tgg aag tct ggc gag cct cag agc gac gat atc gag gca agc 761

Cys Tyr Trp Lys Ser Gly Glu Pro Gln Ser Asp Asp Ile Glu Ala Ser

10 15 20

agg atg aag aga gca gca gcc aag cac ctg atc gag cgg tac tat cac 809

Arg Met Lys Arg Ala Ala Ala Lys His Leu Ile Glu Arg Tyr Tyr His

25 30 35

cag ctg acc gag ggc tgc gga aac gag gcc tgt aca aac gag ttc tgc 857

Gln Leu Thr Glu Gly Cys Gly Asn Glu Ala Cys Thr Asn Glu Phe Cys

40 45 50

gcc tcc tgt cca acc ttt ctg aga atg gat aac aac gcc gcc gcc atc 905

Ala Ser Cys Pro Thr Phe Leu Arg Met Asp Asn Asn Ala Ala Ala Ile

55 60 65 70

aag gcc ctg gag ctg tac aag atc aac gcc aag ctg tgc gac ccc cac 953

Lys Ala Leu Glu Leu Tyr Lys Ile Asn Ala Lys Leu Cys Asp Pro His

75 80 85

cct tct aag aag ggc gcc agc tcc gcc tat ctg gag aac agc aag ggc 1001

Pro Ser Lys Lys Gly Ala Ser Ser Ala Tyr Leu Glu Asn Ser Lys Gly

90 95 100

gcc ccc aac aat agc tgt tcc gag atc aag atg aat aag aag ggc gcc 1049

Ala Pro Asn Asn Ser Cys Ser Glu Ile Lys Met Asn Lys Lys Gly Ala

105 110 115

agg atc gat ttc aag gac gtg acc tac ctg aca gag gag aag gtg tac 1097

Arg Ile Asp Phe Lys Asp Val Thr Tyr Leu Thr Glu Glu Lys Val Tyr

120 125 130

gag atc ctg gag ctg tgc cgg gag aga gag gat tac tcc cct ctg atc 1145

Glu Ile Leu Glu Leu Cys Arg Glu Arg Glu Asp Tyr Ser Pro Leu Ile

135 140 145 150

aga gtg atc gga cgc gtg ttc tct agc gcc gag gcc ctg gtg cag agc 1193

Arg Val Ile Gly Arg Val Phe Ser Ser Ala Glu Ala Leu Val Gln Ser

155 160 165

ttt cgc aag gtg aag cag cac aca aag gag gag ctg aag tcc ctg cag 1241

Phe Arg Lys Val Lys Gln His Thr Lys Glu Glu Leu Lys Ser Leu Gln

170 175 180

gcc aag gac gag gac aag gac gag gac gag aag gag aag gca gct tgt 1289

Ala Lys Asp Glu Asp Lys Asp Glu Asp Glu Lys Glu Lys Ala Ala Cys

185 190 195

tcc gcc gca gca atg gag gag gac tct gag gcc tcc tct agc agg atc 1337

Ser Ala Ala Ala Met Glu Glu Asp Ser Glu Ala Ser Ser Ser Arg Ile

200 205 210

ggc gat tcc tct caa ggc gac aac aat ctg cag aag ctg ggc ccc gac 1385

Gly Asp Ser Ser Gln Gly Asp Asn Asn Leu Gln Lys Leu Gly Pro Asp

215 220 225 230

gac gtg agc gtg gat atc gac gcc atc cgg aga gtg tac aca cgc ctg 1433

Asp Val Ser Val Asp Ile Asp Ala Ile Arg Arg Val Tyr Thr Arg Leu

235 240 245

ctg tct aac gag aag atc gag acc gcc ttc ctg aac gcc ctg gtg tat 1481

Leu Ser Asn Glu Lys Ile Glu Thr Ala Phe Leu Asn Ala Leu Val Tyr

250 255 260

ctg tct cct aat gtg gag tgc gat ctg acc tac cac aac gtg tac agc 1529

Leu Ser Pro Asn Val Glu Cys Asp Leu Thr Tyr His Asn Val Tyr Ser

265 270 275

cgg gac cca aac tac ctg aat ctg ttc atc atc gtg atg gag aac aga 1577

Arg Asp Pro Asn Tyr Leu Asn Leu Phe Ile Ile Val Met Glu Asn Arg

280 285 290

aat ctg cac tct ccc gag tat ctg gag atg gcc ctg cct ctg ttt tgt 1625

Asn Leu His Ser Pro Glu Tyr Leu Glu Met Ala Leu Pro Leu Phe Cys

295 300 305 310

aag gcc atg agc aag ctg cca ctg gca gca cag ggc aag ctg atc agg 1673

Lys Ala Met Ser Lys Leu Pro Leu Ala Ala Gln Gly Lys Leu Ile Arg

315 320 325

ctg tgg tcc aag tac aac gcc gat cag atc agg cgc atg atg gag acc 1721

Leu Trp Ser Lys Tyr Asn Ala Asp Gln Ile Arg Arg Met Met Glu Thr

330 335 340

ttc cag cag ctg atc aca tat aaa gtg atc tcc aac gag ttt aat tct 1769

Phe Gln Gln Leu Ile Thr Tyr Lys Val Ile Ser Asn Glu Phe Asn Ser

345 350 355

aga aac ctg gtg aac gac gac gac gcc atc gtg gcc gcc agc aag tgc 1817

Arg Asn Leu Val Asn Asp Asp Asp Ala Ile Val Ala Ala Ser Lys Cys

360 365 370

ctg aag atg gtg tac tac gcc aac gtg gtg ggc ggc gag gtg gac aca 1865

Leu Lys Met Val Tyr Tyr Ala Asn Val Val Gly Gly Glu Val Asp Thr

375 380 385 390

aac cac aac gag gag gac gac gag gag cca atc ccc gag agc tcc gag 1913

Asn His Asn Glu Glu Asp Asp Glu Glu Pro Ile Pro Glu Ser Ser Glu

395 400 405

ctg acc ctg cag gag ctg ctg gga gag gag cgg aga aat aag aag gga 1961

Leu Thr Leu Gln Glu Leu Leu Gly Glu Glu Arg Arg Asn Lys Lys Gly

410 415 420

cca agg gtg gat cct ctg gag acc gag ctg ggc gtg aag aca ctg gac 2009

Pro Arg Val Asp Pro Leu Glu Thr Glu Leu Gly Val Lys Thr Leu Asp

425 430 435

tgc aga aag cct ctg atc cca ttc gag gag ttt atc aac gag ccc ctg 2057

Cys Arg Lys Pro Leu Ile Pro Phe Glu Glu Phe Ile Asn Glu Pro Leu

440 445 450

aac gag gtg ctg gag atg gat aag gac tac acc ttc ttt aag gtg gag 2105

Asn Glu Val Leu Glu Met Asp Lys Asp Tyr Thr Phe Phe Lys Val Glu

455 460 465 470

aca gag aac aag ttc agc ttt atg acc tgt cct ttc atc ctg aac gcc 2153

Thr Glu Asn Lys Phe Ser Phe Met Thr Cys Pro Phe Ile Leu Asn Ala

475 480 485

gtg acc aag aat ctg ggc ctg tac tac gat aac agg atc cgc atg tac 2201

Val Thr Lys Asn Leu Gly Leu Tyr Tyr Asp Asn Arg Ile Arg Met Tyr

490 495 500

agc gag agg cgc atc acc gtg ctg tat tcc ctg gtg cag ggc cag cag 2249

Ser Glu Arg Arg Ile Thr Val Leu Tyr Ser Leu Val Gln Gly Gln Gln

505 510 515

ctg aat cct tac ctg agg ctg aag gtg cgg aga gac cac atc atc gat 2297

Leu Asn Pro Tyr Leu Arg Leu Lys Val Arg Arg Asp His Ile Ile Asp

520 525 530

gac gcc ctg gtg cgc ctg gag atg atc gcc atg gag aat cca gcc gat 2345

Asp Ala Leu Val Arg Leu Glu Met Ile Ala Met Glu Asn Pro Ala Asp

535 540 545 550

ctg aag aag cag ctg tac gtg gag ttt gag gga gag cag gga gtg gac 2393

Leu Lys Lys Gln Leu Tyr Val Glu Phe Glu Gly Glu Gln Gly Val Asp

555 560 565

gag gga ggc gtg tcc aag gag ttc ttt cag ctg gtg gtg gag gag atc 2441

Glu Gly Gly Val Ser Lys Glu Phe Phe Gln Leu Val Val Glu Glu Ile

570 575 580

ttc aac ccc gat atc ggc atg ttt acc tac gac gag tct aca aag ctg 2489

Phe Asn Pro Asp Ile Gly Met Phe Thr Tyr Asp Glu Ser Thr Lys Leu

585 590 595

ttc tgg ttt aat cct tct tcc ttc gag acc gag ggc cag ttt aca ctg 2537

Phe Trp Phe Asn Pro Ser Ser Phe Glu Thr Glu Gly Gln Phe Thr Leu

600 605 610

atc ggc atc gtg ctg ggc ctg gcc atc tac aac aat tgt atc ctg gac 2585

Ile Gly Ile Val Leu Gly Leu Ala Ile Tyr Asn Asn Cys Ile Leu Asp

615 620 625 630

gtg cac ttc cca atg gtg gtg tat agg aag ctg atg ggc aag aag ggc 2633

Val His Phe Pro Met Val Val Tyr Arg Lys Leu Met Gly Lys Lys Gly

635 640 645

acc ttt cgc gat ctg ggc gac agc cac ccc gtg ctg tac cag tcc ctg 2681

Thr Phe Arg Asp Leu Gly Asp Ser His Pro Val Leu Tyr Gln Ser Leu

650 655 660

aag gat ctg ctg gag tat gag ggc aac gtg gag gat gac atg atg atc 2729

Lys Asp Leu Leu Glu Tyr Glu Gly Asn Val Glu Asp Asp Met Met Ile

665 670 675

acc ttc cag atc tcc cag aca gac ctg ttt ggc aac cca atg atg tac 2777

Thr Phe Gln Ile Ser Gln Thr Asp Leu Phe Gly Asn Pro Met Met Tyr

680 685 690

gat ctg aag gag aac ggc gac aag atc ccc atc aca aac gag aat agg 2825

Asp Leu Lys Glu Asn Gly Asp Lys Ile Pro Ile Thr Asn Glu Asn Arg

695 700 705 710

aag gag ttc gtg aac ctg tac tct gat tat atc ctg aat aag agc gtg 2873

Lys Glu Phe Val Asn Leu Tyr Ser Asp Tyr Ile Leu Asn Lys Ser Val

715 720 725

gag aag cag ttc aag gcc ttt agg cgc ggc ttc cac atg gtg acc aac 2921

Glu Lys Gln Phe Lys Ala Phe Arg Arg Gly Phe His Met Val Thr Asn

730 735 740

gag tct cct ctg aag tat ctg ttt agg cca gag gag atc gag ctg ctg 2969

Glu Ser Pro Leu Lys Tyr Leu Phe Arg Pro Glu Glu Ile Glu Leu Leu

745 750 755

atc tgc ggc agc cgc aat ctg gac ttt cag gcc ctg gag gag acc aca 3017

Ile Cys Gly Ser Arg Asn Leu Asp Phe Gln Ala Leu Glu Glu Thr Thr

760 765 770

gag tac gac ggc ggc tat acc cgg gac tcc gtg ctg atc aga gag ttc 3065

Glu Tyr Asp Gly Gly Tyr Thr Arg Asp Ser Val Leu Ile Arg Glu Phe

775 780 785 790

tgg gag atc gtg cac tct ttt aca gac gag cag aag cgg ctg ttc ctg 3113

Trp Glu Ile Val His Ser Phe Thr Asp Glu Gln Lys Arg Leu Phe Leu

795 800 805

cag ttt acc acc ggc acc gac aga gca cca gtg gga gga ctg ggc aag 3161

Gln Phe Thr Thr Gly Thr Asp Arg Ala Pro Val Gly Gly Leu Gly Lys

810 815 820

ctg aag atg atc atc gcc aag aac ggc cca gac aca gag agg ctg ccc 3209

Leu Lys Met Ile Ile Ala Lys Asn Gly Pro Asp Thr Glu Arg Leu Pro

825 830 835

acc agc cac acc tgt ttc aac gtg ctg ctg ctg ccc gag tac tcc tct 3257

Thr Ser His Thr Cys Phe Asn Val Leu Leu Leu Pro Glu Tyr Ser Ser

840 845 850

aag gag aag ctg aag gag cgc ctg ctg aag gcc atc acc tac gcc aag 3305

Lys Glu Lys Leu Lys Glu Arg Leu Leu Lys Ala Ile Thr Tyr Ala Lys

855 860 865 870

ggc ttt ggc atg ctg tga tga ggtaccagtg aattctacca gtgccatagg 3356

Gly Phe Gly Met Leu

875

atagtgaatt ctaccagtgc catacacgtg agtgaattct accagtgcca tagcatgcag 3416

tgaattctac cagtgccata ggcggccgct tcgagcagac atgataagat acattgatga 3476

gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga 3536

tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg 3596

cattcatttt atgtttcagg ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa 3656

