Expression of products from nucleic acid concatemers

Abstract

Provided are techniques for generating expression products using one or more nucleic acid concatemers that include tandem repeats of a nucleic acid sequence encoding the expression product or products. In one embodiment, different expression products may be co-expressed using a concatemer mixture of a first nucleic acid concatemer and a second nucleic acid concatemer having a predefined ratio to one another.

Description

[0001]SEQUENCE LISTING[0002]The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 15, 2019, is named 324586-1_SL.txt and is 13,566 bytes in size.TECHNICAL FIELD[0003]The disclosure generally relates to expression of recombinant products from nucleic acid concatemers, and, in certain embodiments, relates to a ratiometric co-expression of recombinant products from nucleic acid concatemers. The ratiometric co-expression has been demonstrated in both in vivo and cell free expression systems.BACKGROUND[0004]Recombinant products, including proteins, antibodies, and nucleic acids, have been widely used in clinical applications. Such recombinant products may harness nucleic acid and / or protein expression machinery to generate products of interest with clinical utility. For example, immunotherapy and other cell therapies frequently utilize viral vecto...

AI Technical Summary

Benefits of technology

This approach achieves robust and predictable co-expression of recombinant products with improved purity and reduced production costs, facilitating scalable and cost-effective manufacturing of therapeutic agents like viral vectors and antibodies.

Problems solved by technology

However, certain desired products may involve multiple components that are complexed or that otherwise operate together.

Production of such multi-component expression products may be complex.

However, certain challenges are associated with using multiple plasmid constructs in cells.

One of the challenges is the ratiometric control of co-expression of multiple plasmid constructs.

For example, a single dual promotor plasmid with an internal ribosome entry site (IBES) and a viral 2A peptide can be used to drive co-expression of two or more proteins with reduced heterogeneity, although this requires the expense of laboriously cloning each open reading frame into the multi-cistronic plasmid and consequently increasing the size of the plasmid construct.

In addition, since plasmids contain additional sequences necessary for maintenance in bacteria, the use of plasmids requires post-production purifications and QC analytics to prove absence of bacterial contaminants in the plasmid production, which posts a significant challenge for cGMP (clinical good manufacturing) process.

The latter is especially challenging as the size of the plasmid construct increases and becomes difficult to distinguish from bacterial chromosomal DNA, which complicates post-production purification of plasmid DNA.

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