Adeno-associated virus (AAV)-mediated gene therapy is widely applied to treat numerous hereditary diseases in animal models and humans. The specific expression of AAV-delivered transgenes driven by cell type-specific promoters should further increase the safety of gene therapy. However, current methods for screening cell type-specific promoters are labor-intensive and time-consuming. Herein, we designed a "multiple vectors in one AAV" strategy for promoter construction in vivo. Through this strategy, we truncated a native promoter for Myo15 expression in hair cells (HCs) in the inner ear, from 1,611 bp down to 1,157 bp, and further down to 956 bp. Under the control of these 2 promoters, green fluorescent protein packaged in AAV-PHP.eB was exclusively expressed in the HCs. The transcription initiation ability of the 2 promoters was further verified by intein-mediated otoferlin recombination in a dual-AAV therapeutic system. Driven by these 2 promoters, human otoferlin was selectively expressed in HCs, resulting in the restoration of hearing in treated Otof -/- mice for at least 52 weeks. In summary, we developed an efficient screening strategy for cell type-specific promoter engineering and created 2 truncated Myo15 promoters that not only restored hereditary deafness in animal models but also show great potential for treating human patients in future.
Abstract Hearing loss is one of the most prevalent sensory disorders, but no commercial biological treatments are currently available. Here, we identified an East Asia-specific founder mutation, the homozygous c.220C>T mutation in MPZL2 , that contributes to a significant proportion of hereditary deafness cases in our cohort study. We found that the disease-causing mutation could be targetable by adenine base editors (ABEs) that enable A·T-to-G·C base corrections without DNA double-strand breaks. To demonstrate this, we developed a humanized mouse model ( hMPZL2 Q74X/Q74X ) that recapitulates human MPZL2 deafness and leads to progressive hearing loss. A PAM-flexible ABE variant with reduced bystander and off-target effects (ABE8eWQ-SpRY:sgRNA3) was packaged in dual adeno-associated viruses (AAVs) and injected into the inner ear of hMPZL2 Q74X/Q74X mice and effectively corrected the mutation. This treatment significantly restored hearing function, improved inner ear structural integrity, and reversed altered gene expression. Base editing may hold therapeutic potential for hereditary deafness, including most cases of MPZL2 deafness.
Objective
To investigate the dynamic changes of lymphocyte subsets (lymphocyte count, CD3+ T, CD4+ T, CD8+ T, CD19+ B, CD56+ NK)in children with septic shock.
Methods
Peripheral blood lymphocyte subsets were analysed in 25 cases with severe septic shock and 24 cases with mild septic shock on day 1, 3, 8, and compared with those of 25 healthy volunteers.Children with severe septic shock were divided into survival group and dying group according to the outcome.The detection data were compared.
Results
In the first day of admission, peripheral blood lymphocyte subsets in children with septic shock decreased significantly compared with the normal children, and there was significant difference in the three groups(P 0.05), but children with severe septic shock had statistical increase of CD4+ T, CD3+ T and lymphocyte count(P<0.05). There were 5 cases died who had severe septic shock in day 2 to day 5 after admission.The count of lymphocyte subsets were detected significantly lower in dying group than survival group in the third day of admission(P<0.05).
Conclusion
The continuous low level of lymphocyte subsets indicates bad outcome.The patient's condition improved with increase of CD4+ T, CD3+ T, lymphocyte count.CD4+ T is the most sensitive.Dynamic detection of lymphocyte subsets in the peripheral blood of children with septic shock is of great clinical significance in judging the severity of disease and curative effect.
Key words:
Septic shock; Lymphocyte subsets
Abstract Objective Induced pluripotent stem cells (iPSCs) hold a promising potential for rescuing dopaminergic neurons in therapy for Parkinson's disease (PD). This study clarifies a TREM2‐dependent mechanism explaining the function of iPSC differentiation in neuronal repair of PD. Methods PD‐related differentially expressed genes were screened by bioinformatics analyses and their expression was verified using RT‐qPCR in nigral tissues of 6‐OHDA‐lesioned mice. Following ectopic expression and depletion experiments in iPSCs, cell differentiation into dopaminergic neurons as well as the expression of dopaminergic neuronal markers TH and DAT was measured. Stereotaxic injection of 6‐OHDA was used to develop a mouse model of PD, which was injected with iPSC suspension overexpressing TREM2 to verify the effect of TREM2 on neuronal repair. Results TREM2 was poorly expressed in the nigral tissues of 6‐OHDA‐lesioned mice. In the presence of TREM2 overexpression, the iPSCs showed increased expression of dopaminergic neuronal markers TH and DAT, which facilitated the differentiation of iPSCs into dopaminergic neurons. Mechanistic investigations indicated that TREM2 activated the TGF‐β pathway and induced iPSC differentiation into dopaminergic neurons. In vivo data showed that iPSCs overexpressing TREM2 enhanced neuronal repair in 6‐OHDA‐lesioned mice. Conclusion This work identifies a mechanistic insight for TREM2‐mediated TGF‐β activation in the regulation of neuronal repair in PD and suggests novel strategies for neurodegenerative disorders.
