公开(公告)号：US6130040A

公开(公告)日：2000-10-10

申请号：US4070

申请日：1998-01-08

申请人： Shi-Lung Lin

发明人： Shi-Lung Lin

IPC分类号： C07H21/00 ,  C12Q1/68 ,  C07H19/00 ,  C12P19/34

CPC分类号： C07H21/00

摘要： The present invention provides a fast, simple and direct covalent bond formation between two strands of nucleotide sequences. Non-modified first strand nucleotide sequences are hybridized with second strand nucleotide sequences, of which certain specific base structure(s) is modified by chemical reagents in order to generate covalent bonding with the first strand. While the hybridization of these two strand nucleotide sequences generates double-stranded hybrid duplexes between their homologues, covalent bond formation occurs in the region of modified base-pairs. Since neither a polymerase chain restriction nor a restriction enzyme digestion can be performed with the covalently bonded hybrid duplexes, the present invention can be used to subtract common sequences during subtractive hybridization, to inhibit nonspecific contamination during subcloning and to increase binding stability of antisense probes during in situ hybridization as well as gene therapy.

摘要翻译： 本发明提供了两条核苷酸序列之间的快速，简单和直接的共价键形成。 未修饰的第一链核苷酸序列与第二链核苷酸序列杂交，其中某些特异性碱基结构被化学试剂修饰以产生与第一链的共价键。 虽然这两条链核苷酸序列的杂交在其同源物之间产生双链杂交双链体，但共价键形成发生在修饰的碱基对的区域。 由于不能用共价键合的杂交双链体进行聚合酶链限制和限制性内切酶消化，本发明可用于在减法杂交期间减去常见序列，以抑制亚克隆过程中的非特异性污染，并增加反义探针的结合稳定性 原位杂交以及基因治疗。

公开(公告)号：US11624067B2

公开(公告)日：2023-04-11

申请号：US16135723

申请日：2018-09-19

申请人： Shi-Lung Lin ,  Samantha Chang-Lin ,  Donald Chang

发明人： Shi-Lung Lin ,  Samantha Chang-Lin ,  Donald Chang

IPC分类号： C12N15/113 ,  C12N15/11 ,  C12P19/34 ,  C12N15/70

摘要： This invention generally relates to a composition and its method of use for inducing adult stem cell (ASC) expansion and/or derivation in vitro, using miR-302-like pre-miRNAs, shRNAs and/or siRNAs, all of which contain a shared sequence of 5′-UAAGUGCUUC CAUGUUU-3′ (SEQ ID NO: 7) in the 5′-end, and further in conjunction with the use of some wound-healing-related defined factors, including but not limited to basic fibroblast growth factor (bFGF)/fibroblast growth factor 2 (FGF-2), leukemia inhibitory factor (LIF), insulin-like growth factor (IGF), Epidermal growth factor (EGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), tumor necrosis factor (TNF), stem cell factor (SCF), homeobox proteins (HOX), Notch, GSK, Wnt/beta-Catenin signals, interleukins, and/or bone morphogenetic proteins (BMPs). The principle of the present invention is related to a novel mechanism of inducible symmetric ASC division recently found in a skin wound healing model in vivo. The resulting amplified ASCs are useful for treating a variety of human aging- and cell dysfunction-associated disorders, including but not limited to Alzheimer's disease, Parkinson's disease, motor neuron disease, stroke, diabetes, osteoporosis, myocardial infraction, hemophilia, anemia, AIDS, leukemia, lymphoma and many kinds of cancers as well as aging.

公开(公告)号：US20230099592A1

公开(公告)日：2023-03-30

申请号：US17930292

申请日：2022-09-07

申请人： Shi-Lung LIN ,  Sam Lin ,  Chun-Hung Lin

发明人： Shi-Lung LIN ,  Sam Lin ,  Chun-Hung Lin

IPC分类号： C12N15/10 ,  A61P31/12

摘要： This invention relates to a novel composition and method for RNA/mRNA production as well as amplification using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes and the use of associated RNA/mRNA products thereof. The present invention can be used for manufacturing and amplifying all varieties of RNA/mRNA sequences carrying at least a replicase/RdRp-binding site in the 5′- or 3′-end, or both. The RNA/mRNA so obtained is useful for not only producing mRNA vaccines and/or RNA-based medicines but for generating the mRNA-associated proteins, peptides, and/or antibodies under an in-vitro as well as in-cell translation condition. Principally, the present invention is a novel RNA replicase/RdRp-mediated RNA/mRNA amplification method, namely Replicase Cycling Reaction (RCR). The RNA replicases involved in RCR include but not limited to viral and/or bacteriophage RNA-dependent RNA polymerases (RdRp) in either modified or non-modified mRNA and/or protein compositions, particularly coronaviral (e.g. COVID-19) and hepatitis C viral (HCV) RdRp enzymes.

