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Background—Stem cell factor (SCF), a ligand of the c-kit receptor, is a critical cytokine, which contributes to cell migration, proliferation, and survival. It has been shown that SCF expression increases after myocardial infarction (MI) and may be involved in cardiac repair. The aim of this study was to determine whether gene transfer of membrane-bound human SCF improves cardiac function in a large animal model of MI. Methods and Results—A transmural MI was created by implanting an embolic coil in the left anterior descending artery in Yorkshire pigs. One week after the MI, the pigs received direct intramyocardial injections of either a recombinant adenovirus encoding for SCF (Ad.SCF, n=9) or β-gal (Ad.β-gal, n=6) into the infarct border area. At 3 months post-MI, ejection fraction increased by 12% relative to baseline after Ad.SCF therapy, whereas it decreased by 4.2% (P=0.004) in pigs treated with Ad.β-gal. Preload-recruitable stroke work was significantly higher in pigs after SCF treatment (Ad.SCF, 55.5±11.6 mm Hg versus Ad.β-gal, 31.6±12.6 mm Hg, P=0.005), indicating enhanced cardiac function. Histological analyses confirmed the recruitment of c-kit[superscript +] cells as well as a reduced degree of apoptosis 1 week after Ad.SCF injection. In addition, increased capillary density compared with pigs treated with Ad.β-gal was found at 3 months and suggests an angiogenic role of SCF. Conclusions—Local overexpression of SCF post-MI induces the recruitment of c-kit[superscript +] cells at the infarct border area acutely. In the chronic stages, SCF gene transfer was associated with improved cardiac function in a preclinical model of ischemic cardiomyopathy., National Institutes of Health (U.S.) (R01 HL117505), National Heart, Lung, and Blood Institute (Program of Excellence in Nanotechnology (PEN) Award Contract HHSN268201000045C), P50 HL112324, Leducq Foundation

Despite intensive research effort, the rational design of improved nanoparticulate drug carriers remains challenging, in part due to a limited understanding of the determinants of nanoparticle entry and transport in target cells. Recent studies have shown that Niemann-Pick C1 (NPC1), the lysosome membrane protein that mediates trafficking of cholesterol in cells, is involved in the endosomal escape and subsequent infection caused by filoviruses, and that its absence promotes the retention and efficacy of lipid nanoparticles encapsulating siRNA. Here, we report that NPC1 deficiency results in dramatic reduction in internalization and transfection efficiency mediated by degradable cationic gene delivery polymers, poly(β-amino ester)s (PBAEs). PBAEs utilized cholesterol and dynamin-dependent endocytosis pathways, and these were found to be heavily compromised in NPC1-deficient cells. In contrast, the absence of NPC1 had minor effects on DNA uptake mediated by polyethylenimine or Lipofectamine 2000. Strikingly, stable overexpression of human NPC1 in chinese hamster ovary cells was associated with enhanced gene uptake (3-fold) and transfection (10-fold) by PBAEs. These findings reveal a role of NPC1 in the regulation of endocytic mechanisms affecting nanoparticle trafficking. We hypothesize that in-depth understanding sites of entry and endosomal escape may lead to highly efficient nanotechnologies for drug delivery.

© Springer Science+Business Media New York 2017. An area of active research in the field of cardiac gene therapy aims to achieve high transfection efficiency without eliciting immune or inflammatory reactions. Nanomedicine offers an attractive alternative to traditional viral delivery vehicles because nanoparticle technology can enable safer and more controlled delivery of therapeutic agents. Here we describe the use of lipidoid nanoparticles for delivery of modified mRNA (modRNA) to the myocardium in vivo, with a focus on rodent models that represent a first step toward preclinical studies. Three major procedures are discussed in this chapter: (1) preparation of lipid modRNA nanoparticles, (2) intramyocardial delivery of the lipid modRNA nanoparticles by direct injection with an open chest technique in rats, and (3) intracoronary delivery of the lipid modRNA nanoparticles with open chest and temporary aortic cross clamping in rats.

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., National Heart, Lung, and Blood Institute, National Institutes of Health (U.S.) (Program of Excellence in Nanotechnology (PEN) Award, Contract #HHSN268201000045C)

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., National Heart, Lung, and Blood Institute, National Institutes of Health (U.S.) (Program of Excellence in Nanotechnology (PEN) Award, Contract #HHSN268201000045C), National Institutes of Health (U.S.) (NIH Grant R01-EB000244-27), National Institutes of Health (U.S.) (NIH Grant 5-R01-CA132091-04), National Institutes of Health (U.S.) (NIH Grant R01-DE016516-03), National Science Foundation (U.S.) (Graduate Research Fellowship), Juvenile Diabetes Research Foundation International (Grant 17–2007-1063)

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., Misrock Foundation, American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship, National Institutes of Health (U.S) (Grant EB000244), National Cancer Institute (U.S.) (MIT-Harvard Center of Cancer Nanotechnology Excellence. Grant CA151884), National Science Foundation (U.S.), Massachusetts Institute of Technology (Presidential Fellowships)

Gene delivery has the potential to treat a range of inherited and acquired diseases. Research has primarily focused on the use of viral vectors for this purpose, due to efficient infection of cells with viruses as well as long-term gene expression. However, the use of viral vectors for gene therapy is limited by safety concerns, production/manufacturing challenges, and limited nucleic acid carrying capacity [1, 2]. Thus, increased attention has been focused on biomaterials including cationic polymers as gene transfection agents [3-5] due totheir electrostatic interactions with plasmid DNA to form cationic nanoparticles. Polymers, including polyethylenimine (PEI), are useful in a variety of gene therapy applications [6-10]. One promising class of polymers, poly(β-amino ester)s (PBAEs), are degradable and have enhanced delivery compared to PEI [11-13]. Recent studies show that the amine-terminated polymers are generally more effective at promoting cellular uptake and DNA delivery than their acrylate-capped counterparts [14, 15].

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., Alnylam Pharmaceuticals (Firm), National Institutes of Health (U.S.) (Grant R01-EB000244-27), National Institutes of Health (U.S.) (Grant 5-R01-CA132091-04), National Science Foundation (U.S.). Graduate Research Fellowship, National Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (F32-EB011867)