cctctacaaa tgtggtaaaa tcgataagga tcttcctaga gcatggctac gtagataagt 3716

agcatggcgg gttaatcatt aactacaagg aacccctagt gatggagttg gccactccct 3776

ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct 3836

ttgcccgggc ggcctcagtg agcgagcgag cgcgcag 3873

<210> 6

<211> 875

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 6

Met Glu Lys Leu His Gln Cys Tyr Trp Lys Ser Gly Glu Pro Gln Ser

1 5 10 15

Asp Asp Ile Glu Ala Ser Arg Met Lys Arg Ala Ala Ala Lys His Leu

20 25 30

Ile Glu Arg Tyr Tyr His Gln Leu Thr Glu Gly Cys Gly Asn Glu Ala

35 40 45

Cys Thr Asn Glu Phe Cys Ala Ser Cys Pro Thr Phe Leu Arg Met Asp

50 55 60

Asn Asn Ala Ala Ala Ile Lys Ala Leu Glu Leu Tyr Lys Ile Asn Ala

65 70 75 80

Lys Leu Cys Asp Pro His Pro Ser Lys Lys Gly Ala Ser Ser Ala Tyr

85 90 95

Leu Glu Asn Ser Lys Gly Ala Pro Asn Asn Ser Cys Ser Glu Ile Lys

100 105 110

Met Asn Lys Lys Gly Ala Arg Ile Asp Phe Lys Asp Val Thr Tyr Leu

115 120 125

Thr Glu Glu Lys Val Tyr Glu Ile Leu Glu Leu Cys Arg Glu Arg Glu

130 135 140

Asp Tyr Ser Pro Leu Ile Arg Val Ile Gly Arg Val Phe Ser Ser Ala

145 150 155 160

Glu Ala Leu Val Gln Ser Phe Arg Lys Val Lys Gln His Thr Lys Glu

165 170 175

Glu Leu Lys Ser Leu Gln Ala Lys Asp Glu Asp Lys Asp Glu Asp Glu

180 185 190

Lys Glu Lys Ala Ala Cys Ser Ala Ala Ala Met Glu Glu Asp Ser Glu

195 200 205

Ala Ser Ser Ser Arg Ile Gly Asp Ser Ser Gln Gly Asp Asn Asn Leu

210 215 220

Gln Lys Leu Gly Pro Asp Asp Val Ser Val Asp Ile Asp Ala Ile Arg

225 230 235 240

Arg Val Tyr Thr Arg Leu Leu Ser Asn Glu Lys Ile Glu Thr Ala Phe

245 250 255

Leu Asn Ala Leu Val Tyr Leu Ser Pro Asn Val Glu Cys Asp Leu Thr

260 265 270

Tyr His Asn Val Tyr Ser Arg Asp Pro Asn Tyr Leu Asn Leu Phe Ile

275 280 285

Ile Val Met Glu Asn Arg Asn Leu His Ser Pro Glu Tyr Leu Glu Met

290 295 300

Ala Leu Pro Leu Phe Cys Lys Ala Met Ser Lys Leu Pro Leu Ala Ala

305 310 315 320

Gln Gly Lys Leu Ile Arg Leu Trp Ser Lys Tyr Asn Ala Asp Gln Ile

325 330 335

Arg Arg Met Met Glu Thr Phe Gln Gln Leu Ile Thr Tyr Lys Val Ile

340 345 350

Ser Asn Glu Phe Asn Ser Arg Asn Leu Val Asn Asp Asp Asp Ala Ile

355 360 365

Val Ala Ala Ser Lys Cys Leu Lys Met Val Tyr Tyr Ala Asn Val Val

370 375 380

Gly Gly Glu Val Asp Thr Asn His Asn Glu Glu Asp Asp Glu Glu Pro

385 390 395 400

Ile Pro Glu Ser Ser Glu Leu Thr Leu Gln Glu Leu Leu Gly Glu Glu

405 410 415

Arg Arg Asn Lys Lys Gly Pro Arg Val Asp Pro Leu Glu Thr Glu Leu

420 425 430

Gly Val Lys Thr Leu Asp Cys Arg Lys Pro Leu Ile Pro Phe Glu Glu

435 440 445

Phe Ile Asn Glu Pro Leu Asn Glu Val Leu Glu Met Asp Lys Asp Tyr

450 455 460

Thr Phe Phe Lys Val Glu Thr Glu Asn Lys Phe Ser Phe Met Thr Cys

465 470 475 480

Pro Phe Ile Leu Asn Ala Val Thr Lys Asn Leu Gly Leu Tyr Tyr Asp

485 490 495

Asn Arg Ile Arg Met Tyr Ser Glu Arg Arg Ile Thr Val Leu Tyr Ser

500 505 510

Leu Val Gln Gly Gln Gln Leu Asn Pro Tyr Leu Arg Leu Lys Val Arg

515 520 525

Arg Asp His Ile Ile Asp Asp Ala Leu Val Arg Leu Glu Met Ile Ala

530 535 540

Met Glu Asn Pro Ala Asp Leu Lys Lys Gln Leu Tyr Val Glu Phe Glu

545 550 555 560

Gly Glu Gln Gly Val Asp Glu Gly Gly Val Ser Lys Glu Phe Phe Gln

565 570 575

Leu Val Val Glu Glu Ile Phe Asn Pro Asp Ile Gly Met Phe Thr Tyr

580 585 590

Asp Glu Ser Thr Lys Leu Phe Trp Phe Asn Pro Ser Ser Phe Glu Thr

595 600 605

Glu Gly Gln Phe Thr Leu Ile Gly Ile Val Leu Gly Leu Ala Ile Tyr

610 615 620

Asn Asn Cys Ile Leu Asp Val His Phe Pro Met Val Val Tyr Arg Lys

625 630 635 640

Leu Met Gly Lys Lys Gly Thr Phe Arg Asp Leu Gly Asp Ser His Pro

645 650 655

Val Leu Tyr Gln Ser Leu Lys Asp Leu Leu Glu Tyr Glu Gly Asn Val

660 665 670

Glu Asp Asp Met Met Ile Thr Phe Gln Ile Ser Gln Thr Asp Leu Phe

675 680 685

Gly Asn Pro Met Met Tyr Asp Leu Lys Glu Asn Gly Asp Lys Ile Pro

690 695 700

Ile Thr Asn Glu Asn Arg Lys Glu Phe Val Asn Leu Tyr Ser Asp Tyr

705 710 715 720

Ile Leu Asn Lys Ser Val Glu Lys Gln Phe Lys Ala Phe Arg Arg Gly

725 730 735

Phe His Met Val Thr Asn Glu Ser Pro Leu Lys Tyr Leu Phe Arg Pro

740 745 750

Glu Glu Ile Glu Leu Leu Ile Cys Gly Ser Arg Asn Leu Asp Phe Gln

755 760 765

Ala Leu Glu Glu Thr Thr Glu Tyr Asp Gly Gly Tyr Thr Arg Asp Ser

770 775 780

Val Leu Ile Arg Glu Phe Trp Glu Ile Val His Ser Phe Thr Asp Glu

785 790 795 800

Gln Lys Arg Leu Phe Leu Gln Phe Thr Thr Gly Thr Asp Arg Ala Pro

805 810 815

Val Gly Gly Leu Gly Lys Leu Lys Met Ile Ile Ala Lys Asn Gly Pro

820 825 830

Asp Thr Glu Arg Leu Pro Thr Ser His Thr Cys Phe Asn Val Leu Leu

835 840 845

Leu Pro Glu Tyr Ser Ser Lys Glu Lys Leu Lys Glu Arg Leu Leu Lys

850 855 860

Ala Ile Thr Tyr Ala Lys Gly Phe Gly Met Leu

865 870 875

<210> 7

<211> 3769

<212> DNA

<213> artificial sequence

<220>

<223> vector genome hSyn.hUbe3a-2.GSco.SV40

<220>

<221> repeat_region

<222> (1)..(130)

<223> ITR

<220>

<221> promoter

<222> (213)..(678)

<223> human synapsin promoter

<220>

<221> misc_feature

<222> (690)..(695)

<223> Kozak

<220>

<221> CDS

<222> (696)..(3326)

<223> hUBE3a-2

<220>

<221> misc_feature

<222> (848)..(848)

<223> mutation

<220>

<221> misc_feature

<222> (893)..(893)

<223> mutation

<220>

<221> misc_feature

<222> (1097)..(1097)

<223> mutation

<220>

<221> misc_feature

<222> (1250)..(1250)

<223> mutation

<220>

<221> misc_feature

<222> (1262)..(1262)

<223> mutation

<220>

<221> misc_feature

<222> (1286)..(1286)

<223> mutation

<220>

<221> misc_feature

<222> (1352)..(1352)

<223> mutant SD/AS

<220>

<221> misc_feature

<222> (1784)..(1784)

<223> mutation

<220>

<221> misc_feature

<222> (1790)..(1790)

<223> mutation

<220>

<221> misc_feature

<222> (1835)..(1835)

<223> mutation

<220>

<221> misc_feature

<222> (1874)..(1874)

<223> mutation

<220>

<221> misc_feature

<222> (1883)..(1883)

<223> mutation

<220>

<221> misc_feature

<222> (2060)..(2060)

<223> mutation

<220>

<221> misc_feature

<222> (2132)..(2132)

<223> mutation

<220>

<221> misc_feature

<222> (2180)..(2180)

<223> mutation

<220>

<221> misc_feature

<222> (2363)..(2363)

<223> mutation

<220>

<221> misc_feature

<222> (2508)..(2509)

<223> mutant SD/AS

<220>

<221> misc_feature

<222> (2792)..(2792)

<223> mutation

<220>

<221> misc_feature

<222> (3026)..(3026)

<223> mutation

<220>

<221> misc_feature

<222> (3092)..(3092)

<223> mutation

<220>

<221> misc_feature

<222> (3125)..(3125)

<223> mutant SD/AS

<220>

<221> misc_feature

<222> (3221)..(3221)

<223> mutation

<220>

<221> misc_feature

<222> (3230)..(3230)

<223> mutation

<220>

<221> misc_feature

<222> (3299)..(3299)

<223> mutation

<220>

<221> polyA_Signal

<222> (3344)..(3757)

<223> SV40 polyA

<220>

<221> repeat_region

<222> (3640)..(3769)

<223> ITR

<400> 7

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcc tctagaacta tagctagcat gcctgcagag ggccctgcgt atgagtgcaa 240

