Biomedical subjects

S Connelly

Publications and source records attributed to S Connelly.

At least 19 recordsLinked to original sources

PubMed2001

Adenovirus-mediated factor VIII gene expression results in attenuated anti-factor VIII-specific immunity in hemophilia A mice compared with factor VIII protein infusion.

Hemophilia A patients are typically treated by factor VIII (FVIII) protein replacement, an expensive therapy that induces FVIII-specific inhibitors in approximately 30% of patients with severe hemophilia. FVIII gene therapy has the potential to improve the current treatment protocols. In this report, we used a hemophilia A mouse model to compare the humoral and cellular immune responses between an E1/E2a/E3-deficient adenovirus expressing human FVIII directed by a liver-specific albumin promoter and purified recombinant FVIII protein infusion. Adenovirus-mediated FVIII expression did not elicit detectable CD4+ or CD8+ T cell responses and induced a weak antibody immune response to FVIII. In contrast, FVIII protein administration resulted in a potent anti-FVIII antibody response and moderate CD4+ T cell response. Furthermore, hemophiliac mice preimmunized with FVIII protein infusion to induce anti-FVIII immunity, and subsequently treated by adenovirus-mediated FVIII gene therapy, expressed therapeutic levels of FVIII despite the presence of low levels of anti-FVIII antibodies. No FVIII was detected in the plasma of mice with intermediate or high antibody levels, although anti-FVIII antibody levels in some vector-treated animals declined. The data support the hypothesis that liver-specific gene therapy-mediated expression of FVIII may be less immunogenic than traditional protein replacement therapy.

Adenoviridae↗

PubMed2001

Systemic delivery of an adenoviral vector encoding canine factor VIII results in short-term phenotypic correction, inhibitor development, and biphasic liver toxicity in hemophilia A dogs.

Canine hemophilia A closely mimics the human disease and has been used previously in the development of factor VIII (FVIII) protein replacement products. FVIII-deficient dogs were studied to evaluate an in vivo gene therapy approach using an E1/E2a/E3-deficient adenoviral vector encoding canine FVIII. Results demonstrated a high level of expression of the canine protein and complete phenotypic correction of the coagulation defect in all 4 treated animals. However, FVIII expression was short-term, lasting 5 to 10 days following vector infusion. All 4 dogs displayed a biphasic liver toxicity, a transient drop in platelets, and development of anticanine FVIII antibody. Canine FVIII inhibitor development was transient in 2 of the 4 treated animals. These data demonstrate that systemic delivery of attenuated adenoviral vectors resulted in liver toxicity and hematologic changes. Therefore, the development of further attenuated adenoviral vectors encoding canine FVIII will be required to improve vector safety and reduce the risk of immunologic sequelae, and may allow achievement of sustained phenotypic correction of canine hemophilia A.

Adenoviridae↗

PubMed2001

Generation and characterization of E1/E2a/E3/E4-deficient adenoviral vectors encoding human factor VIII.

The use of adenoviral vectors for gene therapy has been limited due to host immune responses directed toward the vector and/or transgene and vector toxicity. To decrease adenoviral vector immunogenicity and toxicity, we attenuated viral gene expression by eliminating E1, E2a, E3, and E4 early genes from the adenoviral backbone. Two highly attenuated, fourth-generation (Av4) E1/E2a/E3/E4-deficient adenoviral vectors encoding human factor VIII (FVIII) under the control of a liver-specific albumin promoter were generated. One Av4 vector (Av4DeltaE4FVIII) was deficient in the entire E4 coding region and the second vector contained a deletion of the E4 region, except for open reading frame 3 (orf 3; Av4orf3FVIII). The Av4 vectors were compared to an E1/E2a/E3-deficient third-generation vector (Av3H8101) containing an analogous transgene expression cassette in vitro and in vivo following intravenous administration in hemophiliac mice. In vitro transduction of Hep3B cells revealed at all three vectors expressed functional FVIII. However, the Av4DeltaE4FVIII vector could not be scaled-up for in vivo studies. Both Av3H8101 and Av4orf3FVIII initially expressed similar levels of FVIII in hemophiliac mice. However, at 3 months, animals treated with the Av4orf3FVIII vector no longer expressed FVIII while Av3H8101-treated mice displayed persistent FVIII expression. Liver enzyme analyses of plasma samples revealed that the Av4orf3FVIII vector was significantly less hepatotoxic than the Av3H8101 vector. These data demonstrate that further attenuation of the adenoviral vector backbone by removal of the majority of the E4 coding region significantly diminished vector toxicity; however, the duration of transgene expression was reduced.

