To achieve successful gene therapy, safe and efficient gene delivery vectors are needed. As an alternative to viral vectors, non-viral vectors, incorporating compounds such as cationic polymers and lipids have been widely studied. Much effort has been made to enhance transgene delivery efficiency, such as development of more effective cationic lipids or polymers, optimization of transfection formulations, and investigation on structural-activity of delivery vectors. Gemini surfactant, consisting of two surfactant monomers linked by a spacer group, is a thrust research area for gene therapy as non-viral vectors due to their high stability, longer storage on shelves, easiness to produce. A series of phytanyl substituted asymmetric gemini surfactants, phy-3-m (m = 12, 16, and 18) and phy-7NH-m (m = 12, 16, and 18), were rationally designed and synthesized. Due to the bulky nature and increased hydrophobicity of phytanyl branch, phy-3-m surfactants showed much lower values of critical micelle concentration (CMC) compared to their corresponding symmetric m-3-m. Particle size and transmission electron microscopy (TEM) imaging indicate that this type of gemini surfactants tends to form stacked bilayers rather than spherical or rod-like micelles which are typically observed in gemini surfactants with shorter spacers. Phy-3-m surfactants have higher degree of micelle ionization, indicating that the counter ions of the gemini surfactants can be easily replaced by other anionic ions, such as DNA, which is an advantage of phy-3-m used as transgene vectors. To evaluate transfection ability, transfection assays were carried out in OVCAR-3 cells. Transfection complexes formed by a plasmid pVGtelRL, coding enhanced green fluorescence protein (EGFP) gene, phy-3-m, and a neutral lipid, 1,2-Dioleyl-sn-glycerophosphatidylethanolamine (DOPE), at the charge ratios (+/-) of 2:1, 5:1, 10:1, and 20:1, were incubated with OVCAR-3 cells. Treated cells at all charge ratios except 20:1 showed EGFP signals under fluorescence microscopy. Meanwhile, EGFP expression and cell toxicity was quantified using fluorescence-activated cell sorting (FACS). For each gemini surfactant complex, the transfection efficiency and cytotoxicity go through a maximum, occurring at different values of the charge ratio. Considering both transfection efficiency and cytotoxicity, the optimal charge ratio to formulate the complexes containing phy-3-m was found to be 5:1 for in vitro transfection. Compared to a positive control, 16-3-16, phy-3-m showed higher transfection ability and lower cytotoxicity to OVCAR-3 cells. Initial characterization of transfection complexes was investigated by measuring particle size and zeta potential. At all charge ratios, transfection complexes were positively charged, and greater than +30 mV at 5:1 and 10:1, indicating that the complexes would be stable in solution at the ratio above 2:1. Transfection complexes were larger at lower charge ratio, but particle size dropped with increasing charge ratio (+/-). Comparing particle size and zeta potential with transfection efficiency, no correlation between size/zeta potential and transfection ability was observed. The larger particles may enter cells through caveolin-mediated pathway or phagocytosis, and smaller ones through a clathrin-mediated endocytosis. In addition, phase structures of the complexes were investigated using small angle X-ray scattering (SAXS). The complexes containing phy-3-m gemini surfactants were found to be able to adopt multiple phase structures, such as L, HII, and other highly ordered unidentified phase structures. By contrast, L structure was dominant in the transfection complexes formed by 16-3-16. The ability of phy-3-m system to adopt multiple phases appears correlated with their higher transfection efficiency in OVCAR-3 cells.

Breast cancer is the most common cancer in women worldwide. However, the genetic alterations that lead to breast cancer are not fully understood. This thesis aims to identify novel genes of potential mechanistic, diagnostic or therapeutic interest in breast cancers by mutational analysis and whole-genome sequencing. In paper I, sequencing of 36 previously identified candidate genes in 96 breast tumors with patient-matched normal DNA determined the somatic mutation prevalence of these candidate genes and identified additional mutations in Notch, NF-κB, PI3K, and Hedgehog pathways as well as in processes mediating DNA methylation, RNA processing and calcium signaling. In paper II, comparison of massively parallel mate-pair sequencing results of a human genome before and after phi29-mediated multiple displacement amplification (MDA) revealed that MDA introduces structural alteration artifacts, with an emphasis on false positive inversions, and impairs the sensitivity to detect true inversions. Therefore, MDA has limited value in sample preparation for whole-genome sequencing for structural alteration detection. In paper III, massively parallel paired-end sequencing identified gene rearrangements in 15 hormone receptor negative breast cancers. Forty validated rearrangements were predicted to directly affect 30 genes, involved in epigenetic regulation, cell mitosis, signalling transduction and glycolytic flux. RNA interference-based assays revealed the potential roles in cell growth of some affected genes, among which DDX10 was implicated to be involved in apoptosis. In paper IV, a method for statistical evaluation of putative translocations detected by massively parallel paired-end sequencing was proposed. In an application of this method to analyse translocations detected by cancer genome deep paired-end sequencing, 76 putative translocations were classified into four categories, with the majority likely to be caused by mismapping due to repetitive regions. Taken together, this thesis provides insights into genes and pathways mutated in sporadic breast cancer genomes, which broaden our understanding of the genetic basis of breast cancer and may ultimately facilitate the diagnosis and treatment of this disease.

