Site-specific enzymes and methods of use
Inventors
Ostertag, Eric M. • Yeshi, Tseten
Assignees
Transposagen Biopharmaceuticals Inc • Poseida Therapeutics Inc
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Publication Number
US-10844361-B2
Publication Date
2020-11-24
Expiration Date
Abstract
The present invention provides polypeptides related to Ralstonia proteins, nucleic acids encoding the same, compositions comprising the same, kits comprising the same, non-human transgenic animals comprising the same, and methods of using the same.
Core Innovation
The invention relates to a method of modifying genetic material in a cell by administering a fusion protein. The fusion protein comprises at least one amino acid sequence having the sequence of LSTEQVVAIASX1X2GGKQALEAVKAQLLVLRAAPYE (SEQ ID NO: 1), wherein X1X2 comprises a repeat variable diresidue (RVD) consisting of SI, SN, SH, NP, NH, NT, NK, ND, HN, HY, HD, HI-I, RN, RS, NG or GS. The invention uses these modular RVD positions for programmable DNA recognition.
The fusion protein further comprises an effector domain comprising a nuclease, a nickase, a transcriptional activator, a transcriptional repressor, a methyltransferase, a deacetylase, or any functional fragment thereof, and is neither isolated nor derived from a Ralstonia TALEN. The disclosed sequence definitions include variants corresponding to SEQ ID NO: 2 through 19.
The disclosed subject matter includes nucleic acid encoding and delivery through vectors and cells, including transgenic animals and germline mutations. The invention is described in the context of genome engineering, including stimulation of homologous recombination and gene expression modulation, including inducible promoters, and in gene therapy and genome-editing contexts including ex vivo and in vivo delivery.
Claims Coverage
The independent claim coverage centers on administration of a fusion protein that combines a defined LSTEQVVAIASX1X2GGKQALEAVKAQLLVLRAAPYE (SEQ ID NO: 1) sequence with restricted RVD content and a selectable functional effector domain that is not derived from a Ralstonia TALEN. Dependent claims further identify alternative amino-acid sequences and specific nuclease limitations.
Administering a fusion protein with SEQ ID NO: 1 RVD-defined recognition sequence
A method of modifying genetic material in a cell by administering a fusion protein comprising at least one amino acid sequence having the sequence of LSTEQVVAIASX1X2GGKQALEAVKAQLLVLRAAPYE (SEQ ID NO: 1), where X1X2 comprises a repeat variable diresidue (RVD) consisting of SI, SN, SH, NP, NH, NT, NK, ND, HN, HY, HD, HI-I, RN, RS, NG or GS.
Effector domain selection for modification activity
The fusion protein further comprises an effector domain comprising a nuclease, a nickase, a transcriptional activator, a transcriptional repressor, a methyltransferase, a deacetylase, or any functional fragment thereof.
Effector domain not isolated or derived from a Ralstonia TALEN
The effector domain is neither isolated nor derived from a Ralstonia TALEN.
Allowed alternative amino-acid sequences enumerated by specified SEQ ID numbers
The specified amino acid sequence comprises one or more amino acid sequences identified as SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, or 19.
Nuclease narrowed to an explicitly named Fok I endonuclease
The endonuclease used comprises a Fok I endonuclease.
Effector domain includes a zinc-finger nuclease
The effector domain includes a zinc-finger nuclease.
Mammal restricted to human as an organism context
Performed using a mammal that is a human.
The claim coverage is anchored by a fusion protein with a defined SEQ ID NO: 1 amino-acid sequence and RVD restricted to a listed set, together with an effector domain chosen from multiple functional classes while excluding effector domains that are isolated or derived from a Ralstonia TALEN. Dependent claims narrow allowed sequence variants and nuclease types and restrict context to human.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Gene therapy and genome-editing use of vectors encoding engineered TAL effector proteins/RTNs, including ex vivo and in vivo delivery contexts.
Treatment contexts in mammals, including adverse-event management using AZT for a modified tmpk.
Disease examples including blood diseases and neural diseases.
Locus targeting example at Bmpr2 using RTN/XTN TALENs, with mutation assessment described in the examples.
Genome engineering, including homologous recombination stimulation.
Gene expression modulation, including inducible promoters.
Therapeutic or pathogen-directed specificity.
Creation of transgenic animals and germline mutations.
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