Bicistronic Construct Research Articles

62 POSTER Expression and functional activity of interleukin-8 receptors on human malignant melanoma cells

This study aims to determine the effects of curcumin and demethoxycurcumin on the internal ribosome entry site of the amyloid-β precursor protein (APP) and tau protein through a bi-cistronic reporter assay for screening of anti-Alzheimer's disease agents.A bi-cistronic assay was performed wherein the expression of the first cistron, a β-galactosidase gene under the control of a cytomegalovirus promoter, represents the canonical cap-dependent mechanism of translation initiation; while the second cistron involves the utilization of the APP or the tau IRES elements to drive the expression of secreted alkaline phosphatase (SEAP) under a cap-independent mechanism. Bioactive natural products reported to have therapeutic potential for AD such as curcumin and demethoxycurcumin were screened in an murine neuroblastoma (N2A) cell model. Western blot analyses for the expression of APP C-terminal protein, human tau-1, and phosphorylated tau at Serine 262 (p262) and Serine 396 (pS396) were done after treatment of N2A cells with the test compounds.The bi-cistronic reporter assay revealed that curcumin was more effective than demethoxycurcumin, a structural analog of curcumin, in inhibiting both APP and tau IRES-dependent translation initiation. This result was further confirmed by Western blot analysis for the expression of APP C-terminal protein, human tau-1, pS262 and pS396 suggesting that curcumin may play a role in AD pathology alleviation through the inhibition of the APP and tau IRES-mediated translation mechanism. On the other hand, demethoxycurcumin was observed to inhibit the phosphorylation of both tau pS262 and pS396.A novel assay system using the bi-cistronic reporter constructs for the identification of compounds with activity against the translation directed by APP and tau IRES was developed. The results provide novel suggestive insights for the potential use of the mentioned compounds as prophylactic and therapeutic anti-AD agents.

PKC phosphorylation modulates PKA-dependent binding of the R domain to other domains of CFTR

Activity of the CFTR channel is regulated by phosphorylation of its regulatory domain (RD). In a previous study, we developed a bicistronic construct called DeltaR-Split CFTR, which encodes the front and back halves of CFTR as separate polypeptides without the RD. These fragments assemble to form a constitutively active CFTR channel. Coexpression of the third fragment corresponding to the missing RD restores regulation by PKA, and this is associated with dramatically enhanced binding of the phosphorylated RD. In the present study, we examined the effect of PKC phosphorylation on this PKA-induced interaction. We report here that PKC alone enhanced association of the RD with DeltaR-Split CFTR and that binding was further enhanced when the RD was phosphorylated by both kinases. Mutation of all seven PKC consensus sequences on the RD (7CA-RD) did not affect its association under basal (unphosphorylated) conditions but abolished phosphorylation-induced binding by both kinases. Iodide efflux responses provided further support for the essential role of RD binding in channel regulation. The basal activity of DeltaR-Split/7CA-RD channels was similar to that of DeltaR-Split/wild type (WT)-RD channels, whereas cAMP-stimulated iodide efflux was greatly diminished by removal of the PKC sites, indicating that 7CA-RD binding maintains channels in an inactive state that is unresponsive to PKA. These results suggest a novel mechanism for CFTR regulation in which PKC modulates PKA-induced domain-domain interactions.

Use of the viral 2A peptide for bicistronic expression in transgenic mice.

