Philip Felgner - Academia.edu (original) (raw)

Papers by Philip Felgner

Medical Intelligence Unit, 2006

In this chapter, we describe an approach using a peptide nucleic acid (PNA) clamp to directly and... more In this chapter, we describe an approach using a peptide nucleic acid (PNA) clamp to directly and irreversibly modify plasmid DNA, without affecting either its supercoiled conformation or its ability to be efficiently transcribed. This strategy enables investigators to 'functionalize' their gene of interest by direct coupling of ligands (fluorophores, peptide, proteins, sugars or oligonucleotides) to plasmid DNA. This approach provides versatile tools to study the mechanisms of gene delivery and to circumvent some of the main obstacles of synthetic gene delivery systems, such as specific targeting and efficient delivery. The proof-of-principal of PNA-dependent gene chemistry (PDGC) was demonstrated with a fluorescently labeled PNA that allowed generation of a highly fluorescent preparation of plasmid DNA that was functionally and conformationally intact. Fluorescent-PNA/DNA was used to identify critical parameters involved in naked DNA and non-viral gene delivery technology. The greatest potential of PDGC lies in the ability to attach specific ligands (e.g., peptides, proteins) to the plasmid DNA in order to overcome cellular barriers of non-viral gene delivery systems. In this regard, specific examples of ligands coupled to DNA are described and their effect on increasing the efficacy of gene therapy is presented.

The Journal of infectious diseases, Jan 7, 2014

Persons with blood-stage Plasmodium falciparum parasitemia in the absence of symptoms are consid... more Persons with blood-stage Plasmodium falciparum parasitemia in the absence of symptoms are considered to be clinically immune. We hypothesized that asymptomatic subjects with P. falciparum parasitemia would differentially recognize a subset of P. falciparum proteins on a genomic scale. Compared with symptomatic subjects, sera from clinically immune, asymptomatically infected individuals differentially recognized 51 P. falciparum proteins, including the established vaccine candidate PfMSP1. Novel, hitherto unstudied hypothetical proteins and other proteins not previously recognized as potential vaccine candidates were also differentially recognized. Genes encoding the proteins differentially recognized by the Peruvian clinically immune individuals exhibited a significant enrichment of nonsynonymous nucleotide variation, an observation consistent with these genes undergoing immune selection. A limited set of P. falciparum protein antigens was associated with the development of natur...

Journal of Virology, 2012

Despite the eradication of smallpox, orthopoxviruses (OPV) remain public health concerns. Efforts... more Despite the eradication of smallpox, orthopoxviruses (OPV) remain public health concerns. Efforts to develop new therapeutics and vaccines for smallpox continue through their evaluation in animal models despite limited understanding of the specific correlates of protective immunity. Recent monkeypox virus challenge studies have established the black-tailed prairie dog ( Cynomys ludovicianus ) as a model of human systemic OPV infections. In this study, we assess the induction of humoral immunity in humans and prairie dogs receiving Dryvax, Acam2000, or Imvamune vaccine and characterize the proteomic profile of immune recognition using enzyme-linked immunosorbent assays (ELISA), neutralization assays, and protein microarrays. We confirm anticipated similarities of antigenic protein targets of smallpox vaccine-induced responses in humans and prairie dogs and identify several differences. Subsequent monkeypox virus intranasal infection of vaccinated prairie dogs resulted in a significan...

The Journal of Immunology, 2003

Memory B cells are a central component of humoral immunity, and yet little is known about their l... more Memory B cells are a central component of humoral immunity, and yet little is known about their longevity in humans. Immune memory after smallpox vaccination (DryVax) is a valuable benchmark for understanding the longevity of B cell memory in the absence of re-exposure to Ag. In this study, we demonstrate that smallpox vaccinespecific memory B cells last for >50 years in immunized individuals. Virus-specific memory B cells initially declined postimmunization, but then reached a plateau ϳ10-fold lower than peak and were stably maintained for >50 years after vaccination at a frequency of ϳ0.1% of total circulating IgG ؉ B cells. These persisting memory B cells were functional and able to mount a robust anamnestic Ab response upon revaccination. Additionally, virus-specific CD4 ؉ T cells were detected decades after vaccination. These data show that immunological memory to DryVax vaccine is long-lived and may contribute to protection against smallpox.

