Transgene Of Interest Research Articles

Emerging Gene Therapies for Alzheimer's and Parkinson's Diseases: An Overview of Clinical Trials and Promising Candidates.

Gene therapy as a disease-modifying therapeutic approach for neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), is a promising avenue. Promising results in the preclinical studies involving rodents and nonhuman primates utilizing gene therapy have led to multiple clinical trials evaluating various genes of interest for AD and PD. In AD, clinical trials are assessing gene therapy involving brain-derived neurotrophic factor (BDNF) and other targets such as apolipoprotein E2 (APOE2) and human telomerase reverse transcriptase (hTERT). In PD, clinical trials are evaluating gene therapy delivering neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF). Additionally, gene therapy delivering enzymes aromatic L-amino acid decarboxylase (AADC) and glutamic acid decarboxylase (GAD) are also being evaluated for PD. All these trials primarily utilized adeno-associated virus (AAV) to deliver the above transgene of interest. This review summarizes the current clinical trials involving gene therapy for AD and PD. It also discussesthe challenges and opportunities associated with the gene therapy approach in AD and PD and ongoing developments related to increasing the safety and efficacy of the gene therapy for long-term outcomes, which include evaluation of various serotypes and administration routes. This comprehensive review emphasizes translating preclinical findings into clinical trials, further directions, and the potential for this promising therapeutic approach to alleviate neurodegenerative disease.

Genetic modification of cardiac fibroblasts in adult rats using adeno-associated virus serotype 9

Cardiac fibrosis is a process involving pathological extracellular matrix remodeling as well as cardiac fibroblast (CF) activation in response to cardiac injury and stress, which is frequently associated with cardiac dysfunction. Therefore, understanding the molecular mechanisms underlying CF activation is critical to develop successful therapies for treating cardiac fibrosis. CF-specific gene modifications in mouse models have been reported as key tools for cardiac fibrosis research. Although rat cardiac disease models also offer many of the same advantages as the mouse models, there are no rat models that employ CF-specific conditional gene modification. Here, we attempted CF-specific gene introduction in rats using adeno-associated virus serotype 9 (AAV9), allowing us to develop fast and simple rat cardiac disease models rather than establishing transgenic rat lines. AAV9 carrying a CF-specific human TCF21 (hTCF21) promoter and a transgene of interest was generated and introduced into adult rats via tail vein injections. Since we previously reported in cultured CFs that protein kinase D (PKD) activation at the outer mitochondrial membrane (OMM) induces mitochondrial fragmentation via phosphorylation of a mitochondrial fission protein DRP1, we generated an OMM-targeted dominant-negative mutant of PKD (termed mt-PKD-DN) to specifically inhibit PKD activity at the OMM in CFs in vivo. In vivo cardiac function, gene expression, protein expression, and mitochondrial morphology were assessed 2-10 weeks after AAV9 injection by echocardiography, quantitative RT-PCR, immunohistochemistry, and transmission electron microscopy (TEM), respectively. AAV9-hTCF21-injected rats maintained normal cardiac dimensions and contractile function compared to aged-matched control rats. Using AAV9-hTCF21-EGFP, we confirmed organ-specific (i.e., heart) and cell type-specific (i.e., CF-specific) expression of GFP. Next, we injected AAV9-hTCF21-mt-PKD-DN or -luciferase (Luc) as a control in adult rats. TEM images from fixed rat ventricular tissues revealed a significant increase in mitochondrial aspect ratio and mitochondrial perimeter in CFs with AAV9-hTCF21-mt-PKD-DN compared to AAV9-hTCF21-Luc, indicating that PKD inhibition at the OMM induces mitochondrial elongation in CFs in vivo. In contrast, no significant change in mitochondrial morphology was observed in the cardiomyocyte area. In summary, we successfully achieved a CF-specific gene delivery to the adult rat hearts in vivo, demonstrating the potential of a simplified CF-specific rat transgenic model using the AAV9-hTCF21 system. NIH/NHLBI R01HL160699. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Universal ddPCR-based assay for the determination of lentivirus infectious titer and lenti-modified cell vector copy number

