Toward more effective gene delivery

A report on the symposium 'In vivo barriers to gene delivery', Cold Spring Harbor, USA, 26-29 November 2007

The mechanism of transcellular transport of immunoglobulin G in yolk-sac tissue

The route of prenatal antibody transmission in both the rat and rabbit is via the yolk sac (Branbell, 1970), and a method for studying in vitro endocytosis in yolk sacs (Williams et al, 1975) was modified as a model for the process of transcellular immunoglobulin transport (Weisbecker, 1981). In this study, the validity of the model was substantiated and various investigations were performed. The signal mediating specific protection in vitro was shown to reside in the Fc fragment of the IgG molecule, and carbohydrate moieties did not appear to be involved in the interaction. Neuraminidase-treatment of IgG enhanced protection, while formaldehyde destroyed it. The inference was that the signal sequence was polypeptide in nature, contained a basic amino acid group, and was located close to one of the carbohydrate groups of the molecule, probably the asymmetric, hinge-linked one. Various inhibitors were shown to inhibit equally uptake into the protective and degradative routes. The evidence suggested a common uptake stage in the uptake of IgG in both pathways. Release of macromolecular material was found to be sensitive to metabolic inhibitors, but not so much to cytoskeletal ones, EGTA or bulk protein. In contrast to the hypotheses of Brambell (1966) (where specific membrane-bound receptors are thought sterically to protect IgG from lysosomal degradation) and Wild (1975) (specific micropinocytic coated vesicles carry IgG from the plasmalemma to the basal or lateral membranes), it is proposed that IgG for protection enters common sorting vesicles along with other proteins (mainly destined for degradation) and fluid. It is sorted quickly (before the vesicle becomes too acid) and packaged into specific microvesicles that release their contents at the lateral or basal membranes. It is then free to make its way across the subcellular mesenchyme into the vitelline blood capillaries, and thence into the foetus intact

Dynamics of polydisperse irreversible adsorption: a pharmacological example

Many drug delivery systems suffer from undesirable interactions with the host immune system. It has been experimentally established that covalent attachment (irreversible adsorption) of suitable macromolecules to the surface of the drug carrier can reduce such undesirable interactions. A fundamental understanding of the adsorption process is still lacking. In this paper, the classical random irreversible adsorption model is generalized to capture certain essential processes involved in pharmacological applications, allowing for macromolecules of different sizes, partial overlapping of the tails of macromolecules, and the influence of reactions with the solvent on the adsorption process. Working in one dimension, an integro-differential evolution equation for the adsorption process is derived, and the asymptotic behavior of the surface area covered and the number of molecules attached to the surface are studied. Finally, equation-free dynamic renormalization tools are applied to study the asymptotically self-similar behavior of the adsorption statistics

Establishment of a positive-readout reporter system for siRNAs

The use of small interfering RNA molecules for therapeutic applications requires development of improved delivery systems, a process that would be facilitated by a non-invasive positive-readout mouse model for studying siRNA pharmacodynamics. Positive readout would yield better signal/noise ratios than existing negative-readout systems. We have engineered a positive-readout luciferase reporter system, activated by successful delivery of siRNA targeting the lac repressor. Co-transfection of a plasmid expressing lac repressor and a plasmid expressing firefly luciferase under the control of an RSV promoter, containing two lac operator sites, resulted in 5.7-fold lower luciferase activity than luciferase-encoding plasmid alone. Inhibition was reversed following addition of synthetic inducer, IPTG, which elevated luciferase expression to normal levels and confirmed functionality of the lac operon. Delivery of 1nM siRNA targeting lac repressor to repressor/reporter co-transfected cells was sufficient to fully restore luciferase expression to levels observed in the absence of repressor. Maximum expression was observed after 48hr, with a rapid decrease thereafter due to the short half life of luciferase. The luciferase positive-readout reporter system is therefore a dynamic indicator of successful RNAi delivery in vitro and could be adapted to generate a transgenic mouse capable of reporting RNAi activity non-invasively in vivo

Optogenetic control of iPS cell-derived neurons in 2D and 3D culture systems using channelrhodopsin-2 expression driven by the synapsin-1 and calcium-calmodulin kinase II promoters

