Search

The precise mechanism by which many virus-based vectors activate immune responses remains unknown. Dendritic cells (DCs) play key roles in priming T cell responses and controlling virus replication, but their functions in generating protective immunity following vaccination with viral vectors are not always well understood. We hypothesized that highly immunogenic viral vectors with identical cell entry pathways but unique replication mechanisms differentially infect and activate DCs to promote antigen presentation and activation of distinctive antigen-specific T cell responses. To evaluate differences in replication mechanisms, we utilized a rhabdovirus vector (vesicular stomatitis virus; VSV) and an alphavirus-rhabdovirus hybrid vector (virus-like vesicles; VLV), which replicates like an alphavirus but enters the cell via the VSV glycoprotein. We found that while virus replication promotes CD8+ T cell activation by VLV, replication is absolutely required for VSV-induced responses. DC subtypes were differentially infected in vitro with VSV and VLV, and displayed differences in activation following infection that were dependent on vector replication but were independent of interferon receptor signaling. Additionally, the ability of the alphavirus-based vector to generate functional CD8+ T cells in the absence of replication relied on cDC1 cells. These results highlight the differential activation of DCs following infection with unique viral vectors and indicate potentially discrete roles of DC subtypes in activating the immune response following immunization with vectors that have distinct replication mechanisms.

Background: Todays, one of the most important problems in detection of human pathogens, is lack of positive control. The idea of using hybrid vectors, containing genes of different pathogens, can overcome this limitation. We can design specific primers for each region and use the hybrid vector as positive control sample in PCR. In this research we designed a hybrid vector and relevant primers for detection of Variolla and Burkholderia. Materials and Methods: In this study 16srRNA and HA genes were chosen to be located on the vector, to represent of Burkholderia and Variolla, in respectively. The sequence of these genes obtained from NCBI in FASTA format and aligned in BioEdit software for finding conserve region of each gene, then some purposeful changes were applied in the sequence of each gene and the sequences were placed next to each other and the construct was designed. Specific primers designed for each region using Oligo7, BioEdit, GeneRunner softwares, Oligo analyzer website and NCBI database. Finally, the construct cloned in PUC57 in SnapGene and PCRsimulated on hybrid vector using designed primers. Results: Analysis confirmed that conserved region for each gene is located on hybrid vector for each pathogen, and simulation of PCR proved the accuracy of designed primers. Conclusion: Hybrid vectors design contain similar sequence of pathogens genome but they are none-pathogenic. We can use these hybrid vectors as positive control, without any concern.

Background We developed a non-viral vector, a combination of HIV-1 Tat peptide modified with histidine and cysteine (mTat) and polyethylenimine, jetPEI (PEI), displaying the high efficiency of plasmid DNA transfection with little toxicity. Since the highest efficiency of INTERFERin (INT), a cationic amphiphilic lipid-based reagent, for small interfering RNA (siRNA) transfection among six commercial reagents was shown, we hypothesized that combining mTat/PEI with INT would improve transfection efficiency of siRNA delivery. To elucidate the efficacy of the hybrid vector for siRNA silencing, β-actin expression was measured after siRNA β-actin was transfected with mTat/PEI/INT or other vectors in HSC-3 human oral squamous carcinoma cells. Results mTat/PEI/INT/siRNA produced significant improvement in transfection efficiency with little cytotoxicity compared to other vectors and achieved ≈ 100% knockdown of β-actin expression compared to non-treated cells. The electric charge of mTat/PEI/INT/siRNA was significantly higher than INT/siRNA. The particle size of mTat/PEI/INT/siRNA was significantly smaller than INT/siRNA. Filipin III and β-cyclodextrin, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI/INT/siRNA transfection, while chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not inhibit mTat/PEI/INT/siRNA transfection. Furthermore, the transfection efficiency of mTat/PEI/INT at 4 °C was significantly lower than 37 °C. Conclusions These findings demonstrated the feasibility of using mTat/PEI/INT as a potentially attractive non-viral vector for siRNA delivery.

Reconstruction of bone defects and compensation of deficient repair mechanisms represent important goals within the field of regenerative medicine and require novel safe strategies for translation into the clinic. A non-viral osteogenic gene therapeutic vector system (‘hybrid vectors’) was generated, combining an improved bone morphogenetic protein 2 (BMP2) gene cassette and single pro-osteogenic microRNAs (miR-148b-3p, miR-20-5p, miR-590b-5p), driven by the U6 promoter. The vectors were tested in vitro for their osteogenic differentiation potential in C2C12 and C3H/10T1/2 cell lines, using BMP2 alone as control. After confirming BMP2 expression and miRNA transcription, increased osteogenic differentiation was observed by all hybrid vectors, but most consistently by BMP2/miR-590-5p, using alkaline phosphatase enzyme activity assays and osteogenic marker mRNA quantitation, including runt-related transcription factor 2 (Runx2), collagen type 1 (Col1a1) and osteocalcin. To visualise target mRNAs of the respective miRNAs, next generation sequencing was performed, confirming down-regulation of mRNA targets of the hybrid vectors. Since the hybrid vector consisting of BMP2 and miR-590-5p showed the largest increase in osteogenic differentiation in vitro, this was tested in a mouse ectopic-bone model. Mineralisation was more than with BMP2 alone. The present study showed hybrid vectors as a novel non-viral gene therapeutic plasmid system for combining therapeutic effects of recombinant protein expression and miRNA transcription that did not add to the burden of the translation machinery, while improving the therapeutic efficacies. In vivo proof-of-principle in the context of bone regeneration suggested that such hybrid vectors will be applicable in a wide array of gene therapeutic strategies.

