App modification via base editing using the crispr/cas9 system
OwnerJacques P. TREMBLAY, Joël ROUSSEAU +1inventors
InventorsJacques P. TREMBLAY, Joël ROUSSEAU, Antoine GUYON
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Abstract
Methods and products (e.g., guide RNAs or pegRNA and recombinant proteins) are described for decreasing amyloidogenic Aβ peptide levels produced by a cell, or the aggregation of the Aβ peptide, as well as uses of such methods and products, for example for the treatment of Alzheimer's disease and/or age-related cognitive decline in a subject in need thereof.
Claims30
A method of decreasing Amyloid Precursor Protein (APP) processing into amyloidogenic Aβ peptide by a cell or the aggregation of the Aβ peptide, comprising providing said cell with:
- a. a1) at least one guide RNA comprising a seed region of at least 18 consecutive nucleotides of a target sequence in an endogenous APP-encoding polynucleotide sequence present in said cell, wherein the target sequence of the guide RNA guide sequence is contiguous to a protospacer adjacent motif (PAM) in the endogenous APP gene polynucleotide sequence and wherein said PAM is recognized by a ribonucleoprotein complex comprising a Cas9 nickase;
- or
- a2) a vector comprising a nucleic acid sequence corresponding to the guide RNA for expressing the guide RNA;
- and
- b. b1) a Cas nickase-deaminase fusion protein comprising (i) a Cas nickase polypeptide domain and (ii) a cytidine deaminase polypeptide domain or an adenosine deaminase polypeptide domain;
- or
- b2) a vector comprising a nucleic acid sequence encoding the Cas nickase-deaminase fusion protein, for expression of the Cas nickase-deaminase fusion protein;
- and
- wherein at least one modification is introduced into the target sequence of the APP-encoding polynucleotide, wherein the at least one modification comprises modification of a cytidine into a thymidine or an adenosine into an inosine, and wherein the at least one modification decreases the processing of APP into amyloidogenic Aβ peptide by the cell or the aggregation of the Aβ peptide;
- and wherein if the APP-encoding nucleotide is mRNA and the at least one modification comprises deamination of an adenosine into an inosine, the gRNA further comprises a cytidine mismatch with the adenosine to be deaminated to an inosine in the target sequence.
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A method of decreasing Amyloid Precursor Protein (APP) processing into amyloidogenic Aβ peptide by a cell or the aggregation of the Aβ peptide, comprising providing said cell with:
- a. a1) at least one pegRNA comprising a seed region of at least 18 consecutive nucleotides of a target sequence in an endogenous APP-encoding polynucleotide sequence present in said cell, wherein the target sequence of the pegRNA guide sequence is contiguous to a protospacer adjacent motif (PAM) in the endogenous APP gene polynucleotide sequence and wherein said PAM is recognized by a ribonucleoprotein complex comprising a Cas9 nickase;
- or
- a2) a vector comprising a nucleic acid sequence corresponding to the pegRNA for expressing the pegRNA;
- and
- b. b1) a Cas nickase-reverse transcriptase fusion protein comprising (i) a Cas nickase polypeptide domain and (ii) a reverse transcriptase polypeptide domain;
- or
- b2) a vector comprising a nucleic acid sequence encoding the Cas nickase-reverse transcriptase fusion protein, for expression of the Cas nickase-reverse transcriptase fusion protein;
- and
- wherein at least one modification is introduced into the target sequence of the APP-encoding polynucleotide, wherein the at least one modification decreases the processing of APP into amyloidogenic Aβ peptide by the cell or the aggregation of the Aβ peptide.
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Dependencies are inferred from the claim text.