Correction in trans for Fabry disease: expression, secretion and uptake of alpha-galactosidase A in patient-derived cells driven by a high-titer recombinant retroviral vector.

Fabry disease is an X-linked metabolic disorder due to a deficiency of alpha-galactosidase A (alpha-gal A; EC 3.2.1.22). Patients accumulate glycosphingolipids with terminal alpha-galactosyl residues that come from intracellular synthesis, circulating metabolites, or from the biodegradation Of senescent cells. Patients eventually succumb to renal, cardio-, or cerebrovascular disease. No specific therapy exists. One possible approach to ameliorating this disorder is to target corrective gene transfer therapy to circulating hematopoietic cells. Toward this end, an amphotropic virus-producer cell line has been developed that produces a high titer (>10(6) i.p. per ml) recombinant retrovirus constructed to transduce and correct target cells. Virus-producer cells also demonstrate expression of large amounts of both intracellular and secreted alpha-gal A. To examine the utility of this therapeutic vector, skin fibroblasts from Fabry patients were corrected for the metabolic defect by infection with this recombinant virus and secreted enzyme was observed. Furthermore, the secreted enzyme was found to be taken up by uncorrected cells in a mannose-6-phosphate receptor-dependent manner. In related experiments, immortalized B cell lines from Fabry patients, created as a hematologic delivery test system, were transduced. As with the fibroblasts, transduced patient B cell lines demonstrated both endogenous enzyme correction and a small amount of secretion together with uptake by uncorrected cells. These studies demonstrate that endogenous metabolic correction in transduced cells, combined with secretion, may provide a continuous source of corrective material in trans to unmodified patient bystander cells (metabolic cooperativity).

Induction of mucosal immune responses against a heterologous antigen fused to filamentous hemagglutinin after intranasal immunization with recombinant Bordetella pertussis.

Live vaccine vectors are usually very effective and generally elicit immune responses of higher magnitude and longer duration than nonliving vectors. Consequently, much attention has been turned to the engineering of oral pathogens for the delivery of foreign antigens to the gut-associated lymphoid tissues. However, no bacterial vector has yet been designed to specifically take advantage of the nasal route of mucosal vaccination. Herein we describe a genetic system for the expression of heterologous antigens fused to the filamentous hemagglutinin (FHA) in Bordetella pertussis. The Schistosoma mansoni glutathione S-transferase (Sm28GST) fused to FHA was detected at the cell surface and in the culture supernatants of recombinant B. pertussis. The mouse colonization capacity and autoagglutination of the recombinant microorganism were indistinguishable from those of the wild-type strain. In addition, and in contrast to the wild-type strain, a single intranasal administration of the recombinant strain induced both IgA and IgG antibodies against Sm28GST and against FHA in the bronchoalveolar lavage fluids. No anti-Sm28GST antibodies were detected in the serum, strongly suggesting that the observed immune response was of mucosal origin. This demonstrates, to our knowledge, for the first time that recombinant respiratory pathogens can induce mucosal immune responses against heterologous antigens, and this may constitute a first step toward the development of combined live vaccines administrable via the respiratory route.

High affinity type I interleukin 1 receptor antagonists discovered by screening recombinant peptide libraries.

Two families of peptides that specifically bind the extracellular domain of the human type I interleukin I (IL-1) receptor were identified from recombinant peptide display libraries. Peptides from one of these families blocked binding of IL-lalpha to the type I IL-1 receptor with IC50 values of 45-140 microM. Affinity-selective screening of variants of these peptides produced ligands of much higher affinity (IC50 approximately 2 nM). These peptides block IL-1-driven responses in human and monkey cells; they do not bind the human type II IL-1 receptor or the murine type I IL-1 receptor. This is the first example (that we know of) of a high affinity peptide that binds to a cytokine receptor and acts as a cytokine antagonist.

In vitro motility from recombinant dynein heavy chain.

The dyneins are a class of motor protein involved in ciliary and flagellar motility, organelle transport, and chromosome segregation. Because of their large size and subunit complexity, relatively little is known about their mechanisms of force production and regulation. We report here on the expression and analysis of the entire rat cytoplasmic dynein heavy chain (Mr 532,000). Full-length cDNAs were constructed from a series of partial clones and tagged at the C terminus with either a FLAG-epitope tag or a His6-tag. The recombinant polypeptides were expressed either in insect cells by baculovirus infection or in COS-7 cells by transient transfection. The recombinant protein was mostly soluble and showed good microtubule binding. It exhibited a broad sedimentation profile, indicative of the formation of dimers as well as higher order multimers. Good microtubule gliding motility activity was observed in assays of heavy chain expressed in either insect or COS-7 cells. Average microtubule gliding velocities of 1.2-1.8 microm/sec were observed, comparable with the rates determined for calf brain cytoplasmic dynein. These results represent the first indication that recombinant heavy chain alone is capable of force production, and should lead to rapid progress in defining the dynein motor domain.

