Long-term correction of hemophilia A mice following lentiviral mediated delivery of an optimized canine factor VIII gene.

Current therapies for hemophilia A include frequent prophylactic or on-demand intravenous factor treatments which are costly, inconvenient and may lead to inhibitor formation. Viral vector delivery of factor VIII (FVIII) cDNA has the potential to alleviate the debilitating clotting defects. Lentiviral-based vectors delivered to murine models of hemophilia A mediate phenotypic correction. However, a limitation of lentiviral-mediated FVIII delivery is inefficient transduction of target cells. Here, we engineer a feline immunodeficiency virus (FIV) -based lentiviral vector pseudotyped with the baculovirus GP64 envelope glycoprotein to mediate efficient gene transfer to mouse hepatocytes. In anticipation of future studies in FVIII-deficient dogs, we investigated the efficacy of FIV-delivered canine FVIII (cFVIII). Codon-optimization of the cFVIII sequence increased activity and decreased blood loss as compared to the native sequence. Further, we compared a standard B-domain deleted FVIII cDNA to a cDNA including 256 amino acids of the B-domain with 11 potential asparagine-linked oligosaccharide linkages. Restoring a partial B-domain resulted in modest reduction of endoplasmic reticulum (ER) stress markers. Importantly, our optimized vectors achieved wild-type levels of phenotypic correction with minimal inhibitor formation. These studies provide insights into optimal design of a therapeutically relevant gene therapy vector for a devastating bleeding disorder.

Immunochemical analysis of the idiotypes of mouse myeloma proteins with specificity for levan or dextran.

This paper deals solely with idiotypic determinants, the configurations of which are modified when the antibody bearing them interacts with its ligand. This phenomenon is measured as an inhibition of the reaction between anti-idiotype and idiotype. Two points are made: (a) The assay for ligand-modifiable determinants can be used to determine the "size" of the combining site. This is illustrated here with the anti-alpha(1 --> 6) dextran mouse myeloma immunoglobulin W3129. Whether the interaction between a homologous series of alpha(1 --> 6) oligosaccharide ligands and the combining site of W3129 is measured by inhibition of precipitation with alpha(1 --> 6) dextran (4) or of binding of W3129 to anti-W3129 idiotype, the finding is the same. The order of inhibition is isomaltohexaose = isomaltopentaose >> isomaltotetraose > isomaltotriose >>> isomaltose. The combining site is optimally complementary to isomaltopentaose. (b) Cross-idiotypic specificity is closely correlated with cross-combining specificity; the converse is not true. This is illustrated here with three groups of mouse myeloma immunoglobulin, each specific for alpha(1 --> 3) dextran, alpha(1 -->6) dextran, beta(2 --> 1) or beta(2 --> 6) levan. If a given anti-idiotypic serum cross-reacted with several myeloma proteins, they always had similar combining specificity. Thus the three proteins, J558, MOPC 104E, and UPC 102, which cross-react with anti-J558 have combining specificity for alpha(1 --> 3) dextran; cross-reacting W3082, UPC 61, and Y5476 have specificity for levan; and cross-reacting W3129 and W3434 have specificity for alpha(1 --> 6) dextran. This extends previous studies with proteins specific for phosphorylcholine (7) or gamma-globulin (8). As expected, the converse is not true, for proteins may have combining specificity for alpha(1 --> 6) dextran e.g. QUPC 52, or levan e.g. J606, UPC 10 and yet not carry the above-mentioned reference idiotypes. The correlation between cross-idiotypic and combining specificity breaks down when idiotypic determinants which are not modifiable by ligand are studied. The implications of this are pointed out since most investigations deal with ligand-nonmodifiable determinants.

Oncogenic transformation of murine lymphoid cells by in vitro infection with Abelson leukemia virus.

Spleen cell cultures stimulated to DNA synthesis by antigen or mitogen were infected with Abelson virus, a C-type RNA virus inducing nonthymic lymphomas in mice. After 3 days the cells were transferred to mice and caused 100 percent incidence of lymphomas in as few as 29 days. That a number of the tumors were of donor origin, as shown by female karyotypes in recipient male mice, indicated that cells infected by virus in vitro were transformed. The process depended upon both virus and stimulation of lymphocytes in culture. Lymphoid tumors did not develop in mice receiving cells from virus-infected cultures not exposed to antigen or mitogen.

Expression, purification, and characterization of the Kunitz-type proteinase inhibitor domain of the amyloid beta-protein precursor-like protein-2.

