Generation of expression constructs that secrete bioactive alphaMSH and their use in the treatment of experimental autoimmune encephalomyelitis.

alpha Melanocyte-stimulating hormone (alphaMSH) is a 13 amino acid peptide with potent anti-inflammatory effects. We created two DNA expression constructs (miniPOMC and pACTH1-17) that encode bioactive versions of the alphaMSH peptide, and tested these constructs for therapeutic effects in experimental autoimmune encephalomyelitis (EAE). Each construct contained the sequences for alphaMSH, as well as the sequences that are involved in the secretion and processing of the POMC gene with the assumption that these sequences would promote processing and release of the encoded alphaMSH peptide. The differences between the two constructs lie at the C-terminal end where amino acids necessary for amidation of alphaMSH were included in only the pACTH1-17 construct. These two constructs were tested in vitro in bioassays, and in vivo in a mouse model of EAE. The results show that although bioactive peptides are secreted from cells transfected with either construct, there appears to be a significant therapeutic effect only with the pACTH1-17 construct which contains the extra C-terminal amino acids. The data suggest that it is possible to engineer DNA expression vectors encoding small secreted peptides such as alphaMSH, and that similar type constructs may be useful as therapeutics for the treatment of inflammatory diseases.

Protective immune responses elicited in mice by immunization with formulations of poly(lactide-co-glycolide) microparticles.

Parenteral administration of microparticle encapsulated DNA elicits immune responses to the encoded antigens. Experiments were performed to test whether the addition of certain lipophilic agents to such formulations enhanced the activity of a beta-galactosidase (beta-gal) DNA vaccine. Addition of either taurocholic acid (TA) or monomethoxy polyethylene-glycol-distearoylphosphatidylehanolamine (PEG-DSPE) increased the efficiency of DNA encapsulation. Immunization of mice with encapsulated DNA formulations containing either compound significantly increased the number of antibody positive responders over that achieved with non-lipid containing particles. Moreover, responding animals demonstrated trends towards higher antibody titers and increased T cell responses. Tumor protection against the CT26.CL25 tumor cell line was demonstrated with lipid and non-lipid containing formulations. These results are the first demonstration of protection obtained by parenteral administration of PLG encapsulated DNA vaccines.

Two methods for quantifying DNA extracted from poly(lactide-co-glycolide) microspheres.

DNA can be formulated with synthetic polymers such as poly(lactide-co-glycolide) (PLG) to generate microparticles. Researchers have used either UV spectroscopy or fluorometry with PicoGreen((R)) dye to quantify PLG-encapsulated DNA. While the sensitivity of DNA detection and quantification by PicoGreen is higher ( approximately 12 pg/ml) compared to UV ( approximately 0.5 microg/ml), each method as an analytical tool has limitations. The premise of this work addresses the usefulness and limitations of each method to determine encapsulation efficiencies in PLG microspheres post-process, and to quantify release of DNA from microspheres during in vitro release experiments. In addition, assay conditions for accurate and reproducible extraction of DNA from PLG microspheres using a biphasic (aqueous/organic) solvent system are described. It was also determined that residual poly(vinyl alcohol) and DNA isoforms (linear, nicked, supercoiled) affected PicoGreen/DNA fluorescence values.

Gene therapy of chronic inflammatory disease.

Immune mediated inflammation that culminates in severe tissue necrosis is the hallmark of diseases that result from an inappropriate response to antigen. The inflammatory response becomes chronic when antigen is non-limiting and persists until the reactive tissue is destroyed, or the environment is changed and exposure to antigen is eliminated. The purpose of this review is to: (1) briefly outline common features of immune related inflammatory diseases such as rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD), and allergic asthma; (2) provide a rationale for the development of gene based drugs for these indications; and (3) describe current experimental results that support the usefulness of this approach for creating novel DNA based therapeutics.

Biological potency of microsphere encapsulated plasmid DNA.

We evaluated the utility of three in vitro methods to monitor the biological potency of PLGA encapsulated DNA. For each assay we also determined whether the biological activity was influenced by the structural profile of DNA isomers. Collectively, the results indicate that all three methods can be used to evaluate the biological activity of DNA extracted from PLGA microspheres, but they are differentially sensitive to the structural changes of plasmid DNA that can occur during microencapsulation and microsphere storage. More specifically, mammalian cell transfection followed by an enzyme assay affords an accurate determination of DNA potency over time and is less influenced by DNA isoform than bacterial transformation. Cell-free transcription/translation systems can also be utilized, and the results of this assay are influenced by DNA isoform. Finally, bacterial transformation was found to be more sensitive to DNA isoform than the other assay methods.