gtgggtttta ggaccaggat gaggcggggt gggggtgcct acctgacgac cgaccccgac 300

ccactggaca agcacccaac ccccattccc caaattgcgc atcccctatc agagaggggg 360

aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc gcggacagtg 420

ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg cgcgctgacg 480

tcactcgccg gtcccccgca aactcccctt cccggccacc ttggtcgcgt ccgcgccgcc 540

gccggcccag ccggaccgca ccacgcgagg cgcgagatag gggggcacgg gcgcgaccat 600

ctgcgctgcg gcgccggcga ctcagcgctg cctcagtctg cggtgggcag cggaggagtc 660

gtgtcgtgcc tgagagcgca gtcgaattcg ccacc atg gag aag ctg cac cag 713

Met Glu Lys Leu His Gln

1 5

tgc tac tgg aag tct ggc gag cct cag agc gac gat atc gag gca agc 761

Cys Tyr Trp Lys Ser Gly Glu Pro Gln Ser Asp Asp Ile Glu Ala Ser

10 15 20

agg atg aag aga gca gca gcc aag cac ctg atc gag cgg tac tat cac 809

Arg Met Lys Arg Ala Ala Ala Lys His Leu Ile Glu Arg Tyr Tyr His

25 30 35

cag ctg acc gag ggc tgc gga aac gag gcc tgt aca aac gag ttc tgc 857

Gln Leu Thr Glu Gly Cys Gly Asn Glu Ala Cys Thr Asn Glu Phe Cys

40 45 50

gcc tcc tgt cca acc ttt ctg aga atg gat aac aac gcc gcc gcc atc 905

Ala Ser Cys Pro Thr Phe Leu Arg Met Asp Asn Asn Ala Ala Ala Ile

55 60 65 70

aag gcc ctg gag ctg tac aag atc aac gcc aag ctg tgc gac ccc cac 953

Lys Ala Leu Glu Leu Tyr Lys Ile Asn Ala Lys Leu Cys Asp Pro His

75 80 85

cct tct aag aag ggc gcc agc tcc gcc tat ctg gag aac agc aag ggc 1001

Pro Ser Lys Lys Gly Ala Ser Ser Ala Tyr Leu Glu Asn Ser Lys Gly

90 95 100

gcc ccc aac aat agc tgt tcc gag atc aag atg aat aag aag ggc gcc 1049

Ala Pro Asn Asn Ser Cys Ser Glu Ile Lys Met Asn Lys Lys Gly Ala

105 110 115

agg atc gat ttc aag gac gtg acc tac ctg aca gag gag aag gtg tac 1097

Arg Ile Asp Phe Lys Asp Val Thr Tyr Leu Thr Glu Glu Lys Val Tyr

120 125 130

gag atc ctg gag ctg tgc cgg gag aga gag gat tac tcc cct ctg atc 1145

Glu Ile Leu Glu Leu Cys Arg Glu Arg Glu Asp Tyr Ser Pro Leu Ile

135 140 145 150

aga gtg atc gga cgc gtg ttc tct agc gcc gag gcc ctg gtg cag agc 1193

Arg Val Ile Gly Arg Val Phe Ser Ser Ala Glu Ala Leu Val Gln Ser

155 160 165

ttt cgc aag gtg aag cag cac aca aag gag gag ctg aag tcc ctg cag 1241

Phe Arg Lys Val Lys Gln His Thr Lys Glu Glu Leu Lys Ser Leu Gln

170 175 180

gcc aag gac gag gac aag gac gag gac gag aag gag aag gca gct tgt 1289

Ala Lys Asp Glu Asp Lys Asp Glu Asp Glu Lys Glu Lys Ala Ala Cys

185 190 195

tcc gcc gca gca atg gag gag gac tct gag gcc tcc tct agc agg atc 1337

Ser Ala Ala Ala Met Glu Glu Asp Ser Glu Ala Ser Ser Ser Arg Ile

200 205 210

ggc gat tcc tct caa ggc gac aac aat ctg cag aag ctg ggc ccc gac 1385

Gly Asp Ser Ser Gln Gly Asp Asn Asn Leu Gln Lys Leu Gly Pro Asp

215 220 225 230

gac gtg agc gtg gat atc gac gcc atc cgg aga gtg tac aca cgc ctg 1433

Asp Val Ser Val Asp Ile Asp Ala Ile Arg Arg Val Tyr Thr Arg Leu

235 240 245

ctg tct aac gag aag atc gag acc gcc ttc ctg aac gcc ctg gtg tat 1481

Leu Ser Asn Glu Lys Ile Glu Thr Ala Phe Leu Asn Ala Leu Val Tyr

250 255 260

ctg tct cct aat gtg gag tgc gat ctg acc tac cac aac gtg tac agc 1529

Leu Ser Pro Asn Val Glu Cys Asp Leu Thr Tyr His Asn Val Tyr Ser

265 270 275

cgg gac cca aac tac ctg aat ctg ttc atc atc gtg atg gag aac aga 1577

Arg Asp Pro Asn Tyr Leu Asn Leu Phe Ile Ile Val Met Glu Asn Arg

280 285 290

aat ctg cac tct ccc gag tat ctg gag atg gcc ctg cct ctg ttt tgt 1625

Asn Leu His Ser Pro Glu Tyr Leu Glu Met Ala Leu Pro Leu Phe Cys

295 300 305 310

aag gcc atg agc aag ctg cca ctg gca gca cag ggc aag ctg atc agg 1673

Lys Ala Met Ser Lys Leu Pro Leu Ala Ala Gln Gly Lys Leu Ile Arg

315 320 325

ctg tgg tcc aag tac aac gcc gat cag atc agg cgc atg atg gag acc 1721

Leu Trp Ser Lys Tyr Asn Ala Asp Gln Ile Arg Arg Met Met Glu Thr

330 335 340

ttc cag cag ctg atc aca tat aaa gtg atc tcc aac gag ttt aat tct 1769

Phe Gln Gln Leu Ile Thr Tyr Lys Val Ile Ser Asn Glu Phe Asn Ser

345 350 355

aga aac ctg gtg aac gac gac gac gcc atc gtg gcc gcc agc aag tgc 1817

Arg Asn Leu Val Asn Asp Asp Asp Ala Ile Val Ala Ala Ser Lys Cys

360 365 370

ctg aag atg gtg tac tac gcc aac gtg gtg ggc ggc gag gtg gac aca 1865

Leu Lys Met Val Tyr Tyr Ala Asn Val Val Gly Gly Glu Val Asp Thr

375 380 385 390

aac cac aac gag gag gac gac gag gag cca atc ccc gag agc tcc gag 1913

Asn His Asn Glu Glu Asp Asp Glu Glu Pro Ile Pro Glu Ser Ser Glu

395 400 405

ctg acc ctg cag gag ctg ctg gga gag gag cgg aga aat aag aag gga 1961

Leu Thr Leu Gln Glu Leu Leu Gly Glu Glu Arg Arg Asn Lys Lys Gly

410 415 420

cca agg gtg gat cct ctg gag acc gag ctg ggc gtg aag aca ctg gac 2009

Pro Arg Val Asp Pro Leu Glu Thr Glu Leu Gly Val Lys Thr Leu Asp

425 430 435

tgc aga aag cct ctg atc cca ttc gag gag ttt atc aac gag ccc ctg 2057

Cys Arg Lys Pro Leu Ile Pro Phe Glu Glu Phe Ile Asn Glu Pro Leu

440 445 450

aac gag gtg ctg gag atg gat aag gac tac acc ttc ttt aag gtg gag 2105

Asn Glu Val Leu Glu Met Asp Lys Asp Tyr Thr Phe Phe Lys Val Glu

455 460 465 470

aca gag aac aag ttc agc ttt atg acc tgt cct ttc atc ctg aac gcc 2153

Thr Glu Asn Lys Phe Ser Phe Met Thr Cys Pro Phe Ile Leu Asn Ala

475 480 485

gtg acc aag aat ctg ggc ctg tac tac gat aac agg atc cgc atg tac 2201

Val Thr Lys Asn Leu Gly Leu Tyr Tyr Asp Asn Arg Ile Arg Met Tyr

490 495 500

agc gag agg cgc atc acc gtg ctg tat tcc ctg gtg cag ggc cag cag 2249

Ser Glu Arg Arg Ile Thr Val Leu Tyr Ser Leu Val Gln Gly Gln Gln

505 510 515

ctg aat cct tac ctg agg ctg aag gtg cgg aga gac cac atc atc gat 2297

Leu Asn Pro Tyr Leu Arg Leu Lys Val Arg Arg Asp His Ile Ile Asp

520 525 530

gac gcc ctg gtg cgc ctg gag atg atc gcc atg gag aat cca gcc gat 2345

Asp Ala Leu Val Arg Leu Glu Met Ile Ala Met Glu Asn Pro Ala Asp

535 540 545 550

ctg aag aag cag ctg tac gtg gag ttt gag gga gag cag gga gtg gac 2393

Leu Lys Lys Gln Leu Tyr Val Glu Phe Glu Gly Glu Gln Gly Val Asp

555 560 565

gag gga ggc gtg tcc aag gag ttc ttt cag ctg gtg gtg gag gag atc 2441

Glu Gly Gly Val Ser Lys Glu Phe Phe Gln Leu Val Val Glu Glu Ile

570 575 580

ttc aac ccc gat atc ggc atg ttt acc tac gac gag tct aca aag ctg 2489

Phe Asn Pro Asp Ile Gly Met Phe Thr Tyr Asp Glu Ser Thr Lys Leu

585 590 595

ttc tgg ttt aat cct tct tcc ttc gag acc gag ggc cag ttt aca ctg 2537

Phe Trp Phe Asn Pro Ser Ser Phe Glu Thr Glu Gly Gln Phe Thr Leu

600 605 610

atc ggc atc gtg ctg ggc ctg gcc atc tac aac aat tgt atc ctg gac 2585

Ile Gly Ile Val Leu Gly Leu Ala Ile Tyr Asn Asn Cys Ile Leu Asp

615 620 625 630

gtg cac ttc cca atg gtg gtg tat agg aag ctg atg ggc aag aag ggc 2633

Val His Phe Pro Met Val Val Tyr Arg Lys Leu Met Gly Lys Lys Gly

635 640 645

acc ttt cgc gat ctg ggc gac agc cac ccc gtg ctg tac cag tcc ctg 2681

Thr Phe Arg Asp Leu Gly Asp Ser His Pro Val Leu Tyr Gln Ser Leu

650 655 660

aag gat ctg ctg gag tat gag ggc aac gtg gag gat gac atg atg atc 2729

Lys Asp Leu Leu Glu Tyr Glu Gly Asn Val Glu Asp Asp Met Met Ile

665 670 675

acc ttc cag atc tcc cag aca gac ctg ttt ggc aac cca atg atg tac 2777

Thr Phe Gln Ile Ser Gln Thr Asp Leu Phe Gly Asn Pro Met Met Tyr

680 685 690

gat ctg aag gag aac ggc gac aag atc ccc atc aca aac gag aat agg 2825

Asp Leu Lys Glu Asn Gly Asp Lys Ile Pro Ile Thr Asn Glu Asn Arg

695 700 705 710

aag gag ttc gtg aac ctg tac tct gat tat atc ctg aat aag agc gtg 2873

Lys Glu Phe Val Asn Leu Tyr Ser Asp Tyr Ile Leu Asn Lys Ser Val

715 720 725

gag aag cag ttc aag gcc ttt agg cgc ggc ttc cac atg gtg acc aac 2921

Glu Lys Gln Phe Lys Ala Phe Arg Arg Gly Phe His Met Val Thr Asn

730 735 740

gag tct cct ctg aag tat ctg ttt agg cca gag gag atc gag ctg ctg 2969

Glu Ser Pro Leu Lys Tyr Leu Phe Arg Pro Glu Glu Ile Glu Leu Leu

745 750 755

atc tgc ggc agc cgc aat ctg gac ttt cag gcc ctg gag gag acc aca 3017

Ile Cys Gly Ser Arg Asn Leu Asp Phe Gln Ala Leu Glu Glu Thr Thr

760 765 770

gag tac gac ggc ggc tat acc cgg gac tcc gtg ctg atc aga gag ttc 3065

Glu Tyr Asp Gly Gly Tyr Thr Arg Asp Ser Val Leu Ile Arg Glu Phe

775 780 785 790

tgg gag atc gtg cac tct ttt aca gac gag cag aag cgg ctg ttc ctg 3113

Trp Glu Ile Val His Ser Phe Thr Asp Glu Gln Lys Arg Leu Phe Leu

795 800 805

cag ttt acc acc ggc acc gac aga gca cca gtg gga gga ctg ggc aag 3161

Gln Phe Thr Thr Gly Thr Asp Arg Ala Pro Val Gly Gly Leu Gly Lys

810 815 820

ctg aag atg atc atc gcc aag aac ggc cca gac aca gag agg ctg ccc 3209

Leu Lys Met Ile Ile Ala Lys Asn Gly Pro Asp Thr Glu Arg Leu Pro

825 830 835

acc agc cac acc tgt ttc aac gtg ctg ctg ctg ccc gag tac tcc tct 3257

Thr Ser His Thr Cys Phe Asn Val Leu Leu Leu Pro Glu Tyr Ser Ser

840 845 850

aag gag aag ctg aag gag cgc ctg ctg aag gcc atc acc tac gcc aag 3305

Lys Glu Lys Leu Lys Glu Arg Leu Leu Lys Ala Ile Thr Tyr Ala Lys

855 860 865 870

ggc ttt ggc atg ctg tga tga ggtaccggcg gccgcttcga gcagacatga 3356

Gly Phe Gly Met Leu

875

taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta 3416

tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag 3476

ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt 3536

tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatcga taaggatctt cctagagcat 3596

