Comparative analysis of adeno-associated virus serotypes for gene transfer in organotypic heart slices

Background Vectors derived from adeno-associated viruses (AAVs) are widely used for gene transfer both in vitro and in vivo and have gained increasing interest as shuttle systems to deliver therapeutic genes to the heart. However, there is little information on their tissue penetration and cytotoxicity, as well as the optimal AAV serotype for transferring genes to diseased hearts. Therefore, we aimed to establish an organotypic heart slice culture system for mouse left ventricular (LV) myocardium and use this platform to analyze gene transfer efficiency, cell tropism, and toxicity of different AAV serotypes. Methods LV tissue slices, 300 µm thick, were prepared from 15- to 17-day-old transgenic alpha-myosin heavy-chain-mCherry mice using a vibrating microtome. Tissue slice viability in air-liquid culture was evaluated by calcein-acetoxymethyl ester staining, mCherry fluorescence intensity, and the tetrazolium assay. Four recombinant AAV serotypes (1, 2, 6, 8) expressing green fluorescent protein (GFP) under the CAG promoter were added to the slice surface. Gene transfer efficiency was quantified as the number of GFP-positive cells per slice. AAV cell tropism was examined by comparing the number of GFP-positive cardiomyocytes (CMs) and fibroblasts within heart slices. Results Slices retained viability in in vitro culture for at least 5 days. After adding AAV particles, AAV6-infected slices showed the highest number of GFP-expressing cells, almost exclusively CMs. Slice incubation with AAV1, 2, and 8 resulted in fewer GFP-positive cells, with AAV2 having the lowest gene transfer efficiency. None of the AAV serotypes tested caused significant cytotoxicity when compared to non-infected control slices. Conclusions We have established a readily available mouse organotypic heart slice culture model and provided evidence that AAV6 may be a promising gene therapy vector for heart failure and other cardiac diseases.

Effects of Low-Dose X-Ray on Cell Growth, Membrane Permeability, DNA Damage and Gene Transfer Efficiency

Background: We aimed to reveal if low dose X-rays would induce harmful or beneficial effect or dual response on biological cells and whether there are conditions the radiation can enhance gene transfer efficiency and promote cell growth but without damage to the cells. Method: A systematic study was performed on the effects of Kilo-V and Mega-V X-rays on the cell morphology, viability, membrane permeability, DNA damage, and gene transfection of 293 T and CHO cells. Results: The Kilo-V X-rays of very low doses from 0.01 to 0.04 Gray in principle didn’t induce any significant change in cell morphology, growth, membrane permeability, and cause DNA damage. The Mega-V X-ray had a damage threshold between 1.0 and 1.5 Gray. The 0.25 Gray Mega-V-X-ray could promote cell growth and gene transfer, while the 1.5 Gray Mega-V X-ray damaged cells. Conclusion: The very low dose of KV X-rays is safe to cells, while the effects of Mega-V-X-rays are dose-dependent. Mega-V-X-rays with a dose higher than the damage threshold would be harmful, that between 1.0 -1.5 Gray can evoke dual effects, whereas 0.25 Gray MV X-ray is beneficial for both cell growth and gene transfer, thus would be suitable for radiation-enhanced gene transfection.

The Impact of Restriction-Modification Systems on Mating in Haloferax volcanii

Halobacteria have been observed to be highly recombinogenic, frequently exchanging genetic material. Several barriers to mating in the Halobacteria have been examined, such as CRISPR-Cas, glycosylation, and archaeosortases, but these are low barriers that do not drastically reduce the recombination frequency. Another potential barrier could be restriction-modification (RM) systems, which cleave DNA that is not properly methylated, thus limiting the exchange of genetic material between cells which do not have compatible RM systems. In order to examine the role of RM systems on limiting recombination in the Halobacteria, the impact of RM systems on cell-to-cell mating in Haloferax volcanii, a well-characterized method of genetic exchange and recombination in a halobacterial species, was examined. Strains which possessed all naturally-occurring RM system genes in H. volcanii (RM+) and strains without these RM systems (ΔRM) were mated together to compare the efficiency of gene transfer between RM-compatible strains and RM-incompatible strains. The results indicated that mating RM-incompatible strains together resulted in a decrease in gene transfer efficiency compared to mating RM-compatible strains together, suggesting that RM systems limit mating in H. volcanii, but do not act as absolute barriers to recombination. Therefore, RM systems are low barriers to recombination in the Halobacteria, with RM-incompatible strains exchanging genetic material at a lower frequency than those with compatible RM systems, similar to other low recombination barriers in the Halobacteria.

Nonviral Gene Therapy for Cancer: A Review

Although the development of effective viral vectors put gene therapy on the road to commercialization, nonviral vectors show promise for practical use because of their relative safety and lower cost. A significant barrier to the use of nonviral vectors, however, is that they have not yet proven effective. This apparent lack of interest can be attributed to the problem of the low gene transfer efficiency associated with nonviral vectors. The efficiency of gene transfer via nonviral vectors has been reported to be 1/10th to 1/1000th that of viral vectors. Despite the fact that new gene transfer methods and nonviral vectors have been developed, no significant improvements in gene transfer efficiency have been achieved. Nevertheless, some notable progress has been made. In this review, we discuss studies that report good results using nonviral vectors in vivo in animal models, with a particular focus on studies aimed at in vivo gene therapy to treat cancer, as this disease has attracted the interest of researchers developing nonviral vectors. We describe the conditions in which nonviral vectors work more efficiently for gene therapy and discuss how the goals might differ for nonviral versus viral vector development and use.