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Can CRISPR/Cas9 be harnessed to edit out the cccDNA from the hypocyte genome?
Targeting hepatitis B virus cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication.
http://www.ncbi.nlm.nih.gov/pubmed/25843425
They sale the CRISPR/CAS9 self-kit on the internet for about $200...
All we need is to program it with the sequence of our cccDNA and then administer it into the liver cells... Wouldn't it cure hbvers? Is there any biotech company that tries to develop a drug based on this technology?
The micro-organism that this enzyme was extracted for was evolutionary designed to take out the virus from his genome cell... So it sounds like nature answer to retroviruses...
Thoughts?
http://www.ncbi.nlm.nih.gov/pubmed/25843425
They sale the CRISPR/CAS9 self-kit on the internet for about $200...
All we need is to program it with the sequence of our cccDNA and then administer it into the liver cells... Wouldn't it cure hbvers? Is there any biotech company that tries to develop a drug based on this technology?
The micro-organism that this enzyme was extracted for was evolutionary designed to take out the virus from his genome cell... So it sounds like nature answer to retroviruses...
Thoughts?
The hbv cccDNA is a genetic element or extrachromosomal body that exists within the nuclear space but is separate from the 46 human chromosomes.
When we speak of integration, we mean that a stretch of viral dna is actually inserted into one of the human chromosomes. Each time a liver cell divides there is some chance that available extrachromosomal dna pieces are inserted by repair encymes into places that lend them self for such insertion, basically mostly random, but there are places of predeliction.
if such a stretch of dna is later transcribed depends on many factors, most importantly it must contain the chracteristic elements of a functional gene, with a promoter and the place of insertion must be in an active part of the chromatin.
When we speak of integration, we mean that a stretch of viral dna is actually inserted into one of the human chromosomes. Each time a liver cell divides there is some chance that available extrachromosomal dna pieces are inserted by repair encymes into places that lend them self for such insertion, basically mostly random, but there are places of predeliction.
if such a stretch of dna is later transcribed depends on many factors, most importantly it must contain the chracteristic elements of a functional gene, with a promoter and the place of insertion must be in an active part of the chromatin.
Thank you for that.
I am now re-listening to the Arrowhead analysts day broadcast. These are from one of the slides(34):
cccDNR codes for all viral mRNAs and pregenomic RNA (pgRNA)
PgRNA used in viral replication
- converted to relaxed circular DNA (rcDNA)
Another replication product is the double stranded linear (dsl) HBV DNA
- less than full genome length
- replication defective
- 10% or replication products are dslDNA
Both rcDNA and dslDNA transferred to the nucleus after infection
dslDNA can intergrate into the host genome
- propogated as hepatocytes divides
These are what prompted me to say that integrated hbv dna cannot replicate and truncated. In one of Lorncarnini old papers, he also stated that in humans, integrated hbvdna is truncated. It would be interesting to know his new thinking on this issue.
I have no reason to doubt that a mouse model may have integrated hbvdna introduced as a plasmid, but I want to know whether the whole hbv genome can be integrated in humans - after all, the hbv genome is pretty large and why has it not been reported, for example, from HCC tumor samples.
I am now re-listening to the Arrowhead analysts day broadcast. These are from one of the slides(34):
cccDNR codes for all viral mRNAs and pregenomic RNA (pgRNA)
PgRNA used in viral replication
- converted to relaxed circular DNA (rcDNA)
Another replication product is the double stranded linear (dsl) HBV DNA
- less than full genome length
- replication defective
- 10% or replication products are dslDNA
Both rcDNA and dslDNA transferred to the nucleus after infection
dslDNA can intergrate into the host genome
- propogated as hepatocytes divides
These are what prompted me to say that integrated hbv dna cannot replicate and truncated. In one of Lorncarnini old papers, he also stated that in humans, integrated hbvdna is truncated. It would be interesting to know his new thinking on this issue.
I have no reason to doubt that a mouse model may have integrated hbvdna introduced as a plasmid, but I want to know whether the whole hbv genome can be integrated in humans - after all, the hbv genome is pretty large and why has it not been reported, for example, from HCC tumor samples.
The notion that integrated hbv DNA cannot be used as full template for the production of new virions is probably unfortunately not correct. It is a much more rare event than partial truncated integration, but will occur nevertheless to a smaller degree over time.
