Capripoxviruses are of particular interest as live recombinant vectors for use in the veterinary field, since their host-range is restricted to cattle, goats and sheep. The work presented in this thesis is a preliminary study undertaken on the South African Neethling vaccine strain of lumpy skin disease virus (LSDV). As a departure point towards the eventual identification of strong promoter areas in the 143 kb genome of LSDV, a portion of its genome was cloned. Three methods for purification of LSDV DNA were compared, to determine which yielded the best quality DNA for cloning. DNA extracted directly from infected cells was excessively contaminated with bovine host-DNA, complicating the cloning of LSDV DNA. The use of pulsed field gel electrophoresis solved the contamination problem, by separating viral DNA from bovine DNA. However, insufficient amounts of viral DNA for cloning purposes, could be recovered from the gel. Sufficient amounts of good quality LSDV DNA was obtained by extraction from purified virions. Purified LSDV DNA was digested with various restriction enzymes to identify those which yielded several 4-1 0 kb fragments, for cloning into the Bluescribe plasmid transcription vector. Enrichment for large fragments (8-1 0 kb) was achieved by sucrose density centrifugation. Cloned fragments were analysed by Southern blot hybridisation to verify their viral origin. Hybridisation studies indicated that several unique regions of the LSDV genome were cloned as Pst I and Bam HI fragments respectively, i.e. the cloned fragments contained no overlapping regions. In total, 71.25 kb of the DNA of the LSDV Neethling vaccine strain has been cloned, representing approximately 50% of the viral genome. The availability of these clones now paves the way for further molecular investigations of the LSDV Neethling genome, including identification of promoter regions. A trial gene, which will be cloned and expressed in LSDV, namely the cloned VPS-gene of bluetongue virus serotype 4, was prepared and its nucleotide sequence determined. Homopolymer sequences present at the terminal ends of the gene as a result of the original cloning strategy, are known to interfere with expression and were removed by means of the polymerase chain reaction (PCR). The nucleotide sequence of the resulting PCR-tailored BTV4 VPS-genewas determined and used to deduce the amino acid sequence of the protein. The gene is 1638 bp in length and encodes a protein of 526 aa. Conserved sequences, 6 bp in length and unique to the 5'- and 3'terminal ends of all BTV genes, were detected at the termini of the tailored gene, confirming that the original clone was a full-length copy of the gene. Amplification by PCR did not mutate the open reading frame (OAF) of the gene, since it was of similar length to that reported for 5 other BTV serotypes. With a view to future investigations, including the identification of promoter sequences in the LSDV genome, a preliminary investigation of LSDV protein synthesis was undertaken, to acquire some knowledge of the growth cycle of the virus. Eighteen putative virus-specific proteins were identified by radio-labelling infected cells with [³⁵S]-methionine. By pulse-labelling infected cells with [³⁵S]methionine at various times post infection (p.i.), viral proteins were first detected at 16 hr p.i. It is, however, unlikely that the early phase of viral replication commences as late as 16 hr p.i. and these results might be attributed to various problems, such as the low multiplicity of infection used and that host protein shut-down was inefficient, thus masking the presence viral proteins. In conclusion, this investigation resulted in the cloning of 71,25 kb of the LSDV genome, the tailoring and sequencing of the BTV4 VPS gene and the identification of 18 putative LSDV proteins. This now paves the way for further research to develop LSDV as a vaccine vector.

No description available.

Thesis (MSc)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: This study describes: I. The cloning of the CVP 19 gene and construction of the intracellular expression vector pPIC3.5K-CYP19. II. The transformation of the yeast, Pichia pastoris with the constructed vector. III. The expression ofP450arom in Pichia pastoris. IV. The determination of enzyme activity and isolation of the protein from the Pichia pastoris cells. V. The expression of P450c 17 in Pichia pastoris. VI. The determination of kinetic constants for the conversion of progesterone to 170H-progesterone and 160H-progesterone by P450c17. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: I. Die klonering van die CVP 19 geen en die konstruksie van die intrasellulêre uitdrukkingsplasmied, pPIC3.5K-CYPI9. II. Die transformasie van die gis, Pichia pastoris, met die gekonstrueerde plasmied. III. Die uitdrukking van aromatase in Pichia pastoris. IV. Die bepaling van ensiemaktiwiteit en die isolering van die proteïen vanuit Pichia pastoris. V. Die uitdrukking van P450c17 in Pichia pastoris. VI. Die bepaling van kinetiese konstantes vir die omsetting van progesteroon na 170H-progesteroon en 160H-progesteroon deur P450c17.

