Jump to content

Excerpt from Recent Publication by Dr. Cullen on Crispr/Cas9 Antivirals


StayingUpbeat

Recommended Posts

Not exactly an update but in a recent publication co-authored by Dr. Bryan Cullen he talks about how his efforts in genome editing since the FAQ release last year would work for eliminating HSV infections.

http://www.sciencedirect.com/science/article/pii/S0042682215000707

What is clearly needed is an approach that directly targets HSV-1 or HSV-2 episomal DNA for cleavage and elimination from latently infected neurons. AAV-delivered HSV-specific Cas9/sgRNA combinations appear ideal for this purpose (

Chamberlin et al., 1998

). In particular, several AAV serotypes, including AAV8, are able to infect almost all (>95%) of the sensory neurons in the dorsal root ganglion after application of particles of a green fluorescent protein (gfp) expressing AAV8 vector to the rear footpad of the mouse (D. Bloom, personal communication). Latent HSV-1 infections of neurons in the mouse trigeminal ganglia (TGs) can be readily established and it is therefore possible to test whether transduction of these same TGs with AAV8-based vectors encoding HSV-1-specific Cas9/sgRNA combinations will result in a detectable reduction in viral DNA load and an inhibition in the ability of latent HSV-1 to reactivate after explant and culture of the infected TGs. Given the tight localization of HSV-1 and HSV-2 to the trigeminal and sacral ganglia respectively, given the low level of viral DNA genomes present in these cells, and given the ability to efficiently transduce sensory neurons with AAV8-based vectors, this seems like an ideal viral candidate for cure using CRISPR/Cas.

 

---

Edited by StayingUpbeat
Link to comment
Share on other sites

Last time I read, they were going to put different parts into separate vectors and administer them together, which I think would make a lot of sense.  Reading it at the time, they didn't sound too hopeful about what they resorting to, but maybe that's my own personal bias.  Reading so much and everyone has a style of writing where their personality shines out.  Or maybe it was just an intuitive hunch.  Either way, both Dr. Cullen and Bloom, however hopeful and inspiring their research, is a long ways away from being explored in humans.  BUT, God willing, maybe that will change in time.   What we need is a president who has a really bad case of HSV and has overcome it to the point where, in office, he or she vehemently advocates a "cure" or at least a progressive step with treatment for HSV.  That's how the polio vaccine came about.  We had a president who was afflicted by the virus, he got in office and wanted to bring the nation out of its dark depression and whatnot...  And lo n' behold, polio vaccine was created.  Albeit, not a cure for those who already had it but at least stopping it for the time being.

 

My question is that AAV being a non-enveloped virus that it doesn't follow the same path as an enveloped virus like HSV.  I haven't looked any deeper into HSV but when something has a lipid envelope, I think of it being able to just easily pass through lipid membranes, whereas if something lacked that lipid encasing then it'd need to gain access through various cell membrane receptors....  

 

I wish someone would just take The Herpes Simplex Virus and put all these DNA, gene clipping genes in it and try that.  I understand HSV genome is really large so it's hard to tell what alterations in it would do, especially when you have different cellular environments for its proteins to confer to...   It's complicated.  Humans have been pretty crappy about making stuff up.  The most effective treatments we have are derived from the natural world.  Then there's chemo...  But HSV... I understand using AAV, as since it's small they're able to control it as a reliable vector.  I read an article talking about gene therapy using AAV and the results were not good.  They were doing it for a type genetic issue and 2 of the recipients developed an obscure result with having leukemia....   I'm sure that's a concern that pops up when they're doing their safety evaluations for these types of therapies.  But meh, it would be cool if it worked and progressed to a workable phase and data/results just flowed from their lab in ecstatic joy!

Link to comment
Share on other sites

I wonder if he has resolve the size issues with AAV (vectors)? It seems he was having a hard time fitting Cas9/sgRNA into one sole vector?

Should have read article. But still sounds really far off.

In particular, Neisseria meningitidis (Nme) encodes a Cas9 protein with the PAM sequence 5′-NNNNGATT-3′ while Staphylococcus aureus (Sau) encodes a Cas9 with the PAM sequence 5′-NNGRRT-3′, where R is purine ( Esvelt et al., 2013, Hou et al., 2013 and Zhang and Ran, 2014). Both proteins are encoded by genes ~3.2 kb in length, leaving ample room for two sgRNA cassettes, in addition to all required regulatory elements, in an AAV vector context ( Fig. 2). In our hands, Sau Cas9 is at least as active, or possibly more active, than Spy Cas9 on the same DNA target sequence and the sequence specificity of Sau Cas9 appears to be comparable to Spy Cas9 (data not shown). Therefore, it now seems entirely feasible to move to animal experiments that are designed to test the efficacy and safety of AAV-based Cas9/sgRNA vector systems.

