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.

Novel GP64 envelope variants for improved delivery to human airway epithelial cells.

Lentiviral vectors pseudotyped with the baculovirus envelope protein GP64 transduce primary cultures of human airway epithelia (HAE) at their apical surface. Our goal in this study was to harness a directed evolution approach to develop a novel envelope glycoprotein with increased transduction properties for HAE. Using error-prone PCR, a library of GP64 mutants was generated and used to prepare a diverse pool of lentiviral virions pseudotyped with GP64 variants. The library was serially passaged on HAE and three GP64 mutations were recovered. Single-, double- and the triple-combination mutant envelope glycoproteins were compared with wild-type GP64 for their ability to transduce HAE. Our results suggest that lentiviral vectors pseudotyped with evolved GP64 transduced HAE with greater efficiency than wild-type GP64. This effect was not observed in primary cultures of porcine airway epithelial cells, suggesting that the directed evolution protocol was species specific. In summary, our studies indicate that serial passage of a GP64 mutant library yielded specific variants with improved HAE cell tropism, yielding tools with the potential to improve the success of gene therapy for airway diseases.

A novel gene delivery method transduces porcine pancreatic duct epithelial cells.

Gene therapy offers the possibility to treat pancreatic disease in cystic fibrosis (CF), caused by mutations in the CF transmembrane conductance regulator (CFTR) gene; however, gene transfer to the pancreas is untested in humans. The pancreatic disease phenotype is very similar between humans and pigs with CF; thus, CF pigs create an excellent opportunity to study gene transfer to the pancreas. There are no studies showing efficient transduction of pig pancreas with gene-transfer vectors. Our objective is to develop a safe and efficient method to transduce wild-type (WT) porcine pancreatic ducts that express CFTR. We catheterized the umbilical artery of WT newborn pigs and delivered an adeno-associated virus serotype 9 vector expressing green-fluorescent protein (AAV9CMV.sceGFP) or vehicle to the celiac artery, the vessel that supplies major branches to the pancreas. This technique resulted in stable and dose-dependent transduction of pancreatic duct epithelial cells that expressed CFTR. Intravenous (IV) injection of AAV9CMV.sceGFP did not transduce the pancreas. Our technique offers an opportunity to deliver the CFTR gene to the pancreas of CF pigs. The celiac artery can be accessed via the umbilical artery in newborns and via the femoral artery at older ages--delivery approaches that can be translated to humans.

Integrase-defective lentiviral vectors: progress and applications.

Lentiviral vectors (LVs) offer the advantages of a large packaging capacity, broad cell tropism or specific cell-type targeting through pseudotyping, and long-term expression from integrated gene cassettes. However, transgene integration carries a risk of disrupting gene expression through insertional mutagenesis and may not be required for all applications. A non-integrating LV may be beneficial in cases in which transient gene expression is desired. Several recent publications outline the development and initial biological characterization of such vectors. Here, we discuss the potential applications and new directions for the development of integration-defective LVs.

Progress and prospects: prospects of repeated pulmonary administration of viral vectors.

Pulmonary gene therapy may ultimately cure diseases such as cystic fibrosis, alpha1-antitrypsin deficiency, lung cancer and pulmonary hypertension. Efficient expression of delivered genes in target cell types is essential for the achievement of this goal. To this end, re-administration of viral vectors may be required (1) to increase the percentage of transduced airway epithelial cells, (2) to direct gene transfer to individual lobes during successive delivery sessions or (3) to boost attenuated expression over time. Immune responses to viral proteins or viral-encoded proteins are the greatest barrier to repeated vector administration.

Altered fluid transport across airway epithelium in cystic fibrosis.

In cystic fibrosis (CF), absence or dysfunction of a phosphorylation-regulated chloride channel [CF transmembrane conductance regulator (CFTR)] leads to the loss or reduction of chloride secretion into the airways. Active sodium absorption is also increased in CF, and both of these ion transport changes could alter fluid transport across the airways. Under baseline conditions, cultured human airway epithelia from normal individuals absorbed fluid, and this absorption was increased in epithelia from patients with CF. In normal and CF epithelial cultures fluid absorption was inhibited by amiloride. Adenosine 3',5'-monophosphate stimulated fluid secretion in normal epithelial cultures but not in cultures from individuals with CF. In contrast, fluid secretion induced by nucleotide triphosphates (uridine triphosphate or adenosine triphosphate) was unaltered in cultures of epithelia from patients with CF, suggesting an approach to the treatment of CF.

