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Climate change is disrupting historical patterns of adaptation in temperate and boreal tree species, causing local populations to become maladapted. Tree improvement programs typically utilise local base populations and manage adaptation using geographically defined breeding zones. As climates shift, breeding zones are no longer optimal seed deployment zones because base populations are becoming dissociated from their historical climatic optima. In response, climate-based seed transfer (CBST) policies incorporating assisted gene flow (AGF) are being adopted to pre-emptively match reforestation seedlots with future climates, but their implementation requires accurate knowledge of genetic variation in climatically adaptive traits. Here we use lodgepole pine as a case study to evaluate the effects of selective conifer breeding on adaptive traits and their climatic associations to inform CBST and AGF prescriptions. Our approach compared 105 natural stand and 20 selectively bred lodgepole pine seedlots from Alberta and British Columbia grown in a common garden of ∼2200 seedlings. The effects of selection on phenotypic variation and climatic associations among breeding zones were assessed for growth, phenology and cold hardiness. We found substantial differences between natural and selected seedlings in growth traits, but timing of growth initiation was unaffected, growth cessation was delayed slightly (average 4 days, range 0.7 days to 10 days), and cold injury was slightly greater (average 2.5%, range −7% to 11%) in selected seedlings. Phenotypic differentiation among breeding zones and climatic clines were stronger for all traits in selected seedlings. Height gains resulted from both increased growth rate and delayed growth cessation, but negative indirect effects of selection on cold hardiness were weak. Selection, breeding and progeny testing combined have produced taller lodgepole pine seedlings that are not adaptively compromised relative to their natural seedling counterparts. Selective breeding produces genotypes that achieve increased height growth and maintain climate adaptation, rather than reconstituting genotypes similar to populations adapted to warmer climates. While CBST is needed to optimise seedlot deployment in new climates, an absence of systematic indirect selection effects on adaptive traits suggests natural and selected seedlots do not require separate AGF prescriptions.

Rural to urban migrants are the dominant component of population growth in China's coastal cities. China's unique household registration system has excluded migrants from most public housing, resulting in pronounced housing segregation --a social phenomenon that has come to be China's greatest urban social planning challenge. In this paper, we analyze the determinants of migrant housing choices and migrant housing needs following the conventional utility-based approach, but we also emphasize the important role of social capital in migrant decision-making. A case study of Shenzhen shows that quantifiable measures of physical, locational, land use and economic attributes cannot fully explain the congregation of migrants in informal communities and that social capital plays an important role in their housing choices. Social networks, social norms, and trust among neighbors are key factors in the establishment of migrant communities. This study reveals a new aspect of migrant housing needs that will be useful to planners, by paying greater heed to social capital in urban regeneration and migrant housing provision. A more inclusive and humanistic approach to the housing of migrants is suggested.

RNA molecules such as single-stranded RNA (ssRNA) and small interfering RNA (siRNA) duplexes induce Toll-like receptor (TLR)-mediated immune stimulation after intracellular delivery. We have previously shown that selective incorporation of 2'-O-methyl (2'OMe) residues into siRNA abrogates cytokine production without reduction of gene silencing activity. Here we show that 2'OMe-modified RNA acts as a potent inhibitor of RNA-mediated cytokine induction in both human and murine systems. This activity does not require the direct incorporation of 2'OMe nucleotides into the immunostimulatory RNA or that the 2'OMe nucleotide-containing RNA be annealed as a complementary strand to form a duplex. Our results indicate that 2'OMe RNA acts as a potent antagonist of immunostimulatory RNA. We further show that 2'OMe RNA is able significantly to reduce both interferon-alpha (IFN-alpha) and interleukin-6 (IL-6) induction by the small-molecule TLR7 agonist loxoribine in human peripheral blood mononuclear cells (human PBMCs), in murine Flt3L dendritic cells (Flt3L DCs), and in vivo in mice. These results indicate that 2'OMe-modified RNA may have utility as an inhibitor of TLR7 with potential applications in the treatment of inflammatory and autoimmune diseases that involve TLR7-mediated immune stimulation.

