Patent Application
20200113955
This invention relates to treatment of Angelman syndrome. More specifically, the present invention provides therapeutic methods and compositions for treating Angelman syndrome.
Angelman syndrome (AS) is a genetic disorder affecting neurons, estimated to effect about one in every 15,000 births (Clayton-Smith, Clinical research on Angelman syndrome in the United Kingdom: observations on 82 affected individuals. Am J Med Genet. 1993 Apr. 1; 46(1):12-5), though the actual number of diagnosed AS cases is greater likely due to misdiagnosis.
Angelman syndrome is a continuum of impairment, which presents with delayed and reduced intellectual and developmental advancement, most notably regarding language and motor skills. In particular, AS is defined by little or no verbal communication, with some non-verbal communication, ataxia, and disposition that includes frequent laughing and smiling and excitable movement.
More advanced cases result in severe mental retardation, seizures that may be difficult to control that typically begin before or by three years of age, frequent laughter (Nicholls, New insights reveal complex mechanisms involved in genomic imprinting. Am J Hum Genet. 1994 May; 54(5):733-40), miroencephaly, and abnormal EEG. In severe cases, patients may not develop language or may only have use of 5-10 words. Movement is commonly jerky, and walking commonly is associated with hand flapping and a stiff-gait. The patients are commonly epileptic, especially earlier in life, and suffer from sleep apnea, commonly only sleeping for 5 hours at a time. They are social and desire human contact. In some cases, skin and eyes may have little or no pigment, they may possess sucking and swallowing problems, sensitivity to heat, and a fixation to water bodies. Studies in UBE3A-deficient mice show disturbances in long-term synaptic plasticity. There are currently no cures for Angelman syndrome, and treatment is palliative. For example, anticonvulsant medication is used to reduce epileptic seizures, and speech and physical therapy are used to improve language and motor skills.
The gene UBE3A is responsible for AS and it is unique in that it is one of a small family of human imprinted genes. UBE3A, found on chromosome 15, encodes for the homologous to E6AP C terminus (HECT) protein (E6-associated protein (E6AP) (Kishino, et al., UBE3A/E6-AP mutations cause Angelman syndrome. Nat Gen. 1997 Jan. 15.15(1):70-3). UBE3A undergoes spatially-defined maternal imprinting in the brain; thus, the paternal copy is silenced via DNA methylation (Albrecht, et al., Imprinted expression of the murine Angelman syndrome gene, Ube3a, in hippocampal and Purkinje neurons. Nat Genet. 1997 September; 17(1):75-8). As such, only the maternal copy is active, the paternal chromosome having little or no effect on the proteosome of the neurons in that region of the brain. Inactivation, translocation, or deletion of portions of chromosome 15 therefore results in uncompensated loss of function. Some studies suggest improper E3-AP protein levels alter neurite contact in Angelman syndrome patients (Tonazzini, et al., Impaired neurite contract guidance in ubuitin ligase E3a (Ube3a)-deficient hippocampal neurons on nanostructured substrates. Adv Healthc Mater. 2016 April; 5(7):850-62).
The majority of Angelman's syndrome cases (70%) occur through a de novo deletion of around 4 Mb from 15q11-q13 of the maternal chromosome which incorporates the UBE3A gene (Kaplan, et al., Clinical heterogeneity associated with deletions in the long arm of chromosome 15: report of 3 new cases and their possible significance. Am J Med Genet. 1987 September; 28(1):45-53), but it can also occur as a result of abnormal methylation of the maternal copy, preventing its expression (Buiting, et al., Inherited microdeletions in the Angelman and Prader-Willi syndromes define an imprinting centre on human chromosome 15. Nat Genet. 1995 April; 9(4):395-400; Gabriel, et al., A transgene insertion creating a heritable chromosome deletion mouse model of Prader-Willi and Angelman syndrome. Proc Natl Acad Sci U.S.A. 1999 August; 96(16):9258-63) or uniparental disomy in which two copies of the paternal gene are inherited (Knoll, et al., Angelman and Prader-Willi syndromes share a common chromosome 15 deletion but differ in parental origin of the deletion. Am J Med Genet. 1989 Fed; 32(2):285-90; Malcolm, et al., Uniparental paternal disomy in Angelman's syndrome. Lancet. 1991 Mar. 23; 337(8743):694-7). The remaining AS cases arise through various UBE3A mutations of the maternal chromosome or they are diagnosed without a genetic cause (12-15UBE3A codes for the E6-associated protein (E6-AP) ubiquitin ligase. E6-AP is an E3 ubiquitin ligase, therefore it exhibits specificity for its protein targets, which include the tumor suppressor molecule p53 (Huibregtse, et al., A cellular protein mediates association of p53 with the E6 oncoprotein of human papillomavirus types 16 or 18. EMBO J. 1991 December; 10(13):4129-35), a human homologue to the yeast DNA repair protein Rad23 (Kumar, et al., Identification of HHR23A as a substrate for E6-associated protein-mediated ubiquitination. J Biol Chem. 1999 Jun. 25; 274(26):18785-92), E6-AP itself, and Arc, the most recently identified target (Nuber, et al., The ubiquitin-protein ligase E6-associated protein (E6-AP) serves as its own substrate. Eur J Biochem. 1998 Jun. 15; 254(3):643-9; Greer, et al., The Angelman Syndrome protein Ube3A regulates synapse Development by ubiquitinating arc. Cell. 2010 Mar. 5; 140(5): 704-16).
Mild cases are likely due to a mutation in the UBE3A gene at chromosome 15q11-13, which encodes for E6-AP ubiquitin ligase protein of the ubiquitin pathway, and more severe cases resulting from larger deletions of chromosome 15. Commonly, the loss of the UBE3A gene in the hippocampus and cerebellum result in Angelman syndrome, though single loss-of-function mutations can also result in the disorder.
