Dataset Information

Optimization of miR-22 expression cassette for rAAV delivery on diabetes.

ABSTRACT: MicroRNA-22 (miR-22) was suggested to be important for type 2 diabetes but its functions for this disease remained unclear. Recombinant adeno-associated virus (rAAV)-mediated miR delivery is a powerful approach to study miR functions in vivo, however, the overexpression of miR-22 by rAAV remains challenging because it is one of the most abundant miRs in the liver. In this study, a series of expression cassettes were designed and compared. It was shown that different lengths of primary miR-22 were overexpressed in HEK293 and HeLa cells but the longer ones were more efficiently expressed. miR-22 may be placed in either introns or the 3' UTR of a transgene for efficient overexpression. RNA polymerase III or II promoters were successfully utilized for miR expression but the latter showed higher expression levels in cell lines. Specifically, miR-22 was expressed efficiently together with an EGFP gene. After screening, a liver-specific TTR promoter was chosen to overexpress miR-22 in diabetic mice fed a high-fat diet. It was shown that miR-22 was overexpressed 2-3 folds which improved the insulin sensitivity significantly. The approach utilized in this study to optimize miR overexpression is a powerful tool for the creation of efficient rAAV vectors for the other miRs.

SUBMITTER: Yang L

PROVIDER: S-EPMC8727650 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Optimization of miR-22 expression cassette for rAAV delivery on diabetes.

Yang Li L Du Wenya W Zheng Zhaoyue Z Wang Li L Xiao Lin L Yang Qingzhe Q Hao Qiukui Q Zhou Jiao J Du Jintao J Li Jun J Valencia C Alexander CA Dong Birong B Chow Hoi Yee HY Fu Xianghui X Dong Biao B

Molecular biomedicine 20220105 1

MicroRNA-22 (miR-22) was suggested to be important for type 2 diabetes but its functions for this disease remained unclear. Recombinant adeno-associated virus (rAAV)-mediated miR delivery is a powerful approach to study miR functions in vivo, however, the overexpression of miR-22 by rAAV remains challenging because it is one of the most abundant miRs in the liver. In this study, a series of expression cassettes were designed and compared. It was shown that different lengths of primary miR-22 wer ...[more]

PMID: 34984525

Similar Datasets

Project description:Phase 1 and phase 2 gene therapy trials using intramuscular (IM) administration of a recombinant adeno-associated virus serotype 1 (rAAV1) for replacement of serum alpha-1 antitrypsin (AAT) deficiency have shown long-term (5-year) stable transgene expression at approximately 2% to 3% of therapeutic levels, arguing for the long-term viability of this approach to gene replacement of secreted serum protein deficiencies. However, achieving these levels required 100 IM injections to deliver 135 mL of vector, and further dose escalation is limited by the scalability of direct IM injection. To further advance the dose escalation, we sought to bridge the rAAV-AAT clinical development program to regional limb perfusion, comparing two methods previously established for gene therapy, peripheral venous limb perfusion (VLP) and an intra-arterial push and dwell (IAPD) using rAAV1 and rAAV8 in a non-human primate (rhesus macaque) study. The rhesus AAT transgene was used with a c-myc tag to enable quantification of transgene expression. 5 cohorts of animals were treated with rAAV1-IM, rAAV1-VLP, rAAV1-IAPD, rAAV8-VLP, and rAAV8-IAPD (n = 2-3), with a dose of 6 × 1012 vg/kg. All methods were well tolerated clinically. Potency, as determined by serum levels of AAT, of rAAV1 by the VLP method was twice that observed with direct IM injection; 90 μg/mL with VLP versus 38 μg/mL with direct IM injection. There was an approximately 25-fold advantage in estimated vector genomes retained within the muscle tissue with VLP and a 5-fold improvement in the ratio of total vector genomes retained within muscle as compared with liver. The other methods were intermediate in the potency and retention of vector genomes. Examination of muscle enzyme (CK) levels indicated rAAV1-VLP to be equally safe as compared with IM injection, while the IAPD method showed significant CK elevation. Overall, rAAV1-VLP demonstrates higher potency per vector genome injected and a greater total vector retention within the muscle, as compared to IM injection, while enabling a much greater total dose to be delivered, with equivalent safety. These data provide the basis for continuation of the dose escalation of the rAAV1-AAT program in patients and bode well for rAAV-VLP as a platform for replacement of secreted proteins.

Project description:BackgroundThe laccase Lcc9 from Coprinopsis cinerea has optimal catalytic activity at moderate to alkaline pH conditions, making it invaluable for industrial applications. However, C. cinerea naturally secretes Lcc9 at low expression levels, which limits the industrial application of Lcc9 on a large scale. Recombinant production of Lcc9 using Aspergillus niger would be an effective way to achieve its high production.ResultsThis study achieved the secretory production of Lcc9 in A. niger and established an efficient transformation procedure for A. niger by optimizing its protoplast preparation system. The transformation efficiency of A. niger was increased 3.8-fold under the optimal system (cell wall digestion enzyme solution: 2% cellulase, 1% snailase, 1% lyticase, and 0.5% lysozyme; incubation time: 3 h; incubation temperature: 37 ℃; culture time: 48 h). The extracellular yield of Lcc9 was enhanced by optimizing gene expression cassette and bioprocess. First, the strain AnGgcL (containing PgpdA) mediated by the SPCAT, a signal peptide of the extracellular high abundance protein catalase, had an extracellular laccase activity of 10 U/L after shake flask fermentation. Then, by optimizing promoter and signal peptide combinations that regulate lcc9 expression, the strain AnGcgL mediated by PcitA-SPGlaA had an extracellular laccase activity of 20 U/L. Subsequently, the strain AnRcgL1 (containing PcitA-SPGlaA) obtained by random integration had an extracellular laccase activity of 86 U/L. Sequencing revealed that the lcc9 expression cassette was integrated into the citrate synthase gene locus in the AnRcgL1 genome in a 9-copy form. By optimizing the microparticle, osmolyte, and Cu2+ in the fermentation medium, the AnRcgL1 extracellular laccase activity was further increased to 1566.7 U/L, which was 156.7-fold higher than that of AnGgcL. Furthermore, its extracellular laccase activity was increased to 1961 U/L in a 1-L fermenter.ConclusionsTo our knowledge, this study is the first to report the recombinant extracellular production of the C. cinerea laccase Lcc9 in A. niger and to use SPCAT in the A. niger expression system. The results of this study will help accelerate the industrial application of Lcc9. Moreover, the strategy used in this work provides methodological guidance for increasing other exogenous protein yields in A. niger.

Dataset Information

Optimization of miR-22 expression cassette for rAAV delivery on diabetes.

Publications

Optimization of miR-22 expression cassette for rAAV delivery on diabetes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets