New developments in lentiviral vector design, production and purification

1. Introduction

2. Lentiviral vectors

3. Recent improvement in LV

production process

4. Downstream processing of

LVVs

5. Expert opinion

Review

New developments in lentiviralvector design, production andpurificationMaria Mercedes Segura, Mathias Mangion, Bruno Gaillet & Alain Garnier††Chemical Engineering Department, Universite Laval, and Regroupement quebecois Sur la

Fonction, la Structure et l’ ingenierie des proteines (PROTEO), Quebec, QC, Canada

Introduction: Lentiviruses are a very potent class of viral vectors for which

there is presently a rapidly growing interest for a number of gene therapy.

However, their construction, production and purification need to be per-

formed according to state-of-the-art techniques in order to obtain sufficient

quantities of high purity material of any usefulness and safety.

Areas covered: The recent advances in the field of recombinant lentivirus vec-

tor design, production and purification will be reviewed with an eye toward

its utilization for gene therapy. Such a review should be helpful for the

potential user of this technology.

Expert opinion: The principal hurdles toward the use of recombinant lentivirus

as a gene therapy vector are the low titer at which it is produced as well as the

difficulty to purify it at an acceptable level without degrading it. The recent

advances in the bioproduction of this vector suggest these issues are about to

be resolved, making the retrovirus gene therapy a mature technology.

Keywords: gene therapy, lentiviral vectors, production, purification, vectors design

Expert Opin. Biol. Ther. (2013) 13(7):987-1011

1. Introduction

Gene therapy has been part of our reality for more than two decades and is still arapidly evolving field. The first viral vector used for clinical applications has beenconstructed from the Moloney murine leukemia virus (MLV) [1], a g-retrovirus(RV) from the Retroviridae family, also comprising the lentivirus (LV) genus.Recombinant MLV still represents the most prominent gene vector used for genetransfer clinical trials as it combines interesting properties for gene therapy such asa large tropism, an efficient integration and a robust, stable expression in target cells.However, it is hampered by its poor ability to enter nondividing cells. An interestingalternative to this limitation are recombinant LVs, which share many characteristicswith RVs, given their common family, while LV can also transduce nondividingcells. As a result, recombinant LV is one of fastest growing vector [2]. LVs constitutean attractive and promising tool for a wide array of gene transduction applications,notably for therapy [2]. The principal technological hurdle associated with lentiviralvector (LVV) is its low-titer production and the need for more efficient and selectivepurification methods. In the best cases 105 to 107 transducing units per milliliter(TU/mL) of cell culture are produced by standard methods (i.e., a transient trans-fection of a plasmid set or a packaging cell line [PCL]). These titers can be increasedby centrifugation to generate ~ 109 TU/mL, which is sufficient for certain in vitroexperiments and in vivo testing in small animal models. However, these concentra-tions remain too low for gene therapy clinical trials, where the number of functionalparticles needed is at least 1011 -- 1012 per patient [3]. Over the past several years con-siderable improvements have been made in the fields of vector design, productionsystem and purification method in an attempt to develop safe and cost-effective

10.1517/14712598.2013.779249 © 2013 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 987All rights reserved: reproduction in whole or in part not permitted

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processes, amenable to large scale and compatible with goodmanufacturing practices. This review will provide an overviewof the techniques recently made available for the design, pro-duction and downstream processing of LVVs, which shouldbring LV gene therapy to maturity.

2. Lentiviral vectors

2.1 BiologyLV and RV share many common features. As RV, LV genomeconstitutes a single-strand (ss) RNA of 7 -- 12 kb in length.LV is a spherical virus between 80 and 120 nm in diameter [4].The LV particle contains two copies of positive strand RNA,which are complexed with the nucleocapsid protein (NP)and the enzymes reverse transcriptase, integrase and protease(pol) [5]. A second protein shell formed by the capsid protein(gag) encloses the NP and delimits the viral core [6,7]. Matrixproteins form a layer outside the core and interact with acellular-derived lipid envelope, which incorporates viral enve-lope glycoproteins (env), responsible for the interaction withspecific host cell receptors. Two units form these glycopro-teins: transmembrane (TM) that anchors the protein intothe lipid bilayer and surface (SU), which binds to the cellularreceptor.The Retroviridae family can be divided into simple and

complex genera, based on their genome organization. LVsare complex Retroviridae and include primate: human andsimian immunodeficiency virus (HIV and SIV), as well asnon-primate Retroviridae: feline immunodeficiency virus,bovine immunodeficiency virus, caprine arthritis encephalitisvirus and equine infectious anemia virus (EIAV). Similar tothe simple RV, the LV genome is primarily organized aroundthe gag, pol and env genes. In addition, LV is composed of sixother genes: two regulatory genes (tat and rev) and four acces-sory genes (nef, vif, vpr and vpu), that encode proteins playingimportant roles for viral replication, binding, infection andvirus release [8]. Moreover, the retroviral genome containscis-acting sequences such as two long terminal repeats(LTR), together with elements required for gene expression,reverse transcription and integration into the host chromo-somes. Other important sequences are the packaging signal

(psi or y), required for the specific RNA packaging intonewly formed virions [9] and the polypurine tract (PPT),which is the initiation site of the positive strand DNAsynthesis during reverse transcription [10,11].

The LV life cycle can be separated in several steps.i) Binding and entry: virus entry is mediated by the bindingof the viral envelope glycoprotein gp120 to the CD4 receptorexposed on the target cell surface leading to the fusion of theviral envelope with the cell membrane. Subsequently, the viralcore is released into the cytoplasm. ii) Reverse transcription:ssRNA is reverse transcribed into double-strand (ds) DNAwithin the core. iii) Trafficking and nuclear import: dsDNAis actively transported to the nucleus. iv) Integration: viralDNA is integrated into the host DNA as a provirus.v) Gene expression: viral genes are transcribed and spliced;full-length genomic viral RNA (gRNA), as well as viralmRNA are transported to the cytoplasm and translated.vi) Assembly and budding: gRNA and viral proteins translo-cate to the assembly site resulting in virion formation,maturation and budding [12].

2.2 Specific properties and applicationsBesides being able to stably integrate their transgene in divid-ing and nondividing cells [13], LVVs have a lower insertionalmutagenesis induction rate compared to RV vectors (RVV),possibly due to different integration patterns [14,15]. Indeed,several studies have shown that RVs preferentially integratenear the transcriptional start site of genes, while LVs preferen-tially integrate active transcriptional loci. However, LVs andRVs share common characteristics as they have a large packag-ing capacity (up to 10 kb) [16] and they allow long-term geneexpression [17]. LVV are used for many applications such asfor genome-wide function studies of gene expression [18-20],animal transgenesis [21,22], cell engineering (genetic reprogram-ming to generate induced pluripotent stem cells [IPSs]) [23-25],recombinant protein production [26,27] and clinical genetherapy [28-30]. In gene therapy, the major interest of LVV com-pared to RVV in gene therapy is to treat genetic disorders innondividing cells such as in the central nervous system. LVVare used to target several diseases such as adrenoleukodystrophy(ALD) [30], Parkinson’s disease [31], sickle cell anemia andb-thalassemia [32], cystic fibrosis [33], HIV [34] and cancer [35].Current applications and clinical trial status have beenreviewed in Refs. [36,37]. Up to January 2013, when 62 LV-basedgene therapy trials have been performed (Table 1 [2]).

2.3 LVV design evolutionLVV are produced by the trans-complementation of threeplasmids containing different genes from the recombinantLV, transfected in a cell line. This concept was originallydeveloped for the g-RVVs and based on the separation ofcis- and trans-acting sequences in different plasmids. Follow-ing their evolving production technologies, these vectors aredivided into generations according to the HIV-derived LVgenome sequences used in the plasmids. The first generation

Article highlights.

. Retrovirus is one of the principal vectors used for genetherapy.

. Among retroviruses, lentiviruses possess uniqueproperties that make them particularly interesting forgene therapy.

. Recent progress as allowed significant improvement inefficiency and safety of lentiviral vectors.

. Production and purification methods is now allowingmass production of these vectors.

This box summarizes key points contained in the article.

M. M. Segura et al.

988 Expert Opin. Biol. Ther. (2013) 13(7)

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Table 1. LVV-based clinical trials up to January 2012 [2].

Disease Category Phase Date

approved/

initiated

Gene

CD19+ B-lymphoid malignancies Cancer disease I/II 2012 CD19 antigen-specific chimericantigen receptor (CAR)

HIV infection Infectious diseases I 2012 CCR5shRNA/RNA decoy for TAR/TRIM5aCD19+ B-lymphoid malignancies Cancer disease I 2012 CD19 antigen-specific-zeta T-cell receptorThalassemia Monogenic disease I 2012 Human globinPediatric patients with high riskbrain tumors

Cancer disease I 2012 O-6-methylguanine DNAmethyltransferase (MGMT)

Netherton syndrome Monogenic disease I 2012 SPINK5HIV infection Infectious disease I/II 2012 HIV-1 Gag-Pol-NefWiskott-Aldrich syndrome Monogenic diseases I/II 2011 Wiskott Aldrich proteinStargardt macular degeneration (SMD) Other I/II 2011 ABCRX-linked chronic granulomatousdisease (X-CGD)

Monogenic diseases I/II 2011 gp91phox

Retinitis Pigmentosa associated withUsher Syndrome type 1B

Ocular diseases I/II 2011 N/A

Hemophilia A Monogenic diseases I 2011 Factor VIIIFanconi anemia Monogenic diseases I 2011 Fanconi anemia complementation group AHIV infection Infectious diseases I/II 2011 HIV genes C46ADA-deficient individuals Monogenic diseases I/II 2011 ADAMelanoma Cancer diseases I 2011 a- and b-T-cell receptor specific for

tyrosinaseAdvanced myeloma Cancer diseases I 2010 High affinity T-cell receptor specific for

MAGE-A3 or NY-ESO-1Metastatic melanoma Cancer diseases I 2010 High affinity T-cell receptor specific for

MAGE-A3 or NY-ESO-1Age-related macular degeneration (AMD) Ocular disease I 2010 Endostatin angiostatinNon-Hodgkin’s lymphoma Cancer diseases I/II 2010 CD19 antigen-specific CARb-thalassemia Monogenic diseases I 2010 Human g-globin geneSynovial sarcoma Cancer diseases I 2010 High affinity T-cell receptor specific for

NY-ESO-1Childhood cerebral ALD Monogenic diseases II/III 2010 ABCD-1 gene (ALD protein)Ovarian cancer Cancer diseases I 2010 a-folate receptor-scFv with signaling

domains comprising TCR-zeta, and 4-1BBSMD ocular disease I/II 2010 Retina-specific ABC transporter (ABCR)Parkinson’s disease Neurological disease I/II 2010 Dopa decarboxylase tyrosine hydroxylase

GTP-cyclohydrolase 1Metachromatic leukodystrophy Monogenic diseases I/II 2010 Arylsulfatase ASickle cell disease Monogenic diseases I 2010 Human g-globin with g-globin exons geneCD19+ acute lymphoblastic leukemiarelapsed post-allogeneic stemcell transplantation

Cancer diseases I 2010 CD19 antigen-specific-zeta T-cell receptor

Wiskott-Aldrich syndrome (WAS) Monogenic diseases I 2010 Human WAS geneX-linked severe combined immunedeficiency

Monogenic diseases I 2009 g-c common chain receptor

HIV infection Infectious diseases I 2009 HIV-1HXB2 gag, truncated pol, vpr, rev,tat, nef and envelope proteins

Ovarian cancer Cancer diseases I 2009 a-mesothelin-scFv with signaling domainscomprising TCR-zeta, CD28 and 4-1BB

Severe combined immune deficiencydue to adenosine deaminasedeficiency (ADA)

Monogenic diseases I/II 2009 ADA

ALD Monogenic diseases III 2009 ABCD-1 gene (ALD protein)X-linked severe combined immunedeficiency

Monogenic diseases I 2009 g-c common chain receptor

Wiskott Aldrich syndrome Monogenic diseases I 2008 Human WAS geneCD19-CMV+ Non-Hodgkin’s lymphoma Cancer diseases I 2008 CD19 antigen-specific CARFanconi anemia Monogenic diseases I 2008 Fanconi anemia complementation group AGlioma Cancer diseases I 2008 MGMT

New developments in LVV design, production and purification

Expert Opin. Biol. Ther. (2013) 13(7) 989

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consists in i) a plasmid providing all trans-acting sequences(packaging plasmid); ii) a plasmid encoding a heterologousenvelope membrane protein; and iii) a plasmid containingall the cis-acting sequences and the transgene of interest,which expression is driven by a foreign promoter (transferplasmid).Due to its similarity to HIV, this first LVV generation has

undergone several genetic modifications to improve its bio-safety and performance. These improvements were achievedby i) limiting sequence overlap in order to strongly reducespontaneous replication and the generation of replication-competent LV (RCLs) and ii) removing HIV accessory,unnecessary genes such as vif, vpr, vpu and nef [38,39]. This sec-ond LVV generation is produced from a packaging plasmidencoding the essential trans-acting LV proteins (gag-pol, revand tat) and a transfer plasmid containing the transgene ofinterest with the minimal cis-acting sequences. In addition,the transfer plasmid has a self-inactivating (SIN) configura-tion where the homologous enhancer/promoter sequences inthe U3 region of 3¢ LTR are deleted [40]. Consequently thedeletion is reproduced in the 5¢ LTR during reverse transcrip-tion, causing transcriptional inactivation of the provirus. Oth-erwise, the conditional SIN (c-SIN) configurations harboringa minimal tetracycline-inducible promoter in the U3 regionimpart the SIN phenotype in target cells, thereby obviatingvector mobilization [41].Safety was further improved with the third generation LVV

that represents the safest packaging system for clinical appli-cations, since it contains only 10% of the viral genomesequence [42]. It consists of a split-genome packaging system inwhich the rev gene is expressed from a separate plasmid. More-over, a chimeric 5¢ LTRs have been constructed in the transfervector in order to make the LV promoter tat-independent.This has been achieved by replacing the U3 region of the

5¢ LTR by a strong tat-independent constitutive promoter [43].This construction limits both genome mobility and possibilitiesof recombination in the host cell (Figure 1) [40].

The transfer plasmid also underwent several importantmodifications. The design of a safe and efficient LVV requiresboth deletions of non-necessary sequences from the backboneand insertions of elements that are proven to have a positiveeffect on vector titer or transgene expression. The centralPPT--central termination sequence reinsertion into HIV-1-derived vector strongly stimulates gene transfer efficiency indifferent cell types [44]. The post-transcriptional control ele-ment acts with the cytomegalovirus immediate-early (CMV)promoter enhancer to increase transgene expression [45]. Thewoodchuck hepatitis virus post-transcriptional regulatory ele-ment (WPRE) increases transgene expression in mammaliancells from different viral vectors [46,47] and the viral vector titerfrom transiently transfected 293T cells and stable producercells by 7- to 15-fold, as compared to WPRE-lackingretroviral constructs [48,49]. The replacement of the originalpolyA signal in the 3¢ LTR U5 region with a bovine growthhormone polyadenylation sequence significantly increases theefficiency of SIN vectors [46]. The chromatin insulators canhelp maintain long-term expression by suppression of trans-gene silencing [50]. The multimeric copies insertion of cons-titutive transport element (CTE) is able to replace the revresponse element system to export RNA from the nucleus tothe cytoplasm and produce equivalent titers. CTE elementsinto the transfer vector and the packaging plasmid wouldallow removing the cytotoxic rev gene expression duringvector production [51].

Another major LVV modification is pseudotyping, thereplacement of the native envelope protein by a heterolo-gous one, resulting in a tropism change [52]. Pseudotypingcan offer several significant advantages such as i) improved

Table 1. LVV-based clinical trials up to January 2012 [2] (continued).