The anatomy of the mouse and human AS brain shows no major alterations compared to the normal brain, indicating the cognitive deficits may be biochemical in nature as opposed to developmental (Jiang, et al., Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation. Neuron. 1998 October; 21(4):799-811; Davies, et al., Imprinted gene expression in the brain. Neurosci Biobehav Rev. 2005 May; 29(3):421-430). An Angelman syndrome mouse model possessing a disruption of the maternal UBE3A gene through a null mutation of exon 2 (Jiang, et al., Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation. Neuron. 1998 October; 21(4):799-811) was used. This model has been incredibly beneficial to the field of AS research due to its ability in recapitulating the major phenotypes characteristic of AS patients. For example, the AS mouse has inducible seizures, poor motor coordination, hippocampal-dependent learning deficits, and defects in hippocampal LTP. Cognitive deficits in the AS mouse model were previously shown to be associated with abnormalities in the phosphorylation state of calcium/calmodulin-dependent protein kinase II (CaMKII) (Weeber, et al., Derangements of hippocampal calcium/calmodulin-dependent protein kinase II in a mouse model for Angelman mental retardation syndrome. J Neurosci. 2003 April; 23(7):2634-44). There was a significant increase in phosphorylation at both the activating Thr286 site as well as the inhibitory Thr305 site of αCaMKII without any changes in total enzyme level, resulting in an overall decrease in its activity. There was also a reduction in the total amount of CaMKII at the postsynaptic density, indicating a reduction in the amount of active CaMKII. Crossing a mutant mouse model having a point mutation at the Thr305 site preventing phosphorylation with the AS mouse rescued the AS phenotype. i.e. seizure activity, motor coordination, hippocampal-dependent learning, and LTP were restored similar to wildtype levels. Thus, postnatal expression of αCaMKII suggests that the major phenotypes of the AS mouse model are due to postnatal biochemical alterations as opposed to a global developmental defect (Bayer, et al., Developmental expression of the CaM kinase II isoforms: ubiquitous γ- and δ-CaM kinase II are the early isoforms and most abundant in the developing nervous system. Brain Res Mol Brain Res. 1999 Jun. 18; 70(1):147-54).
Deficiencies in Ube3a are also linked in Huntington's disease (Maheshwari, et al., Deficiency of Ube3a in Huntington's disease mice brain increases aggregate load and accelerates disease pathology. Hum Mol Genet. 2014 Dec. 1; 23(23):6235-45).
Matentzoglu noted E6-AP possesses non-E3 activity related to hormone signaling (Matentzoglu, EP 2,724,721 A1). As such, administration of steroids, such as androgens, estrogens, and glucocorticoids, was used for treating various E6-AP disorders, including Angelman syndrome, autism, epilepsy, Prader-Willi syndrome, cervical cancer, fragile X syndrome, and Rett syndrome. Philpot suggested using a topoisomerase inhibitor to demethylate silenced genes thereby correcting for deficiencies in Ube3A (Philpot, et al., P.G. Pub. US 2013/0317018 A1). However, work in the field, and proposed therapeutics, do not address the underlying disorder, as in the use of steroids, or may result in other disorders, such as autism, where demethylation compounds are used. Accordingly, what is needed is a therapeutic that addresses the underlying cause of UBE3A deficiency disorders, in a safe, efficacious manner.
Nash & Weeber (WO 2016/179584) demonstrated that recombinant adeno-associated virus (rAAV) vectors can be an effective method for gene delivery in mouse models. However, only a small population of neurons are successfully transduced and thus express the protein, preventing global distribution of the protein in the brain as needed for efficacious therapy. As such, what is needed is a therapeutic that provides for supplementation of Ube3a protein throughout the entire brain.
While most human disorders characterized by severe mental retardation involve abnormalities in brain structure, no gross anatomical changes are associated with AS. A Ube3a protein has been generated containing an appended to a cellular secretion sequence that allows the secretion of Ube3a from cells and cellular uptake sequence that provides uptake by neighboring neuronal cells. This provides a functional E6-AP protein into the neurons thereby rescuing from disease pathology.
The efficacy of novel plasmid constructs containing a modified Ube3A gene with secretion signals to promote E6-AP secretion and cell-penetrating peptide (CPP) signals to promote E6-AP reuptake in neighboring cells were examined. This allows for a greater global distribution of E6-AP upon transduction into a mouse brain, as a gene therapy for AS.
As such, a UBE3A vector was formed using a transcription initiation sequence, and a UBE construct disposed downstream of the transcription initiation sequence. The UBE construct is formed of a UBE3A sequence, a secretion sequence, and a cell uptake sequence. Nonlimiting examples of the UBE3A sequence include Mus musculus UBE3A, Homo sapiens UBE3A variant 1, variant 2, or variant 3. Nonlimiting examples of the cell uptake sequence include penetratin, R6W3, HIV TAT, HIV TATk and pVEC. Nonlimiting examples of the secretion sequence include insulin, GDNF and IgK.
In some variations of the invention, the transcription initiation sequence is a cytomegalovirus chicken-beta actin hybrid promoter, or human ubiquitin c promoter. The invention optionally includes an enhancer sequence. A nonlimiting example of the enhancer sequence is a cytomegalovirus immediate-early enhancer sequence disposed upstream of the transcription initiation sequence. The vector optionally also includes a woodchuck hepatitis post-transcriptional regulatory element.
