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1.
Mol Cell ; 2024 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-39096898

RESUMO

The IscB proteins, as the ancestors of Cas9 endonuclease, hold great promise due to their small size and potential for diverse genome editing. However, their activity in mammalian cells is unsatisfactory. By introducing three residual substitutions in IscB, we observed an average 7.5-fold increase in activity. Through fusing a sequence-non-specific DNA-binding protein domain, the eIscB-D variant achieved higher editing efficiency, with a maximum of 91.3%. Moreover, engineered ωRNA was generated with a 20% reduction in length and slightly increased efficiency. The engineered eIscB-D/eωRNA system showed an average 20.2-fold increase in activity compared with the original IscB. Furthermore, we successfully adapted eIscB-D for highly efficient cytosine and adenine base editing. Notably, eIscB-D is highly active in mouse cell lines and embryos, enabling the efficient generation of disease models through mRNA/ωRNA injection. Our study suggests that these miniature genome-editing tools have great potential for diverse applications.

2.
Nat Chem Biol ; 2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38553609

RESUMO

Cytosine base editors (CBEs) are effective tools for introducing C-to-T base conversions, but their clinical applications are limited by off-target and bystander effects. Through structure-guided engineering of human APOBEC3A (A3A) deaminase, we developed highly accurate A3A-CBE (haA3A-CBE) variants that efficiently generate C-to-T conversion with a narrow editing window and near-background level of DNA and RNA off-target activity, irrespective of methylation status and sequence context. The engineered deaminase domains are compatible with PAM-relaxed SpCas9-NG variant, enabling accurate correction of pathogenic mutations in homopolymeric cytosine sites through flexible positioning of the single-guide RNAs. Dual adeno-associated virus delivery of one haA3A-CBE variant to a mouse model of tyrosinemia induced up to 58.1% editing in liver tissues with minimal bystander editing, which was further reduced through single dose of lipid nanoparticle-based messenger RNA delivery of haA3A-CBEs. These results highlight the tremendous promise of haA3A-CBEs for precise genome editing to treat human diseases.

3.
BMC Cardiovasc Disord ; 24(1): 106, 2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38355423

RESUMO

AIMS: To explore the role and mechanism of Notch signaling and ERK1/2 pathway in the inhibitory effect of sacubitril/valsartan on the proliferation of vascular smooth muscle cells (VSMCs). MAIN METHODS: Human aortic vascular smooth muscle cells (HA-VSMCs) were cultured in vitro. The proliferating VSMCs were divided into three groups as control group, Ang II group and Ang II + sacubitril/valsartan group. Cell proliferation and migration were detected by CCK8 and scratch test respectively. The mRNA and protein expression of PCNA, MMP-9, Notch1 and Jagged-1 were detected by qRT-PCR and Western blot respectively. The p-ERK1/2 expression was detected by Western blot. KEY FINDINGS: Compared with the control group, proliferation and migration of VSMCs and the expression of PCNA, MMP-9, Notch1, Jagged-1 and p-ERK1/2 was increased in Ang II group. Sacubitril/valsartan significantly reduced the proliferation and migration. Additionally, pretreatment with sacubitril/valsartan reduced the PCNA, MMP-9, Notch1, Jagged-1 and p-ERK1/2 expression.


Assuntos
Aminobutiratos , Compostos de Bifenilo , Sistema de Sinalização das MAP Quinases , Metaloproteinase 9 da Matriz , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Músculo Liso Vascular/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/farmacologia , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Proteína Jagged-1/farmacologia , Células Cultivadas , Valsartana/farmacologia , Proliferação de Células , Miócitos de Músculo Liso/metabolismo , Angiotensina II/metabolismo , Movimento Celular
5.
ACS Nano ; 18(1): 951-971, 2024 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-38146717

RESUMO

Functional remodeling and prolonged anti-inflammatory responses are both vital for repairing damage in the cardiovascular system. Although these aspects have each been studied extensively alone, attempts to fabricate scaffolds that combine these effects have seen limited success. In this study, we synthesized salvianic acid A (SA, danshensu) blocked biodegradable polyurethane (PCHU-D) and enclosed it within electrospun nanofibers to synthesize a durable immunomodulatory nanofiber niche (DINN), which provided sustained SA release during inflammation. Given its excellent processability, mechanical properties, and shape memory function, we developed two variants of the DINN as vascular scaffolds and heart patches. Both these variants exhibited outstanding therapeutic effects in in vivo experiments. The DINN was expertly designed such that it gradually decomposes along with SA release, substantially facilitating cellular infiltration and tissue remodeling. Therefore, the DINN effectively inhibited the migration and chemotaxis of inflammatory cells, while also increasing the expression of angiogenic genes. As a result, it promoted the recovery of myocardial function after myocardial infarction and induced rapid reendothelialization following arterial orthotopic transplantation repair. These excellent characteristics indicate that the DINN holds great potential as a multifunctional agent for repairing cardiovascular tissues.