Tissue distribution and persistence in mice of plasmid DNA encapsulated in a PLGA-based microsphere delivery vehicle.

Information regarding the distribution and persistence of DNA encapsulated in poly-(lactide co-glycolide) microspheres was collected to provide additional information regarding the safety of DNA vaccines and to support the clinical testing of this new delivery system for DNA. Plasmid DNA was encapsulated in poly(lactide co-glycolide) microspheres and the distribution and persistence of plasmid in murine tissues resulting from parenteral administration were examined by a sensitive PCR assay. Encapsulated DNA delivered by intramuscular or subcutaneous injection can be detected for 100 days post-injection and is distributed primarily at the site of injection and the lymphoid organs. Intravenous administration results in more widespread dissemination with long term persistence limited to the lymphoid organs and those of the reticuloendothelial system. Specific cellular uptake of DNA by professional antigen presenting cells (APCs) following injection suggests the utility of microspheres as DNA delivery agents. Distribution and persistence studies support the safety of encapsulated DNA and the specific cellular uptake of DNA by professional APCs following injection suggests the utility of microspheres as DNA delivery agents.

Comparison of process parameters for microencapsulation of plasmid DNA in poly(D,L-lactic-co-glycolic) acid microspheres.

Poly(D,L-lactic-co-glycolic) acid (PLGA) microspheres containing plasmid DNA encoding the firefly luciferase gene were prepared using the water-in-oil-in-water (w/o/w) double emulsion and solvent evaporation method. In this study, we investigated the effects of three process parameters on DNA microencapsulation: (1) emulsification method used to generate the primary emulsion, (2) water/oil ratio during formation of the first emulsion, and (3) surfactant concentration used in the preparation of the second emulsion. The resulting formulations were also analyzed for microsphere size, encapsulation efficiency, and kinetics of DNA release. We found that although each process alteration resulted in encapsulation of biologically active, structurally intact DNA, the surfactant and water/oil ratio significantly affected the size, release kinetics and encapsulation efficiency of plasmid DNA.

Plasmid DNA encoding targeted naturally processed peptides generates protective cytotoxic T lymphocyte responses in immunized animals.

Genetic immunization has been widely applied in efforts to find novel and efficient mechanisms of stimulating the immune response. An effective attack against viral pathogens or tumors often requires activation of T cell-mediated immunity and the generation of cytotoxic T cells. Intramuscular immunization with plasmid DNA containing cDNAs that encode proteins results in expression and secretion of the foreign antigen by muscle cells. T cell activation occurs when peptide fragments of the exogenous protein are presented by major histocompatibility complex class I molecules on the surface of professional antigen-presenting cells. Identification of specific peptide epitopes from a protein antigen presented to T cells during an infectious process or tumor situation would provide all of the antigenic information needed to stimulate effective T cell-mediated immunity. Such peptides represent the naturally processed epitopes selected by the processing machinery of antigen presenting cells. Delivery of this information to the appropriate cells in vivo might be sufficient to stimulate T cell immunity and overcome the difficulties associated with overexpression of large protein antigens or those with potentially toxic side effects. This report describes the use of naturally processed T cell epitopes, administered in plasmid DNA vaccines, to stimulate cytotoxic T cell responses to two viral antigens effectively.

The discovery and use of HLA-associated epitopes as drugs.

MHC receptors "display" peptide fragments to T cells. These peptides are predominantly derived from proteins expressed within or ingested by the presenting cell. Since empty MHC molecules are highly unstable, peptide ligands are bound prior to MHC surface expression and the ensuing t1/2 off rates are often on the order of days. It is the remarkable stability of MHC/peptide complexes, which provide us an opportunity to purify MHC molecules from infected, transfected, or antigen pulsed cells and subsequently identify the naturally processed peptides being presented. On the other hand, the stability of MHC/peptide complexes substantially reduces the potency of parenterally administered peptides in vivo. Using serial immuno-affinity chromatography and mass spectrometry, naturally processed peptides can be identified. When these peptides are then encoded into nucleic acid and delivered parenterally, they are highly immunogenic. Application of these techniques to induce vigorous CTL responses will be discussed.

