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

CRISPR/Cas作为编辑人类疾病相关基因的方法的特征 为创新治疗方法的发展提供了新的机遇。七年前，这个 细菌免疫系统被用来切割人类基因组中的特定区域。目前，有以下几种 已计划或正在进行的十多项癌症、遗传疾病和传染病的临床试验 利用CRISPR编辑。此外，基因编辑可以用来切割和潜在地切除病毒基因组 在受感染的细胞内，这为艾滋病毒-1和乙肝感染的治疗策略以及 其他。大多数临床前和临床研究使用的是来自普通人类的Cas9酶 病原体，以及存在对Cas9的体液和细胞免疫的证据可能会阻碍Cas9的应用 这一编辑适用于部分患者，特别是如果使用病毒载体在体内传递，或者如果需要治疗 重复输液(1，2)。 Cas9内切酶家族的这些和其他限制证明了更多的 不同的基因编辑酶工具箱。最近，几种新的CaS酶被命名为CasX(Cas12e)和 从环境分离株(3)的元基因组测序数据中鉴定出Casy(Cas12d)，我们是 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性 与Cas9相比，它们相对紧凑的大小，以及它们不同的 保护相邻基序(PAM)要求。由引导RNA/CasX核糖核蛋白产生的切割 复合体是不对称的，并产生单链DNA悬垂。这种独特的乳沟切割可以 通过在感兴趣区域两侧使用两个不同的引导RNA来利用来切除目标区域。通过 仔细选择引导RNA，我们预计可以在相反的DNA链上产生DNA突出 它们是相辅相成的，所以它们在DNA修复过程中会退火，实质上是切除了中间的 区域。此外，我们预计这些不对称的悬臂也可以用来退火到一个 外源提供的供体DNA片段，使切除的区域被新的DNA序列取代。 我们建议的研究将开发CRISPR/CasX来用CCR5-取代野生型CCR5基因- 造血干细胞(HSC)中Delta 32等位基因作为一种新一代方法在白血病发生发展中的作用 HIV-1的治疗应用。已经确定的是，来自纯合子的个体的细胞 CCR5-delta32等位基因对R5嗜性HIV感染具有抵抗力，并产生携带两个 这种突变的副本可能会使抗逆转录病毒治疗永久停止。至 为了实现这一目标，我们将开发组合的引导RNA来切除CCR5基因，并取代它 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。一个RNA信标 将用于选择其中两个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HSCs将被用于使免疫缺陷小鼠人性化。数学建模 将定义产生正常数量的免疫细胞所需的最低编辑的HSC数量 在HIV-1挑战之后。最终，我们的方法将导致小说编辑的发展，以取代 HIV-1感染所需的细胞基因与那些传递艾滋病毒抵抗力的基因。

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.