A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus.

Fully deleted adenovirus vectors (FD-AdVs) would appear to be promising tools for gene therapy. Since these vectors are deleted of all adenoviral genes, they require a helper adenovirus for their propagation. The contamination of the vector preparation by the helper limits the utility of currently existing FD-AdVs in gene therapy applications. We have developed an alternative system for the propagation of FD-AdVs, in which the adenoviral genes essential for replication and packaging of the vector are delivered into producer cells by a baculovirus-adenovirus hybrid. A hybrid baculovirus Bac-B4 was constructed to carry a Cre recombinase-excisable copy of the packaging-deficient adenovirus genome. Although the total size of the DNA insert in Bac-B4 was 38 kb, the genetic structure of this recombinant baculovirus was stable. Bac-B4 gave high yields in Sf9 insect cells, with titers of 5 x 10(8)p.f.u./ml before concentration. Transfection of 293-Cre cells with lacZ-expressing FD-AdV plasmid DNA followed by infection by Bac-B4 at a MOI of 2000 p.f.u./ml resulted in rescue of the helper-free vector. Subsequent passaging of the obtained FD-AdV using Bac-B4 as a helper resulted in approximately 100-fold increases of the vector titer at each passage. This resulting vector was completely free of helper virus and was able to transduce cultured 293 cells. However, scaling-up of FD-AdV production was prevented by the eventual emergence of replication-competent adenovirus (RCA). Experiments are underway to optimize this system for the large-scale production of helper virus-free FD-AdVs and to minimize the possibility of generation of replication-competent adenovirus (RCA) during vector production. This baculovirus-based system will be a very useful alternative to current methods for the production of FD-AdVs.

Stabilization of transgenes delivered by recombinant adenovirus vectors through extrachromosomal replication.

BACKGROUND: A major limitation of adenovirus-mediated gene therapy for metabolic and inherited diseases is the instability of transgene expression in vivo. This instability results, at least in part, from the inability of the vector genome to maintain the transgene through replication or integration. In this study we evaluated the possibility of stabilization of an adenovirus-delivered transgene by non-adenovirus replicative elements. METHODS: We have developed a novel system for the maintenance of transgenes delivered by adenovirus vectors through extrachromosomal replication. In its initial configuration, this system combines the Epstein-Barr virus (EBV) replicative elements, a tetracycline (Tc)-inducible expression system, and the Cre-lox recombination system in the context of a single E1/E3/E4-deleted adenovirus vector. Induction of Cre expression initiates a Cre-mediated recombination, resulting in the excision of a fragment of the vector genome and its circularization into an EBV-based episome. RESULTS: In vitro studies have demonstrated that excision of the circular episome can occur in a cell-free system as well as in cultured cells transfected with plasmid DNA or transduced by a virus vector carrying the episome-excising cassette. PCR studies have shown that in proliferating, non-permissive, cultured primate cells the episome generated from the adenovirus vector is maintained much more stably than the genome of the parent vector. This episome was also able to replicate in mammalian cells. CONCLUSION: Together these studies demonstrate the feasibility of this approach for the stabilization of transgenes delivered to dividing cells by adenovirus vectors.

An immunomodulatory procedure that stabilizes transgene expression and permits readministration of E1-deleted adenovirus vectors.

Immune responses against E1-deleted adenovirus vectors and/or their transgene products result in the rapid elimination of vector-transduced cells and the generation of neutralizing antibodies. Different strategies of immunomodulation to stabilize transgene expression at therapeutic levels and to permit productive vector readministration have been examined. Our previous studies have shown that depletion of macrophages from spleen and liver decreases hepatic inflammation, significantly prolongs transgene expression, and delays the onset of humoral immune responses after systemic administration of an E1-deleted adenovirus vector. In the present study, we have examined the effects of macrophage depletion in combination with temporary blockade of CD40 ligation on E1-deleted adenovirus vector-mediated gene transfer. Alone, each of these treatments significantly inhibited the humoral immune response against the transgene product and prolonged its expression. Together, these treatments completely stabilized transgene expression and inhibited the production of neutralizing anti-adenovirus antibodies, permitting successful vector readministration. Animals rendered immunologically unresponsive to vector and transgene antigens regained their ability to mount productive immune responses against the vector after recovery of immune function, but remained unresponsive to the transgene product. These experiments demonstrate that this treatment is transient and antigen-specific.

