Internal ribosome entry site-based vectors for combined gene therapy

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.