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• W103437854 abstract "Lentiviral vectors (LV) have seen considerably increase in use as gene therapy vectors for the treatment of acquired and inherited diseases. This review presents the state of the art of the production of these vectors with particular emphasis on their large-scale production for clinical purposes. In contrast to oncoretroviral vectors, which are produced using stable producer cell lines, clinical-grade LV are in most of the cases produced by transient transfection of 293 or 293T cells grown in cell factories. However, more recent developments, also, tend to use hollow fiber reactor, suspension culture processes, and the implementation of stable producer cell lines. As is customary for the biotech industry, rather sophisticated downstream processing protocols have been established to remove any undesirable process-derived contaminant, such as plasmid or host cell DNA or host cell proteins. This review compares published large-scale production and purification processes of LV and presents their process performances. Furthermore, developments in the domain of stable cell lines and their way to the use of production vehicles of clinical material will be presented. Lentiviral vectors (LV) have seen considerably increase in use as gene therapy vectors for the treatment of acquired and inherited diseases. This review presents the state of the art of the production of these vectors with particular emphasis on their large-scale production for clinical purposes. In contrast to oncoretroviral vectors, which are produced using stable producer cell lines, clinical-grade LV are in most of the cases produced by transient transfection of 293 or 293T cells grown in cell factories. However, more recent developments, also, tend to use hollow fiber reactor, suspension culture processes, and the implementation of stable producer cell lines. As is customary for the biotech industry, rather sophisticated downstream processing protocols have been established to remove any undesirable process-derived contaminant, such as plasmid or host cell DNA or host cell proteins. This review compares published large-scale production and purification processes of LV and presents their process performances. Furthermore, developments in the domain of stable cell lines and their way to the use of production vehicles of clinical material will be presented. With the first marketing authorization of an AAV1 vector for the treatment of lipoprotein lipase deficiency (Glybera®) in Europe,1Salmon F Grosios K Petry H Safety profile of recombinant adeno-associated viral vectors: focus on alipogene tiparvovec (Glybera®).Expert Rev Clin Pharmacol. 2014; 7: 53-65Crossref PubMed Scopus (56) Google Scholar viral vector-based gene therapy is more and more rapidly evolving towards the routine treatment of rare and acquired diseases for which different viral vectors systems are available. Depending on the purpose of the treatment as well as the target cells or tissues to be treated, one or the other vector system is preferable. In case of dividing, tissues or cells integrating vectors are required for the long-term expression of the transgene. Traditionally, retroviral vectors (in a large sense) are the vectors of choice because they lead to a stable integration of the transgene to be expressed. Mainly two different retroviral vector systems have been developed: γ-retroviral vectors derived from murine leukemia viruses (MLV)2Andreadis ST Roth CM Le Doux JM Morgan JR Yarmush ML Large-scale processing of recombinant retroviruses for gene therapy.Biotechnol Prog. 1999; 15: 1-11Crossref PubMed Scopus (88) Google Scholar and lentiviral vectors (LV) mainly derived from HIV-1.3Delenda C Lentiviral vectors: optimization of packaging, transduction and gene expression.J Gene Med. 2004; 6: S125-S138Crossref PubMed Scopus (85) Google Scholar In the past, many clinical trials based on the use of MLV vectors were successful4Cavazzana-Calvo M Hacein-Bey S de Saint Basile G Gross F Yvon E Nusbaum P et al.Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease.Science. 