Prolonged Transgene Expression Research Articles

Evaluation of bird-adapted self-amplifying mRNA vaccine formulations in chickens

Each year, millions of poultry succumb to highly pathogenic avian influenza A virus (AIV) and infectious bursal disease virus (IBDV) infections. Conventional vaccines based on inactivated or live-attenuated viruses are useful tools for disease prevention and control, yet, they often fall short in terms of safety, efficacy, and development times. Therefore, versatile vaccine platforms are crucial to protect poultry from emerging viral pathogens. Self-amplifying (replicon) RNA vaccines offer a well-defined and scalable option for the protection of both animals and humans. The best-studied replicon platform, based on the Venezuelan equine encephalitis virus (VEEV; family Togaviridae) TC-83 vaccine strain, however, displays limited efficacy in poultry, warranting the exploration of alternative, avian-adapted, replicon platforms. In this study, we engineered two Tembusu virus (TMUV; family Flaviviridae) replicons encoding varying capsid gene lengths and compared these to the benchmark VEEV replicon in vitro. The TMUV replicon system exhibited a robust and prolonged transgene expression compared to the VEEV replicon system in both avian and mammalian cells. Moreover, the TMUV replicon induced a lesser cytopathic effect compared to the VEEV replicon RNA in vitro. DNA-launched versions of the TMUV and VEEV replicons (DREP) were also developed. The replicons successfully expressed the AIV haemagglutinin (HA) glycoproteins and the IBDV capsid protein (pVP2). To assess the immune responses elicited by the TMUV replicon system in chickens, a prime-boost vaccination trial was conducted using lipid nanoparticle (LNP)-formulated replicon RNA and DREP encoding the viral (glyco)proteins of AIV or IBDV. Both TMUV and VEEV replicon RNAs were unable to induce a humoral response against AIV. However, TMUV replicon RNA induced IBDV-specific seroconversion in vaccinated chickens, in contrast to VEEV replicon RNA, which showed no significant humoral response. In both AIV and IBDV immunization studies, VEEV DREP generated the highest (neutralizing) antibody responses, which underscores the potential for self-amplifying mRNA vaccine technology to combat emerging poultry diseases.

Antibiotic-Free Gene Vectors: A 25-Year Journey to Clinical Trials.

Until very recently, the major use, for gene therapy, specifically of linear or circular DNA, such as plasmids, was as ancillary products for viral vectors' production or as a genetic template for mRNA production. Thanks to targeted and more efficient physical or chemical delivery techniques and to the refinement of their structure, non-viral plasmid DNA are now under intensive consideration as pharmaceutical drugs. Plasmids traditionally carry an antibiotic resistance gene for providing the selection pressure necessary for maintenance in a bacterial host. Nearly a dozen different antibiotic-free gene vectors have now been developed and are currently assessed in preclinical assays and phase I/II clinical trials. Their reduced size leads to increased transfection efficiency and prolonged transgene expression. In addition, associating non-viral gene vectors and DNA transposons, which mediate transgene integration into the host genome, circumvents plasmid dilution in dividing eukaryotic cells which generate a loss of the therapeutic gene. Combining these novel molecular tools allowed a significantly higher yield of genetically engineered T and Natural Killer cells for adoptive immunotherapies due to a reduced cytotoxicity and increased transposition rate. This review describes the main progresses accomplished for safer, more efficient and cost-effective gene and cell therapies using non-viral approaches and antibiotic-free gene vectors.

AAV-mediated delivery of a Sleeping Beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells.

Engineering cells for adoptive therapy requires overcoming limitations in cell viability and, in the efficiency of transgene delivery, the duration of transgene expression and the stability of genomic integration. Here we report a gene-delivery system consisting of a Sleeping Beauty (SB) transposase encoded into a messenger RNA delivered by an adeno-associated virus (AAV) encoding an SB transposon that includes the desired transgene, for mediating the permanent integration of the transgene. Compared with lentiviral vectors and with the electroporation of plasmids of transposon DNA or minicircle DNA, the gene-delivery system, which we named MAJESTIC (for 'mRNA AAV-SB joint engineering of stable therapeutic immune cells'), offers prolonged transgene expression, as well as higher transgene expression, therapeutic-cell yield and cell viability. MAJESTIC can deliver chimeric antigen receptors (CARs) into T cells (which we show lead to strong anti-tumour activity in vivo) and also transduce natural killer cells, myeloid cells and induced pluripotent stem cells with bi-specific CARs, kill-switch CARs and synthetic T-cell receptors.

