HIV rapidly develops resistance to the "gene-editing healing technique"

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Resistance may be the Achilles heel of the genetic engineering approach

Develop resistance to therapy up to 8 days

One of the "techniques literally drives the virus to resistance counts itself

In a setback for an innovative approach to curing HIV, researchers involved in a technique that uses enzymes to remove viral genes from the DNA of infected cells have found that HIV adapts easily to this action and quickly develops resistance to the leading guide the molecules to the target that carry the correct part of a human cellular DNA sequence. Resistant viruses may also develop a more aggressive strain of the virus and replicate even more rapidly than viruses not exposed to gene therapy (although they are still susceptible to conventional antiretroviral (ARV) drugs).

In addition, researchers suggest how gene therapy works may actually promote the development of resistance because it actively works on creating small mutations at the site where the cellular DNA is cut off. Resistance emerges quickly - within 8 days after the first application of therapy becomes active.

This does not mean that the whole approach to gene editing is doomed to failure, but this implies that the degrading enzyme gene needs to be attached to a wide variety of different "probe genes" designed to attach a greater number of different points of the viral DNA hidden within the human DNA in infected cells.

Genetic technology involves the transport of an enzyme-DNA called CRISPR degrading or cas9 in the heart of the human cell nucleus. The enzyme cas9 which was originally found inside bacteria as a natural defense against the virus, is attached to a single strand length "guide" (sgRNA RNA) that guides the cas9 for the specific part of DNA infiltrated by HIV in its process of integrating the newly converted RNA into DNA that needs to be removed to eliminate HIV "control" of the cell thereby terminating in that cell the HIV life cycle.

The concept is not much different from the versatile gene therapy called short interfering RNA (siRNA), which is being investigated for to a number of diseases including chronic hepatitis B (this link leads to a text on another site, in English, which will also be translated). But considering that siRNA targets and degradation of "messenger RNA" and organ molecules that act as virus replication machines within the main part of the cytoplasm of the cell () sgRNA / cas9 Integrated DNA goal, the Master model for manufacturing retroviruses such as HIV inserts into a nucleus of cells the genetic instructions that exist in the cell nucleus and not in the surrounding cytoplasm.

This study

In this study vitro the T-cell researchers infected with three different "genes probes " sgRNA / cas9 designed for different sections of HIV DNA integrated into the cell nucleus.

One, called T4, is attached to the gag / pol of the HIV genome, which includes space for HIV function equivalent to a "DNA copying machine"; a second gene, T10, is attached to the env / rev, which includes the instructions to form the "viral envelope ". In these two cases the sgRNA itself connected to a single specific point of viral DNA is cleaved into two parts, allowing cas9 to degrade the shredded tips of still Viral DNA.

An intracellular molecular repair mechanism called NHEJ (final non-homologous reassemblers) eventually repairs shredded DNA tips, but DNA is more prone to "Errors" and introduce changes in the DNA chain, some of which may confer resistance to HIV.

The third probe gene, called LTR-B, was similar to that used in a study we reported on at the end of March (Also opens on another website in English and we will try to translate it in a timely manner). This cuts the DNA at the two points that are located in or near the sequence called Litros (Long Term Terminal Repetition in free and interpretive translation - accepted coherent suggestions) that represent the endpoints of the genetic material of HIV. Thus it performs a complete removal of all HIV component materials from the infected cell.

One of the implications of LTR-B is, as always, resistance if the cell remains capable of producing HIV, which in theory it could be able to, because there are some remaining parts of the DNA that, although defective, is still capable of producing infectious virus. It must be because HIV DNA may have undergone a mutation into a form that is resistant to "seizure" by sgRNA in the first place.

Results and implications

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 replication was initially severely impaired in cells infected with sgRNA / cas9 probe gene. In general, peak viral production levels were lower 83% in cells treated with T4, by 95% in those treated with T10, and by about 98% in cells treated with LTR-B.

