A Transposon-Based Transfection System for Primary Cells

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This invention provides a new, efficient transposon-based gene delivery system specifically designed for the genetic modification of primary cells, like T cells, that are typically hard to transfect. The core system uses in vitro transcribed mRNA encoding a transposase (such as Sleeping Beauty) and minicircle DNA containing the transposon, which can simultaneously encode a therapeutic protein and one or more miRNAs. The miRNAs can silence unwanted endogenous genes, increasing the safety and effectiveness of engineered cells, especially for use in therapies such as adoptive T cell therapy and CAR-T/TCR gene therapy for cancers and infectious diseases.

Use CasesContent extracted from patent full text and abstract with AI.

• Engineering T cells for adoptive cell therapy to treat cancer (e.g., CAR-T and TCR therapies).
• Genetic modification of primary human cells for research and therapeutic applications.
• Development of personalized cancer immunotherapies using a patient’s own T cells.
• Gene therapy for chronic viral infections, such as HIV or EBV, by modifying T cell specificity.
• Reducing risk of graft-versus-host disease by silencing endogenous TCRs in engineered T cells.
• Manufacturing modified stem cells or other primary cells for regenerative medicine.

BenefitsContent extracted from patent full text and abstract with AI.

• Allows efficient, high-viability genetic modification of hard-to-transfect primary cells, especially T cells.
• Reduces cell death and improves transfection efficiency compared to conventional DNA or viral methods.
• Enables simultaneous expression of therapeutic proteins and gene-silencing miRNAs from a single construct, increasing safety and efficacy.
• Uses minicircle DNA without bacterial backbone, reducing regulatory and safety concerns associated with antibiotic resistance genes.
• Non-viral delivery avoids risks of insertional mutagenesis and immune reaction linked with viral vectors.
• Streamlines and lowers cost and complexity of gene-engineered cell production, making the process faster and more scalable.

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Patent Abstract

The present invention relates to the field of genetic engineering, in particular, to a transposon-based transfection kit suitable for transfection of primary cells, such as T cells, comprising mRNA encoding a transposase, or reagents for generating mRNA encoding said transposase, as well as minicircle DNA comprising the transposon. The invention also relates to a nucleic acid, preferably, a DNA minicircle, comprising a transposon, wherein the transposon encodes a protein and at least one miRNA, wherein the sequences encoding the miRNA are located in an intron and expression of the protein and the miRNA is regulated by the same promoter. The invention also provides a population of cells obtainable with the method of the invention. Methods of transfection are also provided, as well as medical use, e.g. in immunotherapy, in particular, in adoptive T cell therapy or T cell receptor (TCR) or chimeric antigen receptor (CAR) gene therapy.