Internal ribosome entry site-based vectors for combined gene therapy

Abstract

Gene therapy appears as a promising strategy to treat incurable diseases. In particular, combined gene therapy has shown improved therapeutic efficiency. Internal ribosome entry sites (IRESs), RNA elements naturally present in the 5' untranslated regions of a few mRNAs, constitute a powerful tool to co-express several genes of interest. IRESs are translational enhancers allowing the translational machinery to start protein synthesis by internal initiation. This feature allowed the design of multi-cistronic vectors expressing several genes from a single mRNA. IRESs exhibit tissue specificity, and drive translation in stress conditions when the global cell translation is blocked, which renders them useful for gene transfer in hypoxic conditions occurring in ischemic diseases and cancer. IRES-based viral and non viral vectors have been used successfully in preclinical and clinical assays of combined gene therapy and resulted in therapeutic benefits for various pathologies including cancers, cardiovascular diseases and degenerative diseases.

Keywords: Gene therapy; Gene transfer; Internal ribosome entry site; Vector.

Figure 1

Cap-dependent and internal ribosome entry site-dependent initiation, two alternative mechanisms of translation. A: The so-called cap-dependent ribosome scanning mechanism predicts that ribosome 40S subunit binds to the mRNA 5’ end. Ribosome binding requires the initiation factor 4F (eIF-4F, composed of the three proteins eIF-4E, -4A and -4G). Then the mRNA is unwound under the control of the helicases eIF-4A and -4B, allowing the ribosome to scan the mRNA until recognition of an initiation codon (classically AUG)[11,12]; B: When an Internal ribosome entry site (IRES) is present in the mRNA 5’ untranslated region, IRES trans-acting factors (ITAFs) allow ribosome 40S internal recruitment, independently of the presence of cap and eIF-4F. The IRES-dependent mechanism occurs in the case of picornavirus uncapped mRNAs as well as for cellular capped mRNAs.

Figure 2

Internal ribosome entry site-based multicistronic vector concept. The internal ribosome entry site (IRES)-based expression cassette contains several genes, separated by IRESs, under the control of the same promoter (Pr). This transcription unit gives rise to a single mRNA coding the different genes. Translation initiation occurs at the 5’ end by the cap-dependent mechanism, resulting in translation of the first open reading frame (ORF, Gene A). Internal initiations of translation occur at each IRES, resulting in translation of the other ORFs (Genes B and C). Thus the multicistronic mRNA generates several proteins from a single transcription unit, allowing more stable long term expression and stable transgene ratio[9,48]. For each ORF, initiation (AUG) and termination (STOP) codons are indicated.