**9. Potential application of chitosan-DNA/siRNA nanoparticles**

Gene therapy offers new possibilities for the clinical management of different disease conditions that are difficult to treat by traditional surgical or medical means. In the last decade, extensive improvements have been made to optimize gene therapy and have been tested on several disease conditions. The success of chitosan-DNA nanoparticles for delivery plasmid DNA to mucosal surfaces such as the oral and nasal mucosa has already shown (Bivas-Benita et al., 2003; Chen et al., 2004; Khatri et al., 2008). Oral delivery is most attractive due to easy administration. The oral delivery of peptide, protein, vaccine and nucleic acid-based biotechnology products is the greatest challenge facing the drug delivery industry. Mice were fed with plasmid pCMVβ (containing LacZ gene), whether it was wrapped by chitosan or no. The study demonstrated that oral chitosan-DNA nanoparticles can efficiently deliver genes to enterocytes, and may be used as a useful tool for gene transfer (Chen et al., 2004). Hepatitis B virus infection is a major global health concern and is the most common cause of chronic liver disease, new generation of HBV vaccines are urgently needed in order to overcome problems encountered with the immunization of immunocompromised people and more importantly with the potential of using active immunotherapy in treating chronic patients. DNA vaccines have the potential to eliminate many of the limitations of current vaccine technologies. Chitosan nanoparticles loaded with

Chitosan-DNA/siRNA Nanoparticles for Gene Therapy 469

Clinical trials on gene therapy are limited to naked DNA or plasmid DNAs/siRNA delivered by viral vectors. Among non-viral vectors for DNA and siRNA delivery, chitosan and its derivatives are promising alternatives to viral vectors for targeting DNA and siRNA to specific cells. Chitosan has once been considered as an attractive gene transfer candidate for its superior biocompatibility, superior biodegradability and low cell toxicity, but the low stability, low buffering capacity and low cell-specificity have also hindered its clinical applications. To date, however, no clinical trials of chitosan-DNA or siRNA therapy have been performed. Chitosan-based gene therapy remains in the experimental stage due to low transfection efficacy. Many key challenges were involved in DNA and siRNA delivery to targeted cells using chitosan-based carriers. As a nature resource-based polysaccharide, chitosan has more functional groups that can be chemically modified than do the other cationic polymers, thus has many more potential chemical derivatives to make up these deficiencies. Parameters are critical to achieve favourable transfection efficiency and include degree of deacetylation, molecular weight, pH and N/P ratio. For example, a low molecular weight, high degree of deacetylation, small particle size and a moderate, positive, surface zeta potential along with a high N/P ration are advantageous to achieve high siRNA transfection efficiency. Recent technological advances in the chemical modification of chitosan have instituted improvements of its transfection efficiency without disturbing its

Our work on gene coding for IL-lRa in dogs (Pelletier et al., 1997) and rabbits (Fernandes et al., 1999) was our previous study with the protein itself. We have improved a non-viral intraarticular transfection technique using lipofection and have tested it in osteoarhtritis animal models. These were the very first published articles in the literature that demonstrated the efficacy of gene therapy in osteoarthritis models *in vivo*. Our recent work on polymeric nanoparticles has led us to develop a much safer and effective system for in vitro transfection of embryonic kidney cells, as well as adult mesenchymal stem cells (Corsi et al., 2003). This new system has been successfully tested in muscle and skin tissues *in vivo* in mice and holds great promise for future application on the field of gene therapy and tissue engineering. We developed a second-generation nanovector by successfully coupling folic acid to the polymer (Mansouri et al., 2006). One strategy for improving transfection is to take advantage of the mechanism of folate-mediated uptake by cells to promote targeting and internalization, hence improving transfection efficiency. Folate-mediated transfection has been shown to facilitate DNA internalization into cells via membrane receptors both *in vitro* and *in vivo* (Sudimack & Lee, 2000). Expression of folate receptor (FR)-β in synovial mononuclear cells and CD14+ cells from patients with RA was described by 1999 (Nakashima-Matsushita et al., 1999). Articular macrophages isolated from rats with adjuvant-induced arthritis overexpress FRs and exhibit significantly higher binding capacity for folate conjugates than macrophages obtained from healthy rats (Turk et al., 2002). The wide distribution of FRs at the surface of activated macrophages in rheumatoid arthritis allows the use of folate as potential ligand for folate-targeted chitosan gene therapy. Our laboratory demonstrated that folate-chitosan DNA nanoparticle containing IL-1Ra has been shown to play a role to prevent abnormal osteoblast metabolism and bone damage in this adjuvant-induced arthritis model (Fernandes et al., 2008). It also allows a significant decrease of the inflammation in the rats' paw compared to untreated rats, proving indirectly the efficacy of the IL-1Ra protein treatment. Various inflammation markers (IL-1β and PGE2)