The translation of cell-based therapies from research to clinical setting requires robust analytical methods that successfully adhere to current good manufacturing practices and regulatory guidelines. Lentiviral vectors are commonly used for gene delivery to generate genetically modified therapeutic cell products. For some cell therapy products, standardized characterization assays for potency and safety have gained momentum. Translational applications benefit from assays that can be deployed broadly, such as for lentiviral vectors with various transgenes of interest. Development of a universal method to determine lentivirus infectious titer and vector copy number (VCN) of lenti-modified cells was performed using droplet digital PCR (ddPCR). Established methods relied on a ubiquitous lenti-specific target and a housekeeping gene that demonstrated comparability among flow cytometry-based methods. A linearized plasmid control was used to determine assay linearity/range, sensitivity, accuracy, and limits of quantification. Implementing this assay, infectious titer was assessed for various production runs that demonstrated comparability to the flow cytometry titer. The ddPCR assay described here also indicates suitability in the determination of VCN for genetically modified CAR-T cell products. Overall, the development of these universal assays supports the implementation of standardized characterization methods for quality control.

Quantification of Empty, Partially Filled and Full Adeno-Associated Virus Vectors Using Mass Photometry.

Adeno-associated viruses (AAV) are one of the most commonly used vehicles in gene therapies for the treatment of rare diseases. During the AAV manufacturing process, particles with little or no genetic material are co-produced alongside the desired AAV capsid containing the transgene of interest. Because of the potential adverse health effects of these byproducts, they are considered impurities and need to be monitored carefully. To date, analytical ultracentrifugation (AUC), transmission electron microscopy (TEM) and charge-detection mass spectrometry (CDMS) are used to quantify these subspecies. However, they are associated with long turnaround times, low sample throughput and complex data analysis. Mass photometry (MP) is a fast and label-free orthogonal technique which is applicable to multiple serotypes without the adaption of method parameters. Furthermore, it can be operated with capsid titers as low as 8 × 1010 cp mL-1 with a CV < 5% using just 10 µL total sample volume. Here we demonstrate that mass photometry can be used as an orthogonal method to AUC to accurately quantify the proportions of empty, partially filled, full and overfull particles in AAV samples, especially in cases where ion-exchange chromatography yields no separation of the populations. In addition, it can be used to confirm the molar mass of the packaged genomic material in filled AAV particles.

Systems Biology Approaches for the Improvement of Oncolytic Virus-Based Immunotherapies.

Oncolytic virus (OV)-based immunotherapy is mainly dependent on establishing an efficient cell-mediated antitumor immunity. OV-mediated antitumor immunity elicits a renewed antitumor reactivity, stimulating a T-cell response against tumor-associated antigens (TAAs) and recruiting natural killer cells within the tumor microenvironment (TME). Despite the fact that OVs are unspecific cancer vaccine platforms, to further enhance antitumor immunity, it is crucial to identify the potentially immunogenic T-cell restricted TAAs, the main key orchestrators in evoking a specific and durable cytotoxic T-cell response. Today, innovative approaches derived from systems biology are exploited to improve target discovery in several types of cancer and to identify the MHC-I and II restricted peptide repertoire recognized by T-cells. Using specific computation pipelines, it is possible to select the best tumor peptide candidates that can be efficiently vectorized and delivered by numerous OV-based platforms, in order to reinforce anticancer immune responses. Beyond the identification of TAAs, system biology can also support the engineering of OVs with improved oncotropism to reduce toxicity and maintain a sufficient portion of the wild-type virus virulence. Finally, these technologies can also pave the way towards a more rational design of armed OVs where a transgene of interest can be delivered to TME to develop an intratumoral gene therapy to enhance specific immune stimuli.

TCR and CAR Engineering of Primary Human T Cells.

The efficient expression of T-cell receptors (TCRs) or chimeric antigen receptors (CARs) in primary human T cells is crucial for preclinical testing of receptor properties for adoptive T-cell therapies. Multiple streams of technological platforms have been developed in the recent decades to genetically modify primary T cells including nonviral platforms such as transposon-based systems (PiggyBac, Sleeping Beauty), TALENs, or CRISPR-Cas9). The production of CAR- or TCR-encoding retroviral vectors, however, is still the most commonly used technique both in preclinical as well as in clinical settings.In this chapter we describe a comprehensive 12-day protocol for (a) generating high-titered gamma-retroviral vector particles containing the transgene of interest (e.g., TCR , CAR ), (b) the isolation, activation and rapid expansion of primary T cells and (c) the stable genetic engineering of these T cells with the transgene for subsequent characterization.