A integridade do DNA é ameaçada pelos efeitos lesivos de inúmeros agentes físicos e químicos que podem vir a comprometer sua função. Um dos mais versáteis e estudados mecanismos de reparo de DNA é o reparo por excisão de nucleotídeos (NER). Este mecanismo remove lesões que causam distorções na dupla fita de DNA, incluindo dímeros de pirimidina ciclobutano (CPDs) e 6-4 fotoprodutos (6-4 PPs), provocados pela radiação de luz ultravioleta (UV). Em humanos, a síndrome genética xeroderma pigmentosum (XP) apresenta uma alta sensibilidade à luz solar, resultando em um grande aumento na incidência de tumores em regiões expostas da pele e degeneração neurológica progressiva. O gene xpa parece estar envolvido diretamente no reconhecimento de lesões produzidas pela luz UV, atuando tanto no reparo global (GGR) como no reparo acoplado à transcrição (TCR). A modulação da expressão deste gene deve alterar as taxas de reparo no genoma celular, fornecendo valiosa contribuição para o estudo do reparo de DNA no NER e em outras vias distintas. No entanto, não foi possível a atenuação ou inativação total do transcrito XPA, provavelmente devido ao baixo número de moléculas de mRNA nas células e da relativa estabilidade da proteína XPA. A expressão controlada do cDNA xpa em células deficientes XP12RO foi conseguida através da transfecção do vetor indutível por muristerona A, pINXA. O clone INXA15M mostrou-se eficiente na indução da proteína XPA, complementando células XP12RO. Quantidades reduzidas de XPA foram suficientes para a complementação total de células XP12RO na sobrevivência frente à luz UV, ou para a atividade de reparo de DNA no genoma global. Entretanto, observou-se uma maior incidência de células apoptóticas em períodos de tempo curtos após a UV, quando comparamos com células proficientes para o reparo de DNA (HeLa). O vetor adenoviral portando o cDNA xpa (AdyXPA) mostrou-se eficiente na complementação de fibroblastos derivados de pacientes XPA. Apesar do curto período de expressão do transgene e da conhecida reação imunológica produzida pelos adenovirus, este vetor representa uma potencial ferramenta para testes de complementação, identificação de mutações e busca de sistemas de correção gênica de pacientes XP. / The DNA integrity is always threatened by the damage effects of physical and chemical agents that could jeopardy its function. The nucleotide excision repair (NER) is one of the most known and flexible mechanisms of DNA repair. This mechanism can recognize and remove DNA double-helix distortion, including the cyclobutane pyrimidine dimers (CPDs) and the pyrimidine-pyrimidone (6-4) photoproduct, promoted by ultraviolet light (UV). The human syndrome xeroderma pigmentosum (XP) is clinically characterized chiefly by the early onset of severe photosensitivity of the exposed regions of the skin, a very high incidence of skin cancers and frequent neurological abnormalities. The xpa gene seems to be involved during the UV damage recognition, in both global genome repair (GGR) and transcription-coupled repair (TCR). This gene modulation may modify the DNA repair rate in the cell genome, providing valuable contribution to the NER understanding and other DNA repair pathways. However, the complete inactivation or even the attenuation of the XPA transcript was not possible, mainly because of the low abundance mRNA per cell and the high stability of the XPA protein. The controlled expression of the cDNA xpa in XP12RO deficient cells was achieved through the transfection of a muristerone-A inducible vector, pINXA. The INXA15M clone shows good induction of the XPA protein and total complementation of XP12RO cells deficiency. Small quantities of the XPA protein do not interfere in the cellular UV sensitivity and the DNA repair activity in the global genome. Nevertheless, a higher number of cells in the apoptotic process were detected in short periods of time after UV light when compared to normal cells (HeLa). The adenovirus vector carrying the cDNA xpa (AdyXPA) can efficiently complement XPA patients’ fibroblast cells. In spite of the short period of the transgene expression and the known imunological reaction caused by adenovirus, this vector represents a potential tool for gene complementation diagnostic and gene correction in XP patients.

