Chimeric post-transcriptional regulatory element
Inventors
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10190135-B2
Publication Date
2019-01-29
Expiration Date
Abstract
The present disclosure relates to chimeric post-transcriptional regulatory elements (PRE) and vectors useful for expressing a protein in a cell. The PRE contains alpha, beta and optionally gamma subelements selected from different native PRE sequences and are discovered to be more potent than their native counterparts.
Core Innovation
The invention relates to chimeric post-transcriptional regulatory elements (PRE) made by combining different native PRE subelements, including an alpha subelement and a beta subelement from different native PREs, and optionally a gamma subelement. The disclosed chimeric PREs include defined sequence-identity variants and spacing constraints between PRE fragments.
The disclosed constructs incorporate the chimeric PREs into polynucleotide and vector systems for expressing recombinant proteins in eukaryotic cells. The described construct features include promoter/intron/poly(A) arrangements and the placement of PRE fragments at defined positions, including a 3′-UTR and a poly(A) sequence.
Experimental results reported in the disclosure indicate that chimeric PRE combinations can be more potent than native PREs. The disclosure further states that subelement strength, rather than the number of subelements, drives activity, and that chimeric PREs tolerate removal of other regulatory elements without losing potency as much, based on interaction studies.
Claims Coverage
The partial content provides one independent claim directed to a specific polynucleotide composed of three defined sequence fragments with sequence-identity thresholds. Dependent claims further add construct-level features, specific regulatory elements, and a spacing constraint between fragments, and also specify a cultured cell containing the construct.
Three-fragment polynucleotide defined by SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 3
A polynucleotide comprising a first fragment consisting of the nucleic acid sequence of SEQ ID NO: 7 or a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 7, a second fragment consisting of the nucleic acid sequence of SEQ ID NO: 9 or a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 9, and a third fragment consisting of the nucleic acid sequence of SEQ ID NO: 3 or a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 3.
Spacing-constrained three-fragment arrangement
The polynucleotide having the second fragment between the first and third fragments, with each fragment not more than 20 nucleotides away from a neighboring fragment.
Polynucleotide construct including a protein-coding sequence
A polynucleotide construct that includes the polynucleotide of the three-fragment definition together with a protein-coding sequence.
Polynucleotide construct comprising a 3′-UTR
A polynucleotide construct further including a 3′-UTR.
Polynucleotide construct comprising a poly(A) sequence
A polynucleotide construct further including a poly(A) sequence.
Cultured cell containing the polynucleotide construct
A cultured cell that includes the polynucleotide construct comprising the 3′-UTR and poly(A) sequence.
Overall, the claim coverage centers on a three-fragment polynucleotide defined by SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 3, or at least 95% identity variants, with dependent claims specifying a spacing relationship between fragments, incorporation into a protein-coding polynucleotide construct, addition of a 3′-UTR and poly(A) sequence, and inclusion of the construct in a cultured cell.
Stated Advantages
Chimeric PRE combinations can be more potent than native PREs.
Subelement strength, rather than the number of subelements, drives activity.
Chimeric PREs tolerate removal of other regulatory elements without losing potency as much.
Documented Applications
Use of vectors/polynucleotide constructs for expressing recombinant proteins in eukaryotic cells, including cultured cells (e.g., CHO cells).
Application context includes expressing a recombinant protein such as a Rituximab light chain in the described eukaryotic cell systems.
Interested in licensing this patent?