**8. Acknowledgments**

This work was supported by the Comisión Sectorial de Investigación Cienctífica (CSIC) of the Universidad de la República (UDELAR) and by the Agencia Nacional de Investigación e Innovación (ANII) of the Ministerio de Cultura, Uruguay.

### **9. References**


combination of engineered modular protein vectors with engineered plasmids for long termregulated expression *in vivo* will be essential. For instance, the pEPI DNA vector was the rst prototype of episomal vector whose function relies exclusively on chromosomal elements, replicating autonomously in low copy numbers in all cells tested (Piechaczek *et al.* 1999). This vector was further engineered to show regulated expression and to be removed from transduced cells when transgene expression is no longer needed (Rupprecht *et al.*). Finally, vectors have to be tested *in vivo*, but evaluating biological effects and not only reporter gene expression, and the comparison of different vectors in a same *in vivo* experimental setting will

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**1. Introduction** 

delivery technology.

**26** 

**Peptides as Promising Non-Viral** 

In its most simplistic sense, gene therapy involves the delivery and expression of DNA by target cells so as to produce a therapeutic protein. In the case of RNA interference (RNAi), it is to shut off or silence the expression of a particular target protein. In order to exert its effects, the nucleic acid must first reach its intended site of action. DNA molecules (frequently as plasmids, which are circularised DNA) have to gain nuclear entry to access the transcription machinery. Conversely, RNAi molecules such as small interfering RNA (siRNA), short hairpin RNA (shRNA) and micro RNA (miRNA) will need to accumulate within the cytoplasm, although shRNA-encoding plasmids will require prior nuclear access before transcription into shRNA. However, if administered alone, a great majority of the nucleic acids will be degraded en route, leading to a lost of therapeutic potential. This then necessitates the development of vectors that protect and deliver nucleic acids to their target site. Arguably, it is the lack of safe and efficient delivery systems, rather than suitable

In this chapter, we start by examining how issues at the cellular level have shaped the design of modern, multifunctional vectors. We then briefly review the various types of gene delivery system, focusing on peptides as a promising class of non-viral vector. We will concentrate on the delivery of plasmids since the phenomenon of RNAi is relatively recent (Fire et al., 1998). As such, many strategies for RNAi delivery are adapted from DNA

**2. Intracellular barriers in gene therapy: Problems and potential solutions** 

Ensuring the arrival of a plasmid at its site of action in a transcriptional state is the entire aim of gene delivery systems. However, plasmids face a constant threat of being degraded. The challenge begins as soon as they are introduced into the extracellular milieu (Figure 1). In most experimental setups, cells/tissues are maintained at 37°C in serum-supplemented medium where serum nucleases can extensively damage a naked plasmid. The plasmid therefore needs protection. Next, the plasmid needs to be internalised. However, both DNA (phosphate groups within the backbone) and plasma membrane (glycoproteins with their sialic acid groups, glycerophosphates with their phosphate groups and proteoglycans which contain sulphate groups) are negatively charged. Electrostatic repulsion then ensures that

therapeutic molecules that is limiting the success of gene therapy.

there is little chance of the plasmid being naturally taken up by a cell.

**Vectors for Gene Therapy** 

Wei Yang Seow and Andrew JT George

*Imperial College London, England* 

*United Kingdom* 

Zeng, J., X. Wang and S. Wang. 2007. Self-assembled ternary complexes of plasmid DNA, low molecular weight polyethylenimine and targeting peptide for nonviral gene delivery into neurons. Biomaterials 28, no. 7: 1443-51.