Carriers of genetic material are divided into vectors of viral and non-viral origin. Viral carriers are already successfully used in experimental gene therapies, but despite advantages such as their high transfection efficiency and the wide knowledge of their practical potential, the remaining disadvantages, namely, their low capacity and complex manufacturing process, based on biological systems, are major limitations prior to their broad implementation in the clinical setting. The application of non-viral carriers in gene therapy is one of the available approaches. Poly(amidoamine) (PAMAM) dendrimers are repetitively branched, three-dimensional molecules, made of amide and amine subunits, possessing unique physiochemical properties. Surface and internal modifications improve their physicochemical properties, enabling the increase in cellular specificity and transfection efficiency and a reduction in cytotoxicity toward healthy cells. During the last 10 years of research on PAMAM dendrimers, three modification strategies have commonly been used: (1) surface modification with functional groups; (2) hybrid vector formation; (3) creation of supramolecular self-assemblies. This review describes and summarizes recent studies exploring the development of PAMAM dendrimers in anticancer gene therapies, evaluating the advantages and disadvantages of the modification approaches and the nanomedicine regulatory issues preventing their translation into the clinical setting, and highlighting important areas for further development and possible steps that seem promising in terms of development of PAMAM as a carrier of genetic material.

The exposure rate to air pollution in most urban cities is really a major concern because it results to a life-threatening consequence for human health and wellbeing. Furthermore, the accurate estimation and continuous forecasting of pollution levels is a very complicated task. In this paper, one of the space-temporal models, a vector autoregressive (VAR) with neural network (NN) and genetic algorithm (GA) was proposed and enhanced. The VAR could tackle the issue of multivariate time series, NN for nonlinearity, and GA for parameter estimation determination. Therefore, the model could be used to make predictions, such as the information of series and location data. The applied methods were on the pollution data, including NOX, PM2.5, PM10, and SO2 in Taipei, Hsinchu, Taichung, and Kaohsiung. The metaheuristics genetic algorithm was used to enhance the proposed methods during the experiments. In conclusion, the VAR-NN-GA gives a good accuracy when metric evaluation is used. Furthermore, the methods can be used to determine the phenomena of 10 years air pollution in Taiwan.

We use a tunable commercial liquid-crystal device tuned to a quarter-wave retardance to study the generation and dynamics of different types of hybrid vector beams. The standard situation where the q-plate is illuminated by a Gaussian beam is compared with other cases where the input beam is a vortex or a pure vector beam. As a result, standard hybrid vector beams but also petal-like hybrid vector beams are generated. These beams are analyzed in the near field and compared with the far field distribution, where their hybrid nature is observed as a transformation of the intensity and polarization patterns. Analytical calculations and numerical results confirm the experiments. We include an approach that provides an intuitive physical explanation of the polarization patterns in terms of mode superpositions and their transformation upon propagation based on their different Gouy phase. The tunable q-plate device presents worthy advantages, since it allows a compact and efficient generation of pure and hybrid vector beams to study these effects.

With the expansion of the microbiology field of research, a new genome editing tool arises from the biology of bacteria that holds the promise of achieving precise modifications in the genome with a simplicity and versatility that surpasses previous genome editing methods. This new technique, commonly named CRISPR/Cas9, led to a rapid expansion of the biomedical field; more specifically, cancer characterization and modeling have benefitted greatly from the genome editing capabilities of CRISPR/Cas9. In this paper, we briefly summarize recent improvements in CRISPR/Cas9 design meant to overcome the limitations that have arisen from the nuclease activity of Cas9 and the influence of this technology in cancer research. In addition, we present challenges that might impede the clinical applicability of CRISPR/Cas9 for cancer therapy and highlight future directions for designing CRISPR/Cas9 delivery systems that might prove useful for cancer therapeutics.

A viral vector that can safely and efficiently deliver large and diverse molecular cargos into cells is the holy grail of curing many human diseases. Adeno-associated virus (AAV) has been extensively used but has a very small capacity. The prokaryotic virus T4 has a large capacity but lacks natural mechanisms to enter mammalian cells. Here, we created a hybrid vector by combining T4 and AAV into one nanoparticle that possesses the advantages of both. The small 25 nm AAV particles are attached to the large 120 nm x 86 nm T4 head through avidin-biotin cross-bridges using the phage decoration proteins Soc (small outer capsid protein) and Hoc (highly antigenic outer capsid protein). AAV thus "piggy-backed" on T4 capsid, by virtue of its natural ability to enter many types of human cells efficiently acts as a "driver" to deliver large cargos associated with the T4 head. This unique T4-AAV hybrid vector approach could pave the way for the development of novel therapeutics in the future.

A hybrid biological-biomaterial antigen delivery vector comprised of a polymeric shell encapsulating an Escherichia coli core was previously developed for in situ antigen production and subsequent delivery. Due to the engineering capacity of the bacterial core, the hybrid vector provides unique opportunities for immunogenicity optimization through varying cellular localization (cytoplasm, periplasm, cellular surface) and type (protein or DNA) of antigen. In this work, three protein-based hybrid vector formats were compared in which the pneumococcal surface protein A (PspA) was localized to the cytoplasm, surface, and periplasmic space of the bacterial core for vaccination against pneumococcal disease. Furthermore, we tested the hybrid vector’s capacity as a DNA vaccine against Streptococcus pneumoniae by introducing a plasmid into the bacterial core to facilitate PspA expression in antigen presenting cells (APCs). Through testing these various formulations, we determined that cytoplasmic accumulation of PspA elicited the strongest immune response (antibody production and protection against bacterial challenge) and enabled complete protection at substantially lower doses when compared to vaccination with PspA + adjuvant. We also improved the storage stability of the hybrid vector to retain complete activity after 1 month at 4 °C using an approach in which hybrid vectors suspended in a microbial freeze drying buffer were desiccated. These results demonstrate the flexibility and robustness of the hybrid vector formulation, which has the potential to be a potent vaccine against S. pneumoniae. Keywords: Vaccine delivery, DNA vaccine, Pneumococcal disease, Streptococcus pneumoniae, Pneumococcal surface protein A (PspA)

Virus-mediated gene delivery shows promise for the treatment of chronic pain. However, viral vectors have cytotoxicity. To avoid toxicities and limitations of virus-mediated gene delivery, we developed a novel nonviral hybrid vector: HIV-1 Tat peptide sequence modified with histidine and cysteine residues combined with a cationic lipid. The vector has high transfection efficiency with little cytotoxicity in cancer cell lines including HSC-3 (human tongue squamous cell carcinoma) and exhibits differential expression in HSC-3 (∼45-fold) relative to HGF-1 (human gingival fibroblasts) cells. We used the nonviral vector to transfect cancer with OPRM1, the μ-opioid receptor gene, as a novel method for treating cancer-induced pain. After HSC-3 cells were transfected with OPRM1, a cancer mouse model was created by inoculating the transfected HSC-3 cells into the hind paw or tongue of athymic mice to determine the analgesic potential of OPRM1 transfection. Mice with HSC-3 tumors expressing OPRM1 demonstrated significant antinociception compared with control mice. The effect was reversible with local naloxone administration. We quantified β-endorphin secretion from HSC-3 cells and showed that HSC-3 cells transfected with OPRM1 secreted significantly more β-endorphin than control HSC-3 cells. These findings indicate that nonviral delivery of the OPRM1 gene targeted to the cancer microenvironment has an analgesic effect in a preclinical cancer model, and nonviral gene delivery is a potential treatment for cancer pain.

Fuel cost is the most contributed component in the operational cost of all transportation modes. In the aviation industry, jet fuel cost contributed to a percentage of 33.33% of the total airline operational costs. To increase efficiency in operational costs and the airline should have jet fuel price monitoring systems that can forecast the future price and give some strategy recommendations to airlines. In this research, we propose many multivariate time series-based predictive analytics as a tool for the airline to monitor and forecast the jet fuel price transaction based on jet fuel transaction price. We consider the global crude oil price and also global and local jet fuel prices in each airport. We also consider additional variables for the economical aspect that applied differently for each airport location. We examine two Recurrent Neural Network (RNN) algorithm, Long Short Term Memory (LSTM) and Gate Recurrent Units (GRU). For minimizing the weakness of LSTM and GRU, we combine each methods with Vector Autoregression (VAR). After forecasting results using VAR-LSTM and VAR-GRU, we get forecasting accuracy of 98.98% and 99.40% respectively.

Background: Gene delivery to target cells is crucially important to establish gene therapy and regenerative medicine. Although various virus-based and synthetic molecule-based gene vectors have been developed to date, selective transfection in a site or a cell level is still challenging. For this study, both light-responsive and temperature-responsive synthetic gene vectors were designed for spatiotemporal control of a transfection system. Methods: 11-Mercaptoundecanoic acid-coated gold nanorods were mixed with polyamidoamine dendron-bearing lipids of two types having amino-terminus or ethoxydiethylene glycol-terminus to obtain hybrid vectors. Hybrid vectors were mixed further with pDNA. Then we investigated their physicochemical properties and transfection efficacy with or without near infrared laser irradiation. Results: Hybrid vectors formed complexes with pDNA and exhibited enhanced photothermal property under near infrared laser irradiation compared with parent gold nanorods. Transfection efficacy of complexes was promoted considerably by brief laser irradiation soon after complex application to the cells. Analysis of intracellular distribution revealed that laser irradiation promoted the adsorption of complexes to the cells and cytosolic release of pDNA, which is derived from the change in surface hydrophobicity of complexes through dehydration of temperature-responsive groups. Conclusions: Hybrid vector is promising as a light-activatable transfection system.

Oncolytic viruses represent an exciting new aspect of the evolving field of cancer immunotherapy. We have engineered a novel hybrid vector comprising vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV), named recombinant VSV-NDV (rVSV-NDV), wherein the VSV backbone is conserved but its glycoprotein has been replaced by the hemagglutinin-neuraminidase (HN) and the modified, hyperfusogenic fusion (F) envelope proteins of recombinant NDV. In comparison to wild-type VSV, which kills cells through a classical cytopathic effect, the recombinant virus is able to induce tumor-specific syncytium formation, allowing efficient cell-to-cell spread of the virus and a rapid onset of immunogenic cell death. Furthermore, the glycoprotein exchange substantially abrogates the off-target effects in brain and liver tissue associated with wild-type VSV, resulting in a markedly enhanced safety profile, even in immune-deficient NOD.CB17-prkdc(scid)/NCrCrl (NOD-SCID) mice, which are highly susceptible to wild-type VSV. Although NDV causes severe pathogenicity in its natural avian hosts, the incorporation of the envelope proteins in the chimeric rVSV-NDV vector is avirulent in embryonated chicken eggs. Finally, systemic administration of rVSV-NDV in orthotopic hepatocellular carcinoma (HCC)-bearing immune-competent mice resulted in significant survival prolongation. This strategy, therefore, combines the beneficial properties of the rapidly replicating VSV platform with the highly efficient spread and immunogenic cell death of a fusogenic virus without risking the safety and environmental threats associated with either parental vector. Taking the data together, rVSV-NDV represents an attractive vector platform for clinical translation as a safe and effective oncolytic virus. IMPORTANCE The therapeutic efficacy of oncolytic viral therapy often comes as a tradeoff with safety, such that potent vectors are often associated with toxicity, while safer viruses tend to have attenuated therapeutic effects. Despite promising preclinical data, the development of VSV as a clinical agent has been substantially hampered by the fact that severe neurotoxicity and hepatotoxicity have been observed in rodents and nonhuman primates in response to treatment with wild-type VSV. Although NDV has been shown to have an attractive safety profile in humans and to have promising oncolytic effects, its further development has been severely restricted due to the environmental risks that it poses. The hybrid rVSV-NDV vector, therefore, represents an extremely promising vector platform in that it has been rationally designed to be safe, with respect to both the recipient and the environment, while being simultaneously effective, both through its direct oncolytic actions and through induction of immunogenic cell death.

Oncolytic adenovirus (Ad) holds great promise as a potential gene therapy for cancer. However, intravenously administered Ad may encounter difficulties due to unfavorable host responses, non-specific interactions, and the heterogeneity of the tumor cell population. As an approach to combine the advantages of oncolytic Ad and synthetic polymers and to address the associated difficulties, Ad was physically complexed with a pH-sensitive block copolymer, methoxy poly(ethylene glycol)- b -poly( l -histidine) (mPEG- b -pHis). The in vitro transduction efficiency at an acidic extracellular pH was remarkably enhanced in cancer cells when treated with the Ad expressing green fluorescent protein (GFP) coated with mPEG- b -pHis (c-dE1/GFP) as compared to that of naked Ad (n-dE1/GFP). Time-lapse total internal reflection fluorescence microscopic imaging revealed a significantly enhanced cellular uptake rate of c-dE1/GFP at acidic tumor pH when compared with that at neutral pH or naked cognate Ad (n-dE1/GFP). In addition, c-dE1/GFP remained relatively stable in human serum-containing media, and considerably reduced both the innate and adaptive immune response against Ad. Moreover, the therapeutic efficacy and survival benefit of mPEG- b -pHis-complexed oncolytic Ad (c-H5mT/Luc) by systemic treatment was significantly enhanced compared to that with naked oncolytic Ad (n-H5mT/Luc) in both coxsackie and adenovirus receptor-positive and -negative tumors. Whole-body bioluminescence imaging showed 7.3-fold higher luciferase expression at the tumor site and 23.0-fold less luciferase expression in liver tissue for c-H5mT/Luc relative to that for naked oncolytic Ad (n-H5mT/Luc). Considering the heterogeneity of tumor tissue, these results are important for guiding the development of more potent and specific treatment of devastating metastatic cancers using this viral system. Statement of significance Although adenoviral systems have shown considerable promise and undergone extensive evaluation attempts to specifically target Ad vectors to cancer cells have met limited success. This shortcoming is due to the strong immune response stimulated by Ad and the hepatotoxicity of the viral particles. To overcome restricted vector issues, we generated Ad/mPEG- b -pHis for tumor microenvironment-targeting hybrid vector systems, an oncolytic Ad coated with a pH-responsive polymer, mPEG- b -pHis. The Ad/mPEG- b -pHis exhibited pH-dependent transduction efficiency and cancer-cell killing effects. Moreover, systemic administration of oncolytic Ad/mPEG- b -pHis led to marked suppression of tumor growth and tumor-specific viral replication. Ad successfully avoided the innate and adaptive immune responses and liver accumulation with the help of mPEG- b -pHis on its surface.

X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad.AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK vector via homologous recombination in hematopoietic stem cells.

Vector delivery is still a bottleneck for gene therapy. To overcome some disadvantages of adenoviral and retroviral vectors, we developed a hybrid vector. This hybrid vector, AdLTR-luc, was created by adding two elements from Moloney murine leukemia virus (MoMLV) flanking the luciferase cDNA into an E1/E3-deleted, replication deficient serotype 5 adenovirus vector (Zheng et al., Nature Biotechnol, 2000), and demonstrated that the MoMLV element upstream of the luciferase cDNA was broken during the integration event. The purpose of the current study was to determine if the MoMLV element downstream of the luciferase cDNA was also broken when integration occurred. We used the same A5 cell clones (#10 and 11) from the earlier the paper along with restriction endonuclease digestions, plus Southern hybridization, and PCR. Southern hybridization indicated that the luciferase cDNA was intact in the cloned cells. Results from Xho I and Sal I digestions showed that integration occurred in cloned cells. Southern hybridizations after Nco I digestion suggested that there was a break in both MoMLV elements, upstream and downstream of the luciferase cDNA. After DNA digestion with Not I, hybridization analyses indicated that the MoMLV upstream element was broken during integration. Digestion of genomic DNA with either Xba I/Kpn I, Bam HI/Sac I, or Bam HI/Nco I demonstrated that the MoMLV downstream element was also broken during integration. A PCR assay was unable to amplify the junctional region between the downstream MoMLV element and the adenoviral E2B gene, consistent with a break in that element. Although AdLTR-luc integration is atypical (Zheng et al., Nature Biotechnol, 2000), the present results suggest that both MoMLV elements have important roles in this event.