Using a new controlled mixing process, highly transfection-competent polyplexes were formed and subsequently encapsulated within a lipid bilayer. The resulting "pre-condensed stable plasmid lipid particles" (pSPLPs) have small size (104+/-3 nm) and low surface charge characteristics. The formulation process equally enabled lipid encapsulation of either poly-L-lysine or poly(ethyleneimine) (PEI) condensed DNA, and the endosomolytic benefits of PEI were demonstrated in in vitro gene expression studies. The clearance properties of pSPLP were compared to similar formulations with an uncondensed payload (SPLP) in A/J mice bearing subcutaneous Neuro-2a tumors. Plasma clearance of pSPLP (t(1/2)=6.6 h) was similar to SPLP (t(1/2)=7.1 h), allowing significant accumulation at distal tumor target sites. Gene expression profiles were evaluated in vivo using the Neuro-2a model, and PEI-pSPLP formulations demonstrated a sixfold increase in reporter gene expression in tumors compared to SPLP. No significant gene expression was observed in the liver, lung, or spleen when mice were treated with either SPLP or pSPLP, and both formulations were equally well tolerated. The results support the lipid encapsulation of polyplex plasmid DNA as a means of changing its pharmacologic properties and enabling systemic delivery. The inclusion of endosomolytic DNA-condensing agents such as PEI greatly improves the potency of SPLP.

The systemic application of nucleic acid drugs requires delivery systems that overcome the poor pharmacokinetics, limited biodistribution, and inefficient uptake of nucleic acids. PEGylated liposomes show considerable promise because of their intrinsic ability to accumulate at disease sites and facilitate transfection of target cells. Unlike many viral vectors, PEGylated liposomes are generally considered to be nonimmunogenic. We have developed a PEGylated liposome for the systemic administration of plasmid DNA that achieves high levels of selective gene expression at distal tumor sites. Here we report that the in vivo efficacy and safety of these systems can be severely compromised following repeat administration. This phenomenon is characterized by a loss of disease site targeting, accelerated clearance from the blood, and acute hypersensitivity. These effects are fully attributable to a surprisingly robust, long-lived antibody response generated against polyethylene glycol (PEG) that results from the strong adjuvant effect of the plasmid payload. Importantly, immunogenicity may be substantially reduced by modifying the alkyl chain of the PEG-lipid conjugate, thereby allowing successful repeat dosing of the modified plasmid formulations without adverse side effects. Immunogenicity is a relevant concern for a number of nonviral delivery systems given the potent immunostimulatory properties of many nucleic acid drugs.

Short synthetic small-hairpin RNAS (sshRNAs) (SG220 and SG273) that target the internal ribosome entry site of the hepatitis C virus (HCV) were formulated into lipid nano-particles and administered intravenously to HCV-infected urokinase plasminogen activator–severe combined immunodeficient mice with livers repopulated with human hepatocytes (humanized livers). Weekly administration of 2.5 mg/kg of each sshRNA for 2 weeks resulted in a maximal mean reduction in viral load of 2.5 log10 from baseline. The viral load remained reduced by more than 90% at 14 days after the last dose was given. The sshRNAs were well tolerated and did not significantly increase liver enzyme levels. These findings indicate the in vivo efficacy of a synthetic RNA inhibitor against the HCV genome in reducing HCV infection.

The relatively low levels of transfection that can be achieved by current gene delivery systems have limited the therapeutic utility of gene transfer. This is especially true for non-viral gene delivery systems, where the levels of gene expression achieved are usually well below the levels achieved by viral gene transfer systems. Previous work from our laboratory describes an enhanced dual promoter autogene-based cytoplasmic expression system that gives rise to levels of gene expression 20 fold higher than that of a CMV nuclear expression plasmid control. Here we describe various strategies to increase the levels of autogene based gene expression by changing variables such as the type of nuclear promoter, phage RNAP gene, and IRES sequence. It was found that none of these changes demonstrated a significant increase in gene expression. However determination of the mRNA levels achieved using quantitative RNase protection assays and immunofluorescence experiments, revealed transgene mRNA levels up to 10 times higher than all other mRNA in the transfected cell combined. It follows that mRNA production is a major factor limiting autogene based cytoplasmic expression.

