In Vitro Transcribed Research Articles

PEVL: A Linear Plasmid for Generating mRNA IVT Templates With Extended Encoded Poly(A) Sequences.

Increasing demand for large-scale synthesis of in vitro transcribed (IVT) mRNA is being driven by the increasing use of mRNA for transient gene expression in cell engineering and therapeutic applications. An important determinant of IVT mRNA potency is the 3′ polyadenosine (poly(A)) tail, the length of which correlates with translational efficiency. However, present methods for generation of IVT mRNA rely on templates derived from circular plasmids or PCR products, in which homopolymeric tracts are unstable, thus limiting encoded poly(A) tail lengths to ~120 base pairs (bp). Here, we have developed a novel method for generation of extended poly(A) tracts using a previously described linear plasmid system, pJazz. We find that linear plasmids can successfully propagate poly(A) tracts up to ~500 bp in length for IVT mRNA production. We then modified pJazz by removing extraneous restriction sites, adding a T7 promoter sequence upstream from an extended multiple cloning site, and adding a unique type-IIS restriction site downstream from the encoded poly(A) tract to facilitate generation of IVT mRNA with precisely defined encoded poly(A) tracts and 3′ termini. The resulting plasmid, designated pEVL, can be used to generate IVT mRNA with consistent defined lengths and terminal residue(s).

An RNA Molecule Derived From Sendai Virus DI Particles Induces Antitumor Immunity and Cancer Cell-selective Apoptosis

Inactivated Sendai virus (hemagglutinating virus of Japan; HVJ) envelope (HVJ-E) induces anticancer immunity and cancer cell-selective apoptosis through the recognition of viral RNA genome fragments by retinoic acid-inducible gene-I (RIG-I). Here, we discovered that the "copy-back" type of defective-interfering (DI) particles that exist in the Cantell strain of HVJ induced the human PC3 prostate cancer cell death more effectively than the Sendai/52 strain or Cantell strain, which contain fewer DI particles. DI particle genomic RNA (~550 bases) activated proapoptotic genes such as Noxa and/or TNF-related apoptosis-inducing ligand (TRAIL) in human prostate cancer cells to induce cancer cell-selective apoptosis. DI particle-derived RNA was synthesized by in vitro transcription (in vitro transcribed (IVT)-B2). IVT-B2 RNA, which has a double-stranded region in its secondary structure, promoted a stronger anticancer effect than IVT-HN RNA, which does not have a double-stranded region in its secondary structure. The intratumoral transfection of IVT-B2 significantly reduced the volume of a human prostate tumor and induced tumor cell apoptosis in the xenograft mouse model. Moreover, the involvement of natural killer (NK) cells in IVT-B2-RNA-induced anticancer effects was also suggested. These findings provide a novel nucleic acid medicine for the treatment of cancer.

Optimizing the process of nucleofection for professional antigen presenting cells.

BackgroundIn times of rapidly increasing numbers of immunological approaches entering the clinics, antigen delivery becomes a pivotal process. The genuine way of rendering antigen presenting cells (APC) antigen specific, largely influences the outcome of the immune response. Short peptides bear the demerit of HLA restriction, whereas the proper way of delivery for long peptide sequences is currently a matter of debate. Electroporation is a reliable method for antigen delivery, especially using nucleic acids. The nucleofection process is based on this approach with the twist of further ensuring delivery also into the nucleus. Beside the form of antigen, the type of APC used for immune response induction may be crucial. Dendritic cells (DC) are by far the most commonly used APC; however B cells have entered this field as well and have gained wide acceptance.ResultsIn this study, we compared B cells to DC with regard to nucleofection efficiency and intensity of resulting antigen expression. APC were transfected either with plasmid DNA containing the reporter gene green fluorescent protein (GFP) or directly with in vitro-transcribed (IVT) GPF mRNA as a surrogate antigen. Out of nearly 100 different nucleofection programs tested, the top five for each cell type were identified and validated using cells from cancer patients. Flow cytometric analyses of transfected cells determining GFP expression and viability revealed a reverse correlation of efficiency and viability. Finally, donor dependant variances were analyzed.ConclusionIn summary, nucleofection of both DC and B cells is feasible with plasmid DNA and IVT mRNA. And no differences with regard to nucleofectability were observed between the two cell types. Using IVT mRNA omits the danger of genomic integration and plasmid DNA constructs permit a more potent and longer lasting antigen expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1446-8) contains supplementary material, which is available to authorized users.