In variations, the vector is inserted into a plasmid, such as a recombinant adeno-associated virus serotype 2-based plasmid. In specific variations, the recombinant adeno-associated virus serotype 2-based plasmid lacks DNA integration elements. A nonlimiting example of the recombinant adeno-associated virus serotype 2-based plasmid is a pTR plasmid.
In some variations, the secretion sequence is disposed upstream of the UBE3A sequence. The cell uptake sequence may be disposed upstream of the UBE3A sequence and downstream of the secretion sequence.
Also presented is a method of treating a neurodegenerative disorder characterized by UBE3A deficiency such as Angelman syndrome and Huntington's disease, by administering a therapeutically effective amount of UBE3A vector, as described previously, to the brain of a patient in order to correct the UBE3A deficiency. The vector may be administered by injection into the brain, such as by intrahippocampal or intraventricular injection. In some instances, the vector may be injected bilaterally. Exemplary dosages can range between about 5.55×1011 to 2.86×1012 genomes/g brain mass.
A composition for use in treating a neurodegenerative disorder characterized by UBE3A deficiency is also presented. The composition may be comprised of a UBE3A vector as described above, and a pharmaceutically acceptable carrier. In some instances, the pharmaceutically acceptable carrier can be a blood brain barrier permeabilizer such as mannitol.
For a fuller understanding of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which:
As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a polypeptide” includes a mixture of two or more polypeptides and the like.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference in their entirety to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.
All numerical designations, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied up or down by increments of 1.0 or 0.1, as appropriate. It is to be understood, even if it is not always explicitly stated that all numerical designations are preceded by the term “about”. It is also to be understood, even if it is not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art and can be substituted for the reagents explicitly stated herein.
As used herein, the term “comprising” is intended to mean that the products, compositions and methods include the referenced components or steps, but not excluding others. “Consisting essentially of” when used to define products, compositions and methods, shall mean excluding other components or steps of any essential significance. Thus, a composition consisting essentially of the recited components would not exclude trace contaminants and pharmaceutically acceptable carriers. “Consisting of” shall mean excluding more than trace elements of other components or steps.
As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a vector” includes a plurality of vectors.
As used herein, “about” means approximately or nearly and in the context of a numerical value or range set forth means ±15% of the numerical.
“Adeno-associated virus (AAV) vector” as used herein refers to an adeno-associated virus vector that can be engineered for specific functionality in gene therapy. In some instances, the AAV can be a recombinant adeno-associated virus vector, denoted rAAV. While AAV4 is described for use herein, any suitable AAV known in the art can be used, including, but not limited to, AAV9, AAV5, AAV1 and AAV4.
“Administration” or “administering” is used to describe the process in which compounds of the present invention, alone or in combination with other compounds, are delivered to a patient. The composition may be administered in various ways including injection into the central nervous system including the brain, including but not limited to, intrastriatal, intrahippocampal, ventral tegmental area (VTA) injection, intracerebral, intracerebellar, intramedullary, intranigral, intraventricular, intracisternal, intracranial, intraparenchymal including spinal cord and brain stem; oral; parenteral (referring to intravenous and intraarterial and other appropriate parenteral routes); intrathecal; intramuscular; subcutaneous; rectal; and nasal, among others. Each of these conditions may be readily treated using other administration routes of compounds of the present invention to treat a disease or condition.
“Treatment” or “treating” as used herein refers to any of: the alleviation, amelioration, elimination and/or stabilization of a symptom, as well as delay in progression of a symptom of a particular disorder. For example, “treatment” of a neurodegenerative disease may include any one or more of the following: amelioration and/or elimination of one or more symptoms associated with the neurodegenerative disease, reduction of one or more symptoms of the neurodegenerative disease, stabilization of symptoms of the neurodegenerative disease, and delay in progression of one or more symptoms of the neurodegenerative disease.
“Prevention” or “preventing” as used herein refers to any of: halting the effects of the neurodegenerative disease, reducing the effects of the neurodegenerative disease, reducing the incidence of the neurodegenerative disease, reducing the development of the neurodegenerative disease, delaying the onset of symptoms of the neurodegenerative disease, increasing the time to onset of symptoms of the neurodegenerative disease, and reducing the risk of development of the neurodegenerative disease.
The pharmaceutical compositions of the subject invention can be formulated according to known methods for preparing pharmaceutically useful compositions. Furthermore, as used herein, the phrase “pharmaceutically acceptable carrier” means any of the standard pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier can include diluents, adjuvants, and vehicles, as well as implant carriers, and inert, non-toxic solid or liquid fillers, diluents, or encapsulating material that does not react with the active ingredients of the invention. Examples include, but are not limited to, phosphate buffered saline, physiological saline, water, and emulsions, such as oil/water emulsions. In some embodiments, the pharmaceutically acceptable carrier can be a blood brain permeabilizer including, but not limited to, mannitol. The carrier can be a solvent or dispersing medium containing, for example, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Formulations are described in a number of sources that are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Sciences (Martin E W [1995] Easton Pa., Mack Publishing Company, 19th ed.) describes formulations which can be used in connection with the subject invention.
As used herein “animal” means a multicellular, eukaryotic organism classified in the kingdom Animalia or Metazoa. The term includes, but is not limited to, mammals. Nonlimiting examples include rodents, mammals, aquatic mammals, domestic animals such as dogs and cats, farm animals such as sheep, pigs, cows and horses, and humans. Wherein the terms “animal” or the plural “animals” are used, it is contemplated that it also applies to any animals.
As used herein the phrase “conservative substitution” refers to substitution of amino acids with other amino acids having similar properties (e.g. acidic, basic, positively or negatively charged, polar or non-polar). The following six groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).
As used herein “conservative mutation”, refers to a substitution of a nucleotide for one which results in no alteration in the encoding for an amino acid, i.e. a change to a redundant sequence in the degenerate codons, or a substitution that results in a conservative substitution. An example of codon redundancy is seen in Tables 1 and 2.
