Mice cured of hemophilia
Scientists managed to cure a mouse of hemophilia using CRISPR/Cas9
Anna Stavina, XX2 century, based on Penn Medicine: Penn Scientists Use CRISPR for the First Time to Correct Clotting in Newborn and Adult Mice
CRISPR/Cas9, a powerful genome editing tool, opens up promising prospects for correcting mutations that cause diseases. Scientists from the Perelman School of Medicine at the University of Pennsylvania (Perelman School of Medicine at the University of Pennsylvania) first used this approach to treat hemophilia. They have developed a method for delivering the main components of a CRISPR/Cas9-based gene editing system to the cells of mice suffering from hemophilia B. This disease, also known as "factor IX deficiency", is caused by the absence or defect of a protein that forms blood clots. The results of the study will be presented at the 58th Annual Meeting of the American Society of Hematology (American Society of Hematology) in San Diego.
Many monogenic diseases, such as hemophilia, are caused by various changes in a single gene, and not by any one common mutation. Therefore, scientists faced the task of creating such a vector (gene carrier) that could be used in patients with various mutations. The work carried out became a preclinical test of such an approach to targeted gene exposure using CRISPR/Cas9, which would be applicable in most patients suffering from a certain disease, in particular, hemophilia B. According to the Centers for Disease Control and Prevention (Centers for Disease Control and Prevention), hemophilia occurs on average in 1 in 5,000 newborns.
"In essence, we cured the mouse," said lead author of the study Lili Wang, associate professor of the Gene Therapy Program University of Pennsylvania.
To test the new approach, the research team conducted an experiment on mice in which factor IX synthesis was turned off. Scientists used two vectors. In the first of them, the SaCas9 gene editing system was controlled by a liver-specific promoter. He ensured the delivery of the genetic "tool" to the liver itself, where factor IX is normally synthesized. The principal difference between the new study and previous experiments using CRISPR/Cas9 in the Gene Therapy Program of the University of Pennsylvania was the use of the second vector. It contained an RNA sequence targeting the 5’-end of the second exon of the factor IX gene in mice and a partial sequence of cDNA (complementary DNA) associated with the synthesis of factor IX in humans, which made the method more accurate and powerful.
The researchers used adeno-associated viruses to deliver these components to the liver cells of mice. The strategy developed by the scientists involved the use of homologous recombination based on CRISPR to embed fragments of human cDNA at the location of the gene responsible for the synthesis of factor IX in the mouse genome.
"Precise administration leads to the expression of the hybrid hyperactive factor IX protein under the control of the mouse's own promoters," Wang explained.
The administration of two vectors to newborn and adult mice with a gradual increase in doses led to the appearance of stable activity of factor IX at a normal level and above for four months. Eight weeks after gene therapy, some mice underwent surgery to remove a segment of the liver. All of them survived the procedure without complications and the need for additional interventions. The activity of the factor IX synthesis process in animals remained at the same level.
"This work confirms the effectiveness of hemophilia B treatment in a mouse model using in vivo genome editing," Wang explained.
SaCas9 is a modification of the CRISPR/Cas9 gene editing system, which uses a protein derived from Staphylococcus aureus. The original CRISPR/Cas9 system uses SpyCas9, a Streptococcus pyogenes protein. However, SaCas9 is 25% lighter than SpyCas9, which makes it possible to use adeno-associated viruses for its delivery.
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01.12.2016