**10. Conclusion** 

biocompatibility and biodegradability.

plasmid DNA encoding surface protein of Hepatitis B virus. Nasal administration of such nanoparticles resulted in serum anti-HBsAg titre that was less compared to that elicited by naked DNA and alum adsorbed HBsAg, but the mice were seroprotective within 2 weeks and the immunoglobulin level was above the clinically protective level (Khatri et al., 2008). Particulate mucosal delivery systems that encapsulate protein or plasmid DNA encoding antigens have been widely explored for their ability to induce an immune response. Oral delivery of vaccines using chitosan as a carrier material appears to be beneficial for inducing an immune response against *Toxoplasma gondii*. Chitosan microparticles as carriers for GRA-1 protein vaccine were prepared. It was shown that priming with secreted dense granule protein 1 (GRA1) protein vaccine loaded chitosan particles and boosting with GRA1 pDNA vaccine resulted in high anti-GRA1 antibodies, characterized by a mixed IgG2a/IgG1 ratio (Bivas-Benita et al., 2003). The application of chitosan-based delivery system as ocular gene carriers, there is evidence of their ability to transfect the ocular cells in vitro. This capacity of chitosan nanoparticles to transfect the cells, was found to be highly dependent on the molecular weight of chitosan. Only chitosan of low molecular weight (10-12 kDa) was able to transfect plasmid DNA in both cell lines derived from the human cornea and the conjunctives (De la Fuente et al., 2008). In Utero delivery of chitosan-DNA results in postnatal gene expression, and shows promise for non-viral gene transfer in animal models of fetal gene therapy (Yang et al., 2010). The intravenous and intratracheal solutions and the intratracheal powder of pCMV-Muβ encoding murine interferon-β were administered the day after the inoculation of mice with CT26 cells. Lung weight and the number of pulmonary nodules at day 21 were significantly suppressed by the three formulations at a dose of 10 μg (N/P = 5). Reducing the dose to 1 μg resulted in a loss of effect by the intravenous solution (Okamoto et al., 2010). These findings showed that therapeutic gene powders are promising for gene therapy to treat lung cancer or metastasis.

siRNA gene therapy research has focused on several types of viral vectors: adeno-associated viruses (AAV), adenoviruses, retroviruses, lentiviruses, and herpes simplex viruses. siRNA therapeutics have been assessed in numerous diseases, including genetic and viral diseases, cancer, as well as non-lethal disorders, such as arthritis and osteoporosis. Among these viral vectors, lentiviruses have progressed to clinical trials on metastatic melanoma and HIV infection (Baker, 2010a; 2010b). siRNA-based gene therapy has already been tested in clinical trials dealing with the treatment of age-related macular degeneration, viral infection, skin disorders and cancer. Cancer treatment is by the most important proposed application of gene therapy and many clinical trials using gene therapy are under investigation. Non-viral vectors including chitosan derivatives have been used in animal model, but clinical trials are lagging due to low transfection efficiency. Anderson et al. (Andersen et al., 2008) demonstrated that silencing of pro-inflammatory TNFα in the RAW 246.7 murine macrophage cell line was achieved by using lyophilized chitosan/siRNA. Compared to research in vitro with chitosan-based systems, in vivo research is still in the developmental stage. Only a few studies are avaible which *in vivo* demonstration of chitosan/siRNA nanocomplexes in silencing gene expression in animals. Howard et al. (Howard et al., 2009) demonstrated that chitosan nanopaticles contains an anti-TNFα siRNA knock downed efficiently of TNFα expression in primary peritoneal macrophages in vitro. Downregulation of TNFα-induced inflammatory responses arrested systemic and local inflammation in collagen-induced arthritic mice after intraperitoneal injection of chitosan/anti-TNFα siRNA nanoparticles, thereby presenting a novel strategy for arthritis treatment.

