UBE3a for treatment of angelman syndrome

Abstract

To provide UBE3A for the treatment of Angelman syndrome.SOLUTION: Angelman syndrome is a genetic neurological disorder with characteristics including delayed development, intellectual disability, severe speech impairment, and problems with movement and balance. Provided herein are polynucleotides, vectors, polypeptides, cells, compositions, kits and methods to treat Angelman syndrome. Provided herein are, for example, the following: a recombinant polynucleotide encoding a Ubiquitin Protein Ligase E3A (Ube3a) polypeptide or protein or a biological equivalent thereof, the Ube3a polypeptide or protein or a biological equivalent thereof comprising one or more naturally occurring or non-naturally occurring glycosylation sites.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Nos. 62 / 890,364 and 62 / 945,062, filed August 22, 2019 and December 6, 2019, respectively, the contents of each of which are incorporated herein by reference in their entireties. [Background technology]

[0002] background Angelman syndrome (AS) is a genetic neurological disorder characterized by developmental delay, intellectual disability, severe speech impairment, and problems with movement and balance. Most patients have recurrent epileptic seizures and a smaller head size. Most patients exhibit developmental delay and other common symptoms that manifest in early childhood. Children with AS typically have a playful and excitable demeanor. Other symptoms include hyperactivity, a short attention span, and a strong fascination with water. As patients age, those with Angelman syndrome become less excitable and experience improvement in sleep problems. However, these patients continue to have lifelong intellectual disability, speech impairment, and seizures.

[0003] AS is caused by the loss of function of a gene called UBE3A. People inherit one copy of the UBE3A gene ("Ube3a") from each parent. Both copies of Ube3a are active in many tissues in the body. However, in the brain, only the maternal copy is active. This parent-specific activation of the gene is caused by a process called genomic imprinting. If the maternal copy of UBE3A is lost due to deletion or mutation, the person will lack expression of Ube3a in certain parts of the brain. There are no effective treatments available to treat AS. The present disclosure provides gene therapy to replace or reduce the symptoms and causes of AS. Summary of the Invention [Means for solving the problem]

[0004] Disclosure Overview Thus, in one aspect, provided herein are recombinant polynucleotides encoding ubiquitin-protein ligase E3A (Ube3a) proteins having one or more naturally occurring or non-naturally occurring glycosylation sites, e.g., for use in gene therapy and research. In one aspect, the Ube3a protein has one or more naturally occurring or non-naturally occurring glycosylation sites, or two or more naturally occurring or non-naturally occurring glycosylation sites, or three or more naturally occurring or non-naturally occurring glycosylation sites. In another aspect, the Ube3a protein has four or more naturally occurring or non-naturally occurring glycosylation sites. In another aspect, the Ube3a protein has five or more naturally occurring or non-naturally occurring glycosylation sites. In another aspect, the Ube3a protein has six or more naturally occurring or non-naturally occurring glycosylation sites. In one embodiment, the Ube3a protein has seven or more, or eight or more, naturally occurring or non-naturally occurring glycosylation sites. In one embodiment, the Ube3a protein has naturally occurring and non-naturally occurring glycosylation sites. In one embodiment, the Ube3a protein is non-naturally occurring as disclosed herein, or an equivalent or complement thereof. In one embodiment, the Ube3a protein is naturally occurring but contains one or more naturally occurring or non-naturally occurring glycosylation sites. In another embodiment, the protein is naturally occurring, but in each embodiment, the protein has one or more non-naturally occurring glycosylation sites. In one embodiment, the protein is a non-naturally occurring Ube3a protein or polypeptide having one or more non-naturally occurring glycosylation sites. Additionally or alternatively, one or more of the glycosylation sites are non-naturally occurring.

[0005] Also provided are Ube3a proteins having one or more glycosylation sites, or two or more glycosylation sites, or three or more glycosylation sites. In another embodiment, the Ube3a protein has four or more glycosylation sites. In another embodiment, the Ube3a protein has five or more glycosylation sites. In another embodiment, the Ube3a protein has six or more glycosylation sites. In one embodiment, the Ube3a protein has seven or more, or eight or more glycosylation sites. In one embodiment, the protein is non-naturally occurring and contains one or more glycosylation sites. In another embodiment, the protein is naturally occurring, but in each embodiment, the protein has one or more glycosylation sites. In one embodiment, the protein is a non-naturally occurring Ube3a protein or polypeptide having one or more non-naturally occurring glycosylation sites. Additionally or alternatively, one or more of the glycosylation sites are non-naturally occurring. In a further aspect, the protein further comprises a cell-penetration domain. In a still further aspect, the protein comprises a secretion signal. Additionally or alternatively, the protein further comprises a detectable or purification marker.

[0006] In one aspect, the protein is encoded by a polynucleotide provided herein, for example, in the Sequence Listing section of this document, or its complement or equivalent, and their respective equivalents. In one aspect, the polynucleotide can also optionally include a polynucleotide that is not naturally occurring and encodes a cell-penetrating domain.

[0007] In a further embodiment, the polynucleotide further comprises a promoter operably linked to the polynucleotide. Non-limiting examples of such promoters include pol II promoters selected from the group consisting of MNDU3 promoter, CMV promoter, PGK promoter, and EF1 alpha promoter. The promoter can be operably linked to a coding polynucleotide to drive expression in a suitable host system. In a further embodiment, the polynucleotide further comprises a polynucleotide encoding a secretion signal positioned 5' to the polynucleotide encoding the modified Ube3a protein. Non-limiting examples of secretion signals include a single-chain fragment variable secretion signal, a twin-arginine transport protein secretion signal, an IL-4 secretion signal, an IL-2 secretion signal, and an IL-10 secretion signal. Exemplary secretion signal polynucleotides, along with their equivalents, are listed below. The polynucleotide can further comprise a polynucleotide that is or encodes a detectable or purification marker.

[0008] Also provided are vectors containing the recombinant polynucleotides described herein. Vectors include vectors for expression in prokaryotic and eukaryotic host cell systems, such as plasmids or viral vectors, such as baculovirus, retrovirus, adenovirus, AAV, or lentivirus vectors. Exemplary vector maps are shown in Figures 3A-3D. In some embodiments, the recombinant polynucleotide is flanked by inverted terminal repeats (ITRs) of a viral vector, such as an adenovirus or lentivirus vector.

[0009] Non-viral vectors can include plasmids containing heterologous polynucleotides that can be delivered to target cells either in vitro, in vivo, or ex vivo. The heterologous polynucleotide can include a mutated Ube3a gene (such as a recombinant polynucleotide as disclosed herein) and can be operably linked to one or more regulatory elements that can control the transcription of the mutated Ube3a gene. As used herein, a vector need not be capable of replication in the final target cell or subject. The term vector can include expression vectors and cloning vectors. In one embodiment, the vector is a pCCLc plasmid vector.

[0010] The polynucleotide and vector can be contained in a host cell system for delivery or expression of the polynucleotide. The cell can be a prokaryotic or eukaryotic cell. In one embodiment, the host cell is a mammalian cell, such as a canine, feline, bovine, equine, murine, rat, or human. The mammalian cell can be selected from stem cells, such as induced pluripotent stem cells (iPSCs), embryonic stem cells, adult or somatic stem cells. In one embodiment, the stem cell is a mesenchymal stem cell, such as a hematopoietic stem cell or a neuronal stem cell. In another embodiment, the stem cell is a mesenchymal stem cell, optionally identified by expressing the CD34+ marker.

[0011] Populations of cells, which can be heterogeneous (of different species or with different vectors and polynucleotides) or substantially homogeneous and clonal, are also provided.

[0012] Further provided is a composition comprising one or more of the polynucleotides, proteins or polypeptides, vectors, host cells or populations described herein and a carrier. In one aspect, the carrier is a pharmaceutically acceptable carrier.

[0013] Also provided is a viral packaging system comprising (a) a viral vector as described herein, (b) a packaging plasmid, and (c) an envelope plasmid. In a further aspect, the system further comprises (d) a packaging cell line, such as, for example, a HEK-293 cell line. The packaging system can be used to transduce the packaging cell line under conditions suitable for packaging the viral vector.

[0014] Also provided are methods for expressing secreted modified Ube3a protein, comprising, alternatively, consisting essentially of, or even consisting of, growing host cells as described herein under conditions that allow expression of the Ube3a protein. The methods can be performed in vitro, ex vivo, or in vivo. Further provided are methods for expressing modified Ube3a in a subject, comprising, alternatively, consisting essentially of, or even consisting of, administering to the subject an effective amount of a vector or host cell as described herein, thereby expressing modified Ube3a in the subject. In one aspect, the subject is a mammal, e.g., a human patient. In a further aspect, the subject lacks the Ube3a gene or possesses a defective Ube3a gene. In another aspect, the mammal is asymptomatic for Angelman syndrome. In another aspect, the subject is a fetus, infant, or prepubertal subject, who may or may not be symptomatic for Angelman syndrome (also referred to herein as AS). In additional embodiments, the subject is an adult, optionally an adult, further optionally an adult over the age of 18.

[0015] Further provided are methods for treating Angelman syndrome in a subject carrying a defective Ube3a gene or allele, e.g., comprising, alternatively consisting essentially of, or even consisting of, administering to the subject an effective amount of one or more of a polynucleotide, vector, host cell, or Ube3a protein or polypeptide as described herein, thereby treating Angelman syndrome. In a further aspect, the subject lacks the Ube3a gene or carries a defective Ube3A gene. In one aspect, the subject is a mammal, e.g., a human patient. In one aspect, the mammal is symptomatic for Angelman syndrome. In another aspect, the mammal is asymptomatic for Angelman syndrome. In another aspect, the subject is a fetus, infant, or prepubertal subject, who may or may not be symptomatic for AS. In an additional aspect, the subject is an adult, optionally an adult, and further optionally an adult over the age of 18.

[0016] Further provided are kits comprising one or more of the polynucleotides, proteins or polypeptides, vectors, host cells or populations as described herein and, optionally, instructions for use.

[0017] The foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the present disclosure as claimed. Other objects, advantages and novel features will become readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the present disclosure. [Brief explanation of the drawings]

[0018] [Figure 1]Figures 1A-1F show schematic diagrams of Ube3a-expressing lentiviral vectors. Figure 1A depicts the use of a self-inactivating lentiviral vector, the CCLc-X backbone. This is the parent vector lacking a transgene expressing a Ube3a polynucleotide. An exemplary sequence for the CCLc-MNDU3-X vector is presented as SEQ ID NO: 35. Figure 1B depicts an EGFP vector used as an empty vector control. Figure 1C shows modified mouse Ube3a isoform 3 cloned under the control of the MNDU3 promoter. EGFP was cloned downstream under the control of the PGK promoter. Figure 1D shows modified human isoform 1 cloned under the control of the MNDU3 promoter. EGFP was cloned downstream under the control of the PGK promoter. Figure 1E shows modified human isoform 1 cloned under the control of the MNDU3 promoter without an EGFP reporter gene. Figure 1F shows the exemplary vector of Figure 1E, indicating the secretion signal (ss) within it.

[0019] [Figure 2] Figure 2 shows CFU assays of Ube3a vector-transduced human CD34+ HPCs. Human CD34+ HPCs were either left untransduced (NT, left column of each bar group) or transduced with either an EGFP-only control vector (EGFP, center column of each bar group) or a Ube3a vector (Ube3a, right column of each bar group). Cells were then cultured in methylcellulose medium for 12 days, at which time specific colonies (BFU-E, GM, and GEMM) were counted.

[0020] [Figure 3]Figures 3A-3B show the overexpression and ubiquitination activity of the Ube3a lentivector. Human CD34+ HPCs were transduced with the Ube3a lentivector (Ube3a) and induced to mature macrophages. Cell extracts were then generated and assessed for Ube3a overexpression (Figure 3A) and for the ubiquitination activity of Ube3a S5a target proteins (Figure 3B) by Western blot. Control non-transduced (NT) and EGFP vector alone (EGFP) transduced cells were used as controls. Figure 3B further demonstrates that different mutant forms of Ube3a ubiquitinate their S5a targets. "AS native" indicates that the non-secreted wild-type form of Ube3a naturally found in cells was used as the test sample. "AS WT" indicates that the wild-type form of Ube3a with the addition of an IL-2 secretion signal was used as the test sample. "AS4" indicates that a mutant form of Ube3a with 4x mutation sites to create 4x potential glycosylation sites in addition to an IL-2 secretion signal was used as the test sample. "AS8" indicates that a mutant form of Ube3a with 8x mutation sites to create 8x potential glycosylation sites in addition to an IL-2 secretion signal was used as the test sample. "Ubiquitinated S5A" indicates the band of the ubiquitinated form of S5A, whose molecular weight increases with increasing ubiquitination.

[0021] [Figure 4] FIG. 4 shows an exemplary study design for phenotypic correction in neonatal mice.

[0022] [Figure 5-1]Figures 5A-5H show that locomotor activity, balance, motor coordination, and gait were measured by a tailored motor behavioral battery. Critical assessment of the motor translational phenotype using four standard motor behavioral tests was performed in treated and untreated Ube3a mice irradiated and transplanted as pups with either non-transduced (NT Het, open dots in Figure 5B, dots with a filled left half in other panels) or Ube3a lentivector-transduced (Ube3a Het, dashed lines in Figure 5B, or open dots in other panels) human CD34+ HSCs. Wild-type mice (WT, black dots) were used as controls. Eight weeks after transplantation, mice were subjected to open-field locomotor activity (Figure 5A, horizontal; Figure 5B, vertical; and Figure 5C, total activity), balance beam (Figure 5D and Figure 5E), rotarod (Figure 5F), and treadmill walking and DigiGait analysis (Figure 5G and Figure 5H). In all tests, Ube3a-deficient mice (Ube3a Het) transplanted with Ube3a vector-transduced human CD34+ HSCs exhibited wild-type performance values. Open-field activity increased by both total distance and horizontal activity metrics. Going from rod #1 to rod #2 to rod #3, the balance beam decreased in width, making it more difficult to traverse. The latency to fall from the rotarod was significantly improved in Ube3a-deficient mice (Ube3a Het) transplanted with Ube3a vector-transduced human CD34+ HSCs compared with non-transplanted (NT Het) cell controls. In AS patients, standard AS mice and novel Ube3a mice also exhibit abnormally wide stances. DigiGait analysis showed a narrowing of these wide stances in the treatment groups. *=p<0.05. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above.

[0023] [Figure 6]Figures 6A-6B provide results from a novel object recognition assay. Eleven weeks after transplantation, mice were assessed for learning and memory ability using a novel object recognition test. Figure 6A provides an assessment of the time spent sniffing a novel and familiar object. Figure 6B provides the results of a familiarization test with two identical (familiar) objects. Neonatal BGU mice were transplanted with either non-transduced (NT-HET) or Ube3a vector-transduced (Ube3a-HET) human CD34+ HSCs. Wild-type mice (WT) and non-transplanted BGU mice (HET) were used as controls. Ube3a-HETs showed intact object recognition similar to WTs, while NT-HETs spent no more time with the novel object, indicating a lack of recognition memory. *p<0.05, novel vs. familiar.

[0024] [Figure 7-1] Figures 7A-7B show rescue of elevated delta power detected in Ube3a-HET mice (Figure 7A) compared to human EEG (Figure 7B, reproduced from Anderson, BCM, 2017). Surface EEG was collected in mice using a radiotelemetry device and analyzed for spectral power differences. HET and NT-HET animals show a delta power peak that is absent in either WT or Ube3a-HET-treated animals. The elevated delta phenotype observed in HET animals recapitulates the elevated delta seen clinically, and re-expression of UBE3A rescued it to WT levels. *p<0.0001. [Figure 7-2] Same as above.

[0025] [Figure 8] Figure 8 shows Ube3a expression in the brains of mice transplanted with Ube3a vector-transduced cells. WT, Ube3a- / + BGU (HET), and Ube3a- / + (Ube3a-HET) mice transplanted with Ube3a vector-transduced cells were euthanized and analyzed for Ube3a expression using DAB peroxidase substrate and anti-Ube3a antibody. Brightfield immunohistochemically stained slides were scanned to determine relative Ube3a expression. *p=0.0126.

[0026] [Figure 9] Figures 9A-9C show the engraftment and development of human T cells in NRG mice. Human CD34+ HSCs were left untransduced or transduced with either EGFP control (EGFP) or Ube3a-expressing (hAS8-EGFP) lentiviral vectors. Cells were transplanted into 2- to 5-day-old NRG mice. 16 weeks after transplantation, mice were euthanized, and human T cells were analyzed for CD3, CD4, and CD8 expression in (Figure 9A) peripheral blood, (Figure 9B) spleen, and (Figure 9C) thymus.

[0027] [Figure 10] Figures 10A-10B show human B cell engraftment and development in NRG mice: Human CD34+ HSCs were left untransduced or transduced with either EGFP control (EGFP) or Ube3a-expressing (hAS8-EGFP) lentiviral vectors. Cells were transplanted into 2- to 5-day-old NRG mice. 16 weeks after transplantation, mice were euthanized, and human B cells were analyzed for CD45 and CD19 in (Figure 10A) the spleen and (Figure 10B) the bone marrow.

[0028] [Figure 11] Figure 11 shows the engraftment and development of human macrophages and CD34+ cells in the bone marrow of NRG mice. Human CD34+ HSCs were left untransduced or transduced with either EGFP control (EGFP) or Ube3a-expressing (hAS8-EGFP) lentiviral vectors. Cells were transplanted into 2- to 5-day-old NRG mice. 16 weeks after transplantation, mice were euthanized, and human macrophages and CD34+ cells were analyzed for engraftment in the bone marrow.

[0029] [Figure 12] FIG. 12 shows an exemplary study design for phenotypic correction in adult mice.

[0030] [Figure 13-1]Figures 13A-13G show that locomotor activity, balance, motor coordination, and gait were measured using a tailored motor behavior battery in adult BGU mice transplanted with Ube3a lentiviral vector-transduced human CD34+ HSCs. Four standard motor behavior tests were used to critically assess the translational phenotype of motor activity in treated and untreated Ube3a mice irradiated and transplanted at adulthood with either non-transduced (NT-HET) or Ube3a lentivector (Ube3a-HET)-transduced human CD34+ HSCs. Four- to six-week-old mice were conditioned with busulfan and transplanted intravenously. Six weeks later, mice were subjected to open-field locomotor activity (Figure 13A, horizontal; Figure 13B, vertical, and Figure 13C, total activity), balance beam (Figures 13D and 13E), rotarod (Figure 13F), and treadmill walking (Figure 13G). In all tests, Ube3a-deficient mice transplanted with Ube3a vector-transduced human CD34+ HSCs (Ube3a-HET) exhibited wild-type performance values. Open-field activity increased by both total distance and horizontal activity metrics. Going from rod #1 to rod #2 to rod #3, the balance beam decreased in width, making it more difficult to traverse. Wild-type mice (WT) were used as controls. Latency to fall from the rotarod was significantly improved in Ube3a-deficient mice transplanted with Ube3a vector-transduced human CD34+ HSCs (Ube3a-HET) compared with non-transduced (NT-HET) cells and non-transplanted (HET) controls. In AS patients, standard AS mice and novel Ube3a mice also exhibit abnormally wide stances. DigiGait analysis showed a narrowing of these wide stances in the treatment groups. *=p<0.05. [Figure 13-2] Same as above. [Figure 13-3] Same as above.

[0031] [Figure 14]Figures 14A-14B show a novel object recognition assay using BGU mice transplanted with Ube3a lentiviral vector-transduced human CD34+ HSCs. Six weeks after transplantation in adult mice, subjects were assessed for learning and memory ability using a novel object recognition test. Figure 14A shows the assessment of the time spent sniffing a novel and familiar object. Figure 14B provides the results of a familiarization test with two identical (familiar) objects. Ube3a HET mice were transplanted with either non-transduced (NT Het) or Ube3a vector-transduced (Ube3a Het) human CD34+ HSCs or left untransplanted (HET). Wild-type mice (WT) were used as controls. Transplanted HETs (Ube3a-HET) showed intact object recognition similar to WTs, while NT-HETs and HETs did not spend more time on the novel object, indicating a lack of recognition memory. *p<0.05, novel vs. familiar.

[0032] [Figure 15] Figure 15 shows Ube3a expression in the brains of Ube3a- / + adult mice. WT, Ube3a- / + BGU (HET), and Ube3a- / + mice transplanted with non-transduced (NT-HET) or Ube3a vector-transduced (Ube3a-HET) cells were euthanized and analyzed for Ube3a expression using DAB peroxidase substrate and anti-Ube3a antibody. Brightfield immunohistochemically stained slides were scanned, and Ube3a-positive cells were counted. *p=0.0058.

[0033] [Figure 16-1]Figures 16A-16B provide examples of several glycosylation sites based on a nucleotide fragment (as reproduced in SEQ ID NO: 31) of Homo sapiens ubiquitin protein ligase E3A (UBE3A), transcript variant 5, having an NCBI reference number of NM_001354506, and its translated amino acid sequence (SEQ ID NOs: 32 and 33). The amino acid sequence numbering is based on SEQ ID NO: 8, a fragment of SEQ ID NO: 32. Non-capitalized amino acid residues provide N-glycosylation sites based on an online tool named NetNGlyC. Bold, italicized, and shaded amino acid residues provide examples of S- or T-containing sites, where mutation of the second amino acid at the N-terminus to N results in a potential glycosylation site. Underlined amino acid residues provide examples of N-containing sites, where mutation of the second amino acid at the C-terminus to S / T results in a potential glycosylation site. The boxed amino acid residues indicate some exemplary glycosylation sites that can be created by the mutations identified herein. The coding sequence (CDS) of the nucleotide fragment begins with a start codon (marked as a shaded ATG) and ends with a stop codon (marked as a shaded TAA). The remaining shaded nucleotide residues provide potential mutation sites for creating glycosylation sites. Figure 16B further illustrates how mutations to form glycosylation sites can be made in two fragments of the amino acid sequence shown in Figure 16A. The top fragment is amino acid (aa) 201 to aa220 of SEQ ID NO:32, i.e., aa183 to aa202 of SEQ ID NO:8, while the bottom fragment is aa341 to aa360 of SEQ ID NO:32, i.e., aa323 to aa342 of SEQ ID NO:8. [Figure 16-2] Same as above. [Figure 16-3] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0034] Detailed Description definition It is understood that section or subsection headings, when used herein, are for organizational purposes only and should not be construed as limiting and / or separating the subject matter described.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, preferred methods, devices and materials are described herein. All technical publications and patent disclosures cited herein are incorporated herein by reference in their entirety. Nothing in this specification should be construed as an admission that the present disclosure is not entitled to antedate such disclosure based on prior disclosure.

[0036] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3 rd edition;the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology;the series Methods in Enzymology (Academic Press, Inc., NY);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press);MacPherson et al. (1995) PCR 2: A Practical 方法;哈洛和莱恩编(1999年)《抗体,实验室手册》;弗雷什尼(2005年)《动物细胞培养:基本技术手册》,第5版;盖特编 th (1984年)《寡核苷酸合成》;美国专利第4,683,195号;哈姆斯和希金斯编(1984年)《核酸杂交》;安德森(1999年)《核酸杂交》;哈姆斯和希金斯编(1984年)《转录与翻译》;《固定化细胞与酶》(IRL出版社(1986年));佩尔巴尔(1984年)《分子克隆实用指南》;米勒和卡洛编(1987年)《哺乳动物细胞基因转移载体》(冷泉港实验室);马克里德斯编(2003年)《哺乳动物细胞中的基因转移与表达》;迈耶和沃克编(1987年)《细胞与分子生物学中的免疫化学方法》(伦敦学术出版社);赫岑伯格 等人编(1996年)《韦尔实验免疫学手册》;《操作小鼠胚胎:实验室手册》,第3版 rd ​See, for example, the following publication: (Cold Spring Harbor Laboratory Press (2002)); Sohail (ed.) (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press).

[0037] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations that are varied (+) or (-) by increments of 0.1 or 1.0, as appropriate. It is understood, although not always explicitly stated, that all numerical designations are preceded by the term "about." It is also understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0038] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0039] As used herein, the terms "comprising" or "comprises" are intended to mean that the compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements of any essential importance to the combination for the described purpose. Thus, a composition consisting essentially of elements as defined herein does not exclude trace contaminants from isolation and purification methods, as well as pharmaceutically acceptable carriers, e.g., phosphate-buffered saline, preservatives, and the like. "Consisting of" is intended to mean more than trace elements of other components, and excluding substantial method steps for administering a composition of the present disclosure, or process steps for producing the composition or achieving an intended result. Embodiments defined by each of these transitional phrases are within the scope of the present disclosure.

[0040] "Optionally" or "as needed" means that the situation described after the word may or may not occur, and so the description includes instances where the situation occurs and instances where the situation does not occur.

[0041] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as either / or.

[0042] "Substantially" or "essentially" means almost entirely or completely, e.g., 95% or greater of a given quantity. In some embodiments, "substantially" or "essentially" means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0043] The term "isolated," as used herein with respect to nucleic acids, such as DNA or RNA, refers to a molecule separated from other DNAs or RNAs, respectively, present in the natural source of the macromolecule. The term "isolated nucleic acid" is intended to include nucleic acid fragments that do not naturally occur as fragments and are not found in their natural context. The term "isolated" is also used herein to refer to polypeptides, proteins, and / or host cells isolated from other cellular proteins and is intended to encompass both purified and recombinant polypeptides. In other embodiments, the term "isolated" means separated from cellular and other types of components with which a cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s) thereof is normally associated in nature. For example, an isolated cell is a cell separated from tissues or cells of a different phenotype or genotype. As will be apparent to one of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s) thereof does not require "isolation" to distinguish it from its naturally occurring counterpart.

[0044] In some embodiments, the term "engineered" or "recombinant" refers to having at least one modification that is not normally found in a naturally occurring protein, polypeptide, polynucleotide, strain, wild-type strain, or parent host strain of a reference species. In some embodiments, the term "engineered" or "recombinant" refers to being synthesized by human intervention.

[0045] As known to those skilled in the art, there are six classes of viruses. DNA viruses make up classes I and II. RNA viruses and retroviruses make up the remaining classes. Class III viruses have a double-stranded RNA genome. Class IV viruses have a positive-sense single-stranded RNA genome, with the genome itself acting as mRNA. Class V viruses have a negative-sense single-stranded RNA genome that is used as a template for mRNA synthesis. Class VI viruses have a positive-sense single-stranded RNA genome, but have DNA intermediates not only in replication but also in mRNA synthesis. Retroviruses carry their genetic information in the form of RNA; however, when the virus infects a cell, the RNA is reverse-transcribed into a DNA form, which is integrated into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

[0046] The terms "polynucleotide," "nucleic acid," and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the present disclosure that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0047] A polynucleotide is composed of a specific sequence of the four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and, if the polynucleotide is RNA, uracil (U) in place of thymine. Thus, the term "polynucleotide sequence" refers to the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into a database in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.

[0048] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which can be aligned for comparison purposes. If a position in the sequences being compared is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.

[0049] As used herein, an amino acid (aa) or nucleotide (nt) residue position in a sequence of interest that "corresponds to" an identified position in a reference sequence refers to the residue position being aligned with the identified position in a sequence alignment between the sequence of interest and the reference sequence. Various programs are available for performing such sequence alignments, such as Clustal Omega and BLAST.

[0050] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" to another sequence, meaning that, when aligned, that percentage of bases (or amino acids) are the same when comparing the two sequences. This alignment and percent homology or sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for the alignment. One alignment program is BLAST using default parameters. In particular, the programs are BLASTN and BLASTP using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; matrix=BLOSUM62; description=50 sequences; sorting=by high score; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following internet address: http: / / www.ncbi.nlm.nih.gov / cgi-bin / BLAST. In some embodiments, a polynucleotide as disclosed herein is RNA. In some embodiments, a polynucleotide as disclosed herein is DNA. In some embodiments, a polynucleotide as disclosed herein is a hybrid of DNA and RNA.

[0051] In some embodiments, the equivalent of a reference nucleic acid, polynucleotide, or oligonucleotide encodes the same sequence encoded by the reference. In some embodiments, the equivalent of a reference nucleic acid, polynucleotide, or oligonucleotide hybridizes to the reference, complement reference, reverse reference, and / or reverse complement reference, optionally under high stringency conditions.

[0052] Additionally or alternatively, an equivalent nucleic acid, polynucleotide, or oligonucleotide has at least 70%, or at least 75%, or at least 80% sequence identity to a reference nucleic acid, polynucleotide, or oligonucleotide, or alternatively at least 85% sequence identity, or alternatively at least 90% sequence identity, or alternatively at least 92% sequence identity, or alternatively at least 95% sequence identity, or alternatively at least 97% sequence identity, or alternatively at least 98% sequence identity, or alternatively, the equivalent nucleic acid hybridizes to the reference polynucleotide or its complement under high stringency conditions. In one aspect, the equivalent should encode a functional protein, which can be identified, as appropriate, by one or more assays described herein. In another embodiment, an equivalent has at least 70%, or at least 75%, or at least 80%, or alternatively at least 85% sequence identity, or alternatively at least 90% sequence identity, or alternatively at least 92% sequence identity, or alternatively at least 95% sequence identity, or alternatively at least 97% sequence identity, or alternatively at least 98% sequence identity to a reference nucleic acid, polynucleotide or oligonucleotide; or alternatively, an equivalent nucleic acid hybridizes to a reference polynucleotide or its complement under high stringency conditions, provided that one or more mutant polynucleotides identified herein having one or more non-naturally occurring glycosylation sites are not mutated from the corresponding mutant polynucleotide in the disclosed sequences. For example, an equivalent polynucleotide of modified human isoform #1 has modifications at nucleotide positions selected from 190, 293, 310, 661, 662, 1066, 1067, 1771, 1773, 1870, 1871, 2413, 2414, 2417, 2418, as shown in the sequence listing below, excluding one or more nucleotides.Additionally or alternatively, a polynucleotide equivalent encodes a protein or polypeptide of the same or similar function as the reference or parent polynucleotide.

[0053] The terms "protein," "peptide," and "polypeptide" are used interchangeably and broadly to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In alternative embodiments, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up the sequence of a protein or peptide. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics.

[0054] The terms equivalent and biologically equivalent are used interchangeably, for example, when referring to a protein or polypeptide as a reference. In some embodiments, an equivalent protein or polypeptide is one that has at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a reference protein or polypeptide. In some embodiments, an equivalent protein or polypeptide has at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a polypeptide or protein as disclosed herein. In some embodiments, an equivalent protein or polypeptide has at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a polypeptide or protein encoded by an equivalent polynucleotide as noted herein. Additionally or alternatively, a polynucleotide equivalent encodes a protein or polypeptide of the same or similar function as a reference or parent polynucleotide.

[0055] In some embodiments, equivalents are functional proteins that can be identified, as appropriate, by one or more assays described herein. In another aspect, equivalents have at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a reference protein or polypeptide. Some embodiments provide that one or more amino acids identified herein as mutated into potential glycosylation sites are mutated from the polypeptide or protein in the disclosed sequence. Some embodiments provide that one or more amino acids identified herein as mutated into potential glycosylation sites are not mutated from the polypeptide or protein in the disclosed sequence. For example, an equivalent polypeptide of modified human isoform #1 has modifications at amino acid positions, excluding one or more of the amino acids at positions selected from 64, 98, 104, 221, 356, 591, 624, 805 and 806, as shown in the sequences as disclosed herein.

[0056] In some embodiments, an equivalent protein or polypeptide performs a similar function to the wild-type protein and / or performs at a similar level compared to the wild-type protein. For example, a biological equivalent of a Ube3a protein or polypeptide can have a similar function compared to the wild-type Ube3a protein and / or can have any one or more of the functions of a biological equivalent of a Ube3a protein or polypeptide at a similar level (e.g., have similar activity) compared to the wild-type Ube3a protein or polypeptide. In further embodiments, for example, the function of the equivalent is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 100%, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 5-fold, or at least about 10-fold the level of their wild-type function. Non-limiting examples of such function include ubiquitination activity, such as ubiquitinating the Ube3a target protein S5a. In some embodiments, equivalents have ubiquitination activity that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 100%, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 5-fold, or at least about 10-fold that of wild-type activity. Illustrative methods for assessing such activity are known in the art, and some are exemplified in experimental methods.