An amino acid sequence motif sufficient for subnuclear localization of an arginine/serine-rich splicing factor.

We have identified an amino acid sequence in the Drosophila Transformer (Tra) protein that is capable of directing a heterologous protein to nuclear speckles, regions of the nucleus previously shown to contain high concentrations of spliceosomal small nuclear RNAs and splicing factors. This sequence contains a nucleoplasmin-like bipartite nuclear localization signal (NLS) and a repeating arginine/serine (RS) dipeptide sequence adjacent to a short stretch of basic amino acids. Sequence comparisons from a number of other splicing factors that colocalize to nuclear speckles reveal the presence of one or more copies of this motif. We propose a two-step subnuclear localization mechanism for splicing factors. The first step is transport across the nuclear envelope via the nucleoplasmin-like NLS, while the second step is association with components in the speckled domain via the RS dipeptide sequence.

Assembly and peptide binding of major histocompatibility complex class II heterodimers in an in vitro translation system.

In vitro transcription/translation of HLA-DR1 cDNAs in the presence of microsomal membranes was used to study the association of major histocompatibility complex class II molecules with peptide and invariant chain (Ii) in the endoplasmic reticulum (ER). HLA-DR alpha and HLA-DR beta subunits assembled into SDS-unstable heterodimers in the absence of exogenous peptide. The inclusion of synthetic peptides during the alpha/beta assembly process promoted their conversion to SDS-resistant heterodimers. Addition of Ii RNA during the translation of HLA-DR alpha and HLA-DR beta RNAs resulted in the formation of alpha/beta/Ii complexes. Peptide binding by class II molecules was detected even when excess Ii was present during alpha/beta assembly. These findings indicate that peptides can bind alpha/beta heterodimers in the ER microenvironment and suggest that peptides derived from cytosolic proteins that are presented by class II molecules at the cell surface may have bound to HLA-DR in the ER.

The role of specific protein-RNA and protein-protein interactions in positive and negative control of pre-mRNA splicing by Transformer 2.

We have investigated the function of different structural domains of the Drosophila splicing regulator Transformer 2 (Tra2). We find that the ribonucleoprotein consensus sequence (RNP-CS) of Tra2 is required for male fertility and positive and negative control of alternative splicing in transgenic flies, as well as for in vitro binding of recombinant Tra2 to doublesex and tra2 pre-mRNAs. Thus, all of the known functions of Tra2 require specific protein-RNA interactions. We also show that one of the two arginine-serine (RS)-rich domains of Tra2 is dispensable, while the other is essential for all of the in vivo functions. Part of this domain is also required for RNA binding in vitro. Significantly, the essential RS domain is also required for specific protein-protein interactions. We find that Tra2 interacts with itself, with the splicing regulator Transformer, and with the general splicing factor SF2 in vitro and in the yeast two-hybrid system. These results demonstrate that both protein-RNA and protein-protein interactions are involved in tra2-dependent activation and repression of alternative splicing.

Sex-specific splicing and polyadenylation of dsx pre-mRNA requires a sequence that binds specifically to tra-2 protein in vitro.

Somatic sex determination in Drosophila involves a hierarchy of regulated alternative pre-mRNA processing. Female-specific splicing and/or polyadenylation of doublesex (dsx) pre-mRNA, the final gene in this pathway, requires transformer (tra) and transformer-2 (tra-2) proteins. The mechanisms by which these proteins regulate RNA processing has not been characterized. In this paper we show that tra-2 produced in Escherichia coli binds specifically to a site within the female-specific exon of dsx pre-mRNA. This site, which contains six copies of a 13 nucleotide repeat, is required not only for female-specific splicing, but also for female-specific polyadenylation. These observations suggest that tra-2 is a positive regulator of dsx pre-mRNA processing.

Expression of mutant H-2d proteins encoded by class I genes which alternatively process the 5' end of their transcripts.

