Non-viral gene therapy that targets motor neurons in vivo

A major challenge in neurological gene therapy is safe delivery of transgenes to sufficient cell numbers from the circulation or periphery. This is particularly difficult for diseases involving spinal cord motor neurons such as amyotrophic lateral sclerosis (ALS). We have examined the feasibility of non-viral gene delivery to spinal motor neurons from intraperitoneal injections of plasmids carried by "immunogene" nanoparticles targeted for axonal retrograde transport using antibodies. PEGylated polyethylenimine (PEI-PEG12) as DNA carrier was conjugated to an antibody (MLR2) to the neurotrophin receptor p75 (p75NTR). We used a plasmid (pVIVO2) designed for in vivo gene delivery that produces minimal immune responses, has improved nuclear entry into post mitotic cells and also expresses green fluorescent protein (GFP). MLR2-PEI-PEG12 carried pVIVO2 and was specific for mouse motor neurons in mixed cultures containing astrocytes. While only 8% of motor neurons expressed GFP 72 h post transfection in vitro, when the immunogene was given intraperitonealy to neonatal C57BL/6J mice, GFP specific motor neuron expression was observed in 25.4% of lumbar, 18.3% of thoracic and 17.0% of cervical motor neurons, 72 h post transfection. PEI-PEG12 carrying pVIVO2 by itself did not transfect motor neurons in vivo, demonstrating the need for specificity via the p75NTR antibody MLR2. This is the first time that specific transfection of spinal motor neurons has been achieved from peripheral delivery of plasmid DNA as part of a non-viral gene delivery agent. These results stress the specificity and feasibility of immunogene delivery targeted for p75NTR expressing motor neurons, but suggests that further improvements are required to increase the transfection efficiency of motor neurons in vivo.Mary-Louise Rogers, Kevin S. Smith, Dusan Matusica, Matthew Fenech, Lee Hoffman, Robert A. Rush and Nicolas H. Voelcke

Proteolytic processing of the p75 neurotrophin receptor: A prerequisite for signalling? (vol 33, pg 614, 2011)

The common neurotrophin receptor (p75(NTR)) regulates various functions in the developing and adult nervous system. Cell survival, cell death, axonal and growth cone retraction, and regulation of the cell cycle can be regulated by p75(NTR)-mediated signals following activation by either mature or pro-neurotrophins and in combination with various co-receptors, including Trk receptors and sortilin. Here, we review the known functions of p75(NTR) by cell type, receptor-ligand combination, and whether regulated intra-membrane proteolysis of p75(NTR) is required for signalling. We highlight that the generation of the intracellular domain fragment of p75(NTR) is associated with many of the receptor functions, regardless of its ligand and co-receptor interactions

Electrospun nanodiamond-silk fibroin membranes: a multifunctional platform for biosensing and wound-healing applications

Next generation wound care technology capable of diagnosing wound parameters, promoting healthy cell growth, and reducing pathogenic infections noninvasively would provide patients with an improved standard of care and accelerated wound repair. Temperature is one of the indicating biomarkers specific to chronic wounds. This work reports a hybrid, multifunctional optical material platform-nanodiamond (ND)-silk membranes as biopolymer dressings capable of temperature sensing and promoting wound healing. The hybrid structure was fabricated through electrospinning, and 3D submicron fibrous membranes with high porosity were formed. Silk fibers are capable of compensating for the lack of an extracellular matrix at the wound site, supporting the wound-healing process. Negatively charged nitrogen vacancy (NV-) color centers in NDs exhibit optically detected magnetic resonance (ODMR) and act as nanoscale thermometers. This can be exploited to sense temperature variations associated with the presence of infection or inflammation in a wound, without physically removing the dressing. Our results show that the presence of NDs in the hybrid ND-silk membranes improves the thermal stability of silk fibers. NV- color centers in NDs embedded in silk fibers exhibit well-retained fluorescence and ODMR. Using the NV- centers as fluorescent nanoscale thermometers, we achieved temperature sensing in 25-50 °C, including the biologically relevant temperature window, for cell-grown ND-silk membranes. An enhancement (∼1.5× on average) in the temperature sensitivity of the NV- centers was observed for the hybrid materials. The hybrid membranes were further tested in vivo in a murine wound-healing model and demonstrated biocompatibility and equivalent wound closure rates as the control wounds. Additionally, the hybrid ND-silk membranes exhibited selective antifouling and biocidal propensity toward Gram-negative Pseudomonas aeruginosa and Escherichia coli, while no effect was observed on Gram-positive Staphylococcus aureus.Asma Khalid, Dongbi Bai, Amanda N. Abraham, Amit Jadhav, Denver Linklater, Alex Matusica ... et al

Regulator of Calcineurin 1 helps coordinate whole-body metabolism and thermogenesis

Increasing non-shivering thermogenesis (NST), which expends calories as heat rather than storing them as fat, is championed as an effective way to combat obesity and metabolic disease. Innate mechanisms constraining the capacity for NST present a fundamental limitation to this approach, yet are not well understood. Here, we provide evidence that Regulator of Calcineurin 1 (RCAN1), a feedback inhibitor of the calcium-activated protein phosphatase calcineurin (CN), acts to suppress two distinctly different mechanisms of non-shivering thermogenesis (NST): one involving the activation of UCP1 expression in white adipose tissue, the other mediated by sarcolipin (SLN) in skeletal muscle. UCP1 generates heat at the expense of reducing ATP production, whereas SLN increases ATP consumption to generate heat. Gene expression profiles demonstrate a high correlation between Rcan1 expression and metabolic syndrome. On an evolutionary timescale, in the context of limited food resources, systemic suppression of prolonged NST by RCAN1 might have been beneficial; however, in the face of caloric abundance, RCAN1-mediated suppression of these adaptive avenues of energy expenditure may now contribute to the growing epidemic of obesity.David Rotter, Heshan Peiris, D Bennett Grinsfelder, Alyce M Martin, Jana Burchfield ... Claire F Jessup ... et al

The E. coli pET expression system revisited—mechanistic correlation between glucose and lactose uptake

The final publication is available at Springer via https://doi.org/10.1007/s00253-016-7620-7.Therapeutic monoclonal antibodies are mainly produced in mammalian cells to date. However, unglycosylated antibody fragments can also be produced in the bacterium Escherichia coli which brings several advantages, like growth on cheap media and high productivity. One of the most popular E. coli strains for recombinant protein production is E. coli BL21(DE3) which is usually used in combination with the pET expression system. However, it is well known that induction by isopropyl β-d-1-thiogalactopyranoside (IPTG) stresses the cells and can lead to the formation of insoluble inclusion bodies. In this study, we revisited the pET expression system for the production of a novel antibody single-chain variable fragment (scFv) with the goal of maximizing the amount of soluble product. Thus, we (1) investigated whether lactose favors the recombinant production of soluble scFv compared to IPTG, (2) investigated whether the formation of soluble product can be influenced by the specific glucose uptake rate (qs,glu) during lactose induction, and (3) determined the mechanistic correlation between the specific lactose uptake rate (qs,lac) and qs,glu. We found that lactose induction gave a much greater amount of soluble scFv compared to IPTG, even when the growth rate was increased. Furthermore, we showed that the production of soluble protein could be tuned by varying qs,glu during lactose induction. Finally, we established a simple model describing the mechanistic correlation between qs,lac and qs,glu allowing tailored feeding and prevention of sugar accumulation. We believe that this mechanistic model might serve as platform knowledge for E. coli