CN108853133A - 一种PAMAM与CRISPR/Cas9系统重组质粒递送纳米粒的制备方法 - Google Patents

一种PAMAM与CRISPR/Cas9系统重组质粒递送纳米粒的制备方法 Download PDF

Info

Publication number
CN108853133A
CN108853133A CN201810827456.3A CN201810827456A CN108853133A CN 108853133 A CN108853133 A CN 108853133A CN 201810827456 A CN201810827456 A CN 201810827456A CN 108853133 A CN108853133 A CN 108853133A
Authority
CN
China
Prior art keywords
sgrna
pamam
apt
egfr
cas9
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201810827456.3A
Other languages
English (en)
Inventor
邵敬伟
江佳丽
张冰晨
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fuzhou University
Original Assignee
Fuzhou University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fuzhou University filed Critical Fuzhou University
Priority to CN201810827456.3A priority Critical patent/CN108853133A/zh
Publication of CN108853133A publication Critical patent/CN108853133A/zh
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Inorganic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

本发明公开了一种具有肿瘤靶向性的PAMAM与CRISPR/Cas9系统重组质粒递送纳米粒的制备方法，先将Apt羧基活化，将其与PAMAM表面的氨基反应，合成Apt‑PAMAM运载体，设计sgRNA并构建EGFR‑sgRNA/Cas9重组质粒，重组质粒与运载体通过静电作用形成Apt‑PAMAM/EGFR‑sgRNA/Cas9纳米复合物。该复合物利用其表面所修饰的Apt主动靶向肿瘤细胞表面高表达EpCAM蛋白，增加了对肿瘤组织的靶向性。通过CRISPR/Cas9基因编辑技术设计EGFR‑sgRNA基因的重组质粒，以期敲除肿瘤细胞内高表达的EGFR基因，从而达到抗肿瘤的作用。

