A new type of gene therapy reduced blood clotting times to nearly normal levels in live mice with hemophilia. Described as the next step in gene therapy, “genome editing” precisely targets and corrects mutated DNA. This study is the first to correct DNA in a living animal and achieve “clinically meaningful results”, said the US scientists, who write about their findings in a paper published this week in Nature.
People with hemophilia have inherited a single gene mutation that prevents their bodies being able to produce a blood-clotting protein. The lack of the protein means cuts and injuries that lead to bleeding can be life-threatening.
There are two types of the disease in humans, and occur almost exclusively in men. These are hemophilia A, where the missing protein is clotting factor VIII, and hemophilia B, where the lacking protein is clotting factor IX.
Current treatments involve frequent infusions of clotting proteins. However these can stimulate the body to produce antibodies that undermine successful treatment, and they are expensive.
For this study, the researchers used mice bred to have hemophilia B, modeling the disease in people. Before treatment, they had no detectable levels of clotting factor IX.
Lead researcher Dr Katherine A. High, a hematologist and specialist in gene therapy at The Children’s Hospital of Philadelphia, in Pennsylvania, and colleagues used two versions of adeno-associated virus (AAV, a genetically engineered virus used in gene therapy) to edit genes in the liver cells of the mice.
One version of AAV carried enzymes to cut the DNA in precisely the right spot, and the other carried a replacement gene that was copied into that exact position in the DNA sequence. This gene was a correctly functioning version of F9, that codes for clotting factor IX.
Genome editing is a step forward in the field of gene therapy because of this two-pronged method: it does not just delete or insert a gene, it corrects the disease-causing DNA sequence.
The team in Philadelphia collaborated with scientists from the University of Pennsylvania, and from Sangamo BioSciences, Inc, in Richmond, California.
Together they used genetically engineered enzymes called zinc finger nucleases (ZFNs) to edit the mutated sequences of DNA.
Thanks to recent work done on ZFNs, it is now possible to carry out “efficient genome editing in transformed and primary cells that were previously thought to be intractable to such genetic manipulation”, they write in their Nature paper.
They refer to work where researchers have used cultures to show that ZFNs can carry out efficient genome editing by inducing a site-specific double strand break in the DNA, but it was not clear whether it might be possible to do this at a “clinically meaningful level” in live animals.
High, who has been investigating gene therapy for hemophilia for more than a decade, told the press that:
“Our research raises the possibility that genome editing can correct a genetic defect at a clinically meaningful level after in vivo delivery of the zinc finger nucleases.”
In their paper High and colleagues describe how using ZFNs, custom-matched to a specific gene location, they were able to induce double strand breaks efficiently in cells in the mice’s livers. And:
“… when co-delivered with an appropriately designed gene-targeting vector, [they were able to] … stimulate gene replacement through both homology-directed and homology-independent targeted gene insertion at the ZFN-specified locus.”
By precise targeting of the site on the chromosome, genome editing avoids the risk inherent in conventional gene therapy methods of “insertional mutagenesis”, where the replacement gene ends up in the wrong place and causes an unexpected alteration, such as triggering leukemia.
In this study, the researchers replaced seven different DNA coding sequences, covering 95% of the disease-causing mutations in hemophilia B.
When injected into the mice, the ZFN/gene vectors travelled to the liver, and resulted in reduction in blood clotting times to near normal levels.
A separate group of control mice that received vectors with neither ZFNs nor the F9 minigene, showed no significant improvements in clotting times or the amount of factor IX in their bloodstream.
The improvements were still there eight months later, and the treatment was considered well tolerated because there were no toxic effects on growth, weight gain or liver function.
High, who is also a Howard Hughes Medical Institute Investigator, and director the Center for Cellular and Molecular Therapeutics at Children’s Hospital, said this study was a proof of concept, and they now need to do more work:
“We need to perform further studies to translate this finding into safe, effective treatments for hemophilia and other single-gene diseases in humans, but this is a promising strategy for gene therapy,” she added explaining it has taken a long time, nearly two decades, to move from mice to humans, but we are now seeing some promising results in a range of diseases.
“In vivo genome editing will require time to mature as a therapeutic, but it represents the next goal in the development of genetic therapies,” said High.
Funds from the National Institutes of Health and the Howard Hughes Medical Institute helped pay for the study.
Hojun Li, Virginia Haurigot, Yannick Doyon, Tianjian Li, Sunnie Y. Wong, Anand S. Bhagwat, Nirav Malani, Xavier M. Anguela, Rajiv Sharma, Lacramiora Ivanciu Samuel L. Murphy, Jonathan D. Finn, Fayaz R. Khazi, Shangzhen Zhou, David E. Paschon, Edward J. Rebar, Frederic D. Bushman, Philip D. Gregory, Michael C. Holmes, & Katherine A. High
Nature, Published online 26 June 2011.
DOI:10.1038/nature10177
Additional source: Children’s Hospital of Philadelphia.
Written by: Catharine Paddock, PhD