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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（由申请方提供）：尽管存在有效的四价疫苗，靶向高危人乳头瘤病毒（HPV）血清型6、11、16和18，但HPV诱导的癌症在美国的发病率仍然很高，主要由HPV-16引起的HPV阳性头颈癌和肛门癌的发病率实际上正在增加。已经确定，HPV诱导的癌症中转化状态的维持依赖于两种病毒癌基因E6和E7的持续表达，这两种病毒癌基因分别靶向和抑制细胞肿瘤抑制因子p53和Rb。最近，我们证明了来自化脓性链球菌（Spy）的细菌CRISPR/Cas抗病毒适应性免疫系统可以被重新利用，以有效靶向和抑制培养的HPV转化细胞中的HPV E6或E7基因。Spy Cas9和HPV特异性单向导RNA（sgRNA）的表达分别导致E6或E7的突变失活和p53或Rb活性的诱导，从而导致细胞周期停滞和最终的细胞死亡。在本申请中，我们希望通过证明使用腺相关病毒（AAV）载体递送至HPV-16转化的患者来源的肿瘤、移植到免疫缺陷小鼠中的Cas9/sgRNA组合可以有效且特异性地缩小这些肿瘤来扩展这些研究。为此目的，我们已经鉴定并表征了一种新的Cas9基因，其来源于金黄色葡萄球菌（Sau），其在基因编辑中与Spy Cas9一样有活性，但足够小，约为3.2 kb，以与两种HPV特异性sgRNA和相关转录调控元件一起沿着到AAV载体中。首先使用HPV-16转化的SiHa细胞系测试表达HPV-16特异性Sau Cas9/sgRNA组合的AAV载体在培养物中的有效性，然后注射到携带几种不同的HPV-16转化的患者来源的肿瘤异种移植物的小鼠中，并监测肿瘤大小、生长和活力。我们假设这种方法将证明能够有效地消除这种临床前小鼠模型系统中的HPV-16依赖性肿瘤，从而提供了一种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，而且还可能作为治疗完全依赖于特定前体蛋白的持续功能表达的任何癌症的策略的原理证明。病毒或非病毒来源的致癌基因。