公开(公告)号：US20200165607A1

公开(公告)日：2020-05-28

申请号：US16532353

申请日：2019-08-05

申请人： Shi-Lung Lin ,  Donald C. Chang ,  David TS Wu ,  Hsuan-Hsuan Lu

发明人： Shi-Lung Lin ,  Donald C. Chang ,  David TS Wu ,  Hsuan-Hsuan Lu

IPC分类号： C12N15/113 ,  C12N15/11

摘要： This invention generally relates to a composition and method of using mam-made small RNAs, such as small interfering RNAs (siRNA), microRNAs (miRNA) and their hairpin-like precursors (pre-miRNA), as tumor suppressing anti-cancer drugs for treating human tumors and cancers, in particular, but not limited, for treating skin (melanoma), blood (leukemia), prostate, breast, liver and lung cancers as well as various neoplastic tumors, such as brain tumors and teratocarcinomas that contain a variety of tumorous and cancerous cells derived from all three germ layers of tissues, including ectoderm, mesoderm and endoderm. More specifically, the present invention relates to the use of miR-302-like siRNA (siR-302) and/or miR-302 precursors (pre-miR-302) for developing novel medicines and therapies against a variety of human cancers, in particular lung cancers.

公开(公告)号：US10370658B2

公开(公告)日：2019-08-06

申请号：US15442557

申请日：2017-02-24

申请人： Shi-Lung Lin ,  Donald C. Chang ,  David T S Wu ,  Hsuan-Hsuan Lu

发明人： Shi-Lung Lin ,  Donald C. Chang ,  David T S Wu ,  Hsuan-Hsuan Lu

IPC分类号： C12N15/113 ,  C12N15/11

摘要： This invention generally relates to a composition and method of using mam-made small RNAs, such as small interfering RNAs (siRNA), microRNAs (miRNA) and their hairpin-like precursors (pre-miRNA), as tumor suppressing anti-cancer drugs for treating human tumors and cancers, in particular, but not limited, for treating skin (melanoma), blood (leukemia), prostate, breast, liver and lung cancers as well as various neoplastic tumors, such as brain tumors and teratocarcinomas that contain a variety of tumorous and cancerous cells derived from all three germ layers of tissues, including ectoderm, mesoderm and endoderm. More specifically, the present invention relates to the use of miR-302-like siRNA (siR-302) and/or miR-302 precursors (pre-miR-302) for developing novel medicines and therapies against a variety of human cancers, in particular lung cancers.

公开(公告)号：US20160264974A1

公开(公告)日：2016-09-15

申请号：US15167226

申请日：2016-05-27

申请人： Shi-Lung LIN ,  Donald CHANG ,  David TS WU

发明人： Shi-Lung LIN ,  Donald CHANG ,  David TS WU

IPC分类号： C12N15/113 ,  C12P19/34 ,  C12N15/70

CPC分类号： C12N15/1135 ,  C12N15/111 ,  C12N15/113 ,  C12N15/70 ,  C12N2310/14 ,  C12N2310/141 ,  C12N2320/30 ,  C12N2330/50 ,  C12N2330/51 ,  C12P19/34

摘要： This invention generally relates to a composition and its production method useful for developing drugs/vaccines and/or therapies against a variety of degenerative diseases in humans. Particularly, the present invention teaches the essential steps of production and purification processes necessary for producing small hairpin-like RNA (shRNA) compositions, such as microRNA precursors (pre-miRNA) and short interfering RNAs (siRNA), which are useful for treating human ageing related diseases, such as, but not limited, Alzheimer's diseases, Parkinson's diseases, osteoporosis, diabetes, and cancers. The novelty of the present invention is to create an artificially enhanced adaptation environment for prokaryotic cells to adopt eukaryotic pol-2 and/or pol-2-like promoters for transcribing desired ncRNAs and/or their precursors without going through error-prone prokaryotic promoters, so as to improve the productive efficiency and reading fidelity of the shRNA transcription in the prokaryotic cells. The resulting shRNAs, preferably pre-miRNAs and siRNAs, are useful for developing therapeutic drugs against human degenerative diseases, particularly through a mechanism to induce CD34-positive stem cell expansion and/or regeneration.