In recent years, vector beams (VBs) with spatially varying polarization profiles have been used in many fields. Here we study the transmission properties of a hybrid VB passing through atomic vapor. We observe a four-petal pattern which is induced by the rapid transport of atomic coherence. This intensity distribution can be modified by coherence attenuation and absorption enhancement controlled by an external magnetic field. A second experiment with orthogonal circularly polarized states shows that the quantum coherence can be induced by two spatially separated laser beams and support our interpretation of our results for the hybrid VB experiment. Our results may have applications in optical manipulation, the measurement of magnetic fields, and the study of quantum coherence in atomic ensembles.

The robustness of the polarization spatial distribution of vector beams upon propagation is crucial for a number of applications, including optical communications and materials processing. This study has been commonly centered on Gouy phase effects on focused vector beams. In this work, we present a theoretical and experimental analysis of the Gouy phase's effects on the propagation of pure and hybrid vector beams. Experimental results at various axial planes, before and past the focus, are obtained by using a simplified liquid-crystal spatial light modulator-based optical system that allows the easy generation of these beams. Furthermore, a new alternative optical set-up that is devoid of moving elements is demonstrated, which simplifies this study. We experimentally verify the differences between pure and hybrid vector beams upon propagation. While the first ones remain stable, hybrid vector beams show Gouy phase effects that demonstrate an optical activity where the local polarization states rotate by an angle that depends on the propagation distance. Experimental results agree with the theory.

Adenoviral, retroviral/lentiviral, adeno-associated viral, and herpesviral vectors are the major viral vectors used in gene therapy. Compared with non-viral methods, viruses are highly-evolved, natural delivery agents for genetic materials. Despite their remarkable transduction efficiency, both clinical trials and laboratory experiments have suggested that viral vectors have inherent shortcomings for gene therapy, including limited loading capacity, immunogenicity, genotoxicity, and failure to support long-term adequate transgenic expression. One of the key issues in viral gene therapy is the state of the delivered genetic material in transduced cells. To address genotoxicity and improve the therapeutic transgene expression profile, construction of hybrid vectors have recently been developed. By adding new abilities or replacing certain undesirable elements, novel hybrid viral vectors are expected to outperform their conventional counterparts with improved safety and enhanced therapeutic efficacy. This review provides a comprehensive summary of current achievements in hybrid viral vector development and their impact on the field of gene therapy.

A disease-specific, hybrid vector is developed for pneumococcal disease vaccine., The type and potency of an immune response provoked during vaccination will determine ultimate success in disease prevention. The basis for this response will be the design and implementation of antigen presentation to the immune system. Whereas direct antigen administration will elicit some form of immunological response, a more sophisticated approach would couple the antigen of interest to a vector capable of broad delivery formats and designed for heightened response. New antigens associated with pneumococcal disease virulence were used to test the delivery and adjuvant capabilities of a hybrid biological-biomaterial vector consisting of a bacterial core electrostatically coated with a cationic polymer. The hybrid design provides (i) passive and active targeting of antigen-presenting cells, (ii) natural and multicomponent adjuvant properties, (iii) dual intracellular delivery mechanisms, and (iv) a simple formulation mechanism. In addition, the hybrid format enables device-specific, or in situ, antigen production and consolidation via localization within the bacterial component of the vector. This capability eliminates the need for dedicated antigen production and purification before vaccination efforts while leveraging the aforementioned features of the overall delivery device. We present the first disease-specific utilization of the vector toward pneumococcal disease highlighted by improved immune responses and protective capabilities when tested against traditional vaccine formulations and a range of clinically relevant Streptococcus pneumoniae strains. More broadly, the results point to similar levels of success with other diseases that would benefit from the production, delivery, and efficacy capabilities offered by the hybrid vector.

For efficient delivery of required genetic elements we utilized high-capacity adenoviral vectors in the past allowing high transgene capacities of up to 36 kb. Previously we explored the hyperactive Sleeping Beauty (SB) transposase (HSB5) for somatic integration from the high-capacity adenoviral vectors genome. To further improve this hybrid vector system we hypothesized that the previously described hyperactive SB transposase SB100X will result in significantly improved efficacies after transduction of target cells. Plasmid based delivery of the SB100X system revealed significantly increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5. After optimizing experimental setups for high-capacity adenoviral vectors-based delivery of the SB100X system we observed up to eightfold and 100-fold increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5 and the inactive transposase mSB, respectively. Furthermore, transposon copy numbers per cell were doubled with SB100X compared with HSB5 when using the identical multiplicity of infection. We believe that this improved hybrid vector system represents a valuable tool for achieving stabilized transgene expression in cycling cells and for treatment of numerous genetic disorders. Especially for in vivo approaches this improved adenoviral hybrid vector system will be advantageous because it may potentially allow reduction of the applied viral dose.