The recombinant proregion of transforming growth factor beta1 (latency-associated peptide) inhibits active transforming growth factor beta1 in transgenic mice.

All three isoforms of transforming growth factors beta (TGF-betal, TGF-beta2, and TGF-beta3) are secreted as latent complexes and activated extracellularly, leading to the release of the mature cytokines from their noncovalently associated proregions, also known as latency-associated peptides (LAPs). The LAP region of TGF-beta1 was expressed in a baculovirus expression system and purified to homogeneity. In vitro assays of growth inhibition and gene induction mediated by TGF-beta3 demonstrate that recombinant TGF-beta1 LAP is a potent inhibitor of the activities of TGF-betal, -beta2, and -beta3. Effective dosages of LAP for 50% neutralization of TGF-beta activities range from 4.7- to 80-fold molar excess depending on the TGF-beta isoform and activity examined. Using 125I-labeled LAP, we show that the intraperitoneal application route is effective for systemic administration of LAP. Comparison of concentrations of LAP in tissues shows a homogenous pattern in most organs with the exception of heart and muscle, in which levels of LAP are 4- to 8-fold lower. In transgenic mice with elevated hepatic levels of bioactive TGF-betal, treatment with recombinant LAP completely reverses suppression of the early proliferative response induced by TGF-beta1 in remnant livers after partial hepatectomy. The results suggest that recombinant LAP is a potent inhibitor of bioactive TGF-beta both in vitro and in vivo, after intraperitoneal administration. Recombinant LAP should be a useful tool for novel approaches to study and therapeutically modulate pathophysiological processes mediated by TGF-beta3.

Intercellular transfer of a glycosylphosphatidylinositol (GPI)-linked protein: release and uptake of CD4-GPI from recombinant adeno-associated virus-transduced HeLa cells.

A diverse group of GPI-anchored protein structures are ubiquitously expressed on the external cell membranes of eukaryotes. Whereas the physiological role for these structures is usually defined by their protein component, the precise biological significance of the glycolipid anchors remains vague. In the course of producing a HeLa cell line (JM88) that contained a recombinant adeno-associated virus genome expressing a GPI-anchored CD4-GPI fusion protein on the surface of the cells, we noted the transfer of CD4-GPI to native HeLa cells. Transfer occurred after direct cell contact or exposure to JM88 cell supernatants. The magnitude of contact-mediated CD4-GPI transfer correlated with temperature. Supernatant CD4-GPI also attached to human red blood cells and could be cleaved with phosphatidylinositol-specific phospholipase C. The attached CD4-GPI remained biologically active after transfer and permitted the formation of syncytium when coated HeLa cells were incubated with glycoprotein 160 expressing H9 cells. JM88 cells provide a model for the production, release, and reattachment of CD4-GPI and may furnish insight into a physiologic role of naturally occurring GPI-anchored proteins. This approach may also allow the production of other recombinant GPI-anchored proteins for laboratory and clinical investigation.

Subcellular imaging of intramitochondrial Ca2+ with recombinant targeted aequorin: significance for the regulation of pyruvate dehydrogenase activity.

Specific targeting of the recombinant, Ca2+ -sensitive photoprotein, aequorin to intracellular organelles has provided new insights into the mechanisms of intracellular Ca2+ homeostasis. When applied to small mammalian cells, a major limitation of this technique has been the need to average the signal over a large number of cells. This prevents the identification of inter- or intracellular heterogeneities. Here we describe the imaging in single mammalian cells (CHO.T) of [Ca2+] with recombinant chimeric aequorin targeted to mitochondria. This was achieved by optimizing expression of the protein through intranuclear injection of cDNA and through the use of a charge-coupled device camera fitted with a dual microchannel plate intensifier. This approach allows accurate quantitation of the kinetics and extent of the large changes in mitochondrial matrix [Ca2+] ([Ca2+](m)) that follow receptor stimulation and reveal different behaviors of mitochondrial populations within individual cells. The technique is compared with measurements of [Ca2+](m) using the fluorescent indicator, rhod2. Comparison of [Ca2+](m) with the activity of the Ca2+ -sensitive matrix enzyme, pyruvate dehydrogenase (PDH), reveals that this enzyme is a target of the matrix [Ca2+] changes. Peak [Ca2+](m) values following receptor stimulation are in excess of those necessary for full activation of PDH in situ, but may be necessary for the activation of other mitochondrial dehydrogenases. Finally, the data suggest that the complex regulation of PDH activity by a phosphorylation-dephosphorylation cycle may provide a means by which changes in the frequency of cytosolic (and hence mitochondrial) [Ca2+] oscillations can be decoded by mitochondria.