Disease Category Phase Date

approved/

initiated

Gene

Parkinson’s disease Neurological diseases I/II 2008 Tyrosinase GTP-cyclohydrolase 1 Dopadecarboxylase

b-thalassemia Monogenic diseases I 2007 Human b-globin geneHIV infection Infectious diseases II 2007 HIV-1 envMucopolysaccharidosisType VII (MPS VII)

Monogenic diseases I 2006 b-glucuronidase

Malignant melanoma Cancer diseases I 2006 a- and b-chains of T-cell receptorspecific for MART-1

CD19+ leukemia and lymphoma Cancer diseases I 2006 CD19 antigen-specific-zeta T-cell receptorSickle cell anemia, thalassemia Monogenic diseases I/II 2006 b-globinHIV infection Infectious diseases I 2005 RNAi targeted at HIV tat and rev RNA

decoy for TAR ribozyme targeted atCCR5 cytokine receptor

X-linked ALD Monogenic diseases I/II 2005 ALDHIV infection Infectious diseases I/II 2004 Antisense envHIV infection Infectious diseases I/II 2004 VRX496HIV infection Infectious diseases I 2001 HIV-1 env

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vector safety, due to the elimination of sequence homologywith wild-type virus, ii) widen or more selective specificityof vector tropism toward target cells and iii) improve parti-cle stability allowing virus concentration and long-term virus storage. The G protein of vesicular stomatitisvirus (VSV-G) is the most used envelope protein to pseudo-type LVVs as well as oncoretroviral vectors, because it ishighly stable, allowing the concentration of the vector byultracentrifugation, and because its receptors are ubiqui-tously expressed in mammalian cells conferring a very broadtropism. However, the VSV-G envelope comes with a num-ber of limitations including the susceptibility to inactivationby human complement, toxicity and broad tropism withstill unknown entry receptor(s) [53]. In order to avoidVSV-G drawbacks, several studies report LVV pseudotypedwith other viruses envelop proteins, conferring a moreselective tropism, such as Ebola virus for airway epitheliumand skin [54], rabies virus for neurons [55], baculovirus(BV) [56] and hepatitis C [57] virus for hepatocyte, felineendogenous RV [58] for hematopoietic cells and measlesvirus for B cells and T cells [59]. Despite all of the above,

VSV-G pseudotyping provides the highest titers and themost robust LVV particles [60,61].

The genetic engineering of the viral structural proteinPr55gag has shown a significant improvement of the viral pro-duction. Indeed, retroviral production and infectivity are reg-ulated by the amino-terminal matrix domain (p17MA orMA) of Pr55gag (gag) [62-65]. The myristoylation (myr) signalin the MA domain is critical for efficient viral production byaiding gag trafficking to the plasma membrane (PM). It hasbeen shown that substitution of the HIV-1 gag myr with thephospholipase C-d1 pleckstrin homology (PH) domainincreased the production of the third-generation LVV inwhich the gag-pol was human codon-optimized [66,67]. There-after, it has been discovered that the myr signal motifs of sev-eral heterologous proteins could enhance viral production upto 10-fold and to a greater extent than PH-gag [68].

Non-integrative gene therapy can also be applied usingLVV. This has been achieved with the development of non-integrating LVVs (NILVs) consisting of integrase-deficientLVs obtained by selective mutations within the integrase-coding region of the packaging plasmid. These mutations

A. HIV-derived LV genome C. Transfer plasmid

Non SIN

SIN

attL TAR PBS

U3 U5

DIS, SD, φGag

PolVif

VprTat

RRE PPTPolyA attR

Rev

VpuEnv

Nef

cPPT/CTs

5′LTRR U3 U5

3′LTRR

U3 U55′LTR 3′LTR

R

U5R

GOI

WPRE

U3

ΔU3

PPT

R U5

3′LTR

WPRE

PPT

R U5GOI

Pro

Pro

SA

CMV

PBS RRE

DIS, SD, φ

5′LTR SADIS, SD, φ

cPPT/CTs

PBS RRE cPPT/CTs

Conditional SIN

U5R

ΔU3

3′LTR

WPRE

PPT

RTet-Ind U5GOIPro

Pro VSV-G PolyA

D. Envelope plasmid

B. Packaging plasmid

1st generation

ProGag

PolVif

VprVpu

TatRev

RRE

Nef

PolyA

2nd generation

ProGag

PolTatRev

RRE

PolyA

3rd generation

Pro

Pro

GagPol

RRE

PolyA

PolyARev

CMV5′LTR

SADIS, SD, φ

PBS RRE cPPT/CTs

Figure 1. Evolution of the LVV design starting from HIV-1-derived LV. (A) HIV-1 wild-type containing all cis- and trans-acting

sequences. (B) -- (D) Plasmids used to produce LVVs and based on the trans- and cis-acting sequence split. (B) Packaging

plasmids. First generation of LVVs encoding all HIV proteins except the envelope protein. Second generation of LVVs only

encodes gag/pol, tat and rev. Third generation LVVs split in two plasmids, one encoding gag/pol and the other encoding rev.

(C) Transfer plasmids. Non-SIN configuration plasmid contains the HIV-1 wild-type 5¢ LTR and 3¢ LTR. SIN (self-inactivating)

configuration plasmid contains a CMV promoter in the 5¢ LTR U3 region and a deletion in 3¢ LTR U3 region. The

c-SIN configuration renders the transgene expression tissue specific and/or controllable compared to the SIN configuration.

(D) Envelope plasmid. LVVs can be produced with different viral pseudotypes depending on the envelope protein used.DU3: SIN deletion in U3 region of 3¢ LTR; f: Packaging signal; CMV: Cytomegalovirus immediate-early promoter; cPPT: Central purine tract; CTS: Central

termination sequence; DIS: dimerization signal; GOI: Gene of interest; pA: Polyadenylation signal; PBS: Primer binding site; PPT: Polypurine tract; Pro: Internal

promoter for transgene expression; RRE: Rev response element; SA: Splice acceptor site; SD: Splice donor site; WPRE: Woodchuck hepatitis virus (WHV)

post-transcriptional regulatory element.



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eliminate the integrase activity without affecting reverse tran-scription and transport of the pre-integration complex to thenucleus. The LVV DNA thus remains in the cell nucleus asan episome, leading to sustained expression in post-mitoticcells and tissues such as the retina [69], brain [69,70] and mus-cle [71]. The main advantage of this strategy is to avoid theoncogene activation risk caused by random LVV insertionin the genome [72,73]. Although LVVs have shown distinct tar-get site preferences [74] and less genotoxicity compared tog-RVVs [75], insertional mutagenesis risks still has to beconsidered when developing a clinical application [76].Integrative strategy has to be envisioned when gene repair

or knockdown are desired. In these circumstances, the gener-ation of LV hybrid vectors encompassing non-genotoxicintegration mechanisms combined with NILV is an interest-ing compromise. One of these LV hybrid vectors is the LV-transposon hybrid, using the Sleeping Beauty (SB) transposonsystem, allowing a stable and safe integration of the gene ofinterest in the genome. This transposition phenomenonrequires the interaction between the SB transposon invertedrepeats and the SB transposase [77-79]. Another LV hybridvector used to target desired integration sites is based on theengineered zinc-finger nucleases (ZFNs) homologous recom-bination tool [80,81]. Engineered ZNFs has been coupled toNILV to be delivered to cells [82]. This hybrid LV-ZNF vectorhas shown gene correction at the human IL2RG locus.

3. Recent improvement in LV productionprocess

3.1 Cell linesHEK293 cells and their genetic derivatives are the principalcell lines used for the production of LVVs [83] and RVVs [84]

due to their properties [85]. This cell line is relatively highlytransfectable (leading to 70 -- 90% transfected cells upon LVinfection [86]) and can be modified to stably express theSV40 T-large antigen (T-Ag), which enables replication ofplasmids containing the SV40 replication origin (293T)(Table 2 [86]). It has been shown that 293T cells grow fasterthan their parental cell line and that vector productivity canbe four times higher than with the parental cell line underthe same conditions [87]. Another study reports the productionof LVVs upon chromosomal integration in 293T cells with a10-fold higher titer production compared to HEK293. Itappears that low level of constitutive T-Ag expressionimproves stability and productivity of HEK293 producercell lines [88]. The 293T cells can be adapted to suspensionculture in serum-free medium (SFM) [87,89] which makesthem easily amenable to large-scale production bioreactorsand also, by reducing the complexity of the medium, simpli-fies the number of steps and the cost of downstream process-ing. The use of another HEK cell line derivative, called 293E,for the production of LVV has also been reported [86]. Thiscell line expresses the Epstein--Barr virus (EBV) nuclearantigen-1 and was scaled-up from shake flask to a 3-L

bioreactor for LVV production. These cells promote episomalpersistence of plasmids carrying EBV origin of replicationoriP and consequently increase expression levels [87,89].

3.2 Transient expressionTransient expression is the most commonly used method toproduce LV and consists in the transient expression of oneor more packaging plasmid(s) bearing i) the gag-pol, ii) revand/or tat, an envelope glycoprotein-encoding plasmid andiii) the transfer plasmid containing the transgene and the min-imal cis-acting sequences that are required for viral RNA pro-duction, processing and packaging [12]. Transient transfectionallows avoiding the time-consuming, tedious and cumber-some process of developing stable PCLs and can be used toexpress cytotoxic transgene [90].

Significant improvements in LVV production by transienttransfection have recently been described [86,89,91-94]. Someof these studies report scalable and industrial production sys-tems generating > 1011 LV particles per batch, which is suffi-cient for Phase I clinical trials. Some of these improvementsare the result of a combination of optimized parameterssuch as cell type, cell density, cell culture medium and supple-ments, cell culture process and plasmid transfer reagent. Forinstance, Kuroda et al. proposed a LV production protocolbased on the adherent cell culture in a protein-free mediumtransfected with polyethylenimine (PEI) [95].

Tiscornia et al. described a LV production method withadherent cells, fetal bovine serum (FBS) and calcium phos-phate (CaPO4) transfection [96]. Giry-Laterriere et al. pre-sented a protocol using adherent cells cultured in SFMtransfected with CaCl2 [97]. Segura et al. have shown it ispossible to improve LV production by using suspension culturein SFM additioned with Pluronic� and transfected withPEI [98].

The quality of plasmid DNA preparation and vector insertsize also influence transfection efficiency. LV titer decreaseswith increasing vector size [99] and plasmid DNA used for vec-tor production should be free of bacteria-derived impuritiesand should preferentially be in a supercoiled form [98]. Highyields of good quality DNA preparations are usually achievedusing commercially available kits. The total amount of DNAto be used for transfection (plasmid mix) depends on the cellculture scale and usually ranges between 1 and 10 µg/106

cells. Moreover, a proper ratio of vector, helper(s) andenvelope plasmids must be established.

3.2.1 Transfection agents3.2.1.1 Calcium-phosphate precipitationThe CaPO4 precipitation is traditionally used to produceLVV by transient transfection [13]. Briefly, the protocol con-sists in the transfection of 293T cells using calcium-phos-phate/DNA coprecipitation with several plasmids. Cells aregrown as monolayer in a culture dish in the presence of10% FBS. At days 2 and 3 post-transfection, ~ 106 -- 107

infective viral particles per milliliter (IVP/mL) can be

M. M. Segura et al.


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Table

2.Recentprotoco

lsforLV

Vproduction.

Titerbefore

concentration

Titerafter

concentration

Concentration

method

Celllinecu

lture

LVgeneration

Plasm

id

transferagent

Scale

Adjuvant

medium

Refs.

TransientLV

Vsproduction

1�

106IVP/m

L1010IVPtotal

Ultrafiltration,

heparinaffinity

chromatography

HEK293-EBNA,

suspension,

1�

106cells/m

L

HIV-derived

LV3rd

25-kDalinear

PEI,mass

ratio

3Lbioreactor

Serum

free,

0,1%

pluronic

[86,98]

8�

107TU/m

LHEK293SF-3F6,

suspension,

4,5

�106cells/m

L

HIV-derived

LV3rd

PEI,mass

ratio

3Lbioreactor

Serum

free,5mM

sodium

butyrate

[89]

1�

107IVP/m

LHEK293FT,adherent,

2�

106cells/25cm

2HIV-derived

LV3rd

25-kDabranchedPEI,

N/P

ratio

25cm

2T-flask

10%

FBS

[106]

6�

107TU/m

L2.1

�1010TU/m

LUltracentrifugation

HEK293T,adherent,

1,6

�107

cells/150cm

2

HIV-derived

LV2nd

CaPO4

150cm

2T-flask

10%

FBS,

1%

glutamax,

25µM

chloroquine

[94,100]

2.5

�106TU/m

LHEK293T,adherent

HIV-derived

LV3rd

BV,MOI:250PFU

/cell,

transductionovernight

Serum

free

[116]

1�

106IVP/m

L1�

109IVP/m


HEK293T,adherent,

80--90%

cell

confluence

HIV-derived

LV3rd

CaPO4

15cm

dish

2%

FBS

[96]

2�

108TU/m

LHEK293T,adherent,

2�

108cells

HIV-derived

LV2nd

CaPO4

1720cm

2

HYPERFlask

vessel

10%

FBS,

1%

glutamax,

25µM

chloroquine

[94]

2.2

�106TU/m

L1.1

�109TU/m


HEK293T,

suspension

HIV-derived

LV3rd

BV,MOI:25and

125PFU

/cell,

transduction4h

490cm

2and

850cm

2

rollerbottles

1%

FBS,5mM

sodium

butyrate

[93]

1�

108IVP/m

LHEK293FT,

suspension,

1�

108cells/m

L

HIV-derived

LV3rd

EP,mass

ratio

10Lwave

bioreactor

Serum

free,

10mM

sodium

butyrate

[92]

4�

107TU/m

L6�

109TU/m


HEK293T,adherent,

3�

107cells/15-cm

plate

HIV-derived

LV2nd

40-kDaPEI,N/P

ratio

15cm

plate

serum

free

[95,105]

2�

109IVP/m

LUltrafiltration,

size-exclusion

chromatography

HEK293T,adherent

HIV-derived

LV3rd

CaPO4

Cellfactory

10-traystacks

10%

FBS

[91]

Titerbefore

concentration

Titerafter

concentration

concentration

method

Producercell

linecu

lture

LVgeneration

Regulatory

system

Stability

Scale

Adjuvant

medium

Refs.

Stable

LVVsproduction 1,3

�108TU/m


HEK293T,

8�

106

cells/10-cm

dish

EIAV-derived

LVTet-On

12weeks

without

selective

pressure

10cm

dishes

Serum

free,

10mM

sodium

butyrate

[120,121]



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recovered. Thereafter, supernatant is harvested and pooled,filtered through 0.45 mm membranes, and usually purifiedthrough two rounds of ultracentrifugation in order toimprove vector potency and purity [94,96,100]. This methodappears non-optimal for large LVV production since i) itrequires the presence of serum or albumin to reduce the cyto-toxicity of CaPO4, ii) the culture medium has to be changed16 -- 20 h after transfection to reduce the cytotoxicity ofCaPO4 and iii) this procedure is highly sensitive to smallvariations in pH. It has been demonstrated that theDNA--CaPO4 complex solubility and the transfection effi-ciency are affected by pH and phosphate concentration inthe culture medium [101].

3.2.1.2 Commercial lipid-based approachesCommercial lipid-based approaches can also be used togenerate LVVs [102,103]. It has been described that the use ofSuperFect�, an activated dendrimer-based transfection reagentallowed reproducible and efficient production of high-titer LVV at concentrations greater than 1 � 107 TU/mL priorto concentration step and required less than one-third of thetotal amount of DNA used in traditional CaPO4 transfec-tion [104]. However, the main lipid-based methods are still tooexpensive to use in an industrial context.

3.2.1.3 PolyethylenimineRecently, studies proved that cost-effective PEI-mediated trans-fection were as efficient as CaPO4-based transfection methodsto generate LVVs. LV titer of 107 TU/mL before concentrationwas reported with PEI [105,106]. In contrast to CaPO4, the PEI-based protocol does not involve medium replacement and isless sensitive to pH variations. In addition, a PEI-based trans-fection method resulted in LV particles production 40 to120 times higher than that obtained with the standardCaPO4-based protocol [106]. However, the plasmids/PEI poly-plex may be, like CaPO4, toxic to cells and the DNA:PEI rationeeds to be optimized. Two methods of ratio optimizationhave been described: one is simply based on the DNA:PEImass ratio and the other one is based on the ratio of nitrogencontent of PEI to phosphorous content in DNA [107].

3.2.2 Flow electroporationMost electroporation (EP) systems are designed to transfect rel-atively small volumes of cells. Flow EP addresses this limitationby continuously passing the desired volume of cellular andDNA suspension between two electrodes [108]. The procedurecan be effectively scaled-up for large bioprocessing applications,while maintaining regulatory compliance [109]. EP system cantransfect a higher cell density suspension (1 � 108 cells/mL) [92]

than BV (see Section 3.2.3) (4 � 106 cells/mL) [93] or PEI(1 � 106 cells/mL) [89]. Interestingly, flow EP requires one-third less DNA than the CaPO4 method in a correspondingvolume. LV production in a 2 L volume using EP transfectedcell has been successfully achieved with a titer of 8.8 � 107T

able

2.Recentprotoco

lsforLV

Vproduction(continued).

Titerbefore

concentration

Titerafter

concentration

concentration

method

Producercell

linecu

lture

LVgeneration

Regulatory

system

Stability

Scale

Adjuvant

medium

Refs.