BackgroundTransgenic animals are widely used in biomedical research and biotechnology. Multicistronic constructs, in which several proteins are encoded by a single messenger RNA, are commonly used in genetically engineered animals. This is currently done by using an internal ribosomal entry site to separate the different coding regions. 2A peptides result in the co-translational 'cleavage' of proteins and are an attractive alternative to the internal ribosomal entry site. They are more reliable than the internal ribosomal entry site and lead to expression of multiple cistrons at equimolar levels. They work in a wide variety of eukaryotic cells, but to date have not been demonstrated to function in transgenic mice in an inheritable manner.ResultsTo test 2A function in transgenic mice and uncover any possible toxicity of widespread expression of the 2A peptide, we made a bicistronic reporter construct containing the coding sequence for a membrane localised red fluorescent protein (Myr-TdTomato) and a nuclear localised green fluorescent protein (H2B-GFP), separated by a 2A sequence. When this reporter is transfected into HeLa cells, the two fluorescent proteins correctly localise to mutually exclusive cellular compartments, demonstrating that the bicistronic construct is a reliable readout of 2A function. The two fluorescent proteins also correctly localise when the reporter is electroporated into chick neural tube cells. We made two independent transgenic mouse lines that express the bicistronic reporter ubiquitously. For both lines, transgenic mice are born in Mendelian frequencies and are found to be healthy and fertile. Myr-TdTomato and H2B-GFP segregate to mutually exclusive cellular compartments in all tissues examined from a broad range of developmental stages, ranging from embryo to adult. One transgenic line shows X-linked inheritance of the transgene and mosaic expression in females but uniform expression in males, indicating that the transgene has integrated into the X chromosome in this line.ConclusionThe 2A peptide efficiently mediates co-translational cleavage in transgenic mice in which it has been inherited through the germ-line. Mice expressing it ubiquitously throughout development and into adulthood appear normal. It is therefore a viable tool for use in genetically engineered mice and represents a superior alternative to the widely used internal ribosomal entry site.

Internal translation initiation on the foot-and-mouth disease virus IRES is affected by ribosomal stalk conformation

A human cell line, in which expression of the ribosomal stalk proteins P1 and P2 has been suppressed by RNAi technology, has been used to test how the loss of these proteins affects IRES-dependent translation. Foot-and-mouth disease virus (FMDV) IRES-dependent translation from a bicistronic construct is about three fold higher in the P1/P2-depleted cells than in control cells in the presence of Lb protease. By contrast, no effect on Hepatitis C virus (HCV) IRES translation was observed. These results emphasize the functional heterogeneity of the IRES and they highlight a functional connection between the ribosomal stalk and picornavirus IRES-dependent translation.

The Mammalian Target of Rapamycin Complex 1 Regulates Leptin Biosynthesis in Adipocytes at the Level of Translation: The Role of the 5′-Untranslated Region in the Expression of Leptin Messenger Ribonucleic Acid

Leptin production by adipose cells in vivo is increased after feeding and decreased by food deprivation. However, molecular mechanisms that control leptin expression in response to food intake remain unknown. Here, we test the hypothesis that leptin expression in adipose cells is regulated by nutrient- and insulin-sensitive mammalian target of rapamycin complex 1 (mTORC1)-mediated pathway. The activity of mTORC1 in 3T3-L1 adipocytes was up-regulated by stable expression of either constitutively active Rheb or dominant-negative AMP-activated protein kinase. In both cases, expression of endogenous leptin was significantly elevated at the level of translation. To investigate the role of leptin 5'-untranslated region (UTR) in the regulation of protein expression, we created bicistronic reporter constructs with and without the 5'-UTR. We found that the presence of leptin 5'-UTR renders mRNA resistant to regulation by mTORC1. It appears, therefore, that mTORC1 controls translation of leptin mRNA via a novel mechanism that does not require the presence of either the 5'-terminal oligopyrimidine tract or the 5'-UTR.

Expression and purification of orphan cytochrome P450 4X1 and oxidation of anandamide