Description

一种PAMAM与CRISPR/Cas9系统重组质粒递送纳米粒的制备 方法
技术领域
本发明属于生物技术领域，涉及一种具肿瘤靶向性的纳米复合物Apt-PAMAM/EGFR-sgRNA/Cas9的制备方法。
背景技术
卫生部和科技部发布的全国第三次死因回顾抽样调查报告显示，肺癌已跃居我国居民肿瘤死因的第1位，是我国死亡率上升速度最快、增长幅度最大的恶性肿瘤。肺癌分为小细胞肺癌(SCLC)和非小细胞肺癌（NSCLC），其中NSCLC占所有肺癌的80~85%，且表皮生长因子受体（EGFR）的过度表达是导致肺癌细胞无限增殖的主要原因之一（Moschini I,Dell'Anna C, Losardo PL, Bordi P, D'Abbiero N, Tiseo M. Radiotherapy of non-small-cell lung cancer in the era of EGFR gene mutations and EGF receptortyrosine kinase inhibitors. Future Oncol. 2015;11(16):2329-42.）。
CRISPR-Cas9被称为第三代基因编辑技术，可通过精确定位基因组某一位点，剪断靶标DNA片段并插入、删除或替换新的基因片段高效地改造基因。CRISPR的成功实现通常需要Cas9在一组细胞中引出有效的目标基因并敲除，研究发现CRISPR对目标基因的敲除高度依赖于sgRNA引导序列的效力（Yuen G, Khan FJ, Gao S, Stommel JM, Batchelor E, WuX, et al. CRISPR/Cas9-mediated gene knockout is insensitive to target copynumber but is dependent on guide RNA potency and Cas9/sgRNA thresholdexpression level. Nucleic Acids Res 2017; 45 (20): 12039-53.）。另有研究表明，sgRNA靶向的基因缺失与EGFR蛋白表达密切相关。因此，通过CRISPR-Cas9基因编辑技术敲除sgRNA在下调EGFR蛋白过度表达导致的肿瘤细胞无限增殖具有广泛的应用前景。
聚酰胺-胺型树枝状高分子（Polyamidoamine，PAMAM）是一种新型高分子，其表面富含氨基可与带负电的核酸结合，是目前研究最多的基因载体之一。因其内部存在空腔结构，且在体内外均无毒性，被认为是一种安全的药物载体广泛应用于靶向药物输送系统（Marquez-Miranda V, Abrigo J, Rivera JC, Araya-Duran I, Aravena J, Simon F,et al. The complex of PAMAM-OH dendrimer with Angiotensin (1-7) prevented thedisuse-induced skeletal muscle atrophy in mice. Int J Nanomedicine. 2017;12:1985-99.）。短单链寡核苷酸（Aptamer）是一类具有特殊序列的核酸适配体，能够与EGFR表面抗原特异性结合，从而实现靶向至肿瘤细胞的作用（Kang YR, Byun JS, Kim TJ, ParkHG, Park JC, Barange N, et al. Monitoring of the Binding Between EGFR Proteinand EGFR Aptamer Using In-Situ Total Internal Reflection Ellipsometry. JNanosci Nanotechnol. 2016;16(6):6445-9.）。
本发明制备了具肿瘤靶向性的Apt-PAMAM/EGFR-sgRNA/Cas9纳米复合物，利用短单链寡核苷酸Apt主动靶向EGFR过表达肿瘤细胞，有效地提高药物的生物利用度。通过CRISPR-Cas9基因编辑技术设计sgRNA基因并构建EGFR-sgRNA重组质粒，从基因治疗层面抑制肿瘤细胞增殖，提升其临床应用价值。检索国内外相关文献和专利结果表明：具肿瘤靶向性的Apt-PAMAM/EGFR-sgRNA/Cas9纳米复合物及其制备方法，尚未见报道。
发明内容
本发明的目的在于提供一种具有靶向抗癌作用的适配体修饰树枝状高分子作为运载体介导基因输送在EGFR过表达肺癌中的作用及制备方法。通过基因重组质粒显著抑制EGFR蛋白表达，同时利用纳米载体表面的特异性适配体靶向肺癌细胞，选择性浓集于肿瘤细胞，提高生物利用度。
为达到上述目的，本发明采用如下技术方案：
