Methods for diagnosing, screening and treating an amyloid-associated condition
OwnerRandall HALFMANN, Jianzheng WUinventors
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Abstract
The present disclosure provides, inter alia, methods for preventing, treating or ameliorating the effects of an amyloid-associated condition including Huntington's disease in a subject. Also provided are methods for detecting amyloid nucleation of a protein of interest such as, e.g., the Huntingtin protein.
Claims97
A method for preventing, treating or ameliorating the effects of an amyloid-associated condition in a subject, comprising:
- (a) identifying a target protein whose aggregation causes the amyloid-associated condition in the subject;
- and
- (b) treating the subject by modifying the target protein to induce preemptive oligomerization thereof.
A method for preventing, treating or ameliorating the effects of a polyglutamine (polyQ) disease in a subject, comprising:
- (a) identifying a target protein whose aggregation causes the polyQ disease in the subject;
- (b) obtaining a biological sample from the subject and screening for expanded cytosine-adenine-guanine (CAG) repeats encoding a long polyQ tract in the target protein;
- (c) identifying the subject as having high risk to develop the polyQ disease, if the long polyQ tract in the target protein contains a number of glutamines exceeding a pathogenic threshold for the polyQ disease;
- and
- (d) treating the identified subject by modifying the target protein to induce preemptive oligomerization thereof.
A method for preventing, treating or ameliorating the effects of Huntington's disease (HD) in a subject, comprising:
- (a) obtaining a biological sample from the subject and screening for expanded cytosine-adenine-guanine (CAG) repeats encoding a long polyQ tract in the Huntingtin protein;
- (b) identifying the subject as having high risk to develop HD, if the long polyQ tract in the Huntingtin protein contains 36 or more glutamines;
- and
- (c) treating the identified subject by modifying the Huntingtin protein to induce preemptive oligomerization thereof.
A method for preventing, treating or ameliorating the effects of an amyloid-associated neurodegenerative disease in a subject, comprising:
- (a) identifying an amyloid-forming protein whose aggregation causes the amyloid-associated neurodegenerative disease in the subject;
- (b) obtaining a biological sample from the subject and screening for the amyloid-forming protein;
- (c) identifying the subject as having high risk to develop the amyloid-associated neurodegenerative disease, if the amyloid-forming protein exists in the subject;
- and
- (d) treating the identified subject by modifying the amyloid-forming protein to induce preemptive oligomerization thereof.
A method for preventing, treating or ameliorating the effects of a condition associated with TAR DNA-binding protein 43 (TDP-43) in a subject, comprising:
- (a) obtaining a biological sample from the subject and screening for TDP-43;
- (b) identifying the subject as having high risk to develop the condition, if TDP-43 exists in the subject;
- and
- (c) treating the identified subject by modifying TDP-43 to induce preemptive oligomerization thereof.
A method for detecting amyloid nucleation of a protein of interest, comprising:
- (a) generating a first yeast strain by (i) deleting PDR5 and ATG8 genes from a yeast strain rhy1713, and then (ii) integrating BDFP1.6:1.6 prior to the stop codon of chromosomal PGK1;
- (b) generating a second yeast strain by eliminating the amyloid form of Rnq1 from the first yeast strain;
- (c) transforming the first and second yeast strains with a plasmid encoding the protein of interest as a fusion to a fluorescent tag;
- (d) incubating the transformed strains under suitable conditions and inducing them for a suitable time;
- (e) conducting high-throughput flow cytometry on these induced cells and collecting fluorescence signals;
- and
- (f) analyzing the collected signals to determine amyloid nucleation of the protein of interest.
A method for constructing a biosensor cell that is used to detect aggregates of a specific protein in a biological sample, comprising:
- (a) constructing a reporter gene that encodes the specific protein fused with a fluorescent tag;
- (b) cloning the reporter gene onto a lentiviral transfer plasmid;
- (c) transfecting the lentviral transfer plasmid together with a packaging plasmid and an envelope plasmid into HEK293T cells;
- (d) incubating the transfected cells and collecting viral particles containing the reporter gene;
- (e) incubating the viral particles collected in step (d) with fresh HE293T cells;
- and
- (f) sorting for single cell clone containing the integrated reporter gene and expanding it as the biosensor cell.
A biosensor cell effective to detect aggregates of a specific protein in a biological sample, the biosensor cell comprising a reporter gene that encodes the specific protein fused with a fluorescent tag.
A method for constructing a biosensor cell that is used to detect aggregates of a specific protein in a biological sample, comprising:
- constructing a reporter gene that encodes the specific protein fused with a fluorescent tag;
- transporting the reporter gene inside a cell;
- and
- sorting and/or expanding cells containing the reporter gene as the biosensor cell.
Dependencies are inferred from the claim text.