Luciferase mRNA Research Articles

Selenium nanoparticles (SeNPs) have potent antitumor activity against prostate cancer cells through the upregulation of miR-16

ObjectivesThis research aimed to examine the antitumor mechanisms of selenium nanoparticles (SeNPs) specifically against prostate cancers.MethodsThe antitumor activities of SeNPs against cancer cells were determined via MTT assay. The cell cycle was determined by detecting the DNA content, and apoptosis was determined via annexin V-Fluos staining kit. The microRNA expressions in cancer cells were analyzed via microarray and qRT-PCR. The potential targets of miR-16 were identified via luciferase analysis and mRNA expression determination. miR-16 functions in cancer cells were explored via the transient transfection of miR-16 mimic or inhibitor.ResultsSeNPs were most potent in prostate cancer cells, regardless of whether or not they were androgen-dependent. Furthermore, SeNP stimulation can induce cell cycle arrest and the apoptosis enhancement of prostate cancer cells. Microarray and molecular mechanism studies demonstrated that miR-16 could directly target cyclin D1 and BCL-2 to mediate SeNP apoptosis enhancement. Results show that the serum selenium levels positively correlate with miR-16 expressions, and they correlate with the overall and disease-free survival rates.ConclusionThese results signify the cytotoxic potential of SeNPs in prostate cancer treatment.

Treatment of Hemophilia A Using Factor VIII Messenger RNA Lipid Nanoparticles

Hemophilia A (HemA) patients are currently treated with costly and inconvenient replacement therapy of short-lived factor VIII (FVIII) protein. Development of lipid nanoparticle (LNP)-encapsulated mRNA encoding FVIII can change this paradigm. LNP technology constitutes a biocompatible and scalable system to efficiently package and deliver mRNA to the target site. Mice intravenously infused with the luciferase mRNA LNPs showed luminescence signals predominantly in the liver 4 h after injection. Repeated injections of LNPs did not induce elevation of liver transaminases. We next injected LNPs carrying mRNAs encoding different variants of human FVIII (F8 LNPs) into HemA mice. A single injection of B domain-deleted F8 LNPs using different dosing regimens achieved a wide range of therapeutic activities rapidly, which can be beneficial for various usages in hemophilia treatment. The expression slowly declined yet remained above therapeutic levels up to 5–7 days post-injection. Furthermore, routine repeated injections of F8 LNPs in immunodeficient mice produced consistent expression of FVIII over time. In conclusion, F8 LNP treatment produced rapid and prolonged duration of FVIII expression that could be applied to prophylactic treatment and potentially various other treatment options. Our study showed potential for a safe and effective platform of new mRNA therapies for HemA.

Functional Analysis of RNA Motifs Essential for BC200 RNA-mediated Translational Regulation

Brain cytoplasmic 200 RNA (BC200 RNA) is proposed to act as a local translational modulator by inhibiting translation after being targeted to neuronal dendrites. However, the mechanism by which BC200 RNA inhibits translation is not fully understood. Although a detailed functional analysis of RNA motifs is essential for understanding the BC200 RNA-mediated translation-inhibition mechanism, there is little relevant research on the subject. Here, we performed a systematic domain-dissection analysis of BC200 RNA to identify functional RNA motifs responsible for its translational-inhibition activity. Various RNA variants were assayed for their ability to inhibit translation of luciferase mRNA in vitro. We found that the 111–200-nucleotide region consisting of part of the Alu domain as well as the A/C-rich domain (consisting of both the A-rich and C-rich domains) is most effective for translation inhibition. Surprisingly, we also found that individual A-rich, A/C-rich, and Alu domains can enhance translation but at different levels for each domain, and that these enhancing effects manifest as cap-dependent translation.

An Upstream Open Reading Frame Represses Translation of Chicken PPARγ Transcript Variant 1.

Peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of adipogenesis. The PPARγ gene produces various transcripts with different 5′-untranslated regions (5′ UTRs) because of alternative promoter usage and splicing. The 5′ UTR plays important roles in posttranscriptional gene regulation. However, to date, the regulatory role and underlying mechanism of 5′ UTRs in the posttranscriptional regulation of PPARγ expression remain largely unclear. In this study, we investigated the effects of 5′ UTRs on posttranscriptional regulation using reporter assays. Our results showed that the five PPARγ 5′ UTRs exerted different effects on reporter gene activity. Bioinformatics analysis showed that chicken PPARγ transcript 1 (PPARγ1) possessed an upstream open reading frame (uORF) in its 5′ UTR. Mutation analysis showed that a mutation in the uORF led to increased Renilla luciferase activity and PPARγ protein expression, but decreased Renilla luciferase and PPARγ1 mRNA expression. mRNA stability analysis using real-time RT-PCR showed that the uORF mutation did not interfere with mRNA stability, but promoter activity analysis of the cloned 5′ UTR showed that the uORF mutation reduced promoter activity. Furthermore, in vitro transcription/translation assays demonstrated that the uORF mutation markedly increased the translation of PPARγ1 mRNA. Collectively, our results indicate that the uORF represses the translation of chicken PPARγ1 mRNA.

Ionizable Lipid Nanoparticle-Mediated mRNA Delivery for Human CAR T Cell Engineering.

Chimeric antigen receptor (CAR) T cell therapy relies on the ex vivo manipulation of patient T cells to create potent, cancer-targeting therapies, shown to be capable of inducing remission in patients with acute lymphoblastic leukemia and large B cell lymphoma. However, current CAR T cell engineering methods use viral delivery vectors, which induce permanent CAR expression and could lead to severe adverse effects. Messenger RNA (mRNA) has been explored as a promising strategy for inducing transient CAR expression in T cells to mitigate the adverse effects associated with viral vectors, but it most commonly requires electroporation for T cell mRNA delivery, which can be cytotoxic. Here, ionizable lipid nanoparticles (LNPs) were designed for ex vivo mRNA delivery to human T cells. A library of 24 ionizable lipids was synthesized, formulated into LNPs, and screened for luciferase mRNA delivery to Jurkat cells, revealing seven formulations capable of enhanced mRNA delivery over lipofectamine. The top-performing LNP formulation, C14-4, was selected for CAR mRNA delivery to primary human T cells. This platform induced CAR expression at levels equivalent to electroporation, with substantially reduced cytotoxicity. CAR T cells engineered via C14-4 LNP treatment were then compared to electroporated CAR T cells in a coculture assay with Nalm-6 acute lymphoblastic leukemia cells, and both CAR T cell engineering methods elicited potent cancer-killing activity. These results demonstrate the ability of LNPs to deliver mRNA to primary human T cells to induce functional protein expression, and indicate the potential of LNPs to enhance mRNA-based CAR T cell engineering methods.

Serotonin transporter untranslated regions influence mRNA abundance and protein expression

The serotonin transporter (SERT, SLC6A4) is a Na+-dependent transporter that regulates the availability of serotonin (5-HT, 5-hydroxytryptamine), a key neurotransmitter and hormone in the brain and the intestine. The human SERT gene consists of two alternate promoters that drive expression of an identical SERT protein. However, there are different mRNA transcript variants derived from these two promoters that differ in their 5′ untranslated region (5′UTR), which is the region of the mRNA upstream from the protein-coding region. Two of these transcripts contain exon-1a and are abundant in neuronal tissue, whereas the third transcript contains exon-1c and is abundant in the intestine. The 3′UTR is nearly identical among the transcripts. Current studies tested the hypothesis that the UTRs of SERT influence its expression in intestinal epithelial cells (IECs) by controlling mRNA or protein levels. The SERT UTRs were cloned into luciferase reporter plasmids and luciferase mRNA and activity were measured following transient transfection of the UTR constructs into the model IEC Caco-2. Luciferase activity and mRNA abundance were higher than the empty vector for two of the three 5′UTR variants. Calculation of translation index (luciferase activity divided by the relative luciferase mRNA level) revealed that the exon-1a containing 5′UTRs had enhanced translation when compared to the exon-1c containing 5′UTR which exhibited a low translation efficiency. Compared to the empty vector, the SERT 3′UTR markedly decreased luciferase activity. In silico analysis of the SERT 3′UTR revealed many conserved potential miRNA binding sites that may be responsible for this decrease. In conclusion, we have shown that the UTRs of SERT regulate mRNA abundance and protein expression. Delineating the molecular basis by which the UTRs of SERT influence its expression will lead to an increased understanding of post-transcriptional regulation of SERT in GI disorders associated with altered 5-HT availability.

