**7. Conclusion**

the ability to target, the developed multifunctional vector was able to disrupt endosomal membranes, reach the nucleus by utilizing microtubules, and transfect efficiently while showing no detectable toxicity. McCarthy et al. presented similar results using a DBV for the

Canine et al. described a biopolymer termed FP–(DCE)*n*–NLS–CS–TM that contains repeating units of arginine and histidine to condense pDNA and lyse endosome membranes (DCE), a HER-2 targeting affibody to target cancer cells (TM), a pH responsive fusogenic peptide (FP) H5WYG to destabilize endosome membranes and enhance endosomolytic activity of histidine residues, and a nuclear localization signal (NLS) M9 to enhance translocation of pDNA towards the cell nucleus. A cathepsin D enzyme substrate (CS) was also engineered in between targeting motif and NLS to facilitate dissociation of the targeting motif from the biopolymer inside late endosomes where cathepsin D is abundant [216]. The authors demonstrated the functioning of each motif in the polymer resulting in successful transfection of SKOV-3 and

The production of these recombinant vectors renders it relatively easy to change their charac‐ teristics by sequence modification. Canine et al. further demonstrated that by modifying the sequence of the biopolymer FP–(DCE)*n*–NLS–CS–TM, it was possible to fine tune the vector for either delivery of plasmid DNA to the nucleus or delivery of siRNA to the cytoplasm [217]. It was reported that inclusion of the M9 NLS rendered the biopolymer (FP–(DCE)*n*–NLS–CS– TM) suitable for delivery of plasmid DNA to the nucleus but not for delivery of siRNA. However, exclusion of the NLS from the biopolymer (FP– (DCE)*n*–CS–TM) rendered it more suitable for delivery of siRNA to the cytoplasm but not for nuclear delivery of plasmid DNA in SKOV-3 human ovarian cancer cells. This study demonstrates the possibility of not only targeting specific cells for gene delivery, but also the ability to target intracellular compart‐

Wang et al. presented a novel recombinant protein tetra-H2A (TH) derived from histone H2A that was developed to replace protamine as a conditionally reversible, nucleic acid condensing agent. The recombinant protein comprised of four tandem repeats of human histone H2A peptide, interspersed with cathepsin D cleavage sites and a pH-responsive fusogenic peptide GALA to facilitate the endosome escape of the cargo. The recombinant protein, tetra-H2A (TH), was able to condense siRNA into a stable complex that was in turn coated in a cationic lipid with a high degree of PEGylation, forming Lipid-tetra-H2A-Hyaluronic acid (LHH) nanopar‐ ticles [218]. This design was developed in order to mimic lipid-enveloped viruses to replicate the transfection abilities of viruses in vivo. The histone-containing polymer demonstrated an enhanced intracellular release of the cargo and an increased anti-luciferase siRNA silencing efficiency in vitro compared with the protamine-containing polymer in H460-luc human lung carcinoma cells. Furthermore, in vivo gene silencing by tumour-targeted anti-luciferase siRNA was evaluated in H460-luc xenograft-bearing mice with the histone-containing nanoparticles loaded with anti-luciferase siRNA resulting in ~66% silencing of luciferase expression, significantly higher than that of the protamine-mediated knockdown (34%). This study demonstrates the importance of efficient release of the genetic payload for efficient gene therapy; through optimisation of each component of the multifunctional vector, it may be

delivery of iNOS gene therapy targeted to breast cancer [215].

ments depending on the nature of the therapeutic to be delivered.

GFP transgene expression.

88 Gene Therapy - Principles and Challenges

There is a huge potential for cancer gene therapy, which, in some respect, has yet to be realised. However, the lack of gene therapy products making it onto the market is disappointing when one considers the number of research groups involved and clinical trials underway. It seems that until a suitable delivery system for cancer gene therapy becomes available, the potential of this field will never be reached. The difficulty in this is the number of hurdles to overcome and the extremely high standards of safety and efficiency expected by regulatory authorities. Nevertheless, the development of multifunctional systems and a move towards the creation of artificial viruses may finally help cancer gene therapy to progress to a viable and successful cancer treatment.
