[mRNA vaccines for infectious diseases: research progress and applications].

Messenger RNA (mRNA) vaccines emerge as promising vaccines to prevent infectious diseases. Compared with traditional vaccines, mRNA vaccines present numerous advantages, such as high potency, safe administration, rapid production potentials, and cost-effective manufacturing. In 2020, two COVID-19 vaccines (BNT162b2 and mRNA-1273) were approved by the Food and Drug Administration (FDA). The two vaccines showed high efficiency in combating COVID-19, which indicates the great advantages of mRNA technology in developing vaccines against emergent infectious diseases. Here, we summarize the type, immune mechanisms, modification methods of mRNA vaccines, and their applications in preventing infectious diseases. Current challenges and future perspectives in developing mRNA vaccines are also discussed.

Internally inlaid SaCas9 base editors enable window specific base editing.

Rationale: Base editors composed of catalytic defective Cas9 and cytosine or adenosine deaminase are powerful tools to convert bases in a genome. However, the fixed and narrow editing window of current base editors has impeded their utility. To increase the scope and diversify the editing patterns is quite necessary. Methods and Results: We designed a subset of base editors derived from SaCas9 in which deaminase was inlaid into various locations of the SaCas9 protein. The resulting base editors were characterized with multiple genomic sites and were found to have distinct editing features to the N-terminal SaCas9 CBE (Sa-CBE-N). Among them, Sa-CBE-693, in which a cytosine deaminase was inserted between amino acids 693 and 694, showed an increased editing efficiency and a significantly expanded editing window ranging from bases 2-18. This feature enhanced the editing efficiency of BCL11A enhancer that contains multiple consensus bases in a 15-bp fragment. Another variant, Sa-CBE-125, displayed backward-shifted editing window, which we showed was particularly powerful in editing cytosines that were accompanied with unintended bystander cytosines at their 5' side. Additionally, these editors showed reduced Cas9 independent DNA off-target editing compared with Sa-CBE-N. Conclusion: Our inlaid base editors improved the targeting scope and diversified the editing pattern.

A universal strategy for AAV delivery of base editors to correct genetic point mutations in neonatal PKU mice.

Base editing tools enabled efficient conversion of C:G or A:T base pairs to T:A or G:C, which are especially powerful for targeting monogenic lesions. However, in vivo correction of disease-causing mutations is still less efficient because of the large size of base editors. Here, we designed a dual adeno-associated virus (AAV) strategy for in vivo delivery of base editors, in which deaminases were linked to Cas9 through the interaction of GCN4 peptide and its single chain variable fragment (scFv) antibody. We found that one or two copies of GCN4 peptide were enough for the assembly of base editors and produced robust targeted editing. By optimization of single-guide RNAs (sgRNAs) that target phenylketonuria (PKU) mutation, we were able to achieve up to 27.7% correction in vitro. In vivo delivery of this dual AAV base editing system resulted in efficient correction of PKU-related mutation in neonatal mice and subsequent rescue of hyperphenylalaninemia-associated syndromes. Considering the similarity between Cas9 proteins from different organisms, our delivery strategy will be compatible with other Cas9-derived base editors.

A split cytosine deaminase architecture enables robust inducible base editing.

Directed base substitution with base editing technology enables efficient and programmable conversion of C:G or A:T base pairs to T:A or G:C in the genome. Although this technology has shown great potentials in a variety of basic research, off-target editing is among one of the biggest challenges toward its way to clinical application. Base editing tools, especially the tools converting C to T, caused unpredictable off-target editing throughout the genome, which raise the concern that long-term application of these tools would induce genomic instability or even tumorigenesis. To overcome this challenge, we designed an inducible base editing tool that was active only in the presence of a clinically safe chemical, rapamycin. In the guidance of structural information, we designed four split-human APOBEC3A (A3A) -BE3 base editors in which these A3A deaminase enzymes were split at sites that were opposite to the protein-nucleotide interface. We showed that by inducible deaminase reconstruction with a rapamycin responsible interaction system (FRB and FKBP); three out of four split-A3A-derived base editors showed robust inducible base editing. However, in the absence of rapamycin, their editing ability was dramatically inhibited. Among these split editors, splicing at Aa85 of A3A generated the most efficient inducible editing. In addition, compared to the full-length base editor, the splitting did not obviously alter the editing window and motif preference, but slightly increased the product purity. We also expanded this strategy to another frequently used cytosine deaminase, rat APOBEC1 (rA1), and observed a similar induction response. In summary, these results demonstrated the concept that splitting deaminases is a practicable method for timely controlling of base editing tools.

