The HI virus is an RNA virus belonging to the group of the lentiviruses within the retroviruses family. After penetration into the blood stream, it binds to CD4 receptors of various blood cells such as CD4+-T-cells or -macrophages. Subsequently, the virus fuses with the cell and sets the capsid free in the cytosol. In the next step, the viral RNA is released from the capsid, using reverse transcriptase it gets rewritten in a circular DNA molecule and is ultimately able to be incorporated into the genome of the host cell. In this phase, the HI-virus is also known as provirus. Well protected from attack by antiviral agents it can now survive for several years until the next phase of infection, the production of viral components, starts.
Designer recombinase is efficient and precise
A team led by Frank Buchholz from TU Dresden and Joachim Hauber at the Heinrich Pette-Institut In Hamburg [Paywall] recently had the idea to raise an enzyme which tracks and removes the provirus in the genome of the host cell. However, in order to distinguish viral DNA from the DNA of the host cell, this “gene cutter” requires a recognition sequence, a specific sequence of nucleotides in the DNA molecule of the virus. For this, the team examined the so-called long terminal repeats (LTR)which occur at the two ends of the provirus. These are made up of a DNA sequence of repeating base pairs which is several hundred nucleotides in length. Within the LTR, the researchers discovered a 34 base pair sequence which occurs almost unchanged in more than 80 percent of all HI-viruses, and named it loxBTR.
Starting from the loxBTR sequence, the enzyme structure was adapted during the next step gradually by means of a directed evolution, and thus a recombinase with the name broad-range recombinase 1 – abbreviated as Brec1 – was derived. For its development, the scientists needed all of 145 steps, whereby each step demanded two to three days – this meant a total time of about one to two years. In subsequent tests on E. coli the designed recombinase was tested for its efficiency and specificity. For example Brec1 did not recognise the 34 base pair DNA sequence called loxP, although it demonstrated a 32 percent sequence homology to loxBTR. Viruses are repeatedly found worldwide in which mutations are detected in the recognition sequence. In order to make sure that these would be recognised by the “gene cutter”, the scientists created four loxBTR, each possessing one real existent point mutation. The result: Brec1 also recognised these regions.
From bacteria to mammalian cell
Similar results were obtained by the scientists in experiments with mammalian cells (HeLa cells) in which the HIV-1 genome was stably integrated. Here too Brec1 efficiently and specifically recognised the loxBTR target. Next Brec1 was tested on human PM1-T-lymphocytes which had been infected with replication-competent viruses. This T-cell line is routinely used in HIV-1 research. Whereas in the control group the viral load rose, the Brec1 positive cells unleashed no viral particles.
The team of scientists in further investigations used CD4+T cells isolated from the blood of HIV patients. After transduction using a lentiviral Brec1-positive vector, the cells were cultured for 20 days. While in the control group the virus load increased and at the same time the number of treated cells decreased, the amount of remaining Brec1-positive CD4+cells remained constant and the number of released viral particles fell. According to the authors, this was an indication that the provirus had removed the recombinase from the genome. A subsequent DNA sequencing (deep sequencing) confirmed the assumption. 20 days after transduction the scientists were not able to detect any proviral DNA in the sequence of the DNA molecule.
From the mammalian cell to the mouse
Then the research team administered the Brec1-positive patient material to mice. After 18 to 21 days, the viral load in the plasma of the animals had fallen below the detection limit (<20 HIV-1 RNA copies /mL). However, since T cells have a limited lifespan, the research team in the next step introduced Brec1 into human blood stem cells and then injected these into newborn mice. Since all blood cells which were formed from the Brec1-positive stem cells where equipped with the recombinase, the Brec1 mice, in contrast to the control group, barely demonstrated any HIV load.
No adverse effects
Next Brec1 was studied for its cytotoxic, cytopathic and genotoxic effects. For this purpose the recombinase among other things was transferred to human CD4+ T-lymphocytes and were subsequently examined for their gene expression, apoptosis and immune activity and function. There were no significant differences here between the control cells and Brec1 cells. As evidence of genotoxicity, the scientists determined the six genomes whose DNA sequence showed most similarity to that of loxBTR. These were tested in E. coli bacteria.
Looking at the fact that no recombination took place with any of these nucleotide sections, the authors concluded that the human genome obviously contains no loxBTR-similar DNA sequences recognised by Brec1. Finally, the team of scientists genetically altered the mice such that Brec1 would be expressed via a constitutive promoter. These animals remained healthy over a period of 18 months.
Other genes editing methods
The method which cuts viral genetic material out of the genome of the host cell is not entirely new. Already in 2013 scientists led by Frank Buchholz and Joachim Hauber demonstrated the antiviral effect of Tre-recombinase in humanised mouse models. The enzyme had been produced by the researchers at an earlier point. However, the target sequence in the various HIV-1 classes is apparently not sufficiently well preserved, so that less than one percent of HIV infected patients would benefit from the therapy.
Other gene editing approaches include the CRISPR-Cas9 process, zinc finger nuclease and the TALEN process. CRISPR are sections of repetitive DNA in the genome of, for example, bacteria. They belong to a mechanism, using which bacteria makes foreign DNA harmless. The CRISPR / Cas9 system [Paywall] generates double-strand breaks in the target DNA, through which DNA sequences are removed or others may be added. Zinc finger nucleases are produced artificially. They contain a certain protein domain with a coordinatively bonded zinc ion. This zinc finger domain binds to a specific DNA sequence which gets cut by nucleases. Subsequently, foreign DNA can be inserted. The transcription activator-like effector nucleases (TALEN) are fusion proteins composed of a DNA-cutting endonuclease and a DNA-binding domain.
The use of a recombinase has, according to the authors in their study, in comparison to other gene-editing methods the advantage that there are no double-strand breaks, which would activate the repair mechanisms. This can have unpredictable consequences in the form of rearrangements of the DNA sequence. Recombinases in contrast repair the DNA after cutting it themselves. The disadvantage of Brec1 compared to nucleases is, however, that their production is very time intensive.
The era of genome surgery
With 37 million infected individuals and more than 2 million new infections per year, HIV continues to be a major challenge for world health. In Germany, about 83,000 people are currently living with the HI-virus. Over 72 percent of those affected are infected with the group M, subtype A, B or C forms of HIV-1. 90 percent of these HIV viruses show an exact matching loxBTR sequence. However, since loxBTR also occurs in most other HI-virus (possibly slightly modified), the authors assume that more than 28 million individuals would come into consideration for this therapy.
“This trend [generation of molecular scalpels] would not only benefit HIV patients, but also many other patients with genetically-defined diseases. We are about to usher in an era of genome surgery”, Prof. Frank Buchholz predicts euphorically. However, scientists in their study give food for thought that before its use in humans the potential risks of the gene-editing technologies should be carefully weighed against the benefits. Moreover, it’s quite possible that one single method may not be able to cure HIV in all people.
The authors suggest that future strategies of HIV treatment will combine several antiviral approaches. Examples here would include drugs that activate latent viruses, immuno-modulators and gene therapies. But before things come that far, a few more years will yet come to pass. The next step would be trials involving HIV patients. Whether the method proves itself will have to be shown there. Some of the authors have thus far already applied for a Brec1 patent and would – should the method be successful – not end up being left empty handed.
Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity
Karpinski et al.; Nature Biotechnology, doi: 10.1038/nbt.3467; 2016