3�

107TU/m

LHEK293T,adherent

HIV-derived

LV2nd

Tet-Off

94days

without

selective

pressure

10cm

dishes

Serum

free

[123]

2�

106TU/m

L1.2

�108TU/m


HEK293SF,

suspension,

0,8

�106cells/m

L

HIV-derived

LV3rd

Tet-On+

Cumate

18weeks

without

selective

pressure

3.5

7L

bioreactor

Serum

free

[118]

3.4

�107TU/m

LHEK293SF,

suspension,

1�

106cells/m

L

HIV-derived

LV3rd

Tet-On+

Cumate

Shakeflask

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to 1.3 � 108 TU/mL, under non-optimized conditions,corresponding to a total production of 2 � 1011 TU [92].

3.2.3 Baculovirus infectionBV, an insect virus, is able to transduce mammaliancells [110,111] and has been previously used for the productionof several viruses such as influenza [112], adenovirus [113],adeno-associated virus [114] and viral-like particles [115].Recently, recombinant BVs have been used to generate LVVin suspension or adherent cell cultures [93,116]. Four recombi-nant BVs have been constructed which encode all elementsneeded for the LVV generation in mammalian cells. NoRCL formation was detected. Titers obtained with thismethod were low (2.2 � 106 TU/mL before concentration).However, BV is an interesting alternative to PEI andCaPO4-transfection: Indeed, i) the construction and produc-tion of BV in insect cells are easy to perform at large scaleand high titer, ii) they are a safe transfer agent since they donot have the ability to replicate in mammalian cells, iii) BVtransgene capacity can be as large as 40 kb, iv) they can be cul-tured in suspension under serum-free conditions and v) theBV system has been approved by the Food and Drug Admin-istration and European Medicines Agency for the productionof vaccines [117].

3.3 Packaging and virus-producing cell linesThe major difficulties of transient transfection come fromi) the risk of recombination between the transfected plas-mids, ii) the medium contamination with the transfectedplasmids complicating the purification process and iii) thedifficulty to identify optimal multiple transfection condi-tions leading to variability between different batches. Asan alternative, RVV PCLs and virus-producing cell lines(VPCL) have been developed. The development of a PCLconsists in the generation of a cell line that stably expressesall trans-acting elements and envelops protein (gag-pol, env,rev) for LVV packaging. The final step consists in the trans-fection of the transgene-containing transfer plasmid thatwill be packaged to produce the desired LVV. Typically,the transfer plasmid is transfected using PEI [118], a lipidagent or CaPO4 precipitation [41,119]. The development ofa VPCL consists in the creation of a cell line that stablyexpresses all components used to produce LVVs. This canbe achieved from either transfection of all packagingelements and the transfer vector, or infection with LVVspreviously produced. By doing so, a VPCL can express LVparticles during a prolonged period (superior to5 -- 6 days) [120,121]. However, it has been proven that infec-tion method to generate high titer and stable VPCLs is moreefficient than transfection method [122]. In addition,although a non-SIN VPCL producing stable and highLVV titer (< 107 TU/mL) can be easily generated by infec-tion, it appeared more cumbersome to perform this with aSIN configuration, due to the inactivated LTR. Conse-quently SIN-based LVVs must be produced by transfection.

However to solve this problem, VPCL generation frominfection can be achieved with a c-SIN configuration inwhich LTR enhancers and promoters are regulated.

Ideal PCLs and VPCLs must be stable, produce large quan-tities of LV and grow in i) suspension culture, to simplifyscale-up and ii) SFM, for both safety reasons and downstreamprocessing streamlining. However, PCL present relatively lowyield compared to transient production. LV titers producedwith PCL and VPCL are generally comprised between 105

and 107 TU/mL before concentration [120,121,123]. Moreover,the selection procedure to establish such a stable cell linesexpressing all the vector components at sufficient level is along and tedious process (typically > 6 months). Also, severalof the proteins required to assemble LVVs are toxic to cellswhen overexpressed, including VSV-G, Rev, and protease.This problem has been overcome by the use of inducibleexpression systems for cytotoxic proteins. The tetracycline(Tet)-dependent regulatory system in a TEToff configurationhas been used to generate PCLs and VPCLs by regulatingthe cytotoxic protein expression. LV titers obtained fromthese cell lines are > 107 TU/mL before concentration andafter a 3-day culture [123,124]. However, a long-term analysisof such a system has shown that the cells are genetically [125]

or transcriptionally [83] unstable after 2 -- 3 months of culture.In addition, the TEToff configuration requires the removalof tetracycline or its analog, doxycycline (Dox) from the cul-ture medium to induce gene transcription. For this reason,a TEToff configuration is not compatible for large-scaleproduction of LVVs.

In contrast, the TETon configuration is a scalable regulatingsystem because the induction is triggered by the addition ofDox or tetracycline. A recent study reports that a PCL inwhich VSV-G and gag/pol expressions are regulated by aTETon system can produce a stable titer during 7 weeks with-out selective pressure [121]. In another study, a similar systemcould provide stable LVV during 16 weeks without the useof an antibiotic [120]. Nevertheless, for both these studies,titers were relatively low compared to other transienttransfection systems (4.4 � 105 TU/mL).

An interesting advance for a scalable LVV-producing PCLwas described by Broussau et al. In this study, rev and VSV-G expression were tightly regulated by a double tetracycline/cumate switch. Moreover PCL and VPCL were constructedwith a Tet-inducible c-SIN providing additional safety. ThePCL and VPLC systems, derived from HEK293 (HEK293 SF-PacLV), provided vector titers as high as 2.6 � 107

and 3.4 � 107 TU/mL, respectively. In addition, these PCLand VPCL were stable for 18 weeks without selective pressure.The HEK 293 SF-PacLV cell line was adapted to SFM andcould be cultivated in suspension allowing the easy scale up ofthe process. For example, in a 2.8-L bioreactor, HEK 293 SF-PacLV produced by transfection 2 � 106 TU/mL, 3 days afterinduction [118]. Although not as high as titers obtained in themost productive transient transfection systems, this consists inthe most promising, scalable LVV-producing PCL.



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3.4 Production process, scale-up and culture

adjuvantsCulture process optimization can also lead to substantial LVVyield improvements. Since LVVs can be produce either inadherent or suspension cultures, different scalable processeshave been proposed for the two culture types.

3.4.1 The cell factory systemThe cell factory system (Nunc, ThermoScientific) consists in asuperposition of multiple culture plates in a single unit,200 cm2 per plate and up to 40 plates, for a total culture sur-face area of 24,000 cm2. This system has been successfullyused with CaPO4-mediated transient transfection of adherentculture to produce LVV for clinical ex vivo gene therapy appli-cation [91]. This process was scaled-up to harvest 50 L of viralstock per batch. After a 200-fold volume concentration, 2 �109 IVP/mL were obtained, generating up to 6 � 1011 IVPper batch.

3.4.2 The HYPERFlaskTM (Corning)The HYPERFlaskTM (Corning) consists of 10 interconnectedgrowth surfaces, each containing a membrane, pretreated toallow improved cell adherence. The membrane is gas perme-able, allowing exchange of oxygen and carbon dioxide, result-ing in an improved gas exposure of the culture. The usefulnessof the HYPERFlask for LVV production has been tested forCaPO4-mediated transfection and compared to the produc-tivity of 150 cm2 culture dish. Results indicate that the titersof unconcentrated LVVs produced using HYPERFlask wereup to 2.3 � 108 IVP/mL, while the titers of LVVs producedin 150 cm2 dishes were lower, up to 6.9 � 107 IVP/mL. Thiscorresponds to a productivity per surface available of 7.5 �107 IVP/cm2 for the HYPERFlask, 10-fold more than forthe standard flask, 8 � 106 IVP/cm2. The higher productivityobserved with HYPERFlasks may be related to better gasexchange during LVV production [94].

3.4.3 MicrocarriersMicrocarriers can be used for mass production of LVVs. Thistechnology is currently used to produce recombinantproteins [126-130], adenoviruses and RVs [131,132] and otherviruses [128-131]. However, although microcarriers provide amore scalable means of cell culture, the literature shows thatthey do not allow productivity improvement compared toadherent flask culture (8.5 � 106 TU/mL) [132].

3.4.4 LVV production in cell suspensionLVV production in cell suspension was reported for the firsttime by Segura et al. The process consisted in a PEI-mediated transient transfection of suspension 293E culturein SFM. It was successfully scaled-up from shake flasks to a3-L bioreactor allowing to generate an average of 1.1 � 106

IVP/mL from day 3 to day 6 post-transfection [86].

3.4.5 Bag bioreactorsBag bioreactors have also been described for LVV production.In a 2-L WAVE bioreactor (GE Healthcare) using flow EP, itwas reported that a total of 2 � 1011 IVP was harvested after2 days of production [92]. Compared to T-flask, Cell Factoriesand other adherent cell culture devices, the WAVE bioreactorcan easily be scaled-up by changing the bag size, allowing asingle, closed system, suitable for current good manufacturingpractices (cGMP) production.

3.4.6 Sodium butyrateSodium butyrate inhibits histone deacetylase activity causinghyperacetylation of histone leading to chromatin decondensa-tion which in turn allows higher transcription and expressionof the transfected DNA [133]. It has been reported that thetranscriptional silencing of the transfected plasmid transgeneoccurs in LV production [83,134]. Experiments have shownthat the addition of sodium butyrate in a range of concentra-tion between 2 to 20 mM increases LVV productiv-ity [60,105,135,136]. It appears that sodium butyrate effect isenvelope protein-dependent. For instance, in opposition toother pseudotyping, VSV-G-pseudotyped LV productionwould not be enhanced by sodium butyrate [60]. However,when testing the effect of sodium butyrate on vector produc-tion improvement with a group of surface proteins, the besttiter were obtained with VSV-G (1 � 1010 TU/mL after con-centration). Stewart et al. has argued that the removal ofsodium butyrate would reduce the production cost and thenumber of manipulations required during the manufacturingprocess [121].

3.4.7 ChloroquineChloroquine has been used for the production of LVVs.Chloroquine is an amine that raises the pH of endosomesand lysosomes [137]. The increase in lysosomal pH presumablyinhibits the degradation of transfected DNA by neutralizinglysosomal enzymatic activity [138]. However, long cell incuba-tion periods with chloroquine are described as toxic for thecells causing a decreased virus titer [139]. Otherwise, some pub-lications reveal that chloroquine addition effect is transfectionreagent-dependent. For instance, it has been reported thatLVV titers were not improved by chloroquine when cultureswere transfected using PEI [105,135].

3.4.8 CaffeineCaffeine has been also shown to increase the titer of bothintegrative-competent LV and integrative-deficient LV by 3-to 8-fold with the addition of 2 -- 4 mM for 48 or 72 h duringviral production. Caffeine mechanism of action is not yetknown [140].

4. Downstream processing of LVVs

Downstream processing of LVVs is aimed at removing allimpurities, while maintaining vector activity. Accomplishingthis goal can be a very challenging task considering the fragile

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nature of LV particles, their size and complex structure(Figure 2A) in addition to the variety of contaminants thatmay be present in harvested supernatants. Two types of impu-rities can be distinguished. Product-related impurities aremolecular variants of the product that do not show compara-ble activity, efficacy or safety. These include inactive vectorforms, viral aggregates and soluble envelope proteins(Figure 2B). These impurities are typically difficult and some-times impossible to eliminate since they share commonphysicochemical characteristics with functional LV particles.Process-related impurities are impurities that may derivefrom the culture medium (e.g., serum), the host producercell (e.g., cell debris, cellular nucleic acids and proteins), theproduction process (e.g., plasmid DNA) or the purificationprocess itself (e.g., nucleases, buffers, leachables) (Figure 2C).Contamination can often be avoided or, at least, minimizedusing optimized bioprocessing strategies and/or top qualitypurification reagents/supports, thus facilitating downstreamprocessing operations.

LVVs intended for clinical applications must undergo aseries of purification steps to ensure product quality, safetyand efficacy. Strategic design and step-by-step optimizationof the purification process is crucial to maximize yield and

purity of the final LVV preparation. The best overall purifica-tion results are obtained by selecting steps with the greatestcomplementarity orthogonal process design. This is usuallyachieved by combining purification steps that are based ondistinct separation principles (e.g., one step based on virussize and another one based on virus charge). A general flowscheme comprising the typical steps involved in downstreamprocessing of LVVs is shown in Figure 3A. As an example,several published combinations of the sequence of unitoperations employed for the large-scale manufacturing ofclinical-grade LVVs are depicted in Figure 3B. For more infor-mation about large-scale processes utilized in preparing LVVsfor clinical purposes, the readers are referred to a recent reviewby Schweizer and Merten [141]. Typically, five to six down-stream processing steps are required to bring the product tothe desired level of purity and potency. Overall yields arearound 30 ± 15% depending on the number of steps (whichmay have an impact on the final purity achieved) andmethods selected [141,142].

The use of disposable technology in downstream processing(also known as single-use or limited-use technologies) is gain-ing popularity for recombinant proteins and virus vectors inparticular. In this sense, it is important to consider that viral

Culture medium derived-Proteins, peptides, aminoacids

-Lipids, phospholipids-Salts, buffers

-Sugars (i.e., glucose)-Trace elements, vitamins

-Serum/hydrolysate additives

A. Lentivirus vector particle B. Product-related impurities

Inactive vectorforms

RNAgenome

Polproteins

RTIN

PR

Lipidmembrane

CAMANC

EnvproteinVSV-G

Gagproteins

Viral aggregatesFree viral

components

Broken/disassembledparticles

Env(-) particles

Soluble Env-protein

C. Process-related impurities

Production derived-Polyethilenimine (PEI)

-Plasmid DNA-Transduction enhancers

-Adventitious agents, endotoxins

Purification derived-Nucleases

-Buffers-Detergents

-Extractables, leachables

Host cell derived-Host proteins

-Host nucleic acids-Cell vesicles, debris

-Proteoglycans

Figure 2. LV particle structure and commonly found impurities. (A) LV particles are composed of a lipid bilayer derived from

the host cell, an RNA genome and proteins mainly derived from the proteolytic cleavage of precursor polyproteins (gag, gag-

pol and env) encoded by three viral genes (gag, pol and env). Env may be replaced by the envelope protein of another virus,

typically the VSV-G, in a process known as pseudotyping, (B) product-related impurities, (C) process-related impurities.CA: Capsid protein; Env: Envelope protein; MA: Matrix protein; NC: Nucleocapsid protein; RT: Reverse transcriptase; IN: Integrase; PR: Protease; VSV-G: Vesicular

stomatitis virus glycoprotein.



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vectors cannot be subjected to classical virus inactivation/removal steps used for recombinant proteins and thus, it iscritical to minimize potential sources of contamination withadventitious agents throughout the process. Disposable bagsare commonly used for storage of buffers and intermediateproducts. The adoption of disposable systems for buffer prep-aration is growing. The filters used at different stages of thedownstream process are typically single-use components.Most chromatography supports used for LV purification are

available in disposable format including membrane adsorbers(Sartorius Sartobind�, Pall Mustang�), monoliths (BiaSeparations CIM�) as well as conventional columns (GEReady-to-ProcessTM).

In recent years, a number of kits for the concentrationand purification of LV particles and other viral vectors havebeen introduced in the market (Millipore, Sartorius, Clone-tech, Lamda Biotech, abm, Cell biolabs and Biocat kits,among others) [143]. These commercial kits usually achieve

Microfiltration(0.2 μm)

Microfiltration

Bioreactor

a.

A.

Supernatant harvest

Clarification

Concentration/purification

Nucleic acid digestion∗

Polishing

Sterilization

B.

b.c.

SEC columnchromatography

SEC columnchromatography

AEX columnchromatography

AEX membranechromatography

Ultra/diafiltration

Diafiltration

Ultra/diafiltration

Ultra/diafiltrationBenzonasedigestion

Benzonasedigestion

Benzonasedigestion

Figure 3. Downstream processing of LVVs. (A) General flow scheme of downstream processing steps. *Nuclease digestion may

be introduced at different steps along the purification process as depicted in 1B. (B) Combinations of unit operations

employed at large-scale for the purification of LVVs for clinical trials (Super-ProDesigner). (a, b) Virxys DSP strategies,

approximate overall yields 30% [150] (c) Genethon/MolMed DSP strategy, average overall yield 13% [91].AEX: Anion exchange chromatography; SEC: Size exclusion chromatography.

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concentration by centrifugal ultrafiltration or precipitationmethods, whereas purification is mainly achieved by meansof anion exchange chromatography on membrane adsorbers.Purification of LVVs can be performed in a few hours. How-ever, the kits are intended for small/medium-scale purificationand can be used for research purposes only.

Downstream processing strategies for purification ofg-RVVs [142,144,145], LVVs [141,142] and viral vectors in gen-eral [143,146,147] have been reviewed in depth in the past. Thisreview intends to provide an update of the methods used atthe different stages of the downstream processing of LVVs,highlighting current trends.