摘要翻译： 本发明一般涉及用于开发针对人类各种退行性疾病的药物/疫苗和/或疗法的组合物及其制备方法。 特别地，本发明教导了用于生产小发夹样RNA（shRNA）组合物，例如可用于治疗人类的微小RNA前体（pre-miRNA）和短干扰RNA（siRNA））所需的生产和纯化过程的基本步骤 衰老相关疾病，例如但不限于阿尔茨海默病，帕金森病，骨质疏松症，糖尿病和癌症。 本发明的新颖之处在于为原核细胞产生人造增强的适应环境，以采用真核的pol-2和/或pol-2样启动子来转录所需的ncRNA和/或其前体而不经历易错的原核启动子， 从而提高原核细胞中shRNA转录的生产效率和阅读保真度。 产生的shRNA，优选pre-miRNAs和siRNAs可用于开发针对人退行性疾病的治疗药物，特别是通过诱导CD34阳性干细胞扩增和/或再生的机制。

公开(公告)号：US09422559B2

公开(公告)日：2016-08-23

申请号：US13964705

申请日：2013-08-12

申请人： Shi-Lung Lin ,  David T S Wu

发明人： Shi-Lung Lin ,  David T S Wu

IPC分类号： C12N15/11 ,  C12N15/00 ,  C07H21/02 ,  C12N15/113

CPC分类号： C12N15/1137 ,  C12N15/111 ,  C12N15/113 ,  C12N2310/141 ,  C12N2330/50 ,  C12N2330/51

摘要： This invention generally relates to a design and method for developing novel anti-tumor and/or anti-cancer drugs, vaccines and therapies, using microRNA and/or its shRNA homologs/mimics/derivatives. More specifically, the present invention relates to an use of a prokaryote-produced miRNA precursor (pro-miRNA) composition capable of being delivered into human cells and processed by the cells into mature miRNA effectors to elicit specific silencing effects on mir-302-targeted genes, subsequently leading to a beneficial result of tumor suppression and cancer therapy. The prokaryotic cells do not naturally express or process eukaryotic miRNA precursors (pre-miRNA); meanwhile, the present invention also teaches an inducible method for expressing pre-miRNAs, particularly mir-302 precursors by using the prokaryotic transcription system. Since mir-302 is a known tumor suppressor in human, this novel finding advances the design and method for developing new anti-cancer drugs, vaccines and/or therapies directed against multiple kinds of human tumors and cancers.

摘要翻译： 本发明一般涉及使用微小RNA和/或其shRNA同源物/模拟物/衍生物开发新的抗肿瘤和/或抗癌药物，疫苗和疗法的设计和方法。 更具体地，本发明涉及能够被递送到人细胞中并由细胞加工成成熟miRNA受体的原核生产的miRNA前体（pro-miRNA）组合物的用途，以对mir-302靶向引发特异性沉默效应 基因，随后导致肿瘤抑制和癌症治疗的有益结果。 原核细胞不是天然表达或加工真核miRNA前体（pre-miRNA）; 同时，本发明还教导了通过使用原核转录系统表达前miRNAs，特别是mir-302前体的诱导型方法。 由于mir-302是人类已知的肿瘤抑制因子，因此该新发现推进了开发针对多种人类肿瘤和癌症的新的抗癌药物，疫苗和/或疗法的设计和方法。

公开(公告)号：US08609831B2

公开(公告)日：2013-12-17

申请号：US10663875

申请日：2003-09-16

申请人： Shi-Lung Lin ,  Shao-Yao Ying

发明人： Shi-Lung Lin ,  Shao-Yao Ying

IPC分类号： C07H21/04

CPC分类号： C12N15/113 ,  C12N15/63 ,  C12N2310/14 ,  C12N2310/30

摘要： An isolated RNA comprising an intron RNA that is released in a cell, thereby modulating the function of a target gene. Also disclosed are a composition comprising a chemokine and an isolated RNA of the invention or a DNA template for the isolated RNA, a composition comprising one or more agents that induce RNA-mediated modulation of the functions of two or more target genes in a cell, and methods of using these compositions for modulating the functions of genes in a cell.

摘要翻译： 一种分离的RNA，其包含在细胞中释放的内含子RNA，从而调节靶基因的功能。 还公开了包含本发明的趋化因子和分离的RNA或分离的RNA的DNA模板的组合物，包含诱导RNA介导的调节细胞中两种或更多种靶基因的功能的一种或多种试剂的组合物， 以及使用这些组合物调节细胞中基因功能的方法。

公开(公告)号：US20130210120A1

公开(公告)日：2013-08-15

申请号：US13572263

申请日：2012-08-10

申请人： Shi-Lung Lin ,  Donald C. Chang

发明人： Shi-Lung Lin ,  Donald C. Chang

IPC分类号： C12N15/70

CPC分类号： C12N15/635 ,  C12N1/38 ,  C12N15/70

摘要： Eukaryotic protein-coding messenger RNAs and non-coding microRNAs are naturally transcribed by type II RNA polymerases (pol-2) but not prokaryotic RNA polymerases. As a result, current eukaryotic RNA and protein production is performed either using eukaryotic pol-2 promoters in hybridomas or mammalian cells or using prokaryotic promoters in bacterial cells. However, because prokaryotic RNA transcription tends to be error-prone, frequent mutation is a big problem. Also, growing hybridomas or mammalian cells is relatively laborious and costly. To overcome these problems, the present invention provides a novel inducible composition and method for producing eukaryotic RNAs and/or their related peptides/proteins directly using eukaryotic pol-2 promoter-driven gene expression in fast growing bacteria, without the need of changing to prokaryotic promoters or growing hybridomas/mammalian cells. The RNAs and peptides/proteins so obtained can be used to develop drugs, cure diseases, treat tumors/cancers, produce pluripotent stem (iPS) cells, enhance wound healing, and make foods.

