Multiple Transgenes Research Articles

Non-replicative herpes simplex virus genomic and amplicon vectors for gene therapy - an update.

Two major types of defective vectors have been derived from herpes simplex virus type 1 (HSV-1), non-replicative genomic vectors (nrHSV-1), and amplicon vectors. This review recapitulates the main features of both vector types and summarizes recent improvements in our understanding of virus/vector biology, particularly with regard to the critical role played by the overpowering of antiviral cellular defenses and the epigenetic control of viral gene expression. Over the past years, significant breakthroughs in vector design, genetic engineering, and HSV-1 biology have accelerated the development of nrHSV-1 vectors. The low immunogenicity and enhanced safety profiles allowed the successful translation of these vectors into several clinical trials, with some being approved by the FDA. Regarding amplicons, despite their advantage in carrying very large or multiple transgenes, and their potential to avoid genome dilution in dividing cells, the absence of production procedures capable of generating large amounts of helper-free amplicons at reasonable cost with GMP compliance, still limits the translation of these outstanding vectors to clinical trials.

An efficient AAV vector system of Rec2 serotype for intravenous injection to study metabolism in brown adipocytes in vivo

ObjectiveRecombinant adeno-associated virus (rAAV) vectors are powerful tools for the sustained expression of proteins in vivo and have been successfully used for mechanistic studies in mice. A major challenge associated with this method is to obtain tissue specificity and high expression levels without need of local virus administration. MethodsTo achieve this goal for brown adipose tissue (BAT), we developed a rAAV vector for intravenous bolus injection, which includes an expression cassette comprising an uncoupling protein-1 enhancer-promoter for transcription in brown adipocytes and miR122 target sequences for suppression of expression in the liver, combined with packaging in serotype Rec2 capsid protein. To test tissue specificity, we used a version of this vector expressing Cre recombinase to transduce mice with floxed alleles to knock out MLXIPL (ChREBP) or tdTomato-Cre reporter mice. ResultsWe demonstrated efficient Cre-dependent recombination in interscapular BAT and variable effects in minor BAT depots, but little or no efficacy in white adipose tissues, liver and other organs. Direct overexpression of glucose transporter SLC2A1 (GLUT1) using the rAAV vector in wild type mice resulted in increased glucose uptake and glucose-dependent gene expression in BAT, indicating usefulness of this vector to increase the function even of abundant proteins. ConclusionTaken together, we describe a novel brown adipocyte-specific rAAV method to express proteins for loss-of-function and gain-of-function metabolic studies. The approach will enable researchers to access brown fat swiftly, reduce animal breeding time and costs, as well as enable the creation of new transgenic mouse models combining multiple transgenes.

Prolonged xenokidney graft survival in sensitized NHP recipients by expression of multiple human transgenes in a triple knockout pig.

Genetic modification of porcine donors, combined with optimized immunosuppression, has been shown to improve outcomes of experimental xenotransplant. However, little is known about outcomes in sensitized recipients, a population that could potentially benefit the most from the clinical implementation of xenotransplantation. Here, five highly allosensitized rhesus macaques received a porcine kidney from GGTA1 (α1,3-galactosyltransferase) knockout pigs expressing the human CD55 transgene (1KO.1TG) and were maintained on an anti-CD154 monoclonal antibody (mAb)-based immunosuppressive regimen. These recipients developed de novo xenoreactive antibodies and experienced xenograft rejection with evidence of thrombotic microangiopathy and antibody-mediated rejection (AMR). In comparison, three highly allosensitized rhesus macaques receiving a kidney from GGTA1, CMAH (cytidine monophospho-N-acetylneuraminic acid hydroxylase), and b4GNT2/b4GALNT2 (β-1,4-N-acetyl-galactosaminyltransferase 2) knockout pigs expressing seven human transgenes including human CD46, CD55, CD47, THBD (thrombomodulin), PROCR (protein C receptor), TNFAIP3 (tumor necrosis factor-α-induced protein 3), and HMOX1 (heme oxygenase 1) (3KO.7TG) experienced significantly prolonged graft survival and reduced AMR, associated with dampened post-transplant humoral responses, early monocyte and neutrophil activation, and T cell repopulation. After withdrawal of all immunosuppression, recipients who received kidneys from 3KO.7TG pigs rejected the xenografts via AMR. These data suggest that allosensitized recipients may be suitable candidates for xenografts from genetically modified porcine donors and could benefit from an optimized immunosuppression regimen designed to target the post-transplant humoral response, thereby avoiding AMR.

