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Ricobaraza, A.;  Gonzalez-Aparicio, M.;  Mora-Jimenez, L.;  Lumbreras, S.;  Hernandez-Alcoceba, R. Production of HC-AdVs. Encyclopedia. Available online: https://encyclopedia.pub/entry/26178 (accessed on 03 July 2024).
Ricobaraza A,  Gonzalez-Aparicio M,  Mora-Jimenez L,  Lumbreras S,  Hernandez-Alcoceba R. Production of HC-AdVs. Encyclopedia. Available at: https://encyclopedia.pub/entry/26178. Accessed July 03, 2024.
Ricobaraza, Ana, Manuela Gonzalez-Aparicio, Lucia Mora-Jimenez, Sara Lumbreras, Ruben Hernandez-Alcoceba. "Production of HC-AdVs" Encyclopedia, https://encyclopedia.pub/entry/26178 (accessed July 03, 2024).
Ricobaraza, A.,  Gonzalez-Aparicio, M.,  Mora-Jimenez, L.,  Lumbreras, S., & Hernandez-Alcoceba, R. (2022, August 16). Production of HC-AdVs. In Encyclopedia. https://encyclopedia.pub/entry/26178
Ricobaraza, Ana, et al. "Production of HC-AdVs." Encyclopedia. Web. 16 August, 2022.
Production of HC-AdVs
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This entry is adapted from the peer-reviewed paper 10.3390/ijms21103643

The adaptation of adenoviruses as gene delivery tools has resulted in the development of high-capacity adenoviral vectors (HC-AdVs), also known, helper-dependent or “gutless”. Compared with earlier generations (E1/E3-deleted vectors), HC-AdVs retain relevant features such as genetic stability, remarkable efficacy of in vivo transduction, and production at high titers. The removal of all viral coding genes dictates the unique properties of HC-AdVs in terms of stability of expression and cloning capacity, which differentiate them from early AdV vector versions. The downside is a greater complexity of the production procedures, because stable expression of all adenoviral genes in packaging cells, in the pattern and amount required for trans-complementation, is not feasible.

adenovirus high-capacity adenovirus helper-dependent

1. Viral Rescue and Amplification

The first method for HC-AdV production dates back to 1996, when Kochanek et al. prepared a plasmid containing 28 Kb of non-viral DNA flanked by AdV ITRs, with the packaging signal in one end [1]. The HV was a FGAdV with a 91 bp deletion in the packaging signal, so it would have a competitive disadvantage for encapsidation. When the plasmid was transfected in HEK293 cells and then cells were infected with the HV, they could rescue a small amount of HC-AdV. After several rounds of co-infection in a growing number of cells, the vector was purified by ultracentrifugation in a density gradient. The final contamination with HV was in the range of 1%, but production of HV was problematic because of the inefficiency of packaging. Later, Parks et al. introduced the concept of Cre-mediated excision of the packaging signal [2]. When this sequence is flanked by loxP sites in the HV and the cells express the recombinase, high yields of HC-AdV with less than 0.1% HV contamination can be routinely obtained. Apart from the efficient removal of the packaging signal, an important advantage of this methods is the easy production of the HV in standard HEK293 cells. Although similar results were obtained when the FLP/frt system was used [3], the Cre/loxP system is currently the gold standard, and it is the basis for most improvements developed since then, as described below. The HV is usually an E1/E3-deleted vector, but restoration of the E3 region has been described to increase its helper efficacy. On the other hand, deletion of the E2 region may increase the safety of HVs, although special packaging cells complementing the E1 and E2 genes are required [4]. During HC-AdV amplification, the shut-off of protein synthesis in the cell, imposed by the virus, limits the availability of the Cre recombinase. This is the moment of highest demand for the removal of packaging signal in a growing number of HV genomes [5]. To avoid this limitation, a self-inactivating HV was developed in which the recombinase is inserted in its own genome [6]. Owing to a drug-inducible system and the use of the MerCreMer fusion protein [7], cleavage of ψ can be modulated and this HV can be produced in HEK293 cells. In contrast, this sequence is efficiently cleaved when the expression of MerCreMer is stimulated by doxycycline, and the addition of 4-hydroxy-tamoxifen allows the access of the protein to the nucleus. Increasing the difference in genome size between HV and HC-AdV facilitates the separation of the particles by density, and inverting the orientation of the packaging signal in the HV reduces the risk of productive recombination with the HC-AdV genomes [8]. Deletion of the pIX gene decreases the packaging capacity of HAdV to 35 Kb. If the size of the HV exceeds this limit and harbors this deletion, it can only be produced in specialized HEK293 cells expressing pIX. This phenomenon can be exploited to reduce HV contamination [9]. Flanking the HV packaging signal by attB/attP sequences produces a delay in encapsidation, which can be used to reduce contamination [10]. Other methods rely on the incorporation of the HV genome in other vectors such as Baculovirus [11] or herpes simplex virus (HSV) [12]. More recently, a HV-free method has been described, in which all trans-complementing genes are provided by transfection of a plasmid devoid of packaging signal, in several steps of amplification [13]. This procedure is reminiscent of the initial stages of amplification in pioneering protocols [14], but the new method relies on co-transfection of a plasmid encoding the AdV pre-terminal protein (pTP) to enhance vector yield. This result challenges the notion that TP should be covalently fused to both genome ends in order to promote genome replication [15]. The suitability of this procedure for large-scale production awaits confirmation.

2. Purification

In principle, HC-AdVs can be purified the same as any other AdV. Ultracentrifugation in CsCl density gradients is the traditional method, followed by desalting by size exclusion chromatography (sepharose columns) or dialysis [16][17]. However, iodixanol may have advantages compared with CsCl. On the one hand, it is more biologically compatible and requires shorter centrifugation times. On the other hand, it can preserve particle infectivity during the purification process, and provides better separation of particles with small differences in genome size. In fact, reduction of HV contamination from 2.5 to 0.03% has been reported after two iodixanol purification steps [18]. In principle, these methods allow to discriminate empty and incomplete viral particles from particles containing full vector genomes, improving (reducing) the total to infectious particle ratio. This is especially relevant for HC-AdVs, since crude lysates often present very high ratios. When the difference in genome sizes between HC-AdV and HV is sufficient, selecting the correct fraction can also reduce HV contamination [3]. Anion exchange columns and density gradients can be used sequentially to improve separation [18]. However, ultracentrifugation is not convenient for high-scale production and good manufacturing practices (GMP) adaptation. A combination of chromatographic methods based on capture antibodies, ionic exchange, size exclusion, hydrophobic interaction, and immobilized metal affinity columns has been described [19][20]. Methods that contribute to the enrichment in full vector particles are especially indicated for HC-AdV [21][22].

3. Quantification

Common to all viral vectors, the availability of standardized methods for precise quantification of HC-AdVs is an unmet need. The simplest way to determine the amount of particles (vp) in a purified HC-AdV preparation is based on the absorbance at 260 nm, usually performed after disruption of capsids by detergent (SDS) or enzymatic treatment [23][24]. Similar to other vectors such as those derived from adeno-associated virus (AAV), quantitative PCR can be used to determine the amount of viral genomes (vg) [25], which should provide information equivalent to the spectrophotometer. In both cases, the availability of certified standards could contribute to the reproducibility of results, the comparison of different vector batches, and the uniformity of data across laboratories [26]. In contrast with early generation AdV vectors, HC-AdVs are not replicative even in the packaging cells. Therefore, quantification of plaque forming units (pfu) or infectious units (iu) using end-point dilution methods or commercially available kits is not possible. For determination of iu, permissive cells exposed to the vectors can be lysed a few hours later and viral genomes are quantified by PCR [25][26]. Although this method is more restrictive than direct PCR of particles and provides a closer estimation of transduction potency, it is difficult to standardize, and the values are always relative to the cell line and the culture conditions employed. However, iu quantification can explain apparent inconsistencies in the performance of different vector batches. Only when the production process is perfectly standardized, an equivalent ratio between total and infectious particles can be assumed. This ratio is usually higher in HC-AdVs compared with OAVs or FGAdV vectors, and differences have also been reported among AdV vector platforms. For instance, vectors derived from canine adenovirus type 2 (CAV-2) present ratios of less than 3:1 [27], whereas ratios of 10:1 or higher are common for human vectors, although it can be due to the specific cell lines employed for quantification. Comparing the performance of different vectors such as those derived from AAV and AdV is even more complicated. The few articles taking this challenge usually report the dose of AdV using iu and vp, whereas AAV are quantified in vg [28]. However, a relevant comparison should take into account different parameters such as the balance between safety and efficacy, feasibility of production, and the amount of vector genomes in the target organ needed for the therapeutic effect.

