Enhanced nucleic acid constructs for eukaryotic gene expression
Inventors
Minshull, Jeremy • Welch, Mark • GOVINDRAJAN, SRIDHAR • Caves, Kate
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-9534234-B2
Publication Date
2017-01-03
Expiration Date
Abstract
The present invention provides polynucleotide vectors for high expression of heterologous genes, and methods for constructing such vectors. Some vectors further comprise novel transposons and transposases that further improve expression. Further disclosed are vectors that can be used in a gene transfer system for stably introducing nucleic acids into the DNA of a cell. The gene transfer systems can be used in methods, for example, but not limited to, gene expression, gene therapy, insertional mutagenesis, or gene discovery.
Core Innovation
The invention relates to enhancing eukaryotic gene-expression using optimized nucleic acid and vector configurations that include a transposase and a piggyBac transposon flanked by inverted terminal repeats. The transposase is fused to a heterologous nuclear localization sequence (NLS) and is operably linked to a heterologous promoter in polynucleotide embodiments, with the transposase comprising the amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 46. The system integrates a transposon carrying a non-transposon heterologous DNA sequence into host genomic DNA at a 5′-TTAA-3′ target sequence, where the transposon is flanked by piggyBac ITRs each comprising SEQ ID NO: 42.
The disclosure additionally addresses performance factors affecting transcription, RNA processing/export, integration, replication, and translation by providing enhanced construct elements. RNA export elements, enhancing elements, insulator elements, introns, and polyadenylation signals are described in connection with gene transfer performance, and the content further includes translational coupling elements including IRES and CHYSEL. These elements are used in optimized construct configurations to influence expression outcomes and stable expression.
The described system is presented as enabling improved non-viral gene transfer capabilities and stable expression. It is associated with applications including antibody expression, CAR T-cell engineering, transgenic animals, gene therapy, gene discovery via insertional mutagenesis/genome mapping, and RNA interference via transposon-delivered shRNA/siRNA cassettes. The document also states pharmaceutical composition and kit formulations that include Bombyx mori and Xenopus tropicalis transposase/transposon components.
Claims Coverage
The independent claims cover three closely related inventive concepts: a polynucleotide encoding an NLS-fused piggyBac-like transposase driven by a heterologous promoter; a transposase fused to a heterologous NLS; and an mRNA polynucleotide encoding the NLS-fused transposase. Across the independent claims, the core inventive features are the specified SEQ ID NO: 45 or SEQ ID NO: 46 transposase, piggyBac transposon architecture with ITRs comprising SEQ ID NO: 42, and integration targeting to a 5′-TTAA-3′ site.
NLS-fused SEQ ID NO: 45/46 transposase for 5′-TTAA-3′ integration
A transposase comprising the amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 46, wherein the transposase can integrate into a genomic DNA of a host cell at a 5′-TTAA-3′ target sequence a transposon carrying a non-transposon heterologous DNA sequence.
Heterologous NLS fusion enabling piggyBac ITR-flanked transposon integration
A transposase fused to a heterologous nuclear localization sequence (NLS), where integration is of a transposon comprising a non-transposon heterologous DNA sequence flanked by a pair of inverted terminal repeats (ITR) of a piggyBac transposon.
Heterologous promoter-linked polynucleotide encoding the NLS-fused transposase
A polynucleotide encoding a transposase fused to a heterologous nuclear localization sequence (NLS) and operably linked to a heterologous promoter, where the transposase comprises the amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 46.
mRNA polynucleotide encoding the NLS-fused SEQ ID NO: 45/46 transposase
A polynucleotide is an mRNA encoding a transposase fused to a heterologous NLS, where the transposase comprises the amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 46, and can integrate a piggyBac ITR-flanked transposon into genomic DNA at a 5′-TTAA-3′ target sequence.
Overall, the independent claims are directed to an NLS-fused transposase (SEQ ID NO: 45 or SEQ ID NO: 46) that mediates integration into a host genome at a 5′-TTAA-3′ target sequence using a piggyBac transposon carrying non-transposon heterologous DNA flanked by ITRs each comprising SEQ ID NO: 42, with embodiments specified as a promoter-linked polynucleotide and as an mRNA polynucleotide.
Stated Advantages
Higher expression is stated to result from insertion near polyA signals using 5′-TTAT-3′ or 5′-TTAA-3′ target sequences.
Increased stable expression is stated to occur when the transposase is co-delivered in the described examples.
Improved stable expression from transposon/transposase systems is described.
Improved non-viral gene transfer capabilities are described.
Documented Applications
Gene transfer systems for eukaryotic gene-expression using transposons carrying heterologous genes and an NLS-fused piggyBac-like transposase for stable genomic integration.
Reporters and selection readouts using DasherGFP and selectable markers in CHO and HEK293 cells to demonstrate increased stable expression with co-delivered transposase.
Expression of multi-polypeptide genes such as antibody heavy/light chains using coupling elements for stoichiometry.
Use of kits and pharmaceutical compositions comprising the described transposase/transposon system components.
Antibody expression, including heavy chain/light chain expression using translational coupling elements.
CAR T-cell engineering.
Transgenic animals.
Gene therapy.
Gene discovery via insertional mutagenesis/genome mapping.
RNA interference (RNAi) using transposon-delivered shRNA/siRNA cassettes.
Pharmaceutical composition and kit formulations including Bombyx mori and Xenopus tropicalis transposase/transposon components.
Interested in licensing this patent?