Development of an animal model of progressive vaccinia in nu/nu mice and the use of bioluminescence imaging for assessment of the efficacy of monoclonal antibodies against vaccinial B5 and L1 proteins.

Bioluminescence imaging (BLI) was used to follow dissemination of recombinant vaccinia virus (VACV) expressing luciferase (IHD-J-Luc) in BALB/c nu/nu mice treated post-challenge with monoclonal antibodies (MAbs) against L1 and B5 VACV proteins in a model of Progressive Vaccinia (PV). Areas Under the flux Curve (AUC) were calculated for viral loads in multiple organs in individual mice. Following scarification with 105 pfu, IHD-J-Luc VACV undergoes fast replication at the injection site and disseminates rapidly to the inguinal lymph nodes followed by spleen, liver, and axillary lymph nodes within 2-3 days and before primary lesions are visible at the site of scarification. Extension of survival in nude mice treated with a combination of anti-B5 and anti-L1 MAbs 24 h post challenge correlated with a significant reduction in viral load at the site of scarification and delayed systemic dissemination. Nude mice reconstituted with 104 T cells prior to challenge with IHD-J-Luc, and treated with MAbs post-challenge, survived infection, cleared the virus from all organs and scarification site, and developed anti-VACV IgG and VACV-specific polyfunctional CD8+ T cells that co-expressed the degranulation marker CD107a, and IFNγ and TNFα cytokines. All T cell reconstituted mice survived intranasal re-challenge with IHD-J-Luc (104 pfu) two months after the primary infection. Thus, using BLI to monitor VACV replication in a PV model, we showed that anti-VACV MAbs administered post challenge extended survival of nude mice and protected T cell reconstituted nude mice from lethality by reducing replication at the site of scarification and systemic dissemination of VACV.

HIV latency. Proliferation of cells with HIV integrated into cancer genes contributes to persistent infection.

Antiretroviral treatment (ART) of HIV infection suppresses viral replication. Yet if ART is stopped, virus reemerges because of the persistence of infected cells. We evaluated the contribution of infected-cell proliferation and sites of proviral integration to HIV persistence. A total of 534 HIV integration sites (IS) and 63 adjacent HIV env sequences were derived from three study participants over 11.3 to 12.7 years of ART. Each participant had identical viral sequences integrated at the same position in multiple cells, demonstrating infected-cell proliferation. Integrations were overrepresented in genes associated with cancer and favored in 12 genes across multiple participants. Over time on ART, a greater proportion of persisting proviruses were in proliferating cells. HIV integration into specific genes may promote proliferation of HIV-infected cells, slowing viral decay during ART.

Detection of Mendelian consistent genotyping errors in pedigrees.

Detection of genotyping errors is a necessary step to minimize false results in genetic analysis. This is especially important when the rate of genotyping errors is high, as has been reported for high-throughput sequence data. To detect genotyping errors in pedigrees, Mendelian inconsistent (MI) error checks exist, as do multi-point methods that flag Mendelian consistent (MC) errors for sparse multi-allelic markers. However, few methods exist for detecting MC genotyping errors, particularly for dense variants on large pedigrees. Here, we introduce an efficient method to detect MC errors even for very dense variants (e.g., SNPs and sequencing data) on pedigrees that may be large. Our method first samples inheritance vectors (IVs) using a moderately sparse but informative set of markers using a Markov chain Monte Carlo-based sampler. Using sampled IVs, we considered two test statistics to detect MC genotyping errors: the percentage of IVs inconsistent with observed genotypes (A1) or the posterior probability of error configurations (A2). Using simulations, we show that this method, even with the simpler A1 statistic, is effective for detecting MC genotyping errors in dense variants, with sensitivity almost as high as the theoretical best sensitivity possible. We also evaluate the effectiveness of this method as a function of parameters, when including the observed pattern for genotype, density of framework markers, error rate, allele frequencies, and number of sampled inheritance vectors. Our approach provides a line of defense against false findings based on the use of dense variants in pedigrees.

A statistical framework to guide sequencing choices in pedigrees.

