公开(公告)号：US20170175118A1

公开(公告)日：2017-06-22

申请号：US15442557

申请日：2017-02-24

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

CPC分类号： C12N15/113 ,  C12N15/111 ,  C12N15/1135 ,  C12N2310/14 ,  C12N2310/141 ,  C12N2310/531 ,  C12N2320/30 ,  C12N2320/32 ,  C12N2330/50 ,  C12N2330/51

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：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：US09453219B2

公开(公告)日：2016-09-27

申请号：US12003662

申请日：2007-12-28

申请人： Shi-Lung Lin ,  David TS Wu

发明人： Shi-Lung Lin ,  David TS Wu

IPC分类号： C12N15/113 ,  C12N15/11

CPC分类号： C12N15/111 ,  C12N15/113 ,  C12N2310/111 ,  C12N2310/141 ,  C12N2320/30 ,  C12N2320/50

摘要： The present invention relates to a method and composition for generating a non-naturally occurring intron and its components capable of being processed into small hairpin RNA (shRNA) and/or microRNA (miRNA) molecules by skin cells and thus inducing specific gene silencing effects on skin pigment-related genes and/or aging-causing genes in the cells. The gene silencing effects so obtained are not only useful for lightening and whitening skin colors but also useful for suppressing unwanted aging gene activities in skins.

摘要翻译： 本发明涉及一种用于产生非天然存在的内含子及其组分的方法和组合物，其能够被皮肤细胞加工成小发夹RNA（shRNA）和/或微小RNA（miRNA）分子，从而诱导特定的基因沉默效应 皮肤色素相关基因和/或衰老基因。 如此获得的基因沉默效应不仅可用于减轻和增白皮肤颜色，还可用于抑制皮肤中不需要的老化基因活性。

公开(公告)号：US09567591B2

公开(公告)日：2017-02-14

申请号：US12149725

申请日：2008-05-07

申请人： Shi-Lung Lin ,  Shao-Yao Ying ,  David Ts Wu

发明人： Shi-Lung Lin ,  Shao-Yao Ying ,  David Ts Wu

IPC分类号： C12N15/11 ,  C07H21/02 ,  C07H21/04 ,  C12Q1/68 ,  C12N5/02 ,  C12N15/64 ,  C12N15/63

CPC分类号： C12N15/63

摘要： This invention generally relates to a method for developing, generating and selecting human embryonic stem (hES)-like pluripotent cells using transgenic expression of intronic microRNA-like RNA agents. More particularly, the present invention relates to a method and composition for generating a non-naturally occurring intron and its intronic components capable of being processed into mir-302-like RNA molecules in mammalian cells and thus inducing certain specific gene silencing effects on differentiation-related and fate-determinant genes of the cells, resulting in reprogramming the cells into a pluripotent embryonic stem (ES)-cell-like state. The ES-like cells so obtained are strongly express hES cell markers, such as Oct3/4, SSEA-3 and SSEA-4, and can be guided into various tissue cell types by treating certain hormones and/or growth factors under a feeder-free cell culture condition in vitro, which may be used for transplantation and gene therapies. Therefore, the present invention offers a simple, effective and safe gene manipulation approach for not only reprogramming somatic cells into ES-like pluripotent cells but also facilitating the maintenance of pluripotent and renewal properties of ES cells under a feeder-free cell culture condition, preventing the tedious retroviral insertion of four large transcription factor genes into one single cell as used in the previous iPS methods.

摘要翻译： 本发明一般涉及使用内含子微RNA样RNA试剂的转基因表达来开发，产生和选择人胚胎干（hES）样多能细胞的方法。 更具体地说，本发明涉及一种用于产生非天然存在的内含子及其内含子组分的方法和组合物，其能够在哺乳动物细胞中加工成mir-302样RNA分子，从而诱导对分化 - 相关和命运决定基因，导致将细胞重编程为多能胚胎干（ES）细胞样状态。 如此获得的ES样细胞强烈表达hES细胞标志物，例如Oct3 / 4，SSEA-3和SSEA-4，并且可以通过在饲养层上处理某些激素和/或生长因子而被引导到各种组织细胞类型中， 体外免疫细胞培养条件，可用于移植和基因治疗。 因此，本发明提供了一种简单，有效和安全的基因操作方法，用于不仅将体细胞重编程为ES样多能细胞，而且有助于在无饲养细胞培养条件下维持ES细胞的多能性和更新性质，预防 将以前的iPS方法中使用的四个大转录因子基因的繁琐的逆转录病毒插入一个单细胞中。