Adenovirus E1 Proteins↗

PubMed2001

Phenotypic correction of diabetic mice by adenovirus-mediated glucokinase expression.

Hyperglycemia of diabetes is caused in part by perturbation of hepatic glucose metabolism. Hepatic glucokinase (GK) is an important regulator of glucose storage and disposal in the liver. GK levels are lowered in patients with maturity-onset diabetes of the young and in some diabetic animal models. Here, we explored the adenoviral vector-mediated overexpression of GK in a diet-induced murine model of type 2 diabetes as a treatment for diabetes. Diabetic mice were treated by intravenous administration with an E1/E2a/E3-deleted adenoviral vector encoding human hepatic GK (Av3hGK). Two weeks posttreatment, the Av3hGK-treated diabetic mice displayed normalized fasting blood glucose levels (95 +/- 4.8 mg/dl; P < 0.001) when compared with Av3Null (135 +/- 5.9 mg/dl), an analogous vector lacking a transgene, and vehicle-treated diabetic mice (134 +/- 8 mg/dl). GK treatment also resulted in lowered insulin levels (632 +/- 399 pg/ml; P < 0.01) compared with the control groups (Av3Null, 1,803 +/- 291 pg/ml; vehicle, 1,861 +/- 392 pg/ml), and the glucose tolerance of the Av3hGK-treated diabetic mice was normalized. No significant increase in plasma or hepatic triglycerides, or plasma free fatty acids was observed in the Av3hGK-treated mice. These data suggest that overexpression of GK may have a therapeutic potential for the treatment of type 2 diabetes.

Adenoviridae↗

PubMed2001

Treatment of type 2 diabetes by adenoviral-mediated overexpression of the glucokinase regulatory protein.

The enzyme glucokinase (GK) plays a central role in glucose homeostasis. Hepatic GK activity is acutely controlled by the action of the GK regulatory protein (GKRP). In vitro evidence suggests that GKRP reversibly binds to GK and inhibits its activity; however, less is known about the in vivo function of GKRP. To further explore the physiological role of GKRP in vivo, we used an E1/E2a/E3-deficient adenoviral vector containing the cDNA encoding human GKRP (Av3hGKRP). High fat diet-induced diabetic mice were administered Av3hGKRP or a control vector lacking a transgene (Av3Null). Surprisingly, the Av3hGKRP-treated mice showed a significant improvement in glucose tolerance and had lower fasting blood glucose levels than Av3Null-treated mice. A coincident decrease in insulin levels indicated that the Av3hGKRP-treated mice had sharply improved insulin sensitivity. These mice also exhibited lower leptin levels, reduced body weight, and decreased liver GK activity. In vitro experiments indicated that GKRP was able to increase both GK protein and enzymatic activity levels, suggesting that another role for GKRP is to stabilize and/or protect GK. These data are the first to indicate the ability of GKRP to treat type 2 diabetes and therefore have significant implications for future therapies of this disease.

Adaptor Proteins, Signal Transducing↗

PubMed2000

In vivo dose threshold effect of adenovirus-mediated factor VIII gene therapy in hemophiliac mice.

While much is known about adenovirus biology from its development as a therapeutic gene delivery vehicle, an important question remains regarding the appropriate in vivo vector dose. We describe here an in vivo dose threshold effect with an adenoviral vector expressing human Factor VIII (FVIII) in hemophiliac mice. Upon administration of vector doses between 6 x 10(10) and 2 x 10(10) vector particles per mouse, FVIII was expressed linearly, whereas a dose of 1 x 10(10) vector particles per mouse did not result in detectable levels of FVIII activity. In contrast, in vitro transduction studies demonstrated linear transgene expression over 2 to 3 log units. To further define this dose threshold effect, a vector-mixing study was performed. Mice were injected with a total vector dose of 6 x 10(10) particles containing admixtures of FVIII vector plus a control vector lacking a transgene (null vector). With the admixture, FVIII activity was detected in mice that received 1 3 1010 particles of the FVIII vector, indicating that maintenance of the total viral input at 6 x 10(10) particles per mouse circumvented the threshold dose effect. This threshold dose effect could not be attributed to dose-dependent differences in liver toxicity nor to dose-dependent induction of cellular and humoral immune responses. Southern blot analysis of livers revealed that mice receiving the vector admixture contained FVIII DNA, accounting for the observed FVIII expression, whereas mice receiving 1 x 10(10) particles of FVIII vector had barely detectable FVIII DNA. These results suggest that the threshold effect is an in vivo phenomenon that will have important implications in defining the therapeutic window of adenoviral vectors for clinical applications.

