Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. The rAAV8RSM was produced using transient transfection, and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to 16 laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50×10(11) pt/ml; CI, 4.26×10(11) to 6.75×10(11) pt/ml), vector genomes (mean, 5.75×10(11) vg/ml; CI, 3.05×10(11) to 1.09×10(12) vg/ml), and infectious units (mean, 1.26×10(9) IU/ml; CI, 6.46×10(8) to 2.51×10(9) IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time when the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.

Mounting evidence suggests that human immunodeficiency virus type 1 (HIV-1) Gag-specific T helper cells contribute to effective antiviral control, but their functional characteristics and the precise epitopes targeted by this response remain to be defined. In this study, we generated CD4 ؉ T-cell clones specific for Gag from HIV-1-infected persons with vigorous Gag-specific responses detectable in peripheral blood mononuclear cells. Multiple peptides containing T helper epitopes were identified, including a minimal peptide, VHAGPIAG (amino acids 218 to 226), in the cyclophilin binding domain of Gag. Peptide recognition by all clones examined induced cell proliferation, gamma interferon (IFN-␥) secretion, and cytolytic activity. Cytolysis was abrogated by concanamycin A and EGTA but not brefeldin A or anti-Fas antibody, implying a perforin-mediated mechanism of cell lysis. Additionally, serine esterase release into the extracellular medium, a marker for cytolytic granules, was demonstrated in an antigen-specific, dose-dependent fashion. These data indicate that T helper cells can target multiple regions of the p24 Gag protein and suggest that cytolytic activity may be a component of the antiviral effect of these cells.

We use a novel technique that allows for closed recirculation of vector genomes in the cardiac circulation using cardiopulmonary bypass, referred to here as molecular cardiac surgery with recirculating delivery (MCARD). We demonstrate that this platform technology is highly efficient in isolating the heart from the systemic circulation in vivo. Using MCARD, we compare the relative efficacy of single-stranded (ss) adeno-associated virus (AAV)6, ssAAV9 and self-complimentary (sc)AAV6-encoding enhanced green fluorescent protein, driven by the constitutive cytomegalovirus promoter to transduce the ovine myocardium in situ. MCARD allows for the unprecedented delivery of up to 48 green fluorescent protein genome copies per cell globally in the sheep left ventricular (LV) myocardium. We demonstrate that scAAV6-mediated MCARD delivery results in global, cardiac-specific LV gene expression in the ovine heart and provides for considerably more robust and cardiac-specific gene delivery than other available delivery techniques such as intramuscular injection or intracoronary injection; thus, representing a potential, clinically translatable platform for heart failure gene therapy.

No abstract

Antisense oligodeoxynucleotides (AONs) and short interfering RNAs (siRNAs) effect posttranscriptional gene silencing (PTGS) by hybridizing to an mRNA and then directing its cleavage. To understand the constraints that mRNA structure imposes on AON-vs. siRNA-mediated PTGS, AON-and siRNA-mediated cleavage of defined mRNA structures was monitored in Drosophila embryo whole-cell lysates. We observed that AON-directed cleavage was Ϸ3-fold faster than cleavage with a siRNA directed to the same target site. Furthermore, and unexpectedly, AON-mediated cleavage was found to be much less fastidious with respect to target sequence accessibility, as measured by the presence of unpaired nucleotides, than a corresponding siRNA. Nonetheless, in vivo, siRNAs silenced their mRNA target at least 2-fold more efficiently than the corresponding AON. These seemingly contradictory results suggested that additional, as yet undefined factors play an important role in regulating PTGS efficiency in vivo. We used a well defined RNA-binding protein, ␣CP, and its corresponding highaffinity RNA-binding site to explore this hypothesis. We found that prebound ␣CP effectively blocked AON-mediated cleavage of the RNA-binding site compared with cleavage of the site in the absence of ␣CP. We conclude that higher-order structures formed by RNA and bound proteins play an important role in determining the efficiency of AON-directed PTGS. We hypothesize that strategies aimed at removing RNA-binding proteins might significantly improve AON-mediated PTGS in vivo.antisense ͉ oligodeoxynucleotide ͉ siRNA