The use of large pedigrees is an effective design for identifying rare functional variants affecting heritable traits. Cost-effective studies using sequence data can be achieved via pedigree-based genotype imputation in which some subjects are sequenced and missing genotypes are inferred on the remaining subjects. Because of high cost, it is important to carefully prioritize subjects for sequencing. Here, we introduce a statistical framework that enables systematic comparison among subject-selection choices for sequencing. We introduce a metric "local coverage," which allows the use of inferred inheritance vectors to measure genotype-imputation ability specifically in a region of interest, such as one with prior evidence of linkage. In the absence of linkage information, we can instead use a "genome-wide coverage" metric computed with the pedigree structure. These metrics enable the development of a method that identifies efficient selection choices for sequencing. As implemented in GIGI-Pick, this method also flexibly allows initial manual selection of subjects and optimizes selections within the constraint that only some subjects might be available for sequencing. In the present study, we used simulations to compare GIGI-Pick with PRIMUS, ExomePicks, and common ad hoc methods of selecting subjects. In genotype imputation of both common and rare alleles, GIGI-Pick substantially outperformed all other methods considered and had the added advantage of incorporating prior linkage information. We also used a real pedigree to demonstrate the utility of our approach in identifying causal mutations. Our work enables prioritization of subjects for sequencing to facilitate dissection of the genetic basis of heritable traits.

GIGI: an approach to effective imputation of dense genotypes on large pedigrees.

Recent emergence of the common-disease-rare-variant hypothesis has renewed interest in the use of large pedigrees for identifying rare causal variants. Genotyping with modern sequencing platforms is increasingly common in the search for such variants but remains expensive and often is limited to only a few subjects per pedigree. In population-based samples, genotype imputation is widely used so that additional genotyping is not needed. We now introduce an analogous approach that enables computationally efficient imputation in large pedigrees. Our approach samples inheritance vectors (IVs) from a Markov Chain Monte Carlo sampler by conditioning on genotypes from a sparse set of framework markers. Missing genotypes are probabilistically inferred from these IVs along with observed dense genotypes that are available on a subset of subjects. We implemented our approach in the Genotype Imputation Given Inheritance (GIGI) program and evaluated the approach on both simulated and real large pedigrees. With a real pedigree, we also compared imputed results obtained from this approach with those from the population-based imputation program BEAGLE. We demonstrated that our pedigree-based approach imputes many alleles with high accuracy. It is much more accurate for calling rare alleles than is population-based imputation and does not require an outside reference sample. We also evaluated the effect of varying other parameters, including the marker type and density of the framework panel, threshold for calling genotypes, and population allele frequencies. By leveraging information from existing genotypes already assayed on large pedigrees, our approach can facilitate cost-effective use of sequence data in the pursuit of rare causal variants.

Genome scan in familial late-onset Alzheimer's disease: a locus on chromosome 6 contributes to age-at-onset.

Alzheimer's disease (AD) is a common, genetically complex, fatal neurodegenerative disorder of late life. Although several genes are known to play a role in early-onset AD, identification of the genetic basis of late onset AD (LOAD) has been challenging, with only the APOE gene known to have a high contribution to both AD risk and age-at-onset. Here, we present the first genome-scan analysis of the complete, well-characterized University of Washington LOAD sample of 119 pedigrees, using age-at-onset as the trait of interest. The analysis approach used allows for a multilocus trait model while at the same time accommodating age censoring, effects of APOE as a known genetic covariate, and full pedigree and marker information. The results provide strong evidence for linkage of loci contributing to age-at-onset to genomic regions on chromosome 6q16.3, and to 19q13.42 in the region of the APOE locus. There was evidence for interaction between APOE and the locus on chromosome 6q and suggestive evidence for linkage to chromosomes 11p13, 15q12-14, and 19p13.12. These results provide the first independent confirmation of an AD age-at-onset locus on chromosome 6 and suggest that further efforts towards identifying the underlying causal locus or loci are warranted.

G-protein-coupled receptor independent, immunomodulatory properties of chemokine CXCL9.

Certain chemokines possess anti-angiogenic and antibacterial activity, in addition to their ability to recruit leukocytes. Herein, we demonstrate that CXCL9/MIG induces the expression, by a monocytic cell line and peripheral blood mononuclear cells, of a variety of chemokines including CXCL8/IL-8, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL2/MCP-1 in a pertussis toxin insensitive manner. Similarly, another cationic chemokine CCL20/MIP-3alpha, but not the non-cationic chemokines CCL2 or CCL3, stimulated monocytic cells to produce substantial amounts of CXCL8 and CCL3. Microarray experiments demonstrated that CXCL9, but not CCL2, induced the expression of hundreds of genes, many of which have known or proposed immunomodulatory functions. Induction of CXCL8 required the p38 and ERK1/2 mitogen-activated protein kinases but not NFkappaB, JAK-STAT or JNK signaling pathways. These results collectively demonstrate that CXCL9 has immunomodulatory functions that are not mediated through a G-protein coupled receptor and may possess additional roles in host defenses against infection.

