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1. 发明公开

US20230295627A1 Novel Replicase Cycling Reaction (RCR) and the Related SamRNA Designs Thereof 审中-公开
公开(公告)号：US20230295627A1
公开(公告)日：2023-09-21
申请号：US18156231
申请日：2023-01-18
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： C12N15/113 , C12N9/12 , C12Q1/686
CPC分类号： C12N15/113 , C12N9/127 , C12Q1/686 , C12N2310/141 , C12Y207/07048
摘要： This invention generally relates to a novel composition of RNA/mRNA medicines as well as vaccines produced by using replicase- and/or RNA-dependent RNA polymerase (RdRp)-mediated RNA cycling reaction (RCR). The present invention is useful for developing a variety of self-amplifying RNA/mRNA (samRNA) medicines and vaccines containing at least a replicase/RdRp-binding site in the 5′- or 3′-end, or both, of any desired RNA molecule, including but not limited to antisense RNA (aRNA), small interferring RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA)/miRNA precursor, long non-coding RNA (lnRNA) and mRNA. These RNA molecules can be either in single-stranded or in double-stranded, or mixed, conformation. The samRNA so obtained is useful not only for producing RNA-based vaccines and/or medicines but also for generating the mRNA-associated proteins, peptides, and/or antibodies under a proper in-vitro or in-cell translation condition. The replicase/RdRp-binding sites used in samRNA are derived or modified from coronaviral (e.g. COVID-19) and/or hepatitis C viral (HCV) RNA-dependent RNA polymerases (RdRp) in either single-stranded or double-stranded compositions.

2. 发明申请

US20220411848A1 Novel Replicase Cycling Reaction (RCR) 有权
公开(公告)号：US20220411848A1
公开(公告)日：2022-12-29
申请号：US17648336
申请日：2022-01-19
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： C12Q1/686 , C12N9/12
摘要： This invention generally relates to a novel RNA/mRNA production and amplification method using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes as well as the associated mRNAs thereof. The present invention can be used for manufacturing and amplifying all varieties of RNA/mRNA sequences carrying at least an 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 also 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-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), particularly coronaviral and hepatitis C viral (HCV) RdRp enzymes.

3. 发明申请

US20220396798A1 NOVEL MRNA COMPOSITION AND PRODUCTION METHOD FOR USE IN ANTI-VIRAL AND ANTI-CANCER VACCINES 有权
公开(公告)号：US20220396798A1
公开(公告)日：2022-12-15
申请号：US17489357
申请日：2021-09-29
申请人： Shi-Lung LIN , Samantha CHANG-LIN , Jack SK CHEN , David TS WU , Chia-Ning SHEN , Mei-Jung WANG
发明人： Shi-Lung LIN , Samantha CHANG-LIN , Jack SK CHEN , David TS WU , Chia-Ning SHEN , Mei-Jung WANG
IPC分类号： C12N15/113 , C12N15/85
摘要： This invention relates to a novel mRNA composition and its production method useful for developing and manufacturing RNA-based anti-viral and/or anti-cancer vaccines and medicines. This invention includes two types of mRNA constructs, namely “5′-hairpin messenger RNA (5hmRNA)” and “messenger-hairpin-messenger RNA (mhmRNA)”, respectively. Both of 5hmRNA and mhmRNA contain at least a hairpin-like stem-loop RNA structure. The 5hmRNA contains at least a stem-loop RNA structure in the 5′-UTR of a protein/peptide-coding mRNA, while the mhmRNA contains a middle stem-loop structure flanked with two protein/peptide-coding mRNA sequences on both sides. In mhmRNA, the first 5′-mRNA preferably encodes an RNA replicase, for amplifying the second 3′-mRNA in transfected cells. After transfection into target cells, 5hmRNA and mhmRNA can be further translated into at least a desired protein/peptide. To produce highly structured 5hmRNA and mhmRNA, a novel PCR-IVT methodology has been developed and used with a specially designed RNA polymerase-helicase mixture reaction

