Methods and compositions for enhancing functional myelin production

Abstract

A method of generating a cell that enhances functional myelin production is provided, the method including genetically modifying the cell such that: (i) an endogenous PLP1 gene is modified to decrease its ability to inhibit myelin production; (ii) an endogenous PLP1 genetic regulatory element is modified to decrease its ability to promote PLP1 expression; (iii) an endogenous PLP1 genetic regulatory element is modified to increase its ability to inhibit PLP1 expression; or (iv) an endogenous PLP1 gene product or a PLP1 regulatory element gene product that promotes PLP1 expression is modified to decrease the PLP1 expression level, wherein the cell produces functional myelin.

1 . A method of generating a cell that enhances functional myelin production, the method comprising genetically modifying the cell such that:
 (i) an endogenous PLP1 gene is modified to decrease its ability to inhibit myelin production;   (ii) an endogenous PLP1 genetic regulatory element is modified to decrease its ability to promote PLP1 expression;   (iii) an endogenous PLP1 genetic regulatory element is modified to increase its ability to inhibit PLP1 expression, and/or   (iv) an endogenous PLP1 gene product or a PLP1 regulatory element gene product that promotes PLP1 expression is modified to decrease the PLP1 expression level,   wherein the cell produces functional myelin, or is a progenitor that produces or differentiates into the cell that produces functional myelin.   
     
     
         2 . The method of  claim 1 , wherein said modification of the endogenous PLP1 gene comprises introduction of mutations that reduce the expression of the endogenous PLP1 gene or cause non-sense mediated decay of the PLP1 transcript. 
     
     
         3 . The method of  claim 1 , wherein the endogenous PLP1 gene or the endogenous PLP1 genetic regulatory element comprises a point mutation, and wherein said modification of the endogenous PLP1 gene or the endogenous PLP1 genetic regulatory element comprises correcting the point mutation to wild-type sequence. 
     
     
         4 . The method of  claim 1 , wherein the modification of the endogenous PLP1 genetic regulatory element comprises introduction of indels to alter the activity of the PLP1 genetic regulatory element. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein the modification of the endogenous PLP1 gene or the endogenous PLP1 genetic regulatory element comprises introduction of genetic modification comprising a nucleotide insertion or deletion (indels) in PLP1. 
     
     
         7 .- 8 . (canceled) 
     
     
         9 . The method of  claim 1 , wherein the cell is genetically modified using a nuclease. 
     
     
         10 .- 12 . (canceled) 
     
     
         13 . The method of  claim 1 , wherein modification of the PLP1 gene product comprises delivering to the cell a gene silencing agent. 
     
     
         14 .- 17 . (canceled) 
     
     
         18 . The method of  claim 9 , wherein the method comprises contacting the cell with a delivery vehicle comprising the nuclease or the gene silencing agent. 
     
     
         19 . The method of  claim 18 , wherein the delivery vehicle is an AAV vector, an adenoviral vector, or a lentivirus vector. 
     
     
         20 . The method of  claim 19 , wherein the method comprises:
 (a) contacting the cell with a first AAV vector comprising a nucleic acid encoding a functional Type II CRISPR-Cas9 (such as a Cas9 or a Cas9 ortholog cDNA), and a second AAV vector comprising a guide RNA (sgRNA) sequence specific for a target site in the endogenous PLP1 gene or the endogenous PLP1 genetic regulatory element, and optionally a third AAV vector comprising a donor nucleic acid sequence for correction or replacement of a defective or mutant portion of the endogenous PLP1 gene or the endogenous PLP1 genetic regulatory element; or,   (b) contacting the cell with a first AAV vector comprising a nucleic acid encoding a functional Type II CRISPR-Cas9 (such as a Cas9 or a Cas9 ortholog cDNA), and a guide RNA (sgRNA) sequence encoded in cis and is specific for a target site in the endogenous PLP1 gene or the endogenous PLP1 genetic regulatory element, and optionally a third AAV vector comprising a donor nucleic acid sequence for correction or replacement of a defective or mutant portion of the endogenous PLP1 gene or the endogenous PLP1 genetic regulatory element.   
     
     
         21 . The method of  claim 20 , wherein the first AAV vector further comprises one or more of the following elements, optionally in 5′→3′ orientation:
 i) a 5′ AAV inverted terminal repeat (ITR); 
 ii) a promoter and optional enhancer; 
 iii) a Cas9 cDNA encoding the functional Type II CRISPR-Cas9; 
 iv) a polyadenylation signal; and, 
 v) a 3′ AAV inverted terminal repeat (ITR). 
 
     
     
         22 .- 23 . (canceled) 
     
     
         24 . The method of  claim 19 , wherein the 1 st , 2 nd , and/or 3 rd  AAV vector comprises a VP1, VP2, or VP3 capsid selected from any serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or mixtures, variants or derivatives thereof. 
     
     
         25 . The method of  claim 21 , wherein the 5′ AAV ITR is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, or chimeras or fusions thereof, or wherein the 3′AAV ITR is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, or chimeras or fusions thereof. 
     
     
         26 . (canceled) 
     
     
         27 . A composition comprising the first and the second AAV vectors (and optionally the 3 rd  AAV vector) of  claim 20 . 
     
     
         28 . (canceled) 
     
     
         29 . A genetically modified cell generated by the method of  claim 1 . 
     
     
         30 - 31 . (canceled) 
     
     
         32 . A genetically modified cell descended from the cell of  claim 29 . 
     
     
         33 . A composition comprising the genetically modified cell of any one of  claim 29 . 
     
     
         34 . A method of treating a myelin related disorder in a subject, the method comprising administering to the subject a cell generated by the method of  claim 1 , thereby producing functional myelin in the subject, wherein the myelin related disorder preferably is characterized by abnormal PLP1 gene activity and/or expression. 
     
     
         35 . A method of treating a myelin related disorder in a subject, the method comprising genetically modifying a cell of the subject according to the method of  claim 1 , thereby producing functional myelin in the subject, wherein the myelin related disorder preferably is characterized by abnormal PLP1 gene activity and/or expression. 
     
     
         36 . The method of  claim 34 , wherein the myelin-related disorder is selected from the group consisting of: multiple sclerosis (MS), neuromyelitis optica (NMO), transverse myelitis, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre Syndrome, progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM), adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease (PMD), Wallerian Degeneration, optic neuritis, amylotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, post radiation injury, neurologic complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, AR, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, acute dissmeminated encephalitis, Marie-Charcot-Tooth disease and Bell's palsy. 
     
     
         37 - 44 . (canceled)