Nebulisation of IVT mRNA Complexes for Intrapulmonary Administration.

During the last years the potential role of in vitro transcribed (IVT) mRNA as a vehicle to deliver genetic information has come into focus. IVT mRNA could be used for anti-cancer therapies, vaccination purposes, generation of pluripotent stem cells and also for genome engineering or protein replacement. However, the administration of IVT mRNA into the target organ is still challenging. The lung with its large surface area is not only of interest for delivery of genetic information for treatment of e.g. for cystic fibrosis or alpha-1-antitrypsin deficiency, but also for vaccination purposes. Administration of IVT mRNA to the lung can be performed by direct intratracheal instillation or by aerosol inhalation/nebulisation. The latter approach shows a non-invasive tool, although it is not known, if IVT mRNA is resistant during the process of nebulisation. Therefore, we investigated the transfection efficiency of non-nebulised and nebulised IVT mRNA polyplexes and lipoplexes in human bronchial epithelial cells (16HBE). A slight reduction in transfection efficiency was observed for lipoplexes (Lipofectamine 2000) in the nebulised part compared to the non-nebulised which can be overcome by increasing the amount of Lipofectamine. However, Lipofectamine was more than three times more efficient in transfecting 16HBE than DMRIE and linear PEI performed almost 10 times better than its branched derivative. By contrast, the nebulisation process did not affect the cationic polymer complexes. Furthermore, aerosolisation of IVT mRNA complexes did neither affect the protein duration nor the toxicity of the cationic complexes. Taken together, these data show that aerosolisation of cationic IVT mRNA complexes constitute a potentially powerful means to transfect cells in the lung with the purpose of protein replacement for genetic diseases such as cystic fibrosis or alpha-1-antitrypsin deficiency or for infectious disease vaccines, while bringing along the advantages of IVT mRNA as compared to pDNA as transfection agent.

Abstract 1904: RNA expression based screening of ALK gene fusion from formalin fixed non-small cell lung cancer samples

Abstract The ALK gene fusion results in the constitutive activation of the oncogenic ALK kinase activity through the expression of 3`ALK gene downstream of fusion partners like EML4 and KIF5B. Due to this rearrangement, fusion mRNA show high expression of 3` regions compared to 5` region of ALK gene and can be used as an indicator for presence of gene fusion. However, due to the low intrinsic expression of normal ALK mRNA, detection of the 5′ region of the ALK mRNA requires a highly sensitive assay system. To achieve this high sensitivity, we adapted the quantitative nuclease protection assay to combine biotinylated tiled probes for 3′ and 5′ ALK mRNA with the use of streptavidin conjugated polymeric horseradish peroxidase for detection. This assay can robustly detect baseline levels of ALK mRNA from normal and ALK-negative NSCLC specimen. We then tested the ability of the assay to measure the over expression of 3′ ALK gene by spiking in 3′ and 5′ in vitro transcribed (IVT) ALK RNAs into normal lung samples. Signal over baseline was detected for ALK5` and ALK3` IVT at attomolar concentrations even in the presence of a ratio of 95:5 in the background of normal lung tissue. Known ALK fusion cell lines also showed exquisite sensitivity for detection of fusion by comparing 5′ and 3′ expression ratios with sample input as few as 65 cells per reaction. We screened 293 (222 Adenocarcinoma, 47 Squamous cell carcinoma and 24 other NSCLC subtypes) surgically resected NSCLC FFPE clinical samples from multiple sources for presence of EML4-ALK fusions. Endogenous ALK mRNA expression was detectable in all samples with 12 (4.09 %) samples showing ALK fusion based on aberrant overexpression of 3′ region compared with 5′ region. All the 12 samples were of the adenocarcinoma or large cell subtype consistent with reported ALK fusion epidemiology (table). Confirmation studies with positives show greater sensitivity of detection of ALK fusion than is possible currently by FISH. ALK fusion positive samples epidemiologyALK FUSION POSITIVE SAMPLE IDNSCLC SUBTYPEP63 IHCTTF-1 IHCRACESEXAGEHISTOLOGY041535T2(1)ADENO-+CaucasianFemale69NA042210T2ADENO++CaucasianMale61Poorly differentiatedB610155ADENO++AsianFemale35Undifferentiated carcinomaCU2010/22 15825ADENO++CaucasianNA68Adenosquamous carcinomaCU2010/22 16787ADENO++CaucasianMale71Poorly differentiatedCU2010/22 20035ADENO-+CaucasianMale64Adenosquamous carcinoma with clear cell- and signet cell-like structuresCU2010/22 8119ADENO++CaucasianFemale53NACU2010/24 259313ADENO++CaucasianFemale50NARln020161ADENONANAAsianMale27Mucinous branchioalveolar carcinomaRln020207ADENONANAAsianMale59Mucinous branchioalveolar carcinomaRln060392Large CellNANAAsianFemale45Undifferentiated large cellRln060453Large Cell--AsianMale66Poorly differentiated Citation Format: Krishna Maddula, Mary-Beth Joshi, Vijay Modur, David H. Harpole. RNA expression based screening of ALK gene fusion from formalin fixed non-small cell lung cancer samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1904. doi:10.1158/1538-7445.AM2014-1904

