Gene editing has been used to eliminate all traces of HIV from infected immune cells in a laboratory, with the hope that it could one day be used to target the HIV DNA which acts as a reservoir for the virus in people with the condition.
The gene editing system uses a small piece of genetic material, called guide RNA (gRNA) to direct enzymes, called CRISPR-Cas, to the HIV DNA hiding in cells. These enzymes then act like molecular scissors allowing scientists to cut the DNA very precisely.
One of the big challenges in HIV treatment is the virus’s ability to integrate its DNA into the host’s DNA, making it extremely difficult to eliminate. The authors say they have developed an efficient attack on the HIV virus in various cells and the locations where it can be hidden in reservoirs.
They say these findings represent a “pivotal advancement towards designing a cure strategy,” although they stress much more work is needed.
The endonuclease encoded by Streptococcus pyogenes Cas9 (SpCas9) is the most widely used CRISPR endonuclease for genome engineering, but it has an important limitation because of its relatively large size (∼4.1 kb coding sequence) that may hamper the delivery by means of viral vector systems.
This work aimed to assess the impact of the CRISPR-Cas transgene size on the production of LVs and their ability to transduce cells. We observed that the LV transduction efficiency measured on the SupT1 T cell line dropped dramatically for RNA transcripts approaching 8 kb, e.g. a further increase of 1kb reduced the transduction efficiency by more than 5-fold. As the number of produced virus particles was not affected, this means that the use of larger transgenes may cause the production of more empty virion particles.
Research from the Netherlands shows how the latest CRISPR-Cas gene editing technology can be used to eliminate all traces of the HIV virus from infected cells in the laboratory, raising hopes of a cure.
The studies, led by Dr Elena Herrera-Carrillo and her team at Amsterdam UMC, presents a significant breakthrough in the search for an HIV cure.
CRISPR-Cas gene editing technology is a groundbreaking method in molecular biology that allows for precise alterations to the genomes of living organisms.
This technique, which brought its inventors, Jennifer Doudna and Emmanuelle Charpentier, the Nobel Prize in Chemistry in 2020, enables scientists to accurately target and modify specific segments of an organism’s DNA (genetic code). 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.
The LV can in principle transfer any chosen CRISPR-Cas system into any target cell type. However, LVs have a limited packaging capacity for the transgene RNA transcript and previous studies revealed that the LV titer decreases with increasing transgene size.
Previous research has demonstrated that there is a reduction in transduction titer of approximately 3-fold per kilobases (kb) increase in RNA cargo size for inserts over 5 kb, whereas the titer dropped approximately 100-fold for inserts between 6 and 12.5 kb.
Similarly, the length of the viral RNA is a critical factor that affects the efficiency of LV-mediated gene transfer. Whereas the LV transduction efficiency on human and mouse stem cells dropped dramatically for viral RNAs that approached 6 kb, a modest reduction of their size of 0.6 kb rescued the transduction efficiency by more than threefold.
The LV titer also drops with increasing genome size and in general, genome sizes close to or exceeding that of the natural HIV template (9.8 kb) result in decreased packaging capacity and consequently a low transduction efficiency.
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. Despite their efficacy, lifelong antiviral therapy is essential, as HIV can rebound from established reservoirs when treatment is halted. The authors explain that the CRISPR-Cas genome editing tool provides a new means to target HIV DNA.
The researchers acknowledged that HIV could infect different types of cells and tissues in the body, each with its own unique environment and characteristics. They are searching for a way to target HIV in all these situations.
They emphasised that their work represents proof of concept and will not become a cure for HIV tomorrow.