Plant Synthetic Promoters

This article examines the structure and functions of the plant synthetic promoters frequently used to precisely regulate complex regulatory routes. It details the composition of native promoters and their interacting proteins to provide a better understanding of the tasks associated with synthetic promoter development. The production of synthetic promoters is performed by relatively small libraries produced generally by basic molecular or genetic engineering methods such as cis-element shuffling or domain swapping. The article also describes the preparation of large-scale libraries supported by synthetic DNA fragments, directed evolution, and machine or deep-learning methodologies. The broader application of novel, synthetic promoters reduces the prevalence of homology-based gene silencing or improves the stability of transgenes. A particularly interesting group of synthetic promoters are bidirectional forms, which can enable the expression of up to eight genes by one regulatory element. The introduction and controlled expression of several genes after one transgenic event strongly decreases the frequency of such problems as complex segregation patterns and the random integration of multiple transgenes. These complications are commonly observed during the transgenic crop development enabled by traditional, multistep transformation using genetic constructs containing a single gene. As previously tested DNA promoter fragments demonstrate low complexity and homology, their abundance can be increased by using orthogonal expression systems composed of synthetic promoters and trans-factors that do not occur in nature or arise from different species. Their structure, functions, and applications are rendered in the article. Among them are presented orthogonal systems based on transcription activator-like effectors (dTALEs), synthetic dTALE activated promoters (STAPs) and dCas9-dependent artificial trans-factors (ATFs). Synthetic plant promoters are valuable tools for providing precise spatiotemporal regulation and introducing logic gates into the complex genetic traits that are important for basic research studies and their application in crop plant development. Precisely regulated metabolic routes are less prone to undesirable feedback regulation and energy waste, thus improving the efficiency of transgenic crops.

Enhanced anti-tumor efficacy with multi-transgene armed mesenchymal stem cells for treating peritoneal carcinomatosis

BackgroundMesenchymal stem cells (MSCs) have garnered significant interest for their tumor-tropic property, making them potential therapeutic delivery vehicles for cancer treatment. We have previously shown the significant anti-tumour activity in mice preclinical models and companion animals with naturally occurring cancers using non-virally engineered MSCs with a therapeutic transgene encoding cytosine deaminase and uracil phosphoribosyl transferase (CDUPRT) and green fluorescent protein (GFP). Clinical studies have shown improved response rate with combinatorial treatment of 5-fluorouracil and Interferon-beta (IFNb) in peritoneal carcinomatosis (PC). However, high systemic toxicities have limited the clinical use of such a regime.MethodsIn this study, we evaluated the feasibility of intraperitoneal administration of non-virally engineered MSCs to co-deliver CDUPRT/5-Flucytosine prodrug system and IFNb to potentially enhance the cGAS-STING signalling axis. Here, MSCs were engineered to express CDUPRT or CDUPRT-IFNb. Expression of CDUPRT and IFNb was confirmed by flow cytometry and ELISA, respectively. The anti-cancer efficacy of the engineered MSCs was evaluated in both in vitro and in vivo model. ES2, HT-29 and Colo-205 were cocultured with engineered MSCs at various ratio. The cell viability with or without 5-flucytosine was measured with MTS assay. To further compare the anti-cancer efficacy of the engineered MSCs, peritoneal carcinomatosis mouse model was established by intraperitoneal injection of luciferase expressing ES2 stable cells. The tumour burden was measured through bioluminescence tracking.ResultsFirstly, there was no changes in phenotypes of MSCs despite high expression of the transgene encoding CDUPRT and IFNb (CDUPRT-IFNb). Transwell migration assays and in-vivo tracking suggested the co-expression of multiple transgenes did not impact migratory capability of the MSCs. The superiority of CDUPRT-IFNb over CDUPRT expressing MSCs was demonstrated in ES2, HT-29 and Colo-205 in-vitro. Similar observations were observed in an intraperitoneal ES2 ovarian cancer xenograft model. The growth of tumor mass was inhibited by ~ 90% and 46% in the mice treated with MSCs expressing CDUPRT-IFNb or CDUPRT, respectively.ConclusionsTaken together, these results established the effectiveness of MSCs co-expressing CDUPRT and IFNb in controlling and targeting PC growth. This study lay the foundation for the development of clinical trial using multigene-armed MSCs for PC.

