Steven E. Raper

Recombinant adeno-associated virus for muscle directed gene therapy.

Although gene transfer with adeno-associated virus (AAV) vectors has typically been low, transduction can be enhanced in the presence of adenovirus gene products through the formation of double stranded, non-integrated AAV genomes. We describe the unexpected finding of high level and stable transgene expression in mice following intramuscular injection of purified recombinant AAV (rAAV). The rAAV genome is efficiently incorporated into nuclei of differentiated muscle fibers where it persists as head-to-tail concatamers. Fluorescent in situ hybridization of muscle tissue suggests single integration sites. Neutralizing antibody against AAV capsid proteins does not prevent readministration of vector. Remarkably, no humoral or cellular immune responses are elicited to the neoantigenic transgene product E. coli β-galactosidase. The favorable biology of rAAV in muscle-directed gene therapy described in this study expands the potential of this vector for the treatment of inherited and acquired diseases.

A pilot study of ex vivo gene therapy for homozygous familial hypercholesterolaemia

The outcome of the first pilot study of liver-directed gene therapy is reported here. Five patients with homozygous familial hypercholesterolaemia (FH) ranging in age from 7 to 41 years were enrolled; each patient tolerated the procedure well without significant complications. Transgene expression was detected in a limited number of hepatocytes of liver tissue harvested four months after gene transfer from all five patients. Significant and prolonged reductions in low density lipoprotein (LDL) cholesterol were demonstrated in three of five patients; in vivo LDL catabolism was increased 53% following gene therapy in a receptor negative patient, who realized a reduction in serum LDL equal to ∼150 mg dl−1. This study demonstrates the feasibility of engrafting limited numbers of retrovirus-transduced hepatocytes without morbidity and achieving persistent gene expression lasting at least four months after gene therapy. The variable metabolic responses observed following low-level genetic reconstitution in the five patients studied precludes a broader application of liver-directed gene therapy without modifications that consistently effect substantially greater gene transfer.

A Pilot Study of In Vivo Liver-Directed Gene Transfer with an Adenoviral Vector in Partial Ornithine Transcarbamylase Deficiency

Ornithine transcarbamylase deficiency (OTCD) is an inborn error of urea synthesis that has been considered as a model for liver-directed gene therapy. Current treatment has failed to avert a high mortality or morbidity from hyperammonemic coma. Restoration of enzyme activity in the liver should suffice to normalize metabolism. An E1- and E4-deleted vector based on adenovirus type 5 and containing human OTC cDNA was infused into the right hepatic artery in adults with partial OTCD. Six cohorts of three or four subjects received 1/2 log-increasing doses of vector from 2 x 10(9) to 6 x 10(11) particles/kg. This paper describes the experience in all but the last subject, who experienced lethal complications. Adverse effects included a flu-like episode and a transient rise in temperature, hepatic transaminases, thrombocytopenia, and hypophosphatemia. Humoral responses to the vector were seen in all research subjects and a proliferative cellular response to the vector developed in apparently naive subjects. In situ hybridization studies showed transgene expression in hepatocytes of 7 of 17 subjects. Three of 11 subjects with symptoms related to OTCD showed modest increases in urea cycle metabolic activity that were not statistically significant. The low levels of gene transfer detected in this trial suggest that at the doses tested, significant metabolic correction did not occur.

Preoperative eating behavior, postoperative dietary adherence, and weight loss after gastric bypass surgery

BACKGROUND To investigate the relationship between preoperative eating behavior, postoperative dietary adherence and weight loss after gastric bypass surgery in a major, urban medical center with a comprehensive bariatric surgery program. Despite the significant weight loss and dramatic improvements in co-morbidities associated with bariatric surgery, a significant minority of patients appear to experience suboptimal weight loss. The reasons for this are not well understood, but the suboptimal weight loss is often attributed to preoperative psychosocial characteristics and/or eating behaviors, as well as poor adherence to the recommended postoperative diet. METHODS A prospective investigation was performed of 200 female and male patients who were studied both preoperatively and 20, 40, 66, and 92 weeks postoperatively. All patients underwent either open or laparoscopic Roux-en-Y gastric bypass surgery. The measures were the percentage of weight loss, macronutrient intake, dietary adherence, and eating behavior. RESULTS Gender, baseline cognitive restraint, and self-reported adherence to the postoperative diet at postoperative week 20 were associated with the percentage of weight loss at postoperative week 92. Those high in dietary adherence had lost 4.5% more weight at postoperative week 92 than those low in dietary adherence. CONCLUSION Baseline cognitive restraint and adherence to the recommended postoperative diet were associated with the percentage of weight loss after gastric bypass surgery. These results suggest the potential utility of pre- and/or postoperative dietary counseling interventions to improve the postoperative outcomes.