[0057] In some embodiments, a wild-type polynucleotide, polypeptide, or protein (also referred to herein as wild-type) refers to a naturally occurring polynucleotide, polypeptide, or protein. In further embodiments, a wild-type Ube3a protein or polypeptide comprises, alternatively consists essentially of, or alternatively consists of a sequence selected from any one or more of SEQ ID NOs: 8, 10, 12, 20, 22, and / or 24, or naturally occurring variants thereof. Variable such naturally occurring variants are listed at www.uniprot.org / uniprot / Q05086 and www.uniprot.org / uniprot / O08759, each of which is incorporated herein in its entirety. In some embodiments, a wild-type Ube3a protein or polypeptide is an isoform comprising, alternatively consists essentially of, or alternatively consists of a sequence selected from any one or more of SEQ ID NOs: 8, 10, 12, 20, 22, and / or 24. In further embodiments, the wild-type Ube3a polynucleotide comprises, alternatively consists essentially of, or alternatively consists of a sequence selected from any one or more of SEQ ID NOs: 7, 9, 11, 19, 21, and / or 23, or naturally occurring variants thereof. Such variable naturally occurring variants are listed as transcripts or variants at www.genecards.org / cgi-bin / carddisp.pl?gene=UBE3A, which is incorporated herein in its entirety. In some embodiments, the wild-type Ube3a polynucleotide is an isoform comprising, alternatively consists essentially of, or alternatively consists of a sequence selected from any one or more of SEQ ID NOs: 7, 9, 11, 19, 21, and / or 23.

[0058] As used herein, a naturally occurring variant refers to a variant that is produced naturally (e.g., produced by chance) instead of being produced by artificial means.

[0059] In some embodiments, the naturally occurring variant is functional, e.g., performs a similar function to the wild-type and / or performs at a similar level compared to the wild-type. For example, a naturally occurring variant of a Ube3a protein or polypeptide can have any one or more of the functions of a naturally occurring variant of a Ube3a protein or polypeptide, similarly as compared to the wild-type Ube3a protein and / or at a similar level (e.g., have similar activity) compared to the wild-type Ube3a protein or polypeptide. In further embodiments, for example, the function of the naturally occurring variant is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 100%, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 5-fold, or at least about 10-fold that of the wild-type function. Non-limiting examples of such functions include ubiquitination activity, such as ubiquitinating the Ube3a target protein S5a. In some embodiments, the naturally occurring variant has ubiquitination activity that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 100%, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 5-fold, or at least about 10-fold that of the wild-type. Exemplary methods for assessing such activity can be found in the Experimental Methods.

[0060] In some embodiments, the naturally occurring variants are not functional and are therefore referred to herein as defective variants, genes, or alleles. For example, such defective genes or alleles encode defective protein variants that do not perform a certain wild-type function and / or perform it at a substantially reduced level compared to the wild-type. In some embodiments, the function of the defective protein variants is less than about 50%, less than about 25%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1% of their wild-type function. Non-limiting examples of such functions include ubiquitination activity, such as ubiquitinating the Ube3a target protein S5a. In some embodiments, the defective variants have ubiquitination activity that is less than about 50%, less than about 25%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1% of their wild-type activity. Illustrative methods for assessing such activity can be found in the experimental methods.

[0061] The expression "amplification of polynucleotides" includes methods such as PCR, ligation amplification (or ligase chain reaction, LCR), and amplification methods. These methods are known in the art and widely practiced. See, for example, U.S. Pat. Nos. 4,683,195 and 4,683,202 and Innis et al., 1990 (for PCR); and Wu et al. (1989) Genomics 4:560-569 (for LCR). In general, the PCR procedure describes a method of gene amplification consisting of (i) sequence-specific hybridization of primers to specific genes in a DNA sample (or library), (ii) subsequent amplification with multiple rounds of annealing, extension, and denaturation using DNA polymerase, and (iii) screening of the PCR product for bands of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to initiate polymerization; i.e., each primer is specifically designed to be complementary to each strand of the genomic locus to be amplified.

[0062] Reagents and hardware for carrying out PCR are commercially available. Primers useful for amplifying sequences from specific gene regions are preferably complementary to and specifically hybridize with sequences in the target region or its flanking region. The nucleic acid sequences generated by amplification can be directly sequenced. Alternatively, the amplified sequence(s) can be cloned prior to sequence analysis. Methods for direct cloning and sequence analysis of enzymatically amplified genomic segments are known in the art.

[0063] A "gene" refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated.

[0064] The term "express" refers to the production of a gene product.

[0065] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell.

[0066] "Gene product" or alternatively "gene expression product" refers to the amino acids (eg, peptides or polypeptides) produced when a gene is transcribed and translated.

[0067] "Under transcriptional control" is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, is dependent on the sequence being operably linked to elements that contribute to or facilitate the initiation of transcription. By "operably linked" is intended the polynucleotide be positioned in a manner that allows it to function within the cell.

[0068] The term "encoding," when applied to a polynucleotide, refers to a polynucleotide that is said to "encode" a polypeptide when, in its native context or when manipulated by methods well known to those of skill in the art, it is capable of being transcribed and / or translated to produce mRNA for the polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0069] "Probe," when used in the context of polynucleotide manipulation, refers to an oligonucleotide that serves as a reagent for detecting a target potentially present in a sample of interest by hybridizing with the target. Typically, a probe includes a detectable label or marker, or a means for binding a label or marker either before or after the hybridization reaction. Alternatively, a "probe" can be a biological compound, such as a polypeptide, antibody, or fragment thereof, that can bind to a target potentially present in a sample of interest.

[0070] "Detectable label," "label," "detectable marker," or "marker" are used interchangeably and include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. Detectable labels can also be attached to polynucleotides, polypeptides, antibodies, or compositions described herein.

[0071] As used herein, the term "label" or detectable label refers to a directly or indirectly detectable compound or composition, e.g., an N-terminal histidine tag (N-His), a magnetically active isotope, e.g., a nucleotide sequence ... 115 Sn, 117 Sn and 119 Sn, 13 C and 15Non-radioactive isotopes such as N, polynucleotides, or proteins such as antibodies are contemplated. The term also includes sequences conjugated to polynucleotides that provide a signal after expression of the inserted sequence, such as green fluorescent protein (GFP). The label may be detectable itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze a chemical alteration of a substrate compound or composition that is detectable. The label may be suitable for small-scale detection or more suitable for high-throughput screening. Thus, suitable labels include, but are not limited to, magnetically active isotopes, non-radioactive isotopes, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. Labels can be simply detected or quantified. A simply detected response generally includes a response whose presence is simply confirmed, while a quantified response generally includes a response that has a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other property. In luminescent or fluorescent assays, the detectable response can be generated directly using a luminophore or fluorophore associated with the assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. Examples of luminescent labels that produce a signal include, but are not limited to, bioluminescence and chemiluminescence. A detectable luminescent response generally involves a change in, or the appearance of, a luminescent signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and are described, for example, in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0072] As used herein, the term "immunoconjugate" includes an antibody or antibody derivative associated with or linked to a second agent, such as a cytotoxic agent, a detectable agent, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semi-synthetic antibody, or a multispecific antibody.

[0073] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).

[0074] In another embodiment, the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present within or on the surface of a cell or tissue, such as a cell surface marker. Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which can be used to attach the fluorescent label to a second molecule. The choice of functional group on the fluorescent label depends on the site of attachment to either a linker, drug, marker, or second labeling agent.

[0075] As used herein, a purification tag or marker refers to a tag that can be used in purifying a molecule or component to which it is conjugated, such as an epitope tag (including but not limited to, Myc tag, human influenza hemagglutinin (HA) tag, FLAG tag), an affinity tag (including but not limited to, glutathione-S-transferase (GST), polyhistidine (His) tag, calmodulin-binding protein (CBP) or maltose-binding protein (MBP)), or a fluorescent tag.

[0076] A "primer" is a short polynucleotide, generally having a free 3'-OH group, that hybridizes with the target, thereby binding to a target or "template" potentially present in a sample of interest, and subsequently facilitating the polymerization of a polynucleotide complementary to the target. "Polymerase chain reaction" ("PCR") is a reaction in which replicate copies are made from a target polynucleotide using a "primer pair" or "primer set" consisting of an "upstream" and a "downstream" primer, and a polymerization catalyst such as DNA polymerase and typically a thermostable polymerase enzyme. Methods for PCR are well known in the art and are taught, for example, in MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press). The entire process of producing replicate copies of a polynucleotide, such as PCR or gene cloning, is collectively referred to herein as "replication." Primers can also be used as probes in hybridization reactions, such as Southern or Northern blot analysis. Sambrook and Russell (2001), below.

[0077] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized by hydrogen bonds between the bases of the nucleotide residues. The hydrogen bonds can occur through Watson-Crick base pairing, Hoogsteen binding, or in any other sequence-specific manner. The complex can comprise two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction can constitute a step in a more extensive process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0078] Hybridization reactions can be performed under conditions of different "stringency." Generally, low stringency hybridization reactions are performed at about 40°C, 10xSSC, or a solution of equivalent ionic strength / temperature. Moderate stringency hybridizations are typically performed at about 50°C, 6xSSC, and high stringency hybridization reactions are generally performed at about 60°C, 1xSSC. Hybridization reactions can also be performed under "physiological conditions," as known to those skilled in the art. Non-limiting examples of physiological conditions include the temperature, ionic strength, pH, and Mg normally found in cells. 2+ Concentration.

[0079] When hybridization occurs between two single-stranded polynucleotides in an antiparallel configuration, the reaction is called "annealing," and the polynucleotides are referred to as "complementary." When hybridization can occur between one strand of a first polynucleotide and one strand of a second polynucleotide, the double-stranded polynucleotide can be "complementary" or "homologous" to another polynucleotide. "Complementarity" or "homology" (the degree to which a polynucleotide is complementary to another polynucleotide) can be quantified in terms of the ratio of bases on opposite strands that are expected to form hydrogen bonds with each other according to generally accepted base pairing rules.

[0080] The term "propagating" means to grow a cell or population of cells. The term "growing" also refers to the proliferation of cells in the presence of a support medium, nutrients, growth factors, feeder cells, or any chemical or biological compounds necessary to obtain a desired number of cells or cell type.

[0081] The term "culturing" refers to the in vitro propagation of cells or organisms on or in various types of media. It is understood that the progeny of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.

[0082] Unmodified cells are sometimes referred to as "source cells" or "source stem cells." Cells can be prokaryotic or eukaryotic, including, but not limited to, bacterial cells, yeast cells, plant cells, insect cells, animal cells, and mammalian cells, such as cat, dog, horse, mouse, rat, monkey, cow, pig, and human cells.

[0083] In one embodiment, an "immature cell" refers to a cell that does not possess a desired (adult) phenotype or genotype. For example, in one embodiment, a mature cell is a cell that is being replaced. An immature cell can be subjected to techniques, including physical, biological, or chemical processes, that change, initiate, or alter the phenotype or genotype of the cell to a "mature cell." A "mature cell" refers to a cell that possesses a desired phenotype or genotype.

[0084] A "viral vector" is defined as a recombinantly produced virus or viral particle containing a polynucleotide to be delivered to a host cell either in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, alphaviral vectors, and the like. Alphaviral vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med. 5(7):823-827.

[0085] In embodiments where gene transfer is mediated by a lentiviral vector, the vector construct refers to a polynucleotide comprising a lentiviral genome or a portion thereof and a therapeutic gene. As used herein, "lentiviral-mediated gene transfer" or "lentiviral transduction" have the same meaning and refer to the process by which a gene or nucleic acid sequence is stably transferred into a host cell via a virus that invades the cell and integrates its genome into the host cell genome. The virus can invade a host cell via its normal infection mechanism, or it can be modified to bind to a different host cell surface receptor or ligand and enter the cell. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into a DNA form, which is integrated into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. As used herein, lentiviral vector refers to a viral particle capable of introducing exogenous nucleic acid into cells via a viral or viral-like entry mechanism. A "lentiviral vector" is a type of retroviral vector known in the art that has certain advantages over other retroviral vectors in transducing non-dividing cells. See Trono D. (2002) Lentiviral vectors, New York: Spring-Verlag Berlin Heidelberg.

[0086] The lentiviral vectors of the present disclosure are based on or derived from oncoretroviruses (a subgroup of retroviruses that includes MLV) and lentiviruses (a subgroup of retroviruses that includes HIV). Examples include ASLV, SNV, and RSV, all of which are separated into packaging and vector components for lentiviral vector particle production systems. The lentiviral vector particles of the present disclosure can be based on genetically or otherwise (e.g., by specific selection of packaging cell systems) modified versions of specific retroviruses.

[0087] When a vector particle according to the present disclosure is "based on" a particular retrovirus, it means that the vector is derived from that particular retrovirus. The genome of the vector particle includes components from that retrovirus as a backbone. Vector particles contain essential vector components compatible with the RNA genome, including reverse transcription and integration systems. These typically include the gag and pol proteins from the particular retrovirus. Thus, most of the structural components of a vector particle typically originate from the retrovirus, although they may be genetically or otherwise modified to confer desired useful properties. However, certain structural components, particularly the env protein, can originate from different viruses. The vector host range and cell types infected or transduced can be altered by using different env genes in the vector particle production system to confer different specificities to the vector particles.

[0088] The term "about," when used herein when referring to a measurable value such as, for example, an amount or concentration, is intended to encompass variations of 20%, 10%, 5%, 1%, 0.5% or even 0.1% of the specified amount.

[0089] The terms "acceptable," "effective," or "sufficient," when used to describe the selection of any components, ranges, dosage forms, etc. disclosed herein, are intended to mean that said components, ranges, dosage forms, etc. are suitable for the purpose disclosed.

[0090] The term "adeno-associated virus" or "AAV," as used herein, refers to a member of the class of viruses belonging to the genus Dependoparvovirus in the family Parvoviridae. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes are capable of infecting cells from various tissue types. At least 11 consecutive AAV serotypes are known in the art. Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 serotypes, such as AAV2, AAV8, AAV9, or variant or synthetic serotypes, such as AAV-DJ and AAV PHP.B. AAV particles comprise, alternatively, consist essentially of, or even consist of three major viral proteins: VP1, VP2, and VP3. In one embodiment, AAV refers to one of the serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B, or AAV rh74. These vectors are commercially available or described in the patent or technical literature.

[0091] As used herein, "antibody" includes whole antibodies and any antigen-binding fragments or single chains thereof. Thus, the term "antibody" includes any protein- or peptide-containing molecule comprising at least a portion of an immunoglobulin molecule. Examples of such include, but are not limited to, a heavy or light chain complementarity-determining region (CDR) or ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein, any of which may be incorporated into the antibodies of the present disclosure. The term "antibody" is further intended to encompass digest fragments, designated portions, derivatives, and variants thereof, including antibody mimetics or portions of antibodies that mimic the structure and / or function of an antibody or designated fragment or portion thereof, including single chain antibodies and fragments thereof. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody are V, V- ...L , V H , C L and CH domains; F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by disulfide bridges in the hinge region; V H and C H Fd fragment consisting of domain; V of a single arm of an antibody L and V H Fv fragment consisting of domains, V H dAb fragments comprise the V domain (Ward et al. (1989) Nature 341:544-546); and isolated complementarity-determining regions (CDRs). In addition, two domains of the Fv fragment, the V domain, are isolated. L and V H are encoded by separate genes, but using recombinant methods, V L and V H The regions can be joined by a synthetic linker that allows them to be produced as a single protein chain that pairs to form a monovalent molecule (known as a single-chain Fv (scFv)). Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883. Single-chain antibodies are also intended to be encompassed within the term "antibody fragment." Any of the above-noted antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments are screened for binding specificity and neutralizing activity in the same manner as intact antibodies.

[0092] The term "antibody variant" is intended to include antibodies produced in species other than mouse. It also includes antibodies containing post-translational modifications to the linear polypeptide sequence of the antibody or fragment. It further encompasses fully human antibodies.

[0093] The term "antibody derivative" is intended to encompass molecules that bind the epitopes defined above and that are modifications or derivatives of the native monoclonal antibodies of the present disclosure. Derivatives include, but are not limited to, bispecific, multispecific, heterospecific, trispecific, tetraspecific, polyspecific antibodies, diabodies, chimeric, recombinant, and humanized.

[0094] As used herein, "Ube3a" refers to a gene that encodes a protein called ubiquitin protein ligase E3A. In some embodiments, the abbreviation Ube3a refers to a protein or polypeptide. In some embodiments, the abbreviation Ube3a refers to a polynucleotide. Ubiquitin protein ligases are enzymes that target other proteins for destruction (degradation) within cells. These enzymes attach a small molecule called ubiquitin to the proteins to be degraded. A cellular structure called the proteasome recognizes and digests these ubiquitin-tagged proteins. Protein degradation is a normal process that removes damaged or unnecessary proteins and helps maintain the normal function of cells. See ghr.nlm.nih.gov / gene / UBE3A, last accessed May 23, 2019.

[0095] Research suggests that the ubiquitin protein ligase E3A plays a critical role in the normal development and function of the nervous system. Research suggests that it helps control (regulate) the balance of protein synthesis and degradation (proteostasis) at the junctions between nerve cells (synapses), where cell-to-cell communication occurs. Regulation of proteostasis is important for synapses to change and adapt over time in response to experience, a feature called synaptic plasticity. Synaptic plasticity is crucial for learning and memory. Three isoforms of the gene exist that vary at the 5' end (see NCBI NM_0013545606; NM_000462.5; and NM001354505). Yamamoto et al. (1997) Genomics Apr. 15;41(2):263-266 describe the genomic structure of the E6-AP coding region and the analysis of a set of five E6-AP mRNAs that have the potential to encode three protein isoforms of E6-AP protein (isoforms I, II, and III) that differ in their extreme amino termini.

[0096] As used herein, N-linked glycosylation refers to the attachment of an oligosaccharide or glycan, a carbohydrate consisting of several sugar molecules, to a nitrogen atom, such as the amide nitrogen, of an asparagine (Asn, N) residue of a protein or polypeptide.

[0097] The term "regulatory sequence," "expression control element," or "promoter," as used herein, refers to a polynucleotide that is operably linked to a target polynucleotide to be transcribed and / or replicated, facilitating the expression and / or replication of the target polynucleotide. A promoter is an example of an expression control element or regulatory sequence. A promoter can be placed 5' or upstream of a gene or other polynucleotide, providing a control point for regulated gene transcription. Polymerase II and III are examples of promoters. The sequence of the MNDU3 promoter and the sequence of an exemplary CMV promoter are provided below.

[0098] Polymerase II or "pol II" promoters catalyze the transcription of DNA to synthesize precursors of mRNA and most shRNAs and microRNAs. Examples of Pol II promoters are known in the art and include, but are not limited to, the phosphoglycerate kinase ("PGK") promoter; EF1-alpha; CMV (minimal cytomegalovirus promoter); and LTRs derived from retroviral and lentiviral vectors. Other Pol II promoters can be selected, such as from cell-specific promoters (including, but not limited to, the CD14 promoter, CD3 promoter, and CD19 promoter), any blood cell lineage promoter (including, but not limited to, the promoter of any one of CD2, CD11b, CD11c, CD16, CD24, CD56, CD66b, and CD235), and / or any other promoter capable of directing protein expression in human cells.

[0099] An enhancer is a regulatory element that increases the expression of a target sequence. A "promoter / enhancer" is a polynucleotide containing a sequence capable of providing both promoter and enhancer functions. For example, the long terminal repeat of a retrovirus contains both promoter and enhancer functions. An enhancer / promoter can be "endogenous," "exogenous," or "heterologous." An "endogenous" enhancer / promoter is one that is naturally linked to a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one that is juxtaposed to a gene using genetic manipulation (i.e., molecular biological techniques) so that transcription of the gene is directed by the linked enhancer / promoter.

[0100] Signal peptide, as used herein, refers to a short peptide (usually 16-30 amino acids in length) present at the N-terminus of most newly synthesized proteins destined for the secretory pathway (also sometimes referred to as a signal sequence, targeting signal, localization signal, transit peptide, leader sequence, or leader peptide). In one embodiment, the signal peptide is a secretory signal.

[0101] The term "secretary signal" refers to a secretory signal peptide that enables the export of a protein from the cytosol to the secretory pathway. Proteins can exhibit differential levels of secretion success, and in many cases, a particular signal peptide can achieve lower or higher levels when partnered with a specific protein. In eukaryotes, a signal peptide is a hydrophobic string of amino acids that is recognized by the signal recognition particle (SRP) in the cytosol of eukaryotic cells. After the signal peptide is produced from the mRNA-ribosome complex, the SRP binds the peptide and stops protein translation. The SRP then shuttles the mRNA / ribosome complex to the rough endoplasmic reticulum, where the protein is translated and enters the lumen of the endoplasmic reticulum. The signal peptide is then cleaved from the protein to produce either a soluble or membrane-tagged (if a transmembrane domain is also present) protein in the endoplasmic reticulum. These are known in the art and commercially available from vendors such as Oxford Genetics.

[0102] As used herein, cell-penetrating peptide or cell-penetrating domain (CPP) refers to a short peptide that facilitates cellular uptake of various molecular cargoes (from small chemical molecules to nano-sized particles and large fragments of DNA). The "cargo," such as an engineered protein as disclosed herein, is associated with the peptide through either a covalent chemical linkage or a non-covalent interaction. The function of the CPP is to deliver the cargo into target cells, a process that typically occurs via endocytosis with the cargo delivered to the endosomes of living mammalian cells. In some embodiments, the target cell is a neuron. CPPs typically have an amino acid composition containing a high relative abundance of positively charged amino acids, such as lysine or arginine, or a sequence containing an alternating pattern of polar / charged and non-polar hydrophobic amino acids. It has previously been reported that CPPs can be used to deliver the human immunodeficiency virus transcriptional transactivator (HIV-TAT) protein into cells.

[0103] A CPP may be chemically modified, such as by prenylation, near the C-terminus of the CPP. Prenylation is a post-translational modification that results in the addition of a 15- (farnesyl) or 20- (geranylgeranyl) carbon isoprenoid chain to a peptide. Chemically modified CPPs can be even shorter and still retain cell-penetrating properties. Thus, a CPP according to another embodiment of the present disclosure is a chemically modified CPP having 2 to 35 amino acids, preferably 5 to 25 amino acids, more preferably 10 to 25 amino acids, or even more preferably 15 to 25 amino acids.

[0104] CPPs can be recombinantly linked to proteins by covalent or non-covalent bonds. Recombinant proteins bearing CPP peptides can be prepared in bacteria such as E. coli, mammalian cells such as human HEK293 cells, or any cells suitable for protein expression. Covalent and non-covalent methods have also been developed to form CPP / protein complexes. The CPP, Pep-1, has been shown to form protein complexes and has proven effective for delivery (Kameyama et al. (2006) Bioconjugate Chem. 17:597-602).

[0105] CPPs also include cationic conjugates, which can also be used to facilitate protein delivery into cells or tissues of interest. The cationic conjugates can comprise multiple residues containing amines, guanidines, amidines, N-containing heterocycles, or combinations thereof. In related embodiments, the cationic conjugates can comprise multiple reactive units selected from the group consisting of alpha-amino acids, beta-amino acids, gamma-amino acids, cationically functionalized monosaccharides, cationically functionalized ethylene glycols, ethyleneimines, substituted ethyleneimines, N-substituted spermines, N-substituted spermidines, and combinations thereof. The cationic conjugates can also be oligomers, including oligopeptides, oligoamides, cationically functionalized oligoethers, cationically functionalized oligosaccharides, oligoamines, oligoethyleneimines, and combinations thereof. The oligomers can also be oligopeptides, in which case the amino acid residues of the oligopeptide are capable of forming a positive charge. The oligopeptide can contain 5 to 25 amino acids; preferably 5 to 15 amino acids; more preferably 5 to 10 cationic amino acids or other cationic subunits.

[0106] Recombinant proteins tethering CPPs to proteins can be generated and used for delivery to cells or tissues.

[0107] As used herein, a cleavable peptide, also referred to as a cleavable linker, refers to a peptide that can be cleaved, for example, by an enzyme. A single translated polypeptide containing such a cleavable peptide can produce two end products, thus allowing the expression of more than one polypeptide from a single open reading frame. An example of a cleavable peptide is a self-cleaving peptide, such as the 2A self-cleaving peptide. The 2A self-cleaving peptide is a class of 18-22 aa long peptides that can induce cleavage of recombinant proteins in cells. In some embodiments, the 2A self-cleaving peptide is selected from P2A, T2A, E2A, F2A, and BmCPV2A. See, for example, Wang Y, et al., 2A Self-Cleaving Peptide-Based Multi-Gene Expression System. in the silkworm Bombyx mori. Sci Rep. 2015;5:16273. Published 2015 Nov 5.

[0108] The term "stem cell" refers to a cell in an undifferentiated or partially differentiated state that has the capacity for self-renewal and / or to generate differentiated progeny. Self-renewal is defined as the ability of a stem cell to proliferate and give rise to more such stem cells while maintaining its developmental potential (i.e., totipotency, pluripotency, multipotency, etc.). The term "somatic stem cell" is used herein to refer to any stem cell derived from non-embryonic tissue, including fetal tissue, juvenile tissue, and adult tissue. Natural somatic stem cells have been isolated from a wide variety of adult tissues, including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Exemplary naturally occurring somatic stem cells include, but are not limited to, mesenchymal stem cells (MSCs) and neural or neuronal stem cells (NSCs). In some embodiments, the stem or progenitor cell may be an embryonic stem cell. As used herein, "embryonic stem cells" refer to stem cells derived from tissues formed after fertilization but before the end of gestation, including pre-embryonic tissues (e.g., blastocysts), embryonic tissues, or fetal tissues harvested at any time during gestation, typically, but not necessarily, before approximately 10-12 weeks of gestation. Most frequently, embryonic stem cells are pluripotent cells derived from early embryos or blastocysts. Embryonic stem cells can be obtained directly from suitable tissues, including, but not limited to, human tissues, or from established embryonic cell lines. "Embryonic-like stem cells" refer to cells that share one or more (but not all) characteristics of embryonic stem cells.

[0109] "Differentiation" refers to the process by which an unspecialized cell acquires the characteristics of a specialized cell, such as a heart, liver, or muscle cell. "Directed differentiation" refers to the manipulation of stem cell culture conditions to induce differentiation into a specific cell type. "Dedifferentiated" defines a cell that reverts to a less committed position within a cell's lineage. As used herein, the term "differentiating or differentiated" defines a cell that takes on a more committed ("differentiated") position within a cell's lineage. As used herein, "a cell that differentiates into the mesodermal (or ectodermal or endodermal) lineage" defines a cell that becomes committed to a specific mesodermal, ectodermal, or endodermal lineage, respectively. Examples of cells that differentiate into mesodermal lineages or give rise to specific mesodermal cells include, but are not limited to, cells that are adipogenic, leiomyogenic, chondrogenic, cardiogenic, dermatogenic, hematopoietic, hemangiogenic, myogenic, nephrogenic, urogenitogenic, osteogenic, pericardiogenic, or interstitial.

[0110] As used herein, the term "differentiating or differentiated" defines a cell that assumes a more committed ("differentiated") position within a cell's lineage. "Dedifferentiated" defines a cell that reverts to a less committed position within a cell's lineage. Induced pluripotent stem cells are an example of dedifferentiated cells.

[0111] As used herein, the "lineage" of a cell defines the inheritance of the cell, i.e., its predecessors and progeny. The lineage of a cell places the cell within a genetic scheme of development and differentiation.

[0112] "Multilineage stem cells" or "multipotent stem cells" refer to stem cells that regenerate themselves and at least two further differentiated progeny cells from distinct developmental lineages. The lineages can be from the same germ layer (i.e., mesoderm, ectoderm, or endoderm) or from different germ layers. Examples of two progeny cells from distinct developmental lineages derived from the differentiation of a multilineage stem cell are myogenic cells and adipogenic cells (both of mesodermal origin but which give rise to different tissues). Another example is neurogenic cells (of ectodermal origin) and adipogenic cells (of mesodermal origin).

[0113] "Precursor" or "progenitor cell" is intended to mean a cell that has the ability to differentiate into a specific type of cell. A progenitor cell may be a stem cell. A progenitor cell may be more specific than a stem cell. A progenitor cell may be unipotent or multipotent. Compared to adult stem cells, progenitor cells may be at a later stage of cell differentiation. Examples of progenitor cells include, but are not limited to, progenitor neural cells.

[0114] As used herein, "pluripotent cells" define less differentiated cells that can give rise to at least two distinct (genotypically and / or phenotypically) further differentiated progeny cells. In another aspect, "pluripotent cells" include induced pluripotent stem cells (iPSCs), which are stem cells artificially derived from non-pluripotent cells, typically adult somatic cells, which have historically been produced by inducing the expression of one or more stem cell-specific genes. Such stem cell-specific genes include, but are not limited to, the octamer transcription factor family, i.e., Oct-3 / 4; the Sox gene family, i.e., Sox1, Sox2, Sox3, Sox15, and Sox18; the Klf gene family, i.e., Klf1, Klf2, Klf4, and Klf5; the Myc gene family, i.e., c-myc and L-myc; the Nanog gene family, i.e., OCT4, NANOG, and REX1; or LIN28. Examples of iPSCs are described in Takahashi et al. al. (2007) Cell advance online publication 20 November 2007; Takahashi & Yamanaka (2006) Cell 126:663-76; Okita et al. (2007) Nature 448:260-262; Yu et al. (2007) Science advance online publication 20 November 2007; and Nakagawa et al. (2007) Nat. Biotechnol. Advance online publication 30 November 2007.

[0115] "Embryoid bodies or EBs" are three-dimensional (3D) aggregates of embryonic stem cells formed during culture that facilitate subsequent differentiation. When grown in suspension culture, EB cells form small cell aggregates surrounded by an outer layer of visceral endoderm. After growth and differentiation, EBs become cystic embryoid bodies with a fluid-filled cavity and an inner layer of ectoderm-like cells.

[0116] By "induced pluripotent cells" is intended embryonic-like cells that have been reprogrammed from adult cells to an immature phenotype. Various methods are known in the art, see, for example, "A simple new way to induce pluripotency: Acid," Nature, 29 January 2014, and available at sciencedaily.com / releases / 2014 / 01 / 140129184445, last accessed February 5, 2014, and U.S. Patent Application Publication No. 2010 / 0041054. Human iPSCs also express stem cell markers and are capable of generating cells characteristic of all three germ layers.

[0117] " Parthenogenetic stem cell " refers to the stem cell that arises from the parthenogenetic activation of egg.The method of creating parthenogenetic stem cell is known in the art.For example, see Cibelli et al. (2002) Science 295(5556):819 and Vrana et al. (2003) Proc. Natl. Acad. Sci. USA 100(Suppl.1)11911-6.

[0118] As used herein, the term "pluripotency gene or marker" refers to an expressed gene or protein correlated with an immature or undifferentiated phenotype, e.g., Oct3 / 4, Sox2, Nanog, c-Myc, and LIN-28. Methods for identifying such are known in the art, and systems for identifying such are commercially available, e.g., from EMD Millipore (MILLIPLEX® Map Kit).

[0119] The term "phenotype" refers to a description of an individual's traits or characteristics that are measurable and expressed in only a subset of individuals within a population. In one aspect of the present disclosure, the phenotype of an individual includes the phenotype of a single cell, a substantially homogeneous population of cells, a population of differentiated cells, or a tissue made up of the population of cells.

[0120] The term "pharmaceutically acceptable carrier (or medium), which can be used interchangeably with the term "biologically compatible carrier or medium," refers to reagents, cells, compounds, materials, compositions, and / or dosage forms that are not only compatible with cells and other agents to be administered therapeutically, but also, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other complications, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers suitable for use in the present disclosure include liquids, semi-solid (e.g., gels), and solid materials (e.g., cell scaffolds and matrices, tubes, sheets, and other such materials known in the art and described in further detail herein). These semi-solid and solid materials can be designed to resist degradation within the body (non-biodegradable) or can be designed to degrade within the body (biodegradable, bioerodible). Biodegradable materials may also be bioresorbable or bioabsorbable, i.e., capable of dissolving and being absorbed by bodily fluids (water-soluble implants being one example), or capable of being broken down and eventually eliminated from the body, either by conversion to other materials or by breakdown and elimination by natural pathways.

[0121] A population of cells contemplates a collection of more than one cell that may be phenotypically and / or genotypically identical (clonal) or non-identical. A population may be purified, highly purified, substantially homogenous, or heterogeneous, as described herein.

[0122] The terms effective period (or time) and effective conditions refer to the period of time or other controllable conditions (e.g., temperature, humidity for in vitro methods) necessary or desirable for an agent or composition to achieve its intended result, e.g., differentiation or dedifferentiation of cells into a given cell type.

[0123] "Substantially homogeneous" refers to a population of cells in which greater than about 50%, or alternatively greater than about 60%, or alternatively greater than 70%, or alternatively greater than 75%, or alternatively greater than 80%, or alternatively greater than 85%, or alternatively greater than 90%, or alternatively greater than 95% of the cells are of the same or similar phenotype. Phenotype can be determined by preselected cell surface or other markers.