Cytochrome P450 (P450) 4X1 is one of the so-called 'orphan' P450s without an assigned biological function. Codon-optimized P450 4X1 and a number of N-terminal modified sequences were expressed in Escherichia coli. Native P450 4X1 showed a characteristic P450 spectrum but low expression in E. coli DH5alpha cells (< 100 nmol P450.L(-1)). The highest level of expression (300-450 nmol P450.L(-1) culture) was achieved with a bicistronic P450 4X1 construct (N-terminal MAKKTSSKGKL, change of E2A, amino acids 3-44 truncated). Anandamide (arachidonoyl ethanolamide) has emerged as an important signaling molecule in the neurovascular cascade. Recombinant P450 4X1 protein, co-expressed with human NADPH-P450 reductase in E. coli, was found to convert the natural endocannabinoid anandamide to a single monooxygenated product, 14,15-epoxyeicosatrienoic (EET) ethanolamide. A stable anandamide analog (CD-25) was also converted to a monooxygenated product. Arachidonic acid was oxidized more slowly to 14,15- and 8,9-EETs but only in the presence of cytochrome b(5). Other fatty acids were investigated as putative substrates but showed only little or minor oxidation. Real-time PCR analysis demonstrated extrahepatic mRNA expression, including several human brain structures (cerebellum, amygdala and basal ganglia), in addition to expression in human heart, liver, prostate and breast. The highest mRNA expression levels were detected in amygdala and skin. The ability of P450 4X1 to generate anandamide derivatives and the mRNA distribution pattern suggest a potential role for P450 4X1 in anandamide signaling in the brain.

Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: Eukaryotic clones containing two and three ORF multi-gene cassettes expressed from a single promoter

Two types of eukaryotic operon-type Expression clones were constructed using the Multisite Gateway system employing six types of att signals. These clones harbored a DNA cassette containing two heterologous ORFs (cDNAs) or three heterologous ORFs in tandem downstream of a single promoter. The most promoter-proximal ORF was translated via a Kozak signal and the downstream one or two ORF(s) were translated as directed by internal ribosome entry site(s) (IRES). These clones were observed to produce two or three different proteins at levels that depended on the activities of the translational initiation signals used. With the intention of modulating the expression level of the first ORF, the translational initiation signals including a Kozak sequence and 11 different IRESs were investigated for their efficiency using a single ORF. The translational activity of these signals varied within a 10-fold magnitude. Using these results, expression at pre-described relative levels was achieved from the optional IRES of the respective ORFs in the cassette. Controllable expression at desired levels of two different ORFs directed by optional IRESs on a bicistronic construct, transcribed from a single promoter, was demonstrated.

Alterations in RNA‐binding activities of IRES‐regulatory proteins as a mechanism for physiological variability and pathological dysregulation of IGF‐IR translational control in human breast tumor cells

The type I insulin-like growth factor receptor (IGF-IR) is integrally involved in the control of cellular proliferation and survival. An internal ribosomal entry site (IRES) within the 1,038 nucleotide 5'-untranslated region of the human IGF-IR mRNA helps to provide the tight control of IGF-IR expression necessary for maintenance of normal cellular and tissue homeostasis. The IRES maps to a discrete sequence of 85 nucleotides positioned just upstream of the IGF-IR initiation codon, allowing the ribosome to bypass the highly structured regions of the 5'-UTR as well as the upstream open reading frame. The authenticity of the IGF-IR IRES has been confirmed by its sensitivity to deletion of the promoter from a bicistronic reporter construct, and its resistance in a monocistronic reporter construct to co-expression of a viral 2A protease. We previously characterized HuR as a potent repressor of IGF-IR translation. Here we demonstrate that hnRNP C competes with HuR for binding the IGF-IR 5'-UTR and enhances IRES-mediated translation initiation in a concentration-dependent manner. We observed changes in binding of hnRNP C versus HuR to the IGF-IR 5'-UTR in response to physiological alterations in cellular environment or proliferative status. Furthermore, we have found distinct alterations in the pattern of protein binding to the IGF-IR 5'-UTR in human breast tumor cells in which IGF-IR IRES activity and relative translational efficiency are aberrantly increased. These results suggest that dysregulation of the IGF-IR IRES through changes in the activities of RNA-binding translation-regulatory proteins could be responsible for IGF-IR overexpression in a proportion of human breast tumors.

