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CRISPR Human Trials for HSV1/2?

CRISPR Human Trials for HSV2?  

247 members have voted

  1. 1. Latent HIV was recently eliminated in a murine model (mice) if the same or similar technology was applied to HSV1 and HSV2 would you be willing to become a human guinea pig and try it outside the Country?

    • No - Only FDA clinicals trials starting after the years 2030 with long term safety evidence
      2
    • No - Only FDA clinicals trials starting after the years 2040 with longer term safety evidence
      0
    • No - I don't care
      2
    • No - I'm waiting for live vaccines and may or may not do anything else
      5
    • No - I'm waiting for GEN-003/Admedus and may or may not do anything else
      2
    • No - I fear long term safety
      13
    • Yes - I'd do a Phase 1 and be in the first 10 people
      78
    • Yes - I'd do a Phase 1 and be in the first 10 people but only if asymptomatic measurements are taken (shedding)
      43
    • Yes - But I'd only do a Phase 2 or higher.
      102


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Cas9
18 minutes ago, dont quit!17 said:

There was an excision therapeutics study.

https://www.sciencedaily.com/releases/2017/05/170501112514.htm

HIV would be a more difficult fix imho due to not knowing exactly where latent HIV snoozes at. At the very minimum we know exactly where HSV is at and eventually we will find a way to relax it and penetrate multiple gene sites. 

Yes, that was the problem with HIV that was not an issue with HSV. And that HIV problem has been resolved, at least to some extent. But the HSV problem is an entirely different problem. therefore, success with HIV does not translate to success for HSV.

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Cas9

Has anyone noticed that the Edit functionality is not working again? It was fixed a week ago or so but seems to have returned.

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JJ2017
1 hour ago, Cas9 said:

Has anyone noticed that the Edit functionality is not working again? It was fixed a week ago or so but seems to have returned.

im sure i edited the other day. and hugely frustrating that we cant get to the latent HSV.......it seems most the vaccines focus efforts on killing replicated virus rather then going for the latency. that way we wont get a cure....just a vax we will need to top up that wont 100% reduce shedding. from what i have read only 2 organisations are looking at gene editing for hsv in the whole world......could i be wrong?

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Atrapasueños

Tiene razón  y dicen que han estudiado el virus por años pues no veo que le estudian si siguen con los mismos resultados las vacunas sólo enseñan al sistema inmune como defenderse y en algunos artículos se menciona que al tener el hsv en neuronas es una ventaja para curar el hsv quien les entiende

@JJ2017

He is right and say they have studied the virus for years because I do not see that they study if they continue with the same results vaccines only teach the immune system how to defend themselves and in some articles it is mentioned that having the hsv in neurons is an advantage to cure The hsv who understands

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MoniqueLow
10 minutes ago, JJ2017 said:

im sure i edited the other day. and hugely frustrating that we cant get to the latent HSV.......it seems most the vaccines focus efforts on killing replicated virus rather then going for the latency. that way we wont get a cure....just a vax we will need to top up that wont 100% reduce shedding. from what i have read only 2 organisations are looking at gene editing for hsv in the whole world......could i be wrong?

According to Wikipedia, genom editing  is also used by those two labs:

http://research.fhcrc.org/jerome/en.html

https://sites.duke.edu/cullenlaboratory/

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Cas9
53 minutes ago, JJ2017 said:

im sure i edited the other day. and hugely frustrating that we cant get to the latent HSV.......it seems most the vaccines focus efforts on killing replicated virus rather then going for the latency. that way we wont get a cure....just a vax we will need to top up that wont 100% reduce shedding. from what i have read only 2 organisations are looking at gene editing for hsv in the whole world......could i be wrong?

Try editing feature now and see if it works for you.

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OFMDH
5 hours ago, Cas9 said:

First of all, who specifically are you referring to that is responsible for successfully eliminating HIV in mice? I believe a couple of labs have been successful. But hsv has it's own (unique) problems that do not apply to HIV and therefore I do not believe the method used for HIV applies to hsv.   HSV  DNA for example is too tightly compressed when it is dormant. Also, hsv lives in non-replicating cells. These are issues that don't apply to HIV. HIV has it's own unique issues but apparently, those have been overcome to some extent if not fully. But the issues with latent HSV are still a problem and I don't think the success with HIV translates to HSV.

