Doug Howard, M.Sc.

@article{doi:10.1089/hgtb.2016.180,

title = {Assaying the Stability and Inactivation of AAV Serotype 1 Vectors},

author = {Douglas B Howard and Brandon K Harvey},

url = {https://pubmed.ncbi.nlm.nih.gov/28192678/},

doi = {10.1089/hgtb.2016.180},

year  = {2017},

date = {2017-01-01},

journal = {Human Gene Therapy Methods},

volume = {28},

number = {1},

pages = {39-48},

abstract = {Adeno-associated virus (AAV) vectors are a commonplace tool for gene delivery ranging from cell culture to human gene therapy. One feature that makes AAV a desirable vector is its stability, in regard to both the duration of transgene expression and retention of infectivity as a viral particle. This study examined the stability of AAV serotype 1 (AAV1) vectors under different conditions. First, transducibility after storage at 4$,^circ$C decreased 20% over 7 weeks. Over 10 freeze--thaw cycles, the resulting transduction efficiency became variable at 60--120% of a single thaw. Using small stainless steel slugs to mimic a biosafety cabinet or metal lab bench surface, it was found that an AAV1 vector can be reconstituted after 6 days of storage at room temperature. The stability of AAV is a desired feature, but effective decontamination procedures must be available for safety and experimental integrity. Multiple disinfectants commonly used in the laboratory for ability to inactivate an AAV1 vector were tested, and it was found that autoclaving, 0.25% peracetic acid, iodine, or 10% Clorox bleach completely prevented AAV-mediated transgene expression. These data suggest that peracetic acid should be used for inactivating AAV1 vectors on metal-based surfaces or instruments in order to avoid inadvertent transgene expression in human cells or cross-contamination of instruments.},

note = {PMID: 28192678},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Harvey2011,

title = {Targeted over-expression of glutamate transporter 1 (GLT-1) reduces ischemic brain injury in a rat model of stroke.},

author = {Brandon K Harvey and Mikko Airavaara and Jason Hinzman and Emily M Wires and Matthew J Chiocco and Douglas B Howard and Hui Shen and Greg Gerhardt and Barry J Hoffer and Yun Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/21853027},

doi = {10.1371/journal.pone.0022135},

issn = {1932-6203 (Electronic); 1932-6203 (Linking)},

year  = {2011},

date = {2011-08-09},

urldate = {2011-08-09},

journal = {PLoS One},

volume = {6},

number = {8},

pages = {e22135},

address = {Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland, United States of America. bharvey@mail.nih.gov},

abstract = {Following the onset of an ischemic brain injury, the excitatory neurotransmitter glutamate is released. The excitotoxic effects of glutamate are a major contributor to the pathogenesis of a stroke. The aim of this study was to examine if overexpression of a glutamate transporter (GLT-1) reduces ischemic brain injury in a rat model of stroke. We generated an adeno-associated viral (AAV) vector expressing the rat GLT-1 cDNA (AAV-GLT1). Functional expression of AAV-GLT1 was confirmed by increased glutamate clearance rate in non-stroke rat brain as measured by in vivo amperometry. AAV-GLT1 was injected into future cortical region of infarction 3 weeks prior to 60 min middle cerebral artery occlusion (MCAo). Tissue damage was assessed at one and two days after MCAo using TUNEL and TTC staining, respectively. Behavioral testing was performed at 2, 8 and 14 days post-stroke. Animals receiving AAV-GLT1, compared to AAV-GFP, showed significant decreases in the duration and magnitude of extracellular glutamate, measured by microdialysis, during the 60 minute MCAo. A significant reduction in brain infarction and DNA fragmentation was observed in the region of AAV-GLT1 injection. Animals that received AAV-GLT1 showed significant improvement in behavioral recovery following stroke compared to the AAV-GFP group. We demonstrate that focal overexpression of the glutamate transporter, GLT-1, significantly reduces ischemia-induced glutamate overflow, decreases cell death and improves behavioral recovery. These data further support the role of glutamate in the pathogenesis of ischemic damage in brain and demonstrate that targeted gene delivery to decrease the ischemia-induced glutamate overflow reduces the cellular and behavioral deficits caused by stroke.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Airavaara2010,

title = {Widespread cortical expression of MANF by AAV serotype 7: localization and protection against ischemic brain injury.},

author = {Mikko Airavaara and Matt J Chiocco and Doug B Howard and Katie L Zuchowski and Johan Peranen and Chao Liu and Shengyun Fang and Barry J Hoffer and Yun Wang and Brandon K Harvey},

url = {https://www.ncbi.nlm.nih.gov/pubmed/20685313},

doi = {10.1016/j.expneurol.2010.05.020},

issn = {1090-2430 (Electronic); 0014-4886 (Linking)},

year  = {2010},

date = {2010-09-01},

journal = {Exp Neurol},

volume = {225},

number = {1},

pages = {104--113},

address = {Neural Protection and Regeneration Section, National Institute on Drug Abuse, IRP, NIH, Baltimore, MD 21201, USA.},

