Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and adenosine triphosphate citrate lyase (ACL) in the cytosol1-3. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle (TCA) and fatty acid synthesis4. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis5, the regulation and utilization of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells employ reductive metabolism of alpha-ketoglutarate (αKG) to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase 1 (IDH1) dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived αKG for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel-Lindau (VHL) tumor suppressor protein preferentially utilize reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon utilization in order to produce AcCoA and support lipid synthesis in mammalian cells.

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Materials that provide spatial and temporal control over the delivery of DNA and other nucleic acid-based agents from surfaces play important roles in the development of localized gene-based therapies. This review focuses on a relatively new approach to the immobilization and release of DNA from surfaces: methods based on the layer-by-layer assembly of thin multilayered films (or polyelectrolyte multilayers, PEMs). Layer-by-layer methods provide convenient, nanometer-scale control over the incorporation of DNA, RNA, and oligonucleotide constructs into thin polyelectrolyte films. Provided that these assemblies can be designed in ways that permit controlled film disassembly under physiological conditions, this approach can contribute new methods for spatial and/or temporal control over the delivery of nucleic acid-based therapeutics in vitro and in vivo. We describe applications of layer-by-layer assembly to the fabrication of DNA-containing films that can be used to provide control over the release of plasmid DNA from the surfaces of macroscopic objects and promote surface-mediated cell transfection. We also highlight the application of these methods to the coating of colloidal substrates and the fabrication of hollow micrometer-scale capsules that can be used to encapsulate and control the release or delivery of DNA and oligonucleotides. Current challenges, gaps in knowledge, and new opportunities for the development of these methods in the general area of gene delivery are discussed.

Materials that permit control over the release of DNA from the surfaces of topologically complex implantable devices, such as intravascular stents, could contribute to the development of new approaches to the localized delivery of DNA. We report the fabrication of ultrathin, multilayered polyelectrolyte films that permit both the immobilization and controlled release of plasmid DNA from the surfaces of stainless steel intravascular stents. Our approach makes use of an aqueous-based, layer-by-layer method for the assembly of nanostructured thin films consisting of alternating layers of plasmid DNA and a hydrolytically degradable polyamine. Characterization of coated stents using scanning electron microscopy (SEM) demonstrated that stents were coated uniformly with an ultrathin film ca. 120 nm thick that adhered conformally to the surfaces of stent struts. These ultrathin films did not crack, peel, or delaminate substantially from the surface after exposure to a range of mechanical challenges representative of those encountered during stent deployment (e.g., balloon expansion). Stents coated with eight bilayers of degradable polyamine and a plasmid encoding enhanced green fluorescent protein sustained the release of DNA into solution for up to four days when incubated in phosphate buffered saline at 37 °C, and coated stents were capable of mediating the expression of EGFP in a mammalian cell line without the aid of additional transfection agents. The approach reported here could, with further development, contribute to the development of localized gene-based approaches to the treatment of cardiovascular diseases or related conditions.

Personalized cancer vaccines (PCVs) targeting patient-specific neoantigens are a promising cancer treatment modality; however, neoantigen physicochemical variability can present challenges to manufacturing PCVs in an optimal format for inducing anticancer T cells. Here, we developed a vaccine platform ("SNP-7/8a") based on charge-modified peptide-TLR-7/8a conjugates that are chemically programmed to self-assemble into nanoparticles of uniform size (~20 nm) irrespective of the peptide antigen composition. This approach provided precise loading of diverse peptide neoantigens linked to TLR-7/8a (adjuvant) in nanoparticles that increased uptake by and activation of antigen-presenting cells that promote T cell immunity. Vaccination of mice with SNP-7/8a using predicted neoantigens (n=179) from three tumor models induced CD8 T cells against ~50% of neoantigens with high predicted MHC-I binding affinity and led to enhanced tumor clearance. SNP-7/8a delivering in silico-designed mock neoantigens also induced CD8 T cells in non-human primates. Altogether, SNP-7/8a is a generalizable approach for co-delivering peptide antigens and adjuvants in nanoparticles for inducing anticancer T cell immunity.