German gene-synthesis firm Geneart recently won a patent from the German Patent and Trademark Office for its antiviral drug-screening system based on synthesized, virus-like reporter genes.
Marcus Graf, operations manager and head of the gene-synthesis business unit at Geneart, and his colleagues reengineered the gene from the jellyfish, Aequorea victoria, that encodes the green fluorescent protein so that it transfers its mRNA from the nucleus to the cytoplasm of a cell, in a manner similar to that of the HIV virus.
“That is the basis of our patent,” Ralf Wagner, CEO and CSO of Geneart, told CBA News this week. “We have a reporter gene that behaves like a viral gene and produces an RNA, which needs the cooperation of the viral shuttle and similar proteins that together mediate the export of that GFP reporter from the nucleus to the cytoplasm.”
Small molecules that interfere with this mRNA export machinery and prevent the RNA from being exported from the nucleus into the cytoplasm will result in cells that do not light up in an optical detection system or a fluorescence-activated cell sorter.
It is likely that if a drug candidate effectively blocks this pathway and prevents the synthetic HIV-like gene from transferring its material to the cytoplasm, “you can be sure that you would also have interfered with the viral replication machinery,” said Wagner.
In order to be exported from the nucleus into the cytoplasm, the RNA requires a tertiary structure called the ref responsive element, said Wagner explained. A viral protein, ref, binds to the RNA secondary structure and transports the nuclear RNA into the cytoplasm, where the RNA is translated.
The ref binds to the RNA secondary structure and forms RNA oligomers. In an energy-dependent manner, this RNA is transported from the nucleus to the cytoplasm.
“We learned from our previous work that if we altered the codon usage in the viral genes, for example in the viral gag gene, that the RNA behaved quite differently,” Wagner explained.
He said that the RNA with the altered gene is stable and is constitutively transported from the nucleus to the cytoplasm like conventional mRNA and also like the RNA encoding the GFP protein.
“Our idea was to do a reverse experiment and alter the coding sequence of the GFP so that it looks like a viral sequence,” said Wagner.
Cloning that altered GFP DNA into a cDNA vector and transfecting it into cells results in an RNA that is unstable in the nucleus, Wagner said. “When we used untranslated viral sequences and fused them to the altered modified GFP RNA, this RNA was stable and accumulated in the nucleus,” he added.
“Our idea was to do a reverse experiment and alter the coding sequence of the GFP so that it looks like a viral sequence.”
When the researchers added the 3’ ref responsive element and co-transfected these cells with the ref protein, the ref protein again bound in a cooperative manner to the ref responsive element and exported the RNA from the nucleus to the cytoplasm, he said. “What we achieved was the modification of the RNA of the GFP reporter gene in such a way that it behaves as the viral RNA.”
In other words, after transfection of the modified RNA with the 5’ and the 3’ cis-acting sequence elements into cells, in the absence of ref, the GFP is not active. If co-transfected with the ref protein, the RNA is shuttled from the nucleus to the cytoplasm, translated into the GFP protein, and can be measured by fluorescent techniques or can be monitored in a fluorescence-activated cell sorter.
This technique can be applied to other lentiviruses such as SIV-2, for example, Wagner said. “We did not test, but we are … sure, that this technology is applicable to RNA viruses.”
The reporter construct with the modified GFP gene and the 5’ untranslated sequence and a 3’ cloning hybrid can also be used to do shotgun cloning of pathogen-derived sequences in order to identify the cis-acting sequences that help export pathogen-related RNAase from the nucleus to the cytoplasm, said Wagner.
“That is a reporter system that also helps you to identify regulatory sequences which support the export of pathogen-specific RNAase from the nucleus to the cytoplasm,” he said. “Once you have identified those sequences, you can develop antiviral agents against them.”
Geneart has not yet commercialized this technology, said Wagner. “Following our announcement, two or three biopharma companies contacted us requesting more detailed information, and expressed an interest in starting to negotiate with us.”
Geneart does not intend to develop or screen drugs in-house, Wagner said. “We are more interested in negotiating with and partnering with biopharma companies who may be interested in using our technology for screening purposes.”
The global market for antiretroviral drugs could potentially be worth $500 million to $800 million, said Wagner, though he noted that the tools sector is only a small part of that market. “I would rather stress the fact that this is a basic technology platform that helps you identify novel targets for antiviral therapies.”
Most current antiviral drug-discovery efforts focus on highly specialized viral enzymes, such as reverse transcriptase, protease, integrase, and RNAase, and in the case of influenza, neuraminidase. They are not focused on other components of the viral replication machinery.
Anitvirals targeting the RNA export machinery could become a complementary therapy to those targeting viral enzymes, Wagner said.
Another company that is looking at novel mechanisms of action for antiviral compounds is Trana Discovery. In January, Trana announced that its HIV high-throughput screening assay, designed to identify compounds that inhibit the use of tRNA, which is required for HIV replication, had identified compounds with anti-HIV activity (see CBA News, 1/25/08).
At the time, Trana said it plans to license the assay to pharma companies that will use it to screen “several hundred thousand, maybe even millions, of compounds.”
The biomolecular interaction from Trana’s assay uses two synthetic RNA mimics, CEO Peterson told CBA News at the time: one RNA oligomer comprising 12 bases mimics the human tRNA lysine sequence and the other oligomer mimics the HIV genomic sequence.
He said the assay screens for compounds that inhibit the binding of the fluorescently labeled human tRNA with the biotin-labeled HIV genome in the presence of PerkinElmer AlphaScreen beads.
Wagner and Graf founded Geneart in November 1999. The company employs more than 190 people at its offices in Regensburg, Germany, and Toronto. According to its Web site, Geneart offers services ranging from the production of optimized synthetic genes and the generation of gene variants to the production of DNA-based agents.
The company has been listed on the German stock exchange since 2006.