2000; 288: 669-672Crossref PubMed Scopus (2072) Google Scholar and although these vectors are still used, the general tendency is towards the use of LV vectors. Different reasons can be quoted for this shift: (i) in contrast to γ-retroviral vectors, LVs are able to transduce nondividing cells because they can translocate across the nuclear membrane5Naldini L Blömer U Gallay P Ory D Mulligan R Gage FH et al.In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.Science. 1996; 272: 263-267Crossref PubMed Scopus (3681) Google Scholar; (ii) their integration patterns are different from MLV vectors and seem to be less risky with respect to insertional mutagenesis6Montini E Cesana D Schmidt M Sanvito F Bartholomae CC Ranzani M et al.The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy.J Clin Invest. 2009; 119: 964-975Crossref PubMed Scopus (394) Google Scholar; and (iii) they can be produced at high vector titer. These are the main reasons why there is a clear transition from the use of MLV to LV vectors though the overall manufacturing conditions for LV vectors have not yet reached their maximal potential and the level of those used for MLV vectors. LV vectors have been used successfully in clinical trials, in a first instance for the treatment of rare diseases, in particular, of primary immunodeficiencies7Mukherjee S Thrasher AJ Gene therapy for PIDs: progress, pitfalls and prospects.Gene. 2013; 525: 174-181Crossref PubMed Scopus (96) Google Scholar,8Aiuti A Biasco L Scaramuzza S Ferrua F Cicalese MP Baricordi C et al.Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott–Aldrich syndrome.Science. 2013; 341: 1233151Crossref PubMed Scopus (658) Google Scholar and in neurodegenerative storage diseases.9Cartier N Hacein-Bey-Abina S Bartholomae CC Veres G Schmidt M Kutschera I et al.Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy.Science. 2009; 326: 818-823Crossref PubMed Scopus (1039) Google Scholar,10Biffi A Montini E Lorioli L Cesani M Fumagalli F Plati T et al.Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy.Science. 2013; 341: 1233158Crossref PubMed Scopus (743) Google Scholar However, their application for the treatment of more frequent genetic and acquired diseases, including treatment of β-thalassemia,11Cavazzana-Calvo M Payen E Negre O Wang G Hehir K Fusil F et al.Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia.Nature. 2010; 467: 318-322Crossref PubMed Scopus (856) Google Scholar Parkinson's disease,12Palfi S Gurruchaga JM Ralph GS Lepetit H Lavisse S Buttery PC et al.Long-term safety and tolerability of ProSavin, a lentiviral vector-based gene therapy for Parkinson's disease: a dose escalation, open-label, phase 1/2 trial.Lancet. 2014; 383: 1138-1146Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar and chimeric antigen receptor-based immunotherapy of cancer,13Levine BL Performance-enhancing drugs: design and production of redirected chimeric antigen receptor (CAR) T cells.Cancer Gene Ther. 2015; 22: 79-84Crossref PubMed Scopus (60) Google Scholar has been assessed in clinics with exciting outcomes. This means that manufacturing technology becomes a critical issue in view of the implementation of these novel therapies for routine use. Thus this review, based on publically available sources, presents the actual state of the art of production means for LV vectors, providing information on advantages and short comings of actual protocols (or methods) and devices as well as on maximal manufacturing levels achievable (titer, total vector quantity) and finishing with a perspective of what should come next. The prototype LV vector system is based on HIV-1, a very well-studied human pathogen virus. Besides HIV-1, other lentiviruses have also been developed as gene transfer vectors (TVs) but most of them have not yet reached the clinical study stage, such as HIV-2 (ref. 14Poeschla E Gilbert J Li X Huang S Ho A Wong-Staal F Identification of a human immunodeficiency virus type 2 (HIV-2) encapsidation determinant and transduction of nondividing human cells by HIV-2-based lentivirus vectors.J Virol. 1998; 72: 6527-6536Crossref PubMed Google Scholar) simian immunodeficiency viruses,15Kim SS Kothari N You XJ Robinson Jr, WE Schnell T Uberla K et al.Generation of replication-defective helper-free vectors based on simian immunodeficiency virus.Virology. 