Lee Yiu Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 178-183). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / Abstract --- p.iii / Abstract (Chinese version) --- p.vi / Table of contents --- p.viii / List of Abbreviations --- p.xvi / List of Figures --- p.xviii / List of Tables --- p.xxiv / Chapter Chapter 1 --- INTRODUCTON / Chapter 1.1 --- Cytochrome P450 (P450) --- p.1 / Chapter 1.1.1 --- Cytochrome P450 family --- p.1 / Chapter 1.1.2 --- Role in metabolism --- p.4 / Chapter 1.1.3 --- P450 catalytic cycle --- p.6 / Chapter 1.2 --- NADPH-cytochrome P450 reductase (RED) --- p.6 / Chapter 1.2.1 --- Characterization and distribution --- p.6 / Chapter 1.2.2 --- Structural and functional domains --- p.8 / Chapter 1.2.3 --- Role in P450 catalytic cycle --- p.10 / Chapter 1.3 --- Drug metabolism --- p.10 / Chapter 1.3.1 --- Understanding of drug metabolism is important for drug development --- p.10 / Chapter 1.3.2 --- Role of P450 in drug metabolism --- p.12 / Chapter 1.4 --- Production of RED knockout in vivo mouse model for screening of P450-dependent new drugs --- p.13 / Chapter 1.4.1 --- Background --- p.13 / Chapter 1.4.2 --- Gene targeting --- p.13 / Chapter 1.4.3 --- Gene targeting vector --- p.15 / Chapter 1.4.3.1 --- Classical knockout --- p.15 / Chapter 1.4.3.2 --- Conditional knockout --- p.19 / Chapter 1.4.4 --- Gene knockout mice and its use --- p.21 / Chapter Chapter 2 --- OBJECTIVES --- p.22 / Chapter Chapter 3 --- MATERIALS AND METHODS --- p.24 / Chapter 3.1 --- Preparation of RED cDNA by RT-PCR --- p.24 / Chapter 3.1.1 --- Total RNA isolation --- p.24 / Chapter 3.1.1.1 --- Materials --- p.24 / Chapter 3.1.1.2 --- Methods --- p.24 / Chapter 3.1.2 --- Reverse transcription- polymerase chain reaction (RT-PCR) --- p.25 / Chapter 3.1.2.1 --- Materials --- p.25 / Chapter 3.1.2.2 --- Methods --- p.25 / Chapter 3.1.3 --- T/A cloning of RED cDNA --- p.28 / Chapter 3.1.3.1 --- Materials --- p.28 / Chapter 3.1.3.2 --- Methods --- p.28 / Chapter 3.1.4 --- Midi-preparation of RED cDNA clone --- p.32 / Chapter 3.1.4.1 --- Materials --- p.33 / Chapter 3.1.4.2 --- Methods --- p.33 / Chapter 3.1.5 --- Confirmation of RED cDNA clone --- p.34 / Chapter 3.1.5.1 --- Restriction enzyme mapping --- p.34 / Chapter 3.1.5.1.1 --- Materials --- p.34 / Chapter 3.1.5.1.2 --- Methods --- p.34 / Chapter 3.1.5.2 --- DNA sequencing of RED cDNA sequence --- p.35 / Chapter 3.1.5.2.1 --- Materials --- p.35 / Chapter 3.1.5.2.2 --- Methods --- p.35 / Chapter 3.1.6 --- Preparation and purification of RED cDNA for probe labeling --- p.38 / Chapter 3.1.6.1 --- Materials --- p.38 / Chapter 3.1.6.2 --- Methods --- p.38 / Chapter 3.1.7 --- Non-radioactive random-primed labeling of RED cDNA --- p.39 / Chapter 3.1.7.1 --- Materials --- p.39 / Chapter 3.1.7.2 --- Methods --- p.39 / Chapter 3.2 --- Isolation of RED gene by genomic library screening --- p.40 / Chapter 3.2.1 --- Titering of genomic library --- p.41 / Chapter 3.2.1.1 --- Materials --- p.41 / Chapter 3.2.1.2 --- Methods --- p.41 / Chapter 3.2.2 --- Primary screening of genomic library by RED cDNA probe --- p.42 / Chapter 3.2.2.1 --- Plaque lift --- p.42 / Chapter 