Background Rational design of AAV capsids is a simple method for enhancing AAV transduction efficiency. AAV-DJ is a highly recombinogenic hybrid vector created from DNA shuffling of eight AAV serotypes, which mediates efficient gene expression both in vitro and in vivo. AAV2 and AAV8 are the closest parental vectors of AAV-DJ and it has been reported that mutations on the 137/251/503 ubiquitination or phosphorylation sites of the AAV2 or AAV8 capsid lead to dramatic enhancement of gene delivery. Here, we aimed to find out whether the same point mutations on the AAV-DJ capsid could lead to significant improvement for gene delivery both in vitro and in vivo. Results We constructed three single point mutants (K137R/T251A/S503A) of AAV-DJ and the transduction efficiency of these mutants and AAV-DJ were investigated using two reporter gene systems including green fluorescent protein (GFP) and dual-luciferase (Gaussia luciferase and Firefly luciferase). Data indicated that single point mutations T251A/S503A lead to significant improvement of dual-luciferase expression in vivo after tail vein (TV) injection in mice respectively, despite limited enhancement of GFP expression in 293 T, Hela and HepG2 cells in vitro. Moreover, in vivo bioluminescence image and viral genome DNA copy number in tissue analysis showed that these mutants reserved the liver tropism characteristics, consistent with AAV-DJ. Conclusion Single point mutations on the 251/503 sites of AAV-DJ capsid can lead to a significant improvement for in vivo gene expression. These enhanced AAV vectors have great potential in gene therapy applications.

Inflammatory breast cancer (IBC) is a subtype of breast cancer that has a frequent association with metastatic disease and a poorer prognosis than comparative non-inflammatory breast cancers. While IBC is now considered a distinct subclass of breast cancer, the lack of molecular characterization, both at the genomic and proteomic levels, has hampered the development of rationalized and targeted therapies. Inherent receptor/ligand interactions that can occur on the surface of tumor cells can act as a dynamic molecular address that can enable targeted delivery of drugs and imaging agents to tumors. We hypothesize that such molecular addresses within IBC can be exploited for ligand-based imaging and early detection of disease sites. To this end, it is our goal to generate targeted imaging and therapeutic agents by combining ligand-directed targeting with efficient transduction of IBC cells by hybrid gene delivery vectors. Our strategy utilizes a hybrid vector with genomic elements from adeno-associated virus (AAV) and an M13-derived phage. Ligand-targeted, AAV/phage (AAVP) chimeras can display tumor-homing peptides that mediate selective internalization of viral particles through specific ligand-receptor interactions in vitro and in vivo. Such targeted vehicles are suited for the delivery of different reporter genes that can be used for imaging, diagnosis and therapy of breast cancer As a part of our ongoing studies we have identified, characterized and evaluated a peptide (WIFPWIQL, amino acid sequence) that can target GRP78, a stress-response protein that is expressed in IBC tumors and elevated during metastatic progression. Indeed, we found that this GRP78-targeting peptide can bind to, and internalize within IBC cells. As a result, we sought to characterize the ability of this peptide to mediate the delivery of fluorescent-based compounds and toxic moieties in preclinical models of IBC and breast cancer metastasis. Using amine-based chemical coupling, we conjugated near-infrared dyes on both WIFPWIQL-phage and on a WIFPWIQL-peptide engrafted antibody. When these fluorescent construct were administered into mice bearing IBC or IBC-like tumors, we could visualize tumor-specific targeting of the vectors in vivo. To demonstrate efficacy of GRP78-targeted therapeutics, we conjugated the tumor-homing, GRP78 ligand to a cell-death inducing domain (creating a compound called BMTP-78, βone metastasis targeting peptide-78), which can selectively kill cells upon internalization. We show here that BMTP78 therapy in mice with established GRP78-positive tumors, but not matched GRP78-negative tumors, could effectively reduce tumor growth and metastatic burden. Finally, we demonstrate a WIFPWIQL-AAVP construct that expresses a suicide gene (HSVtk) under the control of either CMV or GRP78 promoter, could sensitize IBC tumor xenografts to the pro-drug ganciclovir. Collectively, our results demonstrate an in vivo receptor/ligand system that has the potential for imaging and therapeutic targeting of IBC and aggressive breast tumors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-10-09.

We would like to thank Huang et al . for comments on our recent report, which assessed the “antitumor effect and safety profile of systemically delivered oncolytic adenovirus complexed with epidermal growth factor receptor (EGFR)-targeted poly(amidoamine) (PAMAM)-based dendrimer (1)”. We read the comments with great interest and acknowledging that this is an area urgently requiring more research to improve safety of nanomaterials in gene therapy for clinical application.