COAD/READ/COADREAD_rnaseq_fpkm.txt files contain TCGA RNA-Seq data in FPKM normalisation for colorectal adenocarcinoma (COAD), rectum adenocarcinoma (READ) or combined (COADREAD). COAD/READ/COADREAD_rnaseq_tpm.txt files contain TCGA RNA-Seq data in TPM normalisation for colorectal adenocarcinoma (COAD), rectum adenocarcinoma (READ) or combined (COADREAD). COAD/READ/COADREAD_clinical_raw.xlsx files contain TCGA clinical data for patients with colorectal adenocarcinoma (COAD), rectum adenocarcinoma (READ) or combined (COADREAD). COAD/READ/COADREAD_rnaseq_clinical_raw.xlsx files contain corresponding information of TCGA clinical data and RNA-Seq data for patients with colorectal adenocarcinoma (COAD), rectum adenocarcinoma (READ) or combined (COADREAD).
Due to the initiation of the priority review program in China, many antitumor drugs have been approved for marketing based on phase II clinical trials and short-term surrogate endpoint indicators. This study used approved targeted drugs for the treatment of non-small-cell lung cancer (NSCLC) in China as an example to evaluate the association between short-term surrogate endpoints [objective response rate (ORR) and disease control rate (DCR)] and median progression-free survival (mPFS) and median overall survival (mOS).
Objective: To investigate effects and functional mechanism of compound Congrongyizhi Capsule (CCRC), a Chinese medicine, on cognitive functions against amnestic mild cognitive impairment (aMCI) patients with functional magnetic resonance imaging (fMRI) based on n-back task. Methods: Forty-one aMCI participants from hospital and local communities in Beijing and randomly divided into treatment (16 patients with CCRC capsule treatment), placebo (12 patients with placebo capsules) and control group (13 patients with no treatment). The duration of intervention lasted for 3 months. Neuropsychological tests and fMRI were applied to assess cognitive ability and brain activation changes after three months treatment. Results: Drug group (n=16) presented increased significance in the MMSE (P=0.008) and digit span (P<0.001) tests, while other scores of neuropsychological tests showed no statistical significance. fMRI results showed an increased brain negative activation in drug group during performing the n-back working-memory task in posterior cingulate (PCC), inferior frontal gyrus and lingual gyrus regions after 3 months; placebo and control group did not show the same effect. Meanwhile, there were negative correlations between left PCC activation levels and changed values of MMSE and digit span separately since increased negative activation was associated with better performance on the scores of MMSE and Digit Span tests. Conclusions: CCRC can increase negative activation degree of PCC under performing working memory tasks while this modulation are associated with better performance on the MMSE and Digit Span. Keywords: BOLD functional MRI, brain activation, compound chinese medicines, mild cognitive impairment.
Abstract Gene therapy is a promising approach for hereditary deafness. We recently showed that unilateral AAV1-hOTOF gene therapy with dual adeno-associated virus (AAV) serotype 1 carrying human OTOF transgene is safe and associated with functional improvements in patients with autosomal recessive deafness 9 (DFNB9). The protocol was subsequently amended and approved to allow bilateral gene therapy administration. Here we report an interim analysis of the single-arm trial investigating the safety and efficacy of binaural therapy in five pediatric patients with DFNB9. The primary endpoint was dose-limiting toxicity at 6 weeks, and the secondary endpoint included safety (adverse events) and efficacy (auditory function and speech perception). No dose-limiting toxicity or serious adverse event occurred. A total of 36 adverse events occurred. The most common adverse events were increased lymphocyte counts (6 out of 36) and increased cholesterol levels (6 out of 36). All patients had bilateral hearing restoration. The average auditory brainstem response threshold in the right (left) ear was >95 dB (>95 dB) in all patients at baseline, and the average auditory brainstem response threshold in the right (left) ear was restored to 58 dB (58 dB) in patient 1, 75 dB (85 dB) in patient 2, 55 dB (50 dB) in patient 3 at 26 weeks, and 75 dB (78 dB) in patient 4 and 63 dB (63 dB) in patient 5 at 13 weeks. The speech perception and the capability of sound source localization were restored in all five patients. These results provide preliminary insights on the safety and efficacy of binaural AAV gene therapy for hereditary deafness. The trial is ongoing with longer follow-up to confirm the safety and efficacy findings. Chinese Clinical Trial Registry registration: ChiCTR2200063181 .
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