A fully integrated hbv genome carrying liver cell has however actually a much better chance to be eliminated by immune detection than the hbsag only producing cells. That is the good news, but the bad is that such elimination can only occur by cell death, not by noncytopathic clearance under the influence of ifn gamma and tnf alpha, the way in which probably most virus is normally cleared.
In Chisaris mouse model a plasmid with the full hbv genome is injected forcefully into the blood stream and enters and integrates into a large percentage of hepatocytes.
And in his transgenic model the hbv genome is permanently integrated in a host liver chromosome.
A fully integrated hbv genome carrying liver cell has however actually a much better chance to be eliminated by immune detection than the hbsag only producing cells. That is the good news, but the bad is that such elimination can only occur by cell death, not by noncytopathic clearance under the influence of ifn gamma and tnf alpha, the way in which probably most virus is normally cleared.
In Chisaris mouse model a plasmid with the full hbv genome is injected forcefully into the blood stream and enters and integrates into a large percentage of hepatocytes.
And in his transgenic model the hbv genome is permanently integrated in a host liver chromosome.
1 Comments
I thought HBV DNA viron cannot reproduce by itself... I thought it only reproduces with it's cccDNA that is integrated in the cell genome. What is "integrated hbv dna"? How does that differ from cccDNA? Isn't the cccDNA code is basically the same between different cells? Or at least most of the cells? Or there are mutations and only partial integration? If it's only partial integration isn't that makes the cccDNA invalid cause it doesn't have the full code? Cause I guess CRISPR is good when you know exactly what is your target... Or if there is at least some common part that is only unique to the virus....
problem is that we know from Arrowhead trials that probably cccDNA amount is very small in compare to integrated HBV DNA in HBeAg- patients
2 Comments
I don't think it is a given that what is observed in chimps apply to humans. According to Arrowhead, integrated hbvdna is also observed in hbeAg+ve patients. BTW, integrated hbvdna is truncated and cannot be used as a template to replicate new virions as cccDNA. Also Crisper can also be used to cleave intergrated hbvdna using a different guide to cccDNA.
A typo: "hbeAg+ve patients" should read "hbeAg+ve CHIMPS"
All im saying is be careful.
If your not in any immediate danger I would wait and see how the trial unfolds first.
No point being a self styled ginipig then you have no support mechanism behind you.
Just a friendly suggestion
If your not in any immediate danger I would wait and see how the trial unfolds first.
No point being a self styled ginipig then you have no support mechanism behind you.
Just a friendly suggestion
Well this is only a proof of concept right now... This technology is still far from clinical application... It's only second generation for the technology... It will probably will need to be improved in accuracy and safety, and find an efficient delivery system to the liver cells. I say 5-10 years, if we don't see a cure before that. The kit being sold on the internet was only to show that overall this technology is pretty affordable.
CRISPR/Cas9 is different than birinapant:
CRISPR/Cas9 only deletes the cccDNA from the cell genome, but it doesn't kill the cell, so it's pretty safe as long as it's accurate in what it deletes, and having the cell system to merge the cut two genome ends.
So for example let's say this is how the genome cell looks:
BLA BLA BLA cccDNA BA BA BA
After you take CRISPR/Cas9 which only cuts the cccDNA from the cell genome this is how it will look:
BLA BLA BLA BA BA BA
CRISPR/Cas9 only deletes the cccDNA from the cell genome, but it doesn't kill the cell, so it's pretty safe as long as it's accurate in what it deletes, and having the cell system to merge the cut two genome ends.
So for example let's say this is how the genome cell looks:
BLA BLA BLA cccDNA BA BA BA
After you take CRISPR/Cas9 which only cuts the cccDNA from the cell genome this is how it will look:
BLA BLA BLA BA BA BA
Correct me if Im wrong and this is just a theory it causes all the infected cells to "commit suicude"
So if the liver has approx 10 trillion cells. If half are infected for example and you take something that makes them "commit suicide" it could be dangerous potentially causing death.
However if there is a way to meaningfully reduce the cccdna then take this stuff might an idea.
I think Biotech tec company working on this theory company name tetra logic birinapant. And they had problems with bells paulsy etc caution would be a wise thing to use
Im no doctor and its just a theory
So if the liver has approx 10 trillion cells. If half are infected for example and you take something that makes them "commit suicide" it could be dangerous potentially causing death.
However if there is a way to meaningfully reduce the cccdna then take this stuff might an idea.
I think Biotech tec company working on this theory company name tetra logic birinapant. And they had problems with bells paulsy etc caution would be a wise thing to use
Im no doctor and its just a theory
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