ggctacgtag ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg 3656

gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc 3716

gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cag 3769

<210> 8

<211> 875

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 8

Met Glu Lys Leu His Gln Cys Tyr Trp Lys Ser Gly Glu Pro Gln Ser

1 5 10 15

Asp Asp Ile Glu Ala Ser Arg Met Lys Arg Ala Ala Ala Lys His Leu

20 25 30

Ile Glu Arg Tyr Tyr His Gln Leu Thr Glu Gly Cys Gly Asn Glu Ala

35 40 45

Cys Thr Asn Glu Phe Cys Ala Ser Cys Pro Thr Phe Leu Arg Met Asp

50 55 60

Asn Asn Ala Ala Ala Ile Lys Ala Leu Glu Leu Tyr Lys Ile Asn Ala

65 70 75 80

Lys Leu Cys Asp Pro His Pro Ser Lys Lys Gly Ala Ser Ser Ala Tyr

85 90 95

Leu Glu Asn Ser Lys Gly Ala Pro Asn Asn Ser Cys Ser Glu Ile Lys

100 105 110

Met Asn Lys Lys Gly Ala Arg Ile Asp Phe Lys Asp Val Thr Tyr Leu

115 120 125

Thr Glu Glu Lys Val Tyr Glu Ile Leu Glu Leu Cys Arg Glu Arg Glu

130 135 140

Asp Tyr Ser Pro Leu Ile Arg Val Ile Gly Arg Val Phe Ser Ser Ala

145 150 155 160

Glu Ala Leu Val Gln Ser Phe Arg Lys Val Lys Gln His Thr Lys Glu

165 170 175

Glu Leu Lys Ser Leu Gln Ala Lys Asp Glu Asp Lys Asp Glu Asp Glu

180 185 190

Lys Glu Lys Ala Ala Cys Ser Ala Ala Ala Met Glu Glu Asp Ser Glu

195 200 205

Ala Ser Ser Ser Arg Ile Gly Asp Ser Ser Gln Gly Asp Asn Asn Leu

210 215 220

Gln Lys Leu Gly Pro Asp Asp Val Ser Val Asp Ile Asp Ala Ile Arg

225 230 235 240

Arg Val Tyr Thr Arg Leu Leu Ser Asn Glu Lys Ile Glu Thr Ala Phe

245 250 255

Leu Asn Ala Leu Val Tyr Leu Ser Pro Asn Val Glu Cys Asp Leu Thr

260 265 270

Tyr His Asn Val Tyr Ser Arg Asp Pro Asn Tyr Leu Asn Leu Phe Ile

275 280 285

Ile Val Met Glu Asn Arg Asn Leu His Ser Pro Glu Tyr Leu Glu Met

290 295 300

Ala Leu Pro Leu Phe Cys Lys Ala Met Ser Lys Leu Pro Leu Ala Ala

305 310 315 320

Gln Gly Lys Leu Ile Arg Leu Trp Ser Lys Tyr Asn Ala Asp Gln Ile

325 330 335

Arg Arg Met Met Glu Thr Phe Gln Gln Leu Ile Thr Tyr Lys Val Ile

340 345 350

Ser Asn Glu Phe Asn Ser Arg Asn Leu Val Asn Asp Asp Asp Ala Ile

355 360 365

Val Ala Ala Ser Lys Cys Leu Lys Met Val Tyr Tyr Ala Asn Val Val

370 375 380

Gly Gly Glu Val Asp Thr Asn His Asn Glu Glu Asp Asp Glu Glu Pro

385 390 395 400

Ile Pro Glu Ser Ser Glu Leu Thr Leu Gln Glu Leu Leu Gly Glu Glu

405 410 415

Arg Arg Asn Lys Lys Gly Pro Arg Val Asp Pro Leu Glu Thr Glu Leu

420 425 430

Gly Val Lys Thr Leu Asp Cys Arg Lys Pro Leu Ile Pro Phe Glu Glu

435 440 445

Phe Ile Asn Glu Pro Leu Asn Glu Val Leu Glu Met Asp Lys Asp Tyr

450 455 460

Thr Phe Phe Lys Val Glu Thr Glu Asn Lys Phe Ser Phe Met Thr Cys

465 470 475 480

Pro Phe Ile Leu Asn Ala Val Thr Lys Asn Leu Gly Leu Tyr Tyr Asp

485 490 495

Asn Arg Ile Arg Met Tyr Ser Glu Arg Arg Ile Thr Val Leu Tyr Ser

500 505 510

Leu Val Gln Gly Gln Gln Leu Asn Pro Tyr Leu Arg Leu Lys Val Arg

515 520 525

Arg Asp His Ile Ile Asp Asp Ala Leu Val Arg Leu Glu Met Ile Ala

530 535 540

Met Glu Asn Pro Ala Asp Leu Lys Lys Gln Leu Tyr Val Glu Phe Glu

545 550 555 560

Gly Glu Gln Gly Val Asp Glu Gly Gly Val Ser Lys Glu Phe Phe Gln

565 570 575

Leu Val Val Glu Glu Ile Phe Asn Pro Asp Ile Gly Met Phe Thr Tyr

580 585 590

Asp Glu Ser Thr Lys Leu Phe Trp Phe Asn Pro Ser Ser Phe Glu Thr

595 600 605

Glu Gly Gln Phe Thr Leu Ile Gly Ile Val Leu Gly Leu Ala Ile Tyr

610 615 620

Asn Asn Cys Ile Leu Asp Val His Phe Pro Met Val Val Tyr Arg Lys

625 630 635 640

Leu Met Gly Lys Lys Gly Thr Phe Arg Asp Leu Gly Asp Ser His Pro

645 650 655

Val Leu Tyr Gln Ser Leu Lys Asp Leu Leu Glu Tyr Glu Gly Asn Val

660 665 670

Glu Asp Asp Met Met Ile Thr Phe Gln Ile Ser Gln Thr Asp Leu Phe

675 680 685

Gly Asn Pro Met Met Tyr Asp Leu Lys Glu Asn Gly Asp Lys Ile Pro

690 695 700

Ile Thr Asn Glu Asn Arg Lys Glu Phe Val Asn Leu Tyr Ser Asp Tyr

705 710 715 720

Ile Leu Asn Lys Ser Val Glu Lys Gln Phe Lys Ala Phe Arg Arg Gly

725 730 735

Phe His Met Val Thr Asn Glu Ser Pro Leu Lys Tyr Leu Phe Arg Pro

740 745 750

Glu Glu Ile Glu Leu Leu Ile Cys Gly Ser Arg Asn Leu Asp Phe Gln

755 760 765

Ala Leu Glu Glu Thr Thr Glu Tyr Asp Gly Gly Tyr Thr Arg Asp Ser

770 775 780

Val Leu Ile Arg Glu Phe Trp Glu Ile Val His Ser Phe Thr Asp Glu

785 790 795 800

Gln Lys Arg Leu Phe Leu Gln Phe Thr Thr Gly Thr Asp Arg Ala Pro

805 810 815

Val Gly Gly Leu Gly Lys Leu Lys Met Ile Ile Ala Lys Asn Gly Pro

820 825 830

Asp Thr Glu Arg Leu Pro Thr Ser His Thr Cys Phe Asn Val Leu Leu

835 840 845

Leu Pro Glu Tyr Ser Ser Lys Glu Lys Leu Lys Glu Arg Leu Leu Lys

850 855 860

Ala Ile Thr Tyr Ala Lys Gly Phe Gly Met Leu

865 870 875

<210> 9

<211> 2562

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid sequence hUbe31a-1

<400> 9

atgaagaggg cagcagcaaa gcacctgatc gagagatact atcaccagct gaccgaggga 60

tgcggaaacg aggcatgtac aaacgagttc tgcgcctcct gtcccacctt tctgaggatg 120

gataacaacg ccgccgccat caaggccctg gagctgtaca agatcaacgc caagctgtgc 180

gacccccacc ctagcaagaa gggcgccagc tccgcctatc tggagaactc caagggcgcc 240

cctaacaata gctgttccga gatcaagatg aataagaagg gcgcccggat cgatttcaag 300

gacgtgacct acctgacaga ggagaaggtg tacgagatcc tggagctgtg ccgggagaga 360

gaggattaca gcccactgat cagagtgatc ggcagagtgt tctctagcgc cgaggccctg 420

gtgcagtcct ttagaaaggt gaagcagcac acaaaggagg agctgaagtc tctgcaggcc 480

aaggacgagg acaaggacga ggacgagaag gagaaggcag cctgttctgc cgcagcaatg 540

gaggaggaca gcgaggcatc ctctagccgg atcggcgatt cctctcaagg cgacaacaat 600

ctgcagaagc tgggccccga cgacgtgagc gtggatatcg acgccatccg gagagtgtac 660

acaagactgc tgagcaacga gaagatcgag accgccttcc tgaacgccct ggtgtatctg 720

agccctaatg tggagtgcga tctgacctac cacaacgtgt actcccggga cccaaactac 780

ctgaatctgt tcatcatcgt gatggagaac agaaatctgc actcccccga gtatctggag 840

atggccctgc ctctgttttg taaggccatg tccaagctgc ctctggcagc acagggcaag 900

ctgatcaggc tgtggtctaa gtacaacgcc gatcagatca ggcgcatgat ggagaccttc 960

cagcagctga tcacatacaa agtgatctct aacgagttta atagccgcaa cctggtgaac 1020

gacgacgacg ccatcgtggc cgcctctaag tgcctgaaga tggtgtacta cgccaacgtg 1080

gtgggcggcg aggtggacac aaaccacaac gaggaggacg acgaggagcc aatccccgag 1140

agctccgagc tgaccctgca ggagctgctg ggagaggagc ggagaaataa gaagggacca 1200

agggtggatc ctctggagac cgagctgggc gtgaagacac tggactgcag aaagcctctg 1260

atcccattcg aggagtttat caacgagccc ctgaacgagg tgctggagat ggataaggac 1320

tacaccttct ttaaggtgga gacagagaac aagttcagct ttatgacctg tcctttcatc 1380

ctgaacgccg tgaccaagaa tctgggcctg tactacgata acaggatccg catgtactcc 1440

gagaggcgca tcaccgtgct gtattctctg gtgcagggcc agcagctgaa tccttacctg 1500

aggctgaagg tgcggagaga ccacatcatc gatgacgccc tggtgcgcct ggagatgatc 1560

gccatggaga atccagccga tctgaagaag cagctgtacg tggagtttga gggagagcag 1620

ggagtggacg agggaggcgt gtctaaggag ttctttcagc tggtggtgga ggagatcttc 1680

aaccccgata tcggcatgtt tacctacgac gagagcacaa agctgttctg gtttaatcct 1740

tcttccttcg agaccgaggg ccagtttaca ctgatcggca tcgtgctggg cctggccatc 1800

tacaacaatt gtatcctgga cgtgcacttc ccaatggtgg tgtataggaa gctgatgggc 1860

aagaagggca cctttcgcga tctgggcgac tcccaccccg tgctgtacca gtctctgaag 1920

gatctgctgg agtatgaggg caacgtggag gatgacatga tgatcacctt ccagatctcc 1980

cagacagacc tgtttggcaa cccaatgatg tacgatctga aggagaacgg cgacaagatc 2040

cccatcacaa acgagaatag aaaggagttc gtgaacctgt acagcgatta tatcctgaat 2100

aagtccgtgg agaagcagtt caaggccttt aggcgcggct tccacatggt gaccaacgag 2160

agccctctga agtatctgtt taggccagag gagatcgagc tgctgatctg cggctcccgc 2220

aatctggact ttcaggccct ggaggagacc acagagtacg acggcggcta taccagggac 2280

tctgtgctga tccgcgagtt ctgggagatc gtgcacagct ttacagacga gcagaagcgg 2340

ctgttcctgc agtttaccac cggcaccgac agagcaccag tgggaggact gggcaagctg 2400

aagatgatca tcgccaagaa cggcccagac acagagaggc tgcccaccag ccacacctgt 2460

ttcaacgtgc tgctgctgcc cgagtactcc tctaaggaga agctgaagga gcgcctgctg 2520

aaggccatca cctacgccaa gggctttggc atgctgtgat ga 2562

<210> 10

<211> 2631

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid sequence hUbe3a-2

<400> 10

atggagaagc tgcaccagtg ctactggaag tctggcgagc ctcagagcga cgatatcgag 60

gcaagcagga tgaagagagc agcagccaag cacctgatcg agcggtacta tcaccagctg 120

accgagggct gcggaaacga ggcctgtaca aacgagttct gcgcctcctg tccaaccttt 180

ctgagaatgg ataacaacgc cgccgccatc aaggccctgg agctgtacaa gatcaacgcc 240

aagctgtgcg acccccaccc ttctaagaag ggcgccagct ccgcctatct ggagaacagc 300

aagggcgccc ccaacaatag ctgttccgag atcaagatga ataagaaggg cgccaggatc 360

gatttcaagg acgtgaccta cctgacagag gagaaggtgt acgagatcct ggagctgtgc 420

cgggagagag aggattactc ccctctgatc agagtgatcg gacgcgtgtt ctctagcgcc 480

gaggccctgg tgcagagctt tcgcaaggtg aagcagcaca caaaggagga gctgaagtcc 540

ctgcaggcca aggacgagga caaggacgag gacgagaagg agaaggcagc ttgttccgcc 600

gcagcaatgg aggaggactc tgaggcctcc tctagcagga tcggcgattc ctctcaaggc 660

gacaacaatc tgcagaagct gggccccgac gacgtgagcg tggatatcga cgccatccgg 720

agagtgtaca cacgcctgct gtctaacgag aagatcgaga ccgccttcct gaacgccctg 780

gtgtatctgt ctcctaatgt ggagtgcgat ctgacctacc acaacgtgta cagccgggac 840

ccaaactacc tgaatctgtt catcatcgtg atggagaaca gaaatctgca ctctcccgag 900

tatctggaga tggccctgcc tctgttttgt aaggccatga gcaagctgcc actggcagca 960

cagggcaagc tgatcaggct gtggtccaag tacaacgccg atcagatcag gcgcatgatg 1020

gagaccttcc agcagctgat cacatataaa gtgatctcca acgagtttaa ttctagaaac 1080

ctggtgaacg acgacgacgc catcgtggcc gccagcaagt gcctgaagat ggtgtactac 1140

gccaacgtgg tgggcggcga ggtggacaca aaccacaacg aggaggacga cgaggagcca 1200

atccccgaga gctccgagct gaccctgcag gagctgctgg gagaggagcg gagaaataag 1260

aagggaccaa gggtggatcc tctggagacc gagctgggcg tgaagacact ggactgcaga 1320

aagcctctga tcccattcga ggagtttatc aacgagcccc tgaacgaggt gctggagatg 1380

gataaggact acaccttctt taaggtggag acagagaaca agttcagctt tatgacctgt 1440

cctttcatcc tgaacgccgt gaccaagaat ctgggcctgt actacgataa caggatccgc 1500

atgtacagcg agaggcgcat caccgtgctg tattccctgg tgcagggcca gcagctgaat 1560

ccttacctga ggctgaaggt gcggagagac cacatcatcg atgacgccct ggtgcgcctg 1620

gagatgatcg ccatggagaa tccagccgat ctgaagaagc agctgtacgt ggagtttgag 1680

ggagagcagg gagtggacga gggaggcgtg tccaaggagt tctttcagct ggtggtggag 1740

gagatcttca accccgatat cggcatgttt acctacgacg agtctacaaa gctgttctgg 1800

tttaatcctt cttccttcga gaccgagggc cagtttacac tgatcggcat cgtgctgggc 1860

ctggccatct acaacaattg tatcctggac gtgcacttcc caatggtggt gtataggaag 1920

ctgatgggca agaagggcac ctttcgcgat ctgggcgaca gccaccccgt gctgtaccag 1980

tccctgaagg atctgctgga gtatgagggc aacgtggagg atgacatgat gatcaccttc 2040

cagatctccc agacagacct gtttggcaac ccaatgatgt acgatctgaa ggagaacggc 2100

gacaagatcc ccatcacaaa cgagaatagg aaggagttcg tgaacctgta ctctgattat 2160

atcctgaata agagcgtgga gaagcagttc aaggccttta ggcgcggctt ccacatggtg 2220

accaacgagt ctcctctgaa gtatctgttt aggccagagg agatcgagct gctgatctgc 2280

ggcagccgca atctggactt tcaggccctg gaggagacca cagagtacga cggcggctat 2340

acccgggact ccgtgctgat cagagagttc tgggagatcg tgcactcttt tacagacgag 2400

cagaagcggc tgttcctgca gtttaccacc ggcaccgaca gagcaccagt gggaggactg 2460

ggcaagctga agatgatcat cgccaagaac ggcccagaca cagagaggct gcccaccagc 2520

cacacctgtt tcaacgtgct gctgctgccc gagtactcct ctaaggagaa gctgaaggag 2580

cgcctgctga aggccatcac ctacgccaag ggctttggca tgctgtgatg a 2631

<210> 11

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> miRNA183 target sequence

<400> 11

agtgaattct accagtgcca ta 22

<210> 12

<211> 466

<212> DNA

<213> artificial sequence

<220>

<223> human synapsin promoter

<400> 12

ctgcagaggg ccctgcgtat gagtgcaagt gggttttagg accaggatga ggcggggtgg 60

gggtgcctac ctgacgaccg accccgaccc actggacaag cacccaaccc ccattcccca 120

aattgcgcat cccctatcag agagggggag gggaaacagg atgcggcgag gcgcgtgcgc 180

actgccagct tcagcaccgc ggacagtgcc ttcgcccccg cctggcggcg cgcgccaccg 240

ccgcctcagc actgaaggcg cgctgacgtc actcgccggt cccccgcaaa ctccccttcc 300

cggccacctt ggtcgcgtcc gcgccgccgc cggcccagcc ggaccgcacc acgcgaggcg 360

cgagataggg gggcacgggc gcgaccatct gcgctgcggc gccggcgact cagcgctgcc 420

tcagtctgcg gtgggcagcg gaggagtcgt gtcgtgcctg agagcg 466

<210> 13

<211> 232

<212> DNA

<213> artificial sequence

<220>

<223> SV40 polyA

<400> 13

cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga 60

aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc 120

tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag 180

atgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaaaat cg 232

<210> 14

<211> 2211

<212> DNA

<213> adeno-associated Virus hu68

<220>

<221> CDS

<222> (1)..(2211)