The piglet as a model for B cell and immune system development.

The ability to identify factors responsible for disease in all species depends on the ability to separate those factors which are environmental from those that are intrinsic. This is particularly important for studies on the development of the adaptive immune response of neonates. Studies on laboratory rodents or primates have been ambiguous because neither the effect of environmental nor maternal factors on the newborn can be controlled in mammals that: (i) transmit potential maternal immunoregulatory factors in utero and (ii) are altricial and cannot be reared after birth without their mothers. Employing the newborn piglet model can address each of these concerns. However, it comes at the price of having first to characterize the immune system of swine and its development. This review focuses on the porcine B cell system, especially on the methods used for its characterization in fetal studies and neonatal piglets. Understanding these procedures is important in the interpretation of the data obtained. Studies on neonatal piglets have (a) provided valuable information on the development of the adaptive immune system, (b) lead to important advances in evolutionary biology, (c) aided our understanding of passive immunity and (d) provided opportunities to use swine to address specific issues in veterinary and biomedical research and immunotherapy. This review summarizes the history of the development of the piglet as a model for antibody repertoire development, thus providing a framework to guide future investigators.

Localization of cystic fibrosis transmembrane conductance regulator mRNA in human fetal lung tissue by in situ hybridization.

The fetal pulmonary epithelium secretes fluid. Cl transport is presumed to provide the driving force for net fluid secretion, although the cellular mechanisms have not been well identified in the fetus. The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP- and nucleoside triphosphate-regulated Cl channel; mutations in CFTR cause cystic fibrosis. We hypothesized that if CFTR is involved in fetal lung fluid transport, the fetal pulmonary epithelium should express CFTR mRNA. We used the technique of in situ hybridization with 3H-anti-sense and, as a control, 3H-sense CFTR cRNA probes to localize CFTR mRNA in human fetal lung tissue and cultured lung explants and determine when in gestation it is expressed. Epithelial cells of both first and second trimester lung tissues expressed CFTR mRNA. A decreasing gradient of CFTR mRNA expression was present from the proximal to the distal pulmonary epithelium. Cultured second trimester lung tissue explants expressed more CFTR mRNA than the uncultured starting tissue, suggesting CFTR gene expression increased during the five days in culture. Furthermore, alveolar type II cells in cultured explants expressed CFTR mRNA, suggesting that these cells are Cl-secretory and may be involved in lung fluid transport. These data confirm that CFTR mRNA is expressed in the human fetal pulmonary epithelium, consistent with the Cl-secretory properties of the fetal lung.

Human beta-defensin-1: an antimicrobial peptide of urogenital tissues.

Antimicrobial peptides are widely distributed mediators of innate host defense in animals and plants. A 36 amino acid antimicrobial peptide belonging to the defensin family, and named human beta-defensin-1 (HBD-1), was purified recently from hemodialysate fluid, but its tissue sources were not identified. By Northern blotting, we found the highest concentrations of HBD-1 mRNA in the kidney and the female reproductive tract. In situ hybridization localized the HBD-1 mRNA in the epithelial layers of the loops of Henle, distal tubules, and the collecting ducts of the kidney and the epithelial layers of the vagina, ectocervix, endocervix, uterus, and fallopian tubes in the female reproductive tract. Using a novel technique designed to detect cationic peptides in urine, we recovered several forms of HBD-1 ranging in length from 36 to 47 amino acid (aa) residues and differing from each other by amino terminal truncation. The total concentration of HBD-1 forms in voided urine was estimated at 10-100 microg/liter, with individual variations in the total amount of HBD-1 peptides and the relative proportion of HBD-1 forms. Multiple forms of HBD-1 (size 36-47 aa) were also found in the blood plasma, bound to carrier macromolecules that released the peptide under acid conditions, and in vaginal mucosal secretions (39, 40, and 44 aa). By immunostaining, HBD-1 was located in the kidney within the lumen of the loops of Henle, but no intracellular storage sites were identified in renal or female reproductive tissues. Recombinant HBD-1 forms (36, 39, and 42 aa) and natural HBD-1 forms were antimicrobial to laboratory and clinical strains of Escherichia coli at micromolar concentrations. HBD-1 activity was not changed appreciably by low pH, but was inhibited by high salt conditions. Some of the HBD-1 peptides retained their activity against E. coli in unconcentrated (low conductance) urine, and the 36 aa form was microbicidal even in normal (high conductance) urine. Production of HBD-1 in the urogenital tract could contribute to local antimicrobial defense.