[0124] As used herein, the terms "treating," "treatment," and the like are used herein to mean obtaining a desired pharmacological and / or physiological effect. In some embodiments, the effect can be prophylactic, in terms of completely or partially preventing a disorder or its signs or symptoms, and / or therapeutic, in terms of partially or completely curing a disorder and / or adverse effects that may result from the disorder. Examples of "treatment" include, but are not limited to, preventing a disorder from occurring in a subject who may be susceptible to the disorder but has not yet been diagnosed with the disorder; inhibiting a disorder, i.e., arresting its onset; and / or alleviating or relieving symptoms of a disorder. In one aspect, treatment is the inhibition of the onset of a disease or disorder, e.g., symptoms of Angelman syndrome. In some embodiments, it refers to (1) preventing a symptom or disease from occurring in a subject who is susceptible to or who has not yet shown symptoms of the disease; (2) inhibiting or arresting the onset of a disease; or (3) relieving or causing regression of a disease or symptoms of a disease. As understood in the art, "treatment" is an approach to obtain beneficial or desired results, including clinical results.For the purpose of the present technology, beneficial or desired results can include, but are not limited to, one or more of the following, whether detectable or undetectable: alleviation or amelioration of one or more symptoms, reduction in the severity of a condition (including a disease), a stabilized (i.e., not worsening) state of a condition (including a disease), delay or slowing of the progression of a condition (including a disease), alleviation or remission of a condition (including a disease), and remission (whether partial or total) of a condition (including a disease).In one embodiment, treatment excludes prevention or prophylaxis.

[0125] In one embodiment, the term "disease" or "disorder" as used herein refers to a disease associated with a defective Ube3a variant or gene, such as Angelman syndrome and / or Prader-Willi syndrome, being diagnosed with, suspected of having, or being at high risk of having such a disease.

[0126] "Administration" or "delivery" of cells or vectors, or other agents and compositions containing same, can be achieved in one dose, continuously or intermittently throughout the course of treatment. Methods for determining the most effective means and dosage of administration are known to those skilled in the art and vary depending on the composition used for treatment, the purpose of the treatment, the target cells being treated, and the subject being treated. Single or multiple administrations can be carried out, with dose levels and patterns selected by the treating physician or, in the case of animals, the treating veterinarian. Appropriate dosage formulations and methods for administering agents are known in the art. Routes of administration can also be determined, and methods for determining the most effective route of administration are known to those skilled in the art and vary depending on the composition used for treatment, the purpose of the treatment, the health or disease stage of the subject being treated, and the target cells or tissues. Non-limiting examples of routes of administration include oral administration, intraperitoneal administration, infusion, intranasal administration, inhalation, injection, and topical application.

[0127] A "pharmaceutical composition" is intended to include a combination of an active polypeptide, polynucleotide, or antibody with a carrier, inert or active, such as a solid support, that makes the composition suitable for diagnostic or therapeutic use in vitro, in vivo, or ex vivo.

[0128] As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, and emulsions such as oil / water or water / oil emulsions, as well as various types of wetting agents. The compositions may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

[0129] The terms "subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, rats, rabbits, monkeys, cows, sheep, pigs, dogs, cats, farm animals, sport animals, pets, horses, and primates, particularly humans. In addition to being useful for treating humans, the present disclosure is also useful for veterinary treatment of mammals, including rodents, companion mammals, exotic animals, and domesticated animals. In one embodiment, mammals include horses, dogs, and cats. In another embodiment of the present disclosure, the human is a fetus, infant, prepubertal subject, adolescent, pediatric patient, or adult. In one aspect, the subject is a pre-symptomatic mammal or human. In another aspect, the subject has minimal clinical symptoms of disease. The subject can be a male or female adult, infant, or pediatric subject. In an additional aspect, the subject is an adult. In some instances, an adult is an adult, for example, an adult over the age of 18.

[0130] "Host cell" refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in successive generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0131] An "enriched population" of cells refers to a substantially homogenous population of cells having a particular defined characteristic, wherein the cells are more than 70%, or alternatively more than 75%, or alternatively more than 80%, or alternatively more than 85%, or alternatively more than 90%, or alternatively more than 95%, or alternatively more than 98% identical in the defined characteristic.

[0132] The term "suffering from" in the context of the term "treatment" refers to a patient or individual who has been diagnosed with or is susceptible to Angelman syndrome. A patient may also be said to be "at risk of suffering from" the disease because they carry one or more genetic mutations. The patient has not yet developed the characteristic disease pathology.

[0133] An "effective amount" is an amount sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages. Such delivery depends on several variables, including the period for which an individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, and the like. However, it is understood that the specific dosage level of the therapeutic agent of the present disclosure for any particular subject will depend on a variety of factors, including the activity of the specific compound being used, the subject's age, weight, general health, sex, and diet, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated, as well as the form of administration. Treatment dosages can generally be titrated to optimize safety and efficacy. Typically, dosage-effect relationships from in vitro and / or in vivo studies can initially provide useful guidance regarding appropriate dosages for patient administration. Generally, it is desirable to administer an amount of compound effective to achieve serum levels corresponding to concentrations found to be effective in vitro. Determination of these parameters is well within the skill of one of ordinary skill in the art. These considerations, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks.

[0134] The term administration is intended to include, without limitation, administration orally, parenterally (e.g., intramuscular, intraperitoneal, intravenous, intracerebroventricular (ICV), intrathecal, intracisternal injection or infusion, subcutaneous injection, or implant), by inhalation, aerosol, nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository), or topical routes of administration (e.g., gels, ointments, creams, aerosols, etc.), and may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration. The present disclosure is not limited by route of administration, formulation, or dosing schedule. ILLUSTRATIVE EMBODIMENTS Polynucleotides and Polypeptides

[0135] The present disclosure provides polynucleotides encoding ubiquitin-protein ligase E3A (Ube3a) proteins, polypeptides, or biological equivalents thereof. In some embodiments, the polynucleotides are recombinant and / or isolated. In some embodiments, the Ube3a proteins, polypeptides, or biological equivalents thereof comprise one or more glycosylation sites.

[0136] Also provided are Ube3a proteins, polypeptides, or biological equivalents thereof, which comprise one or more glycosylation sites. In some embodiments, the Ube3a proteins, polypeptides, or biological equivalents thereof are isolated, engineered, and / or recombinant.

[0137] In some embodiments, the Ube3a protein, polypeptide, or biological equivalent thereof is not a wild-type Ube3a protein, such as a protein comprising a sequence selected from SEQ ID NO: 8, 10, 12, 20, 22, or 24, or any naturally occurring variant thereof. This Ube3a protein, polypeptide, or biological equivalent thereof is also referred to herein as a modified Ube3a protein.

[0138] In some embodiments, the Ube3a protein, polypeptide, or biological equivalent thereof comprises two or more glycosylation sites or three or more glycosylation sites. In some embodiments, the Ube3a protein, polypeptide, or biological equivalent thereof comprises four or more glycosylation sites or five or more glycosylation sites. In some embodiments, the Ube3a protein, polypeptide, or biological equivalent thereof comprises at least four or at least five glycosylation sites. In some embodiments, the Ube3a protein, polypeptide, or biological equivalent thereof comprises six or more or seven or more glycosylation sites. In some embodiments, the Ube3a protein, polypeptide, or biological equivalent thereof comprises eight or more glycosylation sites.

[0139] In some embodiments, any one or more of the glycosylation sites can be naturally occurring. In some embodiments, any one or more of the glycosylation sites may not be naturally occurring. In further embodiments, at least one of the glycosylation sites is not naturally occurring. Additionally or alternatively, any one, or any two, or all three amino acid residues in at least one of the glycosylation sites are mutated compared to a wild-type Ube3a polypeptide or protein, thereby constituting a glycosylation site.

[0140] Recombinant and / or isolated polynucleotides or Ube3a proteins, polypeptides, or biological equivalents thereof can be naturally occurring or can be created, such as by mutating the open reading frame of a wild-type polynucleotide to include one or more glycosylation sites and / or modifying(s) a naturally occurring glycosylation site(s) to a non-naturally occurring sequence. In some embodiments, Ube3a proteins, polypeptides, or biological equivalents thereof include one or more non-naturally occurring glycosylation sites. In some embodiments, non-naturally occurring Ube3a proteins, polypeptides, or biological equivalents thereof are also referred to herein as modified Ube3a proteins or modified proteins.

[0141] In some embodiments of any of the disclosures herein, the Ube3a protein, polypeptide, or biological equivalent thereof contains at least one non-naturally occurring glycosylation site. Such non-naturally occurring glycosylation site does not render the Ube3a protein, polypeptide, or biological equivalent thereof non-functional. For example, as shown in Experiment No. 1, they are still capable of ubiquitinating S5a. Furthermore, as shown in the Experimental Methods, they are capable of effectively treating AS.

[0142] Examples of such Ube3a proteins, polypeptides, or biological equivalents thereof are provided below, where the motif "NXT / S" identifies a glycosylation site, where X is any amino acid residue. In some embodiments, the glycosylation site comprises, alternatively, consists essentially of, or alternatively consists of the consensus sequence NXaaT / S (i.e., NXaaT and / or NXaaS), where Xaa is any amino acid residue. In some embodiments, the glycosylation site comprises, alternatively, consists essentially of, or alternatively consists of the consensus sequence NXaaT / S (i.e., NXaaT and / or NXaaS), where Xaa is any amino acid residue except proline (P). In some embodiments, glycosylation is N-linked.

[0143] In some embodiments, a starting or reference Ube3a protein or polypeptide, such as a wild-type, isoform, naturally occurring variant, or non-naturally occurring variant thereof as disclosed herein, can be mutated to have at least one non-naturally occurring glycosylation site and one or more optionally additional mutated residues that do not constitute a glycosylation site, thereby resulting in an engineered and / or recombinant Ube3a protein, polypeptide, or biological equivalent thereof. In further embodiments, a starting or reference Ube3a protein or polypeptide can have an amino acid residue at any position mutated to N, as appropriate, provided that the second amino acid residue at its C-terminus is T or S. Additionally or alternatively, a starting or reference Ube3a protein or polypeptide can have an amino acid residue at any position mutated to S or T, as appropriate, provided that the second amino acid residue at its N-terminus is N. In some embodiments, any portion of a starting or reference Ube3a protein or polypeptide having the sequence Xaa1Xaa2Xaa3 can be engineered to be NXaaT / S (i.e., NXaaT and / or NXaaS), thereby resulting in a recombinant Ube3a protein, polypeptide, or biological equivalent thereof as disclosed herein, where Xaa1, Xaa2, Xaa3, or Xaa can be any amino acid residue. In further embodiments, either or both of Xaa2 and Xaa are not P. In one embodiment, Xaa3 is S or T, and optionally Xaa1 is not N. In another embodiment, Xaa1 is N, and optionally Xaa3 is neither S nor T. In some embodiments, Xaa1 is not N, and Xaa3 is neither S nor T. Illustrative glycosylation sites and / or their locations can be found in Figures 16A, 16B, and SEQ ID NO: 32.

[0144] In some embodiments, recombinant Ube3a proteins, polypeptides, or biological equivalents thereof can be engineered and / or produced by mutating one or more of the nucleotide residues of the coding sequence of a starting or reference Ube3a protein or polypeptide. In further embodiments, such mutated nucleotide residues encode glycosylation sites. Some illustrative nucleotide mutations can be found in Figure 16A, Figure 16B, and SEQ ID NOs: 13, 15, 17, 25, 27, 29, and 31.

[0145] In some embodiments, the glycosylation site is: aa62 to aa64 of SEQ ID NO:14, aa96 to aa98 of SEQ ID NO:14, aa102 to aa104 of SEQ ID NO:14, aa219 to aa221 of SEQ ID NO:14, aa354 to aa356 of SEQ ID NO:14, aa591 to aa593 of SEQ ID NO:14, aa622 to aa624 of SEQ ID NO:14, aa805 to aa807 of SEQ ID NO:14, or any one of the positions identified herein, shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids towards the C-terminus or N-terminus of a polypeptide, protein, or equivalent; aa85 to aa87 of SEQ ID NO:16, aa119 to aa121 of SEQ ID NO:16, aa125 to aa127 of SEQ ID NO:16, aa242 to aa244 of SEQ ID NO:16, aa377 to aa379 of SEQ ID NO:16, aa614 to aa616 of SEQ ID NO:16, aa645 to aa647 of SEQ ID NO:16, aa828 to aa830 of SEQ ID NO:16, or a position shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids towards the C-terminus or N-terminus of a polypeptide, protein, or equivalent; aa82 to aa84 of SEQ ID NO:18, aa116 to aa118 of SEQ ID NO:18, aa122 to aa124 of SEQ ID NO:18, aa239 to aa241 of SEQ ID NO:18, aa374 to aa376 of SEQ ID NO:18, aa611 to aa613 of SEQ ID NO:18, aa642 to aa644 of SEQ ID NO:18, aa825 to aa827 of SEQ ID NO:18, or any one of the positions identified herein, shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids towards the C-terminus or N-terminus of a polypeptide, protein, or equivalent; aa83 to aa85 of SEQ ID NO:26 or 28, aa117 to aa119 of SEQ ID NO:26 or 28, aa123 to aa125 of SEQ ID NO:26 or 28, aa237 to aa239 of SEQ ID NO:26 or 28, aa372 to aa374 of SEQ ID NO:26 or 28, aa609 to aa611 of SEQ ID NO:26 or 28, aa640 to aa642 of SEQ ID NO:26 or 28, aa823 to aa825 of SEQ ID NO:26 or 28, or a position shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids towards the C-terminus or N-terminus of a polypeptide, protein, or equivalent from any one of the positions identified herein; aa62 to aa64 of SEQ ID NO:30, aa96 to aa98 of SEQ ID NO:30, aa102 to aa104 of SEQ ID NO:30, aa216 to aa218 of SEQ ID NO:30, aa351 to aa353 of SEQ ID NO:30, aa588 to aa590 of SEQ ID NO:30, aa619 to aa621 of SEQ ID NO:30, or aa802 to aa804 of SEQ ID NO:30, or any one of the positions identified herein, shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids to the C-terminus or N-terminus of a polypeptide, protein, or equivalent. and at amino acid (aa) positions of the polypeptide, protein or equivalent thereof corresponding to one or more of the positions selected from:

[0146] In some embodiments of any of the disclosures herein, the shifted position is also referred to herein as a position near the reference position. In further embodiments, the reference position is any one of the positions identified herein. In some embodiments, the shifted position still consists of 3, 2, or 1 amino acid residues. For example, a position shifted by one amino acid C-terminally from the reference position of aa62-aa64 of SEQ ID NO: 14 would result in a position corresponding to aa63-aa65 of SEQ ID NO: 14. In some embodiments, a Ube3a polypeptide, protein, or biological equivalent thereof (e.g., a modified Ube3a protein as used herein) comprising one or more of the glycosylation sites comprises, alternatively consists essentially of, or even consists of the sequence of any one or more of SEQ ID NOs: 14, 16, 18, 26, 28, 30, or fragments thereof. In some embodiments, a Ube3a polypeptide, protein, or biological equivalent thereof comprises one or more of the identified glycosylation sites, but is a variant of the sequence of any one or more of SEQ ID NOs: 14, 16, 18, 26, 28, 30, or fragments thereof. In some embodiments, a Ube3a polypeptide, protein, or biological equivalent thereof further comprises one or more different amino acid residues at positions corresponding to any one or more of SEQ ID NOs: 14, 16, 18, 26, 28, 30, wherein the positions are not glycosylation sites as disclosed herein. In one example, a Ube3a polypeptide, protein, or biological equivalent thereof can be created based on a Ube3a naturally occurring variant by mutating one or more amino acid residues of the variant to form a glycosylation site as disclosed herein.

[0147] In some embodiments, the Ube3a polypeptide, protein, or biological equivalent thereof is one of the following: (a) to (e): (a) a position shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from aa62 to aa64 of SEQ ID NO: 14, aa96 to aa98 of SEQ ID NO: 14, aa102 to aa104 of SEQ ID NO: 14, aa219 to aa221 of SEQ ID NO: 14, aa354 to aa356 of SEQ ID NO: 14, aa591 to aa593 of SEQ ID NO: 14, aa622 to aa624 of SEQ ID NO: 14, aa805 to aa807 of SEQ ID NO: 14, or any one of the positions identified herein, towards the C-terminus or N-terminus of a polypeptide, protein, or equivalent; (b) a position shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from aa85 to aa87 of SEQ ID NO: 16, aa119 to aa121 of SEQ ID NO: 16, aa125 to aa127 of SEQ ID NO: 16, aa242 to aa244 of SEQ ID NO: 16, aa377 to aa379 of SEQ ID NO: 16, aa614 to aa616 of SEQ ID NO: 16, aa645 to aa647 of SEQ ID NO: 16, aa828 to aa830 of SEQ ID NO: 16, or any one of the positions identified herein, towards the C-terminus or N-terminus of a polypeptide, protein, or equivalent; (c) a position shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from aa82 to aa84 of SEQ ID NO: 18, aa116 to aa118 of SEQ ID NO: 18, aa122 to aa124 of SEQ ID NO: 18, aa239 to aa241 of SEQ ID NO: 18, aa374 to aa376 of SEQ ID NO: 18, aa611 to aa613 of SEQ ID NO: 18, aa642 to aa644 of SEQ ID NO: 18, aa825 to aa827 of SEQ ID NO: 18, or any one of the positions identified herein, towards the C-terminus or N-terminus of a polypeptide, protein, or equivalent; (d) a position shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from aa83 to aa85 of SEQ ID NO:26 or 28, aa117 to aa119 of SEQ ID NO:26 or 28, aa123 to aa125 of SEQ ID NO:26 or 28, aa237 to aa239 of SEQ ID NO:26 or 28, aa372 to aa374 of SEQ ID NO:26 or 28, aa609 to aa611 of SEQ ID NO:26 or 28, aa640 to aa642 of SEQ ID NO:26 or 28, aa823 to aa825 of SEQ ID NO:26 or 28, or any one of the positions identified herein, to the C-terminus or N-terminus of a polypeptide, protein, or equivalent; and (e) a position shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from aa62 to aa64 of SEQ ID NO: 30, aa96 to aa98 of SEQ ID NO: 30, aa102 to aa104 of SEQ ID NO: 30, aa216 to aa218 of SEQ ID NO: 30, aa351 to aa353 of SEQ ID NO: 30, aa588 to aa590 of SEQ ID NO: 30, aa619 to aa621 of SEQ ID NO: 30, or aa802 to aa804 of SEQ ID NO: 30, or any one of the positions identified herein, to the C-terminus or N-terminus of a polypeptide, protein, or equivalent. The glycosylation site comprises eight amino acid (aa) positions corresponding to the eight aa positions as identified in any one of

[0148] In some embodiments, the Ube3a polypeptide, protein, or biological equivalent thereof is: aa64 of SEQ ID NO:14, aa98 of SEQ ID NO:14, aa104 of SEQ ID NO:14, aa221 of SEQ ID NO:14, aa356 of SEQ ID NO:14, aa591 of SEQ ID NO:14, aa624 of SEQ ID NO:14, aa805 of SEQ ID NO:14, aa806 of SEQ ID NO:14, or any one of the positions identified herein, shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids towards the C-terminus or N-terminus of a polypeptide, protein, or equivalent; aa87 of SEQ ID NO:16, aa121 of SEQ ID NO:16, aa127 of SEQ ID NO:16, aa244 of SEQ ID NO:16, aa379 of SEQ ID NO:16, aa614 of SEQ ID NO:16, aa647 of SEQ ID NO:16, aa828 of SEQ ID NO:16, aa829 of SEQ ID NO:16, or any one of the positions identified herein, shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids towards the C-terminus or N-terminus of a polypeptide, protein, or equivalent; aa84 of SEQ ID NO:18, aa118 of SEQ ID NO:18, aa124 of SEQ ID NO:18, aa241 of SEQ ID NO:18, aa376 of SEQ ID NO:18, aa611 of SEQ ID NO:18, aa644 of SEQ ID NO:18, aa825 of SEQ ID NO:18, aa826 of SEQ ID NO:18, or any one of the positions identified herein, shifted by about 1 amino acid, about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, or about 10 amino acids towards the C-terminus or N-terminus of a polypeptide, protein, or equivalent; aa85 of SEQ ID NO:26 or 28, aa119 of SEQ ID NO:26 or 28, aa125 of SEQ ID NO:26 or 28, aa239 of SEQ ID NO:26 or 28, aa374 of SEQ ID NO:26 or 28, aa609 of SEQ ID NO:26 or 28, aa610 of SEQ ID NO:26 or 28, aa642 of SEQ ID NO:26 or 28, aa823 of SEQ ID NO:26 or 28, or a position shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids towards the C-terminus or N-terminus of a polypeptide, protein, or equivalent; aa64 of SEQ ID NO:30, aa98 of SEQ ID NO:30, aa104 of SEQ ID NO:30, aa218 of SEQ ID NO:30, aa353 of SEQ ID NO:30, aa588 of SEQ ID NO:30, aa589 of SEQ ID NO:30, aa621 of SEQ ID NO:30 or aa802 of SEQ ID NO:30, or any one of the positions identified herein, shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids to the C-terminus or N-terminus of a polypeptide, protein, or equivalent. and (iii) aa positions corresponding to one or more of the positions selected from the group consisting of:

[0149] In some embodiments, the Ube3a polypeptide, protein, or biological equivalent thereof is one of the following: (a) to (e): (a) a position shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from aa64 of SEQ ID NO:14, aa98 of SEQ ID NO:14, aa104 of SEQ ID NO:14, aa221 of SEQ ID NO:14, aa356 of SEQ ID NO:14, aa591 of SEQ ID NO:14, aa624 of SEQ ID NO:14, aa805 of SEQ ID NO:14, and aa806 of SEQ ID NO:14, or any one of the positions identified herein, towards the C-terminus or N-terminus of a polypeptide, protein, or equivalent; (b) aa87 of SEQ ID NO:16, aa121 of SEQ ID NO:16, aa127 of SEQ ID NO:16, aa244 of SEQ ID NO:16, aa379 of SEQ ID NO:16, aa614 of SEQ ID NO:16, aa647 of SEQ ID NO:16, aa828 of SEQ ID NO:16 and aa829 of SEQ ID NO:16, or any one of the positions identified herein, shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids towards the C-terminus or N-terminus of a polypeptide, protein or equivalent; (c) a position shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from aa84 of SEQ ID NO:18, aa118 of SEQ ID NO:18, aa124 of SEQ ID NO:18, aa241 of SEQ ID NO:18, aa376 of SEQ ID NO:18, aa611 of SEQ ID NO:18, aa644 of SEQ ID NO:18, aa825 of SEQ ID NO:18, and aa826 of SEQ ID NO:18, or any one of the positions identified herein, to the C-terminus or N-terminus of the polypeptide, protein, or equivalent; (d) aa85 of SEQ ID NO:26 or 28, aa119 of SEQ ID NO:26 or 28, aa125 of SEQ ID NO:26 or 28, aa239 of SEQ ID NO:26 or 28, aa374 of SEQ ID NO:26 or 28, aa609 of SEQ ID NO:26 or 28, aa610 of SEQ ID NO:26 or 28, aa642 of SEQ ID NO:26 or 28, and aa823 of SEQ ID NO:26 or 28, or any one of the positions identified herein, shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids to the C-terminus or N-terminus of a polypeptide, protein, or equivalent; or (e) aa64 of SEQ ID NO:30, aa98 of SEQ ID NO:30, aa104 of SEQ ID NO:30, aa218 of SEQ ID NO:30, aa353 of SEQ ID NO:30, aa588 of SEQ ID NO:30, aa589 of SEQ ID NO:30, aa621 of SEQ ID NO:30, and aa802 of SEQ ID NO:30, or any one of the positions identified herein, shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids to the C-terminus or N-terminus of a polypeptide, protein, or equivalent. The present invention contains nine mutated amino acid residues at aa positions corresponding to positions identified in any one of the above, thereby creating eight glycosylation sites.

[0150] As shown in Figure 16A, the online tool NetNGlyC was used to predict potential glycosylation sites. Four N-glycosylation sites were identified, among which only the site starting at amino acid 82 of SEQ ID NO: 8 is at a position corresponding to the glycosylation site created by the mutations described in (a) to (c). Moreover, NetNGlyC does not reveal any mutations as disclosed herein.

[0151] In some embodiments, the Ube3a polypeptide, protein, or biological equivalent thereof is one of the following (a') to (e'): (a') a position shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from aa64 of SEQ ID NO:14, aa98 of SEQ ID NO:14, aa221 of SEQ ID NO:14, aa356 of SEQ ID NO:14, aa591 of SEQ ID NO:14, aa624 of SEQ ID NO:14, aa805 of SEQ ID NO:14, and aa806 of SEQ ID NO:14, or any one of the positions identified herein, to the C-terminus or N-terminus of a polypeptide, protein, or equivalent; (b') a position shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from aa87 of SEQ ID NO:16, aa121 of SEQ ID NO:16, aa244 of SEQ ID NO:16, aa379 of SEQ ID NO:16, aa614 of SEQ ID NO:16, aa647 of SEQ ID NO:16, aa828 of SEQ ID NO:16, and aa829 of SEQ ID NO:16, or any one of the positions identified herein, to the C-terminus or N-terminus of a polypeptide, protein, or equivalent; (c') a position shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from aa84 of SEQ ID NO:18, aa118 of SEQ ID NO:18, aa241 of SEQ ID NO:18, aa376 of SEQ ID NO:18, aa611 of SEQ ID NO:18, aa644 of SEQ ID NO:18, aa825 of SEQ ID NO:18, and aa826 of SEQ ID NO:18, or any one of the positions identified herein, towards the C-terminus or N-terminus of a polypeptide, protein, or equivalent; (d') a position shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from aa85 of SEQ ID NO:26 or 28, aa119 of SEQ ID NO:26 or 28, aa239 of SEQ ID NO:26 or 28, aa374 of SEQ ID NO:26 or 28, aa609 of SEQ ID NO:26 or 28, aa610 of SEQ ID NO:26 or 28, aa642 of SEQ ID NO:26 or 28, and aa823 of SEQ ID NO:26 or 28, or any one of the positions identified herein, to the C-terminus or N-terminus of a polypeptide, protein, or equivalent; and (e') a position shifted by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from aa64 of SEQ ID NO:30, aa98 of SEQ ID NO:30, aa218 of SEQ ID NO:30, aa353 of SEQ ID NO:30, aa588 of SEQ ID NO:30, aa589 of SEQ ID NO:30, aa621 of SEQ ID NO:30, and aa802 of SEQ ID NO:30, or any one of the positions identified herein, to the C-terminus or N-terminus of a polypeptide, protein, or equivalent. The present invention contains eight mutated amino acid residues at aa positions corresponding to positions identified in any one of the above, thereby creating seven glycosylation sites.

[0152] In some embodiments, the glycosylation site formed comprises the consensus sequence NXaaT or NXaaS, where Xaa is any amino acid residue, optionally excluding proline (P).

[0153] In some embodiments, the mutated amino acid residue(s) is threonine (Thr or T) or serine (Ser or S) at aa position corresponding to aa64 of SEQ ID NO:14, T or S at aa position corresponding to aa98 of SEQ ID NO:14, T or S at aa position corresponding to aa104 of SEQ ID NO:14, T or S at aa position corresponding to aa221 of SEQ ID NO:14, T or S at aa position corresponding to aa356 of SEQ ID NO:14, asparagine (Asn or N) at aa position corresponding to aa591 of SEQ ID NO:14, T or S at aa position corresponding to aa624 of SEQ ID NO:14, N at aa position corresponding to aa805 of SEQ ID NO:14, and N at aa position corresponding to aa806 of SEQ ID NO:14; T or S at the aa position corresponding to aa87 of SEQ ID NO:16, T or S at the aa position corresponding to aa121 of SEQ ID NO:16, T or S at the aa position corresponding to aa127 of SEQ ID NO:16, T or S at the aa position corresponding to aa244 of SEQ ID NO:16, T or S at the aa position corresponding to aa379 of SEQ ID NO:16, N at the aa position corresponding to aa614 of SEQ ID NO:16, T or S at the aa position corresponding to aa647 of SEQ ID NO:16, N at the aa position corresponding to aa828 of SEQ ID NO:16, and N at the aa position corresponding to aa829 of SEQ ID NO:16; T or S at the aa position corresponding to aa84 of SEQ ID NO:18, T or S at the aa position corresponding to aa118 of SEQ ID NO:18, T or S at the aa position corresponding to aa124 of SEQ ID NO:18, T or S at the aa position corresponding to aa241 of SEQ ID NO:18, T or S at the aa position corresponding to aa376 of SEQ ID NO:18, N at the aa position corresponding to aa611 of SEQ ID NO:18, T or S at the aa position corresponding to aa644 of SEQ ID NO:18, N at the aa position corresponding to aa825 of SEQ ID NO:18, and N at the aa position corresponding to aa826 of SEQ ID NO:18; a T or S at aa position corresponding to aa85 of SEQ ID NO:26 or 28, a T or S at aa position corresponding to aa119 of SEQ ID NO:26 or 28, a T or S at aa position corresponding to aa125 of SEQ ID NO:26 or 28, a T or S at aa position corresponding to aa239 of SEQ ID NO:26 or 28, a T or S at aa position corresponding to aa374 of SEQ ID NO:26 or 28, an N at aa position corresponding to aa609 of SEQ ID NO:26 or 28, an N at aa position corresponding to aa610 of SEQ ID NO:26 or 28, a T or S at aa position corresponding to aa642 of SEQ ID NO:26 or 28, and an N at aa position corresponding to aa823 of SEQ ID NO:26 or 28; or T or S at the aa position corresponding to aa64 of SEQ ID NO:30, T or S at the aa position corresponding to aa98 of SEQ ID NO:30, T or S at the aa position corresponding to aa104 of SEQ ID NO:30, T or S at the aa position corresponding to aa218 of SEQ ID NO:30, T or S at the aa position corresponding to aa353 of SEQ ID NO:30, N at the aa position corresponding to aa588 of SEQ ID NO:30, N at the aa position corresponding to aa589 of SEQ ID NO:30, T or S at the aa position corresponding to aa621 of SEQ ID NO:30, and N at the aa position corresponding to aa802 of SEQ ID NO:30 The compound is selected from one or more of the following:

[0154] In some embodiments, a Ube3a polypeptide, protein, or biological equivalent thereof comprises an amino acid sequence selected from the following: aa21 to aa872 of SEQ ID NO: 14, aa21 to aa895 of SEQ ID NO: 16, aa21 to aa892 of SEQ ID NO: 18, aa21 to aa890 of SEQ ID NO: 26, aa21 to aa890 of SEQ ID NO: 28, aa21 to aa869 of SEQ ID NO: 30, or a sequence having at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to each thereof. In further embodiments, a Ube3a polypeptide, protein, or biological equivalent thereof comprises one or more glycosylation sites as disclosed herein.

[0155] In some embodiments, the Ube3a polypeptide, protein, or biological equivalent thereof further comprises a signal peptide. In further embodiments, the signal peptide is a secretory signal peptide (also referred to herein as a secretory signal). In some embodiments, the signal peptide or secretory signal is selected from an antibody heavy chain / light chain secretory signal, a twin-arginine transport protein secretory signal, an interleukin-2 (IL2) secretory signal, an interleukin-4 (IL4) secretory signal, an interleukin-10 (IL10) secretory signal, an interleukin-3 (IL3) secretory signal, an interleukin-7 (IL7) secretory signal, a human IL2 secretory signal, a human OSM secretory signal, a VSV-G secretory signal, a mouse Ig kappa secretory signal, a human IgG2 H secretory signal, a BM40 secretory signal, a Secretecon secretory signal, a human IgKVIII secretory signal, a CD33 secretory signal, a tPA secretory signal, a human chymotrypsinogen secretory signal, a human trypsinogen-2 secretory signal, a Gaussia luc secretory signal, an albumin (HSA) secretory signal, an influenza hemagglutinin secretory signal, a human insulin secretory signal, or a silkworm fibroin LC. In one embodiment, the signal peptide or secretion signal comprises the amino acid sequence from aa1 to aa20 of SEQ ID NO: 14. In some embodiments, the signal peptide or secretion signal is positioned at the N-terminus of the Ube3a polypeptide, protein, or biological equivalent thereof. In some embodiments, the Ube3a polypeptide, protein, or biological equivalent thereof begins with a signal peptide or secretion signal at its N-terminus.

[0156] In some embodiments, the Ube3a polypeptide, protein, or biological equivalent thereof comprises an amino acid sequence selected from any one of SEQ ID NOs: 14, 16, 18, 26, 28, and 30, or a sequence having at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto, respectively. In further embodiments, the Ube3a polypeptide, protein, or biological equivalent thereof comprises one or more of the glycosylation sites as disclosed herein.