Maybe I'm misunderstanding something. If so, please elaborate,

I was referring to Chaoran Yin and that group. [link] and measured it in several organs, clearing it, etc.  They used AAV-DJ + four sgRNA's  and SaCas9. AAV-DJ combines numerous capsids of different AAV's together so that transfection rates are increased throughout the body.

What I think you're relying on for HSV latent DNA experiments are models in which they are trying to keep the cells in the lab dish from dividing and are run on a very short time frame and Cas9 basically needs a lot more time for that DNA. But no its not really too tightly wound to be ever accessible. Experiments show that Cas9 does in fact more forays into the heterochromatin DNA at a much slower rate than DNA without histones in chromatin form.  Therefore your reliance on prior lab dish experiments of HSV and whether non-replicating DNA can be edited is misplaced as even Aubert and Jerome last September using mice and homing endonucleases showed that the stage of latency and histones did not interfere with editing.  They did show a low %, but with reduced kinetics it just takes a lot longer to edit the latent DNA that is not reactivating   

I will concede way when it does eventually go into heterochromatin DNA  it has to push things around so to speak and when it finally makes a cut the indels are smaller, however that can be overcome and I know how.  

 

 

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Cas9
59 minutes ago, JJ2017 said:

im sure i edited the other day. and hugely frustrating that we cant get to the latent HSV.......it seems most the vaccines focus efforts on killing replicated virus rather then going for the latency. that way we wont get a cure....just a vax we will need to top up that wont 100% reduce shedding. from what i have read only 2 organisations are looking at gene editing for hsv in the whole world......could i be wrong?

You have a misunderstanding about vaccines and the latent virus. Vaccines are simply the disabled virus in one form or another, that elicits your immune system to attack the virus. That said, your immune system CANNOT kill the latent virus; EVER!!!
That's because our immune system cannot physically enter a cell. That's not how the immune system works. That's where CRISPR comes in. CRISPR is the immune system from a bacteria. Since bacteria are one celled organisms and since bacteria have an immune system, then logically, their immune system lives and operates in the cell. We humans are borrowing this bacterial immune system and placing it into our cells to go after the virus IN THE CELL.; Something our own immune system can not do.

Why they're having problems killing the latent hsv virus is not clear to me. I've read two separate accounts. One stated that there ma be issues with penetrating the neuron because it is a non-replicating cell. I find that odd because as fa as I know, that has already been acheived. Perhaps I am misunderstanding what that article meant. That's certainly possible.

Other articles state that the issue is that the latent virus's DNA, when in dormancy, is too tightly compressed, and so, CRISPR cant's get at it and slice out the desired genomes. If this is the correct reason I would like to know why CRISPR can't  do it's thing when the latent virus comes out of dormancy during replication, This question could be easily answered by one of the research lab scientists.

As far as who's using CRISPR against hsv; in the U.S. Jerome labs is working on it. They have been minimally successful killing he latent virus so far. Cullen Labs at Duke have ceased operation on their research due to funding issues. It's ossible they will be able to secure funding at a later time OR it's possible that Editas make take over that project. The other lab/company is Excision Biotherpeutics. I believe they have been very successful in killing the replicated viruses in the neuron but not the mother of the replicated viruses; aka the latent virus. But even that I'm not fully sure of. In either case they are working on killing the latent virus as part of future trials.

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Cas9
14 minutes ago, OhFuckMyDickHurts said:

I was referring to Chaoran Yin and that group. [link] and measured it in several organs, clearing it, etc.  They used AAV-DJ + four sgRNA's  and SaCas9. AAV-DJ combines numerous capsids of different AAV's together so that transfection rates are increased throughout the body.

What I think you're relying on for HSV latent DNA experiments are models in which they are trying to keep the cells in the lab dish from dividing and are run on a very short time frame and Cas9 basically needs a lot more time for that DNA. But no its not really too tightly wound to be ever accessible. Experiments show that Cas9 does in fact more forays into the heterochromatin DNA at a much slower rate than DNA without histones in chromatin form.  Therefore your reliance on prior lab dish experiments of HSV and whether non-replicating DNA can be edited is misplaced as even Aubert and Jerome last September using mice and homing endonucleases showed that the stage of latency and histones did not interfere with editing.  They did show a low %, but with reduced kinetics it just takes a lot longer to edit the latent DNA that is not reactivating   

I will concede way when it does eventually go into heterochromatin DNA  it has to push things around so to speak and when it finally makes a cut the indels are smaller, however that can be overcome and I know how.  

