**Acknowledgements**

polycistronic gene co-delivery, the expression level of the downstream transgene is not

Another possible strategy to combine several messages for the simultaneous expression of several functions is to generate mRNA for a fusion protein, e.g. the target protein fused with a readily detectable moiety. Fusion proteins can be conveniently 'split up' into individual polypeptide chains using viral 'ribosome skipping' sequences like 2A or, alternatively, using

There is a considerable scope for improvements of the procedures for gene delivery with synthetic mRNA. Further advancements in the field of mRNA-based gene delivery are likely to include the development of more stable, easily deliverable and gene-expression-efficient forms of mRNA vectors, incorporating specialised ligands for cell-specific targeting, cell penetration and intracellular targeting. The vector improvements, the use of potent stimulators of targeted cells' receptivity, the refinement of the mRNA cellular entry procedures and also the employment of optimised modulators of the intercellular environment are expected to increase the efficiency of gene transfer and the efficiency of body-locus-targeting, especially

The potential for advancement of gene delivery with synthetic mRNAs looks strong in comparison to alternative rapid delivery methods. Indeed, high speed and other benefits of non-viral synthetic mRNA vectors are shared by: 1) virally encapsidated RNAs; 2) cellpermeable proteins. Thus, the packaging of mRNA (in positive-strand RNA viral vectors) or a template for mRNA (in vectors containing negative-strand RNA, e.g. Sendai virus-based vectors) within viral capsids is an attractive method of condensing and protecting RNA cargo destined for delivery into the cytosol of target cells. However, there are two unavoidable downsides of viral packaging in RNA transfer. Firstly, viral capsids dictate rigid size con‐ straints for vector RNA. Secondly, this strategy tends to be tedious because the encapsidation of each RNA species requires the laborious insertion of an appropriate viral packaging sequence. Similarly, if compared to protein delivery by protein transduction into cells, gene transfer mediated by synthetic mRNAs is more advantageous since each protein to be delivered through direct cell entry requires time-consuming insertion of an effective PTD sequence. Clearly, various delivery methods can be combined. For example, as protein transduction is a very fast method of increasing the concentration of specific proteins in target cells, so, it can, in principle, be used to augment mRNA transfer. So, as cell viability currently appears to be a critical hurdle in mRNA-based gene delivery, it is possible that most significant future advances in mRNA-mediated gene transfer will be achieved through extensive employment of cell penetrating proteins capable of supporting the viability of cells before, during and after mRNA delivery procedures. The refined mRNA-based transfection techni‐ ques can also be applied to delivery of other medicinally important species of RNA, such as

necessarily equal to the expression level of the upstream transgene.

*bona fide* proteolytic signals for resident proteases in recipient cells.

in clinical applications.

50 Gene Therapy - Principles and Challenges

siRNA.

**3. Future perspectives of mRNA-based gene delivery methods**

The authors are grateful to Dr Nadire N Ali, Dr Tatiana Subkhankulova (Imperial Col‐ lege London, UK) and Ms Lucy Tolmacheva (London City University, UK) for motivat‐ ing discussions.
