Анализ случайной интеграции рекомбинантного аденоассоциированного Вируса-6, упакованного в клетки Sf9 насекомых

Обложка

Цитировать

Полный текст

Открытый доступ Открытый доступ
Доступ закрыт Доступ предоставлен
Доступ закрыт Только для подписчиков

Аннотация

В последнее время растет озабоченность по поводу интеграции в геном вектора рекомбинантного аденоассоциированного вируса (rAAV), используемого для генной терапии. Аденоассоциированный вирус дикого типа (AAV) специфически интегрируется в AAVS1-сайт генома человека, в то время как rAAV случайным образом интегрируется в хромосомы хозяина с низкой частотой. Проанализированы события случайной интеграции конструкции rAAV6-EGFP, упакованной в клетки Sf9 насекомых. Производственная платформа Baculo-Sf9 обладает преимуществами суспензионной культуры клеток насекомых Sf9: высокой плотностью и возможностью крупномасштабного производства векторов rAAV. В проведенном исследовании использованы различные дозы вектора rAAV6-EGFP, продуцируемого Baculo-Sf9, для трансдукции клеток HEK293T и A549-имплантированных опухолей in vitro и in vivo. Методами проточной цитометрии и флуоресцентной микроскопии оценена эффективность экспрессии гена egfp и интенсивность флуоресценции EGFP. С помощью инвертированной гнездовой ПЦР и секвенирования ДНК идентифицированы случайные сайты интеграции генома rAAV6-EGFP в хромосомы человека. По результатам анализа in vitro показано, что эффективность экспрессии репортерной конструкции стабилизировалась через 20 суток, а частота случайной интеграции составляла 0.2–4.2%. В исследованиях как in vitro, так и in vivo выявлено, что случайная интеграция rAAV6 зависела от дозы конструкта. По результатам секвенирования идентифицировано два случайных сайта интеграции, которые находятся на хромосомах человека 8 и 12. На основании полученных данных можно сделать вывод, что для безопасной генной терапии следует использовать как можно более низкие дозы вектора rAAV.

Об авторах

M. H. Zhang

School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine,
University of Science and Technology of China; Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences

Email: chunzhang@sibet.ac.cn
China, 230026, Hefei; China, 215163, Suzhou

X. M. Liu

School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine,
University of Science and Technology of China; Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences

Автор, ответственный за переписку.
Email: liuxm@sibet.ac.cn
China, 230026, Hefei; China, 215163, Suzhou

C. Zhang

School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine,
University of Science and Technology of China; Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences

Автор, ответственный за переписку.
Email: chunzhang@sibet.ac.cn
China, 230026, Hefei; China, 215163, Suzhou

