Cell-specific gene delivery methods for expression and silencing in the lung

用于肺部表达和沉默的细胞特异性基因递送方法

基本信息

批准号：
9199240
负责人：
David A Dean
金额：
$ 38.38万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-01-01 至 2017-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9199240
关键词：
Active Biological Transport Acute Lung Injury Address Adult Respiratory Distress Syndrome Alpha Cell Alveolar Alveolar Cell Alveolar wall Alveolus Animal Model Animals Area Attention Binding Blood Circulation Cell Nucleus Cell division Cell physiology Cell surface Cells Cessation of life Clinical Complex DNA DNA Sequence Development Disease Edema Electroporation Endothelial Cells Endothelium Epithelial Epithelial Cells Family suidae Gene Delivery Gene Expression Gene Silencing Gene Transduction Agent Gene Transfer Genes Goals Human In Vitro Individual Inflammation Injury Interphase Cell Ions Length of Stay Liquid substance Lung Mediating Methods Modeling Molecular Mus NUP214 gene Na(+)-K(+)-Exchanging ATPase Nuclear Nuclear Import Plasmids Proteins Pulmonary Edema RNA Interference Rattus Research Resolution Respiratory physiology Sepsis Small Interfering RNA Specificity Syndrome Techniques Testing Tight Junctions Type I Epithelial Receptor Cell Type II Epithelial Receptor Cell Up-Regulation Water Work alveolar epithelium base cell type clinical application design disorder prevention effective therapy experimental study fluid flow gene delivery system gene therapy gene transfer vector improved improved outcome in vivo injured interest lung injury mortality novel novel strategies overexpression particle plasmid DNA protein complex public health relevance satisfaction small hairpin RNA standard of care tool transcription factor vector

项目摘要

DESCRIPTION (provided by applicant): One of the major problems hindering development of gene therapy approaches, including the use of RNAi for gene silencing, is the relative lack of methods to deliver genes/RNAi to specific cell types. This application addresses this limitation. Our major interest is to develop gene therapy approaches to treat acute lung injury, which accounts for over 50,000 deaths each year in the US. We have developed a highly effective treatment for this disease in mouse and pig models that uses the ubiquitous overexperssion of the Na+,K+-ATPase to increase alveolar fluid clearance from the previously injured lung. Our experiments show that this treatment not only improves edema resolution (and lung function and survival), but also improves alveolar epithelial/endothelial barrier function by upregulating tight junction complexes. However, we do not know in what cells transfer and expression of the Na+,K+-ATPase gene is needed for this protection and treatment, nor whether upregulation of tight junction complexes is needed for maximal effect. In order to understand the mechanisms responsible for this treatment approach, we must be able to deliver genes specifically to the individual cell types in the alveolus and silence gene expression in a similar cell-specific manner. Unfortunately, there are no uniformly effective ways to deliver RNAi to specific cell types in vivo. We have developed a way to overcome this problem. We have shown that the nuclear localization of plasmids in the absence of cell division is sequence-specific and requires transcription factors for nuclear import. We have identified a number of DNA sequences that show cell-specificity of nuclear import because they bind to cell- specific transcription factors. These include sequences that act in endothelial cells, alveolar type 1, and alveolar type 2 epithelial cells in vitro and in living animals. We have used these sequences to overexpress genes in these cells in vivo and will now use them to silence genes by shRNA delivery. This is an entirely new way to direct cell-specific RNAi delivery. Our aims are to (1) Determine in which alveolar cell types gene transfer of the Na+,K+-ATPase is needed for improved alveolar fluid clearance and induction of tight junctions, (2) Test whether tight junctions are needed for Na+,K+-ATPase gene transfer-mediated protection and treatment of acute lung injury by developing a method for cell-specific RNAi delivery, and (3) Determine whether gene transfer of tight junction complex proteins alone can treat lung injury or further enhance Na+,K+- ATPase gene transfer-mediated treatment of acute lung injury.