Effective mRNA pulmonary delivery by dry powder formulation of PEGylated synthetic KL4 peptide

Pulmonary delivery of messenger RNA (mRNA) has considerable potential as therapy or vaccine for a range of lung diseases. Inhaled dry powder formulation of mRNA is particularly attractive as it has superior stability and dry powder inhaler is relatively easy to use. A safe and effective mRNA delivery vector as well as a suitable particle engineering method are required to produce a dry powder formulation that is respirable and mediates robust transfection in the lung. Here, we introduce a novel RNA delivery vector, PEG12KL4, in which the synthetic cationic KL4 peptide is attached to a monodisperse linear PEG of 12-mers. The PEG12KL4 formed nano-sized complexes with mRNA at 10:1 ratio (w/w) and mediated effective transfection on human lung epithelial cells. PEG12KL4/mRNA complexes were successfully formulated into dry powder by spray drying (SD) and spray freeze drying (SFD) techniques. Both SD and SFD powder exhibited satisfactory aerosol properties for inhalation. More importantly, the biological activity of the PEG12KL4 /mRNA complexes were successfully preserved after drying. Using luciferase mRNA, the intratracheal administration of the liquid or powder aerosol of PEG12KL4 /mRNA complexes at a dose of 5μg mRNA resulted in luciferase expression in the deep lung region of mice 24h post-transfection. The transfection efficiency was superior to naked mRNA or lipoplexes (Lipofectamine 2000), in which luciferase expression was weaker and restricted to the tracheal region only. There was no sign of inflammatory response or toxicity of the PEG12KL4 /mRNA complexes after single intratracheal administration. Overall, PEG12KL4 is an excellent mRNA transfection agent for pulmonary delivery. This is also the first study that successfully demonstrates the preparation of inhalable dry powder mRNA formulations with in vivo transfection efficiency, showing the great promise of PEG12KL4 peptide as a mRNA delivery vector candidate for clinical applications.

Ethylcellulose nanoparticles as a new “in vitro” transfection tool for antisense oligonucleotide delivery

Oil-in-water nano-emulsions have been obtained in the HEPES 20 mM buffer solution / [Alkylamidoammonium:Kolliphor EL = 1:1] / [6 wt% ethylcellulose in ethyl acetate] system over a wide oil-to-surfactant range and above 35 wt% aqueous component at 25 °C. The nano-emulsion with an oil-to-surfactant ratio of 70/30 and 95 wt% aqueous component was used for nanoparticles preparation. These nanoparticles (mean diameter around 90 nm and zeta potential of +22 mV) were non-toxic to HeLa cells up to a concentration of 3 mM of cationic species. Successful complexation with an antisense phosphorothioate oligonucleotide targeting Renilla luciferase mRNA was achieved at cationic/anionic charge ratios above 16, as confirmed by zeta potential measurements and an electrophoretic mobility shift assay, provided that no Fetal Bovine Serum is present in the cell culture medium. Importantly, Renilla luciferase gene inhibition shows an optimum efficiency (40%) for the cationic/anionic ratio 28, which makes these complexes promising for “in vitro” cell transfection.

Messenger RNA translation enhancement by immune evasion proteins: a comparative study between EKB (vaccinia virus) and NS1 (influenza A virus)

In this study, we compared vaccinia virus derived monofunctional E3, K3 and B18R (also known as EKB) with influenza A virus derived multifunctional non-structural protein 1 (NS1) based on their ability to enhance mRNA translation. EKB and NS1-TX91 were all found to enhance mRNA translation and suppress interferon production, yet level of enhancement by EKB was much lower than NS1-TX91. Similarly, greater luciferase expression was mediated by co-delivery of unmodified luciferase with NS1 mRNA, compared to co-delivery of unmodified luciferase with either E3, K3 or B18R mRNA, respectively. Different combinations of E3, K3 and/or B18R mRNA were mixed with NS1-TX91 mRNA at varying ratios and co-delivered with luciferase mRNA. However, no synergism was observed as mRNA translation enhancement mediated by NS1-TX91 could not be improved by the inclusion EKB in all tested combinations. Lastly, it was found that E3 was able to rescue mRNA translation enhancement mediated by NS1 PKR knockout mutant (PR8PKR−), suggesting that one of NS1’s multiple immune evasion mechanisms overlapped with E3. Altogether, our data validated mRNA translation enhancement mediated by immune evasion proteins (EKB and NS1) and showed that the multifunctional nature of NS1 accounted for its superior performance.

