Production of viral particles was compared between cells
treated with sgRNA/cas9 and cells treated with cas9 alone. The researchers
found that, as expected, viral reproduction was initially severely impaired in
cells infected with the sgRNA/cas9 gene probe. Overall, peak viral production
levels were 83% lower in cells treated with T4, by 95% in those treated with
T10, and by about 98% in the cells treated with LTR-B.
Viral production started about five days after infection in
control cells but was delayed by about four days in cells treated with
LTR-B and by about ten days in cells treated with T4 or T10. There was,
however, significant viral production in the end. In the LTR-B cells, viral
levels at the peak pf production – which was also delayed by four days – was
still 55% lower than in control cells. But in T10-treated cells it was exactly the
same as in control cells – though delayed by eight days – and was actually
about 20% higher in T4 cells. This suggests that viral
resistance happens rapidly.
In samples of virus, 74% of T4, 70% of T10 and 72% of LTR-B
viruses had had their DNA altered in the way expected by the three different
genetic probes. In the other 16%, 20% and 18%, there were mutations of some
sort, some of which conferred resistance.
The researchers performed the experiment again on cells
infected with HIV viruses taken from the peak of viral production in the
previous experiment, which were all expected to be resistant. The resistant
T4-treated viruses again produced 20% more virus than control cells and in
the case of T10-treated cells, they produced the same amount of virus as
control cells, but actually reached the peak of viral production six days
earlier. This suggests that HIV that had become resistant to T4 or T10 was at
least as reproductively fit as control virus, or fitter.
In the case of the LTR-B treated with resistant virus, the
peak of viral production was also reached about six days earlier than control
virus but viral production levels remained about 30% lower, suggesting that
LTR-B resistant virus might be paying a small reproductive price for its
In T4-resistant viruses, there was a predominant
single-point mutation (a mutation with just one genetic ‘letter’ changed). This
represented 81% of resistant viruses, and another single-point mutation represented 13% of
them. In T10-resistant
virus, while one single-point mutation represented 38% of resistant viruses,
the other resistant viruses had in general more complex 3- or 4-point changes.
The resistance in the LTR-B resistant viruses was more unusual. T4 and T10 remove only small parts of the viral genome; if it is then
repaired inaccurately, this can create resistant strains. In this case, it is the human NHEJ
cellular machinery, that rejoins the frayed DNA inaccurately, that is the core
cause of resistance. But LTR-B should remove the entire viral genome, so the
rejoined viral DNA should not have the chance to even start producing mutated
viruses. Where was the resistance coming from?
The researchers found that a small proportion of the T4 and
T10-resistant viruses, but all the LTR-B resistant viruses, did not have
substitutions of one genetic base for another like most resistant viruses, but
had whole sections either removed or inserted into the genetic code – so-called
What this suggested was that it was the sgRNA/cas9 itself
that was causing the resistance in these cases. The cas9 was deranging the DNA
at the cleavage site in such a way that it was producing resistant viruses
In this case, resistance was not being
caused by viral turnover in the presence of low levels of drug, that exert a
selective evolutionary pressure – it was being caused directly by the drug itself. In short,
sgRNA/cas9 was acting as a mutagen, a direct driver of viral mutation.
Some unconventional antiviral drugs work by being mutagens,
such as ribavirin, but in this case they work on the viral genome. siRNAs can
also introduce mutations directly into cytoplasmic RNA. But this is the first time a proposed HIV treatment has been
seen directly contributing to the production of resistant mutations within the
proviral DNA at the heart of cells.
While the production of resistance would be brought down to
a minimum by the use of multiple sgRNAs, as the researchers suggest, this study shows that unexpected setbacks may lie in wait for researchers
on the way to a cure for HIV and that the novel gene-editing and
other techniques involved could pose risks of their own.