Nanoparticle-motivated gene delivery for ophthalmic application: Nano-vehicle for ocular gene delivery

Ophthalmic gene therapy is an intellectual and intentional manipulation of desired gene expression into the specific cells of an eye for the treatment of ophthalmic (ocular) genetic dystrophies and pathological conditions. Exogenous nucleic acids such as DNA, small interfering RNA (siRNA), micro RNA (miRNA), etc., are used for the purpose of managing expression of the desired therapeutic proteins in ocular tissues. The delivery of unprotected nucleic acids into the cells is limited due to exogenous and endogenous degradation modalities. Nanotechnology, a promising and sophisticated cutting edge tool, works as a protective shelter for these therapeutic nucleic acids. They are able to be safely delivered to the required cells in order to modulate anticipated protein expression. To this end, nanotechnology is seen as a potential and promising strategy in the field of ocular gene delivery. This review focused on current nanotechnology modalities and other promising non-viral strategies being used to deliver therapeutic genes in order to treat various devastating ocular diseases

Nanoparticle-mediated miR200-b delivery for the treatment of diabetic retinopathy

We recently reported that the Ins2Akita mouse is a good model for late-onset diabetic retinopathy. Here, we investigated the effect of miR200-b, a potential anti-angiogenic factor, on VEGF receptor 2 (VEGFR-2) expression and to determine the underlying angiogenic response in mouse endothelial cells, and in retinas from aged Ins2Akita mice. MiR200-b and its native flanking sequences were amplified and cloned into a pCAG-eGFP vector directed by the ubiquitous CAG promoter (namely pCAG-miR200-b-IRES-eGFP). The plasmid was compacted by CK30PEG10K into DNA nanoparticles (NPs) for in vivo delivery. Murine endothelial cell line, SVEC4-10, was first transfected with the plasmid. The mRNA levels of VEGF and VEGFR-2 were quantified by qRT-PCR and showed significant reduction in message expression compared with lipofectamine-transfected cells. Transfection of miR200-b suppressed the migration of SVEC4-10 cells. There was a significant inverse correlation between the level of expression of miR200-b and VEGFR-2. Intravitreal injection of miR200-b DNA NPs significantly reduced protein levels of VEGFR-2 as revealed by western blot and markedly suppressed angiogenesis as evaluated by fundus imaging in aged Ins2Akita mice even after 3 months of post-injection. These findings suggest that NP-mediated miR200-b delivery has negatively regulated VEGFR-2 expression in vivo

Comparative analysis of DNA nanoparticles and AAVs for ocular gene delivery.

Gene therapy is a critical tool for the treatment of monogenic retinal diseases. However, the limited vector capacity of the current benchmark delivery strategy, adeno-associated virus (AAV), makes development of larger capacity alternatives, such as compacted DNA nanoparticles (NPs), critical. Here we conduct a side-by-side comparison of self-complementary AAV and CK30PEG NPs using matched ITR plasmids. We report that although AAVs are more efficient per vector genome (vg) than NPs, NPs can drive gene expression on a comparable scale and longevity to AAV. We show that subretinally injected NPs do not leave the eye while some of the AAV-injected animals exhibited vector DNA and GFP expression in the visual pathways of the brain from PI-60 onward. As a result, these NPs have the potential to become a successful alternative for ocular gene therapy, especially for the multitude of genes too large for AAV vectors

Role of Dichloroacetate in the Treatment of Genetic Mitochondrial Diseases

Dichloroacetate (DCA) is an investigational drug for the treatment of genetic mitochondrial diseases. Its primary site of action is the pyruvate dehydrogenase (PDH) complex, which it stimulates by altering its phosphorylation state and stability. DCA is metabolized by and inhibits the bifunctional zeta-1 family isoform of glutathione transferase/maleylacetoacetate isomerase. Polymorphic variants of this enzyme differ in their kinetic properties toward DCA, thereby influencing its biotransformation and toxicity, both of which are also influenced by subject age. Results from open label studies and controlled clinical trials suggest chronic oral DCA is generally well-tolerated by young children and may be particularly effective in patients with PDH deficiency. Recent in vitro data indicate that a combined DCA and gene therapy approach may also hold promise for the treatment of this devastating condition