Link to comment
Share on other sites

 I read an article talking about gene therapy using AAV and the results were not good.  They were doing it for a type genetic issue and 2 of the recipients developed an obscure result with having leukemia....   I'm sure that's a concern that pops up when they're doing their safety evaluations for these types of therapies.  But meh, it would be cool if it worked and progressed to a workable phase and data/results just flowed from their lab in ecstatic joy!

Safety probably won't be an issue.

Moreover, AAV-based vectors have a well-established record of safety and do not integrate at significant levels into the target cell genome, thus avoiding the potential for insertional activation of deleterious genes ( Kotterman and Schaffer, 2014).

  • Like 1
Link to comment
Share on other sites

I really appreciate your response because it gives me an opportunity to recheck my knowledge. 

I remember reading AAV in the same story but I understand that the cases that induced leukemia were retroviral vectors!!!  That absolutely blows my mind.  I mean, I think it makes sense since they're trying to literally rewrite or overwrite something in the genome.   

So further reading about AAV, is that the DNA it leaves behind is extrachromosomal DNA(at least the lab modified version isn't supposed to integrate into host genome). Meaning that the the viral DNA doesn't go into the nucleus and interfere with the genome but it is in the cytoplasm and makes use of the cell's machinery.  The DNA of HSV does the same.  It is extrachromasomal DNA that isn't with the genome.  So to have a vector that places its DNA in the same area as our target DNA/HSV makes a lot of sense and seems like a very worthwhile avenue to explore.   

 

But here's an excerpt.

http://www.the-scientist.com/?articles.view/articleNo/32141/title/Targeting-DNA/

"Fits and starts

It hasn’t always been such high times for gene therapy, however. The field was booming in its early days, with approvals for gene therapy clinical trials rising exponentially from the first one in 1989 to 116 in 1999. But that year, gene therapy trial participant Jessie Gelsinger, a relatively healthy 18-year-old who had an unusually mild form of liver disease caused by mutations in a gene on the X chromosome, died 4 days after receiving an injection of an adenovirus carrying an unmutated copy of the gene meant to correct his condition. The viral vector apparently triggered a massive immune response that caused multiple organ failure and brain death.

Infographic: Delivering New Genes View full size JPG | PDF
Infographic: Delivering New Genes
View full size JPG | PDF
LUCY READING-IKKANDA

Then, starting in 2002, reports from Paris and London told of patients developing a leukemia-like disease following treatment in clinical trials for a rare autoimmune disorder called severe combined immunodeficiency (SCID), or “bubble-boy” disease. SCID patients lack a functioning immune system, and thus must live in highly sterile conditions to prevent life-threatening infections. The studies started out extremely well: most of the infant boys were able to live relatively normal lives, no longer confined to their “bubbles.” The trials were hailed as the first unequivocal gene therapy success.

But in the years that followed, 5 of the 20 trial patients developed a leukemia-like disease—an effect that was traced to the retroviral vector used to deliver the corrective gene to bone marrow cells ex vivo. The vector had inappropriately inserted the gene into the babies’ genomes close to a proto-oncogene involved in white blood cell proliferation, activating the gene and triggering a flood of T cells. After the second child fell ill, the US Food and Drug Administration suspended 30 US trials using the same retrovirus, or about 15 percent of the 200 gene therapy trials under way at that time—a move the agency called a precautionary measure. Of the five patients that developed leukemia, one died; the rest are in remission.

Events like these had “a big negative impact in the field,” recalls molecular cell biologist Mien-Chie Hung of the University of Texas MD Anderson Cancer Center. Interest in gene therapy started to wane, and treatments that might have been expected to hit the market years ago are still plugging through the clinical trial process."

-----------------

 

Link to comment
Share on other sites

I'm certainly not advocating this to try on a person but at some point we (and I mean the HSV infected with a molec-bio background) really need to just up and develop this for ourselves.  One of the biggest complaints from the medical community about Crispr/Cas9 is that it is too easy.  

CRISPR’s main advantage over its gene-editing predecessors, zinc finger nucleases and TALENS, is that it is extremely easy to use.