4. 发明申请

US20220396778A1 Novel RNA Composition and Production Method for Use in iPS Cell Generation 有权
公开(公告)号：US20220396778A1
公开(公告)日：2022-12-15
申请号：US17648340
申请日：2022-01-19
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： C12N5/074 , C12N15/113 , C12N9/12 , A61K31/713 , A61K38/45
摘要： This invention generally relates to a novel RNA composition and its production method useful for generating and expanding induced pluripotent stem cells (iPS cells; iPSC) as well as adult stem cells (ASC). The RNA composition so defined can be used for producing not only non-transgenic but also tumor-free iPS cells. The defined RNA composition contans at least two types of different RNA constructs; one is “miR-302 precursor RNA (pre-miR-302)” and the other is “RNA-dependent RNA polymerase (RdRp)” mRNA. Both of pre-miR-302 and RdRp mRNA contain highly structured RNA comformations, such as hairpin and stem-loop structures. To produce highly structured RNAs, a novel PCR-IVT methodology has been developed and used with a specially designed RNA polymerase-helicase mixture activity.

5. 发明授权

US09956196B2 Use of monosaccharide-like glycylated sugar alcohol compositions for designing and developing anti-diabetic drugs 有权
公开(公告)号：US09956196B2
公开(公告)日：2018-05-01
申请号：US14585978
申请日：2014-12-30
申请人： Shi-Lung Lin , Samantha Chang-Lin , Yi-Wen Lin , Donald Chang
发明人： Shi-Lung Lin , Samantha Chang-Lin , Yi-Wen Lin , Donald Chang
IPC分类号： A61K31/7012 , A61K31/221 , C12N15/11 , C12N15/113 , A61K9/00 , A61K9/08 , A61K31/7024
CPC分类号： A61K31/221 , A61K9/0053 , A61K9/08 , A61K31/7012 , A61K31/7024 , C12N15/111 , C12N15/113 , C12N2310/141 , C12N2320/32 , C12N2330/50 , C12N2330/51
摘要： This invention is related to a novel sugar-like chemical composition and its use for diabetes therapy. Particularly, the present invention teaches the use of monosaccharide-like glycylated sugar alcohol compounds to block or reduce sugar absorption in diabetes patients, so as to prevent the risk of hyperglycemia symptoms. Glycylation of sugar alcohols is a totally novel reaction that has never been reported before. Therefore, the novelty of the present invention is that for the first time glycylated sugar alcohols not only was found but also was found to be useful for treating Diabetes mellitus. In addition, the present invention teaches a method for producing these glycylated sugar alcohols. In sum, the present invention includes not only a kind of novel sugar-like chemical compositions and its use for treating diabetes but also a state-of-the-art protocol and methodology for producing such a novel composition via glycylation of sugars and sugar alcohols.

6. 发明申请

US20170342418A1 USE OF MICRORNA PRECURSORS AS DRUGS FOR INDUCING CD34-POSITIVE ADULT STEM CELL EXPANSION 审中-公开
公开(公告)号：US20170342418A1
公开(公告)日：2017-11-30
申请号：US15661346
申请日：2017-07-27
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 , C12N15/11 , 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.

7. 发明授权

US09567591B2 Generation of human embryonic stem-like cells using intronic RNA 有权
标题翻译：使用内含子RNA产生人胚胎干细胞样细胞
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。

8. 发明申请

US20160289682A1 PRODUCTION AND UTILIZATION OF A NOVEL ANTI-CANCER DRUG IN THERAPY 审中-公开
公开(公告)号：US20160289682A1
公开(公告)日：2016-10-06
申请号：US15167219
申请日：2016-05-27
申请人： Shi-Lung LIN , David TS WU
发明人： Shi-Lung LIN , David TS WU
IPC分类号： C12N15/113
CPC分类号： C12N15/1135 , C12N2310/141 , C12N2310/531 , 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 homologues/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.