MRNA-based therapeutics--developing a new class of drugs.

In vitro transcribed (IVT) mRNA has recently come into focus as a potential new drug class to deliver genetic information. Such synthetic mRNA can be engineered to transiently express proteins by structurally resembling natural mRNA. Advances in addressing the inherent challenges of this drug class, particularly related to controlling the translational efficacy and immunogenicity of the IVTmRNA, provide the basis for a broad range of potential applications. mRNA-based cancer immunotherapies and infectious disease vaccines have entered clinical development. Meanwhile, emerging novel approaches include in vivo delivery of IVT mRNA to replace or supplement proteins, IVT mRNA-based generation of pluripotent stem cells and genome engineering using IVT mRNA-encoded designer nucleases. This Review provides a comprehensive overview of the current state of mRNA-based drug technologies and their applications, and discusses the key challenges and opportunities in developing these into a new class of drugs.

Rapid detection of equine influenza virus H3N8 subtype by insulated isothermal RT-PCR (iiRT-PCR) assay using the POCKIT™ Nucleic Acid Analyzer

Equine influenza (EI) is an acute, highly contagious viral respiratory disease of equids. Currently, equine influenza virus (EIV) subtype H3N8 continues to be the most important respiratory pathogen of horses in many countries around the world. The need to achieve a rapid diagnosis and to implement effective quarantine and movement restrictions is critical in controlling the spread of EIV. In this study, a novel, inexpensive and user-friendly assay based on an insulated isothermal RT-PCR (iiRT-PCR) method on the POCKIT™, a field-deployable device, was described and validated for point-of-need detection of EIV-H3N8 in clinical samples. The newly established iiRT-PCR assay targeting the EIV HA3 gene was evaluated for its sensitivity using in vitro transcribed (IVT) RNA, as well as ten-fold serial dilutions of RNA extracted from the prototype H3N8 strain A/equine/Miami/1/63. Inclusivity and exclusivity panels were tested for specificity evaluation. Published real-time RT-PCR (rRT-PCR) assays targeting the NP and HA3 genes were used as the reference standards for comparison of RNA extracted from field strains and from nasal swab samples collected from experimentally infected horses, respectively. Limit of detection with a 95% probability (LoD95%) was estimated to be 11copies of IVT RNA. Clinical sensitivity analysis using RNA prepared from serial dilutions of a prototype EIV (Miami 1/63/H3N8) showed that the iiRT-PCR assay was about 100-fold more sensitive than the rRT-PCR assay targeting the NP gene of EIV subtype H3N8. The iiRT-PCR assay identified accurately fifteen EIV H3N8 strains and two canine influenza virus (CIV) H3N8 strains, and did not cross-react with H6N2, H7N7, H1N1 subtypes or any other equine respiratory viral pathogens. Finally, 100% agreement was found between the iiRT-PCR assay and the universal influenza virus type A rRT-PCR assay in detecting the EIV A/equine/Kentucky/7/07 strain in 56 nasal swab samples collected from experimentally inoculated horses. Therefore, the EIV H3N8 subtype specific iiRT-PCR assay along with the portable POCKIT™ Nucleic Acid Analyzer provides a highly reliable, sensitive and specific on-site detection system of both equine and canine influenza viruses.