 

 

My comments were based on the Netherlands study. It was they who mentioned that the DNA was too tightly compressed. Of course, they could be wrong. But even in that scenario, the latent virus must come out of dormancy to start replicating. Since the DNA is not tightly compressed during that state, why can't CRISPR get at it at  that point in time?
As far as Excision Biotherapeutics is concerned, the impression I got was they were successful in killing the viruses spawned by the latent virus inside the neuron. In other words, they could kill the replicated viruses in the neuron, but again, not the latent virus itself. Almost makes me wonder whether it's a timing issue; i.e. with all the newly replicated viruses in the vicinity of CRISPRCRISPR starts killing them. By the time they are all killed, the latent virus has gone back into dormancy, and due to the issues with DNA compression, CRISPR is generally unsuccessful. I know Jerome Labs has been partly successful killing the latent virus.

It was good to hear your thoughts on the latency issue. Perhaps I should give a longer read to what's gong on at Jerome Labs.

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JJ2017
27 minutes ago, Cas9 said:

You have a misunderstanding about vaccines and the latent virus. Vaccines are simply the disabled virus in one form or another, that elicits your immune system to attack the virus. That said, your immune system CANNOT kill the latent virus; EVER!!!
That's because our immune system cannot physically enter a cell. That's not how the immune system works. That's where CRISPR comes in. CRISPR is the immune system from a bacteria. Since bacteria are one celled organisms and since bacteria have an immune system, then logically, their immune system lives and operates in the cell. We humans are borrowing this bacterial immune system and placing it into our cells to go after the virus IN THE CELL.; Something our own immune system can not do.

Why they're having problems killing the latent hsv virus is not clear to me. I've read two separate accounts. One stated that there ma be issues with penetrating the neuron because it is a non-replicating cell. I find that odd because as fa as I know, that has already been acheived. Perhaps I am misunderstanding what that article meant. That's certainly possible.

Other articles state that the issue is that the latent virus's DNA, when in dormancy, is too tightly compressed, and so, CRISPR cant's get at it and slice out the desired genomes. If this is the correct reason I would like to know why CRISPR can't  do it's thing when the latent virus comes out of dormancy during replication, This question could be easily answered by one of the research lab scientists.

As far as who's using CRISPR against hsv; in the U.S. Jerome labs is working on it. They have been minimally successful killing he latent virus so far. Cullen Labs at Duke have ceased operation on their research due to funding issues. It's ossible they will be able to secure funding at a later time OR it's possible that Editas make take over that project. The other lab/company is Excision Biotherpeutics. I believe they have been very successful in killing the replicated viruses in the neuron but not the mother of the replicated viruses; aka the latent virus. But even that I'm not fully sure of. In either case they are working on killing the latent virus as part of future trials.

very informative thanks for taking the time to explain, perhaps its a combination of the 2, the study at the University Medical Center Utrecht also suggest the virus is closed off in non replicating neurons, a model trailed for hiv "kick and kill" was said to force hiv out of latency to allow the bodys immune system to attack it ......could a similar model work here? has kick and kill been trailed with crispr? technically a long period of crispr treatment may potentially eradicate hsv. i think the trouble is in some people the virus may never leave the dormant state.     

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MoniqueLow

Sorry to join your very sophisticated discussion. If virus stay dormant forever, I would not consider it as a trouble at all. I wish mine stay asleep... with a functional cure and possible prophylactic vaccine, asymptomatic shedding should not be problem anymore.

With cure and chance to protect yourself against contracting the virus, HSV won't be any issue anymore. I wish this come soon..

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moialbalushi
13 minutes ago, MoniqueLow said:

Sorry to join your very sophisticated discussion. If virus stay dormant forever, I would not consider it as a trouble at all. I wish mine stay asleep... with a functional cure and possible prophylactic vaccine, asymptomatic shedding should not be problem anymore.

With cure and chance to protect yourself against contracting the virus, HSV won't be any issue anymore. I wish this come soon..

Exacccllltttyyyyy 

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MoniqueLow

The scientists focused on HPV instead because of cancer danger (who knows, some theories say hsv can cause it too), otherwise there might be vaccine against HSV already.

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JJ2017

this is a great video that explain latency and shows why its important that latency it targeted. 