Design, synthesis and biological evaluation of tyrosinase-targeting PROTACs.

The human tyrosinase is the most prominent therapeutic target for pigmentary skin disorders. However, the overwhelming majority efforts have been devoted to search mushroom tyrosinase inhibitors, which show poor inhibitory activity on human tyrosinase and certain side effects that cause skin damage in practical application. Herein, a series of degraders that directly targeted human tyrosinase was firstly designed and synthesized based on newly developed PROTAC technology. The best PROTAC TD9 induced human tyrosinase degradation obviously in dose and time-dependent manner, and its mechanism of inducing tyrosinase degradation has also been clearly demonstrated. Besides, encouraging results that low-toxicity PROTAC TD9 was applied to reduce zebrafish melanin synthesis have been obtained, highlighting the potential to treatment of tyrosinase-related disorders. Moreover, this work has innovatively expanded the application scope of PROTAC technology and laid a solid foundation for further development of novel drugs treating pigmentary skin disorders.

sgBE: a structure-guided design of sgRNA architecture specifies base editing window and enables simultaneous conversion of cytosine and adenosine.

We present a base editing system, in which base editors are attached to different sites of sgRNA scaffold (sgBE). Each independent sgBE has its own specific editing pattern for a given target site. Among tested sgBEs, sgBE-SL4, in which deaminase is attached to the last stem-loop of sgRNA, yields the highest editing efficiency in the window several nucleotides next to the one edited by BE3. sgBE enables the simultaneous editing of adenine and cytosine. Finally, in order to facilitate in vivo base editing, we extend our sgBE system to an AAV-compatible Cas9, SaCas9 (Staphylococcus aureus), and observe robust base editing.

Long-Term Correction of Copper Metabolism in Wilson's Disease Mice with AAV8 Vector Delivering Truncated ATP7B.

Wilson's disease (WD) is an autosomal recessive disorder of copper metabolism caused by mutations in the ATP7B gene encoding a liver active copper transport enzyme. Gene therapy with adeno-associated virus (AAV) carrying full-length ATP7B, which is about 4.4 kb, was shown to rescue copper metabolism disorder in WD mouse model. However, due to its relatively large size, the AAV vector containing full-length ATP7B could be oversized for its packaging capacity, which could lead to inefficient packaging. To this purpose, we engineered a truncated ATP7B mutant (tATP7B) that is about 3.3 kb in length and used for AAV gene therapy for WD mice. In vitro test showed that the excretion of copper outside the cells could be achieved with tATP7B as efficient as the full-length ATP7B. In vivo delivery of tATP7B to WD mice by AAV8 vectors corrected their copper metabolisms and significantly rescued copper accumulation-related syndromes, including reduced urinary copper excretion, increased serum ceruloplasmin, and improved liver damages. Thus, our study demonstrated that AAV gene therapy based on truncated ATP7B is a promising strategy in the treatment of WD.

Monocytic cell junction proteins serve important roles in atherosclerosis via the endoglin pathway.

