Douglas E. Brough

Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals

In the mammalian auditory system, sensory cell loss resulting from aging, ototoxic drugs, infections, overstimulation and other causes is irreversible and leads to permanent sensorineural hearing loss. To restore hearing, it is necessary to generate new functional hair cells. One potential way to regenerate hair cells is to induce a phenotypic transdifferentiation of nonsensory cells that remain in the deaf cochlea. Here we report that Atoh1, a gene also known as Math1 encoding a basic helix-loop-helix transcription factor and key regulator of hair cell development, induces regeneration of hair cells and substantially improves hearing thresholds in the mature deaf inner ear after delivery to nonsensory cells through adenovectors. This is the first demonstration of cellular and functional repair in the organ of Corti of a mature deaf mammal. The data suggest a new therapeutic approach based on expressing crucial developmental genes for cellular and functional restoration in the damaged auditory epithelium and other sensory systems.

Math1 Gene Transfer Generates New Cochlear Hair Cells in Mature Guinea Pigs In Vivo

Hair cell loss in the mammalian cochlea is irreversible and results in permanent hearing loss. Math1, the basic helix-loop-helix transcription factor homolog of the Drosophila atonal gene, is a positive regulator of hair cell differentiation during cochlear development. Developing hair cells express Math1, and nonsensory cells do not. We set out to determine the outcome of overexpression of Math1 in nonsensory cells of the cochlea on the phenotype of these cells. We demonstrate that in vivo inoculation of adenovirus with the Math1 gene insert into the endolymph of the mature guinea pig cochlea results in Math1 overexpression in nonsensory cochlear cells, as evident from the presence of Math1 protein in supporting cells of the organ of Corti and in adjacent nonsensory epithelial cells. Math1 overexpression leads to the appearance of immature hair cells in the organ of Corti and new hair cells adjacent to the organ of Corti in the interdental cell, inner sulcus, and Hensen cell regions. Axons are extended from the bundle of auditory nerve toward some of the new hair cells, suggesting that the new cells attract auditory neurons. We conclude that nonsensory cells in the mature cochlea retain the competence to generate new hair cells after overexpression of Math1 in vivo and that Math1 is necessary and sufficient to direct hair cell differentiation in these mature nonsensory cells.

Pigment Epithelium-Derived Factor Inhibits Retinal and Choroidal Neovascularization

In this study, we investigated whether overexpression of pigment epithelium‐derived factor (PEDF) by gene transfer can inhibit neovascularization by testing its effect in three different models of ocular neovascularization. Intravitreous injection of an adenoviral vector encoding PEDF resulted in expression of PEDF mRNA in the eye measured by RT‐PCR and increased immunohistochemical staining for PEDF protein throughout the retina. In mice with laser‐induced rupture of Bruchs membrane, choroidal neovascularization was significantly reduced after intravitreous injection of PEDF vector compared to injection of null vector or no injection. Subretinal injection of the PEDF vector resulted in prominent staining for PEDF in retinal pigmented epithelial cells and strong inhibition of choroidal neovascularization. In two models of retinal neovascularization (transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors and mice with oxygen‐induced ischemic retinopathy), intravitreous injection of null vector resulted in decreased neovascularization compared to no injection, but intravitreous injection of PEDF vector resulted in further inhibition of neovascularization that was statistically significant. These data suggest that sustained increased intraocular expression of PEDF by gene therapy might provide a promising approach for treatment of ocular neovascularization.

Selectivity of a replication-competent adenovirus for human breast carcinoma cells expressing the MUC1 antigen.

The DF3/MUC1 gene is aberrantly overexpressed in human breast and other carcinomas. Previous studies have demonstrated that the DF3/MUC1 promoter/enhancer confers selective expression of diverse transgenes in MUC1-positive breast cancer cells. In this study, we show that an adenoviral vector (Ad.DF3-E1) in which the DF3/MUC1 promoter drives expression of E1A selectively replicates in MUC1-positive breast cancer cells. We also show that Ad.DF3-E1 infection of human breast tumor xenografts in nude mice is associated with inhibition of tumor growth. In contrast to a replication-incompetent adenoviral vector that infects along the injection track, Ad.DF3-E1 infection was detectable throughout the tumor xenografts. To generate an Ad.DF3-E1 vector with the capacity for incorporating therapeutic products, we inserted the cytomegalovirus (CMV) promoter upstream of the TNF cDNA. Infection with Ad.DF3-E1/CMV-TNF was associated with selective replication and production of TNF in cells that express MUC1. Moreover, treatment of MUC1-positive, but not MUC1-negative, xenografts with a single injection of Ad.DF3-E1/CMV-TNF was effective in inducing stable tumor regression. These findings demonstrate that the DF3/MUC1 promoter confers competence for selective replication of Ad.DF3-E1 in MUC1-positive breast tumor cells, and that the antitumor activity of this vector is potentiated by integration of the TNF cDNA.

ADENOVIRAL VECTORS FOR GENE TRANSFER

Adenoviruses began to be developed into highly effective gene expression vectors in the early 1980s. Recently, the increased interest in utilizing this transfer system in vivo has posed new problems for heterologous gene-transfer, spurring a renewed effort in the field of vector development toward solving the structural, immunological and targeting problems posed by gene therapy applications.

Vestibular hair cell regeneration and restoration of balance function induced by math1 gene transfer.

