Rapid determination of adenoviral vector titers by quantitative real-time PCR.

Replication defective adenoviruses have been used as vectors in a variety of settings including gene transfer, gene manipulation, and functionality studies. A quantitative real-time PCR-based assay is described for rapid determination of physical titers of recombinant adenovirus vectors. This method is based on amplification of a 77 bp fragment located near the left end of the adenovirus type 5 genome. Evaluation of this method demonstrated that it is simple, sensitive and reproducible, and has a dynamic range of quantitation over 5 logs. This assay is applicable to purified adenovirus as well as vectors prepared by simple cell lysis procedure, requiring only a small amount of starting material. The simplicity and short turn-around time of this assay should facilitate rapid titer determination for a large collection of adenoviral vectors.

Recombinant adenovirus vectors with knobless fibers for targeted gene transfer.

Adenoviral vector systems for gene therapy can be much improved by targeting vectors to specific cell types. This requires both the complete ablation of native adenovirus tropism and the introduction of a novel binding affinity in the viral capsid. We reasoned that these requirements could be fulfilled by deleting the entire knob domain of the adenovirus fiber protein and replacing it with two distinct moieties that provide a trimerization function for the knobless fiber and specific binding to the target cell, respectively. To test this concept, we constructed adenoviral vectors carrying knobless fibers comprising the alpha-helix trimerization domain from MoMuLV envelope glycoprotein. Two mimic targeting ligands, a Myc-epitope and a 6His-tag, were attached via a flexible linker peptide. The targeted knobless fiber molecules were properly expressed and imported into the nucleus of adenovirus packaging cells, where they were incorporated as functional trimers into the adenovirus capsid. Both ligands were exposed on the surface of the virion and were available for specific binding to their target molecules. Moreover, the knobless fibers mediated gene delivery into cells displaying receptors for the coupled ligand. Hence, these knobless fibers are prototype substrates for versatile addition of targeting ligands to generate truly targeted adenoviruses.

Keratinocyte growth factor induced epithelial proliferation facilitates retroviral-mediated gene transfer to distal lung epithelia in vivo.

BACKGROUND: Cell proliferation, vector titer and accessibility of target cells represent hurdles for efficient gene transfer to lung epithelia in vivo using recombinant murine leukemia (MuLV)-based retroviruses. We tested the hypothesis that the pulmonary epithelium is susceptible to retroviral-mediated gene transfer when stimulated to proliferate by a mitogen, keratinocyte growth factor (KGF). METHODS: Rats received keratinocyte growth factor (KGF, 2.5 micrograms/g x 4 doses, two consecutive days) intratracheally followed by high titer amphotropic retrovirus expressing beta-galactosidase. Gene transfer was assessed five days later. RESULTS: KGF stimulated transient proliferation in the bronchiolar and alveolar epithelia (30-40% PCNA positive cells at peak) which decreased to background levels seven days after administration. Gene transfer to epithelia (X-Gal positive cells) occurred more frequently in KGF treated rats, but proliferation exceeded the level of gene transfer. X-gal positive cells were noted in the alveolar epithelium and occasionally in the bronchiolar epithelium. In order to understand the discrepancy between the number of proliferating and transduced cells, primary rat tracheal epithelium cultured at the air-liquid interface was infected from either the apical or basolateral side. Gene transfer was achieved only through basolateral application of vector, suggesting that epithelial polarity represents a barrier to MuLV-based lung gene transfer in vivo. CONCLUSIONS: KGF transiently stimulates epithelial proliferation in vivo, facilitating MuLV-based gene transfer. Retroviral vectors may encounter multiple barriers which have evolved to defend the lung from infections.

Influence of cell polarity on retrovirus-mediated gene transfer to differentiated human airway epithelia.

Gene transfer with recombinant murine leukemia viruses (MuLV) provides the potential to permanently correct inherited lung diseases, such as cystic fibrosis (CF). Several problems prevent the application of MuLV-based recombinant retroviruses to lung gene therapy: (i) the lack of cell proliferation in mature pulmonary epithelia, (ii) inefficient gene transfer with a vector applied to the apical surface, and (iii) low titers of many retroviral preparations. We found that keratinocyte growth factor (KGF) stimulated proliferation of differentiated human tracheal and bronchial epithelia. Approximately 50% of epithelia divided in response to KGF as assessed by bromodeoxyuridine histochemistry. In airway epithelia stimulated to divide with KGF, high-titer ampho- and xenotropic enveloped vectors preferentially infected cells from the basal side. However, treatment with hypotonic shock or EGTA transiently increased transepithelial permeability, enhancing gene transfer with the vector applied to the mucosal surfaces of KGF-stimulated epithelia. Up to 35% of cells expressed the transgene after gene transfer. By using this approach, cells throughout the epithelial sheet, including basal cells, were targeted. Moreover, the Cl- transport defect in differentiated CF airway epithelia was corrected. These findings suggest that barriers to apical infection with MuLV can be overcome.