Adeno-associated virus genome quantification with amplification-free CRISPR-Cas12a.

Efficient manufacturing of recombinant Adeno-Associated Viral (rAAV) vectors to meet rising clinical demand remains a major hurdle. One of the most significant challenges is the generation of large amounts of empty capsids without the therapeutic genome. There is no standardized analytical method to accurately quantify the viral genes, and subsequently the empty-to-full ratio, making the manufacturing challenges even more complex. We propose the use of CRISPR diagnostics (CRISPR-Dx) as a robust and rapid approach to determine AAV genome titers. We designed and developed the CRISPR-AAV Evaluation (CRAAVE) assay to maximize sensitivity, minimize time-to-result, and provide a potentially universal design for quantifying multiple transgene constructs encapsidated within different AAV serotypes. We also demonstrate an on-chip CRAAVE assay with lyophilized reagents to minimize end user assay input. The CRAAVE assay was able to detect AAV titers as low as 7e7 vg/mL with high precision (<3% error) in quantifying unknown AAV titers when compared with conventional quantitative PCR (qPCR) method. The assay only requires 30 min of assay time, shortening the analytical workflow drastically. Our results suggest CRISPR-Dx could be a promising tool for efficient rAAV genome titer quantification and has the potential to revolutionize biomanufacturing process analytical technology (PAT).

Physiological basis for xenotransplantation from genetically modified pigs to humans.

The collective efforts of scientists over multiple decades have led to advancements in molecular and cellular biology-based technologies including genetic engineering and animal cloning that are now being harnessed to enhance the suitability of pig organs for xenotransplantation into humans. Using organs sourced from pigs with multiple gene deletions and human transgene insertions, investigators have overcome formidable immunological and physiological barriers in pig-to-nonhuman primate (NHP) xenotransplantation and achieved prolonged pig xenograft survival. These studies informed the design of Revivicor's (Revivicor Inc, Blacksburg, VA) genetically engineered pigs with 10 genetic modifications (10 GE) (including the inactivation of 4 endogenous porcine genes and insertion of 6 human transgenes), whose hearts and kidneys have now been studied in preclinical human xenotransplantation models with brain-dead recipients. Additionally, the first two clinical cases of pig-to-human heart xenotransplantation were recently performed with hearts from this 10 GE pig at the University of Maryland. Although this review focuses on xenotransplantation of hearts and kidneys, multiple organs, tissues, and cell types from genetically engineered pigs will provide much-needed therapeutic interventions in the future.

Gene Doping Control Analysis of Human Erythropoietin Transgene in Equine Plasma by PCR-Liquid Chromatography High-Resolution Tandem Mass Spectrometry.

