Security research of virus vector
一、Introduction
Viral vectors are the most popular delivery methods for cell therapy and gene therapy. In cell and gene therapy, viral vectors that express anti-tumor, cytotoxic, and immunostimulating genes, such as CAR, cytokines, and chemokines, have been approved for the treatment of cancer, metabolic, neurological, and eye diseases. Compared with non-viral vectors, viral vectors have superior gene delivery capabilities, such as high delivery efficiency and little damage to cells, so they play a central role in cell and gene therapy [1].
二、Retroviruses
(1) Integration sites and safety accidents
Retroviruses (RVs) Because of their reverse transcriptional activity, RNA genomes are able to produce dsDNA copies and integrate them into the host genome. Chromosome integration is conducive to long-term transgene expression, in which the integration of gamma retrovirus vectors (gamma-RVV) tends to insert genes near the transcription start site, and the application of gamma-RVV to gene therapy has led to adverse events inducing leukemia in treated SC-D X1 patients [2]. Therefore, the researchers designed a self-inactivated gamma-RV vector (SIN-γRVV), which has been shown to be safe in clinical trials [3]. In severe adenosine deaminase deficiency combined with immune deficiency (ADA-SCID), insertional tumor occurrence after γ-RVV treatment is rare [4], and the underlying technology retrovirus vectors on the market today are self-inactivated vectors with high safety. Lentiviral vectors (LVVS) preferentially integrate into active transcription units, and there have also been reports of adverse events and random insertion leading to tumorigenesis. In June 2021, Bluebird Bio suspended two clinical trials of LentiGlobin (BB305 LV) for sickle cell disease because two patients receiving LentiGlobin were diagnosed with acute myeloid leukemia and myelodysplastic syndrome, respectively [5]. Therefore, both lentiviral vectors and retroviral vectors have the risk of immediate insertion of cancer, but the probability is extremely low (nearly one in 10 million), so far, there is no safety accident caused by random integration in the cell products that have been marketed, whether it is the application of retroviral vectors or lentiviral vectors.
(2)Replication virus risk
Retroviral vectors or lentiviral vectors are also commonly used to efficiently introduce CAR genes into T cells in CAR T cell therapy, but they have the potential to generate Replication Competent Retroviruses, RCR) or the potential risk of Replication Competent Lentivirus (RCL) contamination. RCR/RCL can also be integrated into the cell genome, resulting in the risk of insertion tumors due to the activation of proto-oncogenes, disruption of tumor suppressor genes, or increased expression of factors that promote cell growth. On the other hand, it also increases the risk of integration-induced insertional tumors by producing viruses with the ability to replicate [6]. In vitro transfusions of bone marrow progenitor cells, three out of 10 severely immunodeficient rhesus monkeys treated with gamma-RV developed lymphoma [7]. Analysis showed that RCR was detected in lymphoma tissue, and animals with lymphoma developed gamma-retroviraemia, which was also caused by RCR [8], so the real culprit of lymphoma in animals was RCR and not the gamma-retrovirus vector. Similarly, since the envelope of lentiviral vectors is often replaced with the membrane proteins of other viruses, such as the commonly used VSV-G membrane protein, this may increase the potential risk of RCL contamination [6]. Therefore, the risk of contamination of RCR/RCL is always a safety issue that we should focus on.
三、DNA virus vector
DNA virus vectors mainly include adenovirus (AV) and adeno-associated virus (AVV). Adenovirus is a non-enveloped double-stranded DNA virus, and the gene of the adenovirus vector does not integrate into the genome after introduction, so there is no risk of random insertion. High immunogenicity, cytotoxicity and transient transgene expression still make the study of adenovirus vectors challenging. In 1999, an 18-year-old patient with an ornithine transcarbamylase deficiency died after a large systemic injection of an adenovirus vector due to cytokine release syndrome (CRS) triggered by an innate immune response from the capsid protein. At present, gene therapy based on adenovirus vector is mainly applied to gene vaccine and anti-tumor therapy [5]. AVV is an unenveloped parvovirus with single-stranded DNA. Its vector has the characteristics of wide tendency, low immunogenicity and easy production, which is conducive to clinical application. However, studies have shown that RNA interference (RNAi) therapy delivered by AAV can cause neurotoxicity in the brain of non-human primates [9].
四、Summary
To sum up, the application of viral vectors in cell therapy is very safe, and viral vectors are still reliable, safe and efficient delivery means in cell drug research and development, but the safety of viral vectors should be highly concerned in related gene therapy and stem cell-related therapy. This requires us to continuously optimize the design and production of viral vector technology and control the quality control system to ensure the clinical safety and effectiveness of cell and gene therapy products, and to benefit more patients with minimal safety problems.
