Inženirstvo spreminjanja plazmatk s popravljanjem primarnih človeških celic B na osnovi homologije

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Haemophilia B is a disease caused by a deficiency in the secretion of coagulation factor IX (FIX) which results in the impaired blood coagulation cascade[1]. Thus, the disease is manifested by blood dotting defects. Considering the plasma cells’ ability to produce de novo protein, it opens up an opportunity for the production of deficient protein and thus the potential cure for protein-deficiency diseases. This review article will focus on the methods used to investigate the ability of activated B cells differentiated from primary naive human B cells to produce functioning factor IX by CRISPR/Cas9 genome editing tool and homology-directed repair (HDR) [2].


Optimisation procedure

Prior to testing efficacy of B cells genome editing, respective optimisation of the experimental conditions had to be made. The latter has consisted of four crucial steps, namely:

  1. Optimization of cell expansion conditions – by the cultivation of B cells with the “B cell activation cocktail” approximately 36-fold expansion was achieved
  2. Screening the successfulness of construct insertion – two specific genome regions were selected for editing with RNP complexes containing respective guidance RNAs. Site-specific insertion was confirmed by Illumina sequencing.
  3. Screening the influence of genome editing region on the primary naïve human B cell differentiation – altogether, 5 genomic regions were selected: four of them are important for B cell differentiation (IRF4, PRDM1, PAX5, BACH2). The fifth region, CCR5, has no significant influence on B cell differentiation. By disruption of the listed regions, their function was confirmed and thus appropriate region for insertion of factor IX sequence could be selected. Cell differentiation level was measured via flow cytometry.
  4. Selection of adequate template donor vector – scAAV was designed with inserted GFP sequence driven by MND promoter. GFP expression was measured by flow cytometry, meanwhile, minimal loss of cell viability was observed. B cells transduced with AAV serotype 6 showed the highest mean fluorescence.[2]

Testing the efficacy of genome editing

The genome of primary human B cells was edited in terms of insertion of two genes – one for factor IX (whose production was the main goal of the research) and the other for BAFF (B cell activating factor)[2].

Insertion and expression of factor IX

The region used for genome editing was CCR5 region of the B cells’ genome for which it was shown (in prior experimental conditions testing) that it has no significant effect on B cell differentiation if edited. Two parallel experiments were conducted, the one containing only Cas9 RNP with gRNA for the respective region and AAV with donor template for insertion of FIX and the other, where under the same conditions of FIX insertion, PAX5 region was inhibited to induce a higher rate of B cell differentiation. However, no significant difference in differentiation was shown. The insertion of factor IX nucleotide sequence was successful, as well as the expression of the respective protein in comparison to control. The presence of produced factor IX was determined by ELISA method. The activity of produced factor IX was tested afterwards with chromogenic assay and high-specific-activity has been shown in the vitamin K supplemented culture[2].

Insertion and expression of BAFF

For BAFF it is known that it provides activation and survival of B cells. The idea is that naïve primary B cells would be doubly edited, which means that while expressing functioning factor IX, it would at the same time produce enough BAFF for its own sustainment by autocrine secretion. The expression of BAFF was determined by ELISA method[2].


In this research, conducted by Hung et al., B cells were successfully used as a “cell factory” for production of active factor IX. Since the method of editing of B cells’ genome was CRISPR/Cas9 editing, the significance of the research lies in the opening opportunity for further usage the respective method to edit B cells’ genome for production of some other protein whose deficiency is causing anomalies. Also, optimisation conditions for the successful experiment were determined which sets up the appropriate ground for further research in this field such as studying mutations which may cause autoimmune diseases.


  1. Nathwani et al. (2011). Adenovirusassociated virus vector-mediated gene transfer in hemophilia B. N. Engl. J. Med. 365, 2357–2365
  2. 2.0 2.1 2.2 2.3 2.4 Hung et al. (2018). Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. Mol. Ther. 26(2), 456-467.
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