Second gene expression in bicistronic constructs using short synthetic intercistrons and viral IRES sequences.

In this study, we describe the efficiency of second gene translation in bicistronic constructs containing either a short (36bp) synthetic intercistron or known internal ribosomal entry sites (IRES). Experiments were performed using two different gene combinations: Herpes simplex virus-thymidine kinase (HSV-TK) and neomycine (NEO) or human glucocerebrosidase (hGC) and a methotrexate (MTX) resistant mutant dihydrofolate reductase (DHFR). We demonstrate that upon transfection, second gene translation is efficient using either an IRES or a 36-bp intercistron. Infection with retrovirus carrying the TK and NEO genes linked via a 36-bp intercistron resulted in both G418R (NEO expression) and gancyclovir (GCV) sensitivity (TK expression), indicating that both genes were expressed and thus that the genomic DNA and RNA of this bicistronic construct were intact. Likewise, retrovirus carrying the hGC and mutant DHFR gene separated by a short intercistron was harvested from MTXR murine PsiCRE cells. However, infection of PA317 cells with this virus supernatant did not result in the presence of hGC enzyme activity in these murine cells. Proviral DNA and RNA analyses indicated that the hGC coding region was lost from the original construct in the infected PA317 cells. In contrast, retrovirus carrying the hGC and DHFR cDNAs was linked via an IRES functioned as expected. Based on these results, we conclude that the efficiency of second gene translation using short synthetic intercistrons might prove useful in bicistronic constructs, depending on the gene combination used.

A flow cytometric assay enabling specific detection of the human lysosomal enzyme, beta-glucocerebrosidase.

We have developed a flow cytometric assay specific for human lysosomal beta-glucocerebrosidase (hGC) which is the enzyme deficient in Gaucher disease, a lysosomal storage disorder. The assay is based on the primate-specific monoclonal antibody 8E4 and thus allows detection of endogenous hGC and primate GC protein at a single cell level. We demonstrate that detection of endogenous hGC is possible in rhesus and human cells. Since antibody 8E4 does not bind to rodent GC, hGC detection in murine cell lines and primary cells upon transduction with a retrovirus carrying the hGC cDNA is possible. Comparison of this assay to a flow cytometric method which detects enzymatic GC activity shows that the 8E4-based assay is significantly more sensitive. We also show that multiparameter analyses in combination with hGC detection are feasible. This enables hGC detection in different lineages of complex cell populations. The increased sensitivity in combination with the specificity for hGC makes the 8E4-based flow cytometric assay ideally suited to monitor hGC expression. This assay is therefore of significant value to monitor the success of therapeutic strategies for Gaucher disease such as enzyme supplementation therapy, allogeneic bone marrow transplantation, and gene therapy.

Effects of keratinocyte and hepatocyte growth factor in vivo: implications for retrovirus-mediated gene transfer to liver.

We have previously shown that intravenous administration of keratinocyte growth factor (KGF) induces hepatocyte proliferation, allowing for efficient and noninvasive in vivo gene transfer with high-titer retroviral vectors in mice. The distinctive periportal distribution of transduced cells led us to investigate the ability of virus-sized particles to perfuse the liver adequately after growth factor treatment. We found that perfusion was adequate, and that transduction was limited to the periportal region because only those cells were stimulated to divide. Cells in this region also showed increased expression of Ram-1, the receptor for the murine Moloney leukemia virus (MoMLV) amphotropic envelope, after KGF treatment. In further studies we found that recombinant hepatocyte growth factor (HGF) induces a different population of hepatocytes to divide and upregulate Ram-1. The differential pattern of induction suggested that combining KGF and HGF would improve gene transfer efficiency further. Indeed, simultaneous delivery of both growth factors leads to an overall increase in the number of proliferating cells. Importantly, when coupled with MoMLV delivery, efficiency of gene transfer increased. These results confirm the utility of growth factors for noninvasive hepatic gene transfer in mice, and demonstrate how experiments to define the mechanism of transduction can be taken advantage of to develop improved gene transfer protocols.