A mouse model for the delta F508 allele of cystic fibrosis.

The most common cause of cystic fibrosis is a mutation that deletes phenylalanine 508 in cystic fibrosis transmembrane conductance regulator (CFTR). The delta F508 protein is misprocessed and degraded rather than traveling to the apical membrane. We used a novel strategy to introduce the delta F508 mutation into the mouse CFTR gene. Affected epithelia from homozygous delta F508 mice lacked CFTR in the apical membrane and were Cl-impermeable. These abnormalities are the same as those observed in patients with delta F508 and suggest that these mice have the same cellular defect. 40% of homozygous delta F508 animals survived into adulthood and displayed several abnormalities found in human disease and in CFTR null mice. These animals should provide an excellent model to investigate pathogenesis and to examine therapies directed at correcting the delta F508 defect.

Adenoviral-mediated gene transfer to fetal pulmonary epithelia in vitro and in vivo.

Vector-mediated gene transfer offers a direct method of correcting genetic pulmonary diseases and might also be used to correct temporary abnormalities associated with acquired, nongenetic disorders. Because the fetus or newborn may be a more immune tolerant host for gene transfer using viral vectors, we used replication defective recombinant adenoviral vectors to test the feasibility of gene transfer to the fetal pulmonary epithelium in vitro and in vivo. Both proximal and distal epithelial cells in cultured fetal lung tissues from rodents and humans diffusely expressed the lacZ transgene 3 d after viral infection. In vivo gene delivery experiments were performed in fetal mice and lambs. Delivery of Ad2/CMV-beta Gal to the amniotic fluid in mice produced intense transgene expression in the fetal epidermis and amniotic membranes, some gastrointestinal expression, but no significant airway epithelial expression. When we introduced the adenoviral vector directly into the trachea of fetal lambs, the lacZ gene was expressed in the tracheal, bronchial, and distal pulmonary epithelial cells 3 d after viral infection. Unexpectedly, reactive hyperplasia and squamous metaplasia were noted in epithelia expressing lacZ in the trachea, but not in the distal lung of fetal lambs. 1 wk after infection, adenovirus-treated fetuses developed inflammatory cell infiltrates in the lung tissue with CD4, CD8, IgM, and granulocyte/macrophage positive immune effector cells. Transgene expression faded coincident with inflammation and serologic evidence of antiadenoviral antibody production. While these studies document the feasibility of viral-mediated gene transfer in the prenatal lung, they indicate that immunologic responses to E1-deleted recombinant adenoviruses limit the duration of transgene expression.

Proliferation induced by keratinocyte growth factor enhances in vivo retroviral-mediated gene transfer to mouse hepatocytes.

Retroviral gene transfer to liver without prior injury has not yet been accomplished. We hypothesized that recombinant human keratinocyte growth factor would stimulate proliferation of hepatocytes and allow for efficient in vivo gene transfer with high titer murine Moloney retroviral vectors. This report shows that 48 h after intravenous injection of keratinocyte growth factor, hepatocyte proliferation increased approximately 40-fold compared to non-stimulated livers. When keratinocyte growth factor treatment was followed by intravenous injection of high titer (1 x 10(8) colony forming units/ml) retrovirus coding for the Escherichia Coli beta-galactosidase gene, there was a 600-fold increase in beta-galactosidase expression, with 2% of hepatocytes transduced. Thus, by exploiting the mitogenic properties of keratinocyte growth factor, retrovirus-mediated gene transfer to liver may be accomplished in vivo without the use of partial hepatectomy or pretreatment with other toxins to induce hepatocyte cell division.

Feline immunodeficiency virus vectors persistently transduce nondividing airway epithelia and correct the cystic fibrosis defect.

