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

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

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英文摘要
 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.
期刊论文(1)
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会议论文
DOI: 10.2217/fvl-2018-0010
发表时间: 2018-07
期刊: Future virology
影响因子: 3.1
作者: [Hsu DS, Kornepati AV, Glover W, Kennedy EM, Cullen BR]
通讯作者: Cullen BR
Reversal of epigenetic silencing rescues integrase-deficient HIV-1 replication
  • 批准号:
    10158875
  • 项目类别:
  • 资助金额:
    $23.78万
  • 财政年份:
    2021
  • 负责人:
    BRYAN R. CULLEN
  • 依托单位:
Reversal of epigenetic silencing rescues integrase-deficient HIV-1 replication
  • 批准号:
    10369728
  • 项目类别:
  • 资助金额:
    $19.74万
  • 财政年份:
    2021
  • 负责人:
    BRYAN R. CULLEN
  • 依托单位:
Epitranscriptomic modification of HIV-1 transcripts: Effects of drugs of abuse
  • 批准号:
    10371249
  • 项目类别:
  • 资助金额:
    $32.2万
  • 财政年份:
    2018
  • 负责人:
    BRYAN R. CULLEN
  • 依托单位:
Epitranscriptomic modification of HIV-1 transcripts: Effects of drugs of abuse
  • 批准号:
    9894777
  • 项目类别:
  • 资助金额:
    $32.2万
  • 财政年份:
    2018
  • 负责人:
    BRYAN R. CULLEN
  • 依托单位:
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