Scientists successfully combined the gene-editing tool CRISPR-Cas9 and a special type of virus to deliver corrected genes to restore the function of the coagulation factor IX (FIX) gene in young mice with hemophilia B.
The study, “Long-term correction of hemophilia B using adenoviral delivery of CRISPR/Cas9,” was published in the Journal of Controlled Release.
Hemophilia is a genetic, life-threatening blood disorder that affects the body’s ability to make blood clot to prevent excessive bleeding. In hemophilia B, this inability of the blood to clot is caused by the lack of a specific clotting protein, called factor IX (FIX), due to mutations in the FIX gene.
Because hemophilia B is caused by defects in a single gene (monogenic disorder), it has become one of the primary targets for gene therapy, which encompasses a group of techniques whose main goal is to correct the faulty gene causing a disease.
Despite the success of these gene therapy techniques in clinical trials involving adult patients with hemophilia B (NCT02396342; NCT02484092; NCT00979238), the “application of these strategies to [children and adolescents] is limited due to high cell turnover [cycle] as young patients develop, resulting in vector [corrected gene] dilution and subsequent loss of therapeutic expression.”
There is a way, however, to overcome this limitation: the use of gene-editing tools, such as CRISPR-Cas9, that allow the insertion of the corrected gene into the genome of ill individuals. Once the corrected gene is successfully inserted into the genome (all the genes present in our DNA), it is replicated each time a cell divides, therefore ensuring it will never be “diluted” and its therapeutic effect lost.
In this study, a team of researchers from the Washington University School of Medicine set out to test the efficacy of a special type of virus, called adenovirus, as a platform to deliver the CRISPR-Cas9-corrected FIX gene to mice with hemophilia B.
The advantage of using adenovirus and not other types of virus, such as retrovirus or lentivirus, for gene editing is that the adenovirus allows genome integration in a more specific manner and could be more suitable for therapeutic purposes.
To test the efficacy of CRISPR-Cas9 together with adenovirus gene delivery, investigators injected all the constructs intravenously into young mice (4 weeks old) lacking a functional mFIX (the mouse equivalent of the human FIX gene) and monitored the levels of mFIX in the animals’ plasma continuously for 238 days.
Results showed that animals injected with the constructs maintained high levels of mFIX in the plasma throughout, compared to animals injected with non-editing constructs (untreated controls).
After the follow-up period of 238 days, researchers measured FIX enzymatic activity and the time required to stop bleeding induced by a tail cut in healthy animals, mice injected with non-editing constructs, and mice injected with gene-editing constructs.
Results showed that mice injected with functional gene-editing constructs had higher FIX enzymatic activity compared to the controls, although much lower than healthy animals. In addition, animals injected with gene-editing constructs required less time to stop the bleeding (approximately 15 minutes), while controls bled far longer (approximately 25min) and some animals failed to stop the bleeding. Healthy animals stopped the bleeding much faster than all the other groups (approximately 8 minutes).
Further analysis revealed that some animals injected with gene-editing constructs acquired unintentional mutations in their genome, and others mounted an immune response against the constructs.
“In summary, this proof-of-principle study shows [that] long-term phenotypic [symptoms] correction was achieved using our straightforward approach … supporting the further development of editing-based strategies for treatment of pediatric inherited plasma deficiencies. We highlight that therapeutics involving CRISPR/Cas9 are certainly viable, but preclinical studies should recognize and address potential immune and genetic consequences that may occur post-treatment,” the scientists concluded.