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Gene therapy for Hemophilia A (HA) using the recombinant Adeno-associated virus (rAAV) offers an alternative to classic treatment, which consists of FVIII protein infusions. However, due to limitations associated with rAAV and the FVIII protein itself, the end result is a transgene expression below therapeutic limits. One approach to improving the therapeutic value of rAAV gene therapy for HA is to engineer a more active FVIII protein through genetic modifications. Preliminary testing revealed that canine FVIII Light Chain (kLC) enhances coagulation activity, and that it would be possible to improve FVIII activity through modifications of the light chain. Through the process of engineering, evaluation, and negative selection of kLC, a final construct was engineered. The hLC-K12 is a human Light Chain (hLC) construct containing 12 amino acid changes that work together to enhance coagulation activity. A comparison of the FVIII clotting activity to the amount of protein produced determined that hLC-K12 produced a 3.28 fold increase in specific activity over hLC in vitro. Similar in vitro results were observed when hLC-k12 was tested with the X5 heavy chain (X5HC), a heavy chain that has been genetically modified to enhance production. CD4KO/HA mice were injected with a rAAV vector carrying the hLC-K12 gene in conjugation with a rAAV vector carrying the X5HC gene. Replacing the hLC vector with the hLC-K12 vector produced an average 7.43 fold increase in FVIII clotting activity. An ELISA assay revealed no significant difference between productions of the heavy or light chains at any time point. By comparing the clotting activity to the amount of protein produced, it was determined that the increase in coagulation activity was due to an increase in specific activity. In fact, replacing the hLC vector with the hLC-K12 vector resulted in an average 5.8 fold increase in FVIII specific activity. The K12 modifications were evaluated using a single chain FVIII conformation. In vitro, the addition of the K12 mutations to the human heavy chain, hHCK12BDD, resulted in a 4.3 fold increase in clotting activity, but no increase in protein production. There was however, a 3.3 fold increase in specific activity of the protein. Adding the K12 mutations to the X5 heavy chain, X5K12BDD, in vitro, resulted in a 2.7 fold increase in clotting activity and a 1.42 fold increase in specific activity of the protein. Single chain rAAV vectors were packaged and delivered to CD4KO/HA mice. Compared to mice injected with hFVIIIBDD, the hHCK12BDD produced an average 4.6 fold increase in clotting activity. An ELISA revealed no significant difference in production between these two groups. However, mice injected with hHCK12BDD produced FVIII with an average of 4.13 fold increase in specific activity. Similarly, when compared to mice injected with X5FVIIIBDD, the X5K12BDD produced an average 2.14 fold increase in clotting activity. An ELISA assay demonstrated no significant increase in protein production between these two groups. However, when compared to X5BDD, mice injected with the X5K12BDD vector produced FVIII with an average 1.98 fold increase in specific activity. Results demonstrate that the K12 light chain modifications are able to enhance clotting activity of hFVIII both in vitro and in vivo, using either a dual chain or single chain delivery method. In order to determine the mechanism of enhancement, hFVIIIBDD and hHCK12BDD protein was partially purified and tested for activity. Results demonstrated that the hHCK12BDD protein produced a specific activity of 39,153.69 Units/mg, which is a 6.28 fold increase over hFVIIIBDD specific activity, which was 6,237.92 Units/mg. Measurement of conversion from FX to FXa revealed that the hHCK12BDD protein generated a higher amount of FXa at a quicker rate. In conclusion, these results provide evidence that the K12 modifications enhance specific activity through an increase in FXa generation. / Microbiology and Immunology

Recent advances in plant genetic engineering offer substantial benefits to farmers throughout the world. Genetic research has identified many exogenous genes that could considerably decrease production costs through transgene-mediated resistance to insect, viral, fungal and bacterial pathogens. Potato can be produced from true potato seed (TPS) through a sexual polyploidization step, known as 4x-2x hybridization. Little is known regarding the stability of transgenes through sexual polyploidization in potato, although studies have associated ploidy elevation with transgene silencing in plants such as Arabidopsis thaliana. In the present study, potato was transformed with two different transgenes, cry3Aa and PVYo cp, and transgene expression was analyzed through 4x-2x hybridization. Transgene introgression did not affect fertility or agronomic performance (tuber set, average tuber weight, total tuber yield) of the resulting 4x-2x hybrids; however, reduced seed germination was observed for several transgenic lines in an in vitro study. Ploidy elevation did not affect a highly expressed single copy cry3Aa transgene, simplex or duplex, transmitted through pollen to 4x-2x hybrids. By contrast, multiple copies of cry3Aa triggered significant transgene silencing in diploids and silencing was further pronounced upon pollen transmission to 4x-2x hybrids. Crosses between two, single insert plants demonstrated additional evidence that multiple cry3Aa transgenes resulted in reduced expression, as well as provided evidence for maternal effects on expression of the cry3Aa transgene. Finally, Cry3Aa expression levels of progeny derived from low expressing, multiple copy 4x-2x hybrids indicated that reduction of transgene number in progeny, through meiotic segregation, could increase Cry3Aa expression. The results suggest that 4x-2x hybridization using single copy, male parents can result in high expressing, transgenic 4x-2x hybrids while segregating for a low frequency of non-transgenic hybrids that create a "refuge" to inhibit development of resistance to transgenes in pest populations. / Ph. D.