[0157] In some embodiments, the Ube3a protein, polypeptide, or biological equivalent thereof is encoded by a polynucleotide, or equivalent thereof, as disclosed herein. In one aspect, the polynucleotide equivalent maintains at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight of the glycosylation sites in the encoded Ube3a protein, polypeptide, or biological equivalent thereof. In some embodiments, the encoded Ube3a protein, polypeptide, or biological equivalent thereof comprises eight or more glycosylation sites. In some embodiments, a polynucleotide, as disclosed herein, encodes a biological equivalent of a Ube3a protein or polypeptide having one or more non-naturally occurring glycosylation sites.

[0158] In some embodiments, the Ube3a protein, polypeptide, or biological equivalent thereof is encoded by a polynucleotide selected from any one or more of the following: nt 61 to nt 2619 of SEQ ID NO: 13, nt 61 to nt 2688 of SEQ ID NO: 15, nt 61 to nt 2679 of SEQ ID NO: 17, nt 61 to nt 2673 of SEQ ID NO: 25, nt 61 to nt 2673 of SEQ ID NO: 27, nt 61 to nt 2610 of SEQ ID NO: 29; SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, or a sequence having at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to each thereof. In further embodiments, the Ube3a polypeptide, protein, or biological equivalent thereof comprises one or more of the glycosylation sites as disclosed herein.

[0159] In some embodiments, the recombinant and / or isolated polynucleotide comprises, or alternatively, consists essentially of, or even consists of, a sequence selected from any one or more of the following: nt 61 to nt 2619 of SEQ ID NO: 13, nt 61 to nt 2688 of SEQ ID NO: 15, nt 61 to nt 2679 of SEQ ID NO: 17, nt 61 to nt 2673 of SEQ ID NO: 25, nt 61 to nt 2673 of SEQ ID NO: 27, nt 61 to nt 2610 of SEQ ID NO: 29; SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, or a sequence having at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to each thereof. In further embodiments, the Ube3a polypeptide, protein, or biological equivalent thereof comprises one or more of the glycosylation sites as disclosed herein.

[0160] In some embodiments, a mutated amino acid (aa) or nucleotide (nt) residue refers to an aa or nt residue that differs from the residue at the corresponding position in a reference sequence. In some embodiments, a mutated protein, polypeptide, or polynucleotide comprises a mutated aa or nt residue. In some embodiments, the reference sequence is a naturally occurring and / or wild-type sequence. In one embodiment, the reference sequence is an amino acid sequence that comprises, alternatively consists essentially of, or alternatively consists of, an amino acid sequence selected from one or more of SEQ ID NOs: 8, 10, 12, 20, 22, 24, or their respective naturally occurring variants. In one embodiment, the reference sequence is a nucleotide sequence that comprises, alternatively consists essentially of, or alternatively consists of, an amino acid sequence selected from one or more of SEQ ID NOs: 7, 9, 11, 19, 21, 23, or their respective naturally occurring variants.

[0161] In some embodiments, the polynucleotide further comprises a regulatory sequence directing expression of the Ube3a polypeptide, protein, or biological equivalent thereof. In some embodiments, the regulatory sequence comprises one or more of the following: a promoter, an intron, an enhancer, a polyadenylation signal, a terminator, a silencer, a TATA box, or a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). In further embodiments, the polynucleotide as disclosed herein further comprises a promoter operably linked to the polynucleotide for expression of the polynucleotide. Non-limiting examples of such include a Pol II promoter, e.g., a promoter selected from the MNDU3 promoter, the CMV promoter, the PGK promoter, and the EF1 alpha promoter. Sequences of these and other Pol II promoters are known in the art. The sequence of the MNDU3 promoter and the sequence of an exemplary CMV promoter are provided herein. In one embodiment, the MNDU3 promoter comprises, alternatively, consists essentially of, or additionally consists of the sequence of SEQ ID NO:3. In one embodiment, the CMV promoter comprises, or alternatively consists essentially of, or alternatively consists of, a sequence selected from SEQ ID NO: 1, 2, or 34. In one embodiment, the PKG promoter comprises, or alternatively consists essentially of, or alternatively consists of, the sequence of SEQ ID NO: 4. In one embodiment, the MNDU promoter comprises, or alternatively consists essentially of, or alternatively consists of, the sequence of SEQ ID NO: 5. In one embodiment, the EF1 alpha promoter comprises, or alternatively consists essentially of, or alternatively consists of, the sequence of SEQ ID NO: 6. The polynucleotide can further comprise an enhancer element operably linked to the polynucleotide encoding the mutated Ube3a protein to increase or enhance expression of the polynucleotide.

[0162] In a further embodiment, the polynucleotide further comprises a polynucleotide encoding a signal peptide and / or secretion signal positioned 5' to the polynucleotide encoding the modified Ube3a protein. Non-limiting examples of such signal peptides and / or secretion signals include the single-chain fragment variable signal peptide, the twin-arginine transport protein signal peptide, the IL-4 secretion signal, the IL-2 secretion signal, and the IL-10 secretion signal. An exemplary secretory IL-2 secretion signal polynucleotide is provided herein.

[0163] In some embodiments, the polynucleotide further comprises one or more of the following: a polypurine tract sequence (PPT), a central PPT (cPPT), an R region, a U5, an encapsidation signal (Psi), a Rev-responsive element (RRE), a full-length U3 or a fragment thereof, a detectable or selectable marker, a polynucleotide encoding a detectable or selectable polypeptide, a regulatory sequence directing expression of a detectable or selectable polypeptide, or a coding sequence for a cleavable peptide disposed between the coding sequence for the detectable or selectable polypeptide and the sequence encoding the Ube3a polypeptide or protein or a biological equivalent thereof. In some embodiments, the cleavable peptide is a self-cleaving peptide, optionally a 2A self-cleaving peptide. In some embodiments, the 2A self-cleaving peptide is selected from P2A, T2A, E2A, F2A, and BmCPV2A.

[0164] In some embodiments, the genetic information of a viral vector particle (also referred to herein as a vector genome or viral genome) is RNA that comprises, alternatively consists essentially of, or even consists of minimal LTR regions required for vector integration at the 5' and 3' ends, and a polynucleotide as disclosed herein between the two LTR regions. In some embodiments, the two LTR regions further comprise an encapsidation signal (psi region) required for packaging of the vector RNA into the particle. In some embodiments, the psi region is followed by a Rev-responsive element (RRE) and a central polypurine tract sequence (cPPT), which enhance vector production by transporting the full-length vector transcript out of the nucleus for efficient packaging into vector particles.

[0165] Further provided are polynucleotides that are equivalents, complements, reverse sequences, or reverse complements of modified Ube3a-encoding polynucleotides. In some embodiments, equivalent nucleic acids, polynucleotides, or oligonucleotides have at least 70% sequence identity, or alternatively at least 75% sequence identity, or alternatively at least 80% sequence identity, or alternatively at least 85% sequence identity, or alternatively at least 90% sequence identity, or alternatively at least 91% sequence identity, or alternatively at least 92% sequence identity, or alternatively at least 93% sequence identity, or alternatively at least 94% sequence identity, or alternatively at least 95% sequence identity, or alternatively at least 96% sequence identity, or alternatively at least 97% sequence identity, or alternatively at least 98% sequence identity, or alternatively at least 99% sequence identity to a reference nucleic acid, polynucleotide, or oligonucleotide. Additionally or alternatively, an equivalent nucleic acid, polynucleotide or oligonucleotide hybridizes under high stringency conditions to any one of the reference polynucleotide, its complement or its reverse complement.

[0166] Additionally or alternatively, the equivalent nucleic acid, polynucleotide, or oligonucleotide should encode a functional Ube3a protein, polypeptide, or biological equivalent thereof, which can be identified, as appropriate, by one or more assays described herein. In some embodiments, the equivalent nucleic acid, polynucleotide, or oligonucleotide has at least 70% sequence identity, or alternatively at least 75% sequence identity, or alternatively at least 80% sequence identity, or alternatively at least 85% sequence identity, or alternatively at least 90% sequence identity, or alternatively at least 91% sequence identity, or alternatively at least 92% sequence identity, or alternatively at least 93% sequence identity, or alternatively at least 94% sequence identity, or alternatively at least 95% sequence identity, or alternatively at least 96% sequence identity, or alternatively at least 97% sequence identity, or alternatively at least 98% sequence identity, or alternatively at least 99% sequence identity to the reference nucleic acid, polynucleotide, or oligonucleotide. Additionally or alternatively, an equivalent nucleic acid, polynucleotide or oligonucleotide hybridizes under high stringency conditions to any one of the reference polynucleotide, its complement or its reverse complement.

[0167] Some embodiments provide that one or more polynucleotides identified herein with one or more glycosylation sites are mutated from a polynucleotide selected from any one of SEQ ID NOs: 7, 9, 11, 19, 21, or 23. Some embodiments provide that one or more polynucleotides identified herein with one or more glycosylation sites are mutated not from a polynucleotide selected from any one of SEQ ID NOs: 7, 9, 11, 19, 21, or 23, but from their respective naturally occurring variants. Some embodiments provide that one or more polynucleotides identified herein with one or more glycosylation sites further comprise one or more mutations that do not form a glycosylation site.

[0168] The polynucleotides can further include polynucleotides that are or encode detectable or purification markers.

[0169] Further provided are recombinant and / or isolated polypeptides encoded by the polynucleotides and their respective equivalents. In some embodiments, an equivalent or biologically equivalent protein or polypeptide is one that has at least 70% sequence identity, or alternatively at least 75% sequence identity, or alternatively at least 80% sequence identity, or alternatively at least 85% sequence identity, or alternatively at least 90% sequence identity, or alternatively at least 91% sequence identity, or alternatively at least 92% sequence identity, or alternatively at least 93% sequence identity, or alternatively at least 94% sequence identity, or alternatively at least 95% sequence identity, or alternatively at least 96% sequence identity, or alternatively at least 97% sequence identity, or alternatively at least 98% sequence identity, or alternatively at least 99% sequence identity to a reference protein or polypeptide and / or a polypeptide or protein as disclosed herein (such as any one of the polypeptides or proteins encoded by SEQ ID NOs: 8, 10, 12, 20, 22 or 24, or an equivalent polynucleotide as noted herein).

[0170] In some embodiments, equivalent or biologically equivalent proteins or polypeptides are functional proteins, which can be identified, as appropriate, by one or more of the assays described herein. In some embodiments, an equivalent or biologically equivalent protein or polypeptide has at least 70% sequence identity, or alternatively at least 75% sequence identity, or alternatively at least 80% sequence identity, or alternatively at least 85% sequence identity, or alternatively at least 90% sequence identity, or alternatively at least 91% sequence identity, or alternatively at least 92% sequence identity, or alternatively at least 93% sequence identity, or alternatively at least 94% sequence identity, or alternatively at least 95% sequence identity, or alternatively at least 96% sequence identity, or alternatively at least 97% sequence identity, or alternatively at least 98% sequence identity, or alternatively at least 99% sequence identity to a reference protein or polypeptide and / or a polypeptide or protein as disclosed herein (such as any one of the polypeptides or proteins encoded by SEQ ID NOs: 8, 10, 12, 20, 22 or 24, or an equivalent polynucleotide as noted herein).

[0171] Some embodiments provide that one or more amino acid residues identified herein as mutated to form a potential glycosylation site in a Ube3a protein, polypeptide, or biological equivalent thereof are mutated from a polypeptide selected from any one of SEQ ID NOs: 8, 10, 12, 20, 22, or 24. Some embodiments provide that one or more amino acid residues identified herein as mutated to form a potential glycosylation site in a Ube3a protein, polypeptide, or biological equivalent thereof are mutated not from a polypeptide selected from any one of SEQ ID NOs: 8, 10, 12, 20, 22, or 24, but from their respective naturally occurring variants. Some embodiments provide that one or more amino acid residues identified herein as mutated to form a potential glycosylation site in a Ube3a protein, polypeptide, or biological equivalent thereof further comprise one or more mutations that do not form a glycosylation site. Some embodiments provide that one or more amino acid residues identified herein as mutated to create a potential glycosylation site in a Ube3a protein, polypeptide, or biological equivalent thereof are not mutated from a polypeptide selected from a non-naturally occurring variant of any one of SEQ ID NOs: 8, 10, 12, 20, 22, or 24. In further embodiments, such a non-naturally occurring variant is a Ube3a biological equivalent of the corresponding SEQ ID NO: 8, 10, 12, 20, 22, or 24.

[0172] The polypeptides can further comprise a detectable or purification marker. Polypeptides and proteins can be expressed in any suitable system, e.g., a prokaryotic or eukaryotic system, such as, for example, mammalian or human cells. vector

[0173] The present disclosure also provides vectors comprising, alternatively consisting essentially of, or alternatively consisting of a polynucleotide as disclosed herein, optionally inserted into a viral backbone. In some embodiments, the vector is selected for expression in prokaryotic or eukaryotic cells. In some embodiments, the vector comprises, alternatively consisting essentially of, or alternatively consisting of a polynucleotide as described herein encoding a modified protein. In some embodiments, the vector comprises, alternatively consisting essentially of, or alternatively consisting of a polynucleotide as described herein that enables replication of the polynucleotide (also referred to herein as a modified gene). In further embodiments, the vector further comprises a regulatory sequence operably linked to the modified gene and directing replication of the modified gene. In still further embodiments, the regulatory sequence comprises, alternatively consisting essentially of, or alternatively consisting of one or more of the following: a promoter, an intron, an enhancer, a polyadenylation signal, a terminator, a silencer, a TATA box, or a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE).

[0174] In some embodiments, the vector is a non-viral vector, optionally a plasmid. In some embodiments, the vector is a viral vector, optionally selected from a retroviral vector (such as a lentiviral vector), an adenoviral vector, an adeno-associated viral vector, or a herpes viral vector. In further embodiments, the viral backbone contains essential nucleic acids or sequences for integration of the modified gene into the genome of the target cell. In some embodiments, the essential nucleic acids required for integration into the genome of the target cell include the minimum LTR regions required for vector integration at the 5' and 3' ends.

[0175] In some embodiments, the term "vector" refers to a recombinant vector that retains the ability to infect and transduce non-dividing and / or slowly-dividing cells and integrate into the genome of target cells. In some embodiments, the vector is derived from or based on a wild-type virus. In further embodiments, the vector is derived from or based on a retrovirus, such as a wild-type adenovirus, adeno-associated virus, or lentivirus. Examples of retroviruses include, but are not limited to, human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). Alternatively, it is contemplated that other retroviruses, such as murine leukemia virus (MLV), can be used as the basis for the vector backbone. It should be apparent that viral vectors according to the present disclosure need not be limited to components of a particular virus. Viral vectors can include components from two or more different viruses and can also include synthetic components. Vector components can be engineered to obtain desired characteristics, such as target cell specificity.

[0176] The recombinant vectors of the present disclosure are derived from primates and non-primates. Examples of primate lentiviruses include human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS), and simian immunodeficiency virus (SIV). Non-primate lentiviruses include the prototype "slow virus" Visna / Maedi virus (VMV), as well as related caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV). Prior art recombinant lentiviral vectors are known in the art; see, for example, U.S. Patent Nos. 6,924,123; 7,056,699; 7,419,829, and 7,442,551, which are incorporated herein by reference. In some embodiments, the lentiviral vector is a self-inactivating lentiviral vector. In a further embodiment, the lentiviral vector has a U3 region that lacks a TATA box. Additionally or alternatively, the lentiviral vector has a U3 region that lacks one or more transcription factor binding sites.

[0177] U.S. Patent No. 6,924,123 discloses that certain retroviral sequences facilitate integration into the target cell genome. This patent teaches that each retroviral genome contains genes called gag, pol, and env, which encode virion proteins and enzymes. These genes are flanked at both ends by regions called long terminal repeats (LTRs). LTRs are responsible for proviral integration and transcription. They also function as enhancer-promoter sequences. In other words, LTRs can control viral gene expression. Retroviral RNA encapsidation occurs based on the psi sequence located at the 5' end of the viral genome. The LTRs themselves are identical sequences that can be divided into three elements called U3, R, and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of the three elements can vary considerably among different retroviruses. For the viral genome, the site of poly(A) addition (termination) is at the boundary between R and U5 in the right LTR. U3 contains most of the transcriptional control elements of the provirus, including the promoter and multiple enhancer sequences responsive to cellular and, in some cases, viral transcriptional activator proteins.

[0178] Regarding the structural genes gag, pol, and env themselves, gag encodes the internal structural protein of the virus. The Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid), and NC (nucleocapsid). The pol gene encodes the reverse transcriptase (RT), which contains the DNA polymerase, the associated RNase H, and the integrase (IN), which mediate genome replication.

[0179] For the production of viral vector particles, the vector RNA genome is expressed in a host cell from a DNA construct that encodes it. Components of the particle not encoded by the vector genome are provided in trans by additional nucleic acid sequences ("packaging systems" that typically include either or both of the gag / pol and env genes) expressed in the host cell. The set of sequences required for the production of viral vector particles can be introduced into the host cell by transient transfection, integrated into the host cell genome, or provided in a variety of ways. The techniques involved are known to those skilled in the art.

[0180] Retroviral vectors for use in this disclosure include, but are not limited to, Invitrogen's pLenti series versions 4, 6, and 6.2 "ViraPower" systems, manufactured by Lentigen Corp.; pHIV-7-GFP, laboratory-produced and used by City of Hope Research Institute; "Lenti-X" lentiviral vector, pLVX, manufactured by Clontech; pLKO.1-puro, manufactured by Sigma-Aldrich; pLemiR, manufactured by Open Biosystems; and pLV, laboratory-produced and used by Charite Medical School, Institute of Virology (CBF), Berlin, Germany.

[0181] Thus, in one aspect, a vector is provided comprising a recombinant polynucleotide as disclosed herein encoding a Ube3a protein, polypeptide, or biological fragment thereof having one or more glycosylation sites for use in gene therapy and research. In some embodiments, the Ube3a protein is naturally occurring. In some embodiments, it is recombinantly produced by modifying one or more nucleotides to modify amino acids. In some embodiments, the Ube3a protein, polypeptide, or biological fragment thereof has three or more glycosylation sites. In some embodiments, the Ube3a protein, polypeptide, or biological fragment thereof has four or more glycosylation sites. In some embodiments, the protein, polypeptide, or biological fragment thereof is encoded by a polynucleotide set forth in the Sequence Listing and their respective equivalents. In some embodiments, the equivalents maintain at least one or more identified glycosylation sites. In some embodiments, the Ube3a protein, polypeptide, or biological fragment thereof has eight or more glycosylation sites. In further embodiments, the polynucleotide further comprises a nucleotide sequence encoding a cell-penetrating domain located downstream of the signal sequence.

[0182] In some embodiments, the vector comprises a polynucleotide and a promoter operably linked to the polynucleotide. Non-limiting examples of such promoters include a pol II promoter, optionally selected from the group consisting of the MNDU3 promoter, the minimal cytomegalovirus (CMV) promoter, the phosphoglycerate kinase (PKG) promoter, and the EF1 alpha promoter. In some embodiments, the vector further comprises a polynucleotide encoding a secretion signal located 5' to the polynucleotide encoding the modified Ube3a protein. Non-limiting examples of such secretion signals include a single-chain fragment variable secretion signal, a twin-arginine transport protein secretion signal, an IL-4 secretion signal, an IL-2 secretion signal, and an IL-10 secretion signal. Exemplary secretion signal polynucleotides include, but are not limited to, the polynucleotides set forth in the sequence listing provided below and their equivalents. The vector can further comprise a polynucleotide that is or encodes a detectable or purification marker. Alternative polymerase II promoters include, but are not limited to, LTRs from retroviral and lentiviral vectors.

[0183] In some embodiments, the polynucleotide and / or vector further comprises a marker or detectable label, such as a gene encoding enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), etc., which are commercially available and described in the art. They can be expressed from the same or separate regulatory sequences, such as a promoter driving expression of the modified Ube3a protein. In some embodiments, the promoter is a PGK promoter.

[0184] In some embodiments, the vector comprises a sequence encoding a cell permeability domain, which can include, alternatively consist essentially of, or even consist of, for example, a human immunodeficiency virus transcriptional transactivator (HIV-TAT) peptide.

[0185] A CPP used according to one embodiment of the present disclosure can contain 3 to 35 amino acids, preferably 5 to 25 amino acids, more preferably 10 to 25 amino acids, or even more preferably 15 to 25 amino acids.

[0186] CPPs suitable for carrying out one aspect of the present disclosure can contain at least one basic amino acid, such as arginine, lysine, and histidine. In some embodiments, the CPP can contain more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such basic amino acids, or alternatively, about 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% of the amino acids are basic amino acids. In one embodiment, the CPP contains at least two consecutive basic amino acids, or alternatively, at least three or at least five consecutive basic amino acids. In certain embodiments, the CPP contains at least two, three, four, or five consecutive arginines. In further embodiments, the CPP contains more arginine than lysine or histidine, or preferably more arginine than combined lysine and histidine.

[0187] The CPP may contain acidic amino acids, but the number of acidic amino acids should be less than the number of basic amino acids. In one embodiment, the CPP contains at most one acidic amino acid. In a preferred embodiment, the CPP does not contain any acidic amino acids. In a specific embodiment, a suitable CPP is the HIV-TAT peptide.

[0188] In some embodiments, a vector is provided as shown in any one of Figures 1A-1F, with or without a detectable label or purification marker. In some embodiments, the vector comprises the sequence of SEQ ID NO: 35 and further comprises a polynucleotide sequence as disclosed herein inserted after the MNDU3 promoter in the sequence of SEQ ID NO: 35.

[0189] In some embodiments, vectors (such as retroviral and / or lentiviral vectors) produced by those shown in any one of Figures 1A-1F are provided. In further embodiments, the retroviral and / or lentiviral vectors comprise a polynucleotide (such as an RNA) encoded by any of the vectors shown in any one of Figures 1A-1F. In still further embodiments, the retroviral and / or lentiviral vectors comprise a polynucleotide (such as an RNA) encoded by a vector comprising the sequence of SEQ ID NO:35, and further comprising a polynucleotide (such as an RNA) encoding a Ube3a protein, polypeptide, or biological equivalent thereof, or the reverse complement of a Ube3a-encoding polynucleotide, inserted after the MNDU3 promoter in the sequence of SEQ ID NO:35.

[0190] In some embodiments, a vector (such as a retroviral and / or lentiviral vector) is provided that includes a polynucleotide (such as an RNA) or the reverse complement of a polynucleotide encoding a Ube3a protein, polypeptide, or biological equivalent thereof. In some embodiments, the polynucleotide of the vector (such as a retroviral and / or lentiviral vector) comprises, alternatively, consists essentially of, or even consists of, one or more of the following, optionally from 5' to 3': (1) R region, (2) U5, (3) Psi, (4) RRE, (5) a promoter (such as the MNDU3 promoter), (6) a polynucleotide (such as an RNA) or the reverse complement of a polynucleotide encoding a Ube3a protein, polypeptide, or biological equivalent thereof, (7) U3, (8) R region, and (9) U5.

[0191] Further included are polypeptides encoded by these polynucleotides, vectors, and host cell systems. Packaging System

[0192] The present disclosure also provides a viral packaging system comprising a vector as described herein, optionally with a viral backbone derived from a virus; a packaging plasmid; and an envelope plasmid. The packaging plasmid contains polynucleotides encoding nucleosides, matrix proteins, capsids, and other components necessary for packaging the vector genome into viral particles. Packaging plasmids are described in patent documents, such as U.S. Patent Nos. 7,262,049; 6,995,258; 7,252,991; and 5,710,037, which are incorporated herein by reference.

[0193] The system can also contain a plasmid encoding a pseudotyped envelope protein provided by the envelope plasmid. Pseudotyped viral vectors consist of vector particles that contain glycoproteins from other enveloped viruses, or alternatively contain functional portions. See, for example, U.S. Patent No. 7,262,049, incorporated herein by reference. In some embodiments, the envelope plasmid optionally encodes an envelope protein that does not cause the viral particle to nonspecifically bind to cells or cell populations. Specificity of the viral particle can be conferred by a protein or polypeptide, such as an antibody binding domain, inserted into the particle envelope. Examples of suitable envelope proteins include, but are not limited to, envelope proteins containing VSVG or RD114 domains.

[0194] The present disclosure also provides suitable packaging cell lines.In one embodiment, the packaging cell line is HEK-293 cell line.Other suitable cell lines are known in the art and are described in, for example, U.S. Patent Nos. 7,070,994; 6,995,919; 6,475,786; 6,372,502; 6,365,150 and 5,591,624, each of which is incorporated herein by reference. Viral particles and methods for producing viral particles

[0195] The present disclosure further provides a method for producing viral particles comprising a Ube3a protein, polynucleotide, or biological equivalent thereof, comprising, alternatively, consisting essentially of, or alternatively, consisting of transducing a packaging cell system with the viral system described above under conditions suitable for packaging the viral vector. Such conditions are known in the art and are briefly described herein. Viral particles can be isolated from the cell supernatant using methods known to those skilled in the art, e.g., centrifugation. Such isolated particles are further provided by the present disclosure.

[0196] The present disclosure further provides an isolated viral particle produced by the method, which comprises, alternatively consists essentially of, or even alternatively consists of a polynucleotide as disclosed herein.

[0197] The present disclosure also provides methods for preparing viral particles comprising a polynucleotide as disclosed herein, such as a modified Ube3a gene as disclosed herein, by transducing a packaging cell line as described herein with a vector, an envelope plasmid, and a packaging plasmid under conditions that facilitate packaging of the vector into an enveloped particle. In some embodiments, the viral particles are pseudotyped viral particles. In further embodiments, the particles are isolated from cell supernatants and conjugated to antibodies for cell-specific targeting.

[0198] In some embodiments, the genetic information of a viral vector particle (also referred to herein as a vector genome or viral genome) is RNA that comprises, alternatively consists essentially of, or even consists of minimal LTR regions required for vector integration at the 5' and 3' ends, and a polynucleotide as disclosed herein between the two LTR regions. In some embodiments, the two LTR regions further comprise an encapsidation signal (psi region) required for packaging of the vector RNA into the particle. In some embodiments, the psi region is followed by a Rev-responsive element (RRE) and a central polypurine tract sequence (cPPT), which enhance vector production by transporting the full-length vector transcript out of the nucleus for efficient packaging into vector particles.

[0199] In some embodiments, the vector further comprises a polymerase II promoter, such as MNDU3, that drives expression of the modified Ube3a gene. In some embodiments, the vector comprises a marker, such as an EGFP gene (enhanced green fluorescent protein), optionally driven by a polymerase II promoter, such as the PGK promoter. The EGFP gene is used as a reporter gene to detect transduced cells.

[0200] In some embodiments, the listed genetic elements are transcribed into full-length RNA molecules, which contain all of the genetic information that is packaged into vector particles and incorporated into transduced cells.

[0201] In some embodiments, the full-length RNA transcript is packaged inside the capsid of a vector particle containing a nucleocapsid, capsid, and matrix protein generated from a packaging plasmid such as Delta-8.91. In some embodiments, a reverse transcriptase polymerase generated from the packaging plasmid Delta-8.91 is also disposed within the capsid along with the RNA transcript. In some embodiments, the capsid encases and protects the full-length RNA transcript.

[0202] In some embodiments, packaging cell line cells, such as HEK-293T cells, are plated at 75% confluence in complete DMEM medium 24 hours prior to transfection. At least 24 hours after plating the cells, a transfection mixture is prepared. Three milliliters of serum-free medium is incubated with 150 μl of lipofection reagent for 20 minutes at room temperature. Plasmids are then added to the medium / lipofection reagent mixture at a ratio of 5:5:2 (packaging plasmid:viral vector plasmid:envelope plasmid) and incubated for 30 minutes. After this final incubation period, the medium / lipofection reagent / DNA mixture is then added to the HEK-293T cells and left overnight for transfection to occur. The next day, the transfection medium is removed and fresh complete DMEM is added. After 72 hours, the cell culture supernatant can be collected and concentrated by ultracentrifugation at 20,000 rpm for 1.5 hours.

[0203] Once the vector particles bud from the packaging cells and are released into the supernatant, the vector particles can be isolated and / or purified by antibodies that specifically recognize or bind to the particles and / or by having conjugated antibodies on the particle's envelope as defined herein. Cells and cell populations

[0204] Provided herein are cells that comprise one or more of the following: a recombinant polynucleotide as disclosed herein, a vector as disclosed herein, a recombinant Ube3a protein, polypeptide, or biological equivalent thereof as disclosed herein, and thereby produce the polynucleotide, vector, or recombinant Ube3a protein, polypeptide, or biological equivalent thereof. In some embodiments, the cell is an isolated cell and / or an engineered cell. In some embodiments, the cell is a eukaryotic or prokaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an in vitro and / or ex vivo cell. In some embodiments, the cell is an in vivo cell in a subject.

[0205] Clonal populations of cells as disclosed herein are also provided.

[0206] Additionally provided is a method for expressing a secreted Ube3a protein, polypeptide, or biological equivalent thereof, comprising growing a cell as disclosed herein under conditions that allow expression of the recombinant Ube3a protein or polypeptide, or biological equivalent thereof.

[0207] Also provided are cells or cell populations comprising, alternatively consisting essentially of, or even consisting of one or more of the following: a polynucleotide as disclosed herein, a vector as disclosed herein, and a Ube3a protein, polypeptide, or biological equivalent thereof as disclosed herein. In some embodiments, the vector is a viral particle. In some embodiments, the cell or cell population further comprises a detectable marker.

[0208] In some embodiments, the cell is an isolated cell. In some embodiments, the cell is not a naturally occurring cell. In some embodiments, the cell is also referred to herein as a host cell. In some embodiments, the isolated host cell is a packaging cell line. In some embodiments, the cell is a eukaryotic cell, such as a mammalian cell. In further embodiments, the cell is a murine cell or a human cell.

[0209] Additionally or alternatively, the cell is a progenitor cell or its progeny. In some embodiments, the cell is a stem cell, such as an embryonic stem cell, an induced pluripotent stem cell (iPSC), an adult stem cell, a mesenchymal stem cell, a neural stem cell, a hematopoietic stem cell (HSC), or their respective progeny. In some embodiments, the vector and / or host cell can further comprise a detectable or purification label.

[0210] In some embodiments, the cells are stem cells, such as hematopoietic progenitor cells or hematopoietic stem cells, e.g., CD34+ cells. Alternatively, the stem cells are neural stem cells or iPSCs.

[0211] In some embodiments, the cells are immune cells, optionally selected from B cells, T cells, Natural Killer (NK) cells, dendritic cells, cells of myeloid lineage, neutrophils, monocytes, macrophages, and / or microglia. In further embodiments, the immune cells are derived from progenitor cells (such as hematopoietic progenitor cells), stem cells (e.g., embryonic stem cells, induced pluripotent stem cells (iPSCs), adult stem cells, mesenchymal stem cells, neural stem cells, hematopoietic stem cells (HSCs)), or their respective progeny. In some embodiments, the T cells express CD4, i.e., are CD4+ T cells. In some embodiments, the T cells express CD8, i.e., are CD8+ T cells.

[0212] When used therapeutically, the cells can be allogeneic or autologous to the subject to be treated. The subject can be a mammal, such as a murine, canine, bovine, equine, ovine, feline, or human subject or patient.

[0213] In some embodiments, the cells express and / or secrete a recombinant Ube3a protein or polypeptide, or a biological equivalent thereof, as disclosed herein.

[0214] Populations of cells and / or their progeny as disclosed herein are also provided.

[0215] The present disclosure further provides isolated cells or enriched populations of cells, optionally induced or differentiated from the stem cells described above. In some examples, the induced or differentiated cells or enriched populations of cells comprise, consist essentially of, or even consist of immune cells. In some examples, the immune cells are selected from B cells, T cells, natural killer (NK) cells, dendritic cells, myeloid lineage cells, and / or neutrophils. In some embodiments, the T cells express CD4, i.e., are CD4+ T cells. In some embodiments, the T cells express CD8, i.e., are CD8+ T cells. In some examples, the isolated cells or enriched populations of immune cells comprise, consist essentially of, or even consist of monocytes, macrophages, and / or microglia. In some cases, one or more types of immune cells described herein are modified with a recombinant polynucleotide encoding a Ube3a protein described herein to generate Ube3a-expressing immune cells. In some cases, B cells, T cells, NK cells, dendritic cells, neutrophils, or cells of myeloid lineage are modified (e.g., transduced or transfected) with a recombinant polynucleotide encoding a Ube3a protein described herein to generate modified cells that express a Ube3a protein, polypeptide, or biological equivalent thereof. In some cases, macrophages are modified (e.g., transduced or transfected) with a recombinant polynucleotide encoding a modified Ube3a protein described herein to generate Ube3a-expressing macrophages in vivo and / or in vitro. In some cases, CD34+ HSCs are modified (e.g., transduced or transfected) with a recombinant polynucleotide encoding a modified Ube3a protein described herein to generate Ube3a-expressing HSCs and / or macrophages in vivo and / or in vitro.

