The delivery of exogenous nucleic acids into mammalian cells is a valuable technique for basic research studies on the expression, regulation and function of genes and proteins. At the same time, gene delivery to cultivated mammalian cells has become fundamentally important as a technology for the production of therapeutic proteins for preclinical and clinical applications. Alongside with the applications of gene delivery in biotechnology, gene transfer emerged in the early 1990s as a potential therapeutical method in which a functional copy of the defective gene is introduced to provide the missing function. Polymeric cations are widely used as nonviral transfection agents. The efficiency of these vectors, however, is lower than that of viral vectors and has until now precluded their extensive use in applications where stable and high level gene expression is required. Therefore a better understanding of the parameters that govern transfection efficiency of polycationic vectors is crucial for rational design of novel gene carriers. Thus, the main motivation behind this research was to investigate the role of the architecture and molecular weight of the polycation on the efficiency of transfection. The efficiency of transfection is determined by the cytotoxicity of the gene carrier, its reversible complexation with DNA, and proper intracellular trafficking of the resulting complex. For this purpose the 17 member library was synthesized, containing linear (LPL), hyperbranched (HBPL) and dendritic polylysine (DPL) analogues covering a broad range of molecular weights. HBPL, a new class of cationic polymers prepared by polycondensation of L-lysine, was developed to explore the influence of very high molecular weights and randomly branched structures on cytotoxicity, transfection efficiency, and internalization. The thesis is divided into four chapters. Chapter 1 reviews the current state-of-the-art on gene carriers and the future prospects of the field. Additionally, the current use of polycationic gene carriers for the production of recombinant proteins (r-proteins) via a large-scale transient gene expression (TGE) is discussed. The main goal of the Chapter 2 was to determine the influence of the architecture and molecular weight of the polycations on their in vitro biocompatibility. The extent of acute cell death after short exposure was found to be molecular weight dependent. The acute cytotoxicity of high molecular weight polycations was triggered by the permeabilization of the cell membrane. At high concentrations of small molecular weight polycations, membrane destabilization was caused by increased osmotic pressure. In contrast, delayed cell death was correlated to the collapse of mitochondrial transmembrane potential, triggering caspase-dependent apoptosis after intracellular accumulation of the polycation. The onset and extent of the delayed mode of cell death was shown to be dependent on the molecular weight and degree of branching of the polylysine analogues. Based on these results, the differential long term cytotoxicity reflects the resistance of branched polylysine analogues to proteolytic degradation. Thus the degradability and cumulative cytotoxicty can be predetermined by fine-tuning the frequency of branching points in the architecture of peptidic polycation. Chapter 3 demonstrates the evolution of transfection activity with the molecular weight and degree of branching of polylysine analogues. In summary, the results show that under identical cell culture and transfection conditions, the most critical parameter for high gene expression is the structure of the gene carrier. HBPL is the most efficient gene carrier in comparison to LPL and DPL and results in gene expression levels comparable to polyethyleneimine (PEI). Physico-chemical characterization of complexes revealed that those derived from plasmid DNA and HBPL contained a high quantity of the free polycation in comparison to those formed by LPL and DPL. The presence of the free polycation in the course of endocytosis of the complex may provide an explanation for the high transfection efficiency of HBPL. Chapter 4 describes a feasibility study aimed at proving whether HBPL is potentially applicable to large-scale r-protein production via TGE. Gene delivery efficiency and yield of r-protein were evaluated in serum-free, suspension-adapted CHO-DG44 cells under conditions mimicking the industrial manufacture of r-protein by TGE. High molecular weight HBPL mediates transfection and r-protein expression at levels comparable to the ones obtained with PEI. The main advantage of HBPL is its partial enzymatic degradability, potentially decreasing its cumulative cytotoxicity in the course of TGE. Also, HBPL efficiently transfects cell lines and primary cells in the presence of serum, thus broadening the potential applications of HBPL as a novel gene carrier for basic research applications in molecular and cellular biology. In conclusion, it is possible to obtain an efficient gene carrier by simple rearrangement of the topology of poly-L-lysine.