Molecular correlations in phenylketonuria: mutation patterns and corresponding biochemical and clinical phenotypes in a heterogeneous California population.

We studied 133 California phenylketonuria (PKU) patients and one obligate heterozygote to delineate the molecular basis of PKU in a population with greater ethnic diversity than in previous studies, and to determine whether a correlation exists between genotype and clinical phenotype, with the latter defined by both the diagnostic pretreatment blood phenylalanine (PHE) level and cognitive (IQ) test scores. To determine PAH genotypes, we used PCR-mediated amplification, denaturing gradient gel electrophoresis, and direct sequencing on dried whole blood samples. Where possible, mutation severity was defined according to predicted in vitro PAH enzyme activity estimated by using Cos cell expression analysis for a given mutation. We then asked whether mutation severity, as defined by such expression analysis, correlated with pretreatment PHE levels or with IQ test results. A mutation was identified in 236 (88%) of 267 mutant alleles. Seventeen new mutant alleles were found; A47E, T81P, I102T, E182G, T328D, Y343P, K371R, Y387H, A389E, E422K, IVS9nt5, IVS11nt20, delS70, del364-368/del198-220, delF299, delT323, and -1C/T. In striking contrast to a number of studies in other populations, in this study, based on predicted PAH activity, we observed no correlation between mutation severity and pretreatment PHE levels. There was also no correlation between genotype and IQ. We conclude that in samples collected from an ethnically heterogeneous population, there is no correlation of mutation severity with either pretreatment PHE levels or IQ measurement in treated patients. We caution that genetic counseling in PKU should incorporate the notion that prognosis may not be predicted with precision based on mutation analysis in a given patient.

Haplotypes and mutations of the PAH locus in Egyptian families with PKU.

A high degree of molecular heterogeneneity at the phenylalanine hydroxylase (PAH) locus was established by examining RFLP haplotypes and PAH mutations in the families of 13 Egyptians with phenylketenouria (PKU). Thirteen different haplotypes were unequivocally determined in these kindreds. Haplotypes 1.8, 3.9, 4.3, 7.8, 22.11, 27.6, and 52.8 were found segregating with normal chromosomes, whilst haplotypes 1.8, 5.9, 23.8, 32.8, the newly assigned 73.9, and two as yet incomplete but novel haplotypes were found segregating with the mutant chromosomes. There was no particular preference for a single haplotype among normal or mutant chromosomes. Nine different mutations were also identified among the 26 alleles. IVS 10nt11g (8/26), IVS 2nt5g-c (4/26), R261Q (3/26), R176X (2/26), Y206D (2/26), S231P (2/26), Y198fs [593-614del22bp]; (2/26), G46fs [136/137delG]; (1/26), and E178G (1/26). Six of these mutations (IVS 2nt5g-c, R176X, Y198fs, R261Q, S231P, and IVS 10nt11g) are common to other Mediterranean populations. Two mutations not previously reported in the Mediterranean basin were also observed (Y206D and G46fs). These intriguing preliminary findings confirm IVS 10nt11g as a major mutation among Mediterranean mutations and demonstrate the need for a more comprehensive study of Arab populations to confirm the uniqueness of the two novel mutations to the Egyptian population.

Viral vector-mediated gene therapy for hemophilia B.

Over the past five years, significant advances have been made in the development of novel viral vector systems for the treatment of hemophilia B by somatic gene therapy. At present, both a sustained but partial or a complete but transient correction of the hemophilia B phenotype have been observed in a clinically relevant animal model. Present efforts are being directed toward the development of safe, effective and persistent methods of virally-mediated gene transfer to achieve the complete restoration of normal hemostasis in individuals with hemophilia B.

Hepatic insulin gene expression as treatment for type 1 diabetes mellitus in rats.