4.1 ClarificationClarification is required to eliminate producer cells and celldebris from crude harvested supernatants. Clarification istypically achieved by centrifugation, either in batch or con-tinuous mode. In cases where ultrafiltration or chromato-graphy steps follow, this operation is complemented withmicrofiltration to achieve greater clarification and avoid filteror column clogging. For practical reasons, a single-stepclarification process based on dead-end membrane filtrationis usually preferred at large scale (Figure 3B). Proper mem-brane chemistries should be selected in order to avoid loss ofvirus particles bound to the membrane. Clogging of the poreswith cell debris over time results in reduction of the actualmembrane pore size and consequently, virus entrapment [148].To minimize filter clogging and loss of vector particles, mem-branes with moderately large pore sizes (0.45 -- 1 µm), oftendisposed in series of decreasing pore size, are used [91,148-150].Using suitable membrane supports, yields exceeding 90%are frequently attained.

4.2 Concentration, purification and polishingFollowing clarification, supernatants typically undergo a seriesof processing steps with the objective of concentrating andpurifying the viral vector stock (Figure 3). In the initial con-centration/purification step(s), viral particles are separatedfrom the most abundant contaminants, including water.Concentration of clarified vector stocks at early stages of theprocess is advantageous to reduce the volume of feed and con-sequently, the size of the equipment required in later opera-tions (pumps, filters, columns and vessels). A polishing stepis further introduced to reduce remaining impurities. In prin-ciple, centrifugation, ultrafiltration and chromatographycould be used in these steps since all of these operations allowconcentration and purification of viral particles to differentextents. However, the great majority of large-scale LV purifi-cation protocols are based exclusively on membrane filtrationand chromatography technologies [141,142,146].

4.2.1 CentrifugationUltracentrifugation is the most widely used method for isola-tion of virus particles in standard research laboratories.Although extremely helpful for generation of small quantities

of highly purified material, this purification method is associ-ated with several practical disadvantages that limit its use atthe manufacturing scale. The main drawback of ultracen-trifugation is the limited capacity of commonly availablelaboratory ultracentrifuges. Alternatively, taking advantageof the larger capacity of normal speed centrifuges, long low-speed centrifugation methods (typically, 6,000 -- 7,000 � gfor 16 -- 24 h) can be used to purify and concentrate RV par-ticles by pelleting [142]. This procedure was employed for thelarge-scale purification of LVVs for clinical trials [151]. Impor-tant disadvantages of these technologies include the longprocessing times and the harsh purification conditions, partic-ularly when pelleting of viral particles takes place. These mayresult in disruption of virus particles, loss of viral envelopesurface proteins or virus aggregation, all of which will resultin the concomitant loss of LVV potency [150,152].

4.2.2 UltrafiltrationUltrafiltration is the method of choice for the concentration ofviral particles. Membrane processes are easily scaled-up andadapted to good manufacturing practices (GMP) manufactur-ing. Ultrafiltration allows gentle volume reduction of viralstocks in a relatively short time. This is particularly appealingwhen dealing with labile LV particles. Ultrafiltration processesoffer the possibility of washing off impurities, thus purifyingthe vector stock based on size differences to some extent.The larger the pore size, the greater the purity that can beattained and the shorter the processing time. Typically, poresizes ranging from 100 to 500 kDa are employed for ultrafil-tration of LV particles [142,145]. It is important to empiricallydetermine the optimal membrane pore size, flow rate andTM pressure (TMP) to avoid loss of viral particles [142,145].Membrane fouling is the main problem faced during ultrafil-tration, particularly when used at early stages of the process,since it causes the TMP to increase and the flow rate todecrease over time. Following concentration, the retentatefraction can be diafiltered using the same ultrafiltration unit.Diafiltration provides a fast and convenient means for bufferexchange into a suitable equilibration buffer for subsequentchromatography steps or into a final formulation buffer [91,150].Ultrafiltration can be carried out using different filtrationmodes (dead-end and tangential-flow filtration [TFF]) anddevices (cassettes, hollow fibers, etc.). TFF is widely used forLV particles since it allows effective virus concentration andgood yields with minimal membrane fouling [91,147,153,154].

4.2.3 ChromatographyChromatography is widely used in the industrial setting for thedownstream processing of biological products because it providesan efficient means of achieving the high level of purity requiredfor human therapeutics. Like membrane processes, chromatogra-phy is easily scaled-up and adapted to GMP manufacturing.A number of chromatography methods have been reported forpurification of LVV particles (Table 3). A clear trend towardthe adoption of alternative chromatography supports has been



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observed over the past years. Among them, membrane adsorb-ers and monoliths have been evaluated for the purification ofLVVs (Table 3) [93,94,150,155-157]. This is due to the fact thatmost conventional chromatography resins have been designedfor the purification of proteins rather than large virus particles.Using classical resins, nanoparticle-binding areas are restrictedto the bead surface area, whereas most contaminating proteinswill also have access to the pore area inside the particle [158].As a result, the virus-binding capacity and purification effi-ciency using conventional supports are severely compromised.Binding capacity is a critical parameter since it determines thethroughput and concentrating potential of a chromatographyresin [147]. Using alternative chromatography supports, virusparticles have access to the majority of ligands on the adsorbersurface, which typically results in increased binding capaci-ties [157]. In addition, mass transport through the pores orchannels of membrane adsorbers and monoliths, respectively,takes place mainly by convection overcoming slow nanoparticlediffusion issues encountered with conventional chroma-tography supports [158,159]. This permits the use of higherflow rates, which is particularly attractive for large-scale biopro-cessing [150,157,160]. A further advantage of these chromatogra-phy technologies is that they do not require packing, thuseliminating the problems associated with this procedure.

4.2.3.1 Anion exchange chromatographyLV particles are negatively charged at physiological pH.This feature can be exploited for the isolation of LVVs byanion exchange chromatography. Indeed, anion exchange

chromatography is widely used for the purification of LVVs(Table 3 and Figure 3B) [91,93,94,150,155,156,161]. The methodoffers high selectivity and allows simultaneous concentrationof LV particles. Yields from 30 to 65% of active vector par-ticles have been reported (Table 3). Typically, high salt con-centrations (0.5 -- 1 M NaCl) are necessary to elute virusparticles from the anion exchange columns/filters (Table 3).RV susceptibility to high salt concentrations may explain inpart losses in viral activity observed during anion exchangechromatography. Previous studies with RVVs have shownthat virus activity is negatively affected by NaCl salinity [149].A virus dilution or desalting step immediately after chroma-tography needs to be considered to minimize the time ofexposure of vector particles to high ionic strength [162]. Inter-estingly, some reports have shown that two distinct peaks con-taining active LV particles are obtained upon elution usinglinear salt gradients (Table 3) [93,161]. This chromatographybehavior was observed regardless of the LVV production system(either transiently transfected or BV vector transduced 293Tcells) and regardless of the chromatography support employed(CIM or conventional columns) [93,161]. These research studieshighlight the heterogeneity of LV particles in terms of bindingstrengths to anion exchange matrices. Of note, the majority ofthe active virus particles are recovered from a first peak elutingat lower salt concentrations (Table 3) [93,161]. The nature of virusparticles eluting in a second peak at higher salt concentrations isintriguing. It is tempting to speculate that it comprises either apopulation of virions possessing a higher number of negativelycharged molecules on the surface or small aggregates displaying

Table 3. Chromatography purification methods.

Purification

method

Viral

vector

Phase Chromatography

support

Desorption Yield (%)* Refs.

Ion-exchange chromatographyAnion exchangechromatography

LVLVLVLVLVLVLV

MembraneColumn resinMembraneMonolithMembraneMembraneColumn resin

Fractoflow� 80-6 (Merck)z

HiTrapTM Q (GE Healthcare)Mustang� Q (Pall)CIM� DEAE (Bia Separations)Sartobind� D (Sartorius)LentiSELECT (Sartorius)DEAE resin(Tosoh Bioscience)

2 M NaCl0.5 and 1 M NaCl§

1.5 M NaCl0.45 and0.6 -- 0.7 M NaCl§

--0.75 M NaCl

45 ± 15{

33 and 1765 -- 5665 (first peak)2944 ± 9-

[94,150,155,161]

[91,93,156]

Affinity chromatographyHeparin affinitychromatographyIMACAvidin-biotin affinitychromatography

LVHis6 LVDTB LV

Tentacle resinColumn resinMonolithColumn resin

Fractogel� EMD Heparin(Merck)z

Ni-NTA agarose (Qiagen)CIM�-IDA Ni (Bia Separations)Monomeric avidin (Pierce)

0.35 M NaCl0.25 M imidazol0.15 M imidazol2 mM biotin

53 ± 1> 50-68

[86,157,166,167]

Size exclusion chromatographyLV Column resin Sephacryl S-500

(GE Healthcare)N/A 70 -- 80 [150]

*Refers to step yield published in terms of infectious viral particles, unless otherwise stated.zDiscontinued products.§The authors found two peaks of activity using linear NaCl gradients.{Based on qPCR.

DTB: Desthiobiotin-tagged; His6: Hexahistidine-tagged; N/A: Not applicable.

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a larger number of binding sites accessible for multipoint ligandbinding. In support of the first idea, an interesting study wasreported by Rodrigues et al. showing that surface protein-free virus species (obtained by proteolytic digestion) can be sep-arated from intact g-RV and LV particles by anion exchangechromatography since they bind the chromatography gel lessstrongly [163]. On the other hand, the presence of virus aggre-gates that bind anion exchange supports more strongly thansingle virions cannot be discarded. This phenomenon hasbeen described for monoclonal antibodies [164,165].

4.2.3.2 Affinity chromatographyAffinity chromatography separates biomolecules from com-plex mixtures based on a highly specific interaction betweena target molecule and a selected ligand. The high selectivityof affinity chromatography offers the possibility of reducingthe number of purification steps and increasing productyields. In addition, binding capacities, purity and concentra-tion effects are usually high as there are less contaminantscompeting for binding sites. There are a few reported studiesshowing purification of LVVs by affinity chromatography(Table 3) [86,157,166,167]. The most straightforward approach isto exploit the natural ability of LV particles to interact withspecific ligands. Heparin, an inexpensive generic affinityligand, has shown to be useful for the purification ofLVVs [86]. Using heparin affinity chromatography, LVV par-ticles can be captured directly from clarified supernatantsallowing vector concentration and removal of the great major-ity of impurities. Elution of LV particles from the column isachieved under mild conditions (0.35 M NaCl) resulting ingood recoveries of active vector particles (53%) [86]. Of note,heparin interaction with the vector particle has shown to beindependent of the viral envelope protein and RVV type.Indeed, the same chromatography purification strategy wassuccessfully applied to the purification of g-RVVs pseudo-typed with VSV-G and RD114 envelope proteins, obtainingsimilar yields [149,168]. The evaluation of alternative heparinaffinity supports with comparable or superior binding capac-ities, than the tentacle matrix used in the cited reports, shouldbe explored, as the Fractogel� heparin chromatography resin(Merck) use has been discontinued. Alternatively, LVVs canbe engineered to display affinity tags on their surface enablingpurification by other affinity chromatography methods. Thisis the case of immobilized metal affinity chromatography(IMAC) and avidin-biotin affinity chromatography, bothof which have been successfully used to purify LV par-ticles [157,166,167]. Several functional VSV-G variants contain-ing randomly inserted hexahistidine affinity tags (His6) wereidentified and could be purified by Ni-NTA affinity chro-matography resulting in pronounced reduction of proteinand DNA contaminants with good yields (Table 3) [166].More recently, IMAC monolithic adsorbents were evaluatedfor the purification of VSV-G pseudotyped His6-taggedLVVs [157]. The authors reported that best results in termsof elution efficiency (number of eluted/number of captured

vectors) (69%) and concentration factor (1.3�) were obtainedusing CIM-IDA-Ni2+ adsorbents. Important losses of func-tional RV particles due to vector inactivation during chroma-tography have been repeatedly reported as a concern withIMAC purifications [157,169]. The exact mechanism responsi-ble for LV inactivation was not elucidated but could beattributed to high imidazole concentrations and/or metal-mediated oxidation reactions [157,169]. Alternatively, LVVshave also been metabolically biotinylated, enabling highlyefficient affinity-mediated capture by streptavidin paramag-netic particles [170]. Biotinylated LV particles were generatedby an engineered 293 T PCL (BL15) in the presence of freebiotin in the culture medium. Importantly, because the biotintag is carried by a host protein that is incorporated into LVparticles, the strategy allows the generation of biotinylatedLVVs regardless of the viral envelope glycoprotein used forpseudotyping. This technology was improved by Chen et al.[167] who further modified the BL15 cell line to generatedesthiobiotin-labeled LV particles that could be purified usingmonomeric avidin-coated chromatography columns (Table 3).By using this modified strategy, they eliminated problemsrelated to the contamination with competing free biotin thatreduced the efficiency of viral capture and moderated thebinding strength of the column, allowing for gentle elutionstrategies and high recovery of active LV particles (68%).

4.2.3.3 Size exclusion chromatographySize exclusion chromatography, also known as gel permeationchromatography, profits from the large size of LV particles(~ 100 nm) compared to most contaminating biomoleculesfor separation. Using this method, viral particles are excludedfrom the internal pores of the chromatography gel and recov-ered in the void volume of the column, whereas most contam-inants are retarded inside the pores and elute later. Thischromatography method is commonly introduced in the puri-fication scheme as a polishing step (Table 3 and Figure 3B).Importantly, size exclusion chromatography allows simulta-neous sample desalting and buffer exchange eliminating theneed for an additional formulation step [91,150]. Because novirus binding occurs during the run and no change in thebuffer composition is required for virus elution, this methodoffers a gentle and straightforward method for viral vectorpurification [147]. However, the use of size exclusion chroma-tography is also associated with a few practical disadvantages,most prominently its low loading capacity (< 10% bed vol-ume) when operated in conventional peak mode. Moreover,size exclusion chromatography tends to operate at low linearflow rates and typically results in product dilution [142].Some of these caveats can be minimized by operating in groupmode [171], although at the expense of method selectivity.Careful selection of the chromatography media is requiredsince the use of resins with pore sizes in the range of the virussize may lead to entrapment of virus particles inside thepores [172]. Using suitable chromatography supports, recoveryof ~ 70 -- 80% LV particles have been reported [150].



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4.3 Nucleic acid digestionLVV stocks are typically contaminated with various amountsof nucleic acids. An important source of DNA contaminationis the plasmid used for transient transfection [173]. In addition,cellular DNA and RNA may be released into cell culturesupernatants by broken producer cells. Host nucleic acidconcentrations in the supernatant typically increase withharvest time [149]. Contamination with nucleic acids notonly is undesired from the safety point of view (see Section4.5) but also increases sample viscosity causing difficulties indifferent purification steps. In addition, separation of DNAfrom LV particles is not trivial since they both share commoncharacteristics such as a strong negative charge and a large size.In order to reduce DNA contamination, the use of nucleasessuch as Benzonase� [173] is commonly introduced at sometime during the downstream process (Figure 3B) [91,150]. Opti-mal nuclease treatment conditions affecting nucleic acidremoval (e.g., enzyme concentration, digestion time, temper-ature) need to be determined case by case since the initialamount of contaminating nucleic acids may vary dependingon the particular process being considered. Benzonase reducesDNA to small fragments that along with the enzyme itselfneed to be removed or reduced to acceptable limits in subse-quent purification (residual DNA < 500 bp and residualBenzonase < 100 ng/mL) [151,166]. Introducing the nucleasedigestion step early in the process, prior to any concentrationstep, may be advantageous to maximize the possibility ofremoval of these digested contaminants in subsequent stepssuch as TFF, which proved to be efficient [91]. However, the

amount of enzyme required to treat unconcentrated virusstocks is higher, which will adversely affect the manufacturingcost [141].

4.4 Sterile filtration and storageMembrane filtration through 0.2 µm pores (sterile filtration)is typically the last operation in the generation of clinicalgrade biologicals including LVVs [141]. As with othermembrane-based operations described above, careful selectionof membrane chemistry should be performed in order toavoid virus binding. However, important losses in viral titersmay still be observed upon filtration through 0.2 µm poresand some manufacturers prefer to omit this step and insteadtake every precaution to ensure product sterility at all stepsof the manufacturing process [151]. LVVs are usually storedat -80�C to protect the virus from thermal inactivation. Viralvector stocks should be formulated under conditions thatguarantee maximum stability during storage. Some manufac-turers formulate the purified vector stocks in the protein-containing medium used for ex vivo cell culture [91]. Littlework about these critical aspects has been reported. An inter-esting study reported by Carmo et al. revealed that thehalf-life of LVVs at 4�C can be increased by addition ofrecombinant human albumin and lipoproteins to the storagebuffer [174].

4.5 Quality assessment of purified LVV stocksAny LVV preparation intended for human use must be fullycharacterized to guarantee product quality, safety and efficacy.

Table 4. Quality assessment tools.

Target Example assay(s) Refs.

