Exploring Template-free CRISPR/Cas9 Genome Editing as a Novel Strategy for Personalized Gene Therapy of Monogenic Blood Disorders
Evolutionary Cell and Developmental Biology (Zoology)
Final Report Abstract
In the funding period to be reported here, we developed a method for a safe gene therapy based on the "gene scissors" CRISPR/Cas9. This system is a component of the adaptive immune system of prokaryotes and consists of a DNA-cutting nuclease Cas9, which binds a small RNA - the single guide RNA (sgRNA) - and is thereby guided to its target sequence. Only when these two nucleic acids DNA and sgRNA enter into a complementary base pairing, the nuclease will cut the DNA at this site. This ensures that this CRISPR/Cas9 system only generates DNA double-strand breaks (DSB) at this sequence-specific site. In recent years, this system has been applied to eukaryotic cells. In eukaryotes, DSB are repaired mainly by the most common repair pathway, which is the error-prone non-homology end-joining pathway, so that random changes occur specifically at this site and thus genes can be modified or switched off. Today, this system is a universal gene editing method that has many applications in research. In this project we used CRISPR/Cas9 to correct disease-causing point mutations in cells with the aim of curing life-threatening diseases in patients. To this end, we first generated reporter constructs with which point-mutated model genes (EGFP (fluorescent gene) and Cybb (in case of a mutation triggering chronic granulomatosis)) were introduced into hematopoietic cell lines. Subsequently, we used the CRISPR/Cas9 system to generate DSB at the exact site of this point mutations. The cell's own repair pathway now generated random changes of this sequence, so that in about one third of the cases the point mutations could be successfully corrected, which corresponds to the theoretical capabilities of this repair system. Applied to a patient, this means that in about one third of the cells we can directly correct the mutation of the gene at its genetic locus, and thus we have developed a gene therapy procedure that is safer than the gene therapies currently in use clinically. Unfortunately, we could not transfer this method to a mouse model, because very high lentiviral virus titers have to be transduced into these cells, which led to a high viral toxicity of the cells and cell death. Therefore, we successfully implemented a different approach for gene correction and achieved complete correction by combined infection of adeno-associated viruses and electroporation using CRISPR/Cas9 and synthetically produced oligonucleotides. At the same time, we have developed a novel system that allows vims transduction of Cas9 nuclease with lentiviruses without a viral genome being present in these cells, so that these viruses are only very transiently detectable in the cells and do not have a gene altering effect. Overall, we have developed techniques that should in future allow the safe gene therapeutic treatment of patients directly on the mutated gene. In the future, we will be able to circumvent the risky integration of curative gene copies at a different location in the genome, which is currently the method of choice for gene therapeutic treatment of patients.
Publications
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(2018) CRISPR/Cas9 genome engineering in hematopoietic cells. Drug Discovery Today: Technologies. 28, 33-39
Sürün, D., von Melchner, H., and Schnütgen, F.
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(2018) High Efficiency Gene Correction in Hematopoietic Cells by Donor-Template-Free CRISPR/Cas9 Genome Editing. Mol Ther Nucleic Acids. 10:1-8
Sürün, D., Schwäble, J., Tomasovic, A., Ehling, R., Stein, S., Kurde, N., von Melchner, H., and Schnütgen, F.
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(2019) Metabolic Plasticity of Acute Myeloid Leukemia. Cells. 8, E805
Kreitz, J., Schönfeld, C., Seibert, M., Stolp, V., Alshamleh, I., Oellerich, T., Steffen, B., Schwalbe, H., Schnütgen, F., Kurrie, N., and Serve, H.