<223> AAVhu68 capsid

<400> 14

atg gct gcc gat ggt tat ctt cca gat tgg ctc gag gac aac ctc agt 48

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

gaa ggc att cgc gag tgg tgg gct ttg aaa cct gga gcc cct caa ccc 96

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

aag gca aat caa caa cat caa gac aac gct cgg ggt ctt gtg ctt ccg 144

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

ggt tac aaa tac ctt gga ccc ggc aac gga ctc gac aag ggg gag ccg 192

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

gtc aac gaa gca gac gcg gcg gcc ctc gag cac gac aag gcc tac gac 240

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

cag cag ctc aag gcc gga gac aac ccg tac ctc aag tac aac cac gcc 288

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

gac gcc gag ttc cag gag cgg ctc aaa gaa gat acg tct ttt ggg ggc 336

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

aac ctc ggg cga gca gtc ttc cag gcc aaa aag agg ctt ctt gaa cct 384

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

ctt ggt ctg gtt gag gaa gcg gct aag acg gct cct gga aag aag agg 432

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

cct gta gag cag tct cct cag gaa ccg gac tcc tcc gtg ggt att ggc 480

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Val Gly Ile Gly

145 150 155 160

aaa tcg ggt gca cag ccc gct aaa aag aga ctc aat ttc ggt cag act 528

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

ggc gac aca gag tca gtc ccc gac cct caa cca atc gga gaa cct ccc 576

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

gca gcc ccc tca ggt gtg gga tct ctt aca atg gct tca ggt ggt ggc 624

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

gca cca gtg gca gac aat aac gaa ggt gcc gat gga gtg ggt agt tcc 672

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

tcg gga aat tgg cat tgc gat tcc caa tgg ctg ggg gac aga gtc atc 720

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

acc acc agc acc cga acc tgg gcc ctg ccc acc tac aac aat cac ctc 768

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

tac aag caa atc tcc aac agc aca tct gga gga tct tca aat gac aac 816

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

gcc tac ttc ggc tac agc acc ccc tgg ggg tat ttt gac ttc aac aga 864

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

ttc cac tgc cac ttc tca cca cgt gac tgg caa aga ctc atc aac aac 912

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

aac tgg gga ttc cgg cct aag cga ctc aac ttc aag ctc ttc aac att 960

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

cag gtc aaa gag gtt acg gac aac aat gga gtc aag acc atc gct aat 1008

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

aac ctt acc agc acg gtc cag gtc ttc acg gac tca gac tat cag ctc 1056

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

ccg tac gtg ctc ggg tcg gct cac gag ggc tgc ctc ccg ccg ttc cca 1104

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

gcg gac gtt ttc atg att cct cag tac ggg tat cta acg ctt aat gat 1152

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

gga agc caa gcc gtg ggt cgt tcg tcc ttt tac tgc ctg gaa tat ttc 1200

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

ccg tcg caa atg cta aga acg ggt aac aac ttc cag ttc agc tac gag 1248

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

ttt gag aac gta cct ttc cat agc agc tat gct cac agc caa agc ctg 1296

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

gac cga ctc atg aat cca ctc atc gac caa tac ttg tac tat ctc tca 1344

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

aag act att aac ggt tct gga cag aat caa caa acg cta aaa ttc agt 1392

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

gtg gcc gga ccc agc aac atg gct gtc cag gga aga aac tac ata cct 1440

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

gga ccc agc tac cga caa caa cgt gtc tca acc act gtg act caa aac 1488

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

aac aac agc gaa ttt gct tgg cct gga gct tct tct tgg gct ctc aat 1536

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

gga cgt aat agc ttg atg aat cct gga cct gct atg gcc agc cac aaa 1584

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

gaa gga gag gac cgt ttc ttt cct ttg tct gga tct tta att ttt ggc 1632

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

aaa caa gga act gga aga gac aac gtg gat gcg gac aaa gtc atg ata 1680

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

acc aac gaa gaa gaa att aaa act acc aac cca gta gca acg gag tcc 1728

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

tat gga caa gtg gcc aca aac cac cag agt gcc caa gca cag gcg cag 1776

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590

acc ggc tgg gtt caa aac caa gga ata ctt ccg ggt atg gtt tgg cag 1824

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605

gac aga gat gtg tac ctg caa gga ccc att tgg gcc aaa att cct cac 1872

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

acg gac ggc aac ttt cac cct tct ccg ctg atg gga ggg ttt gga atg 1920

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640

aag cac ccg cct cct cag atc ctc atc aaa aac aca cct gta cct gcg 1968

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

gat cct cca acg gct ttc aac aag gac aag ctg aac tct ttc atc acc 2016

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670

cag tat tct act ggc caa gtc agc gtg gag att gag tgg gag ctg cag 2064

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

aag gaa aac agc aag cgc tgg aac ccg gag atc cag tac act tcc aac 2112

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

tat tac aag tct aat aat gtt gaa ttt gct gtt aat act gaa ggt gtt 2160

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720

tat tct gaa ccc cgc ccc att ggc acc aga tac ctg act cgt aat ctg 2208

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

taa 2211

<210> 15

<211> 736

<212> PRT

<213> adeno-associated Virus hu68

<400> 15

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Val Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 16

<211> 2211

<212> DNA

<213> adeno-associated Virus hu68

<400> 16

atggctgccg atggttatct tccagattgg ctcgaggaca acctcagtga aggcattcgc 60

gagtggtggg ctttgaaacc tggagcccct caacccaagg caaatcaaca acatcaagac 120

aacgctcggg gtcttgtgct tccgggttac aaataccttg gacccggcaa cggactcgac 180

aagggggagc cggtcaacga agcagacgcg gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aggccggaga caacccgtac ctcaagtaca accacgccga cgccgagttc 300

caggagcggc tcaaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gccaaaaaga ggcttcttga acctcttggt ctggttgagg aagcggctaa gacggctcct 420

ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgt gggtattggc 480

aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 540

tcagtccccg accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct 600

cttacaatgg cttcaggtgg tggcgcacca gtggcagaca ataacgaagg tgccgatgga 660

gtgggtagtt cctcgggaaa ttggcattgc gattcccaat ggctggggga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca atcacctcta caagcaaatc 780

tccaacagca catctggagg atcttcaaat gacaacgcct acttcggcta cagcaccccc 840

tgggggtatt ttgacttcaa cagattccac tgccacttct caccacgtga ctggcaaaga 900

ctcatcaaca acaactgggg attccggcct aagcgactca acttcaagct cttcaacatt 960

caggtcaaag aggttacgga caacaatgga gtcaagacca tcgctaataa ccttaccagc 1020

acggtccagg tcttcacgga ctcagactat cagctcccgt acgtgctcgg gtcggctcac 1080

gagggctgcc tcccgccgtt cccagcggac gttttcatga ttcctcagta tggatacctc 1140

accctgaacg acggcagtca ggcggtgggc cgctcatcct tctactgcct ggagtacttc 1200

ccttcgcaga tgctgaggac tggcaacaac ttccagttca gctacgagtt cgagaacgtc 1260

cctttccaca gcagctacgc ccacagccag agtttggacc gcttgatgaa ccctctgatc 1320

gaccagtacc tgtactacct gtcaaagacg atcaacggtt ctggccagaa ccagcagacg 1380

ctgaagttca gcgtggccgg gcctagcaac atggccgtcc agggcagaaa ctacatccct 1440

gggcccagct accggcagca gagagtctca accactgtga ctcagaacaa caacagtgag 1500

ttcgcctggc ctggcgccag ctcttgggcc ctcaacggcc gcaactcgct gatgaaccca 1560

ggcccagcca tggccagtca caaggagggc gaggaccgtt tcttcccttt gtctggctct 1620

ctgatcttcg gcaagcaggg gaccggcaga gacaacgtgg acgcggacaa ggtcatgatc 1680

acgaacgagg aggagatcaa gaccaccaac cctgtggcaa ccgagtccta cggccaggtg 1740

gcaaccaacc accagagcgc ccaggcacag gcgcagactg gctgggtcca gaaccagggg 1800

atcctgcctg gcatggtgtg gcaggaccgt gacgtgtacc tgcagggccc tatctgggca 1860

aagatccctc acacggacgg caacttccac ccttctcctc tgatgggcgg cttcggcatg 1920

aagcacccgc ctcctcagat cctcatcaag aacactccgg tcccggcaga ccctccgacg 1980

gccttcaaca aggacaagct gaactcattc atcactcagt actccactgg ccaggtcagc 2040

gtggagatcg agtgggagct gcagaaggag aacagcaagc gttggaaccc agagatccag 2100

tacacttcca actactacaa gtctaacaac gtggagttcg ccgtcaacac tgagggtgtg 2160

tacagtgagc ctcgccctat cggcacccgg tacctcaccc gaaacttgtg a 2211

<210> 17

<211> 2211

<212> DNA

<213> adeno-associated Virus rh.91

<220>

<221> CDS

<222> (1)..(2211)

<223> AAVrh.91 capsid

<400> 17

atg gct gcc gat ggt tat ctt cca gat tgg ctc gag gac aac ctc tct 48

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

gag ggc att cgc gag tgg tgg gcg ctg aaa cct gga gcc ccg aaa ccc 96

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

aaa gcc aac cag caa aag cag gac gac ggc cgg ggt ctg gtg ctt cct 144

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

ggc tac aag tac ctc gga ccc ttc aac gga ctc gac aag ggg gag ccc 192

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

gtc aac gcg gcg gac gca gcg gcc ctc gag cac gac aag gcc tac gac 240

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

cag cag ctc aaa gcg ggt gac aat ccg tac ctg cgg tat aac cac gcc 288

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

gac gcc gag ttt cag gag cgt ctg caa gaa gat acg tct ttt ggg ggc 336

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

aac ctc ggg cga gca gtc ttc cag gcc aag aag cgg gtt ctc gaa cct 384

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

ttt ggt ctg gtt gag gaa gca gct aag acg gct cct gga aag aaa cgt 432

Phe Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

ccg gta gag cag tcg ccc caa gaa cca gac tcc tcc tcg ggc att ggc 480

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

aaa tca ggc cag cag ccc gcc aaa aag aga ctc aat ttc ggt cag act 528

Lys Ser Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

ggc gac tca gag tca gtc ccc gac cct caa cct ctc gga gaa cct cca 576

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

gaa acc ccc gct gct gtg gga cct act aca atg gct tca ggc ggt ggc 624

Glu Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

gca cca atg gca gac aat aac gaa ggc gcc gac gga gtg ggt aat gcc 672

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

tca gga aat tgg cat tgc gat tcc aca tgg ctg ggc gac aga gtc atc 720

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

acc acc agc acc cga acc tgg gcc ctt cct acc tac aac aac cac ctc 768

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

tac aag caa atc tcc agc gct tca acg ggg gcc agt aac gac aac cac 816

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

tac ttt ggc tac agc acc ccc tgg ggg tat ttt gat ttc aac aga ttc 864

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

cac tgc cac ttc tca cca cgt gac tgg cag cga ctc att aac aac aac 912

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

tgg gga ttc cgg ccc aag aga ctc aac ttc aag ctc ttc aac atc cag 960

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

gtc aag gag gtc acg acg aat gat ggc gtc aca acc atc gct aat aac 1008

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

ctt acc agc acg gtt caa gtg ttc tcg gac tcg gag tac cag ctg ccg 1056

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

tac gtc ctc ggt tct gcg cac cag ggc tgc ctc cct ccg ttc ccg gcg 1104

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

gac gta ttc atg att cct cag tac ggc tac cta acg ctc aac aat ggc 1152

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

agc cag gcc gta gga cgt tca tcc ttt tat tgc ctg gaa tat ttc cca 1200

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

tct caa atg ctg aga acg ggc aac aac ttt acc ttc agc tac acc ttt 1248

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

gaa gat gtg cct ttc cac agc agt tac gcg cac agc cag agc ctg gac 1296

Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

agg cta atg aat cct cta atc gac cag tac ctg tat tac cta aac aga 1344

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

act cag aat caa tcc gga agt gca caa aac aag gac ttg ctg ttt agc 1392

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

cgg ggg tct cca gct ggc atg tct gtt cag ccc aaa aac tgg cta ccc 1440

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

ggg ccc tgt tac cga cag cag cgt gtt tct aaa aca aaa aca gac aac 1488

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

aac aac agc aac ttt acc tgg act ggt gcc tcc aaa tac aat ctg aac 1536

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

gga cgt gaa tcc atc att aac cct ggc acc gct atg gca tcc cac aag 1584

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

gac gac gaa gac aaa ttt ttt ccc atg agc ggt gtt atg att ttt ggc 1632

Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

aaa gaa aat gca gga gca tca aac act gca tta gac aat gtt atg att 1680

Lys Glu Asn Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

aca gat gaa gag gaa att aaa gct acc aac ccc gtg gcc acc gag aga 1728

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

ttt gga act gtg gca gtc aat ctc caa agc agc aat aca gac cct gca 1776

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Asn Thr Asp Pro Ala

580 585 590

aca gga gac gtg cat gtc atg ggg gct tta cct ggc atg gtg tgg caa 1824

Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

gac aga gac gtg tac ctg cag ggt ccc att tgg gcc aag att cct cac 1872

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

acg gat gga cac ttt cac ccg tct cct ctt atg ggc ggc ttt gga ctt 1920

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

aag cac ccg cct cct cag atc ctc atc aaa aac acg cct gtt cct gcg 1968

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

aat cct ccg gca gag ttt tcg gct aca aag ttt gct tca ttc atc acc 2016

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

cag tac tcc aca gga caa gtg agc gtg gaa att gaa tgg gag ctg cag 2064

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

aaa gaa aac agt aag cgc tgg aat cct gaa gtg cag tac acc tcc aac 2112

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

tac gcg aaa tct gcc aac gtt gat ttc act gtg gac aac aat gga ctt 2160

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

tat act gag cct cgc ccc att ggc acc cgt tac ctt acc cgt ccc ctt 2208

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

taa 2211

<210> 18

<211> 736

<212> PRT

<213> adeno-associated Virus rh.91

<400> 18

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Phe Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Ser Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Glu Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Asn Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Asn Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210> 19