 

 

 

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OFMDH
On 5/9/2017 at 11:48 AM, Cas9 said:

My comments were based on the Netherlands study. It was they who mentioned that the DNA was too tightly compressed. Of course, they could be wrong. But even in that scenario, the latent virus must come out of dormancy to start replicating. Since the DNA is not tightly compressed during that state, why can't CRISPR get at it at  that point in time?
As far as Excision Biotherapeutics is concerned, the impression I got was they were successful in killing the viruses spawned by the latent virus inside the neuron. In other words, they could kill the replicated viruses in the neuron, but again, not the latent virus itself.

It was good to hear your thoughts on the latency issue. Perhaps I should give a longer read to what's gong on at Jerome Labs.

I know you were referring to that study and the other ones in his area as there are very few CRISRP HSV1/2 studies out there. About their comment about DNA being latent and inaccessible, that conclusion is really situational,  based on all the things they're doing and only suggestive as they can't conclude as fact that will happen in the animal. I agree, the fact once the DNA starts replicating makes it much easier to insert large indels and stop replication would also prevent future successful reactivations. Thus even if the latent DNA is harder to get, it doesn't matter as eventually the Cas9 protein will cut it. 

Excision Biotherapeutics says on their website they eradicated 90% of HSV1 DNA. 

Quote

Almost makes me wonder whether it's a timing issue; i.e. with all the newly replicated viruses in the vicinity of CRISPR CRISPR starts killing them. By the time they are all killed, the latent virus has gone back into dormancy, and due to the issues with DNA compression, CRISPR is generally unsuccessful. I know Jerome Labs has been partly successful killing the latent virus.

Depends what you mean "generally unsuccessful" as I pointed out already that Cas9 CRISPR does in fact more forays into latent DNA, albeit with much slower kinetics. Thus for timing if a person is just exposed it may just require a month to clear it (which mice experiments have shown), whereas if the person has it for years if it took a year to clear latent HSV DNA I would still call that successful.

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snippy43
28 minutes ago, OhFuckMyDickHurts said:

Excision Biotherapeutics says on their website they eradicated 90% of HSV1 DNA

Depends what you mean "generally unsuccessful" as I pointed out already that Cas9 CRISPR does in fact more forays into latent DNA, albeit with much slower kinetics. Thus for timing if a person is just exposed it may just require a month to clear it (which mice experiments have shown), whereas if the person has it for years if it took a year to clear latent HSV DNA I would still call that successful.

there are varying cas enzymes that can be used in crispr edits, correct? cas9, is considered a large enzyme and the reason for the argument that the latent dna cannot be cut. why not perform curative studies for excise with a smaller cutting tool..?

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OFMDH
3 minutes ago, snippy43 said:

there are varying cas enzymes that can be used in crispr edits, correct? cas9, is considered a large enzyme and the reason for the argument that the latent dna cannot be cut. why not perform curative studies for excise with a smaller cutting tool..?

Yes Cas9 is large and has trouble fitting into a cassette for the AAV vectors so most people make a second AAV strain with the sgRNA's. However having to have two AAV virus enter the same cell to work is not as efficient obviously has 1 virus reaching the cell and being complete in of itself.  The HIV study I references above actually used SaCas9 and 4 sgRNA's, with room for more. Albeit you can't just make a vector with 8+ sgRNA and include targets for other diseases as there is a finite amount of work the promoter will perform and you also don't want to overtax the cell's machinery in making it. 

One theory is that Cas9 came from bacteria cells that did not deal with DNA in chromatin structures, therefore it can't handle these in our cells. However as stated above Cas9 has been found going, albeit with greatly reduced kinetics into the chromatin DNA - other CRISPRs? SaCas9, CjCas9, dCas9, CPF1, etc.  going into the chromatin? I'm not sure.  As for size, since Cas9 is bigger it would have a harder time moving around the chromatin than say a smaller CPF1 or CjCas9 type of CRISPR.

Latent DNA may not be a huge problem because only 7 or so replication centers form inside the nucleus, the first 7 or so virus DNA to enter basically conquer it  and the successive viral DNA shot into the cells (imagine like 100's of sperm into an egg) after that are completely silenced/killed. These silenced DNA strands though still pump out miRNA. My theory is that those miRNA eventually will cause reactivation taking into account the cell's PML protein levels. Basically the cell reaches critical mass and HSV is activated from latency. Anyways, if the average DRG neuron cell has 50-100 copies of HSV2 DNA inside it and only say ~10% will  ever be copied for new virus, you may need to only inert indels in 10% of that latent virus that reactivates. 

Anyways because of CPF1's greater accuracy and ability to make indes below the PAM site its much more desirable to use in my opinion. 

