Quantification of cytosolic plasmid DNA degradation using high‐throughput sequencing: implications for gene delivery

Background Although cytosolic DNA degradation plays an important role in decreasing transgene expression, the plasmid degradation pattern remains largely unexplored. Methods Illumina dye sequencing was employed to provide degradation site information for S1 and cytosolic nucleases. S1 nuclease provided a positive control for a comparison between the agarose gel method and sequencing approaches. Results The poly(A) region between the β‐lactamase gene and the cytomegalovirus (CMV) promoter was identified as the most likely cut site for polyplex‐treated cytosol. The second most likely site, at the 5' end of the β‐lactamase gene, was identified by gel electrophoresis and sequencing. Additional sites were detected in the OriC region, the SV40/poly(A) region, the luciferase gene and the CMV promoter. Sequence analysis of plasmid treated with cytosol from control cells showed the greatest cut activity in the OriC region, the β‐lactamase gene and the poly(A) region following the luciferase gene. Additional regions of cut activity include the SV40 promoter and the β‐lactamase poly(A) termination sequence. Both cytosolic nucleases and the S1 nuclease showed substantial activity at the bacterial origin of replication ( OriC ). Conclusions High‐throughput plasmid sequencing revealed regions of the luciferase plasmid DNA sequence that are sensitive to cytosolic nuclease degradation. This provides new targets for improving plasmid and/or polymer design to optimize the likelihood of protein expression. Copyright © 2014 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106947/1/jgm2761.pd

Nasal Immunization with a Recombinant HIV gp120 and Nanoemulsion Adjuvant Produces Th1 Polarized Responses and Neutralizing Antibodies to Primary HIV Type 1 Isolates

ABSTRACT Epidemiological and experimental data suggest that both robust neutralizing antibodies and potent cellular responses play important roles in controlling primary HIV-1 infection. In this study we have investigated the induction of systemic and mucosal immune responses to HIV gp120 monomer immunogen administered intranasally in a novel, oil-in-water nanoemulsion (NE) adjuvant. Mice and guinea pigs intranasally immunized by the application of recombinant HIV gp120 antigen mixed in NE demonstrated robust serum anti-gp120 IgG, as well as bronchial, vaginal, and serum anti-gp120 IgA in mice. The serum of these animals demonstrated antibodies that cross-reacted with heterologous serotypes of gp120 and had significant neutralizing activity against two clade-B laboratory strains of HIV (HIVBaL and HIVSF162) and five primary HIV-1 isolates. The analysis of gp120-specific CTL proliferation, INF-γ induction, and prevalence of anti-gp120 IgG2 subclass antibodies indicated that nasal vaccination in NE also induced systemic, Th1-polarized cellular immune responses. This study suggests that NE should be evaluated as a mucosal adjuvant for multivalent HIV vaccines.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63251/1/aid.2007.0148.pd

Efficient Transfer of Genes into Murine Cardiac Grafts by Starburst Polyamidoamine Dendrimers

Overview summary Plasmid-mediated gene therapy has been used to deliver immunosuppressive molecules into allografts to prolong graft survival. However, direct injection of naked plasmid DNA is inefficient because transgene expression is low and transient. This study investigated the ability of Starburst dendrimers to augment plasmid-mediated gene transfer efficiency in a murine cardiac transplantation model. The results demonstrate that dendrimers increased the efficiency of transfer and expression of exogenous DNA in cardiac grafts. Improved expression of an immunosuppressive cytokine viral interleukin-10 (vIL-10) by dendrimers significantly prolonged allograft survival. The dose of DNA, the charge ratio of DNA to dendrimer, and the size generation of the dendrimers were all critical for prolongation of allograft survival. Thus, the use of the Starburst dendrimer as a carrier molecule for plasmid-mediated gene transfer improved the efficiency of transfer and expression, providing further therapeutic value for treatment of cardiac allograft rejection.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63156/1/hum.1998.9.4-553.pd

Imaging {Au 0 -PAMAM} Gold-dendrimer Nanocomposites in Cells

Dendrimer nanocomposites (DNC) are hybrid nanoparticles formed by the dispersion and immobilization of guest atoms or small clusters in dendritic polymer matrices. They have a great potential in biomedical applications due to their controlled composition, predetermined size, shape and variable surface functionalities. In this work, d =5–25 nm spherical nanoparticles composed of gold and poly(amidoamine) (PAMAM) dendrimers have been selected to demonstrate this nanoparticle based concept. {Au(0) n -PAMAM} gold dendrimer nanocomposites with a well-defined size were synthesized and imaged by transmission electron microscopy both in vitro and in vivo. DNC have also the potential to be used for imaging and drug delivery vehicles either by utilizing bioactive guests or through the incorporation of radioactive isotopes, such as Au-198.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43295/1/11051_2004_Article_5101884.pd