[0216] In some embodiments, the cell population expresses CD4, CD14, and HLADR. In some embodiments, at least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the cells in the population are CD4+, i.e., optionally express CD4 on the cell surface. Additionally or alternatively, at least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the cells in the population are CD14+, i.e., optionally express CD14 on the cell surface. Additionally or alternatively, at least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the cells in the population are HLA-DR+, i.e., optionally express HLA-DR on the cell surface.

[0217] In some embodiments, the cell population induces macrophages under suitable conditions. See Experimental Methods for examples.

[0218] In some embodiments, the cell populations substantially comprise macrophages, optionally induced from stem cells, such as HSCs. In some embodiments, the cell populations substantially comprise stem cells, such as HSCs, optionally induced into macrophages.

[0219] In some embodiments, the cell population is substantially homogeneous, e.g., at least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the cells in the population are the same.

[0220] These cells are useful for treating and / or preventing Angelman syndrome in a subject in need thereof, or for testing new therapies. The subject may be a fetus, infant, juvenile, or adult. Compositions, Screening and Therapeutic Uses

[0221] The present disclosure provides compositions comprising, alternatively consisting essentially of, or even consisting of any one or more of a polynucleotide as disclosed herein, a Ube3a protein, polypeptide or biological equivalent thereof as disclosed herein, a vector as disclosed herein, a cell as disclosed herein, a cell population as disclosed herein, a clonal population as disclosed herein, and / or a packaging system as disclosed herein, and a carrier. In some embodiments, the carrier is a pharmaceutically acceptable carrier.

[0222] Also provided by the present disclosure are kits comprising, alternatively consisting essentially of, or even consisting of, any one or more of: a probe for detecting a defective Ube3a gene, a polynucleotide as disclosed herein, a Ube3a protein, polypeptide or biological equivalent thereof as disclosed herein, a vector as disclosed herein, a cell as disclosed herein, a cell population as disclosed herein, a clonal population as disclosed herein, a packaging system as disclosed herein, and / or a composition as disclosed herein, optionally with instructions for use. In some embodiments, the instructions are for use in a method as disclosed herein.

[0223] These compositions and / or kits can be used diagnostically or therapeutically as described herein. Additionally or alternatively, these compositions can be used in combination with other known treatments.

[0224] The compositions can be used in vitro to screen for small molecules and other agents that may alter the efficacy of a treatment, alone or in combination with other treatments, by adding various amounts of the agent to be tested to the composition and comparing it to a companion system that does not contain the agent but that exhibits the desired therapeutic effect, as optionally achieved by one or more of the polynucleotides as disclosed herein, Ube3a proteins, polypeptides or biological equivalents thereof as disclosed herein, vectors as disclosed herein, cells as disclosed herein, cell populations as disclosed herein, and / or compositions as disclosed herein.

[0225] When the polynucleotides, vectors, polypeptides, cells and / or compositions are administered to a suitable animal subject, the animal subject can be used as an animal model for testing alternative treatments in the same manner as in vitro screening.

[0226] Also provided are methods for expressing a Ube3a protein, polypeptide, or biological equivalent thereof, comprising, alternatively, consisting essentially of, or alternatively, consisting of growing a host cell as described herein under conditions that allow expression of the Ube3a protein, polypeptide, or biological equivalent thereof. The methods can be performed in vitro, ex vivo, or in vivo. In some embodiments, the expressed Ube3a protein, polypeptide, or biological equivalent thereof is optionally secreted from the cell expressing the protein, polypeptide, or biological equivalent thereof.

[0227] Further provided are methods for expressing a Ube3a protein, polypeptide, or biological equivalent thereof in a subject, e.g., comprising, alternatively, consisting essentially of, or even consisting of, administering to a subject an effective amount of one or more of a polynucleotide as disclosed herein, a vector as disclosed herein, and / or a cell as described herein, thereby expressing Ube3a in the subject. In some embodiments, the polynucleotide encodes a Ube3a protein, polypeptide, or biological equivalent thereof, and the protein, polypeptide, or biological equivalent thereof has one or more glycosylation sites. In further embodiments, the one or more glycosylation sites are not naturally occurring.

[0228] In some embodiments, the expressed Ube3a protein, polypeptide, or biological equivalent thereof is secreted from cells that produce such protein, polypeptide, or biological equivalent. In some embodiments, the expressed Ube3a protein, polypeptide, or biological equivalent thereof is secreted into the blood of a subject. In further embodiments, the expressed Ube3a protein, polypeptide, or biological equivalent thereof is secreted into the peripheral blood of a subject. Additionally or alternatively, the expressed Ube3a protein, polypeptide, or biological equivalent thereof is secreted into the brain of a subject across the blood-brain barrier. In some embodiments, the expressed Ube3a protein, polypeptide, or biological equivalent thereof binds to and, if necessary, enters neuronal cells.

[0229] In some embodiments, the subject is a mammal, e.g., a human patient. In some embodiments, the subject lacks or has a defective Ube3a gene. In some embodiments, the subject is asymptomatic for Angelman syndrome or Prader-Willi syndrome, or symptomatic for these syndromes. In some embodiments, the subject is a fetus, infant, or prepubertal subject. In some embodiments, the subject is an adult.

[0230] Further provided are methods for treating, preventing, arresting or reversing Angelman syndrome in a subject carrying a defective Ube3a gene or allele, e.g., comprising, or alternatively consisting essentially of, or alternatively consisting of, administering to the subject an effective amount of one or more of a polynucleotide as disclosed herein, a vector as disclosed herein, a Ube3a protein, polypeptide or biological equivalent thereof as disclosed herein, a cell as described herein, a cell population as disclosed herein, and / or a composition as disclosed herein, thereby expressing the Ube3a protein, polypeptide or biological equivalent thereof and / or delivering the Ube3a protein, polypeptide or biological equivalent thereof into the brain and / or to neurons of the subject, and / or treating Angelman syndrome and / or Prader-Willi syndrome.

[0231] In some embodiments, the subject lacks the Ube3A gene or has a defective Ube3A gene. In some embodiments, the subject is a mammal, e.g., a human patient. In some embodiments, the subject is asymptomatic for Angelman syndrome. In some embodiments, the subject is a fetus, infant, or prepubertal subject. In some embodiments, the subject is an adult.

[0232] Also provided are methods for enhanced delivery of a Ube3a protein, polypeptide, or biological equivalent thereof, in the brain and / or to neurons, comprising, or alternatively consisting essentially of, or additionally consisting of, e.g., administering to a subject an effective amount of one or more of a polynucleotide as disclosed herein, a vector as disclosed herein, a Ube3a protein, polypeptide, or biological equivalent thereof as disclosed herein, a cell as described herein, a cell population as disclosed herein, and / or a composition as disclosed herein, thereby expressing the Ube3a protein, polypeptide, or biological equivalent thereof, and / or delivering the Ube3a protein, polypeptide, or biological equivalent thereof, in the brain and / or to neurons of the subject, and / or treating Angelman syndrome.

[0233] In some embodiments of any of the methods, compositions, etc. disclosed herein, the subject lacks or possesses a defective Ube3A gene. In further embodiments, the subject comprises and / or expresses a defective Ube3A protein. In one embodiment, the defective Ube3A protein is not a biological equivalent of the Ube3A protein. In one embodiment, the defective Ube3A protein performs a Ube3A function, such as ubiquitination of S5a or another protein, at a level less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.1% of the wild-type level. In yet a further embodiment, the subject comprises and / or expresses a Ube3A protein or a biological equivalent thereof at a reduced level compared to a healthy control. In one embodiment, the healthy control is a subject free of any disease. In another embodiment, the healthy control is a subject free of a disease as disclosed herein. In yet another embodiment, the healthy control is a subject free of AS. In one embodiment, the subject contains and / or expresses Ube3A protein or a biological equivalent thereof at a level less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.1% of that of a healthy control. In some embodiments, the subject is a mammal, e.g., a human patient. In some embodiments, the subject is asymptomatic for Angelman syndrome. In some embodiments, the subject is symptomatic for Angelman syndrome. In some embodiments, the subject is a fetus, infant, or prepubertal subject. In some embodiments, the subject is an adult.

[0234] Additional effective treatments can be combined with the present disclosure and / or added as needed.

[0235] In some embodiments, an "effective amount" is delivered, i.e., an amount sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages. Such delivery depends on several variables, including the period for which an individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. However, it is understood that the specific dosage level of the therapeutic agent of the present disclosure for any particular subject will depend on various factors, including the activity of the specific compound being used, the subject's age, weight, general health, sex, and diet, the time of administration, the excretion rate, the drug combination, and the severity of the particular disorder being treated, and the form of administration. Treatment dosages can generally be titrated to optimize safety and efficacy. Typically, dosage-effect relationships from in vitro and / or in vivo studies can initially provide useful guidance regarding appropriate dosages for patient administration. Generally, it is desired to administer an amount of gene or protein effective to achieve serum levels commensurate with concentrations found to be effective in vitro. Determination of these parameters is well within the skill of one of ordinary skill in the art. These considerations, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks. Consistent with this definition, as used herein, the term "therapeutically effective amount" is an amount sufficient to provide a therapeutic benefit.

[0236] The term administration includes, without limitation, local or systemic administration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, intracerebroventricular (ICV), intrathecal, intracisternal injection or infusion, subcutaneous injection, or implant), by inhalation, aerosol, nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository), intracranial, or topical administration routes (e.g., gels, ointments, creams, aerosols, etc.), and may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each administration route. The present disclosure is not limited by administration route, formulation, or dosing schedule. In some embodiments, administration is local, such as to the bone marrow or within the brain. In some embodiments, administration is systemic. In some embodiments, administration is by infusion over, for example, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 12 hours, or about 1 day.

[0237] In some embodiments, the cells or cell population is about 1.0 x 10 per kg of the subject's body weight. 4 ~Approx. 1×10 15 The cells are administered in a dose of 100 cells.

[0238] In some embodiments, the dose is at least about 1 x 10 per kg of subject body weight. 4 CD34+ cells, or at least approximately 2 x 10 4 CD34+ cells, or at least approximately 3 x 10 4 CD34+ cells, or at least approximately 4 x 10 4 CD34+ cells, or at least approximately 5 x 10 4 CD34+ cells, or at least approximately 6 x 10 4 CD34+ cells, or at least approximately 7 x 10 4 CD34+ cells, or at least approximately 8 x 10 4 CD34+ cells, or at least approximately 9 x 10 4 CD34+ cells, or at least approximately 1 x 10 5 CD34+ cells, or at least approximately 2 x 105 CD34+ cells, or at least approximately 3 x 10 5 CD34+ cells, or at least approximately 4 x 10 5 CD34+ cells, or at least approximately 5 x 10 5 CD34+ cells, or at least approximately 6 x 10 5 CD34+ cells, or at least approximately 7 x 10 5 CD34+ cells, or at least approximately 8 x 10 5 CD34+ cells, or at least approximately 9 x 10 5 CD34+ cells, or at least approximately 1 x 10 6 CD34+ cells, or at least approximately 2 x 10 6 CD34+ cells, or at least approximately 3 x 10 6 CD34+ cells, or at least approximately 4 x 10 6 CD34+ cells, or at least approximately 5 x 10 6 CD34+ cells, or at least approximately 6 x 10 6 CD34+ cells, or at least approximately 7 x 10 6 CD34+ cells, or at least approximately 8 x 10 6 CD34+ cells, or at least approximately 9 x 10 6 CD34+ cells, or at least approximately 1 x 10 7 CD34+ cells, or at least approximately 2 x 10 7 CD34+ cells, or at least approximately 3 x 10 7 CD34+ cells, or at least approximately 4 x 10 7 CD34+ cells, or at least approximately 5 x 10 7 CD34+ cells, or at least approximately 6 x 10 7 CD34+ cells, or at least approximately 7 x 10 7 CD34+ cells, or at least approximately 8 x 10 7 CD34+ cells, or at least approximately 9 x 10 7 CD34+ cells, or at least approximately 1 x 10 8 CD34+ cells, or at least approximately 2 x 10 8CD34+ cells, or at least approximately 3 x 10 8 CD34+ cells, or at least approximately 4 x 10 8 CD34+ cells, or at least approximately 5 x 10 8 CD34+ cells, or at least approximately 6 x 10 8 CD34+ cells, or at least approximately 7 x 10 8 CD34+ cells, or at least approximately 8 x 10 8 CD34+ cells, or at least approximately 9 x 10 8 CD34+ cells, or at least approximately 1 x 10 9 CD34+ cells.

[0239] Additionally or alternatively, the dose may be 1 x 10 per kg of subject body weight. 9 Less than 9 x 10 8 Less than or 8 x 10 8 Less than 7 x 10 8 Less than 6 x 10 8 Less than 5 x 10 8 Less than 4 x 10 8 Less than 3 x 10 8 Less than 2 x 10 8 Less than 1 x 10 8 Less than 9 x 10 7 Less than 8 x 10 7 Less than 7 x 10 7 Less than 6 x 10 7 Less than 5 x 10 7 Less than 4 x 10 7 Less than 3 x 10 7 Less than 2 x 10 7 Less than 1 x 10 7 Less than 9 x 10 6 Less than 8 x 10 6 Less than 7 x 10 6 Less than 6 x 10 6 Less than 5 x 10 6 Less than 4 x 10 6 Less than 3 x 10 6 Less than 2 x 106 Less than 1 x 10 6 Less than 9 x 10 5 Less than 8 x 10 5 Less than 7 x 10 5 Less than 6 x 10 5 Less than 5 x 10 5 Less than 4 x 10 5 Less than 3 x 10 5 Less than 2 x 10 5 Less than 1 x 10 5 These are cells such as CD34+ HSCs.

[0240] Additionally or alternatively, the dose may be 1 x 10 per kg of subject body weight. 9 Less than 9 x 10 8 Less than 8 x 10 8 Less than 7 x 10 8 Less than 6 x 10 8 Less than 5 x 10 8 Less than 4 x 10 8 Less than 3 x 10 8 Less than 2 x 10 8 Less than 1 x 10 8 Less than 9 x 10 7 Less than 8 x 10 7 Less than 7 x 10 7 Less than 6 x 10 7 Less than 5 x 10 7 Less than 4 x 10 7 Less than 3 x 10 7 Less than 2 x 10 7 Less than 1 x 10 7 Less than 9 x 10 6 Less than 8 x 10 6 Less than 7 x 10 6 Less than 6 x 10 6 Less than 5 x 10 6 Less than 4 x 10 6 Less than 3 x 10 6 Less than 2 x 10 6 Less than 1 x 10 6Less than 9 x 10 5 Less than 8 x 10 5 Less than 7 x 10 5 Less than 6 x 10 5 Less than 5 x 10 5 Less than 4 x 10 5 Less than 3 x 10 5 Less than 2 x 10 5 Less than 1 x 10 5 The modified cells are modified cells, such as fewer than 10 modified CD34+ cells. [Example]

[0241] The following examples are intended to illustrate, but not limit, the embodiments disclosed herein. Experimental Method Experiment No. 1 - Vector Construction

[0242] Lentiviral vector design and production

[0243] For the purposes of the next studies in this study, we used the self-inactivating third-generation lentiviral vector backbone CCLc-x to generate all lentiviral vectors (Figure 1A). For in vivo efficacy studies, we used a lentiviral vector expressing a modified form of murine Ube3a isoform 3 because we performed these experiments in a B6-IL2- / -Ube3a- / + mouse model. To generate the murine Ube3a-expressing lentiviral vector, we synthesized a modified form of murine isoform 3 (the modifications included the addition of an N-terminal secretion signal and eight N-glycosylation sites throughout the protein) and cloned it into the CCLc-x vector backbone under the control of the MNDU3 promoter (Figure 1C). An EGFP gene was cloned downstream under the control of the PGK promoter to track transduction and engraftment of transduced cells. Wild-type Ube3a is an intracellular protein and is not secreted from cells. Therefore, a secretion signal was added to enable secretion from transduced cells. N-glycosylation sites are used to bind to the mannose-6-phosphate receptor found on neurons. These sites were added to the mouse Ube3a protein to enable efficient attachment and uptake into neurons after secretion from transduced cells. For in vivo experiments, a lentiviral vector expressing a modified form of human Ube3a isoform 1 was used. Human isoform 1 is the species equivalent of mouse isoform 3. The human Ube3a isoform 1 gene contains the same N-terminal secretion signal and the same eight N-glycosylation site modifications as the mouse Ube3a isoform 3 gene used in in vivo efficacy studies. The modified human Ube3a isoform 1 gene was synthesized and cloned into the CCLc-x vector under the control of the MNDU3 promoter (Figure 1E and Figure 1F). An EGFP gene was cloned downstream from the human Ube3a gene under the control of the PGK promoter (Figure 1D) to be used for tracking during safety / toxicity experiments, but will not be used for clinical use.A control empty vector containing only the EGFP reporter gene was generated by cloning the EGFP gene under the control of the PGK promoter (Figure 1B). Experiments involving recombinant DNA were performed in accordance with NIH guidelines.

[0244] Lentiviral vectors were produced in human embryonic kidney (HEK)-293 cells using GMP-eligible reagents by transfecting the cells with a 1:5:5 ratio of a packaging plasmid (Δ8.9) containing the enveloped vesicular stomatitis virus glycoprotein (VSVG), vector capsid, and reverse transcriptase gene, and one of the transfer plasmids described above: either the Ube3a vector or the control EGFP vector. Forty-eight hours after transfection, vector supernatants were collected and concentrated by ultrafiltration. For vectors containing an EGFP expression cassette transduced into HEK-293 cells, the transducing unit titer of each vector was calculated by analyzing EGFP expression by flow cytometry 48 hours after transduction. For Ube3a vectors without EGFP, total genomic DNA was extracted from transduced cells and analyzed by quantitative PCR using Taqman Real-Time PCR Master Mix with vector-specific psi primers and probe sets.

[0245] Ube3a Vector Functionality - Overexpression and Ubiquitination Activity of Lentivector-Expressed Ube3a in Human CD34+ HPC-Derived Macrophages

[0246] To evaluate the expression and functionality of the human Ube3a lentivector, human CD34+ hPSCs were transduced with the hAS8 vector and induced to mature macrophages in vitro. Human CD34+ HSPCs were isolated from umbilical cord blood obtained from the UC Davis Umbilical Cord Blood Collection Program using a Ficoll-Paque density gradient and further purified by CD34 magnetic bead column separation. Total CD34+ cells were cultured for 48 hours in XVIVO-10 medium supplemented with 50 ng / ml stem cell factor (SCF), thrombopoietin (TPO), and Flt-3 ligand. Forty-eight hours after isolation, CD34+ cells were either left untransduced (NT) or transduced with either the EGFP control vector or the hAS8 vector at an MOI of 20 using 8 mg / ml protamine sulfate for a minimum of 3 hours at 37°C. EGFP control and hAS8 vector-transduced CD34+ cells were then sorted based on EGFP expression for subsequent experiments.

[0247] Colony-forming unit assay (CFU)

[0248] Overexpression of HexA and HexB in CD34+ HSPCs by lentiviral transduction may result in detrimental cell growth and differentiation. To assess this, HSPC CFU assays were performed. CD34+ cells transduced with either NT, fluorescent-activated cell sorting (FACS) EGFP control vector, or FACS-sorted hAS8 vector-transduced cells (500 total cells) were cultured in cytokine-supplemented methylcellulose medium for 12 days. After the culture period, total burst-forming unit-erythroid colonies (BFU-E), granulocyte / macrophage (GM) colonies, and granulocyte / erythroid / megakaryocyte / macrophage (GEMM) colonies were observed and counted by microscopy. Experiments were performed in triplicate.

[0249] As shown in Figure 2, similar levels of the three colony types were observed in Ube3a lentivector-transduced cells compared to control cells.

[0250] Induction of phenotypically normal macrophages

[0251] Cells from the CFU assay were further differentiated in vitro into mature macrophages. CFUs derived from NT and vector-transduced CD34+ cells were further induced into mature macrophages by plating the cells in 6-well plates containing DMEM supplemented with 10% FBS, 10 ng / ml macrophage colony-stimulating factor (M-CSF), and 10 ng / ml granulocyte-macrophage colony-stimulating factor (GM-CSF) for 4 days, with medium changes every 2 days. Cells were observed microscopically to identify macrophage morphology and analyzed by flow cytometry for the expression of normal macrophage cell surface markers. Macrophages were stained with phycoerythrin (PE)-conjugated CD14, PE-conjugated HLA-DR, or PE-conjugated CD4. Flow cytometry was performed using a Beckman Coulter Cytomics FC500 and CXP software. Experiments were performed in triplicate.

[0252] Macrophages were analyzed by flow cytometry using antibodies specific for normal macrophage markers, CD4, CD14, and HLA-DR. Macrophages derived from CD34+ HPCs transduced with the Ube3a lentivector were phenotypically normal, exhibiting, on average, 96.1% CD4%, 99.6% CD14%, and 99.1% HLA-DR%. These levels were similar to those of control NT and EGFP-only macrophages, which exhibited CD4% levels of 95.7% and 96.4%, CD14% levels of 99.3% and 97.6%, and HLA-DR% levels of 98.0% and 97.5%, respectively.

[0253] Western blot

[0254] Total cell extracts were then collected from macrophages using Pierce RIPA buffer supplemented with Halt protease inhibitor and flash-frozen. Total protein concentration was determined using a BCA protein assay kit according to the manufacturer's protocol. Proteins were loaded onto a polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane. The membrane was then incubated with the respective primary antibody: mouse anti-human Ube3a. A goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody was then added. The blot was then developed using SuperSignal West Pico chemiluminescent substrate.

[0255] As shown in Figure 3A, overexpression of Ube3a was detected by the presence of a strong Ube3a band and its splice variants. This was compared with control non-transduced (NT) and control EGFP-only (EGFP) vector-transduced cells. To further assess whether the human Ube3a protein expressed by the lentivector is functional in ubiquitinating its target protein, S5a, we performed a lentivector-expressed Ube3a ubiquitination assay, as described, for example, by Yi et al. (2017). Ubiquitination assays (e.g., R&D Systems Human E6AP / S5a Ubiquitination Kit, cat#K-230) were performed on human CD34+ HPC-derived macrophage cell extracts using the method described in J. Biol. Chem. 28;292(30):12503-12515. As shown in Figure 3B, ubiquitination of S5a protein was detected in Ube3a vector-transduced cells, as indicated by the ladder-like band pattern in lane "AS8."

[0256] Collectively, these results demonstrated the functionality of the human Ube3a lentiviral vector in overexpressing Ube3a and subsequently ubiquitinating its target proteins. The data also demonstrated normal human CD34+ HSPC colony formation and phenotypically normal end-stage macrophage differentiation after transduction with the human Ube3a lentiviral vector in vitro. Experiment No. 2 - Neonatal treatment in the mouse model

[0257] By crossing Ube3a- / + mice with B6-IL2rg- / - knockout mice, we generated a novel immunodeficient Ube3a- / + mouse model (BGU) capable of accepting human CD34+ cells for engraftment. BGU mice retain the phenotype of immunocompetent Ube3a- / + mice, exhibiting deficient motor, behavioral, and cognitive phenotypes, as well as a lower threshold for epileptic seizure induction. This mouse model was generated as a preclinical evaluation of human CD34+ cells transduced with a Ube3a-expressing lentiviral vector.

[0258] Applicants successfully demonstrated the functionality, efficacy, and safety of lentiviral vectors expressing the Ube3a gene in human CD34+ HSCs in humanized AS and NRG mouse models. Restoration of functional enzyme activity was observed in disease-specific cells and HSC-derived immune cells. Significant improvements in motor and behavioral phenotypes were observed in AS mice transplanted with Ube3a vector-transduced human HSCs. Long-term safety of Ube3a lentiviral vector-transduced human CD34+ HSCs was also observed after engraftment and multilineage hematopoiesis in the humanized NRG mouse model.

[0259] Functional efficacy of HSCs after neonatal treatment with HSCs transduced with Ube3a lentiviral vectors

[0260] We generated a humanized immunodeficient Ube3a-deficient mouse model (BGUbe3a) by crossing Ube3a-deficient mice with immunodeficient B6-IL2rg knockout mice. Thus, transplanted human CD34+ hematopoietic stem cells (HSCs) were able to successfully reconstitute mice with a human immune system, enabling the evaluation of a potential therapeutic candidate for clinical use: human CD34+ HSCs transduced with a Ube3a lentiviral vector. BGUbe3a-deficient mice exhibit AS-related phenotypes, including motor, behavioral, and neurological defects. They also exhibit a broader gait and slower movements compared to WT mice. For all experiments described below, a clinically equivalent Ube3a vector (as shown in Figure 1B) was used to transduce the human CD34+ HSCs used for cell transplantation. All in vivo efficacy data presented below are from mice intrahepatically transplanted at 2–5 days of age. At this age, the mice do not exhibit symptoms of AS. The hypothesis of these experiments was that transplantation into mice would prevent the development of AS-associated phenotypes.

[0261] Functional rescue of the AS phenotype in neonatally transplanted BGU mice

[0262] To evaluate the ability of the Ube3a lentiviral vector to ameliorate the AS phenotype, mAS8 vector-transduced human CD34+ HSCs were transplanted into neonatal immunodeficient BGU mice. Untransduced or mAS8 (Ube3a) vector-transduced human CD34+ HSCs (500,000 cells) were intrahepatically transplanted into 2- to 5-day-old BGU pups sublethally irradiated with 100 rads. Eight weeks after transplantation, mice were bled via the tail vein and analyzed for engraftment by flow cytometry using a mouse anti-human CD45 antibody. Successfully engrafted mice were then evaluated for the AS phenotype. The order and age of testing were as follows: (1) open field at 9 weeks of age, (2) balance beam walking at 9 weeks of age, (3) rotarod at 10 weeks of age, (4) DigiGait at 10 weeks of age, and (5) novel object recognition at 11 weeks of age.

[0263] Study cohort

[0264] Four genotype / treatment groups were analyzed for functional rescue in behavioral assays relevant to AS. The groups were control wild-type (WT; with an IL2-null mutation to provide an appropriate control for immune system effects), HET (a novel AS model with maternal deletion of Ube3a, created with an IL2-null mutation), NT-HET (a novel AS model with maternal deletion of Ube3a, created with an IL2-null mutation, and transplanted with untransduced human CD34+ cells to control for the effects of HSCs alone), and Ube3a-HET (a novel AS model with maternal deletion of Ube3a, created with an IL2-null mutation, and transplanted with human CD34+ HSCs transduced with a Ube3a lentiviral vector). Because there were no gender differences in Angelman syndrome preclinical or clinical outcomes, genders were combined.

[0265] Open field activity of BGUbe3a mice transplanted with Ube3a vector-transduced cells

[0266] Ube3a-deficient mice exhibited motor and behavioral defects in open-field assays, with reduced movement and total activity. Therefore, to assess whether human CD34+ HSCs transduced with Ube3a lentiviral vectors prevented these defects, mice were assessed for horizontal, vertical, and total activity. Briefly, as shown in Figures 5A-5C, Ube3a-deficient mice transplanted with Ube3a vector-transduced cells (Ube3a Het) performed significantly (p<0.0001) better than Ube3a-deficient mice transplanted with non-transduced human CD34+ cells (NT Het) in (Figure 5A) horizontal, (Figure 5B) vertical, and (Figure 5C) total activity. Ube3a Het mice performed similarly to wild-type (WT) mice. Further details are described below.

[0267] General exploratory locomotor activity in a novel open-field arena was assessed as previously described (1-8). Briefly, each subject was tested for 30 min in a testing room at approximately 30 lux using a VersaMax animal activity monitoring system. Total distance traveled, horizontal activity, vertical activity, and time spent in the center were automatically measured to assess overall motor performance in mice.

[0268] Neonatal BGU mice transplanted with Ube3a vector-transduced (Ube3a-Het) human CD34+ HSCs and tested for behavior 8 weeks after transplantation performed indistinguishably from WT mice in multiple assays of motor behavioral deficits. Locomotor activity was collected in a novel open field, and total distance traveled and horizontal movement were assessed using a motion-detecting device (beam break) in a novel arena.

[0269] Using a multifactorial repeated-measures ANOVA, we observed group differences in total distance (Figure 5C; F(3,67) = 8.194, p < 0.0001). Holm-Sidak-corrected post hoc analysis for multiple comparisons highlighted that both NT-HET (p < 0.0001) and HET (p < 0.0028) differed from WT, whereas the treated group Ube3a-HET did not differ from WT (p > 0.05). Holm-Sidak-corrected post hoc analysis for multiple comparisons highlighted that both NT-HET (p < 0.0001) and HET (p < 0.0002) differed from WT, whereas the treated group Ube3a-HET did not differ from WT (p = 0.191). The rigor of this post hoc analysis is noteworthy. Ube3a-treated HET did not differ from WT at any time point over the 30-minute assay (0–5 minutes, p=0.389; 6–10 minutes, p=0.5027; 11–15 minutes, p=0.132; 16–20 minutes, p=0.5418; 21–25 minutes, p=0.995; 26–30 minutes, p=0.8991), whereas HET and WT provided Holm-Sidak-derived adjusted p (0–5 minutes, p<0.0052; 6–10 minutes, p<0.007). NT-HET and WT differed in four of the six 5-minute time bins (0-5 min, p<0.0002; 6-10 min, p<0.000; 11-15 min, p<0.0004; 16-20 min, p<0.024; 21-25 min, p<0.0287; 26-30 min, p<0.0003) of the 30-minute task.

[0270] In the confirmation task, horizontal activity counts also showed group differences using a multifactorial repeated measures ANOVA (Figure 5A; F(3,67) = 9.487, p < 0.0001). Holm-Sidak corrected post hoc analysis for multiple comparisons highlighted that both NT-HET (p < 0.0001) and HET (p < 0.0002) differed from WT, whereas the Ube3a-HET treatment group did not differ from WT (p = 0.191). The rigor of the post hoc analysis is noteworthy. Ube3a-treated HET did not differ from WT at any time point over the 30-minute assay (0-5 minutes, p=0.4383; 6-10 minutes, p=0.3297; 11-15 minutes, p=0.0439; 16-20 minutes, p=0.2038; 21-25 minutes, p=0.7833; 26-30 minutes, p=2903), whereas HET and WT provided Holm-Sidak-derived adjusted p (0-5 minutes, p<0.0001; 6-10 minutes, p<0.0001; 11-15 minutes, p=0.0439; 16-20 minutes, p=0.2038; 21-25 minutes, p=0.7833; 26-30 minutes, p=2903). , p<0.0023; 16-20 min, p<0.0002; 21-25 min, p<0.0231; 26-30 min, p<0.0214) differed in five of the six 5-minute time bins, and NT-HET and WT differed in all 5-minute bins of the 30-minute task (0-5 min, p<0.0002; 6-10 min, p<0.0001; 11-15 min, p<0.0001; 16-20 min, p<0.0003; 21-25 min, p<0.0103; 26-30 min, p<0.0001).

[0271] Balance beam walking activity of Ube3a mice transplanted with Ube3a vector-transduced cells

[0272] To further evaluate the ability of Ube3a vector-transduced cells to prevent AS-related phenotypes, transplanted mice were subjected to a balance beam walking assay, measured by the latency to cross three different widths of balance beams. Briefly, as shown in Figures 5D and 5E, Ube3a-deficient mice (Ube3a Het) transplanted with Ube3a vector-transduced human CD34+ HSCs performed significantly (p<0.05) better in balance beam walking activity than Ube3a-deficient mice transplanted with non-transduced (NT Het) cells. Ube3a Het mice performed similarly to WT mice. Further details are described below.

[0273] The balance beam locomotor task was performed as previously described (1–8). A 59-cm-long round rod was suspended 68 cm above a cushioned landing pad. The goal box at the end of the beam consisted of a 12-cm-diameter cylinder, providing the motivation to traverse the beam. Each mouse was placed at one end of the beam, and the time it took to traverse to the goal box at the other end was measured. The test sequence increased in difficulty, moving from the largest diameter rod to the smallest diameter rod. On the day before testing, all animals underwent two practice trials on the largest diameter round beam to familiarize themselves with the procedure. On the test day, each animal was tested sequentially on three different round rods (35, 18, and 13 mm). The test sequence was based on the presentation of decreasing diameters to indicate increasing difficulty. Each mouse underwent two trials on each balance beam, approximately 30 min apart. The time it took to traverse the beam was recorded and averaged across the two trials on each beam. A maximum time of 60 seconds was assigned to individuals that were unable to cross the beam during that period. In the few cases where a mouse fell off the beam, a score of 60 seconds was assigned.