Adenoviridae↗

PubMed1999

In vivo evaluation of an adenoviral vector encoding canine factor VIII: high-level, sustained expression in hemophiliac mice.

Hemophilia A is the most common severe hereditary coagulation disorder and is caused by a deficiency in blood clotting factor VIII (FVIII). Canine hemophilia A represents an excellent large animal model that closely mimicks the human disease. In previous studies, treatment of hemophiliac dogs with an adenoviral vector encoding human FVIII resulted in complete correction of the coagulation defect and high-level FVIII expression [Connelly et al. (1996). Blood 88, 3846]. However, FVIII expression was short term, limited by a strong antibody response directed against the human protein. Human FVIII is highly immunogenic in dogs, whereas the canine protein is significantly less immunogenic. Therefore, sustained phenotypic correction of canine hemophilia A may require the expression of the canine protein. In this work, we have isolated the canine FVIII cDNA and generated an adenoviral vector encoding canine FVIII. We demonstrate expression of canine FVIII in hemophiliac mice at levels 10-fold higher than those of the human protein expressed from an analogous vector. Canine FVIII expression was sustained above human therapeutic levels (50 mU/ml) for at least 1 year in hemophiliac mice.

Adenoviridae↗

PubMed1999

Efficient adenoviral vector transduction and expression of functional human factor VIII in cultured primary human hepatocytes.

Hemophilia A is a severe bleeding disorder caused by a deficiency in blood coagulation factor VIII (FVIII). Adenoviral vectors containing a potent human FVIII expression cassette encoding a truncated FVIII cDNA were developed that mediated sustained FVIII expression in normal and haemophiliac mice and complete phenotypic correction of the bleeding disorder in haemophiliac mice and dogs (Connelly and Kaleko, Haemophilia, 1998; 4: 380-8). Here, we evaluated two E1/E2a/E3-deleted adenoviral vectors encoding human FVIII, one containing the full-length cDNA and the second containing a truncated cDNA lacking the B-domain. Viral vectors encoding the human full-length FVIII cDNA have not been described previously. Hepatocyte transduction was efficient and dose dependent, ranging from 50% to 100%. High levels of functional FVIII were secreted from transduced cells at amounts up to 6000 mU-1 10(6)cells-1 60 h. B-domain deleted FVIII was expressed at levels at least 8-fold higher than the full-length FVIII protein, whereas FVIII RNA levels were similar with both vectors. These data provide the first demonstration of FVIII adenoviral vector function in primary human cells and verify the potential clinical utility of adenoviral vectors for the treatment of haemophilia A.

Adenoviridae↗

PubMed1999

In vivo evaluation of a novel epitope-tagged human factor VIII-encoding adenoviral vector.

Haemophilia A is caused by a deficiency in coagulation factor VIII (FVIII) and is an attractive target for gene therapy. Adenoviral vectors encoding a human B-domain deleted (BDD) FVIII cDNA have been shown previously to mediate expression of high levels of human FVIII and correct the bleeding defect in haemophiliac mice and dogs. While vector assessment in a non-human primate model would have a significant preclinical benefit, a haemophiliac non-human primate model is not available, and assays that distinguish human FVIII from monkey FVIII have not been developed successfully. As a first step to enable vector evaluation in non-human primates, we have constructed an epitope-tagged FVIII molecule by the addition of 16 amino-acids to the carboxy terminus of the BDD protein (BDD-E). Following vector administration to normal mice, therapeutic levels of BDD-E FVIII were expressed for at least 20 weeks. Treatment of haemophiliac mice revealed that the BDD-E protein was biologically active in vivo. To distinguish the BDD-E protein from non-human primate FVIII, a sensitive immunoprecipitation/Western assay was developed that reproducibly detected 1 ng mL-1 of the epitope-tagged human FVIII in the presence of monkey plasma. These data demonstrate that the addition of an epitope tag had no effect on FVIII function or immunogenicity, and suggest that the BDD-E vector will be an effective reagent for non-human primate studies.

Adenoviridae↗

PubMed1999

Evaluation of an adenoviral vector encoding full-length human factor VIII in hemophiliac mice.

Adenoviral vectors provide a promising gene therapy system for the treatment of hemophilia A. Potent vectors encoding a human factor VIII (FVIII) cDNA were developed that mediated sustained FVIII expression in normal and hemophiliac mice and complete phenotypic correction of the bleeding disorder in hemophiliac mice and dogs (Connelly and Kaleko, Haemophilia 1998; 4: 380-8). However, these studies utilized vectors encoding a truncated version of the human FVIII cDNA lacking the B-domain (BDD FVIII). In this work, an adenoviral vector encoding the human full-length (FL) FVIII cDNA was generated and characterized. While functional FL FVIII was secreted in vitro, expression of the FL protein was not detected in the plasma of vector-treated hemophiliac mice. Unexpectedly, the FL FVIII vector-treated animals demonstrated phenotypic correction of the bleeding defect as measured by a tail-clip survival study. FL FVIII protein was visualized in the mouse livers using human FVIII-specific immunohistochemical analyses. These data demonstrate that adenoviral vector-mediated in vivo expression of BDD FVIII is more efficient than that of the FL protein and that phenotypic correction can occur in the absence of detectable levels of FVIII.