[Truncated abstract] The Phase II clinical candidate, [(trans-Pt(NH3)2Cl)2{μ-trans-Pt(NH3)2(H2N(CH2)6NH2)2}]4+ (BBR3464 or 1,0,1/t,t,t) shows a unique binding profile when compared to the anticancer agent cis-[Pt(NH3)2Cl2] (cisplatin) and dinuclear platinum complexes of the general formula [(trans-Pt(NH3)2Cl)2(H2N(CH2)nNH2)]2+. There is evidence that the increased efficacy of 1,0,1/t,t,t results from the presence of the charged central linker, which can alter the mode of binding to DNA. This alternate binding mode may be due to an electrostatic and hydrogen bonding association of the central platinum moiety in the minor groove that occurs prior to covalent binding (termed “pre-association”) . . . This research shows that 0,0,0/t,t,t is an adequate model to study the pre-association process of 1,0,1/t,t,t and that it binds in the minor groove of DNA. Therefore it is likely that 1,0,1/t,t,t pre-associates in the minor groove of DNA prior to covalent binding. This work supports the conclusions reached in NMR studies of the binding of 1,0,1/t,t,t with the 1,4-GG sequence (Qu et al. JBIC. 8, 19-28 (2003)), which showed simultaneous binding in the major and minor groove. The findings of the current work may also explain the observed binding mode of 1,0,1/t,t,t, which can bind to DNA in both the 3',3' and 5',5' directions (Kasparkova et al. JBC. 277, 48076-48086 (2002)). These unique binding characteristics are thought to be responsible for the increased efficacy of 1,0,1/t,t,t, and in light of the current results the observed binding mode most likely stems from the electrostatic pre-association of the central platinum moiety.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Additional advisors: Ada Elgavish, Vithal Ghanta, Hui-Chen Hsu, Thane Wibbels. Description based on contents viewed June 11, 2008; title from title screen. Includes bibliographical references.

Cancer is a serious threat to human health and is now one of major causes of death worldwide. However, the complexity of the cancer makes the development of new and specific diagnostic tools particularly challenging. A number of different strategies have been developed for biomarker discovery in cancer using microarray data. The problem that typically needs to be addressed is the scale of the data sets; we simply do not have (or are likely to obtain) sufficient data for classical machine learning approaches for biomarker discovery to be properly validated. Obtaining a biomarker that is specific to a particular cancer is also very challenging. The initial promise that was held out for gene microarray work for the development of cancer biomarkers has not yet yielded the hoped for breakthroughs. This work discusses the construction of a strategy for a systematic approach to biomarker discovery validation using lung cancer gene expression microarray data based around non-small cell cancer and in patients which either stayed disease free after surgery (a five year window) or in which the disease progressed and re-occurred. As a means of assisting the validation purposes we have therefore looked at new methodologies for using existing biological knowledge to support machine learning biomarker discovery techniques. We employ text mining strategy using previously published literature for correlating biological concepts to a given phenotype. Pathway driven approaches through the use of Web Services and workflows, enabled the large-scale dataset to be analysed systematically. The results showed that it was possible, at least using this specific data set, to clearly differentiate between progressive disease and disease free patients using a set of biomarkers implicated in neuroendocrine signaling. A validation of the biomarkers identified was attempted in three separately published data sets. This analysis showed that although there was support for some of our findings in one of these data sets, this appeared to be a function of the close similarity in experimental design followed rather than through specific of the analysis method developed.

