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RESEARCH ARTICLE

Differential in¯uence of the E4 adenoviral genes

on viral and cellular promoters

Linda Grave

Dominique Dreyer

Annick Dieterle

Pierre Leroy

Anne-Isabelle Michou

Cecile Doderer

Andrea Pavirani

Monika Lusky

Majid Mehtali

Transge

ne S.A., 11 rue de Molsheim.

67085 Strasbourg, France

Crucell BV, Wassenaarseweg 72. PO

Box 2048. 2301 CA Leiden, The

Netherland

*Correspondence to: L. Grave,

Transge

ne S.A., 11 rue de Molsheim,

67000 Strasbourg, France.

E-mail: [email protected]

Received: 7 June 2000

Revised: 7 August 2000

Accepted: 11 August 2000

Published online: 18 September 2000

Abstract

Background Strong and stable transgene expression is fundamental to the

success of recombinant adenovirus vectors in human gene therapy. However,

control of transgene expression is a complex process, involving both viral and

cellular factors. In this study, the in¯uence of the E4 adenoviral region on the

activity of various promoters was investigated in vitro and in vivo.

Methods Pairs of isogenic E1u and E1uE4u vectors were generated and

compared. Levels of transgene expression were determined by Northern blot,

ELISA and FACS analysis. Initiation of transcription was studied by nuclear

run-on assays.

Results Similar to the viral CMV and RSV promoters, the activity of the

ubiquitous cellular PGK promoter required the presence of the E4 genes in

vitro and in vivo. In contrast, transgene expression from selected liver- and

tumor-speci®c promoters did not require E4 functions.

Conclusion Together with the reported low liver toxicity of E1uE4u vectors,

the independence of E4 of liver-speci®c promoters renders such vectors

interesting alternatives to the use of gutless vectors. Copyright # 2000 John

Wiley & Sons, Ltd.

Keywords gene therapy; E1uE4u adenovirus; viral and cellular promoters

Introduction

Replication-de®cient adenoviruses (Ad) constitute an attractive vector system

for the in vivo delivery of therapeutic genes to quiescent or dividing cells

[1±12]. However, the clinical use of such vectors is hampered by signi®cant in

vivo toxicity and in¯ammatory responses, poorly controlled expression of the

transduced therapeutic gene, and an inability to repeatedly administer the

vector. Thus, improvement of the current generation of E1-defective

recombinant vectors is necessary and requires a precise de®nition of the

virological and host immunological factors involved in vector toxicity and

short-term persistence of transgene expression [2,3,13±22].

Despite the absence of the regulatory E1 genes, ®rst-generation vectors still

allow residual expression of early viral proteins [23,24]. It was ®rst

hypothesized that this would be in part responsible for the induction of

speci®c cellular immune responses (cytotoxic T lymphocytes, CTL) and thus

for the elimination of the transduced cells [20,25,26]. However subsequent

studies have established that, provided the transgene product is non-

immunogenic, recombinant Ad vectors can allow long-term in vivo

persistence of transgene expression despite the induction of antiviral humoral

and cellular immune responses [[18,19,22]; L. Grave, unpublished observa-

THE JOURNAL OF GENE MEDICINE

J Gene Med 2000; 2: 433±443.

tions]. We also showed that multiply deleted Ad vectors,

in which residual viral gene expression was markedly

reduced, did not display better viral DNA persistence in

vivo and weaker antiviral immune responses compared to

®rst-generation vectors [24]. Actually Ad-mediated toxi-

city and in¯ammation in the host are mainly implicated in

the loss of viral persistence and the loss of transgene

expression over time [27]. In order to circumvent this, E1-

deleted (E1u) Ad vectors, further crippled in E2 (E1uE2u)

or E4 (E1uE4u) genes [29±33] or helper-dependent

(gutless) vectors fully depleted of viral genes [31±37]

have been generated. The simultaneous deletion of the

viral E1 and E4 regulatory genes led to signi®cant

reduction of vector toxicity in vivo [5,27,29,38]. How-

ever, the absence of the E4 region markedly reduced the

ef®ciency and persistence of transgene expression under

the control of the CMV IE1 promoter [30,32,33,39] and

RSV promoter [28]. This suggests the existence of

complex interactions between viral regulatory gene

products and the transgene control elements incorporated

in the vector [[39±44]; L. Grave, unpublished observa-

tions]. From these studies it appears essential to better

characterize the impact of the genetic organization of the

vector on transgene expression for a rational design of the

expression cassette.

In the present study we investigated the in¯uence of a

speci®c vector genome structure (E1u vs E1uE4u vectors)

on transgene expression controlled by several viral and

cellular genetic elements (promoters, splicing signals).