Reference：
[1] Lundstrom K. Viral Vectors in Gene Therapy: Where Do We Stand in 2023? Viruses. 2023 Mar 7; 15(3):698. doi: 10.3390/v15030698. PMID: 36992407; PMCID: PMC10059137.
[2]Hacein-Bey-Abina S, Garrigue A, Wang GP, Soulier J, Lim A, Morillon E, Clappier E, Caccavelli L, Delabesse E, Beldjord K, Asnafi V, MacIntyre E, Dal Cortivo L, Radford I, Brousse N, Sigaux F, Moshous D, Hauer J, Borkhardt A, Belohradsky BH, Wintergerst U, Velez MC, Leiva L, Sorensen R, Wulffraat N, Blanche S, Bushman FD, Fischer A, Cavazzana-Calvo M. Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J Clin Invest. 2008 Sep; 118(9):3132-42. doi: 10.1172/JCI35700. PMID: 18688285; PMCID: PMC2496963.
[3]Hacein-Bey-Abina S, Pai SY, Gaspar HB, Armant M, Berry CC, Blanche S, Bleesing J, Blondeau J, de Boer H, Buckland KF, Caccavelli L, Cros G, De Oliveira S, Fernandez KS, Guo D, Harris CE, Hopkins G, Lehmann LE, Lim A, London WB, van der Loo JC, Malani N, Male F, Malik P, Marinovic MA, McNicol AM, Moshous D, Neven B, Oleastro M, Picard C, Ritz J, Rivat C, Schambach A, Shaw KL, Sherman EA, Silberstein LE, Six E, Touzot F, Tsytsykova A, Xu-Bayford J, Baum C, Bushman FD, Fischer A, Kohn DB, Filipovich AH, Notarangelo LD, Cavazzana M, Williams DA, Thrasher AJ. A modified gamma -retrovirus vector for X-linked severe combined immunodeficiency. N Engl J Med. 2014 Oct 9; 371(15):1407-17. doi: 10.1056/NEJMoa1404588. PMID: 25295500; PMCID: PMC4274995.
[4] Pai SY. Built to last: gene therapy for ADA SCID. Blood. 2021 Oct 14; 138 (15) : 1287-1288. The doi: 10.1182 / blood. 2021012300. PMID: 34647983; PMCID: PMC8525332.
[5] Li Manqi, Wei Liping, Tao Qiaoyu et al. Gene therapy carrier, research progress and safety applications [J]. China's pharmaceutical industry magazines, does 2022 (12) : 1671-1682. The DOI: 10.16522 / j.carol carroll nki CJPH. 2022.12.001.
[6] 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.
[7]Donahue RE, Kessler SW, Bodine D, McDonagh K, Dunbar C, Goodman S, Agricola B, Byrne E, Raffeld M, Moen R, et al. Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer. J Exp Med. 1992 Oct 1; 176(4):1125-35. doi: 10.1084/ jem.176.4.1125.PMID: 1383375; PMCID: PMC2119385.
[8]Vanin EF, Kaloss M, Broscius C, Nienhuis AW. Characterization of replication-competent retroviruses from nonhuman primates with virus-induced T-cell lymphomas and observations regarding the mechanism of oncogenesis. J Virol. 1994 Jul; 68(7):4241-50. doi: 10.1128/JVI.68.7.4241-4250.1994. PMID: 8207799; PMCID: PMC236347.
[9]Keiser MS, Ranum PT, Yrigollen CM, Carrell EM, Smith GR, Muehlmatt AL, Chen YH, Stein JM, Wolf RL, Radaelli E, Lucas TJ 2nd, Gonzalez-Alegre P, Davidson BL. Toxicity after AAV delivery of RNAi expression constructs into nonhuman primate brain. Nat Med. 2021 Nov; 27(11):1982-1989. doi: 10.1038/s41591-021-01522-3. Epub 2021 Oct 18.PMID: 34663988; PMCID: PMC8605996.
Disclaimer: Shenzhen Cell Valley is committed to the research of cell and gene therapy, in order to promote emerging technologies, so that more people understand the new development of biomedicine. The content of this article is only used for information exchange, and the platform remains neutral on the content, statements and opinions of the article, and does not represent the position and views of Shenzhen Cell Valley. The relevant information in this article should not be used as a diagnosis or treatment, is not a substitute for professional medical advice, and the company's website will not assume any responsibility. The final interpretation of the content of the above statement belongs to the company's website, this statement will apply to the company's website all the time to share the article, thank you for your cooperation! Copyright description: The copyright of the article belongs to Shenzhen Cell Valley, individuals are welcome to forward to the circle of friends, media or institutions without authorization, reproduced in any form to other platforms, will be regarded as infringement. For reprinting, please contact email: contact@duanglink.com
Viral vectors are the most popular delivery methods for cell therapy and gene therapy. In cell and gene therapy, viral vectors that express anti-tumor, cytotoxic, and immunostimulating genes, such as CAR, cytokines, and chemokines, have been approved for the treatment of cancer, metabolic, neurological, and eye diseases. Compared with non-viral vectors, viral vectors have superior gene delivery capabilities, such as high delivery efficiency and little damage to cells, so they play a central role in cell and gene therapy [1].
