Les dysferlinopathies sont des maladies musculaires dues à une déficience en protéine dysferline, codée par le gène DYSF. Dans ce travail de thèse, trois approches thérapeutiques ont été évaluées pour ces pathologies, sur des modèles cellulaires et murins. Un variant transcriptionnel court de la dysferline a été vectorisé dans un AAV8r et injecté dans le modèle murin Bla/J, déficient en dysferline. L’analyse des muscles des animaux traités montre une augmentation de la résistance des fibres musculaires au stress mécanique, mais n’apporte pas de correction histologique. Cette étude souligne également la toxicité de cette miniprotéine. L’anoctamine 5, impliquée dans des pathologies et des activités similaires à la dysferline, a été testée en tant que protéine compensatrice. L’anoctamine 5 surexprimée dans le modèle Bla/J ne permet pas la restauration d’un phénotype normal. La compensation de DYSF par ANO5 n’est donc pas une voie thérapeutique à exploiter pour les dysferlinopathies. Enfin, une thérapie génique par chirurgie de l’ARN dysferline a été évaluée en utilisant le trans-épissage médié par le splicéosome (SMaRT). La preuve de principe de la reprogrammation d’un minigène dysferline a été faite (ARN et protéine trans-épissée obtenus in vitro). L’efficacité du SMaRT dans un contexte endogène s’est en revanche révélée faible, et n’a pas permis la restauration d’une protéine dysferline fonctionnelle dans des myoblastes humains. De plus, l’observation de l’expression de protéines directement à partir du RTM (RNA-trans-splicing molecule) a fait apparaître des limites à l’utilisation du SMaRT pour la thérapie génique, et en particulier pour les dysferlinopathies. / Dysferlinopathies are muscular diseases due to mutations in DYSF gene, inducing dysferlin protein deficiency. In this thesis, three therapeutic approaches have been investigated for these pathologies, on cell or mice models. A short transcriptional dysferlin variant has been injected into Bla/J dysferlin deficient mouse model, using AAV8r vector. Muscle fibers of treated animals displayed an increased resistance to mechanical stress without therapeutic benefit. These experiments also pointed out the toxicity of this strategy. A protein compensation approach has been tested using anoctamin 5, known to be involved in pathologies and activities similar to dysferlin’s ones. AAVr mediated Anoctamin 5 overexpression in Bla/J model does not rescue their muscle phenotype. Overexpression of ANO5 does not seem to be a valuable therapeutic strategy for dysferlin deficiency. Dysferlin RNA surgery was evaluated as a possible genetic therapy using Spliceosome-Mediated RNA Trans-splicing (SMaRT). On a Minigene target, SMaRT is able to induce RNA reprogramming by trans-splicing, and produce the corresponding protein. But efficiency is by far decreased in endogenous context and not good enough to restore functional dysferlin in human myoblasts. Moreover, we described proteins resulting from RNA-trans-splicing molecule (RTM) self-expression, limiting the value of SMaRT as therapeutic strategy, especially for dysferlinopathies.

Due to recent advances in molecular biology and genomic research, numerous diseases have been given their genetic identities for which gene therapy may be a possible prescription. Gradually, the development of viral and non-viral vectors to translocate genes has become a bottleneck. For non-viral vectors, polyethylenimine (PEI) is considered as a potential vector candidate for gene delivery because of its ability to compact DNA and its intrinsic pH buffering capacity. PEI and its derivates have been widely tested in both in-vitro and in-vivo gene transfection experiments. The progress is limited due to the lack of a better understanding of the intracellular mechanism. So far, their cytotoxicity is relatively high and gene transfection efficiency is low. This study was designed to modify PEI and optimize its cytotoxicity and gene transfection efficiency. / During the complexes formation, both LLS and zeta-potential were used to follow the process. The results showed that most of anionic DNA are complexed by cationic PEI-based polymers when the molar ratio of nitrogen from PEI to phosphate from DNA (N:P) reaches ∼3, but the gene transfection reaches the highest efficiency when N:P ∼10. When N:P > 3, there exist two population of PEI chains in the solution mixture: bound to DNA and free in the solution. The bound PEI chains condense and protect DNA. Our current study confirms that it is those free PEI chains that play a vital role in promoting the gene transfection. Our preliminary data shows that the promotion mainly occurs in the intracellular space. The detailed mechanism is still lacking at this moment. Nevertheless, our finding leads to a totally different way in the development of non-viral vectors. / Further, we grafted PEI with polyethylene glycol (PEG), respectively via a reductive disulfide -S-S- and a non-degradable -C-C- bond to form two copolymer vectors. A comparative study shows that the polyplexes formed between the two copolymers and DNA are more stable than that formed between unmodified PEI and DNA under the physiological condition, presumably because the grated PEG chains form a protective hydrophilic shell on the PEI/DNA polyplexes. However, PEGylation reduces the internalization of the copolymer/DNA polyplexes in in-vitro experiments. For the two copolymer vectors, PEG-SS-PEI is 2-8 times more effective than its counterpart (PEG-CC-PEI) in the gene transfection, presumably due to the cleavage of the grafted PEG chains inside the reductive cytosol, which promotes the release and translocation of DNA. Our results demonstrate that using the disulfide as a linker is a promising approach to overcome the PEGylation dilemma in the development of low cytotoxic and high efficient non-viral polymeric vectors. / It has been known that short PEI chains are less toxic, but long chains are more effective in gene transfection. Therefore, we decide to use the disulfide bond (-S-S-) to extend short PEI chains to increase efficiency and also utilize the reductive cytosol environment to cleave such extended PEI chains to reduce their cytotoxicity inside the cell. Laser light scattering (LLS) was used to in-situ monitor the linking reaction between short PEI chains (M w = 2000 g/mol) and dithiobis(succinimidyl propionate) (DSP). The molar mass and crosslinking degree of the extended PEI chains was controlled by either the amounts or the adding rate of DSP. A comparative study of two linked PEI samples (PEI-7K-L and PEI-400K-L, respectively with M w = 6.5 x 103 and 3.8 x 10 5 g/mol) reveals that cytotoxicity and gene transfection efficiency of such extended PEI chains are related to the chain length and structure. Namely, PEI-7K-L with an extended chain structure is less cytotoxic and 2--10 times more effective in the gene transfection than the "golden standard" (PEI25K) and the widely used commercial vector, Lipofectamine 2000RTM. Comparatively, PEI-400K-L with a spherical microgel structure is ineffective in spite of its non-toxicity. Our study clearly demonstrates that a proper control of the chain length and structure is important. / by Deng, Rui. / Adviser: Chi Wu. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Yue, Yanan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 62-63). / Abstract also in Chinese. / ABSTRACT (Chinese) --- p.i / ABSTRACT --- p.iii / CONTENT --- p.v / ACKNOWLEDGMENT --- p.vii / ABBREVIATIONS --- p.viii / Chapter CHAPTER 1 --- Introduction and Background / Chapter 1.1 --- Methods of Gene Delivery --- p.1 / Chapter 1.1.1 --- Viral Delivery Systems --- p.2 / Chapter 1.1.2 --- Non-Viral Delivery Systems --- p.3 / Chapter 1.2 --- The Gene-delivery Problems --- p.7 / Chapter 1.2.1 --- Extracellular Barriers --- p.8 / Chapter 1.2.2 --- Intracellular Barriers --- p.10 / Chapter 1.3 --- Polymer-Mediated Systems for Gene Delivery --- p.13 / Chapter 1.3.1 --- Polyethylenimine (PEI)-Based Vectors --- p.13 / Chapter 1.3.2 --- Cyclodextrin-Based Vectors --- p.15 / Chapter 1.4 --- Objective and Main Achievements --- p.16 / Chapter 1.5 --- References --- p.18 / Chapter CHAPTER 2 --- Effect of Free Polyethylenimine-Mediated Polycations on Gene Delivery: Fundamentals and Vital Factors / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Experimental Section --- p.25 / Chapter 2.3 --- Results and Discussions --- p.29 / Chapter 2.3.1 --- Fundamentals --- p.29 / Chapter 2.3.2 --- Vital Factors for the Efficacy of Free Chains --- p.37 / Chapter 2.4 --- Conclusions --- p.42 / Chapter 2.5 --- References --- p.42 / Chapter CHAPTER 3 --- Effect of Free Polyethylenimine-Mediated Polycations on Gene Delivery: Mechanistic Study / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Experimental Sections --- p.46 / Chapter 3.3 --- Results and Discussion / Chapter 3.3.1 --- Potential Effect of Free PEI Chains on Cellular Uptake --- p.49 / Chapter 3.3.2 --- Potential Effect of Free PEI Chains on Endolysosomal Release --- p.51 / Chapter 3.3.3 --- Exploration on Proton Sponge Hypothesis --- p.53 / Chapter 3.3.4 --- Interactions of PEI-based Polycations and Phospholipid Membranes --- p.55 / Chapter 3.4 --- Conclusions --- p.61 / Chapter 3.5 --- References --- p.62

Yes / The Sphingosine-1-phosphate receptor 1 (S1PR1) and β1-adrenergic receptor (β1AR) are G protein-coupled receptors (GPCRs) expressed in the heart. These two GPCRs have opposing actions on adenylyl cyclase due to differential G protein-coupling. Importantly, both of these receptors can be regulated by the actions of GPCR kinase-2 (GRK2), which triggers desensitization and down-regulation processes. Although, classical signaling paradigms suggest that simultaneous activation of β1ARs and S1PR1s in a myocyte would simply be opposing action on cAMP production, in this report we have uncovered a direct interaction between these two receptors with a regulatory involvement of GRK2. In HEK293 cells overexpressing both β1AR and S1PR1, we demonstrate that β1AR down-regulation can occur after sphingosine 1-phosphate (S1PR1 agonist) stimulation while S1PR1 down-regulation can be triggered by isoproterenol (βAR agonist) treatment. This cross-talk between these two distinct GPCRs appears to have physiological significance since they interact and show reciprocal regulation in mouse hearts undergoing chronic βAR stimulation and also in a rat model of post-ischemic heart failure (HF). We demonstrate that restoring cardiac plasma membrane levels of S1PR1 produce beneficial effects counterbalancing deleterious β1AR overstimulation in HF.

