Glia Exclusive Gene Therapy

胶质细胞独家基因疗法

• 批准号: 10739502
• 负责人: Or Shemesh
• 金额: $ 23.85万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-15 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10739502
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Acute; Address; Affect; Alzheimer' s Disease; Antibodies; Astrocytes; Binding; Biomedical Engineering; Brain; Brain Diseases; Cells; Cholesterol; Clinical; Code; Dependovirus; Development; Disease; Engineering; Ependymal Cell; Exhibits; Gene Delivery; Gene Expression; Gene Targeting; Gene Transduction Agent; Genes; Green Fluorescent Proteins; Hela Cells; Human; Image; Immune response; Intervention; Knock-out; Lentivirus; Lipids; Luciferases; Luminescent Proteins; Malignant Neoplasms; Malignant neoplasm of brain; Medical; Methods; Microglia; Modeling; Modification; Molecular; Mood Disorders; Mus; Mutagenesis; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuroglia; Neurons; Oligodendroglia; Optics; Pain; Pain Disorder; Pathology; Play; Poly(A) Tail; Process; Proteins; RNA; RNA Stability; Rapid screening; Reporter Genes; Research; Role; Specificity; Surface Antigens; Tail; Technology; Testing; Therapeutic; Time; Vaccination; Vascular Diseases; Viral Vector; Virus; analog; aptamer; autism spectrum disorder; cancer therapy; cell type; clinical application; delivery vehicle; design; gene therapy; healing; immunogenicity; in vivo; interest; lipid nanoparticle; nervous system disorder; non-viral gene delivery; novel; prevent; screening; targeted delivery; transmission process; vector

Abstract Glia are supportive cells in the human brain, comprising microglia, oligodendrocytes, astrocytes, and ependymal cells. Glia are deeply involved in diseases of the nervous system such as Alzheimer’s (AD), autism, pain, affective disorders, and cancers. Different glial cell types play different mechanistic roles in disease formation, driven by specific genes. Modulating glial gene expression via a process called gene therapy could thus be studied as a means of preventing deleterious effects of glia in the brain. However, while significant progress has been made in delivering genes exclusively to neurons, such capabilities are lacking for glia, despite their demonstrated role in disease formation, posing a critical medical need. Although gene delivery to neurons can be achieved using viral vectors, their use to transmit genes to glia in-vivo has been unsuccessful. Here, we propose to design a novel nonviral gene delivery vector targeting microglia or astrocytes exclusively by bioengineering Modified RNAs (ModRNAs). ModRNAs are synthetic RNA molecules known not to trigger an immune response and are strongly expressed in target cells. Currently, ModRNAs enable only days- long expression, impeding long-duration medical applications and lacking cell specificity to glia types. We will engineer glia-type-specific, ModRNAs-based constructs, GliaRNAs, as a platform for glia-exclusive gene therapy, with a customizable expression duration. First, ModRNAs that enable robust and prolonged expression (7-14 days) will be developed (Aim 1). For this purpose, existing ModRNA will be altered, through modifications and by inflicting random mutations of structural components of the molecule, including CAP analog, 3’ untranslated region, coding region, 5’ untranslated region, and the poly-A tail. We will test the expression of these novel GliaRNAs in glial cultures from mice. Next, the vector specificity will be optimized (Aim 2). We will screen for molecular manipulations that enable robust and specific delivery of the GliaRNAs into either microglia or astrocytes (GliaRNA-vectors) and select the best gene delivery vectors, specifically either lipid nanoparticles, antibody-lipid conjugates, or aptamers. As a proof of concept, we will use the new GliaRNA-vector technology to express the green fluorescent protein (GFP) in either astrocytes or microglia in mice brains. The GliaRNA-vector platform will pave the way for genetically healing and modifying different types of glia, opening multiple therapeutic and research avenues in humans by targeting neurodegeneration, autism, pain disorders, mood disorders, and brain cancers.

摘要 胶质细胞是人脑中的支持性细胞，由小胶质细胞、少突胶质细胞、星形胶质细胞和室管膜细胞组成。 胶质细胞深度参与神经系统疾病，如阿尔茨海默氏症(AD)、自闭症、疼痛、情感障碍、 和癌症。在特定基因的驱动下，不同类型的胶质细胞在疾病形成中发挥着不同的机制作用。 因此，可以研究通过一种称为基因治疗的过程来调节神经胶质基因的表达，以此作为预防 脑内神经胶质细胞的有害影响。然而，尽管在专门传递基因方面取得了重大进展 对于神经元来说，尽管胶质细胞在疾病形成中发挥了已证明的作用，但神经胶质细胞缺乏这种能力，构成了关键的 医疗需要。虽然基因传递到神经元可以通过病毒载体来实现，但它们用来将基因传递到神经胶质细胞 体内试验一直未获成功。在这里，我们建议设计一种新型的靶向小胶质细胞的非病毒基因传递载体 星形胶质细胞仅通过生物工程修饰的RNA(ModRNAs)。ModRNAs是已知的合成RNA分子 不会引发免疫反应，并在靶细胞中强烈表达。目前，ModRNAs仅启用天数- 表达时间长，阻碍了长期的医学应用，并且缺乏对神经胶质细胞类型的细胞特异性。我们将进行工程设计 胶质细胞类型特异的、基于ModRNAs的构建物，GiliRNAs，作为神经胶质细胞专属基因治疗的平台，具有 可自定义的表达式持续时间。首先，能够实现强健和长期表达(7-14天)的modRNAs将是 已开发(目标1)。为此目的，现有的modRNA将通过修改和施加随机 分子结构成分突变，包括CAP类似物、3‘非翻译区、编码区、5’端。 未翻译区域和Poly-A尾巴。我们将在小鼠的神经胶质细胞培养中测试这些新的Garia RNAs的表达。 接下来，将优化载体的特异性(目标2)。我们将筛选分子操作，使强大的和 将GiliRNAs特异性地导入小胶质细胞或星形胶质细胞(GliaRNA载体)，并选择最佳的基因输送方式 载体，特别是脂质纳米粒、抗体-脂质结合物或适配子。作为概念验证，我们将使用 绿色荧光蛋白(GFP)在星形胶质细胞和小胶质细胞中表达的新技术 老鼠的大脑。Garia RNA载体平台将为基因修复和修改不同类型的基因铺平道路 通过针对神经变性，自闭症，为人类开辟了多种治疗和研究途径， 疼痛障碍、情绪障碍和脑癌。