Histone mediated transduction for gene therapy and novel reverse transfection cell microarrays based on aminomalononitrile self-polymerisation chemistry

Our understanding of the link between gene function and disease is constantly strengthening however gene therapy is currently limited by a lack of safe, efficient and specific vectors for DNA delivery. Histone mediated transduction (HMT) is a promising, efficient non-viral DNA delivery technique utilising histone proteins as non-toxic DNA vectors. The tumour cell enhanced nuclear targeting signal (tNTS) of the Apoptin protein has been fused to histones in Chapter 2 to create novel recombinant histone proteins that specifically accumulate in the nucleus of cancer cells. Nuclear accumulation was characterised in a breast tumour progression model, demonstrating that the tNTS has increased specificity for the latter stages of tumour progression. This study also identified an affinity of histone proteins for cancer cells, leading to recombinant proteins with a >13-fold specificity for cancer cell nuclei, with important implications for future cancer specific gene therapy approaches. Reverse Transfection Cell Microarray (RTCM) technology is designed for the simultaneous high throughput analysis of numerous genes in a live cell format and promises to revolutionise data collection from tissue culture cell lines. RTCM technology however is limited by inefficient transfection and the lack of a simple, efficient and inexpensive 2D cell surface patterning system. These surfaces only allow cells to attach to spots containing arrayed genetic material, facilitating automated analysis and reducing cross-contamination, thereby enhancing the efficiency of the array. Chapter 3 addresses the synthesis and optimisation of a novel, inexpensive and simple 2D cell surface patterning system based on aminomalononitrile (AMN) chemistry. An activated AMN solution was spotted onto a non-fouling (bio-resistant) poly(hydroxyethyl methacrylate) (pHEMA) background, limiting cell attachment to areas where AMN polymer is present. Chapter 4 investigates the addition of positively charged copolymers to the activated AMN and further optimises the transfection solution while chapter 5 investigates histone octamer and tetramer based reverse transfection on RTCMs for the first time. The result is a pHEMA-AMN-5% poly(allylamine) 2D patterned surface that is simpler to produce than any previously described, offering significant advantages over current RTCM technology. HMT was found to provide a benefit to RTCMs utilising Lipofectamine 2000 transfection, with implications for RTCMs utilising that reagent. Due to the simplicity and customisability of the AMN chemistry demonstrated, the 2D surface patterning system generated in this thesis is poised to make a significant contribution to the high-throughput screening of genetic material, drugs and other biomolecules in live cells, facilitating the progress of numerous scientific fields.