Список литературы

  1. Bulcha J.T., Wang Y., Ma H., Tai P.W.L., Gao G. (2021) Viral vector platforms within the gene therapy landscape. Sig. Transduc. Target. Ther. 6, 53.
  2. Lundstrom K. (2018) Viral vectors in gene therapy. D-iseases. 6, 42.
  3. Scott L.J. (2015) Alipogene tiparvovec: a review of its use in adults with familial lipoprotein lipase deficiency. Drugs. 75, 175‒182.
  4. Darrow J.J. (2019) Luxturna: FDA documents reveal the value of a costly gene therapy. Drug Discov. Today. 24, 949‒954.
  5. Urquhart L. (2019) FDA new drug approvals in Q2 2019. Nat. Rev. Drug Discov. 18, 575.
  6. Keam S.J. (2022) Eladocagene exuparvovec: first appro-val. Drugs. 82, 1427‒1432.
  7. Blair H.A. (2022) Valoctocogene roxaparvovec: first approval. Drugs. 82, 1505‒1510.
  8. Rutledge E.A., Russell D.W. (1997) Adeno-associated virus vector integration junctions. J. Virol. 71, 8429‒8436.
  9. Kotin R.M., Menninger J.C., Ward D.C., Berns K.I. (1991) Mapping and direct visualization of a region-specific viral DNA integration site on chromosome 19q13-qter. Genomics. 10, 831‒834.
  10. Linden R.M., Ward P., Giraud C., Winocour E., Berns K.I. (1996) Site-specific integration by adeno-associated virus. Proc. Natl. Acad. Sci. USA. 93, 11288‒11294.
  11. Boftsi M., Whittle F.B., Wang J., Shepherd P., Burger L.R., Kaifer K.A., Lorson C.L., Joshi T., Pintel D.J., Majumder K. (2022) The adeno-associated virus 2 genome and Rep 68/78 proteins interact with cellular sites of DNA damage. Hum. Mol. Genet. 31, 985‒998.
  12. Morales L., Gambhir Y., Bennett J., Stedman H.H. (2020) Broader implications of progressive liver dysfunction and lethal sepsis in two boys following systemic high-dose AAV. Mol. Ther. 28, 1753‒1755.
  13. Palazzi X., Pardo I.D., Sirivelu M.P., Newman L., Kumpf S.W., Qian J., Franks T., Lopes S., Liu J., Monarski L., Casinghino S., Ritenour C., Ritenour H., Dubois C., Olson J., Graves J., Alexander K.E., Coskran T., Lanz T.A., Brady J., McCarty D., Suryanarayan S., Whiteley L.O. (2022) Biodistribution and tolerability of AAV-PHP.B-CBh-SMN1 in Wistar Han rats and cynomolgus macaques reveal different toxicologic profiles. Hum. Gene Ther. 33, 175‒187.
  14. Chandler L.C., Yusuf I.H., McClements M.E., Barnard A.R., MacLaren R.E., Xue K. (2020) Immunomodulatory effects of hydroxychloroquine and chloroquine in viral infections and their potential application in retinal gene therapy. Int. J. Mol. Sci. 21, 4972.
  15. Vasileva A., Linden R.M., Jessberger R. (2006) Homologous recombination is required for AAV-mediated gene targeting. Nucleic Acids Res. 34, 3345‒3360.
  16. Chandler R.J., LaFave M.C., Varshney G.K., Burgess S.M., Venditti C.P. (2016) Genotoxicity in mice following AAV gene delivery: a safety concern for human gene therapy? Mol. Ther. 24, 198‒201.
  17. Rumachik N.G., Malaker S.A., Poweleit N., Maynard L.H., Adams C.M., Leib R.D., Cirolia G., Thomas D., Stamnes S., Holt K., Sinn P., May A.P., Paulk N.K. (2020) Methods matter: standard production platforms for recombinant AAV produce chemically and functionally distinct vectors. Mol. Ther. 18, 98‒118.
  18. Schnepp B.C., Clark K.R., Klemanski D.L., Pacak C.A., Johnson P.R. (2003) Genetic fate of recombinant adeno-associated virus vector genomes in muscle. J. Virol. 77, 3495‒3504.
  19. Li Y., Yin Y., Ma J., Sun Y., Zhou R., Cui B., Zhang Y., Yang J., Yan X., Liu Z., Ma Z. (2020) Combination of AAV-mediated NUPR1 knockdown and trifluoperazine induces premature senescence in human lung adenocarcinoma A549 cells in nude mice. Oncol. Rep. 43, 681‒688.
