This study examines transgene expression and biodistribution of adeno-associated virus (AAV) pseudotyped 1-9 after tail vein (TV) injection in male mice. Using a cytomegalovirus (CMV)-luciferase transgene, the time-course of expression in each animal was tracked throughout the experiment. The animals were imaged at 7, 14, 29, 56, and 100 days after the TV injection. The total number of photons emitted from each animal was recorded, allowing examination of expression level and kinetics for each pseudotyped virus. The bioluminescence imaging revealed three expression levels (i) low-expression group, AAV2, 3, 4, and 5; (ii) moderate-expression group, AAV1, 6, and 8; and (iii) high-expression group, AAV7 and 9. In addition, imaging revealed two classes of kinetics (i) rapid-onset, for AAV1, 6, 7, 8, and 9; and (ii) slow-onset, for AAV2, 3, 4, and 5. We next evaluated protein expression and viral genome copy numbers in dissected tissues. AAV9 had the best viral genome distribution and highest protein levels. The AAV7 protein and genome copy numbers were comparable to those of AAV9 in the liver. Most surprisingly, AAV4 showed the greatest number of genome copies in lung and kidney, and a high copy number in the heart. AAV6 expression was observed in the heart, liver, and skeletal muscle, and the genome distribution corroborated these observations.

Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of preclinical and clinical applications. Many developments including new serotypes as well as self-complementary vectors are now entering the clinic. With these ongoing vector developments, continued effort has been focused on scalable manufacturing processes that can efficiently generate high-titer, highly pure, and potent quantities of rAAV vectors. Utilizing the relatively simple and efficient transfection system of HEK293 cells as a starting point, we have successfully adapted an adherent HEK293 cell line from a qualified clinical master cell bank to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allows for rapid and scalable rAAV production. Using the triple transfection method, the suspension HEK293 cell line generates greater than 1 × 10(5) vector genome containing particles (vg)/cell or greater than 1 × 10(14) vg/l of cell culture when harvested 48 hours post-transfection. To achieve these yields, a number of variables were optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that results in high-purity vector preps of AAV serotypes 1-6, 8, 9 and various chimeric capsids tested. This user-friendly process can be completed within 1 week, results in high full to empty particle ratios (>90% full particles), provides postpurification yields (>1 × 10(13) vg/l) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles. To date, this scalable manufacturing technology has been utilized to manufacture GMP phase 1 clinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B (scAAV8), giant axonal neuropathy (scAAV9), and retinitis pigmentosa (AAV2), which have been administered into patients. In addition, we report a minimum of a fivefold increase in overall vector production by implementing a perfusion method that entails harvesting rAAV from the culture media at numerous time-points post-transfection.

Background-The upregulation of G protein-coupled receptor kinase 2 in failing myocardium appears to contribute to dysfunctional ␤-adrenergic receptor (␤AR) signaling and cardiac function. The peptide ␤ARKct, which can inhibit the activation of G protein-coupled receptor kinase 2 and improve ␤AR signaling, has been shown in transgenic models and short-term gene transfer experiments to rescue heart failure (HF). This study was designed to evaluate long-term ␤ARKct expression in HF with the use of stable myocardial gene delivery with adeno-associated virus serotype 6 (AAV6). Methods and Results-In HF rats, we delivered ␤ARKct or green fluorescent protein as a control via AAV6-mediated direct intramyocardial injection. We also treated groups with concurrent administration of the ␤-blocker metoprolol. We found robust and long-term transgene expression in the left ventricle at least 12 weeks after delivery. ␤ARKct significantly improved cardiac contractility and reversed left ventricular remodeling, which was accompanied by a normalization of the neurohormonal (catecholamines and aldosterone) status of the chronic HF animals, including normalization of cardiac ␤AR signaling. Addition of metoprolol neither enhanced nor decreased ␤ARKct-mediated beneficial effects, although metoprolol alone, despite not improving contractility, prevented further deterioration of the left ventricle. Conclusions-Long-term cardiac AAV6-␤ARKct gene therapy in HF results in sustained improvement of global cardiac function and reversal of remodeling at least in part as a result of a normalization of the neurohormonal signaling axis. In addition, ␤ARKct alone improves outcomes more than a ␤-blocker alone, whereas both treatments are compatible. These findings show that ␤ARKct gene therapy can be of long-term therapeutic value in HF. (Circulation. 2009;119:89-98.)

Background-The incidence of heart failure is ever-growing, and it is urgent to develop improved treatments. An attractive approach is gene therapy; however, the clinical barrier has yet to be broken because of several issues, including the lack of an ideal vector supporting safe and long-term myocardial transgene expression. Methods and Results-Here, we show that the use of a recombinant adeno-associated viral (rAAV6) vector containing a novel cardiac-selective enhancer/promoter element can direct stable cardiac expression of a therapeutic transgene, the calcium (Ca 2ϩ )-sensing S100A1, in a rat model of heart failure. The chronic heart failure-rescuing properties of myocardial S100A1 expression, the result of improved sarcoplasmic reticulum Ca 2ϩ handling, included improved contractile function and left ventricular remodeling. Adding to the clinical relevance, long-term S100A1 therapy had unique and additive beneficial effects over ␤-adrenergic receptor blockade, a current pharmacological heart failure treatment.Conclusions-These findings demonstrate that stable increased expression of S100A1 in the failing heart can be used for long-term reversal of LV dysfunction and remodeling. Thus, long-term, cardiac-targeted rAAV6-S100A1 gene therapy may be of potential clinical utility in human heart failure.

Aldosterone produces a multitude of effects in vivo, including promotion of postmyocardial infarction adverse cardiac remodeling and heart failure progression. It is produced and secreted by the adrenocortical zona glomerulosa (AZG) cells after angiotensin II (AngII) activation of AngII type 1 receptors (AT1Rs). Until now, the general consensus for AngII signaling to aldosterone production has been that it proceeds via activation of Gq/11-proteins, to which the AT1R normally couples. Here, we describe a novel signaling pathway underlying this AT1R-dependent aldosterone production mediated by ␤-arrestin-1 (␤arr1), a universal heptahelical receptor adapter/scaffolding protein. This pathway results in sustained ERK activation and subsequent up-regulation of steroidogenic acute regulatory protein, a steroid transport protein regulating aldosterone biosynthesis in AZG cells. Also, this ␤arr1-mediated pathway appears capable of promoting aldosterone turnover independently of G protein activation, because treatment of AZG cells with SII, an AngII analog that induces ␤arr, but not G protein coupling to the AT1R, recapitulates the effects of AngII on aldosterone production and secretion. In vivo, increased adrenal ␤arr1 activity, by means of adrenal-targeted adenoviral-mediated gene delivery of a ␤arr1 transgene, resulted in a marked elevation of circulating aldosterone levels in otherwise normal animals, suggesting that this adrenocortical ␤arr1-mediated signaling pathway is operative, and promotes aldosterone production and secretion in vivo, as well. Thus, inhibition of adrenal ␤arr1 activity on AT1Rs might be of therapeutic value in pathological conditions characterized and aggravated by hyperaldosteronism.adrenocortical zona glomerulosa cell ͉ G protein-coupled receptor ͉ angiotensin II receptor type I ͉ adrenal steroid hormones ͉ biased agonism