公开(公告)号：US20080293143A1

公开(公告)日：2008-11-27

申请号：US12149725

申请日：2008-05-07

申请人： Shi-Lung Lin ,  Shao-Yao Ying ,  David Ts Wu

发明人： Shi-Lung Lin ,  Shao-Yao Ying ,  David Ts Wu

IPC分类号： C12N15/87 ,  C12N13/00 ,  C12N15/00

CPC分类号： C12N15/63

摘要： This invention generally relates to a method for developing, generating and selecting human embryonic stem (hES)-like pluripotent cells using transgenic expression of intronic microRNA-like RNA agents. More particularly, the present invention relates to a method and composition for generating a non-naturally occurring intron and its intronic components capable of being processed into mir-302-like RNA molecules in mammalian cells and thus inducing certain specific gene silencing effects on differentiation-related and fate-determinant genes of the cells, resulting in reprogramming the cells into a pluripotent embryonic stem (ES)-cell-like state. The ES-like cells so obtained are strongly express hES cell markers, such as Oct3/4, SSEA-3 and SSEA-4, and can be guided into various tissue cell types by treating certain hormones and/or growth factors under a feeder-free cell culture condition in vitro, which may be used for transplantation and gene therapies. Therefore, the present invention offers a simple, effective and safe gene manipulation approach for not only reprogramming somatic cells into ES-like pluripotent cells but also facilitating the maintenance of pluripotent and renewal properties of ES cells under a feeder-free cell culture condition, preventing the tedious retroviral insertion of four large transcription factor genes into one single cell as used in the previous iPS methods.

摘要翻译： 本发明一般涉及使用内含子微RNA样RNA试剂的转基因表达来开发，产生和选择人胚胎干（hES）样多能细胞的方法。 更具体地说，本发明涉及一种用于产生非天然存在的内含子及其内含子组分的方法和组合物，其能够在哺乳动物细胞中加工成mir-302样RNA分子，从而诱导对分化 - 相关和命运决定基因，导致将细胞重编程为多能胚胎干（ES）细胞样状态。 如此获得的ES样细胞强烈表达hES细胞标志物，例如Oct3 / 4，SSEA-3和SSEA-4，并且可以通过在饲养层上处理某些激素和/或生长因子而被引导到各种组织细胞类型中， 体外免疫细胞培养条件，可用于移植和基因治疗。 因此，本发明提供了一种简单，有效和安全的基因操作方法，用于不仅将体细胞重编程为ES样多能细胞，而且有助于在无饲养细胞培养条件下维持ES细胞的多能性和更新性质，预防 将以前的iPS方法中使用的四个大转录因子基因的繁琐的逆转录病毒插入一个单细胞中。

公开(公告)号：US20090203141A1

公开(公告)日：2009-08-13

申请号：US12318806

申请日：2009-01-08

申请人： Shi-Lung Lin ,  David TS Wu

发明人： Shi-Lung Lin ,  David TS Wu

IPC分类号： C12N15/86 ,  C12N15/74

CPC分类号： C12N15/63 ,  C12N2840/102 ,  C12N2840/44 ,  C12N2840/445

摘要： The present invention generally relates to a method for developing, generating and selecting tumor-free embryonic stem (ES)-like pluripotent cells using electroporation delivery of an inducible tumor suppressor mir-302 agent into mammalian cells. More particularly, the present invention relates to a method and composition for generating a Tet-On/Off recombinant transgene capable of expressing a manually re-designed mir-302 microRNA (miRNA)/shRNA agent under the control of doxycyclin (Dox) in human somatic/cancer cells and thus inducing certain specific gene silencing effects on the differentiation-associated genes and oncogenes of the cells, resulting in reprogramming the cells into an ES-like pluripotent state.

