Construct and sequence for enhanced gene expression
Inventors
van der Heijden, Maurice Wilhelmus • Engels, Bart Marinus
Assignees
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Publication Number
US-10889822-B2
Publication Date
2021-01-12
Expiration Date
Abstract
The invention relates to a method for transcription and expression using a nucleic acid construct which is characterized by the presence of a promoter followed by an intronic promoter. The invention further relates to the nucleic acid construct, an expression vector and a cell comprising the construct, and its use. The invention also relates to methods for transcription and optionally expression using a nucleotide sequence. The invention further relates to the nucleotide sequence and a construct, expression vector and cell comprising the nucleotide sequence, and its use.
Core Innovation
The invention relates to nucleic acid constructs for enhanced transcription and/or expression in host cells. The constructs include a first promoter and a second promoter operably linked to a single nucleotide sequence of interest, and the first promoter and the second promoter are constitutive promoters.
The second promoter is an intronic promoter flanked by a first intronic sequence located upstream of the second promoter and a second intronic sequence located downstream of the second promoter. The construct supports formation of different transcripts via promoter-driven alternative splicing, where transcription initiation from either promoter leads to different pre-mRNA and splicing outcomes.
The disclosure further describes expression-enhancing elements defined by sequence identity using SEQ ID sequences, including intronic/promoter-containing forms such as UBC/CCT8-derived sequences and a sequence related to SEQ ID NO:88. The documented constructs are described to increase transcription and/or protein yield in host cells, including mammalian cells.
Claims Coverage
The claim set includes one independent claim centered on a nucleic acid construct with two constitutive promoters operably linked to a single nucleotide sequence of interest, where the second promoter is an intronic promoter flanked by upstream and downstream intronic sequences. The claim set further specifies SEQ ID-defined promoter and intronic sequences, with dependent claims refining additional expression-regulating elements, intron placement, encoded product type, promoter identity, and host or cell contexts.
Two constitutive promoters with intronic-promoter architecture for a nucleotide sequence of interest
A nucleic acid construct comprising a first promoter, a second promoter, and a single nucleotide sequence of interest, wherein the first promoter and second promoter are constitutive promoters and are both operably linked to the single nucleotide sequence of interest, and wherein the second promoter is an intronic promoter flanked by a first intronic sequence located upstream of the second promoter and a second intronic sequence located downstream of the second promoter.
Specific SEQ ID-defined promoter and intronic sequences
The first promoter and the first intronic sequence comprise the sequence of SEQ ID NO: 1 and the second intronic sequence comprises the sequence of SEQ ID NO: 19.
The independent claim requires a nucleic acid construct with two constitutive promoters controlling a nucleotide sequence of interest, with the second promoter implemented as an intronic promoter flanked by upstream and downstream intronic sequences. The claim set further specifies particular SEQ ID-defined promoter and intronic sequence elements, establishing the claimed intron-based promoter architecture for transcription and/or expression.
Stated Advantages
Increases transcription and/or protein yield in host cells.
Enables formation of different transcripts via promoter-driven alternative splicing.
Documented Applications
Gene therapy and nucleic acid-based vaccination applications using nucleic acid constructs, vectors, and host cell expression.
Expression and optional purification of transcript and/or protein from the constructs in host cells, including mammalian cells.
Expression involving noncoding RNA and protein expression applications, as described for the constructs.
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