Viral and non-viral vectors in CAR-T cell therapy
1、Introduction
Currently, the most commonly used CAR-T cell construction methods mainly include three categories: viral vectors represented by retroviruses and lentiviruses, transposon systems and mRNA-mediated non-viral vectors, and CRISPR/Cas9 gene editing technology [1 ]. Both viral vectors and non-viral vectors have their own advantages and disadvantages and suitable disease types. During the preparation process of CAR-T cells, we need to select the optimal vector for gene transduction according to our own needs.
2、Viral vector
γretroviral vectors and lentiviral vectors are two commonly used retroviral vectors. The first CAR-T therapy product approved by the FDA, Kymriah, uses lentiviral vectors, and the second Yescarta (marketed in China as Yikaida) is also the world's highest-selling CAR-T therapy product. are retroviruses. Gammaretroviruses and lentiviruses belong to the family Retroviridae. The main difference between gamma retrovirus and lentivirus is that retrovirus can only transduce cells in the dividing phase, while lentivirus can transduce cells in the non-dividing phase (excluding cells in the G0 phase), but the ability to obtain lentiviral vectors The method is complex and expensive because the lentivirus uses a transient packaging system, and there are also issues such as batch-to-batch variation caused by transient transfection of multiple plasmids [2]. In contrast, retroviruses can produce toxins through stable toxin-producing cell lines, which greatly reduces the time and cost of product production. Whether it is retrovirus or lentivirus, there may be potential safety risks of replicating viruses, so the entire production process needs to be strictly controlled [3]. In summary, transcription viral vectors have high transduction rates and long-term stable transgene expression. However, the number of transgenes they can carry is limited, and clinical safety testing costs are high.
3、Non-viral vector
TTransposons are composed of a DNA segment of the transposase gene and terminal inverted repeats (TIRs) containing transposase binding sites on both sides. During transposition, transposase mediates excision of elements from the donor vector. The transposon is then integrated into the chromosomal site. This feature makes transposons a non-viral gene delivery system that combines viral vectors (stable chromosomal integration and durable transgene expression) with non-viral delivery systems (lower immunogenicity, enhanced safety and reduced GMP manufacturing cost) advantages [4]. However, since transposons can be randomly inserted into transgenes, it brings problems with clinical safety and efficiency. Natural transposons allow transduced genes to repeatedly change their gene positions, making quality control more difficult. In addition, a variety of RNA-based vectors have been optimized for T cell transfection. Joel G. Rurik et al[5] used the LNP-mRNA method to prepare transient anti-fibrosis CAR-T cells. Because mRNA is restricted to the cytoplasm, cannot integrate into the genome, is inherently unstable, and is diluted during cell division, treatment with modified mRNA-targeted lipid nanoparticles reduces post-injury fibrosis and restores cardiac function.
4、Summary
In addition to the viral and non-viral vectors introduced above, the third-generation gene editing technology CRISPR/Cas9 has the characteristics of easy operation, high efficiency and low cost, and multiple genome editing. However, there is a certain off-target rate and potential carcinogenic risks during use. In summary, as an emerging field of cell therapy, CAR-T's structure and preparation methods are still being continuously optimized and improved. It can target tumor cells quickly and accurately at a relatively low cost, and has good efficacy. Safety is the common wish of everyone in the field of cell therapy.
Reference：
[1] Wu Qi, Wang Shaobo. Research progress of CRISPR/Cas9 technology in the treatment of tumor with CAR-T cells [J]. Basic medicine and clinical, lancet, 2023 (8) : 1313-1316. The DOI: 10.16352 / j.i SSN. 1001-6325.2023.08.1313.
[2] ZHANG Ping, ZHAI Jianzhao, Liu Zaishuan et al. Research progress of CAR-T cell construction technology [J]. International Journal of Laboratory Medicine, 2019,42(09):1125-1129. (in Chinese)
[3] Wu Xueling, Zhao Xiang, Meng Shufang. Risk analysis and control of replicative viruses in CAR T cell therapy products [J]. China Pharmaceutical Journal,2018,32(07):879-885.DOI:10.16153/ J.1002-7777.2018.07.006.