High spontaneous integration rates of end-modified linear DNAs upon mammalian cell transfection

In gene therapy, potential integration of therapeutic transgene into host cell genomes is a serious risk that can lead to insertional mutagenesis and tumorigenesis. Viral vectors are often used as the gene delivery vehicle, but they are prone to undergoing integration events. More recently, non-viral delivery of linear DNAs having modified geometry such as closed-end linear duplex DNA (CELiD) have shown promise as an alternative, due to prolonged transgene expression and less cytotoxicity. However, whether modified-end linear DNAs can also provide a safe, non-integrating gene transfer remains unanswered. Herein, we compare the genomic integration frequency upon transfection of cells with expression vectors in the forms of circular plasmid, unmodified linear DNA, CELiDs with thioester loops, and Streptavidin-conjugated blocked-end linear DNA. All of the forms of linear DNA resulted in a high fraction of the cells being stably transfected—between 10 and 20% of the initially transfected cells. These results indicate that blocking the ends of linear DNA is insufficient to prevent integration.

Abstract 5335: circAde: a circRNA-based system for prolonged and more effective treatment of cancer

Abstract In recently performed phase I/II clinical studies, we have demonstrated successful delivery of vectorized transgene-encoding mRNA payloads to solids tumors with substantial clinical benefit. In patients, persistent and prolonged transgene expression correlated with positive clinical outcome, and thus temporal expansion of the transgene expression profile is expected to improve the therapeutic benefit even further. Recently, circular RNA (circRNA) has been discovered as a novel class of endogenously expressed RNA. CircRNAs, in contrast to mRNAs, are resistant to exo-nucleolytic decay which results in high intra-cellular stability and persistent expression within cells. Therefore, circRNA-based systems serve as an interesting new technology towards prolonged effects of treatment. Here, we show that our proprietary vector system, circAde, allows high expression of circRNAs through spliceosome-dependent biogenesis. By comparing the protein output from circAde and mRNA-based vectors, we show that the circAde-system out-performs conventional mRNA-based design, both in terms of transgene yield and temporal sustainability of expression. Moreover, using a panel of proof-of-concept-reporters, we show that certain design-features greatly influence the circRNA production and subsequent protein expression from the vector. Without the 5’cap, circRNAs are dependent on IRESs (internal ribosome entry sequences) for effective translation. By conducting two orthogonal IRES screens on 1000+ putative IRES elements, we have identified the most effective IRES sequence in melanoma and lung-cancer cells supporting effective circRNA production and high-yield protein expression, thereby improving our circAde vector system even further. Our results open a new platform towards development of novel, more effective strategies for the treatment of cancer and other settings in which durable transgene expression is desirable. Citation Format: Eoghan T. O'leary, Sara Vikberg, Emilie Foord, Erik D. Wiklund, Lone Ottesen, Victor Levitsky, Thomas B. Hansen. circAde: a circRNA-based system for prolonged and more effective treatment of cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5335.

Blocking T-cell egress with FTY720 extends DNA vaccine expression but reduces immunogenicity.

Optimal immunogenicity from nucleic acid vaccines requires a balance of antigen expression that effectively engages the host immune system without generating a cellular response that rapidly destroys cells producing the antigen and thereby limiting vaccine antigen expression. We investigated the role of the cellular response on the expression and antigenicity of DNA vaccines using a plasmid DNA construct expressing luciferase. Repeated intramuscular administration led to diminished luciferase expression, suggesting a role for immune‐mediated clearance of expression. To investigate the role of cell trafficking, we used the sphingosine 1‐phosphate receptor (S1PR) modulator, FTY720 (Fingolimod), which traps lymphocytes within the lymphoid tissues. When lymphocyte trafficking was blocked with FTY720, DNA transgene expression was maintained at a constant level for a significantly extended time period. Both continuous and staggered administration of FTY720 prolonged transgene expression. However, blocking lymphocyte egress during primary transgene administration did not result in an increase of transgene expression during secondary administration. Interestingly, there was a disconnect between transgene expression and immunogenicity, as increasing expression by this approach did not enhance the overall immune response. Furthermore, when FTY720 was administered alongside a DNA vaccine expressing the HIV gp140 envelope antigen, there was a significant reduction in both antigen‐specific antibody and T‐cell responses. This indicates that the developing antigen‐specific cellular response clears DNA vaccine expression but requires access to the site of expression in order to develop an effective immune response.