Several problems limit the application of gene transfer to correct the cystic fibrosis (CF) Cl(-) transport defect in airway epithelia. These include inefficient transduction with vectors applied to the apical surface, a low rate of division by airway epithelial cells, failure of transgene expression to persist, and immune responses to vectors or vector-encoded proteins. To address these issues, we used a feline immunodeficiency virus-based (FIV-based) vector. FIV vector formulated with a calcium chelator transduced fully differentiated, nondividing human airway epithelia when applied to the apical surface. FIV-based vector encoding the cystic fibrosis transmembrane conductance regulator cDNA corrected the Cl(-) transport defect in differentiated CF airway epithelia for the life of the culture (>3 months). When this approach was applied in vivo, FIV vector expressing beta-galactosidase transduced 1-14% of adult rabbit airway epithelia. Transduced cells were present in the conducting airways, bronchioles, and alveoli. Importantly, gene expression persisted, and cells with progenitor capacity were targeted. FIV-based lentiviral vectors may be useful for the treatment of genetic lung diseases such as CF. This article may have been published online in advance of the print edition.

Effect of glutathione peroxidase activity on lipid peroxidation in biological membranes.

Results are presented indicating that, although glutathione peroxidase activity inhibits lipid peroxidation in membranes, it does not appear to do so by reducing membrane lipid peroxides to lipid alcohols, as has been shown by others to be the case for free fatty acid peroxides in solution. Lipid peroxidation was studied in an enzymic system (microsomal NADPH oxidase) and in a non-enzymic system (mitochondria plus ascorbate). A study of the fatty acids in the phospholipids of microsomes and mitochondria demonstrated that detectable amounts of hydroxy fatty acids were not formed in the membranes when the latter were incubated in the presence of the glutathione peroxidase system even under conditions known to have generated significant levels of lipid peroxides in the membrane. Fatty acid analyses of the microsomal and mitochondrial particles indicated that glutathione peroxidase activity inhibited loss of polyunsaturated fatty acids when these organelles were exposed to peroxidizing conditions. If glutathione peroxidase activity were inhibiting the formation of malondialdehyde (a product of lipid peroxidation) by converting peroxide groups to alcohols, the loss of the constitutive polyunsaturated fatty acids in the membrane should not have been appreciably affected by addition of the peroxidase system. The protective effect cannot be due to quenching of an autocatalytic type of lipid peroxidation (at least in the microsomal system) since it has been established that the microsomal enzyme system (NADPH oxidase) catalyzes a continuous attack on microsomal polyunsaturated fatty acyl groups during the reaction and that the peroxidative process is not autocatalytic in nature. It appears, therefore, that glutathione peroxidase activity must exert its effect on this system by preventing free radical attack on the polyunsaturated membrane lipids in the first place. A possible mechanism for the interruption of a free radical attack on the lipids is proposed.

Polarity influences the efficiency of recombinant adenoassociated virus infection in differentiated airway epithelia.

To better understand mechanisms that limit rAAV transduction in the lung, we have evaluated several unique features of rAAV infection in polarized primary airway epithelial cultures. rAAV was found to transduce the basolateral surface of airway epithelia 200-fold more efficiently than the apical membrane. These differences in membrane infection correlated with the abundance of apical heparan sulfate proteoglycan (AAV-2 receptor) and virus binding. UV irradiation augmented rAAV transduction greater than 20-fold, only when virus was applied to the apical membrane. Ultrastructural analysis of UV-irradiated primary cultures demonstrated significant changes in microvilli architecture following exposure to 25 J/m2 UV. Although virus binding and the abundance of heparan sulfate proteoglycan were not increased at the apical membrane following UV irradiation, increased receptor-independent endocytosis of fluorescent beads was seen at the apical membrane following UV irradiation. We hypothesize that endocytotic processes associated with apical membrane-specific pathways of viral entry, and/or processing of virus to the nucleus, may be altered following UV irradiation. Interestingly, UV irradiation had an inhibitory effect on rAAV transduction from the basolateral membrane, which correlated with a decrease in the abundance of heparan sulfate proteoglycan at the basal membrane. In summary, these findings suggest that independent pathways of viral transduction may occur in the apical and basolateral compartments of polarized airway epithelia.

Alveolar macrophages inhibit retrovirus-mediated gene transfer to airway epithelia.

