Jiin Felgner

The application of cationic liposome reagents has advanced DNA and mRNA transfection research in vitro, and data are accumulating which show their utility for in vivo gene transfer. However, chemical structure-activity data leading to a better mechanistic understanding of their biological activity is still limited. Most of the cationic lipid reagents in use today for this application are formulated as liposomes containing two lipid species, a cationic amphiphile and a neutral phospholipid, typically dioleoylphosphatidylethanolamine (DOPE). The studies reported here examine the effects of some systematic chemical structural changes in both of these lipid components. Cationic and neutral phospholipids were formulated together as large multilamellar vesicles (MLV) or small sonicated unilamellar vesicles (SUV) in water, and each formulation was assayed quantitatively in 96-well microtiter plates under 64 different assay conditions using COS.7 cells and an RSV-beta-galactosidase expression plasmid. The cationic lipid molecules used for these studies were derived from a novel series of 2,3-dialkyloxypropyl quaternary ammonium compounds containing a hydroxyalkyl moiety on the quaternary amine. A homologous series of dioleylalkyl (C18:1) compounds containing increasing hydroxyalkyl chain lengths on the quaternary amine were synthesized, formulated with 50 mol % DOPE, and assayed for transfection activity. The order of efficacy was ethyl > propyl > butyl > pentyl > 2,3-dioleyloxypropyl-1-trimethyl ammonium bromide (DOTMA). DOTMA, which is commercially available under the trademark Lipofectin Reagent, lacks a hydroxyalkyl moiety on the quaternary amine. A homologous series of hydroxyethyl quaternary ammonium derivatives with different alkyl chain substitutions were synthesized, formulated with 50 mol % DOPE, and assayed in the transfection assay. The order of transfection efficacy was dimyristyl (di-C14:0) > dioleyl (di-C18:1) > dipalmityl (di-C16:0) > disteryl (di-C18:0). The addition of 100 microM chloroquine in the transfection experiment enhanced the activity of the dioleyl compound by 4-fold and decreased the activity of the dimyristyl compound by 70%. For each of the compounds and formulations examined in this report, large multilamellar vesicles (MLV; diameter 300-700 nm) were more active than small unilamellar vesicles (SUV; diameter 50-100 nm). The neutral phospholipid requirements for transfection activity in COS.7 cells with these cationic lipid molecules were examined.(ABSTRACT TRUNCATED AT 400 WORDS)

Direct injection of nonviral, covalently closed circular plasmid DNA into muscle results in expression of the DNA in myofiber cells. We have examined the expression of firefly luciferase DNA constructs injected into adult murine skeletal muscle. Considerable variation in luciferase enzyme expression was noted among constructs with different regulatory elements, among different batches of the same DNA construct, and among similar transfection experiments performed at different times. This variation was minimized by using single batches of plasmid DNA and by performing comparable sets of experiments concurrently. A quantitative experimental protocol was defined for comparing various aspects of the transfection process. We report that a luciferase construct containing the human cytomegalovirus immediate-early gene promoter plus intron A (a construct termed "p-CMVint-lux") showed the highest expression among several constructs tested. Dose-response and time course analyses of p-CMVint-lux DNA injections showed that maximal luciferase expression was achieved with 25 micrograms of DNA at 7-14 days post-injection. Selected manipulations of the transfection process were examined for their influence on luciferase expression. Variations in the rate of DNA injection, needle size, injection volume, and vehicle temperature had no significant effect on luciferase expression. The presence of endotoxin, cationic peptide, muscle stimulants or relaxants, vasoconstrictors, metal chelators, or lysosomal lytic reagents had no significant effect on expression. However, linearization of the DNA, injection of the DNA in water rather than saline, or inclusion of a DNA intercalating agent nearly abolished luciferase expression. And finally, increasing the injection dose by giving multiple injections over a 10-day period increased expression proportionally to the number of injections.

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.

Among the potential nonviral vectors for human gene therapy are DNA–liposome complexes. In a recent clinical study, this delivery system has been utilized. In this report, a novel cationic lipid, dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE), has been substituted into the DNA–liposome complex with dioleoyl phosphatidylethanolamine (DOPE), which both improves transfection efficiencies and allows increased doses of DNA to be delivered in vivo. The safety and toxicity of this DNA–liposome complex has been evaluated in two species, mice and pigs. The efficacy of DMRIE/DOPE in inducing an antitumor response in mice after transfer of a foreign MHC has been confirmed. No abnormalities were detected after administration of up to 1,000-fold higher concentrations of DNA and lipid than could be tolerated in vivo previously. Examination of serum biochemical enzymes, pathologic examination of tissue, and analysis of cardiac function in mice and pigs revealed no toxicities related to this treatment. This improved cationic lipid formulation is well-tolerated in vivo and could therefore allow higher dose administration and potentially greater efficiency of gene transfer for gene therapy. Although several viral vectors have been widely applied to the treatment of human disease, the development of nonviral vectors is still in their infancy. In this report, a novel cationic lipid, DMRIE/DOPE, has been incorporated into the DNA–liposome formulation that improves transfection efficiencies and allows up to 1,000-fold higher concentrations of DNA to be administered in vivo. In this paper, the safety and toxicity of this formulation is described in two species, mice and pigs, suggesting that it may prove useful for human gene therapy.