Gene doping involves the misuse of genetic materials to alter an athlete's performance, which is banned at all times in both human and equine sports. Quantitative polymerase chain reaction (qPCR) assays have been used to control the misuse of transgenes in equine sports. Our laboratory recently developed and implemented duplex as well as multiplex qPCR assays for transgenes detection. To further advance gene doping control, we have developed for the first time a sensitive and definitive PCR-liquid chromatography high-resolution tandem mass spectrometry (PCR-LC-HRMS/MS) method for transgene detection with an estimated limit of detection of below 100 copies/mL for the human erythropoietin (hEPO) transgene in equine plasma. The method involved magnetic-glass-particle-based extraction of DNA from equine plasma prior to PCR amplification with 2'-deoxyuridine 5'-triphosphate (dUTP) followed by treatments with uracil DNA glycosylase and hot piperidine for selective cleavage to give small oligonucleotide fragments. The resulting DNA fragments were then analyzed by LC-HRMS/MS. The applicability of this method has been demonstrated by the successful detection of hEPO transgene in a blood sample collected from a gelding (castrated male horse) that had been administered the transgene. This novel approach not only serves as a complementary method for transgene detection but also paves the way for developing a generic PCR-LC-HRMS/MS method for the detection of multiple transgenes.

Gene Stacking and Stoichiometric Expression of ER-Targeted Constructs Using "2A" Self-Cleaving Peptides.

Simultaneous stoichiometric expression of multiple genes plays a major part in modern research and biotechnology. Traditional methods for incorporating multiple transgenes (or "gene stacking") have drawbacks such as long time frames, uneven gene expression, gene silencing, and segregation derived from the use of multiple promoters. 2A self-cleaving peptides have emerged over the last two decades as a functional gene stacking method and have been used in plants for the co-expression of multiple genes under a single promoter. Here we describe design features of multicistronic polyproteins using 2A peptides for co-expression in plant cells and targeting to the endoplasmic reticulum (ER). We designed up to quad-cistronic vectors that could target proteins in tandem to the ER. We also exemplify the incorporation of self-excising intein domains within 2A polypeptides, to remove residue additions. These features could aid in the design of stoichiometric protein co-expression strategies in plants in combination with targeting to different subcellular compartments.

Comprehensive Comparison of AAV Purification Methods: Iodixanol Gradient Centrifugation vs. Immuno-Affinity Chromatography.

Recombinant adeno-associated viruses (AAVs) have emerged as a widely used gene delivery platform for both basic research and human gene therapy. To ensure and improve the safety profile of AAV vectors, substantial efforts have been dedicated to the vector production process development using suspension HEK293 cells. Here, we studied and compared two downstream purification methods, iodixanol gradient ultracentrifugation versus immuno-affinity chromatography (POROS™ CaptureSelect™ AAVX column). We tested multiple vector batches that were separately produced (including AAV5, AAV8, and AAV9 serotypes). To account for batch-to-batch variability, each batch was halved for subsequent purification by either iodixanol gradient centrifugation or affinity chromatography. In parallel, purified vectors were characterized, and transduction was compared both in vitro and in vivo in mice (using multiple transgenes: Gaussia luciferase, eGFP, and human factor IX). Each purification method was found to have its own advantages and disadvantages regarding purity, viral genome (vg) recovery, and relative empty particle content. Differences in transduction efficiency were found to reflect batch-to-batch variability rather than disparities between the two purification methods, which were similarly capable of yielding potent AAV vectors.

Systematic comparison of nonviral gene delivery strategies for efficient co-expression of two transgenes in human mesenchymal stem cells