IDH1 fine-tunes cap-dependent translation initiation.

The metabolic enzyme isocitrate dehydrogenase 1 (IDH1) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). Its mutation often leads to aberrant gene expression in cancer. IDH1 was reported to bind thousands of RNA transcripts in a sequence-dependent manner; yet, the functional significance of this RNA-binding activity remains elusive. Here, we report that IDH1 promotes mRNA translation via direct associations with polysome mRNA and translation machinery. Comprehensive proteomic analysis in embryonic stem cells (ESCs) revealed striking enrichment of ribosomal proteins and translation regulators in IDH1-bound protein interactomes. We performed ribosomal profiling and analyzed mRNA transcripts that are associated with actively translating polysomes. Interestingly, knockout of IDH1 in ESCs led to significant downregulation of polysome-bound mRNA in IDH1 targets and subtle upregulation of ribosome densities at the start codon, indicating inefficient translation initiation upon loss of IDH1. Tethering IDH1 to a luciferase mRNA via the MS2-MBP system promotes luciferase translation, independently of the catalytic activity of IDH1. Intriguingly, IDH1 fails to enhance luciferase translation driven by an internal ribosome entry site. Together, these results reveal an unforeseen role of IDH1 in fine-tuning cap-dependent translation via the initiation step.

Study of the Inducible Cross-Linking Reaction to mRNA and the Effect on the Translation.

The 4-vinylpyrimidin-2-one nucleoside (T-vinyl) forms a cross-link with the RNA containing uracil at the complementary site at a high reaction rate. To obtain the stable T-vinyl derivative so that its reactivity is protected until it access to the target site, several derivatives were investigated, and the 2-thiopyridinyl- and 2-thiopyrimidinyl T-vinyl derivatives were determined to be good candidates. The 2-thiopyrimidinyl T-vinyl derivative was found to more efficiently cross-link with mRNA albeit having a better stability than the 2-thiopyridinyl T-vinyl derivative. The investigation using the luciferase (Luc) mRNA, the synthetic mRNA and non-cellular translation system revealed that the translation is terminated at the end of the cross-linked duplex between the mRNA and the oligoribonucleotide (ORN). Thus, the 2-thiopyrimidinyl T-vinyl derivative has successfully demonstrated both a good stability and high efficiency for the cross-linking reaction, and expanded its applicability in biological applications.

Abstract 3009: FGFR1 overexpression ER positive breast cancer cells confer the cells resistant to Palbociclib via upregulation of RTK-ER signaling

