Developing novel CRISPR/CasX editors to generate a CCR5/null immune system

开发新型 CRISPR/CasX 编辑器以生成 CCR5/null 免疫系统

• 批准号: 10553152
• 负责人: ALEXANDRA L HOWELL
• 金额: --
• 依托单位: WHITE RIVER JUNCTION VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10553152
• 关键词: Adaptive Immune System; Alleles; Anti-Retroviral Agents; Area; Autologous; Autologous Transplantation; Bacteria; Bacteriophages; Binding; CCR5 gene; CD4 Positive T Lymphocytes; Cell Transplantation; Cell surface; Cells; Cellular Immunity; Clinical Research; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Communicable Diseases; Complex; DNA; DNA Repair; DNA Sequence; DNA annealing; Data; Derivation procedure; Development; Enzymes; Eukaryotic Cell; Event; Excision; Family; Flow Cytometry; Generations; Genes; Genetic Diseases; Genomic DNA; Goals; Guide RNA; HIV; HIV Infections; HIV resistance; HIV-1; Hematopoietic stem cells; Hepatitis B Infection; Hot Springs; Human; Human Genome; Humoral Immunities; Immune; Immune system; Immunodeficient Mouse; In Vitro; Individual; Induced Mutation; Infection; Infusion procedures; Integrase; Interruption; Isoptera; Lentivirus; Location; Malignant Neoplasms; Measures; Mediating; Metagenomics; Methods; Mus; Mutation; Null Lymphocytes; Patients; Pharmaceutical Preparations; Phenotype; Process; Puromycin; RNA; Recombinants; Ribonucleoproteins; Single-Stranded DNA; Site; Sorting; Specificity; System; Technology; Therapeutic; Time; Umbilical Cord Blood; Validation; Viral Genome; Viral Vector; Water; antiretroviral therapy; base; endonuclease; gene transplantation for gene therapy; human disease; human pathogen; hydraulic fracturing; in vivo; innovation; interest; mathematical model; metagenomic sequencing; mutant; next generation; next generation sequencing; novel; nuclease; patient subsets; peripheral blood; preclinical study; receptor; resistance allele; stem cells; tool; trial planning

The characterization of CRISPR/Cas as a method to edit genes that contribute to human diseases provides new opportunities for the development of innovative therapeutic approaches. Seven years ago, this bacterial immune system was adapted to cleave specific regions in the human genome. Currently, there are over a dozen clinical trials planned or in process for cancer, genetic disorders and infectious diseases that utilize CRISPR editing. Moreover, gene editing can be used to cleave and potentially excise viral genomes within infected cells, which offers hope for curative strategies for HIV-1 and hepatitis B infections, as well as others. The majority of pre-clinical and clinical studies use the Cas9 enzyme derived from common human pathogens, and evidence for the existence of humoral and cellular immunity to Cas9 could stymie the utility of this editor in a subset of patients, especially if delivered in vivo using viral vectors, or if therapy requires repeated infusions (1, 2). These and other limitations of the family of Cas9 endonucleases argue for the development of a more diverse toolbox of gene editing enzymes. Recently, several novel Cas enzymes termed CasX (Cas12e) and CasY (Cas12d) were identified in metagenomic sequencing data from environmental isolates (3), and we are actively developing these enzymes for gene editing. The advantages of these enzymes include their derivation from non-pathogenic bacterial species, their relatively compact size compared to Cas9, and their distinct protospacer adjacent motif (PAM) requirements. The cut generated by the guide RNA/CasX ribonucleoprotein complex is asymmetrical and generates single stranded DNA overhangs. This unique cleavage cut could then be leveraged to excise a target region by employing two different guide RNAs that flank the area of interest. By careful selection of guide RNAs, we expect that we can generate DNA overhangs on opposite DNA strands that are complementary to each other so they anneal during DNA repair, essentially excising the intervening region. In addition, we expect that these asymmetrical overhangs can also be used to anneal to an exogenously supplied donor DNA fragment, so that the excised region is replaced by new DNA sequence. Our proposed studies will develop CRISPR/CasX to replace the wild-type CCR5 gene with the CCR5- delta 32 allele in hematopoietic stem cells (HSC) as a next generation approach to the development of a therapeutic application for HIV-1. It is well established that cells from individuals who are homozygous for the CCR5-delta 32 allele are resistant to R5-tropic HIV infection, and generating autologous HSCs carrying two copies of this mutation could potentially allow the permanent cessation of anti-retroviral therapy. To accomplish this goal, we will develop combinations of guide RNAs to excise the CCR5 gene, and replace it with donor DNA carrying the mutant allele. Next generation sequencing approaches (CIRCLE-seq, MTA-seq) will be used to assess the relative efficiency and specificity of CasX both in vitro and in cells. An RNA beacon will be used to select for cells in which both CCR5 alleles are replaced. Finally, various proportions of wild-type and CCR5-delta 32-modified HSCs will be used to humanize immunodeficient mice. Mathematical modeling will define the minimum number of edited HSCs necessary to produce normal numbers of immune cells following HIV-1 challenge. Ultimately, our approach will lead to the development of novel Cas editors to replace cellular genes required for HIV-1 infection with those that impart HIV resistance.