The formation of atherosclerosis is recognized to be caused by multiple factors including pathogenesis in monocytes during inflammation. The current study provided evidence that monocytic junctions were significantly altered in patients with atherosclerosis, which suggested an association between cell junctions and atherosclerosis. Claudin­1, occludin­1 and ZO­1 were significantly enhanced in atherosclerosis, indicating that the tight junction pathway was activated during the pathogenesis of atherosclerosis. In addition, the gene expression of 5 connexin members involved in the gap junction pathway were quantified, indicating that connexin 43 and 46 were significantly up­regulated in atherosclerosis. Furthermore, inflammatory factors including endoglin and SMAD were observed, suggesting that immune regulative factors were down­regulated in this pathway. Silicon­based analysis additionally identified that connexins and tight junctions were altered in association with monocytic inflammation regulations, endoglin pathway. The results imply that reduced expression of the immune regulation pathway in monocytes is correlated with the generation of gap junctions and tight junctions which serve important roles in atherosclerosis.

The Correlation Between 9p21 Chromosome rs4977574 Polymorphism Genotypes and the Development of Coronary Artery Heart Disease.

Our aim is to investigate the correlation between 9p21 chromosome rs4977574 polymorphism genotypes and the development of coronary artery heart disease (CHD). Two hundred and eighty-nine patients with angiography-confirmed CHD were recruited as the CHD group, while 338 subjects without CHD symptoms were enrolled as the control group. For all participating subjects, the genotypes of rs4977574 polymorphism were examined by the real-time PCR analysis. Analyses acquired from single-locus technique showed that genotype distribution of rs4977574 polymorphism was significantly different (p = 0.041) between CHD group and the control group. Logistic regression analysis demonstrated that rs4977574 polymorphism in a dominant mode significantly increased (p = 0.038) the risk of CHD, where odds ratio (OR) was 0.71 and the 95 % confidence interval (CI) 0.58-0.97 was applied. 9p21 chromosome rs4977574 polymorphism genotypes are associated with the incidence and development of CHD. The presence of C allele may reduce the risk of CHD.

Application of adipose-derived stem cells in heart disease.

Therapy with mesenchymal stem cells is one of the promising tools to improve outcomes after myocardial infarction. Adipose-derived stem cells (ASCs) are an ideal source of mesenchymal stem cells due to their abundance and ease of preparation. Studies in animal models of myocardial infarction have demonstrated the ability of injected ASCs to engraft and differentiate into cardiomyocytes and vasculature cells. ASCs secrete a wide array of angiogenic and anti-apoptotic paracrine factors such as vascular endothelial growth factor, hepatocyte growth factor, and insulin-like growth factor 1. ASCs are capable of enhancing heart function, reducing myocardial infarction, promoting vascularization, and reversing remodeling in the ischemically injured hearts. Furthermore, several ongoing clinical trials using ASCs are producing promising results for heart diseases. This article reviews the isolation, differentiation, immunoregulatory properties, mechanisms of action, animal models, and ongoing clinical trials of ASCs for cardiac disease.

Relationship between methylenetetrahydrofolate reductase gene polymorphism and the coronary slow flow phenomenon.

OBJECTIVE: To investigate the correlation between methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism and the coronary slow flow phenomenon (CSFP), and to discover the involvement of genetic factors in CSFP. PARTICIPANTS AND METHODS: Seventy-five patients with normal angiographic coronary arteries were recruited between June 2012 and June 2013. MTHFR C677T genotypes were sequenced by pyrosequencing, whereas the concentration of homocysteine (Hcy) was determined using the enzymatic cycling assay. RESULTS: Compared with the controls, the CSFP patients had higher Hcy concentrations and higher male morbidity. The CSFP patients showed higher frequencies of MTHFR 677(TT+TC) genotypes and the 677T allele compared with the controls. Plasma Hcy levels and male morbidity were correlated positively with the average corrected TIMI frame count. Multiple linear regression and logistic regression analysis indicated that both Hcy and male sex were risk factors for CSFP. MTHFR C677T genotypes and the frequency distribution of 677T allele complied with Hardy-Weinberg equilibrium. CONCLUSION: CSFP was associated with a high level of plasma Hcy, and men were more vulnerable to CSFP. By regulating the plasma Hcy level, the MTHFR C677T gene polymorphism and folic acid level might be involved in the occurrence of CSFP.