2001; 282: 154-167Crossref PubMed Scopus (10) Google Scholar or nonprimate lentiviruses including feline immunodeficiency virus,16Curran MA Kaiser SM Achacoso PL Nolan GP Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors.Mol Ther. 2000; 1: 31-38Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar bovine immunodeficiency virus17Berkowitz RD Ilves H Plavec I Veres G Gene transfer systems derived from Visna virus: analysis of virus production and infectivity.Virology. 2001; 279: 116-129Crossref PubMed Scopus (46) Google Scholar or caprine arthritis-encephalitis virus.18Mselli-Lakhal L Guiguen F Greenland T Mornex JF Chebloune Y Gene transfer system derived from the caprine arthritis-encephalitis lentivirus.J Virol Methods. 2006; 136: 177-184Crossref PubMed Scopus (13) Google Scholar Only equine infectious anemia virus (EIAV)-based vectors19Mitrophanous K Yoon S Rohll J Patil D Wilkes F Kim V et al.Stable gene transfer to the nervous system using a non-primate lentiviral vector.Gene Ther. 1999; 6: 1808-1818Crossref PubMed Scopus (238) Google Scholar have been developed up to clinical use. In the following, this review article will focus on HIV-1-based LV vector system. Essentially guided by safety considerations due to the pathogenicity of HIV-1 in humans, different generations of LV vector systems20Ramezani A Hawley RG Overview of the HIV-1 lentiviral vector system.Curr Protoc Mol Biol. 2002; (Chapter 16: Unit 16.21)Google Scholar have been developed of which the third generation is widely used for R&D and clinical purposes today. It is a four-plasmid system (Figure 1), consisting of three helper plasmids and one TV plasmid. The choice of the helper plasmids was dictated by the principle of the rationale design of a split genome conditional packaging system described by Dull et al.21Dull T Zufferey R Kelly M Mandel RJ Nguyen M Trono D et al.A third-generation lentivirus vector with a conditional packaging system.J Virol. 1998; 72: 8463-8471Crossref PubMed Google Scholar This production system is associated with all required features necessary for a safe use in clinics (use of nonoverlapping split-genome packaging constructs to maximally reducing the potential recombination events, which could lead to the generation of replication-competent lentivirus (RCL)).21Dull T Zufferey R Kelly M Mandel RJ Nguyen M Trono D et al.A third-generation lentivirus vector with a conditional packaging system.J Virol. 1998; 72: 8463-8471Crossref PubMed Google Scholar All accessory genes of HIV-1 (vif, vpr, vpu, and nef),22Kim VN Mitrophanous K Kingsman SM Kingsman AJ Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1.J Virol. 1998; 72: 811-816Crossref PubMed Google Scholar present only in the very first generation of LV, have been removed because they are not necessary. Similarly, the regulatory tat gene, present in the second-generation LV, has been eliminated because its transacting function is dispensable as the U3 promoter of the 5′ long terminal repeat (LTR) in the TV has been replaced by a constitutively active promoter sequence, such as cytomegalovirus (promoter)22Kim VN Mitrophanous K Kingsman SM Kingsman AJ Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1.J Virol. 1998; 72: 811-816Crossref PubMed Google Scholar, 23Miyoshi H Blömer U Takahashi M Gage FH Verma IM Development of a self-inactivating lentivirus vector.J Virol. 1998; 72: 8150-8157Crossref PubMed Google Scholar, 24Zufferey R Dull T Mandel RJ Bukovsky A Quiroz D Naldini L et al.Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery.J Virol. 1998; 72: 9873-9880Crossref PubMed Google Scholar or Rous Sarcoma Virus (promoter)21Dull T Zufferey R Kelly M Mandel RJ Nguyen M Trono D et al.A third-generation lentivirus vector with a conditional packaging system.J Virol. 1998; 72: 8463-8471Crossref PubMed Google Scholar plus an optional enhancer25Schambach A Swaney WP van der Loo JC Design and production of retro- and lentiviral vectors for gene expression in hematopoietic cells.Methods Mol Biol. 2009; 506: 191-205Crossref PubMed Scopus (30) Google Scholar or an inducible/repressible promoter sequence, such as 7tetO.26Throm RE Ouma AA Zhou S Chandrasekaran A Lockey T Greene M et al.Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection.Blood. 2009; 113: 5104-5110Crossref PubMed Scopus (82) Google Scholar This is commonly referred as the pRRL (lentivirus transfer vector construct containing chimeric Rous sarcoma virus (RSV)-HIV 5' LTRs) design or the pCCL design ((CMV)-HIV 5′LTR). These modifications lead to a LV vector system with the helper functions based on the use of gag-pol (encoding for the structural proteins and viral enzymes) and rev (encoding for a post-transcriptional regulator) derived from HIV-1 and env. Although LV vectors can be pseudotyped with different heterologous envelope glycoproteins,27Frecha C Szécsi J Cosset FL Verhoeyen E Strategies for targeting lentiviral vectors.Curr Gene Ther. 2008; 8: 449-460Crossref PubMed Scopus (66) Google Scholar all large-scale (clinical scale) vector preparations have made use of the glycoprotein of the vesicular stomatitis virus (VSV-g) envelope, due to improved stability during downstream processing and its large transduction spectrum. The TV plasmid is the only genetic material transferred to the target cells and consists of the LV backbone containing the transgene expression cassette flanked by cis-acting elements required for encapsidation, reverse transcription, and integration. In view of biosafety improvement, self-inactivating (SIN)-LV vectors have been developed, for which a deletion has been introduced into the U3 element of the 3′LTR.23Miyoshi H Blömer U Takahashi M Gage FH Verma IM Development of a self-inactivating lentivirus vector.J Virol. 1998; 72: 8150-8157Crossref PubMed Google Scholar,24Zufferey R Dull T Mandel RJ Bukovsky A Quiroz D Naldini L et al.Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery.J Virol. 1998; 72: 9873-9880Crossref PubMed Google Scholar This type of vector loses the transcriptional capacity of the viral LTR once transferred to the target cells minimizing the risk of emergence of replication competent recombinants and avoiding problems linked to promoter interference. Considering vector production, HEK293 or HEK293T cells (see below) are transfected with four plasmids encoding the gag-pol genes, the rev gene, the VSV-g envelope gene, as well as a SIN LV TV plasmid with an internal promoter for transgene expression (Figure 1). Further safety improvements of the LV vector system are related to the removal of residual sequence homologies existing between the vector genome because the packaging signal (Ψ) that extends into the first bp of the gag gene28Swanstrom R Wills JW Synthesis, assembly, and processing of viral proteins.in: Coffin J Hughes SH Varmus H Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor/NY1997: 263-334Google Scholar and the gag-pol construct via codon optimization. Given that codon optimization renders the translation of gag-pol proteins rev-independent29Kotsopoulou E Kim VN Kingsman AJ Kingsman SM Mitrophanous KA A Rev-independent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits a codon-optimized HIV-1 gag-pol gene.J Virol. 2000; 74: 4839-4852Crossref PubMed Scopus (181) Google Scholar,30Wagner R Graf M Bieler K Wolf H Grunwald T Foley P et al.Rev-independent expression of synthetic gag-pol genes of human immunodeficiency virus type 1 and simian immunodeficiency virus: implications for the safety of lentiviral vectors.Hum Gene Ther. 2000; 11: 2403-2413Crossref PubMed Scopus (113) Google Scholar the Rev-Responsive-Element (RRE) sequence can be removed from the packaging construct. Since the TV contains a portion of the gag sequence codon optimization of the gag-pol sequence renders the so-called ψ-gag recombination (for instance, reported by Sastry et al.31Sastry L Xu Y Johnson T Desai K Rissing D Marsh J et al.Certification assays for HIV-1-based vectors: frequent passage of gag sequences without evidence of replication-competent viruses.Mol Ther. 2003; 8: 830-839Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) impossible.32Tareen SU Nicolai CJ Campbell DJ Flynn PA Slough MM Vin CD et al.A Rev-independent gag/pol eliminates detectable psi-gag recombination in lentiviral vectors.Biores Open Access. 