二、Retroviruses
(1) Integration sites and safety accidents
Retroviruses (RVs) Because of their reverse transcriptional activity, RNA genomes are able to produce dsDNA copies and integrate them into the host genome. Chromosome integration is conducive to long-term transgene expression, in which the integration of gamma retrovirus vectors (gamma-RVV) tends to insert genes near the transcription start site, and the application of gamma-RVV to gene therapy has led to adverse events inducing leukemia in treated SC-D X1 patients [2]. Therefore, the researchers designed a self-inactivated gamma-RV vector (SIN-γRVV), which has been shown to be safe in clinical trials [3]. In severe adenosine deaminase deficiency combined with immune deficiency (ADA-SCID), insertional tumor occurrence after γ-RVV treatment is rare [4], and the underlying technology retrovirus vectors on the market today are self-inactivated vectors with high safety. Lentiviral vectors (LVVS) preferentially integrate into active transcription units, and there have also been reports of adverse events and random insertion leading to tumorigenesis. In June 2021, Bluebird Bio suspended two clinical trials of LentiGlobin (BB305 LV) for sickle cell disease because two patients receiving LentiGlobin were diagnosed with acute myeloid leukemia and myelodysplastic syndrome, respectively [5]. Therefore, both lentiviral vectors and retroviral vectors have the risk of immediate insertion of cancer, but the probability is extremely low (nearly one in 10 million), so far, there is no safety accident caused by random integration in the cell products that have been marketed, whether it is the application of retroviral vectors or lentiviral vectors.
(2)Replication virus risk
Retroviral vectors or lentiviral vectors are also commonly used to efficiently introduce CAR genes into T cells in CAR T cell therapy, but they have the potential to generate Replication Competent Retroviruses, RCR) or the potential risk of Replication Competent Lentivirus (RCL) contamination. RCR/RCL can also be integrated into the cell genome, resulting in the risk of insertion tumors due to the activation of proto-oncogenes, disruption of tumor suppressor genes, or increased expression of factors that promote cell growth. On the other hand, it also increases the risk of integration-induced insertional tumors by producing viruses with the ability to replicate [6]. In vitro transfusions of bone marrow progenitor cells, three out of 10 severely immunodeficient rhesus monkeys treated with gamma-RV developed lymphoma [7]. Analysis showed that RCR was detected in lymphoma tissue, and animals with lymphoma developed gamma-retroviraemia, which was also caused by RCR [8], so the real culprit of lymphoma in animals was RCR and not the gamma-retrovirus vector. Similarly, since the envelope of lentiviral vectors is often replaced with the membrane proteins of other viruses, such as the commonly used VSV-G membrane protein, this may increase the potential risk of RCL contamination [6]. Therefore, the risk of contamination of RCR/RCL is always a safety issue that we should focus on.
三、DNA virus vector
DNA virus vectors mainly include adenovirus (AV) and adeno-associated virus (AVV). Adenovirus is a non-enveloped double-stranded DNA virus, and the gene of the adenovirus vector does not integrate into the genome after introduction, so there is no risk of random insertion. High immunogenicity, cytotoxicity and transient transgene expression still make the study of adenovirus vectors challenging. In 1999, an 18-year-old patient with an ornithine transcarbamylase deficiency died after a large systemic injection of an adenovirus vector due to cytokine release syndrome (CRS) triggered by an innate immune response from the capsid protein. At present, gene therapy based on adenovirus vector is mainly applied to gene vaccine and anti-tumor therapy [5]. AVV is an unenveloped parvovirus with single-stranded DNA. Its vector has the characteristics of wide tendency, low immunogenicity and easy production, which is conducive to clinical application. However, studies have shown that RNA interference (RNAi) therapy delivered by AAV can cause neurotoxicity in the brain of non-human primates [9].
四、Summary
To sum up, the application of viral vectors in cell therapy is very safe, and viral vectors are still reliable, safe and efficient delivery means in cell drug research and development, but the safety of viral vectors should be highly concerned in related gene therapy and stem cell-related therapy. This requires us to continuously optimize the design and production of viral vector technology and control the quality control system to ensure the clinical safety and effectiveness of cell and gene therapy products, and to benefit more patients with minimal safety problems.
