Dual AAV/IL-10 Plus STAT3 Anti-Inflammatory Gene Delivery Lowers Atherosclerosis in LDLR KO Mice, but without Increased Benefit

Both IL-10 and STAT3 are in the same signal transduction pathway, with IL-10-bound IL10 receptor (R) acting through STAT3 for anti-inflammatory effect. To investigate possible therapeutic synergism, we delivered both full-length wild-type human (h) STAT3 and hIL-10 genes by separate adenoassociated virus type 8 (AAV8) tail vein injection into LDLR KO on HCD. Compared to control Neo gene-treated animals, individual hSTAT3 and hIL-10 delivery resulted in significant reduction in atherogenesis, as determined by larger aortic lumen size, thinner aortic wall thickness, and lower blood velocity (all statistically significant). However, dual hSTAT3/hIL-10 delivery offered no improvement in therapeutic effect. Plasma cholesterol levels in dual hSTAT3/hIL-10-treated animals were statistically higher compared to hIL-10 alone. While no advantage was seen in this case, we consider that the dual gene approach has intrinsic merit, but properly chosen partnered genes must be used

Autocrine, Not Paracrine, Interferon-Gamma Gene Delivery Enhances Ex Vivo Antigen-Specific Cytotoxic T Lymphocyte Stimulation and Killing

The adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTL) shows promise in the treatment of cancer and infectious diseases. We utilize adeno-associated virus-(AAV-) based antigen gene-loaded dendritic cells (DCs) to stimulate such antigen-specific CTL. Yet further improvements in CTL stimulation and killing may result by gene delivery of various Th1-response interferons/cytokines, such as interferon γ (IFN-γ), as the delivered gene can continuously produce that interferon. However which immune cell type should optimally express IFN-γ is unclear as the phenotypes of both DC and T cells are enhanced by it. Here, we used AAV to compare and contrast IFN-γ gene delivery into DC or T cells, and versus the addition of exogenous IFN-γ, for stimulating carcinoembryonic antigen-(CEA-) specific CTL. It was found that AAV/IFN-γ delivery into T cells (autocrine) resulted in T cell populations with the highest CD8(+)/CD4(+) ratio, highest IFN-γ(+)/IL-4(+) ratio, highest CD69(+),CD8(+) levels, and lowest CD4(+)/CD25(+) levels, all consistent with the strongest Th1 response. Most importantly, AAV/IFN-γ transduction of T cells resulted in antigen-specific T cell populations with the highest killing capabilities, 49% above other treatments. These data strongly suggest that AAV/IFN-γ autocrine gene delivery into T cells is worthy of further study towards maximizing the generation of antigen-specific anticancer CTL killers

AAV2/8-hSMAD3 gene delivery attenuates aortic atherogenesis, enhances Th2 response without fibrosis, in LDLR-KO mice on high cholesterol diet

BACKGROUND: Inflammation is a key etiologic component in atherogenesis and transforming growth factor beta 1 (TGFβ1) is a well known anti-inflammatory cytokine which potentially might be used to limit it. Yet TGFβ1 is pleiomorphic, causing fibrosis, cell taxis, and under certain circumstances, can even worsen inflammation. SMAD3 is an important member of TGFβ1′s signal transduction pathway, but is a fully intracellular protein. OBJECTIVES: With the hope of attenuating TGFβ1′s adverse systemic effects (eg. fibrosis) and accentuating its anti-inflammatory activity, we proposed the use of human (h)SMAD3 as an intracellular substitute for TGFβ1. STUDY DESIGN: To test this hypothesis adeno-associated virus type 2/8 (AAV)/hSMAD3 or AAV/Neo (control) was tail vein injected into the low density lipoprotein receptor knockout (LDLR-KO) mice, then placed on a high-cholesterol diet (HCD). RESULTS: The hSMAD3 delivery was associated with significantly lower atherogenesis as measured by larger aortic cross sectional area, thinner aortic wall thickness, and lower aortic systolic blood velocity compared with Neo gene-treated controls. HSMAD3 delivery also resulted in fewer aortic macrophages by immunohistochemistry for CD68 and ITGAM, and quantitative reverse transcriptase polymerase chain reaction analysis of EMR and ITGAM. Overall, aortic cytokine expression showed an enhancement of Th2 response (higher IL-4 and IL-10); while Th1 response (IL-12) was lower with hSMAD3 delivery. While TGFβ1 is often associated with increased fibrosis, AAV/hSMAD3 delivery exhibited no increase of collagen 1A2 or significantly lower 2A1 expression in the aorta compared with Neo-delivery. Connective tissue growth factor (CTGF), a mediator of TGFβ1/SMAD3-induced fibrosis, was unchanged in hSMAD3-delivered aortas. In the liver, all three of these genes were down-regulated by hSMAD3 gene delivery. CONCLUSION: These data strongly suggest that AAV/hSMAD3 delivery gave anti-atherosclerosis therapeutic effect without the expected undesirable effect of TGFβ1-associated fibrosis