Assuntos
Infarto do Miocárdio , Nanofibras , Humanos , Alicerces Teciduais , Miocárdio , Infarto do Miocárdio/tratamento farmacológico , Engenharia Tecidual
6.
JOR Spine ; 7(1): e1283, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38222817

RESUMO

Background: Intervertebral disc degeneration (IDD) is a common musculoskeletal disorder that contributes significantly to disability and healthcare costs. Serum urate concentration has been implicated in the development of various musculoskeletal conditions. While previous observational studies have suggested an association between the two conditions, it might confound the effect of serum urate concentrations on IDD. This Mendelian randomization (MR) study aimed to investigate the causal relationship between serum urate concentration and IDD. Methods: We performed a two-sample MR analysis using summary-level data from genome-wide association studies (GWAS) of serum urate concentration (n = 13 585 994 European ancestry) and IDD (n = 16 380 337 European ancestry). Single nucleotide polymorphisms (SNPs) significantly associated with serum urate concentration (p < 5 × 10-8) were selected as instrumental variables. The associations between genetically predicted serum urate concentration and IDD were estimated using the inverse-variance weighted (IVW) method, with sensitivity analyses employing the weighted median, MR-Egger, and MR-PRESSO approaches to assess the robustness of the findings. Results: In the primary IVW analysis, genetically predicted serum urate concentration was unrelated associated with IDD (odds ratio [OR] = 1.00, 95% confidence interval (CI): 1.00-1.00, p = 0.17)). The results remained consistent across the sensitivity analyses, and no significant directional pleiotropy was detected (MR-Egger intercept: p = 0.15). Conclusions: This MR study provides evidence that there is no causal relationship between serum urate concentration and IDD. It suggests previous observational associations may be confounded. Serum urate levels are unlikely to be an important contributor to IDD.

7.
Bioact Mater ; 37: 477-492, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38698919

RESUMO

Degradable rotator cuff patches, followed over five years, have been observed to exhibit high re-tear rates exceeding 50%, which is attributed to the inability of degradable polymers alone to restore the post-rotator cuff tear (RCT) inflammatory niche. Herein, poly(ester-ferulic acid-urethane)urea (PEFUU) was developed, featuring prolonged anti-inflammatory functionality, achieved by the integration of ferulic acid (FA) into the polyurethane repeating units. PEFUU stably releases FA in vitro, reversing the inflammatory niche produced by M1 macrophages and restoring the directed differentiation of stem cells. Utilizing PEFUU, hierarchical composite nanofiber patch (HCNP) was fabricated, simulating the natural microstructure of the tendon-to-bone interface with an aligned-random alignment. The incorporation of enzymatic hydrolysate derived from decellularized Wharton jelly tissue into the random layer could further enhance cartilage regeneration at the tendon-to-bone interface. Via rat RCT repairing model, HCNP possessing prolonged anti-inflammatory properties uniquely facilitated physiological healing at the tendon-to-bone interface's microstructure. The alignment of fibers was restored, and histologically, the characteristic tripartite distribution of collagen I - collagen II - collagen I was achieved. This study offers a universal approach to the functionalization of degradable polymers and provides a foundational reference for their future applications in promoting the in vivo regeneration of musculoskeletal tissues.

8.
Adv Sci (Weinh) ; 11(31): e2308443, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38922803

RESUMO

Tissue engineering has demonstrated its efficacy in promoting tissue regeneration, and extensive research has explored its application in rotator cuff (RC) tears. However, there remains a paucity of research translating from bench to clinic. A key challenge in RC repair is the healing of tendon-bone interface (TBI), for which bioactive materials suitable for interface repair are still lacking. The umbilical cord (UC), which serves as a vital repository of bioactive components in nature, is emerging as an important source of tissue engineering materials. A minimally manipulated approach is used to fabricate UC scaffolds that retain a wealth of bioactive components and cytokines. The scaffold demonstrates the ability to modulate the TBI healing microenvironment by facilitating cell proliferation, migration, suppressing inflammation, and inducing chondrogenic differentiation. This foundation sets the stage for in vivo validation and clinical translation. Following implantation of UC scaffolds in the canine model, comprehensive assessments, including MRI and histological analysis confirm their efficacy in inducing TBI reconstruction. Encouraging short-term clinical results further suggest the ability of UC scaffolds to effectively enhance RC repair. This investigation explores the mechanisms underlying the promotion of TBI repair by UC scaffolds, providing key insights for clinical application and translational research.