<211> 2211

<212> DNA

<213> adeno-associated Virus rh.91

<400> 19

atggctgctg acggttatct tccagattgg ctcgaggaca acctttctga aggcattcgt 60

gagtggtggg ctctgaaacc tggagcccct aaacccaaag cgaaccaaca aaagcaggac 120

gacggccggg gtcttgtgct tccgggttac aaatacctcg gacccttcaa cggactcgac 180

aaaggagagc cggtcaacgc ggcggacgcg gcagccctcg aacacgacaa agcttacgac 240

cagcagctca aggccggtga caacccgtac ctccggtaca accacgccga cgccgagttt 300

caggagcgtc ttcaagaaga tacgtctttt gggggcaacc ttggcagagc agtcttccag 360

gccaaaaaga gggttcttga gccttttggt ctggttgagg aagcagctaa aacggctcct 420

ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcatcatc tggtattggc 480

aaatcgggcc agcagcctgc caaaaaaaga ctaaatttcg gtcagactgg cgactcagag 540

tcagtccccg accctcaacc tctcggagaa cctccagaaa cccccgctgc tgtgggacct 600

actacaatgg cttcaggcgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660

gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc 720

accaccagca cccgaacctg ggcccttcct acctacaaca accacctcta caagcaaatc 780

tccagcgctt caacgggggc cagtaacgac aaccactact ttggctacag caccccctgg 840

gggtattttg atttcaacag attccactgc cacttctcac cacgtgactg gcagcgactc 900

attaacaaca actggggatt ccggcccaag agactcaact tcaagctctt caacatccag 960

gtcaaggagg tcacgacgaa tgatggcgtc acaaccatcg ctaataacct taccagcacg 1020

gttcaagtgt tctcggactc ggagtaccag ctgccgtacg tcctcggttc tgcgcaccag 1080

ggctgcctcc ctccgttccc ggcggacgta ttcatgattc ctcagtatgg atacctcacc 1140

ctgaacaacg gaagtcaagc ggtgggacgc tcatcctttt actgcctgga gtacttccct 1200

tcgcagatgc taaggactgg aaataacttc accttcagct ataccttcga ggatgtacct 1260

tttcacagca gctacgctca cagccagagt ttggatcgct tgatgaatcc tcttattgat 1320

cagtatctgt actacctgaa cagaacgcaa aatcaatctg gaagtgcaca aaacaaggac 1380

ctgcttttta gccgggggtc tcctgctggc atgtctgttc agcccaaaaa ttggctacct 1440

gggccctgct accggcaaca gagagtttca aagactaaaa cagacaacaa caacagtaac 1500

tttacctgga caggtgccag caaatataat ctcaatggcc gcgaatcgat cattaatcca 1560

ggaaccgcta tggccagtca caaggacgat gaagacaaat ttttccctat gagcggcgtt 1620

atgatatttg gcaaagaaaa tgcaggagca agtaacactg cattagataa tgtaatgatt 1680

acggatgaag aagagattaa agctaccaat cctgtggcaa cagagagatt tggaactgtg 1740

gcagtcaact tgcagagctc aaatacagac cccgcaactg gagacgtcca tgtcatgggg 1800

gccttacctg gcatggtgtg gcaagatcgt gacgtgtacc ttcaaggacc tatctgggca 1860

aagattcctc acacggatgg acactttcat ccttctcctc tgatgggagg ctttggactg 1920

aaacatccgc ctcctcaaat cctcatcaaa aatactccgg taccggcaaa tcctccggca 1980

gagttcagcg ctacaaagtt tgcttcattt atcactcagt actccactgg acaggtcagc 2040

gtggaaattg agtgggagct acagaaagaa aacagcaaac gttggaatcc agaggtgcag 2100

tacacttcca actacgcgaa gtctgccaat gtggacttta ctgtagacaa caatggtctt 2160

tatactgaac ctcgccctat tggaacccgg tatctcacac gacccttgta a 2211

<210> 20

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> miRNA182 target sequence

<400> 20

agtgtgagtt ctaccattgc caaa 24

<210> 21

<211> 872

<212> PRT

<213> artificial sequence

<220>

<223> hUBE3a isoform 3 amino acid sequence

<400> 21

Met Ala Thr Ala Cys Lys Arg Ser Gly Glu Pro Gln Ser Asp Asp Ile

1 5 10 15

Glu Ala Ser Arg Met Lys Arg Ala Ala Ala Lys His Leu Ile Glu Arg

20 25 30

Tyr Tyr His Gln Leu Thr Glu Gly Cys Gly Asn Glu Ala Cys Thr Asn

35 40 45

Glu Phe Cys Ala Ser Cys Pro Thr Phe Leu Arg Met Asp Asn Asn Ala

50 55 60

Ala Ala Ile Lys Ala Leu Glu Leu Tyr Lys Ile Asn Ala Lys Leu Cys

65 70 75 80

Asp Pro His Pro Ser Lys Lys Gly Ala Ser Ser Ala Tyr Leu Glu Asn

85 90 95

Ser Lys Gly Ala Pro Asn Asn Ser Cys Ser Glu Ile Lys Met Asn Lys

100 105 110

Lys Gly Ala Arg Ile Asp Phe Lys Asp Val Thr Tyr Leu Thr Glu Glu

115 120 125

Lys Val Tyr Glu Ile Leu Glu Leu Cys Arg Glu Arg Glu Asp Tyr Ser

130 135 140

Pro Leu Ile Arg Val Ile Gly Arg Val Phe Ser Ser Ala Glu Ala Leu

145 150 155 160

Val Gln Ser Phe Arg Lys Val Lys Gln His Thr Lys Glu Glu Leu Lys

165 170 175

Ser Leu Gln Ala Lys Asp Glu Asp Lys Asp Glu Asp Glu Lys Glu Lys

180 185 190

Ala Ala Cys Ser Ala Ala Ala Met Glu Glu Asp Ser Glu Ala Ser Ser

195 200 205

Ser Arg Ile Gly Asp Ser Ser Gln Gly Asp Asn Asn Leu Gln Lys Leu

210 215 220

Gly Pro Asp Asp Val Ser Val Asp Ile Asp Ala Ile Arg Arg Val Tyr

225 230 235 240

Thr Arg Leu Leu Ser Asn Glu Lys Ile Glu Thr Ala Phe Leu Asn Ala

245 250 255

Leu Val Tyr Leu Ser Pro Asn Val Glu Cys Asp Leu Thr Tyr His Asn

260 265 270

Val Tyr Ser Arg Asp Pro Asn Tyr Leu Asn Leu Phe Ile Ile Val Met

275 280 285

Glu Asn Arg Asn Leu His Ser Pro Glu Tyr Leu Glu Met Ala Leu Pro

290 295 300

Leu Phe Cys Lys Ala Met Ser Lys Leu Pro Leu Ala Ala Gln Gly Lys

305 310 315 320

Leu Ile Arg Leu Trp Ser Lys Tyr Asn Ala Asp Gln Ile Arg Arg Met

325 330 335

Met Glu Thr Phe Gln Gln Leu Ile Thr Tyr Lys Val Ile Ser Asn Glu

340 345 350

Phe Asn Ser Arg Asn Leu Val Asn Asp Asp Asp Ala Ile Val Ala Ala

355 360 365

Ser Lys Cys Leu Lys Met Val Tyr Tyr Ala Asn Val Val Gly Gly Glu

370 375 380

Val Asp Thr Asn His Asn Glu Glu Asp Asp Glu Glu Pro Ile Pro Glu

385 390 395 400

Ser Ser Glu Leu Thr Leu Gln Glu Leu Leu Gly Glu Glu Arg Arg Asn

405 410 415

Lys Lys Gly Pro Arg Val Asp Pro Leu Glu Thr Glu Leu Gly Val Lys

420 425 430

Thr Leu Asp Cys Arg Lys Pro Leu Ile Pro Phe Glu Glu Phe Ile Asn

435 440 445

Glu Pro Leu Asn Glu Val Leu Glu Met Asp Lys Asp Tyr Thr Phe Phe

450 455 460

Lys Val Glu Thr Glu Asn Lys Phe Ser Phe Met Thr Cys Pro Phe Ile

465 470 475 480

Leu Asn Ala Val Thr Lys Asn Leu Gly Leu Tyr Tyr Asp Asn Arg Ile

485 490 495

Arg Met Tyr Ser Glu Arg Arg Ile Thr Val Leu Tyr Ser Leu Val Gln

500 505 510

Gly Gln Gln Leu Asn Pro Tyr Leu Arg Leu Lys Val Arg Arg Asp His

515 520 525

Ile Ile Asp Asp Ala Leu Val Arg Leu Glu Met Ile Ala Met Glu Asn

530 535 540

Pro Ala Asp Leu Lys Lys Gln Leu Tyr Val Glu Phe Glu Gly Glu Gln

545 550 555 560

Gly Val Asp Glu Gly Gly Val Ser Lys Glu Phe Phe Gln Leu Val Val

565 570 575

Glu Glu Ile Phe Asn Pro Asp Ile Gly Met Phe Thr Tyr Asp Glu Ser

580 585 590

Thr Lys Leu Phe Trp Phe Asn Pro Ser Ser Phe Glu Thr Glu Gly Gln

595 600 605

Phe Thr Leu Ile Gly Ile Val Leu Gly Leu Ala Ile Tyr Asn Asn Cys

610 615 620

Ile Leu Asp Val His Phe Pro Met Val Val Tyr Arg Lys Leu Met Gly

625 630 635 640

Lys Lys Gly Thr Phe Arg Asp Leu Gly Asp Ser His Pro Val Leu Tyr

645 650 655

Gln Ser Leu Lys Asp Leu Leu Glu Tyr Glu Gly Asn Val Glu Asp Asp

660 665 670

Met Met Ile Thr Phe Gln Ile Ser Gln Thr Asp Leu Phe Gly Asn Pro

675 680 685

Met Met Tyr Asp Leu Lys Glu Asn Gly Asp Lys Ile Pro Ile Thr Asn

690 695 700

Glu Asn Arg Lys Glu Phe Val Asn Leu Tyr Ser Asp Tyr Ile Leu Asn

705 710 715 720

Lys Ser Val Glu Lys Gln Phe Lys Ala Phe Arg Arg Gly Phe His Met

725 730 735

Val Thr Asn Glu Ser Pro Leu Lys Tyr Leu Phe Arg Pro Glu Glu Ile

740 745 750

Glu Leu Leu Ile Cys Gly Ser Arg Asn Leu Asp Phe Gln Ala Leu Glu

755 760 765

Glu Thr Thr Glu Tyr Asp Gly Gly Tyr Thr Arg Asp Ser Val Leu Ile

770 775 780

Arg Glu Phe Trp Glu Ile Val His Ser Phe Thr Asp Glu Gln Lys Arg

785 790 795 800

Leu Phe Leu Gln Phe Thr Thr Gly Thr Asp Arg Ala Pro Val Gly Gly

805 810 815

Leu Gly Lys Leu Lys Met Ile Ile Ala Lys Asn Gly Pro Asp Thr Glu

820 825 830

Arg Leu Pro Thr Ser His Thr Cys Phe Asn Val Leu Leu Leu Pro Glu

835 840 845

Tyr Ser Ser Lys Glu Lys Leu Lys Glu Arg Leu Leu Lys Ala Ile Thr

850 855 860

Tyr Ala Lys Gly Phe Gly Met Leu

865 870

<210> 22

<211> 3398

<212> DNA

<213> artificial sequence

<220>

<223> expression cassette hSyn. HUbe3a-1.GSco.4XmiRNA183.SV40

<220>

<221> promoter

<222> (1)..(466)

<223> human synaptoprotein

<220>

<221> misc_feature

<222> (478)..(483)

<223> Kozak

<220>

<221> misc_feature

<222> (484)..(3045)

<223> UBE3A-1

<220>

<221> misc_feature

<222> (3052)..(3073)

<223> miRNA183

<220>

<221> misc_feature

<222> (3078)..(3099)

<223> miRNA183

<220>

<221> misc_feature

<222> (3106)..(3127)

<223> miRNA183

<220>

<221> misc_feature

<222> (3134)..(3155)

<223> miRNA183

<220>

<221> polyA_Signal

<222> (3167)..(3398)