Type 1 diabetes mellitus is caused by a lack of insulin that results from the autoimmune destruction of the pancreatic beta-cells. Severe diabetes, if not controlled by periodic insulin injections, can lead to ketoacidosis and death. We have previously shown that sustained low level production of insulin in the liver of diabetic rats prevented their death from complications of diabetes. To test the hypothesis that there is a window of serum insulin concentrations that can prevent ketoacidosis without significant risk of hypoglycemia secondary to hyperinsulinemia, rats were infused with various doses of a recombinant retrovirus encoding an engineered rat preproinsulin-1 gene. The gene was engineered to allow processing into mature insulin by the protease furin. At the lower doses tested, fatal ketoacidosis was prevented, but the rats exhibited nonfasting hyperglycemia. At intermediate doses, which resulted in serum insulin concentrations of 1.6 mg/ml, the rats achieved near-normoglycemia and no serum ketones. These rats did not exhibit hypoglycemia even during a 24-h fast. At high virus doses, the animals achieved nonfasting normoglycemia but exhibited hypoglycemia during the fast. In conclusion, we have defined a therapeutic window of hepatic insulin expression that provides protection against ketoacidosis without significant risk of hypoglycemia. This window of sustained hepatic insulin expression might permit its development into a novel treatment modality for the prevention of ketoacidosis in patients with severe insulin-dependent diabetes mellitus.

Macrophage depletion increases the safety, efficacy and persistence of adenovirus-mediated gene transfer in vivo.

The consequences of macrophage depletion achieved by intravenous infusion of liposome-encapsulated clodronate (dichlormethylene diphosphonate (Cl2MDP)) on adenovirus-mediated transfer of a recombinant human alpha 1-antitrypsin (hAAT) gene were examined in 12-14-week-old male Balb/c mice. The levels of hAAT expression following tail vein infusions of 10(9) p.f.u. of Ad.RSV-hAAT were approximately four-fold higher in macrophage-depleted animals than in control animals pretreated with liposome-encapsulated phosphate-buffered saline (PBS). Clodronate pretreatment also significantly increased the survival of animals injected with high doses of viral vector. Long-term studies performed in animals receiving tail vein infusions of the adenoviral vector also indicated that clodronate pretreatment significantly attenuated the rapid loss of transgene expression usually observed in immunocompetent animals. These findings indicate that the depletion of macrophages before adenovirus-mediated gene transfer may increase the transduction efficiency and reduce the rate of immunologic elimination of the adenovirally transduced cells, thereby increasing the persistence of transgene expression in immunocompetent animals.

Correction of obesity and diabetes in genetically obese mice by leptin gene therapy.

The ob/ob mouse is genetically deficient in leptin and exhibits both an obese and a mild non-insulin-dependent diabetic phenotype. To test the hypothesis that correction of the obese phenotype by leptin gene therapy will lead to the spontaneous correction of the diabetic phenotype, the ob/ob mouse was treated with a recombinant adenovirus expressing the mouse leptin cDNA. Treatment resulted in dramatic reductions in both food intake and body weight, as well as the normalization of serum insulin levels and glucose tolerance. The subsequent diminishment in serum leptin levels resulted in the rapid resumption of food intake and a gradual gain of body weight, which correlated with the gradual return of hyperinsulinemia and insulin resistance. These results not only demonstrated that the obese and diabetic phenotypes in the adult ob/ob mice are corrected by leptin gene treatment but also provide confirming evidence that body weight control may be critical in the long-term management of non-insulin-dependent diabetes mellitus in obese patients.

RFLP discordance in a PKU family due to a deletion in the PAH gene.

We have previously reported preliminary data on a PKU family showing a discordant segregation of Pvu II (b) alleles at the PAH locus. A combination of several restriction enzymes and probe C2.6 (Intron 2) as well as STR typing were used to dissect the molecular structure of the PAH gene around exon 3. In this family, the results of this analysis and a re-examination of the physical map of the 5'-end of the gene provided strong evidence for the occurrence of a deletion removing exon 3. The "Sicilian" (approximately 2.5 kb) and "Yemenite Jew" (6.7 kb) deletions, the latter one also deleting exon 3, are different in terms of both the 5'-end breakpoint and apparent length. This study, besides adding a new member to the long and increasing list of nearly 200 PAH gene mutations, also proposes to undertake a careful evaluation of RFLP discordances incidentally detected at the PAH locus.

