Universal treatment of down syndrome
AssigneeDEI Biopharma
InventorMatthias Magoola
Life of this patent
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Abstract
Allele-specific (AS) CRISPR-Cas9-mediated cleavage to induce selective loss of the extra chromosome, particularly in a trisomic context where the dosage imbalance is inherently deleterious, can treat Down Syndrome, an untreatable disorder. While it is suitable for individualized therapy, the instant invention provides a generalized treatment module that can be used for any Down Syndrome patient.
Claims14
A method for correcting trisomy 21 in a patient with Down syndrome, comprising:
- (a) obtaining somatic cells from the patient;
- (b) reprogramming said somatic cells into induced pluripotent stem cells (iPSCs);
- (c) sequencing the genome of said iPSCs and performing haplotype phasing to identify allele-specific nucleotide sequences unique to the supernumerary chromosome 21;
- (d) designing and validating a set of guide RNAs (gRNAs) that target said allele-specific sequences;
- (e) constructing a vector expressing Cas9, said gRNAs, a puromycin resistance gene, and a TP53-suppressing shRNA;
- (f) transfecting said iPSCs with the vector;
- (g) enriching for transfected cells and verifying loss of the targeted chromosome 21 copy;
- (h) isolating disomic iPSC clones;
- (i) differentiating the corrected iPSCs into therapeutic cell types and
- (j) transplanting said therapeutic cells back into the patient.
A method for in vivo correction of trisomy 21, comprising delivery of Cas9 mRNA and allele-specific gRNAs using lipid nanoparticles or viral vectors to cells within a patient with Down syndrome, wherein said Cas9 and gRNAs selectively induce loss of the supernumerary chromosome 21.
A universal method for correcting trisomy 21 in a patient with Down syndrome, comprising:
- (a) selecting from a pre-validated panel of guide RNAs targeting conserved, non-patient-specific genomic sequences on the supernumerary chromosome 21;
- (b) delivering said guide RNAs along with Cas9 or Cas9 mRNA into patient-derived cells or tissues using a vector or delivery system selected from the group consisting of:
- plasmids, viral vectors, and lipid nanoparticles;
- (c) inducing targeted double-strand breaks in the supernumerary HSA21;
- (d) eliminating the supernumerary chromosome;
- and
- (e) restoring disomy in the patient's cells.
A kit for correcting trisomy 21 in a patient with Down syndrome, comprising:
- (a) a plurality of validated gRNAs targeting conserved sequences on HSA21;
- (b) a Cas9 expression vector or Cas9 mRNA;
- (c) delivery components selected from lipid nanoparticles, electroporation buffers, or viral vectors;
- (d) reagents for validating chromosome elimination, including FISH probes and STR analysis tools.
A composition for the treatment of Down syndrome, comprising a lipid nanoparticle formulation encapsulating Cas9 mRNA and synthetic guide RNAs targeting conserved regions of the supernumerary chromosome 21, formulated for systemic or localized in vivo delivery.
Dependencies are inferred from the claim text.