Purity/Identity Total proteinsTotal nucleic acidsSpecific viral proteins or contaminants(e.g., benzonase, host proteins, BSA)Specific viral sequences or contaminatingnucleic acid sequences from producer cells(e.g., E1A/E1B, SV40 T antigen) or plasmids(e.g., VSV-G, Gag-Pol, Nef )

SDS-PAGE, Colorimetric assays (Bradford, Lowry)Agarose gel electrophoresis, fluorimetry (Picogreen�)ELISA, Western blotqPCR, Southern blot

[91,175]

[91,151]

[91,151,175]

[91,151,175]

Safety RCLAdventitious agentsMycoplasmaEndotoxins/PyrogensSterility

RCL amplification in suitable cell line by serialpassages followed by suitable quantitation assay(e.g., cell-based assay, PCR)In vitro viral testing, PCR-based test for specifichuman pathogensCulture-based assay, PCR-based assaysLAL test, Rabbit pyrogen testBacterial and fungal sterility

[91,151,175]

[91,151,175]

[91,151,175]

[91,151,175]

[91,151,175]

Potency Total virus particlesActive virus particles

p24 ELISA, qRT-PCRGene transfer/gene expression assays insuitable cell line

[91,151,175,176]

[91,151,175,176]

Others Physicochemical characteristics pHOsmolarityAppearance

[91,151,175]

[91,175]

[151,175]

BSA: Bovine serum albumin; ELISA: Enzyme-linked immunosorbant assay; LAL: Limulus amebocyte lysate; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel

electrophoresis; PCR: Polymerase chain reaction; qPCR: Quantitative polymerase chain reaction.

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Characterization tests are required in GMP manufacturingand to enter clinical trials but only a few studies of clinical-grade LVVs have been reported so far [91,151,175]. In all cases,the LVV preparations analyzed were destined to ex vivo genetherapy applications, as is the case with most LVVs being eval-uated in clinical trials. Because of the lack of sufficient infor-mation with novel applications such as the use of LVVs forex vivo treatments, limited regulatory guidelines specific forLVVs are presently available and in most cases the directivesare quite broad [176]. In the reported studies mentioned above,results from purity, safety and potency analyses of LVV lot(s)are shown. The methods used to evaluate these parameters arereviewed in Table 4. Purity tests include determination ofresidual levels of total proteins and nucleic acids. Also, resid-ual levels of specific protein contaminants are reported, suchas benzonase, host 293 proteins and culture medium-derived bovine serum albumin (BSA) as determined byenzyme-linked immunosorbant assay (ELISA). Regardingvector safety, of particular concern is the presence of E1Aand/or SV40 large T-antigen DNA sequences that may bereleased by producer cells as they possess oncogenic treats [91].In addition, the presence of contaminating plasmid-derived sequences, particularly VSV-G or gag/pol, is unde-sired. Quantitative detection of these nucleic acid sequencescan be performed by real-time polymerase chain reaction(PCR). The absence of RCL must also be demonstratedusing a suitable assay. More common safety testing includingadventitious agents, mycoplasma, endotoxins, sterility testsas well as analyses of the physicochemical characteristics ofthe final vector stock (e.g., pH, conductivity, etc.) are carriedout. Finally, to demonstrate vector transducing activity(potency) and lot-to-lot consistency, viral vector concen-tration measurements (functional and non-functional) arereported.

As LVVs progress into clinical trials, more applications arefound (both ex vivo and in vivo) and more knowledge is accu-mulated and regulatory requirements and specifications willmost likely increase. The complex nature of LV particles com-plicates a thorough evaluation of final vector purity. In con-trast with other widely used vectors for gene therapy, thereis no international standard reference material for LVV prod-ucts [91]. Regarding the composition of LV particles, it shouldbe noted that besides known viral proteins (Figure 2A), minuteamounts of host proteins can be incorporated on the virus sur-face or inside the virus particles. Extensive studies have beenperformed to identify host proteins incorporated into wildtype HIV-1 virions [177,178]. More recently, proteomic analy-ses of highly purified g-RVVs [179] and LVVs [180,181] allowedthe identification of cellular HEK 293 proteins associatedwith VSV-G pseudotyped vector particles. The nature andquantity of these proteins will likely vary depending on thecell line used for vector production and/or specific vectorpseudotype being considered. In addition, RV particles areknown to unspecifically package host RNA species [182,183].The presence of these host components associated with LV

particles should be taken into account when evaluating vectorpurity. The possible implications of having these host compo-nents in purified vector preparations (either incorporated oradsorbed to the vector surface) for product quality and safetyare rarely discussed at the moment. Removal of host compo-nents located in the interior of the LVV particles seemsunfeasible using current technologies. Another critical aspectis the availability of suitable quantitation assays. Becauseactive viral titers are affected by multiple assay variables(e.g., target cell, inoculum volume, incubation time, etc.), itis difficult to compare results obtained by different laborato-ries in the absence of standardized quantitation methodsand LV reference materials [91]. The concentration of totalvector particles is usually estimated by an immunosorbentdetermination of p24 concentrations, even though mostp24 ELISA kits cannot distinguish between assembled anddisassembled p24 protein. Other commonly considered totalquantitation methods include assessment of reverse transcrip-tase activity and determination of genomic RNA [184]. Inter-esting tools for determination of total viral particle countand size distributions are available (e.g., ES-DMA, AF4,NanoSight and qViro instruments), although they are stillrarely used for viral vector quantity or quality (e.g., aggre-gates) assessment. The ratio between total and active particlesprovides a good indication of vector quality. LV particles,and RVs in general, are produced with poor ratios (over100:1) that could be further affected during purifica-tion [142,145]. Monitoring this parameter could help identifycritical downstream processing steps.

5. Expert opinion

LVVs are powerful tools to stably modify genome in dividingand nondividing cells. For instance, these vectors have beenused with success to generate IPS cells [185-187] and are prom-ising tools for gene therapy [28,30]. At present, no gene therapyproduct has received the regulatory authority approval yet,but a rapidly growing number of advanced clinical trials usinggene therapy-based LV are initiated [2] and at least onecompany developing these products has emerged (OxfordBioMedica, Ark therapeutics).

Safety has been the first objective in the LVV productionfor therapeutic applications and the vector design has nowled to the third generation of these vectors. However, a singleclinical trial treatment can require as much as 1012 LVV par-ticles [3], which at the average production titer represents hun-dreds of culture liters. Consequently, LVV production processintensification is one of the major challenges to meet for thematuration of LVVs as a relevant gene therapy vector.

Despite improvements made to this day, few new scalableefficient systems to produce LVVs have been developed. Themost efficient ones produce up to 1011 particles per batchafter concentration into relatively low culture volume [91,92].These production systems combine improvements of thedesign vector, the transfection agent, the stable cell line



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selection and the cell culture process resulting in at least a104-fold increase in productivity compared to the averagemethod [13].Stable and transient productions have each their own draw-

backs. The development of stable cell line is still laborious andthe transient production, although undoubtedly faster andcheaper than stable production, is difficult to scale up becauseof important batch-to-batch variability. Consequently stableproduction appears as the best system to produce LVVs atlarge clinical and commercial scale.One of the inconveniences in LVVs stable production is the

expressed viral protein cytotoxicity. At present, to get aroundthis problem, gene regulation mechanisms are used in stableproduction systems [118,121,124]. Nevertheless, current regula-tion systems depend on either addition or removal of aninduction agent complicating the purification process. Yet,other regulating systems, not requiring administration orremoving of an exogenous chemical, could be engineered.Indeed, photon inducible expression systems have beenidentified [188-192] and could be applied to LVV production.One of these systems is based on the red light-inducedbinding of the plant photoreceptor phytochrome to the pro-tein PIF3 and the reversal of this binding by far-red light.Authors have developed a promoter system in yeast cells thatcan be induced, rapidly and reversibly, by short pulses oflight [190]. Another strategy for the photoregulation of geneexpression is the use of photoresponsive DNA binding to aphotoisomerizable molecule. By covalently attaching azoben-zene as a photoswitch to the T7 promoter, the transcriptionalreaction can be photo-controlled based on the reversiblecis-trans photoisomerization of azobenzene [188].Among all the cytotoxic viral proteins in LV, only the pro-

tease is useful to allow the virus packaging. HIV protease pro-motes its cytotoxicity by cleaving and activation of procaspase8 leading to the release of cytochrome c from the mitochon-dria, resulting in caspases 9 and 3 activation followed bynuclear fragmentation (apoptosis) [193,194]. It is now wellestablished that many viruses such as HIV act on molecularmechanisms involved in apoptotic processes. Otherwise,VSG protein used to pseudotype LVVs is also cytotoxic. Con-sequently, cell lines overexpressing antiapoptotic genes shouldbe seriously considered to extend the LVV production. Suchexperiments have already been reported for recombinant pro-tein expression and have shown their potential to increaseproductivity [195-200].

In the same way, metabolic engineering of producingcell lines has shown positive effect on recombinant pro-tein production [201-203] and could be used to optimizeLVVs yield.

It is widely accepted that stirred bioreaction systems usingsuspension cultures offer more advantages compare to adher-ent systems. Furthermore, the most efficient culture systemssuch as bag bioreactor, cell factory system and HYPERFlaskare expensive and destined to single use. These systems cantherefore not be considered to produce LVVs for late phaseclinical trials and commercial applications. At present, biore-actor appears to be the best system to produce LVVs andother biomolecules because it can be used repeatedly, scaledto large volume, operated in fed-batch mode to improveyields and be strictly monitored and controlled.

The cell culture parameters used in the bioreaction mayhave a profound effect on the virus titer by affecting cellularproductivity and vector stability. Moreover, the optimal cellculture parameters have been shown to be producer cell lineand viral vector-dependent. Therefore, identifying and con-trolling these parameters are useful to elaborate a robust pro-duction process. Previous works have already analyzed theimpact of physiochemical parameters [204-209] and mediumcomponents [209-211] mainly for the RVV production. Mostof these publications have reported that the PCL metabolisminfluences the productivity performances, in particular lipidbiosynthesis, thus suggesting it to be an important target forfurther improvement of RVVs and LVVs. However, noLVV production study so far has reported the simultaneousanalysis of multiple parameters. This could be achieved usingstatistical design of experiments, classically used to optimizebiomolecule production [212] or cellular growth [213]. Thiscould lead to much more efficient production and purifica-tion systems in the future, with better characterized andmore stable products.

Acknowledgment

MM Segura and M Mangion contributed equally to thisreview.

Declaration of interest

The authors state no conflict of interest and have received nopayment in preparation of this manuscript.

M. M. Segura et al.


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Bibliography

1. Rosenberg SA, Aebersold P, Cornetta K,

et al. Gene transfer into

humans--immunotherapy of patients with

advanced melanoma, using

tumor-infiltrating lymphocytes modified

by retroviral gene transduction. N Engl

J Med 1990;323(9):570-8

2. Wiley J. Gene therapy clinical trials

worldwide. 2012. Available from:

http://www.wiley.com/legacy/wileychi/

genmed/clinical/

3. MacGregor RR. Clinical protocol.

A phase 1 open-label clinical trial of the

safety and tolerability of single escalating

doses of autologous CD4 T cells

transduced with VRX496 in

HIV-positive subjects. Hum Gene Ther

2001;12(16):2028-9

4. Vogt VM, Simon MN. Mass

determination of rous sarcoma virus

virions by scanning transmission electron

microscopy. J Virol 1999;73(8):7050-5

5. Katz RA, Skalka AM. The retroviral

enzymes. Annu Rev Biochem

1994;63:133-73

6. Adamson CS, Jones IM. The molecular

basis of HIV capsid assembly--five years

of progress. Rev Med Virol

2004;14(2):107-21

7. Jones IM, Morikawa Y. The molecular

basis of HIV capsid assembly.

Rev Med Virol 1998;8(2):87-95

8. Federico M. From lentiviruses to

lentivirus vectors. Methods Mol Biol

2003;229:3-15

9. Watanabe S, Temin HM. Encapsidation

sequences for spleen necrosis virus, an

avian retrovirus, are between the 5’ long

terminal repeat and the start of the gag

gene. Proc Natl Acad Sci USA

1982;79(19):5986-90

10. Charneau P, Alizon M, Clavel F.

A second origin of DNA plus-strand

synthesis is required for optimal human

immunodeficiency virus replication.

J Virol 1992;66(5):2814-20

11. Rattray AJ, Champoux JJ. Plus-strand

priming by Moloney murine leukemia

virus. The sequence features important

for cleavage by RNase H. J Mol Biol

1989;208(3):445-56

12. Pluta K, Kacprzak MM. Use of HIV as a

gene transfer vector. Acta Biochim Pol

2009;56(4):531-95

13. Naldini L, Blomer U, Gallay P, et al. In

vivo gene delivery and stable transduction

of nondividing cells by a lentiviral vector.

Science 1996;272(5259):263-7

14. Hematti P, Hong BK, Ferguson C, et al.

Distinct genomic integration of MLV

and SIV vectors in primate

hematopoietic stem and progenitor cells.

PLoS Biol 2004;2(12):e423

15. Modlich U, Navarro S, Zychlinski D,

et al. Insertional transformation of

hematopoietic cells by self-inactivating

lentiviral and gammaretroviral vectors.

Mol Ther 2009;17(11):1919-28

16. De Meyer SF, Vanhoorelbeke K,

Chuah MK, et al. Phenotypic correction

of von Willebrand disease

type 3 blood-derived endothelial cells

with lentiviral vectors expressing von

Willebrand factor. Blood

2006;107(12):4728-36

17. Quinonez R, Sutton RE. Lentiviral

vectors for gene delivery into cells.

DNA Cell Biol 2002;21(12):937-51

18. Root DE, Hacohen N, Hahn WC, et al.

Genome-scale loss-of-function screening

with a lentiviral RNAi library.

Nat Methods 2006;3(9):715-19

19. Moffat J, Grueneberg DA, Yang X, et al.

A lentiviral RNAi library for human and

mouse genes applied to an arrayed viral

high-content screen. Cell

2006;124(6):1283-98

20. Klinghoffer RA, Frazier J, Annis J, et al.

A lentivirus-mediated genetic screen

identifies dihydrofolate reductase

(DHFR) as a modulator of beta-catenin/

GSK3 signaling. PLoS One

2009;4(9):e6892

21. Gomez MC, Pope CE, Kutner RH, et al.

Generation of domestic transgenic cloned

kittens using lentivirus vectors.

Cloning Stem Cells 2009;11(1):167-76

22. Donaldson ZR, Yang SH, Chan AW,

Young LJ. Production of germline

transgenic prairie voles (Microtus

ochrogaster) using lentiviral vectors.

Biol Reprod 2009;81(6):1189-95

23. Takahashi K, Yamanaka S. Induction of

pluripotent stem cells from mouse

embryonic and adult fibroblast cultures

by defined factors. Cell

2006;126(4):663-76

24. Brambrink T, Foreman R, Welstead GG,

et al. Sequential expression of

pluripotency markers during direct

reprogramming of mouse somatic cells.

Cell Stem Cell 2008;2(2):151-9

25. Chang CW, Lai YS, Pawlik KM, et al.

Polycistronic lentiviral vector for "hit and

run" reprogramming of adult skin

fibroblasts to induced pluripotent stem

cells. Stem Cells 2009;27(5):1042-9

26. Gaillet B, Gilbert R, Broussau S, et al.

High-level recombinant protein

production in CHO cells using lentiviral

vectors and the cumate gene-switch.

Biotechnol Bioeng 2010;106(2):203-15

27. Wurm M, Gross B, Sgodda M, et al.

Improved lentiviral gene transfer into

human embryonic stem cells grown in

co-culture with murine feeder and stroma

cells. Biol Chem 2011;392(10):887-95

28. Cavazzana-Calvo M, Hacein-Bey S,

de Saint Basile G, et al. Gene therapy of

human severe combined

immunodeficiency (SCID)-X1 disease.

Science 2000;288(5466):669-72

29. Wiznerowicz M, Trono D. Harnessing

HIV for therapy, basic research and

biotechnology. Trends Biotechnol

2005;23(1):42-7

30. Cartier N, Hacein-Bey-Abina S,

Bartholomae CC, et al. Hematopoietic

stem cell gene therapy with a lentiviral

vector in X-linked adrenoleukodystrophy.

Science 2009;326(5954):818-23

31. Jarraya B, Boulet S, Ralph GS, et al.

Dopamine gene therapy for Parkinson’s

disease in a nonhuman primate without

associated dyskinesia. Sci Transl Med

2009;1(2):2ra4

32. Bank A, Dorazio R, Leboulch P. A phase

I/II clinical trial of beta-globin gene

therapy for beta-thalassemia. Ann N Y

Acad Sci 2005;1054:308-16

33. Mitomo K, Griesenbach U, Inoue M,

et al. Toward gene therapy for cystic

fibrosis using a lentivirus pseudotyped

with Sendai virus envelopes. Mol Ther

2010;18(6):1173-82

34. Gu Y, Hou W, Xu C, et al. The

enhancement of RNAi against HIV

in vitro and in vivo using H-2K(k)

protein as a sorting method.