Anti-HIV-1 Activity of a New Scorpion Venom Peptide Derivative Kn2-7

For over 30 years, HIV/AIDS has wreaked havoc in the world. In the absence of an effective vaccine for HIV, development of new anti-HIV agents is urgently needed. We previously identified the antiviral activities of the scorpion-venom-peptide-derived mucroporin-M1 for three RNA viruses (measles viruses, SARS-CoV, and H5N1). In this investigation, a panel of scorpion venom peptides and their derivatives were designed and chosen for assessment of their anti-HIV activities. A new scorpion venom peptide derivative Kn2-7 was identified as the most potent anti-HIV-1 peptide by screening assays with an EC50 value of 2.76 µg/ml (1.65 µM) and showed low cytotoxicity to host cells with a selective index (SI) of 13.93. Kn2-7 could inhibit all members of a standard reference panel of HIV-1 subtype B pseudotyped virus (PV) with CCR5-tropic and CXCR4-tropic NL4-3 PV strain. Furthermore, it also inhibited a CXCR4-tropic replication-competent strain of HIV-1 subtype B virus. Binding assay of Kn2-7 to HIV-1 PV by Octet Red system suggested the anti-HIV-1 activity was correlated with a direct interaction between Kn2-7 and HIV-1 envelope. These results demonstrated that peptide Kn2-7 could inhibit HIV-1 by direct interaction with viral particle and may become a promising candidate compound for further development of microbicide against HIV-1

Molecular modeling of solid -fluid equilibrium for complex molecules

This work is concerned with providing the fundamental understanding of solid-fluid phase equilibrium of complex molecules by using Monte Carlo simulation, with a special emphasis on long chain n-alkanes and simple chiral molecule systems. The goal of the work is to look into the solid-fluid equilibrium thermodynamics from perspective view of intermolecular interactions, especially the repulsive interactions. The solid-fluid phase behavior of longer chain n-alkanes can be complicated by a substantial loss of orientational order about the chain axis in the solid phase near the melting point. The solid state formed under these circumstances is called a rotator phase. We have investigated the properties of the solid and fluid phases of a molecular model of n-alkanes with chain lengths in the range C17-- C21, focusing especially on the occurrence of rotator phases. The models we have been using are hard-core united atom models. The bond lengths and bond angles are fixed. Internal rotations are modeled using simple torsional potentials that allow for small fluctuations around the trans and gauche conformations. We have made extensive calculations of the free energy and equation of state of the model for longer chains and have developed order parameters for tracking the degree of orientational order around the chain axis. The model exhibits both a rotator phase and a more orientationally ordered solid phase. Our results indicate that the transition between these two solid phases is first order with a small density change. We also use the models to investigate the solution thermodynamics and solid-fluid phase equilibrium in mixtures of n-alkanes. The phase diagrams show that solid-solid and solid-fluid phase coexistence can occur in those systems. Solid-solid microphase separation was observed directly in the simulations of some binary mixtures. In addition, for some states modulated superstructures were also observed, similar to those seen in experiments on solid phase alkane mixtures. Techniques developed to study chain systems have been extended to chiral molecules with emphasis on the stability of solid phase racemic compounds. The solid-fluid and solid-solid phase equilibrium has been determined for molecular models representative of chiral molecules and enantiomeric mixtures. The models consist of four hard sphere interaction sites of different diameters in a tetrahedral arrangement with the fifth hard sphere interaction site at the center of the tetrahedron. A simulated annealing method has been applied to generate the close packing structure for both the pure components and racemic compounds of the model. The volumetric properties and free energies of the pure enantiomers and binary mixtures were calculated in both fluid and solid phases using isobaric Monte Carlo simulations. The models exhibit ideal solution behavior in the fluid phase with little chiral discrimination. In the solid phase the effects of chirality are much greater. Solid-fluid phase behavior involving the pure enantiomer solids and also racemic compounds was calculated. The calculations indicate that, depending on the relative sizes of the hard sphere interaction sites, packing effects alone can be sufficient to stabilize a racemic compound with respect to the pure enantiomer solids

The X gene of adeno-associated virus 2 (AAV2) is involved in viral DNA replication.

Adeno-associated virus (AAV) (type 2) is a popular human gene therapy vector with a long active transgene expression period and no reported vector-induced adverse reactions. Yet the basic molecular biology of this virus has not been fully addressed. One potential gene at the far 3' end of the AAV2 genome, previously referred to as X (nt 3929 to 4393), overlapping the 3' end of the cap gene, has never been characterized, although we did previously identify a promoter just up-stream (p81). Computer analysis suggested that X was involved in replication and transcription. The X protein was identified during active AAV2 replication using a polyclonal antibody against a peptide starting at amino acid 98. Reagents for the study of X included an AAV2 deletion mutant (dl78-91), a triple nucleotide substitution mutant that destroys all three 5' AUG-initiation products of X, with no effect on the cap coding sequence, and X-positive-293 cell lines. Here, we found that X up-regulated AAV2 DNA replication in differentiating keratinocytes (without helper virus, autonomous replication) and in various forms of 293 cell-based assays with help from wild type adenovirus type 5 (wt Ad5) or Ad5 helper plasmid (pHelper). The strongest contribution by X was seen in increasing wt AAV2 DNA replication in keratinocytes and dl78-91 in Ad5-infected X-positive-293 cell lines (both having multi-fold effects). Mutating the X gene in pAAV-RC (pAAV-RC-3Xneg) yielded approximately a ∼33% reduction in recombinant AAV vector DNA replication and virion production, but a larger effect was seen when using this same X-knockout AAV helper plasmid in X-positive-293 cell lines versus normal 293 cells (again, multi-fold). Taken together these data strongly suggest that AAV2 X encodes a protein involved in the AAV life cycle, particularly in increasing AAV2 DNA replication, and suggests that further studies are warranted

