AAV ancestral reconstruction library enables selection of broadly infectious viral variants.

Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. However, enhanced vectors are required to extend these landmark successes to other indications and protein engineering approaches may provide the necessary vector improvements to address such unmet medical needs. To generate new capsid variants with potentially enhanced infectious properties and to gain insights into AAV's evolutionary history, we computationally designed and experimentally constructed a putative ancestral AAV library. Combinatorial variations at 32 amino acid sites were introduced to account for uncertainty in their identities. We then analyzed the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to iterative selection on a representative cell line panel. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes and, in general, ancestral libraries were broadly infectious across the cell line panel, indicating that they favored promiscuity over specificity. Interestingly, putative ancestral AAVs were more thermostable than modern serotypes and did not use sialic acids, galactose or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19- to 31-fold higher gene expression in the muscle compared with AAV1, a clinically used serotype for muscle delivery, highlighting their promise for gene therapy.

Directed evolution of novel adeno-associated viruses for therapeutic gene delivery.

Gene therapy vectors based on adeno-associated virus (AAV) are currently in clinical trials for numerous disease targets, such as muscular dystrophy, hemophilia, Parkinson's disease, Leber's congenital amaurosis and macular degeneration. Despite its considerable promise and emerging clinical success, several challenges impede the broader implementation of AAV gene therapy, including the prevalence of neutralizing antibodies in the human population, low transduction of a number of therapeutically relevant cell and tissue types, an inability to overcome physical and cellular barriers in vivo and a relatively limited carrying capacity. These challenges arise as the demands we place on AAV vectors are often different from or even at odds with the properties nature bestowed on their parent viruses. Viral-directed evolution-the iterative generation of large, diverse libraries of viral mutants and selection for variants with specific properties of interest-offers an approach to address these problems. Here we outline progress in creating novel classes of AAV variant libraries and highlight the successful isolation of variants with novel and advantageous in vitro and in vivo gene delivery properties.

Multimodal imaging of reversible cerebral vasoconstriction syndrome: a series of 6 cases.

RCVS is a clinical condition of recurrent severe headaches that may be associated with ischemic or hemorrhagic stroke and that is defined by the presence of segmental vasoconstriction in multiple cerebral arteries. The angiographic appearance resembles vasculitis, except that the abnormalities resolve during the course of several months. Because the treatment of RCVS differs from that for vasculitis, radiologists must understand the clinical and radiologic features so as to better guide imaging algorithms and facilitate diagnosis. We present a series of 6 cases of RCVS that highlight the imaging features across multiple modalities.

Secular trends in kidney disease: is the decreased incidence of renal replacement therapy due to a decrease in chronic kidney disease incidence?

AIMS: Little is known about trends in renal replacement therapy among patients with chronic kidney disease (CKD) or about changes in the incidence of CKD. We studied the incidence of renal replacement therapy within the population of a health maintenance organization (HMO) both among the entire HMO population and among those with CKD. METHODS: We calculated yearly incidence rates of renal replacement therapy for each year from 1998 to 2005. We defined CKD using the National Kidney Foundation definition of 2 estimated glomerular filtration rates below 60 ml/min/1.73 m2 90 or more days apart. Poisson regression assessed year-to-year differences. RESULTS: The number of patients with CKD rose consistently from 3,861 in 1998 to 5,242 in 2005. The proportion of patients who had been diagnosed with hypertension rose from 86.7% (starting renal replacement therapy) or 34.5% (with CKD) to 99.1 and 46.9%. The proportion of patients with diabetes changed little throughout the years studied. The mean estimated glomerular filtration rate among CKD patients rose minimally from 38.4 ml/min/1.73 m2 in 1998 to 39.9 ml/min/1.73 m2 in 2005. Age- and sex-adjusted rates of RRT among patients with CKD varied (p=0.0034), but did not follow a consistent pattern over time. CONCLUSIONS: Incidence of renal replacement therapy among patients with CKD changed little between 1998 and 2005, despite an increase in the number of patients diagnosed with CKD. The discrepancy may be due to increased laboratory identification of CKD.