J Virol Methods 2012;182(1-2):9-17

35. Neschadim A, Wang JC, Lavie A,

Medin JA. Bystander killing of malignant

cells via the delivery of engineered

thymidine-active deoxycytidine kinase for



Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f L

aval

on

07/1

5/15

For

pers

onal

use

onl

y.

http://www.wiley.com/legacy/wileychi/genmed/clinical/

http://www.wiley.com/legacy/wileychi/genmed/clinical/

http://informahealthcare.com/action/showLinks?pmid=10398497&crossref=10.1002%2F%28SICI%291099-1654%28199804%2F06%298%3A2%3C87%3A%3AAID-RMV220%3E3.0.CO%3B2-B&coi=1%3ACAS%3A528%3ADyaK1cXkt1KgsL4%253D&isi=000074200600005

http://informahealthcare.com/action/showLinks?pmid=15629857&crossref=10.1016%2Fj.tibtech.2004.11.001&coi=1%3ACAS%3A528%3ADC%252BD2MXktVOl&isi=000226565700011

http://informahealthcare.com/action/showLinks?pmid=19641177&crossref=10.1095%2Fbiolreprod.109.077529&coi=1%3ACAS%3A528%3ADC%252BD1MXhsV2lt7nO&isi=000272030300020

http://informahealthcare.com/action/showLinks?pmid=19672245&crossref=10.1038%2Fmt.2009.179&coi=1%3ACAS%3A528%3ADC%252BD1MXps1agsLw%253D&isi=000271835000013

http://informahealthcare.com/action/showLinks?pmid=12824617&coi=1%3ACAS%3A528%3ADC%252BD3sXkt1Ggtrc%253D

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http://informahealthcare.com/action/showLinks?pmid=2381442&crossref=10.1056%2FNEJM199008303230904&coi=1%3ACAS%3A528%3ADyaK3MXhsl2ls7c%253D&isi=A1990DW21900004

http://informahealthcare.com/action/showLinks?pmid=16904174&crossref=10.1016%2Fj.cell.2006.07.024&coi=1%3ACAS%3A528%3ADC%252BD28Xpt1aktbs%253D&isi=000240276700016

http://informahealthcare.com/action/showLinks?pmid=16478886&crossref=10.1182%2Fblood-2005-09-3605&coi=1%3ACAS%3A528%3ADC%252BD28XlvFCgtr4%253D&isi=000238305400032

http://informahealthcare.com/action/showLinks?pmid=6310558&crossref=10.1073%2Fpnas.79.19.5986&coi=1%3ACAS%3A528%3ADyaL38XlvFeqsrg%253D&isi=A1982PJ71300049

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http://informahealthcare.com/action/showLinks?pmid=12573051&crossref=10.1089%2F104454902762053873&coi=1%3ACAS%3A528%3ADC%252BD3sXhtFahsb4%253D&isi=000180229900007

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http://informahealthcare.com/action/showLinks?pmid=20332767&crossref=10.1038%2Fmt.2010.13&coi=1%3ACAS%3A528%3ADC%252BC3cXjslWiurc%253D&isi=000278545800015

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http://informahealthcare.com/action/showLinks?pmid=16564017&crossref=10.1016%2Fj.cell.2006.01.040&coi=1%3ACAS%3A528%3ADC%252BD28Xjt1Khs7o%253D&isi=000237241400026

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http://informahealthcare.com/action/showLinks?pmid=22401802&crossref=10.1016%2Fj.jviromet.2012.02.007&coi=1%3ACAS%3A528%3ADC%252BC38XkvFWht7s%253D&isi=000303900600002

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http://informahealthcare.com/action/showLinks?pmid=22388453&crossref=10.1038%2Fcgt.2012.4&coi=1%3ACAS%3A528%3ADC%252BC38XjtFOku74%253D&isi=000302978400003

http://informahealthcare.com/action/showLinks?pmid=15027003&crossref=10.1002%2Frmv.418&coi=1%3ACAS%3A528%3ADC%252BD2cXjtVClu7g%253D&isi=000220162800005

http://informahealthcare.com/action/showLinks?pmid=10784449&crossref=10.1126%2Fscience.288.5466.669&coi=1%3ACAS%3A528%3ADC%252BD3cXivFGhtbc%253D&isi=000086727100043

http://informahealthcare.com/action/showLinks?pmid=19203232&crossref=10.1089%2Fclo.2008.0054&coi=1%3ACAS%3A528%3ADC%252BD1MXjt1CmtrY%253D

http://informahealthcare.com/action/showLinks?pmid=15550989&crossref=10.1371%2Fjournal.pbio.0020423&isi=000226099600019

suicide gene therapy of cancer.

Cancer Gene Ther 2012;19(5):320-7

36. D’Costa J, Mansfield SG, Humeau LM.

Lentiviral vectors in clinical trials: current

status. Curr Opin Mol Ther

2009;11(5):554-64

37. Escors D, Breckpot K. Lentiviral vectors

in gene therapy: their current status and

future potential. Arch Immunol Ther

Exp (Warsz) 2010;58(2):107-19

38. Pacchia AL, Adelson ME, Kaul M, et al.

An inducible packaging cell system for

safe, efficient lentiviral vector production

in the absence of HIV-1 accessory

proteins. Virology 2001;282(1):77-86

39. Vigna E, Naldini L. Lentiviral vectors:

excellent tools for experimental gene

transfer and promising candidates for

gene therapy. J Gene Med

2000;2(5):308-16

40. Zufferey R, Dull T, Mandel RJ, et al.

Self-inactivating lentivirus vector for safe

and efficient in vivo gene delivery.

J Virol 1998;72(12):9873-80

41. Cockrell AS, Ma H, Fu K, et al.

A trans-lentiviral packaging cell line for

high-titer conditional self-inactivating

HIV-1 vectors. Mol Ther

2006;14(2):276-84

42. Ramezani A, Hawley RG. Generation of

HIV-1-based lentiviral vector particles.

Curr Protoc Mol Biol

2002;Chapter 16:Unit 16 22

43. Dull T, Zufferey R, Kelly M, et al.

A third-generation lentivirus vector with

a conditional packaging system. J Virol

1998;72(11):8463-71

44. Riviere L, Darlix JL, Cimarelli A.

Analysis of the viral elements required in

the nuclear import of HIV-1 DNA.

J Virol 2010;84(2):729-39

45. Yilmaz A, Fernandez S, Lairmore MD,

Boris-Lawrie K. Coordinate enhancement

of transgene transcription and translation

in a lentiviral vector. Retrovirology

2006;3:13

46. Real G, Monteiro F, Burger C,

Alves PM. Improvement of lentiviral

transfer vectors using cis-acting regulatory

elements for increased gene expression.

Appl Microbiol Biotechnol

2011;91(6):1581-91

47. Dupuy FP, Mouly E, Mesel-Lemoine M,

et al. Lentiviral transduction of human

hematopoietic cells by HIV-1- and

SIV-based vectors containing a bicistronic

cassette driven by various internal

promoters. J Gene Med

2005;7(9):1158-71

48. Schambach A, Bohne J, Baum C, et al.

Woodchuck hepatitis virus

post-transcriptional regulatory element

deleted from X protein and promoter

sequences enhances retroviral vector titer

and expression. Gene Ther

2006;13(7):641-5

49. Higashimoto T, Urbinati F,

Perumbeti A, et al. The woodchuck

hepatitis virus post-transcriptional

regulatory element reduces readthrough

transcription from retroviral vectors.

Gene Ther 2007;14(17):1298-304

50. Hanawa H, Yamamoto M, Zhao H,

et al. Optimized lentiviral vector design

improves titer and transgene expression

of vectors containing the chicken

beta-globin locus HS4 insulator element.

Mol Ther 2009;17(4):667-74

51. Oh T, Bajwa A, Jia G, Park F. Lentiviral

vector design using alternative

RNA export elements. Retrovirology

2007;4:38

52. Cronin J, Zhang XY, Reiser J. Altering

the tropism of lentiviral vectors through

pseudotyping. Curr Gene Ther

2005;5(4):387-98

53. Coil DA, Miller AD. Phosphatidylserine

is not the cell surface receptor for

vesicular stomatitis virus. J Virol

2004;78(20):10920-6

54. Kobinger GP, Weiner DJ, Yu QC,

Wilson JM. Filovirus-pseudotyped

lentiviral vector can efficiently and

stably transduce airway epithelia in

vivo. Nat Biotechnol 2001;19(3):225-30

55. Wong LF, Azzouz M, Walmsley LE,

et al. Transduction patterns of

pseudotyped lentiviral vectors in the

nervous system. Mol Ther

2004;9(1):101-11

56. Kang Y, Xie L, Tran DT, et al.

Persistent expression of factor VIII

in vivo following nonprimate lentiviral

gene transfer. Blood 2005;106(5):1552-8

57. Bartosch B, Dubuisson J, Cosset FL.

Infectious hepatitis C virus

pseudo-particles containing functional

E1-E2 envelope protein complexes.

J Exp Med 2003;197(5):633-42

58. Di Nunzio F, Piovani B, Cosset FL,

et al. Transduction of human

hematopoietic stem cells by lentiviral

vectors pseudotyped with the RD114-TR

chimeric envelope glycoprotein.

Hum Gene Ther 2007;18(9):811-20

59. Frecha C, Levy C, Cosset FL,

Verhoeyen E. Advances in the field of

lentivector-based transduction of T and

B lymphocytes for gene therapy.

Mol Ther 2010;18(10):1748-57

60. Sena-Esteves M, Tebbets JC, Steffens S,

et al. Optimized large-scale production of

high titer lentivirus vector pseudotypes.

J Virol Methods 2004;122(2):131-9

61. Kim YS, Wielgosz MM, Hargrove P,

et al. Transduction of human primitive

repopulating hematopoietic cells with

lentiviral vectors pseudotyped with

various envelope proteins. Mol Ther

2010;18(7):1310-17

62. Bukrinskaya A. HIV-1 matrix protein:

a mysterious regulator of the viral life

cycle. Virus Res 2007;124(1-2):1-11

63. Hearps AC, Jans DA. Regulating the

functions of the HIV-1 matrix protein.

AIDS Res Hum Retroviruses

2007;23(3):341-6

64. Klein KC, Reed JC, Lingappa JR.

Intracellular destinies: degradation,

targeting, assembly, and endocytosis of

HIV Gag. AIDS Rev 2007;9(3):150-61

65. Fiorentini S, Riboldi E, Facchetti F,

et al. HIV-1 matrix protein p17 induces

human plasmacytoid dendritic cells to

acquire a migratory immature cell

phenotype. Proc Natl Acad Sci USA

2008;105(10):3867-72

66. Urano E, Aoki T, Futahashi Y, et al.

Substitution of the myristoylation signal

of human immunodeficiency virus

type 1 Pr55Gag with the phospholipase

C-delta1 pleckstrin homology domain

results in infectious pseudovirion

production. J Gen Virol

2008;89(Pt 12):3144-9

67. Wagner R, Graf M, Bieler K, 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(17):2403-13

68. Aoki T, Shimizu S, Urano E, et al.

Improvement of lentiviral

vector-mediated gene transduction by

genetic engineering of the structural

protein Pr55 Gag. Gene Ther

2010;17(9):1124-33

M. M. Segura et al.


Exp

ert O

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Bio

l. T

her.

Dow

nloa

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from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

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aval

on

07/1

5/15

For

pers

onal

use

onl

y.

http://informahealthcare.com/action/showLinks?pmid=19223867&crossref=10.1038%2Fmt.2009.1&coi=1%3ACAS%3A528%3ADC%252BD1MXhvFKjtL0%253D&isi=000264748000013

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http://informahealthcare.com/action/showLinks?pmid=17824830&crossref=10.1089%2Fhum.2006.138&coi=1%3ACAS%3A528%3ADC%252BD2sXhtFaitbzM&isi=000249843600004

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http://informahealthcare.com/action/showLinks?pmid=20143172&crossref=10.1007%2Fs00005-010-0063-4&coi=1%3ACAS%3A528%3ADC%252BC3cXjtFyjtr4%253D&isi=000275455200004

http://informahealthcare.com/action/showLinks?pmid=19008404&crossref=10.1099%2Fvir.0.2008%2F004820-0&coi=1%3ACAS%3A528%3ADC%252BD1cXhsFWgtrvN&isi=000261527800028

http://informahealthcare.com/action/showLinks?pmid=20736930&crossref=10.1038%2Fmt.2010.178&coi=1%3ACAS%3A528%3ADC%252BC3cXhtVKhtrvL&isi=000282541200005

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http://informahealthcare.com/action/showLinks?pmid=15542136&crossref=10.1016%2Fj.jviromet.2004.08.017&coi=1%3ACAS%3A528%3ADC%252BD2cXpslOks78%253D&isi=000225632900001

http://informahealthcare.com/action/showLinks?pmid=15452212&crossref=10.1128%2FJVI.78.20.10920-10926.2004&coi=1%3ACAS%3A528%3ADC%252BD2cXotlKjuro%253D&isi=000224229000009

http://informahealthcare.com/action/showLinks?pmid=21674167&crossref=10.1007%2Fs00253-011-3392-2&coi=1%3ACAS%3A528%3ADC%252BC3MXhtVKlsr%252FL&isi=000294214900012

http://informahealthcare.com/action/showLinks?pmid=11045424&crossref=10.1002%2F1521-2254%28200009%2F10%292%3A5%3C308%3A%3AAID-JGM131%3E3.0.CO%3B2-3&coi=1%3ASTN%3A280%3ADC%252BD3crgtVSntA%253D%253D&isi=000089667500002

http://informahealthcare.com/action/showLinks?pmid=20410927&crossref=10.1038%2Fgt.2010.61&coi=1%3ACAS%3A528%3ADC%252BC3cXltV2ksLg%253D&isi=000281757900007

http://informahealthcare.com/action/showLinks?pmid=20372106&crossref=10.1038%2Fmt.2010.48&coi=1%3ACAS%3A528%3ADC%252BC3cXkt1Cgurg%253D&isi=000279398900009

http://informahealthcare.com/action/showLinks?pmid=11231554&crossref=10.1038%2F85664&coi=1%3ACAS%3A528%3ADC%252BD3MXhslaqt7k%253D&isi=000167283100021

http://informahealthcare.com/action/showLinks?pmid=15880619&crossref=10.1002%2Fjgm.769&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFegtLbK&isi=000232070400002

http://informahealthcare.com/action/showLinks?pmid=9811723&coi=1%3ACAS%3A528%3ADyaK1cXns1Sgs78%253D&isi=000076892100050

http://informahealthcare.com/action/showLinks?pmid=17210199&crossref=10.1016%2Fj.virusres.2006.07.001&coi=1%3ACAS%3A528%3ADC%252BD2sXhs1Cjtbg%253D&isi=000244924900001

http://informahealthcare.com/action/showLinks?pmid=14741783&crossref=10.1016%2Fj.ymthe.2003.09.017&coi=1%3ACAS%3A528%3ADC%252BD2cXntVOisg%253D%253D&isi=000188659300018

http://informahealthcare.com/action/showLinks?pmid=16355114&crossref=10.1038%2Fsj.gt.3302698&coi=1%3ACAS%3A528%3ADC%252BD28Xis1Gmu7o%253D&isi=000236202500009

http://informahealthcare.com/action/showLinks?pmid=16516556&crossref=10.1016%2Fj.ymthe.2005.12.015&coi=1%3ACAS%3A528%3ADC%252BD28Xms1ahsro%253D&isi=000239249900017

http://informahealthcare.com/action/showLinks?pmid=17411366&crossref=10.1089%2Faid.2006.0108&coi=1%3ACAS%3A528%3ADC%252BD2sXjvVaqsLw%253D&isi=000245740300001

http://informahealthcare.com/action/showLinks?pmid=15886327&crossref=10.1182%2Fblood-2004-11-4358&coi=1%3ACAS%3A528%3ADC%252BD2MXps1OgsL8%253D

http://informahealthcare.com/action/showLinks?pmid=17597793&crossref=10.1038%2Fsj.gt.3302979&coi=1%3ACAS%3A528%3ADC%252BD2sXpt1altbo%253D&isi=000248882800006

http://informahealthcare.com/action/showLinks?pmid=17982940&isi=000250161700003

http://informahealthcare.com/action/showLinks?pmid=12615904&crossref=10.1084%2Fjem.20021756&coi=1%3ACAS%3A528%3ADC%252BD3sXhvFyrtb8%253D&isi=000181411400010

69. Yanez-Munoz RJ, Balaggan KS,

MacNeil A, et al. Effective gene therapy

with nonintegrating lentiviral vectors.