（1）将PAMAM，N-羟基琥珀酰亚胺（NHS）和1-（3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐（EDC）溶于水中，滴加3’端修饰有FITC荧光的Apt（Anti-EpCAM）水溶液，室温低速搅拌反应4小时，超滤后得到Apt-PAMAM聚合物；
（2）根据选定的EGFR基因设计sgRNA，对设计的sgRNA进行退火，Px459质粒进行酶切，再将sgRNA与酶切后的质粒连接，构建EGFR-sgRNA/Cas9重组质粒；
（3）将10mg Apt-PAMAM纳米聚合物溶于10mL二次水中，将水溶液高速搅拌，加入1mgEGFR-sgRNA/Cas9重组质粒，形成Apt-PAMAM/ EGFR-sgRNA/Cas9纳米复合物。
步骤(1)中Apt：EDC：NHS的质量比为15：6：4.5。
步骤(1)中Apt：EDC：NHS的总浓度为1-20 wt%。
步骤(1)中所采用的PAMAM的代数为1代。
步骤(1)中超滤管的孔径为10Kd。
步骤(2)中设计的sgRNA序列为TGAACCGCACGGCGCCATGC；
合成所设计sgRNA与其互补序列，并在首尾端加入酶切位点。合成序列为：
EGFR-sgRNA-F CACCGTGAACCGCACGGCGCCATGC
EGFR-sgRNA-R AAACGCATGGCGCCGTGCGGTTCAC
步骤(2)中sgRNA退火温度梯度设置为：95℃，10min；95℃-85℃，
2.5℃/s；85℃-25℃，0.25℃/s；25℃，5min。
步骤(2)中Px459质粒的酶切条件为37℃，15min。
步骤(2)中sgRNA与酶切后质粒连接的条件为16℃水浴过夜12-24h。
步骤(3)中Apt-PAMAM和EGFR-sgRNA/Cas9重组质粒的反应条件为低速搅拌过夜12-24h。
本发明的原理在于：
第一，利用纳米载体表面的特异性适配体选择性地浓集于肿瘤细胞，有效地提高了药物的生物利用度；
第二，利用CRISPR-Cas9基因编辑技术构建EGFR-sgRNA重组质粒，显著下调EGFR蛋白表达，抑制肿瘤细胞增殖；
第三，利用纳米级运载体PAMAM的被动靶向作用，增强Apt-PAMAM/ EGFR-sgRNA/Cas9纳米复合物在肿瘤组织的滞留效应。
本发明的优点在于：
第一，本发明采用树枝状大分子PAMAM作为载体，它能与能够特异性识别EGFR过表达肿瘤细胞的适配体共价偶联，形成Apt-PAMAM运载体，通过Apt的主动靶向作用提高Apt-PAMAM/ EGFR-sgRNA/Cas9纳米复合物的肿瘤靶向作用。
第二，本发明采用CRISPR-Cas9基因编辑技术设计sgRNA并构建EGFR-sgRNA重组质粒，重组质粒与Apt-PAMAM运载体结合形成Apt-PAMAM/ EGFR-sgRNA/Cas9纳米复合物，达到从基因水平上治疗肿瘤细胞无限增殖的目的。
附图说明
图1 A图为实施例1设计的Anti-EpCAM适配体具有FITC修饰，分别在20nm、50nm、100nm、250nm、500nm的荧光强度检测图；图1中的B图为实施例1设计的Anti-EpCAM适配体20nm、50nm、100nm、250nm、500nm荧光标准曲线。
图2 EGFR-sgRNA/Cas9重组质粒序列对比图。
图3 实施例4中复合纳米的粒径表征。
图4 Apt/PAMAM/ EGFR-sgRNA/Cas9作用于细胞后，EGFR基因sgRNA所在序列与野生型序列对比图。
图5 Apt/PAMAM/ EGFR-sgRNA/Cas9对肿瘤细胞A549的毒性实验结果。
具体实施方式
下面，将通过实施例，对本发明进行详细说明，但发明并不局限于此，在本发明权利要求所阐明的范围内，可进行各种改变或同等替换。
实施例1 Apt-PAMAM偶联物的制备
首先，精确称取15mg的G1 PAMAM于20mL的圆底烧瓶中，然后称取EDC 6mg以及4.5mg的NHS，再取5mL二次水加入圆底烧杯中，使其完全溶解，随后滴加100μL的anti-EpCAM适配体（10mM），反应4 h，然后选用10Kd的超滤管除去未反应的PAMAM，即得Apt-PAMAM偶联物。通过Apt修饰的FITC荧光标曲，计算出每毫克PAMAM纳米载体anti-EpCAM的嫁接量为0.1nmol。
实施例2 EGFR-sgRNA的设计及验证
（1）sgRNA的设计
根据课题选定的EGFR基因及人的种属，在pubmed进行检索其全基因序列，并检索其CDS（Coding sequence）编码区序列，将CDS序列每250碱基一组复制到http://crispr.mit.edu/网站进行sgRNA设计，根据设计结果优先选取设计结果评分在90分以上的，并在全基因中验证设计的合理性。
设计sgRNA为TGAACCGCACGGCGCCATGC，
合成所设计sgRNA与其互补序列，并在首尾端加入酶切位点。合成序列为：EGFR-sgRNA-F：CACCGTGAACCGCACGGCGCCATGC；
