Comparative analysis of molecular strategies attenuating positional effects in lentiviral vectors carrying multiple genes
Introduction
Because of their ability to stably integrate proviral DNA sequences into the genome of host cells, lentivirus-based vectors are suitable tools for permanent genetic modification of cells and tissues. In addition, whereas vectors based on oncoretroviruses can only transduce dividing cells, lentiviral vectors can infect target cells independently of their proliferation status (Trono, 2000). Moreover, lentiviral vectors: (i) have a fairly large cloning capacity, which makes them attractive for simultaneous transduction of multiple genes; (ii) are compatible with pseudotyping strategies, which extend tropism and gene delivery potential; (iii) encode no viral proteins that may evoke an immune response; (iv) appear to be unaffected by gene-silencing alterations commonly occurring to transcription units transduced with murine oncoretroviruses (Svoboda et al., 2000). These optimal features make lentiviral vectors attractive gene transfer tools, targeting proliferating as well as non-dividing cells.
Viral vectors have been usually designed for the expression of a single open reading frame. However, for many gene delivery applications, the expression of multiple transgenes may be desirable. For example, the expression of a reporter gene in addition to the gene of interest is useful in the evaluation of viral titre and transduction efficiency (Reiser et al., 2000). Moreover, transduction of several genes from the same unit may be desirable either if the therapeutic protein consists of different subunits or if the therapeutic strategy is based on co-expression of several proteins for a combined/synergistic effect (Reiser et al., 2000). In fact, complex disorders such as cancer often involve mutations in multiple genes (e.g. oncogenes and tumor suppressor genes) and a combination strategy targeting the different defective genes simultaneously may be more effective. Likewise, the treatment of infectious diseases such as HIV-1 often involves the simultaneous blockade of multiple steps of the viral replication pathway to prevent the emergence of resistant strains of the virus (de Felipe, 2002). Multi-gene lentiviral vectors may also be very useful to co-express genes to reconstruct a metabolic pathway. By using a system based on the equine infectious anemia virus (EIAV), Azzouz et al. (2002) generated a multicistronic lentiviral vector containing cDNAs encoding tyrosine hydroxylase, Aromatic l-amino acid decarboxylase and GTP cyclohydrolase-I. This triple-gene vector efficiently transduced neurons in the striatum of 6-OHDA-lesioned rats; long-term expression of all transduced genes reconstituted a dopamine synthesis pathway in the transduced brain area, causing in turn a significant attenuation of the clinical signs in the 6-OHDA/apomorphine Parkinson disease model.
Efficient, high-level overexpression of multiple genes is a major concern in the construction of lentiviral vectors. It has been frequently observed that the DNA sequences subcloned at the 3′-end of a first transgene are poorly expressed in retroviral systems, i.e. the expression efficiency progressively decreases as we move from the 5′-end of the construct to the 3′-end. This phenomenon, known as positional effect, has been extensively described: Sadaie et al. (1998) showed that in lentiviral vectors the expression of a gene in a dual gene configuration depends on its position in the transcriptional unit; other studies reported that in a dual gene configuration the second protein is produced in a very low amount (about 1%) in relation to the first one (Dirks et al., 1993 and Mizuguchi et al., 2000).
Expression of different transgenes contained in a single construct can be achieved by putting each sequence under the transcriptional control of separate internal promoters. However, interference between promoters may affect gene expression (Kadesch and Berg, 1986, Proudfoot, 1986). Repression of a downstream 3′ unit by an upstream 5′ unit is known as transcriptional interference, whereas repression of a 5′ unit by a 3′ unit is defined as promoter suppression (Villemure et al., 2001). The causes of these phenomena appear to be manifold, depending on the vector design, and there is no univocal understanding of the molecular systems or mechanisms involved. Possible mechanisms include: (i) modification of DNA topology; in particular, the establishment of a transcriptional complex at one promoter may induce changes in supercoiling so that other adjacent transcriptional complexes are not efficiently formed (Hasegawa and Nakatsuji, 2002); (ii) competition for cis- or trans-acting factors, including enhancers or enhancer-binding proteins, or the general transcription apparatus (Eszterhas et al., 2002); (iii) readthrough phenomena, whereby RNA polymerase II complexes initiate transcription at the promoter of the upstream gene and subsequently read through the promoter of the downstream gene, altering the assembly of functional transcription complexes at the downstream promoter. This was demonstrated for the tandem HIV-1 promoters integrated into the genome of HeLa cells, whereby occlusion of the downstream promoter correlated with displacement of the Sp1 transcription factor – bound to the downstream promoter – by an incoming upstream elongation-competent polymerase complex (Greger et al., 1998). Interestingly, insertion between the tandem transcription units of a strong termination signal significantly attenuated transcriptional interference, resulting in elevated levels of transcription from the downstream promoter (Greger et al., 1998).
From these data it is evident that positional effects may hamper effective lentivector-mediated transduction of the transgenes of interest. Different molecular strategies have been adopted to counteract interference between adjacent transgenes in plasmid vectors. One way to prevent occlusion of a downstream promoter is through transcriptional termination (Eggermont and Proudfoot, 1993). It is also possible to create chimeric proteins by in-frame fusing two genes (cDNAs) to guarantee the simultaneous expression of both. Although functional proteins have been successfully created, problems due to peptide misfolding or mistargeting, and to the possibility of losing or lowering the activity of one or both fused proteins have to be taken into account (Thomas and Maule, 2000). Multi-gene lentiviral vectors can also be developed by using the natural splicing signals of HIV, by which multiple RNAs are produced from a single transcript (Zhu et al., 2001). However, this is not a frequently used strategy because of the difficulties in controlling the splicing mechanism. To overcome these shortcomings, bicistronic constructs can be generated. In such a setting, the two genes of interest are under the control of a common upstream promoter and processed as a single transcript. Translation of the two genes is uncoupled: translation initiation of the upstream gene is mediated by cap-dependent mechanisms while the translation initiation of the downstream gene is ensured by an internal ribosome entry site (IRES). Finally, by exploiting the ability of insulator elements to segregate repeated genes into domains that act independently of one another, it is possible to protect gene expression from transcriptional interference. Hasegawa and Nakatsuji (2002) demonstrated that two ORFs in a single construct were expressed in an independent manner and were controlled by their own regulatory elements only when insulators were placed at either sides of one of the two expression units.
Different types of HIV-1-based constructs for the concomitant expression of two foreign proteins were designed and compared to investigate the potential to efficiently express multiple transcriptional units in the context of lentiviral vectors.
Section snippets
Materials and methods
Unless otherwise specified, chemicals and reagents were purchased from Sigma (Milan, Italy).
Lentiviral vector construction
We designed different types of dualgene lentiviral vectors and performed a comparative analysis of these constructs, in order to identify the best strategy in ensuring optimal and simultaneous expression of two model proteins, the rat fatty acid amide hydrolase, and the green fluorescent protein. FAAH is the enzyme responsible for the hydrolysis of anandamide, an endocannabinoid, to arachidonic acid and ethanolamine. It is a membrane-bound protein, localized predominantly in microsomal or
Discussion
The insertion of two or more transgenes in single lentiviral constructs is becoming a commonly used strategy to simultaneously coexpress multiple gene products. However, one consequence of the tandem arrangement of genes within a viral vector is the complete or partial silencing of the sequences placed downstream of a first expression unit. In this study we have constructed and compared different dualgene lentiviral vectors for the concurrent expression of two model proteins (FAAH and GFP), to
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