Person:

Chen, Zheng-Yi

Unlike mammals, the non-mammalian vertebrate inner ear can regenerate the sensory cells, hair cells, either spontaneously or through induction after hair cell loss, leading to hearing recovery. The mechanisms underlying the regeneration are poorly understood. By microarray analysis on a chick model, we show that chick hair cell regeneration involves the activation of proliferation genes and downregulation of differentiation genes. Both MYC and FGF are activated in chick hair cell regeneration. Using a zebrafish lateral line neuromast hair cell regeneration model, we show that the specific inhibition of Myc or Fgf suppresses hair cell regeneration, demonstrating that both pathways are essential to the process. Rapid upregulation of Myc and delayed Fgf activation during regeneration suggest a role of Myc in proliferation and Fgf in differentiation. The dorsal-ventral pattern of fgfr1a in the neuromasts overlaps with the distribution of hair cell precursors. By laser ablation, we show that the fgfr1a-positive supporting cells are likely the hair cell precursors that directly give rise to new hair cells; whereas the anterior-posterior fgfr1a-negative supporting cells have heightened proliferation capacity, likely to serve as more primitive progenitor cells to replenish lost precursors after hair cell loss. Thus fgfr1a is likely to mark compartmentalized supporting cell subtypes with different capacities in renewal proliferation and hair cell regeneration. Manipulation of c-MYC and FGF pathways could be explored for mammalian hair cell regeneration.

Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape, and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains, or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcriptional activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.

The retinoblastoma protein (pRb) is required for cell-cycle exit of embryonic mammalian hair cells but is not required for hair cell fate determination and early differentiation, and this provides a strategy for hair cell regeneration by manipulating the pRb pathway. To reveal the mechanism of pRb functional modification in the inner ear, we compared the effects of attenuated pRb phosphorylation by an inhibitor of the Mitogen-Activated Protein (MAP) kinase pathway and an inhibitor of the Rb–Raf-1 interaction on cultured chicken otocysts. We demonstrated that the activity of pRb is correlated with its phosphorylation state, which is regulated by a newly established cell cycle-independent pathway mediated by the physical interaction between Raf-1 and pRb. The phosphorylation of pRb plays an important role during the early stage of inner ear development, and attenuated phosphorylation in progenitor cells leads to cell cycle arrest and increased apoptosis along with a global down-regulation of the genes involved in cell cycle progression. Our study provides novel routes to modulate pRb function for hair cell regeneration.

Mammalian inner ear harbors diverse cell types that are essential for hearing and balance. Adenovirus is one of the major vectors to deliver genes into the inner ear for functional studies and hair cell regeneration. To identify adenovirus vectors that target specific cell subtypes in the inner ear, we studied three adenovirus vectors, carrying a reporter gene encoding green fluorescent protein (GFP) from two vendors or with a genome editing gene Cre recombinase (Cre), by injection into postnatal days 0 (P0) and 4 (P4) mouse cochlea through scala media by cochleostomy in vivo. We found three adenovirus vectors transduced mouse inner ear cells with different specificities and expression levels, depending on the type of adenoviral vectors and the age of mice. The most frequently targeted region was the cochlear sensory epithelium, including auditory hair cells and supporting cells. Adenovirus with GFP transduced utricular supporting cells as well. This study shows that adenovirus vectors are capable of efficiently and specifically transducing different cell types in the mammalian inner ear and provides useful tools to study inner ear gene function and to evaluate gene therapy to treat hearing loss and vestibular dysfunction.

The role of CD8 T cells in anti-tuberculosis immunity in humans remains unknown, and studies of CD8 T cell–mediated protection against tuberculosis in mice have yielded controversial results. Unlike mice, humans and nonhuman primates share a number of important features of the immune system that relate directly to the specificity and functions of CD8 T cells, such as the expression of group 1 CD1 proteins that are capable of presenting Mycobacterium tuberculosis lipids antigens and the cytotoxic/bactericidal protein granulysin. Employing a more relevant nonhuman primate model of human tuberculosis, we examined the contribution of BCG- or M. tuberculosis-elicited CD8 T cells to vaccine-induced immunity against tuberculosis. CD8 depletion compromised BCG vaccine-induced immune control of M. tuberculosis replication in the vaccinated rhesus macaques. Depletion of CD8 T cells in BCG-vaccinated rhesus macaques led to a significant decrease in the vaccine-induced immunity against tuberculosis. Consistently, depletion of CD8 T cells in rhesus macaques that had been previously infected with M. tuberculosis and cured by antibiotic therapy also resulted in a loss of anti-tuberculosis immunity upon M. tuberculosis re-infection. The current study demonstrates a major role for CD8 T cells in anti-tuberculosis immunity, and supports the view that CD8 T cells should be included in strategies for development of new tuberculosis vaccines and immunotherapeutics.

The in efficient delivery of proteins into mammalian cells remains a major barrier to realizing the therapeutic potential of many proteins. We and others have previously shown that superpositively charged proteins are efficiently endocytosed and can bring associated proteins and nucleic acids into cells. The vast majority of cargo delivered in this manner, however, remains in endosomes and does not reach the cytosol. In this study we designed and implemented a screen to discover peptides that enhance the endosomal escape of proteins fused to superpositively charged GFP (+36 GFP). From a screen of peptides previously reported to disrupt microbial membranes without known mammalian cell toxicity, we discovered a 13-residue peptide, aurein 1.2, that substantially increases cytosolic protein delivery by up to ~5-fold in a cytosolic fractionation assay in cultured cells. Four additional independent assays for non-endosomal protein delivery collectively suggest that aurein 1.2 enhances endosomal escape of associated endocytosed protein cargo. Structure-function studies clarified peptide sequence and protein conjugation requirements for endosomal escape activity. When applied to the in vivo delivery of +36 GFP–Crerecombinase fusions into the inner ear of live mice, fusion with aurein 1.2 dramatically increased non-endosomal Crerecombinase delivery potency, resulting in up to 100% recombined inner hair cellsand 96% recombined outer hair cells, compared to 0-4% recombined hair cells from +36-GFP- Cre without aurein 1.2. Collectively, these findings describe a genetically encodable, endosome escape-enhancing peptide that can substantially increase the cytoplasmic delivery of cationic proteins in vitro and in vivo.

Although genetic factors contribute to almost half of all deafness cases, treatment options for genetic deafness are limited1–5. We developed a genome editing approach to target a dominantly inherited form of genetic deafness. Here we show that cationic lipid-mediated in vivo delivery of Cas9:guide RNA complexes can ameliorate hearing loss in a mouse model of human genetic deafness. We designed and validated in vitro and in primary fibroblasts genome editing agents that preferentially disrupt the dominant deafness-associated allele in the Tmc1 (transmembrane channel-like 1) Beethoven (Bth) mouse model, even though the mutant Bth allele differs from the wild-type allele at only a single base pair. Injection of Cas9:guide RNA:lipid complexes targeting the Bth allele into the cochlea of neonatal Bth/+ mice substantially reduced progressive hearing loss. We observed higher hair cell survival rates and lower auditory brainstem response (ABR) thresholds in injected ears compared with uninjected ears or ears injected with complexes that target an unrelated gene. Enhanced acoustic reflex responses were observed among injected compared to uninjected Bth/+ animals. These findings suggest protein:RNA complex delivery of target gene-disrupting agents in vivo as a potential strategy for the treatment of some autosomal dominant hearing loss diseases.