James J. Lu

Cross-linked Small Polyethylenimines: While Still Nontoxic, Deliver DNA Efficiently to Mammalian Cells in Vitro and in Vivo

No HeadingPurpose.Polyethylenimine (PEI) is among the most efficient nonviral gene delivery vectors. Its efficiency and cytotoxicity depend on molecular weight, with the 25-kDa PEI being most efficient but cytotoxic. Smaller PEIs are noncytotoxic but less efficient. Enhancement in gene delivery efficiency with minimal cytotoxicity by cross-linking of small PEIs via potentially biodegradable linkages was explored herein. The hypothesis was that cross-linking would raise the polycation’s effective molecular weight and hence the transfection efficiency, while biodegradable linkages would undergo the intracellular breakdown after DNA delivery and hence not lead to cytotoxicity. Toward this goal, we carried out cross-linking of branched 2-kDa PEI and its 1:1 (w/w) mixture with a linear 423-Da PEI via ester- and/or amide-bearing linkages; the in vitro and in vivo gene delivery efficiency, as well as toxicity to mammalian cells, of the resultant cross-linked polycations were investigated.Methods.The efficiency of the cross-linked PEIs in delivering in vitro a plasmid containing β-galactosidase gene and their cytotoxicity were investigated in monkey kidney cells (COS-7). Dynamic light scattering was used to compare the relative DNA condensation efficiency of the unmodified and cross-linked PEIs. In vivo gene delivery efficiency was evaluated by intratracheal delivery in mice of the complexes of a luciferase-encoding plasmid and the PEIs and estimating the luciferase expression in the lungs.Results.Cross-linking boosted the gene delivery efficiency of the small PEIs by 40- to 550-fold in vitro; the efficiency of the most potent conjugates even exceeded by an order of magnitude that of the branched 25-kDa PEI. Effective condensation of DNA was evident from the fact that the mean diameter of the complexes of the cross-linked PEIs was some 300 nm with a narrow size distribution, while the complexes of the unmodified small PEIs exhibited a mean size of >700 nm with a very broad size distribution. At concentrations where the 25-kDa PEI resulted in >95% cell death, the conjugates afforded nearly full cell viability. The cross-linked PEIs were 17 to 80 times m ore efficient than the unmodified ones in vivo; furthermore, their efficiencies were up to twice that of the 25-kDa PEI.Conclusions.Cross-linking of small PEIs with judiciously designed amide- and ester-bearing linkers boosts their gene delivery efficiency both in vitro and in vivo without increasing the cytotoxicity. The high efficiency is dependent on the nature of the linkages and the PEIs used.

Non-viral siRNA delivery to the lung☆

Abstract SiRNAs exert their biological effect by guiding the degradation of their cognate mRNA sequence, thereby shutting down the corresponding protein production (gene silencing by RNA interference or RNAi). Due to this property, siRNAs are emerging as promising therapeutic agents for the treatment of inherited and acquired diseases, as well as research tools for the elucidation of gene function in both health and disease. Because of their lethality and prevalence, lung diseases have attracted particular attention as targets of siRNA-mediated cures. In addition, lung is accessible to therapeutic agents via multiple routes, e.g., through the nose and the mouth, thus obviating the need for targeting and making it an appealing target for RNAi-based therapeutic strategies. The clinical success of siRNA-mediated interventions critically depends upon the safety and efficacy of the delivery methods and agents. Delivery of siRNAs relevant to lung diseases has been attempted through multiple routes and using various carriers in animal models. This review focuses on the recent progress in non-viral delivery of siRNAs for the treatment of lung diseases, particularly infectious diseases. The rapid progress will put siRNA-based therapeutics on fast track to the clinic.

Higher-Dimensional Integration with Gaussian Weight for Applications in Probabilistic Design

Higher-dimensional Gaussian weighted integration is of interest in probabilistic simulations. Motivated by the need for variance calculations with functions being at least quadratic, the family of degree 5 formulae is considered. Using an existing formula for the integration over the surface of an n-sphere, an efficient, new formula for Gaussian weighted integration is obtained. Several other formulae that have appeared in the numerical integration literature are also given. The number of function evaluations required by the formulae is compared to a minimal bound result. The degree 5 formulae are applied to simple test problems and the relative errors are compared.

Identification of Novel Superior Polycationic Vectors for Gene Delivery by High-throughput Synthesis and Screening of a Combinatorial Library