Macrophage Depletion via Clodronate Pretreatment Reduces Transgene Expression from AAV Vectors In Vivo

Adeno-associated virus is a popular gene delivery vehicle for gene therapy studies. A potential roadblock to widespread clinical adoption is the high vector doses required for efficient transduction in vivo, and the potential for subsequent immune responses that may limit prolonged transgene expression. We hypothesized that the depletion of macrophages via systemic delivery of liposome-encapsulated clodronate would improve transgene expression if given prior to systemic AAV vector administration, as has been shown to be the case with adenoviral vectors. Contrary to our expectations, clodronate liposome pretreatment resulted in significantly reduced transgene expression in the liver and heart, but permitted moderate transduction of the white pulp of the spleen. There was a remarkable localization of transgene expression from the red pulp to the center of the white pulp in clodronate-treated mice compared to untreated mice. Similarly, a greater proportion of transgene expression could be observed in the medulla located in the center of the lymph node in mice treated with clodronate-containing liposomes as compared to untreated mice where transgene expression was localized primarily to the cortex. These results underscore the highly significant role that the immune system plays in influencing the distribution and relative numbers of transduced cells in the context of AAV-mediated gene delivery.

In Vivo bone tissue induction by freeze-dried collagen-nanohydroxyapatite matrix loaded with BMP2/NS1 mRNAs lipopolyplexes

Messenger RNA (mRNA) activated matrices (RAMs) are interesting to orchestrate tissue and organ regeneration due to the in-situ and sustained production of functional proteins. However, the immunogenicity of in vitro transcribed mRNA and the paucity of proper in vivo mRNA delivery vector need to be overcome to exert the therapeutic potential of RAM. We developed a dual mRNAs system for in vitro osteogenesis by co-delivering NS1 mRNA with BMP2 mRNA to inhibit RNA sensors and enhance BMP-2 expression. Next, we evaluated a lipopolyplex (LPR) formulation platform for in vivo mRNA delivery and adapted the LPRs for RAM preparation. The LPR formulated BMP2/NS1 mRNAs were incorporated into an optimized collagen-nanohydroxyapatite scaffold and freeze-dried to prepare ready-to-use RAMs. The loaded BMP2/NS1 mRNAs lipopolyplexes maintained their spherical morphology in the RAM, thanks to the core-shell structure of LPR. The mRNAs release from RAMs lasted for 16 days resulting in an enhanced prolonged transgene expression period compared to direct cell transfection. Once subcutaneously implanted in mice, the BMP2/NS1 mRNAs LPRs containing RAMs (RAM-BMP2/NS1) induced significant new bone tissue than those without NS1 mRNA, eight weeks post implantation. Overall, our results demonstrate that the BMP2/NS1 dual mRNAs system is suitable for osteogenic engagement, and the freeze-dried RAM-BMP2/NS1 could be promising off-the-shelf products for clinical orthopedic practice.

AAV6 as an effective gene delivery vector for prolonged transgene expression in intervertebral disc cells in vivo

Intervertebral disc degeneration is the main contributor to low back pain, now the leading cause of disability worldwide. Gene transfer, either in a therapeutic attempt or in basic research to understand the mechanisms of disc degeneration, is a fascinating and promising tool to manipulate the complex physiology of the disc. Viral vectors based on the adeno-associated virus (AAV) have emerged as powerful transgene delivery vehicles yet a systematic investigation into their respective tropism, transduction efficiency, and relative toxicity have not yet been performed in the disc in vivo. Herein, we used in vivo bioluminescence imaging to systematically compare multiple AAV serotypes, injection volumes, titers, promoters, and luciferase reporters to determine which result in high transduction efficiency of murine nucleus pulposus (NP) cells in vivo. We find that AAV6 using a CAG promoter to drive transgene expression, delivered into the NP of murine caudal discs at a titer of 1011 GC/mL, provides excellent transduction efficiency/kinetics and low toxicity in vivo. We also show, for the first time, that the transduction of NP cells can be significantly boosted in vivo by the use of small cell permeabilization peptides. Finally, to our knowledge, we are the first to demonstrate the use of optical tissue clearing and three-dimensional lightsheet microscopy in the disc, which was used to visualize fine details of tissue and cell architecture in whole intact discs following AAV6 delivery. Taken together, these data will contribute to the success of using AAV-mediated gene delivery for basic and translational studies of the IVD.

