To express two or more proteins from a single viral or non-viral vector, multiple promoters or an internal ribosome entry site (IRES) sequence are commonly used to drive the individual genes. The use of two promoters within a single vector can result in promoter interference resulting in inefficient expression of both genes. If two genes are linked via an IRES sequence the expression level of the second gene may be significantly reduced (Furler et al., Gene Therapy 8:864-873 (2001)).
The linking of proteins in the form of polyproteins in a single open reading frame is a strategy adopted in the replication of many viruses including picornaviridae. Upon translation, virus-encoded proteinases mediate rapid intramolecular (cis) cleavages of a polyprotein to yield discrete mature protein products. Foot and Mouth Disease viruses (FMDV) are a group within the picornaviridae which express a single, long open reading frame encoding a polyprotein of approximately 225 kD. The full length translation product undergoes rapid intramolecular (cis) cleavage at the C-terminus of a 2A region occurring between the capsid protein precursor (P1-2A) and replicative domains of the polyprotein 2BC and P3, and this cleavage is mediated by proteinase-like activity of the 2A region itself (Ryan et al., J. Gen. Virol. 72:2727-2732 (1991); Vakharia et al., J. Virol. 61:3199-3207 (1987)). Ryan designed constructs identifying the essential amino acid residues for expression of the cleavage activity by the FMDV 2A region. 2A domains have also been characterized from aphthoviridea and cardioviridae of the picornavirus family (Donnelly et al., J. Gen. Virol. 78:13-21 (1997).
The mechanism of action for 2A may involve ribosomal skipping between codons which prevents formation of peptide bonds (de Felipe et al., Human Gene Therapy 11:1921-1931 (2000); Donnelly et al., J. Gen. Virol. 82:1013-1025 (2001); although it has been considered that the domain acts more like an autolytic enzyme (Ryan et al., Virol. 173:35-45 (1989)). Nevertheless, the utility of a 2A domain single gene vector construct for expression of full length polypeptides including antibodies or heterodimeric proteins can be fully appreciated from the embodiments described hereinafter.
Studies in which the FMDV 2A coding region was cloned into expression vectors and transfected into target cells have established that FMDV 2A cleavage of artificial reporter polyproteins is efficient in a broad range of heterologous expression systems (wheat-germ lysate and transgenic tobacco plant (Halpin et al., U.S. Pat. No. 5,846,767 (1998) and Halpin et al., The Plant Journal 17:453-459 (1999)); Hs 683 human glioma cell line (de Felipe et al., Gene Therapy 6:198-208 (1999); hereinafter referred to as “de Felipe II”); rabbit reticulocyte lysate and human HTK-143 cells (Ryan et al., EMBO J. 13:928-933 (1994)); and insect cells (Roosien et al., J. Gen. Virol. 71:1703-1711 (1990)). The FMDV 2A-mediated cleavage of a heterologous polyprotein for a biologically relevant molecule has been shown for IL-12 (p40/p35 heterodimer; Chaplin et al., J. Interferon Cytokine Res. 19:235-241 (1999). In transfected COS-7 cells, FMDV 2A mediated the cleavage of a p40-2A-p35 polyprotein into biologically functional subunits p40 and p35 having activities associated with IL-12.
In a recent report, the FMDV 2A sequence was incorporated into retroviral vectors, alone or combined with different IRES sequences to construct bicistronic, tricistronic and tetracistronic retroviral vectors. These vectors were shown to express drug resistance and reporter genes in producer cell lines and infected cultured fibroblasts. To test the efficiency of 2A-mediated gene expression in animals and the potential for the vector constructs for in vivo gene therapy, Furler (2001) generated recombinant adeno-associated viral (AAV) genomes encoding α-synuclein and EGFP or Cu/Zn superoxide dismutase (SOD-1) and EGFP linked via the FMDV 2A sequence. EGFP and α-synuclein were expressed at substantially higher levels from 2A vectors than from corresponding IRES-based vectors, while SOD-1 was expressed at comparable or slightly higher levels. Furler also demonstrated that the 2A sequence results in bicistronic gene expression in vivo after injection of 2A-dependent AAVs into rat substantia nigra.
Previous attempts to express a full length antibody/immunoglobulin molecule using a single vector have met with limited success, typically resulting in unequal levels of expression of the heavy and light chains of the antibody/immunoglobulin molecule, and more particularly, a lower level of expression for the second gene. In order to express a fully biological functional antibody from a single vector, equimolar expression of the heavy and light chains is required. Additionally, conventional vectors relying on dual promoter regulation of gene expression are invariably affected by promoter interaction (ie, promoter interference) which may compromise equimolar expression of the genes. Other factors that limit the ability to express two or more coding sequences from a single vector include the packaging limitation of the vector itself. For example, in considering the appropriate vector/coding sequence, factors to be considered include: the capacity of the vector (e.g., approx. 4,500 bp for AAV); the duration of expression of the recombinant molecule by a vector-transfected cell (e.g., short term expression for adenoviral vectors); the cell types infected by the vector if a viral vector is used; and the desired expression level of the target gene product(s). The requirement for controlled expression of two or more gene products together with the packaging limitations of viral vectors such as adenovirus and AAV, limits the choices with respect to vector construction and systems for expression of immunoglobulins or fragments thereof.
Accordingly, there remains a need for improved gene expression systems in the context of expression of immunoglobulins or fragments thereof which correct for the deficiencies inherent in currently available technology (e.g., the use of an IRES). The present invention addresses this need.
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