Development of an optogenetically controllable human neural network model in three-dimensional (3D) cultures can provide an investigative system that is more physiologically relevant and better able to mimic aspects of human brain function. Light-sensitive neurons were generated by transducing channelrhodopsin-2 (ChR2) into human induced pluripotent stem cell (hiPSC) derived neural progenitor cells (Axol) using lentiviruses and cell-type specific promoters. A mixed population of human iPSC-derived cortical neurons, astrocytes and progenitor cells were obtained (Axol-ChR2) upon neural differentiation. Pan-neuronal promoter synapsin-1 (SYN1) and excitatory neuron-specific promoter calcium-calmodulin kinase II (CaMKII) were used to drive reporter gene expression in order to assess the differentiation status of the targeted cells. Expression of ChR2 and characterisation of subpopulations in differentiated Axol-ChR2 cells were evaluated using flow cytometry and immunofluorescent staining. These cells were transferred from 2D culture to 3D alginate hydrogel functionalised with arginine-glycine-aspartate (RGD) and small molecules (Y-27632). Improved RGD-alginate hydrogel was physically characterised and assessed for cell viability to serve as a generic 3D culture system for human pluripotent stem cells (hPSCs) and neuronal cells. Prior to cell encapsulation, neural network activities of Axol-ChR2 cells and primary neurons were investigated using calcium imaging. Results demonstrate that functional activities were successfully achieved through expression of ChR2- by both the CaMKII and SYN1 promoters. The RGD-alginate hydrogel system supports the growth of differentiated Axol-ChR2 cells whilst allowing detection of ChR2 expression upon light stimulation. This allows precise and non-invasive control of human neural networks in 3D

Nanomedicines for the delivery of biologics

A special symposium of the Academy of Pharmaceutical Sciences Nanomedicines Focus Group reviewed the current status of the use of nanomedicines for the delivery of biologics drugs. This meeting was particularly timely with the recent approval of the first siRNA-containing product Onpattro™ (patisiran), which is formulated as a lipid nanoparticle for intravenous infusion, and the increasing interest in the use of nanomedicines for the oral delivery of biologics. The challenges in delivering such molecules were discussed with specific emphasis on the delivery both across and into cells. The latest developments in Molecular Envelope Technology® (Nanomerics Ltd, London, UK), liposomal drug delivery (both from an academic and industrial perspective), opportunities offered by the endocytic pathway, delivery using genetically engineered viral vectors (PsiOxus Technologies Ltd, Abingdon, UK), Transint™ technology (Applied Molecular Transport Inc., South San Francisco, CA, USA), which has the potential to deliver a wide range of macromolecules, and AstraZeneca’s initiatives in mRNA delivery were covered with a focus on their uses in difficult to treat diseases, including cancers. Preclinical data were presented for each of the technologies and where sufficiently advanced, plans for clinical studies as well as early clinical data. The meeting covered the work in progress in this exciting area and highlighted some key technologies to look out for in the future

Cytoplasmic expression systems triggered by mRNA yield increased gene expression in post-mitotic neurons

Non-viral vectors are promising vehicles for gene therapy but delivery of plasmid DNA to post-mitotic cells is challenging as nuclear entry is particularly inefficient. We have developed and evaluated a hybrid mRNA/DNA system designed to bypass the nuclear barrier to transfection and facilitate cytoplasmic gene expression. This system, based on co-delivery of mRNA(A64) encoding for T7 RNA polymerase (T7 RNAP) with a T7-driven plasmid, produced between 10- and 2200-fold higher gene expression in primary dorsal root ganglion neuronal (DRGN) cultures isolated from Sprague–Dawley rats compared to a cytomegalovirus (CMV)-driven plasmid, and 30-fold greater expression than the enhanced T7-based autogene plasmid pR011. Cell-free assays and in vitro transfections highlighted the versatility of this system with small quantities of T7 RNAP mRNA required to mediate expression at levels that were significantly greater than with the T7-driven plasmid alone or supplemented with T7 RNAP protein. We have also characterized a number of parameters, such as mRNA structure, intracellular stability and persistence of each nucleic acid component that represent important factors in determining the transfection efficiency of this hybrid expression system. The results from this study demonstrate that co-delivery of mRNA is a promising strategy to yield increased expression with plasmid DNA, and represents an important step towards improving the capability of non-viral vectors to mediate efficient gene transfer in cell types, such as in DRGN, where the nuclear membrane is a significant barrier to transfection

Chronic Rhinosinusitis: Potential Role of Microbial Dysbiosis and Recommendations for Sampling Sites.