Human dendritic cells expressing hepatitis C virus core protein display transcriptional and functional changes consistent with maturation

Hepatitis C virus (HCV) causes a chronic liver infection, which may result in cirrhosis and hepatocellular carcinoma. Impairment of the maturation process in dendritic cells (DCs) may be one of the mechanisms responsible for immune evasion of HCV. The core and NS3 proteins are among the most conserved HCV proteins and play a key role in viral clearance. To evaluate the effects of these proteins on DCs, monocyte-derived immature DCs (iDCs) were transfected with in vitro transcribed (IVT) HCV core or NS3 RNA and treated with maturation factors. Neither core nor NS3 had an inhibitory effect on DC maturation; however, transfection of iDCs with IVT core RNA appeared to result in changes compatible with maturation. To investigate this in more detail, the transcriptional profiles of iDCs transfected with IVT core, NS3 or green fluorescent protein (GFP) RNA were examined using a DC-specific membrane array. Of the 288 genes on the array, 46 genes were distinctively up- or down-regulated by transfection with IVT core RNA in comparison with NS3 or GFP RNA treatments. Forty-two of these genes are involved in DC maturation. The effects of core on maturation of iDCs were confirmed with a significant increase in surface expression of CD83 and HLA-DR, a reduction of phagocytosis, as well as an increase in proliferation and IFN-γ secretion by T cells in a mixed lymphocyte reaction assay. These results show that HCV core does not have an inhibitory effect on human DC maturation, but could be a target for the immune system.

P18-13 LB. Immunogenicity of an autologous dendritic cell anti-HIV therapy in HIV-1 infected individuals

Background The immunogenicity of an autologous dendritic cell (DC) anti-HIV therapy, AGS-004, was evaluated in a multicenter Phase 2 trial. The treatment regiment consisted of four intradermal doses, administered monthly in combination with anti-retroviral therapy (ART) followed by a well-controlled structured treatment interruption (STI). The immunotherapy consisted of monocyte-derived DC co-electroporated with CD40L in vitro transcribed (IVT) RNA along with amplified IVT RNA encoding for 4 HIV-1 antigens (GAG, NEF, REV and VPR).

Development and evaluation of a real-time one step Reverse-Transcriptase PCR for quantitation of Chandipura Virus

BackgroundChandipura virus (CHPV), a member of family Rhabdoviridae was attributed to an explosive outbreak of acute encephalitis in children in Andhra Pradesh, India in 2003 and a small outbreak among tribal children from Gujarat, Western India in 2004. The case-fatality rate ranged from 55–75%. Considering the rapid progression of the disease and high mortality, a highly sensitive method for quantifying CHPV RNA by real-time one step reverse transcriptase PCR (real-time one step RT-PCR) using TaqMan technology was developed for rapid diagnosis.MethodsPrimers and probe for P gene were designed and used to standardize real-time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [in vivo (mice) in ovo (eggs), in vitro (Vero E6, PS, RD and Sand fly cell line)] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which is considered as a gold standard.ResultsReal-time one step RT-PCR was optimized using in vitro transcribed (IVT) RNA. Standard curve showed linear relationship for wide range of 102-1010 (r2 = 0.99) with maximum Coefficient of variation (CV = 5.91%) for IVT RNA. The newly developed real-time RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice) used for the isolation of the virus. Detection limit of real-time one step RT-PCR and nested RT-PCR was found to be 1.2 × 100 PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 × 102 PFU/ml). Vero and PS cell-lines (1.2 × 103 PFU/ml) were least sensitive to CHPV infection. Specificity of the assay was found to be 100% when RNA from other viruses or healthy individual was used.ConclusionOn account of the high sensitivity, reproducibility and specificity, the assay can be used for the rapid detection and quantitation of CHPV RNA from clinical samples during epidemics and from endemic areas. The assay may also find application in screening of antiviral compounds, understanding of pathogenesis as well as evaluation of vaccine.