Abstract Palbociclib is the first CDK4/6 inhibitor approved by the FDA for treating ER+/HER2- breast cancer. Palbociclib resistance is emerging as a clinical challenge. Identification of the factors that determine the sensitivity to Palbociclib is of pivotal significance. Fibroblast Growth Factor Receptor (FGFR) overexpression is frequently detected in breast cancer, which has been associated with poor prognosis. To investigate the impact of FGFR overexpression on Palbociclib resistance, we first established stable sublines of MCF7 and T47D cells that overexpress FGFR1, and examined their responses to Palbociclib. Data from MTT and clonogenic assays demonstrated that overexpression of FGFR1 rendered ER+ MCF7 and T47D cells resistant to Palbociclib significantly. Cell cycle analysis showed that the percentage of cells in S phase in Palbociclib treated MCF7/FGFR1 and T47D/FGFR1 cells was significantly higher than that of the control. FGFR overexpression also significantly attenuated Palbociclib-associated inhibition of cancer cell invasion. We further found that Palbociclib-induced inhibition of cancer cell stemness, as indicated by colony formation in soft agar and mammosphere assays, was significantly reduced in MCF7/FGFR1 and T47D/FGFR1 cells, as compared to control. The data indicate that FGFR1 overexpression in ER+ breast cancer cells are resistant to Palbociclib. We next examined the signaling of the key pathways in the paired cell lines treated with Palbociclib. We found that Palbociclib-induced inhibition of phospho-Rb, E2F, cyclin D3, cyclin B1, cdc-2, c-myc, and cdc-25 was significantly attenuated in MCF-7/FGFR1 and T47D/FGFR1 cells. The protein levels of p-ERK, p-AKT, and p-p38 in MCF-7/FGFR1 and T47D/FGFR1 cells were significantly higher than the control cells with/without Palbociclib treatment. The results also showed that ER alpha phosphorylation, estrogen response element (ERE)-luciferase activity and mRNA levels of ER target genes, including PS2, c-myc, E2F, FGF2 and FGFR2, in MCF-7/FGFR1 and T47D/FGFR1 cells were maintained at higher levels post Palbociclib, as compared to control. We then treated control and FGFR1 overexpressing MCF-7 and T47D cells with Palbociclib in combination with FGFR inhibitor, AZD 4547. The combined drug treatment resulted in remarkably synergistic effect on MCF-7/FGFR1 and T47D/FGFR1 cells, as indicated by MTT and clonogenic assays. Key markers in cell cycle regulation, RTK signaling and ER pathway were significantly inhibited in the cells treated with both Palbociclib and AZD 4547. Taken together, our results demonstrated that FGFR1 overexpression is a critical factor affecting Palbociclib sensitivity. The underlying mechanisms involve FGFR1 overexpression-associated RTK and ER signaling. Combination of Palbociclib with FGFR targeting agents may overcome FGFR-associated Palbociclib resistance. Citation Format: Yujie Shi, Zhikun Ma, Yudan Wu, Amanda B. Parris, Qiong Cheng, Lingfei Kong, Xiaohe Yang. FGFR1 overexpression ER positive breast cancer cells confer the cells resistant to Palbociclib via upregulation of RTK-ER signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3009.

Abstract 1461: mRNA therapeutics against cancers and infectious diseases

Abstract In vitro transcribed (IVT) mRNA is forming a new basis of drug delivery as it can be synthetically engineered to express the desired protein in lesser time and be more specific than the other 2 types of antigens. Using DNA (coding for proto-oncogenes) for immunization poses a risk for host cell transformation into a malignant one and hence proto-oncogenes would express for a longer time. Therefore, mRNA was considered for cancer immunotherapy as it does not integrate into the host cell's genome, so no question of insertional mutagenesis arises. Our main aim is to develop a stable mRNA system working both in vitro and in vivo and then use this system to develop mRNA vaccines. The stability of IVT mRNA can be increased by changing the 3' and 5' UTRs, adding a 5'cap and 3' poly-A tail which could even increase their half-life to a few days. For creating a stable mRNA working system in vitro, EGFP-2A gene was cloned along with the AdsA (Staphylococcus aureusgene important for infection) in a vector backbone with TMV 3’UTR and 5’UTR. The mRNA created in vitro was with modified uridine and cap 1 structure and transfected to the mammalian cell lines. This transfection was further enhanced by using liposome created in the lab (using some cationic and helper lipids in different ratios) by film rehydration method. The expression of luciferase mRNA was then detected in mice by live imaging indicating that the mRNA system is stable in vivo. Further, to direct the mRNA encoded antigens to MHC1 and MHC2 pathways they were tagged with a htPA secretion tag and MHC1 targeting signal along with a 3XFLAG tag. We tried various mode of injections with AdsA mRNA to have a profile of ELISPOT with differently exposed mRNA (in vivo in mice). Now we are focussing on some neo antigens for tumor to be used as. Avaccine in the form of mRNA. Citation Format: Smriti Arya, Qiubin Lin, Nan Zhou, Jiandong Huang. mRNA therapeutics against cancers and infectious diseases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1461.