Serial bone marrow sampling for long-term follow up of human hematopoiesis in NOD/SCID mice.

The study of long-term human hematopoiesis in immunodeficient mice is greatly facilitated by sequential bone marrow (BM) sampling in individual animals. Until now, however, the only way to obtain these samples was by sacrificing the mice. In this paper we describe a novel technique for obtaining BM cells by aspiration from the femur of living mice. The technique is simple and efficient and does not disable the animals. On average 1.6+/-1x10(6) nucleated cells can be collected from one femur at a time, which is sufficient for flow cytometry analysis, cytospin preparations, and polymerase chain reaction assays. The cellular composition of the samples obtained by puncture is identical to that of BM harvested by flushing the femur after sacrificing the animals. We present the results of 81 punctures of the femur in Hu-NOD/SCID chimeras engrafted with Ficoll-separated or CD34bright purified cells from human umbilical cord blood.

Methotrexate selectable retroviral vectors for Gaucher disease.

To develop a gene therapy protocol suitable for the treatment of a benign disease such as Gaucher disease, we have developed two bicistronic vectors that allow transduced cells to be selected for with methotrexate (MTX). The two vectors differ in the presence or absence of a mutant polyoma enhancer (delta Mo + PyF101) replacing the wild-type retroviral enhancer in the LTR. Infection of human TF-1 and K562 cells, Gaucher type II fibroblasts and murine hemopoietic bone marrow cells conferred MTX resistance and glucocerebrosidase (GC) expression. Upon increasing MTX concentrations, the number of proviral copies and GC activity increased, demonstrating in vitro selection of retrovirus-transduced cells. At high MTX selection pressure, up to 140 microM for infected Gaucher type II fibroblasts, no endogenous wild-type DHFR amplification could be detected, indicating that both retroviral constructs provide sufficient DHFR protein levels. Upon transduction, murine bone marrow cells were protected against otherwise lethal MTX concentrations (range 1-5 microM MTX). Flow cytometry specific for human GC (hGC) demonstrated that in vitro selection resulted in increased percentages of hGC-positive murine cells. In conclusion, the generated bicistronic vectors are ideally suited to investigate whether an in vivo selection approach for retrovirus-transduced cells is feasible. Such a strategy might abolish the need for a high initial transduction efficiency and might result in a gene therapy protocol devoid of the undesirable need for marrow ablative treatment of the recipient.

A flow cytometric assay for lysosomal glucocerebrosidase.

A flow cytometric assay is described for the determination of glucocerebrosidase (GC) activity using fluorescein di-beta-glucopyranoside (FDGlu). Fluorescent product is formed upon intracellular hydrolysis of FDGlu and is measured in the FL1 channel of a flow cytometer. We show that the assay is specific for lysosomal beta-glucosidase or glucocerebrosidase (1) by concentration-dependent inhibition of GC activity by conditurol-beta-epoxide (CBE), a specific irreversible inhibitor; (2) by the absence of activity in fibroblasts isolated from patients with Gaucher disease; (3) correction of the biochemical Gaucher phenotype in these cells is detectable following gene transfer and can be inhibited by CBE; (4) murine fibroblasts transfected with the human GC cDNA and expressing 1.5- to 2.5-fold higher levels of human GC in in vitro assays can be distinguished from nontransfected cells in mixing experiments; and (5) preincubation of GC expressing cells with the lysosomotropic compound chloroquine leads to a loss of the GC-mediated increase in fluorescence supporting lysosomal localization of the FDGlu hydrolyzing enzyme. This flow cytometric GC assay will be useful for monitoring GC activity at the single cell level and can be used for monitoring the efficacy of Gaucher patient treatments such as enzyme supplementation and gene therapy. Finally, our findings suggest that other lysosomal enzymes can be measured in this way using alternate fluorescein derivatives.

Retroviral stem cell gene therapy.

Long-term in vivo gene transfer studies in mice have shown that recombinant murine retroviruses are able to infect murine hemopoietic stem cells with high efficiency. Taken together the results indicated that the proviral structure was present at high frequency in circulating hemopoietic cells resulting in significant expression levels. Because of the success of these murine studies, it was believed that gene therapy would soon be applicable to treat a wide variety of congenital or acquired human diseases associated with the hemopoietic system. However, results from gene transfer studies in nonhuman primates and first human clinical trails have indicated that murine retrovirus infection of primate hemopoietic stem cells is inefficient. Although there are essential differences between the murine and primate gene therapy studies with respect to the recombinant viruses and transduction protocols used, these differences cannot solely account for the differences observed in infection efficiency. Therefore, in recent years effort has been spent on the identification of factors limiting retroviral transduction of primate hemopoietic stem cells. Increasing knowledge concerning hemopoiesis and retroviral infection has helped in identifying a number of limiting factors. Novel transduction strategies and tools have been generated which attempt to circumvent these limiting factors. These factors as well as the strategies that showed increased retroviral infection of primate hemopoietic stem cells will be discussed.