A gastrin transcript expressed in gastrointestinal cancer cells contains an internal ribosome entry site

As the hormone gastrin promotes gastrointestinal (GI) cancer progression by triggering survival pathways, regulation of gastrin expression at the translational level was explored. Sequence within the 5′ untranslated region of a gastrin transcript expressed in GI cancer cells was investigated, then cloned into a bicistronic vector upstream of firefly luciferase and transfected into a series of GI cancer cell lines. Firefly luciferase activity was measured relative to that of a cap-dependent Renilla luciferase. A gastrin transcript that was different from that described in Ensembl was expressed in GI cancer cells. Its transcription appears to be initiated within the region designated as the gene's first intron. In GI cancer cells transfected with the bicistronic construct, firefly luciferase activity increased 8–15-fold compared with the control vector, and there was a further induction of the signal (up to 25-fold) following exposure of the cells to genotoxic stress or hypoxia, suggesting that the sequence acts as an internal ribosome entry site. These data suggest that the gastrin transcript within GI cancer cells contains an internal ribosome entry site that may allow continued expression of gastrin peptides when normal translational mechanisms are inactive, such as in hypoxia, thereby promoting cancer cell survival.

Acetylsalicylic acid inhibits hepatitis C virus RNA and protein expression through cyclooxygenase 2 signaling pathways

It has been reported that salicylates (sodium salicylate and aspirin) inhibit the replication of flaviviruses, such as Japanese encephalitis virus and dengue virus. Therefore, we considered it important to test whether acetylsalicylic acid (ASA) had anti-hepatitis C virus (HCV) activity. To this end, we examined the effects of ASA on viral replication and protein expression, using an HCV subgenomic replicon cell culture system. We incubated Huh7 replicon cells with 2-8 mM ASA for different times and measured HCV-RNA and protein levels by northern blot, real-time polymerase chain reaction, and western analysis, respectively. We found that ASA had a suppressive effect on HCV-RNA and protein levels (nearly 58%). ASA-dependent inhibition of HCV expression was not mediated by the 5'-internal ribosome entry site or 3'-untranslated regions, as determined by transfection assays using bicistronic constructs containing these regulatory regions. However, we found that HCV-induced cyclooxygenase 2 (COX-2) messenger RNA and protein levels and activity and these effects were down-regulated by ASA, possibly by a nuclear factor kappa B-independent mechanism. We also observed that the ASA-dependent inhibition of viral replication was due in part to inhibition of COX-2 and activation of p38 and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) mitogen-activated protein kinases (MAPKs). Inhibition of these kinases by SB203580 and U0126, respectively, and by short interfering RNA silencing of p38 and MEK1 MAPK prevented the antiviral effect of ASA. Taken together, our findings suggest that the anti-HCV effect of ASA in the Huh7 replicon cells is due to its inhibitory effect on COX-2 expression, which is mediated in part by the activation of MEK1/2/p38 MAPK. These findings suggest the possibility that ASA could be an excellent adjuvant in the treatment of chronic HCV infection.

Immune responses induced by DNA vaccines encoding Newcastle virus haemagglutinin and/or fusion proteins in maternal antibody-positive commercial broiler chicken

1. The immune responses induced by recombinant plasmids containing Newcastle disease virus (NDV) F (pVAX.nd.f) or HN (pcDNA.nd.hn) genes separately or in combination in bi-cistronic (pIRES.nd.hn.f) constructs were evaluated in maternal antibody-positive commercial chicks. 2. Immunofluorescence and immunoperoxidase tests demonstrated the expression of both F and HN proteins in Vero cells. Real-time PCR analysis revealed the expression of HN and/or F genes in muscle, peripheral blood mononuclear cells (PBMC), spleen and liver after immunisation. 3. Chicks inoculated intramuscularly thrice (two booster doses) with pVAX.nd.f and pcDNA.nd.hn did not develop detectable haemagglutination inhibiting (HI) antibodies. In contrast, an increase in a NDV-specific cell-mediated immune response was demonstrated. 4. After challenge with virulent NDV, chicks immunised with the recombinant plasmids as well as those in control groups succumbed to Newcastle disease. 5. Based on these results, it is concluded that DNA vaccines containing HN and/or F genes fail to protect commercial chicks, possibly due to interference from maternal antibodies.

