**1. Introduction**

#### **1.1. Why messenger-RNA-based vectors are used in gene delivery?**

DNA or RNA fragments of choice can be amplified in bacteria and eukaryotic cells by piggybacking on replicating episomes, called 'cloning vectors'. In contrast to 'cloning vectors', 'gene vectors' are the vehicles that transfer genes into cells. All gene vectors contain nucleic acids or their analogues (e.g. Peptide Nucleic Acid – PNA) as the carriers of genetic informa‐ tion. The complexity of gene vectors ranges from naked DNA or RNA to multi-component nano-devices with a finely ordered internal structure, which can be either virus-derived or purely synthetic. The aim of gene delivery is often the presence of specific proteins in the target cells. One way to achieve this is to transfer an immediate information source for protein biosynthesis, that is, messenger RNA (mRNA), into target cells. Presently, mRNA-based vectors are established multipurpose gene vectors applicable to a diverse range of tasks in gene therapy, gene immunisation and transgene-mediated cell-fate reprogramming [1-4].

The long-term storage of genetic information in cells is mediated by DNA, while short-term cellular memory is stored in RNA. So, if a permanent genetic change in the target cells is desirable, either DNAs or RNA-templates for reverse transcription into DNA are used as carriers of genetic information within the gene vectors. If only a non-permanent genetic change is wanted, then gene vectors containing a translatable 'sense' RNA strand ('positive strand') seem to be particularly suitable. Such vectors, whether based on mRNA generated *in vitro* or cellular mRNAs (including cellular mRNAs isolated through packaging into viral capsids), can reach ribosomes and express genes in the cytosol, without nuclear entry. There are five important implications of the extra-nuclear status of mRNA vectors.

Firstly, as mRNA does not require transfer to nucleosol for expression, mRNA-based vectors can be used in applications where a rapid and transient effect is required, e.g. wound healing or antigen-presenting. The transgene expression is fast because mRNA vectors, as opposed to DNA vectors, do not need to pass through the barrier of the nuclear envelope, which confines the nucleosol in non-dividing cells and do not need to enter the nucleus and then to exit it. In addition, no time is wasted on intra-nuclear transcription in both dividing and non-dividing cells.

Secondly, the fact that gene delivery with mRNA vectors is capable of attaining transgene expression in non-mitotic cells with a closed nuclear envelope is remarkable *per se*. Thus, mRNA vectors can be more efficient than DNA vectors for the transfection of clinically important post-mitotic cells like neurons and cardiomyocytes [5]. In fact, in non-dividing cells there is no dilution of externally delivered mRNAs and their protein products in cell divisions; this circumstance can contribute to longer persistence of mRNA-vector-mediated transgene expression in these cells in comparison to dividing cells.

intercellular environment complicate gene delivery *in vivo*, which, therefore, requires more complex gene transfer procedures than transfection of tissue culture cells. This review is focused on transfection methods for mRNA vectors, which rely either on the forceful propulsion of mRNA inside the target cells (e.g. by electroporation or gene gun) or on the complexing of mRNA with other substances (e.g. polycationic

**Keywords:** Gene therapy, epigenetic reprogramming, gene vaccination, mRNA gene vec‐

DNA or RNA fragments of choice can be amplified in bacteria and eukaryotic cells by piggybacking on replicating episomes, called 'cloning vectors'. In contrast to 'cloning vectors', 'gene vectors' are the vehicles that transfer genes into cells. All gene vectors contain nucleic acids or their analogues (e.g. Peptide Nucleic Acid – PNA) as the carriers of genetic informa‐ tion. The complexity of gene vectors ranges from naked DNA or RNA to multi-component nano-devices with a finely ordered internal structure, which can be either virus-derived or purely synthetic. The aim of gene delivery is often the presence of specific proteins in the target cells. One way to achieve this is to transfer an immediate information source for protein biosynthesis, that is, messenger RNA (mRNA), into target cells. Presently, mRNA-based vectors are established multipurpose gene vectors applicable to a diverse range of tasks in gene

therapy, gene immunisation and transgene-mediated cell-fate reprogramming [1-4].

