臺灣博碩士論文加值系統

B 型肝炎病毒(Hepatitis B virus, HBV)在感染肝細胞後，其基因體會以共價閉合環狀去氧核糖核酸(covalently closed circular DNA, cccDNA)結構或嵌入宿主基因體中存在宿主肝細胞內。現行治療 HBV的藥物雖然可以有效抑制病毒的反轉錄複製，但無法完全清除病毒的複製模板- cccDNA，以至於無法達到根除 HBV 感染。另外，嵌入型的 B 型肝炎基因體(Integrated HBV DNA)有特定嵌入的熱點基因，會導致特定基因的活性上升導致癌化，其中以端粒酶逆轉錄酶基因(TERT gene)為首，若能抑制嵌入型的 B 型肝炎基因體中重要的增強子區域就有機會能降低肝癌的危險因子。在實驗室先前的研究以及其他文獻中，都證明了 HBV 的基因體可以被CRISPR-Cas9 此基因編輯工具破壞，然而CRISPR / Cas系統不可避免地會靶向嵌入型的 B 型肝炎基因體，並誘導宿主基因組的雙股斷裂 ，即伴隨著基因組重組與破壞的風險。因此本篇研究中，我們的目標為利用CRISPR / Cas9 介導的非切割鹼基編輯工具-單鹼基編輯器（base editors）與先導編輯器（prime editors）來修改HBV基因組中增強子的區域，來達到抑制HBV基因組，並評估能否降低HBV嵌入端粒酶啟動子(HBV integrant-fused telomerase promoter)的活性。
當 HBV 在患者體內無法被順利清除時，病毒抗原長存在患者體內循環，導致過度刺激而產生免疫耐受性 (immune tolerance)，但免疫耐受的機制迄今尚不清楚。目前許多研究的實驗證據表明，誘發後天免疫系統產生強而有力的 HBV 抗原專一性毒殺型Tc細胞(cytotoxic T cell)，才是徹底清除病毒完全治癒HBV 慢性感染的關鍵。免疫系統產生具抗原專一性毒殺型 Tc 細胞的過程，主要有幾個關鍵步驟。首先抗原呈現細胞在接受抗原而活化後，透過人類白血球抗原(Human Leukocyte Antigen, HLA)將抗原決定位(epitopes) 呈獻給 CD4+ 與 CD8+ T 細胞，CD4+T 細胞分化成 Th 細胞(Helper T cells)，而CD8+ T 細胞活化成 Tc 細胞的過程除了 APCs 也需要 Th 細胞的協助。我們認為，T細胞調節的保護力對於慢性病毒感染能更有效且持久，為了建立T細胞療法必須瞭解特定HLA呈現的胜肽與表現特定TCR之T細胞之間的關聯。由於HLA具有高度多型性，且等位基因至少表現出三種不同HLA基因，不利於鑑定出特定HLA呈現的胜肽。因此，我們利用CRISPR / Cas9基因編輯工具建立HLA class I null cell lines作為研究的工具，再使HLA class I null cell lines表達單一class I HLA基因，以利我們對於特定class I HLA對應特定T細胞調節免疫進行研究。將來有潛力做為根除 B 型肝炎病毒之抗病毒藥物。

Chronic infection by hepatitis B virus (HBV) is the major contributor to liver disease worldwide. Although HBV replicates via a nuclear episomal DNA (covalently closed circular DNA, cccDNA), integration of HBV DNA into the host cell genome is often observed in the liver of infected patients. In particular, alterations of TERT gene by HBV integrations frequently increase the risk of hepatocarcinogenesis. Current antiviral therapy fails to cure chronic HBV infection because of persistent covalently closed circularDNA (cccDNA) and the integrated HBV genomes, which continuously produce viral antigens. Previous studies, including ours, have shown that the HBV genome can be disrupted by the CRISPR/Cas9 system. However, the CRISPR/Cas system inevitably targets integrated HBV DNA and induces double-strand breaks of host genome, bearing the risk of genomic rearrangement and damage. In this study, we used CRISPR/Cas9-mediated “base editors” (BEs) and “prime editors” (PEs) editing the enhancer regions of the HBV genome to achieve inhibition of the HBV gene expression without cleavage of DNA. Whether it can reduce the activity of HBV integrant-fused telomerase promoter was also evaluated.
Failure of successful clearance of HBV from the patient's body causes persistent viral antigens in the patient's circulation, resulting in excessive stimulation and consequently immune tolerance. Experimental evidence from a number of studies indicates that induction powerful HBV antigen-specific cytotoxic T cells is the key to completely eradicate the virus and cure chronic HBV infection. In order to develop T cell therapy, it is necessary to understand the interactions between specific HLA-restricted peptides and cognate T cell receptors (TCRs). Because HLA is highly polymorphic, and has three different gene loci (HLA A, B and C), it is not conducive to the identification of peptides presented by specific HLAs. Therefore, we used CRISPR/Cas9 gene editing tools to establish HLA class I null cell lines as a research platform, and then generated a single class I HLA expressing cell line in order to understand specific class I HLA corresponding to a specific T cell regulated immunity. This approach should have the potential to develop effective T cell-based antiviral strategy to eradicate chronic HBV infection.