The plant homeodomain 6 gene (PHF6) is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL); however, its specific functional role in leukemia development remains to be established. Here, we show that loss of PHF6 is an early mutational event in leukemia transformation. Mechanistically, genetic inactivation of Phf6 in the hematopoietic system enhances hematopoietic stem cell (HSC) long-term self-renewal and hematopoietic recovery after chemotherapy by rendering Phf6 knockout HSCs more quiescent and less prone to stress-induced activation. Consistent with a leukemia-initiating tumor suppressor role, inactivation of Phf6 in hematopoietic progenitors lowers the threshold for the development of NOTCH1-induced T-ALL. Moreover, loss of Phf6 in leukemia lymphoblasts activates a leukemia stem cell transcriptional program and drives enhanced T-ALL leukemia-initiating cell activity. These results implicate Phf6 in the control of HSC homeostasis and long-term self-renewal and support a role for PHF6 loss as a driver of leukemia-initiating cell activity in T-ALL. Phf6 controls HSC homeostasis, leukemia initiation, and T-ALL leukemia-initiating cell self-renewal. These results substantiate a role for PHF6 mutations as early events and drivers of leukemia stem cell activity in the pathogenesis of T-ALL. Further, in the hematopoietic system stem cell aging is characterized by accumulation HSCs with poor self-renewal capacity and myeloid biased differentiation. Despite improved appreciation of the cell intrinsic and cell extrinsic mechanisms driving age-associated HSC functional exhaustion, no interventions have proven effective in delaying HSC aging to date. Here, we show that genetic inactivation of the Phf6 prevents age- associated HSC functional decline. Immunophenotypic and single cell transcriptomics profiling demonstrated markedly decreased accumulation of immunophenotypically-defined HSCs, reduced myeloid bias and decreased upregulation of transcriptional programs associated with stem cell aging in old hematopoietic-specific Phf6 knockout mice. Functionally, Phf6 knockout HSCs from aged mice demonstrated increased hematopoietic reconstitution capacity and preservation of lymphoid differentiation potential. Mechanistically, analysis of long-term HSCs from old Phf6 knockout mice revealed reduced levels of ongoing DNA damage and downregulation of genotoxic stress-induced transcriptional signaturesconducive of HSC aging. These results identify Phf6 as an important epigenetic regulator of HSC aging, whose inactivation counters the functional deterioration of HSC activity induced with age. Microprotein encoding genes are a class of genes which encode poly-peptide gene products comprised by 100 or fewer amino acids. Until recently, many such genes had been considered of low- or no-coding potential given the technical limitations associated with identification of such small proteins. However, recently prominent examples of microprotein encoding genes have been reported with a wide variety of regulatory functions. Therefore, we hypothesized that novel microprotein genes exist within the human genome with oncogenic and tumor suppressive roles. To test this hypothesis, we developed a pipeline for identification of microproteins based on conservation of the open reading frame. Leveraging PLATE-seq to generate a high-dimensional readout in a loss-of-function screen, we then screened for microproteins with potential tumor suppressive or oncogenic function. From this, we identified a brain- specific, 65 amino-acid microprotein encoded in within LINC00617 (TUNAR) which is conserved at the protein level across vertebrates. We experimentally validated the protein-level expression of the TUNAR microprotein. In vitro and in vivo knockout and overexpression experiments demonstrate a role for TUNAR as a tumor suppressor in glioma. Specifically, we show that loss of Tunar in the mouse brain results in lower expression of Fermt1 and genes in the integrin signaling pathway. Consistently, overexpression of TUNAR in human glioblastoma multiforme cell lines significantly impeded cellular migration suggesting a role of Tunar in glioma cell dissemination. Finally, human glioma sequencing and copy number data were mined to determine the prognostic significance of the loss of TUNAR in human gliomas. These analyses demonstrated that copy number loss of TUNAR is associated with poor outcomes in lower grade gliomas and that TUNAR expression and glioma grade are strongly, negatively correlated suggesting that TUNAR likely has tumor suppressive effects in human glioma.