Gene transfer with integrating vectors such as recombinant retrovirus has the potential to correct inherited lung diseases permanently. As a gene therapy target, the pulmonary epithelium presents several challenges to vector delivery in vivo. Many of the host defenses that have evolved to prevent infection from inhaled bacteria or viruses represent potential barriers to gene transfer to the lung. We performed in vitro studies to determine whether two components of the innate immune system of the lung, airway surface fluid and alveolar macrophages, inhibit retroviral gene transfer to airway epithelia. Human alveolar macrophages obtained by bronchoalveolar lavage from normal subjects were left untreated or activated with lipopolysaccharide (LPS) for 3 hr in the presence of subconfluent human bronchial epithelial cells (HBE); than 4 x 10(5) cfu DA-luciferase retrovirus was added. Three days after infection, luciferase activity was measured in cell lysates. When the epithelial cells were co-cultured with LPS-activated macrophages, retroviral gene transfer to HBE cells was reduced by approximately 60%. Nonactivated macrophages decreased the transfection to approximately 55% of control values. In control experiments with either activated or inactivated macrophages but without epithelia, no luciferase activity was detected, suggesting that terminally differentiated alveolar macrophages are not infected by the recombinant retrovirus. Pretreatment of alveolar macrophages with dexamethasone restored gene transfer to approximately 60% of control values. In contrast, incubation of retrovirus with airway surface fluid had no inhibitory effect on gene transfer. These experiments document that AM inhibit retrovirus-mediated gene transfer to airway epithelia in vitro, and may represent a barrier to retroviral gene transfer in vivo. These barriers may be overcome, at least partially, with pharmacological agents.

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.

Structure and mapping of the human beta-defensin HBD-2 gene and its expression at sites of inflammation.

We cloned a second human beta-defensin gene, HBD-2, and determined its gene structure and expression in inflamed tissue sections. The entire gene spanned about 2 kb with two small exons and one intron. Radiation hybrid studies confirmed the location on chromosome 8p, were consistent with the order HNP-1, HBD-1 and HBD-2, and located HBD-2 as the most centromeric of the genes. By three-color fluorescence in situ hybridization on both free chromatin fiber mapping and interphase mapping, HBD-1, HBD-2 and HNP-1 were mapped to chromosome 8p23. HBD-1 was within 40-100kb of HNP-1, while HBD-2 was about 500-600 kb from HBD-1, with the most likely order HNP-1, HBD-1, HBD-2. The expression of HBD-2 was locally regulated by inflammation. HBD-2 mRNA was markedly increased in the epidermis surrounding inflamed regions, but not detectable in adjacent non-inflamed areas, a distribution that was confirmed at the peptide level by immunostaining with HBD-2 antibody. The HBD-2 gene is the first member of the human defensin family that is locally inducible by inflammation.

Discovery of new human beta-defensins using a genomics-based approach.

Epithelial beta-defensins are broad-spectrum cationic antimicrobial peptides that also act as chemokines for adaptive immune cells. In the human genome, all known defensin genes cluster to a <1 Mb region of chromosome 8p22-p23. To identify new defensin genes, the DNA sequence from a contig of large-insert genomic clones from the region containing human beta-defensin-2 (HBD-2) was analyzed for the presence of defensin genes. This sequence survey identified a novel beta-defensin, termed HBD-3. The HBD-3 gene contains two exons, is located 13 kb upstream from the HBD-2 gene, and it is transcribed in the same direction. A partial HBD-3 cDNA clone was amplified from cDNA derived from IL-1beta induced fetal lung tissue. The cDNA sequence encodes for a 67 amino acid peptide that is approximately 43% identical to HBD-2 and shares the beta-defensin six cysteine motif. By PCR analysis of two commercial cDNA panels, HBD-3 expression was detected in adult heart, skeletal muscle, placenta and in fetal thymus. From RT-PCR experiments, HBD-3 expression was observed in skin, esophagus, gingival keratinocytes, placenta and trachea. Furthermore, in fetal lung explants and gingival keratinocytes, HBD-3 mRNA expression was induced by IL-1beta. Additional sequence analysis identified the HE2 (human epididymis secretory protein) gene 17 kb upstream from the HBD-3 gene. One splice variant of this gene (HE2beta1) encodes a beta-defensin consensus cysteine motif, suggesting it represents a defensin gene product. HE2beta1 mRNA expression was detected in gingival keratinocytes and bronchial epithelia using RT-PCR analysis. The discovery of these novel beta-defensin genes may allow further understanding of the role of defensins in host immunity at mucosal surfaces.

Reye syndrome associated with aspirin therapy for systemic lupus erythematosus.

A 14-year-old girl in whom Reye syndrome developed during aspirin therapy for an inflammatory disorder proven to be systemic lupus erythematosus is reported. This case and similar cases of Reye syndrome in patients with juvenile rheumatoid arthritis suggest that an etiologic relationship exists between salicylate therapy and Reye syndrome in children with collagen vascular disorders.