Evaluation of promoter strength for hepatic gene expression in vivo following adenovirus-mediated gene transfer.

Transgene expression in studies of both gene function and gene therapy may be assisted considerably through the use of transcriptional regulatory elements which permit high-level, and/or tissue-specific gene expression. We have therefore evaluated the transcriptional activities of a series of viral and cellular enhancer/promoter elements, both in vitro and in vivo. The five enhancer/promoter elements showing either high-level or hepatocyte-specific expression following transient transfection into hepatoma cells were incorporated into recombinant adenoviruses expressing human alpha 1-antitrypsin (hAAT) for in vivo studies in the liver of immunodeficient and immunocompetent mice. The human elongation factor 1 alpha gene promoter produced 2 mg/ml serum level of hAAT, which is physiologic in humans and will be therapeutic for patients with AAT deficiency. This and all other enhancer/promoters except that of the CMV-IE gene yielded persistent hAAT expression in SCID mice. These findings demonstrate that adenovirus vectors provide an effective system for studies designed to evaluate enhancer/promoter activities in vivo. Several of the enhancer/promoters examined in this study will have significant utility in adenovirus-mediated gene therapy for alpha 1-antitrypsin deficiency and other genetic disorders.

A simple and efficient method for the concentration and purification of recombinant retrovirus for increased hepatocyte transduction in vivo.

Although recombinant retroviruses have been widely used for the transduction of target organs in vivo, the viral titers achieved by current production methods are often too low to achieve therapeutic levels of gene expression. To overcome this limitation, a simple method for the efficient concentration and purification of amphotropic retrovirus particles was developed. After portal vein infusion into partially hepatectomized rats of 5.5 x 10(7) cfu of a beta-galactosidase (beta-gal)-expressing retrovirus (LX/beta geo) concentrated by this method, up to 25% of hepatocytes stained positive for beta-Gal activity. Measurement of human alpha 1-antitrypsin (hAAT) levels after infusion of various doses of a similarly concentrated retrovirus encoding hAAT (LX/hAAT) demonstrated that viral transduction increased proportionally with titer, up to a dose of 7.5 x 10(7) cfu per rat. The ability to concentrate retroviral virion efficiently from large volumes of supernatant has allowed the further purification of virus particles by sucrose banding ultracentrifugation. This procedure results in a greater than 50% recovery of infectious virus particles, with titers up to 500-fold higher than in the original supernatant. These methods may have significant utility in both ex vivo and in vivo retroviral applications in human gene therapy.

Gene therapy for phenylketonuria.

Classical phenylketonuria (PKU) is an autosomal recessive disorder caused by a deficiency of hepatic phenylalanine hydroxylase (PAH). Limitations of the current dietary treatment for PKU have led to the development of potential treatments based on somatic gene transfer. Three different vector systems have been examined. Vectors derived from a recombinant retrovirus or a DNA/protein complex can efficiently transduce the PAH cDNA into PAH-deficient hepatocytes in vitro, but the application of these vector systems is presently limited by their low transduction efficiency in vivo. In contrast, a vector derived from a recombinant adenovirus can restore 10%-80% of normal hepatic PAH activity into PAH-deficient mice, which completely normalizes serum phenylalanine levels. This treatment is transient and cannot be effectively re-administered due to the presence of neutralizing antibodies directed against the recombinant adenoviral vector. However, these findings suggest that PKU can be completely corrected by somatic gene therapy, and provide some direction for the future development of adenoviral vectors.

Phenylketonuria in Costa Rica: preliminary spectrum of PAH mutations and their associations with highly polymorphic haplotypes.

A preliminary evaluation of the molecular basis of phenylketonuria (PKU) in Costa Rica was made by performing mutational analyses in the six PKU families identified to date. These studies revealed the presence of the previously reported European mutations IVS1nt5, L48S, E221G and IVS12ntl as well as the novel mutation IVS7nt3. The combined use of the STR, VNTR and XmnI polymorphic systems for the PAH gene resulted in a discriminant distribution of haplotypes among normal and mutant chromosomes and suggests its potential usefulness for future diagnostic applications in Costa Rican PKU kindreds. This is the first report of a genetic analysis in a Central American PKU population.