A novel DNA platform designed for vaccine use with high transgene expression and immunogenicity

The development of new, low-cost vaccines and effective gene therapies requires accurate delivery and high-level expression of candidate genes. We developed a plasmid vector, pIDV-II, that allows for both easy manipulation and high expression of exogenous genes in mammalian cells. This plasmid is based upon the pVax1 plasmid and shares a common structure with typical mammalian transcription units. It is composed of a chicken β-actin promoter (CAG), followed by an intron and flanked by two restriction sites, and also includes a post-transcriptional regulatory element, followed by a transcriptional termination signal. While the modification of pVax1 elements either decreased eGFP expression levels or had no effect at all, replacement of the promoter, the poly-A signal, deletion of the T7 and AmpR promoters, and inversion of the ORI-Neo/Kan cassette, significantly increased in vitro eGFP expression with the modified plasmid called pIDV-II. To further evaluate our vector, expression levels of three viral antigens were compared in cell lines transfected either with pVax1 or pCAGGS backbones as controls. Higher transgene expression was consistently observed with pIDV-II. The humoral and cellular responses generated in mice immunized with pIDV-II vs pVax1 expressing each viral antigen individually were superior by 2-fold or more as measured by ELISA and ELISPOT assays. Overall these results indicate that pIDV-II induces robust transgene expression, with concomitant improved cellular and humoral immune responses against the transgene of interest over pVax1. The new vector, pIDV-II, offers an additional alternative for DNA based vaccination and gene therapy for animal and human use.

An Oncolytic Adenovirus Armed with a Nanobody Against CD47 Reprograms Tumor Immune Microenvironment and Drives Durable Antitumor Immunity

Background: Oncolytic virotherapy utilizes natural or engineered viruses to kill tumors selectively, representing a promising cancer immunotherapy. In addition to direct oncolysis, oncolytic viruses (OVs) elicit potent and durable antitumor immune responses by induction of immunogenic cell death of tumors. Membrane protein CD47 overexpressed on tumor cells engages in don't eat me signal that prevents macrophages from engulfing tumor cells. CD47-targeting agents have been tested via preclinical and clinical trials. As ideal gene delivery vectors in cancer therapy, OVs can be engineered to express anti-CD47 antibodies to induce potentiated tumor killing. Importantly, OVs can intratumorally deliver anti-CD47 antibodies without causing uncontrolled off-tumor toxicity. Methods: We developed a recombinant oncolytic adenovirus loaded with a CD47-targeting nanobody fused with the IgG2a Fc protein. An oncolytic adenovirus without transgene of interest was used as control. B16-F10 melanoma, A20 lymphoma, and 4T1 breast cancer mouse models were established to evaluated in vivo antitumor efficacy of oncolytic adenoviruses. Findings: The recombinant oncolytic adenovirus armed with a nanobody against CD47 induced durable suppression of the tumor and long-term survival of tumor-bearing mice. Additionally, the recombinant oncolytic adenovirus elevated the number of tumor-infiltrating immune cells with an activated immunophenotype, suggesting that it could remodel the tumor immune microenvironment. Systemic antitumor effects and immune memory were also observed in mice treated with the anti-CD47 oncolytic adenovirus; tumorigenesis was completely inhibited in these mice after tumor re-challenge. Interpretation: The recombinant anti-CD47 oncolytic adenovirus has an effectual antitumor activity and may be a promising antitumor agent. Funding Statement: This work was supported by the National Science and Technology Major Projects of New Drugs (2018ZX09201018-013), the National Science and Technology Major Project for Infectious Diseases Control (2017ZX10203206-004), the National Natural Science Foundation of China (81101728), Sichuan Regional Innovation Cooperation Project (20QYCX0100), the Innovation Spark Project of Sichuan University (2018SCUH0084). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All animal experiments were approved by the Institutional Animal Care and Use Committee of Sichuan University, Chengdu, China.