References

  1. Kochanek, S.; Clemens, P.R.; Mitani, K.; Chen, H.H.; Chan, S.; Caskey, C.T. A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase. Proc. Natl. Acad. Sci. USA 1996, 93, 5731–5736.
  2. Parks, R.J.; Chen, L.; Anton, M.; Sankar, U.; Rudnicki, M.A.; Graham, F.L. A helper-dependent adenovirus vector system: Removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc. Natl. Acad. Sci. USA 1996, 93, 13565–13570.
  3. Umaña, P.; Gerdes, C.A.; Stone, D.; Davis, J.R.E.; Ward, D.; Castro, M.G.; Lowenstein, P.R. Efficient FLPe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination. Nat. Biotechnol. 2001, 19, 582–585.
  4. Zhou, H.S.; Zhao, T.; Rao, X.M.; Beaudet, A.L. Production of helper-dependent adenovirus vector relies on helper virus structure and complementing. J. Gene Med. 2002, 4, 498–509.
  5. Ng, P.; Evelegh, C.; Cummings, D.; Graham, F.L. Cre levels limit packaging signal excision efficiency in the Cre/loxP helper-dependent adenoviral vector system. J. Virol. 2002, 76, 4181–4189.
  6. Gonzalez-Aparicio, M.; Mauleon, I.; Alzuguren, P.; Bunuales, M.; Gonzalez-Aseguinolaza, G.; San Martín, C.; Prieto, J.; Hernandez-Alcoceba, R. Self-inactivating helper virus for the production of high-capacity adenoviral vectors. Gene Ther. 2011, 18, 1025–1033.
  7. Verrou, C.; Zhang, Y.; Zürn, C.; Schamel, W.W.A.; Reth, M. Comparison of the Tamoxifen Regulated Chimeric Cre Recombinases MerCreMer and CreMer. Biol. Chem. 1999, 380, 1435–1438.
  8. Palmer, D.; Ng, P. Improved system for helper-dependent adenoviral vector production. Mol. Ther. 2003, 8, 846–852.
  9. Sargent, K.; Ng, P.; Evelegh, C.; Graham, F.; Parks, R. Development of a size-restricted pIX-deleted helper virus for amplification of helper-dependent adenovirus vectors. Gene Ther. 2004, 11, 504–511.
  10. Alba, R.; Hearing, P.; Bosch, A.; Chillon, M. Differential amplification of adenovirus vectors by flanking the packaging signal with attB/attP-ΦC31 sequences: Implications for helper-dependent adenovirus production. Virology 2007, 367, 51–58.
  11. Cheshenko, N.; Krougliak, N.; Eisensmith, R.C.; Krougliak, V.A. A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus. Gene Ther. 2001, 8, 846–854.
  12. Kubo, S.; Saeki, Y.; Antonio Chiocca, E.; Mitani, K. An HSV amplicon-based helper system for helper-dependent adenoviral vectors. Biochem. Biophys. Res. Commun. 2003, 307, 826–830.
  13. Lee, D.; Liu, J.; Junn, H.J.; Lee, E.; Jeong, K.; Seol, D. No more helper adenovirus: Production of gutless adenovirus (GLAd) free of adenovirus and replication-competent adenovirus (RCA) contaminants. Exp. Mol. Med. 2019, 51, 1–18.
  14. Alemany, R.; Dai, Y.; Lou, Y.C.; Sethi, E.; Prokopenko, E.; Josephs, S.F.; Zhang, W.W. Complementation of helper-dependent adenoviral vectors: Size effects and titer fluctuations. J. Virol. Methods 1997, 68, 147–159.
  15. Hartigan-O’Connor, D.; Barjot, C.; Crawford, R.; Chamberlain, J.S. Efficient Rescue of Gutted Adenovirus Genomes Allows Rapid Production of Concentrated Stocks Without Negative Selection. Hum. Gene Ther. 2002, 13, 519–531.