EGFR-sgRNA-R：AAACGCATGGCGCCGTGCGGTTCAC。
（2）sgRNA的验证
将(1)设计的sgRNA进行退火。将以上各管按照下表顺序加入到PCR管中，然后轻轻混匀，最后低速离心。按照如下温度梯度设置进行退火： 95℃，10min；95℃-85℃，2.5℃/s；85℃-25℃，0.25℃/s；25℃，5min。等待结束后取样品进行1%琼脂糖凝胶电泳检测。
表1 sgRNA退火体系
实施例3 EGFR-sgRNA/Cas9重组质粒的构建
（1）Px459质粒的酶切：
按照表2的成分及量，将上述混匀后低速离心，37℃反应15min后，取3μL与未使用酶切质粒共同用1%琼脂糖凝胶电泳进行检测。
表2酶切体系
（2）重组质粒的制备：sgRNA与酶切后质粒的连接
按照表3的成分及量，将上述混匀后低速离心。16℃水浴过夜，然后放于4℃冰箱保存。
表3 连接反应体系
（3）质粒的转化
1.5μL离心管中加入感受态细胞50μL，然后温和的加入重组质粒3μL混匀，冰浴30min；42℃水浴，45s之后冰上2min；加入800μL LB；37℃摇床培养1h；室温4000 rpm，离心5min；弃上清750μL；剩余菌液混匀涂板，采用AMP平板；封口，37℃倒置过夜。
（4）质粒提取与测序验证
将40μL 冻存菌液加入4mL具有AMP抗性的LB中，过夜培养14h后细菌菌液加入10mL离心管中；于10000rpm离心1min并弃去上清液；加入250μL Resuspension Buffer重悬细菌体沉淀；加入250μL Lysis Buffer 轻柔颠倒4~6次（勿超过5min否则DNA易会被剪切成小片段失效）；加入350 μL Neutralization Buffer轻柔颠倒4~6次；于13000rpm离心10min；将离心所得上清液转移入spin column内，于6000 rpm离心1min，并弃去移液管内液体；向spincolumn内加入650μl Wash Buffer并于12000g离心1min，离心后弃去移液管内液体，之后重复此步骤1次；空管12000g离心1min，将spin column转移到无菌的1.5mL离心管中；将Elution Buffer提前加热至56℃，向spin column内加30μL Elution Buffe，并于室温中静置1min；12000rpm离心1min，得到的溶液即含有质粒DNA；将提取出的DNA进行浓度检测。提取出的质粒送至公司测序，使用U6引物将所得序列与sgRNA比对，验证sgRNA连接载体，获得EGFR-sgRNA/Cas9质粒。
实施例4 Apt-PAMAM纳米与EGRF-sgRNA/Cas9质粒的靶向递送纳米系统的制备
取Apt-PAMAM纳米粉末10mg，使其超声重悬在10mL二次水中，然后加入2mg的EGFR-sgRNA/Cas9质粒，低速搅拌过夜，即获得所需复合纳米。并测量其粒径电势，如图3复合纳米平均粒径为132nm，电势为-21.8mv。
实施例5 靶向递送质粒的生物学验证
（1）基因水平
在所使用的sgRNA上下游序列设计引物，通过PCR得到纳米复合物Apt-PAMAM/ EGFR-sgRNA/Cas9作用后的目的序列，通过测序与野生型细胞序列对比，验证目的序列在sgRNA序列位点发生碱基突变。通过基因水平检测，这种纳米系统能够成功递送重组质粒，并达到基因敲除的作用。
（2）细胞水平
取对数生长期的细胞，消化后将其铺在96孔板中，每孔细胞约1×104个，置于培养箱中培养，待细胞完全贴壁。设计四个不同实验组别（PBS, PAMAM, Apt-PAMAM, Apt-PAMAM-EGFR-sgRNA/Cas9），作用24h后，每孔加入10ul MTT溶液，4 h后使用DMSO溶解，通过分光光度仪检测OD值，计算各组对细胞的毒性大小。实验结果显示单独载体PAMAM具有较小的细胞毒性，当连接适配体Apt与基因治疗CRISPR/Cas9后复合纳米Apt-PAMAM/EGFR-sgRNA/Cas9有着显著的杀伤肿瘤细胞A549的能力。这种新型复合纳米通过基因治疗的手段为抗肿瘤研究提供了新的思路。
SEQUENCE LISTING
<110> 福州大学
<120> 一种PAMAM与CRISPR/Cas9系统重组质粒递送纳米粒的制备方法
<130> 3
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> 人工序列
<400> 1
tgaaccgcac ggcgccatgc 20
<210> 2
<211> 25
<212> DNA
<213> 人工序列
<400> 2
caccgtgaac cgcacggcgc catgc 25
<210> 3
<211> 25
<212> DNA
<213> 人工序列
<400> 3
aaacgcatgg cgccgtgcgg ttcac 25

