Sex Genotyping of Archival Fixed and Immunolabeled Guinea Pig Cochleas.

For decades, outbred guinea pigs (GP) have been used as research models. Various past research studies using guinea pigs used measures that, unknown at the time, may be sex-dependent, but from which today, archival tissues may be all that remain. We aimed to provide a protocol for sex-typing archival guinea pig tissue, whereby past experiments could be re-evaluated for sex effects. No PCR sex-genotyping protocols existed for GP. We found that published sequence of the GP Sry gene differed from that in two separate GP stocks. We used sequences from other species to deduce PCR primers for Sry. After developing a genomic DNA extraction for archival, fixed, decalcified, immunolabeled, guinea pig cochlear half-turns, we used a multiplex assay (Y-specific Sry; X-specific Dystrophin) to assign sex to tissue as old as 3 years. This procedure should allow reevaluation of prior guinea pig studies in various research areas for the effects of sex on experimental outcomes.

Rescue of Outer Hair Cells with Antisense Oligonucleotides in Usher Mice Is Dependent on Age of Treatment.

The absence of functional outer hair cells is a component of several forms of hereditary hearing impairment, including Usher syndrome, the most common cause of concurrent hearing and vision loss. Antisense oligonucleotide (ASO) treatment of mice with the human Usher mutation, Ush1c c.216G>A, corrects gene expression and significantly improves hearing, as measured by auditory-evoked brainstem responses (ABRs), as well as inner and outer hair cell (IHC and OHC) bundle morphology. However, it is not clear whether the improvement in hearing achieved by ASO treatment involves the functional rescue of outer hair cells. Here, we show that Ush1c c.216AA mice lack OHC function as evidenced by the absence of distortion product otoacoustic emissions (DPOAEs) in response to low-, mid-, and high-frequency tone pairs. This OHC deficit is rescued by treatment with an ASO that corrects expression of Ush1c c.216G>A. Interestingly, although rescue of inner hairs cells, as measured by ABR, is achieved by ASO treatment as late as 7 days after birth, rescue of outer hair cells, measured by DPOAE, requires treatment before post-natal day 5. These results suggest that ASO-mediated rescue of both IHC and OHC function is age dependent and that the treatment window is different for the different cell types. The timing of treatment for congenital hearing disorders is of critical importance for the development of drugs such ASO-29 for hearing rescue.

Antisense oligonucleotide therapy rescues disruptions in organization of exploratory movements associated with Usher syndrome type 1C in mice.

Usher syndrome, Type 1C (USH1C) is an autosomal recessive inherited disorder in which a mutation in the gene encoding harmonin is associated with multi-sensory deficits (i.e., auditory, vestibular, and visual). USH1C (Usher) mice, engineered with a human USH1C mutation, exhibit these multi-sensory deficits by circling behavior and lack of response to sound. Administration of an antisense oligonucleotide (ASO) therapeutic that corrects expression of the mutated USH1C gene, has been shown to increase harmonin levels, reduce circling behavior, and improve vestibular and auditory function. The current study evaluates the organization of exploratory movements to assess spatial organization in Usher mice and determine the efficacy of ASO therapy in attenuating any such deficits. Usher and heterozygous mice received the therapeutic ASO, ASO-29, or a control, non-specific ASO treatment at postnatal day five. Organization of exploratory movements was assessed under dark and light conditions at two and six-months of age. Disruptions in exploratory movement organization observed in control-treated Usher mice were consistent with impaired use of self-movement and environmental cues. In general, ASO-29 treatment rescued organization of exploratory movements at two and six-month testing points. These observations are consistent with ASO-29 rescuing processing of multiple sources of information and demonstrate the potential of ASO therapies to ameliorate topographical disorientation associated with other genetic disorders.

Rescue of peripheral vestibular function in Usher syndrome mice using a splice-switching antisense oligonucleotide.

Usher syndrome type 1C (USH1C/harmonin) is associated with profound retinal, auditory and vestibular dysfunction. We have previously reported on an antisense oligonucleotide (ASO-29) that dramatically improves auditory function and balance behavior in mice homozygous for the harmonin mutation Ush1c c.216G > A following a single systemic administration. The findings were suggestive of improved vestibular function; however, no direct vestibular assessment was made. Here, we measured vestibular sensory evoked potentials (VsEPs) to directly assess vestibular function in Usher mice. We report that VsEPs are absent or abnormal in Usher mice, indicating profound loss of vestibular function. Strikingly, Usher mice receiving ASO-29 treatment have normal or elevated vestibular response thresholds when treated during a critical period between postnatal day 1 and 5, respectively. In contrast, treatment of mice with ASO-29 treatment at P15 was minimally effective at rescuing vestibular function. Interestingly, ASO-29 treatment at P1, P5 or P15 resulted in sufficient vestibular recovery to support normal balance behaviors, suggesting a therapeutic benefit to balance with ASO-29 treatment at P15 despite the profound vestibular functional deficits that persist with treatment at this later time. These findings provide the first direct evidence of an effective treatment of peripheral vestibular function in a mouse model of USH1C and reveal the potential for using antisense technology to treat vestibular dysfunction.

Antisense oligonucleotides delivered to the amniotic cavity in utero modulate gene expression in the postnatal mouse.