I mean I developed and transformed plasmids for my bio courses and with all the CRISPR DIY bio-hacking out there I'm a little bit surprised that someone with an advanced background hasn't simply signed up for a DIYBio lab and gone to town.

Edited by StayingUpbeat
Link to comment
Share on other sites

I have molecular and cell culturing experience but I don't know anything about working with viral vectors.  I mean, you're not dealing with bacteria anymore.  All I know about working with viruses is that one needs chicken eggs to propogate them. Then you gotta sequence it to make sure everything went over as expected.  idk....    It's not as easy as it sounds.  I want to see the data from Cullen and Bloom on how things went when they administer 2 vectors with both containing all the pieces.

 

But I'm all about this underground hsv gene therapy project!  Let's do it!

Link to comment
Share on other sites

It sounds a bit crazy!! (In a good way). One antigen masking others? Insane!

I look forward to hearing some more studies on this one. Specifically - can they expect to make a viable vaccine by ignoring "need" to produce antibodies against gD-2?

Link to comment
Share on other sites

Eliminating only the gene encoding gD is probably too simple/risky for getting through regulatory approval but they have been quietly plugging along with this concept for a few years now.  Since it's a hospital medical center (an not simply a University) there's a better chance of getting to clinical trials but I wouldn't expect 3-5 more years of testing and refining before they can move forward with something like that.

Link to comment
Share on other sites

I know @StayingUpbeat and myself have bumped heads on more than one occasion, however, I do believe its going to take us suffers to bring a cure for this. Has anyone thought about contacting the PEP005 people to see if their drug could possibly bring this virus out of hiding? They know it will HIV and I know their not the same type of virus but it may work. Worth a shot looking into too. 

 

Link to comment
Share on other sites

I know @StayingUpbeat and myself have bumped heads on more than one occasion, however, I do believe its going to take us suffers to bring a cure for this. Has anyone thought about contacting the PEP005 people to see if their drug could possibly bring this virus out of hiding? They know it will HIV and I know their not the same type of virus but it may work. Worth a shot looking into too. 

 

I simply can't say I know enough about this compound to definitively comment.  My gut reaction from reading the paper is that this probably isn't the right molecule for HSV. In HIV research the current cure concept is called "kick and kill."  As the theory goes: since latent HIV kills the infected cell when it reactivates the virus could be eradicated by setting up a situation with medication in which more cells with HIV are dying than can be newly infected and the viral reservoir slowly dies out. 

To summarize my position on using the "kick and kill" approach for Herpes: I think it might work but in addition to being very painful (since neurons will be literally exploding) there is a danger of moving the infection much deeper into the nervous system.

It was hypothesized by the inventor of the HSV-529 vaccine that if a massive enough reactivation could be coupled with a very strong immune response (i.e. through his vaccine) the potential to reduce the HSV reservoir is there.  I can't locate it now but I recall an animal study in which the first of these "kick" drugs (called HDAC inhibitors) was tried in HSV infected guinea pigs and the result simply ended up being a massive increase in the number of copies and lethality of HSV

 

Edited by StayingUpbeat
Link to comment
Share on other sites

@StayingUpbeat you are very informed and knowledgeable about research which is going on. Thanks for your posts. As a side question, how do you embed links into text as you have in the above post? 

Link to comment
Share on other sites

how do you embed links into text as you have in the above post? 

ABOVE, In the box where you type in stuff, next to BoldItalics, and Underline, there's a chain link.   Highlight a word or sentence that you want to link and click on that chain link button and you'll have a prompt show you all you need to know. :)

Link to comment
Share on other sites

There has to be something out there that can cure this virus. It just hasn't been tested. We need to figure it out and make it a reality. By the way, the DRACO campaign is going well. 

Link to comment
Share on other sites

Congratulations on getting the IndyGoGo fund off the ground.  @Broken_14 I look forward to hearing more about your DRACO campaign in the years to come.  Out of curiosity did the Q&A session with Dr. Rider end up happening?

Link to comment
Share on other sites

Unfortunately no, we had to push it back. He did fill out some Q&A's for the campaign and he's been busy with a video and photo shoot as well as gather some other information and filling out all the paperwork to receive the donations. Check out our new Instagram at killingsickness. It has some of his new photos and I'm hoping to post a piece of the video later. This is legit and can't wait to show off all the hard work and hopefully make DRACO a reality. 

  • Like 2
Link to comment
Share on other sites

Join the conversation

You can post now and register later. If you have an account, sign in now to post with your account.

Guest
Reply to this topic...

