Genome editing using the still-experimental CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) method is a potentially faster, easier, cheaper, more accurate, and more flexible therapeutic approach than other DNA editing techniques, and may offer a promising new technology for treating genetic diseases such as hemophilia.

Researchers design guide RNA (gRNA) sequences that pair with the gene they want to alter and use them to program Cas9 enzymes that will function as molecular scissors to cut at the desired spots, adding or removing DNA as needed.

Hemophilia treatment using CRISPR/Cas9

Hemophilia is caused by different mutations in the DNA sequence so, for a CRISPR/Cas9 treatment to be effective, researchers from the Perelman School of Medicine at the University of Pennsylvania had to develop a vector that would be applicable for people with hemophilia with any DNA mutations. They came up with a dual gene therapy approach to deliver the key components of the CRISPR/Cas9 method to hemophilic mice.

Mouse models of the disease were used to confirm this approach. The clotting factor IX was removed from the DNA of these mice. Then, they built a two-vector RNA sequence, where vector 1 expressed the SaCas9 gene driven by a liver-specific promoter, as the liver is where factor IX is produced, and vector 2 contained an RNA sequence that targeted a region at the 5-prime end of exon 2 of the mouse factor IX gene and a partial human factor IX cDNA sequence, for more potency and accuracy. This vector 2 inserted the human sequence of factor IX into the mouse genome.

Researchers then injected the two vectors with increasing doses in newborn and adult mice without the clotting factor IX. These mice had a stable factor IX activity at normal or above for more than four months. Eight weeks after having the double vector treatment, randomly selected mice had a partial liver removal; all of them survived without complications and expressed factor IX at similar levels.

This preclinical proof of concept study demonstrated that the CRISPR/Cas9 method might benefit humans with hemophilia.

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