A Novel Adeno-Associated Viral Variant for Efficient and Selective Intravitreal Transduction of Rat Müller Cells

BACKGROUND:The pathologies of numerous retinal degenerative diseases can be attributed to a multitude of genetic factors, and individualized treatment options for afflicted patients are limited and cost-inefficient. In light of the shared neurodegenerative phenotype among these disorders, a safe and broad-based neuroprotective approach would be desirable to overcome these obstacles. As a result, gene delivery of secretable-neuroprotective factors to Müller cells, a type of retinal glia that contacts all classes of retinal neurons, represents an ideal approach to mediate protection of the entire retina through a simple and innocuous intraocular, or intravitreal, injection of an efficient vehicle such as an adeno-associated viral vector (AAV). Although several naturally occurring AAV variants have been isolated with a variety of tropisms, or cellular specificities, these vectors inefficiently infect Müller cells via intravitreal injection. METHODOLOGY/PRINCIPAL FINDINGS:We have previously applied directed evolution to create several novel AAV variants capable of efficient infection of both rat and human astrocytes through iterative selection of a panel of highly diverse AAV libraries. Here, in vivo and in vitro characterization of these isolated variants identifies a previously unreported AAV variant ShH10, closely related to AAV serotype 6 (AAV6), capable of efficient, selective Müller cell infection through intravitreal injection. Importantly, this new variant shows significantly improved transduction relative to AAV2 (>60%) and AAV6. CONCLUSIONS/SIGNIFICANCE:Our findings demonstrate that AAV is a highly versatile vector capable of powerful shifts in tropism from minor sequence changes. This isolated variant represents a new therapeutic vector to treat retinal degenerative diseases through secretion of neuroprotective factors from Müller cells as well as provides new opportunities to study their biological functions in the retina

Diagnosis of osteoporotic vertebral fractures in children

Osteoporosis is a generalised disorder of the skeleton with reduced bone density and abnormal bone architecture. It increases bone fragility and renders the individual susceptible to fractures. Fractures of the vertebrae are common osteoporotic fractures. Vertebral fractures may result in scoliosis or kyphosis and, because they may be clinically silent, it is imperative that vertebral fractures are diagnosed in children accurately and at an early stage, so the necessary medical care can be implemented. Traditionally, diagnosis of osteoporotic vertebral fractures has been from lateral spine radiographs; however, a small number of studies have shown that dual energy x-ray absorptiometry is comparable to radiographs for identifying vertebral fractures in children, while allowing reduced radiation exposure. The diagnosis of vertebral fractures from dual energy x-ray absorptiometry is termed vertebral fracture assessment. Existing scoring systems for vertebral fracture assessment in adults have been assessed for use in children, but there is no standardisation and observer reliability is variable. This literature review suggests the need for a semiautomated tool that (compared to the subjective and semiquantitative methods available) will allow more reliable and precise detection of vertebral fractures in children

Engineering a serum-resistant and thermostable vesicular stomatitis virus G glycoprotein for pseudotyping retroviral and lentiviral vectors

Vesicular stomatitis virus G glycoprotein (VSV-G) is the most widely used envelope protein for retroviral and lentiviral vector pseudotyping; however, serum inactivation of VSV-G pseudotyped vectors is a significant challenge for in vivo gene delivery. To address this problem, we conducted directed evolution of VSV-G to increase its resistance to human serum neutralization. After six selection cycles, numerous common mutations were present. Based on their location within VSV-G, we analyzed whether substitutions in several surface exposed residues could endow viral vectors with higher resistance to serum. S162T, T230N, and T368A mutations enhanced serum resistance, and additionally K66T, T368A, and E380K substitutions increased the thermostability of VSV-G pseudotyped retroviral vectors, an advantageous byproduct of the selection strategy. Analysis of a number of combined mutants revealed that VSV-G harboring T230N + T368A or K66T + S162T + T230N + T368A mutations exhibited both higher in vitro resistance to human serum and higher thermostability, as well as enhanced resistance to rabbit and mouse serum. Finally, lentiviral vectors pseudotyped with these variants were more resistant to human serum in a murine model. These serum-resistant and thermostable VSV-G variants may aid the application of retroviral and lentiviral vectors to gene therapy