Maintenance of Plasmid Expression in vivo Depends Primarily on the CpG Contents of the Vector and Transgene

Plasmid-mediated gene therapy, being a safe and relatively inexpensive therapeutic strategy, is plagued by a fast silencing of transgene expression. The silencing severely reduces the long-term efficiency of plasmid vectors. We have earlier constructed a low-CpG pMBR2 plasmid vector supporting prolonged expression of transgenes in mesenchymal stem cells in vitro. Long-term expression from the pMBR2 vector was studied for the wild-type mouse secreted alkaline phosphatase gene (mSEAPTwt) and its version devoid of CpGs (mSEAP0) after vector electroporation into mouse hindlimb muscles and hydrodynamic delivery to the liver. The mSEAP levels in the blood were measured over one year. With the pMBR2-mSEAP0 construct, the mSEAP levels in leg muscles increased more than 2.5-fold in the first two months and remained higher than the initial level until the end of the experiment. Far lower expression levels were observed with the control pCDNA3.1-mSEAP0 construct. Expression from pMBR2-mSEAPwt decreased to about 40% after 6 months and remained at similar levels thereafter. In the mouse liver, expression from pMBR2-mSEAP0 was approximately halved within the first 18 weeks and then decrease slowly to the final 17% level. Expression from pMBR2-mSEAPwt initially dropped to 18% and remained at approximately 10% thereafter. In contrast, expression from pCDNA3.1-mSEAP0 sharply dropped to 5% after 2 weeks and remained at nearly zero levels throughout the rest of the experiment. Thus, both vector and transgene should have significantly reduced CpG contents to ensure prolonged plasmid-mediated expression in the liver, while minimizing the vector CpG content is sufficient for expression in skeletal muscles. The results suggested additionally that the localization of S/MAR elements within the transcription unit, in contrast to their outside location, results in significant reduction of the level of secreted, but not cytoplasmic, proteins.

Microvesicle-Mediated Delivery of Minicircle DNA Results in Effective Gene-Directed Enzyme Prodrug Cancer Therapy.

An emerging approach for cancer treatment employs the use of extracellular vesicles, specifically exosomes and microvesicles, as delivery vehicles. We previously demonstrated that microvesicles can functionally deliver plasmid DNA to cells and showed that plasmid size and sequence, in part, determine the delivery efficiency. In this study, delivery vehicles comprised of microvesicles loaded with engineered minicircle (MC) DNA that encodes prodrug converting enzymes developed as a cancer therapy in mammary carcinoma models. We demonstrated that MCs can be loaded into shed microvesicles with greater efficiency than their parental plasmid counterparts and that microvesicle-mediated MC delivery led to significantly higher and more prolonged transgene expression in recipient cells than microvesicles loaded with the parental plasmid. Microvesicles loaded with MCs encoding a thymidine kinase (TK)/nitroreductase (NTR) fusion protein produced prolonged TK-NTR expression in mammary carcinoma cells. In vivo delivery of TK-NTR and administration of prodrugs led to the effective killing of both targeted cells and surrounding tumor cells via TK-NTR-mediated conversion of codelivered prodrugs into active cytotoxic agents. In vivo evaluation of the bystander effect in mouse models demonstrated that for effective therapy, at least 1% of tumor cells need to be delivered with TK-NTR-encoding MCs. These results suggest that MC delivery via microvesicles can mediate gene transfer to an extent that enables effective prodrug conversion and tumor cell death such that it comprises a promising approach to cancer therapy.

Lentiviral Vector-Mediated Expression of Exoenzyme C3 Transferase Lowers Intraocular Pressure in Monkeys.

