Optimised gene delivery using semi-synthetic lentivirus.

Lentiviral vectors offer distinct advantages over retroviral vectors as they have a different integration profile, a tendency to insert in intronic regions and are engineered to be self-inactivating so that the long terminal repeats (LTRs) are transcriptionally inactive. However, compared to retroviral vectors, there are drawbacks to lentiviral vectors, including more limited payload size, reduced expression levels, lack of a splicing intron to stabilise mRNA and a relatively weak polyadenylation [poly(A)] sequence.

Thus, there is a pressing need to re-engineer lentiviral vectors to increase expression through promoter optimisation and the inclusion of sequences to stabilise mRNA transcribed from the internal expression cassette. This proposal aims to address these issues through the generation of semi-synthetic lentivirus.

The concept is to produce viral-like particles (VLPs) from lentivirus and subsequently introduce in vitro transcribed lentiviral genome RNA to the VLPs. As the lentiviral genome RNA will be produced in vitro and will not be subjected to splicing by the spliceosome or polyadenylation by polyadenylate polymerase, it should be possible to engineer previously forbidden elements such as introns and poly(A) sequences into the viral genome.

Elements to be incorporated into the lentiviral genome RNA include a 5' intron such as the chimeric or synthetic intron and a poly(A) signal sequence derived from the human beta-globin gene. The inclusion of an intron at the 5' end of an mRNA has been shown to increase its stability. Similarly, polyadenylation of the 3' end of an mRNA protects it from degradation and increases its stability.

With conventional lentiviral vector production, a strong CMV enhancer/promoter sequence is frequently used to drive transcription of the genome RNA. If an equally strong internal promoter, which drives transcription of the cargo transgenes is used competition between the two promoters can occur, resulting in reduced titres as less of the genome RNA is produced.

If the lentiviral genome RNA is produced in vitro, this is no longer a problem and allows the use of strong internal promoters. Optimisation of the promoter would facilitate increased expression of the cargo transgenes. In the case of chimeric antigen receptor T cell (CAR-T) therapy, a promoter driving strong expression in T cells is desirable.

The generation of semi-synthetic lentiviral vectors should enable re-engineering of the genome RNA to include previously forbidden elements, such as 5' intron and poly(A) signal sequences, and the engineering of strong internal promoters driving transgene expression. If successful, this vector engineering would pave the way to the next generation of gene therapies by enabling the expression of longer transgene cassettes.

This approach would be of particular use to CAR-T cell therapy where dual CARs and additional functional modules are required to enable CAR-T cells to eliminate tumour cells.