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1. (WO2018106782) PROCÉDÉS ET COMPOSITIONS POUR AMÉLIORER LA PRODUCTION DE MYÉLINE FONCTIONNELLE
Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

WHAT IS CLAIMED:

1. A method of generating a cell that enhances functional myelin production, the method comprising genetically modifying the cell such that:

(i) an endogenous PLPl gene is modified to decrease its ability to inhibit myelin production;

(ii) an endogenous PLPl genetic regulatory element is modified to decrease its ability to promote PLPl expression;

(iii) an endogenous PLPl genetic regulatory element is modified to increase its ability to inhibit PLPl expression, and/or

(iv) an endogenous PLPl gene product or a PLPl regulatory element gene product that promotes PLPl expression is modified to decrease the PLPl 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 PLPl gene comprises introduction of mutations that reduce the expression of the endogenous PLPl gene or cause non-sense mediated decay of the PLPl transcript.

3. The method of claim 1, wherein the endogenous PLPl gene or the endogenous PLPl genetic regulatory element comprises a point mutation, and wherein said modification of the endogenous PLPl gene or the endogenous PLPl genetic regulatory element comprises correcting the point mutation to wild-type sequence.

4. The method of any one of claims 1-3, wherein the modification of the endogenous PLPl genetic regulatory element comprises introduction of indels to alter the activity of the PLPl genetic regulatory element.

5. The method of claim 4, wherein the endogenous PLPl genetic regulatory element is a PLPl enhancer or promoter.

6. The method of any one of claims 1-5, wherein the modification of the endogenous PLPl gene or the endogenous PLPl genetic regulatory element comprises

introduction of genetic modification comprising a nucleotide insertion or deletion (indels) in PLPl.

7. The method of any one of claims 1-6, wherein the modification of the endogenous PLPl gene comprises introduction of genetic modification comprising large exonic deletions of PLPl.

8. The method of any one of claims 1-7, wherein the endogenous PLPl gene is a

deleterious disease-causing mutant PLPl gene.

9. The method of any one of claims 1-8, wherein the cell is genetically modified using a nuclease.

10. The method of claim 9, wherein the nuclease comprises a zinc finger nuclease (ZFN), a TALE-effector (TALEN), CRISPR/Cas system or NgAgo system.

11. The method of claim 9, wherein the endogenous PLPl gene is modified with a

CRISPR/Cas system nuclease at exon 1 or exon 3.

12. The method of claim 11, wherein the endogenous PLPl gene is modified with a

CRISPR/Cas system nuclease at the 5' end of exon 3.

13. The method of any one of claims 1-12, wherein modification of the PLPl gene

product comprises delivering to the cell a gene silencing agent.

14. The method of claim 13, wherein the gene silencing agent comprises an RNAi

construct {e.g., an siRNA, shRNA, or an miRNA).

15. The method of claim 13, wherein the gene silencing agent comprises an antisense oligonucleotide (ASO).

16. The method of any one of claims 1-15, wherein the cell that is genetically modified exhibit enhanced myelin production {e.g., due to reduced PLPl -related toxicity in the cell).

17. The method of any one of claims 1-16, wherein modification of the endogenous PLPl gene or PLPl genetic regulatory element alleviates PLPl related cell stress in the cell.

18. The method of any one of claims 9-15, 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. The method of claim 20 and 21, wherein the second or the third AAV vector (when present) further comprises one or more of the following elements, optionally in 5' ->3' orientation:

i) a 5' AAV ITR;

ii) a promoter and optional enhancer;

iii) the guide RNA sequence;

iv) a stuffer or filler nucleic acid sequence; and,

v) a 3 ' AAV ITR.

23. The method of any of claims 20-22, wherein the third AAV vector (when present) further comprises one or more of the following elements, optionally in 5' ->3' orientation:

i) a 5' AAV ITR;

ii) a 5' slice acceptor site;

iii) the donor nucleic acid sequence;

iv) a polyadenylation signal; and,

v) an AAV 3' ITR.

24. The method of any of claims 19-23, wherein the 1st, 2nd, and/or 3rd AAV vector

comprises a VPl, VP2, or VP3 capsid selected from any serotype of AAVl, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVl l, or mixtures, variants or derivatives thereof.

25. The method of any of claims 21-24, wherein the 5' AAV ITR is selected from any one of AAVl, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAVl 1, or chimeras or fusions thereof, or wherein the 3'AAV ITR is selected from any one of AAVl, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAVl l, or chimeras or fusions thereof.

26. The method of any one of claims 18-25, wherein the cell is contacted in vitro, in vivo, or ex vivo.

27. A composition comprising the first and the second AAV vectors (and optionally the 3rd AAV vector) of any one of claims 20-26.

28. A pharmaceutical composition comprising the composition of claim 27.

29. A genetically modified cell generated by the method of any one of claims 1-26.

30. The cell of claim 29, wherein the cell is selected from a neural stem cell (NSC),

oligodendrocyte progenitor cell (OPC), or oligodendrocyte cell.

31. The cell of claim 30, wherein the cell is a NSC or OPC.

32. A genetically modified cell descended from the cell of any one of claims 29-31.

33. A composition comprising the genetically modified cell of any one of claims 29-32.

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 any one of claims 1-26, 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 any one of claims 1-26, 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 or 35, the myelin-related disorder selected from 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. The method of claim 34 or 35, wherein the myelin-related disorder comprises a

leukodystrophy.

38. The method of claim 34 or 35, wherein the myelin-related disorder comprises

Pelizaeus-Merzbacher disease (PMD).

39. The method of claim 38, wherein the method restores the lifespan of the subject to at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100% of a control subject without the myelin related disorder.

40. The method of claim 38 or 39, wherein the method alleviates at least one symptom(s) of the subject associated with said myelin related disorder.

41. The method of any one of claims 38-40, wherein the method restores a function of the subject to at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100% of a control subject without the myelin related disorder, preferably, the function is motor coordination, locomotion, or axon conduction velocity.

42. The method of any one of claims 34-41, wherein the cell is selected from the group consisting of a genetically modified NSC, OPC, and oligodendrocyte.

43. The method of any one of claims 34-42, wherein the endogenous PLP1 gene or genetic regulatory element thereof, or a portion thereof (such as a portion no more than 4.8, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, or 1.0 kb), is inactivated, disrupted, corrected or replaced.

44. The method of any one of claims 34-43, wherein the subject is a mammal, such as a human {e.g. , a human younger than 20 years old, 15 years old, 10 years old, 5 years old, 3 years old, 2 years old, 1 year old, 6 months old, 3 months old, 1 month old, 2 weeks old, 1 week old, 3 days old, or 1 day old).