Pulmonary arterial hypertension is a rare, chronic disease characterized by progressive remodeling of the pulmonary vasculature. Historically, prognosis has been very poor with relatively low 3-year survival rates. Common symptoms include fatigue and shortness of breath upon exercise, chest pain, and syncope. Patients exhibit increased pressure and resistance in pulmonary arteries due to fibrosis, vessel narrowing, and elevated levels of vasoconstrictive agents; diagnosis is confirmed by right heart catheterization. Reduced blood flow through the pulmonary vasculature not only reduces the amount of oxygenated blood available for the systemic circulation, but increases afterload on the right ventricle and, if left untreated, ultimately causes right ventricular heart failure. In the past, few medications were available to pulmonary arterial hypertension patients. However, recent advancements in our molecular understanding of the disease have led to the development of new therapeutic options that show promise of slowing, or in some cases reversing, disease progression. Currently available treatments have been shown to significantly improve 3-year survival rates and help promote a better quality of life for patients. While an exact molecular or genetic mechanism of disease progression is not yet known, several studies have noted the presence of dysfunctional endothelial cells and an imbalance in molecular modulators of the pulmonary vasculature. Specifically, patients exhibit chronically low levels of vasodilating agents such as prostacyclin and nitric oxide. In addition, there is a heightened vasoconstrictive effect due to elevated endothelin-1 and thromboxane A2. Drugs have been developed to target these signaling pathways and show considerable promise and efficacy for managing pulmonary hypertension in patients. Although these therapeutics have been shown to significantly improve survival rates and symptoms, many have complex and inconvenient administration protocols and a host of adverse side effects. Moreover, many require monitoring or frequent follow up visits due to their off-target effects. Recent innovative advancements in pulmonary arterial hypertension pharmaceuticals hope to deliver safe, efficacious treatment options to patients debilitated by this chronic disease.

惡性腫瘤嚴重威脅著人類的身體健康，其治療也成為人類關注的焦點。傳統的化學療法和放射療法由於缺乏特異性，取得療效的同時往往也帶來較大的毒副作用。隨著對腫瘤發生發展分子機制認識的不斷深入，腫瘤的基因治療已成為攻克和治愈腫瘤最具希望和挑戰的研究領域。近年來研究發現骨髓間充斥幹細胞(MSCs)可被募集至腫瘤或損傷部位并參與腫瘤生長或組織修復，研究證明間充斥幹細胞通過靜脈注入帶瘤鼠（比如乳腺癌、膠質瘤、結腸癌及黑色素瘤）體內后，特異性的分佈于生長中的腫瘤中。這種特異性向腫瘤組織趨化轉移的特性使得骨髓間充斥幹細胞成為腫瘤基因靶向治療的載體的理想細胞。酶蛋白基因如單純皰疹病毒胸苷激酶(HSV-TK)可以使一些無毒或低毒的前藥轉化為強細胞毒性物質，殺死腫瘤細胞。我們前期實驗結果表明，通過遺傳改造后的表達TK基因的MSCs在GCV的存在下，具有殺傷腫瘤細胞抑制腫瘤生長的能力。但沒有改造的MSCs遷移至腫瘤之後可能會分化成成纖維細胞或者腫瘤基質細胞等支持腫瘤生長，但其命運和影響到底如何，我們怎麼樣進一步促進其向腫瘤的遷移以提高殺傷腫瘤的效率是本研究需要解決的問題。 / 本研究擬採用免疫螢光組織化學技術和分子生物學等技術研究和觀察MSCs對腫瘤（以乳腺癌，前列腺癌為例）的趨化過程及其在腫瘤生長中的作用，在在此基礎上研究促進攜帶HSV-TK自殺基因的MSCs的腫瘤靶向性細胞治療策略，採用分子和細胞生物學等方法評估其對荷瘤鼠體內腫瘤殺傷的原理，為利用TK-MSCs腫瘤的靶向治療奠定基礎。 / 研究結果顯示體外共培養的條件下，小鼠骨髓間充斥幹細胞可促進小鼠乳腺癌細胞增長，且增長速度同培養體系中間充斥幹細胞數目呈正相關。將兩種細胞混合注射于裸鼠體內，相比共注射小鼠皮膚成纖維細胞，間充斥幹細胞可促進體內腫瘤生長。使用人胚胎骨髓間充斥幹細胞和前列腺癌細胞可得出類似的效果。將腫瘤組織切片分析發現間充斥幹細胞促進體內腫瘤細胞增殖的同時，提高了腫瘤組織內血管密度。體外實驗發現共培養前列腺癌細胞和間充斥幹細胞可促進血管生成且在間充斥細胞內同血管增生相關的蛋白表達量都有相應提高，進一步證實間充斥幹細胞可能通過促進血管增生從而促進腫瘤生長。另外，我們利用人胚胎來源的骨髓間充斥幹細胞建立了穩定表達TK自殺基因的細胞系，且在GCV的存在下具有抑制腫瘤生長的能力。為了促進它們向腫瘤遷移的能力，我們用多柔比星預處理腫瘤細胞，和沒處理過的對照組相比，能增強對表達TK的間充斥幹細胞的招募能力。且在聯合利用多柔比星和TK的條件下，腫瘤生長能得到較大程度的抑制，這種抑制作用强於單獨使用多柔比星和表達自殺基因的間充斥幹細胞系統。初步認為是多柔比星的處理能增強腫瘤組織內炎性介質的分泌從而增強間充斥幹細胞的遷移達到增強自殺基因系統殺死腫瘤細胞的目的。 / 總的來說，雖然間充質幹細胞對腫瘤的生長存在一定的促進作用，但我們仍能對其進行遺傳改造，且在其它抗腫瘤藥的配合下達到最大的抗腫瘤效果。 / Eradication of cancer, especially when it has metastasized is extremely difficult and conventional cancer therapies are simply unable to specifically target tumors/cancers, thus causing unwanted side effects and complications. Recently, it has been shown that bone marrow mesenchymal stem cells (MSCs) are able to migrate specifically to tumors and contribute to the formation of tumor-associated stroma. These properties make MSCs good candidates as anti-tumor agent delivery vehicles and lead to a great deal of interest in the possibility of genetically modifying MSCs to express anticancer molecules and using them as specific targeted anticancer agents. We and others have showed that MSCs have the ability to migrate towards various cancer cells including breast, colon, fibrosarcoma and prostate cancer cells. Suicide gene therapy is widely used in cancer gene therapy. When stably infected with herpes simplex virus thymidine kinase gene by lentivirus, TK-MSCs maintained their MSCs characters and tumor tropism potential and significantly inhibited tumor growth, in the presence of the pro-drug ganciclovir (GCV). Improve MSCs homing to tumor tissue as anti-tumor gene therapy vehicles and maximizing their tumor killing effects is highly warranted. Furthermore, MSCs interact with tumor cells in numerous ways, which have the potential to support or suppress tumor growth. Therefore the fate and role of MSCs engrafted in tumor sites need to be clarified in order to making better use of these cells as anti-cancer agent delivery vehicles. / The aims of the current study are: (1) to study the role and fate of MSCs homed into the tumors; (2) to establish human bone marrow MSCs that stably express the TK genes; (3) to investigate the methods that enhance the anti-tumor efficiency of TK-MSCs. / In this study, bone marrow-derived mesenchymal stem cells from mice or human fetus were isolated and characterized. Effects of BM-MSCs on tumor cell proliferation in vitro were analyzed in a co-culture system with mouse breast cancer cell 4T1 cells. Both co-culture with BM-MSCs and treatment with MSC-conditioned medium led to enhanced growth of 4T1 cells. Co-injection of 4T1 cells and MSCs into nude mice led to increased tumor size compared with injection of 4T1 cells alone. Identical experiments using human prostate cancer cell DU145 cells and hBM-MSCs instead of 4T1 cells and mBM-MSCs yielded similar results. Compared with tumors induced by injection of cancer cells alone, tumor vessel area was greater in tumors from co-injection of 4T1 or DU145 with BM-MSCs, which correlated with decreased central tumor necrosis and increased tumor cell proliferation. Furthermore, both conditioned medium from co-cultures of hBM-MSCs and DU145 cells or hBM-MSCs alone was able to induce angiogenesis in human umbilical vein endothelial cells (HUVEC). When hBM-MSCs exposed to DU145 cells environment, the expression of markers associated with neovascularization (α-SMA, VEGF, TGF-β and IL6) were increased. Together, these results indicate that MSCs promote tumor growth both in vitro and in vivo and suggest that tumor promotion in vivo may be attributable in part to enhanced angiogenesis. / Immortalized human fetal bone marrow-derived MSCs (hfBMSCs) expressing herpes simplex virus thymidie kinase was established by conventional lentiviral transduction method. Functional expression of TK was evaluated by cytotoxicity in the presence of its prodrug GCV. SV40-TK-hfBMSCs exhibited comparable proliferation, surface phenotype expression, multi-differentiation potential and tumor-tropic migration ability as hfBMSCs. By measurement of tumor volume, repeated injection of the SV40-TK-hfBMSCs and subsequent consecutive GCV administration could suppress tumor growth in DU145 or PC3 human prostate tumor