p53 as a target for cancer vaccines: recombinant canarypox virus vectors expressing p53 protect mice against lethal tumor cell challenge.

The p53 protein is an attractive target for immunotherapy, because mutations in the p53 gene are the most common genetic alterations found in human tumors. These mutations result in high levels of p53 protein in the tumor cell, whereas the expression level of wild-type p53 in nonmalignant tissue is usually much lower. Several canarypox virus recombinants expressing human or murine p53 in wild-type or mutant form were constructed. Immunization with these viruses protected BALB/c mice from a challenge with an isogenic and highly tumorigenic mouse fibroblast tumor cell line expressing high levels of mutant p53. The tumor protection was equally effective regardless of whether wild-type or mutant p53 was used for the immunization, indicating that the immunologic response was not dependent on any particular p53 mutation and that immunization with this live virus vaccine works effectively against mutant p53 protein expressed in a tumor cell. In tumors escaping immunologic rejection, the expression of the p53 protein was commonly down-regulated.

Human immunodeficiency virus type 1 infection despite prior immunization with a recombinant envelope vaccine regimen.

With efforts underway to develop a preventive human immunodeficiency virus type 1 (HIV-1) vaccine, it remains unclear which immune responses are sufficient to protect against infection and whether prior HIV-1 immunity can alter the subsequent course of HIV-1 infection. We investigated these issues in the context of a volunteer who received six HIV-1LAI envelope immunizations and 10 weeks thereafter acquired HIV-1 infection through a high-risk sexual exposure. In contrast to nonvaccinated acutely infected individuals, anamnestic HIV-1-specific B- and T-cell responses appeared within 3 weeks in this individual, and neutralizing antibody preceded CD8+ cytotoxic responses. Despite an asymptomatic course and an initial low level of detectable infectious virus, a progressive CD4+ cell decline and dysfunction occurred within 2 years. Although vaccination elicited immunity to HIV-1 envelope, which was recalled upon HIV-1 exposure, it was insufficient to prevent infection and subsequent immunodeficiency.

Transposon tools for recombinant DNA manipulation: characterization of transcriptional regulators from yeast, Xenopus, and mouse.

Transposon Tn1000 has been adapted to deliver novel DNA sequences for manipulating recombinant DNA. The transposition procedure for these "tagged" Tn1000s is simple and applicable to most plasmids in current use. For yeast molecular biology, tagged Tn1000s introduce a variety of yeast selective markers and replication origins into plasmids and cosmids. In addition, the beta-globin minimal promoter and lacZ gene of Tn(beta)lac serve as a mobile reporter of eukaryotic enhancer activity. In this paper, Tn(beta)lac was used to localize a mouse HoxB-complex enhancer in transgenic mice. Other tagged transposons create Gal4 DNA-binding-domain fusions, in either Escherichia coli or yeast plasmids, for use in one- and two-hybrid tests of transcriptional activation and protein-protein interaction, respectively. With such fusions, the Saccharomyces cerevisiae Swi6 G1/S-phase transcription factor and the Xenopus laevis Pintallavis developmental regulator are shown to activate transcription. Furthermore, the same transposon insertions also facilitated mapping of the Swi6 and Pintallavis domains responsible for transcriptional activation. Thus, as well as introducing novel sequences, tagged transposons share the numerous other applications of transposition such as producing insertional mutations, creating deletion series, or serving as mobile primer sites for DNA sequencing.

Recombinant human uteroglobin suppresses cellular invasiveness via a novel class of high-affinity cell surface binding site.