BackgroundHuman mesenchymal stem cells (hMSCs) are being researched for cell-based therapies due to a host of unique properties, however, genetic modification of hMSCs, accomplished through nonviral gene delivery, could greatly advance their therapeutic potential. Furthermore, expression of multiple transgenes in hMSCs could greatly advance their clinical significance for treatment of multifaceted diseases, as individual transgenes could be expressed that target separate pathogenic drivers of complex diseases. Expressing multiple transgenes can be accomplished by delivering multiple DNA vectors encoding for each transgene, or by delivering a single poly-cistronic vector that encodes for each transgene and accomplishes expression through either use of multiple promoters, an internal ribosome entry site (IRES), or a 2A peptide sequence. These different transgene expression strategies have been used to express multiple transgenes in various mammalian cells, however, they have not been fully evaluated in difficult-to-transfect primary cells, like hMSCs. This study systematically compared four transgene expression and delivery strategies for expression of two reporter transgenes in four donors of hMSCs from two tissue sources using lipid- and polymer-mediate transfection, as follows: (i) delivery of separate DNA vectors in separate nanoparticles; (ii) delivery of separate DNA vectors combined in the same nanoparticle; (iii) delivery of a bi-cistronic DNA vector with an IRES sequence via nanoparticles; and (iv) delivery of a bi-cistronic DNA vector with a dual 2A peptide sequence via nanoparticles.ResultsOur results indicate that expression of two transgenes in hMSCs, independent of expression or delivery strategy, is inefficient compared to expressing a single transgene. However, delivery of separate DNA vectors complexed in the same nanoparticle, or delivery of a bi-cistronic DNA vector with a dual 2A peptide sequence, significantly increased the number of hMSCs expressing both transgenes compared to other conditions tested.ConclusionSeparate DNA vectors delivered in the same nanoparticle and bi-cistronic DNA vectors with dual 2A peptide sequences are highly efficient at simultaneously expressing two transgenes in multiple donors of hMSCs from different tissue sources. The data presented in this work can guide the development of hMSC transfection systems for delivery of multiple transgenes, with the goal of producing clinically relevant, genetically modified hMSCs.

API-192, an Allogeneic CD19/20 CAR-NKT Cell Product Derived from Cord Blood CD34+ HSPCs for the Treatment of B-Cell Malignancies

Autologous chimeric antigen receptor (CAR)-T cell therapies induce durable remissions and improve survival in patients with advanced B cell malignancies. However, challenges such as manufacturing variability, cost, and time to treatment highlight the need for an allogeneic, off-the-shelf approach that preserves the efficacy of autologous CAR-T. Additionally, antigen escape is one of the established mechanisms of resistance to current CAR-T therapies which target a single antigen. Classical natural killer T (NKT) cells express an invariant T cell receptor (iTCR) that is restricted to the conserved CD1d protein, making it a promising cell type for allogeneic therapies as they do not require TCR gene editing to avoid graft-vs-host (GvH) disease. Furthermore, NKT cells mediate dendritic cell cross-priming, target immunosuppressive myeloid cells/macrophages in the tumor microenvironment, and directly kill CD1d + tumor cells through the iTCR. Endogenous NKT cells are an extremely rare population in the blood (&amp;lt;1% of lymphocytes), posing manufacturing challenges. API-192 is a human cord blood CD34 + hematopoietic stem and progenitor cell-derived NKT cell product engineered to express dual CARs targeting CD19 and CD20 and armored with soluble IL-15 for improved expansion and persistence. The Appia Cells Utilized for Allogeneic (ACUA) platform enables the production of large numbers of product NKT cells co-expressing multiple transgenes at high purity (&amp;gt;90% iTCR + 19CAR + 20CAR +) with no purification step or gene editing. Phenotypically, API-192 cells are predominantly CD4 -CD8 - or CD4 -CD8 +, express major activating NK receptors, and harbor intracellular stores of cytotoxic molecules, resembling endogenous NKT cells. API-192 kills Raji and Nalm6 tumor cells in a CAR-specific and dose-dependent manner and can control multiple rounds of tumor challenge in vitro. API-192 secretes pro-inflammatory cytokines and proliferates in response to tumor antigen . In human tumor xenograft models in immunocompromised mice, API-192 robustly expands, suppresses tumor growth, and exhibits prolonged persistence, enabling control of a tumor rechallenge 70 days after initial dosing. API-192 expresses lower levels of MHC I and MHC II molecules relative to conventional CAR-T cells and elicits less IFN-γ release by NK and T cells in mixed lymphocyte reaction (MLR) assays, indicative of a more favorable immunogenic profile. Compared to conventional CAR-T cells, API-192 also secretes minimal levels of IFN-γ in response to co-culture with healthy donor peripheral blood mononuclear cells, consistent with the expression of a non-HLA restricted TCR and low GvH risk. These results demonstrate the feasibility, potency, and potential safety of API-192, warranting further development as a novel allogeneic therapy.

