Methods to genetically modify cells for delivery of therapeutic proteins
Inventors
Juillerat, Alexandre • Duchateau, Philippe • HONG, Patrick • Poirot, Laurent • BUSSER, Brian • BOYNE, Alex
Assignees
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Publication Number
US-12534744-B2
Publication Date
2026-01-27
Expiration Date
Abstract
The present disclosure provides methods to genetically modify cells by insertion of an artificial exon (ArtEx) for delivery of therapeutic proteins in specific cell types and more particularly engineered cells for expression of a transgene into the brain of a patient.
Core Innovation
The invention relates to integrating an exogenous coding sequence into an endogenous intronic genomic region at an insertion site by introducing a polynucleotide template into cells comprising an endogenous intronic genomic region and inducing integration via homologous recombination. The template includes first and second homologous polynucleotide sequences, splice-site elements comprising a branch point, a splice acceptor, and a splice donor, and one or more 2A self-cleaving peptide sequences positioned around an exogenous sequence coding for a protein of interest.
The exogenous coding sequence is transcribed at the endogenous locus along with the first exon(s) or a copy thereof, so that a protein of interest is expressed through the endogenous transcriptional context while preserving endogenous exon expression by incorporating exon copies. The ordered template arrangement uses splice acceptance and splicing signals combined with 2A self-cleaving peptide sequences to separate or generate protein products from the inserted genetic material at the intronic integration site.
The disclosure further characterizes an AAV insertion vector and an engineered cell constructed by the same intronic insertion architecture. The AAV insertion vector comprises an exogenous polynucleotide sequence for insertion at an endogenous locus using homologous sequences upstream and downstream of the insertion site together with splice-site sequences and 2A self-cleaving peptides, and includes a copy of the coding sequence of a first exon.
Claims Coverage
The document provides three independent claim sets covering 3 inventive features: a method of integrating an exogenous coding sequence into an endogenous intronic genomic region, an AAV insertion vector with an intronic insertion cassette design, and an engineered cell characterized by an intronic insertion architecture.
Intronic homologous insertion with ordered splice-site and 2A cassette
A method for integrating an exogenous coding sequence into an endogenous intronic genomic region at an insertion site comprising providing cell(s) comprising an endogenous intronic genomic region, introducing into said cell(s) a polynucleotide template comprising, in order, first and second homologous polynucleotide sequences flanking the insertion site, first and second strong splice site sequences comprising a branch point and a splice acceptor and a splice donor, first and second sequences encoding 2A self-cleaving peptide, an exogenous sequence coding for a protein of interest, and a copy of the coding sequence of the first exon(s), and inducing integration of said exogenous polynucleotide into said intronic sequence by homologous recombination so said exogenous coding sequence being transcribed at said endogenous locus along with the first exon(s) or a copy thereof.
AAV insertion vector with intronic splice-site and 2A exon-copy cassette
An AAV insertion vector characterized in that it comprises an exogenous polynucleotide sequence for insertion at an endogenous locus comprising, in order, a first homologous polynucleotide sequence homologous to the intronic sequence upstream of the insertion site, a first strong splice site sequence comprising a branch point and a splice acceptor, a first sequence encoding 2A self-cleaving peptide, an exogenous sequence coding for a protein of interest, a second sequence encoding 2A self-cleaving peptide, a copy of the coding sequence of the first exon, a second strong splice site sequence comprising a splice donor, and a second homologous polynucleotide sequence homologous to the intronic sequence downstream of the insertion site.
Engineered cell with exogenous intronic insertion arranged for endogenous exon co-transcription
An engineered cell characterized in that an exogenous polynucleotide sequence has been inserted into an intron at an endogenous locus, said polynucleotide sequence comprising, in order, a first strong splice site sequence comprising a branch point and an acceptor site, a first sequence encoding 2A self-cleaving peptide, an exogenous sequence coding for a protein of interest, a second sequence encoding 2A self-cleaving peptide, a copy of the coding sequence of the preceding exon endogenous to said locus, and a second strong splice donor site.
Across the independent claims, the core claim coverage is directed to an intronic integration architecture in which an exogenous coding sequence is introduced as a template into an endogenous intron using homologous sequences and splice-site elements, together with 2A self-cleaving peptide sequences and an endogenous exon coding sequence copy. The same ordered intronic cassette is claimed both in a method of integration, an AAV insertion vector, and an engineered cell characterized by having the inserted intronic polynucleotide arrangement.
Stated Advantages
Avoidance of polar effects.
Induces expression of an exogenous coding sequence at the endogenous locus via transcription along with endogenous exon(s) or a copy thereof.
Documented Applications
In vitro GFP reporting of intron artificial-exon insertion while preserving endogenous gene expression.
In vitro enhancement of IDUA secretion after insertion at S100A9/CD11B.
In vivo engraftment in NSG mice with brain-derived microglia containing edits, including measured IDUA secretion and microglial marker enrichment.
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