京都大学 / 新制・課程博士 / 博士(情報学) / 甲第24730号 / 情博第818号 / 新制||情||138(附属図書館) / 京都大学大学院情報学研究科知能情報学専攻 / (主査)教授 阿久津 達也, 教授 山本 章博, 教授 岡部 寿男 / 学位規則第4条第1項該当 / Doctor of Informatics / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / In this thesis, I investigated the preclinical utility and antitumor efficacy of TRAIL delivered by Ad-IU2, a prostate-specific replication-competent adenovirus (PSRCA), against androgen-independent prostate cancer. Through transcriptional control of adenoviral early genes E1a, E1b and E4, as well as TRAIL by two bidirectional prostate-specific enhancing sequences (PSES), expression of TRAIL as well as adenoviral replication was limited to prostate-specific antigen and prostate-specific membrane antigen (PSA/PSMA)-expressing cells. Ad-IU2 replicated efficiently in and was restricted to PSA/PSMA-positive prostate cancer cells and induced 5-fold greater apoptosis in androgen-independent CWR22rv and C4-2 prostate cancer cells than the PSRCA control not expressing TRAIL. Ad-IU2 exhibited superior killing efficiency in PSA/PSMA-positive prostate cancer cells at doses 5 to 8-fold lower than that required by a non-TRAIL expressing PSRCA to produce a similar effect. This enhanced cytotoxic effect was not observed in non-prostatic cells, however. As an enhancement of its therapeutic efficacy, Ad-IU2 exerted a bystander effect through either direct cell-to-cell contact or soluble factors present in conditioned media from Ad-IU2-infected cells. In vivo, Ad-IU2, as compared to a control PSRCA, markedly suppressed the growth of subcutaneous CWR22rv xenografts at six weeks post-treatment (3.1 vs. 17.1-fold growth of tumor). The treatment of androgen-independent prostate cancer with Ad-IU2 prior to external beam radiation therapy (EBRT) significantly reduced clonogenic survival with dose reduction factors of 4.91 and 2.43 for CWR22rv and C4-2 cells, respectively. Radio-sensitization by Ad-IU2 was restricted to PSA/PSMA-positive cells. Combinatorial radio-gene therapy resulted in accumulation of cells in G1 phase and a perturbation of the radiation-induced G2 phase arrest. This multi-modal approach combining viral lysis, apoptosis-inducing gene therapy, and radiation therapy could have great impact in achieving complete local tumor control while reducing radiation dose and associated treatment morbidities. This would result in improvement of the clinical outcome of patients with high risk prostate cancer.

MicroRNAs (miRNAs) are short non-coding RNAs that function in post-transcriptional gene silencing and mRNA regulation. Although the number of nucleotides of miRNAs ranges from 17 to 27, they are mostly made up of 22 nucleotides. The expression of miRNAs changes significantly in cancer, causing protein alterations in cancer cells by preventing some genes from being translated into proteins. In this research, a structural analysis of 587 miRNAs that are differentially expressed in myeloid cancer was carried out. Length distribution studies revealed a mean and median of 22 nucleotides, with an average of 21.69 and a variance of 1.65. We performed nucleotide analysis for each position where Uracil was the most observed nucleotide and Adenine the least observed one with 27.8% and 22.6%, respectively. There was a higher frequency of Adenine at the beginning of the sequences when compared to Uracil, which was more frequent at the end of miRNA sequences. The purine content of each implicated miRNA was also assessed. A novel motif analysis script was written to detect the most frequent 3–7 nucleotide (3–7n) long motifs in the miRNA dataset. We detected CUG (42%) as the most frequent 3n motif, CUGC (15%) as a 4n motif, AGUGC (6%) as a 5n motif, AAGUGC (4%) as a 6n motif, and UUUAGAG (4%) as a 7n motif. Thus, in the second part of our study, we further characterized the motifs by analyzing whether these motifs align at certain consensus sequences in our miRNA dataset, whether certain motifs target the same genes, and whether these motifs are conserved within other species. This thorough structural study of miRNA sequences provides a novel strategy to study the implications of miRNAs in health and disease. A better understanding of miRNA structure is crucial to developing therapeutic settings.

Simple Summary Gene therapy is a novel approach to treat diseases by introducing corrective genetic information into target cells. Adeno-associated virus vectors are the most frequently applied gene delivery tools for in vivo gene therapy and are also studied as part of innovative anticancer strategies. Here, we report on the natural preference of AAV2 vectors for hepatocellular carcinoma (HCC) compared to nonmalignant liver cells in mice and human tissue. This preference in transduction is due to the improved intracellular processing of AAV2 vectors in HCC, resulting in significantly more vector genomes serving as templates for transcription in the cell nucleus. Based on this natural tropism for HCC, novel therapeutic strategies can be designed or existing therapeutic approaches can be strengthened as they currently result in only a minor improvement of the poor prognosis for most liver cancer patients. Abstract Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current—or develop novel—strategies for treating HCC.