### **10. Conclusion**

468 Non-Viral Gene Therapy

plasmid DNA encoding surface protein of Hepatitis B virus. Nasal administration of such nanoparticles resulted in serum anti-HBsAg titre that was less compared to that elicited by naked DNA and alum adsorbed HBsAg, but the mice were seroprotective within 2 weeks and the immunoglobulin level was above the clinically protective level (Khatri et al., 2008). Particulate mucosal delivery systems that encapsulate protein or plasmid DNA encoding antigens have been widely explored for their ability to induce an immune response. Oral delivery of vaccines using chitosan as a carrier material appears to be beneficial for inducing an immune response against *Toxoplasma gondii*. Chitosan microparticles as carriers for GRA-1 protein vaccine were prepared. It was shown that priming with secreted dense granule protein 1 (GRA1) protein vaccine loaded chitosan particles and boosting with GRA1 pDNA vaccine resulted in high anti-GRA1 antibodies, characterized by a mixed IgG2a/IgG1 ratio (Bivas-Benita et al., 2003). The application of chitosan-based delivery system as ocular gene carriers, there is evidence of their ability to transfect the ocular cells in vitro. This capacity of chitosan nanoparticles to transfect the cells, was found to be highly dependent on the molecular weight of chitosan. Only chitosan of low molecular weight (10-12 kDa) was able to transfect plasmid DNA in both cell lines derived from the human cornea and the conjunctives (De la Fuente et al., 2008). In Utero delivery of chitosan-DNA results in postnatal gene expression, and shows promise for non-viral gene transfer in animal models of fetal gene therapy (Yang et al., 2010). The intravenous and intratracheal solutions and the intratracheal powder of pCMV-Muβ encoding murine interferon-β were administered the day after the inoculation of mice with CT26 cells. Lung weight and the number of pulmonary nodules at day 21 were significantly suppressed by the three formulations at a dose of 10 μg (N/P = 5). Reducing the dose to 1 μg resulted in a loss of effect by the intravenous solution (Okamoto et al., 2010). These findings showed that therapeutic gene

powders are promising for gene therapy to treat lung cancer or metastasis.

nanoparticles, thereby presenting a novel strategy for arthritis treatment.

siRNA gene therapy research has focused on several types of viral vectors: adeno-associated viruses (AAV), adenoviruses, retroviruses, lentiviruses, and herpes simplex viruses. siRNA therapeutics have been assessed in numerous diseases, including genetic and viral diseases, cancer, as well as non-lethal disorders, such as arthritis and osteoporosis. Among these viral vectors, lentiviruses have progressed to clinical trials on metastatic melanoma and HIV infection (Baker, 2010a; 2010b). siRNA-based gene therapy has already been tested in clinical trials dealing with the treatment of age-related macular degeneration, viral infection, skin disorders and cancer. Cancer treatment is by the most important proposed application of gene therapy and many clinical trials using gene therapy are under investigation. Non-viral vectors including chitosan derivatives have been used in animal model, but clinical trials are lagging due to low transfection efficiency. Anderson et al. (Andersen et al., 2008) demonstrated that silencing of pro-inflammatory TNFα in the RAW 246.7 murine macrophage cell line was achieved by using lyophilized chitosan/siRNA. Compared to research in vitro with chitosan-based systems, in vivo research is still in the developmental stage. Only a few studies are avaible which *in vivo* demonstration of chitosan/siRNA nanocomplexes in silencing gene expression in animals. Howard et al. (Howard et al., 2009) demonstrated that chitosan nanopaticles contains an anti-TNFα siRNA knock downed efficiently of TNFα expression in primary peritoneal macrophages in vitro. Downregulation of TNFα-induced inflammatory responses arrested systemic and local inflammation in collagen-induced arthritic mice after intraperitoneal injection of chitosan/anti-TNFα siRNA Clinical trials on gene therapy are limited to naked DNA or plasmid DNAs/siRNA delivered by viral vectors. Among non-viral vectors for DNA and siRNA delivery, chitosan and its derivatives are promising alternatives to viral vectors for targeting DNA and siRNA to specific cells. Chitosan has once been considered as an attractive gene transfer candidate for its superior biocompatibility, superior biodegradability and low cell toxicity, but the low stability, low buffering capacity and low cell-specificity have also hindered its clinical applications. To date, however, no clinical trials of chitosan-DNA or siRNA therapy have been performed. Chitosan-based gene therapy remains in the experimental stage due to low transfection efficacy. Many key challenges were involved in DNA and siRNA delivery to targeted cells using chitosan-based carriers. As a nature resource-based polysaccharide, chitosan has more functional groups that can be chemically modified than do the other cationic polymers, thus has many more potential chemical derivatives to make up these deficiencies. Parameters are critical to achieve favourable transfection efficiency and include degree of deacetylation, molecular weight, pH and N/P ratio. For example, a low molecular weight, high degree of deacetylation, small particle size and a moderate, positive, surface zeta potential along with a high N/P ration are advantageous to achieve high siRNA transfection efficiency. Recent technological advances in the chemical modification of chitosan have instituted improvements of its transfection efficiency without disturbing its biocompatibility and biodegradability.

