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Targeting HPV16 DNA using CRISPR/Cas inhibits anal cancer growth in vivo
 
 
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Short Communication
 
Published Online:12 Jun 2018
David S Hsu1, Anand VR Kornepati1, Wayne Glover1, Edward M Kennedy1 & Bryan R Cullen*,1
1Departments of Medicine & Molecular Genetics & Microbiology, Duke Cancer Institute, Duke University Medical Center, Durham,
NC 27710, USA
*Author for correspondence: Tel.: +1 919 684 3369; bryan.cullen(at)duke.edu
 
OPEN ACCESS
https://www.futuremedicine.com/doi/full/10.2217/fvl-2018-0010
 
Aim: The goal of this study was to determine if a single AAV vector, encoding Cas9 and guide RNAs specific for the HPV16 E6 and E7 genes, could inhibit the growth of an HPV16-induced tumor in vivo.
 
Materials & methods: We grew HPV16+, patient-derived anal cancer explants in immunodeficient mice and then challenged these by injection of AAV-based vectors encoding Cas9 and control or HPV16-specific guide RNAs.
 
Results & conclusion: We observed a significant and selective reduction in tumor growth when the HPV16 E6 and E7 genes were targeted using Cas9. These studies provide proof of principle for the hypothesis that CRISPR/Cas has the potential to be used to selectively treat HPV-induced tumors in humans.
 
Although anal cancer makes up only 1.5% of all gastrointestinal malignancies, its incidence has been rising over the past few years at a rate of approximately 3% per year [1]. Thus, in 2003 there were an estimated 4000 new cases of anal cancer in the USA and 10 years later, in 2013, there were an estimated 6500 new cases [1]. For patients with locally advanced anal cancer, standard of care treatment consists of a combination of chemotherapy and radiation and with this approach 5-year survival is about 75% [2,3]. For patients who recur, salvage surgery is an option, but this can lead to significant postoperative complications including bacterial infections, poor wound healing, urinary retention and slow recovery. For patients who are not candidates for surgery, treatment consists of palliative chemotherapy for disease control. Unfortunately, few therapies have been shown to be clinically beneficial in this setting and therefore there is an unmet clinical need to develop new therapeutics for the treatment of anal cancer.
 
Human papillomaviruses (HPVs) are the etiologic agents responsible for almost all cervical carcinomas and an increasing percentage of both anal and head-and-neck cancers [4-6]. Only a subset of the many different HPV serotypes present a high risk for malignant transformation, with HPV16 and HPV18 accounting for approximately 70% of cervical cancers, while HPV16 alone accounts for >80% of HPV+ anal and head-and-neck cancers. A unique aspect of HPV-induced tumors is that their growth and survival requires the ongoing expression of two HPV-encoded oncogenes, E6 and E7, which induce the degradation of several host cell tumor suppressor proteins, including p53 and the retinoblastoma protein [4-9]. Therefore, one potential approach to the elimination of HPV-induced cancers is to block the expression of E6 and/or E7, which results in cell cycle arrest and apoptosis. Indeed, several groups have reported that the introduction into HPV18- or HPV16-transformed cervical carcinoma cell lines, such as HeLa or SiHa, of a vector encoding the Streptococcus pyogenes (Spy) Cas9 protein, together with single guide RNAs (sgRNAs) specific for either the HPV E6 or E7 gene, results not only in the efficient inactivation of E6 and/or E7 expression due to gene editing but also in the apoptotic death of the HPV-transformed cell culture, while cells transformed by factors other than HPV are unaffected [10-12]. This suggests that the expression of Cas9 and an HPV E6- or E7-specific sgRNA might represent an effective and specific approach to the elimination of HPV-transformed cells in vivo. Here, we describe the construction of adeno-associated virus (AAV) vectors expressing the short Staphylococcus aureus (Sau) Cas9 protein and two guide RNAs specific for both the HPV16 E6 and E7 gene and show that these can significantly reduce the growth of patient-derived xenografts (PDXs) of HPV16+ anal tumors in vivo in immunodeficient mice.
 
