Selection of an Efficient AAV Vector for Robust CNS Transgene Expression

Adeno-associated virus (AAV) capsid libraries have generated improved transgene delivery vectors. We designed an AAV library construct, iTransduce, that combines a peptide library on the AAV9 capsid with a Cre cassette to enable sensitive detection of transgene expression. After only two selection rounds of the library delivered intravenously in transgenic mice carrying a Cre-inducible fluorescent protein, we flow sorted fluorescent cells from brain, and DNA sequencing revealed two dominant capsids. One of the capsids, termed AAV-F, mediated transgene expression in the brain cortex more than 65-fold (astrocytes) and 171-fold (neurons) higher than the parental AAV9. High transduction efficiency was sex-independent and sustained in two mouse strains (C57BL/6 and BALB/c), making it a highly useful capsid for CNS transduction of mice. Future work in large animal models will test the translation potential of AAV-F

Versatile Role of Rab27a in Glioma: Effects on Release of Extracellular Vesicles, Cell Viability, and Tumor Progression

Introduction: Glioma cells exert influence over the tumor-microenvironment in part through the release of extracellular vesicles (EVs), membrane-enclosed structures containing proteins, lipids, and RNAs. In this study, we evaluated the function of Ras-associated protein 27a (Rab27a) in glioma and evaluated the feasibility of assessing its role in EV release in glioma cells in vitro and in vivo. Methods: Rab27a was knocked down via a short hairpin RNA (shRNA) stably expressed in mouse glioma cell line GL261, with a scrambled shRNA as control. EVs were isolated by ultracentrifugation and quantified with Nanoparticle Tracking Analysis (NTA) and Tunable Resistive Pulse Sensing (TRPS). CellTiter-Glo viability assays and cytokine arrays were used to evaluate the impact of Rab27a knockdown. GL261.shRab27a cells and GL261.shControl were implanted into the left striatum of eight mice to assess tumor growth and changes in the tumor microenvironment. Results: Knockdown of Rab27a in GL261 glioma cells decreased the release of small EVs isolated at 100,000 × g in vitro (p = 0.005), but not the release of larger EVs, isolated at 10,000 × g. GL261.shRab27a cells were less viable compared to the scramble control in vitro (p < 0.005). A significant increase in CCL2 expression in shRab27a GL261 cells was also observed (p < 0.001). However, in vivo there was no difference in tumor growth or overall survival between

Changes in reflectin protein phosphorylation are associated with dynamic iridescence in squid

Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of The Royal Society for personal use, not for redistribution. The definitive version was published in Journal of The Royal Society Interface 6 (2010): 549-560, doi:10.1098/rsif.2009.0299.Many cephalopods exhibit remarkable dermal iridescence, a component of their complex, dynamic camouflage and communication. In the species Euprymna scolopes, the light-organ iridescence is static and is due to reflectin protein-based platelets assembled into lamellar thin-film reflectors called iridosomes, contained within iridescent cells called iridocytes. Squid in the family Loliginidae appear to be unique in that the dermis possesses a dynamic iridescent component, with reflective, colored structures that are assembled and disassembled under the control of the muscarinic cholinergic system and the associated neurotransmitter acetylcholine (Mathger et al. 2004). Here we present the sequences and characterization of three new members of the reflectin family associated with the dynamically changeable iridescence in Loligo and not found in static Euprymna iridophores. In addition, we show that application of genistein, a protein tyrosine kinase inhibitor, suppresses acetylcholine- and calcium-induced iridescence in Loligo. We further demonstrate that two of these novel reflectins are extensively phosphorylated in concert with the activation of iridescence by exogenous acetylcholine. This phosphorylation and the correlated iridescence can be blocked with genistein. Our results suggest that tyrosine phosphorylation of reflectin proteins is involved in the regulation of dynamic iridescence in Loligo.We gratefully acknowledge support from Anteon contract F33615-03-D-5408 to the Marine Biological Laboratory, Woods Hole, MA and grant # W911NF-06-1-0285 from the Army Research Office to D.E.M

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1000 cases of unexplained pediatric hepatitis in children have been reported worldwide, including 278 cases in the UK. Here we report investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator subjects, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in liver, blood, plasma or stool from 27/28 cases. We found low levels of Adenovirus (HAdV) and Human Herpesvirus 6B (HHV-6B), in 23/31 and 16/23 respectively of the cases tested. In contrast, AAV2 was infrequently detected at low titre in blood or liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T-cells and B-lineage cells. Proteomic comparison of liver tissue from cases and healthy controls, identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and in severe cases HHV-6B, may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children.</p