Adenoviruses, Human↗

PubMed1999

Adenoviral vectors for liver-directed gene therapy.

Adenoviral vectors currently represent one of the most efficient means of in vivo hepatocyte gene delivery. Consequently, liver-directed gene transfer has been increasingly explored as a promising approach for the treatment of a diverse range of genetic and acquired diseases. Numerous demonstrations of efficacious adenoviral vector-mediated delivery of a wide array of transgenes in several animal species and humans have been reported. In general, transgene expression was efficient, but transient, in many cases lasting < 1 month. Currently, efforts in the field are focused on the development of highly attenuated adenoviral vectors designed to prolong transgene expression by reducing vector immunogenicity and hepatoxicity. Vector optimization strategies include the development of vectors devoid of all viral coding regions, the generation of chimeric vectors engineered to capitalize on favorable aspects of the component viral systems, the development of tissue-specific regulated gene expression, and the development of strategies to circumvent the host immune system. The use of adenoviral vectors for gene therapy of hereditary, malignant and infectious diseases of the liver, and the vector optimization strategies outlined above are discussed in this work.

Adenoviridae↗

PubMed1998

Sustained phenotypic correction of murine hemophilia A by in vivo gene therapy.

Hemophilia A is caused by a deficiency of blood coagulation factor VIII (FVIII) and has been widely discussed as a candidate for gene therapy. While the natural canine model of hemophilia A has been valuable for the development of FVIII pharmaceutical products, the use of hemophiliac dogs for gene therapy studies has several limitations such as expense and the long canine generation time. The recent creation of two strains of FVIII-deficient mice provides the first small animal model of hemophilia A. Treatment of hemophiliac mice of both genotypes with potent, human FVIII-encoding adenoviral vectors resulted in expression of biologically active human FVIII at levels, which declined, but remained above the human therapeutic range for over 9 months. The duration of expression and FVIII plasma levels achieved were similar in both hemophiliac mouse strains. Treated mice readily survived tail clipping with minimal blood loss, thus showing phenotypic correction of murine hemophilia A by in vivo gene therapy.

Adenoviridae↗

PubMed1998

Haemophilia A gene therapy.

Gene therapy for haemophilia A would represent a significant improvement over the current treatment by providing prophylactic expression of FVIII and correction of the coagulation defect. Furthermore, a gene therapy protocol allowing simple, infrequent vector administration may extend haemophilia treatment to remote locations world-wide that currently lack access to FVIII replacement therapy. Within the last half decade, significant progress has been made on the development of gene therapy for the treatment of haemophilia A. Recent achievements include high level clotting factor expression in mice, dogs, and monkeys as well as phenotypic correction in haemophiliac mice and dogs. With the efforts that are currently directed toward the improvement of gene transfer vectors and the development of technologies to enable sustained clotting factor expression, gene therapy for haemophilia A will ultimately become a reality.

Animals↗

PubMed1997

Hepatic transplantation in children under 3 months of age: a single centre's experience.

Liver transplantation in neonates represents a major medical and technical challenge particularly in babies weighing less than 5 kg. The authors report the experience of 10 liver transplants in 9 babies (6 boys and 3 girls), mean age, 6 weeks (range, 2 to 11); median weight, 3.7 kg (range, 2.4 to 5). All had fulminant hepatic failure caused by neonatal haemochromatosis (n = 3), non-A non-B hepatitis (n = 3), total parenteral nutrition induced (n = 1), hepatitis B (n = 1), and hepatic haemangio-endothelioma (n = 1). One child underwent retransplantation for hepatic artery thrombosis. Immunosuppression was by Cyclosporine A-based triple therapy in all cases. All received a reduced size graft consisting of left lobe (n = 1), left lateral segment (n = 6) and monosegment III (n = 3). In nine cases the donor hepatic artery was anastomosed to an iliac artery conduit from the infrarenal aorta, and a Roux loop was used for bile duct reconstruction. Primary abdominal wound closure was possible in six patients, skin closure alone in one, and a silastic patch was used in three. Complications included infection (n = 5), bowel perforation (n = 2), diaphragmatic perforation (n = 2), bile leak (n = 1), hepatic artery thrombosis (n = 1), and portal vein thrombosis (n = 1). None of the babies experienced acute rejection. Currently five of the nine recipients are alive with good graft function at a mean follow-up of 22 months (range, 5 to 58). Of the four deaths, two were related to infection (one in combination with portal vein thrombosis), one to multiorgan failure and fluid overload, and one to early graft dysfunction and sepsis after undergoing retransplantation for hepatic artery thrombosis. From our experience liver transplantation offers a promising option for the treatment of severe liver disease in children less than 3 months old.

