Transduction of Adeno-Associated Virus Vectors Targeting Hair Cells and Supporting Cells in the Neonatal Mouse Cochlea

Adeno-associated virus (AAV) is the preferred vector for gene therapy of hereditary deafness, and different viral serotypes, promoters and transduction pathways can influence the targeting of AAV to different types of cells and the expression levels of numerous exogenous genes. To determine the transduction and expression patterns of AAV with different serotypes or promoters in hair cells and supporting cells in the neonatal mouse cochlea, we examined the expression of enhanced green fluorescent protein (eGFP) for five different types of AAV vectors [serotypes 2, 9, and Anc80L65 with promoter cytomegalovirus (CMV)-beta-Globin and serotypes 2 and 9 with promoter chicken beta-actin (CBA)] in in vitro cochlear explant cultures and we tested the transduction of AAV2/2-CBA, AAV2/9-CBA, and AAV2/Anc80L65-CMV by in vivo microinjection into the scala media of the cochlea. We found that each AAV vector had its own transduction and expression characteristics in hair cells and supporting cells in different regions of the cochlea. There was a tonotopic gradient for the in vitro transduction of AAV2/2-CBA, AAV2/9-CBA, AAV2/2-CMV, and AAV2/9-CMV in outer hair cells (OHCs), with more OHCs expressing eGFP at the base of the cochlea than at the apex. AAV2/2-CBA in vitro and AAV2/Anc80L65-CMV in vivo induced more supporting cells expressing eGFP at the apex than in the base. We found that AAV vectors with different promoters had different expression efficacies in hair cells and supporting cells of the auditory epithelium. The CMV-beta-Globin promoter could drive the expression of the delivered construct more efficiently in hair cells, while the CBA promoter was more efficient in supporting cells. The in vitro and in vivo experiments both demonstrated that AAV2/Anc80L65-CMV was a very promising vector for gene therapy of deafness because of its high transduction rates in hair cells. These results might be useful for selecting the appropriate vectors for gene delivery into different types of inner ear cells and thus improving the effectiveness of gene therapy

Loss of ARHGEF6 Causes Hair Cell Stereocilia Deficits and Hearing Loss in Mice

ARHGEF6 belongs to the family of guanine nucleotide exchange factors (GEFs) for Rho GTPases, and it specifically activates Rho GTPases CDC42 and RAC1. Arhgef6 is the X-linked intellectual disability gene also known as XLID46, and clinical features of patients carrying Arhgef6 mutations include intellectual disability and, in some cases, sensorineural hearing loss. Rho GTPases act as molecular switches in many cellular processes. Their activities are regulated by binding or hydrolysis of GTP, which is facilitated by GEFs and GTPase-activating proteins, respectively. RAC1 and CDC42 have been shown to play important roles in hair cell (HC) stereocilia development. However, the role of ARHGEF6 in inner ear development and hearing function has not yet been investigated. Here, we found that ARHGEF6 is expressed in mouse cochlear HCs, including the HC stereocilia. We established Arhgef6 knockdown mice using the clustered regularly interspaced short palindromic repeat-associated Cas9 nuclease (CRISPR-Cas9) genome editing technique. We showed that ARHGEF6 was indispensable for the maintenance of outer hair cell (OHC) stereocilia, and loss of ARHGEF6 in mice caused HC stereocilia deficits that eventually led to progressive HC loss and hearing loss. However, the loss of ARHGEF6 did not affect the synapse density and did not affect the mechanoelectrical transduction currents in OHCs at postnatal day 3. At the molecular level, the levels of active CDC42 and RAC1 were dramatically decreased in the Arhgef6 knockdown mice, suggesting that ARHGEF6 regulates stereocilia maintenance through RAC1/CDC42

Mutations in TUBB8 and Human Oocyte Meiotic Arrest

BACKGROUND Human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. METHODS We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, early embryos, sperm cells, and several somatic tissues by means of a quantitative reverse- transcriptase–polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. RESULTS We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. CONCLUSIONS TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility. (Funded by the National Basic Research Program of China and others.

Efficient, glucose responsive, and cell-specific transgene expression in pancreatic islets.