Pairs of isogenic E1u and E1uE4u viruses coding for

secreted or intracellular human proteins were compared

in vitro (established and primary animal cells) and in vivo

(immunode®cient mouse model). The regulatory

sequences selected for the present study were chosen

from viral or mammalian transcriptional and post-

transcriptional control elements commonly used to

generate expression plasmids and recombinant viral

vectors [30,33,45±47]. Since the activity of a given

regulatory sequence is often modulated in a tissue-

speci®c manner [48], the in¯uence of the transduced cell

type and target tissue on pCMV-driven transgene expres-

sion was also investigated.

Materials and methods

Viral vectors

Nucleotide numbering throughout this paper is according

to Chroboczek et al. [49]. All viral genomes were

constructed as infectious plasmids by homologous

recombination in Escherichia coli as described by Chartier

et al. [50]. In brief, all vectors (Tables 1 and 2) contain a

deletion in E1 from nucleotide (nt) 459 to nt 3327 and in

E3 from nt 28592 to nt 30470. The E4 deletion in the

E1uE4u vectors is identical to the H2dl808 deletion in Ad2

[51], removing most of the E4 coding sequences (nt

32994 to nt 34998) with the exception of ORF1 [24]. For

the generation of viruses, the viral genomes were released

from the respective recombinant plasmids by PacI

digestion and transfected into the appropriate comple-

mentation cell lines as described previously [50]. The E1u

and E1uE4u vectors were grown on 293 cells [52] and

293-E4ORF6/7 cells [24], respectively. Virus propaga-

tion, puri®cation and titration of infectious units (IU/ml)

by indirect immuno¯uorescence of the DNA Binding

Protein (DBP) were as described previously [24]. Puri®ed

viruses were stored in viral storage buffer [1 M sucrose,

10 mM Tris-HCl pH 8.5, 1 mM MgCl

, 150 mM NaCl,

0.005% (vol/vol) Tween 80]. Viral particle concentration

(P/ml) of each vector preparation was calculated using

the optical density [53].

All viral vectors involved in the present study are

described in Tables 1 and 2. The expression cassettes

encoded the human interleukin 2 (hIL2), the human

clotting factor IX (hFIX), the human cystis ®brosis

transmembrane conductance regulator (hCFTR) or the

enhanced green ¯uorescence protein (eGFP). The ubi-

quitous promoters tested were the immediate early

promoter of the human cytomegalovirus (pCMV) [45],

the long terminal repeat (LTR) sequences of the Rous

sarcoma virus (pRSV) [54,55] and the promoter of the

mouse phosphoglycerate kinase gene (pPGK) [56,57].

Different tissue-speci®c promoters were also involved

in the study: the minimal promoter of the human

a1-antitrypsin (x348; +15), shown to be liver-speci®c

(phAAT) [58,59], the H19 regulatory transcrip-

tional sequences involved in fetal development processes

(pH19) [60] or the MUC1-speci®c promoter active in

some of human tumor breast cells (pMUC1) [61].

Splicing signals were used to test whether addition of

an intron can increase the amount of cytoplasmic

transgene transcripts in recombinant adenoviral vectors

[62,63]. The selected sequences were either the ®rst

intron of the rabbit b-globin gene [64,65] or a chimeric

intron generated by a fusion between the splice donor site

of the intron I of the human b-globin gene and the splice

acceptor site of a human IgG gene [66].

Cell culture

All human and animal cells were maintained in Dulbecco

Modi®ed Eagle Medium (DMEM) supplemented with

10% fetal calf serum (FCS) (Gibco-BRL, Cergy-Pontoise,

France), with the exception of the human and canine

primary myoblasts which were maintained in HAMS14

(Gibco-BRL) with 20% dialyzed fetal bovine serum (FBS)

(Hyclone, Logan, Utah, USA) supplemented with 10 mg/

ml insulin (Sigma, St Louis, MO, NJ USA), 10 ng/ml

epidermal growth factor (EGF) (Sigma) and 10 ng/ml

bFGF (Perotech, Rocky Hill, NJ, USA). Most human

(A549, lung carcinoma; HeLa, cervix epithelioõ

d carci-

noma; Wish, amnion HeLa markers; 293, Ad5 trans-

formed embryonic kidney; HepG2, liver epithelioõ

carcinoma; Hep3B, liver epithelioõ

d carcinoma; T47d,

breast epithelioid carcinoma), simian (Vero, African

green monkey kidney epithelioõ

d; Cos-1, African green

monkey kidney epithelioid SV40 transformed), murine

434 L. Grave et al.

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