Deeper Dive in Cell Stem Cell: Confirming Function of CRISPR-Edited Cells with IsoPlexis’ Functional Immune Landscaping

  • CRISPR is a gene editing method that uses the Cas9 protein and guide RNAs to cut and insert sequences at precise spots in DNA and has quickly become the standard in gene editing.
  • One challenge in developing CRISPR-edited therapies is that the technology doesn’t have a way to confirm function post-edit. IsoPlexis’ functional proteomics characterizes the cytokines released by cells and their effect on the surrounding immune response before and after CRISPR edits, vastly improving researchers’ ability to evaluate the function of CRISPR-edits.
  • IsoPlexis also helps researchers identify if a potential therapy is highly potent, low in cytotoxicity or exhibiting undesired effects. In a study published in Cell Stem Cell, researchers Zhu, et al. used IsoPlexis’ single-cell proteomics to evaluate the function of CRISPR-edited NK cells, where they found that the edited cells showed improved in vivo persistence, potency, polyfunctionality, and anti-tumor activity against acute myeloid leukemia.

Why Functionally Profiling CRISPR-Edited and Engineered Cells is Critical

Gene-editing is a powerful tool for developing cell therapies, with applications across a wide range of research areas. CRISPR is a gene editing method that uses the Cas9 protein (CRISPR associated protein 9) and guide RNAs to cut or insert sequences at precise spots within DNA. Compared to other commonly used gene editing techniques, CRISPR offers a cheaper and more accessible alternative and has quickly become the standard in gene editing.

While CRISPR is an impactful tool that facilitates critical research discoveries, one challenge in the development of CRISPR-edited therapies is that the technology doesn’t have a way to confirm the function of the gene edits. While genomics is typically used to analyze edited and engineered cells, it can only confirm that the edit was made properly. Thus, if you are evaluating the potential of CRISPR-edited cells for use in cell therapies, functional profiling is necessary to determine if the cells are functioning as predicted prior to using them as a therapy product. Single-cell functional proteomics is the only way to obtain this type of direct cellular information, rather than estimating with traditional technologies such as ELISA and flow cytometry.

IsoPlexis’ functional proteomics technology uniquely connects each individual cell to a full range of relevant, secreted cytokines. With the ability to measure the direct function of individual immune cells in response to a CRISPR-edited therapy, researchers can predict patient outcomes, identify additional potential therapies, and develop novel therapies using data obtained from the IsoPlexis platform.

Furthermore, IsoPlexis’ Polyfunctional Strength Index, or PSI, has helped researchers identify highly functional and potent immune cell subsets which have correlated to in vivo outcome. PSI multiplies the proportion of polyfunctional cells in a sample with the average signal intensity of the secreted proteins from individual functional groups within each cell. This powerful and unique metric has a wide range of applications in engineered immune cell therapy research, development, and beyond. In CRISPR-edited cell therapies, the metric is being used in pre-clinical discovery to understand correlations with in vivo response and confirm the ability of a therapy to maintain potency without undesired functional responses, post-edits. This metric can also identify the most potent and effective cells, allowing researchers to make informed decisions within the development of their cell therapy programs and accelerating the advancement of their therapies.

Polyfunctional CRISPR-Edited NK Cells Demonstrate Increased Anti-Tumor Response Against Leukemia

In a Cell Stem Cell publication, researchers Zhu, et al. used IsoPlexis’ single-cell proteomics to test the anti-tumor effect of CRISPR-edited NK cells against leukemia. NK cells have the ability to kill tumor cells or cells infected with viruses without any stimulation from antigens, making them ideal to use in immune therapies. However, due to their tendency to not last very long in vivo, their anti-tumor response is short lived. In order to overcome this challenge, researchers created a sustainable supply of NK cells by deriving them from induced pluripotent stem cells (iPSCs). They then used CRISPR-editing to engineer the NK cells to further improve their anti-tumor function before using IsoPlexis’ single-cell proteomics to characterize the function of the CRISPR edit.

Researchers Zhu, et al. identified a gene that they predicted would improve the function of the engineered NK cells. Using CRISPR, they deleted CISH, the human gene for cytokine-inducible SH2-containing protein (CIS), which is a critical negative regulator for IL-15 signaling within NK cells. In order to confirm the function of these CRISPR-edited cells, Zhu, et al. utilized IsoPlexis’ single-cell proteomics to analyze these novel cells and discover if the CRISPR edits really did improve cellular function. These CRISPR-engineered NK cells were found to have increased persistence compared to wild type NK cells.1  When these cells were tested against acute myeloid leukemia, the CISH-KO iPSC-NK cells also demonstrated enhanced anti-tumor function and persistence in vivo compared to the wild type NK cells. The CISH-KO iPSC-NK cells were more polyfunctional and functionally potent than the wild type and PB-NK cells, correlating with increased anti-tumor activity. 1 IsoPlexis’ technology was also able to verify that the observed increased function resulted from the CRISPR edits.

IsoPlexis’ Platform Identifies Functional Changes Due to Gene Edits to Accelerate Therapy Development

This study demonstrates the need for single-cell functional proteomics to confirm function after CRISPR edits. Aside from toxicity and rejection, engineered and gene-edited cells could function differently than intended and expected after initial modification. The IsoPlexis platform builds upon the well-validated ELISA method, adding single-cell capabilities which detect functionally heterogeneous and potent cells within a phenotypically identical sample. By using IsoPlexis’ functional immune landscaping system, which can analyze the true function of each cell, researchers can better understand how their potential immunotherapies will affect patients. With a growing number of studies in allogeneic therapies, IsoPlexis’ platform can help improve these types of therapies by detecting the functionally heterogeneous and potent cells that other technologies miss, meaning more accurate functional information leading to more targeted and effective therapies. This study highlights the functional differences within subtle gene edits that remain hidden when using traditional technologies.

For these reasons, IsoPlexis’ ability to uniquely identify the functional changes due to gene edits helps accelerate edited and engineered cellular therapy development. IsoPlexis’ platforms provide the essential functional data needed to develop and test next generation immunotherapies which other technologies are unable to provide, accelerating the process of getting critical therapies to the market.

To learn more about how IsoPlexis’ single-cell proteomics can be used to empower gene-edited and engineered therapies, download our CRISPR eBook.

Reference:

  1. Zhu H et al. Metabolic Reprogramming via Depletion of CISH in Human iPSC-Derived NK Cells Promotes In Vivo Persistence and Enhances Anti-tumor Activity. Cell Stem Cell 27: 1-14, 2020.
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