2013; 2: 421-430Crossref PubMed Google Scholar An additional advantage of codon optimization is about 100-fold reduced frequency of potential recombination events.30Wagner R Graf M Bieler K Wolf H Grunwald T Foley P et al.Rev-independent expression of synthetic gag-pol genes of human immunodeficiency virus type 1 and simian immunodeficiency virus: implications for the safety of lentiviral vectors.Hum Gene Ther. 2000; 11: 2403-2413Crossref PubMed Scopus (113) Google Scholar A complete rev-independence of the LV vector system characterized by the absence of the RRE sequence in the TV has not been shown for the moment. The function of RRE (which is part of the unspliced vector transcript) consists in fact in the assembly of multiple molecules of Rev to form an oligomeric complex stabilizing and mediating the export of the vector transcripts from the nucleus to the cytoplasm during the production of the vector, but not after integration of the TV in target cells.33Fernandes J Jayaraman B Frankel A The HIV-1 Rev response element: an RNA scaffold that directs the cooperative assembly of a homo-oligomeric ribonucleoprotein complex.RNA Biol. 2012; 9: 6-11Crossref PubMed Scopus (37) Google Scholar Alternative export sequences, such as the constitutive transport elements or the RNA transport element, were shown to be functional in the context of the gag-pol production,34Zolotukhin AS Valentin A Pavlakis GN Felber BK Continuous propagation of RRE(−) and Rev(−)RRE(−) human immunodeficiency virus type 1 molecular clones containing a cis-acting element of simian retrovirus type 1 in human peripheral blood lymphocytes.J Virol. 1994; 68: 7944-7952PubMed Google Scholar, 35Gasmi M Glynn J Jin MJ Jolly DJ Yee JK Chen ST Requirements for efficient production and transduction of human immunodeficiency virus type 1-based vectors.J Virol. 1999; 73: 1828-1834Crossref PubMed Google Scholar, 36Nappi F Schneider R Zolotukhin A Smulevitch S Michalowski D Bear J et al.Identification of a novel posttranscriptional regulatory element by using a rev- and RRE-mutated human immunodeficiency virus type 1 DNA proviral clone as a molecular trap.J Virol. 2001; 75: 4558-4569Crossref PubMed Scopus (40) Google Scholar but they have not yet been shown to be as efficient as RRE sequence in context of the TV.3Delenda C Lentiviral vectors: optimization of packaging, transduction and gene expression.J Gene Med. 2004; 6: S125-S138Crossref PubMed Scopus (85) Google Scholar,22Kim VN Mitrophanous K Kingsman SM Kingsman AJ Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1.J Virol. 1998; 72: 811-816Crossref PubMed Google Scholar,35Gasmi M Glynn J Jin MJ Jolly DJ Yee JK Chen ST Requirements for efficient production and transduction of human immunodeficiency virus type 1-based vectors.J Virol. 1999; 73: 1828-1834Crossref PubMed Google Scholar Thus, today, LV vector systems still make use of Rev/RRE for efficient vector genome export to the cytoplasm. A rev-independent vector technology was only successfully implemented for the EIAV system in which only three plasmids (two helper plasmids coding for the gag-pol and the env functions and the TV plasmid) are required37Rohll JB Mitrophanous KA Martin-Rendon E Ellard FM Radcliffe PA Mazarakis ND et al.Design, production, safety, evaluation, and clinical applications of nonprimate lentiviral vectors.Methods Enzymol. 2002; 346: 466-500Crossref PubMed Scopus (62) Google Scholar (Table 1).Table 1Large-scale cell culture GMP productions of LV vectorsCompany/institutionCell line, vector systemCulture system (number of culture units)Number of plasmidsMaximal production scale (l)Titer (before purification)bIt has to be kept in mind that the titers between the different laboratories cannot be compared because different transgenes, different promoters, and non standardized analytical methods have been used.Virxsys52Slepushkin V Chang N Cohen R Gan Y Jiang B Deausen E et al.Large-scale purification of a lentiviral vector by size exclusion chromatography or Mustang Q ion exchange chromatography.Bioproc J. 2003; 2: 89-95Crossref Google Scholar293, HIV-1NC236–522.02 × 107 TU/mlGénéthon42Merten OW Charrier S Laroudie N Fauchille S Dugué C Jenny C et al.Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application.Hum Gene Ther. 2011; 22: 343-356Crossref PubMed Scopus (103) Google Scholar/MolMed105Bellintani F Piacenza L Sciarretta R Martelli L Massa S Vallanti G et al.Large scale process for the production and purification of lentiviral vectors for clinical applications.Hum Gene Ther. 2008; 19: 1089Google Scholar293T, HIV-1CF-10 (12–24)424–501–5 × 107 IG/mlBeckman Research In-stitut (City of Hope/CA)43Ausubel LJ Hall C Sharma A Shakeley R Lopez P Quezada V et al.Production of CGMP-grade lentiviral vectors.Bioprocess Int. 2012; 10: 32-43PubMed Google Scholar,61Couture LA Vector Production in Support of Early Clinical Trials. 2008; (Presented at the ASGT Meeting, 28 May–1 June 2008, Boston, MA)Google Scholar293T, HIV-1CF-10 (12 × 10 = 120)41200.5–1 × 106 TU/mlOxford Biomedica/Henogen[55Dupont F Large Scale Manufacturing of a Lentiviral Vector (ProSavin®) for Phase I/II Clinical Trial. 2008; (Presented at the CONSERT Labcourse, Evry/F, 29 June–1 July 2008)Google Scholar, Mitrophanous, personal communication]293T, EIAVCF-10 (24 per run, 3 campaigns)3720.2–2 × 106 TU/mlGénéthon63Marceau N Gasmi M Scalable lentiviral vector production system compatible with industrial pharmaceutical applications. 2013; (WO 2013076309 A1)Google Scholar,123Hebben M Pre-industrial Manufacturing of Lentiviral Vectors by Transient Transfection in Single Use Systems. 2015; (Presented at the Spring Meeting of ISBiotech, 9 March–11 March 2015, Washington/DC)Google Scholar293T, HIV-1STR (50/ 200 l)450/2005 × 107 IG/mlSt. Jude Children's Hospital56Greene MR Lockey T Mehta PK Kim YS Eldridge PW Gray JT et al.Transduction of human CD34+ repopulating cells with a self-inactivating lentiviral vector for SCID-X1 produced at clinical scale by a stable cell line.Hum Gene Ther Methods. 2012; 23: 297-308Crossref PubMed Scopus (27) Google ScholarStable 293T, tet-off, HIV-150-l WAVE reactor with HEK293T cells immobilized on Fibra-celInduction by removal of doxycyclineAbout 138 per batch0.5–1 × 107 TU/mlUniversity of California Davis School of Medicine62Sheu J Beltzer J Fury B Wilczek K Tobin S Falconer D et al.Large-scale production of lentiviral vector in a closed system hollow fiber bioreactor.Mol Ther Methods Clin Dev. 2015; 2: 15020Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar293T, HIV-1Hollow fiber systemaEquivalent with three CF-10.4NC1.0–2.8 × 108 vg/mlLV, lentiviral vectors; NC, not communicated; STR, stirred tank reactor.a Equivalent with three CF-10.b It has to be kept in mind that the titers between the different laboratories cannot be compared because different transgenes, different promoters, and non standardized analytical methods have been used. Open table in a new tab LV, lentiviral vectors; NC, not communicated; STR, stirred tank reactor. In the past, Virxsys has developed a two-plasmid system for the treatment of HIV infection using a conditionally replicating LV vector.38Levine BL Humeau LM Boyer J MacGregor RR Rebello T Lu X et al.Gene transfer in humans using a conditionally replicating lentiviral vector.Proc Natl Acad Sci USA. 2006; 103: 17372-17377Crossref PubMed Scopus (387) Google Scholar This system is characterized by the assembly of all helper functions (gag-pol, rev, tat, and VSV-g) on one plasmid.39Lu X Humeau L Slepushkin V Binder G Yu Q Slepushkina T et al.Safe two-plasmid production for the first clinical lentivirus vector that achieves >99% transduction in primary cells using a one-step protocol.J Gene Med. 2004; 6: 963-973Crossref PubMed Scopus (70) Google Scholar Due to the use of full LTRs, this system is tat-dependent and therefore by definition a second-generation LV vector system. The maintenance of full LTRs is retained to allow the transcription of the antisense against the HIV envelope gene39Lu X Humeau L Slepushkin V Binder G Yu Q Slepushkina T et al.Safe two-plasmid production for the first clinical lentivirus vector that achieves >99% transduction in primary cells using a one-step protocol.J Gene Med. 2004; 6: 963-973Crossref PubMed Scopus (70) Google Scholar only in the HIV-infected target cells that express Tat. Though such a production system is easier to produce and less expensive in its application and leads to higher vector titer than the three or four