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Complete Spectrum of CRISPR/Cas9-induced Mutations on HBV cccDNA

another interesting news.

http://www.nature.com/mt/journal/vaop/ncurrent/full/mt201694a.html
12 Responses
Avatar universal
It may be interesting but it is way above my head. Can somebody translate that to English?
Avatar universal
Hepatitis B virus (HBV) causes chronic infections that cannot yet be cured. The virus persists in infected hepatocytes, because covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs, is stable in nondividing cells. Antiviral therapies with nucleoside analogues inhibit HBV DNA synthesis in capsids in the cytoplasm of infected hepatocytes, but do not destroy nuclear cccDNA. Because over 200 million people are still infected, a cure for chronic hepatitis B (CHB) has become one of the major challenges in antiviral therapy. As a first step toward the development of curative therapies, we previously demonstrated that the CRISPR/Cas9 system can be used to functionally inactivate cccDNA derived from infectious HBV. Moreover, some evidence suggests that certain cytokines might induce an APOBEC-mediated cascade leading to the destruction of cccDNA. In this report we investigated whether a combination of the two mechanisms could act synergistically to inactivate cccDNA. Using next generation sequencing (NGS), we determined the complete spectrum of mutations in cccDNA following Cas9 cleavage and repair by nonhomologous end joining (NHEJ). We found that over 90% of HBV DNA was cleaved by Cas9. In addition our results showed that editing of HBV DNA after Cas9 cleavage is at least 15,000 times more efficient that APOBEC-mediated cytosine deamination following treatment of infected cells with interferon alpha (IFNα). We also found that a previously used method to detect cytosine deaminated DNA, termed 3D-PCR, overestimates the amount and frequency of edited HBV DNA. Taken together, our results demonstrated that the CRISPR/Cas9 system is so far the best method to functionally inactivate HBV cccDNA and provide a cure for CHB.
Avatar universal
Wow! This is really a good news! Thanks for info!
Avatar universal
So cccDNA also mutates that's why Cas9 can not reach it fully ? But I guess 90% removal would indicate functional cure or at least inactive state ?
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It's in the article abstract studyforhope shared. I'm not very knowledgeable in this either, but the most impressive part is that it can functionally inactivate most cccDNA which other treatments can't do.
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Welcome! Hoping the cure will soon be available with these advances.
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Hoping the same with the 90% removal
1 Comments
A 90% removal  of cccDNA would not be  sufficient to induce a functional cure.
Avatar universal
I guess in functional cure there's usually no 100% removal ? So how much ?
Avatar universal
i think that removal needs to be close to 99% or little more to allow hbsag to be so low and pegintf or future immune modulators to wake up immune response and clear and nobody got close to this except replicor
6359077 tn?1441486304
Go Replicor!!!!!!!
Avatar universal
I thought Replicor clears only HBsAg from the body but does not touch cccDNA.
2 Comments
Replicor blocks the formation of surface antigen particles in the hepatocytes,  but not the production of the protein monomers into the ER membrane.

The retention of hbsag in the infected cells might have some positive effect in clearing such cells.

The key effect of almost complete clearance of circulating hbsag is an activation of immune mechanisms that had been blocked by the presence of hbsag. This can lead to reductions of cccDNA or lysis of infected cells.

Furthermore an existing antibody typically becomes visible or can be further enhanced by immunotherapy, causing a reduction of reinfection by trapping exiting virions into immune complexes.

All resulting reductions in cccDNA must however be finally stabilized by a permanent, effective t cell activation that blocks respreading of the virus after external therapy ends.
Replicor blocks the formation of surface antigen particles in the hepatocytes,  but not the production of the protein monomers into the ER membrane.

The retention of hbsag in the infected cells might have some positive effect in clearing such cells.

The key effect of almost complete clearance of circulating hbsag is an activation of immune mechanisms that had been blocked by the presence of hbsag. This can lead to reductions of cccDNA or lysis of infected cells.

Furthermore an existing antibody typically becomes visible or can be further enhanced by immunotherapy, causing a reduction of reinfection by trapping exiting virions into immune complexes.

All resulting reductions in cccDNA must however be finally stabilized by a permanent, effective t cell activation that blocks respreading of the virus after external therapy ends.
Avatar universal
Thank you very much for the clarifications
3 Comments
thanks
You need liver targeted gene therapy to introduce the crispr and the cas9 gene/proteins into the hepatocyte.
This would introduce a new foreign antigen into the liver cell, for which no tolerance exists. It can be expected, that after some time, an immune response will be raised and then you might have an additional endless source of hepatitis.
Also the efficacy of this system at this time is way too low to reduce the cccDNA to a level, where the surface antigen would seroconvert or the replicor type secondary effects might kick in.
https://biotechin.asia/2016/06/25/u-s-gives-green-signal-for-the-first-crispr-human-clinical-trial/
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