RU2016104674A - ERYTHROCYT MODIFICATION DEVICE WITH DIRECTED MEDICINAL TRANSPORT MECHANISM FOR CRISPR / CAS9 GENE THERAPY FUNCTIONS - Google Patents

ERYTHROCYT MODIFICATION DEVICE WITH DIRECTED MEDICINAL TRANSPORT MECHANISM FOR CRISPR / CAS9 GENE THERAPY FUNCTIONS Download PDF

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RU2016104674A
RU2016104674A RU2016104674A RU2016104674A RU2016104674A RU 2016104674 A RU2016104674 A RU 2016104674A RU 2016104674 A RU2016104674 A RU 2016104674A RU 2016104674 A RU2016104674 A RU 2016104674A RU 2016104674 A RU2016104674 A RU 2016104674A
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suspension
blood cells
red blood
cells according
washed
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RU2016104674A
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Анатолий Викторович Зазуля
Владимир Анатольевич Зазуля
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Анатолий Викторович Зазуля
Владимир Анатольевич Зазуля
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1. Лекарственное средство в форме жидкой суспензии отмытых модифицированных эритроцитов, предназначенной для инъекционного применения, с замедленным регулируемым высвобождением в организме лекарственного препарата и обеспечивающих эффективную направленность лекарства, отличающееся тем, что производят проведение трехкратной отмывки эритроцитов в физиологическом растворе, ресуспензирование эритроцитов в изотоническом растворе низкой ионной силы нанокапсулированного желатина, содержащего в середине микроРНК (sgRNAs), белок Cas9 и последовательность ДНК для внедрения, суккуссию суспензии эритроцитов в закрытой пробирке с температурным и тензометрическим датчиком посредством ударов о твердую эластичную поверхность, добавление антител определенной специфичности, инкубацию пробирки в термостате, проведение трехкратной отмывки эритроцитов в физиологическом растворе, ресуспензирование модифицированных эритроцитов в изотоническом растворе натрия хлорида.1. The drug is in the form of a liquid suspension of washed modified red blood cells, intended for injection use, with a slow controlled release in the body of the drug and providing an effective drug focus, characterized in that they carry out three-time washing of red blood cells in physiological saline, and the red blood cells are resuspended in isotonic solution ionic strength of nanocapsulated gelatin containing in the middle of microRNAs (sgRNAs), Cas9 protein and after ovatelnost DNA for introduction, succussion erythrocyte suspension in a sealed tube with a temperature and a strain gauge sensor by means of bumps on a hard elastic surface, the addition of antibodies of a particular specificity, incubation tubes in a thermostat holding washing three times erythrocytes in saline, resuspension modified erythrocytes in isotonic sodium chloride solution. 2. Суспензия отмытых модифицированных эритроцитов по п. 1, отличающаяся тем, что в центре нанокапсулированного желатина заключаеться упаковываная в вирус CRISPR/Cas9 - кассета, включающая молекулы микроРНК (sgRNAs), белок Cas9 и последовательность ДНК для внедрения.2. Suspension of washed modified red blood cells according to claim 1, characterized in that in the center of the nanocapsulated gelatin is a CRISPR / Cas9 virus-encapsulated cassette comprising microRNA molecules (sgRNAs), Cas9 protein and a DNA sequence for insertion. 3. Суспензия отмытых модифицированных эритроцитов по п. 1, отличающаяся тем, что в пробирку с эритроцитарной массой добавляют 8% раствор нанокапсулированного желатина в изотоническом растворе низкой ионной силы Low Ionic Strength Solution (Liss) при температуре 36,3-36,9°C, предпочтительно 36,6°C, рН раствора должно быть равным 7,00-7,40, предпочтительно рН 7,35.3. A suspension of washed modified red blood cells according to claim 1, characterized in that an 8% solution of nanocapsulated gelatin in a low ionic strength Low Ionic Strength Solution (Liss) is added to a test tube with an erythrocyte mass at a temperature of 36.3-36.9 ° C preferably 36.6 ° C, the pH of the solution should be between 7.00-7.40, preferably pH 7.35. 4. Суспензия отмытых модифицированных эритроцитов по п. 1, отличающаяся тем, что пробирку заполнят по объему на 1/2-2/3 суспензией эритроцитов.4. A suspension of washed modified red blood cells according to claim 1, characterized in that the tube is filled in volume by 1 / 2-2 / 3 with a suspension of red blood cells. 