[0274] A balance beam locomotor task was performed. All groups showed, as expected, longer latencies to traverse the rod-shaped balance beam as it became thinner and more difficult to cross. Group differences were confirmed using a multifactorial repeated-measures ANOVA (Figure 5D; F(3,67) = 17.02, p < 0.0001). Interestingly, Ube3a-deficient mice transplanted with Ube3a lentivector-transduced human CD34+ HSCs (Ube3a-Het) exhibited wild-type performance on the balance beam, as shown by Holm-Sidak post-hoc analysis. Ube3a-treated HET did not differ from WT on any of rods #3 (rod #3, p > 0.999), whereas HET (p < 0.0001) and NT-HET (p < 0.227) had significantly slower latencies to traverse rod #3, highlighting the improved motor coordination of the treatment groups. Figure 5E shows the fastest groups traversing the rod, highlighting WT and Ube3a-HET as indistinguishable from each other by rod crossing time, highlighting the substantial improvement in motor coordination.

[0275] Rotarod and Digigait activities of Ube3a mice transplanted with Ube3a vector-transduced cells

[0276] As another test to evaluate the ability of Ube3a vector-transduced cells to prevent AS-related phenotypes, transplanted mice were subjected to rotarod and DigiGait assays. The rotarod assay determines the latency to fall from a rotating rod with gradually increasing acceleration. As shown in Figure 5F, Ube3a-deficient mice (Ube3a Het) transplanted with Ube3a vector-transduced human CD34+ HSCs performed significantly (p<0.05) better in the rotarod assay than Ube3a-deficient mice transplanted with non-transduced (NT Het) cells. Ube3a Het mice performed similarly to WT mice. The DigiGait assay measures the gait width of the mice being tested. As shown in Figure 5G, Ube3a-deficient mice (Ube3a Het) transplanted with Ube3a vector-transduced human CD34+ HSCs performed significantly better in the rotarod assay than non-transduced (NT Het) mice. Compared with Ube3a-deficient mice transplanted with Ube3a (Het) cells, both forelimbs and hindlimbs showed significantly (p<0.05) improved gait compared with Ube3a-deficient mice transplanted with Ube3a (Het) cells. The gait of Ube3a Het mice appeared similar to that of WT mice. Further details are provided below.

[0277] Rotarod: Motor coordination, balance, and motor learning were tested with the accelerating rotarod from Ugo Basile as previously described (1-8). Mice were placed on a rotating cylinder that gradually accelerated from 5 to 40 revolutions per minute over a 5-minute period. Mice underwent three trials per day with a 60-minute intertrial rest interval, for a total of nine trials over three consecutive days. Performance was scored as the latency to fall off the cylinder at a maximum latency of 5 minutes.

[0278] Gait was analyzed using a DigiGait analyzer from Mouse Specifics Inc. The DigiGait is a treadmill equipped with a ventral-plane camera positioned under a motorized transparent belt. Mice were allowed to acclimate to the walking track for 1 minute before starting the belt to capture images. The belt speed was set at 20 cm / s, and each subject's limb was recorded for 5 seconds. 900 video frames were collected over a 5-second video at 180 frames per second. Captured frames were digitized, and relevant gait parameters were analyzed by the DigiGait analysis software. Left and right fore and hind paws were averaged together for each subject. Animals unable to walk at the target speed for 5 seconds were rested and retested. Subjects were removed from the study if they were unable to complete this criterion after three attempts.

[0279] Because mice have dramatic deficits in this task, a secondary assay to confirm motor coordination is the rotarod assay. As expected, HET mice differed from WT mice in the latency to fall from an accelerating rod on days 2 (p<0.0098) and 3 (p<0.0133) (Figure 5F; F(3,67)=8.395, p<0.0001), indicating poor motor coordination and impaired motor learning. Strikingly, Ube3a-deficient mice transplanted with Ube3a lentivector-transduced human CD34+ HSCs performed similarly to wild-type mice across all 3 days of testing using Holm-Sidak post-hoc analysis (day 1; p=0.8356), (day 2; p=0.9572), and (day 3; p=9979). Group differences were detected using multifactorial ANOVA (Figure 4F; F(3,65) = 5.782, p < 0.002).

[0280] Several studies have reported that AS patients exhibit a wide stance in the Zenowalk Way (1-8). Digigait analysis showed that HET (p<0.0026) and NT-HET (p<0.002) differed from wild-type, while the Ube3a-treated HET group showed a narrowing of these wide stances (p=0.3486).

[0281] Novel object recognition (NOR) in Ube3a mice transplanted with Ube3a vector-transduced cells

[0282] Ube3a-deficient mice exhibit a deficit in novel object recognition due to their neurological deficits. Therefore, to assess whether transplantation of Ube3a vector-transduced human CD34+ HSCs improved these neurological deficits, we performed the NOR assay. This version of the NOR began with a 30-minute habituation period for the animals to the test arena. 24 hours later, subjects were given a 10-minute familiarization session, in which the time spent sniffing each object was recorded. The objects were then cleaned, and after a 1-hour interval, the mice were returned to the arena with the familiar and novel objects. As shown in Figures 6A-6B, Ube3a-deficient mice transplanted with Ube3a vector-transduced human CD34+ HSCs (Ube3a Het) performed significantly better (p<0.05) than Ube3a-deficient mice transplanted with non-transduced (NT Het) cells. Ube3a Het mice performed similarly to WT mice. Further details are described below.

[0283] The novel object recognition test was performed in an opaque matte white (P95 White, Tap Plastics, Sacramento, CA, USA) arena (41 cm long x 41 cm wide x 30 cm high) as previously described (1–8). The assay consisted of four sessions: a 30-min habituation session, a second 10-min habituation phase, a 10-min familiarization session, and a 5-min recognition test. On day 1, each subject was habituated to a clean, empty arena for 30 min. 24 h later, each subject was returned to the empty arena for an additional 10-min habituation session. Mice were then removed from the test arena and placed in a clean, temporary holding cage, while two identical objects were placed in the arena. Subjects were returned to the test arena for a 10-min familiarization period, during which they had time to investigate the two identical objects. After the familiarization phase, subjects were returned to their holding cages for a 1-h interval period. One familiar object and one novel object were placed in the arena, which contained two identical objects during the familiarization phase. After a 1-hour interval, each subject returned to the arena for a 5-minute recognition test. The familiarization session and recognition test were recorded using Ethovision XT video tracking software (version 9.0, Noldus Information Technologies, Leesburg, VA, USA). Sniffing was defined as a head movement toward an object with the tip of the nose within 2 cm or less of the object. The time spent sniffing each object was scored by a researcher blinded to both genotype and treatment. Recognition memory was defined as spending significantly more time sniffing the novel object compared to the familiar object. The total time spent sniffing both objects was used as a measure of general exploration. The time spent sniffing the two identical objects during the familiarization phase confirmed the lack of an innate side bias. Novel object recognition was analyzed using repeated measures ANOVA within genotype, using novel versus familiar objects as comparison. F, degrees of freedom, and p-values ​​are reported.

[0284] AS Ube3a-deficient mice exhibit learning and memory deficits in novel object recognition assays (1–8). As shown in Figures 6A–6B, functional reversal of cognitive and behavioral impairments was observed in BGU mice transplanted with human CD34+ HSCs transduced with Ube3a lentiviral vectors. All scores were initially performed using automated Ethovision software and confirmed by manual scoring by highly trained observers blinded to genotype and treatment group. As expected, WT mice spent more time investigating the novel object compared with the familiar object. In contrast, the HET and NT-HET groups did not exhibit the typical novel object preference (Figure 6A: WT; t(15) = 8.714, p < 0.0001; HET; t(14) = 44.10, p < 0.0002; NT-HET; t(19) = 4.544, p < 0.0060). In contrast to the HET and NT-HET groups, we observed cognitive rescue in the Ube3a-HET-treated group (Figure 6A: Ube3a-HET; t(16) = 3.271, p < 0.003). All groups explored the two identical objects similarly during the familiarization phase (Figure 6B: WT; HET; NT-HET; Ube3a-HET).

[0285] Electroencephalogram (EEG) analysis

[0286] Several EEG abnormalities, including increased delta power, have been described in AS. These observations are also present in the Ube3a- / + BGU mouse model. Therefore, we performed EEG analysis to assess whether transplantation of Ube3a vector-transduced cells improved the EEG phenotype and reduced the delta wave spectrum.

[0287] EEG Implantation: Wireless EEG transmitters were implanted into anesthetized test animals using continuous isoflurane. The implants were placed in subcutaneous pockets lateral to the spine to avoid animal discomfort and displacement due to movement. Each implant contained two channels, including signal and reference leads made of a nickel-cobalt (Colbalt)-based alloy insulated in medical-grade silicone. EEG, EMG, temperature, activity, and signal strength data were collected with each implant. To collect EEG data, two 1.0 mm burr holes were drilled relative to bregma (1.0 mm anterior and 1.0 mm lateral; -3.0 mm posterior and 1.0 mm lateral), and the biopotential leads were secured using stainless steel skull screws. Once in place, dental cement was used to secure the skull screws and lead connections. To collect EMG data, leads were placed in the animal's trapezius muscle. Mice were given Carpofen (5 mg / kg; i.p.) immediately after surgery and 24 hours postoperatively as an analgesic. Subjects were individually caged with free access to food and water and monitored daily for 1 week before EEG acquisition to ensure proper incision healing and recovery.

[0288] EEG Data Acquisition, Processing, and Analysis: After 1 week of recovery from surgical implantation, individually housed mice were assigned to a PhysioTel RPC receiver plate, which transmitted data from the EEG implant to a computer via a data exchange matrix using Ponemah software (Data Sciences International). EEG and EMG data were collected at a sampling rate of 500 Hz with a 0.1 Hz high-pass and a 100 Hz low-pass bandpass filter. Activity, temperature, and signal intensity were collected at a sampling rate of 200 Hz. Data acquired in Ponemah were read in Python and further processed with a 0-50 Hz bandpass filter to focus on frequencies of interest. For spectral analysis, frequency bands were defined as delta 0.5-4 Hz, theta 5-9 Hz, alpha 9-12 Hz, beta 13-30 Hz, and gamma 30-50 Hz. Spectral power was analyzed using Welch's method, which windowed across the signal and averaged across the spectral sample. Relative delta frequency was calculated by dividing the mean delta density by the total density per animal and averaging across genotypes. Power spectral densities between genotypes were analyzed using a two-way repeated measures ANOVA, and significance at each frequency point was tested using Sidak's multiple comparison test. F, degrees of freedom, and p-values ​​are reported.

[0289] As shown in Figure 7A, Applicants observed a decrease in delta power in the Ube3a-HET cohort that was similar to the values ​​in WT mice (F(25,300) = 0.223, p > 0.999). Ube3a-HET mice also exhibited significantly lower delta power than HET (non-transplanted Ube3a- / +) mice (F(25,475) = 3.249, p < 0.0001). Applicants observed an increase in delta power in the HET group compared to WT littermate controls, consistent with mice with the Ube3a- / + genotype (F(25,362) = 4.312, p < 0.0001). Applicants did not detect a significant change in delta power in NT-HET animals (F(25,150) = 0.651, p = 0.896), but since the mean line correlated with that of Ube3a- / +HET mice, this could be due to the higher error rate detected in that group.

[0290] Expression of Ube3a in the CNS of transplanted Ube3a- / + mice

[0291] To assess whether Ube3a could be detected in the CNS of Ube3a- / + BGU mice transplanted with Ube3a vector-transduced cells, brain sections obtained from the mice were stained using the 3,3'-diaminobenzidine (DAB) method.

[0292] Immunohistochemical Labeling and Analysis: After assessing the functional improvement of the AS phenotype, mice were euthanized and bilateral midline sagittal brain sections (40 μm) were obtained. Tissues were labeled with Vector Labs' ImmPACT DAB peroxidase substrate using the Vectastain ABC kit, following the manufacturer's included protocols. Tissues were quenched with 0.3% aqueous hydrogen peroxide for 30 minutes, followed by immersion in 10% blocking solution in PBS (SEA BLOCK blocking buffer) for 1 hour, followed by immersion in primary antibody UBE3a (monoclonal anti-UBE3A antibody raised in mouse, SAB1404508) at a concentration of 1:500 with overnight incubation at 4°C. On day 2, tissues were immersed in a biotinylated secondary antibody solution (goat anti-mouse IgG antibody, Vector Labs) at a concentration of 1:200 for 1 hour of incubation, followed by a 30-minute Vectastain ABC reagent incubation (Vector Labs) and completed by an 8-minute immersion in ImmPACT DAB peroxidase substrate (Vector Labs) at the manufacturer's recommended concentration. Between each step, all tissues were washed for approximately 15 minutes using PBST (0.1% Triton®). Serial sections were mounted and coverslipped onto uncharged slides using Permount mounting medium. Brightfield immunohistochemically stained slides were scanned using a 20x objective (0.8, M27) ​​with brightfield illumination on an Axio Scan (Zeiss).

[0293] As shown in Figure 8, a significant (p = 0.0126) increase in Ube3a expression was observed in the brains of Ube3a- / + BGU mice transplanted with Ube3a vector-transduced cells compared to non-transplanted Ube3a- / + mice (HET). The level of Ube3a expression in the brains of mice transplanted with Ube3a vector-transduced cells was similar to that in WT mice and was not significantly different (p = 0.1923). Statistical analysis was performed using Tukey's multiple comparison test.

[0294] Taken together, the above data strongly demonstrate that improvements in motor, behavioral, and cognitive function were observed after transplantation of neonatal Ube3a- / BGU mice with human CD34+ HSCs transduced with Ube3a-expressing lentiviral vectors, similar to those observed in WT mice. Normal EEG delta waves were also observed in Ube3a- / + mice transplanted with Ube3a vector-transduced cells. Ube3a expression similar to that observed in WT mice was detected in Ube3a- / + mice transplanted with Ube3a vector-transduced cells. These results demonstrate that the AS phenotype can be prevented by restoring Ube3a expression by transplanting therapeutic cells early in the life of mice, prior to the onset of clinical AS symptoms. These results also demonstrate the successful engraftment and functionality of human CD34+ HSCs transduced with Ube3a-expressing lentiviral vectors, as they can be detected in peripheral blood, and demonstrate Ube3a expression in the brains of transplanted mice. Experiment No. 3 - In vivo safety and multilineage hematopoietic and in vitro immortalization assays of Ube3a vector-transduced human CD34+ HSCs

[0295] It is possible that transduction of human CD34+ HSCs with Ube3a-expressing lentiviral vectors and subsequent Ube3a overexpression may impair the in vivo engraftment and multilineage hematopoietic potential of these cells. Therefore, an in vivo model system capable of mimicking human CD34+ HSC engraftment and multilineage hematopoiesis should be used. The NOD-RAG1- / -IL2rg- / - (NRG) immunodeficient mouse model is ideal for assessing these properties of human CD34+ HSCs. Due to the deletion of the RAG1 and IL2 gamma receptor genes, this model allows the engraftment of human CD34+ HSCs and the long-term development of mature human cells, including T cells, B cells, and macrophages, in peripheral blood and lymphoid organs, including the spleen, thymus, and bone marrow. Due to these characteristics of the NRG mouse, we used this model to evaluate the safety of lentiviral vector transduction and Ube3a overexpression in human CD34+ HSCs.

[0296] NOD-RAG1- / -IL2rg- / - (NRG) mice (strain number 007799) were obtained from The Jackson Laboratory. (9) Two- to five-day-old NRG mice were sublethally irradiated with 100 rads and transplanted with human CD34+ HSCs (300,000 total cells) that were untransduced (N=8), transduced with an EGFP control vector (Figure 1B), or transduced with the hAS8 Ube3a lentiviral vector (N=8) (Figure 1D) (MOI 20 with 8 μg / ml protamine sulfate). As described above, the hAS8 vector expresses human Ube3a isoform 1, modified to contain a secretion signal at the N-terminus and eight N-glycosylation sites throughout the protein. Human Ube3a isoform 1 is the species equivalent of mouse Ube3a isoform 3, which was used in preclinical studies in the BGU mouse model. A vector containing an EGFP reporter gene was used to distinguish Ube3a vector-transduced cells from non-transduced cells, and these cells were specifically gated when flow cytometry analysis was performed. This gating strategy allowed the applicant to select vector-transduced cells and specifically focus their development and differentiation into the various immune cells analyzed. To determine engraftment levels, mice were bled via the tail vein 3 months after transplantation and analyzed by flow cytometry using a PE-CY7-conjugated anti-human CD45 antibody. Flow cytometry was performed using a Beckman Coulter FC-500. Mice were used in accordance with institutional and IACUC guidelines. Once successful engraftment was observed, mice were kept for an additional 3 months (total 6 months after transplantation) before euthanasia to evaluate multilineage hematopoietic and lymphoid organ engraftment. Human T cell analysis (CD3, CD4, and CD8 cell markers) was performed in blood, spleen, and thymus. Human B cell analysis (CD19) was performed in spleen and bone marrow. Human macrophage (CD14) analysis was performed in bone marrow. Human CD34+ analysis (CD34) was performed in bone marrow. Cells from spleen, thymus, peripheral blood, and bone marrow were labeled with antibodies specific for human immune cell markers and analyzed by flow cytometry.Flow cytometry was performed using a Beckman Coulter FC500. Cells were first gated on EGFP to identify vector-transduced cells and then analyzed for human cell-specific markers to identify the occurrence of specific cell types.

[0297] Analysis determined that normal engraftment of hAS8 Ube3a lentiviral vector-transduced CD34+ cells and the generation of human T cells were demonstrated in the peripheral blood of engrafted NRG mice. As shown in Figure 9A, no significant differences (p>0.05) were observed in the generation of CD3+ / CD4+ T cells, CD3+ / CD8+ T cells, or CD4+ / CD8+ double-positive T cells from hAS8 vector-transduced human CD34+ cells compared with EGFP control vector-transduced or non-transduced human CD34+ cells. Similar levels of all analyzed T cell populations were observed in the peripheral blood of all mouse cohorts. On average, mice transplanted with Ube3a vector-transduced cells exhibited lower CD3+ / CD4+ (76.1%), CD3+ / CD8+ (38.2%), and CD3+ / CD4+ / CD8+ (24.4%) levels in peripheral blood compared with mice transplanted with non-transduced cells (CD3+ / CD4+ (72.1%), CD3+ / CD8+ (47.5%), and CD3+ / CD4+ / CD8+ (19.6%)) and EGFP-only vector-transduced cells (CD3+ / CD4+ (68.2%), CD3+ / CD8+ (37.9%), and CD3+ / CD4+ / CD8+ (13.1%)). Similar to peripheral blood, no significant differences (p>0.05) were observed in the development of CD3+ / CD4+ T cells, CD3+ / CD8+ T cells, or CD4+ / CD8+ double-positive T cells in the spleens of engrafted mice from hAS8 vector-transduced human CD34+ cells compared with EGFP control vector-transduced or non-transduced human CD34+ cells, as shown in Figure 9B. On average, mice transplanted with Ube3a vector-transduced cells exhibited increased CD3+ / CD4+ (70.9%), CD3+ / CD8+ (46.3%), and CD3+ / CD4+ / CD8+ (15.3%) levels in the spleen compared to mice transplanted with untransduced cells (CD3+ / CD4+ (67.8%), CD3+ / CD8+ (58.5%), and CD3+ / CD4+ / CD8+ (26.4%)) and EGFP-only vector-transduced cells (CD3+ / CD4+ (57.9%), CD3+ / CD8+ (52.0%), and CD3+ / CD4+ / CD8+ (19.1%)). Applicants next analyzed the levels of T cells in the thymus of engrafted mice.As shown in Figure 9C, no significant differences (p>0.05) were observed in the development of CD3+ / CD4+ T cells, CD3+ / CD8+ T cells, or CD4+ / CD8+ double-positive T cells in the thymus of engrafted mice from hAS8 vector-transduced human CD34+ cells compared with EGFP control vector-transduced or non-transduced human CD34+ cells. On average, mice transplanted with Ube3a vector-transduced cells showed lower CD3+ / CD4+ (66.1%), CD3+ / CD8+ (54.4%), and CD3+ / CD4+ / CD8+ (27.7%) levels in the thymus compared with mice transplanted with non-transduced cells (CD3+ / CD4+ (74.3%), CD3+ / CD8+ (53.6%), and CD3+ / CD4+ / CD8+ (28.0%)) and EGFP-only vector-transduced cells (CD3+ / CD4+ (65.2%), CD3+ / CD8+ (57.1%), and CD3+ / CD4+ / CD8+ (22.3%)). These results demonstrate that human CD34+ HSCs transduced with hAS8 Ube3a-expressing lentiviral vectors were able to engraft in NRG mice and differentiate into normal T cells in the peripheral blood, spleen, and thymus of engrafted mice.

[0298] As a next step in assessing the safety of Ube3a vector-transduced cells, human B cell analysis was performed in the spleen and bone marrow of engrafted NRG mice. As shown in Figure 10A, no significant difference (p>0.05) was observed in the development of CD45+ / CD19+ B cells in the spleens of engrafted mice from hAS8 vector-transduced human CD34+ cells compared with EGFP control vector-transduced or non-transduced human CD34+ cells. On average, mice transplanted with Ube3a vector-transduced cells exhibited higher CD45+ / CD19+ (38.5%) levels in the spleen compared with mice transplanted with non-transduced cells (CD45+ / CD19+ (51.6%)) and EGFP-only vector-transduced cells (CD45+ / CD19+ (45.8%)). Similarly, as shown in Figure 10B, hAS8 vector-transduced human CD34+ cells exhibited significantly higher levels of CD45+ / CD19+ (38.5%) in the spleen compared with EGFP control vector-transduced or non-transduced human CD34+ cells. No significant difference (p>0.05) was observed in the development of CD45+ / CD19+ B cells in the bone marrow of engrafted mice from hAS8 cells. On average, mice transplanted with Ube3a vector-transduced cells showed lower CD45+ / CD19+ (32.6%) levels in the bone marrow compared to mice transplanted with untransduced cells (CD45+ / CD19+ (47.9%)) and EGFP-only vector-transduced cells (CD45+ / CD19+ (39.8%)). These results demonstrate that human CD34+ HSCs transduced with hAS8 Ube3a-expressing lentiviral vectors were able to engraft in NRG mice and differentiate into normal B cells in the spleen and bone marrow of engrafted mice.

[0299] Applicants next evaluated the levels of human macrophages and human CD34+ cells engrafted in the bone marrow of NRG mice transplanted with hAS8 Ube3a vector-transduced cells. As shown in Figure 11, no significant differences (p>0.05) were observed in the development of CD45+ / CD14+ macrophages or CD45+ / CD34+ cells in the bone marrow of engrafted mice from hAS8 vector-transduced human CD34+ cells compared with EGFP control vector-transduced or non-transduced human CD34+ cells. On average, mice transplanted with Ube3a vector-transduced cells showed significantly lower CD45 / CD14 (19.2%) and CD45 / CD34 (18.5%) levels in the bone marrow compared with mice transplanted with untransduced cells (CD45 / CD14 (24.5%) and CD45 / CD34 (13.0%)) and EGFP-only vector-transduced cells (CD45 / CD14 (14.6%) and CD45 / CD34 (9.5%)). These results demonstrate that human CD34 HSCs transduced with hAS8 Ube3a-expressing lentiviral vectors were able to engraft in NRG mice and differentiate into normal macrophages in the bone marrow of engrafted mice. These results also demonstrate that human CD34 cells transduced with Ube3a lentiviral vectors were still present in the bone marrow of engrafted mice 6 months after transplantation.

[0300] In vitro immortalization assay

[0301] To assess whether transduction of human CD34+ cells with the hAS8 Ube3a-expressing lentiviral vector resulted in any immortalization of the cells, an in vitro immortalization assay was performed. Briefly, human CD34+ cells were left untransduced or transduced with either an EGFP-only control vector, a hAS8-GFP vector, or a hAS8 lentiviral vector (Figure 1). These cells were cultured for 14 days in IMDM medium containing 10% FBS and supplemented with 50 ng / ml SCF, Flt-3 ligand, and TPO. Every 3 days, the cell density was increased to 5 × 10 5The density was adjusted to 100 cells / ml. After this expansion, cells were plated in 3 x 96-well plates per cell group at a density of 100 cells / ml and allowed to sit for 14 days. The wells were then counted for immortalization frequency. A total of 288 wells were plated per cell group and counted for immortalization frequency. Table 1. In vitro immortalization assay using Ube3a lentiviral vector-transduced cells [Table 1] Experiment No. 4 – Functional efficacy of HSCs after adult treatment with Ube3a lentiviral vector-transduced HSCs

[0302] As a next step in evaluating the efficacy of Ube3a lentiviral vector-transduced human CD34+ HSCs, we transplanted the cells into adult BGU mice after the AS phenotype had developed. To evaluate adult mice, we transplanted the cells intravenously at 4–5 weeks of age, after the AS phenotype had already developed in BGU mice.

[0303] Functional rescue of the AS phenotype in adult BGU mice transplanted with Ube3a vector-transduced cells

[0304] To evaluate the ability of the Ube3a lentiviral vector to ameliorate the AS phenotype after its appearance, mAS8 vector-transduced human CD34+ HSCs were transplanted into adult immunodeficient BGU mice. Adult mice were treated intraperitoneally with 20 mg / kg busulfan 48 and 24 hours before intravenous transplantation of 500,000 total cells / mouse of either non-transduced (NT) or Ube3a (Ube3a-HET) lentiviral vector-transduced human CD34+ HSCs at 4–5 weeks of age. Six weeks after transplantation, mice were bled via the tail vein and analyzed for engraftment by flow cytometry using a mouse anti-human CD45 antibody. Successfully engrafted mice were then evaluated for behavioral phenotypes. The test order and ages were as follows: (1) open field at 11–12 weeks of age, (2) balance beam walking at 11–12 weeks of age, (3) rotarod at 12–13 weeks of age, (4) digigait at 12–13 weeks of age, and (5) novel object recognition at 14–15 weeks of age.

[0305] Methods for open field, balance beam walking, rotarod, DigiGait and novel object recognition assays were performed as described above.

[0306] Study cohort

[0307] The same four test cohorts were used for adult efficacy experiments: WT (wild-type expression of Ube3a), HET (BGU mice deficient in Ube3a), NT-HET (BGU mice deficient in Ube3a transplanted with non-transduced human CD34+ HSCs), and Ube3a-HET (BGU mice deficient in Ube3a transplanted with Ube3a lentiviral vector-transduced human CD34+ HSCs). Genders were combined because there were no gender differences in Angelman syndrome preclinical or clinical outcomes.

[0308] Adult HET mice treated with either non-transduced HSCs (NT-Het) or Ube3a vector-transduced (Ube3a-HET) human CD34+ HSCs were tested for behavior 6 weeks after transplantation. Similar to the neonatal studies disclosed herein, Applicants performed multiple assays of a tailored motor behavior battery.

[0309] Locomotor activity in the novel open field was collected by assessing total distance traveled and horizontal movement using a motion detector (beam break) in the novel arena. Group differences in total distance traveled were observed using a multifactorial repeated-measures ANOVA (Figure 13C; F(3,63) = 9.650, p < 0.0001) followed by a Holm-Sidak post-hoc test (p = 0.3465). Similar to the treated pups, in the adult treatment groups, the Holm-Sidak corrected for multiple comparisons and still clearly highlighted that both NT-HET (p < 0.0397) and HET (p < 0.0001) were different from WT, while the Ube3a-HET treatment group was not different from WT (p = 0.3465). Holm-Sidak corrected post-hoc analysis for multiple comparisons highlighted that both NT-HET (p<0.0001) and HET (p<0.0002) differed from WT, while the treatment group Ube3a-HET did not differ from WT (p=0.191). Ube3a-treated HET did not differ from WT at any time point across the 30-minute assay (0-5 minutes, p=0.0122; 6-10 minutes, p=0.4324; 11-15 minutes, p=0.6395; 16-20 minutes, p=>0.9999; 21-25 minutes, p=0.5583; 26-30 minutes, p=0.7356), whereas HET differed in four of the six five-minute time bins (0-5 minutes, p<0.0001; 6-10 minutes, p<0.0001; 11-15 minutes, p<0.0001; 16-20 minutes, p<0.0067; 21-25 minutes, p<0.0002; 26-30 minutes, p<0.0059). NT-HET differed in three of six 5-minute time bins (0-5 min, p<0.0013; 6-10 min, p<0.0316; 11-15 min, p<0.017; 16-20 min, p=0.7957; 21-25 min, p=0.1765; 26-30 min, p=0.7682).

[0310] In the confirmation task, horizontal activity counts also showed group differences using a multifactorial repeated measures ANOVA (Figure 13A; F(3,61) = 11.78, p < 0.0001). Holm-Sidak corrected post hoc analysis for multiple comparisons highlighted that both NT-HET (p < 0.0001) and HET (p < 0.0002) differed from WT, whereas the Ube3a-HET treatment group did not differ from WT (p = 0.191). Ube3a-treated HET did not differ from WT at any time point over the 30-minute assay (0–5 minutes, p = 0.4383; 6–10 minutes, p = 0.3297; 11–15 minutes, p = 0.0439; 16–20 minutes, p = 0.2038; 21–25 minutes, p = 0.7833; 26–30 minutes, p = 2903), whereas in HET, the Holm-Sidak-derived adjusted p (0–5 minutes, p < 0.0001; 6–10 minutes, p < 0.0001; 11–15 minutes, p NT-HET differed in five of the six 5-minute time bins (0-5 min, p<0.0002; 6-10 min, p<0.0001; 11-15 min, p<0.0001; 16-20 min, p<0.0003; 21-25 min, p<0.0103; 26-30 min, p<0.0214) and in all 5-minute bins of the 30-minute task (0-5 min, p<0.0002; 6-10 min, p<0.0001; 11-15 min, p<0.0001; 16-20 min, p<0.0003; 21-25 min, p<0.0103; 26-30 min, p<0.0001).

[0311] The balance beam locomotor task performed similarly across pup cohorts, and as expected, all groups showed longer latencies to traverse the rod-shaped balance beam as it became thinner and more difficult to cross (F(2,124) = 11.73, p<0.0001). Group differences were confirmed using a multifactorial repeated measures ANOVA (Figures 13D-13E; F(3,62) = 11.24, p<0.0001). Surprisingly, Ube3a-deficient mice transplanted with Ube3a lentivector-transduced human CD34+ HSCs (Ube3a-HET) exhibited wild-type performance on the balance beam as shown by Holm-Sidak post-hoc analysis. While Ube3a-treated HET did not differ from WT on rod #3 (rod #3, p=0.9931), HET (p<0.0164) and NT-HET (p<0.0002) had much slower latencies to traverse rod #3, highlighting the improved motor coordination of the treatment groups. Figure 13E shows the fastest group to traverse the rod, highlighting WT and Ube3a-HET as indistinguishable from each other by rod crossing time, highlighting the substantial improvement in motor coordination.

[0312] In the rotarod, a confirmatory coordination assay, HET mice differed from WT in the latency to fall from an accelerating rod on day 3 (p<0.05) (Figure 13F; F(3,59)=3.30, p<0.05), indicating poor motor coordination and impaired motor learning in Ube3a- / + HET mice. Strikingly, Ube3a- / + mice transplanted with Ube3a lentivector-transduced human CD34+ HSCs were similar to wild-type mice across all 3 days of testing (day 1; p=0.9321), (day 2; p=0.9691), and (day 3; p=0.9514) using Holm-Sidak post-hoc analysis. Group differences were detected using multifactorial ANOVA (Figure 20F; F(3,65)=5.782, p<0.002). As shown in Figure 13G, DigiGait analysis demonstrated that HET (p<0.0026) and NT-HET (p<0.002) differed from wild-type, while the Ube3a-treated HET group showed a narrowing of these wide stances (p=0.3486).

[0313] The data showed functional reversal of cognitive-behavioral impairments in Figures 14A-14B 6 weeks after transplantation with human CD34+ HSCs transduced with a lentiviral vector expressing Ube3a. To reach the desired sample size, cognitive performance as measured by the NOR task was tested in several independent cohorts, with each subcohort containing each treatment group and following the standards of the experimental design. The design maximized the overall score. All scoring was initially performed by automated Ethovision software and confirmed by manual scoring by highly trained observers blinded to genotype and treatment group. As expected, WT spent more time investigating the novel object compared to the familiar object. In contrast, the HET and NT-HET groups did not exhibit the typical novel object preference (Figure 14A: WT; t(14) = 2.626, p < 0.0139; HET; t(16) = 0.1392, p > 0.8464; NT-HET; t(12) = 0.4597, p > 0.6553). Strikingly, Applicants observed cognitive rescue in the Ube3a-HET-treated group (Figure 23A: Ube3a-HET; t(14) = 3.271, p < 0.0026). All groups explored the two identical objects similarly during the familiarization phase (Figure 14B: WT; t(14) = 0.3698, p = 0.7193; HET; t(16) = 0.2968, p = 0.8903; NT-HET; t(12) = 0.1331, p = 0.8952; Ube3a-HET; t(14) = 0.1392, p = 0.6488).