[4] Irving M, Lanitis E, Migliorini D, Ivics Z, Guedan S. Choosing the Right Tool for Genetic Engineering: Clinical Lessons from Chimeric Antigen Receptor-T Cells. Hum Gene Ther. 2021 Oct; 32(19-20):1044-1058. doi: 10.1089/hum.2021.173. PMID: 34662233; PMCID: PMC8697565.
[5] Rurik JG, Tombacz I, Yadegari A, Mendez Fernandez PO, Shewale SV, Li L, Kimura T, Soliman OY, Papp TE, Tam YK, Mui BL, Albelda SM, Pure E, June CH, Aghajanian H, Weissman D, Parhiz H, Epstein JA. CAR T cells produced in vivo to treat cardiac injury. Science. 2022 Jan 7; 375(6576):91-96. doi: 10.1126/ science.abm0594.Epub 2022 Jan 6. PMID: 34990237; PMCID: PMC9983611.
Disclaimer: Shenzhen Cell Valley is committed to the research of cell and gene therapy, in order to promote emerging technologies and let more people understand the new developments in biomedicine.The content of this article is for information exchange only. This platform remains neutral with respect to the content, statements, and opinion judgments in the article, and does not represent the position and opinions of Shenzhen Cell Valley.The relevant information in this article should not be used for diagnosis or treatment, and cannot replace professional medical advice. Our website will not assume any responsibility.The final interpretation of the above statement belongs to our company’s website. This statement will apply to articles shared on our website at all times. Thank you for your cooperation! Copyright statement: The copyright of the article belongs to Shenzhen Cell Valley. Individuals are welcome to forward it to friends, media or Any unauthorized reproduction by the organization to other platforms will be regarded as infringement.If you need to reprint, please contact email: contact@sz-cell.com
Currently, the most commonly used CAR-T cell construction methods mainly include three categories: viral vectors represented by retroviruses and lentiviruses, transposon systems and mRNA-mediated non-viral vectors, and CRISPR/Cas9 gene editing technology [1 ]. Both viral vectors and non-viral vectors have their own advantages and disadvantages and suitable disease types. During the preparation process of CAR-T cells, we need to select the optimal vector for gene transduction according to our own needs.
2、Viral vector
γretroviral vectors and lentiviral vectors are two commonly used retroviral vectors. The first CAR-T therapy product approved by the FDA, Kymriah, uses lentiviral vectors, and the second Yescarta (marketed in China as Yikaida) is also the world's highest-selling CAR-T therapy product. are retroviruses. Gammaretroviruses and lentiviruses belong to the family Retroviridae. The main difference between gamma retrovirus and lentivirus is that retrovirus can only transduce cells in the dividing phase, while lentivirus can transduce cells in the non-dividing phase (excluding cells in the G0 phase), but the ability to obtain lentiviral vectors The method is complex and expensive because the lentivirus uses a transient packaging system, and there are also issues such as batch-to-batch variation caused by transient transfection of multiple plasmids [2]. In contrast, retroviruses can produce toxins through stable toxin-producing cell lines, which greatly reduces the time and cost of product production. Whether it is retrovirus or lentivirus, there may be potential safety risks of replicating viruses, so the entire production process needs to be strictly controlled [3]. In summary, transcription viral vectors have high transduction rates and long-term stable transgene expression. However, the number of transgenes they can carry is limited, and clinical safety testing costs are high.