Single-Nucleotide-Specific siRNA Targeting in a Dominant-Negative Skin Model

RNA interference offers a novel approach for developing therapeutics for dominant-negative genetic disorders. The ability to inhibit expression of the mutant allele without affecting wild-type gene expression could be a powerful new treatment option. Targeting the single-nucleotide keratin 6a (K6a) N171K mutation responsible for the rare monogenic skin disorder pachyonychia congenita (PC), we demonstrate that small interfering RNAs (siRNAs) can potently and selectively block expression of mutant K6a. To test whether lead siRNAs could discriminate mutant mRNA in the presence of both wild-type and mutant forms, a dominant-negative PC cell culture model was developed. As predicted for a dominant-negative disease, simultaneous expression of both wild-type and mutant K6a resulted in defective keratin filament formation. Addition of mutant-specific siRNAs allowed normal filament formation, suggesting selective inhibition of mutant K6a. The effectiveness of our siRNA in skin was tested by co-delivering a firefly luciferase/mutant K6a bicistronic reporter construct and mutant-specific siRNAs to mouse footpads. Potent inhibition of the fluorescent reporter was demonstrated using the Xenogen IVIS200 in vivo imaging system. Additionally, wild type-specific siRNAs knocked down the expression of pre-existing endogenous K6a in human keratinocytes. These results suggest that efficient delivery of these "designer siRNAs" may allow effective treatment of numerous genetic disorders including PC.

A Dormant Internal Ribosome Entry Site Controls Translation of Feline Immunodeficiency Virus

The characterization of internal ribosome entry sites (IRESs) in virtually all lentiviruses prompted us to investigate the mechanism used by the feline immunodeficiency virus (FIV) to produce viral proteins. Various in vitro translation assays with mono- and bicistronic constructs revealed that translation of the FIV genomic RNA occurred both by a cap-dependent mechanism and by weak internal entry of the ribosomes. This weak IRES activity was confirmed in feline cells expressing bicistronic RNAs containing the FIV 5' untranslated region (UTR). Surprisingly, infection of feline cells with FIV, but not human immunodeficiency virus type 1, resulted in a great increase in FIV translation. Moreover, a change in the cellular physiological condition provoked by heat stress resulted in the specific stimulation of expression driven by the FIV 5' UTR while cap-dependent initiation was severely repressed. These results reveal the presence of a "dormant" IRES that becomes activated by viral infection and cellular stress.

RNAi-mediated abrogation of cathepsin B and MMP-9 gene expression in a malignant meningioma cell line leads to decreased tumor growth, invasion and angiogenesis

Malignant meningiomas are highly aggressive and frequently recur after surgical resection of the tumor. Earlier studies have reported that the cysteine protease cathepsin B and the matrix metalloproteinase MMP-9 play important roles in tumor progression. In the present study, we made an attempt to evaluate the roles of these proteases in the malignant meningioma tumor microenvironment and determined the effectiveness of using single or bicistronic siRNA constructs for cathepsin B and MMP-9, in both in vitro and in vivo models. Transfection of a plasmid vector expressing double-stranded RNA for cathepsin B and MMP-9 significantly inhibited mRNA and protein levels of cathepsin B and MMP-9. The migration and invasion of meningioma cells were decreased after treatment with single or bicistronic siRNA constructs for cathepsin B and MMP-9 compared to controls and vector controls. Inhibition of angiogenesis was observed when the cells were transfected with single or bicistronic constructs for cathepsin B and MMP-9, when compared to controls or empty vector controls. Our study revealed that abrogation of cathepsin B and MMP-9 expression decreased the activation of major proteins involved in MAP kinase and PI3 kinase pathways indicating that targeting these proteases may hinder intracellular signaling and thus decrease cell survival and proliferation in malignant meningiomas. In addition to the in vitro evidence, we observed a significant regression of pre-established orthotopic tumors after treatment with RNAi plasmid vectors targeting cathepsin B and MMP-9. Furthermore, these observations demonstrate that the simultaneous RNAi-mediated targeting of cathepsin B and MMP-9 has potential application for the treatment of human meningiomas.