Presence and homology of AAV2 X with that of the possible X proteins encoded by other AAV clades and types.

<p>Shown are the results of NCBI ProteinBLAST software analysis comparing the indicated proteins derived from ORFs in the same relative position as AAV2 X within the AAV2 genome.</p><p>*Refers to the combined amino acids of cy.5R4 X ORFs fused A before B.</p

pSM620-3Xneg, without <i>X</i>, displays weaker DNA replication in Ad5-infected 293 cells.

<p><b>A</b> shows a <i>Pst</i> I restriction digestion analysis of wt pSM620 and pSM620-3Xneg (<i>X-</i>). <b>B</b> shows the Southern blot of DNA replication, using ³²P-<i>rep</i> pribe, of pSM620 and pSM620-3Xneg relative to each other in Ad5-infected 293 cells. Note that pSM620 replicated to a slightly higher level than pSM620-3Xneg (<i>p</i> = 0.057). <b>C</b> shows a “2^nd plate analysis” where equal aliquots of virus stock from plates identical to those of <b>B</b> where heated to 56°C (to kill Ad5), and then used to infect a second plate of Ad5-infected 293 cells. Shown is the Southern blot of DNA replication, using P32-<i>rep</i> probe, of pSM620 and pSM620-3Xneg replication from resulting first plate generated virus infection (<i>p</i><0.05). <b>D</b> shows a Southern blot analysis comparison of pSM620-3Xneg replication in Ad5-infected unaltered 293 cells, 293-X-B, and 293-X-K, probed with ³²P-<i>rep</i> DNA. Note that pSM620-3Xneg replicates to higher levels in the 293 cells which contain the <i>X</i> gene (complementation) compared to 293 cells without <i>X</i> (<i>p</i><0.05). <b>E</b> shows another “2^nd plate analysis” where equal aliquots of virus stock from plates identical to those of <b>D</b>, which where heated to 56°C (to kill Ad5), and then used to infect a second plate of Ad5-infected 293 (normal) cells. Shown is the Southern blot of DNA replication, using ³²P-<i>rep</i> probe, of pSM620-3Xneg replication from resulting first plate generated virus infection. Note that, pSM620-3Xneg replicated to higher levels in the 2^nd plate (<i>p<</i>0.05) due to higher levels of virus produced in the first plate.</p

Deletion of <i>X</i> gives lower DNA replication of AAV2.

<p><b>A</b> shows the structure of AAV2 deletion mutants dl63-78 and dl78-91, with wild type (wt) AAV2 shown at the top, including <i>Pst</i> I restriction sites. <b>B</b> shows a <i>Pst</i> I, <i>Bgl</i> II dual digestion of dl63-78 and dl78-91. <b>C</b> shows a Southern blot analysis comparison of dl63-78 and dl78-91 DNA replication in Ad5-infected 293 cells, probed with 32P-<i>rep</i> DNA, and densitometrically quantitated in <b>D</b>. Note that dl63-78 replicates approximately 2.5 fold higher than the dl78-91(<i>p</i><0.05). <b>E</b> shows a comparison of dl78-91 DNA replication upon co-transfection with either AAV/Neo or AAV/X/Neo into Ad5-infected 293 cells (<i>p<</i>0.05). <b>F</b> shows a Southern blot analysis comparison of dl78-91 DNA replication in Ad5-infected unaltered 293 cells, 293-X-B, and 293-X-K, probed with 32P-<i>rep</i> DNA. An analysis of the level of copy numbers of <i>X</i> in these cells is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104596#pone-0104596-g004" target="_blank">Figure 4 C</a>. Note that dl78-91 replicates to higher levels in the 293 cells which contain the <i>X</i> gene (complementation) compared to unaltered 293 cells without <i>X</i> (<i>p</i><0.05).</p

Identification of X protein.

<p>Shown is a Western blot of protein from 293 cells infected with Ad5 and transfected with pSM620 (wt AAV2) plus either AAV/Neo or AAV/X/Neo. The Western blot was probed with polyclonal rabbit antibodies directed against a peptide derived from aa 98–111of AAV2 <i>X</i>. While polyclonal antibodies are well known for having cross-reactivity, note that a protein of approximately 18 kDa, the predicted size of X, is seen strongly enhanced in cells transfected with AAV/X/Neo, consistent with X.</p