High-Efficiency Lentiviral Genetic Modification of Primary Human Natural Killer Cells

Natural killer (NK) cells have the unique capacity to lyse tumor cells without the need for antigen specific receptors. Modulation of NK cell cytokine, chemokine, and activating and inhibitory receptor expression using gene regimens represents an attractive strategy to further bolster NK cell anti-tumor activity for the purpose of NK cell immunotherapy. Current efforts in clinical-grade genetic modification of patient-derived cells, used in immunotherapy protocols, rely on stable transduction mediated by viral vectors. However, primary NK cells are notoriously difficult to transduce using viral vectors. Here we describe a lentiviral vector-based approach that results in an efficient, robust, and highly reproducible method of stable gene transfer into primary human peripheral blood-derived NK cells, which can subsequently be expanded ex vivo for adoptive infusion into humans. In vitro experiments established highly efficient transduction of ex vivo expanded NK cells could be achieved by activation of primary NK cells in vitro for 2-4 days in cytokine containing media followed by transduction of activated NK cells using LV vectors 2-3 days before integration into our existing ex vivo expansion protocol using irradiated EBV-LCL feeder cells. Unless NK cells were first activated in cytokine-containing media, LV transduction was unsuccessful. Cytokine activation in media containing IL-2 for 2-4 days was found to be sufficient to achieve high transduction efficiency, while additional supplementation of the media with IL-15 or IL-21 had negligible effects. NK cell transduction efficiency and viability with the use of RetroNectin® was uniformly superior to polycation-based transduction protocols. In experiments where NK cells were transduced with identical lentiviral constructs with 8 different promoter sequences driving expression of EGFP, PGK, EFS, and SV40 promoters consistently demonstrated the highest transduction efficiencies reproducibly in the range of 25-60%. Remarkably, when transduced NK cells were stimulated ex vivo using a clinical grade irradiated EBV-LCL feeder cell line, 100-1000 fold expansions of transduced NK cells could be achieved with sustained transgene expression over two weeks. Transduced and expanded primary NK cells remained highly cytotoxic against K562 tumor targets without developing functional deficiencies in degranulation, IFNγ, or TNFα production. These data establish a simple method to produce large numbers (>1011) of genetically modified clinical grade NK cells suitable for infusion. The protocol described herein overcomes previous difficulties described with common transduction techniques opening new avenues to investigate the impact of adoptive infusion of primary NK cells stably transduced to express a variety of different transgenes of interest. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Novel Branched DNA-Based Flowcytometric Method for Single-Cell Characterization of Gene Therapy Products and Expression of Therapeutic Genes.

Many preclinical and clinical studies of hematopoietic stem cell-based gene therapy (GT) are based on the use of lentiviruses as the vector of choice. Assessment of the vector titer and transduction efficiency of the cell product is critical for these studies. Efficacy and safety of the modified cell product are commonly determined by assessing the vector copy number (VCN) using qPCR. However, this optimized and well-established method in the GT field is based on bulk population averages, which can lead to misinterpretation of the actual VCN per transduced cell. Therefore, we introduce here a single cell-based method that allows to unmask cellular heterogeneity in the GT product, even when antibodies are not available. We use Invitrogen’s flow cytometry-based PrimeFlow™ RNA Assay with customized probes to determine transduction efficiency of transgenes of interest, promoter strength, and the cellular heterogeneity of murine and human stem cells. The assay has good specificity and sensitivity to detect the transgenes, as shown by the high correlations between PrimeFlow™-positive cells and the VCN. Differences in promoter strengths can readily be detected by differences in percentages and fluorescence intensity. Hence, we show a customizable method that allows to determine the number of transduced cells and the actual VCN per transduced cell in a GT product. The assay is suitable for all therapeutic genes for which antibodies are not available or too cumbersome for routine flow cytometry. The method also allows co-staining of surface markers to analyze differential transduction efficiencies in subpopulations of target cells.

PCYOs: Binary vectors for simple visible selection of transformants using an albino-cotyledon mutant in Arabidopsis thaliana.

Several selection markers for the screening of transformants have been developed; however, simple and reliable methods are generally preferred. We have developed a novel visible selection system for the identification of transformants in Arabidopsis thaliana that does not require any special reagent and/or equipment except using the albino-cotyledon mutant cyo1. In this system, the pCYO vector carrying the CYO1 genomic fragment as a selection marker is introduced into the cyo1 mutant. Transformation is performed by the Agrobacterium-mediated floral dip method and resultant T1 seeds are sown in soil. Seedlings with green cotyledons, not albino, are expected to be 'complemented' transformants with the transgene of interest. This system provides a very simple selection method that can be performed without any special equipment, reagent, sterile conditions, or UV illumination. We have constructed three vectors, (1) pCYO1, an empty vector; (2) pCYO2, an overexpression vector carrying CaMV35S promoter; and (3) pCYO3, a vector for genome editing, carrying the CRISPR/Cas9 cassette. Example transformation experiments using these vectors, including genome editing, are shown.

