Compositions and Methods for the Treatment of Conditions Associated with Nucleotide Repeat Expansion
AssigneeThe General Hospital Corporation
InventorsJeannie T. Lee, Hun-goo Lee
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Abstract
Described herein are compositions and methods that can be used to contract nucleotide repeat expansion (e.g., CGG) in a gene (e.g., FMR1) in cells of subjects having a condition associated with nucleotide repeat expansion, e.g., subjects with Fragile X syndrome (FXS), FXTAS-Parkinsonism, myotonic dystrophy, and other repeat disorders, by treating the cells using an inactive Cas9 protein and a guide RNA that directs the Cas9 to the gene or by treating the cells using a reactivation cocktail comprising small molecules.
Claims41
A method of contracting an expansion of nucleotide repeats in a gene in a cell, the method comprising contacting the cell with or expressing in the cell an inactive Cas9 protein and a guide RNA (gRNA) that directs the Cas9 to the gene and/or the nucleotide repeat, in an amount sufficient to reduce the number of nucleotide repeats in the cell, and optionally contacting the cell with or expressing in the cell a DNMT inhibitor.
A method of treating a subject who has a condition associated with nucleotide repeat expansion in a gene, the method comprising:
- obtaining iPSC derived from differentiated somatic cells obtained from the subject;
- exposing the iPSC to, or expressing in the iPSC, an inactive Cas9 protein and a guide RNA (gRNA) that directs the dCas9 to the gene and/or the nucleotide repeat, in an amount sufficient to reduce the number of nucleotide repeats in the cell for a time and under conditions sufficient for reduction of the number of nucleotide repeats;
- optionally, exposing the iPSC to, or expressing in the iPSC, a DNMT inhibitor;
- promoting differentiation of the reactivated cells to neural precursor cells or other neuronal cell types;
- and administering the cells to the brain or spinal cord of the subject with a condition associated with nucleotide repeat expansion.
A method of treating a subject who has a condition associated with nucleotide repeat expansion in a gene, the method comprising administering to the subject a therapeutically effective amount of an inactive Cas9 protein and a guide RNA that directs the dCas9 to the gene and/or the nucleotide repeat, in an amount sufficient to reduce the number of nucleotide repeats in the cell, and optionally, administering to the subject a therapeutically effective amount of a DNMT inhibitor.
A method of contracting nucleotide repeats in a gene a living cell, e.g., a cell having a number of repeats above a reference number or in a reference range, the method comprising contacting the cell with an inactive Cas9 protein and a guide RNA that directs the dCas9 to the gene and/or the nucleotide repeat, in an amount sufficient to reduce the number of nucleotide repeats in the cell, and optionally, contacting the cell with a DNMT inhibitor.
A method of preparing a population of neural precursor cells or other neuronal cell type with decreased nucleotide repeats, the method comprising
- obtaining iPSC derived from differentiated somatic cells obtained from a subject who has a condition associated with nucleotide repeat expansion;
- exposing the iPSC to an inactive Cas9 protein and a guide RNA that directs the dCas9 to the gene and/or the nucleotide repeat for a time and under conditions sufficient for reduction of the number of nucleotide repeats;
- optionally, exposing the iPSC to a DNMT inhibitor;
- and
- promoting differentiation of the iPSC to neural precursor cells or other neuronal cell type,
- thereby promoting differentiation of the reactivated cells to neural precursor cells or other neuronal cell types.
A method of reactivating an inactive gene in a living cell, wherein the gene is inactive due to the presence of expansion of nucleotide repeats, optionally wherein the inactive gene is fragile X mental retardation 1 (FMR1) and the FMR1 gene is inactive due to the presence of expansion of nucleotide repeats (e.g., CGG nucleotide repeats in the 5′-UTR of the FMR1 gene), the method comprising contacting the cell with a reactivation cocktail with active factors comprising or consisting of:
- (i) a MEK inhibitor and a Raf inhibitor;
- (ii) a MEK inhibitor, a Raf inhibitor, and a ROCK1 inhibitor;
- (iii) a MEK inhibitor, a Raf inhibitor, and a GSK-3β inhibitor;
- (iv) a MEK inhibitor, a Raf inhibitor, a ROCK1 inhibitor, and a GSK-3β inhibitor;
- or
- (v) a MEK inhibitor, a Raf inhibitor, a ROCK1 inhibitor, a GSK-3β inhibitor, and a Src Inhibitor, for a time and under conditions sufficient for reactivation of the gene, and optionally contacting the cell with or expressing in the cell a DNMT inhibitor.
