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细胞，包括小胶质细胞、少突胶质细胞、星形胶质细胞和室管膜细胞。 神经胶质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，不同的神经胶质细胞类型在疾病形成中发挥着不同的机制作用。 因此，通过一种称为基因治疗的过程来调节神经胶质基因表达，可以作为预防 神经胶质细胞对大脑的有害影响。然而，尽管在专门递送基因方面取得了重大进展， 对于神经元来说，神经胶质细胞缺乏这种能力，尽管它们在疾病形成中起着重要作用， 医疗需求。虽然基因传递到神经元可以实现使用病毒载体，它们的使用传递基因到神经胶质细胞， 体内试验不成功。在这里，我们建议设计一种新的非病毒基因载体靶向小胶质细胞或 星形胶质细胞专门通过生物工程修饰的RNA（ModRNA）。ModRNA是已知的合成RNA分子， 不会引发免疫反应，并且在靶细胞中强烈表达。目前，ModRNA只能在几天内- 长表达，阻碍了长期的医学应用，并且缺乏对神经胶质类型的细胞特异性。我们将工程师 胶质细胞类型特异性的，基于ModRNA的构建体，GliaRNA，作为胶质细胞排他性基因治疗的平台， 可自定义表达式持续时间。首先，能够实现稳健和延长表达（7-14天）的ModRNA将被 发展（目标1）。为此目的，现有的ModRNA将通过修饰和施加随机干扰来改变。 分子的结构组分的突变，包括CAP类似物、3'非翻译区、编码区、5'非翻译区和5'非翻译区。 非翻译区和poly-A尾。我们将测试这些新的GliaRNA在小鼠神经胶质细胞培养物中的表达。 接下来，将优化载体特异性（目标2）。我们将筛选分子操作，使强大的， 将GliaRNA特异性递送到小胶质细胞或星形胶质细胞（GliaRNA载体）中，并选择最佳基因递送 载体，特别是脂质纳米颗粒、抗体-脂质缀合物或适体。作为概念验证，我们将使用 新的GliaRNA载体技术在星形胶质细胞或小胶质细胞中表达绿色荧光蛋白（GFP）， 老鼠的大脑GliaRNA载体平台将为基因治疗和修改不同类型的 通过靶向神经退行性疾病、自闭症 疼痛障碍情绪障碍和脑癌