Includes bibliographical references (p. 130-147).Diabetes mellitus is increasing in prevalence, affecting more than 5% of the population throughout the world. Novel therapeutic strategies, including new medications, islet transplantation, and gene therapy, are vigorously being sought to treat diabetes. Direct in vivo pancreatic gene delivery targeting the islets is a key approach for diabetic gene therapy. So far studies have shown that the adenovirus, adeno-associated virus, lentivirus, and herpes simplex virus-1 vectors can be used for efficient gene transfer to the islets in vivo, but suffered from host immune responses and vector cytotoxicity. Non-viral gene delivery systems, including naked DNA and DNA complexes, have also demonstrated islet cell transfection at much lower levels and with transient transgene expression over time. However, it seems likely that non-viral vector systems will more easily satisfy bio-safety concerns in clinical trials. Our new gene therapy approach, ultrasound-targeted micro-bubble destruction (UTMD), has shown that it is able to deliver genes to the pancreas in vivo. Briefly, genes are incorporated into cationic liposome and then attached to the phospholipids shell of gas-filled micro-bubble, which are then injected intravenously and destroyed within the microcirculation of intact islets by ultrasound. This approach enables transfection of the entire islet core, wherein most beta cells reside. Our current study focuses on how to construct gene therapy vectors that can drive functional gene expression in beta cells efficiently. We now demonstrate differential efficiency of gene expression by varying the length of the RIP segments. We attempted to expand the insulin gene promoter from the traditional area to the non-coding region in exon1, intron1 and part of exon2 of the rat insulin gene 1 with a downstream reporter gene in the plasmid construction. We report that UTMD delivers this rat insulin promoter driving the reporter vectors to intact islets of adult, living animals, providing a relative safety, tissue-specific, high efficient and regulated gene expression for diabetic gene therapy.by Renjie Chai.Ph.D

Insm1a Is Required for Zebrafish Posterior Lateral Line Development

Insulinoma-associated 1 (Insm1), a zinc-finger transcription factor, is widely expressed in the developing nervous system and plays important roles in cell cycle progression and cell fate specification. However, the functions of Insm1 in the embryonic development of the sensory system and its underlying molecular mechanisms remain largely unexplored. Here, through whole-mount in situ hybridization, we found that the zebrafish insm1a gene was expressed in the posterior lateral line (pLL) system, including both the migrating pLL primordium and the deposited neuromast cells. In order to decipher the specific roles of insm1a in zebrafish pLL development, we inhibited insm1a expression by using a morpholino knockdown strategy. The insm1a morphants exhibited primordium migration defects that resulted in reduced numbers of neuromasts. The inactivation of insm1a reduced the numbers of hair cells in neuromasts, and this defect could be a secondary consequence of disrupting rosette formation in the pLL primordium. Additionally, we showed that insm1a knockdown decreased the proliferation of pLL primordium cells, which likely contributed to these pLL defects. Furthermore, we showed that loss of insm1a resulted in elevated Wnt/β-catenin signaling and downregulation of Fgf target genes in the primordium. Insm1a knockdown also perturbed the expression patterns of chemokine signaling genes. Taken together, this study reveals a pivotal role for Insm1a in regulating pLL development during zebrafish embryogenesis

TARJETA DE VISITA [Material gráfico]

EUROPACopia digital. Madrid : Ministerio de Educación, Cultura y Deporte, 201

Designs of Biomaterials and Microenvironments for Neuroengineering

Recent clinical research on neuroengineering is primarily focused on biocompatible materials, which can be used to provide electroactive and topological cues, regulate the microenvironment, and perform other functions. Novel biomaterials for neuroengineering have been received much attention in the field of research, including graphene, photonic crystals, and organ-on-a-chip. Graphene, which has the advantage of high mechanical strength and chemical stability with the unique electrochemical performance for electrical signal detection and transmission, has significant potential as a conductive scaffolding in the field of medicine. Photonic crystal materials, known as a novel concept in nerve substrates, have provided a new avenue for neuroengineering research because of their unique ordered structure and spectral attributes. The “organ-on-a-chip” systems have shown significant prospects for the developments of the solutions to nerve regeneration by mimicking the microenvironment of nerve tissue. This paper presents a review of current progress in the designs of biomaterials and microenvironments and provides case studies in developing nerve system stents upon these biomaterials. In addition, we compose a conductive patterned compounded biomaterial, which could mimic neuronal microenvironment for neuroengineering by concentrating the advantage of such biomaterials