Pyramiding stacking of multigenes (PSM): a simple, flexible and efficient multigene stacking system based on Gibson assembly and gateway cloning.

Genetic engineering of complex metabolic pathways and multiple traits often requires the introduction of multiple genes. The construction of plasmids carrying multiple DNA fragments plays a vital role in these processes. In this study, the Gibson assembly and Gateway cloning combined Pyramiding Stacking of Multigenes (PSM) system was developed to assemble multiple transgenes into a single T-DNA. Combining the advantages of Gibson assembly and Gateway cloning, the PSM system uses an inverted pyramid stacking route and allows fast, flexible and efficient stacking of multiple genes into a binary vector. The PSM system contains two modular designed entry vectors (each containing two different attL sites and two selectable markers) and one Gateway-compatible destination vector (containing four attR sites and two negative selection markers). The target genes are primarily assembled into the entry vectors via two parallel rounds of Gibson assembly reactions. Then, the cargos in the entry constructs are integrated into the destination vector via a single tube Gateway LR reaction. To demonstrate PSM's capabilities, four and nine gene expression cassettes were respectively assembled into the destination vector to generate two binary expression vectors. The transgenic analysis of these constructs in Arabidopsis demonstrated the reliability of the constructs generated by PSM. Due to its flexibility, simplicity and versatility, PSM has great potential for genetic engineering, synthetic biology and the improvement of multiple traits.

CRISPR-Cas12a-integrated transgenes in genomic safe harbors retain high expression in human hematopoietic iPSC-derived lineages and primary cells

Discovery of genomic safe harbor sites (SHSs) is fundamental for multiple transgene integrations, such as reporter genes, chimeric antigen receptors (CARs), and safety switches, which are required for safe cell products for regenerative cell therapies and immunotherapies. Here we identified and characterized potential SHS in human cells. Using the CRISPR-MAD7 system, we integrated transgenes at these sites in induced pluripotent stem cells (iPSCs), primary T and natural killer (NK) cells, and Jurkat cell line, and demonstrated efficient and stable expression at these loci. Subsequently, we validated the differentiation potential of engineered iPSC toward CD34+ hematopoietic stem and progenitor cells (HSPCs), lymphoid progenitor cells (LPCs), and NK cells and showed that transgene expression was perpetuated in these lineages. Finally, we demonstrated that engineered iPSC-derived NK cells retained expression of a non-virally integrated anti-CD19 CAR, suggesting that several of the investigated SHSs can be used to engineer cells for adoptive immunotherapies.

SynLight: a bicistronic strategy for simultaneous active zone and cell labeling in the Drosophila nervous system.

