Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs.

Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here, we tested the ability of cell-penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection-enhanced delivery system. These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9. The production of ultra-low endotoxin Cas9 protein manufactured at scale further improved innate immunity. We conclude that injection-based delivery of minimally immunogenic CRISPR genome editing RNPs into the CNS provides a valuable alternative to virus-mediated genome editing.

Results of a 10-year hearing screening using automated auditory brainstem response in newborns: The two-step AABR method.

OBJECTIVES: Approximately 1-6 in every 1000 children are born with hearing loss. An automated auditory brainstem response (AABR) test is essential for screening newborns for abnormal hearing. At the tertiary hospital, we have been using a two-step AABR protocol for newborn hearing assessment since 2005. This study aimed to report the 10-year hearing screening results of newborns at the institution, and prove the efficacy of the two-step AABR protocol. METHODS: From August 2005 to January 2015, 3059 newborns were screened through AABR testing using the MASTER ABaer system. The first screening test was performed after the first 24 h of life. If a newborn was referred, the test was performed within 1 month after discharge from the hospital. The results were regarded as pass when the point optimized variance ratio was >3.5, using a stimulus level of 35 dB HL. When newborns were referred for the second AABR, they received follow-up tests including tympanometry, ABR, auditory steady-state response, and otoacoustic emission within 3 months. RESULTS: A total of 3059 newborns underwent newborn hearing screening tests over a period of 10 years. One hundred and twenty (3.9%) newborns were referred with the initial AABR, and 104 (3.4%) were referred with a subsequent AABR. Of the newborns, 42 (1.37%) were confirmed to have a bilateral hearing impairment. CONCLUSIONS: It is known that the referral rate for the AABR test is 3-4%, as recommended by the Joint Committee on Infant Hearing. Our data showed a referral rate of 3.4%. The two-step AABR test has been useful for screening hearing loss in newborns at tertiary hospital.

Use of model peptide reactions for the characterization of kinetically controlled ligation.

Since the introduction of kinetically controlled ligation (KCL), a chemoselective reaction between a peptide-(α)thioarylester and a Cys-peptide-(α)thioalkylester, KCL has been utilized for the total chemical synthesis of large proteins (i.e., lysozyme and HIV-protease) by providing fully convergent synthetic routes. Although KCL has the potential to become an important chemistry for protein synthesis, the principle of KCL is not fully characterized. In particular, prior work on KCL has focused on the reactivity difference of the two different -(α)thioester forms-alkyl vs aryl. Another equally important feature of KCL, Xaa-Cys ligation sites, has not been investigated. The work reported here describes combinatorial KCL reactions using model peptides to dissect the interplay of the Xaa(1), Xaa(2), -(α)thioarylester, and -(α)thioalkylester. Results from these studies provide fundamental insights into the KCL reaction, and will lead to the optimal synthetic route for the routine chemical synthesis of large target protein molecules.