Retroviral Vector

Abstract

A cell for producing retroviral vectors comprising nucleic acid sequences encoding: i) gag-pol; ii) env; iii) the RNA genome of the retroviral vector; and iv) optionally rev, or a functional substitute thereof, wherein at least two nucleic acid sequences are located at the same genetic locus; and wherein the at least two nucleic acid sequences are in reverse and / or alternating orientations.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of priority of European Patent Application No. 17210359.0, filed on Dec. 22, 2017, the entire contents of which are incorporated by reference herein.FIELD OF THE INVENTION[0002]The invention relates to the production of retroviral vectors. In particular, it relates to modular constructs comprising retroviral gene components.BACKGROUND TO THE INVENTION[0003]Gene therapy broadly involves the use of genetic material to treat disease. It includes the supplementation of cells with defective genes (e.g. those harbouring mutations) with functional copies of those genes, the inactivation of improperly functioning genes and the introduction of new therapeutic genes.[0004]Therapeutic genetic material may be incorporated into the target cells of a host using vectors to enable the transfer of nucleic acids. Such vectors can be generally divided into viral and non-viral categories.[0005]Viruses naturally introduce t...

AI Technical Summary

Benefits of technology

The present invention has found that using modular constructs containing multiple nucleic acid components necessary for viral vector production can lead to the generation of vector production cells. These modular constructs can also be used to reduce the use of transfection agents and evaluate the optimal stoichiometry of the various retroviral vector components to yield the most advantageous vector production cell. Additionally, the use of these modular constructs can improve the process of generating stable packaging and producer cell lines, reduce production time and costs, and offer the advantage of reducing the number of selectable markers and thus selection steps required for stable cell line generation. The fact that at least some of the components necessary for vector production will be integrated at the same site within the production cell genome can further mean that such a process can also overcome problems such as gene silencing, which can occur when retroviral vector genes are integrated randomly and at different locations within the production cell genome. Overall, the present invention provides a stable cell for producing retroviral vectors.

Problems solved by technology

Previously, it had been thought that this would be ineffective due to modular constructs, such as those of the present invention, being too large to be held by bacterial plasmids which were to be used for retroviral production, as oversized plasmids can lead to both genetic instability and poor expression.

Additionally, the size of these constructs could negate the efficiency of cellular entry in transient and stable vector production systems, and integration in stable vector production cells using typical transfection methods.

The safety and efficacy of using such plasmids for the generation of vector production cells could also be questioned as it may lead to an increased chance of the formation of replication-competent viral particles.

Images