Pre-Clinical Evaluation of a Novel Nanoemulsion-Based Hepatitis B Mucosal Vaccine

Hepatitis B virus infection remains an important global health concern despite the availability of safe and effective prophylactic vaccines. Limitations to these vaccines include requirement for refrigeration and three immunizations thereby restricting use in the developing world. A new nasal hepatitis B vaccine composed of recombinant hepatitis B surface antigen (HBsAg) in a novel nanoemulsion (NE) adjuvant (HBsAg-NE) could be effective with fewer administrations.Physical characterization indicated that HBsAg-NE consists of uniform lipid droplets (349+/-17 nm) associated with HBsAg through electrostatic and hydrophobic interactions. Immunogenicity of HBsAg-NE vaccine was evaluated in mice, rats and guinea pigs. Animals immunized intranasally developed robust and sustained systemic IgG, mucosal IgA and strong antigen-specific cellular immune responses. Serum IgG reached > or = 10(6) titers and was comparable to intramuscular vaccination with alum-adjuvanted vaccine (HBsAg-Alu). Normalization showed that HBsAg-NE vaccination correlates with a protective immunity equivalent or greater than 1000 IU/ml. Th1 polarized immune response was indicated by IFN-gamma and TNF-alpha cytokine production and elevated levels of IgG(2) subclass of HBsAg-specific antibodies. The vaccine retains full immunogenicity for a year at 4 degrees C, 6 months at 25 degrees C and 6 weeks at 40 degrees C. Comprehensive pre-clinical toxicology evaluation demonstrated that HBsAg-NE vaccine is safe and well tolerated in multiple animal models.Our results suggest that needle-free nasal immunization with HBsAg-NE could be a safe and effective hepatitis B vaccine, or provide an alternative booster administration for the parenteral hepatitis B vaccines. This vaccine induces a Th1 associated cellular immunity and also may provide therapeutic benefit to patients with chronic hepatitis B infection who lack cellular immune responses to adequately control viral replication. Long-term stability of this vaccine formulation at elevated temperatures suggests a direct advantage in the field, since potential excursions from cold chain maintenance could be tolerated without a loss in therapeutic efficacy

Mucosal Immunization with a Novel Nanoemulsion-Based Recombinant Anthrax Protective Antigen Vaccine Protects against Bacillus anthracis Spore Challenge▿

The currently available commercial human anthrax vaccine requires multiple injections for efficacy and has side effects due to its alum adjuvant. These factors limit its utility when immunizing exposed populations in emergent situations. We evaluated a novel mucosal adjuvant that consists of a nontoxic, water-in-oil nanoemulsion (NE). This material does not contain a proinflammatory component but penetrates mucosal surfaces to load antigens into dendritic cells. Mice and guinea pigs were intranasally immunized with recombinant Bacillus anthracis protective antigen (rPA) mixed in NE as an adjuvant. rPA-NE immunization was effective in inducing both serum anti-PA immunoglobulin G (IgG) and bronchial anti-PA IgA and IgG antibodies after either one or two mucosal administrations. Serum anti-PA IgG2a and IgG2b antibodies and PA-specific cytokine induction after immunization indicate a Th1-polarized immune response. rPA-NE immunization also produced high titers of lethal-toxin-neutralizing serum antibodies in both mice and guinea pigs. Guinea pigs nasally immunized with rPA-NE vaccine were protected against an intradermal challenge with ∼1,000 times the 50% lethal dose (∼1,000× LD50) of B. anthracis Ames strain spores (1.38 × 103 spores), which killed control animals within 96 h. Nasal immunization also resulted in 70% and 40% survival rates against intranasal challenge with 10× LD50 and 100× LD50 (1.2 × 106 and 1.2 × 107) Ames strain spores. Our results indicate that NE can effectively adjuvant rPA for intranasal immunization. This potentially could lead to a needle-free anthrax vaccine requiring fewer doses and having fewer side effects than the currently available human vaccine