xenograft nude mice model without causing weight loss. However, its clinical applications are still limited. Alternative strategies have been pursued in this study by the use of combination therapy with cytotoxic chemotherapy to improve the overall efficacy of the TK-hfBMSCs/GCV system. / TK-hfBMSCs/GCV was evaluated alone or combined with low-dose doxorubicin in human prostate carcinoma DU145 xenografts in nude mice, testing for effects on local growth and overall survival. Tissues were evaluated through immunofluorescence and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining (TUNEL) for treatment effects on tumor cell proliferation and apoptosis. Transwell migration assay was used to access the migration ability of TK-hfBMSCs to tumor cells upon doxorubicin treatment and caspase-3 activity was conducted for test the tumor cells apoptosis under TK-hfBMSCs/GCV, doxorubicine, or combination of the two compound treatments respectively. Only minimal growth inhibition was observed in DU145 after treatment with TK-hfBMSCs/GCV or doxorubicin alone at doses and time points as indicated. In contrast, the combination of both agents resulted in a significant growth inhibition. Caspase-3, plays a central role in the execution-phase of cell apoptosis, was increased by TK-hfBMSCs/GCV or doxorubicine and also to a much greater extent by the combination treatment. Treatment by TK-hfBMSCs/GCV resulted in only a slight decrease in tumor growth compared with controls. Treatment with low-dose doxorubicin alone resulted in a small, nonstatistically significant decrease in tumor growth; In contrast, combined low-dose doxorubicin and TK-hfBMSCs/GCV was markedly inhibitory compared with control, doxorubicin alone, or TK-hfBMSCs/GCV alone. During the whole treatment process, no significant weight loss was observed. Furthermore, combined therapy induced increased area of necrosis, significant apoptosis and decreased tumor cell proliferation in treated tumors. Taken together, low dosage of doxorubicin could be used in combination with TK-hfBMSCs based suicide gene therapy. / In conclusion, we have demonstrated that BM-MSCs could increase the growth of human prostate cancer and mouse breast cancer. The promotion effect may partly attribute to the increased expression of pro-angiogenic factors in BM-MSCs in tumor microenvironment and subsequent enhancement in angiogenesis and tumor growth. The current study also suggests combination of TK-hfBMSCs/GCV and doxorubicin was more effective in inhibiting prostate cancer cells growth than TK-hfBMSCs/GCV or doxorubicin alone. Although many problems need to be resolved for further application, our study provided the possibility of a new strategy of suicide gene-based therapy accompanied by low dosage of chemotherapy in treating prostate cancer. Therefore MSCs were described as a “double-edged sword in their interaction with tumors. However, if MSCs are suitably engineered with anticancer genes they could be employed as a valuable “single-edged sword“ against cancers. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Ting. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 120-158). / Abstracts also in Chinese. / ACKNOWLEDGEMENT --- p.ii / PUBLICATIONS --- p.vii / ABSTRACT --- p.xiii / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1 --- Mesenchymal stem cells (MSCs) --- p.2 / Chapter 1.2 --- Tumor microenvironment and involvement of MSCs in tumor establishment --- p.5 / Chapter 1.3 --- Tumors-tropic characteristics of MSCs --- p.15 / Chapter 1.4 --- Impact of MSCs on in vivo tumors --- p.21 / Chapter 1.5 --- In vivo imaging demonstrating MSCs tumor-homing potentials --- p.25 / Chapter 1.6 --- Evidence for use of MSCs as anti-tumor agents delivery vehicles --- p.26 / Chapter 1.7 --- Homing strategies to enhance efficacy and safety of MSCs therapy --- p.32 / Chapter 1.8 --- Summary --- p.35 / Chapter CHAPTER 2 --- Hypotheses, Objectives and Study Design --- p.35 / Chapter 2.1 --- Hypothesis --- p.35 / Chapter 2.2 --- Objective --- p.36 / Chapter 2.3 --- Study design --- p.37 / Chapter CHAPTER 3 --- Bone Marrow-derived Mesenchymal Stem Cells Promote Growth and Angiogenesis of Breast and Prostate Tumors (Study I) --- p.40 / Chapter 3.1 --- Materials and Methods --- p.40 / Chapter 3.2 --- Results --- p.49 / Chapter 3.3 --- Discussion --- p.64 / Chapter 3.4 --- Conclusions --- p.67 / Chapter CHAPTER 4 --- Immortalized human fetal bone marrow-derived mesenchymal stem cell expressing anti-tumor suicide gene for anti-tumor therapy in vitro and in vivo (Study II) --- p.68 / Chapter 4.1 --- Materials and Methods --- p.68 / Chapter 4.2 --- Results --- p.73 / Chapter 4.3 --- Discussion --- p.85 / Chapter CHAPTER 5 --- Enhanced antitumor effects by combination therapy using mesenchymal stem cell expressing anti-tumor suicide gene and Doxorubicin in a xenograft mouse model (Study III) --- p.89 / Chapter 5.1 --- Materials and Methods --- p.89 / Chapter 5.2 --- Results --- p.97 / Chapter 5.3 --- Discussion --- p.111 / Chapter CHAPTER 6 --- General discussion and conclusions --- p.116 / Chapter 6.1 --- General discussion --- p.116 / Chapter 6.2 --- General conclusions --- p.119 / FUNDING --- p.120 / REFERENCE --- p.120

全球人類健康的頭號殺手--腫瘤目前仍是難以攻克的醫學難題。腫瘤的發生是一個復雜的過程，主要由促癌基因的異常增多或激活及抑癌基因（TSG）的缺失或功能喪失的累積效果導致。近年來基於非基因序列改變所致基因表達水平變化的表觀遺傳學的研究進展表明，啟動子區CpG島甲基化所致的表觀遺傳沉默是抑癌基因轉錄失活的重要機制。Ras和Wnt信號轉導通路在癌病的發生和發展過程中均起到重要的作用，因此針對該兩種信號通路的拮抗因子的表觀遺傳調控及功能學研究將為我們提供有研究及應用前景的候選抑癌基因。 / 作為一種重要的原癌基因，Ras家族基因具有致癌活性的點突變及其導致的過度激活的Ras信號通路被發現廣泛存在於大約30%的人類腫瘤中。然而在一些缺乏Ras基因突變的腫瘤類型中，持續激活的Ras信號通路仍然普遍存在並具有重要作用，昭示著除了Ras基因點突變以外的信號轉導異常激活的機制。與GTP的結合可激活Ras，而RasGAP家族蛋白可通過水解GTP達到使Ras失活的作用。通過采用微陣列比較基因組雜交(aCGH)的實驗手段我們發現6p21.3染色體區具有半接合子缺失， 並於此區域發現了候選抑癌基因RASA5。在以往的研究報道中，RASA5被命名為SynGAP且其功能研究僅限於神經系統。我們的研究發現不同於RasGAP家族的其它基因RASA2-4，RASA5廣泛表達於人類正常器官組織中，並特異性地在腫瘤細胞，特別是鼻咽癌(NPC)，食管鱗狀上皮細胞癌(ESCC)和乳腺癌這些具有野生型Ras基因但Ras信號通路仍被過度激活的細胞中被表觀遺傳沉默。RASA5的異位表達可有效促進腫瘤細胞的雕亡，抑制腫瘤細胞的生長、遷移及“幹性（stemness）“。同時，使用siRNA敲除內源性RASA5可以激發細胞的克隆形成及上皮-間質(EMT)轉化。RASA5的抑癌功能是通過調低Ras-GTP水平並進而抑制其下遊信號通路的活性實現的。過量表達具有致癌活性的點突變的Ras或RasGAP結構域缺失均可部分逆轉這種抑癌作用。此項研究首次證明了RASA5的抑癌功能。 / Wnt/Dvl/β-catenin信號轉導通路在人類腫瘤中存在廣泛的異常激活。我們發現DACT (Dpr/Frodo)家族成員TUSC-T2的表觀遺傳沉默是一種普遍存在於人類腫瘤中的現象。TUSC-T2編碼一種胞質蛋白，外源性表達TUSC-T2可促進腫瘤細胞雕亡並導致腫瘤細胞的克隆形成能力下降。TUSC-T2可與Dvl蛋白結合並下調其活化水平，從而保護GSK-3β蛋白不被Dvl蛋白抑制。GSK-3β可與Axin及APC蛋白形成蛋白質復合物，該復合物可捕捉並降解細胞內信號分子β-catenin。TUSC-T2的過量表達可以抑制β-catenin的激活及其向細胞核內的富集，並進一步阻止β-catenin在細胞核內與Lef/Tcf轉錄因子家族的作用及下遊特定原癌基因，例如c-Myc, CCND1及Fibronectin的表達。因此TUSC-T2具有抑制腫瘤細胞增殖、遷移及上皮-間質(EMT)轉化的作用。 / 綜上所述，我們的研究結果表明RASA5及TUSC-T2是具有抑癌功能的Ras或Wnt/Dvl/β-catenin信號轉導通路抑制因子，其表觀遺傳沉默導致的轉錄失活對於腫瘤的發生發展具有重要意義。同時，針對這兩種抑癌基因的進一步研究將為我們提供富有應用前景的腫瘤標記物。值得註意的是，RASA5課題的研究開創性地闡明了Ras信號通路的拮抗因子的表觀遺傳沉默是一種Ras信號轉導通路於腫瘤細胞中異常激活的新機制。 / Cancer is the top killer of the world, as well as the medical problem difficult to overcome. The conversion of a normal cell to a cancer cell is usually caused by upregulation of oncogenes and downregulation of tumor suppressor genes (TSGs). Epigenetic silencing has been proved to be important in TSGs inactivation, often through methylation of CpG-rich promoter regions. Ras and Wnt signaling pathways are both important for the tumorigenesis, epigenetic and functional studies of antagonists to Ras and Wnt signaling would provide us with candidate TSGs. / Ras is a well-known oncogene. Aberrant mutations of Ras genes occur in approximately 30% of human tumors, causing constitutively activated Ras signaling. However, in certain types of tumors with wild type Ras genes, abnormally activated Ras signaling is still a common and critical event, suggesting alternative mechanisms for Ras signaling hyperactivation. Ras is active when it is bound to GTP, while the hydrolysis of bound GTP and inactivation of Ras is catalyzed by Ras GTPase activating proteins (RasGAPs). Using 1-Mb array CGH (aCGH), we refined a small hemizygous deletion at the 6p21.3 chromosome region that contains a RasGAP family member gene RASA5, which used to be named as SynGAP and studied only in the neuron systems. We demonstrated that RASA5, rather than other RasGAP family members RASA2-4, is broadly expressed in human normal tissues while frequently epigenetically silenced in multiple tumors, especially in certain tumor types such as nasopharyngeal (NPC), esophageal (ESCC) and breast carcinomas (BrCa) with wild-type Ras while Ras cascade is still constitutively active. Ectopic expression of RASA5 led to apoptosis, growth and migration inhibition, as well as ‘stemness’ repression of tumor cells. Meanwhile, knockdown of RASA5 by siRNA promoted the tumor cell colony formation as well as epithelial-mesenchymal transition (EMT). The tumor-suppressive function of RASA5 was exerted through downregulating Ras-GTP level and further inactivating Ras signaling. Such an inhibitory effect could be partially abrogated in the presence of mutated, activated Ras or by deletion of the RasGAP domain. For the first time, our study refined the role of RASA5 as a tumor suppressor. / Wnt/DVL/β-catenin signaling pathway is aberrantly activated in a wide range of human cancers. We identified a DACT (Dpr/Frodo) family member TUSC-T2 as an epigenetically downregulated gene in human tumors. TUSC-T2 encodes a punctate cytoplasmic protein. Ectopic expression of TUSC-T2 dramatically inhibited tumor cell colony formation in silenced tumor cell lines, mainly through inducing apoptosis. TUSC-T2 interacts and downregulates Dishevelled (Dvl) protein, thus protecting glycogen synthase kinase 3β (GSK-3β) from inactivation by Wnt/Dvl and allowing GSK-3β to form a complex with Axin and APC to promote the phosphorylation and proteasomal degradation of β-catenin. Overexpression of TUSC-T2 disrupted β-catenin activation and accumulation in nuclei, thus preventing its binding to transcription factors of the Lef/Tcf family. This caused the downregulation of β-catenin target oncogenes such as c-Myc, CCND1 and Fibronectin as well as the inhibition of tumor cell proliferation and migration. We also observed that TUSC-T2 could inhibit tumor cell EMT. / Taken together, our data demonstrate that RASA5 and TUSC-T2 are functional tumor suppressors epigenetically silenced in multiple tumors through acting as negative regulators of the Ras or Wnt/Dvl/β-catenin cancer pathways, and could be developed as promising biomarkers for human tumors. Of note, our study reveals that epigenetic silencing of the Ras antagonist represents a new mechanism responsible for Ras aberrant activation in cancers with wild-type Ras. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Fan, Yichao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 184-216). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / List of abbreviations --- p.ii-iii / List of tables --- p.iv / List of Figures --- p.v-vii / List of Publications --- p.viii-ix / Abstract in English --- p.x-xii / Abstract in Chinese --- p.xiii-xiv / Table of Contents --- p.xv / Chapter Chapter 1 --- Introduction and Literature Review --- p.1 / Chapter 1.1 --- Cancer epigenetics --- p.4 / Chapter 1.1.1 --- Epigenetic modifications --- p.5 / Chapter 1.1.1.1 --- DNA Methylation --- p.5 / Chapter 1.1.1.2 --- Histone modifications --- p.10 / Chapter 1.1.1.3 --- RNA interference --- p.14 / Chapter 1.1.1.4 --- Nucleosome positioning --- p.15 / Chapter 1.1.2 --- Epigenetic alteration induced Tumor suppressor genes (TSGs) silencing during carcinogenesis --- p.17 / Chapter 1.2 --- Epigenetic alterations in cancer pathways --- p.23 / Chapter 1.2.1 --- Brief introduction of cancer pathways --- p.23 / Chapter 1.2.2 --- Ras pathway --- p.25 / Chapter 1.2.2.1 --- Ras pathway and carcinogenesis --- p.25 / Chapter 1.2.2.2 --- Epigenetic regulation of RasGAP proteins in carcinogenesis --- p.28 / Chapter 1.2.2.3 --- Epigenetic silencing of other negative regulators of Ras signaling --- p.30 / RAS association domain family (RASSF) proteins --- p.30 / PTEN --- p.32 / Sprouty (SPRY) proteins --- p.33 / Chapter 1.2.2.4 --- Hypomethylation induced Ras oncogenes activation --- p.35 / Chapter 1.2.2.5 --- Ras mediates epigenetic regulation through feedback loop --- p.36 / Chapter 1.2.3 --- Wnt pathway --- p.43 / Chapter 1.2.3.1 --- Wnt signaling pathway and carcinogenesis --- p.43 / Chapter 1.2.3.2 --- Epigenetic silencing of negative regulators of Wnt signaling --- p.45 / Chapter 1.2.3.3 --- DACT family proteins and carcinogenesis --- p.48 / Chapter 1.3 --- Application of tumor specific epigenetic alterations as tumor biomarkers and therapeutic targets --- p.49 / Chapter 1.3.1 --- The potential and advantage of tumor specific epigenetic alterations used as tumor biomarkers and therapeutic targets --- p.49 / Chapter 1.3.2 --- Epigenetic-disrupted regulators of Ras signaling as tumor biomarkers and therapeutic targets --- p.50 / Chapter 1.3.3 --- Epigenetic-disrupted regulators of Wnt signaling as tumor biomarkers and therapeutic targets --- p.52 / Chapter Chapter 2 --- Aims of this study --- p.54 / Chapter 2.1 --- To identify epigenetically silenced candidate TSGs as antagonists to Ras or Wnt signaling --- p.55 / Chapter 2.2 --- To elucidate the functional of candidate TSGs --- p.56 / Chapter Chapter 3 --- Materials and Methods --- p.57 / Chapter 3.1 --- Cell lines, tumor samples and routine cell line maintenance --- p.57 / Chapter 3.2 --- Drug and stress treatments --- p.59 / Chapter 3.3 --- DNA and RNA extraction --- p.59 / Chapter 3.4 --- Semi-quantitative RT-PCR and Real time PCR --- p.60 / Chapter 3.5 --- Direct sequencing of PCR products --- p.67 / Chapter 3.6 --- CpG island analysis --- p.67 / Chapter 3.7 --- Bisulfite treatment --- p.67 / Chapter 3.8 --- Methylation-specific PCR (MSP) and bisulfite genomic sequencing --- p.68 / Chapter 3.9 --- Plasmid extraction --- p.69 / Chapter 3.9.1 --- Bacteria culture --- p.69 / Chapter 3.9.2 --- Mini-scale preparation of plasmid DNA --- p.70 / Chapter 3.9.3 --- Large-scale endotoxin-free plasmids extraction --- p.71 / Chapter 3.10 --- Construction of expression plasmids --- p.71 / Chapter 3.10.1 --- Gene cloning and plasmids construction of RASA5 --- p.71 / Chapter 3.10.2 --- Gene cloning and plasmids construction of TUSC-T2 --- p.74 / Chapter 3.11 --- Immunofluorescence Staining --- p.74 / Chapter 3.12 --- Colony formation assay --- p.76 / Chapter 3.13 --- Apoptosis assay --- p.77 / Chapter 3.14 --- Luciferase reporter assay --- p.78 / Chapter 3.15 --- Protein preparation and Western blot --- p.79 / Chapter 3.16 --- Ras Activity Assay --- p.80 / Chapter 3.17 --- Wound healing assay --- p.81 / Chapter 3.18 --- Matrigel invasion assay --- p.81 / Chapter 3.19 --- RNA Interference --- p.81 / Chapter 3.20 --- Statistical analysis --- p.82 / Chapter Chapter 4: --- Epigenetic disruption of Ras signaling