Transduction profiles in minipig following MRI guided delivery of AAV-5 into thalamic and corona radiata areas.

BACKGROUND: As a step towards clinical use of AAV-mediated gene therapy, brains of large animals are used to settle delivery parameters as most brain connections, and relative sizes in large animals and primates, are reasonably common. Prior to application in the clinic, approaches that have shown to be successful in rodent models are tested in larger animal species, such as dogs, non-human primates, and in this case, minipigs. NEW METHOD: We evaluated alternate delivery routes to target the basal ganglia by injections into the more superficial corona radiata, and, deeper into the brain, the thalamus. Anatomically known connections can be used to predict the expression of the transgene following infusion of AAV5. For optimal control over delivery of the vector with regards to anatomical location in the brain and spread in the tissue, we have used magnetic resonance image-guided convection-enhanced diffusion delivery. RESULTS: While the transduction of the cortex was observed, only partial transduction of the basal ganglia was achieved via the corona radiata. Thalamic administration, on the other hand, resulted in widespread transduction from the midbrain to the frontal cortex COMPARISON WITH EXISTING METHODS: Compared to other methods, such as delivery directly to the striatum, thalamic injection may provide an alternative when for instance, injection into the basal ganglia directly is not feasible. CONCLUSIONS: The study results suggest that thalamic administration of AAV5 has significant potential for indications where the transduction of specific areas of the brain is required.

Self-assembly of bovine serum albumin (BSA)-dextran bio-nanoconjugate: structural, antioxidant and in vitro wound healing studies.

Glycation of proteins is often considered as a method to improve their functional properties for promising applications in wound healing. Furthermore, a marked increase in percentage of radical scavenging activity of the conjugates makes it an effective antioxidant synthetic strategy. A simple conjugation process was employed to develop bovine serum albumin-dextran conjugates (BSA-dextran) using Maillard reaction. Higher electrophoretic mobility and surface charge in the prepared conjugates was observed in native PAGE electrophoresis and zeta potential. Moreover, the fluorescence, FTIR and Raman analysis of the BSA-dextran conjugates shows significant shift in the fluorescence and wavelength as a consequence of conjugate formation. In vitro wound healing assay showed increased cell proliferation and migration effect. These finding suggests that BSA-dextran conjugate could open up a new practical way for exploration in the area of wound healing.

Correction: Self-assembly of bovine serum albumin (BSA)-dextran bio-nanoconjugate: structural, antioxidant and in vitro wound healing studies.

[This corrects the article DOI: 10.1039/D0RA09301G.].

Polyethylenimine based magnetic nanoparticles mediated non-viral CRISPR/Cas9 system for genome editing.

Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas9) system has become a revolutionary tool for gene editing. Since viral delivery systems have significant side effects, and naked DNA delivery is not an option, the nontoxic, non-viral delivery of CRISPR/Cas9 components would significantly improve future therapeutic delivery. In this study, we aim at characterizing nanoparticles to deliver plasmid DNA encoding for the CRISPR-Cas system in eukaryotic cells in vitro. CRISPR/Cas9 complexed polyethylenimine (PEI) magnetic nanoparticles (MNPs) were generated. We used a stable HEK293 cell line expressing the traffic light reporter (TLR-3) system to evaluate efficient homology- directed repair (HDR) and non-homologous end joining (NHEJ) events following transfection with NPs. MNPs have been synthesized by co-precipitation with the average particle size around 20 nm in diameter. The dynamic light scattering and zeta potential measurements showed that NPs exhibited narrow size distribution and sufficient colloidal stability. Genome editing events were as efficient as compared to standard lipofectamine transfection. Our approach tested non-viral delivery of CRISPR/Cas9 and DNA template to perform HDR and NHEJ in the same assay. We demonstrated that PEI-MNPs is a promising delivery system for plasmids encoding CRISPR/Cas9 and template DNA and thus can improve safety and utility of gene editing.

Cks overexpression enhances chemotherapeutic efficacy by overriding DNA damage checkpoints.

Cdc kinase subunit (Cks) proteins Cks1 and Cks2 are adaptor-like proteins that bind many cyclin-dependent kinases. A wealth of clinical data has shown that Cks proteins are overexpressed in many types of human cancers and this often correlates with increased tumor aggressiveness. Previously, we showed that Cks overexpression abrogates the intra-S-phase checkpoint, a major barrier to oncogene-mediated transformation. Interestingly, the intra-S-phase checkpoint is crucial for the cellular response to replication stress, a major pathway of apoptosis induction by many chemotherapeutic agents. Here, we demonstrate cancer cells that overexpress Cks1 or Cks2 override the intra-S-phase checkpoint in the presence of replication stress-inducing chemotherapies such as 5-Fluorouracil (5-FU) and methotrexate (MTX) leading to enhanced sensitivity in vitro and in vivo. Furthermore, enforced expression of Cks1 in an MTX-resistant breast cancer cell line was found to restore drug sensitivity. Our results suggest that Cks proteins are important determinants of apoptosis induction of replication stress-inducing chemotherapies such as 5-FU.