5. Суспензия отмытых модифицированных эритроцитов по п. 1, отличающаяся тем, что производят суккуссию суспензии посредством ударов о твердую эластичную поверхность при температуре 36,3-36,9°С, предпочтительно 36,6°С, величину повышения давления при гидравлическом ударе о дно сосуда суспензии эритроцитов контролируют тензометрическим датчиком, работающим в комплекте со шлейфовым осциллографом, давление составляет 2,2-2,4 кПа, предпочтительно 2.3 кПа, время суккуссии суспензии эритроцитов от 30 секунд до 2 минут, предпочтительно 30 секунд.5. A suspension of washed modified red blood cells according to claim 1, characterized in that they succuss the suspension by striking a hard elastic surface at a temperature of 36.3-36.9 ° C, preferably 36.6 ° C, the magnitude of the pressure increase during hydraulic shock o the bottom of the erythrocyte suspension vessel is monitored by a strain gauge that works with a loop oscilloscope, the pressure is 2.2-2.4 kPa, preferably 2.3 kPa, the succussion time of the erythrocyte suspension is from 30 seconds to 2 minutes, preferably 30 seconds. 6. Суспензия отмытых модифицированных эритроцитов по п. 1, отличающаяся тем, что в изотонический раствор низкой ионной силы 8% нанокапсулированного желатина добавляют антитела определенной специфичности, инкубируют в термостате от 15 минут до 1 часа, предпочтительно 30 минут при температуре 36,6°С.6. A suspension of washed modified red blood cells according to claim 1, characterized in that antibodies of a specific specificity are added to the isotonic solution of low ionic strength of 8% nanocapsulated gelatin, incubated in an incubator for 15 minutes to 1 hour, preferably 30 minutes at a temperature of 36.6 ° C . 7. Суспензия отмытых модифицированных эритроцитов по п. 1, отличающаяся тем, что отмывают эритроциты по три раза: центрифугируют в физиологическом растворе в течение 10 минут при 3000 об/мин, при температуре 36,3-36,9°С, предпочтительно 36,6°С, рН раствора должно быть равным 7,00-7,40, предпочтительно рН 7,35.7. A suspension of washed modified red blood cells according to claim 1, characterized in that the red blood cells are washed three times: centrifuged in saline for 10 minutes at 3000 rpm, at a temperature of 36.3-36.9 ° C, preferably 36, 6 ° C, the pH of the solution should be 7.00-7.40, preferably pH 7.35. 8. Суспензия отмытых модифицированных эритроцитов по п. 1, отличающаяся тем, что инфузия пациенту производится при температуре суспензии 36,6°С.8. The suspension of washed modified red blood cells according to claim 1, characterized in that the patient is infused at a suspension temperature of 36.6 ° C. 9. Суспензия отмытых модифицированных эритроцитов по любому из пп. 1-6, отличающаяся тем, что применяют аутологичные эритроциты.9. A suspension of washed modified red blood cells according to any one of paragraphs. 1-6, characterized in that the use of autologous red blood cells.
RU2016104674A 2016-02-11 2016-02-11 ERYTHROCYT MODIFICATION DEVICE WITH DIRECTED MEDICINAL TRANSPORT MECHANISM FOR CRISPR / CAS9 GENE THERAPY FUNCTIONS RU2016104674A (en)

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US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
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US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

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US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US11920181B2 (en) 2013-08-09 2024-03-05 President And Fellows Of Harvard College Nuclease profiling system
US10954548B2 (en) 2013-08-09 2021-03-23 President And Fellows Of Harvard College Nuclease profiling system
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US10912833B2 (en) 2013-09-06 2021-02-09 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US11299755B2 (en) 2013-09-06 2022-04-12 President And Fellows Of Harvard College Switchable CAS9 nucleases and uses thereof
US9999671B2 (en) 2013-09-06 2018-06-19 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US11053481B2 (en) 2013-12-12 2021-07-06 President And Fellows Of Harvard College Fusions of Cas9 domains and nucleic acid-editing domains
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US11124782B2 (en) 2013-12-12 2021-09-21 President And Fellows Of Harvard College Cas variants for gene editing
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US11578343B2 (en) 2014-07-30 2023-02-14 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
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US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11932884B2 (en) 2017-08-30 2024-03-19 President And Fellows Of Harvard College High efficiency base editors comprising Gam
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