Vaccination with polymerase chain reaction-generated linear expression cassettes protects mice against lethal influenza A challenge.

The feasibility of a linear expression cassette (LEC)-based influenza A DNA vaccine was demonstrated in mice, using a lethal dose (LD90) of a mouse-adapted A/Hong Kong/8/68 (H3N2) influenza strain. LECs expressing hemagglutinin (HA) from either the homotypic H3N2 or the heterotypic H1N1 (A/Puerto Rico/8/34) influenza virus were produced by polymerase chain reaction and either phosphodiester- or phosphorothioate-modified oligonucleotide primers. Survival subsequent to lethal viral challenge was used as a primary end point; weight loss was the secondary end point.

Plasmid vaccines and therapeutics: from design to applications.

In the late 1980s, Vical and collaborators discovered that the injection into tissues of unformulated plasmid encoding various proteins resulted in the uptake of the plasmid by cells and expression of the encoded proteins. After this discovery, a period of technological improvements in plasmid delivery and expression and in pharmaceutical and manufacturing development was quickly followed by a plethora of human clinical trials testing the ability of injected plasmid to provide therapeutic benefits. In this chapter, we summarize in detail the technologies used in the most recent company-sponsored clinical trials and discuss the potential for future improvements in plasmid design, manufacturing, delivery, formulation and administration.

Vaxfectin enhances antigen specific antibody titers and maintains Th1 type immune responses to plasmid DNA immunization.

Antigen specific immune responses were characterized after intramuscular immunization of BALB/c mice with 5 antigen encoding plasmid DNAs (pDNAs) complexed with Vaxfectin, a cationic lipid formulation. Vaxfectin increased IgG titers for all of the antigens with no effect on the CTL responses to the 2 antigens for which CTL assays were performed. Both antigen specific IgG1 and IgG2a were increased, although IgG2a remained greater than IgG1.

Vaxfectin enhances the humoral immune response to plasmid DNA-encoded antigens.

This report characterizes Vaxfectin, a novel cationic and neutral lipid formulation which enhances antibody responses when complexed with an antigen-encoding plasmid DNA (pDNA). In mice, intramuscular injection of Vaxfectin formulated with pDNA encoding influenza nucleoprotein (NP) increased antibody titers up to 20-fold, to levels that could not be reached with pDNA alone. As little as 1 microg of pDNA formulated with Vaxfectin per muscle resulted in higher anti-NP titers than that obtained with 25 microg naked pDNA.

Sodium phosphate enhances plasmid DNA expression in vivo.

Intramuscular injection of plasmid DNA results in myofiber cell expression of proteins encoded by the DNA. The preferred vehicle for plasmid DNA injections has been saline (154 mM sodium chloride) or PBS (154 mM NaCl plus 10 mM sodium phosphate). Here, it is shown that injection of luciferase or beta-galactosidase encoding plasmid DNA in a 150 mM sodium phosphate vehicle into murine muscle resulted in a two- to seven-fold increase in transgene expression compared with DNA injected in saline or PBS.

Adjuvants for plasmid DNA vaccines.

In the late 1980s, Jon Wolff of the University of Wisconsin and Phil Felgner here at Vical were screening cationic lipids for their ability to encapsulate and deliver purified plasmid DNA into mouse tissues. They discovered that direct injection of lipid-DNA complexes into muscle resulted in measurable protein expression. A belated control experiment without lipid led to the serendipitous discovery that "naked" plasmid DNA was taken up and expressed in muscle to a greater extent than DNA-lipid complexes (1).

Development and characterization of lyophilized DNA vaccine formulations.

The potential applications of using plasmid DNA for immunization and other gene therapy approaches have been discussed in an increasing number of publications in the past few years. Injection of mouse muscle with naked DNA (plasmid DNA in saline) resulted in significant episomal expression from a number of encoded reporter genes such as firefly luciferase, chloramphenicol acetyltransferase, and β-galactosidase (1). DNA vaccination has been shown to induce neutralizing antibodies against the gene product, helper T-cell responses of the Th1 phenotype, and cytotoxic T lymphocyte responses (2).

Enhancer and promoter chimeras in plasmids designed for intramuscular injection: a comparative in vivo and in vitro study.

