公开(公告)号：US20080305142A1

公开(公告)日：2008-12-11

申请号：US11792800

申请日：2005-12-12

申请人： Yin Chen ,  Frederic Kendirgi ,  Malcolm Skolnick

发明人： Yin Chen ,  Frederic Kendirgi ,  Malcolm Skolnick

IPC分类号： A61F2/04 ,  C12P19/34 ,  A01N43/00 ,  A61K39/00 ,  A61P31/12 ,  A61P31/10 ,  A61K31/7052 ,  A61K9/12

CPC分类号： C12P19/34

摘要： The invention provides an improved cell free amplification method capable of producing large quantities of therapeutic-quality nucleic acids and methods of using the synthesized nucleic acid in research, therapeutic and other applications—The methods combine several different state-of-the-art procedures and coordinate their applications to affordably synthesize nucleic acids for therapeutic purposes. It combines in vitro rolling circle amplification, high fidelity polymerases, high affinity primers, and streamlined template specifically designed for particular applications. For expression purposes, the templates contain an expression cassette including a eukaryotic promoter, the coding sequence for the gene of interest, and a eukaryotic termination sequence. Following amplification, concatamers are subsequently processed according to their intended use and may include: restriction enzyme digestion for the production of short expression cassettes (SECs); ligation steps to circularize the SEC (CNAs); and/or supercoiling steps to produce sCNAs. The final product contains nearly non-detectable levels of bacterial endotoxin.

摘要翻译： 本发明提供了能够产生大量治疗质量核酸的改进的无细胞扩增方法和在研究，治疗和其它应用中使用合成核酸的方法 - 该方法结合了几种不同的最先进的方法和 协调其应用，以便为治疗目的负担得起的合成核酸。 它结合体外滚环扩增，高保真聚合酶，高亲和力引物和专为特定应用设计的流线型模板。 为了表达目的，模板含有包含真核启动子，目的基因的编码序列和真核终止序列的表达盒。 扩增后，随后根据其预期用途处理连接体，并且可以包括：用于产生短表达盒（SEC）的限制酶消化; 结转步骤，循环SEC（CNA）; 和/或超螺旋步骤以产生sCNA。 最终产品含有几乎不可检测的细菌内毒素水平。

公开(公告)号：US07419964B2

公开(公告)日：2008-09-02

申请号：US10313828

申请日：2002-12-06

申请人： Yin Chen ,  Malcolm Skolnick

发明人： Yin Chen ,  Malcolm Skolnick

IPC分类号： A01N43/04 ,  C12Q1/68 ,  C12N15/00 ,  C07H21/02 ,  C07H21/04

CPC分类号： C12N15/1133 ,  A61K2121/00 ,  C12N15/10 ,  C12N15/113 ,  C12N15/85 ,  C12N2310/11 ,  C12N2310/111 ,  C12N2310/12 ,  C12N2310/15 ,  C12N2310/16 ,  C12N2310/53 ,  C12N2840/20

摘要： A composition for treatment of HSV-related pathologies including an expression vector for altering expression of a target sequence in an HSV-infected cell by production of single-stranded cDNA (ssDNA) in the cell in vivo suspended for topical application to an affected site in a suitable delivery vehicle. The expression vector is comprised of a cassette comprising a sequence of interest, an inverted tandem repeat, and a primer binding site 3′ to the inverted tandem repeat, and a reverse transcriptase/RNAse H coding gene, and is transfected into the infected cells for inhibition of HSV replication. The resulting ssDNA binds to the target sequence to alter expression of the target sequence for such purposes as gene activation or inactivation using duplex or triplex binding of nucleic acids, site-directed mutagenesis, interruption of cellular function by binding to specific cellular proteins, or interfering with RNA splicing functions.

摘要翻译： 一种用于治疗HSV相关病状的组合物，其包括用于通过在体内悬浮用于局部施用于受影响部位的细胞中的单链cDNA（ssDNA）产生来改变HSV感染细胞中靶序列表达的表达载体 合适的运送工具。 表达载体由包含感兴趣序列，反向串联重复序列和反向串联重复序列的引物结合位点3'和逆转录酶/ RNA酶H编码基因的盒组成，并转染入感染的细胞中 抑制HSV复制。 所得的ssDNA结合靶序列以改变靶序列的表达，用于诸如基因激活或失活的目的，使用核酸的双链或三重结合，定点突变，通过结合特异性细胞蛋白中断细胞功能或干扰 具有RNA拼接功能。

公开(公告)号：US20080312173A1

公开(公告)日：2008-12-18

申请号：US12009825

申请日：2008-01-22

申请人： Yin Chen ,  Malcolm Skolnick

发明人： Yin Chen ,  Malcolm Skolnick

IPC分类号： A61K31/711 ,  A61P31/12

CPC分类号： C12N15/1133 ,  A61K2121/00 ,  C12N15/10 ,  C12N15/113 ,  C12N15/85 ,  C12N2310/11 ,  C12N2310/111 ,  C12N2310/12 ,  C12N2310/15 ,  C12N2310/16 ,  C12N2310/53 ,  C12N2840/20

摘要： A composition for treatment of HSV-related pathologies including an expression vector for altering expression of a target sequence in an HSV-infected cell by production of single-stranded cDNA (ssDNA) in the cell in vivo suspended for topical application to an affected site in a suitable delivery vehicle. The expression vector is comprised of a cassette comprising a sequence of interest, an inverted tandem repeat, and a primer binding site 3′ to the inverted tandem repeat, and a reverse transcriptase/RNAse H coding gene, and is transfected into the infected cells for inhibition of HSV replication. The resulting ssDNA binds to the target sequence to alter expression of the target sequence for such purposes as gene activation or inactivation using duplex or triplex binding of nucleic acids, site-directed mutagenesis, interruption of cellular function by binding to specific cellular proteins, or interfering with RNA splicing functions.