Nat Med 2006;12(3):348-53

70. Philippe S, Sarkis C, Barkats M, et al.

Lentiviral vectors with a defective

integrase allow efficient and sustained

transgene expression in vitro and in vivo.

Proc Natl Acad Sci USA

2006;103(47):17684-9

71. Apolonia L, Waddington SN,

Fernandes C, et al. Stable gene transfer

to muscle using non-integrating lentiviral

vectors. Mol Ther 2007;15(11):1947-54

72. Hacein-Bey-Abina S, von Kalle C,

Schmidt M, et al. A serious adverse event

after successful gene therapy for X-linked

severe combined immunodeficiency.

N Engl J Med 2003;348(3):255-6

73. Howe SJ, Mansour MR,

Schwarzwaelder K, et al. Insertional

mutagenesis combined with acquired

somatic mutations causes leukemogenesis

following gene therapy of

SCID-X1 patients. J Clin Invest

2008;118(9):3143-50

74. Mitchell RS, Beitzel BF, Schroder AR,

et al. Retroviral DNA integration: ASLV,

HIV, and MLV show distinct target site

preferences. PLoS Biol 2004;2(8):E234

75. Montini E, Cesana D, Schmidt 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(4):964-75

76. Dropulic B. Lentiviral vectors: their

molecular design, safety, and use in

laboratory and preclinical research.

Hum Gene Ther 2011;22(6):649-57

77. Mates L, Chuah MK, Belay E, et al.

Molecular evolution of a novel hyperactive

Sleeping Beauty transposase enables robust

stable gene transfer in vertebrates.

Nat Genet 2009;41(6):753-61

78. VandenDriessche T, Ivics Z, Izsvak Z,

Chuah MK. Emerging potential of

transposons for gene therapy and

generation of induced pluripotent stem

cells. Blood 2009;114(8):1461-8

79. Staunstrup NH, Moldt B, Mates L, et al.

Hybrid lentivirus-transposon vectors with

a random integration profile in human

cells. Mol Ther 2009;17(7):1205-14

80. Cathomen T, Joung JK. Zinc-finger

nucleases: the next generation emerges.

Mol Ther 2008;16(7):1200-7

81. Dreyer AK, Cathomen T. Zinc-finger

nucleases-based genome engineering to

generate isogenic human cell lines.

Methods Mol Biol 2012;813:145-56

82. Lombardo A, Genovese P,

Beausejour CM, et al. Gene editing in

human stem cells using zinc finger

nucleases and integrase-defective lentiviral

vector delivery. Nat Biotechnol

2007;25(11):1298-306

83. Ni Y, Sun S, Oparaocha I, et al.

Generation of a packaging cell line for

prolonged large-scale production of

high-titer HIV-1-based lentiviral vector.

J Gene Med 2005;7(6):818-34

84. Qi P, Han J, Lu Y, et al. A transient

three-plasmid expression system for the

production of hepatocytes targeting

retroviral vectors. Acta Biochim Biophys

Sin (Shanghai) 2007;39(8):567-74

85. Stacey GN, Merten OW. Host cells and

cell banking. Methods Mol Biol

2011;737:45-88

86. Segura MM, Garnier A, Durocher Y,

et al. Production of lentiviral vectors by

large-scale transient transfection of

suspension cultures and affinity

chromatography purification.


87. Van Craenenbroeck K, Vanhoenacker P,

Haegeman G. Episomal vectors for gene

expression in mammalian cells.

Eur J Biochem 2000;267(18):5665-78

88. Gama-Norton L, Botezatu L,

Herrmann S, 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(10):1269-79

89. Ansorge S, Lanthier S, Transfiguracion J,

et al. Development of a scalable process

for high-yield lentiviral vector production

by transient transfection of

HEK293 suspension cultures.

J Gene Med 2009;11(10):868-76

90. Diaz RM, Bateman A, Emiliusen L,

et al. A lentiviral vector expressing a

fusogenic glycoprotein for cancer gene

therapy. Gene Ther 2000;7(19):1656-63

91. Merten OW, Charrier S, Laroudie N,

et al. Large-scale manufacture and

characterization of a lentiviral vector

produced for clinical ex vivo gene

therapy application. Hum Gene Ther

2011;22(3):343-56

92. Witting SR, Li LH, Jasti A, et al.

Efficient large volume lentiviral vector

production using flow electroporation.

Hum Gene Ther 2012;23(2):243-9

93. Lesch HP, Laitinen A, Peixoto C, et al.

Production and purification of lentiviral

vectors generated in 293T suspension

cells with baculoviral vectors. Gene Ther

2011;18(6):531-8

94. Kutner RH, Puthli S, Marino MP,

Reiser J. Simplified production and

concentration of HIV-1-based lentiviral

vectors using HYPERFlask vessels and

anion exchange membrane

chromatography. BMC Biotechnol

2009;9:10

95. Kuroda H, Marino MP, Kutner RH,

Reiser J. Production of lentiviral vectors

in protein-free media. Curr Protoc

Cell Biol 2011;Chapter 26:Unit 26 8

96. Tiscornia G, Singer O, Verma IM.

Production and purification of lentiviral

vectors. Nat Protoc 2006;1(1):241-5

97. Giry-Laterriere M, Verhoeyen E,

Salmon P. Lentiviral vectors.


98. Segura MM, Garnier A, Durocher Y,

et al. New protocol for lentiviral vector

mass production. Methods Mol Biol

2010;614:39-52

99. al Yacoub N, Romanowska M,

Haritonova N, Foerster J. Optimized

production and concentration of

lentiviral vectors containing large inserts.

J Gene Med 2007;9(7):579-84

100. Kutner RH, Zhang XY, Reiser J.

Production, concentration and titration

of pseudotyped HIV-1-based lentiviral

vectors. Nat Protoc 2009;4(4):495-505

101. Jordan M, Kohne C, Wurm FM.

Calcium-phosphate mediated

DNA transfer into HEK-293 cells in

suspension: control of physicochemical

parameters allows transfection in stirred

media. Transfection and protein

expression in mammalian cells.

Cytotechnology 1998;26(1):39-47

102. Shin KJ, Wall EA, Zavzavadjian JR,

et al. A single lentiviral vector platform

for microRNA-based conditional

RNA interference and coordinated

transgene expression. Proc Natl Acad

Sci USA 2006;103(37):13759-64

103. Kosaka Y, Kobayashi N, Fukazawa T,

et al. Lentivirus-based gene delivery in

mouse embryonic stem cells.

Artif Organs 2004;28(3):271-7

104. Coleman JE, Huentelman MJ,

Kasparov S, et al. Efficient large-scale



Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f L

aval

on

07/1

5/15

For

pers

onal

use

onl

y.

http://informahealthcare.com/action/showLinks?pmid=21248790&crossref=10.1038%2Fgt.2010.162&coi=1%3ACAS%3A528%3ADC%252BC3MXnvVKmsQ%253D%253D&isi=000291438900001

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production and concentration of

HIV-1-based lentiviral vectors for use in

vivo. Physiol Genomics

2003;12(3):221-8

105. Kuroda H, Kutner RH, Bazan NG,

Reiser J. Simplified lentivirus vector

production in protein-free media using

polyethylenimine-mediated transfection.

J Virol Methods 2009;157(2):113-21

106. Toledo JR, Prieto Y, Oramas N,

Sanchez O. Polyethylenimine-Based

Transfection Method as a Simple and

Effective Way to Produce Recombinant

Lentiviral Vectors. Appl Biochem

Biotechnol 2009;157(3):538-44

107. Reed SE, Staley EM, Mayginnes JP,

et al. Transfection of mammalian cells

using linear polyethylenimine is a simple

and effective means of producing

recombinant adeno-associated virus

vectors. J Virol Methods

2006;138(1-2):85-98

108. Rols MP, Coulet D, Teissie J. Highly

efficient transfection of mammalian cells

by electric field pulses. Application to

large volumes of cell culture by using a

flow system. Eur J Biochem

1992;206(1):115-21

109. Li LH, Shivakumar R, Feller S, et al.

Highly efficient, large volume flow

electroporation. Technol Cancer

Res Treat 2002;1(5):341-50

110. Aucoin MG, Mena JA, Kamen AA.

Bioprocessing of baculovirus vectors:

a review. Curr Gene Ther

2010;10(3):174-86

111. Kost TA, Condreay JP. Recombinant

baculoviruses as expression vectors for

insect and mammalian cells.

Curr Opin Biotechnol

1999;10(5):428-33

112. Poomputsa K, Kittel C, Egorov A, et al.

Generation of recombinant influenza

virus using baculovirus delivery vector.

J Virol Methods 2003;110(1):111-14

113. Cheshenko N, Krougliak N,

Eisensmith RC, Krougliak VA. A novel

system for the production of fully deleted

adenovirus vectors that does not require

helper adenovirus. Gene Ther

2001;8(11):846-54

114. Sollerbrant K, Elmen J, Wahlestedt C,

et al. A novel method using

baculovirus-mediated gene transfer for

production of recombinant

adeno-associated virus vectors.

J Gen Virol 2001;82(Pt 9):2051-60

115. Chen YH, Wu JC, Wang KC, et al.

Baculovirus-mediated production of

HDV-like particles in BHK cells using a

novel oscillating bioreactor. J Biotechnol

2005;118(2):135-47

116. Lesch HP, Turpeinen S, Niskanen EA,

et al. Generation of lentivirus vectors

using recombinant baculoviruses.

Gene Ther 2008;15(18):1280-6

117. Paavonen J, Jenkins D, Bosch FX, et al.

Efficacy of a prophylactic adjuvanted

bivalent L1 virus-like-particle vaccine

against infection with human

papillomavirus types 16 and 18 in young

women: an interim analysis of a phase III

double-blind, randomised controlled

trial. Lancet 2007;369(9580):2161-70

118. Broussau S, Jabbour N, Lachapelle G,

et al. Inducible packaging cells for

large-scale production of lentiviral vectors

in serum-free suspension culture.

Mol Ther 2008;16(3):500-7

119. Naldini L, Blomer U, Gallay P, et al. In

vivo gene delivery and stable transduction

of nondividing cells by a lentiviral vector.

Science 1996;272(5259):263-7

120. Stewart HJ, Fong-Wong L, Strickland I,

et al. A stable producer cell line for the

manufacture of a lentiviral vector for

gene therapy of Parkinson’s disease.

Hum Gene Ther 2011;22(3):357-69

121. Stewart HJ, Leroux-Carlucci MA,

Sion CJ, et al. Development of inducible

EIAV-based lentiviral vector packaging

and producer cell lines. Gene Ther

2009;16(6):805-14

122. Ikeda Y, Takeuchi Y, Martin F, et al.

Continuous high-titer HIV-1 vector

production. Nat Biotechnol

2003;21(5):569-72

123. Lee CL, Chou M, Dai B, et al.

Construction of stable producer cells to

make high-titer lentiviral vectors for

dendritic cell-based vaccination.


124. Throm RE, Ouma AA, Zhou S, 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(21):5104-10

125. Farson D, Witt R, McGuinness R, et al.

A new-generation stable inducible

packaging cell line for lentiviral vectors.

Hum Gene Ther 2001;12(8):981-97

126. Kong D, Gentz R, Zhang J. Long-term

stable production of monocyte-colony

inhibition factor (M-CIF) from CHO

microcarrier perfusion cultures.

Cytotechnology 1998;26(2):131-8

127. Hu X, Xiao C, Huang Z, et al. Pilot

production of u-PA with porous

microcarrier cell culture. Cytotechnology

2000;33(1-3):13-19

128. Rourou S, van der Ark A, Majoul S,

et al. A novel animal-component-free

medium for rabies virus production in

Vero cells grown on Cytodex

1 microcarriers in a stirred bioreactor.

Appl Microbiol Biotechnol

2009;85(1):53-63

129. Trabelsi K, Rourou S, Loukil H, et al.

Optimization of virus yield as a strategy

to improve rabies vaccine production by

Vero cells in a bioreactor. J Biotechnol

2006;121(2):261-71

130. Rourou S, van der Ark A,

van der Velden T, Kallel H.

A microcarrier cell culture process for

propagating rabies virus in Vero cells

grown in a stirred bioreactor under fully

animal component free conditions.

Vaccine 2007;25(19):3879-89

131. Genzel Y, Olmer RM, Schafer B,

Reichl U. Wave microcarrier cultivation

of MDCK cells for influenza virus

production in serum containing and

serum-free media. Vaccine

2006;24(35-36):6074-87

132. Wu SC, Huang GY, Liu JH. Production

of retrovirus and adenovirus vectors for

gene therapy: a comparative study using

microcarrier and stationary cell culture.

Biotechnol Prog 2002;18(3):617-22

133. Davie JR. Inhibition of histone

deacetylase activity by butyrate. J Nutr

2003;133(7 Suppl):2485S-93S

134. Kafri T, van Praag H, Ouyang L, et al.

A packaging cell line for lentivirus

vectors. J Virol 1999;73(1):576-84

135. Karolewski BA, Watson DJ,

Parente MK, Wolfe JH. Comparison of

transfection conditions for a lentivirus

vector produced in large volumes.

Hum Gene Ther 2003;14(14):1287-96

136. Jaalouk DE, Crosato M, Brodt P,

Galipeau J. Inhibition of histone

deacetylation in 293GPG packaging cell

line improves the production of

self-inactivating MLV-derived retroviral

vectors. Virol J 2006;3:27

137. Wibo M, Poole B. Protein degradation

in cultured cells. II. The uptake of

chloroquine by rat fibroblasts and the

M. M. Segura et al.


Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

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info

rmah

ealth

care

.com

by

Uni

vers

ity o

f L

aval

on

07/1

5/15

For

pers

onal

use

onl

y.

http://informahealthcare.com/action/showLinks?pmid=17602732&crossref=10.1016%2FS0140-6736%2807%2960946-5&coi=1%3ACAS%3A528%3ADC%252BD2sXntF2lsLg%253D&isi=000247644200027

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http://informahealthcare.com/action/showLinks?pmid=11423932&crossref=10.1038%2Fsj.gt.3301459&coi=1%3ACAS%3A528%3ADC%252BD3MXktFOrtrw%253D&isi=000169119800004

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http://informahealthcare.com/action/showLinks?pmid=1316837&crossref=10.1111%2Fj.1432-1033.1992.tb16908.x&coi=1%3ACAS%3A528%3ADyaK38XksVWmsro%253D

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http://informahealthcare.com/action/showLinks?pmid=18463689&crossref=10.1038%2Fgt.2008.76&coi=1%3ACAS%3A528%3ADC%252BD1cXhtVGgtb%252FF&isi=000258915300004

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http://informahealthcare.com/action/showLinks?pmid=16781022&crossref=10.1016%2Fj.vaccine.2006.05.023&coi=1%3ACAS%3A528%3ADC%252BD28XotV2ntb4%253D&isi=000240398000006

http://informahealthcare.com/action/showLinks?pmid=19286997&crossref=10.1182%2Fblood-2008-11-191049&coi=1%3ACAS%3A528%3ADC%252BD1MXmslaltbk%253D&isi=000266404500015

inhibition of cellular protein degradation

and cathepsin B1. J Cell Biol

1974;63(2 Pt 1):430-40

138. Luthman H, Magnusson G. High

efficiency polyoma DNA transfection of

chloroquine treated cells.

Nucleic Acids Res 1983;11(5):1295-308

139. Ciftci K, Levy RJ. Enhanced plasmid

DNA transfection with lysosomotropic

agents in cultured fibroblasts.

Int J Pharm 2001;218(1-2):81-92

140. Ellis BL, Potts PR, Porteus MH.

Creating higher titer lentivirus with

caffeine. Hum Gene Ther

2011;22(1):93-100

141. Schweizer M, Merten OW. Large-scale

production means for the manufacturing

of lentiviral vectors. Curr Gene Ther

2010;10(6):474-86

142. Segura MM, Kamen A, Garnier A.

Downstream processing of oncoretroviral

and lentiviral gene therapy vectors.

Biotechnol Adv 2006;24(3):321-37

143. Wolff M, Reichl U. Downstream

processing of cell culture-derived virus

particles. Expert Rev Vaccines

2011;10(10):1451-75

144. Andreadis ST, Roth CM, Le Doux JM,

et al. Large-scale processing of

recombinant retroviruses for gene

therapy. Biotechnol Prog

1999;15(1):1-11

145. Rodrigues T, Carrondo MJ, Alves PM,

Cruz PE. Purification of retroviral vectors

for clinical application: biological

implications and technological challenges.

J Biotechnol 2007;127(3):520-41

146. Morenweiser R. Downstream processing

of viral vectors and vaccines. Gene Ther

2005;12(Suppl 1):S103-10

147. Segura MM, Kamen AA, Garnier A.

Overview of current scalable methods for

purification of viral vectors.