摘要翻译： 真核生物蛋白质编码的信使RNA和非编码微小RNA由II型RNA聚合酶（pol-2）天然转录而不是原核RNA聚合酶。 结果是，目前的真核RNA和蛋白质生产在杂交瘤或哺乳动物细胞中使用真核的pol-2启动子，或者在细菌细胞中使用原核启动子。 然而，由于原核RNA转录往往容易出错，频繁突变是一个大问题。 此外，生长中的杂交瘤或哺乳动物细胞相对费力和费用高昂。 为了克服这些问题，本发明提供了一种用于在快速生长的细菌中直接使用真核的pol-2启动子驱动的基因表达来生产真核RNA和/或其相关肽/蛋白质的新型诱导组合物和方法，而不需要改变为原核生物 启动子或生长杂交瘤/哺乳动物细胞。 如此获得的RNA和肽/蛋白质可用于开发药物，治愈疾病，治疗肿瘤/癌症，产生多能干细胞（iPS）细胞，增强伤口愈合和制备食物。

公开(公告)号：US20100298416A1

公开(公告)日：2010-11-25

申请号：US12740334

申请日：2008-10-29

申请人： Shao-Yao Ying ,  Shi-Lung Lin

发明人： Shao-Yao Ying ,  Shi-Lung Lin

IPC分类号： C12Q1/68 ,  A61K31/7105

CPC分类号： C12N15/1135 ,  C12N2310/141 ,  C12Q1/6886 ,  C12Q2600/112 ,  C12Q2600/178

摘要： Patterns of microRNA (miRNA) expression are correlated to the degrees of tumor cell differentiation in human prostate cancer. MiRNAs can complementarily bind to either oncogenes or tumor suppressor genes, resulting in targeted gene silencing and thus changes of cellular tumorigenecity. Using miRNA microarray analysis, 8 down-regulated and 3 up-regulated known miRNAs in androgen-independent human prostate cancer cell lines, such as LNCaP C4-2B and PC3, compared to those androgen-dependent cell lines, such as LNCaP and PC3-AR9 were consistently detected. Fluorescent in-situ hybridization assays in human prostate cancer tissue arrays containing sixty patients at different stages also showed the same miRNA expression patterns in hormone-refractory prostate carcinomas (HRPC) compared to androgen-sensitive non-cancerous prostate epithelium. In-vitro tumorigenecity assays using one of the identified miRNAs, mir-146a, were performed to provide validation of its function in prostate cancer. Gain-of-function transfection of mir-146a markedly suppressed its targeted ROCK1 gene expression in androgen-independent PC3 cells, consequently resulting in reduced cancer cell proliferation, invasion and metastasis to human bone marrow endothelial cell monolayers. Since ROCK1 is the key kinase for activating hyaluronan-mediated HRPC transformation in vivo and in PC3 cells, mir-146a should function as a tumor-suppressor gene in modulating the ROCK1-associated tumorigenecity.

摘要翻译： microRNA（miRNA）表达的模式与人类前列腺癌的肿瘤细胞分化程度相关。 MiRNA可以互补结合癌基因或肿瘤抑制基因，导致靶向基因沉默，从而导致细胞致瘤性的变化。 与雄激素依赖性细胞系（如LNCaP和PC3）相比，使用miRNA微阵列分析，8个下调和3个上调已知的与雄激素非依赖性前列腺癌细胞系（如LNCaP C4-2B和PC3）的miRNA相比， 一直检测到AR9。 与雄激素敏感的非癌前列腺上皮相比，含有60位不同阶段患者的人类前列腺癌组织阵列中的荧光原位杂交测定也显示与激素难治性前列腺癌（HRPC）相同的miRNA表达模式。 使用鉴定的miRNA之一的mir-146a进行体外肿瘤发生测定，以提供其在前列腺癌中的功能的验证。 mir-146a功能的功能转染显着抑制雄激素依赖性PC3细胞中靶向的ROCK1基因表达，从而导致癌细胞增殖，侵袭和转移到人类骨髓内皮细胞单层。 由于ROCK1是在体内和PC3细胞中激活透明质酸介导的HRPC转化的关键激酶，因此mir-146a应该作为调节ROCK1相关致瘤性的肿瘤抑制基因。