A method of treating a subject who has a condition associated with nucleotide repeat expansion in a gene, the method comprising:
- obtaining iPSC derived from differentiated somatic cells obtained from the subject;
- exposing the iPSC to a reactivation cocktail with active factors comprising or consisting of:
- (i) a MEK inhibitor and a Raf inhibitor;
- (ii) a MEK inhibitor, a Raf inhibitor, and a ROCK1 inhibitor;
- (iii) a MEK inhibitor, a Raf inhibitor, and a GSK-3β inhibitor;
- (iv) a MEK inhibitor, a Raf inhibitor, a ROCK1 inhibitor, and a GSK-3β inhibitor;
- (v) a MEK inhibitor, a Raf inhibitor, a ROCK1 inhibitor, a GSK-3β inhibitor, and a Src Inhibitor, for a time and under conditions sufficient for reduction of the number of nucleotide repeats;
- optionally, exposing the iPSC to, or expressing in the iPSC, a DNMT inhibitor;
- promoting differentiation of the reactivated cells to neural precursor cells or other neuronal cell types;
- and administering the cells to the brain or spinal cord of the subject with a condition associated with nucleotide repeat expansion.
A method of treating a subject who has a condition associated with nucleotide repeat expansion, the method comprising administering to the subject a therapeutically effective amount of a reactivation cocktail with active factors comprising or consisting of:
- (i) a MEK inhibitor and a Raf inhibitor;
- (ii) a MEK inhibitor, a Raf inhibitor, and a ROCK1 inhibitor;
- (iii) a MEK inhibitor, a Raf inhibitor, and a GSK-3β inhibitor;
- (iv) a MEK inhibitor, a Raf inhibitor, a ROCK1 inhibitor, and a GSK-3β inhibitor;
- (v) a MEK inhibitor, a Raf inhibitor, a ROCK1 inhibitor, a GSK-3β inhibitor, and a Src Inhibitor, and optionally, administering to the subject a therapeutically effective amount of a DNMT inhibitor.
A method of contracting nucleotide repeats in a living cell, e.g., a cell having a number of repeats above a reference number or in a reference range, the method comprising contacting the cell with a reactivation cocktail with active factors comprising or consisting of:
- (i) a MEK inhibitor and a Raf inhibitor;
- (ii) a MEK inhibitor, a Raf inhibitor, and a ROCK1 inhibitor;
- (iii) a MEK inhibitor, a Raf inhibitor, and a GSK-3β inhibitor;
- (iv) a MEK inhibitor, a Raf inhibitor, a ROCK1 inhibitor, and a GSK-3β inhibitor;
- or
- (v) a MEK inhibitor, a Raf inhibitor, a ROCK1 inhibitor, a GSK-3β inhibitor, and a Src Inhibitor, for a time and under conditions sufficient for contraction of the nucleotide repeats, and optionally, contacting the cell with a DNMT inhibitor.
A method of preparing a population of neural precursor cells or other neuronal cell type with decreased nucleotide repeats, the method comprising
- obtaining iPSC derived from differentiated somatic cells obtained from a subject who has a condition associated with nucleotide repeat expansion;
- exposing the iPSC to a reactivation cocktail with active factors comprising or consisting of:
- (i) a MEK inhibitor and a Raf inhibitor;
- (ii) a MEK inhibitor, a Raf inhibitor, and a ROCK1 inhibitor;
- (iii) a MEK inhibitor, a Raf inhibitor, and a GSK-3β inhibitor;
- (iv) a MEK inhibitor, a Raf inhibitor, a ROCK1 inhibitor, and a GSK-3β inhibitor;
- (v) a MEK inhibitor, a Raf inhibitor, a ROCK1 inhibitor, a GSK-3β inhibitor, and a Src Inhibitor, for a time and under conditions sufficient for reduction of the number of nucleotide repeats,
- optionally, exposing the iPSC to a DNMT inhibitor;
- and
- promoting differentiation of the iPSC to neural precursor cells or other neuronal cell type, thereby promoting differentiation of the reactivated cells to neural precursor cells or other neuronal cell types.
Dependencies are inferred from the claim text.