A Massive Library of AAVs to Target Transcriptionally-Defined Primate Cell Types

针对转录定义的灵长类细胞类型的庞大 AAV 库

• 批准号: 9804256
• 负责人: William Richard Stauffer
• 金额: $ 228.7万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9804256
• 关键词: Adult; Anatomy; Behavior; Behavioral; Brain; Brain region; Breeding; Capsid; Cells; Clinical; Code; Cognition; Cognitive; Complex; Consensus; Corpus striatum structure; DNA; Data Set; Databases; Decision Making; Dependovirus; Development; Dimensions; Disease; Dopamine Receptor; Engineering; Enhancers; Equipment and supply inventories; Evolution; Expression Profiling; Foundations; Gene Delivery; Gene Expression; Gene Expression Profile; Gene Transfer Techniques; Genes; Genetic; Genetic Transcription; Genomics; Goals; Human; Individual; Investigation; Investigational Therapies; Learning; Libraries; Macaca; Macaca mulatta; Maps; Mediating; Mental disorders; Methods; Midbrain structure; Modeling; Modification; Monitor; Monkeys; Moods; Motor Cortex; Mus; Mutate; Neurologic; Neurons; Neurosciences; Opsin; Outcome; Photophobia; Photosensitivity; Physiological; Prefrontal Cortex; Primates; Property; Regulatory Element; Retina; Retinal Degeneration; Retinal Ganglion Cells; Retinal gene therapy; Rewards; Role; Specificity; Structure; System; Thalamic structure; Transgenes; Tropism; Tyrosine 3-Monooxygenase; Update; Validation; Variant; Viral Vector; Vision; adeno-associated viral vector; base; brain cell; cell type; cost; dopaminergic neuron; experimental study; functional restoration; gene therapy clinical trial; high throughput screening; human disease; innovation; minimally invasive; multidisciplinary; nervous system disorder; neural circuit; nonhuman primate; optogenetics; promoter; reference genome; relating to nervous system; single-cell RNA sequencing; synthetic biology; therapeutic gene; therapeutic transgene; tool; transcriptome; transgene expression; validation studies; vector; visual processing

Here we will identify nonhuman primate (NHP) neuron types and build an extensive toolbox of vectors for circuit- based neuroscience studies. NHPs share substantial neuroanatomical, genetic, and behavioral homology with humans, and therefore they are indispensable for investigating the neural circuit basis of cognition and devising therapies to treat neurological and psychiatric disorders. Despite the importance of NHPs, we lack the tools to analyze and manipulate complex circuits in the primate brain. This lack severely limits the use of genetically- coded neuroscience tools to examine circuit specific functions and hinders development of targeted gene therapeutics. Current methods for achieving transgenesis in small model species, such as the creation of genetically modified strains, are prohibitively expensive in NHP and not applicable to human disease. AAVs are the leading alternative to germline modification and selective breeding. AAVs infect adult neurons, confer stable transgene expression, and have proven safe in gene therapy clinical trials. AAVs do not have natural cell-types specific properties, but when altered or combined with cell type specific regulatory sequences (enhancers/promoters) they have been able to achieve cell type-specific transgenesis. This has made possible, for example, our previous optogenetic investigation of midbrain dopamine neurons for learning and decision making. However, before AAV-mediated gene delivery can be generalized to circuits across the brain and for multiple behavioral functions, we must create currently lacking vectors and promoters that permit efficient and specific gene delivery to all required cell types. Here, we will combine single-cell RNA-Seq (scRNA-Seq) with high-throughput screening of engineered adeno-associated viruses (AAVs) to create a complete toolbox of viral vectors and promoters enabling minimally invasive monitoring and manipulation of neurons in NHP brain. We have devised a transdisciplinary approach to classify individual neurons according to their gene expression profile and simultaneously screen for adeno-associated virus (AAV) vectors (capsids and regulatory sequences) capable of specific and efficient transgene delivery to classified neurons. First, we will synthesize massive libraries of mutated AAV vectors and synthetic promoters, in which each variant is paired with a unique DNA barcode. We will then scRNA-Seq to capture the transcriptome for each cell and quantify the AAV and promoter- specific barcodes in every cell’s expression profile. Preliminary experiments in Rhesus monkeys have fully validated and demonstrated the promise of this innovative approach. The outcomes of our Specific Aims will include (1) an inventory of cell types in the retina, prefrontal cortex, primary motor cortex, and striatum, (2) cell type-specific AAVs and promoters targeting all defined cell types, (3) AAVs with broad tropisms, (4) a publicly available dataset of transcription profiles for millions of NHP brain cells, (5) an updated and comprehensive Rhesus macaque reference genome, and (6) anatomical, physiological, and functional validation of cell type- specific circuits tools and their function in the NHP brain.

在这里，我们将识别非人类灵长类动物（NHP）神经元类型，并建立一个广泛的工具箱向量电路-