Therapeutic twin prime editing of hematopoietic stem cells for TINF2 dyskeratosis congenita

Background

Dyskeratosis congenita (DC) is a rare, genetic form of bone marrow (BM) failure. It is estimated that one in one million people has this condition. About 50% of the people who have DC have changes in the genes that are important in telomere maintenance, e.g. DKC1, TERT, TERC or TINF2. TINF2, which encodes the TIN2 protein, is mutated in 12% of patients and thereby the second most frequently altered gene in DC cases. Variants in TINF2 have been associated with a cancer predisposition syndrome (CPS) in which melanoma, thyroid cancer, and sarcoma predominate. The technology developed in this study, if applied, may also assist the early intervention and treatment of certain types of CPS.

The classic form of DC is characterized by mucocutaneous abnormalities, BM failure, and a predisposition to cancer. The leading cause of death in DC patients is BM failure, and hematopoietic stem cell transplantation is the only definitive intervention to restore hematopoiesis, with the risk of graft-versus-host disease, general morbidity and frequent difficulties in finding matched donors.

Recently developed CRISPR/Cas9 technology facilitates targeted, template-guided correction of disease-causing genetic variants, making ex vivo Cas9-directed gene correction a potential treatment of various blood disorders. However, the complexity of ex vivo handling of stem cells in combination with gene editing renders this technology an expensive and time-consuming process. However, methods for in vivo genome editing lack a safe, targeted, and potent delivery system. Safety of in vivo gene manipulation is further compromised by excess by-product formation (indels, translocations, and inversions), potential chromothripsis, extensive off-target effects, and toxicity related to Cas9-induced DNA double-stranded break (DSB).

The CRISPR/Cas9 system based prime editing (PE) technology was reported in 2019. It is capable of installing all small types of alterations in the genome without relying on donor templates or DSBs, therefore resulting in little cellular toxicity and virtually no indel formation, off-target editing, or by-stander mutations. Although several generations of improvements have been applied to the PE technology, it is still challenged by the complexity of designing and synthesizing longer engineered pegRNAs. The present application improves the current PE technology by introducing paired pegRNAs, thus allowing longer range of genome editing without using long engineered pegRNAs. The use of paired pegRNAs also enhances editing efficiency and precision, making it better than the existing PE2 sytem which edits only one DNA strand and PE3 system which incorporates a nick-single-guide RNA (sgRNA) targeting the opposite DNA strand.

Technology Overview

Dr. Bauer’s group at Boston Children’s Hospital employed gene editing to restore the TINF2 gene function and hence cure the diseases. In particular, the TINF2 gene encodes for the central component of the shelterin complex, a protein complex that protects telomeres, and dominant gain-of-function mutations in a small region of exon 6 encoding for 30 amino acids. The group developed a mutation-agnostic twin prime editing (TwinPE) method to recode the 30 amino acids region known as the DC cluster, which could theoretically correct any pathogenic mutations causing this severe telomere biology disorder. Different pairs of engineered prime editing guide RNAs (epegRNAs) have been designed and the codons of the DC cluster optimized to decrease the homology between the prime editing DNA flaps and the targeted genomic locus, allowing highly efficient TwinPE to recode the DC cluster and restore the original TINF2 amino acid sequence.

Applications

• Treatment of dyskeratosis congenita.

Advantages

• Currently, treatment for this condition is mainly supportive but the presented method proposes a cure to restore function of the original gene.

Publications

• Yan, J., Oyler-Castrillo, P., Ravisankar, P., Ward, C. C., Levesque, S., Jing, Y., Simpson, D., Zhao, A., Li, H., Yan, W., Goudy, L., Schmidt, R., Solley, S. C., Gilbert, L. A., Chan, M. M., Bauer, D. E., Marson, A., Parsons, L. R., & Adamson, B. (2024). Improving prime editing with an endogenous small RNA-binding protein. Nature, 628(8008), 639–647. https://doi.org/10.1038/s41586-024-07259-6
• Jonas Holst Wolff 1, Jacob Giehm Mikkelsen (2023). Prime editing in hematopoietic stem cells—From ex vivo to in vivo CRISPR-based treatment of blood disorders. Front Genome Ed. 2023 Mar 10;5:1148650. doi: 10.3389/fgeed.2023.1148650.