Ahmed A. Eltoukhy

Non-viral vectors for gene-based therapy

Gene-based therapy is the intentional modulation of gene expression in specific cells to treat pathological conditions. This modulation is accomplished by introducing exogenous nucleic acids such as DNA, mRNA, small interfering RNA (siRNA), microRNA (miRNA) or antisense oligonucleotides. Given the large size and the negative charge of these macromolecules, their delivery is typically mediated by carriers or vectors. In this Review, we introduce the biological barriers to gene delivery in vivo and discuss recent advances in material sciences, nanotechnology and nucleic acid chemistry that have yielded promising non-viral delivery systems, some of which are currently undergoing testing in clinical trials. The diversity of these systems highlights the recent progress of gene-based therapy using non-viral approaches.

Rapid Discovery of Potent siRNA-Containing Lipid Nanoparticles Enabled by Controlled Microfluidic Formulation

The discovery of potent new materials for in vivo delivery of nucleic acids depends upon successful formulation of the active molecules into a dosage form suitable for the physiological environment. Because of the inefficiencies of current formulation methods, materials are usually first evaluated for in vitro delivery efficacy as simple ionic complexes with the nucleic acids (lipoplexes). The predictive value of such assays, however, has never been systematically studied. Here, for the first time, by developing a microfluidic method that allowed the rapid preparation of high-quality siRNA-containing lipid nanoparticles (LNPs) for a large number of materials, we have shown that gene silencing assays employing lipoplexes result in a high rate of false negatives (~90%) that can largely be avoided through formulation. Seven novel materials with in vivo gene silencing potencies of >90% at a dose of 1.0 mg/kg in mice were discovered. This method will facilitate the discovery of next-generation reagents for LNP-mediated nucleic acid delivery.

Lipidoid-Coated Iron Oxide Nanoparticles for Efficient DNA and siRNA delivery

The safe, targeted and effective delivery of gene therapeutics remains a significant barrier to their broad clinical application. Here we develop a magnetic nucleic acid delivery system composed of iron oxide nanoparticles and cationic lipid-like materials termed lipidoids. Coated nanoparticles are capable of delivering DNA and siRNA to cells in culture. The mean hydrodynamic size of these nanoparticles was systematically varied and optimized for delivery. While nanoparticles of different sizes showed similar siRNA delivery efficiency, nanoparticles of 50-100 nm displayed optimal DNA delivery activity. The application of an external magnetic field significantly enhanced the efficiency of nucleic acid delivery, with performance exceeding that of the commercially available lipid-based reagent, Lipofectamine 2000. The iron oxide nanoparticle delivery platform developed here offers the potential for magnetically guided targeting, as well as an opportunity to combine gene therapy with MRI imaging and magnetic hyperthermia.

Small‐Molecule End‐Groups of Linear Polymer Determine Cell‐type Gene‐Delivery Efficacy

End-modified polymers are promising for the nonviral delivery of genes to cancer cells, immune cells, and human stem cells and point to polymer end-groups as regulators for cell-type specificity. A library of polymers has been synthesized and, although some polymers are strong transfection agents overall, for each cell type, a particular polymer is most effective.

Effect of molecular weight of amine end-modified poly(β-amino ester)s on gene delivery efficiency and toxicity.

Amine end-modified poly(β-amino ester)s (PBAEs) have generated interest as efficient, biodegradable polymeric carriers for plasmid DNA (pDNA). For cationic, non-degradable polymers, such as polyethylenimine (PEI), the polymer molecular weight (MW) and molecular weight distribution (MWD) significantly affect transfection activity and cytotoxicity. The effect of MW on DNA transfection activity for PBAEs has been less well studied. We applied two strategies to obtain amine end-modified PBAEs varying in MW. In one approach, we synthesized four amine end-modified PBAEs with each at 15 different monomer molar ratios, and observed that polymers of intermediate length mediated optimal DNA transfection in HeLa cells. Biophysical characterization of these feed ratio variants suggested that optimal performance was related to higher DNA complexation efficiency and smaller nanoparticle size, but not to nanoparticle charge. In a second approach, we used preparative size exclusion chromatography (SEC) to obtain well-defined, monodisperse polymer fractions. We observed that the transfection activities of size-fractionated PBAEs generally increased with MW, a trend that was weakly associated with an increase in DNA binding efficiency. Furthermore, this approach allowed for the isolation of polymer fractions with greater transfection potency than the starting material. For researchers working with gene delivery polymers synthesized by step-growth polymerization, our data highlight the potentially broad utility of preparative SEC to isolate monodisperse polymers with improved properties. Overall, these results help to elucidate the influence of polymer MWD on nucleic acid delivery and provide insight toward the rational design of next-generation materials for gene therapy.

Degradable terpolymers with alkyl side chains demonstrate enhanced gene delivery potency and nanoparticle stability.

Degradable, cationic poly(β-amino ester)s (PBAEs) with alkyl side chains are developed for non-viral gene delivery. Nanoparticles formed from these PBAE terpolymers exhibit significantly enhanced DNA transfection potency and resistance to aggregation. These hydrophobic PBAE terpolymers, but not PBAEs lacking alkyl side chains, support interaction with PEG-lipid conjugates, facilitating their functionalization with shielding and targeting moieties and accelerating the in vivo translation of these materials.

Alkane-modified short polyethyleneimine for siRNA delivery.

RNA interference (RNAi) is a highly specific gene-silencing mechanism triggered by small interfering RNA (siRNA). Effective intracellular delivery requires the development of potent siRNA carriers. Here, we describe the synthesis and screening of a series of siRNA delivery materials. Short polyethyleneimine (PEI, Mw 600) was selected as a cationic backbone to which lipid tails were conjugated at various levels of saturation. In solution these polymer-lipid hybrids self-assemble to form nanoparticles capable of complexing siRNA. The complexes silence genes specifically and with low cytotoxicity. The efficiency of gene knockdown increased as the number of lipid tails conjugated to the PEI backbone increased. This is explained by reducing the binding affinity between the siRNA strands to the complex, thereby enabling siRNA release after cellular internalization. These results highlight the importance of complexation strength when designing siRNA delivery materials.

Dendrimeric siRNA for Efficient Gene Silencing

Programmable molecular self-assembly of siRNA molecules provides precisely controlled generation of dendrimeric siRNA nanostructures. The second-generation dendrimers of siRNA can be effectively complexed with a low-molecular-weight, cationic polymer (poly(β-amino ester), PBAE) to generate stable nanostructures about 160 nm in diameter via strong electrostatic interactions. Condensation and gene silencing efficiencies increase with the increased generation of siRNA dendrimers due to a high charge density and structural flexibility.

Lipid‐Like Nanomaterials for Simultaneous Gene Expression and Silencing In Vivo

New lipid-like nanomaterials are developed to simultaneously regulate expression of multiple genes. Self-assembled nanoparticles are capable of efficiently encapsulating pDNA and siRNA. These nanoparticles are shown to induce simultaneous gene expression and silencing both in vitro and in vivo.

Transmetalation of tetranuclear copper complexes. VIII: Transmetalation of tetranuclear copper(I) complexes with a Co(NS)3 reagent

Transmetallation des complexes (DENC) 4 Cu 4 X 4 (DENC=diethylnicotinamide), par le tris(S-methyl isopropylidenehydrazinocarbodithioate)-Co(III) dans les solvants aprotiques sous N 2