It has been previously shown that integrase-defective HIV-1-based gene vectors can serve, with moderate efficiency, as substrate for DNA transposition by a transiently expressed Sleeping Beauty (SB) transposase. Here, we describe the enhanced gene transfer properties of a HIV-1/SB hybrid vector that allows efficient DNA transposition, facilitated by the hyperactive SB100X transposase, from vector DNA intermediates in primary human cells. Potent transposase-dependent integration of genetic cargo carried by the hybrid HIV-1/SB vector (up to 160-fold above background) is reported in human cell lines as well as in primary human fibroblasts and keratinocytes. The efficiency of transgene integration in context of the newly developed hybrid vector is comparable with that of conventional lentiviral vectors (LVs). Integration profiles of integrating HIV-1-derived vectors and SB transposons mobilized from LVs are investigated by deep sequencing of a large number of integration sites. A significant bias of lentiviral integrations in genes is reported, confirming that biological properties of the viral integration machinery facilitate preferred insertion into actively transcribed genomic regions. In sharp contrast, lentiviral insertions catalyzed by the SB100X transposase are far more random with respect to genes. Based on these properties, HIV-1/SB vectors may become valuable tools for genetic engineering and therapeutic gene transfer.

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors have a transgene capacity of up to 150 kbp and can efficiently transduce many different cell types in culture and in vivo without causing cytopathic effects. However, these vectors do not support long-term transgene expression. Adeno-associated virus type 2 (AAV-2) has the capacity to integrate its genome into a specific site on human chromosome 19, but AAV-2-derived gene therapy vectors have a transgene capacity of only 4.5 kb. To combine the large transgene capacity of HSV-1 with the potential for site-specific genomic integration and long-term transgene expression of AAV-2, HSV/AAV hybrid vectors have been developed. This review describes the design, applications and limitations of these hybrid vectors. However, as HSV-1 is a full helper virus for AAV-2 replication, the main focus is the analysis of the molecular mechanisms of interaction between the two viruses. The knowledge of these interactions will have direct implications on the design of novel HSV/AAV hybrid vectors.

Adenoviral vectors offer many advantages for cancer gene therapy, including high transduction efficiency, but safety concerns related to severe immunogenicity and other side effects have led to careful reconsideration of their use in human clinical trials. To overcome these issues, a strategy of generating hybrid vectors that combine viral and non-viral elements as more intelligent gene carriers has been employed. Here, we coated adenovirus (Ad) with an arginine-grafted bioreducible polymer (ABP) via electrostatic interaction. We examined the effect of ABP-coated Ad complex at various ABP molecules/Ad particle ratios. Enhanced transduction efficiency was observed in cells treated with cationic ABP polymer-coated Ad complex compared to naked Ad. We also examined the coating of Ad with ABP polymers at the optimal polymer ratio using dynamic light scattering and transmission electron microscopy. In both high and low coxsackie virus and adenovirus receptor (CAR)-expressing cells, ABP-coated Ad complex produced higher levels of transgene expression than cationic polymer 25 K PEI. Notably, high cytotoxicity was observed with 25 K PEI-coated Ad complex treatment, but not with ABP-coated Ad complex treatment. In addition, ABP-coated Ad complex was not significantly inhibited by serum, in contrast to naked Ad. Moreover, ABP-coated Ad complex significantly reduced the innate immune response relative to naked Ad, as assessed by interleukin-6 (IL-6) cytokine release from macrophage cells. Overall, our studies demonstrate that Ad complex formed with ABP cationic polymer may improve the efficiency of Ad and be a promising tool for cancer gene therapy.

We developed hybrid vectors employing high-capacity adenovirus as a first-stage carrier encoding all the components required for in situ production of a second-stage lentivirus, thereby achieving stable transgene expression in secondary target cells. Such vectors have never previously been tested in normal tissues, because of the scarcity of suitable in vivo systems permissive for second-stage lentivirus assembly. Here we employed a novel murine model in which endogenous liver tissue is extensively reconstituted with engrafted human hepatocytes, and successfully achieved stable transduction by the second-stage lentivirus produced in situ from first-stage adenovirus. This represents the first demonstration of the functionality of adenoviral-lentiviral hybrid vectors in a normal parenchymal organ in vivo.

The turbulent history of clinical trials in viral gene therapy has taught us important lessons about vector design and safety issues. Much effort was spent on analyzing genotoxicity after somatic integration of therapeutic DNA into the host genome. Based on these findings major improvements in vector design including the development of viral hybrid vectors for somatic integration have been achieved. This review provides a state-of-the-art overview of available hybrid vectors utilizing viruses for high transduction efficiencies in concert with various integration machineries for random and targeted integration patterns. It discusses advantages but also limitations of each vector system.

Lentiviral vectors enter cells with high efficiency and deliver stable transduction through integration into host chromosomes, but their preference for integration within actively transcribing genes means that insertional mutagenesis following disruption of host proto-oncogenes is a recognized concern. We have addressed this problem by combining the efficient cell and nuclear entry properties of HIV-1–derived lentiviral vectors with the integration profile benefits of Sleeping Beauty (SB) transposase. Importantly, this integration enzyme does not exhibit a preference for integration within active genes. We generated integrase-deficient lentiviral vectors (IDLVs) to carry SB transposon and transposase expression cassettes. IDLVs were able to deliver transient transposase expression to target cells, and episomal lentiviral DNA was found to be a suitable substrate for integration via the SB pathway. The hybrid vector system allows genomic integration of a minimal promoter-transgene cassette flanked by short SB inverted repeats (IRs) but devoid of HIV-1 long terminal repeats (LTRs) or other virus-derived sequences. Importantly, integration site analysis revealed redirection toward a profile mimicking SB-plasmid integration and away from integration within transcriptionally active genes favored by integrase-proficient lentiviral vectors (ILVs).