Age Factors↗

PubMed1997

Multiplex sequencing of 1.5 Mb of the Mycobacterium leprae genome.

The nucleotide sequence of 1.5 Mb of genomic DNA from Mycobacterium leprae was determined using computer-assisted multiplex sequencing technology. This brings the 2.8-Mb M. leprae genome sequence to approximately 66% completion. The sequences, derived from 43 recombinant cosmids, contain 1046 putative protein-coding genes, 44 repetitive regions, 3 tRNAs, and 15 tRNAs. The gene density of one per 1.4 kb is slightly lower than that of Mycoplasma (1.2 kb). Of the protein coding genes, 44% have significant matches to genes with well-defined functions. Comparison of 1157 M. leprae and 1564 Mycobacterium tuberculosis proteins shows a complex mosaic of homologous genomic blocks with up to 22 adjacent proteins in conserved map order. Matches to known enzymatic, antigenic, membrane, cell wall, cell division, multidrug resistance, and virulence proteins suggest therapeutic and vaccine targets. Unusual features of the M. leprae genome include large polyketide synthase (pks) operons, inteins, and highly fragmented pseudogenes.

Amino Acid Sequence↗

PubMed1997

Gene therapy for hemophilia A.

Significant progress has been made on the development of gene therapy for the treatment of hemophilia A, a common bleeding disorder caused by subnormal levels of blood coagulation factor VIII (FVIII). Recent advances in gene transfer technology have enabled the expression of therapeutic to physiological levels of human FVIII in normal animals as well as hemophiliac mice and dogs. However, the in vivo persistence of FVIII expression was variable, ranging from one day to over five months. Despite recent advances in the development of hemophilia A gene transfer vectors, each still faces limitations to its clinical utility. Current research is focused on improving gene transfer vehicles and delivery methods to enable sustained clotting factor expression, treatment readministration, and circumvention of the host immune response to the treatment.

Cells, Cultured↗

PubMed1996

Complete short-term correction of canine hemophilia A by in vivo gene therapy.

Hemophilia A is a severe bleeding disorder caused by a deficiency in clotting factor VIII (FVIII). A canine model that closely mimics the human disease was used to determine if an adenoviral vector expressing a human FVIII cDNA could be used to correct the hemophilia A phenotype. Within 48 hours after peripheral vein administration of the vector to FVIII-deficient dogs, the hemophilic phenotype was corrected, based on determination of the activated clotting time, the activated partial thromboplastin time, and the cuticle bleeding time. Direct measurement of human FVIII in the dog plasma showed FVIII expression at amounts well above the human therapeutic level. FVIII expression in treated dogs was short-term, lasting 1 to 2 weeks, due to the development of a human FVIII-specific inhibitor antibody response. These data provide the first demonstration of in vivo gene therapy of hemophilia A.

Adenoviridae↗

PubMed1996

Screening for abdominal aortic aneurysms in a hypertensive patient population.

BACKGROUND: Abdominal aortic aneurysm (AAA) is an important cause of preventable death in the elderly. OBJECTIVE: To search for an effective screening program for the detection of AAA. METHODS: We consecutively and prospectively screened 240 male, predominantly white, patients, aged 60 to 75 years, by ultrasound for the presence of AAA in 2 phases: in screen 1, hypertension (diastolic blood pressure reading > 95 mm Hg) was the only selection criterion; in screen 2, we included hypertensive patients with a systolic blood pressure level higher than 175 mm Hg with or without antihypertensive therapy, transient ischemic attacks and/or stroke, and claudication. RESULTS: The yield of AAAs in screens 1 and 2 was 3% and 11%, respectively (P < .02, (chi)2 analysis). No AAAs were found in patients with uncomplicated hypertension. Of the cardiovascular complications, only claudication was independently associated with the presence of AAA (relative risk, 5.8; confidence interval, 1.8-18.6; P = .004). CONCLUSIONS: Screening for AAA is recommended for elderly white patients with claudication. Uncomplicated hypertension by itself is not an indication for screening.

Aged↗