DNA damage response signature guided rational design of CRISPR-based systems and therapies
Inventors
Ben-David, Uri • Golub, Todd • BEROUKHIM, RAMEEN • ENACHE, Oana • RENDO, Veronica
Assignees
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Publication Number
US-12297426-B2
Publication Date
2025-05-13
Expiration Date
Abstract
Described herein are embodiments of methods to rationally design CRISPR-Cas system-based therapeutics and therapies based on expression of a DNA-damage response signature in a cell. In some embodiments, the methods include screening a set of CRISPR-Cas systems by expressing each CRISPR-Cas system in a test cell population and modifying one or more target sequences in the test cell population; screening in the test cell population for each CRISPR-Cas system and expression of a DNA-damage response signature; and selecting one or more CRISPR-Cas systems that do not result in expression of a DNA-damage response signature.
Core Innovation
The invention relates to developing or designing a CRISPR-Cas based therapy or therapeutic by expressing a Cas-protein and optionally a CRISPR-Cas system in an initial cell or initial cell population to modify one or more target sequence, thereby generating a modified cell or modified cell population. The modified cell or modified cell population is clonally expanded to obtain an expanded cell population.
Cells from the expanded cell population are analyzed for expression of a Cas-induced DNA damage response protein signature. Clones are selected that do not express the Cas-induced DNA-damage response signature and that do not have a p53 inactivating mutation.
A related approach expresses a set of CRISPR-Cas systems or components thereof in a test cell population and modifies one or more target sequence in the test cell population. Each system or component is screened for expression of a DNA-damage response signature and for the presence of p53 inactivating mutations, and one or more systems or components are selected that do not result in both expression of a Cas-induced DNA-damage response signature and a p53 inactivating mutation.
Claims Coverage
The independent claims cover two CRISPR-Cas therapeutic development methods. Across the claims, the inventive focus is on detecting a Cas-induced DNA-damage response protein signature or DNA-damage response signature and selecting clones or CRISPR-Cas systems/components that do not show that signature and do not have p53 inactivating mutations.
Signature-guided clone selection
Modifying one or more target sequence in an initial cell or initial cell population by expressing a Cas-protein and optionally a CRISPR-Cas system, generating a modified cell or modified cell population; clonally expanding the modified cell or modified cell population to obtain an expanded cell population; detecting, in cells from the expanded cell population, expression of a Cas-induced DNA damage response protein signature; and selecting clones from the expanded cell population that do not express the Cas-induced DNA-damage response signature and that do not have a p53 inactivating mutation.
Set-based CRISPR-Cas screening and selection
Expressing a set of CRISPR-Cas systems or components thereof in a test cell population and modifying one or more target sequence in the test cell population; screening, in the test cell population for each CRISPR-Cas system or components thereof, for expression of a DNA-damage response signature and the presence of p53 inactivating mutations; and selecting one or more CRISPR-Cas systems or components thereof that do not result in: (i) expression of a Cas-induced DNA-damage response signature and (ii) a p53 inactivating mutation.
Both independent claims cover CRISPR-Cas based therapeutic development workflows that assess a Cas-induced DNA damage response protein signature or DNA-damage response signature and exclude systems or clones associated with both that signature and p53 inactivating mutations.
Stated Advantages
Avoids clones that express the Cas-induced DNA-damage response signature.
Avoids clones that have a p53 inactivating mutation.
Identifies and selects CRISPR-Cas systems or components that do not result in Cas-induced DNA-damage response signature expression.
Identifies and selects CRISPR-Cas systems or components that do not result in p53 inactivating mutations.
Documented Applications
Developing or designing a CRISPR-Cas based therapy or therapeutic by modifying target sequences in initial or test cell populations, expanding or screening modified cells, and selecting CRISPR-Cas candidates based on DNA-damage response signature and p53 inactivating mutation status.
Adoptive cell therapy context where CRISPR-designed cells are edited ex vivo, screened to lack a DNA-damage response signature, and administered after selection of suitable clones.
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