Viruses such as HIV and herpes simplex virus (HSV) are especially pernicious because they are able to “hide” in our bodies, persisting only in DNA form. Approaches to treat or prevent viral infections exist, but to cure an existing infection of this type means permanently altering the viral DNA hidden in human cells. VIDD senior staff scientist Dr. Martine Aubert, along with other researchers in VIDD associate member Dr. Keith Jerome’s group, are working on a novel technique to do just that. Using enzymes that are specially engineered to recognize and cleave viral DNA sequences, the researchers hope to develop a lifelong cure for HIV and other viral infections.
“For viruses like HIV, herpes or hepatitis B virus, there are treatments for the infections but because the virus can hide in the body, sooner or later it can come back,” Aubert said. “These treatments cause the viruses to go latent, or silent, but they don’t remove the virus from your body. Our goal is to find a therapeutic cure for latent diseases.”
Viruses that establish lifelong infections leave behind copies of their own genome in human cells, where our cellular machinery will later make new viral particles from these DNA instructions. In the case of HIV, the virus’ DNA is woven into our own chromosomes. HSV’s DNA is self-contained in a ring inside human cells’ nuclei, but it is still able to hijack the host’s protein-making abilities. Any successful cure of viral infection must either kill off all the human cells that contain viral DNA, which would likely be extremely toxic or lethal to the patient, or slice out or permanently damage this DNA while leaving the human DNA intact.
Aubert is pursuing this latter approach using homing endonucleases, proteins that target and cleave a very specific DNA sequence – each homing endonuclease type targets one specific sequence. In nature, these proteins exist in microbes and insert a copy of their own DNA into the gap that results from cleavage. To target viral DNA, Aubert and Jerome are working with collaborators Dr. Barry Stoddard, member in the Basic Sciences division, and Seattle Children’s researcher Dr. Andrew Scharenberg to engineer homing endonuclease proteins to specifically recognize HIV, HSV, or HBV DNA. In human cells, when these enzymes cut DNA, our cellular repair machinery will try to rejoin the broken ends. This repair is prone to error, and eventually this rejoining process will introduce a mutation into the viral sequence.
“Our strategy is to attack the latent virus and introduce mutations into its genome that will cripple the virus when it starts to wake up,” Aubert said. “In theory you would no longer need the antiviral treatment because the virus is no longer functional.”
To prove that this could be done, Aubert created a vector derived from HIV containing a target sequence for an existing homing endonuclease. She “infected” human cells in the laboratory with this modified vector, which also contained a fluorescent reporter to indicate whether the desired sequence had been cut, and then introduced the homing endonuclease. She found that the enzyme could cripple the virus sufficiently so that it could no longer produce proteins, without harming the cells. Aubert is currently completing experiments testing the technique in a similar in vitro model for HSV infection.
To date, Aubert’s experiments have involved modifying viral DNA to include the target sequence of an existing homing endonuclease. Next up, she said, is creating the modified enzymes to recognize a conserved region of HIV or HSV. Since HIV mutates very rapidly, a successful approach to disable this virus will most likely require a handful of homing endonucleases; each engineered to target a different segment of the genome. HSV mutates more slowly, but also contains more non-essential genes, Aubert said. The approach to disable HSV will have to make sure the target gene will completely cripple the virus without causing toxic side-effects to the human cells. Soon, the group hopes to begin testing these modified enzymes in animal models of the different viral infections, Aubert said. Scientists in Jerome’s group are also exploring two other types of modified enzymes, zinc finger nucleases and TAL effector nucleases, in a similar approach.
The group’s work to cure HIV and other viral infections using targeting enzymes has garnered a lot of attention: last summer, Jerome and Clinical Research Division member Dr. Hans-Peter Kiem received a $20 million grant from the National Institutes of Health to explore the applications of this technology to cure HIV. See more about this project at http://defeathiv.org/.