The long-term storage of genetic information in cells is mediated by DNA, while short-term cellular memory is stored in RNA. So, if a permanent genetic change in the target cells is desirable, either DNAs or RNA-templates for reverse transcription into DNA are used as carriers of genetic information within the gene vectors. If only a non-permanent genetic change is wanted, then gene vectors containing a translatable 'sense' RNA strand ('positive strand') seem to be particularly suitable. Such vectors, whether based on mRNA generated *in vitro* or cellular mRNAs (including cellular mRNAs isolated through packaging into viral capsids), can reach ribosomes and express genes in the cytosol, without nuclear entry. There are five

Firstly, as mRNA does not require transfer to nucleosol for expression, mRNA-based vectors can be used in applications where a rapid and transient effect is required, e.g. wound healing or antigen-presenting. The transgene expression is fast because mRNA vectors, as opposed to DNA vectors, do not need to pass through the barrier of the nuclear envelope, which confines the nucleosol in non-dividing cells and do not need to enter the nucleus and then to exit it. In addition, no time is wasted on intra-nuclear transcription in both dividing and non-dividing

transfection reagents) for delivery via endocytic pathways.

**1.1. Why messenger-RNA-based vectors are used in gene delivery?**

important implications of the extra-nuclear status of mRNA vectors.

tors, transfection methods, gene transfer

**1. Introduction**

38 Gene Therapy - Principles and Challenges

cells.

Thirdly, the major mechanisms of transgene silencing, e.g. chromatin remodelling and genomic DNA methylation [6], are entirely intra-nuclear and, thus, are irrelevant for the desired expression of exogenous mRNA.

Fourthly, for the successful implementation of many therapeutic strategies, it is important that gene delivery with mRNA vectors cannot cause potentially deleterious mutations via inser‐ tional gene inactivation or undesired position effects like gene activation in the neighbourhood of a chromosomally integrated transgene. Indeed, in many cases the full long-term conse‐ quences of the genomic insertions are difficult to predict and so any permanent genetic change is often unwanted. As any gene delivery with mRNA-based vectors does not leave an unde‐ sired genomic trace, gene transfer with mRNA vectors *in vivo* benefits from the absence of the safety risks and ethical controversies of vector elements being incorporated into the human germ line and subsequently being transmitted vertically through future generations.

Fifthly, as only extra-nuclear localisation of externally delivered mRNA is required for transgene expression, 'milder' transfection conditions (e.g. shorter electric field pulses during electroporation) might be sufficient for delivery of mRNA into its 'expression *milieu*'. Indeed, 'milder' conditions increase the cell survival rate and, hence, offer higher transfection effi‐ ciency with mRNA vectors in comparison to DNA vectors [7].

Another advantage of mRNA-based vectors is the flexibility to combine several mRNAs into a single multi-gene cocktail. In addition, a number of proteins can be expressed from a single mRNA using internal ribosome entry sites (IRESes), ribosome skipping sequences or *bona fide* proteolytic signals. The ease of transgene reshuffling makes mRNA-based vectors partic‐ ularly convenient in the complex tasks of epigenetic engineering, where multiple combinations of transgenes need to be screened to assess their cell-fate reprogramming effectiveness. However, on the downside, uncontrolled extracellular and intracellular decay of mRNA can be a substantial hurdle for mRNA-mediated gene transfer.

#### **1.2. What strategies are used to deliver mRNA-based vectors to cells?**

Methods for mRNA delivery are similar to DNA transfer procedures, which are well-estab‐ lished. Overall, there are three actors in the gene delivery play, namely, the vector, the cell and the transfer environment. The desired outcome, the efficient transfer of a gene to a target cell population and its installation as a functional transgene depends on the productive interaction of all three parties. Thus, the vector should ideally be targeted to reach the desired cells selectively and efficiently and also presented in a form that is resistant to the aggressive factors in the delivery *milieu*. At the same time, the delivery environment should be adapted to be better vector-accommodating and better cell-accommodating. The recipient cells should be subjected to a specific set of treatment procedures or artificially modified to become receptive to gene transfer with a particular vector and resistant to the environment.

In general, mRNA-based vectors can be delivered to cells in tissue culture (*in vitro*) and intracorporeally (*in vivo*). *In vitro* gene delivery is a necessary step in *ex vivo* strategies of gene immunisation [8, 9], gene therapy [10] and therapeutic cell-fate reprogramming [4]. As a rule, barriers outside tissues (e.g. mucus) and an aggressive intercellular environment complicate gene delivery *in vivo*, which, therefore, requires more complex transfection procedures than transfection *in vitro*. The standard transfection routes rely either on the forceful propulsion of mRNA inside the target cells (e.g. by electroporation or gene gun) or on the complexing of mRNA with other substances (e.g. polycationic transfection reagents) for delivery via endo‐ cytic pathways.