描述（由申请人提供）：阻碍基因治疗方法（包括使用RNAi进行基因沉默）发展的主要问题之一是相对缺乏将基因/RNAi递送至特定细胞类型的方法。该应用程序解决了这个限制。我们的主要兴趣是开发基因治疗方法来治疗急性肺损伤，美国每年有超过 50,000 人因急性肺损伤而死亡。我们在小鼠和猪模型中开发了一种针对这种疾病的高效治疗方法，该方法利用无处不在的 Na+,K+-ATP 酶过度表达来增加先前受伤肺部的肺泡液清除率。我们的实验表明，这种治疗不仅可以改善水肿消退（以及肺功能和生存），还可以通过上调紧密连接复合物来改善肺泡上皮/内皮屏障功能。然而，我们不知道这种保护和治疗需要在哪些细胞中转移和表达 Na+,K+-ATPase 基因，也不知道是否需要上调紧密连接复合物才能获得最大效果。为了了解这种治疗方法的机制，我们必须能够将基因特异性地递送到肺泡中的各个细胞类型，并以类似的细胞特异性方式沉默基因表达。不幸的是，没有统一有效的方法将 RNAi 传递到体内特定的细胞类型。我们已经开发出一种方法来克服这个问题。我们已经证明，在没有细胞分裂的情况下，质粒的核定位是序列特异性的，并且需要转录因子进行核输入。我们已经鉴定出许多 DNA 序列，这些序列显示出核输入的细胞特异性，因为它们与细胞特异性转录因子结合。这些包括在体外和活体动物体内的内皮细胞、1 型肺泡和 2 型肺泡上皮细胞中起作用的序列。我们已经使用这些序列在体内这些细胞中过度表达基因，现在将使用它们通过 shRNA 传递来沉默基因。这是一种指导细胞特异性 RNAi 传递的全新方法。我们的目标是 (1) 确定哪些肺泡细胞类型需要 Na+,K+-ATP 酶的基因转移，以改善肺泡液清除和诱导紧密连接，(2) 测试 Na+,K+-ATP 酶是否需要紧密连接通过开发细胞特异性 RNAi 递送方法，通过基因转移介导的保护和治疗急性肺损伤，以及 (3) 确定仅紧密连接复合蛋白的基因转移是否可以治疗肺损伤或进一步增强 Na+,K+- ATP 酶基因转移介导的急性肺损伤治疗。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Ouabain Enhances Cell-Cell Adhesion Mediated by beta 1 Subunits of the Na ( + ) , K ( + )-ATPase in CHO Fibroblasts

哇巴因增强 CHO 成纤维细胞中 Na ( ) , K ( )-ATP 酶 β1 亚基介导的细胞间粘附

DOI：
10.1042/bj1720509
发表时间：
2024-09-14
期刊：
The Biochemical journal
影响因子：
0
作者：
C. A. Vilchis;Luisa Rold;Angelina Leonardi;Juan G. Navea;Teresita Padilla;L. Shoshani
通讯作者：
L. Shoshani

Caveolin-1 gene therapy inhibits inflammasome activation to protect from bleomycin-induced pulmonary fibrosis.

Caveolin-1 基因治疗可抑制炎症小体激活，从而防止博来霉素诱导的肺纤维化。

DOI：
发表时间：
2019
期刊：
影响因子：
4.6
作者：
Lin, Xin;Barravecchia, Michael;Matthew Kottmann, R;Sime, Patricia;Dean, David A
通讯作者：
Dean, David A

Pulmonary gene delivery-Realities and possibilities.

肺部基因传递——现实与可能性。

DOI：
发表时间：
2021-02
期刊：
影响因子：
0
作者：
Baliga, Uday K;Dean, David A
通讯作者：
Dean, David A

RNAi therapeutic strategies for acute respiratory distress syndrome.

急性呼吸窘迫综合征的 RNAi 治疗策略。

DOI：
发表时间：
2019-12
期刊：
影响因子：
0
作者：
Jagrosse, Melissa L;Dean, David A;Rahman, Arshad;Nilsson, Bradley L
通讯作者：
Nilsson, Bradley L

Changes in lung immune cell infiltrates after electric field treatment in mice.

小鼠电场治疗后肺部免疫细胞浸润的变化。

DOI：
发表时间：
2021
期刊：
影响因子：
4.6
作者：
Eliseeva, Sophia I;Knowlden, Zackery A;Lester, Gillian MSchiralli;Dean, David A;Georas, Steve N;Chapman, Timothy J
通讯作者：
Chapman, Timothy J

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David A Dean其他文献

Tubulin acetylation and histone deacetylase 6 activity in the lung under cyclic load.

循环负荷下肺中微管蛋白乙酰化和组蛋白脱乙酰酶 6 的活性。

DOI：
10.1165/rcmb.2007-0307oc
发表时间：
2024-09-14
期刊：
American journal of respiratory cell and molecular biology
影响因子：
6.4
作者：
R. C. Geiger;Christopher D. Kaufman;Christopher D. Kaufman;A. P. Lam;G. R. S. Budinger;David A Dean;David A Dean
通讯作者：
David A Dean

Microtubule acetylation through HDAC6 inhibition results in increased transfection efficiency.

通过 HDAC6 抑制实现微管乙酰化可提高转染效率。

DOI：
10.1038/mt.2008.190
发表时间：
2008-11-01
期刊：
Molecular therapy : the journal of the American Society of Gene Therapy
影响因子：
0
作者：
Erin E Vaughan;R. C. Geiger;Aaron M Miller;Phoebe L Loh;Takayoshi Suzuki;N. Miyata;David A Dean
通讯作者：
David A Dean