through silencing of a Ras GTPase-activating protein RASA5 in human cancers --- p.83 / Chapter 4.1 --- Identification of RASA5 as a downregulated gene residing in the 6p21.3 deletion region --- p.86 / Chapter 4.2 --- RASA5 is widely expressed in human normal tissues but downregulated in tumor cell lines --- p.91 / Chapter 4.3 --- The tumor-specific downregulation pattern of RASA5 is unique in the RASA family genes --- p.95 / Chapter 4.4 --- RASA5 promoter CpG methylation resulted in its transcription inactivation --- p.96 / Chapter 4.5 --- Frequent methylation of RASA5 promoter in multiple primary tumors --- p.101 / Chapter 4.6 --- Cloning and characterization of human RASA5 --- p.104 / Chapter 4.7 --- RASA5 inhibits tumor cell clonogenicity through inducing apoptosis --- p.108 / Chapter 4.8 --- RasGAP domain is required for the tumor suppressive function of RASA5 --- p.111 / Chapter 4.9 --- Certain cancer types harbor wild type Ras but active Ras signaling, with RASA5 epigenetically silenced --- p.114 / Chapter 4.10 --- RASA5 antagonizes Ras signaling pathway --- p.117 / Chapter 4.10.1 --- RASA5 represses Ras signaling through downregulating Ras-GTP level --- p.117 / Chapter 4.10.2 --- Oncogenic mutant form of Ras abrogated colony formation inhibitory effect of RASA5 on tumor cells --- p.120 / Chapter 4.10.3 --- Knockdown of RASA5 promoted the tumor cell colony formation and Ras signaling activation --- p.122 / Chapter 4.10.4 --- RASA5 inhibits ERK1/2 nuclei translocation and activation --- p.123 / Chapter 4.10.5 --- RASA5 negatively regulates Ras target gene expression --- p.125 / Chapter 4.11 --- RASA5 inhibits tumor cell migration and invasion through the Ras/Rac/cofilin signaling --- p.127 / Chapter 4.12 --- RASA5 suppresses tumor cell epithelial-mesenchymal transition (EMT) and stemness --- p.133 / Chapter 4.13 --- RASA5 appears in the cellcell interaction region nanotubes --- p.139 / Chapter 4.14 --- Discussion --- p.141 / Chapter Chapter 5: --- The Wnt/Dvl signaling antagonist TUSC-T2 is a pro-apoptotic tumor suppressor epigenetically silenced in tumors and inhibits tumor cell proliferation and migration --- p.150 / Chapter 5.1 --- Expression of TUSC-T2 is downregulated in human tumors --- p.150 / Chapter 5.2 --- TUSC-T2 promoter methylation results in its transcriptional inactivation --- p.151 / Chapter 5.3 --- Cloning and characterization of TUSC-T2 --- p.155 / Chapter 5.4 --- TUSC-T2 inhibits tumor cell clonogenicity through inducing apoptosis --- p.157 / Chapter 5.5 --- TUSC-T2 inhibits Wnt/Dvl/β-catenin pathway --- p.161 / Chapter 5.6 --- TUSC-T2 suppresses cell migration and EMT through upregulating E-cadherin --- p.165 / Chapter 5.7 --- Discussion --- p.171 / Chapter Chapter 6: --- Conclusions --- p.176 / Chapter 6.1. --- RasGAP family member RASA5 is epigenetically silenced in human cancers, acting as a tumor suppressor through negatively regulating Ras signaling --- p.177 / Chapter 6.2. --- DACT family member TUSC-T2 functions as a candidate TSG silenced by promoter methylation and inhibits Wnt/Dvl/β-catenin pathway --- p.178 / Chapter Chapter 7: --- Future Studies --- p.181 / Chapter 7.1. --- Further functional study of RASA5 and TUSC-T2 --- p.181 / Chapter 7.2. --- Clinical application of epigenetic silenced candidate TSGs --- p.182 / Chapter 7.3. --- Further screening of candidate TSGs as antagonists to cancer pathways --- p.183 / Reference list --- p.184

Orientador: Erich Vinícius de Paula / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T18:53:53Z (GMT). No. of bitstreams: 1 Faiotto_VanessaBoury_M.pdf: 2777980 bytes, checksum: 26068f6f5813665c531b744d7a8c8f60 (MD5) Previous issue date: 2015 / Resumo: A sepse representa uma condição potencialmente fatal em que a resposta do organismo a uma infecção resulta em lesão em seus próprios tecidos. A terapia gênica (TG) consiste na modificação do repertório de células somáticas com fins terapêuticos, substituindo genes defeituosos que causam doenças. Na sepse, vetores não virais podem representar uma estratégia excelente para a transferência do gene terapêutico, uma vez que não provoca resposta imune significativa e são expressos apenas transitoriamente. Além disso, a sua produção é mais simples. Métodos: O estudo foi dividido em três etapas. Em primeiro lugar, dois genes repórter (lacZ e F9) foram testados em dois modelos experimentais de sepse (endotoxemia e ligadura e punção cecal, CLP) para confirmar a viabilidade da transferência gênica no contexto da sepse. A expressão foi avaliada por métodos qualitativos (histoquímica) e quantitativos (avaliação funcional; métodos coagulométricos). Em seguida avaliou-se a eficácia terapêutica da transferência do gene de sFlt-1, um antagonista de VEGF natural, no modelo de endotoxemia. A expressão foi avaliada por ELISA, e a eficácia foi avaliada através de uma curva de sobrevida. Os camundongos foram tratados com o plasmídeo DNA (pDNA) contendo o cDNA do gene Flt1 (tratamento) lacZ (controle), 6 horas após o desafio com LPS. A propósito, sFlt-1 foi capaz de proteger da sepse experimental murina em outros estudos, devido a sua propriedade estabilizadora da barreira endotelial (BE). Na última etapa, avaliamos os níveis séricos de quatro proteínas envolvidas na modulação da integridade BE, utilizando amostras de soro de indivíduos diagnosticados com sepse grave e choque séptico (n = 53), através de um kit Multiplex comercial. Resultados: No primeiro passo, a transferência gênica por vetores não virais foi demonstrada no modelo de endotoxemia, pois tanto a expressão de ?-galactosidase quanto de F IX mostraram-se aumentadas nos animais tratados neste modelo. A expressão não foi confirmada no modelo CLP, embora o número reduzido de animais por grupo não permita uma conclusão definitiva sobre o assunto. Em relação à segunda etapa, apenas 4/14 animais tratados apresentaram níveis detectáveis de sFlt-1 por Elisa. Além disso, não houve diferença na sobrevida entre os animais tratados e controles. Juntos, estes resultados confirmam que a transferência gênica com vetores não virais é possível no contexto de uma inflamação grave, mas apresenta baixa eficiência e previsibilidade. Por fim, observamos diferenças significativas nos níveis séricos de endoglina (maior expressão) e HB-EGF (menor expressão) em pacientes com choque séptico, em comparação ao grupo controle. Os níveis de BMP-9 e FGF-2 não foram significativamente diferentes no choque séptico. Conclusões: Nosso estudo nos permite concluir que, apesar de factível, a utilização de vetores não-virais não parece representar uma estratégia terapêutica eficaz na sepse experimental. Maiores estudos são necessários para validar o uso de endoglina e HB-EGF como biomarcadores de sepse grave / Abstract: Sepsis represents a potentially fatal condition that occurs when the body's response to infection results in injury to its own tissues. Gene therapy (GT) consists in modification of the repertory of somatic cells with therapeutic purposes, replacing defective genes that cause diseases. In sepsis, non-viral vectors, could represent an excellent strategy for therapeutic gene transfer, since they do not elicit intense immune responses in the individual and are expressed only transiently. Besides, their production is easy and inexpensive. Methods: The study was divided in 3 steps. First, two