3.2.2.1.1 --- Materials --- p.42 / Chapter 3.2.2.1.2 --- Methods --- p.42 / Chapter 3.2.2.2 --- Proteinase K treatment --- p.43 / Chapter 3.2.2.2.1 --- Materials --- p.43 / Chapter 3.2.2.2.2 --- Methods --- p.43 / Chapter 3.2.2.3 --- "Pre-hybridization, hybridization and detection" --- p.44 / Chapter 3.2.2.3.1 --- Materials --- p.44 / Chapter 3.2.2.3.2 --- Methods --- p.44 / Chapter 3.3 --- Isolation of RED by hybridization screening by Genome System Inc. --- p.45 / Chapter 3.4 --- Characterization of BAC clones containing RED genomic DNA fragments commercially obtained from Genome System Inc. --- p.45 / Chapter 3.4.1 --- Large scale preparation of BAC DNA --- p.45 / Chapter 3.4.1.1 --- Materials --- p.47 / Chapter 3.4.1.2 --- Methods --- p.47 / Chapter 3.4.2 --- Restriction enzyme mappings and Southern blotting analysis of BAC DNA fragments --- p.47 / Chapter 3.4.2.1 --- Materials --- p.48 / Chapter 3.4.2.2 --- Methods --- p.48 / Chapter 3.4.3 --- Shot-gun sub-cloning of RED genomic DNA fragments from BAC clone in pGEM®-3Z vector --- p.49 / Chapter 3.4.3.1 --- Preparation of cloning vector and DNA insert for ligation --- p.50 / Chapter 3.4.3.1.1 --- Materials --- p.50 / Chapter 3.4.3.1.2 --- Methods --- p.50 / Chapter 3.4.3.1.2.1 --- Cloning vectors --- p.50 / Chapter 3.4.3.1.2.2 --- DNA inserts --- p.52 / Chapter 3.4.3.2 --- Preparation of competent cells and transformation --- p.52 / Chapter 3.4.3.2.1 --- Materials --- p.52 / Chapter 3.4.3.2.2 --- Methods --- p.53 / Chapter 3.4.3.3 --- Screening for positive recombinant clones --- p.54 / Chapter 3.4.3.3.1 --- Picking of colonies randomly from the agar plates (method 1) --- p.54 / Chapter 3.4.3.3.1.1 --- Materials --- p.54 / Chapter 3.4.3.3.1.2 --- Methods --- p.54 / Chapter 3.4.3.3.2 --- Colony lifts and hybridization with RED cDNA probes (method 2) --- p.55 / Chapter 3.4.3.3.2.1 --- Materials --- p.55 / Chapter 3.4.3.3.2.2 --- Methods --- p.55 / Chapter 3.5 --- Restriction enzyme mappings and Southern blotting analysis of RED gene subcloned in pGEM®-3Z vector --- p.56 / Chapter 3.5.1 --- Materials --- p.56 / Chapter 3.5.2 --- Methods --- p.56 / Chapter 3.6 --- Exon mappings of the RED genomic DNA fragments by PCR --- p.57 / Chapter 3.6.1 --- Materials --- p.57 / Chapter 3.6.2 --- Methods --- p.57 / Chapter 3.7 --- Construction of gene targeting vector --- p.57 / Chapter 3.7.1 --- Gene targeting vectors la and lb derived from clone H (strategy 1) --- p.60 / Chapter 3.7.1.1 --- Sub-cloning 3.65 kb Hind Ill/Hind III RED gene fragment to pGEM®-3Z vector --- p.60 / Chapter 3.7.1.1.1 --- Materials --- p.62 / Chapter 3.7.1.1.2 --- Methods --- p.62 / Chapter 3.7.1.2 --- Deletion of exonic sequence of RED gene and modification of the digested restriction end to Xho I site --- p.62 / Chapter 3.7.1.2.1 --- Materials --- p.63 / Chapter 3.7.1.2.2 --- Methods --- p.63 / Chapter 3.7.1.3 --- Preparation of neo cassette --- p.63 / Chapter 3.7.1.3.1 --- Materials --- p.64 / Chapter 3.7.1.3.2 --- Methods --- p.64 / Chapter 3.7.1.4 --- Cloning of neo cassette --- p.66 / Chapter 3.7.1.4.1 --- Methods --- p.66 / Chapter 3.7.1.5 --- Sub-cloning the neo cassette containing RED genomic