Recombinant adeno-associated viruses as a gene delivery vehicle for the use in molecular medicine

Breast cancer (BC) is a cancer with a high prevalence and mortality among women worldwide. With the current diagnostics methods, BC may remain undetected at its early stages, and the therapies developed for the disease are associated with severe side effects. Oncolytic viruses can be the basis of the new, effective BC treatment approaches. The viruses destroy tumor cells directly and launch the antitumor immune response; this dual action supports their efficacy. It is possible to make the oncolytic virus therapy more effective by designing genetically modified viruses that can target BC cells better and/or induce a stronger antitumor immune response. This review outlines the directions of development of oncolytic viruses in BC treatment, covers the optimal ways of delivering viruses to the tumor and the efficacy of their use in combination with other therapeutic agents (methods) and presents the prospects of using oncolytic viruses in antitumor vaccines.

Recombinant adeno-associated viruses as a gene delivery vehicle for the use in molecular medicine

Viral mechanisms for the delivery of genetic material are widely used in molecular medicine. Recombinant adeno-associated viruses (rAAV) represent a promising tool for in vivo gene delivery. The review considers nosological spectrum, molecular mechanisms, the choice of drug administration route depending on target structures, the choice of serotype, and the methods of active ingredient manufacturing for rAAV-mediated gene therapy.

Abstract 16565: Intramyocardial Tbx18 Gene Delivery in a Radiopaque Medium Enables Real-Time Assessment of Gene Transfer, and is Compatible With Biological Pacemaker Function

Introduction: The efficacy and specificity of myocardial gene transfer depends on retention of the injectate at the site of gene delivery. However, no direct method exists to confirm myocardial retention of a biologic delivered via an intracardiac transvenous catheter. We tested feasibility of a radiographic contrast media as a gene delivery vehicle, and fluoroscopic assessment of intramyocardial retention as well as proper function of a transgene. Methods and Results: Synthetic mRNA expressing GFP in saline was mixed with an iodine-based radiopaque contrast agent, iopamidol, at 2, 5, 20, 30 and 40%. The minimum content of iopamidol that allowed unequivocal radiographic identification ex vivo was 20%. To validate functional expression of the transgene, GFP mRNA was injected focally into the left ventricular apex of the rat heart upon thoracotomy at 0, 2, 5 or 20% iopamidol solution. On day 5, the hearts were harvested and examined for GFP expression, which illustrated comparable and focal GFP expression in all animals. To test whether gene delivery in a contrast media is compatible with intended disease-modifying activity, we employed TBX18 gene transfer via the right femoral vein in a porcine model of complete heart block. We have previously demonstrated that TBX18 could convert ventricular myocytes to pacemaker cells, and create de novo ventricular pacing in situ. Intracardiac delivery of TBX18 or GFP (n=6 or 2 pigs, respectively) mRNA dissolved in 20% iopamidol to the high His bundle region revealed clear, real-time visualization of the injectate in all pigs. In one of the 8 pigs, the injectate diffused rapidly into the ventricular circulation, which necessitated a repeat injection. The maximum heart rate of TBX18-injected pigs was faster than that of GFP-injected pigs upon β-adrenergic stimulation at 77±12 vs. 43±13 BPM at week 2 ( P =0.08), and at 93±19 vs. 48±3BPM at week 4 ( P <0.05), respectively. Conclusion: Radiographic contrast agent can serve as an effective gene delivery vehicle, allowing real-time and non-invasive assessment of the retention of intramyocardial gene delivery. This approach may be generalizable to other focal gene therapy applications, and minimize suboptimal gene delivery.

Intestinal DCs global gene expression dynamics affected by recombinant baker’s yeasts saccharomyces cerevisiae

Background The baker’s yeast, saccharomyces cerevisiae, has been widely used throughout our daily life in diverse aspects for thousands of years. The saccharomyces cerevisiae was found to specifically target the dendritic cells (DCs) in mammalian with a manner of antigen-receptor interaction as described previously. It is necessary to investigate the effect of the baker’s yeasts on global gene expression dynamics of intestinal DCs and explore the possibilities of using baker’s yeast as gene delivery vehicle to modulate animal’s immune functions Results with a murine oral delivery model in vivo, we confirmed the feasibility of using budding yeast as gene delivery vehicle to the intestinal DCs using the Western blots. We then examined the transcriptome profile of the mouse intestinal DCs upon yeast stimulus. The enrichment analysis of unique transcripts indicated the beneficial role of yeast in modulating the DC-mediated adaptive immunity. Compared with previous study, we also found that a large fraction of the regulated genes is coincident with the response induced by other fungus, suggesting that the budding yeast induces a similar tailored unique genetic re-programming of DCs. Another analysis of transcriptome profile indicated that expression of β-catenin gene significantly changes DCs gene expression related to inflammatory response and cell adhesion. Conclusions Here, we defined the role of budding yeast on global gene expression of intestinal DCs, and confirmed the important role of β-catenin gene on the DCs-related inflammatory response, which provides a framework for the development of mucosa yeast-based DNA vaccine.

