Lentiviral vectors for efficient transduction of isolated primary quiescent hepatocytes

doi:10.1016/S0168-8278(01)00308-7

Journal of Hepatology

Volume 36, Issue 4, April 2002, Pages 459-465

https://doi.org/10.1016/S0168-8278(01)00308-7 Get rights and content

Abstract

Background/Aims: Lentiviral vectors were designed to obtain efficient transduction of primary quiescent hepatocytes.

Methods: A hepatitis B virus (HBV) fragment containing enhancers and posttranscriptional regulatory element was used to increase expression levels. The human immunodeficiency virus (HIV) central polypurine tract (PPT) was used to increase transduction of quiescent cells. HBV elements were incorporated downstream and the HIV PPT was incorporated upstream of green fluorescent protein expression cassettes in third generation self inactivating lentiviral vectors.

Results: The HBV fragment increased mean fluorescence of transduced HepG2 hepatoma cells 4.3±1.7-fold and 2.3–6.0-fold in various other cell types. A role of HBV×protein in the function of the HBV element was excluded. The HBV element increased the number of transducing units per pg of HIV p24 twofold. The unmodified lentiviral vector transduced 5±1% of cultured quiescent primary rat hepatocytes, HBV elements increased transduction to 54±13% and increased fluorescence 2.8±0.6-fold. The PPT increased transduction to 47±11% and increased fluorescence 2.3±0.4-fold. The vector with PPT and HBV elements transduced 68±10% of hepatocytes and increased fluorescence synergistically, 17±6 fold.

Conclusions: This study shows that HBV elements or HIV PPT are required for efficient transduction of primary hepatocytes.

Introduction

Stable expression of therapeutic proteins in the liver would be beneficial for patients suffering from a wide variety of disorders. Vectors based on adeno-associated virus (AAV) are able to transduce quiescent hepatocytes in vivo and mediate long-term expression [1], [2]. Murine retroviruses can transduce hepatocytes stimulated by mitogens such as epidermal-, hepatocyte-, or keratinocyte growth factor [3], [4], [5], [6]. However, this transduction is inefficient and the high cost of growth factors makes it unattractive for human applications.

Vectors based on lentiviruses such as human immunodeficiency virus (HIV) are, unlike murine retroviral vectors, able to transduce quiescent cells [7], [8], [9]. This property makes lentiviral vectors attractive candidates for the development of gene transfer into cell types that are normally not dividing such as hepatocytes. The ability of lentiviral vectors to transduce quiescent hepatocytes in vivo is controversial; an initial report documented efficient transduction in vivo [10] but a more recent study showed that cell cycling of hepatocytes was required for efficient transduction [11]. No published studies on lentiviral transduction of isolated quiescent primary hepatocytes are available.

Recent studies show that the central polypurine tract (PPT) and termination sequence from HIV strongly improve vector performance [12], [13], especially the transduction of non-dividing cells. We therefore investigated whether inclusion of the HIV PPT increased transduction efficiency of primary quiescent rat hepatocytes.

Human hepatitis B virus (HBV) enhancers I and II are able to increase expression from heterologous promoters in an orientation independent and liver specific fashion [14], [15], [16]. This property has been exploited to construct liver specific promoter elements for gene therapy [17].

The HBV posttranscriptional regulatory element (PRE) is a cis acting element able to increase expression of transgenes when included in the transcribed region of the gene [18], [19], probably by stimulating export of unspliced mRNA from the nucleus [20].

We investigated whether the inclusion of HBV PRE plus enhancers I and II in a lentiviral vector was able to specifically increase expression in liver cells.

The PRE element contains the start codon and most of the coding region of the HBVx protein (HBVx) [21]. HBVx is essential for viral replication and is implicated in the development of hepatocellular carcinoma associated with HBV infection [21]. Because HBVx can transactivate heterologous promoters and carboxy terminally truncated HBVx mutants retain this transactivation function [21], a role of HBVx in the function of these elements must be excluded. We therefore also investigated whether the interruption of the HBVx reading frame would compromise the function of the HBV element.

Section snippets

Production of recombinant virus

To obtain a fragment from HBV containing the enhancers I, II and the PRE, a 1237 bp HBV fragment was amplified by PCR with the following primers: GACGGAAATTGCACCTGTA, CATGGTGCTGGTGCGCAGA. The fragment was sequenced and cloned as a SacI/SalI fragment containing 1142 bp HBV sequence (HBV subtype AYW accession J02203, bases 682–1818) into the lentiviral vector backbones pRRLpgkgfpsin (PGK) and pRRLcmvgfpsin (CMV) [22]. The lentiviral vectors pRRLpgkgfpsin and pRRLcmvgfpsin contain a

Construction of lentiviral vectors

Diagrams of the different lentiviral transfer vectors described in this study are shown in Fig. 1. Several studies have mapped the PRE and enhancer elements in the HBV genome, largely with identical results but with some differences in the 5′ and 3′ ends of the different subelements. For the initial studies, we therefore used a large HBV fragment, positions 682–1818 of HBV. Fig. 1 shows a schematic drawing of the positions of the HBV enhancers and PRE, in the fragment used in our study.

Discussion

Safe third generation lentiviral vectors were improved by introduction of HBV elements and the HIV PPT. These improvements were shown to be essential for the efficient transduction of quiescent hepatocytes.

Our results indicate that HBV elements increase expression levels of a transgene delivered by lentiviral vectors. The amount of transducing units per pg of HIV p24 is increased in vectors with the HBV element, this might be an important property for in vivo gene transfer because viral

Acknowledgements

We are grateful to Drs D. Trono and R. Zufferey (University of Geneva, Geneva, Switzerland) for providing us with the third generation lentiviral vector system. We wish to thank Dr B. Berkhout (Academic Medical Center, Amsterdam, the Netherlands) for SupT1 cells, for helpful discussions and for providing us with a fragment of the HIV clone LAI-1, Dr J. van Wamel (Academic Medical Center, Amsterdam, the Netherlands) for help with the p24 ELISA, Dr B. van Montfrans (Academic Medical Center,

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Lentiviral vectors for efficient transduction of isolated primary quiescent hepatocytes

Abstract

Introduction

Section snippets

Production of recombinant virus

Construction of lentiviral vectors

Discussion

Acknowledgements

Gastroenterology

Blood

Curr Opin Biotechnol

Cell

Biochim Biophys Acta

FEBS Lett

J Hepatol

Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors

Nat Med

Persistent expression of human clotting factor IX from mouse liver after intravenous injection of adeno-associated virus vectors

Proc Natl Acad Sci USA

Retrovirus-mediated transduction of adult hepatocytes

Proc Natl Acad Sci USA

Effects of keratinocyte and hepatocyte growth factor in vivo: implications for retrovirus-mediated gene transfer to liver

Hum Gene Ther

Hepatocyte growth factor induces hepatocyte proliferation in vivo and allows for efficient retroviral-mediated gene transfer in mice

Hepatology

Lentiviral vectors: turning a deadly foe into a therapeutic agent

Gene Ther

Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors

Nat Genet

Efficient lentiviral transduction of liver requires cell cycling in vivo

Nat Genet

Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences

Nat Genet

Hepatitis B virus (HBV) promoters are regulated by the HBV enhancer in a tissue-specific manner

J Virol