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Veterinary CAR Packaging and Transfection
Chimeric antigen receptor (CAR) T cell therapy is a type of adoptive cell therapy that has demonstrated positive results, especially in treating hematologic malignancies such as leukemia. CAR-modified cell therapies also hold significant potential in the treatment of companion animal tumors. A critical step in developing CAR T cell therapies involves CAR constructs into T cells, enabling them to identify and eliminate tumor cells with high specificity. In addition to disease target discovery, CAR development, and CAR characterization, BioVenic also provide services for the critical step of veterinary CAR gene transduction. Our services include the construction of viral vectors or non-viral vectors, the transfection of genes into the target cell genome using methods such as lentivirus or electroporation, and the induction of their expression.
Fig.1. Summary of the Current Vectors Used to Genetically Modify T Cells to Express CARs.1,2
Background
Genetic modification of T cells to express CAR has demonstrated significant potential in treating B cell malignancies. Beyond the development of personalized co-engineering techniques and enhanced CAR designs, the selection of genetic engineering tools is a vital factor for the effective application of CAR-related cellular products. Key factors to consider when choosing an engineering tool include the efficiency and stability of transgene expression (except when transient expression is intended), the genetic cargo capacity, safety (emphasizing minimal vector-related genotoxicity), and the capability to scale up production swiftly and cost-effectively for clinical applications.
Viral Vector Packaging
CAR-T cells generated using gamma-retroviral or lentiviral vectors have demonstrated remarkable antitumor effects, with no observed secondary effects such as replication-competent lentivirus (RCL) presence or T cell transformation. Among available viral vectors, the use of lentiviral vectors is typically favored over AAV or adenovirus due to their distinct advantages, including efficient host cell integration, high transduction efficiency, and a significant genetic payload capacity. BioVenic provides a wide array of lentivirus preparation services to support the development of veterinary CAR-related therapies. We excel in producing lentiviral vectors characterized by high titer, superior transduction efficiency, and enhanced safety. The technical workflow encompasses vector construction, cell culture, transfection processes, virus collection, and purification, followed by titer detection.
Gene Editing for Developing Veterinary CAR-X Cells
Advancements in research have enabled the use of genome editing for the streamlined production of CAR-T cell products. The gene editing technology we employ extends beyond its traditional use in gene knockout to enable non-viral CAR integration through HDR repair. This approach enhances the effectiveness of CAR-T cell therapies by ensuring stable CAR expression and increased potency. Additionally, it streamlines complex manufacturing procedures and reduces the preparation time for CAR-T cells. Our gene editing service employs various advanced techniques to conduct gene editing across diverse animal cell types, supporting the development of veterinary CAR-related cell therapies.
Optimization of Transfection/Transduction
Lentiviral transduction plays a pivotal role in creating veterinary CAR-X cells, directly influencing their quality. Hence, comprehensive optimization of numerous parameters within the lentiviral transduction process is essential. Variables such as multiplicity of infection (MOI), cell incubation density, pre-activation duration, and the transduction system's configuration significantly affect the quality of CAR-T cells. Our experienced technical team aids researchers by optimizing transduction parameters and conducting functional assessments, evaluating aspects like proliferation capacity, transduction efficiency, cell viability, and the in vitro cytotoxicity of veterinary CAR-X cells, to ensure high-quality results.
Comparison of CAR Transfection Methods
| Method | Description |
| Viral Vector Transfection | γ-Retroviral and lentiviral vectors are frequently employed in the generation of CAR-T cells because they enable high transduction rates and ensure long-term stable expression of transgenes. However, they are expensive and require rigorous safety monitoring. Moreover, they have a limited capacity for additional transgenes, restricting the number of extra genes that CAR-T cells can express. |
| Transposon Systems | As the simplest natural gene delivery vehicles, transposons are highly effective tools in genetic engineering applications, including gene therapy. Despite their versatility, there is a potential risk of oncogenic activity. |
| RNA Transfection | Typically, mRNA is synthesized in vitro and then transfected via electroporation. This method leads to temporary expression of the CAR construct, with RNA translated into CAR proteins that remain expressed on the cell surface for up to 7 days. This approach provides an opportunity to assess the safety of previously untested scFvs or CAR constructs, though it may potentially reduce anti-tumor effects. |
| Genome Editing | Various engineered nucleases, including transcription activator-like effector nucleases (TALENs) and other advanced systems, are employed in genome editing. One of these systems, known for its adaptability and rapid processing, holds significant promise in enhancing CAR therapy. It facilitates the quick evaluation of new targets for knockout or knock-in in T cells through the easy generation of guide RNA (gRNA). |
Why Choose Us?
Multiple Choices
With our viral or non-viral vector building and transduction services, researchers can choose suitable methods in the development of veterinary CAR cell therapy.
Focus on Animals
Our technical team continually learns and tracks the forefront of veterinary cell therapy and is committed to helping advance disease therapy research related to companion animals.
Integrated Technology Platform
We integrate genome editing, plasmid construction and transduction, and cell function research services to assist researchers throughout the entire process of developing veterinary CAR cell therapy.
As a targeted therapy for tumors, the application of CAR-T cells and other immune cells expressing CAR holds broad prospects in veterinary medicine. BioVenic's viral vector packaging, gene editing, and transfection/transduction optimization services can assist with CAR gene expression issues in the development of veterinary CAR-X cells. If you are interested in our Veterinary CAR Packaging and Transfection services, please feel free to contact us. We are pleased to offer customized services according to your research needs.
References
- Image retrieved from Figure 1 "Summary of the current vectors used to genetically modify T cells to express CARs." Irving et al., 2021, used under [CC BY 4.0] (https://creativecommons.org/licenses/by/4.0/). Without modification.
- Irving, Melita, et al. "Choosing the right tool for genetic engineering: clinical lessons from chimeric antigen receptor-T cells." Human Gene Therapy 32.19-20 (2021): 1044-1058.