Mutation of the 3' splice sites bordering exon 2 of the H-2Dd and H-2Kd genes generated alternatively spliced transcripts when the constructs were transfected into L cells (J. Immunol. 143:1018). The H-2Dd transcripts contained an additional 84 nucleotides derived from the first intervening sequence, whereas 60 extra bases were included in the H-2Kd mRNA. Proteins derived from these transcripts were recognized by mAb. Moreover, both Ag served as recognition elements for CTL, and the mutant H-2Kd molecule functioned as a restricting element for an Ag peptide. As a result of alternative splicing, the mutant proteins should have additional residues at their NH2 termini to increase their lengths by 28 (Dd) or 20 (Kd) amino acids. Immunoprecipitation and analysis on SDS-PAGE demonstrated that the mutant H-2Kd molecule was indeed larger than the normal H-2Kd protein, but the mutant and wild-type H-2Dd Ag were the same size. In addition, treatment of H-2Dd mutant and normal Ag with N-glycanase produced molecules of equal size, demonstrating that the mutant protein was completely glycosylated. Limited amino acid sequencing of this Ag indicated that it was normal H-2Dd. Therefore, before its transfer to the cell surface, post-translational modifications remove the additional NH2-terminal residues of the mutant Dd but not Kd protein.

Mutation of 3' splice sites in two different class I genes results in different usage of cryptic splice sites.

To determine the pattern of alternative splicing at the 5' end of class I genes, the 3' splice sites bordering exon 2 of the H-2Dd and H-2Kd genes were mutated from AG to GG (H-2Dd) or CG (H-2Kd). The mutant genes were transfected into L cells, and RNA from clones expressing these Ag was used for analysis by RNase and S1 nuclease mapping techniques. The first intervening sequence of both class I genes contains several potential 3' splice acceptor sites. However, a clear preference for only one site was detected in each of the H-2Dd and H-2Kd mRNA. Examination of the endogenous H-Dd and H-2Kd class I transcripts in normal murine tissues and in tumors demonstrated that the alternatively spliced mRNAs were produced, but at a low frequency. Infection of transfected L cells or tumor lines with vesicular stomatitis virus altered the level of differentially spliced message in these cells.

Differential expression of the class I MHC genes in the embryo and placenta during midgestational development in the mouse.

A sensitive RNase mapping technique was used to investigate the expression of individual class I mRNA in the embryo and placenta of the mouse from day 7.5 through day 13 of gestation. Transcripts of the H-2Kd and -Dd genes appeared in both placental and embryonic tissues as early as day 7.5 post coitum and continued to be expressed thereafter. In contrast, H-2Ld mRNA was barely detectable in embryonic RNA until day 18 of pregnancy, although it was present in placental RNA samples on days 10 to 13 of gestation. Qa-region genes demonstrated a different pattern of expression than H-2Dd, -Kd, and -Ld. Transcripts of Q7, one of the genes encoding Qa-2 surface Ag, were detected in the developing embryo on days 9 to 11 post coitum but decreased thereafter. However, Q7d transcripts were not detected in placental tissues at any stage. Q6 mRNA was not detected in any of the tissues, and Q10 mRNA was detected only in day 12 to 13 embryos, where expression is known to be restricted to the fetal liver. Transcripts from T13, a Tla-region gene, and D2d, a D-region gene, were not detected in any of the samples tested. This study has revealed a complex pattern of class I gene regulation both within the genes of the MHC and between the embryo and placental lineages during the midgestational stages of development.

Analysis of D2d: a D-region class I gene.

The mouse major histocompatibility complex is composed of several genes arranged into the K, D, Qa, and Tla regions. The D region of the BALB/c mouse includes genes D2d, D3d, and D4d, in addition to H-2Dd and H-2Ld. We have determined the DNA sequence of the D2d gene and compared it with the known sequences of several class I genes. The exon/intron structure of the D2d gene is similar to other class I genes. It also contains similar 5' regulatory elements. A frameshift occurs in exon seven, resulting in a gene product with a truncated cytoplasmic tail. To examine the surface expression of the D2d molecule, we generated an exon-shuffled construct containing the promoter and exons 1-3, encoding the signal peptide, alpha 1, and alpha 2 external domains of the D2d gene linked to exons 4-8, encoding the alpha 3, transmembrane and cytoplasmic domains, of the H-2Dd gene. The construct was transfected into mouse L cells, and a protein was detected at the cell surface by a monoclonal antibody (mAb) specific for the alpha 3 domain of H-2Dd, as well as by other class I-specific mAbs. Although D2d is expressed at low levels, it may be a functional class I gene that most probably evolved from a Qa region gene.