Thus, according to Table 2, conservative mutations to the codon UUA include UUG, CUU, CUC, CUA, and CUG.
As used herein, the term “homologous” means a nucleotide sequence possessing at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, and even more preferably at least 98% sequence identity to the target sequence. Variations in the nucleotide sequence can be conservative mutations in the nucleotide sequence, i.e. mutations in the triplet code that encode for the same amino acid as seen in the Table 2.
As used herein, the term “therapeutically effective amount” refers to that amount of a therapy (e.g., a therapeutic agent or vector) sufficient to result in the amelioration of Angelman syndrome or other UBE3A-related disorder or one or more symptoms thereof, prevent advancement of Angelman syndrome or other UBE3A-related disorder, or cause regression of Angelman syndrome or other UBE3A-related disorder. In accordance with the present invention, a suitable single dose size is a dose that is capable of preventing or alleviating (reducing or eliminating) a symptom in a patient when administered one or more times over a suitable time period. One of skill in the art can readily determine appropriate single dose sizes for systemic administration based on the size of a mammal and the route of administration.
The dosing of compounds and compositions of the present invention to obtain a therapeutic or prophylactic effect is determined by the circumstances of the patient, as known in the art. The dosing of a patient herein may be accomplished through individual or unit doses of the compounds or compositions herein or by a combined or prepackaged or pre-formulated dose of a compounds or compositions. An average 40 g mouse has a brain weighing 0.416 g, and a 160 g mouse has a brain weighing 1.02 g, a 250 g mouse has a brain weighing 1.802 g. An average human brain weighs 1508 g, which can be used to direct the amount of therapeutic needed or useful to accomplish the treatment described herein.
Nonlimiting examples of dosages include, but are not limited to: 5.55×1011 genomes/g brain mass, 5.75×1011 genomes/g brain mass, 5.8×1011 genomes/g brain mass, 5.9×1011 genomes/g brain mass, 6.0×1011 genomes/g brain mass, 6.1×1011 genomes/g brain mass, 6.2×1011 genomes/g brain mass, 6.3×1011 genomes/g brain mass, 6.4×1011 genomes/g brain mass, 6.5×1011 genomes/g brain mass, 6.6.×1011 genomes/g brain mass, 6.7×1011 genomes/g brain mass, 6.8×1011 genomes/g brain mass, 6.9.×1011 genomes/g brain mass, 7.0×1011 genomes/g brain mass, 7.1×1011 genomes/g brain mass, 7.2×1011 genomes/g brain mass, 7.3×1011 genomes/g brain mass, 7.4×1011 genomes/g brain mass, 7.5×1011 genomes/g brain mass, 7.6×1011 genomes/g brain mass, 7.7×1011 genomes/g brain mass, 7.8×1011 genomes/g brain mass, 7.9×1011 genomes/g brain mass, 8.0×1011 genomes/g brain mass, 8.1×1011 genomes/g brain mass, 8.2×1011 genomes/g brain mass, 8.3×1011 genomes/g brain mass, 8.4×1011 genomes/g brain mass, 8.5×1011 genomes/g brain mass, 8.6×1011 genomes/g brain mass, 8.7×1011 genomes/g brain mass, 8.8×1011 genomes/g brain mass, 8.9×1011 genomes/g brain mass, 9.0×1011 genomes/g brain mass, 9.1×1011 genomes/g brain mass, 9.2×1011 genomes/g brain mass, 9.3×1011 genomes/g brain mass, 9.4×1011 genomes/g brain mass, 9.5×1011 genomes/g brain mass, 9.6×1011 genomes/g brain mass, 9.7×1011 genomes/g brain mass, 9.80×1011 genomes/g brain mass, 1.0×1012 genomes/g brain mass, 1.1×1012 genomes/g brain mass, 1.2×1012 genomes/g brain mass, 1.3×1012 genomes/g brain mass, 1.4×1012 genomes/g brain mass, 1.5×1012 genomes/g brain mass, 1.6×1012 genomes/g brain mass, 1.7×1012 genomes/g brain mass, 1.8×1012 genomes/g brain mass, 1.9×1012 genomes/g brain mass, 2.0×1012 genomes/g brain mass, 2.1×1012 genomes/g brain mass, 2.2×1012 genomes/g brain mass, 2.3×1012 genomes/g brain mass, 2.40×1012 genomes/g brain mass, 2.5×1012 genomes/g brain mass, 2.6×1012 genomes/g brain mass, 2.7×1012 genomes/g brain mass, 2.75×1012 genomes/g brain mass, 2.8×1012 genomes/g brain mass, or 2.86×1012 genomes/g brain mass.
The compositions used in the present invention may be administered individually, or in combination with or concurrently with one or more other therapeutics for neurodegenerative disorders, specifically UBE3A deficient disorders.
As used herein “patient” is used to describe an animal, preferably a human, to whom treatment is administered, including prophylactic treatment with the compositions of the present invention.
“Neurodegenerative disorder” or “neurodegenerative disease” as used herein refers to any abnormal physical or mental behavior or experience where the death or dysfunction of neuronal cells is involved in the etiology of the disorder. Further, the term “neurodegenerative disease” as used herein describes “neurodegenerative diseases” which are associated with UBE3A deficiencies. Exemplary neurodegenerative diseases include Angelman's Syndrome, Huntington's disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, autistic spectrum disorders, epilepsy, multiple sclerosis, Prader-Willi syndrome, Fragile X syndrome, Rett syndrome and Pick's Disease.
“UBE3A deficiency” as used herein refers to a mutation or deletion in the UBE3A gene.