The mechanism(s) that regulates invasion of trophoblasts through the uterine epithelium during embryo implantation and nidation in hemochorial placental mammals is poorly understood. While limited trophoblast invasion is essential for the establishment of normal pregnancy, dysregulation of this process may contribute to the pathogenesis of choriocarcinoma, a highly invasive and lethal form of cancer arising from the trophoblasts. We have previously demonstrated that rabbit uteroglobin (UG), a cytokine-like, antiinflammatory protein, produced by the endometrial epithelium during pregnancy, has a potent antichemotactic effect on neutrophils and monocytes in vitro. Here, we report that recombinant human UG (hUG) dramatically suppresses invasion of human trophoblasts and NIH 3T3 cells through an artificial basement membrane (Matrigel) in vitro but has no effect on that of human choriocarcinoma cells. We identified a previously unreported high-affinity, high molecular weight (approximately 190 kDa), nonglycosylated hUG-binding protein, readily detectable on human trophoblasts and NIH 3T3 cells but totally lacking on choriocarcinoma cells. Taken together, these results raise the possibility that (i) hUG plays a critical role in regulating cellular invasiveness, at least in part, via its previously unrecognized cell surface binding site, and (ii) some of the numerous biological activities of proteins of the UG family, reported so far, may be mediated via this binding site.

A recombinant antibody with the antigen-specific, major histocompatibility complex-restricted specificity of T cells.

Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might lead to novel approaches in immunotherapy. However, it has proven difficult to generate antibodies with the specificity of T cells by conventional hybridoma techniques. Here we report that the phage display technology is a feasible alternative to generate antibodies recognizing specific, predetermined peptide/MHC complexes.

Z-membranes: artificial organelles for overexpressing recombinant integral membrane proteins.

We have expressed a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme beta-glucuronidase in transgenic tobacco cells. Electron microscope images of such cells demonstrate that overexpression of this fusion protein gives rise to a type of endoplasmic reticulum membrane domain in which adjacent membranes become zippered together apparently as a consequence of the oligomerizing action of beta-glucuronidase. These zippered (Z-) membranes lack markers of the endoplasmic reticulum (NADH cytochrome c reductase and ribosomes) and accumulate in the cells in the form of multilayered scroll-like structures (up to 2 micrometers in diameter; 20-50 per cell) without affecting plant growth. The discovery of Z-membranes has broad implications for biology and biotechnology in that they provide a means for accumulating large quantities of recombinant membrane proteins within discrete domains of native membranes.

Improved antitumor activity of a recombinant anti-Lewis(y) immunotoxin not requiring proteolytic activation.

B1(dsFv)-PE33 is a recombinant immunotoxin composed of a mutant form of Pseudomonas exotoxin (PE) that does not need proteolytic activation and a disulfide-stabilized Fv fragment of the anti-Lewis(y) monoclonal antibody B1, which recognizes a carbohydrate epitope on human carcinoma cells. In this molecule, amino acids 1-279 of PE are deleted and domain Ib (amino acids 365-394) is replaced by the heavy chain variable region (VH) domain of monoclonal antibody B1. The light chain (VL) domain is connected to the VH domain by a disulfide bond. This recombinant toxin, termed B1(dsFv)-PE33, does not require proteolytic activation and it is smaller than other immunotoxins directed at Lewis(y), all of which require proteolytic activation. Furthermore, it is more cytotoxic to antigen-positive cell lines. B1(dsFv)-PE38 has the highest antitumor activity of anti-Lewis(y) immunotoxins previously constructed. B1(dsFv)-PE33 caused complete regression of tumors when given at 12 micrograms/kg (200 pmol/kg) every other day for three doses, whereas B1(dsFv)-PE38 did not cause regressions at 13 micrograms/kg (200 pmol/kg). By bypassing the need for proteolytic activation and decreasing molecular size we have enlarged the therapeutic window for the treatment of human cancers growing in mice, so that complete remissions are observed at 2.5% of the LD50.

Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.

An efficient method of constructing recombinant adenoviruses (Ads) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique Swa I site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected into human embryonic kidney 293 cells together with the Ad DNA-terminal protein complex digested at several sites with Eco T22I or Ase I/EcoRI. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the above restriction digestion drastically reduced regeneration of the parent virus. Several hundred virus clones were readily obtained in each experiment, and about 70% of the clones were the desired recombinant viruses. Furthermore, because the cassette contained the full-length Ad genome, any position of the genome could be easily modified to develop a new vector design. We established construction systems for two types of Ad vectors, the E1-substitution type and the E4-insertion type. This method may greatly facilitate the application of recombinant Ads and should be useful for further improvement of Ad vectors.