Journal of Biological Chemistry, 2001

There are many very effective methods to introduce transcriptionally active DNA into viable cells... more There are many very effective methods to introduce transcriptionally active DNA into viable cells but approaches to deliver functional proteins are limited. We have developed a lipid-mediated delivery system that can deliver functional proteins or other bioactive molecules into living cells. This delivery system is composed of a new trifluoroacetylated lipopolyamine (TFA-DODAPL) and dioleoyl phosphatidylethanolamine (DOPE). This cationic formulation successfully delivered antibodies, dextran sulfates, phycobiliproteins, albumin, and enzymes (beta-galactosidase and proteases) into the cytoplasm of numerous adherent and suspension cells. Two systems were used to demonstrate that the proteins were delivered in a functionally active form. First, intracellular beta-galactosidase activity was clearly demonstrated within X-gal-stained cells after TFA-DODAPL:DOPE-mediated delivery of the enzyme. Second, the delivery system mediated delivery of several caspases (caspase 3, caspase 8, and granzyme B) into cultured cell lines and primary cells triggering apoptosis. Mechanistic studies showed that up to 100% of the protein mixed with the lipid formulation was captured into a lipid-protein complex, and up to 50% of the input protein associated with cells. This lipid-mediated transport system makes protein delivery into cultured cells as convenient, effective, and reliable as DNA transfection.

Human Gene Therapy, 1998

Five analytical assays are described that provide a platform for systematically evaluating the ef... more Five analytical assays are described that provide a platform for systematically evaluating the effect of formulation variables on the physical properties of cationic lipid-DNA complexes (lipoplexes). The assays are for (i) lipid recovery, (ii) total DNA, (iii) free DNA, (iv) nuclease sensitivity, and (v) physical stability by filtration. Lipid recovery was determined by measuring lipid primary amino groups labeled with the fluorescamine reagent in the presence of the detergent Zwittergent. Zwittergent was effective at disrupting lipoplexes, making the primary amine accessible to the fluorescamine reagent. Total DNA was determined with the PicoGreen reagent, also in the presence of Zwittergent. The PicoGreen assay in the absence of Zwittergent gave the percentage of the total DNA that was not complexed with cationic lipid. The results of this assay for free DNA agreed well with the amount of DNA that could be separated from complexes by centrifugation as well as with the amount of DNA that was accessible to DNase I digestion. Monitoring the lipid and DNA recoveries after filtration through polycarbonate membranes provided a quantitative method for assessing changes in lipoplex physical characteristics. Together, these assays provide a convenient high-throughput approach to assess physical properties of lipoplexes, allowing systematic evaluation of different formulations.

Human Gene Therapy, 1999

We describe an effective approach using a peptide nucleic acid (PNA) "clamp" to... more We describe an effective approach using a peptide nucleic acid (PNA) "clamp" to directly and irreversibly modify plasmid DNA, without affecting either its supercoiled conformation or its ability to be efficiently transcribed. To demonstrate this approach a highly fluorescent preparation of plasmid DNA was generated by hybridizing a fluorescently labeled PNA to the plasmid. Fluorescent plasmid prepared in this way was neither functionally nor conformationally altered. PNA binding was sequence specific, saturable, extremely stable, and did not influence the nucleic acid intracellular distribution. This method was utilized for the first time to study the biodistribution of conformationally and functionally intact plasmid DNA in living cells after cationic lipid-mediated transfection. A fluorescent plasmid expressing green fluorescent protein (GFP) enabled simultaneous colocalization of both plasmid and expressed protein in living cells and in real time. GFP was shown to be expressed in cells containing detectable nuclear fluorescent plasmid. The fluorescent PNA-labeled plasmid revealed a marked difference in the nuclear uptake between oligonucleotide and plasmid, suggesting that nuclear entry of plasmid may require cell division. This detection method provides a way to simultaneously monitor the intracellular localization and expression of plasmid DNA in living cells, and to elucidate the mechanism of plasmid delivery and its nuclear import with synthetic gene delivery systems.

Gene Therapy, 1998

A stable single vial lipoplex formulation has been of a T-connector. Homogenous cationic liposome... more A stable single vial lipoplex formulation has been of a T-connector. Homogenous cationic liposome prepdeveloped that can be stored frozen without losing either arations were prepared by extrusion in two different size biological activity or physical stability. This formulation was ranges of either 400 or 100 nm. Extruded liposomes proidentified by systematically controlling several formulation duced more monodisperse and physically stable lipoplex variables and without introducing either stabilizers or sur-formulations than unextruded liposomes, but the formufactants. Analytical assays were used to unambiguously lations prepared with 100 nm liposomes were less active characterize the formulations. The critical formulation para-in in vitro transfection assays than either the 400 nm or meters were: (1) the size of the cationic liposomes; (2) the unextruded liposomes. Low ionic strength and 5% sorbitol rate and method of DNA and cationic liposome mixing; and were required for the lipoplex formulations to survive freez-(3) the ionic strength of the suspending vehicle. The mixing ing and thawing. A frozen lipoplex formulation stored for conditions were precisely controlled by using a novel, spe-more than a year maintained its biological activity. These cially designed continuous flow pumping system in which results have broad implications for the pharmaceutical the DNA and liposome solutions were mixed at the junction development of lipoplex formulations for gene delivery.