‘Molecular scissors’ successfully remove HIV genes from all tissues in infected monkeys

The top story from the Conference on Retroviruses and Opportunistic Infections (CROI 2019) in Seattle this week has been a likely second HIV cure. However, the cure involved an expensive and risky therapy – a bone-marrow transplant – that would never be broadly applicable.

Just as significant in the long term may be a study reported in the same session that used much more benign technology to achieve what may be a cure in monkeys.

A team of researchers at Temple University in Philadelphia, USA, has removed the retroviral genes from the cells of monkeys infected with SIV, the monkey analogue of HIV. The researchers found that the gene-snipping enzyme they used, contained within the shell of a common cold-type virus so that it could simulate an infection and enter cells, successfully removed the SIV genes from a majority – and possibly all – cells in all the monkeys’ organs where levels were measured, including hard-to-access ones such as the brain.

As the monkeys in this experiment were euthanised after the intervention so that all tissues could be biopsied, we cannot tell if what the researchers achieved amounts to an SIV cure. But the comprehensive nature of the results is impressive, and any attempt to culture SIV from the immune cells in the blood of the treated monkeys, in what is called a viral outgrowth assay, produced no virus. The next step will be to give the same therapy to SIV-positive monkeys on antiretroviral therapy (ART) and then interrupt treatment to find out if the SIV reappears.

The experiment

The gene-splicing technology was originally applied to SIV in studies reported in 2016 by the same research team. The active components in the viral vector (shell) are twofold. Firstly, the vector contains lengths of guide RNA (gRNA), whose job is to find the SIV sequences within the monkeys’ genome – it can detect and link to specific sequences in SIV that are highly conserved, i.e. don’t change much from one variety of SIV to another. Secondly, it also contains an enzyme called CRISPR/Cas9 which ‘snips out’ the SIV genetic material and rejoins the cut ends, replacing the long SIV sequence with a short genetic sequence that serves as a signal that the gene excision has occurred.

Further studies in 2016 found that the SIV DNA soon developed mutations that led to resistance – it found a way to become ‘invisible’ to the gRNA. We commented at the time that the gene-degrading Cas9 would need to be attached to a variety of different gRNAs to avoid this, and this is what was done in the new study. The gRNAs enabled the excision of three different sections of SIV, allowing for a very low probability of the development of escape mutants. Two of the gene edits involved removing almost the entire SIV genome; the third involved removing a shorter but still significant length of about 10% of the viral genome and its replacement with a sequence designed to be a ‘flag’ indicating that successful excision had occurred.

Just three monkeys were used in the experiment. All were infected with SIV and then twelve weeks after infection were put on ART. Eight weeks later, immune cells were taken from their blood and the gRNA/Cas9 therapy was performed in lab-dish experiments. These produced positive results, with ‘excision product’ (the short length of ‘flag’ DNA that replaced the excised length) being readily detectable as well as other genetic markers that indicated successful excision.

Four weeks later the gRNA/Cas9 therapy was given to two out of the three monkeys, with the third receiving no therapy and serving as a control. The therapy was given as an infusion over 100 minutes and altogether about 100 trillion of the adenovirus particles containing the therapy were transfused.

Three weeks later, the monkeys who received the therapy were euthanised and many different body tissues were sampled to see if the SIV DNA had been excised from infected cells. Lymphocytes taken from their blood were also cultured in the lab dish to see if virus could be cultured from them. The same viral outgrowth assay and tissue sampling was also performed on the control monkey.

Results

Cells from the control monkey readily produced virus in the lab dish but none was produced by cells from the treated monkeys.

Tissue sampling found the DNA ‘flag’ product in cells taken from every organ sampled. This excision product was found in 42% of individual tissue samples from one monkey and 76% of samples from the other. The density of excision product ranged from one copy per 100 to 1000 cells in the brain and spiral cord samples, to 20 to 40 copies per cell for samples taken from the spleen and liver.

The researchers also looked for indicators of actual full-length genome removal. They are still in the process of doing this, but found the presence of excision products indicating full genome removal in all samples taken from the spleen and lungs of the two monkeys, and the absence of any viral DNA from the spleen of one and the left lung of another, indicating complete viral removal. They are still in the process of sampling lymph nodes but found the absence of any viral DNA in the bronchial lymph nodes of one monkey and the inguinal lymph nodes of the other.

Viral DNA was still detected from some other tissues, but given the failure of the cells to produce virus, these could be lengths of ‘dead’, non-productive DNA. However Dr Tricia Burdo, presenting, emphasised that standard practice was to repeat the viral outgrowth assay using immune stimulants to encourage any remaining cells to start producing virus and this had not yet been done.

One of the concerns around this kind of therapy is that it could damage useful genes, or switch on harmful ones, that are located near to the site of insertion of the viral DNA – so called ‘off-target’ effects. So far there is no evidence of any such accidental gene alterations.

At a press conference, Burdo was asked if the two monkeys could be considered cured. She answered that it would take another experiment treating multiple monkeys with the gRNA/Cas9 therapy and then seeing if their SIV re-appeared after they were taken off ART before they could start talking about a cure. But the fact that cure was being talked about in the context of a highly experimental therapy that was three years ago of debatable efficacy is a sign of how far we have come.