Molecular basis of phenylketonuria and a correlation between genotype and phenotype in a heterogeneous southeastern US population.

OBJECTIVE: To determine the molecular basis of phenylketonuria (PKU) and related hyperphenylalaninemia (HPA) and to establish correlations between phenylalanine hydroxylase (PAH) genotypes and biochemical and clinical phenotypes in an ethnically diverse US population, PAH genotypes were determined in 35 patients with PKU or HPA and 1 carrier from the Medical Genetics Clinic of the Emory University School of Medicine. METHODOLOGY: Patients were identified through Georgia's population-based newborn screening program. PAH genotypes in these individuals were determined from dried blood spots or whole-blood samples using a combination of polymerase chain reaction-mediated amplification, denaturing gradient gel electrophoresis, and direct-sequence analysis. The phenotypic severity of patients with PKU and HPA was based on pretreatment serum phenylalanine (PHE) levels during the neonatal period and on dietary tolerance of PHE later in life. RESULTS: Sixty-eight (96%) of 71 mutant alleles were identified. Major mutations in this population included R408W (11 of 71), I65T (11 of 71), Y414C (6 of 71), L348V (4 of 71), and IVS10 (4 of 71). Five new nucleotide substitutions, E76A (1 of 71), R241L (1 of 71), Q304R (2 of 71), C334S (1 of 71), and R400R (2 of 71) were also detected. Thirty-two of the thirty-five nonrelated patients examined in this study were completely genotyped. Strong correlations were observed between the level of PAH activity predicted from the genotype, when known from previous in vitro expression studies of the mutant proteins, and pretreatment serum PHE levels (r = .841) or clinical severity (Kendall rank-order correlation coefficient, .936). CONCLUSIONS: These results demonstrate strong correlations between PAH genotype and biochemical and clinical phenotypes in this heterogeneous American sample population, extending our previous findings from relatively homogeneous European populations. These correlations further demonstrate the clinical utility of genotype analysis in the treatment of patients with PKU and HPA.

Lack of persistence of E1- recombinant adenoviral vectors containing a temperature-sensitive E2A mutation in immunocompetent mice and hemophilia B dogs.

Two recombinant adenoviruses expressing either human alpha 1-antitrypsin (hAAT) or canine factor IX (cFIX) were modified so that they also contained a temperature-sensitive mutation (ts125) in the DNA binding protein encoded within the viral E2A region. The effects of the inclusion of the ts125 mutation on transgene expression in vivo were evaluated in Balb/c mice and hemophilia B dogs by comparison with adenoviral vectors containing the same transgene but lacking the ts125 mutation. No significant differences in the duration of transgene expression were observed in either animal model. Insufficiency of the ts125 mutation in the prolongation of transgene expression in these two animal models suggests that further modification of the vector backbone may be required to achieve long-term gene expression in a wide variety of applications. Additionally, humoral immune response to transgene products has been demonstrated in immunocompetent animal models, which will also need to be surmounted for long-term efficacy in disease treatment by gene therapy.

Adenovirus-mediated gene transfer into dissociated and explant cultures of rat hippocampal neurons.

Genetic manipulation offers great potential for studying the molecular and cellular processes which control or regulate the complex developmental properties of neurons. Gene transfer into neurons, however, is notoriously difficult. In this study we have used a replication-defective adenovirus (Adv/RSV beta gal), expressing beta-galactosidase (beta-gal) as a reporter gene, to infect dissociated cultures of rat hippocampal neurons and hippocampal slice cultures. Because future studies will require either long-term (e.g., developmental) or short-term (e.g., electrophysiological) expression of recombinant genes in neuronal cultures, we have optimized infection conditions for each situation. The Adv/RSV beta gal construct infects neurons and glial cells equally well, with no apparent alterations in cellular morphology. In slice cultures, the same efficiency and temporal control of beta-gal expression following Adv/RSV beta gal infection was achieved. Focal application of the adenoviruses, by microinjection, permitted infection of discrete subregions within the hippocampal explants. Whole cell recordings of dissociated hippocampal neurons and field recordings from the explant cultures, infected with Adv/RSV beta gal at low multiplicities of infection, indicated no significant alteration in the electrophysiological profiles of neurons in these cultures. The results demonstrate the utility of adenoviruses as gene transfer vectors for primary cultures of neurons. Adenovirus-mediated gene transfer into slice cultures also provides an opportunity to study development or plasticity in an environment where the circuitry and cytoarchitecture of the tissue are preserved and the areas of genetic manipulation can be spatially isolated.

