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

Pharmacological characterization of muscarinic receptors in the uterus of oestrogen-primed and pregnant rats

1. Radioligand binding and contractility studies were undertaken to determine the subtype/s of muscarinic receptors present in uteri of oestrogen-treated and late pregnant rats. 2. Competition binding studies with uterine membrane preparations and [(3)H]-QNB (quinuclidinyl benzilate) provided negative log dissociation constants (pK(i)) for each antagonist as follows; oestrogen-treated – atropine (7.98)⩾himbacine (7.83)>methoctramine (7.52)⩾hexahydrosiladiphenidol (HHSiD; 7.32)⩾5,11-dihydro-11-[[[2-[2 - [(dipropylamino)methyl] - 1piperidinyl]ethyl]amino] - carbonyl] - 6H-pyrido- [2,3 - b][1,4] - benzodiazepin - 6-one (AF - DX 384; 7.10)>11 - [[2 - [(diethylamino)methyl]-1-piperidinyl]- acetyl]5,11-dihydro-6H-pyridol]2,3,-b][1,4]benzodiazepin-6-one (AF-DX 116, 6.77)>pirenzepine (6.17); late pregnant – atropine (8.05)⩾methoctramine (7.95)⩾himbacine (7.71)⩾HHSiD (7.52)⩾AF-DX 384 (7.34)>AF-DX 116 (6.72)>pirenzepine (6.18). 3. The potency of carbachol in causing uterine contraction was similar in preparations from pregnant and non-pregnant animals (pD(2)=5.57 and 5.46, respectively). Each muscarinic antagonist caused parallel, rightward shifts of carbachol concentration-response curves. The pA(2) estimates were: oestrogen-treated – atropine (9.42)>himbacine (8.73)⩾HHSiD (8.68)⩾methoctramine (8.49)⩾AF-DX 384 (7.91)⩾AF-DX 116 (7.36)⩾pirenzepine (7.26); late pregnant – atropine (9.48)>himbacine (8.37)⩾HHSiD (8.22)⩾methoctramine (8.01)⩾AF-DX 116 (7.73)⩾AF-DX 384 (7.44)⩾pirenzepine (6.92). 4. The relative pK(i) estimates for antagonists obtained in membrane preparations from oestrogen-treated rats suggest the presence of muscarinic M(2) subtypes. In functional studies pA(2) values indicated the additional presence of muscarinic M(3) receptor or, possibly an atypical receptor subtype. The similarity between pK(i) and pA(2) estimates obtained in uteri from oestrogen-treated and pregnant animals, respectively, indicates that pregnancy does not affect myometrial muscarinic receptors in the rat