We previously identified short synthetic shRNAs (sshRNAs) that target a conserved hepatitis C virus (HCV) sequence within the internal ribosome entry site (IRES) of HCV and potently inhibit HCV IRES-linked gene expression. To assess in vivo liver delivery and activity, the HCV-directed sshRNA SG220 was formulated into lipid nanoparticles (LNP) and injected i.v. into mice whose livers supported stable HCV IRES-luciferase expression from a liver-specific promoter. After a single injection, RNase protection assays for the sshRNA and (3)H labeling of a lipid component of the nanoparticles showed efficient liver uptake of both components and long-lasting survival of a significant fraction of the sshRNA in the liver. In vivo imaging showed a dose-dependent inhibition of luciferase expression (90% 1 day after injection of 2.5 mg/kg sshRNA) with t1/2 for recovery of about 3 weeks. These results demonstrate the ability of moderate levels of i.v.-injected, LNP-formulated sshRNAs to be taken up by liver hepatocytes at a level sufficient to substantially suppress gene expression. Suppression is rapid and durable, suggesting that sshRNAs may have promise as therapeutic agents for liver indications.Molecular Therapy-Nucleic Acids (2013) 2, e123; doi:10.1038/mtna.2013.50; published online 17 September 2013.

RNA interference (RNAi) represents a powerful, naturally occurring biological strategy for inhibition of gene expression that has demonstrated utility in the inhibition of viral replication. However, the challenges associated with the effective in vivo delivery of siRNAs has been a major obstacle to their use. Here we describe the development of an effective siRNA based therapy for hepatitis virus infection.

Ebola virus infection causes a severe and frequently fatal hemorrhagic fever that is refractory to treatment with currently available antiviral therapeutics. Ebola and the related Marburg virus are of concern as potential bioweapon (BW) threats since they have the potential for aerosol dissemination and weaponization. RNA interference (RNAi) represents a powerful, naturally occurring biological strategy for inhibition of gene expression that has demonstrated utility in the inhibition of viral replication. However, the challenges associated with the effective in vivo delivery of siRNAs has been a major obstacle to their use. Here we describe the development of an siRNA based therapy for Ebola virus (EBOV) infection. Multiple siRNAs were designed targeting the viral polymerase (L) gene. When used either individually or in combination, siRNA inhibited EBOV replication in vitro in Vero and Vero E6 cells. A 60-99% reduction in production of infectious EBOV and 75-100% reduction in numbers of cells expressing EBOV protein was observed. Promising siRNA candidates demonstrating significant inhibition of viral replication in vitro were selected for formulation in Stable Nucleic Acid Particles (SNALP). SNALP consist of siRNA fully encapsulated in a lipid bilayer containing a diffusible polyethylene glycol (PEG)-lipid conjugate. The PEG-lipid conjugates in the SNALP particle play an essential role during the formulation process, stabilizing the nascent particle and preventing aggregation in the vial. In the blood, the PEG-lipid shields the positive surface charge, preventing rapid clearance following intravenous injection. Following administration the PEG conjugate dissociates from the SNALP, revealing the positive charge and an increasingly fusogenic lipid bilayer, transforming the particle into a transfection-competent entity. EBOV is known to replicate in the tissues of the reticuloendothelial system, specifically in Kupffer cells of liver, and the monocytes, macrophages and dendritic cells of the peripheral blood and lymphoid tissues. Pharmacokinetics and biodistribution studies utilizing radiolabelled SNALP in mice demonstrated accumulation of more than 30 percent of the injected dose in the liver and 10 percent in the spleen 24 hours after intravenous administration. Fluorescent microscopy of tissue sections showed that SNALP containing Cy3 labelled siRNA were concentrated in the Kupffer cells and resident macrophages of the liver. Promising formulations were selected for evaluation in a guinea pig model of EBOV hemorrhagic fever. The ability of SNALP delivered siRNA to inhibit EVOV plasma viremia is compared to that of PEI polyplex. Further development and optimization of this technology has the potential to yield effective treatments against EBOV hemorrhagic fever (HF), Marburg virus (MARV) HF and other BW threat agents.

Short interfering double-stranded RNA (siRNA) that mediate specific gene silencing through RNA interference (RNAi) are widely used to study gene function in vitro and this technology is now being applied to animal models in vivo . Synthetic siRNA are also being developed for a wide variety of clinical applications although to date, approved clinical trials are restricted to local administration in occular indications due largely to the lack of systemic delivery vehicles appropriate for use with therapeutic siRNA.

Systemic delivery of DNA to disease sites has been previously demonstrated with Stable Plasmid Lipid Particles (SPLP) containing a single plasmid molecule encapsulated with a lipid bilayer stabilized by poly(ethylene glycol) (PEG) lipids. This technology is the subject of an ongoing Phase I clinical trial.