We newly prepared lipopolyplexes containing N-lauroylsarcosine (LS) as a hybrid vector for pulmonary gene delivery via the systemic route. Lipopolyplexes were composed of polyethylenimine (PEI), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethlylammonium chloride (DOTMA), LS, and plasmid DNA (pDNA). The particle size of lipopolyplex of PEI, DOTMA, and pDNA (lipopolyplex 2P-2D) was not largely changed by the addition of LS, although the addition of LS decreased the high zeta potential. Lipopolyplexes containing LS with low zeta potential showed little aggregation with erythrocytes and low cytotoxicity. In HepG2 cells, lipopolyplexes containing LS showed high transgene efficiency comparable to lipopolyplex 2P-2D. After intravenous injection of the complexes into mice, lipopolyplexes containing LS showed high transgene efficiency, comparable to lipopolyplex 2P-2D. In particular, lipopolyplexes containing LS showed extremely high transgene efficiency in the lung. As a result of the analysis to identify optimum formulations, we discovered that LS contributed to the high transgene efficiency in the lung as 76.7% of the contribution index. These results suggest that lipopolyplexes containing LS are safe and useful gene delivery vectors with lung directivity., Journal of Controlled Release, 136(3), pp. 213-219

Autonomous parvoviruses possess an intrinsic oncotropism based on viral genetic elements controlling gene expression and genome replication. We constructed a hybrid vector consisting of the H1 parvovirus-derived expression cassette comprising the p4 promoter, the ns1 gene and the p38 promoter flanked by the adeno-associated viruses 2 (AAV2) inverted terminal repeats and packaged into AAV2 capsids. Gene transduction using this vector could be stimulated by coinfection with adenovirus, by irradiation or treatment with genotoxic agents, similar to standard AAV2 vectors. However, the latter were in most cases less efficient in gene transduction than the hybrid vector. With the new vector, tumor cell-selective increase in transgene expression was observed in pairs of transformed and non-transformed cells, leading to selective killing of the transformed cells after expression of a prodrug-converting enzyme. Preferential gene expression in tumor versus normal liver tissue was also observed in vivo in a syngeneic rat model. Comparative transduction of a panel of different tumor cell lines with the H1 and the H1/AAV hybrid vector showed a preference of each vector for distinct cell types, probably reflecting the dependence of the viral tropism on capsid determinants.

The trans-splicing (ts) and overlapping (ov) vectors expand the packaging capacity of adeno-associated virus (AAV). But their application depends on the inherent properties of the target gene. The ts vectors require an optimal gene-splitting site and the ov vectors require a highly recombinogenic domain. In order to overcome these limitations, we developed a hybrid dual (hd) vector system. In the hd vectors, we inserted a highly recombinogenic alkaline phosphatase (AP) sequence in the ts vectors to allow for transgene-independent reconstitution through homologous recombination of the AP sequences. We first tested the hybrid system with the LacZ gene. Both in the cell line (in vitro) and in the mouse muscle (in vivo), the hd vectors significantly outperformed the ts and ov vectors. In muscle, the transduction efficiency of the hybrid vectors reached 80% of that from the single intact vector. Southern blot confirmed AP sequence-mediated transgene reconstitution. In order to validate the hybrid system, we split the 6 kilobase (kb) mini-dystrophin gene at the exon 55/56 junction, a predicted poor site for the ts approach. In dystrophic mdx mouse muscle, the hd vectors yielded 5.6-fold higher transduction than the ts vectors did. Taken together, these data suggest that the hybrid system efficiently expresses large therapeutic genes that are poor candidates for the ts and ov approaches.

Antipathogen immune responses create a balance between immunity, tolerance, and immune evasion. However, during gene therapy most viral vectors are delivered in substantial doses and are incapable of expressing gene products that reduce the host's ability to detect transduced cells. Gene transfer efficacy is also modified by the in vivo transduction of dendritic cells (DC), which notably increases the immunogenicity of virions and vector-encoded genes. In this study, we evaluated parameters that are relevant to the use of canine adenovirus serotype 2 (CAV-2) vectors in the clinical setting by assaying their effect on human monocyte-derived DC (hMoDC). We compared CAV-2 to human adenovirus (HAd) vectors containing the wild-type virion, functional deletions in the penton base RGD motif, and the CAV-2 fiber knob. In contrast to the HAd type 5 (HAd5)-based vectors, CAV-2 poorly transduced hMoDC, provoked minimal upregulation of major histocompatibility complex class I/II and costimulatory molecules (CD40, CD80, and CD86), and induced negligible morphological changes indicative of DC maturation. Functional maturation assay results (e.g., reduced antigen uptake; tumor necrosis factor alpha, interleukin-1β [IL-1β], gamma interferon [IFN-γ], IL-10, IL-12, and IFN-α/β secretion; and stimulation of heterologous T-cell proliferation) were also significantly lower for CAV-2. Our data suggested that this was due, in part, to the use of an alternative receptor and a block in vesicular escape. Additionally, HAd5 vector-induced hMoDC maturation was independent of the aforementioned cytokines. Paradoxically, an HAd5/CAV-2 hybrid vector induced the greatest phenotypical and functional maturation of hMoDC. Our data suggest that CAV-2 and the HAd5/CAV-2 vector may be the antithesis ofAdenoviridaeimmunogenicity and that each may have specific clinical advantages.