[0314] Expression of Ube3a in adult Ube3a- / + mice transplanted with Ube3a vector-transduced cells

[0315] To assess whether Ube3a was expressed in the brains of adult Ube3a- / + mice transplanted with Ube3a vector-transduced cells, a DAB / anti-Ube3a antibody detection assay was performed. The method for this assay is described above. As shown in Figure 15, a significant increase in Ube3a expression (p = 0.0058 and p = 0.0124) was observed in the brains of Ube3a- / + mice transplanted with Ube3a vector-transduced cells (Ube3a-HET) compared with non-transplanted Ube3a- / + mice (HET) and Ube3a- / + mice transplanted with non-transduced cells (NT-HET), respectively. The level of Ube3a expression in the Ube3a-HET cohort was similar to that in WT BGU mice (p = 0.9867). Statistical analysis was performed using Tukey's multiple comparison test.

[0316] Taken together, the above data strongly demonstrate that correction of the established AS phenotype was achieved after intravenous transplantation of adult Ube3a- / + BGU mice with human CD34+ HSCs transduced with a Ube3a-expressing lentiviral vector. Mice receiving Ube3a-expressing cells showed improved motor, behavioral, and cognitive function to levels similar to those of WT mice. Ube3a expression levels in the brains of Ube3a- / + mice were also restored to WT levels after transplantation of Ube3a vector-transduced cells. These results demonstrate that transplantation of therapeutic cells can correct AS phenotypes after their onset. These results also demonstrate the successful engraftment and functionality of human CD34+ HSCs transduced with a Ube3a-expressing lentiviral vector, as they can be detected in peripheral blood, and demonstrate Ube3a expression in the brains of mice transplanted via the intravenous route. Experiment Number 5 - Clinical

[0317] Applicants also demonstrated that transduction of human CD34+ HSCs and subsequent Ube3a overexpression had no adverse effects on CD34+ cell function, engraftment, or differentiation into normal immune cells as shown by CFU assays, on the further differentiation of in vitro-derived phenotypically normal macrophages, and on the in vivo engraftment and further differentiation of T cells, B cells, and macrophages in NRG mice.

[0318] HSC mobilization and peripheral stem cell collection

[0319] Insertion of a central venous catheter (CVC) is required for all apheresis procedures. Patients undergo stem cell mobilization using subcutaneous injections of 10 μg / kg G-CSF for 5 consecutive days. Plerixafor 240 μg / kg / dose has been shown to improve collection outcomes at the discretion of the treating physician and is administered 4-6 hours before apheresis begins. Routine monitoring, patient supportive care, vital signs, and CBC monitoring during collection follow institutional guidelines. On collection day, subjects receive a subcutaneous injection of plerixafor (240 μg / kg) 5 hours before collection.

[0320] Subjects may undergo up to two mobilization cycles to achieve an adequate cell dose. Insertion of a temporary apheresis catheter is highly recommended to facilitate HSPC collection.

[0321] The drug product cell dose will be selected for each subject from the following and will meet all release criteria: at least about 1 x 10 4 CD34+ cells / kg, or at least approximately 2 x 10 4 CD34+ cells / kg, or at least approximately 3 x 10 4 CD34+ cells / kg, or at least approximately 4 x 10 4 CD34+ cells / kg, or at least approximately 5 x 10 4 CD34+ cells / kg, or at least approximately 6 x 10 4 CD34+ cells / kg, or at least approximately 7 x 10 4CD34+ cells / kg, or at least approximately 8 x 10 4 CD34+ cells / kg, or at least approximately 9 x 10 4 CD34+ cells / kg, or at least approximately 1 x 10 5 CD34+ cells / kg, or at least approximately 2 x 10 5 CD34+ cells / kg, or at least approximately 3 x 10 5 CD34+ cells / kg, or at least approximately 4 x 10 5 CD34+ cells / kg, or at least approximately 5 x 10 5 CD34+ cells / kg, or at least approximately 6 x 10 5 CD34+ cells / kg, or at least approximately 7 x 10 5 CD34+ cells / kg, or at least approximately 8 x 10 5 CD34+ cells / kg, or at least approximately 9 x 10 5 CD34+ cells / kg, or at least approximately 1 x 10 6 CD34+ cells / kg, or at least approximately 2 x 10 6 CD34+ cells / kg, or at least approximately 3 x 10 6 CD34+ cells / kg, or at least approximately 4 x 10 6 CD34+ cells / kg, or at least approximately 5 x 10 6 CD34+ cells / kg, or at least approximately 6 x 10 6 CD34+ cells / kg, or at least approximately 7 x 10 6 CD34+ cells / kg, or at least approximately 8 x 10 6 CD34+ cells / kg, or at least approximately 9 x 10 6 CD34+ cells / kg, or at least approximately 1 x 10 7 CD34+ cells / kg, or at least approximately 2 x 10 7 CD34+ cells / kg, or at least approximately 3 x 10 7 CD34+ cells / kg, or at least approximately 4 x 10 7 CD34+ cells / kg, or at least approximately 5 x 10 7 CD34+ cells / kg, or at least approximately 6 x 107 CD34+ cells / kg, or at least approximately 7 x 10 7 CD34+ cells / kg, or at least approximately 8 x 10 7 CD34+ cells / kg, or at least approximately 9 x 10 7 CD34+ cells / kg, or at least approximately 1 x 10 8 CD34+ cells / kg, or at least approximately 2 x 10 8 CD34+ cells / kg, or at least approximately 3 x 10 8 CD34+ cells / kg, or at least approximately 4 x 10 8 CD34+ cells / kg, or at least approximately 5 x 10 8 CD34+ cells / kg, or at least approximately 6 x 10 8 CD34+ cells / kg, or at least approximately 7 x 10 8 CD34+ cells / kg, or at least approximately 8 x 10 8 CD34+ cells / kg, or at least approximately 9 x 10 8 CD34+ cells / kg, or at least approximately 1 x 10 9 CD34+ cells / kg body weight.

[0322] HSC dose

[0323] In some embodiments, the dose is 1×10 9 Less than 9 x 10 8 Less than 8 x 10 8 Less than 7 x 10 8 Less than 6 x 10 8 Less than 5 x 10 8 Less than 4 x 10 8 Less than 3 x 10 8 Less than 2 x 10 8 Less than 1 x 10 8 Less than 9 x 10 7 Less than 8 x 10 7 Less than 7 x 10 7 Less than 6 x 10 7 Less than 5 x 10 7 Less than 4 x 107 Less than 3 x 10 7 Less than 2 x 10 7 Less than 1 x 10 7 Less than 9 x 10 6 Less than 8 x 10 6 Less than 7 x 10 6 Less than 6 x 10 6 Less than 5 x 10 6 Less than 4 x 10 6 Less than 3 x 10 6 Less than 2 x 10 6 Less than 1 x 10 6 Less than 9 x 10 5 Less than 8 x 10 5 Less than 7 x 10 5 Less than 6 x 10 5 Less than 5 x 10 5 Less than 4 x 10 5 Less than 3 x 10 5 Less than 2 x 10 5 Less than 1 x 10 5 Fewer than 1 CD34+ HSC / kg body weight.

[0324] In some embodiments, the dose is 1×10 9 Less than 9 x 10 8 Less than 8 x 10 8 Less than 7 x 10 8 Less than 6 x 10 8 Less than 5 x 10 8 Less than 4 x 10 8 Less than 3 x 10 8 Less than 2 x 10 8 Less than 1 x 10 8 Less than 9 x 10 7 Less than 8 x 10 7 Less than 7 x 10 7 Less than 6 x 10 7 Less than 5 x 10 7 Less than 4 x 10 7 Less than 3 x 10 7 Less than 2 x 107 Less than 1 x 10 7 Less than 9 x 10 6 Less than 8 x 10 6 Less than 7 x 10 6 Less than 6 x 10 6 Less than 5 x 10 6 Less than 4 x 10 6 Less than 3 x 10 6 Less than 2 x 10 6 Less than 1 x 10 6 Less than 9 x 10 5 Less than 8 x 10 5 Less than 7 x 10 5 Less than 6 x 10 5 Less than 5 x 10 5 Less than 4 x 10 5 Less than 3 x 10 5 Less than 2 x 10 5 Less than 1 x 10 5 Fewer than 10 genetically modified CD34+ / kg body weight.

[0325] Cells can be administered between 72 and 84 hours after the last dose of IV busulfan or myoablative therapy.

[0326] treatment

[0327] The preparation regimen set forth in Table 2 is exemplary of the treatment protocol. Table 2. [Table 2] *BU pharmacokinetics will be performed routinely after the first and third doses. For daily dosing, a 3-hour infusion starting at 0600 will allow for the dispatch of plasma samples for BU PK studies at the reference site. Samples will be obtained at the end of the 3-hour infusion, then 15 minutes, 1 hour, 2 hours, and 4 hours after completion of the infusion.

[0328] Infection prevention

[0329] Patients may receive infection prophylaxis and nutritional support. Infection prophylaxis includes, but is not limited to, agents or strategies (e.g., PCR screening and preemptive treatment) to reduce the risk of bacterial, herpes simplex, CMV, HHV-6, EBV, Pneumocystis jiroveci, and fungal infections.

[0330] Indwelling central venous catheter

[0331] A double-lumen central venous catheter is inserted at the time of apheresis and maintained during transplantation to allow for the administration of IV medications, transfusion of blood products, and administration of stem cells. This catheter can be removed and replaced if clinically indicated. However, the graft must be infused through the central line.

[0332] Preparatory regimen for transplantation

[0333] Patients can receive myeloablative conditioning therapy with intravenous (IV) busulfan. Single-agent busulfan is administered intravenously through a central venous catheter at 3.2 mg / kg once daily for 4 days as a myeloablative conditioning regimen. This regimen has been used in multiple gene therapy stem cell transplant trials due to its successful engraftment and tolerable toxicity.

[0334] An illustrative transplant timeline is provided below: 1. Single-agent busulfan from days -5 to -3. 2. Genetically modified hematopoietic stem cells will be thawed if frozen and infused on day 0, at least 72 hours after the last dose of busulfan. 3. Backup hematopoietic stem cells can be infused after day 30 in patients with ANC<500 and on or after day 20 in patients with ANC<500 and life-threatening complications. equivalent

[0335] While the invention has been described in conjunction with the above-described embodiments, it should be understood that the foregoing description and examples are intended to illustrate, rather than limit, the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0336] While the present disclosure has been particularly disclosed by specific embodiments and optional features, it is to be understood that modifications, improvements, and variations of the embodiments therein disclosed herein may be employed by those skilled in the art, and that such modifications, improvements, and variations are deemed to be within the scope of the present disclosure. The materials, methods, and examples provided herein are representative and illustrative of particular embodiments and are not intended as limitations on the scope of the disclosure.

[0337] The scope of the present disclosure is described broadly and generically herein. Each of the narrower species and subgeneric groupings that fall within the generic disclosure also form part of this disclosure. This includes conditional generic descriptions or negative limitations that remove any subject matter from the genus, regardless of whether the excised material is specifically recited herein.

[0338] Additionally, where features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also hereby described with respect to any individual member or subgroup of members of the Markush group.

[0339] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated herein by reference in their entirety to the same extent as if each were individually incorporated by reference. In case of conflict, the present specification, including definitions, will control. References [ka] [ka] [ka] Sequence Listing

[0340] SEQ ID NO: 1, CMV promoter sequence 1: [ka]

[0341] SEQ ID NO: 2, CMV promoter sequence 2: [ka]

[0342] SEQ ID NO: 3, MNDU3 promoter [ka]

[0343] SEQ ID NO: 4, PGK promoter [ka] [ka]

[0344] SEQ ID NO: 5, MNDU promoter sequence: [ka]

[0345] SEQ ID NO: 6, EF1 alpha promoter sequence: [ka]

[0346] SEQ ID NO: 7, WT human Ube3a isoform 1 [ka] [ka] [ka]

[0347] SEQ ID NO: 8, WT human Ube3a isoform 1 aa sequence [ka] [ka]

[0348] SEQ ID NO: 9, WT human Ube3a isoform 2 [ka] [ka]

[0349] SEQ ID NO: 10, WT human Ube3a isoform 2 aa sequence [ka] [ka]

[0350] SEQ ID NO: 11, WT human Ube3a isoform 3 [ka] [ka] [ka]

[0351] SEQ ID NO: 12, WT human Ube3a isoform 3 aa sequence [ka] Modified human isoform #1 Nucleotide positions 190, 293, 310, 661, 662, 1066, 1067, 1771, 1773, 1870, 1871, 2413, 2414, 2417 and 2418 AA positions 64, 98, 104, 221, 356, 591, 624, 805 and 806

[0352] SEQ ID NO: 13, human Ube3a isoform 1 with 8x N-glycan sites (bold) and an IL2 secretion signal (underlined). Bold, capitalized font provides examples of mutations to create glycosylation sites. [ka] [ka]

[0353] SEQ ID NO: 14, human Ube3a isoform 1 aa sequence with 8x N-glycan sites (bold) and an IL2 secretion signal (first 20 amino acids underlined). Italics indicate mutation sites for glycosylation. [ka] Modified human isoform #2 Nucleotide positions 259, 362, 379, 730, 731, 1135, 1136, 1840, 1842, 1939, 1940, 2482, 2483, 2486 and 2487 AA positions 87, 121, 127, 244, 379, 614, 647, 828 and 829

[0354] SEQ ID NO: 15, human Ube3a isoform 2 with 8x N-glycan sites (bold) and an IL2 secretion signal (underlined). Bold, capitalized font provides examples of mutations to create glycosylation sites. [ka] [ka]

[0355] SEQ ID NO: 16, human Ube3a isoform 2 aa sequence with 8x N-glycan sites (bold) and IL2 secretion signal (underlined) [ka] Modified human isoform #3 Nucleotide positions 250, 353, 370, 721, 722, 1126, 1127, 1831, 1833, 1930, 1931, 2473, 2474, 2477 and 2478 AA positions 84, 118, 124, 241, 376, 611, 644, 825 and 826

[0356] SEQ ID NO: 17, human Ube3a isoform 3 with 8x N-glycan sites (bold) and an IL2 secretion signal (underlined). Bold, capitalized font provides examples of mutations to create glycosylation sites. [ka] [ka] [ka]

[0357] SEQ ID NO: 18, human Ube3a isoform 3 aa sequence with 8x N-glycan sites (bold) and IL2 secretion signal (underlined) [ka] [ka]

[0358] SEQ ID NO: 19, WT mouse Ube3a isoform 1 [ka] [ka] [ka]

[0359] SEQ ID NO: 20, WT mouse Ube3a isoform 1 aa sequence [ka]

[0360] SEQ ID NO: 21, WT mouse Ube3a isoform 2 [ka] [ka]

[0361] SEQ ID NO: 22, WT mouse Ube3a isoform 2 aa sequence [ka] [ka]

[0362] SEQ ID NO: 23, WT mouse Ube3a isoform 3 [ka] [ka] [ka]

[0363] SEQ ID NO: 24, WT mouse Ube3a isoform 3 aa sequence [ka] Modified Mouse Isoform #1 Nucleotide positions 253, 356, 373, 715, 716, 1120, 1121, 1825, 1827, 1828, 1829, 1924, 1925, 2467 and 2468 AA positions 85, 119, 125, 239, 374, 609, 610, 642 and 823

[0364] SEQ ID NO: 25, mouse Ube3a isoform 1 with 8x N-glycan sites (bold) and secretion signal (underlined). Bold, capitalized font provides examples of mutations to create glycosylation sites. [ka] [ka]

[0365] SEQ ID NO: 26, mouse Ube3a isoform 1 aa sequence with 8x N-glycan sites (bold) and secretion signal (underlined) [ka] Modified Mouse Isoform #2 Nucleotide positions 253, 356, 373, 715, 716, 1120, 1121, 1825, 1827, 1828, 1829, 1924, 1925, 2467 and 2468 AA positions 85, 119, 125, 239, 374, 609, 610, 642 and 823

[0366] SEQ ID NO: 27, mouse Ube3a isoform 2 with 8x N-glycan sites (bold) and secretion signal (underlined). Bold, capitalized font provides examples of mutations to create glycosylation sites. [ka] [ka] [ka]

[0367] SEQ ID NO: 28, mouse Ube3a isoform 2 aa sequence with 8x N-glycan sites (bold) and secretion signal (underlined) [ka] [ka] Modified Mouse Isoform #3 Nucleotide positions 190, 293, 310, 652, 653, 1057, 1058, 1762, 1764, 1765, 1766, 1861, 1862, 2404 and 2405 AA positions 64, 98, 104, 218, 353, 588, 589, 621 and 802

[0368] SEQ ID NO: 29, mouse Ube3a isoform 3 with 8x N-glycan sites (bold) and secretion signal (underlined). Bold, capitalized font provides examples of mutations to create glycosylation sites. [ka] [ka] [ka]

[0369] SEQ ID NO: 30, mouse Ube3a isoform 3 aa sequence with 8x N-glycan sites (bold) and secretion signal (underlined) [ka] [ka]

[0370] The nucleotide sequence shown in Figure 16A, which has SEQ ID NO: 31, NCBI Reference Number NM_001354506, is a fragment of Homo sapiens ubiquitin-protein ligase E3A (UBE3A), transcript variant 5, containing the wild-type Ube3a CDS (i.e., SEQ ID NO: 7) beginning with capitalized ATG and ending with capitalized TAA. Other capitalized nucleotide residues provide potential mutation sites to create glycosylation sites. [ka] [ka]

[0371] The amino acid sequence shown in Figure 16A includes SEQ ID NO:32, the wild-type Ube3a protein of SEQ ID NO:8. Non-capitalized font indicates NetNGlyC predicted N-glycosylation sites (marked in Figure 16A by 82, 579, 700, 719, which are provided as the residue numbers of the beginning of glycosylation sites in SEQ ID NO:8). Bold font provides some potential glycosylation sites that can be created by the mutations identified herein. Italic font indicates potential glycosylation sites that can be created by mutating the two amino acids preceding the S or T. Underlines represent potential glycosylation sites that can be created by mutating the two amino acids following the N. [ka] [ka]

[0372] SEQ ID NO: 33, the amino acid sequence shown in Figure 16A. [ka]

[0373] SEQ ID NO: 34, CMV promoter sequence 3. [ka]

[0374] SEQ ID NO: 35, CCLc-MNDU3-X vector, in which the CMV promoter of SEQ ID NO: 34 is shown in bold, italic, and capitalized font, and the MNDU3 promoter of SEQ ID NO: 3 is shown in bold, italic, and non-capitalized font.

