Bitoque, Diogo

Oliveira, Ana

Silva, Gabriela A.

Molecular Therapy

English

Sapientia

 Molecular Therapy Volume 20, Supplement 1, May 2012  Copyright © The American Society of  Gene & Cell TherapyS160CHEMICAL AND MOLECULAR CONJUGATES Isimultaneously. Thus, arsonium-containing cationic lipids appear as promising versatile multi-faceted agents that could fi nd various applications for the treatment of inherited as well as acquired diseases.410. pDMAEMA-Based Polyplexes as Vectors for Retinal Gene TherapyDiogo B. Bitoque,1,2 Andreia P. Silva,1 Margarita D. Romero,3 Ana V. Oliveira,1,2,5 Eduardo M. Lopes,1,2 Ana M. Costa,4 Gabriela A. Silva.1,21Centre for Molecular and Structural Biomedicine/Institute for Biotechnology and Bioengineering (CBME/IBB)), University of Algarve, Faro, Portugal; 2Department of Biomedical Sciences and Medicine, University of Algarve, Faro, Portugal; 3Department of Cell Therapy and Regenerative Medicine, Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Sevilla, Spain; 4Department of Chemistry and Pharmacy, University of Algarve, Faro, Portugal; 5PhD Program in Biomedical Sciences, Department of Biomedical Sciences and Medicine, University of Algarve, Faro, Portugal.Methacrylate polymers have been described as highly biocompatible and are successfully used in medical applications1. Due to their cationic nature, these polymers can be used to form polyplexes with DNA for its delivery. This work aims to understand how feasible pDMAEMA (poly(2-(N,N’-dimethylamino)ethyl methacrylate)) is as a non viral gene delivery system to the retina. pDMAEMA-DNA polyplexes were prepared in PBS at 5, 7.5, 10, 12.5 and 20 nitrogen/phosphorous (N/P) ratio. Dynamic light scattering and zeta potential measurements confi rmed positively charged nanosized polyplexes for all ratios. pDMAEMA was able to effi ciently complex and protect DNA from DNase I degradation. The cytotoxicity was evaluated using two retinal pigment epithelium (RPE) cell lines: ARPE-19 and D407 and the HEK293 cell line (control). We have found that cytotoxicity of the uncomplexed polymer is concentration and time dependent, as expected, and negligible for the pDMAEMA-DNA polyplexes. Furthermore, for the concentrations to be used in vivo, the cytotoxicity was negligible, as shown by the absence of infl ammation upon injection in the subretinal space of C57Bl6 mice. Transfection efficiency, as evaluated by fluorescence microscopy and flow cytometry, showed that the retinal cell lines were in fact transfected by these polyplexes, with the effi ciency varying according to the ratio of N:P. These results suggest that pDMAEMA is a feasible candidate for non-viral gene delivery to the retina. 1. Ji Hoon Jeong, Sung Wan Kim, Tae Gwan Park (2007). Molecular design of functional polymers for gene therapy. Progress in Polymer Science, Volume 32, Issue 11: 1239 – 1274. Acknowledgements IBB/CBME, LA, FEDER/POCI 2010; Fundação para a Ciência e Tecnologia (PTDC-SAU-BEB-2008 to G.A.Silva, SFRH/BD/70318/2010 to A.V.Oliveira) and Marie Curie Reintegration Grant (PIRG-GA-2009-249314) under the FP7 program.411. Chitosan-Hyaluronic Acid Hybrid Vectors for Retinal Gene TherapyAna V. Oliveira,1,2,3 Diogo M. Bitoque,1,3 Gabriela A. Silva.1,31Centre for Molecular and Structural Biomedicine (CBME/IBB, LA), University of Algarve, Faro, Portugal; 2PhD Program in Biomedical Sciences, Department of Biomedical Sciences and Medicine, University of Algarve, Faro, Portugal; 3Department of Biomedical Sciences and Medicine, University of Algarve, Faro, Portugal.Our goal is to develop chitosan-based non-viral vectors optimized for ocular gene therapy. Therefore, chitosan-DNA polyplexes were prepared using a NH3:PO4 ratio of 5:1. These polyplexes were characterized regarding their morphology, stability and transfection effi ciency on retinal pigment epithelial cells. The method to produce the polyplexes proved to be reproducible, consistently yielding polyplexes with a spherical morphology and sizes below 500 nm. These polyplexes have long-term stability and are capable of effi ciently protecting DNA from DNase I degradation. However, transfection effi ciency, evaluated by fl uorescence microscopy and fl ow cytometry, showed these polyplexes to fall below commercial transfection reagents. The lower transfection effi ciency observed for chitosan-DNA polyplexes is most likely due to reduced cell penetration and defi cient plasmid release. Our endeavor to improve this lower transfection effi ciency encompassed the preparation of polyplexes with different NH3:PO4 ratios and combining these with hyaluronic acid. The results for these hybrid polyplexes show a marked increase in the transfection effi ciency. In addition, we are studying the effect of chemical modifi cation of chitosan to promote timely and effi cient DNA release. Overall, our hybrid systems show an improvement of the effi ciency of chitosan-based nonviral vectors for gene therapy. Acknowledgements: IBB/CBME, LA, FEDER/POCI 2010; Fundação para a Ciência e Tecnologia (PTDC-SAU-BEB-2008 to G.A.Silva, SFRH/BD/70318/2010 to A.V.Oliveira) and Marie Curie Reintegration Grant (PIRG-GA-2009-249314) under the FP7 program.412. Novel Nanoparticles To Encapsulate microRNA for the Treatment of StrokeLi-Fang Wang,1 Jenn-Shing Chen,2 Yu-Lun Lo.11Kaohsiung Medical University, Kaohsiung, Taiwan; 2National University of Kaohsiung, Kaohsiung, Taiwan.microRNAs cannot be directly applied for clinical use in that they can be rapidly degraded and their cellular internalization is poor due to its negative surface charge. A variety of delivery systems, either viral or non-viral, have been investigated for microRNA delivery. Non-viral vectors have received growing interests due to many advantages such as they offer over the viral counterparts including improved safety, low immune responses, enabling repeated uses, and ease of production. In this report, an anionic natural polysaccharide-chondroitin sulphate (CS) was chemically linked to polyethylenimine (PEI) with a lower molecular weight of 10 kDa to minimize its cytotoxicity as a microRNA carrier for the treatment of ischemia stroke. It is noted the cytotoxicity of PEI reduced dramatically after modifi cation as compared to the original PEI of molecular weight of 10K and 25K Da. The TEM image of CS-PEI and miRNA shows a spherical structure with a uniform size distribution. The size falls in a perfect range (about 100 nm), larger enough to escape the renal fi ltration (< 10 nm) but smaller enough to prevent uptake by the reticuloendothelial system (> 200 nm).

Chitosan-Hyaluronic acid hybrid vectors for retinal gene therapy

Bitoque, Diogo M.

Universidade do Algarve

https://sapientia.ualg.pt/bitstream/10400.1/11714/1/H11714.pdf

Chitosan-Hyaluronic acid hybrid vectors for retinal gene therapy

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