Chitinase-3-like-1 deficiency attenuates ethanol-induced liver injury by inhibition of sterol regulatory element binding protein 1-dependent triglyceride synthesis

ObjectiveAlcohol overconsumption and abuse lead to alcoholic liver disease (ALD), which is a major chronic liver disease worldwide. Chitinase-3-like protein 1 (CHI3L1) have an important role in the pathogenesis of inflammatory disease. However, the role of CHI3L1 in ALD has not yet been reported. In the present study, we investigated the effect of CHI3L1 on chronic plus binge ethanol-induced liver injury. MethodsCHI3L1 knock out (KO) mice and their littermate control mice based on C57BL/6 (10–12 weeks old) were fed on a Lieber-DeCarli diet containing 6.6% ethanol for 10 days. And, CHI3L1 siRNA or CHI3L1 expressing vector was transfected HepG2 cells were treated with ethanol or without. ResultsEthanol-induced hepatic triglyceride (TG) levels and the mRNA levels of TG synthesis-related genes such as acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 (SCD1) were decreased in the liver of CHI3L1 knock out (KO) mice and the HepG2 cells transfected with CHI3L1 siRNA. Increased mRNA level and activation of SREBP1 which is transcription factor of ACC, FAS and SCD1 by ethanol feeding were reduced in the liver of ethanol-fed CHI3L1 KO mice. Moreover, ethanol-induced SREBP1 luciferase activity and mRNA level of SREBP1, ACC, FAS and SCD1 were also decreased in the HepG2 cells transfected with CHI3L1 siRNA, while those were further increased in the HepG2 cells treated with recombinant human CHI3L1. Furthermore, oxidative stress and up-regulated pro-inflammatory cytokines by ethanol were recovered in the liver of ethanol-fed CHI3L1 KO mice. ConclusionOur finding suggest that inhibition of CHI3L1 suppressed ethanol-induced liver injury through inhibition of TG synthesis, and the blocking of oxidative stress and hepatic inflammation induced SREBP1 activity could be significant.

Preparation of Messenger RNA Nanomicelles via Non-Cytotoxic PEG-Polyamine Nanocomplex for Intracerebroventicular Delivery: A Proof-of-Concept Study in Mouse Models.

The specific delivery of messenger RNA (mRNA) is an excellent alternative to plasmid DNA, due to the latter’s potential risk for random integration into the host genome. In this study, we propose the use of specially tailored polyplex nanomicelles for the intravenous delivery of mRNA into the brain of mice. In brief, along the backbone of a polyaspartamide polymer that is terminated with a 42k Polyethylene glycol chain (PEG), aminoethylene-repeating groups (two, three, and four units, respectively) were conjugated to side-chains to promote electrostatic interactions with mRNA. This structural configuration would ultimately condense into a polyplex nanomicelle ranging between 24 and 34 nm, as was confirmed by transmission electron microscopy (TEM) and dynamic light scattering (DLS) while the chemistry of the synthesis was validated through NMR analysis. Subsequently, we hypothesized an important correlation pertaining to the role of hydrogen bonding between the interaction of polyamine and mRNA in due course. As a proof of concept, we encapsulated the luciferase (Luc2) mRNA as a reporter gene through in vitro transcription (IVT) and subsequently infused the polyplex nanomicelles into mouse brains via an intracerebroventricular (ICV) injection to bypass the blood–brain barriers (BBB). Data revealed that PEGylated polyplex nanomicelles possessing four repeating units of aminoethylene groups had exhibited the best Luc2 mRNA delivery efficiency with no significant immune response registered.

Inhaled Nanoformulated mRNA Polyplexes for Protein Production in Lung Epithelium.

Noninvasive aerosol inhalation is an established method of drug delivery to the lung, and remains a desirable route for nucleic-acid-based therapeutics. In vitro transcribed (IVT) mRNA has broad therapeutic applicability as it permits temporal and dose-dependent control of encoded protein expression. Inhaled delivery of IVT-mRNA has not yet been demonstrated and requires development of safe and effective materials. To meet this need, hyperbranched poly(beta amino esters) (hPBAEs) are synthesized to enable nanoformulation of stable and concentrated polyplexes suitable for inhalation. This strategy achieves uniform distribution of luciferase mRNA throughout all five lobes of the lung and produces 101.2 ng g-1 of luciferase protein 24 h after inhalation of hPBAE polyplexes. Importantly, delivery is localized to the lung, and no luminescence is observed in other tissues. Furthermore, using an Ai14 reporter mouse model it is identified that 24.6% of the total lung epithelial cell population is transfected after a single dose. Repeat dosing of inhaled hPBAE-mRNA generates consistent protein production in the lung, without local or systemic toxicity. The results indicate that nebulized delivery of IVT-mRNA facilitated by hPBAE vectors may provide a clinically relevant delivery system to lung epithelium.