Development of safe and efficient retroviral vectors for Gaucher disease.

We have generated amphotropic and Gibbon ape leukemia (GaLV) viruses carrying either a full-length (IG-GC2) or a shortened glucocerebrosidase cDNA (IG-GC4). For all recombinant retroviruses, a single infection was sufficient to augment glucocerebrosidase activity in unselected Gaucher type I and type II fibroblasts to levels which can be considered therapeutic. Transfer efficiency of the glucocerebrosidase cDNA into normal human and Gaucher type I CD34+ cells, using supernatant transduction, ranged from 4 to 50% as established on vector-positive CFU-GM. In these experiments, GaLV and amphotropic virus were equally efficient in transducing early human progenitors. Importantly, mixing amphotropic and GaLV pseudotyped retroviruses resulted in significantly higher transduction efficiencies as compared with single infections, up to 70% vector-positive CFU-GM. Glucocerebrosidase activity, measured in the progeny of human CD34+ cells, increased up to 460% compared with mock-infected CD34+ cells. Upon transduction of Gaucher CD34+ bone marrow cells the glucocerebrosidase deficiency was reversed.

Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdr1 gene.

Expression of the pfmdr1-encoded Pgh1 protein of Plasmodium falciparum in CHO cells confers a phenotype of increased sensitivity to chloroquine due to an increased Pgh1-mediated accumulation of this antimalarial. Pgh1 carrying amino acid substitutions associated with chloroquine resistance in P. falciparum does not confer this phenotype. Here, we present studies on the underlying mechanism of Pgh1 mediated chloroquine influx into CHO cells. First, we measured intralysosomal pH using FITC-labelled dextran and found the intralysosomal pH in Pgh1 expressing cells to be decreased. A decreased lysosomal pH was not observed in cells expressing Pgh1 carrying the S1034C and N1042D double substitution found in some chloroquine-resistant P. falciparum parasites. Secondly, Pgh1-mediated uptake of chloroquine was abolished in the presence of bafilomycin A1, a specific inhibitor of vacuolar [H+]ATPases and was nearly abrogated in the presence of NH4Cl. Finally, cells expressing wild-type Pgh1 showed increased uptake of both (+)- and (-)[3H]chloroquine enantiomers, indicating that Pgh1-mediated uptake of chloroquine is not enantioselective and in agreement with a pH-driven process. We conclude from these studies that Pgh1 does not transport chloroquine, but instead influences chloroquine accumulation by modulating the pH of acidic organelles. This function is abolished in Pgh1 carrying amino acid substitutions S1034C and N1042D. We speculate that the pfmdr1 gene encodes a vacuolar chloride channel.

Expression of the plasmodial pfmdr1 gene in mammalian cells is associated with increased susceptibility to chloroquine.

Chloroquine (CQ)-resistant (CQR) Plasmodium falciparum malaria parasites show a strong decrease in CQ accumulation in comparison with chloroquine-sensitive parasites. Controversy exists over the role of the plasmodial pfmdr1 gene in the CQR phenotype. pfmdr1 is a member of the superfamily of ATP-binding cassette transporters. Other members of this family are the mammalian multidrug resistance genes and the CFTR gene. We have expressed the pfmdr1-encoded protein, Pgh1, in CHO cells and Xenopus oocytes. CHO cells expressing the Pgh1 protein demonstrated an increased, verapamil-insensitive susceptibility to CQ. Conversely, no increase in drug susceptibility to primaquine, quinine, adriamycin, or colchicine was observed in Pgh1-expressing cells. CQ uptake experiments revealed an increased, ATP-dependent accumulation of CQ in Pgh1-expressing cells over the level in nonexpressing control cells. The increased CQ accumulation in Pgh1-expressing cells coincided with an enhanced in vivo inhibition of lysosomal alpha-galactosidase by CQ. CHO cells expressing Pgh1 carrying two of the CQR-associated Pgh1 amino acid changes (S1034C and N1042D) did not display an increased CQ sensitivity. Immunofluorescence experiments revealed an intracellular localization of both mutant and wild-type forms of Pgh1. We conclude from our results that wild-type Pgh1 protein can mediate an increased intracellular accumulation of CQ and that this function is impaired in CQR-associated mutant forms of the protein. We speculate that the Pgh1 protein plays an important role in CQ import in CQ-sensitive malaria parasites.