reporter genes (lacZ and F9) were tested in two experimental models of sepsis (endotoxemia and cecal ligation and puncture, CLP) to confirm the feasibility of gene transfer in the context of sepsis. We assessed expression by qualitative (histochemistry) and quantitative methods (functional evaluation, through coagulometric methods). Next we evaluated the efficacy of the therapeutic gene transfer of sFlt-1, a natural VEGF antagonist, in the endotoxemia model. Expression was evaluated by ELISA, and efficacy was evaluated by a survival curve. Mice were treated with pDNA containing the Flt1 (treatment) or lacZ (control) cDNA, 6 hours after challenge with LPS. Of note, sFlt-1 has been previously shown to protect from experimental sepsis due to its endothelial barrier (EB) stabilizing properties. In the last step, we evaluated serum levels of 4 proteins involved in the modulation of EB integrity, using serum samples of subjects diagnosed with severe sepsis and septic shock (n=53), through a commercial Multiplex kit. Results: In the first step, we could confirm the expression of both reporter genes by non-viral vectors in the endotoxemia model. Of note, expression of ?-galactosidase showed a notable increase when compared with control group. Similarly, we noted increased expression of factor IX in the mice which were submitted to gene transfer with pDNA, when compared with the controls that received the lacZ pDNA (111,4 ± 16,10 vs 64,73 ± 12,34; p<0,001) in the endotoxemia model, and similar result in the group without sepsis induction by endotoxemia (163,4± 73,46 vs 79,88 ± 9,39; p=0,0006). Expression was not confirmed in the CLP model, although the limited number of animals per group does not allow a definite conclusion on this matter. In relation to the second step, only 4/14 treated animals presented detectable levels of sFlt-1 by Elisa. In addition, no difference could be observed in the survival between treated and control animals. Together, these results confirme that gene transfer with non-viral vectors is feasible in the context of severe inflammation, but with a low efficiency and predictability. Finally, we demonstrated significant differences in serum levels of endoglin (higher expression) and HB-EGF (lower expression) in patients with septic shock, compared to controls. BMP-9 and FGF-2 levels were not significantly different in septic shock. Conclusions: Our study allows us to conclude that although feasible, the use of non-viral vectors does not seem to represent an effective therapeutic strategy in experimental sepsis. Larger studies are needed to validate the use of endoglin and HB-EGF as biomarkers of severe sepsis / Mestrado / Clinica Medica / Mestra em Ciências

目前，骨形成低下的骨代謝異常在臨床中面臨巨大挑戰。治療這些疾病的途徑之一可通過小干擾核酸沉默骨形成抑制的基因。隨著核酸干擾技術的快速發展，採用核酸干擾策略進行治療的很多問題已被解決。然而，小干擾核酸的安全和有效遞送仍然是核酸干擾治療進行臨床轉化的瓶頸。其主要問題在於促進骨形成治療所需的小干擾核酸劑量較大，其系統給藥後可能對其他非骨組織產生副作用。所以，亟需針對具有促進成骨潛力的小干擾核酸開發安全有效的遞送系統。本研究的目的就是針對具有促進成骨潛力的小干擾核酸開發特定的遞送系統，以便應用於核酸干擾治療中的促進骨形成。策略之一是利用靶向骨形成表面的遞送系統攜載小干擾核酸到富集于骨形成表面的成骨系細胞。策略之二是直接把小干擾核酸遞送到成骨細胞，使其具有高度的細胞選擇性。在該研究中，我們採用具有成骨潛能的酪蛋白激酶2相互作用蛋白1小干擾核酸作為模型小干擾核酸以考察基因沉默效率。 / 靶向骨形成表面的(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體-小干擾核酸遞送系統：首先對多肽序列(天門冬氨酸-絲氨酸-絲氨酸)₆靶向骨形成表面的特性進行鑒定。進一步將(天門冬氨酸-絲氨酸-絲氨酸)₆作為靶向分子與以DOTAP為主要成分的陽離子脂質體進行連接製備（天門冬氨酸-絲氨酸-絲氨酸)6-脂質體遞送系統。採用凍幹/再水化方法對小干擾核酸進行包裹並對其粒徑，ζ電位，包封率以及穩定性進行考察。最後分別在體外和體內模型對該遞送系統遞送效果以及其攜載小干擾核酸的基因沉默效率進行評價。 / 實驗結果證實(天門冬氨酸-絲氨酸-絲氨酸)₆是一種在體內可以有效靶向骨形成表面的多肽。(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體的平均粒徑為140 nm左右，其包封率可高達80%。該遞送系統較穩定，可使攜載的小干擾核酸具有較高的基因沉默效率，而且沒有明顯的細胞毒性。體內試驗表明，該遞送系統在促進小干擾核酸在骨組織的分佈同時降低其被肝組織的攝取。該遞送系統所攜帶的酪蛋白激酶2相互作用蛋白1小干擾核酸可選擇性地沉默骨組織中的酪蛋白激酶2相互作用蛋白1基因，且對其他組織並沒有明顯影響。該結果表明(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體可促進小干擾核酸靶向骨組織並在骨組織沉默攜載小干擾核酸相應的基因。免疫化學分析結果顯示(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體可攜載小干擾核酸選擇性地到達骨形成表面的成骨系細胞，避免被前破骨細胞/破骨細胞吞噬。大鼠骨髓細胞採用Alp，Stro-1和Oscar抗體分選後的酪蛋白激酶2相互作用蛋白1 mRNA表達水平顯示該遞送系統可選擇性地沉默成骨系細胞。 / 靶向成骨細胞的L6適配子-脂質納米顆粒-小干擾核酸遞送系統：將針對大鼠成骨細胞（ROS 17/2.8細胞系）進行正向篩選，大鼠肝細胞（BRL-3A細胞系）和外周血細胞進行負向篩選的L6適配子與以DLin-KC2-DMA為主要成分的脂質納米顆粒採用膠束形式插入的方法進行連接製備L6適配子-脂質納米顆粒-小干擾核酸遞送系統。並對其粒徑，ζ電位，包封率和形態學進行考察。在體外評價實驗中，考察了該遞送系統的選擇性，細胞毒性，基因沉默效率以及細胞攝取機制。在體內實驗中，對小干擾核酸的組織分佈以及其攜載小干擾核酸在成骨細胞和肝細胞的分佈進行了評價。 / 實驗結果顯示L6適配子-脂質納米顆粒-小干擾核酸的平均粒徑為84.0±5.3 nm，其電勢為-23 ± 2 mV，包封率為80.8 ± 3.4%. 脂質納米顆粒表面的L6適配子可促進小干擾核酸在ROS 17/2.8細胞系（靶向細胞）中的攝取， 然而在BRL-3A 細胞系（非靶向細胞）中攝入很少。該遞送系統沒有明顯細胞毒性，在10 nM小干擾核酸的低濃度下，體外基因沉默效率可高達50 % 以上。由L6適配子引起的巨胞被證實是成骨細胞攝取L6適配子-脂質納米顆粒所攜載小干擾核酸的主要機制。體內實驗顯示該遞送系統可促進小干擾核酸在骨組織的分佈，降低其被肝組織的攝取。在肝组织冰凍切片中，肝血竇和肝細胞中沒有明顯的小干擾核酸分佈，進一步說明該遞送系統可降低對肝組織的影響。免疫化學分析結果顯示L6適配子-脂質納米顆粒-小干擾核酸可攜載小干擾核酸選擇性地到達成骨細胞，避免被前破骨細胞/破骨細胞吞噬。 / 重要意義：本研究中的兩種新型小干擾核酸系統可分別選擇性地遞送小干擾核酸靶向骨形成表面和成骨細胞。 (天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體-小干擾核酸遞送系統開拓了全新的途徑，實現選擇性地遞送小干擾核酸到骨形成表面從而降低對骨吸收的影響。 L6適配子-脂質納米顆粒-小干擾核酸遞送系統在成骨細胞表面特徵蛋白未知的情況下，首次採用適配子技術在細胞水準實現成骨細胞的選擇性遞送。該研究中的兩種遞送系統為核酸干擾治療的促進骨形成策略提供了強而有力的工具，為實現肌肉骨骼疾病相關領域的核酸干擾治療策略從基礎科學向臨床應用的轉化建立了堅實的基礎。 / Metabolic skeletal disorders that are associated with impaired bone formation are a major clinical challenge. One approach to treat these diseases was to silence bone formation-inhibitory genes by small interference RNAs (siRNAs). With the rapid development of RNA interference (RNAi) technology, more issues of RNAi-based therapy strategies have been addressed. However, the safe and effective delivery of siRNAs is still the bottleneck for its translation from bench to bedside. One major concern was that the large therapeutic doses of systemically administered siRNA to stimulate sufficient bone formation may carry a high risk for adverse effects on non-skeletal tissues. Therefore, development of specific siRNA delivery systems for safe and efficient transporting osteogenic siRNAs is highly desirable. The objective of the present study was to explore siRNA delivery systems for osteogenic siRNAs in RNAi-based bone anabolic therapy. One strategy was to develop siRNA delivery system targeting bone formation surfaces to facilitate delivery of siRNAs to osteogenic cells. Another approch was to develop siRNA delivery system targeting osteoblasts directly. Plekho1 siRNA targeting casein kinase-2 interacting protein-1 (Ckip-1) with osteogenic potential was employed as a representative siRNA in our current study. / (AspSerSer)6-liposome-siRNA for targeting bone formation surfaces: (AspSerSer)6 for targeting bone formation surfaces was firstly identified. Then, (AspSerSer)6 was conjugated with DOTAP-based liposome to produce (AspSerSer)6-liposome. (AspSerSer)6-liposome-siNRA was prepared by lyophilization/rehydration method and characterized in terms of particle size, zeta potential, encapsulation efficiency and the stability in serum. Finally, the delivery of siRNA and the corresponding gene silencing mediated by (AspSerSer)6-liposome-siRNA were evaluated in the in vitro and in vivo models. / The results indicated that the novel (AspSerSer)₆ was a promising peptide for targeting bone formation surfaces in vivo. (AspSerSer)₆-liposome with the average particle size of 140 nm encapsulating Plekho1 siRNA exhibited more than 80% encapsulation efficiency and good stability against enzymatic degradation. It demonstrated high knockdown efficiency without obvious cytotoxicity. In in vivo study, the result of tissue distribution experiment indicated that (AspSerSer)6-liposome-siRNA enhanced the distribution of