Multifunctional pancreatic islet encapsulation barriers achieved via multilayer PEG hydrogels.

The diverse requirements for a successful islet encapsulation barrier suggest the benefit of a barrier system that presents differing functionalities to encapsulated cells and host cells. Initially, multifunctional hydrogels were synthesized via the sequential photopolymerization of PEG hydrogel layers, each with different isolated functionalities. The ability to achieve localized biological functionalities was confirmed by immunostaining of different entrapped antibodies within each hydrogel layer. Survival of murine islets macroencapsulated within the interior gel of two-layer hydrogel constructs was then assessed. Maintenance of encapsulated islet survival and function was observed within multilayer hydrogels over 28 days in culture. Additionally, the functionalization of the islet-containing interior PEG gel layer with cell-matrix moieties, with either 100 microg/ml laminin or 5 mM of the adhesive peptide IKVAV found in laminin, resulted in increased insulin secretion from encapsulated islets similar to that in gels without an exterior hydrogel layer. Finally, through cell seeding experiments, the ability of an unmodified, exterior PEG layer to prevent interactions, and thus attachment, between nonencapsulated fibroblasts and entrapped ECM components within the interior PEG layer was demonstrated. Together the presented results support the potential of multilayer hydrogels for use as multifunctional islet encapsulation barriers that provide a localized biologically active islet microenvironment, while presenting an inert, immunoprotective exterior surface to the host environment, to minimize graft-host interactions.

Cellular delivery of TGFbeta1 promotes osteoinductive signalling for bone regeneration.

Administration of osteoinductive growth factors to wound sites, alone or in conjunction with a delivery vehicle, is an appealing treatment option for critical bone defects. The delivery of cells transfected with genes encoding for osteoinductive growth factors, such as TGFbeta(1), represents an attractive option to locally deliver constant levels of these growth factors to stimulate new bone formation at the defect site. Using non-viral transfection methods, we showed that osteoblasts can be genetically modified in vitro to secrete sustained therapeutic levels of TGFbeta(1) in its active form through control of the transfected cell environment. In addition, delivery of TGFbeta(1) produced by genetically modified cells that contained the proper post-translational modifications provided a more robust cellular response compared to administration of bacterially-derived recombinant TGFbeta(1). Migration and subsequent proliferation of osteoblasts are critical aspects of the initial steps in the cascade of new bone tissue formation. Exposure to mammalian-derived TGFbeta(1) induced a more pronounced chemotactic response upon administration of 10 pg/ml TGFbeta(1), whereas osteoblasts showed enhanced levels of metabolic activity at 100 pg/ml, which is indicative of greater levels of cellular proliferation when compared to addition of the same levels of recombinant TGFbeta(1). This increased efficacy of cell-derived TGFbeta(1) over recombinant forms of TGFbeta(1), combined with provision of a continual source of TGFbeta(1), highlights the advantages of delivering genetically modified cells over exogenous protein delivery for bone tissue engineering.

Multifunctional Pancreatic Islet Encapsulation Barriers Achieved via Multilayer PEG Hydrogels.

The diverse requirements for a successful islet encapsulation barrier suggest the benefit of a barrier system that presents differing functionalities to encapsulated cells and host cells. Initially, multifunctional hydrogels were synthesized via the sequential photopolymerization of PEG hydrogel layers, each with different isolated functionalities. The ability to achieve localized biological functionalities was confirmed by immunostaining of different entrapped antibodies within each hydrogel layer. Survival of murine islets macroencapsulated within the interior gel of two-layer hydrogel constructs was then assessed. Maintenance of encapsulated islet survival and function was observed within multilayer hydrogels over 28 days in culture. Additionally, the functionalization of the islet-containing interior PEG gel layer with cell-matrix moieties, with either 100 µg/ml laminin or 5 mM of the adhesive peptide IKVAV found in laminin, resulted in increased insulin secretion from encapsulated islets similar to that in gels without an exterior hydrogel layer. Finally, through cell seeding experiments, the ability of an unmodified, exterior PEG layer to prevent interactions, and thus attachment, between nonencapsulated fibroblasts and entrapped ECM components within the interior PEG layer was demonstrated. Together the presented results support the potential of multilayer hydrogels for use as multifunctional islet encapsulation barriers that provide a localized biologically active islet microenvironment, while presenting an inert, immunoprotective exterior surface to the host environment, to minimize graft-host interactions.

Synthesis of immunoisolation barriers that provide localized immunosuppression for encapsulated pancreatic islets.