Congenital diseases account for a large portion of pediatric illness. Prenatal screening and diagnosis permit early detection of many genetic diseases. Fetal therapeutic strategies to manage disease processes in utero represent a powerful new approach for clinical care. A safe and effective fetal pharmacotherapy designed to modulate gene expression ideally would avoid direct mechanical engagement of the fetus and present an external reservoir of drug. The amniotic cavity surrounding the fetus could serve as an ideal drug reservoir. Antisense oligonucleotides (ASOs) are an established tool for the therapeutic modulation of gene expression. We hypothesize that ASOs administered to the amniotic cavity will gain entry to the fetus and modulate gene expression. Here, we show that an ASO targeting MALAT1 RNA, delivered by transuterine microinjection into the mouse amniotic cavity at embryonic day 13-13.5, reduces target RNA expression for up to 4 weeks after birth. A similarly delivered ASO targeting a causal splice site mutation for Usher syndrome corrects gene expression in the inner ear, a therapeutically relevant target tissue. We conclude that intra-amniotic delivery of ASOs is well tolerated and produces a sustained effect on postnatal gene expression. Transuterine delivery of ASOs is an innovative platform for developing fetal therapeutics to efficaciously treat congenital disease.

Disruption of the lamin A and matrin-3 interaction by myopathic LMNA mutations.

The nuclear face of the nuclear membrane is enriched with the intermediate filament protein lamin A. Mutations in LMNA, the gene encoding lamin A, lead to a diverse set of inherited conditions including myopathies that affect both the heart and skeletal muscle. To gain insight about lamin A protein interactions, binding proteins associated with the tail of lamin A were characterized. Of 130 nuclear proteins found associated with the lamin A tail, 17 (13%) were previously described lamin A binding partners. One protein not previously linked to lamin A, matrin-3, was selected for further study, because like LMNA mutations, matrin-3 has also been implicated in inherited myopathy. Matrin-3 binds RNA and DNA and is a nucleoplasmic protein originally identified from the insoluble nuclear fraction, referred to as the nuclear matrix. Anti-matrin-3 antibodies were found to co-immunoprecipitate lamin A, and the lamin-A binding domain was mapped to the carboxy-terminal half of matrin-3. Three-dimensional mapping of the lamin A-matrin-3 interface showed that the LMNA truncating mutation Δ303, which lacks the matrin-3 binding domain, was associated with an increased distance between lamin A and matrin-3. LMNA mutant cells are known to have altered biophysical properties and the matrin-3-lamin A interface is positioned to contribute to these defects.

Nesprin-1 mutations in human and murine cardiomyopathy.

Mutations in LMNA, the gene encoding the nuclear membrane proteins, lamins A and C, produce cardiac and muscle disease. In the heart, these autosomal dominant LMNA mutations lead to cardiomyopathy frequently associated with cardiac conduction system disease. Herein, we describe a patient with the R374H missense variant in nesprin-1alpha, a protein that binds lamin A/C. This individual developed dilated cardiomyopathy requiring cardiac transplantation. Fibroblasts from this individual had increased expression of nesprin-1alpha and lamins A and C, indicating changes in the nuclear membrane complex. We characterized mice lacking the carboxy-terminus of nesprin-1 since this model expresses nesprin-1 without its carboxy-terminal KASH domain. These Delta/DeltaKASH mice have a normally assembled but dysfunctional nuclear membrane complex and provide a model for nesprin-1 mutations. We found that Delta/DeltaKASH mice develop cardiomyopathy with associated cardiac conduction system disease. Older mutant animals were found to have elongated P wave duration, elevated atrial and ventricular effective refractory periods indicating conduction defects in the myocardium, and reduced fractional shortening. Cardiomyocyte nuclei were found to be elongated with reduced heterochromatin in the Delta/DeltaKASH hearts. These findings mirror what has been described from lamin A/C gene mutations and reinforce the importance of an intact nuclear membrane complex for a normally functioning heart.

Eya4-deficient mice are a model for heritable otitis media.

Otitis media is an extremely common pediatric inflammation of the middle ear that often causes pain and diminishes hearing. Vulnerability to otitis media is due to eustachian tube dysfunction as well as other poorly understood factors, including genetic susceptibility. As EYA4 mutations cause sensorineural hearing loss in humans, we produced and characterized Eya4-deficient (Eya4(-/-)) mice, which had severe hearing deficits. In addition, all Eya4(-/-) mice developed otitis media with effusion. Anatomic studies revealed abnormal middle ear cavity and eustachian tube dysmorphology; thus, Eya4 regulation is critical for the development and function of these structures. We suggest that some human otitis media susceptibility reflects underlying genetic predisposition in genes like EYA4 that regulate middle ear and eustachian tube anatomy.

Mutation in the transcriptional coactivator EYA4 causes dilated cardiomyopathy and sensorineural hearing loss.

We identified a human mutation that causes dilated cardiomyopathy and heart failure preceded by sensorineural hearing loss (SNHL). Unlike previously described mutations causing dilated cardiomyopathy that affect structural proteins, this mutation deletes 4,846 bp of the human transcriptional coactivator gene EYA4. To elucidate the roles of eya4 in heart function, we studied zebrafish embryos injected with antisense morpholino oligonucleotides. Attenuated eya4 transcript levels produced morphologic and hemodynamic features of heart failure. To determine why previously described mutated EYA4 alleles cause SNHL without heart disease, we examined biochemical interactions of mutant Eya4 peptides. Eya4 peptides associated with SNHL, but not the shortened 193-amino acid peptide associated with dilated cardiomyopathy and SNHL, bound wild-type Eya4 and associated with Six proteins. These data define unrecognized and crucial roles for Eya4-Six-mediated transcriptional regulation in normal heart function.