Reference：
[1] Lundstrom K. Viral Vectors in Gene Therapy: Where Do We Stand in 2023? Viruses. 2023 Mar 7; 15(3):698. doi: 10.3390/v15030698. PMID: 36992407; PMCID: PMC10059137.
[2]Hacein-Bey-Abina S, Garrigue A, Wang GP, Soulier J, Lim A, Morillon E, Clappier E, Caccavelli L, Delabesse E, Beldjord K, Asnafi V, MacIntyre E, Dal Cortivo L, Radford I, Brousse N, Sigaux F, Moshous D, Hauer J, Borkhardt A, Belohradsky BH, Wintergerst U, Velez MC, Leiva L, Sorensen R, Wulffraat N, Blanche S, Bushman FD, Fischer A, Cavazzana-Calvo M. Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J Clin Invest. 2008 Sep; 118(9):3132-42. doi: 10.1172/JCI35700. PMID: 18688285; PMCID: PMC2496963.
[3]Hacein-Bey-Abina S, Pai SY, Gaspar HB, Armant M, Berry CC, Blanche S, Bleesing J, Blondeau J, de Boer H, Buckland KF, Caccavelli L, Cros G, De Oliveira S, Fernandez KS, Guo D, Harris CE, Hopkins G, Lehmann LE, Lim A, London WB, van der Loo JC, Malani N, Male F, Malik P, Marinovic MA, McNicol AM, Moshous D, Neven B, Oleastro M, Picard C, Ritz J, Rivat C, Schambach A, Shaw KL, Sherman EA, Silberstein LE, Six E, Touzot F, Tsytsykova A, Xu-Bayford J, Baum C, Bushman FD, Fischer A, Kohn DB, Filipovich AH, Notarangelo LD, Cavazzana M, Williams DA, Thrasher AJ. A modified gamma -retrovirus vector for X-linked severe combined immunodeficiency. N Engl J Med. 2014 Oct 9; 371(15):1407-17. doi: 10.1056/NEJMoa1404588. PMID: 25295500; PMCID: PMC4274995.
[4] Pai SY. Built to last: gene therapy for ADA SCID. Blood. 2021 Oct 14; 138 (15) : 1287-1288. The doi: 10.1182 / blood. 2021012300. PMID: 34647983; PMCID: PMC8525332.
[5] Li Manqi, Wei Liping, Tao Qiaoyu et al. Gene therapy carrier, research progress and safety applications [J]. China's pharmaceutical industry magazines, does 2022 (12) : 1671-1682. The DOI: 10.16522 / j.carol carroll nki CJPH. 2022.12.001.
[6] 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.
[7]Donahue RE, Kessler SW, Bodine D, McDonagh K, Dunbar C, Goodman S, Agricola B, Byrne E, Raffeld M, Moen R, et al. Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer. J Exp Med. 1992 Oct 1; 176(4):1125-35. doi: 10.1084/ jem.176.4.1125.PMID: 1383375; PMCID: PMC2119385.
[8]Vanin EF, Kaloss M, Broscius C, Nienhuis AW. Characterization of replication-competent retroviruses from nonhuman primates with virus-induced T-cell lymphomas and observations regarding the mechanism of oncogenesis. J Virol. 1994 Jul; 68(7):4241-50. doi: 10.1128/JVI.68.7.4241-4250.1994. PMID: 8207799; PMCID: PMC236347.
[9]Keiser MS, Ranum PT, Yrigollen CM, Carrell EM, Smith GR, Muehlmatt AL, Chen YH, Stein JM, Wolf RL, Radaelli E, Lucas TJ 2nd, Gonzalez-Alegre P, Davidson BL. Toxicity after AAV delivery of RNAi expression constructs into nonhuman primate brain. Nat Med. 2021 Nov; 27(11):1982-1989. doi: 10.1038/s41591-021-01522-3. Epub 2021 Oct 18.PMID: 34663988; PMCID: PMC8605996.
Disclaimer: Shenzhen Cell Valley is committed to the research of cell and gene therapy, in order to promote emerging technologies, so that more people understand the new development of biomedicine. The content of this article is only used for information exchange, and the platform remains neutral on the content, statements and opinions of the article, and does not represent the position and views of Shenzhen Cell Valley. The relevant information in this article should not be used as a diagnosis or treatment, is not a substitute for professional medical advice, and the company's website will not assume any responsibility. The final interpretation of the content of the above statement belongs to the company's website, this statement will apply to the company's website all the time to share the article, thank you for your cooperation! Copyright description: The copyright of the article belongs to Shenzhen Cell Valley, individuals are welcome to forward to the circle of friends, media or institutions without authorization, reproduced in any form to other platforms, will be regarded as infringement. For reprinting, please contact email: contact@duanglink.com
