Resistance may be the Achilles heel of the genetic engineering approach
Develop resistance to therapy up to 8 days
One of the “techniques literally leads the virus to resistance against itself
In a setback for an innovative approach to healing the HIV, researchers involved in a technique that uses enzymes to remove viral genes from the DNA of infected cells have found that HIV easily adapts to this action and rapidly develops resistance to the guide that drives target molecules that carry the right part of a sequence. of human cellular DNA. Resistant viruses may also develop a more aggressive strain of the virus and replicate even faster than viruses not exposed to gene therapy (although they are still susceptible to conventional antiretroviral drugs (ARVs)).
In addition, the researchers suggest how gene therapy can actually promote the development of resistance, as it actively works to create small mutations at the site where cell DNA is cut. 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 within bacteria as a natural defense against the virus, is attached to a single length of the “guide” strand (sgRNA RNA) that guides the cas9 for the specific part of HIV-infiltrated DNA in its process of integrating newly converted RNA into DNA that needs to be removed to eliminate HIV “control” over the cell, thus ending that cell cycle. life of HIV.
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 like HIV inserts the genetic instructions that exist in the cell nucleus and not in the surrounding cytoplasm into a cell nucleus.
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 PF production - which was also postponed for four days - was still 55% lower in control cells. But in cells treated with T10 it was exactly the same in control cells - although delayed by eight days - and it was actually 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” o 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 viruses resistant to T4 and T10, but all viruses resistant to LTR-B, 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 viral business transaction in the presence of low levels of drugs, which exert selective evolutionary pressure - what was being caused was generated directly by the drug itself. In short, sgRNA / cas9 has been acting as a mutagenic agent, a direct driver of the viral mutation towards the strain resistant to sgRNA / cas9 itself. Editor's note. In good Portuguese it reminds me of a backfire. This type of "shot", which backfires, kills "only" one person, if he gets out of control and goes 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 that I can give my readers good news, and the more I go on 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 thoughtful of what I publish and I have not seen, in my sources, that I consider sensible, no mention of this cure that has arisen in London. I hate to look like Cassandra announcing the apocalypse and all I ask is attention and caution. I would love to be absolutely sure that a particular study has actually found a cure for HIV, but I know that among the application of this possible cure, there must be a protocol of application that should, at short notice, determine a treatment of X time and a period of time. “DELTA” time waiting for evidence of complete remission, elimination of reservoirs (the intestines are an unbelievably large area where HIV can hide and if it is hidden there forever, this may be acceptable as a medical condition leading to the person to a life without ART but with a complex schedule of collecting exam material for the rest of my life and this one, even though it is a cure, has got me in the face of a disease, because I am here, 02:22 am simply because I had a great day and believed that the very suspicious flu that caught me (I got the vaccine) was gone and it was enough that I lay down so that after ten minutes I was sure I would not sleep -0 I am not cured of a flu for which I got vaccinated and I am not at this point in my life - 52 years old, 22 of them with HIV, given to abuse and, to be honest, I only go to the street if I have to make a commitment that cannot 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 walked a long road and although i can give you many truths, my book will leave you thinking about how i got through everything you will read. I say this was the easy part. There is a difficult part, and I dare not even think about pola in a Word document ... What if I put them in a book ... lol Until next time.
Cau. Está é uma maneira carinhosa que uma baiana deliciosamente amorosa escolheu para se referir a mim. Quando uma mulher me chama de Cau, meu coração bate mais forte, a respiração se confunde e tudo o que eu posso lembrar é da frase, depois de posto o condom:
"Now I can…"
What came next…. It was chaos ... (...) ... Almost twenty years ago and, sometimes, I still feel the bitterness of gall that was poured over me. And the dismal question: How will you manage when the tide is high? I didn't answer anything, but I thought: swimming.
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
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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