×   Pasted as rich text.   Paste as plain text instead

  Only 75 emoji are allowed.

×   Your link has been automatically embedded.   Display as a link instead

×   Your previous content has been restored.   Clear editor

×   You cannot paste images directly. Upload or insert images from URL.

  • Donate

    If Honeycomb has helped you, please help us by making a donation so we can provide you with even better features and services.

  • The Hive is Thriving!

    • Total Topics
      72.1k
    • Total Posts
      486.1k
  • Posts

    • WilsoInAus
      I really suggest that the best thing is for both you and your boyfriend to obtain the Westernblot HSV test. He has a 50%+ chance of being positive and you have a 50%+ chance of being negative. Only the Westernblot can sort this out for you.
    • WilsoInAus
      Hi @kpn the first thing to note is that it is all OK to have HSV-1 or indeed any HSV type. It is not negligence, it is just being human. Oral HSV-1 is not an STD in the sense that the primary transmission is non sexual and the majority of it occurs between parents to children. If any of your children contracted HSV, you would know it. It wouldn't be a silent infection for children. HSV-2 tends not to shed from the oral region for people who have it there in any event. At age 73, about 80% of the population has HSV-1. There is no reason to believe that your mother isn't one of those people. About half of all carriers of oral HSV-1 do not realise they have it and have no living memory of cold sores as they were infected when very young. The most logical explanation is that your mother has oral HSV-1 from her childhood and that your daughter doesn't have oral HSV. Not that it is relevant to anyone but yourself, but your wife might find she actually has genital HSV-1 having had an untyped swab when she was diagnosed.
    • WilsoInAus
      Hi @Dylan86 and welcome to the website. First note that you cannot pragmatically become infected with HSV-1 from sharing a drink. If you carry HSV-1, it did not come from that episode. As such it is extremely unlikely HSV-1 is the primary cause of your issues. Can the WB miss a HSV-1 infection? Rarely but its feasible in less than 1% of carriers who test with WB. Note that the WB does not have values, it has positive or negative as you say. It is way more accurate than IgG as it looks for all 30+ antibodies that are in your blood for HSV. Could you have HSV-1? Yes it is feasible, but it would be a very old childhood infection. Could HSV-1 be causing your oral issues? No herpes will not cause the burning mouth syndrome you describe. Could some of the lesions be herpes related? It is feasible if you are a carrier. But it is unlikely to be the primary cause of the issues, but its an opportunistic virus that can cause issues when something else is taxing your immune system. The best thing you can do is the PCR test on a oral lesion and that will be pretty definitive. Either way, in summary. I'd suggest there is <1% chance you are part of about 70% of the population that has HSV-1. Further there is less than a 1% chance that herpes is the primary cause of your issues.   
    • kpn
      My wife has had hsv 2 for around 8 years. We have two young children 3 years old and 18 months old. My wife only had one outbreak when she was first infected so we didn't worry too much about passing it on to our children. I understand the risk to be pretty low under those circumstances. I don't believe my wife took antivirals during either pregnancy. My younger child has diaper rashes pretty often and has had what I thought was hand foot and mouth disease. That was going around the daycare a while back. I haven't really given it much concern though.  About a week ago, my mother kissed my youngest on the lips. My kid was congested at the time but they pretty much always are. About 3 days later, my mother developed a cold sore on her mouth. She has never had cold sores in her life and she is 73 and happily married so she is not going around messing with anyone. She pointed to the fact she had kissed my youngest and presumed that is where she was infected. At first I said that's not possible since my child has never had any cold sores but since then I have really started to consider that maybe it is possible she contracted it from my daughter. This has me worried that my daughter does indeed have hsv2 and was shedding in her mouth. Does anyone have any experience with this? I am really losing sleep about this. Two people I care so much about got hsv from my negligence. I am fine if I were to contract it, I am not worried about what others think at this point in my life. I just don't want anyone else to have it. 
    • CHT
      Hello DavidGua.... based on the two pictures, I'm not seeing anything that resembles a typical herpes outbreak.  I am not sure what those spots are on your penis.  Please have a doctor take a look and I'm sure you'll get a proper diagnosis.  Have you ever received an HSV antibody test (IgG)?  Again, I doubt your symptoms are herpes-related but, if you'd like some peace of mind, you could request the IgG antibody test for HSV2.   Best of luck.... let us know if you have any other questions/concerns.
×
×
  • Create New...

Important Information

We have placed cookies on your device to help make this website better. You can adjust your cookie settings, otherwise we'll assume you're okay to continue.