Preparation of Genomic DNA from Mouse Tails and Other Small Samples.

This protocol is used for extracting DNA from fragments of tissue for genotyping transgenic and knockout mice. Digestions of mouse tail fragments are performed overnight at 55°C. Each tail snippet generates 50-100 µg of DNA that can be used in dot or slot blotting to detect a transgene of interest, in Southern hybridization to detect DNA fragments that are <20 kb in size, and, more expediently, as a template in polymerase chain reactions (PCRs).

English

Cassava production in India is drastically affected by cassava mosaic disease (CMD) caused by the Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV). An attempt was done to develop transgenic cassava lines resistant to SLCMV through RNAi vector targeting a conserved 440 bp of 5’ end of SLCMV Rep (AC1) gene which also overlaps with part of AC4 gene, and functions as a viral RNAi suppressor protein. The partial Rep gene of SLCMV was cloned in sense and anti-sense orientations in the RNAi intermediate vector, pHANNIBAL and finally mobilised into binary vector pART27, to construct pSCR1 which contains the kanamycin-resistance gene as a plant selectable marker. In order to use hygromycin as selection agent in cassava genetic transformation, Rep-RNAi gene cassettes of SLCMV was cloned into pCAMBIA1305.2 and the constructs was named pSCR2. Agrobacterium mediated cocultivation of cassava embryogenic calli was done with the developed RNAi constructs using two different explants namely, immature leaf lobes and somatic cotyledons. In total, 48 putative transgenic cassava shoots were regenerated on a regeneration medium containing 30 mgl/l hygromycin, of which 2 putative transgenic plants were transferred for hardening. All the putative transgenic cassava plants were PCR-positive for hph gene and Rep gene indicating integration of transgenes of interest. Key words: RNAi constructs, cocultivation, cassava mosaic disease (CMD).

Mifepristone-inducible transgene expression in neural progenitor cells in vitro and in vivo.

Numerous gene and cell therapy strategies are being developed for the treatment of neurodegenerative disorders. Many of these strategies use constitutive expression of therapeutic transgenic proteins, and although functional in animal models of disease, this method is less likely to provide adequate flexibility for delivering therapy to humans. Ligand-inducible gene expression systems may be more appropriate for these conditions, especially within the central nervous system (CNS). Mifepristone's ability to cross the blood-brain barrier makes it an especially attractive ligand for this purpose. We describe the production of a mifepristone-inducible vector system for regulated expression of transgenes within the CNS. Our inducible system used a lentivirus-based vector platform for the ex vivo production of mifepristone-inducible murine neural progenitor cells that express our transgenes of interest. These cells were processed through a series of selection steps to ensure that the cells exhibited appropriate transgene expression in a dose-dependent and temporally controlled manner with minimal background activity. Inducible cells were then transplanted into the brains of rodents, where they exhibited appropriate mifepristone-inducible expression. These studies detail a strategy for regulated expression in the CNS for use in the development of safe and efficient gene therapy for neurological disorders.