fragment to pMCI-Thymidine kinase (TK) Poly A vector --- p.67 / Chapter 3.7.1.5.1 --- Materials --- p.67 / Chapter 3.7.1.5.2 --- Methods --- p.67 / Chapter 3.7.2 --- "Gene targeting vectors 2a/2b, 3a/3b and 4a derived from clone X8 (strategy 2,3 and 4 respectively)" --- p.67 / Chapter 3.8 --- Preparation and testing the genomic probes for screening recombinant embryonic stem (ES) cells --- p.73 / Chapter 3.8.1 --- Cloning of genomic probes --- p.73 / Chapter 3.8.1.1 --- Materials --- p.73 / Chapter 3.8.1.2 --- Methods --- p.73 / Chapter 3.8.2 --- Purification of DNA for labeling --- p.78 / Chapter 3.8.2.1 --- Materials --- p.78 / Chapter 3.8.2.2 --- Methods --- p.78 / Chapter 3.8.3 --- ECF random prime labeling of genomic probes --- p.79 / Chapter 3.8.3.1 --- Materials --- p.79 / Chapter 3.8.3.2 --- Methods --- p.79 / Chapter 3.8.4 --- Restriction enzyme digestion of genomic DNA and Southern blotting --- p.80 / Chapter 3.8.4.1 --- Materials --- p.80 / Chapter 3.8.4.2 --- Methods --- p.80 / Chapter 3.8.5 --- Testing the specificity of genomic probes --- p.80 / Chapter 3.8.5.1 --- Materials --- p.80 / Chapter 3.8.5.2 --- Methods --- p.80 / Chapter Chapter 4 --- RESULTS --- p.86 / Chapter 4.1 --- Total RNA isolation and RT-PCR of RED cDNAs --- p.86 / Chapter 4.2 --- Confirmation of the RT-PCR RED cDNA clone --- p.86 / Chapter 4.2.1 --- Restriction enzyme mapping --- p.86 / Chapter 4.2.2 --- DNA sequencing --- p.86 / Chapter 4.3 --- Genomic library screening of RED gene --- p.90 / Chapter 4 4 --- Restriction enzyme mappings and Southern blotting analysis of RED Gene containing BAC clone from Genome System Inc. --- p.90 / Chapter 4.5 --- Shot-gun sub-cloning of RED gene containing genomic DNA fragments to pGEM®-3Z vectors --- p.93 / Chapter 4.5.1 --- Cloning of Hind III cut RED gene fragment --- p.93 / Chapter 4.5.2 --- Cloning of Xba I cut RED gene fragment --- p.93 / Chapter 4.5.3 --- Cloning of EcoR I cut RED gene fragment --- p.95 / Chapter 4.6 --- Identification of RED exons in the shot-gun sub-cloning clones by PCR --- p.95 / Chapter 4.7 --- Construction of restriction enzyme maps of the RED gene containing clones --- p.100 / Chapter 4.7.1 --- Clone H --- p.100 / Chapter 4.7.1.1 --- Single restriction enzyme digestions and Southern blotting --- p.100 / Chapter 4.7.1.2 --- Double restriction enzyme digestions and Southern blotting --- p.100 / Chapter 4.7.1.3 --- Restriction enzyme map --- p.101 / Chapter 4.7.2 --- Clone X8 --- p.101 / Chapter 4.7.2.1 --- Single restriction enzyme digestions and Southern blotting --- p.101 / Chapter 4.7.2.2 --- Double restriction enzyme digestion and Southern blotting --- p.104 / Chapter 4.7.2.3 --- Restriction enzyme map --- p.104 / Chapter 4.7.3 --- Clone El4 --- p.105 / Chapter 4.7.3.1 --- Single restriction enzyme digestions and Southern blotting --- p.105 / Chapter 4.7.3.2 --- Double restriction enzyme digestion and Southern blotting --- p.108 / Chapter 4.7.3.3 --- Restriction enzyme map --- p.108 / Chapter 4.8 --- Construction of gene targeting vector --- p.108 / Chapter 4.8.1 --- Gene targeting vector based on the clone H (strategy 1) with deletion of RED exon 16 --- p.113 / Chapter 4.8.1.1 --- Cloning a smaller RED genomic DNA into pGEM®-3Z vectors --- p.113 / Chapter 4.8.1.2 --- Replacement of exon of RED gene by neo cassette --- p.113 / Chapter 4.8.1.3 --- Cloning to TK vector --- p.113 / Chapter 4.8.2 --- Targeting vector based on the clone X8 --- p.124 / Chapter 4.8.2.1 --- Strategy 2 (deletion of RED exon 4) --- p.124 / Chapter 4.8.2.1.1 --- Cloning 3.9 kb Kpn I/Hinc II RED genomic DNA into pGEM®-3Z vectors --- p.124 / Chapter 4.8.2.1.2 --- Replacement of exon of RED gene by neo cassette --- p.124 / Chapter 4.8.2.1.3 --- Cloning to TK vector --- p.124 / Chapter 4.8.2.2 --- Strategy 3 (deletion of RED exon 5-8) --- p.136 / Chapter 4.8.2.2.1 --- Cloning the genomic DNA into pGEM®-3Z vectors --- p.136 / Chapter 4.8.2.2.2 --- Replacement of exon of RED gene by neo cassette --- p.136 / Chapter 4.8.2.2.3 --- Cloning to TK vector --- p.136 / Chapter 4.8.2.3 --- Strategy 4 (deletion of RED exon 7-10) --- p.136 / Chapter 4.8.2.3.1 --- Cloning the genomic DNA into pGEM®-3Z vectors --- p.136 / Chapter 4.8.2.3.2 --- Replacement of exon of RED gene by neo cassette --- p.152 / Chapter 4.8.2.3.3 --- Cloning to TK vector --- p.152 / Chapter 4.9 --- Testing for the specificity of genomic DNA probes --- p.152 / Chapter 4.9.1 --- Preparation of restriction enzyme digested genomic DNA --- p.152 / Chapter 4.9.2 --- Hybridization of the probes to genomic DNA --- p.163 / Chapter Chapter 5 --- DISCUSSION --- p.167 / Chapter 5.1 --- Proposed significant of RED knockout mice for new drug screening --- p.167 / Chapter 5.2 --- Experimental problems --- p.168 / Chapter 5.2.1 --- Genomic library screening --- p.168 / Chapter 5.2.2 --- Cloning --- p.168 / Chapter 5.3 --- RED gene targeting vector construction / Chapter 5.3.1 --- Isolation of RED gene for gene targeting vectors construction --- p.169 / Chapter 5.3.2 --- Deletion of different exons in different RED gene targeting vectors --- p.169 / Chapter 5.3.3 --- Components in the targeting vectors --- p.170 / Chapter 5.3.4 --- Enhancements of homologous recombination --- p.171 / Chapter Chapter 6 --- CONCLUSIONS --- p.173 / Chapter Chapter 7 --- FUTURE STUDIES --- p.175 / Chapter 7.1 --- Identification of the sizes of RED gene introns --- p.175 / Chapter 7.2 --- Production of RED knockout mice --- p.175 / Chapter 7.3 --- Characterization of RED knockout mice --- p.175 / Chapter 7.4 --- Conditional gene knockout for RED gene --- p.177 / REFERENCES --- p.178 / APPENDIX --- p.184

Bibliography: leaves 104-130. The aim of this thesis is to characterise subgenomic RNAs of cucumoviruses and the functions of their encoding genes. Strains of cucumber mosaic virus (CMV) are classified into two major subgroups (I and II) on the basis of nucleotide sequence homology. The V strain of tomato aspermy virus (V-TAV) and a subgroup I CMV strain (WAII) are chosen to determine whether the 2b genes encoded by these viruses are expressed 'in vivo'. For further investigation of the 2b gene function, cDNA clones of three genomic RNAs of V-TAV are constructed. Using the infectious cDNA clones of V-TAV, a mutant virus containing only one of the two repeats is constructed.