High transfection efficiency, gene expression, and viability of monocyte-derived human dendritic cells after nonviral gene transfer

Dendritic cells (DCs) are bone marrow-originated, professional antigen-capturing cells and APCs, which can function as vaccine carriers. Although efficient transfection of human DCs has been achieved with viral vectors, viral gene products may influence cellular functions. In contrast, nonviral methods have generally resulted in inefficient gene transfer, low levels of gene expression, and/or low cell viability. Monocyte-derived DCs are the most common source of DCs for in vitro studies and for in vivo applications. We hypothesized that reduction of the time to generate immature DCs (iDCs) might result in higher viability after transfection. Therefore, we established a protocol to generate human iDCs from CD14(+) monocytes within 3 days. These "fast" iDCs were phenotypically and functionally indistinguishable from conventional iDCs, showing high endocytic ability and low antigen-presenting capacity. Furthermore, the fast iDCs matured normally and had similar antigen-presenting capacity to conventional mature DCs. To optimize transfection of iDCs, we compared nonviral transfection of plasmid DNA and in vitro-transcribed (IVT) RNA with transfection reagents, electroporation, and nucleofection. Nucleofection of IVT RNA with the X1 program of an Amaxa Co. Nucleofector resulted in the most efficient transfection, with an average of 93% transfected iDCs, excellent long-term viability, and strong protein expression. Furthermore, the IVT RNA-transfected iDCs retained all phenotypic and functional characteristics of iDCs. This method is applicable to most purposes, including in vitro functional assays, in vivo DC immunotherapy, and DC-based vaccines.

RNA Loading of Leukemic Antigens into Cord Blood–Derived Dendritic Cells for Immunotherapy

The manipulation of dendritic cells (DCs) ex vivo to present tumor-associated antigens for the activation and expansion of tumor-specific cytotoxic T lymphocytes (CTLs) attempts to exploit these cells’ pivotal role in immunity. However, significant improvements are needed if this approach is to have wider clinical application. We optimized a gene delivery protocol via electroporation for cord blood (CB) CD34 + DCs using in vitro–transcribed (IVT) mRNA. We achieved > 90% transfection of DCs with IVT-enhanced green fluorescent protein mRNA with > 90% viability. Electroporation of IVT-mRNA up-regulated DC costimulatory molecules. DC processing and presentation of mRNA-encoded proteins, as major histocompatibility complex/peptide complexes, was established by CTL assays using transfected DCs as targets. Along with this, we also generated specific antileukemic CTLs using DCs electroporated with total RNA from the Nalm-6 leukemic cell line and an acute lymphocytic leukemia xenograft. This significant improvement in DC transfection represents an important step forward in the development of immunotherapy protocols for the treatment of malignancy.

Generation of Antigen Specific Cytotoxic T Lymphocytes (CTL) by Dendritic Cells (DCs) Transfected with In Vitro Transcribed (IVT) SART-1 and WT-1 mRNA.