242 HIGHLY EFFICIENT SLEEPING BEAUTY TRANSPOSON-MEDIATED TRANSGENESIS IN BOVINE FETAL FIBROBLASTS

Active transposon-mediated transgenesis is an emerging tool for basic and applied research in livestock. We have demonstrated the effectiveness of a helper-independent piggyBac transposon (pGENIE-3) for gene transfer into the genome of bovine cells (Alessio et al. 2014 Reprod. Domest. Anim. 49, 8). Here, we extend our previous research by examining the suitability of a Sleeping Beauty (SB) transposon-based methodology to deliver transgenes into the genome of bovine fetal fibroblasts (BFF), and the ability of these cells to support in vitro embryo development upon somatic cell nuclear transfer (SCNT). In a first experiment, BFF were chemically cotransfected (JetPRIME®, Polyplus-transfection, Illkirch, France) with a helper plasmid (pCMV-SB100X), which carries an expression cassette for the SB transposase, and the donor vector (pT2/Venus/RMCE) harboring an expression cassette for a fluorescent protein (Venus) flanked by the SB inverted terminal repeats (ITR). Three different ratios of helper and donor plasmids were studied: 1 : 2, 1 : 1 and 2 : 1. After 15 days of culture, the number of fluorescent colonies was counted on an inverted microscope. When vectors were used at ratios of 1 : 1 and 2 : 1, a 78-fold and 88-fold increase (P ≤ 0.05) in the number of fluorescent colonies compared with that in the no-transposase control were calculated. In a second experiment, BFF were chemically cotransfected with the helper vector pCMV-SB100X, and 2 donor transposons: pT2/Venus/RMCE and pT2/SV40-Neo. The former harbors a neo resistance cassette framed by SB ITRs. Different ratios of helper:donors (1 : 1 : 1, 2 : 1 : 1 and 2 : 0.5 : 0.5) were studied, and each ratio compared with a no-transposase control. After 15 days of antibiotic selection, the number of G418-resistant colonies was determined. Every time a functional SB transposase vector was included, the number of fluorescent and G418-resistant colonies was markedly higher compared with that in the respective control without transposase (P ≤ 0.001). Interestingly, all G418-resistant colonies expressed Venus. Molecular characterisation of genomic insertions in 6 monoclonal cell lines was performed by PCR and splinkerette PCR. PCR analysis confirmed presence of the Venus transgene in all cell lines. Splinkerette PCR results revealed at least 15 transposase-catalyzed genomic insertions of the transgene. Individual cells from a polyclonal SB transgenic fibroblast culture were used as nuclear donors to produce zona-free SCNT embryos. Of the reconstructed embryos, 33% reached blastocyst stage and about half of them expressed Venus. In conclusion, SB transposase is able to actively transpose monomeric copies of transgenes into the genome of bovine cells, which can be reprogrammed upon nuclear transfer to generate morphologically normal embryos expressing the transgene of interest.

Targeted Integration of Single-Copy Transgenes in Drosophila melanogaster Tissue-Culture Cells Using Recombination-Mediated Cassette Exchange.

Transfection of transgenes into Drosophila cultured cells is a standard approach for studying gene function. However, the number of transgenes present in the cell following transient transfection or stable random integration varies, and the resulting differences in expression level affect interpretation. Here we developed a system for Drosophila cell lines that allows selection of cells with a single-copy transgene inserted at a specific genomic site using recombination-mediated cassette exchange (RMCE). We used the φC31 integrase and its target sites attP and attB for RMCE. Cell lines with an attP-flanked genomic cassette were transfected with donor plasmids containing a transgene of interest (UAS-x), a dihydrofolate reductase (UAS-DHFR) gene flanked by attB sequences, and a thymidine kinase (UAS-TK) gene in the plasmid backbone outside the attB sequences. In cells undergoing RMCE, UAS-x and UAS-DHFR were exchanged for the attP-flanked genomic cassette, and UAS-TK was excluded. These cells were selected using methotrexate, which requires DHFR expression, and ganciclovir, which causes death in cells expressing TK. Pure populations of cells with one copy of a stably integrated transgene were efficiently selected by cloning or mass culture in ∼6 weeks. Our results show that RMCE avoids the problems associated with current methods, where transgene number is not controlled, and facilitates the rapid generation of Drosophila cell lines in which expression from a single transgene can be studied.

Abstract 2498: The MVA viral platform for the treatment of cancer and chronic infectious diseases: Clinical experience from four randomized controlled phase II studies