The term “normal” or “control” as used herein refers to a sample or cells or patient which are assessed as not having Angelman syndrome or any other neurodegenerative disease or any other UBE3A deficient neurological disorder.
Generally, a UBE3A vector was formed using a transcription initiation sequence, and a UBE construct disposed downstream of the transcription initiation sequence. The UBE construct is formed of a UBE3A sequence, a secretion sequence, and a cell uptake sequence. Nonlimiting examples of the UBE3A sequence are SEQ ID No: 4, SEQ ID No: 9, SEQ ID No: 14, SEQ ID No:15, SEQ ID NO: 17, a cDNA of SEQ ID No: 10, a cDNA of SEQ ID No: 16, or a homologous sequence. Variations of the DNA sequence include conservative mutations in the DNA triplet code, as seen in Tables 1 and 2. In specific variations, the UBE3A sequence is Mus musculus UBE3A, Homo sapiens UBE3A variant 1, variant 2, or variant 3.
Nonlimiting examples of the secretion sequence are SEQ ID No: 2, SEQ ID No: 5, SEQ ID No: 11, SEQ ID No: 12, a cDNA of SEQ ID No: 3, a cDNA of SEQ ID NO: 7, a cDNA of SEQ ID NO: 18. A cDNA of SEQ ID NO: 19, or a homologous sequence, with variations of the DNA sequence that include the aforementioned conservative mutations.
Nonlimiting examples of the cell uptake sequence are SEQ ID No: 6, a cDNA of SEQ ID No. 8, a cDNA of SEQ ID No: 13, a cDNA of SEQ ID No: 20, a cDNA of SEQ ID No: 21, a cDNA of SEQ ID No: 22, or a homologous sequence. Variations of the DNA sequence include the aforementioned conservative mutations.
In specific variations of the invention, the secretion sequence is disposed upstream of the UBE3A sequence, and more specifically is optionally is disposed upstream of the UBE3A sequence and downstream of the secretion sequence. Other possible uptake proteins include penetratin, TATk, pVEC, transportan, MPG, Pep-1, polyarginines, MAP, and R6W3.
In some variations of the invention, the transcription initiation sequence is a cytomegalovirus chicken-beta actin hybrid promoter, or human ubiquitin c promoter. The invention optionally includes an enhancer sequence. A nonlimiting example of the enhancer sequence is a cytomegalovirus immediate-early enhancer sequence disposed upstream of the transcription initiation sequence. The vector optionally also includes a woodchuck hepatitis post-transcriptional regulatory element. The listed promotors, enhancer sequence and post-transcriptional regulatory element are well known in the art. (Garg S. et al., The hybrid cytomegalovirus enhancer/chicken beta-actin promotor along with woodchuck hepatitis virus posttranscriptional regulatory element enhances the protective efficacy of DNA vaccines, J. Immunol., Jul. 1, 2004; 173(1):550-558; Higashimoto, T. et al., The woodchuck hepatitis virus post-transcriptional regulatory element reduces readthrough transcription from retroviral vectors, September 2007; 14(17): 1298-304; Cooper, A. R. et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter, Nucleic Acids Res., January 2015; 43(1):682-90).
In variations, the vector is inserted into a plasmid, such as a recombinant adeno-associated virus serotype 2-based plasmid. In specific variations, the recombinant adeno-associated virus serotype 2-based plasmid lacks DNA integration elements. A nonlimiting example of the recombinant adeno-associated virus serotype 2-based plasmid is a pTR plasmid.
A method of synthesizing the UBE3A vector includes inserting a UBE3A construct into a backbone plasmid having a transcription initiation sequence. The TBE3A construct is formed of a UBE3A sequence, a secretion sequence, and a cell uptake sequence as described above. For example, Ube3a gene was cloned and fused in frame to the 3′ DNA sequence (N-terminus with two other peptide sequences), signal peptide and HIV TAT sequences, which were cloned into a recombinant adeno-associated viral vector for expression of the secreted E6-AP protein in the brain and spinal cord of AS patients. The UBE construct is optionally inserted by cleaving the backbone plasmid with at least one endonuclease, and the UBE3A construct ligated to the cleaved ends of the backbone plasmid.
The vector was then optionally inserted into an amplification host, possessing an antibiotic resistance gene, and subjected to an antibiotic selection corresponding to the antibiotic resistance gene. The amplification host was then expanded in a medium containing the antibiotic selection and the expanded amplification host collected. The vector was then isolated from the amplification host. In specific variations of the invention, the antibiotic resistance gene is an ampicillin resistance gene, with the corresponding antibiotic selection, ampicillin.
In a preferred embodiment, a UBE3A vector is formed from cDNA cloned from a Homo sapiens UBE3A gene to form the UBE3A, version 1 gene (SEQ ID No: 9) which is fused to a gene encoding a secretion signaling peptide, such as GDNF, insulin or IgK. In a preferred embodiment, GDNF is used. The construct is inserted into the hSTUb vector, under a CMV chicken-beta actin hybrid promoter (preferred) or a human ubiquitin c promoter. Woodchuck hepatitis post-transcriptional regulatory element (WPRE) is present to increase expression levels.
The UBE3A-seretion signal construct is then attached to a cellular uptake peptide (cell penetrating peptide or CPP) such as HIV TAT or HIV TATk (preferred). The human UBE3A vector is then transformed into an amplification host such as E. coli using the heat shock method described in Example 2. The transformed E. coli were expanded in broth containing ampicillin to select for the vector and collect large amounts of vector. Therapeutically effective doses of vector can then the administered to a patient as a gene therapy for treating Angelman syndrome or another neurological disorder having UBE3A deficiency. The vector may be administered via injection into the hippocampus or ventricles, in some cases, bilaterally. Dosages of the therapeutic can range between about 5.55×1011 to 2.86×1012 genomes/g brain mass.