Viral production started about five days after infection in control cells, but was delayed for about four days in LTR-B treated cells and for about ten days in cells treated with T4 or T10. However, there was significant viral production at the end of LTR in B cells, viral levels at peak production of FP - which was also postponed for four days - was still lower 55 in control cells. But in cells treated with T10 was exactly the same in control cells - although delayed by eight days - and actually was about 20% higher in T4 cells. This suggests that viral resistance happens quickly.

In virus samples, 74% of T4, 70% of T10 and 72% of LTR-B viruses had their DNA altered in the manner expected by three different genetic probes. In the other 16%, 20% and 18%, there were mutations of some kind and some of which were attributed to resistance generation.

The researchers performed the experiment again on cells infected with the HIV virus taken from the peak viral production in the previous experiment that was expected at all to become resistant. The resistant viruses treated with T4 again produced 20% more viruses than the control cells and in the case of cells treated with T10, they produced the same amount of virus as cell control, but actually reached the viral production peak six days early. This suggests that the HIV that had become resistant to T4 or T10 was at least how to apply a virus replication control, or a installer.

In the case of LTR-B treated with resistant virus, peak viral production was also reached about six days earlier than the control virus, but viral production levels remained lower 30%, suggesting that LTR-resistant viruses -B may be paying the price of a small resistance to your breeder.

In T4-resistant virus, there was a predominance of a single mutation point (a genetic mutation with only one Letter changed). This represented a quota of 81% of resistant viruses and another single point mutation represented 13% of them. In virus resistant to T10, whereas a single point mutation represented 38% of virus resistant to other resistant viruses had, in general, a more complex mutation of 3 or 4 points of changes.

The resistance of LTR-B resistant viruses was more unusual. T4 and T10 extracted only small parts of the viral genome; if inaccurately repaired, this can create resistant strains. In this case it is the NHEJ cellular machinery, which goes back to and inaccurately "edited" DNA, which is the core of the resistance. But the LTR-B must remove the entire viral genome, so the viral DNA junction should not have the chance to even begin to produce mutant viruses. Where was the resistance? From what or what?

The researchers found that a small proportion of the T4 and T10-resistant viruses, but all LTR-B resistant viruses, did not have substitutions from one genetic base to another, like most resistant viruses, but had entire sections removed or inserted in the genetic code - the so-called "Indels".

This suggests that it was the sgRNA / cas9 itself that was causing resistance in these cases. Cas9 disrupted the DNA at the cleavage site such that it was producing the resistant viruses.

In this case, the resistance was not caused by the business viral transaction in the presence of low levels of drug exerting selective evolutionary pressure - what was being caused was generated directly by the drug itself. In summary, sgRNA / cas9 was acting as a mutagenic agent, a direct driver of the viral mutation toward the strain resistant to sgRNA / cas9 itself. Editor's note. In Portuguese that reminds me of shooting backwards. This type of "shot", which backfires, kills "only" one person, if you escape from control and go out into the streets ...

Some unconventional antiviral drugs generate problems and work because they are mutagenic, such as ribavirin, but in this case they work on the viral genome. siRNAs may also display mutations directly in cytoplasmic RNA. But this is the first time that a proposal for HIV treatment has been seen directly contributing to the production of resistant mutations within the proviral DNA in the heart cells.

While resistance production would be brought down to a minimum by the use of multiple sgRNAs, as the researchers suggest, this study shows that unexpected setbacks may be waiting for researchers on how to cure HIV and that the new gene edition and other techniques involved may pose risks of its own research or life, depending on how it is conducted and in what type of study, in vitro or human, and can generate serious problems.

