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

 描述(由申请人提供)：尽管存在针对高危人乳头瘤病毒(HPV)6、11、16和18型的有效四价疫苗，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阳性头颈部和肛门癌的发病率实际上正在上升。众所周知，在HPV诱导的癌症中，转化状态的维持依赖于两个病毒癌基因E6和E7的持续表达，这两个基因分别靶向和失活细胞肿瘤抑制基因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定位并在HPV转化的培养细胞中有效地靶向和灭活HPVE6或E7基因。Spy Cas9和HPV特异性单引导RNA(SgRNAs)的表达分别导致E6或E7突变失活和P53或Rb活性诱导，导致细胞周期停滞和最终细胞死亡。在本申请中，我们希望通过证明将Cas9/sgRNA组合传递到HPV-16转化的患者来源的肿瘤，使用腺相关病毒(AAV)载体移植到免疫缺陷小鼠体内，可以有效和特异性地缩小这些肿瘤，从而扩展这些研究。为此，我们从金黄色葡萄球菌(Sau)中鉴定了一个新的Cas9基因，它在基因编辑方面与Spy Cas9一样活跃，但足够小，约3.2kb，与两个HPV特异性sgRNAs和相关的转录调控元件一起适合AAV载体。表达HPV-16特异性Sau Cas9/sgRNA组合的AAV载体将首先使用HPV-16转化的SIHA细胞系在培养中测试有效性，然后注射到携带几种不同HPV-16转化的患者来源的肿瘤移植瘤的小鼠体内，并监测肿瘤的大小、生长和存活率。我们假设，这种方法将被证明能够有效地消除这个临床前小鼠模型系统中依赖HPV-16的肿瘤，从而提供原理证明，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，而且可能作为一种策略来治疗任何完全依赖于病毒或非病毒来源的特定致癌基因的持续功能表达的癌症。

 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.