Pancreatic islet encapsulation into synthetic, passive material matrixes can provide protection for transplanted islets from destruction via cell-contacted mediated interactions with autoreactive immune cells for treatment of Type I diabetes mellitus. However, one of the fundamental deficiencies with current encapsulation technology is that passive material barriers cannot protect islets from exposure to cytokines and other small, diffusible cytotoxic molecules produced by activated immune cells, subsequently leading to beta-cell destruction. Preparation of material matrixes that can actively provide localized immunosuppression of autoreactive immune cells may prolong the viability, and hence function, of encapsulated islet grafts. We have demonstrated the ability to conjugate apoptosis-inducing anti-Fas monoclonal antibodies (MAbs) to the surfaces of poly(ethylene glycol)-modified hydrogels, providing a surface that actively attempts to locally down-regulate the autoimmune response by destroying autoreactive T cells against pancreatic islet cells. We have conjugated anti-Fas MAbs to a high degree to the surface of these hydrogels, with retention of anti-Fas recognition of the Fas antigen as shown by ELISA testing. Apoptosis induction of Fas-sensitive Jurkat T cells was enhanced in the presence of anti-Fas conjugated hydrogels. In addition, this apoptosis induction was specific to anti-Fas MAbs, with no apoptosis induction with control antibodies or with Fas-insensitive T cells. These experiments promote the concept that surface-conjugated hydrogel constructs can provide localized immunosuppression for encapsulated grafted tissue.

Detection of antigens in biologically complex fluids with photografted whole antibodies.

A highly sensitive (pM), efficient (t < 20 min) detection assay was developed by designing surfaces with grafted antibodies. Through this approach, a short half-life antigen, glucagon, was rapidly detected in a biologically complex plasma/blood environment. Tailoring of graft composition eliminates the need for time-consuming blocking steps, significantly reducing antigen-antibody incubation times, while maintaining antibody specificity and activity toward target antigen. Grafted antibodies were bound through solvated, mobile polymer chains, thereby circumventing problems associated with antibody accessibility, analyte diffusion, and steric limitations. The efficiency of this assay is provided through grafting synthesized, acrylated antibodies in the presence of PEG monoacrylate. This procedure eliminates the need for blocking steps, due to a decrease in nonspecific protein interactions. These polymerizable antibodies were tethered with a range of densities while retaining biological activity. Moreover, biological activity of acrylated antibodies was compared to that of unmodified antibodies and remained comparable. The acrylated antibodies were grafted from substrate surfaces using controlled radical photopolymerization, maintaining the advantages of classical antibody immobilization techniques while providing improved detection. Through integrating this antibody conjugation chemistry and immunoassay approach with photolithographic techniques, construction of spatial patterns on a microfluidic device was demonstrated for efficient, parallel screening of multiple antigens.

Poly(propylacrylic acid)-mediated serum stabilization of cationic lipoplexes.

The serum instability associated with cationic lipoplexes represents one of the major obstacles for the in vivo delivery of nonviral gene therapy vectors. Recently, we have shown that poly(propylacrylic acid) (PPAA), a pH-sensitive polyanionic polymer, can significantly improve the in vitro serum stability of DOTAP lipoplexes and enhance transfection (Cheung et al., Bioconjug. Chem. 12, 906 (2001)). We investigated this serum-stabilizing effect provided by PPAA using methods to identify the specific serum proteins that interact with DOTAP/DNA and DOTAP/DNA/PPAA lipoplexes and determined their modes of interaction with these lipoplexes. Studies showed that only low-density lipoprotein (LDL) caused significant decondensation of DNA from lipoplexes lacking PPAA, but that fully condensed DNA was retained within lipoplexes incorporating PPAA. Another major factor in the loss of transfection activity was due to the reduced cellular uptake of DOTAP lipoplexes upon exposure to serum, with bovine serum albumin (BSA) and high-density lipoprotein (HDL) acting as major contributors to this reduction in vector internalization. In contrast, lipoplexes containing PPAA maintained high levels of uptake into cells in the presence of these proteins. Transfection results generally concurred with the mechanistic studies, suggesting that maintaining effective cellular delivery of intact lipoplexes in the presence of serum proteins is important to retain high transfection efficiencies. These results indicate that the addition of PPAA as a ternary component in DOTAP lipoplexes can overcome some of the serum-related deficiencies encountered with these lipoplexes to provide efficient transfection.

Formulation of chitosan-DNA nanoparticles with poly(propyl acrylic acid) enhances gene expression.

