A new method of gene therapy – genome editing –
cures hemophilia on an animal model of the disease
LifeSciencesToday based on the materials of The Children's Hospital of Philadelphia:
Genome Editing, A Next Step in Genetic Therapy, Corrects Hemophilia in AnimalsUsing an innovative method of gene therapy, known as genome editing, which allows to influence a strictly defined section of mutated DNA, scientists cured blood clotting disease – hemophilia – in mice.
This is the first example of how genome editing – a method that precisely targets a specific target in a DNA molecule and corrects a specific genetic defect – has been applied to animals and has shown clinically significant results.
This is an important step forward in the long history of the development of gene therapy – methods of treatment based on the correction of the DNA sequence that is the cause of the disease. In the new study, scientists used two variants of a genetically modified adenoassociated virus (AAV), one of which was a carrier of enzymes that cut the DNA molecule exactly in the right place, and the other was a correcting gene that needed to be embedded in the DNA sequence for further synthesis of a normal protein. Scientists managed to do all this in the liver cells of living mice.
"Our study increases the likelihood that the genome editing method is able to correct a genetic defect at a clinically significant level after delivery of zinc fingers nucleases to the body," comments the results of her work, Katherine High, MD, hematologist and specialist in gene therapy from The Children's Hospital of Philadelphia (The Children's Hospital of Philadelphia). Dr. High, a researcher at the Howard Hughes Medical Institute Investigator, heads the Center for Cellular and Molecular Therapy at the Children's Hospital of Philadelphia and has been engaged in gene therapy for hemophilia for more than ten years.
Dr. Hai's research, conducted in collaboration with scientists from Sangamo BioSciences, Inc., used genetically modified zinc finger nucleases (ZFNs), a kind of molecular word processors that edit mutated DNA sequences. Scientists have learned how to create ZFNs that interact with genes that have a strictly defined localization. To restore the normal function of the gene lost in hemophilia, ZFNs specific to the blood clotting factor IX (F9) gene were used in combination with a normal DNA sequence.
By precisely hitting their target, a specific section of the chromosome, zinc finger nucleases demonstrate a clear advantage over traditional methods of gene therapy, which can accidentally deliver the correcting gene to an undesirable position bypassing normal biological regulatory mechanisms. Such inaccurate targeting carries the risk of so-called "insertion mutagenesis" (insertional mutagenesis), in which a correcting gene can, for example, initiate the development of leukemia.
In hemophilia, a hereditary mutation in one gene deprives the body of the ability to produce one of the blood-clotting proteins, which leads to spontaneous, sometimes life-threatening, bleeding. The two main forms of the disease that occur almost exclusively in men – hemophilia A and hemophilia B – are caused by the absence of coagulation factors VIII and IX, respectively. Treatment of patients consists in frequent intravenous infusion of coagulating proteins – expensive and, moreover, sometimes stimulating the production of antibodies by the body, which make further use of this method impossible.
Using genetic engineering, scientists have obtained mice with a model of hemophilia in humans. Prior to the start of treatment, coagulation factor IX in the blood of animals was not determined.
Previous experiments by other research groups have shown that ZFNs can edit the genome of cultured stem cells, which are then injected into mice with a sickle cell anemia model. However, such an ex vivo approach is not possible for many human genetic diseases that affect entire organ systems. Therefore, in this study, the effectiveness of genome editing was tested in vivo, that is, directly in a living organism.
Dr. Hai and her colleagues created two versions of the vector, or gene delivery vehicle, using an adeno-associated virus. One AAV vector carried ZFNs for editing, the other delivered a properly functioning variant of the F9 gene. Since the cause of hemophilia can be different mutations of the same gene, seven different coding sequences covering 95 percent of the mutations causing hemophilia B were replaced.
Mice that received a combination of "ZFNs + correcting gene" began to produce clotting factor IX in an amount sufficient to reduce blood clotting time to almost normal levels. In control mice receiving vectors either without ZFNs or without F9, there was no significant increase in the level of clotting factor or a decrease in blood clotting time.
The improvement persisted for eight months of follow–up, while the scientists did not reveal any toxic effects on the height, weight or liver function of the animals - an indicator that the treatment is well tolerated by a living organism.
"We have confirmed the very concept that genome editing performed directly in vivo allows us to obtain stable and clinically significant results," sums up Dr. Hai. "In order to translate these results into a safe and effective method of treating hemophilia and other monogenic human diseases, further research is needed, but the strategy of gene therapy seems promising. The transition from mouse models to the clinical use of gene therapy methods was a long process that stretched for almost two decades, but now we are seeing positive results in a number of diseases – from hereditary retinal lesions to hemophilia."
The development of in vivo genome editing as an effective and reliable therapeutic method will take time, but this is another milestone in the development of gene therapy.
The study was published in the online edition of the journal Nature: In vivo genome editing restores haemostasis in a mouse model of haemophilia.
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