Chemotherapy of malaria: a battle against all odds?

The most important treatment of malaria to date is chemotherapy using a number of different natural and synthetic drugs. Antifolates, such as pyrimethamine, inhibit the parasite's dihydrofolatereductase, whereas the aminoquinolines, such as chloroquine, have the digestive vacuole as their major site of action. The use of these drugs in chemotherapy of malaria infections is strongly hampered by widespread resistance and in this review current knowledge on the molecular mechanisms of resistance is discussed. Finally, new antimalarial drugs and treatment strategies are considered which may offer new possibilities for the treatment of malaria resistant to the drugs currently in use.

Assignment of the human UDP glucuronosyltransferase gene (UGT1A1) to chromosome region 2q37.

UDP glucuronosyltransferases (UGTs) comprise a multigene family of drug-metabolizing enzymes. The sub-family of UGTs that conjugate bilirubin and phenolic compounds with glucuronic acid has been termed UGT1A1. In man, UGT1A1 isoforms are encoded by a single gene, UGT1A1. Protein isoforms encoded by UGT1A1 originate by alternative splicing. In the present study, we used the cDNA of UGT1A1*4, a bilirubin-conjugating isoform, to localize the UGT1A1 locus in the human genome. The UGT1A1 gene was assigned by in situ hybridization to chromosome region 2q37.

Mechanisms of inherited deficiencies of multiple UDP-glucuronosyltransferase isoforms in two patients with Crigler-Najjar syndrome, type I.

Crigler-Najjar syndrome, type I (CN-I) is a potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from a recessively inherited deficiency of hepatic UDP-glucuronosyl-transferase (UGT) activity toward bilirubin (B-UGT). Two forms of B-UGT exist in human liver. mRNAs for these two forms and that for another isoform with activity toward simple phenols (P-UGT) have unique 5' regions, but their 3' regions are identical. The three mRNA species are derived from a single locus; the unique 5' regions are encoded by single unique exons and the identical 3' regions consist of four consecutive exons that are shared by all three isoforms. In this paper, we determined genetic lesions in two CN-I patients with deficiency of hepatic B-UGT and P-UGT activities. In one patient, there was a C----T substitution in exon 4 (common region) predicting the substitution of a serine residue with a phenylalanine residue; this mutation was present in the identical region of B-UGT and P-UGT mRNAs. In the other patient, a C----T substitution in exon 2 (common region) of the B-UGT/P-UGT locus resulted in a premature stop codon. This exon (132 nt) was absent in heptic B-UGT and P-UGT mRNAs of this patient due to exon skipping during pre-mRNA processing. Sequence abnormality of three distinct mRNA species explains the abnormality of multiple UGT isoforms in these patients. Presence of identical abnormalities in the common regions of the three mRNAs is consistent with the finding that the common 3' regions of the two B-UGT mRNAs and the P-UGT mRNA are encoded by four shared exons.

Immunochemical analysis of uridine diphosphate-glucuronosyltransferase in four patients with the Crigler-Najjar syndrome type I.

The functional heterogeneity of uridine diphosphate-glucuronosyltransferase (UDPGT) and its deficiency in human liver were investigated. The monoclonal antibody (MAb) WP1, which inhibits bilirubin and phenol-glucuronidating activity, was used to immunopurify UDPGTs from human liver. Purified UDPGTs were injected into mice to obtain new MAbs. Immunoblotting of microsomes with MAb HEB7 revealed at least three polypeptides in liver (56, 54, and 53 kD) and one in kidney (54 kD). In liver microsomes from four patients (A, B, C, and D) with Crigler-Najjar syndrome type I (CN type I), UDPGT activity towards bilirubin was undetectable (A, B, C, and D) and activity towards phenolic compounds and 5-hydroxytryptamine either reduced (A and B) or normal (C and D). UDPGT activity toward steroids was normal. Immunoblot studies revealed that the monoclonal antibody WP1 recognized two polypeptides (56 and 54 kD) in liver microsomes from patient A and none in patient B. With HEB7 no immunoreactive polypeptides were seen in these two patients. Patient C showed a normal banding pattern and in patient D only the 53-kD band showed decreased intensity. These findings suggest considerable heterogeneity with regard to the expression of UDPGT isoenzymes among CN type I patients.