Direct interaction of human serum proteins with AAV virions to enhance AAV transduction: immediate impact on clinical applications.

Recent hemophilia B clinical trials using adeno-associated virus (AAV) gene delivery have demonstrated much lower coagulation factor IX (FIX) production in patients compared with the high levels observed in animal models and AAV capsid-specific cytotoxic T lymphocyte response elicited at high doses of AAV vectors. These results emphasize the necessity to explore effective approaches for enhancement of AAV transduction. Initially, we found that incubation of all AAV vectors with human serum enhanced AAV transduction. Complementary analytical experiments demonstrated that human serum albumin (HSA) directly interacted with the AAV capsid and augmented AAV transduction. The enhanced transduction was observed with clinical grade HSA. Mechanistic studies suggest that HSA increases AAV binding to target cells, and that the interaction of HSA with AAV does not interfere with the AAV infection pathway. Importantly, HSA incubation during vector dialysis also increased transduction. Finally, HSA enhancement of AAV transduction in a model of hemophilia B displayed greater than a fivefold increase in vector-derived circulating FIX, which improved the bleeding phenotype correction. In conclusion, incubation of HSA with AAV vectors supports a universal augmentation of AAV transduction and, more importantly, this approach can be immediately transitioned to the clinic for the treatment of hemophilia and other diseases.

Efficient and stable transduction of cardiomyocytes after intramyocardial injection or intracoronary perfusion with recombinant adeno-associated virus vectors.

BACKGROUND: The delivery of recombinant genes to cardiomyocytes holds promise for the treatment of a variety of cardiovascular diseases. Previous gene transfer approaches that used direct injection of plasmid DNA or replication-defective adenovirus vectors have been limited by low transduction frequencies and transient transgene expression due to immune responses, respectively. In this report, we have tested the feasibility of using intramyocardial injection or intracoronary infusions of recombinant adeno-associated virus (rAAV) vectors to program transgene expression in murine cardiomyocytes in vivo. METHODS AND RESULTS: We constructed an rAAV containing the LacZ gene under the transcriptional control of the cytomegalovirus (CMV) promoter (AAVCMV-LacZ). We then injected 1x10(8) infectious units (IU) of this virus into the left ventricular myocardium of adult CD-1 mice. Control hearts were injected with the AdCMV-LacZ adenovirus vector. Hearts harvested 2, 4, and 8 weeks after AAVCMV-LacZ injection demonstrated stable beta-galactosidase (beta-gal) expression in large numbers of cardiomyocytes without evidence of myocardial inflammation or myocyte necrosis. In contrast, the AdCMV-LacZ-injected hearts displayed transient beta-gal expression, which was undetectable by 4 weeks after injection. Explanted C57BL/6 mouse hearts were also perfused via the coronary arteries with 1.5x10(9) IU of AAVCMV-LacZ and assayed 2, 4, and 8 weeks later for beta-gal expression. beta-Gal expression was detected in <1% of cardiomyocytes at 2 weeks after perfusion but was detected in up to 50% of cardiomyocytes 4 to 8 weeks after perfusion. CONCLUSIONS: Direct intramyocardial injection or coronary artery perfusion with rAAV vectors can be used to program stable transgene expression in cardiomyocytes in vivo. rAAV appears to represent a useful vector for the delivery of therapeutic genes to the myocardium.

Distribution of Isolates of Sclerotium rolfsii Tolerant to Pentachloronitrobenzene in Texas Peanut Fields.

The tolerance to pentachloronitrobenzene (PCNB) of an isolate of Sclerotium rolfsii collected in 1985 was quantified, and a survey of tolerance to PCNB in 377 other isolates of the fungus collected from Texas peanut fields from 1990 through 1994 was conducted. The effective dose (ED)50 of the previously collected PCNB-tolerant isolate was 11.07 µg PCNB/ml and was more than 5-fold greater than the ED50 of PCNB-sensitive isolates. The distribution of tolerance to PCNB among all isolates was slightly skewed, with 18 of the 377 isolates identified as having greater (P ≤ 0.05) tolerance to PCNB than the standard sensitive isolate. No isolate of S. rolfsii collected during the period of 1990 to 1994 had as high an ED50 value as did the 1985 isolate, even among those isolates collected from the same field from which the 1985 isolate was collected. ED50 values of two PCNB-sensitive and five PCNB-tolerant isolates were unchanged after 15 generations on potato dextrose agar amended with 10 µg PCNB/ml or on unamended media. The PCNB-tolerant isolate collected in 1985 was less aggressive than other isolates in greenhouse and microplot tests, but no correlation was observed between ED50 values and disease incidence in these tests for other PCNB-sensitive and tolerant isolates. These data suggest that even though high levels of tolerance to PCNB can be confirmed in some isolates of S. rolfsii, this phenomenon is likely to remain a rare event.

Packaging effective community service delivery: the utility of mandates and contracts in obtaining administrative cooperation.

Voluntary agreements, mandates, and contracts integrate networks of social service organizations, allowing them to function as coordinated wholes. The author reviews the history of contracting and mandating in the public sector. It is hypothesized that contracted relationships formalize agreements between local organizations dependent on others. Mandated relationships are perceived to be important by policy-makers at a state or federal level. The differential acceptance and rejection of these relationships in the community is explored. Data from social service agencies are used to compare administrators' assessments of the effectiveness of mandated and contracted relationships used to coordinate a group of agencies delivering services to children. When a mandated relationship has been formalized into a contract by a local administrator the perceived effectiveness of that relationship is higher than any other relationship in the community. If the mandated relationship has not been formalized by a contract this relationship is perceived to be the least effective. Important mandated inter-organizational ties without monetary incentives are less likely to work. Local administrators having developed the contracted ties see these ties as producing a higher level of performance.

A surgical technique for the correction of the hypermobile anterior maxillary ridge.

Surgical treatment of the hypermobile anterior maxillary ridge has had varied success. The most frequent problem of the prosthodontist has been that the loss of specialized masticatory mucosa has been too extensive. A surgical approach is presented that combines the removal of the flabby anterior maxillary tissue with an approximately 100% expansion of the zone of masticatory mucosa along the anterior maxilla.