Tumor necrosis factor α plays a central role in immune-mediated clearance of adenoviral vectors

Adenovirus (Ad) gene transfer vectors are rapidly cleared from infected hepatocytes in mice. To determine which effector mechanisms are responsible for elimination of the Ad vectors, we infected mice that were genetically compromised in immune effector pathways [perforin, Fas, or tumor necrosis factor α (TNF-α)] with the Ad vector, Ad5-chloramphenicol acetyl transferase (CAT). Mice were sacrificed at 7–60 days postinfection, and the levels of CAT expression in the liver determined by a quantitative enzymatic assay. When the livers of infected mice were harvested 28 days postinfection, the levels of CAT expression revealed that the effectors most important for the elimination of the Ad vector were TNF-α > Fas > perforin. TNF-α did not have a curative effect on infected hepatocytes, as the administration of TNF-α to infected severe combined immunodeficient mice or to infected cultures in vitro had no specific effect on virus persistence. However, TNF-α-deficient mice demonstrated a striking reduction in the leukocytic infiltration early on in the infection, suggesting that TNF-α deficiency resulted in impaired recruitment of inflammatory cells to the site of inflammation. In addition, the TNF-deficient mice had a significantly reduced humoral immune response to virus infection. These results demonstrate a dominant role of TNF-α in elimination of Ad gene transfer vectors. This result is particularly important because viral proteins that disable TNF-α function have been removed from most Ad vectors, rendering them highly susceptible to TNF-α-mediated elimination.

Geometry of circular vectors and pattern recognition of shape of a boundary

This paper deals with pattern recognition of the shape of the boundary of closed figures on the basis of a circular sequence of measurements taken on the boundary at equal intervals of a suitably chosen argument with an arbitrary starting point. A distance measure between two boundaries is defined in such a way that it has zero value when the associated sequences of measurements coincide by shifting the starting point of one of the sequences. Such a distance measure, which is invariant to the starting point of the sequence of measurements, is used in identification or discrimination by the shape of the boundary of a closed figure. The mean shape of a given set of closed figures is defined, and tests of significance of differences in mean shape between populations are proposed.

Noncytopathic Sindbis virus RNA vectors for heterologous gene expression

Infection of vertebrate cells with alphaviruses normally leads to prodigious expression of virus-encoded genes and a dramatic inhibition of host protein synthesis. Recombinant Sindbis viruses and replicons have been useful as vectors for high level foreign gene expression, but the cytopathic effects of viral replication have limited their use to transient studies. We recently selected Sindbis replicons capable of persistent, noncytopathic growth in BHK cells and describe here a new generation of Sindbis vectors useful for long-term foreign gene expression based on such replicons. Foreign genes of interest as well as the dominant selectable marker puromycin N-acteyltransferase, which confers resistance to the drug puromycin, were expressed as subgenomic transcripts of noncytopathic replicons or defective-interfering genomes complemented in trans by a replicon. Based on these strategies, we developed vectors that can be initiated via either RNA or DNA transfection and analyzed them for their level and stability of foreign gene expression. Noncytopathic Sindbis vectors express reasonably high levels of protein in nearly every cell. These vectors should prove to be flexible tools for the rapid expression of heterologous genes under conditions in which cellular metabolism is not perturbed, and we illustrate their utility with a number of foreign proteins.

Sustained secretion of human alpha-1-antitrypsin from murine muscle transduced with adeno-associated virus vectors

Recombinant adeno-associated virus (AAV) vectors have been used to transduce murine skeletal muscle as a platform for secretion of therapeutic proteins. The utility of this approach for treating alpha-1-antitrypsin (AAT) deficiency was tested in murine myocytes in vitro and in vivo. AAV vectors expressing the human AAT gene from either the cytomegalovirus (CMV) promoter (AAV-C-AT) or the human elongation factor 1-α promoter (AAV-E-AT) were examined. In vitro in C2C12 murine myoblasts, the expression levels in transient transfections were similar between the two vectors. One month after transduction, however, the human elongation factor 1 promoter mediated 10-fold higher stable human AAT expression than the CMV promoter. In vivo transduction was performed by injecting doses of up to 1.4 × 1013 particles into skeletal muscles of several mouse strains (C57BL/6, BALB/c, and SCID). In vivo, the CMV vector mediated higher levels of expression, with sustained serum levels over 800 μg/ml in SCID and over 400 μg/ml in C57BL/6 mice. These serum concentrations are 100,000-fold higher than those previously observed with AAV vectors in muscle and are at levels which would be therapeutic if achieved in humans. High level expression was delayed for several weeks but was sustained for over 15 wk. Immune responses were dependent upon the mouse strain and the vector dosage. These data suggest that recombinant AAV vector transduction of skeletal muscle could provide a means for replacing AAT or other essential serum proteins but that immune responses may be elicited under certain conditions.