CRISPR/Cas 作为一种编辑导致人类疾病的基因方法的特征 为创新治疗方法的发展提供了新的机遇。七年前，这个 细菌免疫系统适应切割人类基因组中的特定区域。目前，有 计划或正在进行的针对癌症、遗传性疾病和传染病的十多项临床试验 利用 CRISPR 编辑。此外，基因编辑可用于切割并可能切除病毒基因组 在受感染的细胞内，这为 HIV-1 和乙型肝炎感染的治疗策略以及 其他的。大多数临床前和临床研究使用源自普通人类的 Cas9 酶 病原体，以及 Cas9 体液和细胞免疫存在的证据可能会阻碍其效用 该编辑器在一部分患者中使用，特别是如果使用病毒载体在体内递送，或者如果治疗需要 重复输注 (1, 2)。 Cas9 核酸内切酶家族的这些和其他局限性表明需要开发更多的 基因编辑酶的多样化工具箱。最近，几种新型 Cas 酶被称为 CasX (Cas12e) 和 CasY (Cas12d) 在环境分离株的宏基因组测序数据中被鉴定出来 (3)，我们正在 积极开发这些用于基因编辑的酶。这些酶的优点包括它们的衍生 来自非致病性细菌物种，与 Cas9 相比，它们的尺寸相对紧凑，并且具有独特的特性 原型间隔子相邻基序 (PAM) 要求。指导RNA/CasX核糖核蛋白产生的切割 复合物是不对称的并产生单链 DNA 突出端。这种独特的乳沟切割可以 通过使用位于感兴趣区域两侧的两种不同的向导RNA来切除目标区域。经过 仔细选择引导RNA，我们期望能够在相反的DNA链上产生DNA突出端 彼此互补，因此它们在 DNA 修复过程中退火，本质上是切除干扰物 地区。此外，我们期望这些不对称突出也可以用于退火到 外源提供的供体 DNA 片段，以便切除的区域被新的 DNA 序列取代。 我们提出的研究将开发 CRISPR/CasX 以用 CCR5- 取代野生型 CCR5 基因 造血干细胞 (HSC) 中的 delta 32 等位基因作为下一代开发方法 HIV-1的治疗应用。众所周知，来自纯合子个体的细胞 CCR5-delta 32等位基因对R5-tropic HIV感染有抵抗力，并产生携带两种病毒的自体HSC 这种突变的拷贝可能会导致抗逆转录病毒治疗永久停止。到 为了实现这一目标，我们将开发指导RNA的组合来切除CCR5基因，并替换它 供体 DNA 携带突变等位基因。下一代测序方法（CIRCLE-seq、MTA-seq） 将用于评估 CasX 在体外和细胞内的相对效率和特异性。 RNA信标 将用于选择两个 CCR5 等位基因都被替换的细胞。最后，不同比例的野生型 CCR5-delta 32修饰的HSC将用于使免疫缺陷小鼠人源化。数学建模 将定义产生正常数量免疫细胞所需的编辑 HSC 的最小数量 HIV-1挑战后。最终，我们的方法将导致新型 Cas 编辑器的开发来取代 HIV-1 感染所需的细胞基因和赋予 HIV 抵抗力的细胞基因。