In this report we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the Kunitz-type proteinase inhibitor (KPI) domain of the amyloid beta-protein precursor-like protein-2 (APLP-2). The expression plasmid for the KPI domain of APLP-2 encoded amino acids 305-364 of the APLP-2 cDNA (Slunt et al. (1994) J. Biol. Chem. 269, 2637-2644). The secreted 60 amino-acid product was purified to homogeneity and biochemically characterized. Amino-acid sequencing of the expressed KPI domain of APLP-2 verified its integrity. The proteinase inhibitory properties of the KPI domain of APLP-2 were compared to those of the KPI domain of proteinase nexin-2/amyloid beta-protein precursor (PN-2/A beta PP). Both KPI domains potently inhibited trypsin and, to a lesser extent, chymotrypsin, plasmin, and coagulation factors XIa and IXa. However, the KPI domain of APLP-2 was a approximately 20-fold less effective inhibitor of coagulation factor XIa compared to the KPI domain of PN-2/A beta PP. Similarly, the KPI domain of APLP-2 was a less effective anticoagulant in coagulation based assays than the KPI domain of PN-2/A beta PP. These studies indicate that the KPI domains of PN-2/A beta PP and APLP-2 form a family of proteinase inhibitors although the former is a better inhibitor of factor XIa and a more potent anticoagulant than the latter.

Murine T-lymphoma 'immunoglobulin' is identical to leukemia virus gp 70.

We have used chicken anti-mouse immunoglobulin antiserum to precipitate molecules from mouse T-lymphoma cells that had been radioiodinated. We analysed the immunoprecipitates by two-dimensional gel electrophoresis and compared the results with immunoprecipitates generated by other antisera. We found that the molecule precipitated by chicken anti-mouse immunoglobulin from T-lymphoma cells was identical to the mouse leukemia virus envelope glycoprotein (gp 70) produced by the T-lymphoma. Mouse IgM myeloma proteins block precipitation of T-lymphoma molecules by chicken anti-mouse immunoglobulin. We conclude that mouse IgM and gp 70 share antigenic determinants. This may lead to erroneous conclusions about the presence of immunoglobulin on T-cells.

Stable expression of the mouse lymphocyte T200 antigen in L-cells after transfection with lymphoma DNA.

L-cells, which normally do not express the mouse lymphocyte T200 antigen, were transfected with DNA from the mouse T-cell lymphoma, BW5147, and a T200+ L-cell line isolated. The detection and enrichment of T200+ L-cells from a pool of transfectants was accomplished using monoclonal anti-T200 antibody and fluorescence-activated cell sorting. A subclone has been selected that is stable for expression of T200. The T200 molecules expressed by BW5147 and the T200+ L-cell are similar in size, around 190 Kd, compared to the 220-Kd B-cell form of T200. The BW5147 T200 molecule is 3000 daltons larger than the molecule expressed by the transfected L-cell, a size difference due to glycosylation moieties, since treatment of the molecules with Endoglycosidase F or treatment of cells with tunicamycin yields T200 molecules of the same size from the 2 cell sources. In comparison, the B-cell form of T200 retains a size difference of 25,000 daltons from the T-cell form after these treatments. Monoclonal antibodies specific for the B-cell form of T200 do not recognize the T200+ L-cell, providing further evidence that the T-cell form of T200 is expressed by the transfected L-cell.

Expression of CD45 isoforms lacking exons 7, 8 and 10.

The CD45 exon usage pattern of various CD8+ and CD4+ T cell lines was studied. By using the reverse transcription-polymerase chain reaction (RT PCR) and Southern analysis with exon specific or exon junction probes, we showed that all of the cytotoxic T cell lines and the majority of the helper T cells expressed the 789 isoform as a major splice variant. Expression of the splice product lacking exons 4-7 (isoform 89) was not as ubiquitous. All Th lines produced mRNA encoding this isoform, but in only three of the Tc lines was the 89 isoform detectable by RT/PCR. RNase protection assays with RNA isolated from normal CD8+ splenic cells demonstrated the 89 splice product was present in low abundance. The relative abundance of the different isoforms in the thymic lymphoma, BW5147, was determined through RNase protection analysis. The 789 isoform predominates, representing approximately 75% of the CD45 mRNA whereas the 89 form constitutes about 24%. In addition, an isoform lacking exons 4-8 (isoform 9) also was detected and comprises approximately 1% of the total CD45 mRNA in this cell line. Finally, these studies demonstrate that exon 10 is also used as an alternatively spliced exon.

Murine mast cells and monocytes express distinctive sets of CD45 isoforms.

CD45 isoform expression patterns were examined in various mast and monocyte cell populations. The reverse transcription-polymerase chain reaction (RT/PCR) and Southern analysis showed these myeloid cells express characteristic sets of CD45 isoforms. Mast cells produce mRNA for two splice variants, one containing exons 5, 7 and 8 of the alternatively expressed exons (therefore lacking exons 4 and 6) and another containing variable exons 7 and 8. Monocytes express three prominent CD45 mRNA species, one which includes exons 5, 7 and 8, another with exons 7 and 8 and the third containing only exon 8 of the variable exons. These results show that there are clear differences within the myeloid lineage sub-populations with respect to CD45 exon usage which appear to delineate mast cell and monocyte specific patterns.