Abstract The modified vaccinia strain Ankara (MVA) is attenuated and non-propagative but retains infectivity and immunogenicity, its large DNA genome allows the insertion of several full-length coding sequences for disease associated antigens or other transgenes of interest. MVA based targeted immunotherapeutics are designed to induce a cytolytic cellular immune response. TG4010 expresses the full-length sequences of MUC1 and IL2 and was tested in combination with first line platin-based chemotherapy in stage IV non-small cell lung cancer (NSCLC) versus chemotherapy alone (NCT00415818 and NCT01383148); 108 plaque forming units (pfu) was given S/C weekly for 6 weeks then every three weeks up to progression. TG4001 expressing E6 and E7 of HPV16, and IL2 was assessed versus placebo in women with cervical intra-epithelial neoplasia (CIN2/3) (NCT01022346) at the dose of 5.107 pfu, three S/C injections a week apart. TG4040 expresses the HCV antigens NS3, NS4 and NS5B and was evaluated in combination with peg-interferon-α and ribavirin versus the same treatment alone in treatment-naïve patients with chronic hepatitis C, (107 pfu). Two distinct schedules of administration were evaluated: with or without immunotherapy run-in phase (NCT01055821). A total of 729 patients were included and randomized in these four studies: 443 in active arms (185 NSCLC, 136 CIN2/3, 122 HCV) and 286 in control arms. In all studies the repeated S/C administration of the MVA vectors alone or in combination therapies appeared feasible and well tolerated, neither dose reduction nor modification of the schedules of administration have been necessary. Injection site reactions have been the most frequent adverse events associated with treatment, mild to moderate in the majority of cases. Despite lower doses they seemed more prevalent for vectors expressing xenoantigens (TG4001 99% pts, TG4040 42% pts) than for TG4010 (31%). The phase II studies with TG4010 met their primary endpoints based respectively on 6 month progression free survival (PFS) and overall PFS. The primary endpoint of improvedcomplete early viral response (cEVR) was also achieved in the study with TG4040. These studies had in common 1/ to combine the MVA from the beginning of standard of care and 2/ to use a schedule of administration with a run-in phase of 6 or more weekly injections followed by at least monthly injections. The study with TG4001 did not meet its threshold-based primary endpoint of complete response but significantly more women in the experimental arm had a clearance of their cervical lesions at a conization performed 6 months later. Three MVA-based immunotherapeutics have shown meaningful clinical activity in different settings. Their favorable safety profile allows the combination of these products with standard of care therapies and also with immune checkpoint inhibitors (ongoing). Citation Format: Jean-Marc LIMACHER, Elisabeth QUOIX, Heiner WEDEMEYER, Francisco GARCIA, Pekka NIEMINEN, Gisèle LACOSTE, Delphine AGATHON, Geraldine HONNET, Elizabeth CALLEJA, Isabelle DIDILLON, Berangère MARIE-BASTIEN. The MVA viral platform for the treatment of cancer and chronic infectious diseases: Clinical experience from four randomized controlled phase II studies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2498. doi:10.1158/1538-7445.AM2015-2498

Murine granulocyte-macrophage colony-stimulating factor expressed from a bicistronic simian immunodeficiency virus-based integrase-defective lentiviral vector does not enhance T-cell responses in mice.

As a prelude to immunization studies in nonhuman primates, we compared in mice the immunogenicity of a simian immunodeficiency virus (SIV)-based integrase (IN)-defective lentiviral vector (IDLV) encoding the model antigen-enhanced green fluorescence protein (eGFP) in the presence or absence of the murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) expressed from an internal ribosomal entry site (IRES) sequence. BALB/c mice were immunized once intramuscularly with IDLV expressing eGFP alone or eGFP and mGM-CSF and immune responses were evaluated up to 90 days from the single intramuscular immunization. Results indicated that the mGM-CSF was unable to improve the magnitude and quality of the immune response against the eGFP transgene in the context of the SIV-based IDLV, as evaluated by enzyme-linked immunosorbent spot (ELISPOT) assays for interferon-γ (IFN-γ) and by intracellular cytokine staining for IFN-γ, interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-α). These findings suggest that for vaccination purposes, the presence of mGM-CSF expressed after the IRES in a SIV-based IDLV system does not favor the improvement of the immunological response against the transgene of interest. Further studies should investigate whether the selection of a different cytokine gene might improve the immune response against the transgene.

A toolkit for transgenesis at the ROSA26 locus by recombinase-mediated cassette exchange

We describe a toolkit to perform transgenesis at the ROSA26 locus by recombinase-mediated cassette exchange that will eliminate the inherent problem of random insertion via traditional pronuclear injection. A recombination site-tagged embryonic stem (ES) cell line and two cloning vectors were constructed to facilitate the generation of targeted ES cells with the transgene of interest at ROSA26. The experimental procedure is simple and efficient, and can be readily adapted in many laboratories for rapid generation of transgenic mice.

Streamlining the Genotyping Workflow with the MultiGene OptiMax

Streamlining the Genotyping Workflow with the MultiGene OptiMax