  20. Carbone L., Carbone E.T., Yi E.M., Bauer D.B., Lindstrom K.A., Parker J.M., Austin J.A., Seo Y., Gandhi A.D., Wilkerson J.D. (2012) Assessing cervical dislocation as a humane euthanasia method in mice. J. Am. Assoc. Lab. Anim. Sci. 51, 352‒356.
  21. Tu T., Jilbert A.R. (2017) Detection of hepatocyte clones containing integrated hepatitis B virus DNA using inverse nested PCR. Methods Mol. Biol. 1540, 97‒118.
  22. Saito S., Ohno S.I., Harada Y., Oikawa K., Fujita K., Mineo S., Gondo A., Kanno Y., Kuroda M. (2019) rAAV6-mediated miR-29b delivery suppresses renal fibrosis. Clin. Exp. Nephrol. 23, 1345‒1356.
  23. Tu L., Sun L., Xue J., Zhang Y., Lu Y. (2012) Efficient and durable gene delivery of self complementary adeno-associated virus 6 vector and impact of pre-existing immunity. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 29, 1150‒1155 (in Chenese).
  24. Wang D., Zhou Q., Qiu X., Liu X., Zhang C. (2022) Optimizing rAAV6 transduction of primary T cells for the generation of anti-CD19 AAV-CAR-T cells. Biomed. Pharmacother. 150, 113027.
  25. Bak R.O., Dever D.P., Porteus M.H. (2018) CRISPR-/Cas9 genome editing in human hematopoietic stem cells. Nat. Protoc. 13, 358‒376.
  26. Wilkinson A.C., Dever D.P., Baik R., Camarena J., Hsu I., Charlesworth C.T., Morita C., Nakauchi H., Porteus M.H. (2021) Cas9-AAV6 gene correction of β‑globin in autologous HSCs improves sickle cell disease erythropoiesis in mice. Nat. Commun. 12, 686.
  27. Bengtsson N.E., Tasfaout H., Hauschka S.D., Chamberlain J.S. (2021) Dystrophin gene-editing stability is dependent on dystrophin levels in skeletal but not cardiac muscles. Mol. Ther. 29, 1070‒1085.
  28. Eyquem J., Mansilla-Soto J., Giavridis T., van der Stegen S.J., Hamieh M., Cunanan K.M., Odak A., Gonen M., Sadelain M. (2017) Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. N-ature. 543, 113‒117.
  29. Ciuffi A., Ronen K., Brady T., Malani N., Wang G., Berry C.C., Bushman F.D. (2009) Methods for integration site distribution analyses in animal cell genomes. Methods. 47, 261‒268.
  30. Nakai H., Yant S.R., Storm T.A., Fuess S., Meuse L., Kay M.A. (2001) Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo. J. Virol. 75, 6969‒6976.
  31. Miller D.G., Trobridge G.D., Petek L.M., Jacobs M.A., Kaul R., Russell D.W. (2005) Large-scale analysis of adeno-associated virus vector integration sites in normal human cells. J. Virol. 79, 11434‒11442.
  32. Malik P., McQuiston S.A., Yu X.J., Pepper K.A., Krall W.J., Podsakoff G.M., Kurtzman G.J., Kohn D.B. (1997) Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line. J. Virol. 71, 1776‒1783.
  33. Duan D., Sharma P., Dudus L., Zhang Y., Sanlioglu S., Yan Z., Yue Y., Ye Y., Lester R., Yang J., Fisher K.J., Engelhardt J.F. (1999) Formation of adeno-associated virus circular genomes is differentially regulated by adenovirus E4 ORF6 and E2a gene expression. J. Virol. 73, 161‒169.
  34. Kaeppel C., Beattie S.G., Fronza R., van Logtenstein R., Salmon F., Schmidt S., Wolf S., Nowrouzi A., Glimm H., von Kalle C., Petry H., Gaudet D., Schmidt M. (2013) A largely random AAV integration profile after LPLD gene therapy. Nat. Med. 19, 889‒891.
  35. McAlister V.J., Owens R.A. (2007) Preferential integration of adeno-associated virus type 2 into a polypyrimidine/polypurine-rich region within AAVS1. J. Virol. 81, 9718‒9726.
  36. Bijlani S., Pang K.M., Sivanandam V., Singh A., Chatterjee S. (2021) The role of recombinant AAV in precise genome editing. Fron. Genome Ed. 3, 799722.