Assuntos
Modelos Animais de Doenças , Lesões do Manguito Rotador , Alicerces Teciduais , Cicatrização , Animais , Cães , Lesões do Manguito Rotador/cirurgia , Cicatrização/efeitos dos fármacos , Alicerces Teciduais/química , Humanos , Engenharia Tecidual/métodos , Masculino , Feminino , Manguito Rotador/cirurgia , Cordão Umbilical/citologia , Resultado do Tratamento
9.
Bioact Mater ; 39: 354-374, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38846529

RESUMO

Osteoporosis is majorly caused by an imbalance between osteoclastic and osteogenic niches. Despite the development of nationally recognized first-line anti-osteoporosis drugs, including alendronate (AL), their low bioavailability, poor uptake rate, and dose-related side effects present significant challenges in treatment. This calls for an urgent need for more effective bone-affinity drug delivery systems. In this study, we produced hybrid structures with bioactive components and stable fluffy topological morphology by cross-linking calcium and phosphorus precursors based on mesoporous silica to fabricate nanoadjuvants for AL delivery. The subsequent grafting of -PEG-DAsp8 ensured superior biocompatibility and bone targeting capacity. RNA sequencing revealed that these fluffy nanoadjuvants effectively activated adhesion pathways through CARD11 and CD34 molecular mechanisms, hence promoting cellular uptake and intracellular delivery of AL. Experiments showed that small-dose AL nanoadjuvants effectively suppress osteoclast formation and potentially promote osteogenesis. In vivo results restored the balance between osteogenic and osteoclastic niches against osteoporosis as well as the consequent significant recovery of bone mass. Therefore, this study constructed a drug nanoadjuvant with peculiar topological structures and high bone targeting capacities, efficient intracellular drug delivery as well as bone bioactivity. This provides a novel perspective on drug delivery for osteoporosis and treatment strategies for other bone diseases.

10.
Cell Stem Cell ; 31(8): 1187-1202.e8, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38772378

RESUMO

Cell-based ex vivo gene therapy in solid organs, especially the liver, has proven technically challenging. Here, we report a feasible strategy for the clinical application of hepatocyte therapy. We first generated high-quality autologous hepatocytes through the large-scale expansion of patient-derived hepatocytes. Moreover, the proliferating patient-derived hepatocytes, together with the AAV2.7m8 variant identified through screening, enabled CRISPR-Cas9-mediated targeted integration efficiently, achieving functional correction of pathogenic mutations in FAH or OTC. Importantly, these edited hepatocytes repopulated the injured mouse liver at high repopulation levels and underwent maturation, successfully treating mice with tyrosinemia following transplantation. Our study combines ex vivo large-scale cell expansion and gene editing in patient-derived transplantable hepatocytes, which holds potential for treating human liver diseases.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Terapia Genética , Hepatócitos , Hepatopatias , Hepatócitos/metabolismo , Hepatócitos/transplante , Sistemas CRISPR-Cas/genética , Humanos , Animais , Hepatopatias/terapia , Hepatopatias/genética , Hepatopatias/patologia , Camundongos , Terapia Genética/métodos , Tirosinemias/terapia , Tirosinemias/genética , Proliferação de Células , Hidrolases
11.
Fundam Res ; 2(6): 829-835, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-38933375

RESUMO

Atrial fibrillation (AF) is a common cardiac disease with high prevalence in the general population. Despite a mild manifestation at the onset stage, it causes serious consequences, including sudden death, when the disease progresses to the late stage. Most available treatments of AF focus on symptom management or alleviation, due to a lack of fundamental knowledge and the fact that considerable variations of AF exist. With the popularisation of the next-generation sequencing technology, several causal genetic factors, including MYL4, have been discovered to contribute to AF, giving hope to developing its gene therapies. In this study, we attempted to treat a previously established rat AF model, which carried Myl4E11K/E11K loss of function mutation, via overexpression of exogenous wild-type Myl4 by AAV9 vectors. Our results showed that delivery of Myl4 expressing AAV9 to postnatal rat models rescued the symptoms of AF, indicating the therapeutic potential that early gene therapy intervention can achieve long-term effects in treating cardiac arrhythmias caused by gene mutations.

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