Primary open-angle glaucoma (POAG) is considered a lifelong disease characterized by optic nerve deterioration and visual field damage. Although the disease progression can usually be controlled by lowering the intraocular pressure (IOP), therapeutic effects of current approaches do not last long. Gene therapy could be a promising method for persistent treatment of the disease. Our previous study demonstrated that gene transfer of exoenzyme C3 transferase (C3) to the trabecular meshwork (TM) to inhibit Rho GTPase (Rho), the upstream signal molecule of Rho-associated kinase (ROCK), resulted in lowered IOP in normal rodent eyes. In the present study, we show that the lentiviral vector (LV)-mediated C3 expression inactivates RhoA in human TM cells by ADP ribosylation, resulting indisruption of the actin cytoskeleton and altered cell morphology. In addition, intracameral delivery of the C3 vector to monkey eyes leads to persistently lowered IOP without obvious signs of inflammation. This is the first report of using a vector to transduce the TM of an alive non-human primate with a gene that alters cellular machinery and physiology. Our results in non-human primates support that LV-mediated C3 expression in the TM may have therapeutic potential forglaucoma, the leading cause of irreversible blindness in humans.

Attenuate Newcastle disease virus by codon modification of the glycoproteins and phosphoprotein genes

A codon modification strategy was used to attenuate the avian pathogenicity of an oncolytic mesogenic Newcastle disease virus (NDV) by targeting the three major virulence factors: the fusion (F) protein, hemagglutinin neuraminidase (HN) and phosphoprotein (P). Recoding the F and HN genes with rare codons greatly reduced expression of both F and HN proteins and resulted in their low incorporation into virions. The F and HN recoded virus was partially attenuated in chickens even when the F protein cleavage site was modified. Full attenuation was achieved when the 5′ portion of the P gene was recoded. The recoded P, F and HN triple gene mutant exhibited delayed cell death in human cancer cells with prolonged expression of a GFP transgene. While this engineered attenuated NDV strain has lower oncolytic potency, its capacity for prolonged transgene expression may allow its use as a vaccine or gene delivery vector.

An efficient, non-viral dendritic vector for gene delivery in tissue engineering.

Recent developments within the field of tissue engineering (TE) have shown that biomaterial scaffold systems can be augmented via the incorporation of gene therapeutics. The objective of this study was to assess the potential of the activated polyamidoamine dendrimer (dPAMAM) transfection reagent (SuperfectTM) as a gene delivery system to mesenchymal stem cells (MSCs) in both monolayer and 3D culture on collagen based scaffolds. dPAMAM-pDNA polyplexes at a mass ratio (M:R) 10:1 (dPAMAM : pDNA) (1 ug pDNA) were capable of facilitating prolonged reporter gene expression in monolayer MSCs which was superior to that facilitated using polyethylenimine (PEI)-pDNA polyplexes (2 ug pDNA). When dPAMAM-pDNA polyplexes (1 ug pDNA) were soak loaded onto a collagen-chondroitin sulphate (CS) scaffold prolonged transgene expression was facilitated which was higher than that obtained for a PEI-pDNA polyplex (2 ug pDNA) loaded scaffold. Transgene expression was dependent on the composite nature of the collagen scaffold with varying expression profiles obtained from a suite of collagen constructs including a collagen alone, collagen-CS, collagen-hydroxyapatite, collagen-nanohydroxyapatite and collagen-hyaluronic acid scaffold. Therefore, the dPAMAM vector described herein represents a biocompatible, effective gene delivery vector for TE applications which, via matching with a particular composite scaffold type, can be tailored for regeneration of various tissue defects.

Enzyme-crosslinked gene-activated matrix for the induction of mesenchymal stem cells in osteochondral tissue regeneration.

In this study, a gene-activated matrix (GAM) to promote the growth of both cartilage and subchondral bone within a single integrated construct is developed. It has the capacity to promote transfection of human mesenchymal stem cells (hMSCs) and the functional osteochondral tissues formation. The results show that the sustained release of plasmids including TGF-beta and BMP-2 from GAM could promote prolonged transgene expression and stimulate hMSCs differentiation into the osteogenic and chondrogenic lineages by spatial control manner. This improved delivery method should enhance the functionalized composite graft to accelerate healing process in vitro and in vivo for osteochondral tissue regeneration.

Sustained viral gene delivery from a micro-fibrous, elastomeric cardiac patch to the ischemic rat heart.

