- Gene editing using CRISPR-Cas9 is a powerful tool to reduce Kaposi's sarcoma-associated herpesvirus (KSHV) latency in infected epithelial and endothelial cell lines (Vero219 type).
- This in-vitro study uses for the first time a KSHV latency-associated nuclear antigen (LANA) targeted CRISPR-Cas9 and adenoviral delivery system to disrupt KSHV latency.
- KSHV latency is a major challenge to clearing infection and preventing Kaposi’s sarcoma (KS) development.
- Given the safety record of adenovirus as vaccine or delivery vectors, this approach represents a viable strategy against other tumorigenic viruses in immunodeficient patients.
CRISPR-Cas9 gene editing is effective in reducing Kaposi's sarcoma-associated herpesvirus (KSHV) latency in infected epithelial and endothelial cell lines and could potentially help millions of infected individuals worldwide at risk of developing Kaposi's sarcoma (KS).
The replication cycle of KSHV involves both a lytic and a latent phase. During latency, a reduced repertoire of viral genes involved in immune evasion and maintenance of the viral episome are expressed and enable KSHV to establish a life-long infection that is correlated with KS development.
The latency-associated nuclear antigen (LANA) encoded by ORF73 plays a critical role in the maintenance and replication of the viral episome during mitosis and interacts with tumour suppressor genes such as p53 and pRb.
CRISPR-Cas9 has been tested against several latency-prone viruses such as Herpes Simplex virus, Human Papillomavirus, Epstein-Barr virus and even HIV-1.
Among KSHV gene products, LANA is an ideal target for CRISPR-Cas9 editing. Researchers designed a gRNA specifically targeting the N-terminus of LANA gene. This region was selected since any mutation or deletion can truncate or lead to frame-shift of ORF73.
In this study, the researchers used Vero 219, a kidney epithelial line from an African green monkey, as the first in vitro model for efficacy testing. Vero219 cells are stably infected with KSHV and maintain the KSHV episome in a latent state under puromycin selection.
As expected, after a replication-incompetent adenovirus type 5 delivering LANA-specific Cas9 system, into various KSHV latent target cells, was applied to the sample, overall KSHV episome burden in the cells decreased with time due to the lack of puromycin selection for the viral genome.
The use of an adenovirus vector might confer potential in-vivo applications of LANA-specific Cas9 against KSHV infection and Kaposi’s sarcoma.