To test the efficacy of the secretion signal, GFP (SEQ ID No: 1) (XM 013480425.1) was cloned in frame with human insulin, GDNF (SEQ ID No: 2) (AB675653.1) or IgK signal peptides.
fused with a secretion protein based on GDNF;
The construct was inserted into a pTR plasmid and transfected into HEK293 cells (American Type Culture Collection, Manassas, Va.). HEK293 cells were grown at 37° C. 5% CO2 in Dulbecco's Modified Essential Medium (DMEM) with 10% FBS and 1% Pen/Strep and subcultured at 80% confluence.
The vector (2 μg/well in a 6-well plate) was transfected into the cells using PEI transfection method. The cells were subcultured at 0.5×106 cells per well in a 6-well plate with DMEM medium two days before the transfection. Medium was replaced the night before transfection. Endotoxin-free dH2O was heated to at around 80° C., and polyethylenimine (Sigma-Aldrich Co. LLC, St. Louis, Mo.) dissolved. The solution was cooled to around 25° C., and the solution neutralized using sodium hydroxide. AAV4-STUb vector or negative control (medium only) was added to serum-free DMEM at 2 μg to every 200 μL for each well transfected, and 9 μL of 1 μg/L polyethylenimine added to the mix for each well. The transfection mix was incubated at room temperature for 15 minutes, then added to each well of cells at 210 μL per well and incubated for 48 hours.
Media was collected from each culture well and 2 μL spotted onto a nitrocellulose membrane using a narrow-tipped pipette. After the samples dried, the membrane was blocked applying 5% BSA in TBS-T to the membrane and incubating at room temperature for 30 minutes to 1 hour, followed by incubating the membrane with chicken anti-GFP (5 μg/mL, Abcam PLC, Cambridge, UK; # ab13970) in BSA/TBS-T for 30 min at room temperature. The membrane was washed with TBS-T 3 times, 5 minutes for each wash. The membrane was incubated with anti-chicken HRP conjugate secondary antibody (Southern Biotechnology, Thermo Fisher Scientific, Inc., Waltham, Mass.; #6100-05, 1/3000) conjugated with HRP for 30 minutes at room temperature, followed by washing the membrane three times with TBS-T, once for 15 minutes, and subsequent washed at 5 minutes each. The membrane was washed with TBS for 5 minutes at room temperature, and incubated with luminescence reagent for 1 minute (Millipore, Merck KGaA, Darmstadt, DE; # WBKLS0100). The membrane was recorded on a GE Amersham Imager 600 (General Electric, Fairfield, Calif.), shown in
As seen from
A mouse-UBE3A vector construct was generated using a pTR plasmid. The mouse (Mus musculus) UBE3A gene was formed from cDNA (U82122.1);
The cDNA was subcloned and sequenced. The mouse UBE3A gene (SEQ ID No. 4) was fused to DNA sequences encoding the secretion signaling peptide GDNF (SEQ ID No. 5) and cell uptake peptide HIV TAT sequence (SEQ ID No: 6). The secretion signaling peptide has the DNA sequence;
while HIV TAT sequence is;
The construct sequence of SEQ ID No: 4 fused with SEQ ID No: 5 and SEQ ID No: 6 was inserted into a pTR plasmid. The plasmid was cleaved using Age I and Xho I endonucleases and the construct sequence ligated using ligase. The vector contains AAV serotype 2 terminal repeats, CMV-chicken-beta actin hybrid promoter and a WPRE, seen in
The vector (AAV4-STUb vector) was then transformed into Escherichia coli (E. coli, Invitrogen, Thermo Fisher Scientific, Inc., Waltham, Mass.; SURE2 cells). Briefly, cells were equilibrated on ice and 1 pg to 500 ng of the vector were added to the E. coli and allowed to incubate for about 1 minute. The cells were electroporated with a BioRad Gene Pulser in a 0.1 cm cuvette (1.7V, 200 Ohms). The E. Coli were then grown in media for 60 min prior to being plated onto agar, such as ATCC medium 1065 (American Type Culture Collection, Manassas, Va.), with ampicillin (50 μg/mL). E. coli was expanded in broth containing ampicillin to collect large amounts of vector.
The mouse vector properties of the construct generated in Example 2 were tested in HEK293 cells (American Type Culture Collection, Manassas, Va.). HEK293 cells were grown at 37° C. 5% CO2 in Dulbecco's Modified Essential Medium (DMEM) with 10% FBS and 1% Pen/Strep and subcultured at 80% confluence.
The vector (2 μg/well in a 6-well plate) was transfected into the cells using PEI transfection method. The cells were subcultured at 0.5×106 cells per well in a 6-well plate with DMEM medium two days before the transfection. Medium was replaced the night before transfection. Endotoxin-free dH2O was heated to at around 80° C., and polyethylenimine (Sigma-Aldrich Co. LLC, St. Louis, Mo.) dissolved. The solution was allowed to cool to around 25° C., and the solution neutralized using sodium hydroxide. AAV4-STUb vector or negative control (medium only) was added to serum-free DMEM at 2 μg to every 200 μl for each well transfected, and 9p of 1 μg/μl polyethylenimine added to the mix for each well. The transfection mix was incubated at room temperature for 15 minutes, then added to each well of cells at 210 μl per well and incubated for 48 hours.
