The scientists in this study used a genetic ‘missile’ that
combined two different modules. The first is a probe consisting of two pieces of
so-called ‘guide RNA’ (gRNA). Their job is to sensitively detect and home in on
the two ends of the HIV genome, the so-called long terminal repeats (LTRs) that act
as the viral genome’s ‘frame’.
The second is a nuclease, an enzyme called Cas9 that
removes the viral genetic material and rejoins the two cut ends of the human
DNA together. In the process, it adds some ‘filler’ DNA of its own.
Cas9 is a development of CRISPR, an enzyme already used in
another HIV cure approach, to extract and ‘re-engineer’ CD4 cells outside the
body to make them immune to HIV and then re-introduce them; continued research into
this strategy is also underway, though so far it has proved difficult to turn the
HIV-resistant cells into the majority of immune cells once they are re-introduced.
The present team used a lentiviral vector – the shell of a
virus of the same family as HIV, containing the gRNA/Cas9 as a ring or plasmid of
nucleic acid – to infect T-lymphocyte cells (of which CD4 cells are a subset) in
the test tube.
In the first set of experiments they used 2D10 cells,
laboratory-created immune cells containing a specially engineered ‘fake’ HIV
genetic sequence consisting of the HIV genome with most of its replication genes
removed and a fluorescence gene inserted. These cells therefore, instead of HIV viral particles,
spit out green fluorescent protein (GFP) when
stimulated, which shows up in microphotographs.
The 2D10 cells were infected with the vector and then
stimulated with an HDAC inhibitor to see if they expressed GFP. Cells infected
only with the Cas9 nuclease produced one unit of GFP unstimulated but 94 units
of GFP when stimulated, while ones infected with the full gRNA/Cas9 probe produced
less than one unit of GFP whether stimulated or not.
One of the issues with HIV infection is that the virus
inserts its genome at random into the human genome, wherever it will fit,
though some sites are more likely than others. Genetic analysis of cells used in the study found a complete HIV genome, consisting of 6130 base pairs (units of
the DNA chain) on chromosome 1 of the cell's 23 chromosomes, and a
near-complete genome of 5467 base pairs in chromosome 16. In the
gRNA/Cas9-treated cells, these had been replaced by smaller DNA ‘fillers’ consisting
of 909 and 759 base pairs respectively.
These ‘fillers’ were not just inactive
DNA: they acted as genes and actively expressed the gRNA and Cas9 nucleic acid
The researchers then investigated whether the infection of
the cells by the gRNA/Cas9 vector had any adverse effects on other genes in chromosomes
1 and 16 and on cellular health in general. They found no indication of
significant mutations in other genes, or in the viability or lifespan of cells.
They next looked at whether it was possible to infect
T-cells with HIV if they had already been infected with the gRNA/Cas9 vector. They infected HIV-negative T-cells with the vector,
and selected four clonal lines of T-cells. One line produced gRNA but not Cas9, one Cas9
but not gRNA, and two both, one of which expressed more Cas9 than the other.
When these cells
were cultivated with HIV, the cells expressing either gRNA or Cas9 could be
infected, with 20-50% infected in the lab dish, but cells expressing both were
more resistant to infection, with 3-4% of cells with the lower level of Cas9
infected and only 1% of cells with the higher level.
Two different strains of HIV were in fact used. With one the
reduction in infections in cells expressing both gRNA and Cas9 was 48%; but with
the other strain the reduction was 100% and no HIV replication was seen at all.
The researchers found that the expression of the gRNA and Cas9 products by
cells diminished over time and eventually disappeared, but that as long as they
were integrated within the cell, the cells were protected from infection.
The researchers then looked at the ability of the gRNA/Cas9 vector
to suppress replication in cells taken from people with HIV. They took T-cells
from four patients who were all on antiretroviral therapy (ART) but had
different responses: case one had a low viral load and high CD4 count but a low
CD4 percentage (11%); cases two and three had undetectable viral loads and high
CD4 counts, and case four a low but detectable viral load and a low CD4 count (53
Their CD4 cells were cultured in the test tube without ART
and with the gRNA/Cas9 vector. Four days after the vector was introduced, they tested lab dish fluid
and individual cells for HIV viral loads. Cells cultured with the gRNA/Cas9 vector had
lower levels of the p24 HIV protein (71, 62, 39 and 54% less than control cells from the four patients respectively); cases
one and two also had levels of the HIV gag protein measured too and this decreased
by 92% and 56% respectively.