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In 1904, two scientists, Henry Wheeler and Treat Johnson, synthesised a variant of cytosine, one of the building blocks of DNA, in the laboratory.
The variant they synthesised contained a chemical tag called a methyl group (-CH3) attached to cytosine, so they called it 5-methylcytosine. Their motivation for doing so was based on the observation that two other building blocks of DNA and its chemical cousin RNA, called thymine and uracil, differed only by the presence of a methyl group.
So Wheeler and Johnson figured that a methylated version of cytosine might also exist in nature.
In 1925, Johnson, while working with another scientist named Robert Coghill, finally discovered 5-methyl-cytosine in the DNA of the bacterium that causes tuberculosis. Two more decades later, Rollin Hotchkiss found 5-methyl-cytosine in the DNA of cows. Soon, scientists found that it existed in nearly all animals.
However, nobody knew why it existed — or why some of the cytosines in DNA were methylated while others were not.
Genes on and off
That insight came in 1975, when Robin Holliday and John Pugh proposed that the methylation of the cytosine could be a way for the body to use the information stored in DNA.
DNA is segmented into genes, and each gene contains the information necessary to make a particular protein. These proteins perform their specific functions in the cells. Since every cell in the body has two copies — one from each parent — of all human DNA, all cells technically have the blueprint to make a whole organism.
However, cells also have specialised functions. A brain cell will not need the genes for the proteins used to digest food. So Holliday and Pugh suggested that chemical modifications such as the methylation of cytosine could help establish and activate genes that a cell wants while silencing those it does not.
This idea, that cells could use chemical tags on DNA to control which genes were active, has come to be called epigenetics. (The British biologist Conrad Waddington had actually coined the term in 1942 itself.) Soon, scientists found enzymes that could add and remove these chemical tags to and from DNA.
Today, we know that cells use many other chemical tags, not only on DNA itself but also on the proteins around which DNA is wrapped like a vine around a stick.
Together, all these chemical marks help control which genes are switched on or off. Problems in this system can lead to changes in gene activity, which in turn can lead to diseases like cancers.
Functional cure
Now, according to a paper that appeared in Nature Biomedical Engineering on September 21, scientists have used this epigenetic control to treat a hepatitis B infection.
This is a remarkable development because, instead of targeting the virus directly, the work silenced the viral DNA. This is promising because current treatments rarely eliminate the DNA, which allows the virus to rebound. By silencing this residue, the treatment could offer a path to a lasting functional cure for hepatitis B.
The hepatitis B virus mainly infects liver cells. After entering a cell, the virus’s DNA serves as the template to make new viral particles. In many people, the immune system clears the infection, but if it does not, the viral DNA can persist for years or even decades in the host’s liver.
In this time, the virus can remain silent, meaning the viral DNA is still present in the cell but its genes are not active to produce new viruses. This allows the virus to ‘hide’ from the immune system. Then, at a later point, the virus can activate again and produce new virus particles.
And studies have shown that one of the mechanisms by which the virus stays silent is by methylating the cytosines.
Locator and effector
The authors of the new study have reported a new experimental therapy called CRMA-1001, which, they have claimed, can silence the DNA of the hepatitis B virus by selectively adding methylation marks to the viral genome.
The study is innovative because it uses several different technologies to overcome three major challenges. To epigenetically silence viral DNA, scientists first need to be able to target the virus’s DNA without methylating any of the cytosines in the host’s DNA. Then, the silencing mechanism must switch off the virus’s DNA effectively. Finally, all of this should happen without causing any unwanted side-effects to the host.
The study authors solved the first two problems by combining two substances called a ‘locator’ and an ‘effector’. The locator used the CRISPR-Cas9 gene-editing tool to find and bind to a specific DNA sequence: in this case, that was a sequence within the viral DNA. This allowed the CRISPR system to take the ‘effector’, the methylation machinery, to the correct location. The effector had proteins that could add methyl groups to nearby cytosines in the viral DNA and proteins that help switch off genes.
Finally, the researchers packed the entire system and delivered it on an mRNA platform (similar to the mRNA vaccines during the COVID-19 pandemic). This made the system transient: once a cell had read the instructions to make the required proteins, it degraded them, stopping the production of those proteins too.
Novel therapy
The researchers gave mice infected with hepatitis B virus either a single dose of CRMA-1001 or three doses, one month apart, and regularly tested them for six months for the signs of infection. They reported that after one dose, the levels of several hepatitis B virus markers, including the viral DNA and two viral proteins called HBsAg and HBeAg, dropped significantly. When they tested the viral DNA, they found new methyl groups, suggesting that the treatment was working as intended. The effect was even stronger after three doses: six months after the first dose, as many as 90% of the animals had less viral DNA than could be detected.
CRMA-1001 is not a cure in the traditional sense of the word. The hepatitis B virus DNA is still present in the liver cells. Instead, the study demonstrated a functional cure: when the virus is not necessarily eliminated but is kept so thoroughly suppressed that, for all intents and purposes, the patient has no active signs of disease.
In this study, the team followed up with the mice for six months after treatment, and the researchers found sustained suppression of the virus in that period. However, the study did not establish whether the virus could eventually become active again after those six months, since the viral DNA was still present.
Further studies are needed to examine the long-term effects of CRMA-1001, including whether the silencing effect can persist over many years and whether the virus can ever become active again after treatment. An ongoing phase I/II clinical trial includes these among other questions.
If CRMA-1001 is eventually approved as a therapy for the market, it will represent a novel way to tackle a viral infection: where instead of fighting the threat, the cell ignores it.
Arun Panchapakesan is an assistant professor at the Y.R. Gaitonde Centre for AIDS Research and Education, Chennai.