At synapses, chemical neurotransmission mediates the exchange of information between neurons, leading to complex movement, behaviors, and stimulus processing. The immense number and variety of neurons within the nervous system make discerning individual neuron populations difficult, necessitating the development of advanced neuronal labeling techniques. In Drosophila, Bruchpilot-Short and mCD8-GFP, which label presynaptic active zones and neuronal membranes, respectively, have been widely used to study synapse development and organization. This labeling is often achieved via the expression of 2 independent constructs by a single binary expression system, but expression can weaken when multiple transgenes are expressed by a single driver. Recent work has sought to circumvent these drawbacks by developing methods that encode multiple proteins from a single transcript. Self-cleaving peptides, specifically 2A peptides, have emerged as effective sequences for accomplishing this task. We leveraged 2A ribosomal skipping peptides to engineer a construct that produces both Bruchpilot-Short-mStraw and mCD8-GFP from the same mRNA, which we named SynLight. Using SynLight, we visualized the putative synaptic active zones and membranes of multiple classes of olfactory, visual, and motor neurons and observed the correct separation of signal, confirming that both proteins are being generated separately. Furthermore, we demonstrate proof of principle by quantifying synaptic puncta number and neurite volume in olfactory neurons and finding no difference between the synapse densities of neurons expressing SynLight or neurons expressing both transgenes separately. At the neuromuscular junction, we determined that the synaptic puncta number labeled by SynLight was comparable to the endogenous puncta labeled by antibody staining. Overall, SynLight is a versatile tool for examining synapse density in any nervous system region of interest and allows new questions to be answered about synaptic development and organization.

A multiplex qPCR assay for transgenes detection: A novel approach for gene doping control in horseracing using conventional laboratory setup.

Illicit administration of transgene into horses is a form of gene doping that has been a key concern in equine sports. The large number of potential performance-enhancing transgenes has demanded a cost-effective and reliable detection method. Multiplex qPCR is a relevant technique, but the cross-talking between fluorophores and high background noise limits the method sensitivity and specificity. This study reports a simpler multiplexing approach by using the same fluorophore for four hydrolysis probes each targeting one of the four transgenes: human growth hormone, insulin-like growth factor 1, equine erythropoietin and interleukin-10. Any positive findings from this multiplex qPCR assay can then be confirmed by individual qPCR assays to identify potential transgene(s). This has effectively eliminated the cross-talking issue and allowed an improved signal-to-noise than conventional multiplex qPCR assay. It has also removed the limitation imposed by the available choice of fluorophores and optical channels of qPCR instruments on the number of transgenes that can be analysed in a multiplex qPCR assay. This novel multiplex qPCR has been successfully validated. The estimated limits of detection were ~1500-2500 copies/mL of blood, thus demonstrating comparable sensitivity with the corresponding duplex qPCR assays. Concurring results were obtained by analysing hundreds of official blood samples provided by racehorses with this multiplex qPCR assay and the accredited individual duplex qPCR assays. This novel multiplex qPCR assay for detecting multiple transgenes is a cost-effective screening method using a conventional laboratory setup and has opened up the potential to include the testing of additional transgenes in a single assay.

Self-cleaving peptides for expression of multiple genes in Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum is a model for a wide range of biological processes including chemotaxis, cell-cell communication, phagocytosis, and development. Interrogating these processes with modern genetic tools often requires the expression of multiple transgenes. While it is possible to transfect multiple transcriptional units, the use of separate promoters and terminators for each gene leads to large plasmid sizes and possible interference between units. In many eukaryotic systems this challenge has been addressed through polycistronic expression mediated by 2A viral peptides, permitting efficient, co-regulated gene expression. Here, we screen the most commonly used 2A peptides, porcine teschovirus-1 2A (P2A), Thosea asigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth disease virus 2A (F2A), for activity in D. discoideum and find that all the screened 2A sequences are effective. However, combining the coding sequences of two proteins into a single transcript leads to notable strain-dependent decreases in expression level, suggesting additional factors regulate gene expression in D. discoideum that merit further investigation. Our results show that P2A is the optimal sequence for polycistronic expression in D. discoideum, opening up new possibilities for genetic engineering in this model system.

CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX.