plasmid systems,39Lu X Humeau L Slepushkin V Binder G Yu Q Slepushkina T et al.Safe two-plasmid production for the first clinical lentivirus vector that achieves >99% transduction in primary cells using a one-step protocol.J Gene Med. 2004; 6: 963-973Crossref PubMed Scopus (70) Google Scholar the presence of all helper genes located on one plasmid might be a concern with respect to the formation of RCLs. However, none of the vector lots or of the transduced cell products were shown to contain RCLs.40Schonely K Afable C Slepushkin V Lu X Andre K Boehmer J et al.QC release testing of an HIV-1 based lentiviral vector lot and transduced cellular product.Bioproc J. 2003; 2: 39-47Google Scholar Most of the current LV vector production methods involve cotransfection of, preferentially, HEK293T or HEK293 cells. The reason for the preference of HEK293T cells is that the presence of the SV40 T-antigen in the producer cells renders them more efficient for vector production. In addition, HEK293T cells show increased cell growth and transfection efficiency in comparison to HEK293 cells.41Gama-Norton L Botezatu L Herrmann S Schweizer M Alves PM Hauser H et al.Lentivirus production is influenced by SV40 large T-antigen and chromosomal integration of the vector in HEK293 cells.Hum Gene Ther. 2011; 22: 1269-1279Crossref PubMed Scopus (19) Google Scholar, 42Merten OW Charrier S Laroudie N Fauchille S Dugué C Jenny C et al.Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application.Hum Gene Ther. 2011; 22: 343-356Crossref PubMed Scopus (103) Google Scholar, 43Ausubel LJ Hall C Sharma A Shakeley R Lopez P Quezada V et al.Production of CGMP-grade lentiviral vectors.Bioprocess Int. 2012; 10: 32-43PubMed Google Scholar A thorough explanation for the improvement of the “cellular context” cannot be given today, however, Gama-Norton et al.41Gama-Norton L Botezatu L Herrmann S Schweizer M Alves PM Hauser H et al.Lentivirus production is influenced by SV40 large T-antigen and chromosomal integration of the vector in HEK293 cells.Hum Gene Ther. 2011; 22: 1269-1279Crossref PubMed Scopus (19) Google Scholar could show that, in the context of stable HEK293-based producer cell lines, clones containing the SV40 T-antigen showed higher vector production rates than those not expressing the SV40 T-antigen. The only difference between the clones was the expression/nonexpression of this antigen. The positive effect of the presence of SV40 T-antigen during LV vector production was also shown by Smith and Shioda44Smith SL Shioda T Advantages of COS-1 monkey kidney epithelial cells as packaging host for small-volume production of high-quality recombinant lentiviruses.J Virol Methods. 2009; 157: 47-54Crossref PubMed Scopus (11) Google Scholar in the context of CV-1 (SV40 T-antigen negative) – COS-1 (SV40 T-antigen positive) cells. The latter cells are of high interest for screening purposes related to LV vectors as well as for automated manufacturing at small scale because of improved vector quality (improved infectious to total vector ratio; reduced level of contaminating cellular proteins) in comparison to HEK293T cells.44Smith SL Shioda T Advantages of COS-1 monkey kidney epithelial cells as packaging host for small-volume production of high-quality recombinant lentiviruses.J Virol Methods. 2009; 157: 47-54Crossref PubMed Scopus (11) Google Scholar However, the main drawback when using COS-1 cells is that these cells are obligatory adherent cells, whereas HEK293T cells can be adapted to suspension growth in serum-free medium which is of particular interest for large-scale vector productions. The following section compares the production processes that have been described in the literature from bench scale to industrial manufacturing. The comparison focuses mainly on the technical methods developed by the producers. It would not be appropriate to discuss the performance and prod" @default.
• W103437854 created "2016-06-24" @default.
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• W103437854 date "2016-01-01" @default.
• W103437854 modified "2023-10-16" @default.
• W103437854 title "Production of lentiviral vectors" @default.
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