Gene Repair Breakthrough Led by Seattle Children’s Research Institute

Imagine a prowler casing a neighborhood, looking for a way into a home. That’s essentially what HIV, the human immunodeficiency virus, does:  It moves through the bloodstream trying to gain entry to T-cells — the primary warrior of the immune system. A special receptor on the T-cell’s surface (called CCR5) is the open door it seeks. Once it gains entry, the virus hampers a T-cell’s ability to do its job, leaving people vulnerable to infection and disease — and enabling HIV to spread.

Now imagine you can lock that door forever. The virus can’t enter the T-cells and interfere with the immune system and the body can fight off the infection.

Drs. Dave Rawlings, Andy Scharenberg and a team at Seattle Children’s are getting close to making that vision a reality. Working with colleagues at University of Washington and Fred Hutchinson Cancer Research Center in the Northwest Genome Engineering Consortium, they have figured out how to modify genes and knock the CCR5 receptor off T-cells.

Dr. Dave Rawlings, Dr. Andy Scharenberg (right)

In the latest breakthrough, the research team found a way to modify or disrupt the genes at an increased rate that is up to 25 times more efficient than existing technologies. The breakthrough has implications not only for the Research Institute, but also for scientists around the world who hope to cure chronic, debilitating diseases like AIDS.

Dr. Rawlings recently talked with On the Pulse about the study, “Coupling endonucleases with DNA end-processing enzymes to drive gene disruption,” which was published in Nature Methods in September 2012. He also discussed current funding challenges.

Q:  What is important to note about this discovery?

A:  It’s a major advance in the efficiency of being able to edit genes.  With genetic diseases, there’s a mutation in a gene.  If you revert that mutation back to the normal sequence, you essentially fix the gene and it becomes normal.  Alternatively, if a specific gene is involved in a disease, it may be beneficial to disrupt it. Genes are the basic building blocks of DNA.  They tell a cell how to behave and what to do.  Over the last five years, we have developed methods to try to manipulate the DNA so that we can edit genes.

A striking feature of our new paper is that it shows that our enhanced editing technology works not only for the homing endonucleases (HE) platform that we use here at the Research Institute, but also for the other designer endonucleases including zinc-finger and TALENS (transcription activator like effector nucleases).  The zinc-finger platform is currently being used by biotech companies including Sangamo BioSciences in human clinical trials to test modification of the gene encoding CCR5.

We think the tools created here in Seattle are more efficient and potentially safer than these other platforms.  We started with ‘nature’s scissor,’ a naturally occurring enzyme that evolved in fungi over millions of years to build our homing endonucleases.  The sole purpose of these natural enzymes is to cut a DNA sequence and insert its own DNA within the genome of these fungi.  Our paper describes using a second enzyme (along with these HEs) that chews away just a bit of the DNA so that a gene becomes permanently modified.  This allows levels of gene editing that are unprecedented.

Q: What does it mean, exactly, to disrupt genes at a rate that is 25 times more efficient?

A: This increased efficiency should allow us to edit genes in cells much more efficiently, allowing us to translate this approach into clinical practice.

Q:  Based on your research, what applications do you see for use of the technology?

A:  We know that this will work well in T-cell engineering. In cancer immunotherapy, researchers likely need to disrupt two or more genes that might limit the effectiveness of the engineered T-cells.  We call that multiplexing.  To be successful in editing multiple genes, we need a high level of efficiency at each step. Currently, scientists working on editing genes are not achieving very high levels of efficiency.  Adding this technology will increase the efficiency and the gene disruption in T-cell or stem cell engineering, in plant engineering and all of the other scientific realms for gene editing.

Q:  What’s next?

A: The tools we have developed have the potential to improve the immune system’s ability to fight many diseases, including leukemia and brain cancer and to treat patients with HIV.

In terms of what we found in the published paper, we are already focusing on using this technology to edit the CCR5 gene. We believe that our work will provide a more efficient, clinically viable way to treat patients with HIV.  We are, in fact, already working on that.

We’re going to deliver the enzyme in two different ways.  One is RNA transfection, in which we put in the RNA that codes for the enzyme directly in the cells.  In the other method, we use a virus to shuttle in the enzyme and express it for a short period of time in the cell.  We have already identified and made recombinant virus that effectively does this in hematopoietic cells, which form all blood cells and T-cells.  So from there, we’ll aim to show that we can do this with high efficiency, and show that those cells are resistant to HIV.  We will do that work collaboratively with a lab at the Fred Hutchinson Cancer Research Center.

Finally, we also aim to continue taking what we’ve learned to begin to fix mutant genes.  While this is technically much harder to do than disrupting genes, it has enormous potential. If you can fix genes, there are hundreds of additional diseases that you can treat.

Q:  What is the current funding situation for your work?

A:  We’re actively continuing this work now, even though the National Institutes of Health no longer funds us for these studies.

Like many researchers in the United States, we are at a precarious financial juncture.  Our research team at the Northwest Genome Engineering Consortium was not allowed to renew our five-year $25 million grant from the NIH.  So we are now applying on an individual basis from each of our labs, and that takes an incredible amount of time.  It’s also very frustrating, given the gains that we’ve made, though we know we’re not alone in the scientific community in feeling that frustration.

One key goal is to move towards building clinical trials.  We will closely parallel what is currently being done in the Sangamo HIV trial except that we will use our dual-enzyme technology and a different delivery system. We expect to have a higher efficiency of gene disruption and greater clinical benefit using our new technology.  Our timeline for human clinical trials is in the range of two to four years from now.

Dr. Michael Certo, Seattle Children’s Research Institute, is the lead author of the Nature Methods paper. While a graduate student at the University of Washington, Dr. Certo and a team of researchers invented a technology called Traffic Light, which has the ability to measure different DNA repair pathways by turning the cells different fluorescent colors.  Traffic Light allows researchers to understand how cells repair DNA, and importantly, how to control the system to achieve efficient and precise genome editing following the induction of a DNA break.  The Traffic Light and a novel coupling of two enzyme types (e.g., endonucleases with exonucleases) is described in this recent Nature Methods paper.

Dr. Certo was supported in part by Public Health Service, National Research Service Award, T32GM07270, from the U.S. National Institute of General Medical Sciences.  Additional funding for this research was from NIH (RL1CA13382, UL1DE019582, RO1-HL092557, RL1-HL092553, RL1-HL92554 and U19-AI96111) and Seattle Children’s Center for Immunity and Immunotherapies.

Dr. Scharenberg is an employee of Cellectis, and receives a salary and equity compensation. He is also co-founder and a board member of Pregenen, Inc., a genome engineering company.