"During acute infection, HIV actively replicates," explained co-senior study investigator Kamel Khalili, Ph.D., professor and chair of the department of neuroscience at LKSOM. "With EcoHIV mice, we were able to investigate the ability of the CRISPR/Cas9 strategy to block viral replication and potentially prevent systemic infection." The excision efficiency of their strategy reached 96% in EcoHIV mice, providing the first evidence for HIV-1 eradication by prophylactic treatment with a CRISPR/Cas9 system.
In the third animal model, a latent HIV-1 infection was recapitulated in humanized mice engrafted with human immune cells, including T cells, followed by HIV-1 infection. "These animals carry latent HIV in the genomes of human T cells, where the virus can escape detection, Dr. Hu explained. Amazingly, after a single treatment with CRISPR/Cas9, viral fragments were successfully excised from latently infected human cells embedded in mouse tissues and organs.
In all three animal models, the researchers employed a recombinant adeno-associated viral (rAAV) vector delivery system based on a subtype known as AAV-DJ/8. "The AAV-DJ/8 subtype combines multiple serotypes, giving us a broader range of cell targets for the delivery of our CRISPR/Cas9 system," remarked Dr. Hu. Additionally, the researchers re-engineered their previous gene-editing apparatus to now carry a set of four guide RNAs, all designed to efficiently excise integrated HIV-1 DNA from the host cell genome and avoid potential HIV-1 mutational escape.
To determine the success of the strategy, the team measured levels of HIV-1 RNA and used a novel and cleverly designed live bioluminescence imaging system. "The imaging system, developed by Dr. Won-Bin Young while at the University of Pittsburgh, pinpoints the spatial and temporal location of HIV-1-infected cells in the body, allowing us to observe HIV-1 replication in real time and to essentially see HIV-1 reservoirs in latently infected cells and tissues," stated Dr. Khalili.
The researchers were excited by their findings and are optimistic about their next steps. The next stage would be to repeat the study in primates, a more suitable animal model where HIV infection induces disease, in order to further demonstrate the elimination of HIV-1 DNA in latently infected T cells and other sanctuary sites for HIV-1, including brain cells," Dr. Khalili concluded. "Our eventual goal is a clinical trial in human patients."
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CRISPR Eliminates HIV in Live Animals - Genetic Engineering & Biotechnology News
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