 

 

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OFMDH

Anyways as mentioned above, with an AAV vector the latent virus is not an issue as much, because if it were to ever reactivate, the CRISPR protein would still be there to stop it, thus mooting whether it can or cannot edit latent DNA. Others are concerned about latent DNA because they also want to do, in simple terms,  lipo-nano therapies with CRISPR systems that would insert indels when used. I've seen quite low (6%) in getting those into animal cells. 

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dont quit!17
10 hours ago, OhFuckMyDickHurts said:

Anyways as mentioned above, with an AAV vector the latent virus is not an issue as much, because if it were to ever reactivate, the CRISPR protein would still be there to stop it, thus mooting whether it can or cannot edit latent DNA. Others are concerned about latent DNA because they also want to do, in simple terms,  lipo-nano therapies with CRISPR systems that would insert indels when used. I've seen quite low (6%) in getting those into animal cells. 

How long is the Crispr protein viable for?

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Atrapasueños

http://onlinelibrary.wiley.com/doi/10.1111/cmi.12694/full

Therefore, CRISPR/Cas9 targeting of the latent HSV-1 genome would be an appealing strategy to mutate or clear the virus from latent cells, thereby preventing future virus reactivation events beneficial for treatment of recurrent infections of the cornea or genital herpes. However, van Diemen et al. (2016) showed that the CRISPR/Cas9 system was not able to target the HSV-1 genome in quiescent HSV-1-infected MRC5 cells, whereas newly generated viral genomes from reactivated HSV-1 were readily inactivated. This suggests that the repressed state of the quiescent genome may interfere with Cas9 activity (van Diemen et al., 2016). Indeed, latent HSV-1 genomes are present in a severely repressed state and are closely associated with nucleosomes (Nicoll, Proença, & Efstathiou, 2012; Paulus, Nitzsche, & Nevels, 2010); recent studies show that Cas9 binding and cleavage is directly inhibited by the presence of nucleosomes at a target site (Chen et al., 2016; Horlbeck et al., 2016). Therefore, additional studies are needed to assess whether Cas9 cleavage can occur at sites in the HSV-1 genome, which have a naturally low occupancy of nucleosomes, or that display destabilized or repositioned nucleosomes due to the activity of chromatin remodelers provided in trans, thereby potentially exposing previously occluded target sites. In addition, studies are needed to assess whether HSV-1 latency can be targeted in vivo as a potential therapeutic strategy. Alternatively, sensory neurons may be equipped with anti-HSV-1 CRISPRs that interfere with virus replication upon reactivation, limiting spread of the virus to, for example, the cornea

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OFMDH
13 hours ago, dont quit!17 said:

How long is the Crispr protein viable for?

Depends as they can be there for a week to years. CRISPR proteins are foreign and thus do not stay forever, however when they're in a vector it keeps coming back. Thus it depends on the vector, what cells they're in and promoters are being used. Some Cas9 experiments with HSV-1 vector show duration as short as 8 days and with longer expressing designs up to 3 years. An AAV vector would theoretically last a long time. 

Status:  83 members have voted. 

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dont quit!17
1 hour ago, OhFuckMyDickHurts said:

Depends as they can be there for a week to years. CRISPR proteins are foreign and thus do not stay forever, however when they're in a vector it keeps coming back. Thus it depends on the vector, what cells they're in and promoters are being used. Some Cas9 experiments with HSV-1 vector show duration as short as 8 days and with longer expressing designs up to 3 years. An AAV vector would theoretically last a long time. 

Status:  83 members have voted. 

Just give patients a high dose of glucocorticoid steroids (dexamethasone, solumedrol) in order to temporarily cause immunosuppression in patients to bait activation of HSV out and clip the son of a bitch virus with CRISPR. One can dream!

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moialbalushi

...... im excited to see what you are going to do 

Vote guys. Vooooottteee

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Ajuda
On 2017-5-6 at 4:39 PM, OhFuckMyDickHurts said:

O HIV latente foi recentemente eliminado num modelo murino (ratinhos) se a mesma ou semelhante tecnologia fosse aplicada ao HSV1 e ao HSV2, estaria disposto a tornar-se uma cobaia humana e tentar fazê-lo fora do País?

It is the first time that I see someone taking a real initiative to seek a functional cure here. Congratulations OFMDH for your willingness and courage. What we need are people like that, willing to do something. Let us go in search of our cure. If they knew how much we want this, they will certainly speed up the process.

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