Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase

I'm JohE Hvar and I'm heading to Department of Cell Biology and Biology, the HE Pet Institute in Hamburg, Germany. The HE PET Institute is basic research institute focusing exclusively on human pe, pathogenic viruses and yeah, and connected issues, I would say. So our department is primarily interested in do investigating the regulation of HIV gene expression, and on the interaction in the particularly intracellular interaction of HIV with components of the host cell.

On, on basis of this data, we also try to develop experi new and experimental therapies for HIV Disease. Problem With HIV is, HIV is a retrovirus or lentivirus and like every retrovirus, HIV integrates upon infection of the host cell, the viral genome into the host cell genome. So far, one cannot get rid of the integrated provirus anymore.

That means with, in terms of therapy that we are talking about a chronic disease with a lifelong therapy and basically with all the problems connected to lifelong therapies like side effects of the drugs, occurrence of resistance and so on. Well, for many years in, in, in basic research, we know so-called recombinase, naturally occurring in recombinations like in yeast or in bacteria, for example. And these recombinations are recognized specific DNA sequences and shuffle genes and rearranged the genome and so on.

And particularly one recombinase called decre recombination is used in basic research and the situ in basic research. And CRE is basically recognizing its specific sequence. And if you have a sequence located between two recognition sites, CRE is able to excise the intervening sequence basically.

So the idea already since 10 or 15 years is from the medicine to eradicate the virus and on the other side, to use like recombinase like grief, for example, to excise the profile. DNA, this is should be in theory possible because upon infection, HIV reverse transcribes, its RA genome into DNA and, and due to this reverse transcription process sequence called the long terminal repeats are generated and they mark basically the five bris or the beginning and the free brime border, the end of the integrated profile D.So in theory, if one could come up with a three recombination, that re recognizes specifically in a sequence in the LTR, so it would, the disagree would basically attack the five prime end and the free BRI end of the integrated profile, DNA excise the DNA, and basically eradicate the virus From disinfected cell. Now, the problem with HIV Is that we have different isolates maybe also in, in, in a, in a single person, a single patient, however, particularly as we know so far, the sequence is in the beginning and at the end of the virus of the viral genome.

So in other words, the LTRs are more conserved than, for example, the HIV genes. So I think there's a good chance that we can get away with just a few numbers of recombinance that basically recognize the vast maturity of all HIV isolates. And if not, basically then we have to generate a new one by molecular evolution, which can be in the future semi autotomized.

And it will, the process will be much faster than doing it In the lab. In our Colleague, Dr.Frank from the Max Splunk Institute came into end of the picture. Basically he is an expert on recombinase and everything.

Basically what he used is what we call a molecular evolution method. But the method is based on PCR polymer chain reaction technology. And he takes the gene of the naturally occurring recombinase degree combs and sets up a PCR or multiple PCR reactions at conditions that introduce in a random fashion mutations.

And the trick is now to select the new versions of the recombinase that have acquired a new capacity or new activity, namely recognizing HIV specific sequences. And he's doing this in reiterate cycles. He, he, he took about 120 or over 120 cycles to doing this molecular, this type of molecular evolution method.

When he finally came up with this specific, now called three for LTR specific recombinance with this new recombinance that is now specifically recognizing HIV sequences. So the initial Study here was the, the, the experiments with the recombinants were performed here at the HE PET Institute in Hamburg. And what we did is especially we delivered this recombinance with vectors, gene vectors into human cell culture that had been infected with a more or less a clinical isolate of H HIV V one.

And over time, over six to eight weeks, we had been able to demonstrate that. Now the recombinant is exciting quantitatively, they integrated HIV profile DA, basically the whole culture is cured by getting rid of HIV. There are certainly numerous problems with this technology.

First of all, we have to make sure that the expression of recombinase is not causing toxic side effects. We don't, we don't expect this because for naturally occurring recombinations like gre, there are so-called transgenic animals existing. They express this GRE recombination and okay, so there are some problems, but not too significant.

I may say so at this point. So there is hope that there are no toxic side effects, but you never know when you have to do the experiments. And there are also problems that me, we maybe will not recognize every clinical isolate with it so that we get, can get rid of, of the dominant isolate, HIV isolate in a patient.

But we will select from minor, minor isolate over time. This is, we don't know, we have to do these experiments. Then there's a big problem with the gene delivery, with the vector technology.

We have to use so-called lentiviral vectors or also retroviral vectors to deliver the gene or the recombinase gene. So are there side effects? There are, are these vectors causing toxic effects?

These are all the questions we have. Also, we hope to improve the activity of the combs. They, recombination is acting just faster than we see it over now, couple weeks, the activity.

So these are all open questions we have to develop over the next years. And This is now certainly the problem because recombinase are not orally available. So there will be no pill, recombinase pill just well, and you get rid of the virus.

So, but speculating about future therapy, we have to think about gene therapy approaches. So maybe one can conceive that we isolate so-called hematopoietic stem cells or CD 34 positive cells from the blood of patients of HIV infected patients. And then in the laboratory we introduce the gene for the combs into this patient cells.

Finally, we reintroduce, we reinject the patient, we rein, infuse the patient cells and now they genetically modified patient cells into the same patient's back. So what should happen is that the immune system or the hematopoietic system of the patient is over time at least partially reconstituting with cells that upon infection with HIV, they can get rid of the virus because they now express a recombinase. So effect would be that a viral load will drop in the patient but will improve the quality of life, certainly.

And we can also hope that the immune system is re reconstituting in this patients at least partially. So now the, the, the function, the, the immune cells that are fully functional, they will be able hopefully to attack the remaining lengthly infected cells in these patients. Whether or not we can really completely, totally eradicate HIV from a patient that we, we cannot tell at this time.