Final Report Summary - POLEXGENE (Biocompatible non-viral polymeric gene delivery systems for the ex vivo treatment of ocular and cardiovascular diseases with high unmet medical need)
The objective of the POLEXGENE project was to develop a non-viral ex vivo gene therapy that will be applied for both ocular and cardiovascular diseases with high unmet medical need. The research of the project focused on improving polymeric DNA-delivery by combining polyplexes with cell penetrating peptides (CPP). To circumvent the toxic and immunogenic problems related to viral vectors, the gene vectors developed in this project will be a non-toxic and non-immunogenic, biodegradable polymeric carrier based on multifunctional poly-a-aminoacids. The potential of the CPP-containing polyplexes regarding their transfection efficiency and the absence of any toxic or immunogenic side effects was evaluated using two gene transfer approaches.
In a first approach, cells were transfected with polyplexes (i.e. polymer-DNA complexes) and then seeded on a polymer membrane prior to implantation. Alternatively, the polymer membrane was surface coated with polyplexes prior to cell seeding and implantation. In order to enhance the internalisation efficiency, the polyplexes were functionalised using CPP including Penetratin and others. In order to improve the membrane-cell interaction and to enhance the cell proliferation and differentiation, the polymer membrane were functionalised with cell interacting peptides (CIP).
A series of water soluble cationic (co)polymers based on poly-a-amino acids were developed and characterised for their chemical structure and molecular weight. The polymers developed contained various side groups including tertiary amines, primary amines, hydroxyl groups, imidazole groups, guanidine functions or combinations thereof. All the polymers developed had in common that they are positively charged and can thus interact with DNA through electrostatic binding, forming polyelectrolyte complexes (PEC).
As second polyplex component, a series of therapeutic plasmids were also developed. The plasmids developed included among other an EBNA1-ORIP plasmid with the SEAP marker under the viral CMV promoter or a human tyrosinase promoter. For therapeutic use, VEGF-R1, VEGF-R2 and VEGF-R3 were cloned into these plasmids. Additionally, these genes have been cloned in replicating Ebstain-Barr virus nuclear antigen (EBNA) plasmids. Finally, cloning of IK-17 or Mertk into the pCDNA3 Plasmid was realised.
The last polyplex constituent which was developed included different types of CPP. As an example, the following CPP were developed and characterised: Penetratin, poly-arginine, TAT and KALA. To test the biological behaviour of the CPP developed, an impedance chip with fluidic system was developed. The chip can be used to study the effects of CPP on single cell level. A similar chip can also be applied to measure the polyplex-cell interactions. Using the materials developed, a variety of techniques were used to study the interaction between DNA and the (co)polymers developed. The DNA condensation capacity was studied using ethidium bromide exclusion tests and agarose gel electrophoresis. The studies indicated that all polymers studied are able to condense DNA. Dynamic light scattering and zeta potential measurements further indicated that the polyplexes formed possess a net positive charge at charge ratios where DNA condensation occurs. In addition, the polyplexes formed are small enough to be taken up through endocytosis. Addition of CPP to pre-formed polyplexes does not alter the stability of the polyplexes. After the polymer development and the physico-chemical evaluation of the polyplexes, a biological evaluation of the materials was performed. This covered a series of transfection studies using a variety of cells: mammalian epithelial cell line (CV1), a human retinal pigment epithelium (RPE) cell line (ARPE19) and primary RPE cells. The final results show that some polymer derivatives developed within the framework of POLEXGENE possess a higher transfection efficiency compared to PEI while the toxicity is lower or similar compared to PEI.
During the first term of the project, the website was developed and set up. The website had two objectives. On the one hand, it was intended to disseminate the project among the scientific community as well as the general public. On the other hand, this web served to exchange information among the partners of the project. After the end of the POLEXGENE project, the website will be kept active for at least one year.
In a first approach, cells were transfected with polyplexes (i.e. polymer-DNA complexes) and then seeded on a polymer membrane prior to implantation. Alternatively, the polymer membrane was surface coated with polyplexes prior to cell seeding and implantation. In order to enhance the internalisation efficiency, the polyplexes were functionalised using CPP including Penetratin and others. In order to improve the membrane-cell interaction and to enhance the cell proliferation and differentiation, the polymer membrane were functionalised with cell interacting peptides (CIP).
A series of water soluble cationic (co)polymers based on poly-a-amino acids were developed and characterised for their chemical structure and molecular weight. The polymers developed contained various side groups including tertiary amines, primary amines, hydroxyl groups, imidazole groups, guanidine functions or combinations thereof. All the polymers developed had in common that they are positively charged and can thus interact with DNA through electrostatic binding, forming polyelectrolyte complexes (PEC).
As second polyplex component, a series of therapeutic plasmids were also developed. The plasmids developed included among other an EBNA1-ORIP plasmid with the SEAP marker under the viral CMV promoter or a human tyrosinase promoter. For therapeutic use, VEGF-R1, VEGF-R2 and VEGF-R3 were cloned into these plasmids. Additionally, these genes have been cloned in replicating Ebstain-Barr virus nuclear antigen (EBNA) plasmids. Finally, cloning of IK-17 or Mertk into the pCDNA3 Plasmid was realised.
The last polyplex constituent which was developed included different types of CPP. As an example, the following CPP were developed and characterised: Penetratin, poly-arginine, TAT and KALA. To test the biological behaviour of the CPP developed, an impedance chip with fluidic system was developed. The chip can be used to study the effects of CPP on single cell level. A similar chip can also be applied to measure the polyplex-cell interactions. Using the materials developed, a variety of techniques were used to study the interaction between DNA and the (co)polymers developed. The DNA condensation capacity was studied using ethidium bromide exclusion tests and agarose gel electrophoresis. The studies indicated that all polymers studied are able to condense DNA. Dynamic light scattering and zeta potential measurements further indicated that the polyplexes formed possess a net positive charge at charge ratios where DNA condensation occurs. In addition, the polyplexes formed are small enough to be taken up through endocytosis. Addition of CPP to pre-formed polyplexes does not alter the stability of the polyplexes. After the polymer development and the physico-chemical evaluation of the polyplexes, a biological evaluation of the materials was performed. This covered a series of transfection studies using a variety of cells: mammalian epithelial cell line (CV1), a human retinal pigment epithelium (RPE) cell line (ARPE19) and primary RPE cells. The final results show that some polymer derivatives developed within the framework of POLEXGENE possess a higher transfection efficiency compared to PEI while the toxicity is lower or similar compared to PEI.
During the first term of the project, the website was developed and set up. The website had two objectives. On the one hand, it was intended to disseminate the project among the scientific community as well as the general public. On the other hand, this web served to exchange information among the partners of the project. After the end of the POLEXGENE project, the website will be kept active for at least one year.