Cancer and hepatitis viruses are two of the leading causes of death worldwide. Recombinant Interferon alpha2b (IFNa2b) is used as an immunotherapeutic drug for cancers, hepatitis viruses and several other viral diseases. Interferons are produced in low quantity naturally and production cost for recombinant IFNa2b in E.coli is very high. Since prokaryotes cannot form disulfide bonds, additional techniques have to be employed to create a functional form of IFNa2b. The average cost per patient for treatment with recombinant IFNa2b is $26,000 per year. Around 800 million people in the world are infected with Hepatitis C virus and most of them cannot afford the treatment costs. Producing recombinant IFNa2b in tobacco chloroplasts will overcome these problems and make the drug affordable for many people. A recombinant IFNa2b chloroplast vector was introduced into the tobacco cultivars Petit Havana (model) and LAMD-609 (low nicotine hybrid plants) in the Daqjell lab by particle gun bombardment. In this research, second-generation transgenic plants with the IFNa2b gene are subjected to various experiments to study the levels of IFNa2b expression. The psbA regulatory sequences present in the chloroplast vector are known to enhance protein expression in the presence of light. To analyze this effect and to find optimal growth conditions for maximal IFN a2b production, continuous light studies were performed. These results can be vital for mass production of IFNa2b.

A polymerase chain reaction (PCR) technique was used to detect transgene presence after pronuclear microinjection of mouse zygotes cultured to various stages of development. The transgene was detected in 88% of 1-cell, 88% of 2-cell, 44% of 4- cell, 40% of morula, and 29% of blastocysts. By comparison, the integration frequency for transgenic mice made using the same DNA construct was 22%. After 5 days of in vitro culture, the injected construct was detected in 83% of arrested 1-cell, 85% of arrested 2-cell, and 85% of fragmented embryos. Only 28% of zygotes cultured after microinjection of DNA developed to the blastocyst stage compared to 74% of non-injected zygotes. When DNA buffer alone was injected, 63% of zygotes developed to the blastocyst stage. These data suggest that pronuclear microinjection of DNA is highly detrimental to subsequent embryonic development. Also, most injected DNA that is either unintegrated or that will not be integrated into the genome has been degraded by the blastocyst stage such that it can no longer be detected by PCR. The production of transgenic mice by cytoplasmic injection of DNA mixed with poly-L-lysine is also described. The effects of DNA concentration and stoichiometric ratio of positive charges provided by the polycation to negative charges provided by DNA on transgenic frequency and embryonic viability were studied. The highest transgenic frequency (13% of pups born were transgenic) was obtained when a polylysine/DNA complex having a stoichiometric charge ratio of one to one (equal positive charges as negative charges) at a DNA concentration of 50 ug/ml was used. The transgenic frequency by pronuclear injection of the same DNA construct was 22%. The percentage of zygotes, cultured in vitro, reaching the blastocyst stage which were injected cytoplasmicly was not different (p>0.05) than that of control zygotes that were not microinjected (65% versus 74%, respectively). The percentage of zygotes reaching the blastocyst stage after pronuclear microinjection with DNA at a concentration of 1.5 ug/ml was significantly lower (p<0.05) than control embryos (28% versus 74%, respectively). The overall transgenic pup production efficiency (percent of transgenic pups per embryos transferred) by cytoplasmic injection was 2.4% compared to 3.5% by pronuclear microinjection. / Ph. D.

<p>Small populations are facing a particular risk of extinction due to a lack of appropriate genetic diversity and associated negative effects, factors dealt with in the discipline of conservation genetics. Many orchid species exhibit characteristics that make them a perfect study object in the scope of conservation genetics. The aim with this thesis was to investigate genetic structure at different levels in two orchid species <i>Gymnadenia conopsea</i>, geographically widespread, although diminishing and <i>G. odoratissima</i> with a long history of being rare. Microsatellite markers, developed in and used in studies of <i>G. conopsea</i> were also used in the study of <i>G. odoratissima</i>.</p><p>Populations of <i>G. conopsea</i> expressed high levels of genetic variation and a certain amount of gene flow, although investigated mating pattern in a small population indicated non-random mating among individuals, with the majority of pollen exchange between near neighbours, and noticeable levels of geitonogamous pollinations. Further a pronounced year to year variation in flowering frequency among individuals was found. </p><p>It was also discovered that flowering time variants (early and late) within the species <i>G. conopsea</i> were highly differentiated and seem to have had a more ancient historical separation than the separation between the two different species, <i>G. conopsea</i> and <i>G. odoratissima. </i></p><p>Levels of genetic variation in the rare congener, <i>G. odoratissima</i> differed between island and mainland populations where the more numerous island populations expressed larger levels of genetic variation and were less differentiated compared to the few remaining and genetically depauperated mainland populations.</p><p>Uppsala University Library, Box 510, 75120, Uppsala, Sweden </p>

Small populations are facing a particular risk of extinction due to a lack of appropriate genetic diversity and associated negative effects, factors dealt with in the discipline of conservation genetics. Many orchid species exhibit characteristics that make them a perfect study object in the scope of conservation genetics. The aim with this thesis was to investigate genetic structure at different levels in two orchid species Gymnadenia conopsea, geographically widespread, although diminishing and G. odoratissima with a long history of being rare. Microsatellite markers, developed in and used in studies of G. conopsea were also used in the study of G. odoratissima. Populations of G. conopsea expressed high levels of genetic variation and a certain amount of gene flow, although investigated mating pattern in a small population indicated non-random mating among individuals, with the majority of pollen exchange between near neighbours, and noticeable levels of geitonogamous pollinations. Further a pronounced year to year variation in flowering frequency among individuals was found. It was also discovered that flowering time variants (early and late) within the species G. conopsea were highly differentiated and seem to have had a more ancient historical separation than the separation between the two different species, G. conopsea and G. odoratissima. Levels of genetic variation in the rare congener, G. odoratissima differed between island and mainland populations where the more numerous island populations expressed larger levels of genetic variation and were less differentiated compared to the few remaining and genetically depauperated mainland populations. Uppsala University Library, Box 510, 75120, Uppsala, Sweden

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