9. 发明申请

US20140350085A1 NOVEL SUGAR ALCOHOL-BASED COMPOSITIONS FOR DELIVERING NUCLEIC ACID-BASED DRUGS IN VIVO AND IN VITRO 有权
标题翻译：用于在血液和体内输送基于核酸的药物的新型基于酒精的基于醇的组合物
公开(公告)号：US20140350085A1
公开(公告)日：2014-11-27
申请号：US14457829
申请日：2014-08-12
申请人： Shi-Lung Lin , David TS Wu
发明人： Shi-Lung LIN , Yi-Wen LIN
IPC分类号： A61K47/26 , A61K31/713 , A61K31/7105
CPC分类号： A61K47/26 , A61K31/7105 , A61K31/713 , C12N15/111 , C12N15/113 , C12N2310/141 , C12N2320/32 , C12N2330/50 , C12N2330/51 , C12N2740/15041
摘要： This invention relates to a composition and its use for formulating nucleic acid-based drugs/vaccines with sugar alcohol compositions into complexes for both in-vitro and in-vivo delivery. Particularly, the present invention includes the ingredients and processes necessary for formulating therapeutic and pharmaceutical nucleic acid compositions, such as miRNA, microRNA precursors, shRNAs, siRNAs, ribozymes, antisense RNAs/DNAs, RNA-DNA hybrids and DNA vectors/vaccines, with glycylated sugar alcohols/sugars into delivery complexes, which can then be absorbed by cells in vivo and in vitro via active endocytosis. Also, the present invention discloses that chemical compounds containing sugar alcohol- and/or sugar-like structures can protect nucleic acids, in particular miRNAs, shRNAs, siRNAs and ribozymes, from degradation in vivo as well as in vitro. Therefore, the present invention is also a formula and method for preserving the structural integrity and functional efficacy of these nucleic acid-based drugs and/or vaccines in vivo and in vitro.
摘要翻译：本发明涉及一种组合物及其用于将糖醇组合物配制成用于体外和体内递送的复合物的基于核酸的药物/疫苗的用途。特别地，本发明包括配制具有糖基化的治疗和药物核酸组合物如miRNA，微RNA前体，shRNA，siRNA，核酶，反义RNA / DNA，RNA-DNA杂交体和DNA载体/疫苗所必需的成分和方法糖醇/糖进入递送复合物，其然后可以通过活性内吞作用在体内和体外被细胞吸收。此外，本发明公开了含有糖醇和/或糖类结构的化合物可以保护核酸，特别是miRNA，shRNA，siRNA和核酶免受体内和体外降解。因此，本发明也是用于在体内和体外保持这些基于核酸的药物和/或疫苗的结构完整性和功能功效的配方和方法。

10. 发明申请

US20140141470A1 PRODUCTION AND EXTRACTION OF MicroRNA PRECURSOR AS DRUG FOR CANCER THERAPY 有权
标题翻译：作为药物治疗的微RNA前体的生产和提取
公开(公告)号：US20140141470A1
公开(公告)日：2014-05-22
申请号：US14142512
申请日：2013-12-27
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
CPC分类号： C12N15/113 , C12N15/111 , C12N2310/141 , C12N2330/50 , C12N2330/51
摘要： This invention generally relates to a composition for developing novel anti-cancer drugs and/or vaccines and producing microRNA precursor (pre-miRNA) and/or its shRNA homologues/mimics/derivatives, and a method thereof. The present invention also relates to a use of a composition in producing novel prokaryote-produced microRNA precursor (pro-miRNA) capable of being delivered into human cells and processed by the cells into microRNA-like effectors to elicit specific silencing effects on certain targeted oncogenes, subsequently leading to a therapeutic result of tumor suppression and cancer therapy. Specifically, the method of the present invention includes inducing an expression of the pre-miRNA/pro-miRNAs, particularly human pre-miR-302, in prokaryotes through pol-2 or pol-2-like RNA promoter. Most importantly, the composition of the present invention is further a novel pre-miRNA-based drug that is capable of reprogramming the malignant properties of high-grade human liver cancers into a low-grade benign or even relatively normal stage—a mechanism called “Cancer Reversion”.
摘要翻译：本发明一般涉及用于开发新的抗癌药物和/或疫苗并产生微小RNA前体（pre-miRNA）和/或其shRNA同源物/模拟物/衍生物的组合物及其方法。本发明还涉及一种组合物在生产新型原核生产的微小RNA前体（pro-miRNA）中的用途，其能够被递送到人类细胞中并被细胞加工成微小RNA样效应物以引发对某些靶向癌基因的特异性沉默效应，随后导致肿瘤抑制和癌症治疗的治疗结果。具体地说，本发明的方法包括通过pol-2或pol-2样RNA启动子在原核生物中诱导前miRNA /前miRNAs，特别是人pre-miR-302的表达。最重要的是，本发明的组合物还是一种新的基于前体miRNA的药物，其能够将高级别人类肝癌的恶性特征重新编程成低级良性或甚至相对正常的阶段 - 称为“ 癌症逆转“。

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