Thesis (Ph. D.)--University of Florida, 2003. / Title from title page of source document. Includes vita. Includes bibliographical references.

The discovery of RNA interference and the increasing understanding of disease genetics have created a new class of potential therapeutics based on oligonucleotides. This therapeutic class includes antisense molecules, small interfering RNA (siRNA), and microRNA modulators such as antagomirs (antisense directed against microRNA) and microRNA mimics, all of which function by altering gene expression at the translational level. While these molecules have the promise of treating a host of diseases from neurological disorders to cancer, a major hurdle is their inability to enter cells on their own, where they may render therapeutic effect. Nanotechnology is the engineering of materials at the nanometer scale and has gained significant interest for nucleic acid delivery due to its biologically relevant length-scale and amenability to multifunctionality. While a number of nanoparticle vehicles have shown promise for oligonucleotide delivery, there remains a lack of understanding of how nanoparticle coating and size affect these delivery processes. This dissertation seeks to elucidate some of these factors by evaluating oligonucleotide delivery efficiencies of a panel of iron oxide nanoparticles with varying cationic coatings and sizes. A panel of uniformly-sized nanoparticles was prepared with surface coatings comprised of various amine groups representing high and low pKas. A separate panel of nanoparticles with sizes of 40, 80, 150, and 200 nm but with the same cationic coating was also prepared. Results indicated that both nanoparticle surface coating and nanoparticle hydrodynamic size affect transfection efficiency. Specific particle coatings and sizes were identified that gave superior performance. The intracellular fate of iron oxide nanoparticles was also tracked by electron microscopy and suggests that they function via the proton sponge effect. The research presented in this dissertation may aid in the rational design of improved nanoparticle delivery vectors for nucleic acid-based therapy.

Human T cells may be genetically modified to express targeted chimeric antigen receptors (CARs). We have previously demonstrated that T cells modified to express a CAR specific to the B cell tumor antigen CD19, termed 19-28z, successfully eradicate systemic human CD19+ tumors in SCID-Beige mice. While these results are encouraging, this xenogeneic tumor model fails to address potential limitations of this therapeutic approach in the clinical setting wherein these modified T cells encounter a hostile tumor microenvironment. Specifically, these models fail to address potential effector T cell inhibition mediated by endogenous regulatory T cells (Tregs). To investigate the role of inhibitory Tregs, we initially assessed the in vitro function of CAR-modified T cells in the context of Tregs. We found that CD19-targeted T cell proliferation and cytotoxicity were inhibited by purified natural Tregs. To further assess the role of these Tregs in vivo, we isolated and genetically modified Tregs to express the CD19-targeted 19z1 CAR. We verified specific trafficking of targeted Tregs to CD19+ tumors in vivo, and demonstrate that 19z1 Tregs wholly inhibit anti-tumor function of subsequently injected 19-28z effector T cells even at low Treg to effector T cell ratios (1:8). In order to overcome this limitation, we assessed whether the addition of a pro-inflammatory cytokine in vitro could overcome Treg inhibition. Indeed, the addition of exogenous IL-12 mediated resistance of 19-28z T cells to Treg inhibition. In light of this data we generated a bicistronic retroviral vector containing both the 19-28z CAR as well as the murine IL-12 fusion gene (19-28z IRES IL-12). Significantly, we found that 19-28z/IL-12+ T cells when compared to 19-28z+ T cells exhibited enhanced proliferation in vitro as well as resistance to Treg mediated inhibition. Finally, we demonstrate that 19-28z/IL-12+ T cells overcome Treg inhibition in vivo in our SCID-Beige Treg tumor model. In conclusion, tumor targeted T cells modified to express IL-12 demonstrate significantly enhanced in vivo anti-tumor efficacy in the presence of Tregs that are similarly targeted to the site of tumor. These results validate utilization of IL-12 secreting tumor targeted T cells in future clinical trials.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 112-133).