Long-term expression of γ-globin mRNA in mouse erythrocytes from retrovirus vectors containing the human γ-globin gene fused to the ankyrin-1 promoter

Gene therapy for patients with hemoglobin disorders has been hampered by the inability of retrovirus vectors to transfer globin genes and their cis-acting regulatory sequences into hematopoietic stem cells without rearrangement. In addition, the expression from intact globin gene vectors has been variable in red blood cells due to position effects and retrovirus silencing. We hypothesized that by substituting the globin gene promoter for the promoter of another gene expressed in red blood cells, we could generate stable retrovirus vectors that would express globin at sufficient levels to treat hemoglobinopathies. Recently, we have shown that the human ankyrin (Ank) gene promoter directs position-independent, copy number-dependent expression of a linked γ-globin gene in transgenic mice. We inserted the Ank/Aγ-globin gene into retrovirus vectors that could transfer one or two copies of the Ank/Aγ-globin gene to target cells. Both vectors were stable, transferring only intact proviral sequences into primary mouse hematopoietic stem cells. Expression of Ank/Aγ-globin mRNA in mature red blood cells was 3% (single copy) and 8% (double copy) of the level of mouse α-globin mRNA. We conclude that these novel retrovirus vectors may be valuable for treating a variety of red cell disorders by gene replacement therapy including severe β-thalassemia if the level of expression can be further increased.

The asymptotic distribution of canonical correlations and vectors in higher-order cointegrated models

The study of the large-sample distribution of the canonical correlations and variates in cointegrated models is extended from the first-order autoregression model to autoregression of any (finite) order. The cointegrated process considered here is nonstationary in some dimensions and stationary in some other directions, but the first difference (the “error-correction form”) is stationary. The asymptotic distribution of the canonical correlations between the first differences and the predictor variables as well as the corresponding canonical variables is obtained under the assumption that the process is Gaussian. The method of analysis is similar to that used for the first-order process.

The function of herpes simplex virus genes: a primer for genetic engineering of novel vectors.

Herpes simplex virus vectors are being developed for delivery and expression of human genes to the central nervous system, selective destruction of cancer cells, and as carriers for genes encoding antigens that induce protective immunity against infectious agents. Vectors constructed to meet these objectives must differ from wild-type virus with respect to host range, reactivation from latency, and expression of viral genes. The vectors currently being developed are (i) helper free amplicons, (ii) replication defective viruses, and (iii) genetically engineered replication competent viruses with restricted host range. Whereas the former two types of vectors require stable, continuous cell lines expressing viral genes for their replication, the replication competent viruses will replicate on approved primary human cell strains.

Epstein-Barr virus vectors for gene delivery to B lymphocytes.

Basic research in Epstein-Barr virus (EBV) molecular genetics has provided means to maintain episomes in human cells, to efficiently deliver episomes with up to 150 kbp of heterologous DNA to human B lymphocytes, and to immortalize human B lymphocytes with EBV recombinants that can maintain up to 120 kbp of heterologous DNA. Episome maintenance requires an EBV nuclear protein, EBNA1, whereas immortalization of cells with EBV recombinants requires EBNA1, EBNA2, EBNA3A, EBNA3C, EBNALP, and LMP1. EBV-derived vectors are useful for experimental genetic reconstitution in B lymphocytes, a cell type frequently used in establishing repositories of human genetic deficiencies. The ability of EBV-infected cells to establish a balanced state of persistence in normal humans raises the possibility that cells infected with EBV recombinants could be useful for genetic reconstitution, in vivo.

Applications of pox virus vectors to vaccination: an update.

Recombinant pox viruses have been generated for vaccination against heterologous pathogens. Amongst these, the following are notable examples. (i) The engineering of the Copenhagen strain of vaccinia virus to express the rabies virus glycoprotein. When applied in baits, this recombinant has been shown to vaccinate the red fox in Europe and raccoons in the United States, stemming the spread of rabies virus infection in the wild. (ii) A fowlpox-based recombinant expressing the Newcastle disease virus fusion and hemagglutinin glycoproteins has been shown to protect commercial broiler chickens for their lifetime when the vaccine was administered at 1 day of age, even in the presence of maternal immunity against either the Newcastle disease virus or the pox vector. (iii) Recombinants of canarypox virus, which is restricted for replication to avian species, have provided protection against rabies virus challenge in cats and dogs, against canine distemper virus, feline leukemia virus, and equine influenza virus disease. In humans, canarypox virus-based recombinants expressing antigens from rabies virus, Japanese encephalitis virus, and HIV have been shown to be safe and immunogenic. (iv) A highly attenuated vaccinia derivative, NYVAC, has been engineered to express antigens from both animal and human pathogens. Safety and immunogenicity of NYVAC-based recombinants expressing the rabies virus glycoprotein, a polyprotein from Japanese encephalitis virus, and seven antigens from Plasmodium falciparum have been demonstrated to be safe and immunogenic in early human vaccine studies.