Biodegradable and elastomeric patches have been applied to the surface of infarcted hearts as temporary mechanical supports to effectively alter adverse left ventricular remodeling processes. In this report, recombinant adeno-associated virus (AAV), known for its persistent transgene expression and low pathogenicity, was incorporated into elastomeric polyester urethane urea (PEUU) and polyester ether urethane urea (PEEUU) and processed by electrospinning into two formats (solid fibers and core-sheath fibers) designed to influence the controlled release behavior. The extended release of AAV encoding green fluorescent protein (GFP) was assessed invitro. Sustained and localized viral particle delivery was achieved over 2 months invitro. The biodegradable cardiac patches with or without AAV-GFP were implanted over rat left ventricular lesions three days following myocardial infarction to evaluate the transduction effect of released viral vectors. AAV particles were directly injected into the infarcted hearts as a control. Cardiac function and remodeling were significantly improved for 12 weeks after patch implantation compared to AAV injection. More GFP genes was expressed in the AAV patch group than AAV injection group, with both α-SMA positive cells and cardiac troponin T positive cells transduced in the patch group. Overall, the extended release behavior, prolonged transgene expression, and elastomeric mechanical properties make the AAV-loaded scaffold an attractive option for cardiac tissue engineering where both gene delivery and appropriate mechanical support are desired.

Enhanced critical-size calvarial bone healing by ASCs engineered with Cre/loxP-based hybrid baculovirus.

Calvarial bone repair remains challenging for adults. Although adipose-derived stem cells (ASCs) hold promise to heal bone defects, use of ASCs for critical-size calvarial bone repair is ineffective. Stromal cell-derived factor 1 (SDF-1) is a chemokine capable of triggering stem cell migration. Although recombinant SDF-1 protein is co-delivered with other molecules including BMP-2 to facilitate calvarial bone repair, these approaches did not yield satisfactory healing. This study aimed to exploit a newly developed Cre/loxP-based hybrid baculovirus for efficient gene delivery and prolonged transgene expression in ASCs. We demonstrated that transduction of rat ASCs with the hybrid Cre/loxP-based baculovirus enabled robust and sustained expression of functional BMP-2 and SDF-1. Expression of BMP-2 or SDF-1 alone failed to effectively induce rat ASCs osteogenesis and healing of critical-size calvarial bone defects. Nonetheless, prolonged BMP-2/SDF-1 co-expression in ASCs synergistically activated both Smad and ERK1/2 pathways and hence potentiated the osteogenesis. Consequently, transplantation of the hybrid baculovirus-engineered, BMP-2/SDF-1-expressing ASCs/scaffold constructs potently healed the critical-size (6mm) calvarial bone defects (filling ≈70% of defect volume), which considerably outperformed the calvarial bone repair using BMP-2/SDF-1 delivered with biomaterial-based scaffolds. These data implicated the potential of Cre/loxP-based hybrid baculovirus vector for ASCs engineering and calvarial bone healing.

Minicircle microporation-based non-viral gene delivery improved the targeting of mesenchymal stem cells to an injury site.

Genetic engineering approaches to improve the therapeutic potential of mesenchymal stem cells (MSCs) have been made by viral and non-viral gene delivery methods. Viral methods have severe limitations in clinical application because of potential oncogenic, pathogenic, and immunogenic risks, while non-viral methods have suffered from low transfection efficiency and transient weak expression as MSCs are hard-to-transfect cells. In this study, minicircle, which is a minimal expression vector free of bacterial sequences, was employed for MSC transfection as a non-viral gene delivery method. The conventional cationic liposome method was not effective for MSC transfection as it resulted in very low transfection efficiency (less than 5%). Microporation, a new electroporation method, greatly improved the transfection efficiency of minicircles by up to 66% in MSCs without any significant loss of cell viability. Furthermore, minicircle microporation generated much stronger and prolonged transgene expression compared with plasmid microporation. When MSCs microporated with minicircle harboring firefly luciferase gene were subcutaneously injected to mice, the bioluminescence continued for more than a week, whereas the bioluminescence of the MSCs induced by plasmid microporation rapidly decreased and disappeared in mice within three days. By minicircle microporation as a non-viral gene delivery, MSCs engineered to overexpress CXCR4 showed greatly increased homing ability toward an injury site as confirmed through invivo bioluminescence imaging in mice. In summary, the engineering of MSCs through minicircle microporation is expected to enhance the therapeutic potential of MSCs in clinical applications.

