Enhancement of Motor Function Recovery after Spinal Cord Injury in Mice by Delivery of Brain-Derived Neurotrophic Factor mRNA.

Spinal cord injury (SCI) is a debilitating condition that can cause impaired motor function or full paralysis. In the days to weeks following the initial mechanical injury to the spinal cord, inflammation and apoptosis can cause additional damage to the injured tissues. This secondary injury impairs recovery. Brain-derived neurotrophic factor is a secreted protein that has been shown to improve a variety of neurological conditions, including SCI, by promoting neuron survival and synaptic plasticity. This study treated a mouse model of contusion SCI using a single dose of brain-derived neurotrophic factor (BDNF) mRNA nanomicelles prepared with polyethylene glycol polyamino acid block copolymer directly injected into the injured tissue. BDNF levels in the injured spinal cord tissue were approximately doubled by mRNA treatment. Motor function was monitored using the Basso Mouse Scale and Noldus CatWalk Automated Gait Analysis System for 6 weeks post-injury. BDNF-treated mice showed improved motor function recovery, demonstrating the feasibility of mRNA delivery to treat SCI.

Metabolically stabilized double-stranded mRNA polyplexes.

The metabolic instability of mRNA currently limits its utility for gene therapy. Compared to plasmid DNA, mRNA is significantly more susceptible to digestion by RNase in the circulation following systemic dosing. To increase mRNA metabolic stability, we hybridized a complementary reverse mRNA with forward mRNA to generate double-stranded mRNA (dsmRNA). RNase A digestion of dsmRNA established a 3000-fold improved metabolic stability compared to single-stranded mRNA (ssmRNA). Formulation of a dsmRNA polyplex using a PEG-peptide further improved the stability by 3000-fold. Hydrodynamic dosing and quantitative bioluminescence imaging of luciferase expression in the liver of mice established the potent transfection efficiency of dsmRNA and dsmRNA polyplexes. However, hybridization of the reverse mRNA against the 5' and 3' UTR of forward mRNA resulted in UTR denaturation and a tenfold loss in expression. Repeat dosing of dsmRNA polyplexes produced an equivalent transient expression, suggesting the lack of an immune response in mice. Co-administration of excess uncapped dsmRNA with a dsmRNA polyplex failed to knock down expression, suggesting that dsmRNA is not a Dicer substrate. Maximal circulatory stability was achieved using a fully complementary dsmRNA polyplex. The results established dsmRNA as a novel metabolically stable and transfection-competent form of mRNA.

Combined CatWalk Index: an improved method to measure mouse motor function using the automated gait analysis system.

OBJECTIVE: Measuring motor function in mice is important for studying models of spinal cord injury (SCI) or other diseases. Several methods exist based on visual observation of mice moving in an open field. Though these methods require very little equipment, observers must be trained, and the possibility of human error or subjectivity cannot be eliminated. The Noldus CatWalk XT Automated Gait Analysis system assesses mouse motor function by taking high-resolution videos of the mice, with specialized software to measure several aspects of the animal's gait. This instrument reduces the possibility of human error, but it is not always clear what data is important for assessing motor function. This study used data collected during mouse SCI experiments to create a simple mathematical model that combines the data collected by the CatWalk system into a single score, the Combined CatWalk Index or CCI. RESULTS: The CCI system produces similar results to the Basso Mouse Scale or the CatWalk's Step Sequence Regularity Index. However, the CCI has a significantly smaller coefficient of variation than either other method. Additionally, CCI scoring shows slightly better correlation with impact force. The CCI system is likely to be a useful tool for SCI research.

Structure-Activity Relationship of PEGylated Polylysine Peptides as Scavenger Receptor Inhibitors for Non-Viral Gene Delivery.

PEGylated polylysine peptides of the general structure PEG30 kDa-Cys-Trp-LysN (N = 10 to 30) were used to form fully condensed plasmid DNA (pGL3) polyplexes at a ratio of 1 nmol of peptide per µg of DNA (ranging from N:P 3:1 to 10:1 depending on Lys repeat). Co-administration of 5 to 80 nmols of excess PEG-peptide with fully formed polyplexes inhibited the liver uptake of (125)I-pGL3-polyplexes. The percent inhibition was dependent on the PEG-peptide dose and was saturable, consistent with inhibition of scavenger receptors. The scavenger receptor inhibition potency of PEG-peptides was dependent on the length of the Lys repeat, which increased 10-fold when comparing PEG30 kDa-Cys-Trp-Lys10 (IC50 of 20.2 µM) with PEG30 kDa-Cys-Trp-Lys25 (IC50 of 2.1 µM). We hypothesize that PEG-peptides inhibit scavenger receptors by spontaneously forming small 40 to 60 nm albumin nanoparticles that bind to and saturate the receptor. Scavenger receptor inhibition delayed the metabolism of pGL3-polyplexes, resulting in efficient gene expression in liver hepatocytes following delayed hydrodynamic dosing. PEG-peptides represent a new class of scavenger inhibitors that will likely have broad utility in blocking unwanted liver uptake and metabolism of a variety of nanoparticles.

"Evolving nanoparticle gene delivery vectors for the liver: What has been learned in 30 years".

Nonviral gene delivery to the liver has been under evolution for nearly 30years. Early demonstrations established relatively simple nonviral vectors could mediate gene expression in HepG2 cells which understandably led to speculation that these same vectors would be immediately successful at transfecting primary hepatocytes in vivo. However, it was soon recognized that the properties of a nonviral vector resulting in efficient transfection in vitro were uncorrelated with those needed to achieve efficient nonviral transfection in vivo. The discovery of major barriers to liver gene transfer has set the field on a course to design biocompatible vectors that demonstrate increased DNA stability in the circulation with correlating expression in liver. The improved understanding of what limits nonviral vector gene transfer efficiency in vivo has resulted in more sophisticated, low molecular weight vectors that allow systematic optimization of nanoparticle size, charge and ligand presentation. While the field has evolved DNA nanoparticles that are stable in the circulation, target hepatocytes, and deliver DNA to the cytosol, breaching the nucleus remains the last major barrier to a fully successful nonviral gene transfer system for the liver. The lessons learned along the way are fundamentally important to the design of all systemically delivered nanoparticle nonviral gene delivery systems.

Miniaturization of gene transfection assays in 384- and 1536-well microplates.

The miniaturization of gene transfer assays to either 384- or 1536-well plates greatly economizes the expense and allows much higher throughput when transfecting immortalized and primary cells compared with more conventional 96-well assays. To validate the approach, luciferase and green fluorescent protein (GFP) reporter gene transfer assays were developed to determine the influence of cell seeding number, transfection reagent to DNA ratios, transfection time, DNA dose, and luciferin dose on linearity and sensitivity. HepG2, CHO, and NIH 3T3 cells were transfected with polyethylenimine (PEI)-DNA in both 384- and 1536-well plates. The results established optimal transfection parameters in 384-well plates in a total assay volume of 35µl and in 1536-well plates in a total assay volume of 8µl. A luciferase assay performed in 384-well plates produced a Z' score of 0.53, making it acceptable for high-throughput screening. Primary hepatocytes were harvested from mouse liver and transfected with PEI DNA and calcium phosphate DNA nanoparticles in 384-well plates. Optimal transfection of primary hepatocytes was achieved on as few as 250cellsperwell in 384-well plates, with CaPO4 proving to be 10-fold more potent than PEI.