Abstract
Gene therapy can be defined as the introduction of exogenous nucleic acids into cells with the intention of altering gene expression to prevent, halt or reverse a pathological process. It forms an attractive approach for therapeutic intervention of a wide range of diseases, including genetic diseases, metabolic disorders, infectious diseases,
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chronic illnesses, cardiovascular diseases and cancer. In order for gene-based therapeutics to become effective, the therapeutic nucleic acids must be delivered into target cells and have to reach their site of action within the cell. However, due to the high charge density and large molecular weight, nucleic acids are generally impermeable to cellular membranes and require assistance in order to reach their target site. To facilitate the uptake by target cells and delivery of nucleic acids at their target site, a sophisticated delivery system is required which must be capable of targeting the diseased cell, facilitate uptake and intracellular trafficking of the nucleic acid cargo to their site-of-action. Gene delivery systems can be divided into two broad classes: viral vectors and non-viral (synthetic) vectors. Viral delivery systems are derived from viruses, whereas non-viral systems are based on macromolecular complexes. Viruses are highly complex and are adepted to infecti cells and deliver their RNA/DNA cargo. The transfection efficiencies of viral vectors remain unprecedented and outperform their non-viral counterparts. However, viral vectors have several drawbacks, including their immunotoxicity and the chances for insertional mutagenesis. Subsequently, non-viral delivery systems have emerged as potential alternatives to viral vectors. In general, non-viral vectors lack the major safety issues associated with their viral counterparts. However, compared to viral vectors, the gene delivery efficiency of non-viral vectors is poor. Peptide-based gene delivery systems may offer a versatile platform for efficient gene delivery. Peptides are biodegradable, biocompatible and various peptides have been identified that can perform several basic functions for gene delivery, such as DNA condensation or membrane disruption. By assembling different functional peptides required for effective gene delivery into a single-chain, the ideal gene delivery system can be created, thereby eliminating compositional variations, facilitate pharmaceutical formulation, and achieve reproducibility at the molecular level. The aim of this thesis is to set up a high-throughput screening method to select out of a large library of multimodular peptides those candidates that are able to efficiently deliver therapeutic nucleic acids into target cells at their site of action. To achieve this, we propose a design strategy that follows a random, integrative approach selecting multimodular peptides containing combinations of functional traits that are optimal for efficient gene transfer. By randomly combining peptides with properties needed for gene delivery (e.g. DNA condensing peptides and membrane disrupting peptides), a combinatorial library encoding multimodular peptides will be generated. This library will be used for the recombinant production and screening of these multimodular peptides for their transfection efficiency. Several rounds of screening and selection will be performed to obtain multimodular peptides for effective gene delivery
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