摘要翻译： 本发明一般涉及使用诱导型抑制剂mir-302试剂向哺乳动物细胞中电穿孔递送来开发，产生和选择无肿瘤胚胎干（ES）样多能细胞的方法。 更具体地说，本发明涉及一种能够在人的多西环素（Dox）控制下表达手工重新设计的mir-302微小RNA（miRNA）/ shRNA试剂的Tet-On / Off重组转基因的方法和组合物。 体细胞/癌细胞，从而对细胞的分化相关基因和癌基因诱导某些特异性基因沉默效应，导致将细胞重新编程成ES样多能状态。

公开(公告)号：US20090170204A1

公开(公告)日：2009-07-02

申请号：US12003662

申请日：2007-12-28

申请人： Shi-Lung Lin ,  David Ts Wu

发明人： Shi-Lung Lin ,  David Ts Wu

IPC分类号： C07H21/02 ,  C12N15/64

CPC分类号： C12N15/111 ,  C12N15/113 ,  C12N2310/111 ,  C12N2310/141 ,  C12N2320/30 ,  C12N2320/50

摘要： The present invention relates to a method and composition for generating a non-naturally occurring intron and its components capable of being processed into small hairpin RNA (shRNA) and/or microRNA (miRNA) molecules by skin cells and thus inducing specific gene silencing effects on skin pigment-related genes and/or aging-causing genes in the cells. The gene silencing effects so obtained are not only useful for lightening and whitening skin colors but also useful for suppressing unwanted aging gene activities in skins.

摘要翻译： 本发明涉及一种用于产生非天然存在的内含子及其组分的方法和组合物，其能够被皮肤细胞加工成小发夹RNA（shRNA）和/或微小RNA（miRNA）分子，从而诱导特定的基因沉默效应 皮肤色素相关基因和/或衰老基因。 如此获得的基因沉默效应不仅可用于减轻和增白皮肤颜色，还可用于抑制皮肤中不需要的老化基因活性。

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：US09394538B2

公开(公告)日：2016-07-19

申请号：US12792413

申请日：2010-06-02

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 ,  C12Q1/68 ,  C12N5/02 ,  C07H21/04 ,  C12N15/113 ,  C12N15/63

CPC分类号： C12N15/113 ,  C12N15/63 ,  C12N15/635 ,  C12N2310/141 ,  C12N2330/51

摘要： This invention generally relates to a design and method for developing novel anti-tumor/cancer drugs, vaccines and therapies, using microRNA (miRNA) and its shRNA homologues/derivatives. More particularly, the present invention relates to the use of a nucleic acid composition capable of expressing mir-302-like gene silencing effectors upon delivery into human cells and then silencing mir-302-targeted cell cycle regulators and oncogenes, resulting in an inhibitory effect on tumor/cancer cell growth and metastasis. Mir-302 is the most predominant miRNA found in human embryonic stem (hES) and induced pluripotent stem (iPS) cells, yet its function is unclear. The present invention establishes that in humans mir-302 concurrently suppressed both cyclin-E-CDK2 and cyclin-D-CDK4/6 pathways and eventually blocked over 70% of the G1-S transition. Simultaneously, mir-302 also silences BMI-1, a cancer stem cell marker, and subsequently promotes the tumor suppressor functions of p16Ink4a and p14/p19Arf in inhibiting CDK4/6-mediated cell proliferation. Therefore, the present invention for the first time reveals the tumor suppressor function of mir-302 in humans. This novel finding advances the design and method for developing new cancer drugs, vaccines and therapies directed against multiple kinds of human tumors and cancers, in particular including, but not limited, malignant skin, prostate, breast and liver cancers as well as various tumors.

摘要翻译： 本发明一般涉及使用微小RNA（miRNA）及其shRNA同源物/衍生物开发新的抗肿瘤/癌症药物，疫苗和疗法的设计和方法。 更具体地说，本发明涉及在递送至人细胞后能够表达mir-302样基因沉默效应物，然后使mir-302靶向的细胞周期调节因子和致癌基因沉默的核酸组合物的用途，从而产生抑制作用 对肿瘤/癌细胞的生长和转移。 Mir-302是人类胚胎干细胞（hES）和诱导多能干（iPS）细胞中最主要的miRNA，但其功能尚不清楚。 本发明确定人mir-302同时抑制细胞周期蛋白E-CDK2和细胞周期蛋白-D-CDK4 / 6途径，并最终阻断70％以上的G1-S转换。 同时，mir-302还沉默BMI-1，一种癌症干细胞标记物，随后促进p16Ink4a和p14 / p19Arf抑制CDK4 / 6介导的细胞增殖的肿瘤抑制功能。 因此，本发明首次揭示了人类mir-302的肿瘤抑制功能。 该新发现推进了针对多种人类肿瘤和癌症开发新的癌症药物，疫苗和疗法的设计和方法，特别是包括但不限于恶性皮肤，前列腺癌，乳腺癌和肝癌以及各种肿瘤。