Media was collected from AAV4-STUb vector transfected cells, medium-only transfected control cells, and untransfected control cells. The medium was run on Western blot and stained with rabbit anti-E6-AP antibody (A300-351A, Bethyl Labs, Montgomery, Tex.), which is reactive against human and mouse E6-AP, at 0.4 μg/ml. Secondary conjugation was performed with rabbit-conjugated horseradish peroxidase (Southern Biotechnology, Thermo Fisher Scientific, Inc., Waltham, Mass.). The results were determined densiometrically, and show the HEK293 cells transfected with AAV4-STUb secrete E6-AP protein into the medium, as seen in
Transgenic mice were formed by crossbreeding mice having a deletion in the maternal UBE3A (Jiang, et al., Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation. Neuron. 1998 October; 21(4):799-811; Gustin, et al., Tissue-specific variation of Ube3a protein expression in rodents and in a mouse model of Angelman syndrome. Neurobiol Dis. 2010 September; 39(3):283-91; Heck, et al., Analysis of cerebellar function in Ube3a-deficient mice reveals novel genotype-specific behaviors. Hum Mol Genet. 2008 Jul. 15; 17(14):2181-9) and GABARB3. Mice were housed in a 12-hour day-light cycle and fed food and water ad libitum. Three month old mice were treated with the vector.
Mice were anesthetized with isoflurane and placed in the stereotaxic apparatus (51725D Digital Just for Mice Stereotaxic Instrument, Stoelting, Wood Dale, Ill.). An incision was made sagittally over the middle of the cranium and the surrounding skin pushed back to enlarge the opening. The following coordinates were used to locate the left and right hippocampus: AP 22.7 mm, L 62.7 mm, and V 23.0 mm. Mice received bilateral intrahippocampal injections of either AAV4-STUb particles at a concentration of 1×1012 genomes/mL (N=2) in 10 μL of 20% mannitol or vehicle (10 μL of 20% mannitol) using a 10 mL Hamilton syringe in each hemisphere. The wound was cleaned with saline and closed using Vetbond (NC9286393 Fisher Scientific, Pittsburgh, Pa.). Control animals included uninjected AS mice and littermate wild type mice (n=2). Mice recovered in a clean, empty cage on a warm heating pad and were then singly housed until sacrificed. The mice were monitored over the course of the experiment.
At day 30 after treatment, the mice were euthanized by injecting a commercial euthanasia solution, Somnasol®, (0.22 ml/kg) intraperitoneally. After euthanizing the animals, CSF was collected and the animals were perfused with PBS and the brain removed. The brain was fixed in 4% paraformaldehyde solution overnight prior to cryoprotection in sucrose solutions. Brains were sectioned at 25 m using a microtome.
Most recombinant adeno-associated virus vector studies inject the vector directly into the parenchymal, which typically results in limited cellular transduction (Li, et al., Intra-ventricular infusion of rAAV-1-EGFP resulted in transduction in multiple regions of adult rat brain: a comparative study with rAAV2 and rAAV5 vectors. Brain Res. 2006 Nov. 29; 1122(1):1-9). However, appending a secretion signaling sequence and TAT sequence to the Ube3A protein allows for secretion of the HECT protein (i.e., UBE3A) from transfected cells and uptake of the peptide by adjacent neurons, allowing injection into a discrete site to serve as a supply of protein for other sites throughout the brain.
Brains from sacrificed mice were sliced using a microtome and stained for E6-AP protein using anti-E6-AP antibody (A300-351A, Bethyl Labs, Montgomery, Tex.) with a biotinylated anti-rabbit secondary antibody (Vector Labs # AB-1000). Staining was completed with ABC (Vector Labs) and DAB reaction. Sections were mounted and scanned using Zeiss Axio Scan microscope. Percentage area staining was quantified using IAE-NearCYTE image analysis software (University of Pittsburgh Starzl Transplant Institute, Pittsburgh, Pa.).
Nontransgenic (Ntg) control mice shows the level of UBE3a expression in a normal mouse brain, which was about 40%, as seen in
Immunohistochemical analysis of brain slices indicate nontransgenic mice possess relatively high levels of E6-AP, with region-specific staining, seen in
Detailed analysis of the coronal cross sections of Angelman syndrome-model mice confirmed that administration of the UBE3A construct increased levels of E6-AP in and around the lateral ventricle, as seen in
A human vector construct was generated using a pTR plasmid. A Homo sapiens UBE3A gene was formed from cDNA (AH005553.1);
The cDNA was subcloned and sequenced. The UBE3A, version 1 gene (hUBEv1) (SEQ ID No: 9) was fused to one of three genes encoding a secretion signaling peptide, based on GDNF;
from insulin protein;
or from IgK;
The construct was inserted into the hSTUb vector, under a CMV chicken-beta actin hybrid promoter or human ubiquitin c promoter. Woodchuck hepatitis post-transcriptional regulatory element (WPRE) is present to increase expression levels.
The UBE3A-seretion signal construct was then attached to a cellular uptake peptide (cell penetrating peptide); either a
The human UBE3A vector, seen in
Other sequences of UBE3A include variants 1, 2, or 3, seen below;
H sapiens UBE3A variant 1:
H sapiens UBE3A variant 2;
H sapiens UBE3A variant 3
Human vector properties were tested in HEK293 cells (American Type Culture Collection, Manassas, Va.), grown at 37° C. 5% CO2 in DMEM with 10% FBS and 1% Pen/Strep and subcultured at 80% confluence.