abstract = {Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a secreted protein which reduces endoplasmic reticulum (ER) stress and has neurotrophic effects on dopaminergic neurons. Intracortical delivery of recombinant MANF protein protects tissue from ischemic brain injury in vivo. In this study, we examined the protective effect of adeno-associated virus serotype 7 encoding MANF in a rodent model of stroke. An AAV vector containing human MANF cDNA (AAV-MANF) was constructed and verified for expression of MANF protein. AAV-MANF or an AAV control vector was administered into three sites in the cerebral cortex of adult rats. One week after the vector injections, the right middle cerebral artery (MCA) was ligated for 60min. Behavioral monitoring was conducted using body asymmetry analysis, neurological testing, and locomotor activity. Standard immunohistochemical and western blotting procedures were conducted to study MANF expression. Our data showed that AAV-induced MANF expression is redistributed in neurons and glia in cerebral cortex after ischemia. Pretreatment with AAV-MANF reduced the volume of cerebral infarction and facilitated behavioral recovery in stroke rats. In conclusion, our data suggest that intracortical delivery of AAV-MANF increases MANF protein production and reduces ischemic brain injury. Ischemia also caused redistribution of AAV-mediated MANF protein suggesting an injury-induced release.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{HOWARD200824,

title = {Tropism and toxicity of adeno-associated viral vector serotypes 1, 2, 5, 6, 7, 8, and 9 in rat neurons and glia in vitro},

author = {Douglas B Howard and Kathleen Powers and Yun Wang and Brandon K Harvey},

url = {https://pubmed.ncbi.nlm.nih.gov/18035387/},

doi = {https://doi.org/10.1016/j.virol.2007.10.007},

issn = {0042-6822},

year  = {2008},

date = {2008-01-01},

journal = {Virology},

volume = {372},

number = {1},

pages = {24-34},

abstract = {Recombinant adeno-associated viral (rAAV) vectors are frequently used for gene delivery to the central nervous system and are capable of transducing neurons and glia in vitro. In this study, seven serotypes of a rAAV vector expressing green fluorescent protein (GFP) were characterized for tropism and toxicity in primary cortical cells derived from embryonic rat brain. At 2 days after transduction, serotypes 1 and 5 through 8 expressed GFP predominately in glia, but by 6 days post-transduction expression was neuronal except for AAV5. AAV2 and 9 produced minimal GFP expression. Using cell viability assays, toxicity was observed at higher multiplicities of infection (MOI) for all serotypes except AAV2 and 9. The toxicity of AAV1 and 5--8 affected mostly glia as indicated by a loss of glial-marker immunoreactivity. A frameshift mutation in the GFP gene reduced overall toxicity for serotypes 1, 5 and 6, but not 7 and 8 suggesting that the toxicity was not solely due to the overexpression of GFP. Collectively, a differential tropism and toxicity was observed among the AAV serotypes on primary cortical cultures with an overall preferential glial transduction and toxicity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{doi:10.3727/000000007783464984,

title = {An Immortalized Rat Ventral Mesencephalic Cell Line, RTC4, Is Protective in a Rodent Model of Stroke},

author = {B K Harvey and G J Chen and C J Schoen and C T Lee and D B Howard and O Dillon-Carter and M Coggiano and W J Freed and Y Wang and B J Hoffer and J F Sanchez},

url = {https://pubmed.ncbi.nlm.nih.gov/17708338/},

doi = {10.3727/000000007783464984},

year  = {2007},

date = {2007-01-01},

journal = {Cell Transplantation},

volume = {16},

number = {5},

pages = {483-491},

abstract = {One therapeutic approach to stroke is the transplantation of cells capable of trophic support, reinnervation, and/or regeneration. Previously, we have described the use of novel truncated isoforms of SV40 large T antigen to generate unique cell lines from several primary rodent tissue types. Here we describe the generation of two cell lines, RTC3 and RTC4, derived from primary mesencephalic tissue using a fragment of mutant T antigen, T155c (cDNA) expressed from the RSV promoter. Both lines expressed the glial markers vimentin and S100β, but not the neuronal markers NeuN, MAP2, or β-III-tubulin. A screen for secreted trophic factors revealed substantially elevated levels of platelet-derived growth factor (PDGF) in RTC4, but not RTC3 cells. When transplanted into rat cortex, RTC4 cells survived for at least 22 days and expressed PDGF. Because PDGF has been reported to reduce ischemic injury, we examined the protective functions of RTC4 cells in an animal model of stroke. RTC4 or RTC3 cells, or vehicle, were injected into rat cortex 15--20 min prior to a 60-min middle cerebral artery ligation. Forty-eight hours later, animals were sacrificed and the stroke volume was assessed by triphenyl-tetrazolium chloride (TTC) staining. Compared to vehicle or RTC3 cells, transplanted RTC4 cells significantly reduced stroke volume. Overall, we generated a cell line with glial properties that produces PDGF and reduces ischemic injury in a rat model of stroke.},

note = {PMID: 17708338},

keywords = {},

pubstate = {published},

tppubtype = {article}

}