593. Anti-Angiogenic Therapy for Pancreatic Cancer by Systemic Delivery of Messenger RNA Using Polyplex Nano Micelle

In pancreatic cancer, the access of therapeutic reagents to cancer cells was restricted by thick fibrotic tissue, and thus anti-angiogenic therapy targeting endothelial cells, which are more accessible, is a promising strategy. Gene therapy based on messenger RNA (mRNA) delivery allows for sustainable introduction of anti-angiogenic factors without the risks associated with DNA delivery, such as insertion mutagenesis, and uncontrollably prolonged transgene expression. Systemic intravenous injection is a proper delivery route for mRNA to access endothelial cells from blood vessels with a simple injection procedure. Because mRNA is highly susceptible to enzymatic degradation in the blood circulation, mRNA carrier with high stability is required. In this study, we used polyplex nanomicelles, prepared from mRNA and poly(ethylene glycol) (PEG)-polycation block copolymers (Adv Drug Deliv Rev 2001, 47, 113-131), and a cholesterol moiety (Chol) was introduced to the block copolymer to stabilize the nanomicelles by hydrophobic interaction. In the cationic segment of the block copolymer, a polyaspartamide with four aminoethylene repeating units in the side chain (PAsp(TEP)) was used, which has high endosomal escaping capability and mRNA stabilizing effect (J Am Chem Soc 2014, 136, 12396-12405). In quantitative PCR (qPCR) analysis of nuclease resistance after in vitro incubation in 50% serum, Chol introduction failed to increase mRNA stability. In contrast, after intravenous injection to mice, the nanomicelles with Chol showed significantly enhanced mRNA retention in the blood circulation in qPCR analysis, when compared to those without Chol. These results indicate that the stabilizing effect of Chol is obvious especially in the harsh environment of blood, where nanomicelles are exposed to large amount of polyanion, such as proteoglycans on the cell surface. Indeed, nanomicelles with Chol showed enhanced resistance to dissociation by dextran sulfate compared to those without Chol. Eventually, when luciferase mRNA was introduced to the mice subcutaneously inoculated with human pancreatic adenocarcinoma (BxPC3), the nanomicelles with Chol showed enhanced luciferase expression in the tumor tissue, compared to those without Chol. Then, anti-angiogenic treatment was performed to this BxPC3 model, using mRNA encoding sFlt-1, a soluble form of vascular endothelial growth factor (VEGF) receptor, which inhibits VEGF signaling by entrapping VEGF protein. In the evaluation of tumor volume, the nanomicelles with Chol exhibited significant growth inhibitory effect, whereas those without Chol failed to show detectable effect. In immunohistochemical staining of vascular endothelial cells, the nanomicelles with Chol induced significant reduction of vascular density in the tumor tissue, indicating that the anti-angiogenic effect contributed to the inhibition of tumor growth after injection the nanomicelles with Chol. In conclusion, we succeeded in anti-angiogenic treatment of intractable pancreatic cancer by systemic mRNA delivery using stabilized nanomicelles. This mRNA delivery system can also be applied to the treatment of various diseases in the future.

Minicircle DNA Provides Enhanced and Prolonged Transgene Expression Following Airway Gene Transfer.

Gene therapy for cystic fibrosis using non-viral, plasmid-based formulations has been the subject of intensive research for over two decades but a clinically viable product has yet to materialise in large part due to inefficient transgene expression. Minicircle DNA give enhanced and more persistent transgene expression compared to plasmid DNA in a number of organ systems but has not been assessed in the lung. In this study we compared minicircle DNA with plasmid DNA in transfections of airway epithelial cells. In vitro, luciferase gene expression from minicircles was 5–10-fold higher than with plasmid DNA. In eGFP transfections in vitro both the mean fluorescence intensity and percentage of cells transfected was 2–4-fold higher with minicircle DNA. Administration of equimolar amounts of DNA to mouse lungs resulted in a reduced inflammatory response and more persistent transgene expression, with luciferase activity persisting for 2 weeks from minicircle DNA compared to plasmid formulations. Transfection of equal mass amounts of DNA in mouse lungs resulted in a 6-fold increase in transgene expression in addition to more persistent transgene expression. Our findings have clear implications for gene therapy of airway disorders where plasmid DNA transfections have so far proven inefficient in clinical trials.