Editor's note: I am very happy when I feel I can give good news to my readers, and the more I go about researching and translating these texts, which sometimes make me go and study "a little genetics so I can translate them ", Make me increasingly careful and considerate of what I publish and I have not seen, in my sources, that I regard as sensible, no mention of this cure arising in London. I hate to sound like Cassandra announcing the apocalypse, and all I ask for is attention and caution. I would love to be absolutely sure that particular study has been able to find a cure for HIV, but I know that between the application of this possible cure, that it should have an application protocol that should, when little, determine a treatment of X time and a period of "DELTA" time waiting for confirmation of complete remission, elimination of reservoirs (the intestines are an unbelievably large area where HIV can be hidden and if it is hidden there, forever, perhaps this is acceptable as a medical condition leading the person to a life without ART but with a complex agenda of collecting material for exams for the rest of the life and this, even being a cure, has the face to me of an illness, therefore I am here, 02: 22 of the morning simply because I had an excellent day and I thought that the very suspicious flu that got me (I took the vaccine) was gone and it was enough that I lay down, after ten minutes I made sure that I did not sleep -0 I am not cured of an influenza for which I have vaccinated myself and I am not, at this point in life - 52 years of age, 22 of them with HIV, given to abuses and to be honest I'm just going to the street if you are forced by an appointment that can not be resolved right here.

I was very selective in choosing people who could come into my house and, after being treated like an idiot by ESPM students who tricked me with hateful and deplorable cynicism (all I asked them was not to change my name or that they hid my face and that's exactly what they did. Moral of the story: Interview, even for fundamental students, will have to be remunerated and, donate to anyone who hurts I'll look at the camera's adjustment and make any interested to use my work for your professional training to be subject to a contract with my non-negotiable terms or nothing. Thank all the students of ESPM because it was childish, immoral, ladino and cheating behavior that led me to these lessons and it will not be changed until ' for the day of my death.

In this way, in the name of prudence. We are gathered around a problem that, speaking generically speaking, we are engulfed by pure, exquisite and foolish recklessness. How many of us, and even myself, do not stop at a time when the health crisis settles, feels like going back in time and giving a baseball bat in the head of that "our reckless me" and leave a message for him that would be, I believe, more or less like that. Use his / her condom.

I'm investigating this supposed cure in England. I have good sources there and nothing was sent to me. Maybe there's nothing to send and all this boasting is a kind of "click-hunting." Or, who could know (???) I fell in disgrace with them and will have to search deeper and deeper.

Well, the flu is not quiet and my nasal passages are closed. Who can sleep like this?

I'm going to Netflix, watch Luke Cage, because I'm dying of curiosity about what's next.

Yes, people, I play hard, but it's because life has given me no choice and characters like Daredevil, The Punisher, Luck Cage and Jessica Jones bring me, at least for a few minutes, the feeling that there may be justice, or at least, according to one who is far off in the night of time, be fair in the accounts, and if I can not charge mine, let me rave with joy by seeing "righteous men" doing something I would very much like to be able to do.

Yes, smart girl, I've walked a long road and, although I can give you many truths, my book will leave you wondering how I could go through what you will read. I say this was the easy part. There is a difficult part, and I dare not even think about a Word document ... What if I put them in a book ... lol. Until next time.

Cau. This is an affectionate way that a deliciously loving Baiana chose to refer to me. When a woman calls me Cau, my heart beats harder, my breath is confused, and all I can remember is the phrase, after I put the condom on:

"Now I can…"

What came after .... It was chaos ... (...) ... It's been almost twenty years and sometimes I still feel the bitterness of the gall that was poured over me. And the baneful question: How are you going to turn around when the tide fills up? I did not answer anything, but I thought: swimming.

Gus Cairns

Posted in: 17 May of 2016

Translated by Claudio Souza do Original in HIV rapidly develops resistance to gene-editing cure technology by Gus Cairms for AIDSMAP reviewed by Mara Macedo

Reference

Wang Z et al. CRISPR / Cas9-derived mutations both inhibit HIV-1 replication and accelerate viral escape. Cell Reports, 15, 1-9 pages. IT HURTS: http://dx.doi.org/10.1016/j.celrep.2016.03.042. April 2016.

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