Poly(propyl acrylic acid) (PPAA) is a polymer specifically designed to disrupt lipid bilayer membranes within a sharply defined pH range. The pH sensitivity can be used to enhance the release of endocytosed drugs into the cytoplasmic compartment of the cell. By incorporating this polymer in a polymeric gene carrier, chitosan, the release of plasmid DNA from the endosomal compartment was enhanced. In vitro transfection studies confirmed that the incorporation of PPAA into the chitosan-DNA nanoparticles enhanced gene expression in both HEK293 and HeLa cells compared to chitosan nanoparticles alone. The dose and time at which PPAA was incorporated during the complex formation affected the release of DNA and transfection efficiency. The optimal dose of PPAA incorporated into the chitosan nanoparticles was determined to be 10 microg, corresponding to a PPAA/DNA weight ratio of 1:1. At this dose, the ternary complexes are approx. 400 nm in size with a net negative surface charge of -17.4 mV. Intracellular trafficking studies confirmed the association of PPAA, DNA and chitosan at 24 h post-transfection and the subsequent release of DNA and PPAA from the chitosan at 48 h. The diffuse appearance of the majority of the DNA and the PPAA at later time points suggests that the PPAA triggered membrane disruption resulting in the release of DNA from the endosomal compartment. Finally, the lack of colocalization between PPAA and Lysotracker indicated that the PPAA-loaded nanoparticles were not trafficked through a lysosomal pathway. This study suggests the promising strategy of including PPAA in the formulation of polymer-DNA complexes for non-viral gene delivery.

Poly(2-alkylacrylic acid) polymers deliver molecules to the cytosol by pH-sensitive disruption of endosomal vesicles.

The permeability barrier posed by cell membranes represents a challenge for the delivery of hydrophilic molecules into cells. We previously proposed that poly(2-alkylacrylic acid)s are endocytosed by cells into acidified vesicles and are there triggered by low pH to disrupt membranes and release the contents of endosomes/lysosomes to the cytosol. If this hypothesis is correct, these polymers could be valuable in drug-delivery applications. The present paper reports functional comparisons of a family of three poly(2-alkylacrylic acid)s. Poly(2-propylacrylic acid) (PPAA), poly(2-ethylacrylic acid) (PEAA) and poly(2-methylacrylic acid) (PMAA) were compared in red-blood-cell haemolysis assays and in a lipoplex (liposome-DNA complex) assay. We also directly examined the ability of these polymers to disrupt endosomes and lysosomes in cultured human cells. Our results show that: (i) unlike membrane-disruptive peptides, the endosomal-disruptive ability of poly(2-alkylacrylic acid)s cannot necessarily be predicted from their haemolytic activity at low pH, (ii) PPAA (but not PEAA or PMAA) potently facilitates gene transfection by cationic lipoplexes and (iii) endocytosed poly(2-alkylacrylic acid)s are triggered by luminal acidification to selectively disrupt endosomes (not lysosomes) and release their contents to the cytosol. These results will facilitate the rational design of future endosomal-disrupting polymers for drug delivery.

pH-sensitive polymers that enhance intracellular drug delivery in vivo.

Cytosolic delivery from endosomes is critical for those drugs that are susceptible to attack by lysosomal enzymes, such as DNA, RNA, oligonucleotides, proteins and peptides. Therefore, we have designed pH-sensitive, membrane-disruptive polymers to enhance the release of drugs from the acidic endosomal compartment to the cytoplasm. We have found that one polymer in particular, poly(propylacrylic acid) (PPAA), is very effective at membrane disruption at pHs below 6.5, based on hemolysis studies. PPAA also significantly enhances in vitro transfections of lipoplex formulations in cell culture, and does so in the presence of as much as 50% serum. In this study, we have extended our in vitro hemolysis and cell culture studies to an in vivo murine excisional wound healing model. A pilot study with a green fluorescent protein (GFP)-encoding plasmid indicated that injection of formulations containing PPAA into healing wounds resulted in increased GFP expression. Subsequently, by administering sense and antisense DNA for the angiogenesis inhibitor thrombospondin-2 (TSP2), we were able to alter the wound healing response in TSP2-null and wild type mice, respectively. Our findings showed that when PPAA was added to lipoplex formulations, expression of TSP2 was enhanced in TSP2-null mice compared to control formulations. These results show that PPAA can enhance in vivo transfections and that inhibition of TSP2 expression may lead to improved wound healing. These results suggest that PPAA can provide significant improvements in the in vivo efficacy of drugs such as DNA.