Changes in quality of life and body image after gastric bypass surgery

BACKGROUND Improvements in psychosocial status are an important aspect of successful outcomes after bariatric surgery. Relatively few studies have investigated the changes in psychosocial functioning at a number of points in the first few postoperative years. The present study was undertaken to assess the changes in quality of life and body image after gastric bypass surgery. The present study was performed at an academic medical center. METHODS A total of 200 men and women were enrolled in the study and completed psychometric measures of quality of life and body image before surgery and again 20, 40, and 92 weeks postoperatively. RESULTS The participants reported significant improvements in several domains of health- and weight-related quality of life, as well as changes in body image, after surgery. These changes were correlated with the percentage of weight loss. CONCLUSION Those who undergo gastric bypass surgery experienced significant improvements in quality of life and body image within the first few months after surgery. These changes were, with few exceptions, maintained into the second postoperative year.

Comparison of Psychosocial Status in Treatment-Seeking Women with Class III vs. Class I–II Obesity

Objective: This study compared the psychosocial status and weight loss expectations of women with extreme (class III) obesity who sought bariatric surgery with those of women with class I–II obesity who enrolled in a research study on behavioral weight control.

Gene Transfer into the Liver of Nonhuman Primates with E1-Deleted Recombinant Adenoviral Vectors: Safety of Readministration

Preclinical studies were designed to investigate the safety of recombinant adenoviruses infused into the portal vein of adult rhesus monkeys, as well as the safety and efficacy of readministration of these agents. The vectors used were recombinant adenoviruses, the E1 region of which was replaced with a marker gene expression cassette. Four 3- to 5-kg rhesus monkeys underwent portal vein cannulation, and infusion of escalating doses of recombinant first-generation vector. Serial sequential liver biopsies were performed, and necropsies were performed out to 14 months. X-Gal histochemical analysis of the liver showed evidence of dose-dependent increased gene transfer throughout the liver. Quantitative analysis of histopathology showed that portal inflammation was also present in transduced livers, and occurred in a dose-dependent manner. Severe toxicity, including mortality, was noted at the highest dose of vector. Readministration of a second vector was associated with the same degree of toxicity as the first vector, but prompted a much more vigorous neutralizing antibody response. The data suggest that intraportal administration and readministration of recombinant adenoviral E1-deleted vectors are feasible and safe. Vector administration at the highest dose (1 x 10(13) particles/kg) was associated with severe clinical and biochemical toxicity, and significant gene expression was associated with transaminitis. Readministration of vector is safe, but gene transfer is limited by the presence of neutralizing antibody.

Gene transfer to the thymus. A means of abrogating the immune response to recombinant adenovirus.

ObjectiveThe authors investigated whether adenoviral gene transfer to the thymus could be accomplished in vivo and whether immunologic unresponsiveness to recombinant adenovirus could be induced by intrathymic inoculation. Summary Background DataA major barrier to the clinical application of adenovirus-mediated gene therapy for diseases requiring long-lasting gene expression is the immunogenicity of adenoviral vectors, which limits the duration of its effects. In other experimental models, intrathymic inoculation of foreign proteins or cells has proven to be an effective means to induce immunologic tolerance. MethodsThe efficiency of gene transfer to the mouse thymus after direct inoculation of recombinant adenovirus was compared with that of several other vectors. Animals inoculated with adenovirus-infected pancreatic islets into the thymus were tested for unresponsiveness to the virus with a subsequent challenge of adenovirus administered into the liver by intravenous injection. ResultsAdenovirus accomplished highly efficient gene transfer to the thymus, unlike plasmid DNA, DNA-liposome complexes, retrovirus, and adeno-associated virus. Adenoviral transgene expression was transient in the thymus of immunocompetent mice but persistent in CD8+ T-cell-deficient and severe combined immunodeficiency (SCID) mice, implicating the role of cytotoxic T lymphocytes in viral clearance. Intrathymic transplantation of syngeneic pancreatic islet cells infected with adenovirus impaired the normal antiviral cytotoxic T-lymphocyte response and prolonged hepatic transgene expression after an intravenous challenge with adenovirus. ConclusionsRecombinant adenovirus accomplishes highly efficient gene transfer to the thymus in vivo. Intrathymic inoculation of adenovirus-infected islets can be used to induce immunologic unresponsiveness to the adenoviral vector and, potentially, to other proteins that it might be engineered to encode.

