Thomas M. Daly

Overview of adeno-associated viral vectors.

The use of adeno-associated virus (AAV) as a gene transfer vector has been steadily increasing over the past several years. AAV vectors have been particularly useful for applications where sustained gene expression is required. Prolonged in vivo expression following AAV treatment has been seen in the liver (1,2), brain (3,4), skeletal muscle (5,6), lung (7,8), and hematopoietic stem cells (9,10) of animal models. Therapeutic benefit from AAV treatment has been shown in a number of preclinical models of disease, including animal models of coagulopathies (11,12), lysosomal storage diseases (13,14), vision defects (15,16), and amino acid disorders. Clinical trials using AAV for the treatment of hemophilia B have begun, and early reports from these trials have been promising. In this introductory chapter to AAV, we will provide a brief overview of the molecular biology of this virus, an overview of methods of vector production, and a brief summary of the use of alternate AAV serotypes. The following chapters will then focus on specific methods and techniques for AAV transduction of the organs listed previously.

Utilization, Reliability, and Clinical Impact of Point-of-Care Testing during Critical Care Transport: Six Years of Experience

The use of point-of-care testing (POCT) has been reported in the setting of critical care transport (1)(2)(3)(4)(5)(6), although the overall benefits have not been evaluated in depth. In addition, problems with testing reliability may be uncovered only after an extended period of field use. This report describes the use of POCT by our critical care transport program over 6 years. All critical care transports made from January 1996 to December 2001 were reviewed. Transport vehicles were ambulances or twin-engine jets. The transport teams consisted of a physician on transport or with radio contact, a respiratory therapist, and a registered nurse. All transports were equipped with i-STAT® portable analyzers (i-STAT® Corporation) and disposable cartridges for testing. The analyzer and cartridges were stored in an insulated bag for temperature control during the trip. The analytical performance verification protocol (electronic controls) recommended in the i-STAT System Manual was followed before each patient test. Liquid controls were run monthly. Proficiency testing was completed in accordance with the requirements of the College of American Pathologists. The manufacturer’s test cartridges were the G3, 6+, EG7+, and glucose. Tests included pH, P co2, P o2, calculated bicarbonate, total CO2, base excess, oxygen saturation, sodium, potassium, chloride, urea, glucose, hematocrit, and calculated hemoglobin and glucose. Each cartridge requires 65 μL of whole blood for testing. The blood was drawn and analyzed by physician order. From 1997 through 2001, the team filled out an evaluation form for quality review after cases where POCT was performed. Patient test results and charts for each POCT episode were reviewed retrospectively to identify changes in treatment linked to test results. Other data were extracted from transport department and quality-control records. This research was approved by …

AAV-Mediated Gene Transfer to the Liver

The liver is a frequent target of gene-transfer experiments, because of its central role in many metabolic and synthetic pathways. For applications where prolonged expression of genes in the liver is required, adeno-associated virus (AAV) has proven to be an effective tool for in vivo gene transfer. High-level, persistent hepatic expression has been achieved in a number of experimental systems following a single treatment with AAV in murine and larger animal models. This prolonged expression is particularly useful for the treatment of genetic diseases such as the inborn errors of metabolism, where lifelong expression of the deficient enzyme may be required. Therapeutic benefits using AAV vectors have been demonstrated in animal models of amino acid disorders, lysosomal storage diseases, and coagulopathies (3-5), and Phase I clinical trials are proposed for the treatment of hemophilia B (6). Gene transfer to the murine liver using AAV is achieved by intravenous (iv) injection of recombinant virus, either via a peripheral or portal vein. The liver is the primary organ transduced following intravenous injection of AAV, although other tissues such as heart and lung may also take up virus to a lesser extent when peripheral injection sites are used (7). Portal-vein injection can reduce the amount of extra-hepatic transduction, and allows a larger dose of virus to be delivered to the liver. However, this technique requires surgical expertise, and can only be performed on adult mice.

Adeno-associated virus-mediated gene transfer to the neonatal brain.

For many metabolic diseases, early treatment is necessary to prevent irreversible developmental damage. This is particularly true for childhood diseases that affect the central nervous system (CNS). The development of effective techniques for gene transfer to the neonatal brain would provide a new set of therapeutic options for many of these disorders. Vectors based on adeno-associated virus (AAV) have shown promise as agents for neonatal CNS transduction. In preclinical animal models, a single treatment with AAV vectors at birth has been shown to produce persistent CNS expression of transduced genes into adulthood. Transduction of the neonatal brain has been accomplished by a variety of methods, including direct intraparenchymal injection, intraventricular infusion, and intravenous administration. Of these methods, intraparenchymal injection provides the highest levels of localized activity, while intraventricular infusion results in a more widespread distribution of activity when performed in the neonate. Here we describe a method for direct, intraparenchymal injection of AAV into the neonatal brain. This technique provides a method for investigators to evaluate the effects of in vivo expression of exogenous genes on the process of early brain development.

The Impact of Analytical Sensitivity on Screening Algorithms for Syphilis

To the Editor: Assays for syphilis serology can be broadly divided into 2 categories; treponemal tests that detect antibodies against the infectious agent Treponema pallidum , and nontreponemal tests that measure antibodies against nonspecific antigens, such as cardiolipin. Historically, syphilis diagnosis has used a nontreponemal assay such as rapid plasma reagin (RPR) for screening, followed by a treponemal assay to confirm syphilis infection in screen-positive patients. With the increased availability of automated assays for treponemal antibodies, however, many laboratories have shifted to a “treponemal-first” algorithm for syphilis testing. The merits of the 2 approaches have been debated in the literature in terms of clinical utility and cost-effectiveness (1, 2). The main difference between the 2 approaches is the identification of treponemal-positive,RPR-negative patients when a treponemal-first algorithm is used (3). Discriminating between latent syphilis infection and a false-positive screening test result can be challenging for patients without a clear history of prior disease. In such cases, the CDC has recommended retesting samples with a second treponemal assay to confirm the presence of antitreponemal antibodies (4). For such an algorithm to be valid, the analytical sensitivity of the confirmatory treponemal …