Somatic gene therapy for phenylketonuria and other hepatic deficiencies.

Gene therapy is the delivery of genetic material to specific cell types of an organism to alter its physiology or function. This technology is being explored as a means of treating diseases caused by deficiencies of hepatic gene products. The two diseases being used as models for hepatic gene therapy are classical phenylketonuria (PKU) and haemophilia B. Vectors derived from adenoviruses can be used to completely correct these diseases in animal models. The phenotypic correction generated in these studies is transient, and cannot be duplicated by vector readministration. The transient nature of transgene expression results from the destruction of the virally-transduced cells by a cellular immune response directed against the late viral gene products that are also expressed in the target cells. The inability to repeatedly administer virus is caused by a humoral immune response directed against viral proteins present at the time of infusion. If the host immune response is suppressed, transgene expression can persist for 6 months or more. These findings suggest that host immunomodulation in combination with further modification of the adenoviral vector to reduce or eliminate late viral gene expression may permit long-term expression of potentially therapeutic gene products in mammalian liver.

Gene therapy for hemophilia B: host immunosuppression prolongs the therapeutic effect of adenovirus-mediated factor IX expression.

Hemophilia B is caused by a deficiency of blood clotting factor IX (FIX). Previous studies have shown that the delivery of a recombinant adenoviral vector expressing canine FIX (cFIX) resulted in a complete correction of hemophilia B in FIX-deficient dogs, but that cFIX expression decreased to only about 1-2% of normal levels 3 weeks after treatment. In the present study, therapeutic levels of cFIX expression capable of producing a partial correction of hemophilia B were maintained for at least 6 months after the coadministration of the cFIX-expressing adenovirus and the immunosuppressive agent cyclosporin A (CsA). These findings support a recent report (Yang et al., 1994) that host T-cell-mediated immunity against virally transduced cells is a major contributing factor to the transient nature of adenovirus-mediated gene expression in immunocompetent animals. Although a second administration of the cFIX-expressing adenovirus 6 months after the first infusion had only a minimal effect on plasma FIX levels in a dog that had been continuously treated with CsA, the prolonged expression of the transgene indicates that immunosuppression may be applicable in attaining long-term treatment of clinically relevant disorders.

In vivo gene therapy for hyperlipidemia: phenotypic correction in Watanabe rabbits by hepatic delivery of the rabbit LDL receptor gene.

Elevations of plasma total or LDL cholesterol are major risk factors for cardiovascular disease. Efforts directed at preventing and treating cardiovascular disease have often focused on reducing the levels of these substances in the blood. The Watanabe Heritable Hyperlipidemic Rabbit, which has exceedingly high plasma cholesterol levels resulting from an LDL receptor deficiency, provides an excellent animal model for testing new treatments. A recombinant adenoviral vector containing the rabbit LDL receptor cDNA was administered to Watanabe rabbits. Plasma total cholesterol levels in the treated animals were reduced from 825.5 +/- 69.8 (mean +/- SD) to 247.3 +/- 61.5 mg/dl 6 d after infusion. These animals also demonstrated a 300-400% increase in plasma levels of HDL cholesterol and apo AI 10 d after treatment. As a result, the LDL:HDL ratio exhibited a dramatic decrease. Because only the rabbit LDL receptor gene was used for treatment, the results strongly suggest that the elevations of plasma HDL cholesterol and apo AI were secondary to a reduction in plasma total cholesterol in the treated animals. These results suggest an inverse relationship between plasma LDL and HDL cholesterol levels and imply that reduction of LDL cholesterol levels may have a beneficial effect on plasma HDL cholesterol.