Dendrimer-Based Lipid Nanoparticles Deliver Therapeutic FAH mRNA to Normalize Liver Function and Extend Survival in a Mouse Model of Hepatorenal Tyrosinemia Type I.

mRNA-mediated protein replacement represents a promising concept for the treatment of liver disorders. Children born with fumarylacetoacetate hydrolase (FAH) mutations suffer from Hepatorenal Tyrosinemia Type 1 (HT-1) resulting in renal dysfunction, liver failure, neurological impairments, and cancer. Protein replacement therapy using FAH mRNA offers tremendous potential to cure HT-1, but is currently hindered by the development of effective mRNA carriers that can function in diseased livers. Structure-guided, rational optimization of 5A2-SC8 mRNA-loaded dendrimer lipid nanoparticles (mDLNPs) increases delivery potency of FAH mRNA, resulting in functional FAH protein and sustained normalization of body weight and liver function in FAH-/- knockout mice. Optimization using luciferase mRNA produces DLNP carriers that are efficacious at mRNA doses as low as 0.05 mg kg-1 in vivo. mDLNPs transfect > 44% of all hepatocytes in the liver, yield high FAH protein levels (0.5 mg kg-1 mRNA), and are well tolerated in a knockout mouse model with compromised liver function. Genetically engineered FAH-/- mice treated with FAH mRNA mDLNPs have statistically equivalent levels of TBIL, ALT, and AST compared to wild type C57BL/6 mice and maintain normal weight throughout the month-long course of treatment. This study provides a framework for the rational optimization of LNPs to improve delivery of mRNA broadly and introduces a specific and viable DLNP carrier with translational potential to treat genetic diseases of the liver.

Delivery of nucleic acids to ex vivo porcine airways using electrospray

Aim of the Study: Nucleic acid-based therapies have the potential to provide clinically meaningful benefit across a wide spectrum of lung disease. However, in vivo delivery remains a challenge. Here we examined the feasibility of using electrospray to deliver nucleic acids to both porcine tracheal tissue sections and whole lung ex vivo. Materials and Methods: The effect of electrospray solution, emitter gauge, flow rate and voltage on plasmid DNA integrity was examined by analyzing supercoiled:open circle structure ratio by gel electrophoresis. Optimal parameters were used to deliver luciferase DNA and mRNA and siRNA-FITC to tracheal tissue sections. Luciferase mRNA was delivered to whole porcine lungs ex vivo using a catheter and bronchoscope system. Luciferase activity and fluorescence were analyzed by luminometry and microscopy respectively. Results: The incidence of DNA plasmid nicking was greatest in a low salt solution without ethanol compared with 1% and 20% ethanol with salt. From a range of emitters tested, a 32 gauge emitter produced the best supercoiled:open circle structure ratio, likely because less voltage was required to produce a stable electrospray with this emitter. Lower flow rates also showed a trend towards reduced DNA nicking. GFP DNA electrosprayed at 5 kV and 6 kV resulted in lower levels of GFP expression in A549 lung cells following lipofection compared with 3 kV and 4 kV. Optimised parameters of 20% ethanol solution, 32 gauge emitter, low flow rates and voltages of 3–5 kV, nucleic acid molecules were successful for delivery of luciferase DNA and mRNA as well as siRNA-FITC to porcine tracheal tissue sections and for delivery of luciferase mRNA to whole porcine lungs via bronchoscope. Conclusions: We report ex vivo delivery of nucleic acids to porcine lung tissue via electrospray and bronchoscopic electrospray delivery of nucleic acid to an ex vivo porcine lung model.

