Applications of Fluorescence In Situ Hybridization (FISH) in Cell & Gene Therapy

The rapid advancements in the field of cell and gene therapy have revolutionized the treatment of various genetic and degenerative diseases. As researchers and scientists strive to develop innovative methods to improve the efficacy and safety of these therapies, fluorescence in situ hybridization (FISH) has emerged as a powerful tool for the visualization and analysis of genetic material within cells and tissues.

Fig. 1 A simplified diagram depicting FISH. (Tingey M, et al., 2022)

Biodistribution Studies in Cell & Gene Therapy

Cell and gene therapy have garnered significant attention due to their potential to treat a wide range of diseases, including cancer, genetic disorders, and autoimmune conditions. Chimeric Antigen Receptor (CAR) T-cell therapy has shown promise in the treatment of certain types of leukemia and lymphoma. AAV (adeno-associated virus)-based therapies are effective for some types of genetic diseases. Currently, cell and gene therapy research is focused on optimizing the delivery, targeting, and efficacy of gene therapy. As this field continues to evolve, there is a growing need for advanced tools and methods to assess the biodistribution of therapeutic genes and cells within organisms.

Biodistribution (BD) studies play a pivotal role in evaluating the dissemination and localization of gene therapy vectors and cellular therapeutics within the body, according to guidelines for biodistribution studies developed by different regulatory agencies and international scientific organizations. In a guidance document from the Food and Drug Administration (FDA), the BD study is viewed as one of the considerations for the determination of the cell fate, which refers to the conditions of cells after administration determined by assessment of survival/engraftment and distribution, differentiation/integration, and tumorigenicity of cells. Accurate assessment of biodistribution is crucial for ensuring the effective targeting of therapeutic genes and cells to the intended tissues and organs. Furthermore, it aids in monitoring potential off-target effects and the overall safety profile of the treatment.

Applications of FISH in Cell & Gene Therapy

FISH is a molecular cytogenetic technique that enables the visualization and mapping of specific DNA or RNA sequences within cells and tissues. The technique utilizes labeled nucleic acid probes that hybridize to complementary target sequences, allowing for the precise localization and detection of genetic material. The history of FISH dates back to the 1980s. Since then, it has undergone remarkable advancements, including the development of RNAscope, Basescope, and miRNAscope, which can accurately detect very short sequences.

Applications of FISH in gene and cellular localization
FISH technology plays a crucial role in the precise localization of therapeutic genes, vectors, and cellular components within target tissues. By leveraging FISH probes tailored to specific genetic sequences, researchers can visualize the spatial distribution of genetic material, providing invaluable insights into the intracellular and intravascular localization of gene therapy vectors and transduced cells. This capability is instrumental in optimizing the delivery and targeting of gene therapy products, ultimately enhancing their therapeutic efficacy.

Applications of FISH in gene diagnosis and gene efficacy assessment
FISH facilitates the visualization of therapeutic gene vectors, such as AAV and lentivirus, within tissues by employing specific probes designed to target and identify the transferred genetic material. This enables researchers to trace the spatial distribution of the therapeutic genes and precisely determine their localization within the target tissues.

Fig. 2 Detection of rAAV viral DNA injected in the rat striatum using the RNAscope technique (Grabinski TM, et al., 2015); The distribution of SaCas9 in the retina was examined using RNAscope (Maeder ML, et al., 2019).

FISH can be used to quantitatively assess the expression of therapeutic genes within the target tissues, providing valuable insights into the extent and efficiency of gene delivery. By detecting and counting the copies of the inserted gene sequences, FISH enables the measurement of transgene expression levels, thereby contributing to the evaluation of gene therapy efficacy.

Application of FISH in cell therapy development
FISH allows for the visualization and precise localization of transplanted cells within living organisms. By utilizing cell-specific FISH probes, researchers can track the cellular biodistribution and monitor the spatial distribution of transplanted cells within the target tissues over time.

Fig. 3 In situ detection of anti-CD19-CAR T cells in mouse xenografts of NSG mice after anti-CD19 CAR T cell infusion (Eichholz K, et al., 2021).

FISH technology enables the assessment of cell engraftment, retention, and proliferation within the target tissues. Researchers can analyze the dynamics of transplanted cell localization and integration, providing critical insights into the efficacy of cell therapy interventions.

Advantages of FISH in Cell and Gene Therapy

• High sensitivity and specificity. FISH provides high sensitivity and specificity for detecting specific DNA or RNA sequences within cells and tissues, enabling precise localization and visualization of gene delivery and transplanted cells in cell and gene therapy research.
• Quantitative Assessment. FISH can provide quantitative data regarding the abundance and localization of specific DNA or RNA sequences within cells. This quantitative analysis is valuable for evaluating the efficiency of gene transfer, the expression of transgenes, and the determination of gene copy numbers in modified cells.
• Spatial resolution. FISH provides high spatial resolution, enabling researchers to accurately pinpoint the location of therapeutic genes or transplanted cells within the target tissues. This is crucial for assessing biodistribution and engraftment in cell and gene therapy interventions.
• Multiplexing capabilities. Advanced FISH techniques allow for multiplexing, which means researchers can simultaneously visualize and analyze multiple genetic targets or cellular components within the same sample. This is beneficial for comprehensive assessments in cell and gene therapy research.

References

1. Tingey M, et al. (2022). "Technologies Enabling Single-Molecule Super-Resolution Imaging of mRNA." Cells. 11 (19): 3079.
2. Kamiyama Y, et al. (2021). "Biodistribution studies for cell therapy products: Current status and issues." Regen Ther. 18: 202-216.
3. Grabinski TM, et al. (2015). "A method for combining RNAscope in situ hybridization with immunohistochemistry in thick free-floating brain sections and primary neuronal cultures." PLoS One. 10 (3): e0120120.
4. Maeder ML, et al. (2019). "Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10." Nat Med. 25 (2): 229-233.
5. Eichholz K, et al. (2021). "A CAR RNA FISH assay to study functional and spatial heterogeneity of chimeric antigen receptor T cells in tissue." Sci Rep. 11 (1): 12921.