Inducible expression of a heterologous protein in Hansenula polymorpha using the alcohol oxidase 1 promoter of Pichia pastoris.

Pichia pastoris (Pp) and Hansenula polymorpha (Hp) are methylotrophic yeasts commonly used for industrial purposes. Growth of either of these yeasts in the presence of methanol as the carbon source results in high-level induction of alcohol oxidase expression. The respective alcohol oxidase genes, AOX1 in Pp and MOX in Hp, have similar regulatory characteristics. Our studies show that the Pp AOX1 promoter (AOX1p) can be used for methanol-induced expression of a heterologous gene in Hp. Furthermore, the size of an AOX1p-heterologous gene-AOX1 terminator cassette transcript synthesized in Hp is indistinguishable from that synthesized in Pp suggesting that transcription both initiates and terminates at the same sites in both yeast species. Induction of AOX1p in Hp demonstrates that the methanol-inducible regulatory mechanism in Hp is able to recognize and activate the Pp promoter in spite of extensive sequence variations between AOX1p and MOXp.

Recent advances in the expression of foreign genes in Pichia pastoris.

The Pichia pastoris heterologous gene expression system has been utilized to produce attractive levels of a variety of intracellular and extracellular proteins of interest. Recent advances in our understanding and application of the system have improved its utility even further. These advances include: (1) methods for the construction of P. pastoris strains with multiple copies of AOX1-promoter-driven expression cassettes; (2) mixed-feed culture strategies for high foreign protein volumetric productivity rates; (3) methods to reduce proteolysis of some products in high cell-density culture media; (4) tested procedures for purification of secreted products; and (5) detailed information on the structures of N-linked oligosaccharides on P. pastoris secreted proteins. In this review, these advances along with basic features of the P. pastoris system are described and discussed.

Cloned murine T200 (Ly-5) cDNA reveals multiple transcripts within B- and T-lymphocyte lineages.

The murine T200 family of cell-surface glycoproteins is expressed on hematopoietic lineage cell types in a developmentally regulated manner with different lymphocyte subpopulations expressing cross-reactive but structurally distinct forms. To investigate the differences between these forms and the regulation of these differences, murine T200 cDNA clones were isolated by using a probe obtained by subtractive hybridization. This procedure made use of a T200+ L-cell transfectant and the parent Ltk- cell line. The 1.9-kilobase (kb) cDNA clone was sequenced and found to contain the coding region of the COOH-terminal 331 amino acids and 0.9 kb of 3' untranslated region. Where the sequence overlapped with the rat sequence, 80-90% homology was observed. RNA blot analysis revealed that B-lineage cell lines express either a 5.6-kb or a 6.5-kb mRNA correlating to the size difference of the T200 glycoprotein synthesized. Similarly, in the T-cell lineage a helper T-cell clone and a cytotoxic T-cell clone express T200 transcripts of 5.6 kb and 5.9 kb, respectively, which correlate with the distinct sizes of their T200 glycoproteins. A T200-negative mutant of a T-cell line was found to express full-length T200 mRNA, although at a diminished level.

Transformation by Abelson murine leukemia virus: properties of the transformed cells.

Ab-MuLV transforms mouse 3T3 fibroblasts, macrophages, and lymphocytes, which have properties of immature cells in the B-lymphocyte differentiation pathway. Differences in expression of intracellular immunoglobulin and surface antigens the lymphoma lines suggest that more than one B-lymphocyte differentiation stage may be transformed, each of which must be before the B lymphocyte acquires cell-surface immunoglobulin receptors for antigen and antigen sensitivity. The plasmacytomas induced by Ab-MuLV plus pristane tested thus far show no evidence of infection by Ab-MuLV. The transformation of these immunoglobulin-secreting cells may be due to causes other than the transforming elements of the Ab-MuLV genome.

Differentiation antigens on normal and Abelson virus transformed lymphocytes.

Rabbit antisera to Abelson leukemia virus (A-MuLV)-induced murine lymphomas have been analyzed by absorption with a variety of murine lymphoma lines. Antibody binding to a panel of cell lines and normal lymphocytes was visualized by using hapten-sandwich indirect membrane immunofluorescence. Novel membrane antigens thereby detected are shared between lymphosarcomas, B lymphomas, normal B lymphocytes, and normal membrane immunoglobulin negative (sIg-) bone marrow cells, but are not found on T cells, thymic lymphomas, plasmacytoid lymphomas, or myelomas. The existence of such shared differentiation antigens suggests that sIg- A-MuLV-induced lymphosarcomas may be transformed B cell precursors. Since differences in the expression of these antigens on individual plasma-cytoid lymphoma lines were found, this category of lymphomas may include cells at a variety of differentiation states.