Injury-related factors and conditions down-regulate the thrombin receptor (PAR-1) in a human neuronal cell line.

Previous studies have demonstrated that thrombin can induce potent effects on neural cell morphology, biochemistry, and viability. Nearly all of these effects are mediated by proteolytic activation of the thrombin receptor (PAR-1). Mechanisms of PAR-1 regulation in several nonneural cell types have been shown to be novel and cell type specific; however, little is known about PAR-1 regulation in neural cells. In the present study, PAR-1 cell surface expression and regulation were examined in a transformed retinoblast (Ad12 HER 10) cell line using radioiodinated anti-PAR-1 monoclonal antibodies ATAP2, which recognizes intact and cleaved receptors, and SPAN12, which is specific for the intact form of the receptor. Scatchard analysis revealed high-affinity, specific binding to a single affinity class of receptors: K(D) = 3.13 and 5.25 nM, Bmax = 190.1 and 67.8 fmol/mg of protein for 125I-ATAP2 and 125I-SPAN12, respectively. Specificity for PAR-1 was confirmed by demonstrating rapid and near complete decreases for both antibodies following treatment with thrombin or PAR-1 activating peptide (SFLLRN). Differential antibody binding was used to demonstrate rapid and near complete thrombin-induced PAR-1 cleavage and internalization, with protein synthesis-dependent replacement of intact receptors occurring over longer time intervals, but only minimal recycling of cleaved receptors. A variety of factors and conditions were screened for their effects on PAR-1 expression. Significant decreases in PAR-1 expression were induced by the protein kinase C activator phorbol 12-myristate 13-acetate (87% at 3 h), the phospholipid inflammatory mediator lysophosphatidic acid (32% at 3 h), and the injury-related condition hypoglycemia (64 and 100% at 24 h in the absence and presence of dibutyryl cyclic AMP, respectively). The effect of hypoglycemia was shown by RNase protection to be at least partially pretranslational. Finally, thrombin's ability to enhance hypoglycemia-induced cell killing correlated temporally with PAR-1 cell surface expression.

Cellular localization of thrombin receptor mRNA in rat brain: expression by mesencephalic dopaminergic neurons and codistribution with prothrombin mRNA.

Cell culture studies demonstrating that the serine protease thrombin can induce neuronal and glial process retraction, glial proliferation, and changes in gene expression suggest a role for thrombin in CNS development, plasticity, and response to injury. Most cellular responses to thrombin are mediated by proteolytic activation of the cloned thrombin receptor (TR), a member of the seven transmembrane domain, G-protein-coupled receptor superfamily. As a step toward understanding the role of thrombin and its receptor in the CNS, Northern blot, in situ hybridization, and immunohistochemical techniques were used to analyze the cellular localization of TR mRNA in weanling-age rat brain. TR mRNA was broadly distributed across the neuraxis, although expression was very focal and often anatomically limited within specific neural structures. The greatest hybridization was associated with individual neurons in neocortex, cingulate/retrosplenial cortex, and subiculum, subsets of nuclei in hypothalamus, thalamus, pretectum, and ventral mesencephalon, and discrete cell layers in the hippocampus, cerebellum, and olfactory bulb. Patterns of hybridization included neuronal, glial, and ependymal cells, although white matter was uniformly negative, as were most cerebrovascular endothelial cells. Expression of TR mRNA by astroglia and dopaminergic neurons was confirmed by colocalization with immunoreactivity for glial fibrillary acidic protein (GFAP) in hippocampus and tyrosine hydroxylase in the substantia nigra. Comparison between TR and prothrombin (thrombin's precursor) cRNA hybridization demonstrated distinct but overlapping brain distributions of these transcripts, most clearly evident in postnatally developing, laminated structures. These results suggest widespread utilization of, and multiple physiologic, and possibly pathophysiologic, functions for, the thrombin/TR cell signaling system in the CNS.