AAV and Compacted DNA Nanoparticles for the Treatment of Retinal Disorders: Challenges and Future Prospects

Gene therapy based on delivery of viral and nonviral vectors has shown great promise for the treatment of human ocular diseases; however, limitations have consistently prevented its widespread clinical application. Viral vectors have generally been better in terms of efficiency but have safety concerns. Nonviral vectors, on the other hand, offer safety but have often been disappointing in terms of efficiency of nuclear delivery and gene expression. Extensive animal studies have reported significant progress using both systems, but thus far only a few studies have shown promise in human clinical trials. This article reviews both viral and nonviral work with focus on two candidates for clinical ocular application—AAV and nanoparticles. Of particular interest are various requirements for successful clinical application of these technologies including vector trafficking, delivery, specific gene expression, and treatment safety, and tolerance

Riemerella anatipestifer GldM is required for bacterial gliding motility, protein secretion, and virulence

International audienceAbstractRiemerella anatipestifer is a major pathogenic agent of duck septicemic and exudative diseases. Genetic analyses suggest that this pathogen has a novel protein secretion system, known as the “type IX secretion system” (T9SS). We previously reported that deletion of the AS87_RS08465 gene significantly reduced the bacterial virulence of the R. anatipestifer strain Yb2, but the mechanism remained unclear. The AS87_RS08465 gene is predicted to encode the gliding motility protein GldM (GldM) protein, a key component of the T9SS complex. In this study, Western blotting analysis demonstrated that R. anatipestifer GldM was localized to the cytomembrane. Further study revealed that the adhesion and invasion capacities of the mutant strain RA2281 (designated Yb2ΔgldM) in Vero cells and the bacterial loads in the blood of infected ducks were significantly reduced. RNA-Seq and PCR analyses showed that six genes were upregulated and five genes were downregulated in the mutant strain Yb2ΔgldM and that these genes were mainly involved in the secretion of proteins. Yb2ΔgldM was also found to be defective in gliding motility and protein secretion. Liquid chromatography–tandem mass spectrometry analysis revealed that nine of the proteins had a conserved T9SS C-terminal domain and were differentially secreted by Yb2ΔgldM compared to Yb2. The complementation strain cYb2ΔgldM recovered the adhesion and invasion capacities in Vero cells and the bacterial loads in the blood of infected ducks as well as the bacterial gliding motility and most protein secretion in the mutant strain Yb2ΔgldM to the levels of the wild-type strain Yb2. Taken together, these results indicate that R. anatipestifer GldM is associated with T9SS and is important in bacterial virulence

Cu2O Mediated Synthesis of Metal Organic Framework UiO-66 in Nanometer Scale

Controlled synthesis of metal organic frameworks (MOFs) in nanometer scale is highly desired for the optimization of properties and the extending of applications. In this work, Cu2O was used as additive for the first time in the synthesis of zirconium-based MOF UiO-66. We found that the amount of Cu2O additive had an important impact on downsizing the crystal size of UiO-66. Cu2O additive plays a role of rate controller of crystal growth of UiO-66 crystals by their competitive coordination behavior with the linker H2BDC between the [Zr-O] clusters. This generates a depleted H2BDC concentration for the coordination with the [Zr-O] clusters for constructing the UiO-66 framework, which favors the production of small crystals. As a result, well-dispersed nanometer scale UiO-66 crystals with mean crystal size of 40 nm can be easily synthesized by the addition of Cu2O. The as-synthesized nanometer scale UiO-66 crystals have a pure crystal structure with high crystallinity and superior porosity