Enhancers and promoters from various muscle-specific genes were substituted for or combined with the enhancer/promoter of the human cytomegalovirus (CMV) IE gene in a luciferase reporter gene plasmid in an effort to identify new promoter chimeras with increased expression activity after direct intramuscular injection. The regulatory sequence substitutions or additions varied in content, location, and orientation relative to the CMV regulatory sequences. The expression activities of the derivative and parent plasmids were compared quantitatively in vivo using a standard mouse intramuscular injection assay, and in vitro by transfection of differentiated C2C12 mouse myoblasts and BHK hamster kidney cells, to test whether cultured cell transfection could substitute for at least some animal experimentation.

Immunotherapy of established tumors in mice by intratumoral injection of interleukin-2 plasmid DNA: induction of CD8+ T-cell immunity.

Intratumoral (i.t.) injection of a plasmid DNA vector encoding the murine interleukin-2 (IL-2) gene was used to treat established renal cell carcinoma (Renca) tumors in BALB/c mice.

Spatial-temporal patterns of gene expression in mouse skeletal muscle after injection of lacZ plasmid DNA.

Gene therapy for muscular diseases requires the efficient transfection of a large proportion of myofiber cells within a given muscle. In the present experiments, patterns of beta-galactosidase expression were examined in mouse rectus femoris muscles at various time-points after a single injection of lacZ encoded plasmid DNA. beta-Galactosidase expression was detected 3 h after injection and rose to peak levels at 3-14 days, and then stabilized at lower levels.

Development of improved vectors for DNA-based immunization and other gene therapy applications.

Optimizing gene expression and delivery are necessary steps in the production of vectors for DNA-based immunization as well as for other gene therapy applications. A mouse muscle/reporter gene assay system was used to systematically improve a plasmid DNA vector. The optimized vector VR1255 contained: (1) CMV promoter and enhancer; (2) CMV IE Intron A; (3) kanamycin resistance gene; (4) deleted SV40 origin of replication; (5) optimized lux coding region; and (6) a minimal synthetic terminator from the rabbit beta globin gene, mRBG.

Synthetic NGF peptide derivatives prevent neuronal death via a p75 receptor-dependent mechanism.

Cyclized peptides corresponding to beta-loop regions of NGF were purified by HPLC and assayed for neurotrophic activity using DRG neurons. Peptides with the highest activity corresponded to loop region 29-35, a domain likely to interact with the p75 receptor. Unexpectedly, activity was confined to late-eluting HPLC fractions containing peptide multimers and primarily promoted neuronal survival without neurite outgrowth.

A novel cationic lipid greatly enhances plasmid DNA delivery and expression in mouse lung.

Effective gene therapy for lung tissue requires the use of efficient vehicles to deliver the gene of interest into lung cells. When plasmid DNA encoding chloramphenicol acetyltransferase (CAT) was administered intranasally to BALB/c mice without carrier lipids, CAT activity was detected in mouse lung extracts. Plasmid DNA delivered with optimally formulated commercially available transfection reagents expressed up to 10-fold more CAT activity in lung than observed with naked DNA alone.

An improved plasmid DNA expression vector for direct injection into skeletal muscle.

In previous work, the direct injection of 50 micrograms of a plasmid DNA vector encoding firefly luciferase (VR1205) into murine quadriceps muscle produced an average of 6.5 ng of luciferase per muscle at 7 days postinjection. In this report, various elements of the VR1205 vector were modified to increase gene expression levels or to eliminate undesired viral sequences.

Plasmid DNA gene therapy: studies with the human interleukin-2 gene in tumor cells in vitro and in the murine B16 melanoma model in vivo.

The plasmid DNA vector pVCL-1102 containing the coding sequence for the human IL-2 gene was evaluated for expression in tumor cells in vitro and in vivo. In vitro transfection of murine B16 tumor cells with pVCL-1102 resulted in the expression of 36,000 IU (5.7 micrograms) of biologically active IL-2/10(6) cells/48 h.

Converting an alcohol to an amine in a cationic lipid dramatically alters the co-lipid requirement, cellular transfection activity and the ultrastructure of DNA-cytofectin complexes.