Chronic rhinosinusitis (CRS) is an inflammatory condition that affects up to 12% of the human population in developed countries. Previous studies examining the potential role of the sinus bacterial microbiota within CRS infections have found inconsistent results, possibly because of inconsistencies in sampling strategies. The aim of this study was to determine whether the sinus microbiome is altered in CRS and additionally if the middle meatus is a suitable representative site for sampling the sinus microbiome. Swab samples were collected from 12 healthy controls and 21 CRS patients, including all eight sinuses for CRS patients and between one and five sinuses for control subjects. The left and right middle meatus and nostril swabs were also collected. Significant differences in the sinus microbiomes between CRS and control samples were revealed using high-throughput 16S rRNA gene sequencing. The genus Escherichia was over-represented in CRS sinuses, and associations between control patients and Corynebacterium and Dolosigranulum were also identified. Comparisons of the middle meatuses between groups did not reflect these differences, and the abundance of the genus Escherichia was significantly lower at this location. Additionally, intra-patient variation was lower between sinuses than between sinus and middle meatus, which together with the above results suggests that the middle meatus is not an effective representative sampling site

The High Angular Resolution Multiplicity of Massive Stars

We present the results of a speckle interferometric survey of Galactic massive stars that complements and expands upon a similar survey made over a decade ago. The speckle observations were made with the KPNO and CTIO 4 m telescopes and USNO speckle camera, and they are sensitive to the detection of binaries in the angular separation regime between 0.03" and 5" with relatively bright companions (Delta V < 3). We report on the discovery of companions to 14 OB stars. In total we resolved companions of 41 of 385 O-stars (11%), 4 of 37 Wolf-Rayet stars (11%), and 89 of 139 B-stars (64%; an enriched visual binary sample that we selected for future orbital determinations). We made a statistical analysis of the binary frequency among the subsample that are listed in the Galactic O Star Catalog by compiling published data on other visual companions detected through adaptive optics studies and/or noted in the Washington Double Star Catalog and by collecting published information on radial velocities and spectroscopic binaries. We find that the binary frequency is much higher among O-stars in clusters and associations compared to the numbers for field and runaway O-stars, consistent with predictions for the ejection processes for runaway stars. We present a first orbit for the O-star Delta Orionis, a linear solution of the close, apparently optical, companion of the O-star Iota Orionis, and an improved orbit of the Be star Delta Scorpii. Finally, we list astrometric data for another 249 resolved and 221 unresolved targets that are lower mass stars that we observed for various other science programs.Comment: 76 pages, 6 figures, 11 table

A versatile reducible polycation-based system for efficient delivery of a broad range of nucleic acids

Synthetic vectors based on reducible polycations consisting of histidine and polylysine residues (HIS RPCs) were evaluated for their ability to deliver nucleic acids. Initial experiments showed that RPC-based vectors with at least 70% histidine content mediated efficient levels of gene transfer without requirement for the endosomolytic agent chloroquine. Significant gene transfer was observed in a range of cell types achieving up to a 5-fold increase in the percentage of transfected cells compared to 25 kDa PEI, a gold standard synthetic vector. In contrast to 25 kDa PEI, HIS RPCs also mediated efficient transfer of other nucleic acids, including mRNA encoding green fluorescent protein in PC-3 cells and siRNA directed against the neurotrophin receptor p75(NTR) in post-mitotic cultures of rat dorsal root ganglion cell neurons. Experiments to elevate intracellular glutathione and linear profiling of cell images captured by multiphoton fluorescent microscopy highlighted that parameters such as the molecular weight and rate of cleavage of HIS RPCs were important factors in determining transfection activity. Altogether, these results demonstrate that HIS RPCs represent a novel and versatile type of vector that can be used for efficient cytoplasmic delivery of a broad range of nucleic acids. This should enable different or a combination of therapeutic strategies to be evaluated using a single type of polycation-based vector