Our work on gene coding for IL-lRa in dogs (Pelletier et al., 1997) and rabbits (Fernandes et al., 1999) was our previous study with the protein itself. We have improved a non-viral intraarticular transfection technique using lipofection and have tested it in osteoarhtritis animal models. These were the very first published articles in the literature that demonstrated the efficacy of gene therapy in osteoarthritis models *in vivo*. Our recent work on polymeric nanoparticles has led us to develop a much safer and effective system for in vitro transfection of embryonic kidney cells, as well as adult mesenchymal stem cells (Corsi et al., 2003). This new system has been successfully tested in muscle and skin tissues *in vivo* in mice and holds great promise for future application on the field of gene therapy and tissue engineering. We developed a second-generation nanovector by successfully coupling folic acid to the polymer (Mansouri et al., 2006). One strategy for improving transfection is to take advantage of the mechanism of folate-mediated uptake by cells to promote targeting and internalization, hence improving transfection efficiency. Folate-mediated transfection has been shown to facilitate DNA internalization into cells via membrane receptors both *in vitro* and *in vivo* (Sudimack & Lee, 2000). Expression of folate receptor (FR)-β in synovial mononuclear cells and CD14+ cells from patients with RA was described by 1999 (Nakashima-Matsushita et al., 1999). Articular macrophages isolated from rats with adjuvant-induced arthritis overexpress FRs and exhibit significantly higher binding capacity for folate conjugates than macrophages obtained from healthy rats (Turk et al., 2002). The wide distribution of FRs at the surface of activated macrophages in rheumatoid arthritis allows the use of folate as potential ligand for folate-targeted chitosan gene therapy. Our laboratory demonstrated that folate-chitosan DNA nanoparticle containing IL-1Ra has been shown to play a role to prevent abnormal osteoblast metabolism and bone damage in this adjuvant-induced arthritis model (Fernandes et al., 2008). It also allows a significant decrease of the inflammation in the rats' paw compared to untreated rats, proving indirectly the efficacy of the IL-1Ra protein treatment. Various inflammation markers (IL-1β and PGE2)

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showed a significant decrease in muscle and serum after the injection of the IL-1Ra protein demonstrating by direct evidence the efficacy of the administration technique to deliver efficient nanoparticles. Therefore, we have already shown it is possible to do gene therapy with IL-1Ra to decrease arthritis and have a positive effect on inflammation.

### **11. Acknowledgments**

This work was supported by the grants from National Natural Science Foundation of China (No. 30811120440 and 30911120261), The Ministry of Science and Technology of China (No. S2011GR0323), Shanghai International Collaboration Foundation (No. 08410701800) and Canadian Institutes of Health Research (CCI-92212, CCL-99636 and CCM 104888). Dr Fernandes and Dr Benderdour are research scholars of *Fonds de la Recherche en santé du Québec* (FRSQ). Dr Tiera holds a post-PH D scholarship from UNESP - Brazil.