Post-transcriptional regulation of chicken PPARγ transcript variant 3 by upstream open reading frame

Peroxisome proliferator-activated receptor gamma (PPARγ) is a critical regulator of adipogenesis. Our previous study showed that unlike human and mouse PPARγ transcripts, several chicken PPARγ transcript variants contain upstream open reading frames (uORFs) in their 5'untranslated region (5'TR). To decipher the role of uORFs in post-transcriptional regulation of chicken PPARγ gene, we constructed wild-type (psiCHECK2-cPPARγ3-5'UTR-WT) and a uORF mutant (the upstream ATG (uATG) was mutated to stop codon TGA) 5'UTR reporters (psiCHECK2-cPPARγ3- 5'UTR-Mut) of chicken PPARγ transcript variant 3 (cPPARγ3). These two reporters were individually transfected into immortalized chicken pre-adipocytes (ICPA) and DF1 cells, and the renilla luciferase (hRluc) activity and mRNA expression level were detected by reporter assay and qRT-PCR. The results showed that the hRluc activity of the mutated 5'UTR was significantly higher than that of the wild-type 5'UTR in ICPA cells (P<0.01), and the hRluc activity of the mutated 5'UTR tended to be higher than that of the wild-type 5'UTR in DF1 cells, but this difference did not reach statistical significance (P>0.05). The qRT-PCR analysis showed, in ICPA cells, the hRluc mRNA expression was significantly lower in the cells transfected with the mutated 5'UTR construct than in the cells transfected with the wild-type 5'UTR construct (P<0.01). In DF1 cells, the hRluc mRNA expression tended to be lower in the cells transfected with the mutated 5'UTR construct than in the cells transfected with the wild-type 5'UTR construct, but this difference did not reach statistical significance (P>0.05). To further gain insight into the post-transcriptional regulation of cPPARγ3 by the uORF, we constructed the expression plasmids bearing the full-length coding region of chicken PPARγ gene plus either wild-type or mutant uORF 5'UTR (pcDNA3.1-cPPARγ3-WT and pcDNA3.1-cPPARγ3-Mut). These two constructed PPARγ expression plasmids were individually transiently transfected into both ICPA and DF1 cells, and PPARγ mRNA and protein levels were assayed by qRT-PCR and western blotting. The result showed that in both cell lines, PPARγ mRNA expression was significantly lower in the cells transfected with pcDNA3.1-cPPARγ3-Mut than in the cells transfected with pcDNA3.1-cPPARγ3-WT (P<0.05). In contrast, western blot analysis showed that PPARγ protein level was significantly higher in the cells transfected with pcDNA3.1-cPPARγ3-Mut than in the cells transfected with pcDNA3.1-cPPARγ3-WT (P<0.001). Taken together, our results demonstrate that the uORF in 5'UTR of the cPPARγ3 inhibits its translation.

MiR-204 enhances p27 mRNA stability by targeting Brd4 in head and neck squamous cell carcinoma.

The present study aimed to explore the function of microRNA (miR)-204 in modulating cyclin-dependent kinase inhibitor 1B (p27) mRNA stability in head and neck squamous cell carcinoma (HNSCC). Briefly, reverse transcription quantitative polymerase chain reaction and western blot analysis were used to detect miR-204 and Brd4 level. Cell viability, cell cycle and cell apoptosis were used to investigate the effects of miR-204. Additional luciferase reporter and mRNA stability assays were used to explore the mechanisms contributing to miR-204 effects. Here, miR-204 was downregulated in HNSCC tissues compared with the adjacent normal tissues. The expression levels of miR-204 and bromodomain-containing protein 4 (Brd4) were negatively associated in HNSCC tissues. Ectopic expression of miR-204 inhibited HNSCC cell proliferation, promoted cell cycle arrest at the G1/S phase and promoted cell apoptosis compared with control cells. Additionally, upregulation of miR-204 expression levels enhanced p27 mRNA stability. Notably, Brd4 was identified as a target of miR-204, and the co-expression of Brd4 with miR-204 mimics attenuated the inhibitory effects of miR-204 on cell proliferation and enhanced p27 mRNA stability compared with control cells. Thus, it was concluded that miR-204 functions as a tumor suppressor by enhancing p27 mRNA stability through targeting Brd4 in HNSCC.