Author(s): Sousa, Ângela Maria Almeida de
Date: 2011
Persistent ID: http://hdl.handle.net/10400.6/2774
Origin: uBibliorum
Project/scholarship: info:eu-repo/grantAgreement/FCT/FARH/SFRH%2FBD%2F41390%2F2007/PT;
Subject(s): Eficiência de transfecção; DNA plasmídico
The discovery of disease-related genes and the possibility to manipulate the gene set-up in some organisms has fostered the development of innovative human DNA therapeutics over the last years. Although viral vectors are used in the majority of the trials, non-viral vectors, particularly plasmid DNA (pDNA) vectors, are attracting considerable attention as biotherapeutics both in gene therapy or DNA vaccination, due to their lower immunogenicity, toxicity and also the economic, safer and easier manufacture. Nevertheless, it is well known that the success of gene transfer to cells and subsequent expression is strictly affected by the pDNA manufacturing process. The use of pDNA-based therapeutics relies on procedures that efficiently purify the most biologically active and effective topology, the supercoiled (sc) plasmid conformation. However, chromatographic purification of sc pDNA has specific problems which are mostly related to the structural nature of this biomolecule, the resemblance between pDNA topologies and also with some host impurities, as well as the lack of capacity and selectivity of the traditional bead adsorbents. Recently, sc pDNA purification strategies that use amino acids as immobilized ligands have yielded interesting results. Thus, the present work intends to explore and understand the underlying interactions responsible for the biorecognition of sc conformation by the amino acids ligands already used for pDNA purification as well as to establish the elution conditions that favor the prevalence of some interactions on other. By performing some fundamental studies with oligonucleotides it was observed the involvement of several elementary interactions with the amino acids matrices studied, such as hydrophobic, ring-stacking, cation-π, water-mediated bonds, multiple hydrogen bonds, van der Waals and electrostatic interactions. Although hydrophobic interactions easily appear with histidine matrix or ionic interactions with arginine matrix, it was verified the presence of other interactions in function of the elution conditions used. These results were useful for the implementation of a new affinity chromatographic strategy with the amino acid lysine for efficiently and selectively purify the sc pDNA isoform. Lysineaffinity matrix allowed the elimination of the E. coli impurities as well as other ineffective plasmid isoforms present in a complex clarified lysate meeting all the regulatory requirements. The preferential retention of the nucleic acids with higher bases exposure indicates that this matrix can be more suitable for RNA purification. In accordance with the traditional supports limitations, the alternative monolithic chromatography revealed satisfactory affinity properties with excellent mass transfer and capacity characteritics, allowing a fast and efficient plasmid isoforms separation without flow rate dependence. The similar elution conditions with histidine-agarose and the presence of canbonyldiimidazole groups suggested that the imidazole ring present in this monolithic disk is the major responsible in the specific biorecognition of sc pDNA isoform. Integration of monolithic chromatography in the pDNA manufacturing process also increase the global yield of pDNA xviiirecovery for 89%, with a purity degree according to the recommendations of the regulatory agencies that was reflected in the high transfection efficiency of sc pDNA sample on eukaryotic cells (59% in COS-7 cells). Overall, this doctoral research work revealed that amino acid-based affinity chromatography is a powerful and versatile methodology for nucleic acids purification, mainly the sc pDNA topology, guaranteeing suitable purity degree for DNA-based therapies. Besides the selectivity and specificity obtained with amino acids affinity ligands, the application of the innovative monolithic technology in the pDNA purification field brought a great improvement of the speed, resolution and capacity to the chromatographic performance, being a promising association for plasmid purification technology. Hence, this work provided valuable and helpful information concerning amino acid-affinity chromatography and chromatographic supports that can be useful in the future pDNA bioseparation either for preparative and analytical processes.
Work financed by the Portuguese Foundation for Science and Technology (SFRH/BD/41390/2007) under the programe QREN - POPH - Type 4.1 – Advanced Training, cofunded by the European Social Fund and by national funds from the MCTES.