148. Reeves L, Cornetta K. Clinical retroviral

vector production: step filtration using

clinically approved filters improves titers.

Gene Ther 2000;7(23):1993-8

149. Segura MM, Kamen A, Trudel P,

Garnier A. A novel purification strategy

for retrovirus gene therapy vectors using

heparin affinity chromatography.


150. Slepushkin V, Chang N, Cohen R, et al.

Large-scale purification of a lentiviral

vector by size exclusion chromatography

or mustang Q ion exchange capsule.

Bioprocessing J 2003;2:89-95

151. Ausubel L, Hall C, Sharma A, et al.

Production of cGMP-Grade Lentiviral

Vectors. Bio Process Int

2012;10(2):32-43

152. Burns JC, Friedmann T, Driever W,

et al. Vesicular stomatitis virus G

glycoprotein pseudotyped retroviral

vectors: concentration to very high titer

and efficient gene transfer into

mammalian and nonmammalian cells.

Proc Natl Acad Sci USA

1993;90(17):8033-7

153. Cooper AR, Patel S, Senadheera S, et al.

Highly efficient large-scale lentiviral

vector concentration by tandem

tangential flow filtration.

J Virol Methods 2011;177(1):1-9

154. Geraerts M, Michiels M, Baekelandt V,

et al. Upscaling of lentiviral vector

production by tangential flow filtration.

J Gene Med 2005;7(10):1299-310

155. Scherr M, Battmer K, Eder M, et al.

Efficient gene transfer into the CNS by

lentiviral vectors purified by anion

exchange chromatography. Gene Ther

2002;9(24):1708-14

156. Zimmermann K, Scheibe O,

Kocourek A, et al. Highly efficient

concentration of lenti- and retroviral

vector preparations by membrane

adsorbers and ultrafiltration.

BMC Biotechnol 2011;11(1):55

157. Cheeks MC, Kamal N, Sorrell A, et al.

Immobilized metal affinity

chromatography of histidine-tagged

lentiviral vectors using monolithic

adsorbents. J Chromatogr A

2009;1216(13):2705-11

158. Jungbauer A, Hahn R. Polymethacrylate

monoliths for preparative and industrial

separation of biomolecular assemblies.

J Chromatogr A 2008;1184(1-2):62-79

159. Stadler J, Lemmens R, Nyhammar T.

Plasmid DNA purification. J Gene Med

2004;6(Suppl 1):S54-66

160. Kramberger P, Peterka M, Boben J, et al.

Short monolithic columns--a

breakthrough in purification and fast

quantification of tomato mosaic virus.

J Chromatogr A 2007;1144(1):143-9

161. Yamada K, McCarty DM, Madden VJ,

Walsh CE. Lentivirus vector purification

using anion exchange HPLC leads to

improved gene transfer. Biotechniques

2003;34(5):1074-8; 80

162. Slepushkin V. Chromatography Methods

for Purification of Lentiviral Vectors.

2011.Available from: http://

biotechexpertsnet/wordpress/?p=59

163. Rodrigues T, Alves A, Lopes A, et al.

Removal of envelope protein-free

retroviral vectors by anion-exchange

chromatography to improve product

quality. J Sep Sci 2008;31(20):3509-18

164. Eriksson K, Ljunglof A, Rodrigo G,

Brekkan E. MAb contaminant removal

with a multimodal anion exchanger:

a platform step to follow protein A.

Bioprocess Int 2009;7:52-6

165. Vazquez-Rey M, Lang DA. Aggregates in

monoclonal antibody manufacturing

processes. Biotechnol Bioeng

2011;108(7):1494-508

166. Yu JH, Schaffer DV. Selection of novel

vesicular stomatitis virus glycoprotein

variants from a peptide insertion library

for enhanced purification of retroviral

and lentiviral vectors. J Virol

2006;80(7):3285-92

167. Chen R, Folarin N, Ho VH, et al.

Affinity recovery of lentivirus by

diaminopelargonic acid mediated

desthiobiotin labelling. J Chromatogr B

Analyt Technol Biomed Life Sci

2010;878(22):1939-45

168. Segura MM, Kamen A, Lavoie MC,

Garnier A. Exploiting heparin-binding

properties of MoMLV-based retroviral

vectors for affinity chromatography.

J Chromatogr B Analyt Technol Biomed

Life Sci 2007;846(1-2):124-31

169. Ye K, Jin S, Ataai MM, et al. Tagging

retrovirus vectors with a metal binding

peptide and one-step purification by

immobilized metal affinity

chromatography. J Virol

2004;78(18):9820-7

170. Nesbeth D, Williams SL, Chan L, et al.

Metabolic biotinylation of lentiviral

pseudotypes for scalable paramagnetic

microparticle-dependent manipulation.

Mol Ther 2006;13(4):814-22

171. Kalbfuss B, Wolff M, Morenweiser R,

Reichl U. Purification of cell

culture-derived human influenza A virus

by size-exclusion and anion-exchange

chromatography. Biotechnol Bioeng

2007;96(5):932-44

172. Huyghe BG, Liu X, Sutjipto S, et al.

Purification of a type 5 recombinant

adenovirus encoding human p53 by



Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f L

aval

on

07/1

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For

pers

onal

use

onl

y.

http://biotechexpertsnet/wordpress/?p=59

http://biotechexpertsnet/wordpress/?p=59

http://informahealthcare.com/action/showLinks?pmid=20599176&crossref=10.1016%2Fj.jchromb.2010.05.019&coi=1%3ACAS%3A528%3ADC%252BC3cXosVyhtbw%253D&isi=000280210300005

http://informahealthcare.com/action/showLinks?pmid=17097098&crossref=10.1016%2Fj.chroma.2006.10.055&coi=1%3ACAS%3A528%3ADC%252BD2sXit1Gltr0%253D&isi=000244881700018

http://informahealthcare.com/action/showLinks?pmid=21784103&crossref=10.1016%2Fj.jviromet.2011.06.019&coi=1%3ACAS%3A528%3ADC%252BC3MXhtVGrs7jJ&isi=000295113000001

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http://informahealthcare.com/action/showLinks?pmid=15331716&crossref=10.1128%2FJVI.78.18.9820-9827.2004&coi=1%3ACAS%3A528%3ADC%252BD2cXnvVars74%253D&isi=000223701100027

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http://informahealthcare.com/action/showLinks?pmid=18830959&crossref=10.1002%2Fjssc.200800195&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVKit7zM&isi=000261237500005

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http://informahealthcare.com/action/showLinks?pmid=8573613&crossref=10.1089%2Fhum.1995.6.11-1403&coi=1%3ACAS%3A528%3ADyaK28XhslSkug%253D%253D&isi=A1995TE48600005

http://informahealthcare.com/action/showLinks?pmid=21480193&crossref=10.1002%2Fbit.23155&coi=1%3ACAS%3A528%3ADC%252BC3MXmtFyltr8%253D&isi=000291467600002

http://informahealthcare.com/action/showLinks?pmid=18241874&crossref=10.1016%2Fj.chroma.2007.12.087&coi=1%3ACAS%3A528%3ADC%252BD1cXivVynt70%253D&isi=000254429600009

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http://informahealthcare.com/action/showLinks?pmid=16537595&crossref=10.1128%2FJVI.80.7.3285-3292.2006&coi=1%3ACAS%3A528%3ADC%252BD28Xjt1Kjurg%253D&isi=000236305200015

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http://informahealthcare.com/action/showLinks?pmid=16950534&crossref=10.1016%2Fj.jbiotec.2006.07.028&coi=1%3ACAS%3A528%3ADC%252BD28Xht1KlsrrP&isi=000243697400021

http://informahealthcare.com/action/showLinks?pmid=6298741&crossref=10.1093%2Fnar%2F11.5.1295&coi=1%3ACAS%3A528%3ADyaL3sXhsFyltrw%253D&isi=A1983QF97800007

column chromatography.

Hum Gene Ther 1995;6(11):1403-16

173. Sastry L, Xu Y, Cooper R, et al.

Evaluation of plasmid DNA removal

from lentiviral vectors by benzonase

treatment. Hum Gene Ther

2004;15(2):221-6

174. Carmo M, Alves A, Rodrigues AF, et al.

Stabilization of gammaretroviral and

lentiviral vectors: from production to

gene transfer. J Gene Med

2009;11(8):670-8

175. Schonely K, Afable C, Slepushkin V,

et al. QC release testing of an

HIV-1 based lentiviral vector lot and

transduced cellular product.

Bioprocessing J 2003;2:39-47

176. Agency EEM. Guideline on development

and manufacture of lentiviral vectors.

CHMPBWP245803; 2005

177. Santos S, Obukhov Y, Nekhai S, et al.

Virus-producing cells determine the host

protein profiles of HIV-1 virion cores.

Retrovirology 2012;9:65

178. Chertova E, Chertov O, Coren LV, et al.

Proteomic and biochemical analysis of

purified human immunodeficiency virus

type 1 produced from infected

monocyte-derived macrophages. J Virol

2006 Sep;80(18):9039-52

179. Segura MM, Garnier A, Di Falco MR,

et al. Identification of host proteins

associated with retroviral vector particles

by proteomic analysis of highly purified

vector preparations. J Virol

2008;82(3):1107-17

180. Denard J, Rundwasser S, Laroudie N,

et al. Quantitative proteomic analysis of

lentiviral vectors using 2-DE. Proteomics

2009;9(14):3666-76

181. Wheeler JX, Jones C, Thorpe R, Zhao Y.

Proteomics analysis of cellular

components in lentiviral vector

production using Gel-LC-MS/MS.

Proteomics Clin Appl 2007;1(2):224-30

182. Muriaux D, Mirro J, Harvin D, Rein A.

RNA is a structural element in retrovirus

particles. Proc Natl Acad Sci USA

2001;98(9):5246-51

183. Valley-Omar Z, Meyers AE,

Shephard EG, et al. Abrogation of

contaminating RNA activity in

HIV-1 Gag VLPs. Virol J 2011;8:462

184. Geraerts M, Willems S, Baekelandt V,

et al. Comparison of lentiviral titration

methods. BMS Biotechnol 2006;6:34

185. Fan Y, Luo Y, Chen X, et al. Generation

of Human beta-thalassemia Induced

Pluripotent Stem Cells from Amniotic

Fluid Cells Using a Single Excisable

Lentiviral Stem Cell Cassette.

J Reprod Dev 2012;58(4):404-9

186. Somers A, Jean JC, Sommer CA, et al.

Generation of transgene-free lung

disease-specific human induced

pluripotent stem cells using a single

excisable lentiviral stem cell cassette.

Stem Cells 2010;28(10):1728-40

187. Sommer CA, Stadtfeld M, Murphy GJ,

et al. Induced pluripotent stem cell

generation using a single lentiviral stem

cell cassette. Stem Cells

2009;27(3):543-9

188. Liang X, Fujioka K, Tsuda Y, et al.

Construction of a photo-switchable gene

for turning on and off gene expression

with light irradiation. Nucleic Acids

Symp Ser (Oxf) 2008(52):19-20

189. Liu M, Komiyama M, Asanuma H.

Design of light-switchable phage

promoter for efficient photo-regulation of

gene-expression. Nucleic Acids Symp Ser

(Oxf) 2005(49):283-4

190. Shimizu-Sato S, Huq E, Tepperman JM,

Quail PH. A light-switchable gene

promoter system. Nat Biotechnol

2002;20(10):1041-4

191. Quail PH. Phytochrome-interacting

factors. Semin Cell Dev Biol

2000;11(6):457-66

192. Martinez-Garcia JF, Huq E, Quail PH.

Direct targeting of light signals to a

promoter element-bound transcription

factor. Science 2000;288(5467):859-63

193. Nie Z, Bren GD, Vlahakis SR, et al.

Human immunodeficiency virus

type 1 protease cleaves procaspase 8 in

vivo. J Virol 2007;81(13):6947-56

194. Nie Z, Phenix BN, Lum JJ, et al.

HIV-1 protease processes procaspase 8 to

cause mitochondrial release of

cytochrome c, caspase cleavage and

nuclear fragmentation. Cell Death Differ

2002;9(11):1172-84

195. Choi SS, Rhee WJ, Kim EJ, Park TH.

Enhancement of recombinant protein

production in Chinese hamster ovary

cells through anti-apoptosis engineering

using 30Kc6 gene. Biotechnol Bioeng

2006;95(3):459-67

196. Vieira HL, Pereira AC, Peixoto CC,

et al. Improvement of recombinant

protein production by an anti-apoptotic

protein from hemolymph of Lonomia

obliqua. Cytotechnology

2010;62(6):547-55

197. Majors BS, Betenbaugh MJ,

Pederson NE, Chiang GG. Enhancement

of transient gene expression and culture

viability using Chinese hamster ovary

cells overexpressing Bcl-x(L).


198. Figueroa B Jr, Ailor E, Osborne D, et al.

Enhanced cell culture performance using

inducible anti-apoptotic genes E1B-19K

and Aven in the production of a

monoclonal antibody with Chinese

hamster ovary cells. Biotechnol Bioeng

2007;97(4):877-92

199. Majors BS, Betenbaugh MJ,

Pederson NE, Chiang GG.

Mcl-1 overexpression leads to higher

viabilities and increased production of

humanized monoclonal antibody in

Chinese hamster ovary cells.

Biotechnol Prog 2009;25(4):1161-8

200. Wang XH, Xu J, Zhang Y, et al.

Inducible expression of Bcl-XL inhibits

sodium butyrate-induced apoptosis in

hybridoma, resulting in enhanced

antibody production. Cell Biol Int

2004;28(3):185-91

201. Warner TG. Enhancing therapeutic

glycoprotein production in Chinese

hamster ovary cells by metabolic

engineering endogenous gene control

with antisense DNA and gene targeting.

Glycobiology 1999;9(9):841-50

202. Wullschleger S, Loewith R, Hall MN.

TOR signaling in growth and

metabolism. Cell 2006;124(3):471-84

203. Zhao X, Guo J, Yu Y, et al.

Overexpression of survivin and cyclin

D1 in CHO cells confers apoptosis

resistance and enhances growth in

serum-free suspension culture.

Biotechnol Lett 2011;33(7):1293-300

204. Ye K, Dhiman HK, Suhan J, Schultz JS.

Effect of pH on infectivity and

morphology of ecotropic moloney

murine leukemia virus. Biotechnol Prog

2003;19(2):538-43

205. Le Doux JM, Davis HE, Morgan JR,

Yarmush ML. Kinetics of retrovirus

production and decay. Biotechnol Bioeng

1999;63(6):654-62

206. Beer C, Meyer A, Muller K, Wirth M.

The temperature stability of mouse

retroviruses depends on the cholesterol

levels of viral lipid shell and cellular

M. M. Segura et al.


Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

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rmah

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.com

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ity o

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pers

onal

use

onl

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plasma membrane. Virology

2003;308(1):137-46

207. Coroadinha AS, Silva AC, Pires E, et al.

Effect of osmotic pressure on the

production of retroviral vectors:

enhancement in vector stability.


208. Kotani H, Newton PB III, Zhang S,

et al. Improved methods of retroviral

vector transduction and production for

gene therapy. Hum Gene Ther

1994;5(1):19-28

209. Merten OW. State-of-the-art of the

production of retroviral vectors.

J Gene Med 2004;6(Suppl 1):S105-24

210. Rodrigues AF, Amaral AI, Verissimo V,

et al. Adaptation of retrovirus producer

cells to serum deprivation: implications

in lipid biosynthesis and vector

production. Biotechnol Bioeng

2012;109(5):1269-79

211. Coroadinha AS, Ribeiro J, Roldao A,

et al. Effect of medium sugar source on

the production of retroviral vectors for

gene therapy. Biotechnol Bioeng

2006;94(1):24-36

212. Li P, Xu L, Mou Y, et al. Medium

Optimization for Exopolysaccharide

Production in Liquid Culture of

Endophytic Fungus Berkleasmium sp.

Dzf12. Int J Mol Sci

2012;13(9):11411-26

213. Yao CL, Chu IM, Hsieh TB,

Hwang SM. A systematic strategy to

optimize ex vivo expansion medium for

human hematopoietic stem cells derived

from umbilical cord blood mononuclear

cells. Exp Hematol 2004;32(8):720-7

AffiliationMaria Mercedes Segura1, Mathias Mangion2,

Bruno Gaillet2 & Alain Garnier†2

†Author for correspondence1Chemical Engineering Department,

Universitat Autonoma de Barcelona,

Campus Bellaterra, Cerdanyola del Valles

(08193), Barcelona, Spain2Chemical Engineering Department,

Universite Laval, and Regroupement quebecois

sur la fonction, la structure et l’ingenierie des

proteines (PROTEO), Quebec,

QC, G1V 0A6, Canada

E-mail: [email protected]



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