Cytofectins are positively charged lipophilic molecules that readily form complexes with DNA and other anionic polynucleotides. Normally, cytofectins are combined with an activity-augmenting phospholipid such as dioleoylphosphatidylethanolamine (DOPE), and a film of dried, mixed lipid is prepared and hydrated to form cationic liposomes. The liposome solution is then mixed with a plasmid DNA solution to afford cytofectin-DNA complexes which, when presented to living cells, are internalized and the transgene is expressed.

Novel, high expressing and antibiotic-controlled plasmid vectors designed for use in gene therapy.

The promise of effective gene therapy can only be accomplished by high-level expression and regulatable delivery of gene products. To achieve this end, a eukaryotic expression plasmid was modified to make transcription dependent on a tetracycline(Tc)-regulated chimeric transactivator. Mouse muscle injected with this two plasmid cis/trans control system expressed reporter proteins at levels five- to 10-fold greater than the cytomegalovirus immediate-early promoter-controlled parental plasmid.

Improved cationic lipid formulations for in vivo gene therapy.

The problem of assessing in vivo activity of gene delivery systems is complex. The reporter gene must be carefully chosen depending on the application. Plasmids with strong promoters, enhancers and other elements that optimize transcription and translation should be employed, such as the CMVint and pCIS-CAT constructs.

Cancer gene therapy using plasmid DNA: safety evaluation in rodents and non-human primates.

To evaluate the safety of a plasmid DNA-lipid complex, a series of good laboratory practice (GLP) safety studies were conducted with VCL-1005, a plasmid DNA expression vector containing both the human class I MHC HLA-B7 heavy-chain and the beta 2-microglobulin (beta 2m) light-chain genes formulated with the cationic lipid, DMRIE/DOPE. In mice, the repeated intravenous injection of VCL-1005 at plasmid DNA doses of 0.1, 1.

Immunization with plasmid DNA using a pneumatic gun.

We characterize a method by which the Med-E-Jet pneumatic vaccination gun can be used to propel intact, supercoiled plasmid DNA through skin and into skeletal muscles of mice. Intramuscular injection of plasmids containing the firefly luciferase gene linked to the human cytomegalovirus promoter resulted in the expression of several hundred picograms of luciferase enzyme in quadriceps muscles. Intramuscular injections of a plasmid containing the influenza A nuclear protein gene regulated by the same promoter resulted in the generation of potent and specific anti-nuclear protein humoral and cellular immune responses.

Gene therapy by intramuscular injection of plasmid DNA: studies on firefly luciferase gene expression in mice.

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.

The localization of nerve growth factor-like immunoreactivity in the adult rat basal forebrain and hippocampal formation.

The role of nerve growth factor (NGF) as a target derived neurotrophic agent for specific cell populations in the peripheral nervous system has been well documented and much evidence suggests that NGF may serve a similar neurotrophic role in the CNS supporting the cholinergic neurons of the basal forebrain. Previous attempts to localize NGF by immunocytochemical methods, however, have not yielded evidence confirming the regional distribution expected based upon reported levels of extractable NGF. In the present study, affinity purified polyclonal antibodies to beta-NGF and a modified immunohistochemical protocol were used to demonstrate specific NGF-like immunoreactivity in the adult rat hippocampal formation and basal forebrain.

Nerve growth factor receptor immunoreactivity in neurons of the normal adult rat spinal cord and its modulation after peripheral nerve lesions.

Motoneurons of the rat spinal cord express low-affinity nerve growth factor receptor (LNGFR) and corresponding mRNA during development, and re-express it after their axotomy by peripheral nerve injury. The present study establishes the anatomical and quantitative baseline of LNGFR immunoreactive (LNGFR-IR) neurons of the entire normal adult female rat and then investigates the temporal course for the re-expression of LNGFR-IR in lumbar motoneurons after either a crush lesion (which is followed by regeneration and reconnection to the muscle) or a cut lesion with removal of the distal stump (where a neuroma but no reconnection is formed). In the normal adult spinal cord, two types of LNGFR-IR neurons were recognized: (1) small populations of large motoneurons located in the ventral horn mainly in correspondence to the regions of the phrenic, cremasteric and dorsolateral nuclei, and (2) a more numerous and more dorsally located population of small neurons.