Hypothesis: Delivery of math1 using an adenovector (Admath1.11D) results in vestibular hair cell regeneration and recovery of balance function in ototoxin-treated adult mice. Background: Loss of peripheral vestibular function is associated with disease processes such as vestibular neuronitis, aminoglycoside ototoxicity, and aging. Loss of vestibular hair cells is one of the mechanisms underlying balance dysfunction in all of these disorders. Currently, recovery from these diseases relies on central vestibular compensation rather than on local tissue recovery. Overexpression of the mammalian atonal homologue math1 has been demonstrated to induce generation of hair cells in neonatal organ of Corti cultures and in the guinea pig cochlea in vivo and could thus provide an approach to local tissue recovery. Methods: Admath1.11D was applied to cultures of aminoglycoside-treated macular organs or in vivo in a mouse aminoglycoside ototoxicity model. Outcome measures included histologic examination, immunohistochemistry, swim testing, and evaluation of the horizontal vestibulo-ocular reflex. Results: Delivery of math1 resulted in the generation of vestibular hair cells in vitro after aminoglycoside-mediated loss of hair cells. Math1-treated mice showed recovery of the vestibular neuroepithelium within 8 weeks after Admath1.11D treatment. Assessment of animals after vector infusion demonstrated a recovery of vestibular function compared with aminoglycoside-only-treated mice. Conclusion: Molecular replacement of math1 may provide a therapeutic means of restoring vestibular function related to vestibular hair cell loss.

Selective atonal gene delivery improves balance function in a mouse model of vestibular disease

Loss of balance is often due to loss of vestibular hair cells. In mammals, regeneration of functional hair cells in the mature sensory epithelium is limited; therefore, loss of sensory cells can lead to debilitating balance problems. Delivery of the transcription factor atonal (atoh1) after aminoglycoside ototoxicity has previously been shown to induce the transdifferentiation of supporting cells into new hair cells and restore function. A problem with mouse aminoglycoside models is that the partial loss of hair cells seen in human disease is difficult to establish consistently. To more closely mirror human clinical balance dysfunction, we have used systemic application of 3,3′-iminodipropionitrile (IDPN), a vestibulotoxic nitrile compound known to cause vestibular hair cell loss, to induce a consistent partial loss of vestibular hair cells. To determine if balance function could be restored, we delivered atoh1 using a new adenovirus vector, based on Ad28. The Ad28 adenovector is based on a human serotype with a low seroprevalence that appears to target gene delivery to vestibular supporting cells. To further provide cell type selectivity of gene delivery, we expressed atoh1 using the supporting cell-specific glial fibrillary acid protein promoter. Delivery of this vector to IDPN-damaged vestibular organs resulted in a significant recovery of vestibular hair cells and restoration of balance, as measured by time on rotarod compared with untreated controls.

Bcl-2 Gene Therapy Prevents Aminoglycoside-Induced Degeneration of Auditory and Vestibular Hair Cells

To evaluate the protective effects of bcl-2, we have developed an in vivo model of gentamicin ototoxicity in C57BL/6 mice using intratympanic delivery of gentamicin. Hair cell survival was evaluated using myosin VIIa immunohistochemistry, cytocochleogram and auditory brainstem response (ABR) testing. At 10 days after gentamicin application, a consistent loss of outer hair cells was seen. Mice were pretreated with an adenovector expressing human bcl-2 (Ad.11D.bcl-2) or a control vector (Ad.11D). Seventy-two hours after vector delivery mice were treated with intratympanic gentamicin and evaluated at 10 days after ototoxin delivery. Pretreatment with Ad.11D.bcl-2 resulted in morphologic protection of hair cells and preservation of hearing thresholds measured by ABR.

Replication-Defective Adenovirus Vectors with Multiple Deletions Do Not Induce Measurable Vector-Specific T Cells in Human Trials

ABSTRACT The magnitude and character of adenovirus serotype 5 (Ad5)-specific T cells were determined in volunteers with and without preexisting neutralizing antibodies (NAs) to Ad5 who received replication-defective Ad5 (rAd5)-based human immunodeficiency virus vaccines. There was no correlation between T-cell responses and NAs to Ad5. There was no increase in magnitude or activation state of Ad5-specific CD4+ T cells at time points where antibodies to Ad5 and T-cell responses to the recombinant gene products could be measured. These data indicate that rAd5-based vaccines containing deletions in the E1, E3, and E4 regions do not induce appreciable expansion of vector-specific CD4+ T cells.

Improved Production of Adenovirus Vectors Expressing Apoptotic Transgenes

Adenovirus vectors expressing gene products that can induce apoptosis have potential utility in gene therapy applications ranging from the treatment of proliferative diseases to transplantation. However, adenovirus vectors carrying proapoptotic gene products are difficult to produce, as the apoptotic environment is not conducive to adenovirus gene expression and replication. Production of AdFasL/G, an adenovirus vector that expresses high levels of Fas ligand, was severely reduced in the 293 packaging cell line. Increased yields of AdFasL/G were achieved by inclusion of peptide-based caspase inhibitors in the growth medium. However, use of these inhibitors for large-scale production would be difficult and expensive. A screen for gene products that increase the yield of AdFasL/G in 293 cells revealed that the poxvirus serpin CrmA and the adenovirus 14.7K product were able to increase virus yields significantly. Apoptosis induced by AdFasL/G was attenuated in 293CrmA cell lines and virus titers were increased dramatically. However, serial passage of AdFasL/G on 293CrmA cells resulted in the generation of replication-competent adenovirus. To resolve this problem, the CrmA gene was introduced into AE25 cells, an E1-complementing cell line that has limited sequence identity with the vectors. AdFasL/G titers were increased 100-fold on AE25CrmA cells relative to the AE25 cells and RCA contamination was not detectable. In addition, adenovirus vectors that express FADD, caspase 8, and Fas/APO1 were produced efficiently in AE25CrmA and 293CrmA.