摘要翻译： 一种用于治疗HSV相关病状的组合物，其包括用于通过在体内悬浮用于局部施用于受影响部位的细胞中的单链cDNA（ssDNA）产生来改变HSV感染细胞中靶序列表达的表达载体 合适的运送工具。 表达载体由包含感兴趣序列，反向串联重复序列和反向串联重复序列的引物结合位点3'和逆转录酶/ RNA酶H编码基因的盒组成，并转染入感染的细胞中 抑制HSV复制。 所得的ssDNA结合靶序列以改变靶序列的表达，用于诸如基因激活或失活的目的，使用核酸的双链或三重结合，定点突变，通过结合特异性细胞蛋白中断细胞功能或干扰 具有RNA拼接功能。

公开(公告)号：US5012808A

公开(公告)日：1991-05-07

申请号：US258785

申请日：1988-10-17

申请人： Ron Stubbers ,  Chen Chen ,  Malcolm Skolnick

发明人： Ron Stubbers ,  Chen Chen ,  Malcolm Skolnick

IPC分类号： G01R13/34 ,  G01R19/25 ,  G01R23/10 ,  G01R29/02

CPC分类号： G01R13/342 ,  G01R19/25 ,  G01R23/10 ,  G01R29/02

摘要： A pulse generation and tracking system for connection to an object or a subject under treatment therewith, the waveform being a train of repetitive complex pulses each pulse comprising a large amplitude, short duration positive polarity portion and a small amplitude, long duration negative polarity portion, and a zero value portion. The prescribed pulse train for a subject is delivered by a delivery device, which also provides monitoring of the averaged voltage amplitude of the positive portion in terms of either voltage or current value. The impedance of the subject is expected to change over time and cause the individual pulses to vary from predetermined limits. When this occurs, then a subsequent generation of pulses will be changed so that the pulses are again delivered within the predetermined value limits or boundary. Tracking and generation can be operationally crossed, if desired, so that a voltage out of boundary will cause the current of a subsequent pulse to be changed or vice versa.

摘要翻译： 一种脉冲产生和跟踪系统，用于连接到被处理的对象或对象，该波形是重复的复杂脉冲序列，每个脉冲包括大振幅，短持续时间的正极性部分和小振幅，持续时间长的负极性部分， 和零值部分。 用于被摄体的规定的脉冲串由传送装置传送，传送装置还根据电压或电流值提供对正部分的平均电压幅度的监视。 受试者的阻抗预期随着时间的推移而改变，并导致各个脉冲从预定的限制变化。 当发生这种情况时，随后的脉冲产生将被改变，使得脉冲在预定值限制或边界内再次传送。 如果需要，跟踪和产生可以在操作上交叉，使得超出边界的电压将导致随后的脉冲的电流被改变，反之亦然。

公开(公告)号：US4779044A

公开(公告)日：1988-10-18

申请号：US95009

申请日：1987-09-08

申请人： Malcolm Skolnick ,  Ron Stubbers ,  Chris Hymel ,  Chen Chen

发明人： Malcolm Skolnick ,  Ron Stubbers ,  Chris Hymel ,  Chen Chen

IPC分类号： G01R13/34 ,  G01R19/25 ,  G01R23/10 ,  G01R23/16

CPC分类号： G01R19/25 ,  G01R13/342 ,  G01R23/10

摘要： A regularly repetitive, almost periodic but non-regularly shaped waveform is amplitude sampled at an interval different from that of the waveform under investigation. For a voltage measurement, the positive samples are averaged for a predetermined count number (one set) and the negative samples are likewise averaged. Zero value samples are ignored. The highest absolute value of the two is taken as a meaningful measurement. Alternatively, one of these values can be selected. For a current measurement, a running average for a predetermined number of sets can also be developed. Finally, frequency can be developed by sensing the transitions and operating a counter on/off at the beginning and ending of a waveform cycle. The count of regular interval counts is then multiplied by the interval time to give the period of the waveform. The reciprocal of the period is a measure of the frequency.

摘要翻译： 定期重复，几乎周期性但非规则形状的波形以与正在研究的波形的间隔不同的幅度采样。 对于电压测量，将正样本平均为预定计数（一组），并且负样本同样平均。 零值样本被忽略。 两者的最高绝对值作为有意义的测量。 或者，可以选择这些值之一。 对于当前的测量，也可以开发预定数量的集合的运行平均值。 最后，可以通过在波形周期的开始和结束时感测转换并运行计数器开/关来开发频率。 然后将定期间隔计数的计数乘以间隔时间以给出波形的周期。 期间的倒数是频率的度量。