Systemic Delivery of CRISPR/Cas9 Targeting HPV Oncogenes Is Effective at Eliminating Established Tumors.

The recent advancements in CRISPR/Cas9 engineering have resulted in the development of more targeted and potentially safer gene therapies. The challenge in the cancer setting is knowing the driver oncogenes responsible, and the translation of these therapies is hindered by effective and safe delivery methods to target organs with minimal systemic toxicities, on-target specificity of gene editing, and demonstrated lack of long-term adverse events. Using a model system based on cervical cancer, which is driven by the ongoing expression of the human papillomavirus E6 and E7 proteins, we show that CRISPR/Cas9 delivered systemically in vivo using PEGylated liposomes results in tumor elimination and complete survival in treated animals. We compared treatment and editing efficiency of two Cas9 variants, wild-type (WT) Cas9 and the highly specific FokI-dCas9, and showed that the latter was not effective. We also explored high-fidelity repair but found that repair was inefficient, occurring in 6%-8% of cells, whereas non-homologous end joining (NHEJ) was highly efficient, occurring in ∼80% of the cells. Finally, we explored the post gene-editing events in tumors and showed that cell death is induced by apoptosis. Overall, our work demonstrates that in vivo CRISPR/Cas editing treatment of preexisting tumors is completely effective despite the large payloads.

Short interfering RNA induced generation and translation of stable 5' mRNA cleavage intermediates.

Sequence-specific degradation of homologous mRNA is the main mechanism by which short-interfering RNAs (siRNAs) suppress gene expression. Generally, it is assumed that the mRNA fragments resulting from Ago2 cleavage are rapidly degraded, thus making the transcript translation-incompetent. However, the molecular mechanisms involved in the post-cleavage mRNA decay are not completely understood and the fate of cleavage intermediates has been poorly studied. Using specific siRNAs and short-hairpin RNAs (shRNAs) we show that the 5' and 3' mRNA cleavage fragments of human papilloma virus type 16 (HPV-16) E6/7 mRNA, over-expressed in cervical malignancies, are unevenly degraded. Intriguingly, the 5' mRNA fragment was more abundant and displayed a greater stability than the corresponding 3' mRNA fragment in RNAi-treated cells. Further analysis revealed that the 5' mRNA fragment was polysome-associated, indicating its active translation, and this was further confirmed by using tagged E7 protein to show that C-terminally truncated proteins were produced in treated cells. Overall, our findings provide new insight into the degradation of siRNA-targeted transcripts and show that RNAi can alter protein expression in cells as a result of preferential stabilization and translation of the 5' cleavage fragment. These results challenge the current model of siRNA-mediated RNAi and provide a significant step forward towards understanding non-canonical pathways of siRNA gene silencing.

CRISPR/Cas9-loaded stealth liposomes effectively cleared established HPV16-driven tumours in syngeneic mice.

Gene-editing has raised the possibility of being able to treat or cure cancers, but key challenges remain, including efficient delivery, in vivo efficacy, and its safety profile. Ideal targets for cancer therapy are oncogenes, that when edited, cause cell death. Here, we show, using the human papillomavirus (HPV) type 16 cancer cell line TC1, that CRISPR/Cas9 targeting the E7 oncogene and packaged in PEGylated liposomes cleared established tumours in immunocompetent mice. Treatment caused no significant toxicity in the spleen or liver. An ideal therapeutic outcome would be the induction of an immunogenic cell death (ICD), such that recurrent tumours would be eliminated by the host immune system. We show here for the first time that CRISPR/Cas9-mediated cell death via targeting E7 did not result in ICD. Overall, our data show that in vivo CRISPR/Cas targeting of oncogenes is an effective treatment approach for cancer.

The Therapeutic Potential of CRISPR/Cas9 Systems in Oncogene-Addicted Cancer Types: Virally Driven Cancers as a Model System.

