CRISPR-Cas gene editing eliminates HIV in lab

New research presented ahead of this year’s European Congress of Clinical Microbiology and Infectious Diseases (ECCMID 2024), to be held in Barcelona from 27–30 April, shows how the latest CRISPR-Cas gene editing technology can be used to eliminate all traces of HIV from infected cells in the laboratory. The studies, conducted at Amsterdam University Medical Center (Amsterdam UMC), present a significant breakthrough in the search for a cure for HIV.

CRISPR-Cas gene editing technology is a groundbreaking technique that allows for precise alterations to the genomes of living organisms, enabling scientists to accurately target and modify specific segments of an organism’s DNA. Functioning like molecular ‘scissors’ with the guidance of guide RNA (gRNA), CRISPR-Cas can cut the DNA at designated spots. This action facilitates either the deletion of unwanted genes or the introduction of new genetic material into an organism’s cells, paving the way for advanced therapies.

One of the significant challenges in HIV treatment is the virus’s ability to integrate its genome into the host’s DNA, making it extremely difficult to eliminate. Numerous potent antiviral drugs are currently in use for treating HIV infection — but despite their efficacy, lifelong antiviral therapy is essential, as HIV can rebound from established reservoirs when treatment is halted. CRISPR-Cas genome editing thus provides a new means to target HIV DNA.

“Our aim is to develop a robust and safe combinatorial CRISPR-Cas regimen, striving for an inclusive ‘HIV cure for all’ that can inactivate diverse HIV strains across various cellular contexts,” the Amsterdam team said. With HIV able to infect different types of cells and tissues in the body, each with its own unique environment and characteristics, the researchers are searching for a way to target HIV in all of these situations.

The scientists deployed CRISPR-Cas molecular scissors and two gRNAs against ‘conserved’ HIV sequences, meaning they focused on parts of the virus genome that stay the same across all known HIV strains, and achieved cure of HIV-infected T cells. By focusing on these conserved sections, the approach aims to provide a broad-spectrum therapy capable of combating multiple HIV variants effectively.

However, the team found that the size of the vehicle or ‘vector’ used to transport the cassette encoding the therapeutic CRISPR-Cas reagents into the cells presented logistical challenges, as it was too large. Thus, they trialled various techniques to reduce the size of the cassette — and therefore the vector system itself. In simpler terms, they’re attempting to pack oversized luggage into a compact car for a journey to the infected cell, leading them to find ways to downsize the ‘luggage’ (cassette) for easier transport. Another issue they wanted to overcome was reaching the HIV reservoir cells that ‘rebound’ when HIV antiretroviral treatment is stopped.

The researchers further evaluated various CRISPR-Cas systems from different bacteria to determine their effectiveness and safety in treating CD4+ T cells infected with HIV. They shared results from two systems, saCas9 and cjCas. SaCas9 showed outstanding antiviral performance, managing to completely inactivate HIV with a single guide RNA (gRNA) and excise (cut out) the viral DNA with two gRNAs. The strategy of minimising the vector size was successful, enhancing its delivery to HIV-infected cells. Moreover, they were able to target ‘hidden’ HIV reservoir cells by focusing on specific proteins found on the surfaces of these cells (CD4+ and CD32a+).

“We have developed an efficient combinatorial CRISPR-attack on the HIV virus in various cells and the locations where it can be hidden in reservoirs, and demonstrated that therapeutics can be specifically delivered to the cells of interest,” the scientists stated. They said their findings represent a pivotal advancement towards designing a cure strategy for HIV, while acknowledging that their work at this stage is only a proof of concept.

“Our next steps involve optimising the delivery route to target the majority of the HIV reservoir cells,” the researchers said. “We will combine the CRISPR therapeutics and receptor-targeting reagents and move to preclinical models to study in detail the efficacy and safety aspects of a combined cure strategy. This will be instrumental to achieving preferential CRISPR-Cas delivery to the reservoir cells and avoiding delivery into non-reservoir cells. This strategy is to make this system as safe as possible for future clinical applications.

“We hope to achieve the right balance between efficacy and safety of this cure strategy. Only then can we consider clinical trials of ‘cure’ in humans to disable the HIV reservoir. While these preliminary findings are very encouraging, it is premature to declare that there is a functional HIV cure on the horizon.”

Image credit: iStock.com/Artem_Egorov