  16. Peixoto, C.; Ferreira, T.B.; Sousa, M.F.Q.; Carrondo, M.J.T.; Alves, P.M. Towards purification of adenoviral vectors based on membrane technology. Biotechnol. Prog. 2008, 24, 1290–1296.
  17. Kratzer, R.F.; Kreppel, F. Production, Purification, and Titration of First-Generation Adenovirus Vectors. In Functional Genomics: Methods in Molecular Biology; Kaufmann, M., Klinger, C., Savelsbergh, A., Eds.; Springer: New York, NY, USA, 2017; Volume 1654, pp. 377–388. ISBN 978-1-4939-7230-2.
  18. Dormond, E.; Chahal, P.; Bernier, A.; Tran, R.; Perrier, M.; Kamen, A. An efficient process for the purification of helper-dependent adenoviral vector and removal of helper virus by iodixanol ultracentrifugation. J. Virol. Methods 2010, 165, 83–89.
  19. Ma, J.; Su, C.; Wang, X.; Shu, Y.; Hu, S.; Zhao, C.; Kuang, Y.; Chen, Y.; Li, Y.; Wei, Y.; et al. A novel method to purify adenovirus based on increasing salt concentrations in buffer. Eur. J. Pharm. Sci. 2020, 141, 105090.
  20. Nestola, P.; Silva, R.J.S.; Peixoto, C.; Alves, P.M.; Carrondo, M.J.T.; Mota, J.P.B. Robust design of adenovirus purification by two-column, simulated moving-bed, size-exclusion chromatography. J. Biotechnol. 2015, 213, 109–119.
  21. Lee, D.; Kim, B.; Seol, D. Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography. Biochem. Biophys. Res. Commun. 2009, 378, 640–644.
  22. Bo, H.; Chen, J.; Liang, T.; Li, S.; Shao, H.; Huang, S. Chromatographic purification of adenoviral vectors on anion-exchange resins. Eur. J. Pharm. Sci. 2015, 67, 119–125.
  23. Mittereder, N.; March, K.L.; Trapnell, B.C. Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy. J. Virol. 1996, 70, 7498–7509.
  24. Sweeney, J.A.; Hennessey, J.P. Evaluation of Accuracy and Precision of Adenovirus Absorptivity at 260 nm under Conditions of Complete DNA Disruption. Virology 2002, 295, 284–288.
  25. Puntel, M.; Curtin, J.F.; Zirger, J.M.; Muhammad, A.K.M.; Xiong, W.; Liu, C.; Hu, J.; Kroeger, K.M.; Czer, P.; Sciascia, S.; et al. Quantification of High-Capacity Helper-Dependent Adenoviral Vector Genomes in Vitro and in Vivo, Using Quantitative TaqMan Real-Time Polymerase Chain Reaction. Hum. Gene Ther. 2006, 17, 531–544.
  26. Palmer, D.J.; Ng, P. Physical and infectious titers of helper-dependent adenoviral vectors: A method of direct comparison to the adenovirus reference material. Mol. Ther. 2004, 10, 792–798.
  27. Kremer, E.J.; Boutin, S.; Chillon, M.; Danos, O. Canine Adenovirus Vectors: An Alternative for Adenovirus-Mediated Gene Transfer. J. Virol. 2000, 74, 505–512.
  28. Montenegro-Miranda, P.S.; Pichard, V.; Aubert, D.; Ten Bloemendaal, L.; Duijst, S.; De Waart, D.R.; Ferry, N.; Bosma, P.J. In the rat liver, Adenoviral gene transfer efficiency is comparable to AAV. Gene Ther. 2014, 21, 168–174.
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Subjects: Biotechnology & Applied Microbiology
Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :
Ana Ricobaraza
,
Manuela Gonzalez-Aparicio
,
Lucia Mora-Jimenez
,
Sara Lumbreras
,
Ruben Hernandez-Alcoceba
View Times: 410
Entry Collection: Biopharmaceuticals Technology
Revisions: 2 times (View History)
Update Date: 16 Aug 2022
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