Analysis of IgG heavy chain to light chain ratio with mutant Encephalomyocarditis virus internal ribosome entry site

Immunoglobulin G (IgG) is a heterotetrameric protein assembled from two identical heavy chain (HC) and two identical light chain (LC) polypeptides. The HC and LC folding and assembly are a crucial step for IgG production. It is affected by the ratio of HC to LC expression (HC:LC). To date, the HC:LC ratio was analysed mainly by cotransfection of different amounts of two monocistronic HC and LC expression plasmids, an approach biased by different transfection efficiencies. To circumvent this problem, a series of Encephalomyocarditis virus internal ribosome entry site (EMCV IRES) variants with different translation efficiencies were created and used to mediate HC translation in bicistronic constructs. HC and LC were translated from the same mRNA, which provides a more accurate method for the evaluation of the optimal ratio of HC:LC. The results show that the IgG optimal expression levels were obtained when the IRES mediated translation efficiency of the HC was about 50% compared to the cap-dependent translation of the LC. A surprisingly sharp transition to low production was shown when the ratios were below 40%. This study provides a new method to investigate the production of heterodimeric proteins in mammalian cells and adds understanding to the mechanisms of IgG folding and assembly.

Development of a bicistronic vector for multimodality imaging of estrogen receptor activity in a breast cancer model: preliminary application

The aim of this study was to develop a cellular model for the concurrent imaging of reporter genes expression by using positron emission tomography (PET) and bioluminescence imaging (BLI) for the assessment of estrogen receptor activity in vivo in a breast cancer model. Two reporters were chosen: a mutated form of the dopaminergic D2 receptor (D(2)R80A) for PET imaging, and the Firefly Luciferase for BLI. The presence of an IRES sequence between the two reporters ensured the coordinated expression driven by the same regulatory sequence containing an estrogen responsive element (ERE). To prevent chromatin effects on reporter expression, the construct was flanked by insulator sequences (Matrix Attachment Region, MAR). In vitro studies showed that the vector was efficient in coordinating the expression of the two genes. Moreover, stably transfected cells implanted in recipient animals maintained their capacity to express the reporters and react to systemic treatments permitting the in vivo study of ERs activity by PET and BLI imaging. In vitro expression analysis after long-term treatments showed different behaviour of the two reporter proteins in monitoring estrogen-dependent transcription outlining the importance of multi-reporter systems. With this model, PET and BLI can be applied to the concurrent evaluation of gene expression induced by estrogen and its analogues by using a bicistronic construct. The combined features of rapid, sensitive, sequential BLI and tomographic and quantitative PET imaging will allow the use of this strategy for the in vivo evaluation of molecular processes also for pharmacodynamic studies.

Minor contribution of an internal ribosome entry site in the 5′-UTR of ornithine decarboxylase mRNA on its translation

The mechanism of synthesis of ornithine decarboxylase (ODC) at the level of translation was studied using cell culture and cell-free systems. Synthesis of firefly luciferase (Fluc) from the second open reading frame (ORF) in a bicistronic construct transfected into FM3A and HeLa cells was enhanced by the presence of the 5′-untranslated region (5′-UTR) of ODC mRNA between the two ORFs. However, cotransfection of the gene encoding 2A protease inhibited the synthesis of Fluc. Synthesis of Fluc from the second cistron in the bicistronic mRNA in a cell-free system was not affected significantly by the 5′-UTR of ODC mRNA. Synthesis of ODC from ODC mRNA in a cell-free system was inhibited by 2A protease and cap analogue (m 7GpppG). Rapamycin inhibited ODC synthesis by 40–50% at both the G 1/S boundary and the G 2/M phase. These results indicate that an IRES in the 5′-UTR of ODC mRNA does not function effectively.