[0375] [ka] [ka] [ka] The present invention provides, for example, the following items. (Item 1) A recombinant polynucleotide encoding a ubiquitin-protein ligase E3A (Ube3a) polypeptide or protein, or a biological equivalent thereof, wherein said Ube3a polypeptide or protein, or a biological equivalent thereof, comprises one or more naturally occurring or non-naturally occurring glycosylation sites. (Item 2) 2. The recombinant polynucleotide of item 1, wherein the glycosylation is N-linked glycosylation and the glycosylation site comprises a consensus sequence of NXaaT or NXaaS, where Xaa is any amino acid residue, optionally excluding proline (P). (Item 3) The glycosylation site is: aa62 to aa64 of SEQ ID NO: 14, aa96 to aa98 of SEQ ID NO: 14, aa102 to aa104 of SEQ ID NO: 14, aa219 to aa221 of SEQ ID NO: 14, aa354 to aa356 of SEQ ID NO: 14, aa591 to aa593 of SEQ ID NO: 14, aa622 to aa624 of SEQ ID NO: 14, aa805 to aa807 of SEQ ID NO: 14; aa85 to aa87 of SEQ ID NO: 16, aa119 to aa121 of SEQ ID NO: 16, aa125 to aa127 of SEQ ID NO: 16, aa242 to aa244 of SEQ ID NO: 16, aa377 to aa379 of SEQ ID NO: 16, aa614 to aa616 of SEQ ID NO: 16, aa645 to aa647 of SEQ ID NO: 16, aa828 to aa830 of SEQ ID NO: 16; aa82 to aa84 of SEQ ID NO: 18, aa116 to aa118 of SEQ ID NO: 18, aa122 to aa124 of SEQ ID NO: 18, aa239 to aa241 of SEQ ID NO: 18, aa374 to aa376 of SEQ ID NO: 18, aa611 to aa613 of SEQ ID NO: 18, aa642 to aa644 of SEQ ID NO: 18, aa825 to aa827 of SEQ ID NO: 18; aa83 to aa85 of SEQ ID NO: 26 or 28, aa117 to aa119 of SEQ ID NO: 26 or 28, aa123 to aa125 of SEQ ID NO: 26 or 28, aa237 to aa239 of SEQ ID NO: 26 or 28, aa372 to aa374 of SEQ ID NO: 26 or 28, aa609 to aa611 of SEQ ID NO: 26 or 28, aa640 to aa642 of SEQ ID NO: 26 or 28, aa823 to aa825 of SEQ ID NO: 26 or 28; aa62 to aa64 of SEQ ID NO: 30, aa96 to aa98 of SEQ ID NO: 30, aa102 to aa104 of SEQ ID NO: 30, aa216 to aa218 of SEQ ID NO: 30, aa351 to aa353 of SEQ ID NO: 30, aa588 to aa590 of SEQ ID NO: 30, aa619 to aa621 of SEQ ID NO: 30, or aa802 to aa804 of SEQ ID NO: 30 at amino acid (aa) positions of said polypeptide, protein or equivalent thereof corresponding to one or more of the positions selected from Optionally, any one or any two or all three amino acid residues in at least one of the glycosylation sites are mutated compared to a wild-type Ube3a polypeptide or protein, thereby constituting the glycosylation site. 3. The recombinant polynucleotide according to item 1 or 2. (Item 4) The Ube3a polypeptide, protein, or biological equivalent thereof is selected from the group consisting of the following (a) to (e): (a) aa62 to aa64 of SEQ ID NO: 14, aa96 to aa98 of SEQ ID NO: 14, aa102 to aa104 of SEQ ID NO: 14, aa219 to aa221 of SEQ ID NO: 14, aa354 to aa356 of SEQ ID NO: 14, aa591 to aa593 of SEQ ID NO: 14, aa622 to aa624 of SEQ ID NO: 14, and aa805 to aa807 of SEQ ID NO: 14; (b) aa85 to aa87 of SEQ ID NO: 16, aa119 to aa121 of SEQ ID NO: 16, aa125 to aa127 of SEQ ID NO: 16, aa242 to aa244 of SEQ ID NO: 16, aa377 to aa379 of SEQ ID NO: 16, aa614 to aa616 of SEQ ID NO: 16, aa645 to aa647 of SEQ ID NO: 16, and aa828 to aa830 of SEQ ID NO: 16; (c) aa82 to aa84 of SEQ ID NO: 18, aa116 to aa118 of SEQ ID NO: 18, aa122 to aa124 of SEQ ID NO: 18, aa239 to aa241 of SEQ ID NO: 18, aa374 to aa376 of SEQ ID NO: 18, aa611 to aa613 of SEQ ID NO: 18, aa642 to aa644 of SEQ ID NO: 18, and aa825 to aa827 of SEQ ID NO: 18; (d) aa83 to aa85 of SEQ ID NO: 26 or 28, aa117 to aa119 of SEQ ID NO: 26 or 28, aa123 to aa125 of SEQ ID NO: 26 or 28, aa237 to aa239 of SEQ ID NO: 26 or 28, aa372 to aa374 of SEQ ID NO: 26 or 28, aa609 to aa611 of SEQ ID NO: 26 or 28, aa640 to aa642 of SEQ ID NO: 26 or 28, and aa823 to aa825 of SEQ ID NO: 26 or 28; (e) aa62 to aa64 of SEQ ID NO: 30, aa96 to aa98 of SEQ ID NO: 30, aa102 to aa104 of SEQ ID NO: 30, aa216 to aa218 of SEQ ID NO: 30, aa351 to aa353 of SEQ ID NO: 30, aa588 to aa590 of SEQ ID NO: 30, aa619 to aa621 of SEQ ID NO: 30, or aa802 to aa804 of SEQ ID NO: 30 4. The recombinant polynucleotide of any one of items 1 to 3, comprising eight glycosylation sites at amino acid (aa) positions corresponding to the eight aa positions as identified in any one of (Item 5) The Ube3a polypeptide, protein or biological equivalent thereof is aa64 of SEQ ID NO:14, aa98 of SEQ ID NO:14, aa104 of SEQ ID NO:14, aa221 of SEQ ID NO:14, aa356 of SEQ ID NO:14, aa591 of SEQ ID NO:14, aa624 of SEQ ID NO:14, aa805 of SEQ ID NO:14, aa806 of SEQ ID NO:14; aa87 of SEQ ID NO:16, aa121 of SEQ ID NO:16, aa127 of SEQ ID NO:16, aa244 of SEQ ID NO:16, aa379 of SEQ ID NO:16, aa614 of SEQ ID NO:16, aa647 of SEQ ID NO:16, aa828 of SEQ ID NO:16, aa829 of SEQ ID NO:16; aa84 of SEQ ID NO:18, aa118 of SEQ ID NO:18, aa124 of SEQ ID NO:18, aa241 of SEQ ID NO:18, aa376 of SEQ ID NO:18, aa611 of SEQ ID NO:18, aa644 of SEQ ID NO:18, aa825 of SEQ ID NO:18, aa826 of SEQ ID NO:18; aa85 of SEQ ID NO:26 or 28, aa119 of SEQ ID NO:26 or 28, aa125 of SEQ ID NO:26 or 28, aa239 of SEQ ID NO:26 or 28, aa374 of SEQ ID NO:26 or 28, aa609 of SEQ ID NO:26 or 28, aa610 of SEQ ID NO:26 or 28, aa642 of SEQ ID NO:26 or 28, aa823 of SEQ ID NO:26 or 28; aa64 of SEQ ID NO:30, aa98 of SEQ ID NO:30, aa104 of SEQ ID NO:30, aa218 of SEQ ID NO:30, aa353 of SEQ ID NO:30, aa588 of SEQ ID NO:30, aa589 of SEQ ID NO:30, aa621 of SEQ ID NO:30, or aa802 of SEQ ID NO:30 5. The recombinant polynucleotide of any one of items 1 to 4, wherein the polynucleotide comprises one or more mutated amino acid residues at aa positions corresponding to one or more of the positions selected from the group consisting of: (Item 6) The Ube3a polypeptide, protein, or biological equivalent thereof is selected from the group consisting of the following (a) to (e): (a) aa64 of SEQ ID NO:14, aa98 of SEQ ID NO:14, aa104 of SEQ ID NO:14, aa221 of SEQ ID NO:14, aa356 of SEQ ID NO:14, aa591 of SEQ ID NO:14, aa624 of SEQ ID NO:14, aa805 of SEQ ID NO:14, and aa806 of SEQ ID NO:14; (b) aa87 of SEQ ID NO:16, aa121 of SEQ ID NO:16, aa127 of SEQ ID NO:16, aa244 of SEQ ID NO:16, aa379 of SEQ ID NO:16, aa614 of SEQ ID NO:16, aa647 of SEQ ID NO:16, aa828 of SEQ ID NO:16, and aa829 of SEQ ID NO:16; (c) aa84 of SEQ ID NO:18, aa118 of SEQ ID NO:18, aa124 of SEQ ID NO:18, aa241 of SEQ ID NO:18, aa376 of SEQ ID NO:18, aa611 of SEQ ID NO:18, aa644 of SEQ ID NO:18, aa825 of SEQ ID NO:18, and aa826 of SEQ ID NO:18; (d) aa85 of SEQ ID NO:26 or 28, aa119 of SEQ ID NO:26 or 28, aa125 of SEQ ID NO:26 or 28, aa239 of SEQ ID NO:26 or 28, aa374 of SEQ ID NO:26 or 28, aa609 of SEQ ID NO:26 or 28, aa610 of SEQ ID NO:26 or 28, aa642 of SEQ ID NO:26 or 28, and aa823 of SEQ ID NO:26 or 28; or (e) aa64 of SEQ ID NO: 30, aa98 of SEQ ID NO: 30, aa104 of SEQ ID NO: 30, aa218 of SEQ ID NO: 30, aa353 of SEQ ID NO: 30, aa588 of SEQ ID NO: 30, aa589 of SEQ ID NO: 30, aa621 of SEQ ID NO: 30, and aa802 of SEQ ID NO: 30 6. The recombinant polynucleotide of any one of items 1 to 5, comprising nine mutated amino acid residues at aa positions corresponding to positions identified in any one of (Item 7) The Ube3a polypeptide, protein, or biological equivalent thereof is selected from the group consisting of the following (a) to (e): (a) aa64 of SEQ ID NO:14, aa98 of SEQ ID NO:14, aa221 of SEQ ID NO:14, aa356 of SEQ ID NO:14, aa591 of SEQ ID NO:14, aa624 of SEQ ID NO:14, aa805 of SEQ ID NO:14, and aa806 of SEQ ID NO:14; (b) aa87 of SEQ ID NO:16, aa121 of SEQ ID NO:16, aa244 of SEQ ID NO:16, aa379 of SEQ ID NO:16, aa614 of SEQ ID NO:16, aa647 of SEQ ID NO:16, aa828 of SEQ ID NO:16, and aa829 of SEQ ID NO:16; (c) aa84 of SEQ ID NO:18, aa118 of SEQ ID NO:18, aa241 of SEQ ID NO:18, aa376 of SEQ ID NO:18, aa611 of SEQ ID NO:18, aa644 of SEQ ID NO:18, aa825 of SEQ ID NO:18, and aa826 of SEQ ID NO:18; (d) aa85 of SEQ ID NO:26 or 28, aa119 of SEQ ID NO:26 or 28, aa239 of SEQ ID NO:26 or 28, aa374 of SEQ ID NO:26 or 28, aa609 of SEQ ID NO:26 or 28, aa610 of SEQ ID NO:26 or 28, aa642 of SEQ ID NO:26 or 28, and aa823 of SEQ ID NO:26 or 28; or (e) aa64 of SEQ ID NO: 30, aa98 of SEQ ID NO: 30, aa218 of SEQ ID NO: 30, aa353 of SEQ ID NO: 30, aa588 of SEQ ID NO: 30, aa589 of SEQ ID NO: 30, aa621 of SEQ ID NO: 30, and aa802 of SEQ ID NO: 30 6. The recombinant polynucleotide of any one of items 1 to 5, comprising eight mutated amino acid residues at aa positions corresponding to positions identified in any one of (Item 8) 8. The recombinant polynucleotide of any one of Items 5 to 7, wherein the formed glycosylation site comprises a consensus sequence of NXaaT or NXaaS (wherein Xaa is any amino acid residue, optionally excluding proline (P)). (Item 9) The mutated amino acid residue is T or S at the aa position corresponding to aa64 of SEQ ID NO:14, T or S at the aa position corresponding to aa98 of SEQ ID NO:14, T or S at the aa position corresponding to aa104 of SEQ ID NO:14, T or S at the aa position corresponding to aa221 of SEQ ID NO:14, T or S at the aa position corresponding to aa356 of SEQ ID NO:14, N at the aa position corresponding to aa591 of SEQ ID NO:14, T or S at the aa position corresponding to aa624 of SEQ ID NO:14, N at the aa position corresponding to aa805 of SEQ ID NO:14, and N at the aa position corresponding to aa806 of SEQ ID NO:14; T or S at the aa position corresponding to aa87 of SEQ ID NO:16, T or S at the aa position corresponding to aa121 of SEQ ID NO:16, T or S at the aa position corresponding to aa127 of SEQ ID NO:16, T or S at the aa position corresponding to aa244 of SEQ ID NO:16, T or S at the aa position corresponding to aa379 of SEQ ID NO:16, N at the aa position corresponding to aa614 of SEQ ID NO:16, T or S at the aa position corresponding to aa647 of SEQ ID NO:16, N at the aa position corresponding to aa828 of SEQ ID NO:16, and N at the aa position corresponding to aa829 of SEQ ID NO:16; T or S at the aa position corresponding to aa84 of SEQ ID NO:18, T or S at the aa position corresponding to aa118 of SEQ ID NO:18, T or S at the aa position corresponding to aa124 of SEQ ID NO:18, T or S at the aa position corresponding to aa241 of SEQ ID NO:18, T or S at the aa position corresponding to aa376 of SEQ ID NO:18, N at the aa position corresponding to aa611 of SEQ ID NO:18, T or S at the aa position corresponding to aa644 of SEQ ID NO:18, N at the aa position corresponding to aa825 of SEQ ID NO:18, and N at the aa position corresponding to aa826 of SEQ ID NO:18; a T or S at aa position corresponding to aa85 of SEQ ID NO:26 or 28, a T or S at aa position corresponding to aa119 of SEQ ID NO:26 or 28, a T or S at aa position corresponding to aa125 of SEQ ID NO:26 or 28, a T or S at aa position corresponding to aa239 of SEQ ID NO:26 or 28, a T or S at aa position corresponding to aa374 of SEQ ID NO:26 or 28, an N at aa position corresponding to aa609 of SEQ ID NO:26 or 28, an N at aa position corresponding to aa610 of SEQ ID NO:26 or 28, a T or S at aa position corresponding to aa642 of SEQ ID NO:26 or 28, and an N at aa position corresponding to aa823 of SEQ ID NO:26 or 28; or T or S at the aa position corresponding to aa64 of SEQ ID NO:30, T or S at the aa position corresponding to aa98 of SEQ ID NO:30, T or S at the aa position corresponding to aa104 of SEQ ID NO:30, T or S at the aa position corresponding to aa218 of SEQ ID NO:30, T or S at the aa position corresponding to aa353 of SEQ ID NO:30, N at the aa position corresponding to aa588 of SEQ ID NO:30, N at the aa position corresponding to aa589 of SEQ ID NO:30, T or S at the aa position corresponding to aa621 of SEQ ID NO:30, and N at the aa position corresponding to aa802 of SEQ ID NO:30 9. The recombinant polynucleotide according to any one of items 5 to 8, selected from one or more of: (Item 10) 10. The recombinant polynucleotide of any one of items 1 to 6 and 8 to 9, wherein the Ube3a polypeptide, protein or biological equivalent thereof comprises one or more non-naturally occurring glycosylation sites. (Item 11) The Ube3a polypeptide, protein or biological equivalent thereof is aa21 to aa872 of SEQ ID NO: 14, aa21 to aa895 of SEQ ID NO: 16, aa21 to aa892 of SEQ ID NO: 18, aa21 to aa890 of SEQ ID NO: 26, aa21 to aa890 of SEQ ID NO: 28, aa21 to aa869 of SEQ ID NO: 30, or a sequence having at least about 90%, or at least about 95%, or at least about 99% identity thereto, respectively. 11. The recombinant polynucleotide according to any one of items 1 to 10, comprising an amino acid sequence selected from the group consisting of: (Item 12) 12. The recombinant polynucleotide according to any one of items 1 to 11, further comprising a polynucleotide encoding a signal peptide. (Item 13) 13. The recombinant polynucleotide of item 12, wherein the signal peptide is a secretion signal. (Item 14) 14. The recombinant polynucleotide of item 12 or 13, encoding a signal peptide or secretion signal selected from the following: an antibody heavy chain / light chain secretion signal, a twin-arginine transport protein secretion signal, an interleukin-2 (IL2) secretion signal, an interleukin-4 (IL4) secretion signal, an interleukin-10 (IL10) secretion signal, an interleukin-3 (IL3) secretion signal, an interleukin-7 (IL7) secretion signal, a human IL2 secretion signal, a human OSM secretion signal, a VSV-G secretion signal, a mouse Ig kappa secretion signal, a human IgG2 H secretion signal, a BM40 secretion signal, a secretion signal, a human IgKVIII secretion signal, a CD33 secretion signal, a tPA secretion signal, a human chymotrypsinogen secretion signal, a human trypsinogen-2 secretion signal, a Gaussia luc secretion signal, an albumin (HSA) secretion signal, an influenza hemagglutinin secretion signal, a human insulin secretion signal, or a silkworm fibroin LC. (Item 15) Item 16. The recombinant polynucleotide according to any one of Items 12 to 14, wherein the signal peptide or the secretory signal comprises the amino acid sequence of aa1 to aa20 of SEQ ID NO: 14. 16. The recombinant polynucleotide of any one of items 1 to 15, wherein the Ube3a polypeptide, protein, or biological equivalent thereof comprises an amino acid sequence selected from any one of SEQ ID NOs: 14, 16, 18, 26, 28, and 30, or a sequence having at least about 90%, or at least about 95%, or at least about 99% identity thereto, respectively. (Item 17) Next: nt61 to nt2619 of SEQ ID NO: 13, nt61 to nt2688 of SEQ ID NO: 15, nt61 to nt2679 of SEQ ID NO: 17, nt61 to nt2673 of SEQ ID NO: 25, nt61 to nt2673 of SEQ ID NO: 27, nt61 to nt2610 of SEQ ID NO: 29; SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, or a sequence having at least about 90%, or at least about 95%, or at least about 99% identity to each thereof. 17. The recombinant polynucleotide according to any one of items 1 to 16, comprising a nucleotide (nt) sequence selected from any one of the following: (Item 18) 18. The recombinant polynucleotide according to any one of items 1 to 17, wherein the Ube3a polypeptide, protein or biological equivalent thereof is derived from wild-type human Ube3a protein or wild-type mouse Ube3a protein. (Item 19) 19. The recombinant polynucleotide of Item 18, wherein the wild-type human Ube3a protein comprises the amino acid sequence of any one of SEQ ID NOs: 8, 10, or 12, and the wild-type mouse Ube3a protein comprises the amino acid sequence of any one of SEQ ID NOs: 20, 22, or 24. (Item 20) 20. The recombinant polynucleotide of any one of items 1 to 19, further comprising a regulatory sequence directing expression of the Ube3a polypeptide, protein or biological equivalent thereof. (Item 21) 21. The recombinant polynucleotide of item 20, wherein the regulatory sequence comprises one or more of the following: a promoter, an intron, an enhancer, a polyadenylation signal, a terminator, a silencer, a TATA box, or a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). (Item 22) 22. The recombinant polynucleotide of item 21, wherein the promoter is selected from the following: MNDU3 promoter, CMV promoter, PGK promoter, MNDU promoter, or EF1 alpha promoter. (Item 23) 23. The recombinant polynucleotide of item 21 or 22, wherein the promoter is the MNDU3 promoter. (Item 24) 24. The recombinant polynucleotide of claim 22 or 23, wherein the MNDU3 promoter comprises the sequence of SEQ ID NO: 3, the CMV promoter comprises a sequence selected from SEQ ID NO: 1, 2, or 34, the PKG promoter comprises the sequence of SEQ ID NO: 4, the MNDU promoter comprises the sequence of SEQ ID NO: 5, and the EF1 alpha promoter comprises the sequence of SEQ ID NO: 6. (Item 25) Next: Polypurine tract sequence (PPT), central PPT (cPPT), R region, U5, encapsidation signal (Psi), Rev-responsive element (RRE), full-length U3 or a fragment thereof, a detectable or selectable marker, a polynucleotide encoding a detectable or selectable polypeptide, a regulatory sequence directing expression of said detectable or selectable polypeptide, or a coding sequence for a self-cleaving peptide located between the coding sequence for said detectable or selectable polypeptide and the sequence encoding said Ube3a polypeptide or protein or a biological equivalent thereof. 25. The recombinant polynucleotide of any one of items 1 to 24, further comprising one or more of: (Item 26) 26. A recombinant polynucleotide which is the reverse or complement or reverse complement of the recombinant polynucleotide of any one of items 1 to 25. (Item 27) 27. A vector comprising the recombinant polynucleotide according to any one of items 1 to 26. (Item 28) 28. The vector according to item 27, which is a viral vector or a non-viral vector. (Item 29) 29. The vector of item 28, wherein the viral vector is selected from a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a herpes viral vector. (Item 30) 30. The vector of item 29, wherein the retroviral vector is a lentiviral vector. (Item 31) 31. The vector of item 30, wherein the lentiviral vector is a self-inactivating lentiviral vector optionally having a U3 region lacking a TATA box and, optionally, one or more transcription factor binding sites. (Item 32) 32. The vector of item 30 or 31, wherein the lentiviral vector is derived from the human immunodeficiency virus (HIV). (Item 33) 33. The vector according to item 32, wherein the non-viral vector is a plasmid. (Item 34) 34. The vector according to any one of items 27 to 33, further comprising a regulatory sequence operably linked to the recombinant polynucleotide and directing replication of the recombinant polynucleotide. (Item 35) A recombinant Ube3a protein, polypeptide, or biological equivalent thereof comprising one or more, or two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or ten or more, or eleven or more naturally occurring or non-naturally occurring glycosylation sites, wherein said recombinant Ube3a protein or polypeptide, or biological equivalent thereof, is not a wild-type Ube3a protein. (Item 36) 36. The recombinant Ube3a protein, polypeptide, or biological equivalent thereof of item 35, wherein the glycosylation is N-linked glycosylation and the glycosylation site comprises the consensus sequence NXaaT or NXaaS, where Xaa is any amino acid residue, optionally excluding proline (P). (Item 37) The glycosylation site is: aa62 to aa64 of SEQ ID NO: 14, aa96 to aa98 of SEQ ID NO: 14, aa102 to aa104 of SEQ ID NO: 14, aa219 to aa221 of SEQ ID NO: 14, aa354 to aa356 of SEQ ID NO: 14, aa591 to aa593 of SEQ ID NO: 14, aa622 to aa624 of SEQ ID NO: 14, aa805 to aa807 of SEQ ID NO: 14; aa85 to aa87 of SEQ ID NO: 16, aa119 to aa121 of SEQ ID NO: 16, aa125 to aa127 of SEQ ID NO: 16, aa242 to aa244 of SEQ ID NO: 16, aa377 to aa379 of SEQ ID NO: 16, aa614 to aa616 of SEQ ID NO: 16, aa645 to aa647 of SEQ ID NO: 16, aa828 to aa830 of SEQ ID NO: 16; aa82 to aa84 of SEQ ID NO: 18, aa116 to aa118 of SEQ ID NO: 18, aa122 to aa124 of SEQ ID NO: 18, aa239 to aa241 of SEQ ID NO: 18, aa374 to aa376 of SEQ ID NO: 18, aa611 to aa613 of SEQ ID NO: 18, aa642 to aa644 of SEQ ID NO: 18, aa825 to aa827 of SEQ ID NO: 18; aa83 to aa85 of SEQ ID NO: 26 or 28, aa117 to aa119 of SEQ ID NO: 26 or 28, aa123 to aa125 of SEQ ID NO: 26 or 28, aa237 to aa239 of SEQ ID NO: 26 or 28, aa372 to aa374 of SEQ ID NO: 26 or 28, aa609 to aa611 of SEQ ID NO: 26 or 28, aa640 to aa642 of SEQ ID NO: 26 or 28, aa823 to aa825 of SEQ ID NO: 26 or 28; aa62 to aa64 of SEQ ID NO: 30, aa96 to aa98 of SEQ ID NO: 30, aa102 to aa104 of SEQ ID NO: 30, aa216 to aa218 of SEQ ID NO: 30, aa351 to aa353 of SEQ ID NO: 30, aa588 to aa590 of SEQ ID NO: 30, aa619 to aa621 of SEQ ID NO: 30, or aa802 to aa804 of SEQ ID NO: 30 37. The recombinant Ube3a protein, polypeptide or biological equivalent thereof according to item 35 or 36, wherein the amino acid (aa) positions of said polypeptide or protein correspond to one or more of the positions selected from: (Item 38) The Ube3a polypeptide or protein, or a biological equivalent thereof, is one of the following (a) to (e): (a) aa62 to aa64 of SEQ ID NO: 14, aa96 to aa98 of SEQ ID NO: 14, aa102 to aa104 of SEQ ID NO: 14, aa219 to aa221 of SEQ ID NO: 14, aa354 to aa356 of SEQ ID NO: 14, aa591 to aa593 of SEQ ID NO: 14, aa622 to aa624 of SEQ ID NO: 14, and aa805 to aa807 of SEQ ID NO: 14; (b) aa85 to aa87 of SEQ ID NO: 16, aa119 to aa121 of SEQ ID NO: 16, aa125 to aa127 of SEQ ID NO: 16, aa242 to aa244 of SEQ ID NO: 16, aa377 to aa379 of SEQ ID NO: 16, aa614 to aa616 of SEQ ID NO: 16, aa645 to aa647 of SEQ ID NO: 16, and aa828 to aa830 of SEQ ID NO: 16; (c) aa82 to aa84 of SEQ ID NO: 18, aa116 to aa118 of SEQ ID NO: 18, aa122 to aa124 of SEQ ID NO: 18, aa239 to aa241 of SEQ ID NO: 18, aa374 to aa376 of SEQ ID NO: 18, aa611 to aa613 of SEQ ID NO: 18, aa642 to aa644 of SEQ ID NO: 18, and aa825 to aa827 of SEQ ID NO: 18; (d) aa83 to aa85 of SEQ ID NO: 26 or 28, aa117 to aa119 of SEQ ID NO: 26 or 28, aa123 to aa125 of SEQ ID NO: 26 or 28, aa237 to aa239 of SEQ ID NO: 26 or 28, aa372 to aa374 of SEQ ID NO: 26 or 28, aa609 to aa611 of SEQ ID NO: 26 or 28, aa640 to aa642 of SEQ ID NO: 26 or 28, and aa823 to aa825 of SEQ ID NO: 26 or 28; (e) aa62 to aa64 of SEQ ID NO: 30, aa96 to aa98 of SEQ ID NO: 30, aa102 to aa104 of SEQ ID NO: 30, aa216 to aa218 of SEQ ID NO: 30, aa351 to aa353 of SEQ ID NO: 30, aa588 to aa590 of SEQ ID NO: 30, aa619 to aa621 of SEQ ID NO: 30, or aa802 to aa804 of SEQ ID NO: 30 38. The recombinant Ube3a protein, polypeptide, or biological equivalent thereof according to items 35 to 37, comprising eight glycosylation sites at amino acid (aa) positions corresponding to the eight aa positions as identified in any one of (Item 39) Next: aa64 of SEQ ID NO:14, aa98 of SEQ ID NO:14, aa104 of SEQ ID NO:14, aa221 of SEQ ID NO:14, aa356 of SEQ ID NO:14, aa591 of SEQ ID NO:14, aa624 of SEQ ID NO:14, aa805 of SEQ ID NO:14, aa806 of SEQ ID NO:14; aa87 of SEQ ID NO:16, aa121 of SEQ ID NO:16, aa127 of SEQ ID NO:16, aa244 of SEQ ID NO:16, aa379 of SEQ ID NO:16, aa614 of SEQ ID NO:16, aa647 of SEQ ID NO:16, aa828 of SEQ ID NO:16, aa829 of SEQ ID NO:16; aa84 of SEQ ID NO:18, aa118 of SEQ ID NO:18, aa124 of SEQ ID NO:18, aa241 of SEQ ID NO:18, aa376 of SEQ ID NO:18, aa611 of SEQ ID NO:18, aa644 of SEQ ID NO:18, aa825 of SEQ ID NO:18, aa826 of SEQ ID NO:18; aa85 of SEQ ID NO:26 or 28, aa119 of SEQ ID NO:26 or 28, aa125 of SEQ ID NO:26 or 28, aa239 of SEQ ID NO:26 or 28, aa374 of SEQ ID NO:26 or 28, aa609 of SEQ ID NO:26 or 28, aa610 of SEQ ID NO:26 or 28, aa642 of SEQ ID NO:26 or 28, aa823 of SEQ ID NO:26 or 28; aa64 of SEQ ID NO:30, aa98 of SEQ ID NO:30, aa104 of SEQ ID NO:30, aa218 of SEQ ID NO:30, aa353 of SEQ ID NO:30, aa588 of SEQ ID NO:30, aa589 of SEQ ID NO:30, aa621 of SEQ ID NO:30, or aa802 of SEQ ID NO:30 wherein the amino acid sequence is a sequence selected from the group consisting of: Optionally, any one or any two or all three amino acid residues in at least one of the glycosylation sites are mutated compared to a wild-type Ube3a polypeptide or protein, thereby constituting the glycosylation site. 39. The recombinant Ube3a protein, polypeptide or biological equivalent thereof according to any one of items 35 to 38. (Item 40) The following (a) to (e): (a) aa64 of SEQ ID NO:14, aa98 of SEQ ID NO:14, aa104 of SEQ ID NO:14, aa221 of SEQ ID NO:14, aa356 of SEQ ID NO:14, aa591 of SEQ ID NO:14, aa624 of SEQ ID NO:14, aa805 of SEQ ID NO:14, and aa806 of SEQ ID NO:14; (b) aa87 of SEQ ID NO:16, aa121 of SEQ ID NO:16, aa127 of SEQ ID NO:16, aa244 of SEQ ID NO:16, aa379 of SEQ ID NO:16, aa614 of SEQ ID NO:16, aa647 of SEQ ID NO:16, aa828 of SEQ ID NO:16, and aa829 of SEQ ID NO:16; (c) aa84 of SEQ ID NO:18, aa118 of SEQ ID NO:18, aa124 of SEQ ID NO:18, aa241 of SEQ ID NO:18, aa376 of SEQ ID NO:18, aa611 of SEQ ID NO:18, aa644 of SEQ ID NO:18, aa825 of SEQ ID NO:18, and aa826 of SEQ ID NO:18; (d) aa85 of SEQ ID NO:26 or 28, aa119 of SEQ ID NO:26 or 28, aa125 of SEQ ID NO:26 or 28, aa239 of SEQ ID NO:26 or 28, aa374 of SEQ ID NO:26 or 28, aa609 of SEQ ID NO:26 or 28, aa610 of SEQ ID NO:26 or 28, aa642 of SEQ ID NO:26 or 28, and aa823 of SEQ ID NO:26 or 28; or (e) aa64 of SEQ ID NO: 30, aa98 of SEQ ID NO: 30, aa104 of SEQ ID NO: 30, aa218 of SEQ ID NO: 30, aa353 of SEQ ID NO: 30, aa588 of SEQ ID NO: 30, aa589 of SEQ ID NO: 30, aa621 of SEQ ID NO: 30, and aa802 of SEQ ID NO: 30 40. The recombinant Ube3a protein, polypeptide or biological equivalent thereof of any one of items 35 to 39, comprising 9 mutated amino acid residues at aa positions corresponding to positions identified in any one of (Item 41) The following (a) to (e): (a) aa64 of SEQ ID NO:14, aa98 of SEQ ID NO:14, aa221 of SEQ ID NO:14, aa356 of SEQ ID NO:14, aa591 of SEQ ID NO:14, aa624 of SEQ ID NO:14, aa805 of SEQ ID NO:14, and aa806 of SEQ ID NO:14; (b) aa87 of SEQ ID NO:16, aa121 of SEQ ID NO:16, aa244 of SEQ ID NO:16, aa379 of SEQ ID NO:16, aa614 of SEQ ID NO:16, aa647 of SEQ ID NO:16, aa828 of SEQ ID NO:16, and aa829 of SEQ ID NO:16; (c) aa84 of SEQ ID NO:18, aa118 of SEQ ID NO:18, aa241 of SEQ ID NO:18, aa376 of SEQ ID NO:18, aa611 of SEQ ID NO:18, aa644 of SEQ ID NO:18, aa825 of SEQ ID NO:18, and aa826 of SEQ ID NO:18; (d) aa85 of SEQ ID NO:26 or 28, aa119 of SEQ ID NO:26 or 28, aa239 of SEQ ID NO:26 or 28, aa374 of SEQ ID NO:26 or 28, aa609 of SEQ ID NO:26 or 28, aa610 of SEQ ID NO:26 or 28, aa642 of SEQ ID NO:26 or 28, and aa823 of SEQ ID NO:26 or 28; or (e) aa64 of SEQ ID NO: 30, aa98 of SEQ ID NO: 30, aa218 of SEQ ID NO: 30, aa353 of SEQ ID NO: 30, aa588 of SEQ ID NO: 30, aa589 of SEQ ID NO: 30, aa621 of SEQ ID NO: 30, and aa802 of SEQ ID NO: 30 40. The recombinant Ube3a protein, polypeptide or biological equivalent thereof of any one of items 35 to 39, comprising eight mutated amino acid residues at aa positions corresponding to positions identified in any one of (Item 42) 42. The recombinant Ube3a protein, polypeptide, or biological equivalent thereof of any one of items 39 to 41, wherein the formed glycosylation site comprises the consensus sequence NXaaT or NXaaS, where Xaa is any amino acid residue, optionally excluding proline (P). (Item 43) The mutated amino acid residue is T or S at the aa position corresponding to aa64 of SEQ ID NO:14, T or S at the aa position corresponding to aa98 of SEQ ID NO:14, T or S at the aa position corresponding to aa104 of SEQ ID NO:14, T or S at the aa position corresponding to aa221 of SEQ ID NO:14, T or S at the aa position corresponding to aa356 of SEQ ID NO:14, N at the aa position corresponding to aa591 of SEQ ID NO:14, T or S at the aa position corresponding to aa624 of SEQ ID NO:14, N at the aa position corresponding to aa805 of SEQ ID NO:14, and N at the aa position corresponding to aa806 of SEQ ID NO:14; T or S at the aa position corresponding to aa87 of SEQ ID NO:16, T or S at the aa position corresponding to aa121 of SEQ ID NO:16, T or S at the aa position corresponding to aa127 of SEQ ID NO:16, T or S at the aa position corresponding to aa244 of SEQ ID NO:16, T or S at the aa position corresponding to aa379 of SEQ ID NO:16, N at the aa position corresponding to aa614 of SEQ ID NO:16, T or S at the aa position corresponding to aa647 of SEQ ID NO:16, N at the aa position corresponding to aa828 of SEQ ID NO:16, and N at the aa position corresponding to aa829 of SEQ ID NO:16; T or S at the aa position corresponding to aa84 of SEQ ID NO:18, T or S at the aa position corresponding to aa118 of SEQ ID NO:18, T or S at the aa position corresponding to aa124 of SEQ ID NO:18, T or S at the aa position corresponding to aa241 of SEQ ID NO:18, T or S at the aa position corresponding to aa376 of SEQ ID NO:18, N at the aa position corresponding to aa611 of SEQ ID NO:18, T or S at the aa position corresponding to aa644 of SEQ ID NO:18, N at the aa position corresponding to aa825 of SEQ ID NO:18, and N at the aa position corresponding to aa826 of SEQ ID NO:18; a T or S at aa position corresponding to aa85 of SEQ ID NO:26 or 28, a T or S at aa position corresponding to aa119 of SEQ ID NO:26 or 28, a T or S at aa position corresponding to aa125 of SEQ ID NO:26 or 28, a T or S at aa position corresponding to aa239 of SEQ ID NO:26 or 28, a T or S at aa position corresponding to aa374 of SEQ ID NO:26 or 28, an N at aa position corresponding to aa609 of SEQ ID NO:26 or 28, an N at aa position corresponding to aa610 of SEQ ID NO:26 or 28, a T or S at aa position corresponding to aa642 of SEQ ID NO:26 or 28, and an N at aa position corresponding to aa823 of SEQ ID NO:26 or 28; or T or S at the aa position corresponding to aa64 of SEQ ID NO:30, T or S at the aa position corresponding to aa98 of SEQ ID NO:30, T or S at the aa position corresponding to aa104 of SEQ ID NO:30, T or S at the aa position corresponding to aa218 of SEQ ID NO:30, T or S at the aa position corresponding to aa353 of SEQ ID NO:30, N at the aa position corresponding to aa588 of SEQ ID NO:30, N at the aa position corresponding to aa589 of SEQ ID NO:30, T or S at the aa position corresponding to aa621 of SEQ ID NO:30, and N at the aa position corresponding to aa802 of SEQ ID NO:30 43. The recombinant Ube3a protein, polypeptide or biological equivalent thereof according to any one of items 39 to 42, selected from one or more of: (Item 44) 45. The recombinant Ube3a protein, polypeptide, or biological equivalent thereof according to any one of claims 35 to 43, comprising one or more non-naturally occurring glycosylation sites. Next: aa21 to aa872 of SEQ ID NO: 14, aa21 to aa895 of SEQ ID NO: 16, aa21 to aa892 of SEQ ID NO: 18, aa21 to aa890 of SEQ ID NO: 26, aa21 to aa890 of SEQ ID NO: 28, aa21 to aa869 of SEQ ID NO: 30, or a sequence having at least about 90%, or at least about 95%, or at least about 99% identity thereto, respectively. 45. The recombinant Ube3a protein, polypeptide, or biological equivalent thereof according to any one of items 35 to 44, comprising an amino acid sequence selected from: (Item 46) 46. ​​The recombinant Ube3a protein, polypeptide or biological equivalent thereof of any one of items 35 to 45, further comprising a signal peptide. (Item 47) 47. The recombinant Ube3a protein, polypeptide or biological equivalent thereof according to item 46, wherein the signal peptide is a secretion signal. (Item 48) 48. The recombinant Ube3a protein, polypeptide or biological equivalent thereof according to item 46 or 47, wherein the signal peptide or the secretory signal is selected from the following: an antibody heavy chain / light chain secretory signal, a twin-arginine transport protein secretory signal, an interleukin-2 (IL2) secretory signal, an interleukin-4 (IL4) secretory signal, an interleukin-10 (IL10) secretory signal, an interleukin-3 (IL3) secretory signal, an interleukin-7 (IL7) secretory signal, a human IL2 secretory signal, a human OSM secretory signal, a VSV-G secretory signal, a mouse Ig kappa secretory signal, a human IgG2 H secretory signal, a BM40 secretory signal, a secretory signal, a human IgKVIII secretory signal, a CD33 secretory signal, a tPA secretory signal, a human chymotrypsinogen secretory signal, a human trypsinogen-2 secretory signal, a Gaussia luc secretory signal, an albumin (HSA) secretory signal, an influenza hemagglutinin secretory signal, a human insulin secretory signal or a silkworm fibroin LC. (Item 49) 49. The recombinant Ube3a protein, polypeptide, or biological equivalent thereof according to any one of items 46 to 48, wherein the signal peptide or the secretory signal comprises the amino acid sequence of aa1 to aa20 of SEQ ID NO: 14. (Item 50) 50. The recombinant Ube3a protein, polypeptide, or biological equivalent thereof according to items 35 to 49, wherein the Ube3a polypeptide or protein, or biological equivalent thereof, comprises an amino acid sequence selected from any one of SEQ ID NOs: 14, 16, 18, 26, 28, and 30, or a sequence having at least about 90%, or at least about 95%, or at least about 99% identity thereto, respectively. (Item 51) 51. A recombinant Ube3a protein or polypeptide or a biological equivalent thereof according to items 35 to 50, further comprising an optional self-cleaving peptide and a detectable or selection polypeptide. (Item 52) 51. An isolated or engineered cell comprising one or more of the following: a recombinant polynucleotide according to any one of items 1 to 26, a vector according to any one of items 27 to 34, or a recombinant Ube3a protein, polypeptide or biological equivalent thereof according to any one of items 35 to 51, thereby expressing and secreting the recombinant Ube3a protein or polypeptide or biological equivalent thereof according to any one of items 35 to 51. (Item 53) 53. The isolated or engineered cell of item 52, which is a mammalian cell. (Item 54) 54. The isolated or engineered cell of item 53, wherein the mammalian cell is a mouse cell or a human cell. (Item 55) 55. The isolated or manipulated cell of any one of paragraphs 52 to 54, wherein the cell is selected from a stem cell, a progenitor cell, an induced pluripotent stem cell (iPSC), an embryonic stem cell, an adult or somatic stem cell, a mesenchymal stem cell, a neural stem cell, or their respective progeny. (Item 56) 56. The isolated or engineered cell of any one of paragraphs 52 to 55, which is a hematopoietic stem cell or its progeny. (Item 57) 57. The isolated or engineered cell of paragraph 56, which is CD34+. (Item 58) 58. The isolated or engineered cell of any one of paragraphs 52 to 57, further comprising a detectable marker. (Item 59) 59. An isolated cell population comprising the cell of any one of items 52 to 58, or a progeny thereof. (Item 60) 60. The isolated cell population of paragraph 59, which expresses CD4, CD14 and HLADR. (Item 61) 61. The isolated cell population of paragraph 60, wherein at least 90% of the cells in the population are CD4+, at least 95% of the cells in the population are CD14+, and at least 95% of the cells in the population are HLADR+. (Item 62) 62. The isolated cell population of any one of items 59 to 61, which is induced to produce macrophages under suitable conditions. (Item 63) 63. The isolated cell population of any one of items 59 to 62, which substantially comprises macrophages. (Item 64) 64. The isolated cell population of any one of items 59 to 63, which is substantially homogeneous. (Item 65) 65. The isolated cell population of any one of items 59 to 64, wherein the cells of the population further comprise a detectable marker. (Item 66) 67. A method for treating, preventing, halting, or reversing Angelman syndrome in a subject carrying a defective Ube3a allele, comprising the step of locally or systemically administering to the subject one or more of the following: a recombinant polynucleotide of any one of Items 1 to 26, a vector of any one of Items 27 to 34, a recombinant Ube3a protein, polypeptide, or biological equivalent thereof of any one of Items 35 to 51, a cell of any one of Items 52 to 58, or a cell population of any one of Items 59 to 65, thereby treating Angelman syndrome in the subject. (Item 67) 6. A method for expressing a Ube3a protein or polypeptide or a biological equivalent thereof in a subject, the method comprising the step of administering to the subject one or more of the following: a recombinant polynucleotide according to any one of Items 1 to 26, a vector according to any one of Items 27 to 34, a recombinant Ube3a protein, polypeptide or biological equivalent thereof according to any one of Items 35 to 51, a cell according to any one of Items 52 to 58, or a cell population according to any one of Items 59 to 65, thereby expressing Ube3a in the subject. (Item 68) 68. The method of claim 67, wherein the subject has a defective Ube3a gene. (Item 69) the cells or cell population are at least about 1.0 x 10 per kg of the subject's body weight 4 ~Approx. 1×10 15 69. The method of any one of items 66 to 68, wherein the cells are administered in a dose of 100 cells. (Item 70) 70. The method of any one of items 66 to 69, wherein the subject is symptomatic or asymptomatic for Angelman syndrome. (Item 71) 71. The method according to any one of items 66 to 70, wherein the subject is a mammal. (Item 72) 72. The method according to any one of items 66 to 71, wherein the subject is a human. (Item 73) 73. The method of any one of items 66 to 72, wherein the subject is a fetus. (Item 74) 73. The method of any one of items 66 to 72, wherein the subject is an infant or a prepubertal subject. (Item 75) 73. The method of any one of items 66 to 72, wherein the subject is an adult. (Item 76) An isolated or engineered cell comprising one or more of the following: a recombinant polynucleotide according to any one of items 1 to 26, a vector according to any one of items 27 to 34, a recombinant Ube3a protein, polypeptide or biological equivalent thereof according to any one of items 35 to 51, thereby producing said polynucleotide, said vector, or said recombinant Ube3a protein or polypeptide or biological equivalent thereof. (Item 77) 77. The isolated or engineered cell of item 76, which is a eukaryotic or prokaryotic cell. (Item 78) 78. The isolated or engineered cell of item 76 or 77, which is a mammalian cell. (Item 79) A clonal population of cells according to any one of items 76 to 78. (Item 80) A virus packaging system comprising: (a) the vector according to any one of Items 27 to 34; (b) a packaging plasmid; and (c) an envelope plasmid. (Item 81) (d) The viral packaging system of item 80, further comprising a packaging cell line. (Item 82) 82. The viral packaging system of claim 81, wherein the packaging cell line is a HEK-293 cell line. (Item 83) 81. A method for producing viral particles, comprising the step of transducing a packaging cell line with the system of paragraph 80 under conditions suitable for packaging said viral particles. (Item 84) Item 85. The method of Item 83, wherein the packaging cell line is a HEK-293 cell line. 79. A method for expressing a secreted Ube3a protein, polypeptide or biological equivalent thereof, comprising growing the cell of any one of items 52-58 and 76-78 under conditions that allow expression of the recombinant Ube3a protein or polypeptide or biological equivalent thereof. (Item 86) Item 87. The method of Item 85, wherein the cell is in vitro or in vivo. A composition comprising a carrier and one or more of the recombinant polynucleotide of any one of items 1 to 26, the vector of any one of items 27 to 34, the recombinant Ube3a protein, polypeptide or biological equivalent thereof of any one of items 35 to 51, the cell of any one of items 52 to 58 and 76 to 78, the cell population of any one of items 59 to 65, or the clonal population of item 79. (Item 88) 88. The composition of claim 87, wherein the carrier is a pharmaceutically acceptable carrier. (Item 89) A kit comprising one or more of the recombinant polynucleotide of any one of Items 1 to 26, the vector of any one of Items 27 to 34, the recombinant Ube3a protein, polypeptide or biological equivalent thereof of any one of Items 35 to 51, the cell of any one of Items 52 to 58 and 76 to 78, the cell population of any one of Items 59 to 65, or the clonal population of Item 79, and optionally instructions for use.

Claims

[Claim 1] A recombinant polynucleotide encoding a ubiquitin protein ligase E3A (Ube3a) polypeptide or protein or a biological equivalent thereof, wherein the Ube3a polypeptide or protein or a biological equivalent thereof comprises one or more naturally occurring or non-naturally occurring glycosylation sites.

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