Discussion
 
Treatment of anal cancer has evolved over the past few decades from surgery to chemoradiation. Efforts to improve treatment have focused on altering dosage and frequency of radiation in addition to evaluating different types of chemotherapy. Unfortunately, minimal improvement has been made using these treatment modalities and new, more specific and more effective therapies are clearly needed.
 
Although mouse models of cancer have been used in front-line preclinical studies to study the efficacy and toxicity of anticancer drugs [17,18], currently, there are limited preclinical models of anal cancer. For example, a mouse model of K14E6 and K14E7 transgenic mice, in which the HPV16 E6 and E7 genes are directed in their expression to stratified squamous epithelia, has been developed [18]. Recently, we and others demonstrated that an approach of rapid engraftment of patient tumor tissue into immunodeficient mice to develop PDXs can provide a more clinically applicable murine model to study drug sensitivities [15,19-23]. In this study, we used the same method to develop three anal cancer PDXs, one of which was found to be HPV16+. More importantly, these anal cancer PDXs can now be used as preclinical models to further study and identify new therapies for anal cancer.
 
In our current work, we are the first to demonstrate that targeting of the HPV16 genome in primary human anal cancer cells in vivo using CRISPR/Cas can significantly slow tumor growth (Figure 5). However, a complete response was not observed. Nevertheless, tumor growth inhibition by HPV-AAV was both readily detectable and statistically significant. We hypothesize that the lack of complete tumor clearance reflects the fact that only a subset of tumor cells actually took up the AAV vector and expressed Cas9, although it is also possible that the editing efficiency of Sau Cas9 in these in vivo experiments was insufficient to introduce indels into all copies of the HPV16 genome. Of note, sequencing of the HPV16 E6 gene present in the anal tumor PDX at day 14, when the experiment was terminated and the mice sacrificed, did not reveal any indels at the predicted Cas9 cleavage site in E6 (data not shown), thus demonstrating that tumor growth was not due to acquisition of resistance to the specific sgRNAs used. We hypothesize that tumor cells that underwent Cas9-mediated gene editing of the HPV16 E6 or E7 gene were rapidly lost from the tumor, thus explaining both the reduced tumor volume and our inability to observe any viral indels. Nevertheless, these data do suggest that CRISPR/Cas has the potential to eliminate HPV16+ tumors in vivo if delivery to tumor cells could be made more efficient.
 
Conclusion
 
The results presented in this manuscript demonstrate that AAV vectors designed to deliver the Sau Cas9 gene and cognate sgRNAs into cells in vivo have the potential to induce the efficient editing of chromosomal genes.
 
Future perspective
 
Given the intense efforts currently ongoing to generate the tools required to move the CRISPR/Cas from the laboratory into the clinic, it seems very likely that this technology will soon be in clinical trials for a range of human diseases. Chronic disease caused by DNA viruses, including HPV-induced cancers, seem to be an ideal area for this approach especially as potential off-target editing is of less consequence if the idea is to kill the cell that is edited and if the patient already has late stage cancer. Clearly, these experiments need to repeated with a range of HPV16+ anal and head-and-neck PDXs in mice, and with a range of different AAV-vector constructs, using mouse numbers that would allow a clearer definition of whether high levels of selective tumor cell killing can be achieved in vivo. However, the current data do provide proof of concept that CRISPR/Cas indeed has the potential to selectively deplete HPV-transformed cells in vivo.
 
Executive summary
 
o We generated a single AAV8-based vector expressing the Sau Cas9 protein and single guide RNAs specific for the HPV16 E6 and E7 genes.
o We established patient-derived explants (PDXs) from a human anal cancer that was HPV16+ and showed that these PDXs can grow and be passaged in mice.
o We demonstrated that AAV8-based vectors efficiently infect anal tumor PDXs after direct injection in vivo in mice.
o We showed that direct injection of the AAV vector expressing Sau Cas9 and HPV16-specific single guide RNAs markedly and significantly inhibits the growth of human anal tumor PDXs in mice.

 
 
 
 
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