Transfection with tumor antigen RNA is one of the promising tools not only because of a possible sufficient amplification of tumor antigen RNA but also because of the absence of antigen peptides-associated MHC restriction. Several succeeded experiments about generation of CTLs using DCs transfeced in vitro transcribed (IVT) cancer specific antigen mRNA such as PSA, CEA, hTERT and MUC-1 have been reported in these a few years. In addition, recent reports about the simultaneous presentation of peptides in both MHC class I and class II molecules on DCs after mRNA electroporation show another superiority of mRNA transfection into DCs. In this presentation, we demonstrate successful generation of tumor antigen specific CTLs using with DCs transfected with IVT mRNA such as SART-1 and WT-1 by electroporation. This is the first report about the generation of SART-1 and WT-1 specific CTLs by using mRNA transfected DCs. [Methods] HLA-A24 positive human PB CD14+ cell-derived DCs were transfected with IVT mRNA (SART-1and WT-1) by electroporation. MRNA transfected DCs were co-cultured with autologous lymphocytes. The bulk co-cultures were re-stimulated several times with same DCs. CD8+ cells were separated and CTL activity was evaluated by 51chromium release assay. To determine whether the induced CTL cells could recognize the target cells in an HLA class I restricted manner, anti-HLA class I monoclonal antibodies were utilized to block the cytotoxicity of effectors. [Results] Electroporation of mRNA showed no effect on the surface phenotypes and antigen presenting ability of DCs. In addition to the demonstration of efficient transfection of M1 mRNA into DCs by using RT-PCR, which eliminated the amplification of transfected mRNA by the treatment with RNase before RNA extraction from the transfected cells, we identified the definite expression of WT-1 protein in the cytoplasm of DCs by using immunoblotting. CTL assay indicated that 1) DCs transfected with mRNA stimulated the generation of antigen-specific CTLs which are capable of lysing autologous DCs transfected with the same mRNA. 2) CTLs also demonstrated cytotoxic ability against cell lines such as KE-4 presenting SART-1 peptides on HLA-A24, MEGO1 presenting WT-1 peptides on MHC class I, and HLA-A24 cDNA transfected T2 which were used as target cells after co- incubation with 9 mer SART-1 peptides with strong affinity to HLA-A24. 3) Each cytotoxicities were markedly blocked after co-incubation of target cells with anti-MHC class I antibody and not inhibited with anti-MHC class II antibody. [Conclusion] Our results showed that IVT mRNA-transfected DCs which is constructed non-virally have a highly efficient ability to stimulate specific T-cell immunity against tumor. Unlike peptide- or tumor cells extract-pulsed DCs based vaccines, anti-tumor immunotherapy using the DCs transfected with antigen mRNA could be extended to a wide range of patients who have previously been excluded from clinical trials for the reason of the un-identification of tumor specific antigens, for the reason of the impossibility of obtaining sufficient tumor specimens, or for the reason of MHC restriction of the tumor specific antigens.

Bestowing Transient Cytotoxic Capability on Dendritic Cells by Transfection with In Vitro Transcribed (IVT) TRAIL mRNA.

Bestowing transient cytotoxic capability on dendritic cells could expand the applicability of dendritic cells in the fields of the treatment for autoimmune disorders and anti-tumor immunotherapy. If it would be possible to construct dendritic cells, which present antigens associated with autoimmune disorders and possess transient cytotoxic ability, these dendritic cells could be used for the therapy of the disorders by eliminating active lymphocytes reactive to autologous etiological antigens specifically. As to the applicability for anti-tumor immunotherapy, dendritic cells with transient cytotoxicity could be administered into the tumor region directly for efficient tumor antigen presentation by effective killing tumor cells and up taking apoptotic/necrotic tumor cells. In the present study, the transfection of IVT TRAIL mRNA into dendritic cells was applied to induce a transient cytotoxic ability in functional dendritic cells. By using microbeads with anti-CD34 monoclonal antibody, human CD34+ cells were separated from cord blood with informed consent. In order to generate mature dendritic cells, CD34+ cells were cultured with GM-CSF, SCF and TNF-α for 8-10 days. CD34+ cell-derived dendritic cells were transfected with TRAIL mRNA by square wave pulse-electroporation and cultured in the medium containing the same cytokines for 1 day. TRAIL mRNA was in vitro transcribed from the T7 promoter transcription vector containing TRAIL cDNA by using mMESSAGE mMACHINE kit. As a positive control of TRAIL protein expression, CD34+ cell-derived dendritic cells were cultured with IFN-γ at the concentration of 1,000 U/ml for 1 day, which was the best cytokine/culture condition to induce the expression of TRAIL protein in dendritic cells. The expression of TRAIL protein and dendritic cell-associated surface phenotypes on the dendritic cells transfected with IVT TRAIL mRNA or cultured with IFN-γ was evaluated by flow cytometry using anti-TRAIL monoclonal antibody and the other antibodies. Cytotoxic capability of dendritic cells transfected with IVT TRAIL mRNA or cultured with IFN-g was evaluated by 51Cr-release assay in which TRAIL receptor (DR4 and/or DR5) -expressing cell lines such as HL-60 and Jurkat were used as target cells. By the transfection of IVT TRAIL mRNA, CD34+ cell-derived dendritic cells were demonstrated to express TRAIL proteins and showed no change of dendritic cell-associated surface phenotypes. The dendritic cells cultured with IFN-γ also showed the similar results. The expression of TRAIL protein on dendritic cells lasted only for a few days. Dendritic cells gained the cytotoxic capability against cell lines like HL-60 after the transfection with IVT TRAIL mRNA. The present study demonstrated that trans-membrane protein like TRAIL could be expressed and function as a molecule generating death signals transiently in the dendritic cells by the transfection with IVT mRNA. These findings revealed that the dendritic cells with bestowed transient cytotoxic capability by RNA transfection could be applied for the treatment of autoimmune disorders and anti-tumor immunotherapy.

