Breaching the barrier with matrix biology.

突破基质生物学的障碍。

• 批准号: 10582940
• 负责人: WILLIAM J BRUNKEN
• 金额: $ 24.45万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10582940
• 关键词: Ablation; Animal Model; Astrocytes; Back; Basement membrane; Biological; Biological Assay; Biological Process; Biology; Blood Vessels; Bypass; Cell Therapy; Cell Transplantation; Cell-Matrix Junction; Cells; Cicatrix; Collaborations; Cre lox recombination system; Data; Dependovirus; Deposition; Development; Dyes; Effectiveness; Engineering; Engraftment; Excision; Extravasation; Eye; Foundations; Future; Gene Transfer; Genes; Genetic; Goals; Immunologic Surveillance; In Vitro; Inner Limiting Membrane; Knowledge; Label; Laboratories; Laminin; Mediating; Membrane; Methods; Microspheres; Molecular Structure; Movement; Mus; Nature; Penetrance; Penetration; Physiological; Polymers; Procedures; Proteins; Publishing; Reporter; Retina; Retinal Diseases; Site; Stem cell transplant; Techniques; Testing; Therapeutic Intervention; Tissues; Transfection; Vertebral column; Viral; Viral Vector; Virus; Virus Integration; Wild Type Mouse; Work; cell type; clinically relevant; conditional knockout; depolymerization; experimental study; gene therapy; genetic approach; high risk; in vivo; in vivo evaluation; infection rate; intravitreal injection; molecular rearrangement; mouse model; nerve stem cell; novel; polymerization; prevent; reconstitution; response; retinal progenitor cell; reverse genetics; stem cells; success; theories; therapeutic gene; tool

Basement membranes (BMs) are non-cellular protein layers that simultaneously provide parenchymal cell-attachment sites and also barriers between tissue compartments. The inner limiting membrane (ILM) is a special BM that prevents movement into the retina from the vitreous as such it is a formidable barrier to intravitreal viral-mediated gene transfer or stem cell transplantation. Current methods to circumvent the ILM involve its physical or enzymatic removal with limited effectiveness. The consequence has been that successful cell- and gene- based therapies have targeted a subretinal approach, an effective approach for the outer retina, but of extremely limited use for the inner retina. The goal of this exploratory project is to manipulate the molecular structure of the ILM to promote integration of neural stem cells or viral transfection. ILM formation is dependent on the deposition of laminins, particularly those containing the β2 subunit. We propose two different manipulations of the laminin polymer forming the backbone of the ILM: one, genetic and a second, employs a small biomolecule to disrupt the laminin polymer. Our first approach will be to remove genetically a key component of the ILM, Lamb2. Our prior studies show this disrupts the sheet-like nature of the ILM. We will test if this molecular rearrangement of the ILM renders these retinae more permeant to cell and virus entry. Second approach will be to disrupt laminin polymerizing; netrins, small matrix molecules with laminin homology, are known to disrupt laminin polymerization during normal development and in experimental conditions. We will produce netrin-induced focal disruptions of the ILM and assay for facilitated gene and cell integration. We will perform our experiments, in vitro using organotypic cultures of retina as well as in vivo murine models. The in vitro approach has the advantage that we can add back laminin and reconstitute the ILM and thereby proving that laminin manipulation is the key step. The in vivo approach is more clinically relevant. While in this exploratory project, we will use murine models future successor applications could employ more clinically relevant animal models. Together, the experiments in this project will assess the barrier that the ILM presents to biologic-based engraftment therapies. They will determine if specifically disrupting the laminin backbone is enough to promote engraftment. These experiments if successful will provide a powerful tool to promote advanced matrix-based approach to manipulation of retinal BMs to promote gene-based and cell-based therapeutics in the inner retinal disease.

基底膜（BM）是非细胞蛋白层，同时提供 实质细胞附着位点以及组织隔室之间的屏障。内 界膜（ILM）是一种特殊的BM，它阻止从视网膜进入视网膜的运动。 玻璃体本身是玻璃体内病毒介导的基因转移或干细胞 移植目前规避ILM的方法涉及其物理或酶促 去除效果有限。结果是成功的细胞和基因 基于视网膜下的治疗方法，一种针对外层视网膜的有效方法， 但是对于内层视网膜的用途极其有限。这个探索性项目的目标是 操纵ILM的分子结构以促进神经干细胞或病毒的整合， 转染ILM的形成依赖于层粘连蛋白的沉积，特别是那些 含有β2亚基。我们提出了两种不同的操作层粘连蛋白聚合物 形成ILM的主干：第一，遗传，第二，利用小生物分子， 破坏层粘连蛋白聚合物。 我们的第一种方法是从基因上去除ILM的一个关键组成部分Lamb 2。我们 先前的研究表明，这破坏了ILM的片状性质。我们将测试这种分子 ILM的重排使得这些视网膜对细胞和病毒的进入更具渗透性。二 方法将是破坏层粘连蛋白聚合; netrins，具有层粘连蛋白的小基质分子 同源性，已知在正常发育过程中破坏层粘连蛋白聚合， 实验条件我们将产生netrin诱导的ILM局灶性破坏， 促进基因和细胞的整合。 我们将进行我们的实验，在体外使用视网膜的器官型培养物，以及在 体内鼠模型。体外方法的优点是，我们可以添加回层粘连蛋白， 重组ILM，从而证明层粘连蛋白操作是关键步骤。体内 这种方法更具有临床意义。在这个探索性的项目中，我们将使用小鼠模型， 未来的后续应用可以采用更多临床相关的动物模型。 总之，本项目中的实验将评估ILM对 基于生物学的移植治疗。他们将确定是否特异性地破坏层粘连蛋白 中坚力量足以促进移植。这些实验如果成功， 一个强大的工具，以促进先进的矩阵为基础的方法来操纵视网膜BM， 促进基于基因和基于细胞的疗法治疗视网膜内疾病。