3、Non-viral vector
TTransposons are composed of a DNA segment of the transposase gene and terminal inverted repeats (TIRs) containing transposase binding sites on both sides. During transposition, transposase mediates excision of elements from the donor vector. The transposon is then integrated into the chromosomal site. This feature makes transposons a non-viral gene delivery system that combines viral vectors (stable chromosomal integration and durable transgene expression) with non-viral delivery systems (lower immunogenicity, enhanced safety and reduced GMP manufacturing cost) advantages [4]. However, since transposons can be randomly inserted into transgenes, it brings problems with clinical safety and efficiency. Natural transposons allow transduced genes to repeatedly change their gene positions, making quality control more difficult. In addition, a variety of RNA-based vectors have been optimized for T cell transfection. Joel G. Rurik et al[5] used the LNP-mRNA method to prepare transient anti-fibrosis CAR-T cells. Because mRNA is restricted to the cytoplasm, cannot integrate into the genome, is inherently unstable, and is diluted during cell division, treatment with modified mRNA-targeted lipid nanoparticles reduces post-injury fibrosis and restores cardiac function.
4、Summary
In addition to the viral and non-viral vectors introduced above, the third-generation gene editing technology CRISPR/Cas9 has the characteristics of easy operation, high efficiency and low cost, and multiple genome editing. However, there is a certain off-target rate and potential carcinogenic risks during use. In summary, as an emerging field of cell therapy, CAR-T's structure and preparation methods are still being continuously optimized and improved. It can target tumor cells quickly and accurately at a relatively low cost, and has good efficacy. Safety is the common wish of everyone in the field of cell therapy.
Reference：
[1] Wu Qi, Wang Shaobo. Research progress of CRISPR/Cas9 technology in the treatment of tumor with CAR-T cells [J]. Basic medicine and clinical, lancet, 2023 (8) : 1313-1316. The DOI: 10.16352 / j.i SSN. 1001-6325.2023.08.1313.
[2] ZHANG Ping, ZHAI Jianzhao, Liu Zaishuan et al. Research progress of CAR-T cell construction technology [J]. International Journal of Laboratory Medicine, 2019,42(09):1125-1129. (in Chinese)
[3] Wu Xueling, Zhao Xiang, Meng Shufang. Risk analysis and control of replicative viruses in CAR T cell therapy products [J]. China Pharmaceutical Journal,2018,32(07):879-885.DOI:10.16153/ J.1002-7777.2018.07.006.
[4] Irving M, Lanitis E, Migliorini D, Ivics Z, Guedan S. Choosing the Right Tool for Genetic Engineering: Clinical Lessons from Chimeric Antigen Receptor-T Cells. Hum Gene Ther. 2021 Oct; 32(19-20):1044-1058. doi: 10.1089/hum.2021.173. PMID: 34662233; PMCID: PMC8697565.
[5] Rurik JG, Tombacz I, Yadegari A, Mendez Fernandez PO, Shewale SV, Li L, Kimura T, Soliman OY, Papp TE, Tam YK, Mui BL, Albelda SM, Pure E, June CH, Aghajanian H, Weissman D, Parhiz H, Epstein JA. CAR T cells produced in vivo to treat cardiac injury. Science. 2022 Jan 7; 375(6576):91-96. doi: 10.1126/ science.abm0594.Epub 2022 Jan 6. PMID: 34990237; PMCID: PMC9983611.
Disclaimer: Shenzhen Cell Valley is committed to the research of cell and gene therapy, in order to promote emerging technologies and let more people understand the new developments in biomedicine.The content of this article is for information exchange only. This platform remains neutral with respect to the content, statements, and opinion judgments in the article, and does not represent the position and opinions of Shenzhen Cell Valley.The relevant information in this article should not be used for diagnosis or treatment, and cannot replace professional medical advice. Our website will not assume any responsibility.The final interpretation of the above statement belongs to our company’s website. This statement will apply to articles shared on our website at all times. Thank you for your cooperation! Copyright statement: The copyright of the article belongs to Shenzhen Cell Valley. Individuals are welcome to forward it to friends, media or Any unauthorized reproduction by the organization to other platforms will be regarded as infringement.If you need to reprint, please contact email: contact@sz-cell.com