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1000 cases of unexplained pediatric hepatitis in children have been reported worldwide, including 278 cases in the UK 1. Here we report investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator subjects, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in liver, blood, plasma or stool from 27/28 cases. We found low levels of Adenovirus (HAdV) and Human Herpesvirus 6B (HHV-6B), in 23/31 and 16/23 respectively of the cases tested. In contrast, AAV2 was infrequently detected at low titre in blood or liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T-cells and B-lineage cells. Proteomic comparison of liver tissue from cases and healthy controls, identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and in severe cases HHV-6B, may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

A Novel Retinal Ganglion Cell Promoter for Utility in AAV Vectors

Significant advances in gene therapy have enabled exploration of therapies for inherited retinal disorders, many of which are in preclinical development or clinical evaluation. Gene therapy for retinal conditions has led the way in this growing field. The loss of retinal ganglion cells (RGCs) is a hallmark of a number of retinal disorders. As the field matures innovations that aid in refining therapies and optimizing efficacy are in demand. Gene therapies under development for RGC-related disorders, when delivered with recombinant adeno associated vectors (AAV), have typically been expressed from ubiquitous promoter sequences. Here we describe how a novel promoter from the murine Nefh gene was selected to drive transgene expression in RGCs. The Nefh promoter, in an AAV2/2 vector, was shown to drive preferential EGFP expression in murine RGCs in vivo following intravitreal injection. In contrast, EGFP expression from a CMV promoter was observed not only in RGCs, but throughout the inner nuclear layer and in amacrine cells located within the ganglion cell layer (GCL). Of note, the Nefh promoter sequence is sufficiently compact to be readily accommodated in AAV vectors, where transgene size represents a significant constraint. Moreover, this promoter should in principle provide a more targeted and potentially safer alternative for RGC-directed gene therapies

AAV-S: A versatile capsid variant for transduction of mouse and primate inner ear

Gene therapy strategies using adeno-associated virus (AAV) vectors to treat hereditary deafnesses have shown remarkable efficacy in some mouse models of hearing loss. Even so, there are few AAV capsids that transduce both inner and outer hair cells—the cells that express most deafness genes—and fewer still shown to transduce hair cells efficiently in primates. AAV capsids with robust transduction of inner and outer hair cells in primate cochlea will be needed for most clinical trials. Here, we test a capsid that we previously isolated from a random capsid library, AAV-S, for transduction in mouse and non-human primate inner ear. In both mice and cynomolgus macaques, AAV-S mediates highly efficient reporter gene expression in a variety of cochlear cells, including inner and outer hair cells, fibrocytes, and supporting cells. In a mouse model of Usher syndrome type 3A, AAV-S encoding CLRN1 robustly and durably rescues hearing. Overall, our data indicate that AAV-S is a promising candidate for therapeutic gene delivery to the human inner ear

Optimisation of AAV-NDI1 Significantly Enhances Its Therapeutic Value for Correcting Retinal Mitochondrial Dysfunction

AAV gene therapy for ocular disease has become a reality with the market authorisation of LuxturnaTM for RPE65-linked inherited retinal degenerations and many AAV gene therapies currently undergoing phase III clinical trials. Many ocular disorders have a mitochondrial involvement from primary mitochondrial disorders such as Leber hereditary optic neuropathy (LHON), predominantly due to mutations in genes encoding subunits of complex I, to Mendelian and multifactorial ocular conditions such as dominant optic atrophy, glaucoma and age-related macular degeneration. In this study, we have optimised the nuclear yeast gene, NADH-quinone oxidoreductase (NDI1), which encodes a single subunit complex I equivalent, creating a candidate gene therapy to improve mitochondrial function, independent of the genetic mutation driving disease. Optimisation of NDI1 (ophNdi1) substantially increased expression in vivo, protected RGCs and increased visual function, as assessed by optokinetic and photonegative response, in a rotenone-induced murine model. In addition, ophNdi1 increased cellular oxidative phosphorylation and ATP production and protected cells from rotenone insult to a significantly greater extent than wild type NDI1. Significantly, ophNdi1 treatment of complex I deficient patient-derived fibroblasts increased oxygen consumption and ATP production rates, demonstrating the potential of ophNdi1 as a candidate therapy for ocular disorders where mitochondrial deficits comprise an important feature