ABSTRACT
Large Proteins .......................................................... 400
22.5.2.2 Pep-1-Mediated Transduction of Biologically
Active Molecules .................................................... 400
22.5.2.3 Application of Pep-1-Mediated PNA and Protein
Transduction In Vivo .............................................. 401
22.6 Conclusions and Perspectives ................................................................ 401
References ........................................................................................................ 402
Over the past 10 years, substantial progress has been made in the design of new
technologies to improve cellular uptake of therapeutic compounds.1-6 This
evolution has been directly correlated with the dramatic acceleration in the
production of new therapeutic molecules, as cell delivery systems described until
then were restricted by very specific issues. However, only a few nonviral
technologies are efficiently applied in vivo at either preclinical or clinical states.1,5,6
Their major limitations are the poor stability of the formulations, the rapid
degradation of the cargo, as well as its insufficient capability to reach its target. Cell-
penetrating peptides (CPPs) constitute one of the most promising generations of
tools for delivering biologically active molecules into cells and can thereby have a
major impact on the future of treatments.4,7,8 CPPs have been shown to improve
intracellular delivery of various biomolecules efficiently, including plasmid DNA,
oligonucleotides, short interfering RNA (siRNA), PNA, proteins, peptides, as well
as liposomes into cells both in vivo and in vitro.7-12 Short synthetic CPPs able to
overcome both extracellular and intracellular limitations have been designed. These
peptides can trigger the movement of a cargo across the cell membrane into the
cytoplasm of the cells and improve its intracellular routing, thereby facilitating
the interaction with the target.7-13 Most of the technologies described to date require
the attachment of a CPP to the target cargo, which is achieved by either chemical
cross-linking or cloning and expression of a protein fused to the CPP.14-17
Conjugation methods offer several advantages for in vivo applications, including
rationalization, reproducibility of the procedure, together with the control of the
stoichiometry of the protein transduction domain (PTD) cargo. However, the
covalent PTD technology is limited from the chemical point of view as it is mainly
performed via a synthetic disulfide linkage and risks altering the biological activity
of the cargoes. In order to offer an alternative to covalent strategies we have
proposed a new potent strategy for the delivery of biomolecules into mammalian
cells, based on the short amphipathic peptide carriers, MPG and Pep-1.18-23 MPG
and Pep-1 form stable nanoparticles with cargoes without the need for cross-linking
or chemical modifications. MPG efficiently delivers nucleic acids (plasmid DNA,
oligonucleotides, siRNA) and Pep-1 improves the delivery of proteins and peptides
in a fully biologically active form into a variety of cell lines and in vivo.21-23
The mechanism through which MPG or Pep-1 delivers active macromolecules
does not involve the endosomal pathway and therefore allows control of the release
of the cargo in the appropriate target subcellular compartment.24,25 In this chapter
we will describe the characteristics of the noncovalent MPG and Pep-1 strategies,
and their applications for nucleic acid and peptide transduction, both in vitro
and in vivo.