siRNA in bone, meanwhile reduced the uptake of siRNA in liver. The Plekho1 protein and mRNA expression in various tissues demonstrated that (AspSerSer)₆-liposome-siRNA could facilitate gene silencing in a bone-selective manner. The results of immunochemistry analyses indicated (AspSerSer)₆-liposome-siRNA facilitated delivering siRNA to osteogenic cells at bone formation surfaces and avoided siRNA to pre-osteoclast/osteoclast. Plekho1 mRNA expression in rat bone marrow cells sorted by fluorescence activated cell sorting (FACS) using Alp, Stro-1 and Oscar antibody, respectively, further suggested (AspSerSer)₆-liposome-siRNA could silence gene in a cell-selective manner in vivo. / L6-LNPs-siRNA for targeting osteoblasts: L6 aptamer for targeting osteoblasts (ROS 17/2.8 cell line) and using rat hepatocyte (BRL-3A cell line) and peripheral blood cells in negative selection was conjugated to DLin-KC2-DMA-based lipid nanoparticles (LNPs) to generate L6-LNPs-siRNA by post-insertion method in the form of micelles. L6-LNPs-siRNA was characterized with particle size, zeta potential, encapsulation efficiency and morphology. Its selectivity, cytotoxicity and knockdown efficiency were evaluated in vitro. The mechanism of L6-LNPs-mediated siRNA cellular uptake was further investigated. The tissue distribution of the injected siRNA and the localization of the siRNA with osteoblasts as well as hepatocytes were also evaluated in vivo. / The results showed L6-LNPs-siRNA have the average particle size of 84.0 ± 5.3 nm and zeta potential of -23 ± 2 mV. Its encapsulation efficiency was 80.8 ± 3.4%. The L6 aptamer on the surface of LNPs facilitated the cellular uptake of Plekho1 siRNA in ROS 17/2.8 cell line (target cells) but no uptake in BRL-3A cell line (non-target cells) in vitro. L6-LNPs-siRNA with low cytotoxicity exhibited above 50% knockdown efficiency at a low concentration of 10 nM in vitro. Macropinocytosis induced by L6 was demonstrated to be the predominant mechanism of L6-LNPs mediated siRNA uptake in osteoblasts. In in vivo study, it was shown that L6-LNPs-siRNA facilitated the distribution of siRNA in bone and decreased the hepatic uptake. No obvious siRNA fluorescent signals in sinus and hepatocyte was observed in liver cryosection further indicated the reducing influence on liver after administration of L6-LNPs-siRNA. Co-localization of fluorescence-labeled siRNA with Alp-positive cells was dominantly documented, whereas there were no instances of such overlapping staining with Oscar-positive cells after L6-LNPs-siRNA treatment, which suggested L6-LNPs-siRNA facilitated delivering siRNA in a cell-selective manner in vivo. / Significance: These two innovative siRNA delivery systems in the present study selectively targeted bone formation surfaces and osteoblasts, respectively. (AspSerSer)₆-liposome-siRNA opened up a new avenue to specifically deliver therapeutic siRNAs to bone formation surfaces without affecting bone resorption. L6-LNPs-siRNA achieved the osteoblast-specific delivery for siRNA at cellular level by aptamer technology for the first time, even without knowledge of characteristic protein on the surface of osteoblasts. The two delivery systems provided the powerful tools for RNAi-based bone anabolic strategy and established a solid foundation for translating RNAi-based therapies from basic science to clinic applications in the musculoskeletal field. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wu, Heng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 130-142). / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 論文摘要 --- p.vi / Table of contents --- p.ix / Publications --- p.xiv / List of tables --- p.xvi / List of figures --- p.xvii / List of abbreviations --- p.xxi / Chapter One Introduction --- p.1 / Chapter 1.1 --- Great challenges in skeletal disorders --- p.2 / Chapter 1.2 --- RNA interference (RNAi) as therapeutic strategy --- p.3 / Chapter 1.2.1 --- Mechanism of RNAi --- p.3 / Chapter 1.2.2 --- Potential triggers of RNAi-mediated gene silencing --- p.4 / Chapter 1.2.3 --- Current clinical trials using RNAi as therapeutic strategy --- p.7 / Chapter 1.2.4 --- Current application of therapeutic siRNAs in skeletal disorders --- p.11 / Chapter 1.3 --- Challenges of siRNA in vivo delivery for targeting bone --- p.12 / Chapter 1.3.1 --- General challenges of siRNA delivery in vivo --- p.13 / Chapter 1.3.2 --- Challenges of siRNA delivery to bone --- p.15 / Chapter 1.3.2.1 --- Physiological property --- p.15 / Chapter 1.3.2.2 --- Targeting ligands for approaching bone --- p.16 / Chapter 1.4 --- Strategies of siRNAs in vivo delivery after systemic administration --- p.18 / Chapter 1.4.1 --- Naked siRNA and naked siRNA with chemical conjugation --- p.18 / Chapter 1.4.2 --- Nanoparticle delivery systems --- p.20 / Chapter 1.4.2.1 --- Liposome and lipid-like materials --- p.20 / Chapter 1.4.2.2 --- Polymers --- p.22 / Chapter 1.4.2.3 --- Targeted delivery system --- p.23 / Chapter 1.5 --- Strategies of osteogenic siRNAs delivery for stimulating bone formation --- p.24 / Chapter 1.6 --- Objective of present study --- p.25 / Chapter Chapter Two --- Preparation and characterization of (AspSerSer)₆-liposome-siRNA for targeting bone formation surfaces --- p.26 / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.2 --- Materials and Methods --- p.28 / Chapter 2.2.1 --- Materials --- p.28 / Chapter 2.2.2 --- Identification of (AspSerSer)₆ --- p.29 / Chapter 2.2.3 --- Development of formulation --- p.30 / Chapter 2.2.3.1 --- Selection of the molar ratio of DOTAP --- p.30 / Chapter 2.2.3.2 --- Selection of the molar ratio of siRNA to lipids --- p.30 / Chapter 2.2.4 --- Preparation of (AspSerSer)6-liposome-siRNA --- p.30 / Chapter 2.2.5 --- Characterization of (AspSerSer)₆-liposome --- p.33 / Chapter 2.2.5.1 --- Particle Size and Zeta Potential --- p.33 / Chapter 2.2.5.2 --- Encapsulation Efficiency --- p.33 / Chapter 2.2.5.3 --- Stability in serum --- p.33 / Chapter 2.3 --- Results --- p.34 / Chapter 2.3.1 --- (AspSerSer)₆ as a targeting moiety --- p.34 / Chapter 2.3.2 --- Development of formulation --- p.37 / Chapter 2.3.3 --- Particle size, Zeta Potential and Encapsulation Efficiency --- p.38 / Chapter 2.3.4 --- Stability in serum --- p.38 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.5 --- Conclusion --- p.42 / Chapter Chapter Three --- Evaluation of (AspSerSer)₆-liposome-siRNA for cell-specific delivery and gene silencing in vitro and in vivo --- p.43 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods --- p.45 / Chapter 3.2.1 --- Materials --- p.45 / Chapter 3.2.2 --- Biological evaluation in vitro --- p.46 / Chapter 3.2.2.1 --- Binding affinity with hydroxyapatite --- p.46 / Chapter 3.2.2.2 --- Cell culture --- p.46 / Chapter 3.2.2.3 --- Cellular uptake --- p.47 / Chapter 3.2.2.4 --- Knockdown efficiency in vitro --- p.47 / Chapter 3.2.2.5 --- Total RNA extraction, reverse transcription and quantitative real-time PCR --- p.48 / Chapter 3.2.3 --- Cytotoxicity --- p.49 / Chapter 3.2.4 --- Tissue distribution --- p.50 / Chapter 3.2.4.1 --- Experimental design --- p.50 / Chapter 3.2.4.2 --- Fluorescence image analysis --- p.50 / Chapter 3.2.4.3 --- Quantitative Analysis --- p.50 / Chapter 3.2.5 --- Localization of siRNA in liver --- p.51 / Chapter 3.2.5.1 --- Experimental design --- p.51 / Chapter 3.2.5.2 --- Histochemisty analysis --- p.51 / Chapter 3.2.6 --- Gene silencing in tissues --- p.52 / Chapter 3.2.6.1 --- Experimental design --- p.52 / Chapter 3.2.6.2 --- Determination of mRNA expression --- p.52 / Chapter 3.2.6.3 --- Western blot analysis --- p.52 / Chapter 3.2.7 --- Localization of siRNA with Osteoblasts/Osteoclasts --- p.53 / Chapter 3.2.7.1 --- Experimental design --- p.53 / Chapter 3.2.7.2 --- Immunohistochemistry analysis --- p.53 / Chapter 3.2.8 --- Gene silencing at cellular levels --- p.54 / Chapter 3.2.8.1 --- Experimental design --- p.54 / Chapter 3.2.8.2 --- Flow cytometry cell sorting --- p.54 / Chapter 3.2.9 --- Statistical analysis --- p.55 / Chapter 3.3 --- Results --- p.56 / Chapter 3.3.1 --- Binding affinity with hydroxyapatite --- p.56 / Chapter 3.3.2 --- Cellular uptake --- p.57 / Chapter 3.3.3 --- Knockdown efficiency in vitro --- p.57 / Chapter 3.3.4 --- Cytotoxicity --- p.59 / Chapter 3.3.5 --- Tissue distribution by imaging analysis --- p.60 / Chapter 3.3.6 --- Quantitative analysis of tissue distribution --- p.62 / Chapter 3.3.7 --- Localization of siRNA in liver --- p.63 / Chapter 3.3.8 --- Plekho1 mRNA and protein expressions --- p.64 / Chapter 3.3.9 --- Immunohistochemistry analysis --- p.65 / Chapter 3.3.10 --- Gene silencing at cellular level --- p.71 / Chapter 3.4 --- Discussion --- p.74 / Chapter 3.5 --- Conclusion --- p.77 / Chapter Chapter Four --- Preparation and characterization of aptamer-functionalized lipid nanoparticle for siRNA cell-specific delivery --- p.78 / Chapter 4.1 --- Introduction --- p.79 / Chapter 4.2 --- Materials and Methods --- p.80 / Chapter 4.2.1 --- Materials --- p.80 / Chapter 4.2.2 --- Synthesis of 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-di- oxolane (DLin-KC2-DMA) --- p.80 / Chapter 4.2.2.1 --- Synthesis of Linoleyl alcohol (1) --- p.81 / Chapter 4.2.2.2 --- Synthesis of Linoleyl bromide (2) --- p.81 / Chapter 4.2.2.3 --- Synthesis of Dilinoleylmethyl formate (3) --- p.82 / Chapter 4.2.2.4 --- Synthesis of Dilinoleyl Methanol (4) --- p.82 / Chapter 4.2.2.5 --- Synthesis of Dilinoleyl Ketone (5) --- p.83 / Chapter 4.2.2.6 --- Synthesis of 2, 2- Dilinoleyl- 4- (2-hydroxyethyl)-[1,3]-dioxolane (6) --- p.83 / Chapter 4.2.2.7 --- Synthesis of DLin-KC2-DMA --- p.83 / Chapter 4.2.3 --- Development of formulation --- p.84 / Chapter 4.2.3.1 --- Selection of the molar ratio of lipids --- p.84 / Chapter 4.2.3.2 --- Selection of the mass ratios of siRNA to lipids --- p.85 / Chapter 4.2.3.3 --- Selection of the molar ratios of L6-PEG2000-DSPE on L6-LNPs-siRNA --- p.85 / Chapter 4.2.4 --- Binding affinity with osteoblasts --- p.86 / Chapter 4.2.5 --- Preparation of L6-LNPs-siRNA --- p.86 / Chapter 4.2.5.1 --- Synthesis of L6-PEG2000-DSPE --- p.87 / Chapter 4.2.5.2 --- Preparation of LNPs-siRNA --- p.87 / Chapter 4.2.5.3 --- Post-insertion of aptamers on the surface of LNPs-siRNA --- p.88 / Chapter 4.2.6 --- Characterization of L6-LNPs-siRNA --- p.88 / Chapter 4.2.6.1 --- Particle size and Zeta Potential --- p.88 / Chapter 4.2.6.2 --- Encapsulation Efficiency (EE) --- p.88 / Chapter 4.2.6.3 --- Cryo-Transmission electron microscope --- p.89 / Chapter 4.3 --- Results --- p.90 / Chapter 4.3.1 --- Synthesis of DLin-KC2-DMA --- p.90 / Chapter 4.3.2 --- Formulation development --- p.93 / Chapter 4.3.3 --- Preparation of L6-LNPs --- p.95 / Chapter 4.3.4 --- Characterization of L6-LNPs-siRNA --- p.96 / Chapter 4.4 --- Discussion --- p.98 / Chapter 4.5 --- Conclusion --- p.101 / Chapter Chapter Five --- Evaluation of L6 aptamer functionalized lipid nanoparticles (L6-LNPs-siRNA) for osteoblast-specific delivery in vitro and in vivo --- p.102 / Chapter 5.1 --- Introduction --- p.103 / Chapter 5.2 --- Materials and Methods --- p.103 / Chapter 5.2.1 --- Materials --- p.103 / Chapter 5.2.2 --- Biological evaluation in vitro --- p.104 / Chapter 5.2.2.1 --- Cell culture --- p.104 / Chapter 5.2.2.2 --- Binding affinity with target/non-target cells --- p.105 / Chapter 5.2.2.3 --- Cellular uptake of siRNA in target/non-target cells --- p.105 / Chapter 5.2.2.4 --- Knockdown efficiency in vitro --- p.105 / Chapter 5.2.3 --- Cytotoxicity --- p.106 / Chapter 5.2.4 --- Mechanism of cellular uptake --- p.106 / Chapter 5.2.4.1 --- Spectral bio-imaging for endocytic pathways --- p.106 / Chapter 5.2.4.2 --- Chemical inhibition for endocytic pathways --- p.107 / Chapter 5.2.4.3 --- Determination of membrane ruffling --- p.107 / Chapter 5.2.5 --- Evaluation of specific delivery in vivo --- p.107 / Chapter 5.2.5.1 --- Experimental design --- p.107 / Chapter 5.2.5.2 --- Tissue distribution --- p.108 / Chapter 5.2.5.3 --- Localization of siRNA in liver --- p.108 / Chapter 5.2.5.4 --- Localization of siRNA with osteoblast/osteoclast --- p.108 / Chapter 5.2.6 --- Statistical analysis --- p.109 / Chapter 5.3 --- Results --- p.109 / Chapter 5.3.1 --- Binding selectivity of L6-LNPs-siRNA --- p.109 / Chapter 5.3.2 --- Selectivity of siRNA cellular uptake --- p.111 / Chapter 5.3.3 --- Knockdown efficiency in vitro --- p.112 / Chapter 5.3.4 --- Cytotoxicity --- p.113 / Chapter 5.3.5 --- Mechanism of cellular uptake --- p.113 / Chapter 5.3.6 --- Tissue distribution --- p.118 / Chapter 5.3.7 --- Localization of siRNA in liver --- p.119 / Chapter 5.3.8 --- Localization of siRNA with Osteoblasts/Osteoclasts --- p.120 / Chapter 5.4 --- Discussion --- p.123 / Chapter 5.5 --- Conclusion --- p.125 / Chapter Chapter Six --- Summary of the study and future research --- p.126 / Chapter 6.1 --- Summary of the study --- p.127 / Chapter 6.2 --- Future research --- p.128 / References --- p.130

Canavan’s Disease is a fatal Central Nervous System disorder caused by genetic defects in the enzyme – aspartoacylase and currently has no effective treatment options. We report additional phenotypes in a stringent preclinical aspartoacylase knockout mouse model. Using this model, we developed a gene therapy strategy with intravenous injections of the aspartoacylase gene packaged in recombinant adeno associated viruses (rAAVs). We first investigated the CNS gene transfer abilities of rAAV vectors that can cross the blood-brain-barrier in neonatal and adult mice and subsequently used different rAAV serotypes such as rAAV9, rAAVrh.8 and rAAVrh.10 for gene replacement therapy. A single intravenous injection rescued lethality, extended survival and corrected several disease phenotypes including motor dysfunctions. For the first time we demonstrated the existence of a therapeutic time window in the mouse model. In order to limit off-target effects of viral delivery we employed a synthetic strategy using microRNA mediated posttranscriptional detargeting to restrict rAAV expression in the CNS. We followed up with another approach to limit peripheral tissue distribution. Strikingly, we demonstrate that intracerebroventricular administration of a 50-fold lower vectors dose can rescue lethality and extend survival but not motor functions. We also study the contributions of several peripheral tissues in a primarily CNS disorder and examine several molecular attributes behind pathogenesis of Canavan’s disease using primary neural cell cultures. In summary, this thesis describes the potential of novel rAAV-mediated gene replacement therapy in Canavan’s disease and the use of rAAVs as a tool to tease out its pathological mechanism.