Obstructive sleep apnea in patients undergoing bariatric surgery--a tertiary center experience.

BackgroundThe patient population that is evaluated for bariatric surgery is characterized by a very high body mass index (BMI). Since obesity is the most important risk factor for obstructive sleep apnea (OSA), sleep disordered breathing is highly prevalent in this population. If undiagnosed before bariatric surgery, untreated OSA can lead to perioperative and postoperative complications. Debate exists whether all patients that are considered for bariatric surgery should undergo polysomnography (PSG) evaluation and screening for OSA as opposed to only those patients with clinical history or examination concerning sleep disordered breathing. We examined the prevalence and severity of OSA in all patients that were considered for bariatric surgery. We hypothesized that, by utilizing preoperative questionnaires (regarding sleepiness and OSA respiratory symptoms) in combination with menopausal status and BMI data, we would be able to predict which subjects did not have sleep apnea without the use of polysomnography. In addition, we hypothesized that we would be able to predict which subjects had severe OSA (apnea–hypopnea index (AHI) > 30).MethodsThree hundred forty-two consecutive subjects, evaluated for bariatric surgery from November 1, 2005 to January 31, 2007 underwent overnight polysomnography and completed questionnaires regarding sleepiness, menopausal status, and respiratory symptoms related to OSA. Apneas and hypopneas were classified as follows: mild apnea 5 ≤ AHI ≤ 15, moderate apnea 15 < AHI ≤ 30, and severe apnea AHI > 30.ResultsThe overall sample prevalence of OSA was 77.2%. Of these, 30.7% had mild OSA; 19.3% had moderate OSA, and 27.2% had severe OSA. Among men, the prevalence of OSA was 93.6% and 73.5% among women. The mean AHI (events per hour) for men with OSA was 49.2 ± 35.5 and 26.3 ± 28.3 for women with OSA. Separate logistic regression models were developed for the following three outcomes: AHI ≥ 5 events per hour, AHI > 15 events per hour, and AHI > 30 events per hour. When predicting these three levels of OSA severity, the area under the curve (AUC) values were: 0.8, 0.72, and 0.8, respectively. The negative predictive value for the presence of sleep apnea (AHI ≥ 5) was 75% when using the most stringent possible cutoff for the prediction model.ConclusionsThe prevalence of OSA in all patients considered for bariatric surgery was greater than 77%, irrespective of OSA symptoms, gender, menopausal status, age, or BMI. The prediction model that we developed for the presence of OSA (AHI ≥ 5 events per hour) has excellent discriminative ability (evidenced by an AUC value of 0.8). However, the negative prediction values for the presence of OSA were too low to be clinically useful due to the high prevalence of OSA in this high-risk group. We demonstrated that, by utilizing even the most stringent possible cutoff values for the prediction model, OSA cannot be predicted with enough certainty. Therefore, we advocate routine PSG testing for all patients that are considered for bariatric surgery.

Readministration of Adenovirus Vector in Nonhuman Primate Lungs by Blockade of CD40-CD40 Ligand Interactions

ABSTRACT The interaction between CD40 on B cells and CD40 ligand (CD40L) on activated T cells is important for B-cell differentiation in T-cell-dependent humoral responses. We have extended our previous murine studies of CD40-CD40L in adenoviral vector-mediated immune responses to rhesus monkeys. Primary immune responses to adenoviral vectors and the ability to readminister vector were studied in rhesus monkeys in the presence or absence of a transient treatment with a humanized anti-CD40 ligand antibody (hu5C8). Adult animals were treated with hu5C8 at the time vector was instilled into the lung. Immunological analyses demonstrated suppression of adenovirus-induced lymphoproliferation and cytokine responses (interleukin-2 [IL-2], gamma interferon, IL-4, and IL-10) in hu5C8-treated animals. Animals treated with hu5C8 secreted adenovirus-specific immunoglobulin M (IgM) levels comparable to control animals, but did not secrete IgA or develop neutralizing antibodies; consequently, the animals could be readministered with adenovirus vector expressing alkaline phosphatase. A second study was designed to examine the long-term effects on immune functions of a short course of hu5C8. Acute hu5C8 treatment resulted in significant and prolonged inhibition of the adenovirus-specific humoral response well beyond the time hu5C8 effects were no longer significant. These studies demonstrate the potential of hu5C8 as an immunomodulatory regimen to enable administration of adenoviral vectors, and they advocate testing this model in humans.