Using bacterial CRISPR/Cas endonucleases to selectively eliminate HPV-transformed cells in vivo

使用细菌 CRISPR/Cas 核酸内切酶选择性消除体内 HPV 转化细胞

基本信息

批准号：
9136078
负责人：
BRYAN R. CULLEN
金额：
$ 17.29万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-02 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9136078
关键词：
Antiviral Agents Anus Applications Grants Biological Models Cancer Patient Cell Cycle Arrest Cell Death Cell Line Cells Cervical Cervix carcinoma Cessation of life Cleaved cell Clinical Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Cultured Cells DNA Deoxyribonuclease I Dependovirus Early Diagnosis Effectiveness Family Foundations Future Genes Genetic Transcription Genome Grant Growth Guide RNA Head and Neck Cancer Health Histology Human Human Papilloma Virus Vaccine Human Papillomavirus Human papillomavirus 16 Human papillomavirus 18 Immune system Immunodeficient Mouse Incidence Injection of therapeutic agent Laboratories Lead Left Local Therapy Maintenance Malignant Epithelial Cell Malignant Neoplasms Malignant neoplasm of anus Malignant neoplasm of cervix uteri Monitor Morbidity - disease rate Mus Normal Cell Oncogenic Operative Surgical Procedures Pap smear Patients Postoperative Period Pre-Clinical Model Preparation Primary Neoplasm Protein p53 Proteins Radiation Recurrence Regulator Genes Serotyping Solid Neoplasm Staphylococcus aureus Streptococcus pyogenes System TP53 gene Testing Therapeutic Agents Tumor Suppressor Proteins Tumor-Derived Ursidae Family Vaccines Viral Genes Viral Oncogene Work Xenograft Model Xenograft procedure adeno-associated viral vector base cancer cell cell transformation chemotherapy design efficacy testing endonuclease expression vector high risk immunodeficient mouse model implantation in vivo killings mouse model novel pathogen pre-clinical tumor tumor growth tumor xenograft vector vector control

项目摘要

DESCRIPTION (provided by applicant): Despite the existence of an effective quadrivalent vaccine, targeting the high risk human papillomavirus (HPV) serotypes 6, 11, 16 and 18, the incidence of HPV-induced cancers in the USA remains high and the incidence of HPV-positive head and neck and anal cancers, which are primarily caused by HPV-16, is actually increasing. It is well established that the maintenance of the transformed state in HPV-induced cancers is dependent on the continued expression of two viral oncogenes, E6 and E7, that target and inactivate the cellular tumor suppressors p53 and Rb, respectively. Recently, we demonstrated that the bacterial CRISPR/Cas antiviral adaptive immune system from Streptococcus pyogenes (Spy) could be repurposed to effectively target and inactivate either the HPV E6 or E7 gene in cultured HPV-transformed cells. Expression of Spy Cas9 and HPV-specific single guide RNAs (sgRNAs) resulted in the mutational inactivation of E6 or E7 and in the induction of p53 or Rb activity, respectively, leading to cell cycle arrest and eventual cell death. In this application, e wish to extend these studies by demonstrating that Cas9/sgRNA combinations delivered to HPV-16-transformed, patient-derived tumors, explanted into immunodeficient mice, using adeno-associated virus (AAV) vectors, can effectively and specifically shrink these tumors. For this purpose, we have identified and characterized a novel Cas9 gene, derived from Staphylococcus aureus (Sau), that is as active as Spy Cas9 in gene editing yet sufficiently small, at ~3.2 kb, to fit into an AAV vector along with two HPV-specific sgRNAs and relevant transcriptional regulatory elements. AAV vectors expressing HPV-16-specific Sau Cas9/sgRNA combinations will first be tested for effectiveness in culture, using the HPV-16 transformed SiHa cell line, then injected into mice carrying several different HPV-16-transformed, patient derived tumor xenografts and monitored for tumor size, growth and viability. We hypothesize that this approach will prove able to effectively eliminate HPV-16-dependent tumors in this preclinical murine model system, thus providing proof-of-principle that a similar approach could work not only as a clinical approach to the treatment of HPV-16-induced human tumors but also possibly as a strategy to treat any cancer that is entirely dependent on the continued functional expression of a specific pro-oncogenic gene of viral or non-viral origin.

描述（由适用提供）：尽管存在有效的四价疫苗，针对高风险人乳头瘤病毒（HPV）血清型6、11、16和18，但HPV诱导的癌症在美国仍然很高，而HPV阳性的头部和颈部和颈部和肛门癌症的事件仍然是由HPV造成的，实际上是HPV-16，这实际上增加了。众所周知，HPV诱导的癌症中转化状态的维持取决于分别靶向和失活细胞肿瘤补充剂p53和rb的两个病毒癌基因E6和E7的持续表达。最近，我们证明了来自链霉菌（SPY）的细菌CRISPR/CAS抗病毒自适应免疫系统可以重新定位，以有效靶向培养的HPV转换细胞中的HPV E6或E7基因。间谍Cas9和HPV特异性单引导RNA（SGRNA）的表达导致E6或E7的突变失活以及p53或Rb活性的诱导，导致细胞周期停滞和最终的细胞死亡。在此应用中，E希望通过证明使用腺相关病毒（AAV）载体可以有效地有效地缩小了这些肿瘤，以证明将Cas9/SGRNA组合传递到HPV-16转化的患者衍生的肿瘤中，这些肿瘤可依赖于免疫缺陷的小鼠。为此，我们已经确定并表征了一种新型的Cas9基因，该基因源自金黄色葡萄球菌（SAU），在基因编辑中与间谍Cas9一样活跃，但在〜3.2 kb中却足够小，以〜3.2 kb的形式适合AAV矢量，以适合两个HPV特异性的SGRNAS和相关的转录调节元素。使用HPV-16转化后的SIHA细胞系，将首先对表达HPV-16特异性SAU Cas9/SGRNA组合的AAV矢量进行培养的有效性，然后注入携带几种不同的HPV-16转化的，患者衍生的Tumor Xenografts的小鼠，并以肿瘤的大小和生长量，并监测。我们假设这种方法将证明能够在这个临床前鼠模型系统中有效地消除依赖HPV-16的依赖性肿瘤，从而提供原始证明，即类似的方法不仅可以作为治疗HPV-16诱导的人类肿瘤的临床方法起作用，而且可以作为一种完全依赖于某种特定功能的概念的策略，可以作为一种策略来处理或依赖于特定型的表达。