<223> SV40 late polyA

<400> 22

ctgcagaggg ccctgcgtat gagtgcaagt gggttttagg accaggatga ggcggggtgg 60

gggtgcctac ctgacgaccg accccgaccc actggacaag cacccaaccc ccattcccca 120

aattgcgcat cccctatcag agagggggag gggaaacagg atgcggcgag gcgcgtgcgc 180

actgccagct tcagcaccgc ggacagtgcc ttcgcccccg cctggcggcg cgcgccaccg 240

ccgcctcagc actgaaggcg cgctgacgtc actcgccggt cccccgcaaa ctccccttcc 300

cggccacctt ggtcgcgtcc gcgccgccgc cggcccagcc ggaccgcacc acgcgaggcg 360

cgagataggg gggcacgggc gcgaccatct gcgctgcggc gccggcgact cagcgctgcc 420

tcagtctgcg gtgggcagcg gaggagtcgt gtcgtgcctg agagcgcagt cgaattcgcc 480

accatgaaga gggcagcagc aaagcacctg atcgagagat actatcacca gctgaccgag 540

ggatgcggaa acgaggcatg tacaaacgag ttctgcgcct cctgtcccac ctttctgagg 600

atggataaca acgccgccgc catcaaggcc ctggagctgt acaagatcaa cgccaagctg 660

tgcgaccccc accctagcaa gaagggcgcc agctccgcct atctggagaa ctccaagggc 720

gcccctaaca atagctgttc cgagatcaag atgaataaga agggcgcccg gatcgatttc 780

aaggacgtga cctacctgac agaggagaag gtgtacgaga tcctggagct gtgccgggag 840

agagaggatt acagcccact gatcagagtg atcggcagag tgttctctag cgccgaggcc 900

ctggtgcagt cctttagaaa ggtgaagcag cacacaaagg aggagctgaa gtctctgcag 960

gccaaggacg aggacaagga cgaggacgag aaggagaagg cagcctgttc tgccgcagca 1020

atggaggagg acagcgaggc atcctctagc cggatcggcg attcctctca aggcgacaac 1080

aatctgcaga agctgggccc cgacgacgtg agcgtggata tcgacgccat ccggagagtg 1140

tacacaagac tgctgagcaa cgagaagatc gagaccgcct tcctgaacgc cctggtgtat 1200

ctgagcccta atgtggagtg cgatctgacc taccacaacg tgtactcccg ggacccaaac 1260

tacctgaatc tgttcatcat cgtgatggag aacagaaatc tgcactcccc cgagtatctg 1320

gagatggccc tgcctctgtt ttgtaaggcc atgtccaagc tgcctctggc agcacagggc 1380

aagctgatca ggctgtggtc taagtacaac gccgatcaga tcaggcgcat gatggagacc 1440

ttccagcagc tgatcacata caaagtgatc tctaacgagt ttaatagccg caacctggtg 1500

aacgacgacg acgccatcgt ggccgcctct aagtgcctga agatggtgta ctacgccaac 1560

gtggtgggcg gcgaggtgga cacaaaccac aacgaggagg acgacgagga gccaatcccc 1620

gagagctccg agctgaccct gcaggagctg ctgggagagg agcggagaaa taagaaggga 1680

ccaagggtgg atcctctgga gaccgagctg ggcgtgaaga cactggactg cagaaagcct 1740

ctgatcccat tcgaggagtt tatcaacgag cccctgaacg aggtgctgga gatggataag 1800

gactacacct tctttaaggt ggagacagag aacaagttca gctttatgac ctgtcctttc 1860

atcctgaacg ccgtgaccaa gaatctgggc ctgtactacg ataacaggat ccgcatgtac 1920

tccgagaggc gcatcaccgt gctgtattct ctggtgcagg gccagcagct gaatccttac 1980

ctgaggctga aggtgcggag agaccacatc atcgatgacg ccctggtgcg cctggagatg 2040

atcgccatgg agaatccagc cgatctgaag aagcagctgt acgtggagtt tgagggagag 2100

cagggagtgg acgagggagg cgtgtctaag gagttctttc agctggtggt ggaggagatc 2160

ttcaaccccg atatcggcat gtttacctac gacgagagca caaagctgtt ctggtttaat 2220

ccttcttcct tcgagaccga gggccagttt acactgatcg gcatcgtgct gggcctggcc 2280

atctacaaca attgtatcct ggacgtgcac ttcccaatgg tggtgtatag gaagctgatg 2340

ggcaagaagg gcacctttcg cgatctgggc gactcccacc ccgtgctgta ccagtctctg 2400

aaggatctgc tggagtatga gggcaacgtg gaggatgaca tgatgatcac cttccagatc 2460

tcccagacag acctgtttgg caacccaatg atgtacgatc tgaaggagaa cggcgacaag 2520

atccccatca caaacgagaa tagaaaggag ttcgtgaacc tgtacagcga ttatatcctg 2580

aataagtccg tggagaagca gttcaaggcc tttaggcgcg gcttccacat ggtgaccaac 2640

gagagccctc tgaagtatct gtttaggcca gaggagatcg agctgctgat ctgcggctcc 2700

cgcaatctgg actttcaggc cctggaggag accacagagt acgacggcgg ctataccagg 2760

gactctgtgc tgatccgcga gttctgggag atcgtgcaca gctttacaga cgagcagaag 2820

cggctgttcc tgcagtttac caccggcacc gacagagcac cagtgggagg actgggcaag 2880

ctgaagatga tcatcgccaa gaacggccca gacacagaga ggctgcccac cagccacacc 2940

tgtttcaacg tgctgctgct gcccgagtac tcctctaagg agaagctgaa ggagcgcctg 3000

ctgaaggcca tcacctacgc caagggcttt ggcatgctgt gatgaggtac cagtgaattc 3060

taccagtgcc ataggatagt gaattctacc agtgccatac acgtgagtga attctaccag 3120

tgccatagca tgcagtgaat tctaccagtg ccataggcgg ccgcttcgag cagacatgat 3180

aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat 3240

ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt 3300

taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt gggaggtttt 3360

ttaaagcaag taaaacctct acaaatgtgg taaaatcg 3398

<210> 23

<211> 3294

<212> DNA

<213> artificial sequence

<220>

<223> expression cassette hSyn.hUbe3a-1.GSco.SV40

<220>

<221> promoter

<222> (1)..(466)

<223> human synapsin promoter

<220>

<221> misc_feature

<222> (478)..(483)

<223> Kozak

<220>

<221> misc_feature

<222> (484)..(3045)

<223> UBE3A-1

<220>

<221> polyA_Signal

<222> (3063)..(3294)

<223> SV40 late polyA

<400> 23

ctgcagaggg ccctgcgtat gagtgcaagt gggttttagg accaggatga ggcggggtgg 60

gggtgcctac ctgacgaccg accccgaccc actggacaag cacccaaccc ccattcccca 120

aattgcgcat cccctatcag agagggggag gggaaacagg atgcggcgag gcgcgtgcgc 180

actgccagct tcagcaccgc ggacagtgcc ttcgcccccg cctggcggcg cgcgccaccg 240

ccgcctcagc actgaaggcg cgctgacgtc actcgccggt cccccgcaaa ctccccttcc 300

cggccacctt ggtcgcgtcc gcgccgccgc cggcccagcc ggaccgcacc acgcgaggcg 360

cgagataggg gggcacgggc gcgaccatct gcgctgcggc gccggcgact cagcgctgcc 420

tcagtctgcg gtgggcagcg gaggagtcgt gtcgtgcctg agagcgcagt cgaattcgcc 480

accatgaaga gggcagcagc aaagcacctg atcgagagat actatcacca gctgaccgag 540

ggatgcggaa acgaggcatg tacaaacgag ttctgcgcct cctgtcccac ctttctgagg 600

atggataaca acgccgccgc catcaaggcc ctggagctgt acaagatcaa cgccaagctg 660

tgcgaccccc accctagcaa gaagggcgcc agctccgcct atctggagaa ctccaagggc 720

gcccctaaca atagctgttc cgagatcaag atgaataaga agggcgcccg gatcgatttc 780

aaggacgtga cctacctgac agaggagaag gtgtacgaga tcctggagct gtgccgggag 840

agagaggatt acagcccact gatcagagtg atcggcagag tgttctctag cgccgaggcc 900

ctggtgcagt cctttagaaa ggtgaagcag cacacaaagg aggagctgaa gtctctgcag 960

gccaaggacg aggacaagga cgaggacgag aaggagaagg cagcctgttc tgccgcagca 1020

atggaggagg acagcgaggc atcctctagc cggatcggcg attcctctca aggcgacaac 1080

aatctgcaga agctgggccc cgacgacgtg agcgtggata tcgacgccat ccggagagtg 1140

tacacaagac tgctgagcaa cgagaagatc gagaccgcct tcctgaacgc cctggtgtat 1200

ctgagcccta atgtggagtg cgatctgacc taccacaacg tgtactcccg ggacccaaac 1260

tacctgaatc tgttcatcat cgtgatggag aacagaaatc tgcactcccc cgagtatctg 1320

gagatggccc tgcctctgtt ttgtaaggcc atgtccaagc tgcctctggc agcacagggc 1380

aagctgatca ggctgtggtc taagtacaac gccgatcaga tcaggcgcat gatggagacc 1440

ttccagcagc tgatcacata caaagtgatc tctaacgagt ttaatagccg caacctggtg 1500

aacgacgacg acgccatcgt ggccgcctct aagtgcctga agatggtgta ctacgccaac 1560

gtggtgggcg gcgaggtgga cacaaaccac aacgaggagg acgacgagga gccaatcccc 1620

gagagctccg agctgaccct gcaggagctg ctgggagagg agcggagaaa taagaaggga 1680

ccaagggtgg atcctctgga gaccgagctg ggcgtgaaga cactggactg cagaaagcct 1740

ctgatcccat tcgaggagtt tatcaacgag cccctgaacg aggtgctgga gatggataag 1800

gactacacct tctttaaggt ggagacagag aacaagttca gctttatgac ctgtcctttc 1860

atcctgaacg ccgtgaccaa gaatctgggc ctgtactacg ataacaggat ccgcatgtac 1920

tccgagaggc gcatcaccgt gctgtattct ctggtgcagg gccagcagct gaatccttac 1980

ctgaggctga aggtgcggag agaccacatc atcgatgacg ccctggtgcg cctggagatg 2040

atcgccatgg agaatccagc cgatctgaag aagcagctgt acgtggagtt tgagggagag 2100

cagggagtgg acgagggagg cgtgtctaag gagttctttc agctggtggt ggaggagatc 2160

ttcaaccccg atatcggcat gtttacctac gacgagagca caaagctgtt ctggtttaat 2220

ccttcttcct tcgagaccga gggccagttt acactgatcg gcatcgtgct gggcctggcc 2280

atctacaaca attgtatcct ggacgtgcac ttcccaatgg tggtgtatag gaagctgatg 2340

ggcaagaagg gcacctttcg cgatctgggc gactcccacc ccgtgctgta ccagtctctg 2400

aaggatctgc tggagtatga gggcaacgtg gaggatgaca tgatgatcac cttccagatc 2460

tcccagacag acctgtttgg caacccaatg atgtacgatc tgaaggagaa cggcgacaag 2520

atccccatca caaacgagaa tagaaaggag ttcgtgaacc tgtacagcga ttatatcctg 2580

aataagtccg tggagaagca gttcaaggcc tttaggcgcg gcttccacat ggtgaccaac 2640

gagagccctc tgaagtatct gtttaggcca gaggagatcg agctgctgat ctgcggctcc 2700

cgcaatctgg actttcaggc cctggaggag accacagagt acgacggcgg ctataccagg 2760

gactctgtgc tgatccgcga gttctgggag atcgtgcaca gctttacaga cgagcagaag 2820

cggctgttcc tgcagtttac caccggcacc gacagagcac cagtgggagg actgggcaag 2880

ctgaagatga tcatcgccaa gaacggccca gacacagaga ggctgcccac cagccacacc 2940

tgtttcaacg tgctgctgct gcccgagtac tcctctaagg agaagctgaa ggagcgcctg 3000

ctgaaggcca tcacctacgc caagggcttt ggcatgctgt gatgaggtac cggcggccgc 3060

ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt 3120

gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa 3180

gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg 3240

agatgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa atcg 3294

<210> 24

<211> 3467

<212> DNA

<213> artificial sequence

<220>

<223> expression cassette hSyn. HUbe3a-2.GSco.4XmiRNA183.SV40

<220>

<221> promoter

<222> (1)..(466)

<223> human synaptoprotein

<220>

<221> misc_feature

<222> (478)..(483)

<223> Kozak

<220>

<221> misc_feature

<222> (484)..(3114)

<223> hUBE3A-2

<220>

<221> misc_feature

<222> (3121)..(3142)

<223> miRNA183

<220>

<221> misc_feature

<222> (3147)..(3168)

<223> miRNA183

<220>

<221> misc_feature

<222> (3175)..(3196)

<223> miRNA183

<220>

<221> misc_feature

<222> (3203)..(3224)

<223> miRNA183

<220>

<221> polyA_Signal

<222> (3236)..(3467)

