Development of STAR Editors (CRISPR-Cas9/lgRNA-ssDNA) for the cure of chronic hepatitis B

摘要 SBIR项目的第一阶段是研究创新的CRISPR-Cas9无疤痕编辑的可行性。 技术永久性地将B型肝炎病毒（HBV）共价闭合环状DNA（cccDNA） 以及整合DNA用于治疗慢性B型肝炎（CH B）。虽然目前可用的抗病毒剂， 包括核苷（酸）类似物、病毒DNA聚合酶抑制剂和聚乙二醇化α-干扰素， 有效地抑制HBV复制，并防止大多数治疗患者的疾病进展， 慢性HBV感染的治疗很少实现，因此需要终身抗病毒治疗。治疗的失败 这是由于目前的抗病毒方案不能从感染的肝细胞的细胞核中消除cccDNA。 cccDNA是病毒RNA的转录模板，是最稳定的HBV复制中间体， 抗病毒治疗中断后病毒复制反弹的来源。此外，尽管没有 对于病毒复制至关重要，来自细胞染色体中整合的HBV DNA的转录物最近被 已被证明支持HBeAg阴性CHB中大部分HBV表面抗原（HBsAg）的分泌 患者HBsAg长期过量表达诱导病毒抗原特异性T和B耗竭 细胞，有利于HBV的持续感染。显然，cccDNA的消除或永久失活 以及整合的HBV DNA对于实现CHB的治愈至关重要。CRISPR-Cas9基因编辑 迄今为止，技术是实现这一治疗目标的最有希望的方法。不过虽然 经典的CRISPR-Cas9基因编辑技术已被证明可以在培养细胞中切割和编辑cccDNA 和动物模型，它们对整合的HBV DNA的切割可能导致染色体断裂和非预期的 突变，导致遗传不稳定和遗传毒性。最近，CRISPR-Cas9基础编辑器技术已经 它被用来克服这种局限性。遗憾的是，编辑效率低，导赏率高， 慢病毒载体的独立编辑和使用阻碍了其进一步发展。在这 项目，我们建议开发我们专有的基于连接指导RNA（lgRNA）的星星（Seek-Tag-Amend- 释放）编辑器技术，用于通过以下方式有效和准确地灭活两种形式的核HBV DNA： 在重叠的HBsAg和病毒DNA聚合酶基因中引入终止密码子。具体来说，我们将首先 体外验证星星编辑器的切割活性和特异性，以优化lgRNA的结构 作为单链DNA（ssDNA）的结合位点（编辑的模板）（目的1）。我们将评估 星星编辑器在人肝癌细胞系中的编辑效率和特异性， HBV DNA和cccDNA（目的2）。成功完成第一阶段项目将使我们能够申请 评价选定的多重星星编辑器对整合HBV的灭活活性的II期研究 体内HBV转基因小鼠中的DNA和HBV感染的人源化uPA-SCID小鼠中的cccDNA。

Abstract This phase 1 SBIR project is to investigate the feasibility of an innovative CRISPR-Cas9 scar-less editing technology to permanently inactivate both hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and integrated DNA for the cure of chronic hepatitis B (CHB). Although the currently available antiviral agents, including nucleos(t)ide analogue viral DNA polymerase inhibitors and pegylated alpha-interferon, can efficiently inhibit HBV replication and prevent disease progression in the majority of treated patients, the cure of chronic HBV infection is rarely achieved and life-long antiviral therapy is thus required. The failure of a cure is due to the current antiviral regimens cannot eliminate cccDNA from the nuclei of infected hepatocytes. cccDNA, the transcription template of viral RNA, is the most stable HBV replication intermediate and the resource of viral replication rebound after disruption of antiviral therapies. Moreover, despite not being essential for viral replication, the transcripts from integrated HBV DNA in cellular chromosomes have recently been proven to support the secretion of majority of HBV surface antigen (HBsAg) in HBeAg-negative CHB patients. Prolonged excessive expression of HBsAg induces the exhaustion of viral antigen-specific T and B cells and favors the persistent infection of HBV. Apparently, elimination or permanent inactivation of cccDNA as well as integrated HBV DNA is essential to achieve the cure of CHB. CRISPR-Cas9 gene editing technology is thus far the most promising approach to achieve this therapeutic goal. However, although the classic CRISPR-Cas9 gene editing technologies had been proven to cleave and edit cccDNA in cultured cells and animal models, their cleavage of integrated HBV DNA may lead to chromosome break and unintended mutations, causing genetic instability and genotoxicity. Recently, CRISPR-Cas9 base editor technology had been developed to overcome this limitation. Unfortunately, the low editing efficiency, high rate of guide- independent editing and usage of lentiviral vector for delivery hampered its further development. In this project, we propose to develop our proprietary ligated-guide RNA (lgRNA)-based STAR (Seek-Tag-Amend- Release) editor technology for efficient and accurate inactivation of both forms of nuclear HBV DNA by introducing stop codons into overlapping HBsAg and viral DNA polymerase genes. Specifically, we will first verify the cleavage activity and specificity of STAR editors in vitro to optimize the structures of lgRNA as well as the conjugation sites of single strand DNA (ssDNA) (the template of editing) (Aim 1). We will then evaluate the editing efficiency and specificity of STAR editors in human hepatoma cell line harboring both integrated HBV DNA and cccDNA (Aim 2). Successful completion of this Phase 1 project will enable us to apply for a Phase 2 study to evaluate selected multiplexing STAR editors for their inactivation activity of integrated HBV DNA in HBV transgenic mice and cccDNA in HBV infected humanized uPA-SCID mice in vivo.