Generation of Ag-specific cytotoxic T lymphocytes by DC transfected with in vitro transcribed influenza virus matrix protein (M1) mRNA.

Application of DC transfected with tumor Ag RNA is promising for DC-based tumor immunotherapy. In this study, Ag-specific cytotoxic T lymphocytes (CTL) were generated by priming lymphocytes with DC transfected with in vitro transcribed (IVT) influenza virus matrix protein M1 (M1) mRNA. Human UC blood-CD34+ cell-derived DC were transfected with IVT mRNA encoding either the enhanced green fluorescence protein (EGFP), or M1 by square-wave electroporation. DC were confirmed to have typical morphology and phenotype. DC transfected with IVT EGFP mRNA were analyzed with the FACScan flow cytometer, to confirm the efficiency of this transfection method. On Days 7, 14, 21 and 28 after the start of DC culture, DC were harvested and electroporated with M1 mRNA. The transfected DC were co-cultured with autologous UC blood CD34- cells. One week after the fourth priming of autologous CD34 negative cells with M1 mRNA electroporated DC, Ag-specific CTL activity was evaluated. To prepare target cells, M1 mRNA was added to autologous DC 48 h prior to CTL assays. Our CTL assays results indicate that UC blood CD34+ cell-derived DC transfected with M1 mRNA by electroporation stimulated Ag-specific CTL responses that are capable of recognizing and lysing autologous DC loaded with M1 mRNA. M1 mRNA transfected DC-primed CTL showed a significant cytotoxic activity against M1 mRNA loaded autologous DC, while nearly baseline cytotoxic activity was recorded for the M1 mRNA unloaded DC. Our results showed that mRNA-transfected DC are potent stimulators of T-cell immunity in vitro. In addition, mRNA-loaded DC can function as targets in CTL cytotoxicity assays, which offer a practical substitute for tumor cells in assays to test the immunological effects of specific Ags.

Strand-scission of Sarcoma 180 tumor cell DNA induced by 1-formylisoquinoline thiosemicarbazone

The potential clinical applications of the powerful in vitro-transcribed (IVT)-mRNAs, to restore defective protein functions, strongly depend on their successful intracellular delivery and transient translation through the development of safe and efficient delivery platforms. In this study, an innovative (international patent-pending) methodology was developed, combining the IVT-mRNAs with the protein transduction domain (PTD) technology, as an efficient delivery platform. Based on the PTD technology, which enables the intracellular delivery of various cargoes intracellularly, successful conjugation of a PTD to the IVT-mRNAs was achieved and evaluated by band-shift assay and NMR spectroscopy. In addition, the PTD-IVT-mRNAs were applied and evaluated in two protein-disease models, including the mitochondrial disorder fatal infantile cardioencephalomyopathy and cytochrome c oxidase (COX) deficiency (attributed to SCO2 gene mutations) and β-thalassemia. The PTD-IVT-mRNA of SCO2 was successfully transduced and translated to the corresponding Sco2 protein inside the primary fibroblasts of a SCO2/COX-deficient patient, whereas the PTD-IVT-mRNA of β-globin was transduced and translated in bone marrow cells, derived from three β-thalassemic patients. The transducibility and the structural stability of the PDT-IVT-mRNAs, in both cases, were confirmed at the RNA and protein levels. We propose that our novel delivery platform could be clinically applicable as a protein therapy for metabolic/genetic disorders.