<223> SV40 late polyA

<400> 24

ctgcagaggg ccctgcgtat gagtgcaagt gggttttagg accaggatga ggcggggtgg 60

gggtgcctac ctgacgaccg accccgaccc actggacaag cacccaaccc ccattcccca 120

aattgcgcat cccctatcag agagggggag gggaaacagg atgcggcgag gcgcgtgcgc 180

actgccagct tcagcaccgc ggacagtgcc ttcgcccccg cctggcggcg cgcgccaccg 240

ccgcctcagc actgaaggcg cgctgacgtc actcgccggt cccccgcaaa ctccccttcc 300

cggccacctt ggtcgcgtcc gcgccgccgc cggcccagcc ggaccgcacc acgcgaggcg 360

cgagataggg gggcacgggc gcgaccatct gcgctgcggc gccggcgact cagcgctgcc 420

tcagtctgcg gtgggcagcg gaggagtcgt gtcgtgcctg agagcgcagt cgaattcgcc 480

accatggaga agctgcacca gtgctactgg aagtctggcg agcctcagag cgacgatatc 540

gaggcaagca ggatgaagag agcagcagcc aagcacctga tcgagcggta ctatcaccag 600

ctgaccgagg gctgcggaaa cgaggcctgt acaaacgagt tctgcgcctc ctgtccaacc 660

tttctgagaa tggataacaa cgccgccgcc atcaaggccc tggagctgta caagatcaac 720

gccaagctgt gcgaccccca cccttctaag aagggcgcca gctccgccta tctggagaac 780

agcaagggcg cccccaacaa tagctgttcc gagatcaaga tgaataagaa gggcgccagg 840

atcgatttca aggacgtgac ctacctgaca gaggagaagg tgtacgagat cctggagctg 900

tgccgggaga gagaggatta ctcccctctg atcagagtga tcggacgcgt gttctctagc 960

gccgaggccc tggtgcagag ctttcgcaag gtgaagcagc acacaaagga ggagctgaag 1020

tccctgcagg ccaaggacga ggacaaggac gaggacgaga aggagaaggc agcttgttcc 1080

gccgcagcaa tggaggagga ctctgaggcc tcctctagca ggatcggcga ttcctctcaa 1140

ggcgacaaca atctgcagaa gctgggcccc gacgacgtga gcgtggatat cgacgccatc 1200

cggagagtgt acacacgcct gctgtctaac gagaagatcg agaccgcctt cctgaacgcc 1260

ctggtgtatc tgtctcctaa tgtggagtgc gatctgacct accacaacgt gtacagccgg 1320

gacccaaact acctgaatct gttcatcatc gtgatggaga acagaaatct gcactctccc 1380

gagtatctgg agatggccct gcctctgttt tgtaaggcca tgagcaagct gccactggca 1440

gcacagggca agctgatcag gctgtggtcc aagtacaacg ccgatcagat caggcgcatg 1500

atggagacct tccagcagct gatcacatat aaagtgatct ccaacgagtt taattctaga 1560

aacctggtga acgacgacga cgccatcgtg gccgccagca agtgcctgaa gatggtgtac 1620

tacgccaacg tggtgggcgg cgaggtggac acaaaccaca acgaggagga cgacgaggag 1680

ccaatccccg agagctccga gctgaccctg caggagctgc tgggagagga gcggagaaat 1740

aagaagggac caagggtgga tcctctggag accgagctgg gcgtgaagac actggactgc 1800

agaaagcctc tgatcccatt cgaggagttt atcaacgagc ccctgaacga ggtgctggag 1860

atggataagg actacacctt ctttaaggtg gagacagaga acaagttcag ctttatgacc 1920

tgtcctttca tcctgaacgc cgtgaccaag aatctgggcc tgtactacga taacaggatc 1980

cgcatgtaca gcgagaggcg catcaccgtg ctgtattccc tggtgcaggg ccagcagctg 2040

aatccttacc tgaggctgaa ggtgcggaga gaccacatca tcgatgacgc cctggtgcgc 2100

ctggagatga tcgccatgga gaatccagcc gatctgaaga agcagctgta cgtggagttt 2160

gagggagagc agggagtgga cgagggaggc gtgtccaagg agttctttca gctggtggtg 2220

gaggagatct tcaaccccga tatcggcatg tttacctacg acgagtctac aaagctgttc 2280

tggtttaatc cttcttcctt cgagaccgag ggccagttta cactgatcgg catcgtgctg 2340

ggcctggcca tctacaacaa ttgtatcctg gacgtgcact tcccaatggt ggtgtatagg 2400

aagctgatgg gcaagaaggg cacctttcgc gatctgggcg acagccaccc cgtgctgtac 2460

cagtccctga aggatctgct ggagtatgag ggcaacgtgg aggatgacat gatgatcacc 2520

ttccagatct cccagacaga cctgtttggc aacccaatga tgtacgatct gaaggagaac 2580

ggcgacaaga tccccatcac aaacgagaat aggaaggagt tcgtgaacct gtactctgat 2640

tatatcctga ataagagcgt ggagaagcag ttcaaggcct ttaggcgcgg cttccacatg 2700

gtgaccaacg agtctcctct gaagtatctg tttaggccag aggagatcga gctgctgatc 2760

tgcggcagcc gcaatctgga ctttcaggcc ctggaggaga ccacagagta cgacggcggc 2820

tatacccggg actccgtgct gatcagagag ttctgggaga tcgtgcactc ttttacagac 2880

gagcagaagc ggctgttcct gcagtttacc accggcaccg acagagcacc agtgggagga 2940

ctgggcaagc tgaagatgat catcgccaag aacggcccag acacagagag gctgcccacc 3000

agccacacct gtttcaacgt gctgctgctg cccgagtact cctctaagga gaagctgaag 3060

gagcgcctgc tgaaggccat cacctacgcc aagggctttg gcatgctgtg atgaggtacc 3120

agtgaattct accagtgcca taggatagtg aattctacca gtgccataca cgtgagtgaa 3180

ttctaccagt gccatagcat gcagtgaatt ctaccagtgc cataggcggc cgcttcgagc 3240

agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa 3300

atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa 3360

taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggagatgtg 3420

ggaggttttt taaagcaagt aaaacctcta caaatgtggt aaaatcg 3467

<210> 25

<211> 3363

<212> DNA

<213> artificial sequence

<220>

<223> expression cassette hSyn.hUbe3a-2.GSco.SV40

<220>

<221> promoter

<222> (1)..(466)

<223> human synaptoprotein

<220>

<221> misc_feature

<222> (478)..(483)

<223> Kozak

<220>

<221> misc_feature

<222> (484)..(3114)

<223> hUBE3A-2

<220>

<221> polyA_Signal

<222> (3132)..(3363)

<223> SV40 PolyA

<400> 25

ctgcagaggg ccctgcgtat gagtgcaagt gggttttagg accaggatga ggcggggtgg 60

gggtgcctac ctgacgaccg accccgaccc actggacaag cacccaaccc ccattcccca 120

aattgcgcat cccctatcag agagggggag gggaaacagg atgcggcgag gcgcgtgcgc 180

actgccagct tcagcaccgc ggacagtgcc ttcgcccccg cctggcggcg cgcgccaccg 240

ccgcctcagc actgaaggcg cgctgacgtc actcgccggt cccccgcaaa ctccccttcc 300

cggccacctt ggtcgcgtcc gcgccgccgc cggcccagcc ggaccgcacc acgcgaggcg 360

cgagataggg gggcacgggc gcgaccatct gcgctgcggc gccggcgact cagcgctgcc 420

tcagtctgcg gtgggcagcg gaggagtcgt gtcgtgcctg agagcgcagt cgaattcgcc 480

accatggaga agctgcacca gtgctactgg aagtctggcg agcctcagag cgacgatatc 540

gaggcaagca ggatgaagag agcagcagcc aagcacctga tcgagcggta ctatcaccag 600

ctgaccgagg gctgcggaaa cgaggcctgt acaaacgagt tctgcgcctc ctgtccaacc 660

tttctgagaa tggataacaa cgccgccgcc atcaaggccc tggagctgta caagatcaac 720

gccaagctgt gcgaccccca cccttctaag aagggcgcca gctccgccta tctggagaac 780

agcaagggcg cccccaacaa tagctgttcc gagatcaaga tgaataagaa gggcgccagg 840

atcgatttca aggacgtgac ctacctgaca gaggagaagg tgtacgagat cctggagctg 900

tgccgggaga gagaggatta ctcccctctg atcagagtga tcggacgcgt gttctctagc 960

gccgaggccc tggtgcagag ctttcgcaag gtgaagcagc acacaaagga ggagctgaag 1020

tccctgcagg ccaaggacga ggacaaggac gaggacgaga aggagaaggc agcttgttcc 1080

gccgcagcaa tggaggagga ctctgaggcc tcctctagca ggatcggcga ttcctctcaa 1140

ggcgacaaca atctgcagaa gctgggcccc gacgacgtga gcgtggatat cgacgccatc 1200

cggagagtgt acacacgcct gctgtctaac gagaagatcg agaccgcctt cctgaacgcc 1260

ctggtgtatc tgtctcctaa tgtggagtgc gatctgacct accacaacgt gtacagccgg 1320

gacccaaact acctgaatct gttcatcatc gtgatggaga acagaaatct gcactctccc 1380

gagtatctgg agatggccct gcctctgttt tgtaaggcca tgagcaagct gccactggca 1440

gcacagggca agctgatcag gctgtggtcc aagtacaacg ccgatcagat caggcgcatg 1500

atggagacct tccagcagct gatcacatat aaagtgatct ccaacgagtt taattctaga 1560

aacctggtga acgacgacga cgccatcgtg gccgccagca agtgcctgaa gatggtgtac 1620

tacgccaacg tggtgggcgg cgaggtggac acaaaccaca acgaggagga cgacgaggag 1680

ccaatccccg agagctccga gctgaccctg caggagctgc tgggagagga gcggagaaat 1740

aagaagggac caagggtgga tcctctggag accgagctgg gcgtgaagac actggactgc 1800

agaaagcctc tgatcccatt cgaggagttt atcaacgagc ccctgaacga ggtgctggag 1860

atggataagg actacacctt ctttaaggtg gagacagaga acaagttcag ctttatgacc 1920

tgtcctttca tcctgaacgc cgtgaccaag aatctgggcc tgtactacga taacaggatc 1980

cgcatgtaca gcgagaggcg catcaccgtg ctgtattccc tggtgcaggg ccagcagctg 2040

aatccttacc tgaggctgaa ggtgcggaga gaccacatca tcgatgacgc cctggtgcgc 2100

ctggagatga tcgccatgga gaatccagcc gatctgaaga agcagctgta cgtggagttt 2160

gagggagagc agggagtgga cgagggaggc gtgtccaagg agttctttca gctggtggtg 2220

gaggagatct tcaaccccga tatcggcatg tttacctacg acgagtctac aaagctgttc 2280

tggtttaatc cttcttcctt cgagaccgag ggccagttta cactgatcgg catcgtgctg 2340

ggcctggcca tctacaacaa ttgtatcctg gacgtgcact tcccaatggt ggtgtatagg 2400

aagctgatgg gcaagaaggg cacctttcgc gatctgggcg acagccaccc cgtgctgtac 2460

cagtccctga aggatctgct ggagtatgag ggcaacgtgg aggatgacat gatgatcacc 2520

ttccagatct cccagacaga cctgtttggc aacccaatga tgtacgatct gaaggagaac 2580

ggcgacaaga tccccatcac aaacgagaat aggaaggagt tcgtgaacct gtactctgat 2640

tatatcctga ataagagcgt ggagaagcag ttcaaggcct ttaggcgcgg cttccacatg 2700

gtgaccaacg agtctcctct gaagtatctg tttaggccag aggagatcga gctgctgatc 2760

tgcggcagcc gcaatctgga ctttcaggcc ctggaggaga ccacagagta cgacggcggc 2820

tatacccggg actccgtgct gatcagagag ttctgggaga tcgtgcactc ttttacagac 2880

gagcagaagc ggctgttcct gcagtttacc accggcaccg acagagcacc agtgggagga 2940

ctgggcaagc tgaagatgat catcgccaag aacggcccag acacagagag gctgcccacc 3000

agccacacct gtttcaacgt gctgctgctg cccgagtact cctctaagga gaagctgaag 3060

gagcgcctgc tgaaggccat cacctacgcc aagggctttg gcatgctgtg atgaggtacc 3120

ggcggccgct tcgagcagac atgataagat acattgatga gtttggacaa accacaacta 3180

gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa 3240

ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg 3300

ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa 3360

tcg 3363

Claims

1. A composition comprising a stock of recombinant adeno-associated virus (rAAV) useful for treating Angeman Syndrome (AS), the rAAV comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising:

(a) AAV 5' Inverted Terminal Repeats (ITRs);

(b) A UBE3A nucleic acid sequence comprising SEQ ID No. 9 or a sequence encoding a UBE3A isoform 1 protein (SEQ ID No. 2) which is at least 95% identical thereto, wherein said nucleic acid sequence is operably linked to regulatory elements which regulate the expression of a UBE3A protein in a human cell;

(c) A regulatory element that directly expresses UBE3A of (b); and

(d)AAV 3'ITR。

2. the composition of claim 1, wherein the regulatory element comprises a neuron-specific promoter.

3. The composition of claim 2, wherein the neuron-specific promoter is a synapsin promoter.

4. The composition of claim 3, wherein the synapsin promoter is a shortened promoter having the nucleic acid sequence of SEQ ID No. 12.

5. The composition of claim 1, wherein the regulatory element comprises a constitutive promoter.

6. The composition of any one of claims 1 to 5, wherein the regulatory element further comprises one or more enhancers and one or more introns.

7. The composition of any one of claims 1-6, wherein the regulatory sequences further comprise one or more target sequences for mirs selected from miR182 and/or miR183 in the dorsal root ganglion, the target sequences being operably linked to a UBE3A nucleic acid sequence.

8. The composition of claims 1-7, wherein the regulatory sequences further comprise one or more target sequences for a miR selected from miR182 and/or miR183 in the dorsal root ganglion, downstream of the UBE3A nucleic acid sequence.

9. The composition of claims 1-8, wherein the regulatory sequence further comprises four target sequences for miR183, the target sequences being downstream of the UBE3A nucleic acid sequence.

10. The composition of claims 1 to 9, wherein the regulatory sequence comprises four copies of SEQ ID No. 11.

11. The composition of any one of claims 1 to 10, wherein the AAV capsid is an AAVhu68 capsid.

12. The composition of any one of claims 1 to 10, wherein the AAV capsid is an AAVhu68 capsid resulting from expression of the nucleic acid sequence of SEQ ID No. 14 or SEQ ID No. 16.

13. The composition of any one of claims 1 to 10, wherein the AAV capsid is an AAVrh91 capsid.

14. The composition of any one of claims 1 to 10, wherein the AAV capsid is an AAVrh91 capsid resulting from expression of the nucleic acid sequence of SEQ ID No. 17 or SEQ ID No. 19.

15. The composition according to any one of claims 1 to 14, which is an aqueous suspension further comprising a physiologically compatible carrier, buffer, adjuvant and/or diluent.

16. Use of a composition according to any one of claims 1 to 15 for treating a patient suffering from angermann syndrome.

17. A composition according to any one of claims 1 to 15 for use in the treatment of one or more symptoms of angermann syndrome, optionally wherein the symptoms are selected from one or more of the following: developmental delay, mental disability, severe language disorder, ataxia and/or epilepsy.

18. A method of treating angemann syndrome, comprising administering to a patient in need thereof the composition of any one of claims 1 to 15.

19. A method for treating one or more symptoms of angermann syndrome in a patient having defective UBE3A expression in neurons, the method comprising delivering a composition according to any one of claims 1 to 15.

20. The method of claim 18 or 19, wherein the symptom is selected from one or more of: developmental delay, mental disability, severe language disorder, ataxia and/or epilepsy.

21. The composition of any one of claims 1 to 15, the use of claim 16 or 17, or the method of claims 18 to 20, wherein the composition is delivered intrathecally to the patient.

22. The composition of any one of claims 1 to 15, the use of claim 16 or 17, or the method of claims 18 to 20, wherein the patient is injected at least 1x 10 ¹⁰ To 1x 10 ¹³ GC/kg of the rAAV.