Vectors and methods for targeted integration in loci comprising constitutively expressed genes
Inventors
STANLEY, Ed • ELEFANTY, Andrew • Elliott, David • LABONNE, Tatiana
Assignees
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Publication Number
US-10745717-B2
Publication Date
2020-08-18
Expiration Date
Abstract
The invention relates to a vector comprising: a 5′ nucleic acid that is homologous to a genomic sequence 5′ of a stop codon of a constitutively expressed gene; an exogenous nucleic acid; a 3′ nucleic acid that is homologous to a genomic sequence 3′ of the stop codon of the constitutively expressed gene; a translation interruption-reinitiation signal operably linked to the 5′ nucleic acid and the exogenous nucleic acid, wherein the translation interruption-reinitiation signal is capable of replacing the stop codon of the constitutively expressed gene.
Core Innovation
The invention relates to genome-engineering vectors for targeted integration of one or more exogenous nucleic acids into a constitutively expressed gene. The vector includes a 5′ nucleic acid homologous to a genomic sequence 5′ of a genomic stop codon of the constitutively expressed gene and a 3′ nucleic acid homologous to a genomic sequence 3′ of the genomic stop codon.
A translation interruption-reinitiation signal is operably linked to the 5′ nucleic acid and the exogenous nucleic acid, and the translation interruption-reinitiation signal is capable of replacing the stop codon of the constitutively expressed gene. In a specific embodiment, the constitutively expressed gene comprises GAPDH, and the translation interruption-reinitiation signal replaces the GAPDH stop codon to express the exogenous nucleic acid.
The translation interruption-reinitiation signal is capable of being provided as a 2A peptide or 2A-like peptide, including T2A, P2A, E2A, and F2A, and vector architectures include an optional internal ribosomal entry site to operably link a second exogenous nucleic acid for expressing more than one payload from the targeted locus. The disclosed approach is used with targeting or cleavage at or near the locus to achieve stable targeted integration.
Functional outcomes are described as persistent reporter expression during hESC differentiation, retention in co-cultures, and performance of inducible variants such as a doxycycline-inducible GT-iCherry and a Tamoxifen-inducible GT-MYC:ER, with corresponding effects described in differentiation and colony assays.
Claims Coverage
The independent claim covers a vector architecture with at least five core inventive elements: 5′ and 3′ homology to a genomic stop codon region of a constitutively expressed gene, an exogenous nucleic acid payload, a translation interruption-reinitiation signal positioned to replace the stop codon, specificity that the constitutively expressed gene comprises GAPDH, and optional support for more than one payload.
Targeted integration vector into a constitutively expressed gene
A vector for targeted integration of one or more exogenous nucleic acids into a constitutively expressed gene using 5′ and 3′ nucleic acids homologous to genomic sequences flanking a genomic stop codon, together with a translation interruption-reinitiation signal operably linked to the 5′ nucleic acid and the exogenous nucleic acid.
Stop codon replacement by translation interruption-reinitiation signal
The translation interruption-reinitiation signal is capable of replacing the stop codon of the constitutively expressed gene to enable expression of the exogenous nucleic acid.
GAPDH as the constitutively expressed gene
The constitutively expressed gene comprises GAPDH, wherein the translation interruption-reinitiation signal replaces the GAPDH stop codon.
2A peptide translation interruption-reinitiation signal
The translation interruption-reinitiation signal is provided as a 2A peptide or 2A-like peptide, including T2A, P2A, E2A, and F2A.
Optional IRES-driven second payload
The vector further comprises an internal ribosomal entry site and a second exogenous nucleic acid operably linked to the IRES for expressing more than one payload from the targeted locus.
Coverage centers on integrating exogenous nucleic acids into a constitutively expressed GAPDH locus by replacing the genomic stop codon with a translation interruption-reinitiation signal, with optional IRES-based support for a second payload.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Persistent reporter expression during hESC differentiation.
Retention in co-cultures without transfer.
Performance of inducible variants in differentiation and colony assays, including a doxycycline-inducible GT-iCherry and a Tamoxifen-inducible GT-MYC:ER.
Cell identification/tracking.
Drug screening.
Therapeutic use including medicament use.
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