口試委員審定書 ii
致謝 iii
中文摘要 iv
英文摘要 vi
1 Introduction p.1
1.1 Epidemiology of HBV infection p.1
1.2 Genome structure and organization of HBV p.1
1.3 Control of hepatitis B virus at the level of transcription p.2
1.4 The replication cycle of HBV p.3
1.5 Integrated HBV DNA contributes to hepatocellular carcinoma p.5
1.6 Limitations of current treatment for HBV infection p.6
1.7 Application of the genome editing tool - CRISPR/Cas to
treatment of HBV p.6
1.7.1 Introduction of CRISPR/Cas technology p.6
1.7.2 Disruption of HBV genome by CRISPR/Cas9 system p.7
1.8 Concerns and solutions of the CRISPR/Cas system targeting HBV p.8
1.8.1 The risk of cutting HBV DNA by Cas-associated nucleases p.8
1.8.2 Inactivation of HBV genomes by non-cleavage base editing p.9
1.8.3 Prime editing: search and replace genome editing without DSB p.9
1.9 Introduction of immunopeptidomics approaches p.11
1.9.1 T-cell epitope discovery technologies p.11
1.9.2 MS–based identification of MHC-bound peptides p.12
1.9.3 Single-allelic HLA class I cells enable more accurate epitope
discovery and prediction prediction p.13
1.9.4 Establishment the HLA class I null artificial antigen presenting
cells (AAPCs) p.14
2 Specific Aims p.15
3 Materials and Methods p.16
3.1 Plasmids p.16
3.2 Site-directed mutagenesis p.17
3.3 Base editing gRNAs screening p.18
3.4 pegRNA design and cloning p.18
3.5 HLA gRNA design and cloning p.19
3.6 Cell lines and culture conditions p.20
3.7 DNA transfection p.20
3.8 Dual-luciferase reporter assay (DLR™) p.21
3.9 Lentiviral transduction of HEK293T p.22
3.10 Flow cytometry p.23
3.11 FACS sorting p.23
3.12 Single cell polymerase chain reaction (PCR) and sequencing p.24

4 Results p.25
PART 1
4.1 Identification of the critical region of enhancer I that contributes to HBV
core promoter activity p.25
4.2 Point mutations caused by the base editor have no significant effect on
enhancer I p.26
4.3 pegRNA targeting HBV conserved sequences did not induce 82 b.p-length
deletion p.27
PART 2
4.4 Establishment of HLA class I negative cell clones p.29
4.5 Genetic and flow cytometric analysis of HLA class I negative
cell clones p.30
4.6 Generation of single-HLA class I-expressing cell lines p.31
5 Discussion p.32
5.1 Utility of non-cleavage editing in the treatment of HBV
infection p.32
5.2 The establishment of HLA null 293T and its application as AAPCs p.33
6 Figures p.36
PART 1
Figure 1. The reporter of the HBV promoter/enhancer and an array of mutants with different parts of enhancer deletion and detect promoter activity p.36
Figure 2. Validation of the effect of point mutations on HBV enhancer I
activity p.38
Figure 3. Generation of the vectors for prime editing on HBV p.39
PART 2
Figure 4. The workflow for the establishment of human leukocyte antigens (HLA) class I null HEK 293T cell line using the multiplex CRPSIR/Cas9 p.41
Figure 5. Screening and identification of class I HLA-deficient HEK 293T
cell lines p.44
Figure 6. Mapping of the deletions in HLA null 293T clone #157 p.45
Figure 7. The expression of HLA class I molecules in single HLA-expressing
cell lines p.46
7 References p.47
8 Supplementary Information p.51
Table 1. pegRNA/gRNA sequences for prime editing p.51
Table 2. gRNA sequences for HLA-A, HLA-B, and HLA-C target editing p.51
Table 3. Sequences of HLA-A, HLA-B, and HLA-C specific primers p.52
Figure S1. Genetic and flow cytometric analysis of HLA class I negative cell pools which editing by conserved gRNAs p.54
Figure S2. Clone #157 nucleotide sequences alignment p.55

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