Intestinal DCs global gene expression dynamics affected by recombinant baker’s yeasts saccharomyces cerevisiae

Background The baker’s yeast, saccharomyces cerevisiae, has been widely used throughout our daily life in diverse aspects for thousands of years. The saccharomyces cerevisiae was found to specifically target the dendritic cells (DCs) in mammalian with a manner of antigen-receptor interaction as described previously. It is necessary to investigate the effect of the baker’s yeasts on global gene expression dynamics of intestinal DCs and explore the possibilities of using baker’s yeast as gene delivery vehicle to modulate animal’s immune functions Results with a murine oral delivery model in vivo, we confirmed the feasibility of using budding yeast as gene delivery vehicle to the intestinal DCs using the Western blots. We then examined the transcriptome profile of the mouse intestinal DCs upon yeast stimulus. The enrichment analysis of unique transcripts indicated the beneficial role of yeast in modulating the DC-mediated adaptive immunity. Compared with previous study, we also found that a large fraction of the regulated genes is coincident with the response induced by other fungus, suggesting that the budding yeast induces a similar tailored unique genetic re-programming of DCs. Another analysis of transcriptome profile indicated that expression of β-catenin gene significantly changes DCs gene expression related to inflammatory response and cell adhesion. Conclusions Here, we defined the role of budding yeast on global gene expression of intestinal DCs, and confirmed the important role of β-catenin gene on the DCs-related inflammatory response, which provides a framework for the development of mucosa yeast-based DNA vaccine.

Intestinal DCs global gene expression dynamics affected by recombinant baker’s yeasts saccharomyces cerevisiae

Background The baker’s yeast, saccharomyces cerevisiae, has been widely used throughout our daily life in diverse aspects for thousands of years. The saccharomyces cerevisiae was found to specifically target the dendritic cells (DCs) in mammalian with a manner of antigen-receptor interaction as described previously. It is necessary to investigate the effect of the baker’s yeasts on global gene expression dynamics of intestinal DCs and explore the possibilities of using baker’s yeast as gene delivery vehicle to modulate animal’s immune functions Results with a murine oral delivery model in vivo, we confirmed the feasibility of using budding yeast as gene delivery vehicle to the intestinal DCs using the Western blots. We then examined the transcriptome profile of the mouse intestinal DCs upon yeast stimulus. The enrichment analysis of unique transcripts indicated the beneficial role of yeast in modulating the DC-mediated adaptive immunity. Compared with previous study, we also found that a large fraction of the regulated genes is coincident with the response induced by other fungus, suggesting that the budding yeast induces a similar tailored unique genetic re-programming of DCs. Another analysis of transcriptome profile indicated that expression of β-catenin gene significantly changes DCs gene expression related to inflammatory response and cell adhesion. Conclusions Here, we defined the role of budding yeast on global gene expression of intestinal DCs, and confirmed the important role of β-catenin gene on the DCs-related inflammatory response, which provides a framework for the development of mucosa yeast-based DNA vaccine.

Cardiomyocyte Progenitor Cells as a Functional Gene Delivery Vehicle for Long-Term Biological Pacing

Sustained pacemaker function is a challenge in biological pacemaker engineering. Human cardiomyocyte progenitor cells (CMPCs) have exhibited extended survival in the heart after transplantation. We studied whether lentivirally transduced CMPCs that express the pacemaker current If (encoded by HCN4) can be used as functional gene delivery vehicle in biological pacing. Human CMPCs were isolated from fetal hearts using magnetic beads coated with Sca-1 antibody, cultured in nondifferentiating conditions, and transduced with a green fluorescent protein (GFP)- or HCN4-GFP-expressing lentivirus. A patch-clamp analysis showed a large hyperpolarization-activated, time-dependent inward current (−20 pA/pF at −140 mV, n = 14) with properties typical of If in HCN4-GFP-expressing CMPCs. Gap-junctional coupling between CMPCs and neonatal rat ventricular myocytes (NRVMs) was demonstrated by efficient dye transfer and changes in spontaneous beating activity. In organ explant cultures, the number of preparations showing spontaneous beating activity increased from 6.3% in CMPC/GFP-injected preparations to 68.2% in CMPC/HCN4-GFP-injected preparations (P < 0.05). Furthermore, in CMPC/HCN4-GFP-injected preparations, isoproterenol induced a significant reduction in cycle lengths from 648 ± 169 to 392 ± 71 ms (P < 0.05). In sum, CMPCs expressing HCN4-GFP functionally couple to NRVMs and induce physiologically controlled pacemaker activity and may therefore provide an attractive delivery platform for sustained pacemaker function.

DNA Condensation Study of Fully Synthesized Lipopeptide-Based Transfection Agent for Gene Delivery Vehicle

The main requirement of transfection agent has to condense DNA in nanoparticle size, protect the DNA from nucleases and other degrading enzymes during its transport in cell cytoplasm and nucleus and should not toxic to target cells. In this research, lipopeptide composed of palmitoyl (C-16) and short peptide sequence have been designed fully synthesized and tested to DNA condensation capability and toxicity. The DNA condensation study was performed using EtBr exclusion assay and cytotoxicity determination was carried out by colorimetric MTT assay. It was revealed that lipopeptide-based transfection agent of Pal-CKKHH and Pal-CKKHH-YGRKKRRQRRR-PKKKRKV condensed DNA molecules efficiently. The lipopeptide was less toxic compared to Lipofectamine and Poly-L-Lysine, that shown by 90% of CHO-K1 cells remained viable when they were treated with 4.36 µM Pal-CKKHHYGRKKRRQRRR-PKKKRKV. Meanwhile, there were only ~75% and 80% of CHO-K1 viable cells when it was treated with PLL and Lipofectamine®2000, respectively. Moreover, cell viability of HepG2 was ~ 75% after treated with 2.18 µM of Pal-CKKHH-YGRKKRRQRRR-PKKKRKV and decreased to ~65% when the lipopeptide concentration increased to 8.72 M. In summary, the synthesized lipopeptide condenses DNA molecules efficiently, less toxic than Lipofectamine®2000 and PLL and has possibility to be explored as a non-viral gene delivery vehicle.