Ectropis obliqua picorna-like virus IRES-driven internal initiation of translation in cell systems derived from different origins

Ectropis obliqua picorna-like virus (EoPV) is an insect RNA virus that causes a lethal granulosis infection of larvae of the tea looper (Ectropis obliqua). An internal ribosome entry site (IRES) mediates translation initiation of EoPV RNA. Here, bicistronic constructs were used to examine the 5' untranslated region (UTR) of EoPV for IRES activity. The capacities of the EoPV 5' UTR IRES and another insect virus IRES, the cricket paralysis virus intergenic region IRES, to mediate internal translation initiation in a variety of translation systems were also compared. The results demonstrated that the EoPV IRES functioned efficiently not only in mammalian cell-derived systems, but also in an insect cell-derived translation system. However, it functioned inefficiently in a plant cell-derived translation system. This study reveals the host preferences of the EoPV IRES and important differences in IRES function between the EoPV IRES and other characterized picorna-like insect viral IRESs.

A Ferritin-responsive Internal Ribosome Entry Site Regulates Folate Metabolism

Cytoplasmic serine hydroxymethyltransferase (cSHMT) enzyme levels are elevated by the expression of the heavy chain ferritin (H ferritin) cDNA in cultured cells without corresponding changes in mRNA levels, resulting in enhanced folate-dependent de novo thymidylate biosynthesis and impaired homocysteine remethylation. In this study, the mechanism whereby H ferritin regulates cSHMT expression was determined. cSHMT translation is shown to be regulated by an H ferritin-responsive internal ribosome entry site (IRES) located within the cSHMT mRNA 5'-untranslated region (5'-UTR). The cSHMT 5'-UTR exhibited IRES activity during in vitro translation of bicistronic mRNA templates, and in MCF-7 and HeLa cells transfected with bicistronic mRNAs. IRES activity was depressed in H ferritin-deficient mouse embryonic fibroblasts and elevated in cells expressing the H ferritin cDNA. H ferritin was shown to interact with the mRNA-binding protein CUGBP1, a protein known to interact with the alpha and beta subunits of eukaryotic initiation factor eIF2. Small interference RNA-mediated depletion of CUGBP1 decreased IRES activity from bicistronic templates that included the cSHMT 3'-UTR in the bicistronic construct. The identification of this H ferritin-responsive IRES represents a mechanism that accounts for previous observations that H ferritin regulates folate metabolism.

Novel Adeno-Associated Virus Serotypes Efficiently Transduce Murine Photoreceptors

Severe inherited retinal diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are caused by mutations in genes preferentially expressed in photoreceptors. While adeno-associated virus (AAV)-mediated gene transfer can correct retinal pigment epithelium (RPE) defects in animal models, approaches for the correction of photoreceptor-specific diseases are less efficient. We evaluated the ability of novel AAV serotypes (AAV2/7, AAV2/8, AAV2/9, AAV2rh.43, AAV2rh.64R1, and AAV2hu.29R) in combination with constitutive or photoreceptor-specific promoters to improve photoreceptor transduction, a limiting step in photoreceptor rescue. Based on a qualitative analysis, all AAV serotypes tested efficiently transduce the RPE as well as rod and cone photoreceptors after subretinal administration in mice. Interestingly, AAV2/9 efficiently transduces Müller cells. To compare photoreceptor transduction from different AAVs and promoters in both a qualitative and quantitative manner, we designed a strategy based on the use of a bicistronic construct expressing both enhanced green fluorescent protein and luciferase. We found that AAV2/8 and AAV2/7 mediate six- to eightfold higher levels of in vivo photoreceptor transduction than AAV2/5, considered so far the most efficient AAV serotype for photoreceptor targeting. In addition, following subretinal administration of AAV, the rhodopsin promoter allows significantly higher levels of photoreceptor expression than the other ubiquitous or photoreceptor-specific promoters tested. Finally, we show that AAV2/7, AAV2/8, and AAV2/9 outperform AAV2/5 following ex vivo transduction of retinal progenitor cells differentiated into photoreceptors. We conclude that AAV2/7 or AAV2/8 and the rhodopsin promoter provide the highest levels of photoreceptor transduction both in and ex vivo and that this may overcome the limitation to therapeutic success observed so far in models of inherited severe photoreceptor diseases.