  37. Miller D.G., Petek L.M., Russell D.W. (2004) Adeno-associated virus vectors integrate at chromosome breakage sites. Nat. Genet. 36, 767‒773.
  38. Schultz B.R., Chamberlain J.S. (2008) Recombinant adeno-associated virus transduction and integration. Mol. Ther. 16, 1189‒1199.
  39. Nakai H., Iwaki Y., Kay M.A., Couto L.B. (1999) Isolation of recombinant adeno-associated virus vector-cellular DNA junctions from mouse liver. J. Virol. 73, 5438‒5447.
  40. Yang C.C., Xiao X., Zhu X., Ansardi D.C., Epstein N.D., Frey M.R., Matera A.G., Samulski R.J. (1997) Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro. J. Virol. 71, 9231‒9247.
  41. Sabatino D.E., Bushman C.F.D., Chandler R.J., Crystal R.G., Davidson B.L., Dolmetsch R., Eggan K.C., Gao G., Gil-Farina I., Kay M.A., McCarty D.M., Montini E., Ndu A., Yuan J. (2022) Evaluating the state of the science for adeno-associated virus (AAV) integration: an integrated perspective. Mol. Ther. 30, 2646‒2663.
  42. Game J.C. (1993) DNA double-strand breaks and the RAD50-RAD57 genes in Saccharomyces. Semin. Cancer Biol. 4, 73‒83.
  43. Smih F., Rouet P., Romanienko P.J., Jasin M. (1995) Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells. Nucleic Acids Res. 23, 5012‒5019.
  44. Miller D.G., Petek L.M., Russell D.W. (2003) Human gene targeting by adeno-associated virus vectors is enhanced by DNA double-strand breaks. Mol. Cell. Biol. 23, 3550‒3557.
  45. Donsante A., Vogler C., Muzyczka N., Crawford J.M., Barker J., Flotte T., Campbell-Thompson M., Daly T., Sands M.S. (2001) Observed incidence of tumorigenesis in long-term rodent studies of rAAV vectors. Gene Ther. 8, 1343‒1346.
  46. Bell P., Moscioni A.D., McCarter R.J., Wu D., Gao G., Hoang A., Sanmiguel J.C., Sun X., Wivel N.A., Raper S.E., Furth E.E., Batshaw M.L., Wilson J.M. (2006) Analysis of tumors arising in male B6C3F1 mice with and without AAV vector delivery to liver. Mol. Ther. 14, 34‒44.
  47. Chandler R.J., LaFave M.C., Varshney G.K., Trivedi N.S., Carrillo-Carrasco N., Senac J.S., Wu W., Hoffmann V., Elkahloun A.G., Burgess S.M., Venditti C.P. (2015) Vector design influences hepatic genotoxicity after ade-no-associated virus gene therapy. J. Clin. Invest. 125, 870‒880.
  48. Senís E., Mosteiro L., Wilkening S., Wiedtke E., Nowrouzi A., Afzal S., Fronza R., Landerer H., Abad M., Niopek D., Schmidt M., Serrano M., Grimm D. (2018) AAV vector-mediated in vivo reprogramming into pluripotency. Nat. Commun. 9, 2651.
  49. Gil-Farina I., Fronza R., Kaeppel C., Lopez-Franco E., Ferreira V., D’Avola D., Benito A., Prieto J., Petry H., Gonzalez-Aseguinolaza G., Schmidt M. (2016) Recombinant AAV integration is not associated with hepatic genotoxicity in nonhuman primates and patients. Mol. Ther. 24, 1100‒1105.
  50. Zen Z., Espinoza Y., Bleu T., Sommer J.M., Wright J.F. (2004) Infectious titer assay for adeno-associated virus vectors with sensitivity sufficient to detect single infectious events. Hum. Gene Ther. 15, 709‒715.
  51. Mietzsch M., Grasse S., Zurawski C., Weger S., Bennett A., Agbandje-McKenna M., Muzyczka N., Zolotukhin S., Heilbronn R. (2014) OneBac: platform for scalable and high-titer production of adeno-associated virus serotype 1‒12 vectors for gene therapy. Hum. Gene Ther. 25, 212‒222.
  52. Dalwadi D.A., Calabria A., Tiyaboonchai A., Posey J., Naugler W.E., Montini E., Grompe M. (2021) AAV integration in human hepatocytes. Mol. Ther. 29, 2898‒2909.

Дополнительные файлы

Доп. файлы
Действие
1. JATS XML
Скачать

© M.H. Zhang, X.M. Liu, C. Zhang, 2023