Poxvirus vectors represent versatile modalities for engineering novel vaccines and cancer immunotherapies. In addition to their oncolytic capacity and immunogenic influence, they can be readily engineered to express multiple large transgenes. However, the integration of multiple payloads into poxvirus genomes by traditional recombination-based approaches can be highly inefficient, time-consuming and cumbersome. Herein, we describe a simple, cost-effective approach to rapidly generate and purify a poxvirus vector with multiple transgenes. By utilizing a simple, modular CRISPR/Cas9 assisted-recombinant vaccinia virus engineering (CARVE) system, we demonstrate generation of a recombinant vaccinia virus expressing three distinct transgenes at three different loci in less than 1 week. We apply CARVE to rapidly generate a novel immunogenic vaccinia virus vector, which expresses a bacterial diadenylate cyclase. This novel vector, STINGPOX, produces cyclic di-AMP, a STING agonist, which drives IFN signaling critical to the anti-tumor immune response. We demonstrate that STINGPOX can drive IFN signaling in primary human cancer tissue explants. Using an immunocompetent murine colon cancer model, we demonstrate that intratumoral administration of STINGPOX in combination with checkpoint inhibitor, anti-PD1, promotes survival post-tumour challenge. These data demonstrate the utility of CRISPR/Cas9 in the rapid arming of poxvirus vectors with therapeutic payloads to create novel immunotherapies.

Therapy with oncolytic viruses: progress and challenges.

Oncolytic viruses (OVs) are an emerging class of cancer therapeutics that offer the benefits of selective replication in tumour cells, delivery of multiple eukaryotic transgene payloads, induction of immunogenic cell death and promotion of antitumour immunity, and a tolerable safety profile that largely does not overlap with that of other cancer therapeutics. To date, four OVs and one non-oncolytic virushave been approved for the treatment of cancer globally although talimogene laherparepvec (T-VEC) remains the only widely approved therapy. T-VEC is indicated for the treatment of patients with recurrent melanoma after initial surgery and was initially approved in 2015. An expanding body of data on the clinical experience of patients receiving T-VEC is now becoming available as are data from clinical trials of various other OVs in a range of other cancers. Despite increasing research interest, a better understanding of the underlying biology and pharmacology of OVs is needed to enable the full therapeutic potential of these agents in patients with cancer. In this Review, we summarize the available data and provide guidance on optimizing the use of OVs in clinical practice, with a focus on the clinical experience with T-VEC. We describe data on selected novel OVs that are currently in clinical development, either as monotherapies or as part of combination regimens. We also discuss some of the preclinical, clinical and regulatory hurdles that have thus far limited the development of OVs.

Sulfadiazine and phosphinothricin selection systems optimised for the transformation of tobacco BY-2 cells.

We extended the applicability of the BY-2 cell line as a model by introducing two new selection systems. Our protocol provides guidelines for optimising Basta selection in other recalcitrant models. Tobacco BY-2 cell line is the most commonly used cytological model in plant research. It is uniform, can be simply treated by chemicals, synchronised and easily transformed. However, only a few selection systems are available that complicate advanced studies using multiple stacked transgenes and extensive gene editing. In our work, we adopted for BY-2 cell line two other selection systems: sulfadiazine and phosphinothricin (PPT, an active ingredient of Basta herbicide). We show that sulfadiazine can be used in a wide range of concentrations. It is suitable for co-transformation and subsequent double selection with kanamycin or hygromycin, which are standardly used for BY-2 transformation. We also have domesticated the sulfadiazine resistance for the user-friendly GoldenBraid cloning system. Compared to sulfadiazine, establishing selection on phosphinothricin was considerably more challenging. It did not work in any concentration of PPT with standardly cultured cells. Since the selection is based on blocking glutamine synthetase and consequent ammonium toxicity and deficiency of assimilated nitrogen, we tried to manipulate nitrogen availability. We found that the PPT selection reliably works only with nitrogen-starved cells with reduced nitrate reserves that are selected on a medium without ammonium nitrate. Both these adjustments prevent the release of large amounts of ammonium, which can toxify the entire culture in the case of standardly cultured cells. Since high nitrogen reserves can be a common feature of in vitro cultures grown on MS media, nitrogen starvation could be a key step in establishing phosphinothricin resistance in other plant models.