The field of gene editing is undergoing unprecedented growth. The first ex vivo human clinical trial in China started in 2016, more than 1000 US patents have been filed, and there is exponential growth in publications. The ability to edit genes with high fidelity is promising for the development of new treatments for a range of diseases, particularly inherited conditions, infectious diseases, and cancers. For cancer, a major issue is the identification of driver mutations and oncogenes to target for therapeutic effect, and this requires the development of robust models with which to prove their efficacy. The challenge is that there is rarely a single critical gene. However, virally driven cancers, in which cells are addicted to the expression of a single viral oncogene in some cases, may serve as model systems for CRISPR/Cas therapies, as they did for RNAi. These models and systems offer an excellent opportunity to test both preclinical models and clinical conditions to examine the effectiveness of gene editing, and here we review the options and offer a way forward.

Critical risk-benefit assessment of the novel anti-cancer aurora a kinase inhibitor alisertib (MLN8237): A comprehensive review of the clinical data.

BACKGROUND: Many current anticancer chemotherapeutics suffer from significant side effects, which have led to the exploration of more targeted therapies. This resulted in the exploration of inhibitors of Aurora A kinase as a potential anti-cancer treatment. Alisertib (MLN8237) has proven to be a potent Aurora A kinase inhibitor that had the highest safety profile among its therapeutic family. Phase I/II/III clinical trials with Alisertib have been carried out and reported promising efficacy, yet serious side effects. This article attempts to assess the clinical effect of Alisertib administration in various cancer phenotypes while describing the reported side effects. METHODS: Alisertib clinical data were systematically retrieved from Medline, CINAHL, PubMed, and Cochrane Central Register of Controlled Trials and analyzed for quality, relevance, and originality in three stages prior to inclusion. RESULTS: Overall, seven studies met inclusion criteria and enrolled a total of 630 patients. The reported "potential" clinical effect of Alisertib in various tumours is promising as it improved time to disease progression, progression-free survival, and the duration of disease stability. The achieved improvement therefore rationalizes its further investigation as a novel anticancer therapy. However, the administration of the drug was associated with serious haematological disturbances in a relatively high percentage of patients. CONCLUSION: The evidence of the anti-tumour effect of Alisertib administration is compelling in various types of malignancies. The reported side effects were serious but manageable in many cases. Topical or more targeted routes of administration are suggested when possible to overcome off-target events with systematic administration of the drug.

Bright light therapy for nonseasonal depression: Meta-analysis of clinical trials.

BACKGROUND: Bright light therapy (BLT) is a well-established treatment for seasonal depression. In the last two decades, the interest in BLT has expanded to involve other nonseasonal types of depression. The role of BLT for nonseasonal depression remains unsettled. In view of the growing number of studies in this area, this review aimed to assess the efficacy of BLT in nonseasonal depression. METHODS: We searched Pubmed; Scopus; PsychINFO; Evidence Based Medicine Guidelines and Cochrane Library until December 2015. The Standardized mean difference was calculated to assess the efficacy of BLT in nonseasonal depression. Data were subgrouped according to different study characteristics. Heterogeneity was assessed by examining the I(2) index. RESULTS: Nine trials met the inclusion criteria. After employing the more conservative random-effects model, the overall model showed a significant reduction of depressive symptoms after BLT administration (SMD=-0.62, P<0.001, I(2)=37%). In particular, BLT appears to be efficacious when administered for 2-5 weeks (SMD=-0.78, P<0.001, I(2)=0%), and as monotherapy (SMD=-0.71, P<0.001, I(2)=18%). Studies of BLT for perinatal depression have found statistically insignificant improvement (SMD=-0.17, P>0.05, I(2)=44%). LIMITATIONS: The overall heterogeneity of the included trials was moderate. The participants were not adequately blinded to the intervention. The sample size was small for certain subgroups. The long-term effect of BLT on depression was not explored. CONCLUSIONS: BLT appears to be efficacious, particularly when administered for 2-5 weeks' duration and as monotherapy. There is an obvious need to optimize the duration and intensity of exposure, the timing and the duration of treatment sessions.