Overloaded adeno-associated virus as a novel gene therapeutic tool for otoferlin-related deafness

Hearing impairment is the most common sensory disorder in humans. So far, rehabilitation of profoundly deaf subjects relies on direct stimulation of the auditory nerve through cochlear implants. However, in some forms of genetic hearing impairment, the organ of Corti is structurally intact and therapeutic replacement of the mutated gene could potentially restore near natural hearing. In the case of defects of the otoferlin gene (OTOF), such gene therapy is hindered by the size of the coding sequence (~6 kb) exceeding the cargo capacity (<5 kb) of the preferred viral vector, adeno-associated virus (AAV). Recently, a dual-AAV approach was used to partially restore hearing in deaf otoferlin knock-out (Otof-KO) mice. Here, we employed in vitro and in vivo approaches to assess the gene-therapeutic potential of naturally-occurring and newly-developed synthetic AAVs overloaded with the full-length Otof coding sequence. Upon early postnatal injection into the cochlea of Otof-KO mice, overloaded AAVs drove specific expression of otoferlin in ~30% of all IHCs, as demonstrated by immunofluorescence labeling and polymerase chain reaction. Recordings of auditory brainstem responses and a behavioral assay demonstrated partial restoration of hearing. Together, our results suggest that viral gene therapy of DFNB9—using a single overloaded AAV vector—is indeed feasible, reducing the complexity of gene transfer compared to dual-AAV approaches

Sensory Communication

Contains table of contents for Section 2, an introduction, reports on nine research projects and a list of publications.National Institutes of Health Grant 5 R01 DC00117National Institutes of Health Grant 2 R01 DC00270National Institutes of Health Grant 1 P01 DC00361National Institutes of Health Grant 2 R01 DC00100National Institutes of Health Grant FV00428National Institutes of Health Grant 5 R01 DC00126U.S. Air Force - Office of Scientific Research Grant AFOSR 90-200U.S. Navy - Office of Naval Research Grant N00014-90-J-1935National Institutes of Health Grant 5 R29 DC0062

Ralstonia pickettii bacteremia in a cardiac surgery patient in Belgrade, Serbia

Ralstonia pickettii is an opportunistic bacterium found in the water environment with an increasing incidence as a nosocomial pathogen. The objectives of this study were to describe R. pickettii bacteremia in a cardiac surgery patient and to evaluate its ability to grow in a saline solution and to form biofilm. The patient in this study underwent mitral and aortic valve replacement surgery with two aortocoronary bypasses. She developed signs of respiratory and renal failure, therefore hemodialysis was started. After 25 days in an intensive care unit, the patient had recurrent episodes of fever with signs of bacteremia. R. pickettii was identified from blood cultures by MALDI-TOF MS. Antimicrobial susceptibility testing was performed using disc diffusion and broth microdilution methods in accordance with EUCAST methodology and results were interpreted following clinical breakpoints for Pseudomonas spp. The isolate was susceptible to all tested antimicrobial agents except aminoglycosides and colistin. Survival of R. pickettii was analyzed in saline solution with four different starting concentrations at 25 degrees C and 37 degrees C for six days. Biofilm capacity was tested using the microtiter plate method. R. pickettii showed substantial growth in saline solution, with starting concentration of 2 CFU ml(-1) reaching 107 CFU ml(-1) after six days. There was no significant difference between growth at 25 degrees C and 37 degrees C. This indicates that storage of contaminated solutions at room temperature can enhance the count of R. pickettii. Our strain did not show the capacity to form biofilm. The patient responded well to adequate treatment with ceftazidime, and after 48 days in ICU she was discharged to convalesce

Structure based evolution of a novel series of positive modulators of the AMPA receptor

Starting from compound 1, we utilized biostructural data to successfully evolve an existing series into a new chemotyope with a promising overall profile, exemplified by

Sensory Communication

Contains table of contents for Section 2, an introduction, reports on ten research projects and a list of publications.National Institutes of Health Grant 5 R01 DC00117National Institutes of Health Grant 5 R01 DC00270National Institutes of Health Grant 5 P01 DC00361National Institutes of Health Grant 2 R01 DC00100National Institutes of Health Grant 7 R29 DC00428National Institutes of Health Grant 2 R01 DC00126U.S. Air Force - Office of Scientific Research Grant AFOSR 90-0200U.S. Navy - Office of Naval Research Grant N00014-90-J-1935National Institutes of Health Grant 5 R29 DC00625U.S. Navy - Office of Naval Research Grant N00014-91-J-145

Development of an in-vivo active reversible butyrylcholinesterase inhibitor

Alzheimer’s disease (AD) is characterized by severe basal forebrain cholinergic deficit, which results in progressive and chronic deterioration of memory and cognitive functions. Similar to acetylcholinesterase, butyrylcholinesterase (BChE) contributes to the termination of cholinergic neurotransmission. Its enzymatic activity increases with the disease progression, thus classifying BChE as a viable therapeutic target in advanced AD. Potent, selective and reversible human BChE inhibitors were developed. The solved crystal structure of human BChE in complex with the most potent inhibitor reveals its binding mode and provides the molecular basis of its low nanomolar potency. Additionally, this compound is noncytotoxic and has neuroprotective properties. Furthermore, this inhibitor moderately crosses the blood-brain barrier and improves memory, cognitive functions and learning abilities of mice in a model of the cholinergic deficit that characterizes AD, without producing acute cholinergic adverse effects. Our study provides an advanced lead compound for developing drugs for alleviating symptoms caused by cholinergic hypofunction in advanced AD

Sensory Communication

Contains table of contents on Section 2, an introduction, reports on eleven research projects and a list of publications.National Institutes of Health Grant 5 R01 DC00117National Institutes of Health Grant 5 R01 DC00270National Institutes of Health Contract 2 P01 DC00361National Institutes of Health Grant 5 R01 DC00100National Institutes of Health Contract 7 R29 DC00428National Institutes of Health Grant 2 R01 DC00126U.S. Air Force - Office of Scientific Research Grant AFOSR 90-0200U.S. Navy - Office of Naval Research Grant N00014-90-J-1935National Institutes of Health Grant 5 R29 DC00625U.S. Navy - Office of Naval Research Grant N00014-91-J-1454U.S. Navy - Office of Naval Research Grant N00014-92-J-181

Sensory Communication

Contains table of contents for Section 2, an introduction and reports on twelve research projects.National Institutes of Health Grant R01 DC00117National Institutes of Health Grant R01 DC02032National Institutes of Health/National Institute of Deafness and Other Communication Disorders Grant 2 R01 DC00126National Institutes of Health Grant 2 R01 DC00270National Institutes of Health Contract N01 DC-5-2107National Institutes of Health Grant 2 R01 DC00100U.S. Navy - Office of Naval Research Grant N61339-96-K-0002U.S. Navy - Office of Naval Research Grant N61339-96-K-0003U.S. Navy - Office of Naval Research Grant N00014-97-1-0635U.S. Navy - Office of Naval Research Grant N00014-97-1-0655U.S. Navy - Office of Naval Research Subcontract 40167U.S. Navy - Office of Naval Research Grant N00014-96-1-0379U.S. Air Force - Office of Scientific Research Grant F49620-96-1-0202National Institutes of Health Grant RO1 NS33778Massachusetts General Hospital, Center for Innovative Minimally Invasive Therapy Research Fellowship Gran