PurposeLow efficiency and toxicity are two major drawbacks of current non-viral gene delivery vectors. Since DNA delivery to mammalian cells is a multi-step process, generating and searching combinatorial libraries of vectors employing high-throughput synthesis and screening methods is an attractive strategy for the development of new improved vectors because it increases the chance of identifying the most overall optimized vectors.Materials and MethodsBased on the rationale that increasing the effective molecular weight of small PEIs, which are poor vectors compared to the higher molecular weight homologues but less toxic, raises their transfection efficiency due to better DNA binding, we synthesized a library of 144 biodegradable derivatives from two small PEIs and 24 bi- and oligo-acrylate esters. A 423-Da linear PEI and its 1:1 (w/w) mixture with a 1.8-kDa branched PEI were cross-linked with the acrylates at three molar ratios in DMSO. The resulting polymers were screened for their efficiency in delivering a β-galactosidase expressing plasmid to COS-7 monkey kidney cells. Selected most potent polymers from the initial screen were tested for toxicity in A549 human lung cancer cells, and in vivo in a systemic gene delivery model in mice employing a firefly luciferase expressing plasmid.ResultsSeveral polycations that exhibited high potency and low toxicity in vitro were identified from the library. The most potent derivative of the linear 423-Da PEI was that cross-linked with tricycle-[5.2.1.0]-decane-dimethanol diacrylate (diacrylate 14), which exhibited an over 3,600-fold enhancement in efficiency over the parent. The most potent mixed PEI was that cross-linked with ethylene glycol diacrylate (diacrylate 4) which was over 850-fold more efficient than the physically mixed parent PEIs. The relative efficiencies of these polymers were even up to over twice as high as that of the linear 22-kDa PEI, considered the “gold standard” for in vitro and systemic gene delivery. The potent cross-linked polycations identified were also less toxic than the 22-kDa PEI. The optimal vector in vivo was the mixed PEI cross-linked with propylene glycol glycerolate diacrylate (diacrylate 7); it mediated the highest gene expression in the lungs, followed by the spleen, with the expression in the former being 53-fold higher compared to the latter. In contrast, the parent PEIs mediated no gene expression at all under similar conditions, and injection of the polyplexes of the 22-kDa PEI at its optimal N/P of 10 prepared under identical conditions killed half of the mice injected.ConclusionsHigh-throughput synthesis and transfection assay of a cross-linked library of biodegradable PEIs was proven effective in identifying highly transfecting vectors. The identified vectors exhibited dramatically superior efficiency compared to their parents both in vitro and in an in vivo systemic gene delivery model. The majority of these vectors mediated preferential gene delivery to the lung, and their in vivo toxicity paralleled that in vitro.

Drug Delivery–mediated Control of RNA Immunostimulation

RNA interference (RNAi) has generated significant interest as a strategy to suppress viral infection, but in some cases antiviral activity of unmodified short-interfering RNA (siRNA) has been attributed to activation of innate immune responses. We hypothesized that immunostimulation by unmodified siRNA could mediate both RNAi as well as innate immune stimulation depending on the mode of drug delivery. We investigated the potential of immunostimulatory RNAs (isRNAs) to suppress influenza A virus in vivo in the mouse lung. Lipidoid 98N12-5(1) formulated with unmodified siRNA targeting the influenza nucleoprotein gene exhibited antiviral activity. Formulations were optimized to increase antiviral activity, but the antiviral activity of lipidoid-delivered siRNA did not depend on sequence homology to the influenza genome as siRNA directed against unrelated targets also suppressed influenza replication in vivo. This activity was primarily attributed to enhancement of innate immune stimulation by lipidoid-mediated delivery, which indicates increased toll-like receptor (TLR) activation by siRNA. Certain chemical modifications to the siRNA backbone, which block TLR7/8 activation but retain in vitro RNAi activity, prevented siRNA-mediated antiviral activity despite enhanced lipidoid-mediated delivery. Here, we demonstrate that innate immune activation caused by unmodified siRNA can have therapeutically relevant effects, and that these non-RNAi effects can be controlled through chemical modifications and drug delivery.

An Exact Dual Adjoint Solution Method for Turbulent Flows on Unstructured Grids

An algorithm for solving the discrete adjoint system based on an unstructured-grid discretization of the Navier-Stokes equations is presented. The method is constructed such that an adjoint solution exactly dual to a direct differentiation approach is recovered at each time step, yielding a convergence rate which is asymptotically equivalent to that of the primal system. The new approach is implemented within a three-dimensional unstructured-grid framework and results are presented for inviscid, laminar, and turbulent flows. Improvements to the baseline solution algorithm, such as line-implicit relaxation and a tight coupling of the turbulence model, are also presented. By storing nearest-neighbor terms in the residual computation, the dual scheme is computationally efficient, while requiring twice the memory of the flow solution. The scheme is expected to have a broad impact on computational problems related to design optimization as well as error estimation and grid adaptation efforts.

Polycation-mediated delivery of siRNAs for prophylaxis and treatment of influenza virus infection

Influenza A virus causes one of the most prevalent infections in humans. In a typical year, 10 – 20% of the population of the US is infected by influenza virus, resulting in up to 40,000 deaths and 200,000 hospitalisations. Vaccination is the most effective preventative measure that can protect 70 – 90% of healthy adults aged < 65; however, the protection rate is much lower in those most susceptible to infection, namely infants, the elderly and individuals with weakened immune systems. Although four drugs have been approved by the FDA for use as prophylaxis and/or treatment of influenza, concerns about their side effects and the emergence of drug-resistant viruses persist. RNA interference (RNAi), an emerging method of post transcriptional gene silencing, appears ideal for the prevention and treatment of influenza. RNAi in mammals can be mediated by short interfering RNAs (siRNAs) of ∼21– 27 nucleotides in length. The authors have previously shown that si-RNAs specific for conserved regions of the influenza virus genome are potent inhibitors of influenza virus replication in both cell lines and chicken embryos. This review discusses the recent progress in the invivo inhibition of influenza virus by the delivery of siRNAs mediated by non-viral vectors, and the prospects of this strategy for prophylaxis and treatment of influenza infection in humans.

Adaptive precision methodology for flow optimization via discretization and iteration error control

An adaptive precision optimization methodology based on discretization and iteration error estimates is presented. Issues of efficient concurrent flow-adjoint solution and consistency between PDE and discrete adjoint for the Discontinious Galerkin (DG) discretization of the Euler compressible equations are developed. Optimization results for quasi-one dimensional channel flow are presented to demonstrate the methodology.