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Gene Editing Services

StemEdit – Clinical Gene Editing Service

Thanks to the discovery of CRISPR-Cas9, gene editing is more accessible than ever before. However, some genetic modifications remain challenging and there are even more factors to consider if your cells are intended for clinical use. By outsourcing your clinical gene editing to REPROCELL, you can achieve the genotype you need before moving on to expensive cell bank manufacturing processes.

Our StemEdit service uses advanced CRISPR-SNIPER* gene editing technology to develop your engineered stem cells. Due to the increased screening specificity of CRISPR-SNIPER, we can successfully achieve complex genetic edits with high accuracy at an early stage. By evaluating the percentage of target cells and determining their transfection efficiency (go/no-go decision point) StemEdit saves time as the SNIPER pre-screen is performed before laborious clone selection.


*SNIPER = Specification of Newly Integrated Position and Exclusion of Random-integration. Note: CRISPR-SNIPER modified cells are developed, manufactured or supplied by GenAhead Bio Inc. under license from ERS Genomics Limited and Broad Institute.

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Case study:
HLA Disruption in clinical Seed Stock iPSCs

One limitation of using iPSCs clinically is immunogenicity caused by Human Leucocyte Antigen (HLA) mismatching, which can reduce the in vivo survival and therapeutic efficacy of these cells.1,2 The immunogenicity of iPSCs can be reduced by disrupting the genes responsible for immune recognition using CRISPR-Cas9. One approach is to knock out the B2M (β2 microglobulin) and CIITA (major histocompatibility complex [MHC] II transactivator) genes.

In humans, the B2M protein is required for the presentation of HLA protein A-G on the cell surface, while CIITA is essential for HLA-II transcription.2,3 Dual knock-out of the B2M and CIITA genes disrupts the presentation of MHC-I/MHC-II proteins in iPSCs, improving their therapeutic potential while maintaining pluripotency.1StemEdit -- clinical B2M figure 1a

StemEdit -- clinical B2M figure 1b

Role of B2M and CIITA in the regulation of the immune response. B2M regulates the activation of effector T cells. B2M knockouts do not activate effector T cells, leading to a reduced immune response. CIITA regulates the expression of the HLA-II complex. Knockout of CIITA leads to a reduced expression of HLA-II, and a reduced immune response.

References

  1. Wang X et al. Diminished expression of major histocompatibility complex facilitates the use of human induced pluripotent stem cells in monkey. Stem Cell Research & Therapy 11:334 (2020).
  2. Xu H et al. Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility. Cell Stem Cell 24 pp566-578 (2019).
  3. Deuse et alHypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipientsNature Biotechnology 37 pp 252–258 (2019).

At REPROCELL, we offer this knock-out above as a service. The diagram below shows our workflow for the double knock-out of B2M and CIITA genes, with phase contrast images of the Seed-Stock iPSCs at each stage.

Workflow for Clinical Gene Editing

iPSCsiPSC line
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DNA manipulation plastic modelGene editing CIITA & B2M
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B2M CIITA knockout iPSC ineB2M/CIITA knockout iPSC line

HLA expression was measured by flow cytometry in iPS cell lines with double knockout of B2M and CIITA genes. Since the regulation of Class II expression occurs in post-differentiated cells, the expression of Class II is compared in macrophages induced to differentiate from iPS cells (right: using anti-HLA-DR antibody). The expression of HLA of both Classes is suppressed in the B2M/CIITA knockout line. These results indicate that the generation of iPS cells to suppress the immune rejection is possible with genome editing technology. Other gene editing services are also available, so please contact us if you have any requests using the inquiry form below.

Since HLA's mediate the immunological response to therapeutic cells, these B2M/CIITA knockout iPSCs are a perfect place to start the development of cellular therapeutics.

Creating hypoimmune clinical iPSCs by B2M/CIITA double knock-out

Clinical Stem Cells - B2M CIITA Knock Out Macrophages

HLA Class I and II expression in B2M/CIITA double knock-out hypoimmune cells. HLA expression was measured by flow cytometry in unedited parental iPSC line and after knock out of B2M and CIITA genes. Expression of HLA I and II is suppressed in B2M/CIITA knockout lines. Results indicate that the generation of iPSC for the purpose of suppressing an immune rejection is feasible with Crispr SNIPER gene editing technology.
A) HLA Class I expression was measured in the parental iPSC-derived macrophages by flow cytometry. 
B) Undifferentiated iPSCs (“Parental iPSCs”) do not express HLA II. Expression of HLA Class II occurs only in cells post differentiation. Expression of HLA II gene HLA-DR was therefore measured in iPSC-derived macrophages.

Interested in finding out more about StemEdit?

If you are interested in finding out more about REPROCELL's StemEdit Clinical Gene Editing Service, please make an inquiry using the form below. Our stem cell experts will be happy to arrange a free consultation to discuss your project in more detail. For further information about our clinical iPSC services, please refer to our Clinical iPSC FAQ page