Claims (10)

1.一种具有肿瘤靶向性的Apt-PAMAM/ EGFR-sgRNA/Cas9纳米复合物的制备方法，其特征在于：先将Apt表面的羧基活化，再将Apt与PAMAM表面的氨基通过酰胺键连接形成Apt-PAMAM运载体，设计EGFR-sgRNA并构建重组质粒，并将其装载在Apt-PAMAM纳米中制备获得Apt-PAMAM/EGFR-sgRNA/Cas9纳米复合物。
2.根据权利要求1所述的Apt-PAMAM/ EGFR-sgRNA/Cas9纳米复合物的制备方法，其特征在于：具体包括以下步骤：
（1）将PAMAM，N-羟基琥珀酰亚胺和1-（3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶于滴加Apt的水溶液中，室温低速搅拌反应4小时，随后用超滤管滤去未反应的PAMAM，得到Apt-PAMAM聚合物；
（2）根据选定的EGFR基因设计sgRNA，对设计的sgRNA进行退火，Px459质粒进行酶切，再将sgRNA与酶切后的质粒连接，构建EGFR-sgRNA/Cas9重组质粒；
（3）将Apt-PAMAM纳米聚合物溶于水中，将水溶液高速搅拌，加入EGFR-sgRNA/Cas9重组质粒，形成Apt-PAMAM/ EGFR-sgRNA/Cas9纳米复合物。
3.根据权利要求2所述的Apt-PAMAM/ EGFR-sgRNA/Cas9纳米复合物的制备方法，其特征在于：步骤(1)中所采用的PAMAM的代数为1代；Apt与PAMAM的摩尔比为1：10。
4.根据权利要求2所述的Apt-PAMAM/ EGFR-sgRNA/Cas9纳米复合物的制备方法，其特征在于:步骤(1)中Apt：EDC：NHS的质量比为15：6：4.5。
5.根据权利要求2所述的Apt-PAMAM/ EGFR-sgRNA/Cas9纳米复合物的制备方法，其特征在于：步骤(1)中超滤管的孔径为10Kd。
6.根据权利要求2所述的Apt-PAMAM/ EGFR-sgRNA/Cas9纳米复合物的制备方法，其特征在于：步骤(2)中设计的sgRNA序列为TGAACCGCACGGCGCCATGC。
7.根据权利要求2所述的Apt-PAMAM/ EGFR-sgRNA/Cas9纳米复合物的制备方法，其特征在于：步骤(2)中sgRNA退火温度梯度设置为： 95℃，10min；95℃-85℃，2.5℃/s；85℃-25℃，0.25℃/s；25℃，5min。
8.根据权利要求2所述的Apt-PAMAM/ EGFR-sgRNA/Cas9纳米复合物的制备方法，其特征在于：步骤(2)中Px459质粒的酶切条件为37℃，15min；sgRNA与酶切后质粒连接的条件为16℃水浴过夜12-24h。
9.根据权利要求2所述的Apt-PAMAM/ EGFR-sgRNA/Cas9纳米复合物的制备方法，其特征在于：步骤(3)中Apt-PAMAM和EGFR-sgRNA/Cas9重组质粒的反应条件为低速搅拌过夜12-24h。
10.如权利要求1-9任一所述的方法制备获得Apt-PAMAM/ EGFR-sgRNA/Cas9纳米复合物。
CN201810827456.3A 2018-07-25 2018-07-25 一种PAMAM与CRISPR/Cas9系统重组质粒递送纳米粒的制备方法 Pending CN108853133A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810827456.3A CN108853133A (zh) 2018-07-25 2018-07-25 一种PAMAM与CRISPR/Cas9系统重组质粒递送纳米粒的制备方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810827456.3A CN108853133A (zh) 2018-07-25 2018-07-25 一种PAMAM与CRISPR/Cas9系统重组质粒递送纳米粒的制备方法

Publications (1)

Publication Number Publication Date
CN108853133A true CN108853133A (zh) 2018-11-23

Family

ID=64304928

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810827456.3A Pending CN108853133A (zh) 2018-07-25 2018-07-25 一种PAMAM与CRISPR/Cas9系统重组质粒递送纳米粒的制备方法

Country Status (1)

Country Link
CN (1) CN108853133A (zh)

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors 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
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
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
US12006520B2 (en) 2011-07-22 2024-06-11 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US12031126B2 (en) 2023-12-08 2024-07-09 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107130000A (zh) * 2017-05-12 2017-09-05 浙江卫未生物医药科技有限公司 一种同时敲除KRAS基因和EGFR基因的CRISPR‑Cas9系统及其应用
CN107998083A (zh) * 2017-12-11 2018-05-08 福州大学 一种具肿瘤靶向性的纳米复合物Apt-PAMAM/ERL/SUV及其制备和应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107130000A (zh) * 2017-05-12 2017-09-05 浙江卫未生物医药科技有限公司 一种同时敲除KRAS基因和EGFR基因的CRISPR‑Cas9系统及其应用
CN107998083A (zh) * 2017-12-11 2018-05-08 福州大学 一种具肿瘤靶向性的纳米复合物Apt-PAMAM/ERL/SUV及其制备和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
AYATOLLAHI S: "Aptamer-targeted delivery of Bcl-xL shRNA using alkyl modified PAMAM dendrimers into lung cancer cells", 《INT J BIOCHEM CELL BIOL.》 *

Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12006520B2 (en) 2011-07-22 2024-06-11 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10954548B2 (en) 2013-08-09 2021-03-23 President And Fellows Of Harvard College Nuclease profiling system
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
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
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
US11299755B2 (en) 2013-09-06 2022-04-12 President And Fellows Of Harvard College Switchable CAS9 nucleases and uses thereof
US11124782B2 (en) 2013-12-12 2021-09-21 President And Fellows Of Harvard College Cas variants for gene editing
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
US11578343B2 (en) 2014-07-30 2023-02-14 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11702651B2 (en) 2016-08-03 2023-07-18 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11999947B2 (en) 2016-08-03 2024-06-04 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11820969B2 (en) 2016-12-23 2023-11-21 President And Fellows Of Harvard College Editing of CCR2 receptor gene to protect against HIV infection
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
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
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)
US11932884B2 (en) 2017-08-30 2024-03-19 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11795452B2 (en) 2019-03-19 2023-10-24 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11643652B2 (en) 2019-03-19 2023-05-09 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
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
US12031126B2 (en) 2023-12-08 2024-07-09 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

Similar Documents

Publication Publication Date Title
CN108853133A (zh) 一种PAMAM与CRISPR/Cas9系统重组质粒递送纳米粒的制备方法
Sung et al. Recent advances in the development of gene delivery systems
Ding et al. A self-assembled RNA-triple helix hydrogel drug delivery system targeting triple-negative breast cancer
US11235071B2 (en) Compositions of nucleic acid-containing nanoparticles for in vivo delivery
US20160375050A1 (en) Methods and compositions involving chitosan nanoparticles
CN107001627B (zh) 用于将核酸引入细胞的组合物
Cheng et al. Stearyl polyethylenimine complexed with plasmids as the core of human serum albumin nanoparticles noncovalently bound to CRISPR/Cas9 plasmids or siRNA for disrupting or silencing PD-L1 expression for immunotherapy
CN111511405A (zh) 用于细胞内递送mRNA的肽和纳米颗粒
Chen et al. Adoptive cellular immunotherapy of tumors via effective CpG delivery to dendritic cells using dendrimer-entrapped gold nanoparticles as a gene vector
Atabi et al. Doxorubicin loaded DNA aptamer linked myristilated chitosan nanogel for targeted drug delivery to prostate cancer
US20200291394A1 (en) Conjugation of peptides to spherical nucleic acids (snas) using traceless linkers
CN106929508B (zh) 一种激活PTPRO基因表达的saRNA及其转运载体
CN110168090B (zh) mRNA的功能化方法
CN107137718A (zh) 一种肽修饰的多壁碳纳米管载体及其制备方法和应用
CN106467915B (zh) 小干扰RNA及其应用和抑制plk1基因表达的方法
Shao et al. Phenylboronic acid-functionalized polyaminoglycoside as an effective CRISPR/Cas9 delivery system
Guo et al. Enhancement of thermal damage to adenocarcinoma cells by iron nanoparticles modified with MUC1 aptamer
Pressnall et al. Glatiramer acetate enhances tumor retention and innate activation of immunostimulants
CN106727323B (zh) 一种透明质酸纳米囊泡及其制备方法和应用
Wang et al. Mannose-functionalized star polycation mediated CRISPR/Cas9 delivery for lung cancer therapy
US20230135432A1 (en) Polymeric nanoparticles and derivatives thereof for nucleic acid binding and delivery
Chen et al. Farnesylthiosalicylic acid-derivatized PEI-based nanocomplex for improved tumor vaccination
JP2022525866A (ja) 粘膜癌の処置のための、単独の、またはｉ型ｉｆｎインデューサーと組み合わせた、キトサンポリプレックスベースのｉｌ－１２の局部発現
WO2016210098A1 (en) Dual assembly nanoparticles
WO2005104785A3 (en) Methods and compositions for specifically targeting human hepatocellular carcinoma cells

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20181123