The vector (2 μg/well in a 6-well plate) was transfected into the cells using PEI transfection method. The cells were subcultured at 0.5×106 cells per well in a 6-well plate with DMEM medium two days before the transfection. Medium was replaced the night before transfection. Endotoxin-free dH2O was heated to at around 80° C., and polyethylenimine (Sigma-Aldrich Co. LLC, St. Louis, Mo.) dissolved. The solution was allowed to cool to around 25° C., and the solution neutralized using sodium hydroxide. AAV4-STUb vector or negative control (medium only) was added to serum-free DMEM at 2 μg to every 200 μl for each well transfected, and 9p of 1 μg/μl polyethylenimine added to the mix for each well. The transfection mix was incubated at room temperature for 15 minutes, then added to each well of cells at 210 μl per well and incubated for 48 hours. Cells and media were harvested by scraping the cells from the plates. The medium and cells were then centrifuged at 5000×g for 5 minutes.
For Western blotting of the extracts, cell pellets were resuspended in 50 μL of hypo-osmotic buffer and the cells lysed by three repeated freeze/thaws. 15 μL of lysate was heated with Lamelli sample buffer and run on a BioRad 4-20% acrylamide gel. Transferred to nitrocellulose membrane using a TransBlot. The blot was blocked with 5% milk and protein detected using an anti-E6AP antibody.
As seen in
The efficacy of secretion peptides in promoting extracellular secretion of the protein by neurons was measured by creating plasmid constructs containing the various secretion signals, GFP or a human Ube3A version 1 (hUbev1) gene, and the CPP TATk, as seen in
The plasmid constructs containing the various secretion signals were generated and gel electrophoresis run to confirm successful gene insertion for each plasmid. As seen in
The efficacy of the select CPP signals in inducing reuptake of the protein by neurons was measured by creating plasmid constructs containing the secretion signal (GDNF), the hUbev1 gene, and the various CPP signals, outlined below, and transfecting them into HEK293 cells.
The cell lyses from these cells was then taken and added to new cell cultures of HEK293 cells and the concentration of E6-AP in these cells after incubation measured via Western blot. Results of the uptake for the CPP signals penetratin, TATk, R6RW, and pVEC are seen in
To ensure that the Ube3A gene modified to include secretion and reuptake signals maintained its ability to improve cognitive deficits associated with AS, a plasmid construct (hSTUb) containing human Ube3A version 1 (hUbev1), a secretion signal, and the CPP TATk was transduced via an rAAV vector into mouse models of AS. Long-term potentiation of the murine brain was measured via electrophysiology post-mortem and compared to GFP-transfected AS model control mice and wild-type control mice. The results indicate that the hSTUb plasmid successfully rescued LTP deficits, as seen in
The potential of secretion and CPP signal peptides were analyzed for their ability to promote greater global distribution of E6-AP in neurons for use in a gene therapy for AS. Rescue of LTP by the hSTUb plasmid in the mouse model suggests that the UBE3A gene retains its efficacy in treating cognitive deficits in AS following the addition of secretion and CPP signals, supporting the potential of the construct in a gene therapy. The GDNF signal presents as the optimal signal for utilization in this proposed therapy as indicated by its plasmid construct showing the most secretion of E6-AP into media following transduction. Failure of the CPP signals to induce measurable reuptake of E6-AP after the application of cell lyses to the cells may be due to several factors, including insufficient concentration of E6-AP in the lyses.
A human child presents with severe developmental delay that becomes apparent around the age of 12 months. The child later presents with absent speech, seizures, hypotonia, ataxia and mricrocephaly. The child moves with a jerky, puppet like gait and displays an unusually happy demeanor that is accompanied by laughing spells. The child has dysmorphic facial features characterized by a prominent chin, an unusually wide smile and deep-set eyes. The child diagnoses with Angelman's Syndrome. The child is treated with a therapeutically effective amount of UBE3A vector which is injected bilaterally into the left and right hippocampal hemispheres of the brain. Improvement is seen in the symptoms after treatment with a decrease in seizures, increased muscle tone, increased coordination of muscle movement and improvement in speech.
The UBE3A vector is formed from cDNA cloned from a Homo sapiens UBE3A gene. The UBE3A, version 1 gene (SEQ ID No: 9) is fused to a gene encoding a secretion signaling peptide, in this case GDNF, although insulin or IgK may also be used. The construct is inserted into the hSTUb vector, under a CMV chicken-beta actin hybrid promoter or human ubiquitin c promoter. Woodchuck hepatitis post-transcriptional regulatory element (WPRE) is present to increase expression levels.
The UBE3A-seretion signal construct is attached to a cellular uptake peptide (cell penetrating peptide or CPP) such as HIV TAT or HIV TATk. The human UBE3A vector is then transformed into E. coli using the heat shock method described in Example 2. The transformed E. coli were expanded in broth containing ampicillin to select for the vector and collect large amounts of vector.
In the preceding specification, all documents, acts, or information disclosed does not constitute an admission that the document, act, or information of any combination thereof was publicly available, known to the public, part of the general knowledge in the art, or was known to be relevant to solve any problem at the time of priority.
The disclosures of all publications cited above are expressly incorporated herein by reference, each in its entirety, to the same extent as if each were incorporated by reference individually.
While there has been described and illustrated specific embodiments of a method of treating UBE3A deficiencies, it will be apparent to those skilled in the art that variations and modifications are possible without deviating from the broad spirit and principle of the present invention. It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween.
This application is a continuation of and claims priority to International Patent Application No. PCT/US2018/039980, entitled “Modified UBE3A Gene for a Gene Therapy Approach for Angelman Syndrome”, filed Jun. 28, 2018 which claims priority to U.S. Provisional Patent Application Ser. No. 62/525,787, entitled “Modified UBE3A Gene for a Gene Therapy Approach for Angelman Syndrome”, filed Jun. 28, 2017, the contents of each of which are hereby incorporated by reference into this disclosure.
| Number | Date | Country | |
|---|---|---|---|
| 62525787 | Jun 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2018/039980 | Jun 2018 | US |
| Child | 16716785 | US |
