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Luqman Jubair1, Sora Fallaha1, Nigel A J McMillan1
1School of Medical Sciences, Griffith University, Gold Coast, QLD 4222 Australia; Menzies Health Institute Queensland, Griffith University, Gold Coast, QLD 4222 Australia.
Abstract:
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.
Insights
Systemic CRISPR/Cas9 gene editing effectively eliminated cervical tumors in vivo using PEGylated liposomes, leading to complete animal survival. This approach shows promise for cancer gene therapy, inducing apoptosis and utilizing non-homologous end joining repair.
Area of Science:
- CRISPR/Cas9 gene editing
- Cancer gene therapy
- Viral oncology
Background:
- CRISPR/Cas9 advancements offer targeted gene therapies, but challenges remain in cancer treatment, including identifying driver oncogenes and ensuring safe, effective delivery.
- Translating gene therapies requires overcoming hurdles like systemic toxicity, on-target specificity, and demonstrating long-term safety.
- Cervical cancer, driven by human papillomavirus (HPV) E6 and E7 oncoproteins, serves as a relevant model for evaluating in vivo gene editing strategies.
Purpose of the Study:
- To evaluate the efficacy of systemically delivered CRISPR/Cas9 gene editing for treating existing cervical tumors in vivo.
- To compare the effectiveness of wild-type (WT) Cas9 and FokI-dCas9 variants for gene editing in this cancer model.
- To investigate the efficiency of different DNA repair pathways (high-fidelity repair vs. non-homologous end joining) and the mechanism of tumor cell death post-editing.
Main Methods:
- Systemic in vivo delivery of CRISPR/Cas9 using PEGylated liposomes in a cervical cancer model.
- Comparison of gene editing and therapeutic outcomes between WT Cas9 and FokI-dCas9 variants.
- Assessment of DNA repair pathway usage (high-fidelity repair and NHEJ) and analysis of post-editing cellular events, including apoptosis.
Main Results:
- Systemic CRISPR/Cas9 delivery via PEGylated liposomes resulted in complete tumor elimination and 100% survival in treated animals.
- Wild-type Cas9 demonstrated effective treatment, while the FokI-dCas9 variant showed no efficacy.
- Non-homologous end joining (NHEJ) was the predominant repair pathway (∼80% efficiency), whereas high-fidelity repair was inefficient (6%-8%).
- Post-gene editing analysis revealed that tumor cell death was induced via apoptosis.
Conclusions:
- Systemic in vivo CRISPR/Cas9 gene editing is a highly effective strategy for eliminating established cervical tumors, achieving complete survival.
- The choice of Cas9 variant and understanding DNA repair mechanisms are crucial for optimizing CRISPR-based cancer therapies.
- This study demonstrates the potential of CRISPR/Cas9 delivered via liposomes as a viable therapeutic approach for cancer, even with large gene editing payloads.
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