**7. Summary**

448 Non-Viral Gene Therapy

Lastly, the route of transmission and target gene delivery are the major factors which

Table 4 summarises some attempts to modify the chitosan-nucleic acid complexes for target

The transfection efficiency was higher than PEI when transfected in KB cell line, which over expressed the folate receptor (FR) in presence of 10% foetal bovine serum

(FBS).

for broad applications in human disease.

RGD-CH-NP is a novel and highly selective delivery system for siRNA with the potential

Targeted antisense agent would be a potential approach to overcome tumour drug resistance.

Transduction efficiency of Ad/chitosan-PEG-FA was 57% higher than Ad/chitosan. This system aims for development of

systemic

lesion.

administration of the vectors to target

complexes Conclusion Study design Reference

*In vitro* (KB cell line)

Orthotopic mouse models of ovarian carcinoma

*In vitro* (KB cell line)

*In vitro*  (KB cell line) Morris & Sharma, 2010

Han et al*.*, 2010

Wang et al., 2010

Park et al., 2010

contribute to the success in gene therapy, which still requires further investigation.

gene therapy.

Folate mediated targeting induced by conjugating poly(ethylene glycol)-folate (PEG-FA) with arginine modified

chitosan

Arg-Gly-Asp (RGD) peptidelabelled chitosan nanoparticle (RGD-CH-NP) as a novel tumour targeted delivery system for short interfering RNA (siRNA).

Antisense oligodeoxynucleotides (asODN), using folic acid (FA) conjugated hydroxypropylchitosan

Tumour- of adenoviral complexes targeting of Adenovirus (Ad)/chitosan-PEG-FA nanocomplexes formed by electrospining

Modification Chitosan

PEG-FAchitosan-DNA

RGD-CH— SiRNA nanoparticles

FA-ChtosanasODN nanoparticles

Ad/chitosan-PEG-FA nanocomplexes

Table 4. Some modifications of chitosan for target gene therapy

An efficient gene delivery system is very important for gene therapy. Currently, the most efficient of these systems is a viral vector, which usually yields a transfection efficiency of more than 90%. However, by using a viral vector for gene therapy, there is a concern about the host versus vector immunological response, mutation and oncogenic effects; hence the need to develop a non-viral vector. There are many non-viral vectors; the high efficient one is cationic lipid, which gives high transfection efficiency, especially in tissue culture or *in vitro* conditions. *In vivo*, the intravenous administration of cationic lipid/DNA complexes presented significant problems, as these reagents can be quite toxic. PEI is another non-viral transfection material that has been used for some time, but due to its toxicity and the variable results, it has not been widely accepted.

Chitosan (poly[β-(1-4)-2-amino-2deoxy-D-glucopyranose]), a nontoxic biodegradable biopolymer, has been broadly studied as a promising non-viral vector for gene delivery. This cationic polysaccharide has been produced by partial deacetylation of chitin, a naturally polymer from crustacean shells. However, the transfection efficiency of chitosan itself is not efficient enough and depends on many factors such as Mw, DDA, DNA complexes charge ratio, pH and particle sizes, as well as the type of cells. There have been many attempts to modify chitosan in order to improve transfection efficiency. Some studies have revealed that low Mw chitosan, especially the product of oxidative depolymerisation from higher Mw chitosan with NaNO2, had low cytotoxicity and improved solubility properties, as well as having potential for gene delivery both *in vitro* and *in vivo*. However, some studies have reported decreased transfection efficiency with lower Mw chitosan.

There have been other attempts modifying the chemical structure of chitosan. These have included introducing a hydrophilic group, such as coupling dextran, as well as incorporating poly (vinyl pyrolidone) into the galactosylated chitosan, which can reduce the aggregation of particles and increase transfection efficiency. Some studies have also using hydrophobic modification of chitosan, such as deoxycholic acid-modified chitosan, in order to increase transfection efficiency through enhancement of complex interaction with cells and cellular uptake of the particles. Chitosan can be modified by conjugation of chitosan-DNA complexes with ligands to target specific cell surface receptors, but these attempts have had variable results. Many factors may have to be considered for improving transfection efficiency of chitosan, not just ligand binding, but also the method of binding or conjugation, the size and morphology of the particles, the aggregation of the complexes, and especially the chitosan itself, as Mw, DDA and charge ratio may have to be adjusted.

The design criteria of the effective vector for non-viral gene therapy should also consider cost–effectiveness in synthesis and purification steps, serum stability and efficient packaging of large amount of the vector-nucleic acid complexes. Moreover, the route of administration of this vector to the target cells or tumour lesion, high transfection efficiency, specific target gene delivery should also be considered. Once the complexes enter the target cells, they have to escape from enzyme degradation. The complexes then release the therapeutic gene/ nucleic acid to the target organelle, such as DNA, which has to enter the nucleus, while siRNA functions in cytoplasm. This release has to occur without too many difficulties, which means that the bonding between the vectors and nucleic acid should not be too strong. Most importantly, these non-viral vectors have to be

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## **8. Acknowledgment**

The authors wish to acknowledge the support of the World Safety Organization Collaborating Centre within the Anton Breinl Centre for Public Health and Tropical Medicine, James Cook University, Townsville, Queensland, Australia, for their kind support of this project.
