CRISPR-Cas gene therapy for monogenic blood disorders

This project aims to optimize CRISPR-Cas9 technology for therapeutic use in genetic blood diseases. The goal is to develop a personalized therapy that can correct patient-specific mutations in autologous harvested stem cells, which will then be given back to the patient.


Monogenic blood diseases present with variable clinical symptoms, such as infection susceptibility, autoimmune disease, bleeding diasthesis, and early cancer. They predominantly affect children. Allogeneic hematopoietic stem cell transplant (HSCT) can cure most of the conditions, but as the procedure carries significant treatment-related mortality, most patients are treated symptomatically.

Gene therapy can circumvent most problems related to HSCT. In conventional gene therapy, a functional copy of the defective gene is inserted by a viral vector to harvested patient stem cells, which are then infused back after little or no chemotherapy conditioning. Virus-based gene therapy can, however, only treat selected gene defects, whereas the current number of causative genes for monogenic blood diseases is >500. Although very effective in certain conditions, it is not a cure for the majority of the patients.

Figure 1. CRISPR-Cas9 system. The Cas9 nuclease scans the DNA with the help of the guide RNA (gRNA). Once the pairing with DNA and gRNA is perfect, Cas9 cuts the DNA. The resulting double-stranded break is corrected by the cell’s own DNA repair mechanisms. Non-homologous end-joining (NHEJ) “glues” the DNA ends together, resulting in a small deletion. Homologous recombination (HR), in contrast, can make precise changes to DNA if a repair DNA template is provided. HR is the desired outcome for most gene therapy applications.

CRISPR-Cas is a novel gene editing technology which can correct patient-specific mutations (“precision genome editing”), and is currently used in clinical trials for HIV and cancer immunotherapy5. A single CRISPR-Cas – platform can potentially treat a large number of conditions and mutations, thus vastly outperforming the viral approach. CRISPR-Cas involves a Cas nuclease attached to a guide RNA (gRNA) that leads the nuclease to a defined genomic location, which the nuclease then cuts. The cut induces the cellular DNA damage response. The cell can repair the cut either by homology directed repair (HR) or non-homologous end joining (NHEJ) (See Figure 1). Generally, repair by NHEJ is the most common outcome, causing a small deletion and gene knockdown. Repair by HR is less frequent, but allows the incorporation of a specific mutation, if a repair DNA template is provided.

Study hypothesis

The CRISPR-Cas gene therapy technology has developed significantly in the last two years. Currently, the most efficient hematologic gene therapy system utilizes Cas9-sgRNA complex delivered as ribonucleoprotein (RNP), and repair template via adeno-associated virus (AAV) (see Figure 2A). Repair template delivery as naked DNA results in low editing efficiencies, which is thought to be due to type I interferon response triggered by the DNA and the CRISPR-Cas9 RNP.

We are developing an alternative, cheaper approach that –unlike virus-based technologies- is easy to scale into high-throughput clinical use (Figure 2B). This system utilizes a modified Cas9 nuclease that is more efficient in inducing HR and precision genome editing than wild-type Cas9 (see Fig. 3C). In addition, as HR-mediated mutation correction happens exclusively in the synthesis (S) phase of the cell cycle, we will use an inhibitor that allows Cas9 only to be active in the S phase, thus increasing safety of the technology. Furthermore, we have recently shown that the stem cell death is mainly due to p53-mediated apoptosis and DNA damage response that is caused by Cas9 cutting. Therefore, we will transiently inhibit p53 during genome editing.



Figure 2. Schematic representation of the study. Currently, the most successful gene therapy approach for hematopoietic stem cells utilizes Cas9-gRNA delivered as ribonucleoprotein complex (RNP) and repair template within adeno-associated virus (AAV) (A). In our study, we will utilize Cas9 that is engineered to induce HR in high efficiency (green dot) and carry the repair template (black). Furthermore, an inhibitor (red) is noncovalently attached to the Cas9. When the cell enters the synthesis phase, the inhibitor is degraded, the Cas9 activated and the mutation corrected. MDM2 (violet) is used to transiently inhibit p53 and the mutation corrected.


Study aims


  1. Improve CRISPR-Cas9 editing by a) fusing editing-enhancing mammalian proteins to Cas9; b) binding the repair DNA template to Cas9; c) timing editing with cell cycle and d) transiently inhibiting the cellular danger signaling to allow efficient repair instead of apoptosis and cell cycle arrest;
  2. Optimize CRISPR/Cas delivery to hematopoietic cells using electroporation;
  3. Transplant the edited stem cells to a SCID mice to determine their capacity to form a functional bone marrow;
  4. Test the system in patients with a genetic blood disorder who are ineligible for HSCT and have a poor prognosis with conservative treatment.

Work tasks

The study in preclinical stage and has multiple subprojects. The work will be tailored based on the interests of the particular student. Examples of projects include:

• Combine the already developed improvements (see Fig. 2B) and test these in hematopoietic cells

• Screen novel proteins that, when fused to Cas9, improve editing in model cell lines and eventually in primary cells

• Screen proteins and compounds that attenuate CRISPR-mediated cell danger signaling in different primary and stem cell types

• Collect cells from patients with monogenic blood diseases, edit the cells both with conventional and modified CRISPR-Cas9 system, and assess the editing outcome to see if certain conditions are particularly easy or hard to edit

• Edit healthy blood stem cells, and transplant the edited cells to SCID mouse models to assess their capacity to form a functional hematopoiesis


Research environment

We are a new laboratory which currently consists of a medically trained PI, a PhD student and a Msc student. Another PhD student and a senior scientist are about to start during spring 2019. We are located in Norsk senter för molekularmedisin (NCMM), which houses internationally recruited research groups in cutting-edge research infrastructure. The environment is very international and the working language is English. Vi har et godt samarbeid med complementary research groups in Karolinska Institute (Sweden), Sanger Institute (UK), and University of Helsinki (Finland), and the student has a possiblity visit these groups in later stages of their project. En student vil få opplæring og veiledning i et meget bredt spekter av cellebiologiske metoder og teknikker, og deltar i forskning på et basalt prosjekt som samtidig er meget klinisk relevant og interessant.

Supervisor: Emma Haapaniemi,


Centre for Molecular Medicine Norway

Gaustadalléen 21,

0349 Oslo


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Publisert 14. jan. 2020 14:29 - Sist endret 20. jan. 2020 12:48

Omfang (studiepoeng)