Gene therapy has great potential to treat a range of different diseases, such as cancer. For that therapeutic gene can be inserted into a plasmid vector and delivered specifically to tumor cells. The most frequently used applications utilize lipoplex and polyplex approaches where DNA is non-covalently condensed into nanoparticles. However, lack of in vivo efficacy is the major concern that hinders translation of such gene therapeutic applications into clinics. In this work we introduce a novel method for in vivo delivery of plasmid DNA (pDNA) and efficient tumor-specific gene induction using intravenous (i.v) administration route. To achieve this, we utilize a cell penetrating peptide (CPP), PepFect14 (PF14), double functionalized with polyethylene glycol (PEG) and a matrix metalloprotease (MMP) substrate. We show that this delivery vector effectively forms nanoparticles, where the condensed CPP and pDNA are shielded by the PEG, in an MMP-reversible manner. Administration of the complexes results in efficient induction of gene expression specifically in tumors, avoiding normal tissues. This strategy is a potent gene delivery platform that can be used for tumor-specific induction of a therapeutic gene.

The successful applicability of gene therapy approaches will heavily rely on the development of efficient and safe nonviral gene delivery vectors, for example, cell-penetrating peptides (CPPs). CPPs can condense oligonucleotides and plasmid DNA (pDNA) into nanoparticles, thus allowing the transfection of genetic material into cells. However, despite few promising attempts, CPP-mediated pDNA delivery has been relatively inefficient due to the unfavorable nanoparticle characteristics or the nanoparticle entrapment to endocytic compartments. In many cases, both of these drawbacks could be alleviated by modifying CPPs with a stearic acid residue, as demonstrated in the delivery of both the pDNA and the short oligonucleotides. In this study, PepFect14 (PF14) peptide, previously used for the transport of shorter oligonucleotides, is demonstrated to be suited also for the delivery of pDNA. It is shown that PF14 forms stable nanoparticles with pDNA with a negative surface charge and size of around 130-170 nm. These nanoparticles facilitate efficient gene delivery and expression in a variety of regular adherent cell lines and also in difficult-to-transfect primary cells. Uptake studies indicate that PF14/pDNA nanoparticles are utilizing class A scavenger receptors (SCARA) and caveolae-mediated endocytosis as the main route for cellular internalization. Conclusively, PF14 is an efficient nonviral vector for gene delivery.

No abstract

Non-viral gene delivery systems have gained considerable attention as a promising alternative to viral delivery to treat diseases associated with aberrant gene expression. However, regardless of extensive research, only a little is known about the parameters that underline in vivo use of the nanoparticle-based delivery vectors. The modest efficacy and low safety of non-viral delivery are the two central issues that need to be addressed. We have previously characterized an efficient cell penetrating peptide, PF14, for in vivo applications. In the current work, we first develop an optimized formulation of PF14/pDNA nanocomplexes, which allows removal of the side-effects without compromising the bioefficacy in vivo. Secondly, based on the physicochemical complex formation studies and biological efficacy assessments, we develop a series of PF14 modifications with altered charge and fatty acid content. We show that with an optimal combination of overall charge and hydrophobicity in the peptide backbone, in vivo gene delivery can be augmented. Further combined with the safe formulation, systemic gene delivery lacking any side effects can be achieved.

Global cancer prevalence has continuously increased in the last decades despite substantial progress achieved for patient care. Cancer is no longer recognized as a singular disease but as a plurality of different ones, leading to the important choice of the drug administration route and promoting the development of novel drug-delivery systems (DDS). Due to their structural diversity, therapeutic cancer drugs present specific challenges in physicochemical properties that can adversely affect their efficacy and toxicity profile. These challenges are addressed by innovative DDS to improve bioavailability, pharmacokinetics and biodistribution profiles. Here, we define the drug delivery challenges related to oral, intravenous, subcutaneous or alternative routes of administration, and review innovative DDS, marketed or in development, that answer those challenges.