CAR-NK/Transgene/Cytokine/Biomarker Protein Biodistribution Analysis

Creative Bioarray offers the RNA ISH assay combining IHC assay to visualize and monitor the delivery and biodistribution of the CAR-NK vectors, transgenes, and related proteins to access the specificity and efficacy of these therapies.

In the context of cell therapy CAR-NK/transgene/Cytokine/Biomarker RNA Biodistribution Analysis, RNA In situ Hybridization (RNA ISH) assay combined with Immunohistochemistry (IHC) assay have several advantages:

• Advanced Visualization: RNA ISH and IHC assay allow for direct visualization of specific RNA or proteins in specific cells within a tissue, including their precise location in the cellular structure.
• Improved Sensitivity: RNA ISH assay is highly sensitive and can detect target RNA molecules in low abundance. Similarly, IHC assay can identify specific proteins at the cellular level, even if they are present in small amounts.
• Simultaneous detection: Both techniques can be simultaneously applied to the same sample, providing a 'double labeling' effect. This allows for the simultaneous detection of mRNA and protein, and the relationship between them can be better understood.
• Better characterization: Using RNA ISH assay in conjunction with IHC assay provides a more comprehensive characterization of individual cells. For instance, it can explain how gene expression and protein synthesis in the same cell are related.
• High Specificity: Both techniques have high specificity. RNA ISH assay can differentiate between highly similar RNA molecules, thus identifying specific gene transcripts. Similarly, IHC assay can discriminate proteins based on post-translational modifications.
• Translational insights: Using RNA ISH assay and IHC assay provides valuable insights into the mechanism of action of CAR-NK/transgene/Cytokine, helping to optimize therapeutic strategies and monitor treatment response.

In conclusion, RNA ISH combined with IHC provides a powerful tool for precise, reliable, and comprehensive biomarker detection and cell therapy research.

Fig. 1. RNA ISH assay for detection. RNA ISH probes were designed to target the 3' UTR of the CAR vector.

Figure 2. RNAscope ISH-IHC in primary neuron cultures, dual RNAscope ISH detection, and detection of ectopic viral vector DNA.
A) Primary rat neurons were processed for tau ISH and βIII-tubulin IHC. Clear puncta are present for the tau ISH signal (red), while neuronal cell bodies and processes are clearly labeled with tubulin IHC (blue). B-E) Color thresholding (using the YUV index) allows clear separation of ISH signal (red puncta) from the IHC signal (blue staining) in primary neurons. Here, the YUV index was used and Y was 0–135, U was 0–255 and V was 135–255. The pixels within the threshold limits are shown by the bright red overlay. F) Dual ISH for tau (red) and βIII-tubulin (blue/green) was performed on 40μm thick rat brain sections (cortex depicted). G-J) Rats were unilaterally injected in the striatum with recombinant adeno-associated viruses (rAAV, serotype 2/5). RNAscope was used to detect rAAV DNA. Robust rAAV DNA ISH signal is detected in the injected striatum (G) compared to the uninjected hemisphere (I). ISH signal was also detected in the SN ipsilateral to the injected striatum because rAAV particles are retrogradely transported to the SN (H), no signal is detected in the contralateral SN (J). Scale bars: A = 50μm, C and E = 20μm, F = 25μm, and J = 50μm.

CAR-NK, transgene, Cytokine, and Biomarker RNA Biodistribution Analysis is vital in cell therapy by giving valuable insights into the pharmacokinetics and pharmacodynamics of the drug compounds. This plays a pivotal role in the safety, efficacy, duration of action, dose, and route of administration determination of the therapy. The regulatory agencies such as FDA or EMA require us to understand the biodistribution and persistence of the administered cells.

Features of CAR-NK/Transgene/Cytokine/Biomarker Protein Biodistribution Analysis (RNA ISH + IHC method)

(1) Detection of activated CAR-NK cells in the tissue context
(2) Codon-optimized transgene and biomarker expression analysis
(3) Biomarker protein analysis
(4) Custom probes designed within 1-2 weeks
(5) Fastest turnaround time
(6) Experienced scientists for accurate data analysis
(7) Standard and customized experiment plan

Benefits of CAR-NK/Transgene/Cytokine/Biomarker Protein Biodistribution Analysis (RNA ISH + IHC method)

(1) Detect low levels of gene expression across all normal tissues in animal models and human samples
(2) Assess cell-specific target expression and biomarker protein in normal tissues and expression heterogeneity in tumor tissues
(3) Visualize and quantify CAR-NK cell infiltration and activation in tumor and off-tumor tissues
(4) Simultaneously detect CAR/TCR vector and cytokines, T cell markers, or other cell-type markers

Creative Bioarray offers CAR-NK/Transgene/Cytokine/Biomarker Protein Biodistribution Analysis for you as follows:

• Probe design
• Probe synthesis
• ISH + IHC staining
• Imaging
• Data analysis

Quotation and ordering

Our customer service representatives are available 24hr a day! We thank you for considering Creative Bioarray as your CAR-NK/Transgene/Cytokine/Biomarker Protein Biodistribution Analysis partner.

References

1. Wright JH, Huang L-Y, Weaver S et al. (2021) Detection of engineered T cells in FFPE tissue by multiplex in situ hybridization and immunohistochemistry. Journal of Immunological Methods 492:112955.
2. Gauthier, L., et al. (2019). Multifunctional Natural Killer Cell Engagers Targeting NKp46 Trigger Protective Tumor Immunity. Cell. 177(7):1701-1713
3. Ramachandran I., et al. (2019). Systemic and local immunity following adoptive transfer of NY-ESO-1 SPEAR T cells in synovial sarcoma. J Immunother Cancer 7:276
4. Tang N, Cheng C, Zhang X et al. (2020) TGF-β inhibition via CRISPR promotes the long-term efficacy of CAR T cells against solid tumors. JCI insight 5 (4). doi:10.1172/jci.insight.133977
5. Wang Y, Tong C, Dai H et al. (2021) Low-dose decitabine priming endows CAR T cells with enhanced and persistent antitumour potential via epigenetic reprogramming. Nature Communications 12 (1):409. doi:10.1038/s41467-020-20696-x
6. Barber-Axthelm IM, Barber-Axthelm V, Sze KY et al. (2021) Stem cell–derived CAR T cells traffic to HIV reservoirs in macaques. JCI insight 6 (1). doi:10.1172/jci.insight.141502
7. Grabinski, Tessa M., et al. "A method for combining RNAscope in situ hybridization with immunohistochemistry in thick free-floating brain sections and primary neuronal cultures." PloS one 10.3 (2015): e0120120.