VIROLOGY
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2023
·
IF 3.7
Xiaoping Huang,Xiao Wang,Yaqi Sun,Ling Li,Anna Li,Wentao Xu,Xiaolan Xie,Yong Diao
Because it is safe and has a simple genome, recombinant adeno-associated virus (rAAV) is an extremely appealing vector for delivery in in vivo gene therapy. However, its low transduction efficiency for some cells, limits its further application in the field of gene therapy. Bleomycin is a chemotherapeutic agent approved by the FDA whose effect on rAAV transduction has not been studied. In this study, we systematically investigated the effect of Bleomycin on the second-strand synthesis and used CRISPR/CAS9 and RNAi methods to understand the effects of Bleomycin on rAAV vector transduction, particularly the effect of DNA repair enzymes. The results showed that Bleomycin could promote rAAV2 transduction both in vivo and in vitro. Increased transduction was discovered to be a direct result of decreased cytoplasmic rAAV particle degradation and increased second-strand synthesis. TDP1 , PNKP , and SETMAR are required to repair the DNA damage gap caused by Bleomycin, TDP1, PNKP, and SETMAR promote rAAV second-strand synthesis. Bleomycin induced DNA-PKcs phosphorylation and phosphorylated DNA-PKcs and Artemis promoted second-strand synthesis. The current study identifies an effective method for increasing the capability and scope of in-vivo and in-vitro rAAV applications, which can amplify cell transduction at Bleomycin concentrations. It also supplies information on combining tumor gene therapy with chemotherapy.
研究领域:
分子生物学
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基因治疗
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癌症研究
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药理学
✓ 已验证
DOI
PubMed
高影响因子
Molecular Therapy-Methods & Clinical Development
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2019
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IF 4.875
Zhilong Chen,Guoqing Fan,Anan Li,Jing Yuan,Tonghui Xu
The motor system controls muscle movement through lower motor neurons in the spinal cord and brainstem. Lower motor neurons are efferent neurons in the central nervous system (CNS) characterized by axonal projections that reach specific targets in the periphery. Lower motor neuron lesions result in the denervation and dysfunction of peripheral skeletal muscle. Great progress has been made to develop therapeutic strategies to transduce lower motor neurons with genes. However, the widespread distribution of lower motor neurons makes their specific, extensive, and efficient transduction a challenge. In this study, we demonstrated that, compared to the other tested recombinant adeno-associated virus (rAAV) serotypes, rAAV2-retro mediated the most efficient retrograde transduction of lower motor neurons in the spinal cord following intramuscular injection in neonatal mice. A single injection of rAAV2-retro in a single muscle enabled the efficient and extensive transduction of lower motor neurons in the spinal cord and brainstem rather than transducing only the lower motor neurons connected to the injected muscle. rAAV2-retro achieved the extensive transduction of lower motor neurons by the cerebrospinal fluid pathway. Our work suggests that gene delivery via the intramuscular injection of rAAV2-retro represents a promising tool in the development of gene therapy strategies for motor neuron diseases.
研究领域:
神经科学
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基因治疗
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分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
International Journal of Medical Sciences
·
2025
·Vol. 22
·期 4
·775-789
·
IF 3.2
Gong Xueqi,Gao Haitong,Wang Wenyuan,Xu Tonghui
Recombinant adeno-associated viral vectors (rAAVs) can effectively deliver transgene to the nervous system. The selection of AAV serotype and promoter significantly influences the dynamics of the transgene expression, including its strength and cell-specificity. Previous studies demonstrated that in neonatal mice, the intramuscular (IM) injection of the rAAV2-retro vector could efficiently deliver transgene to lower motor neurons (LMNs) of the brainstem and spinal cord. However, the best promoter for the expression of transgene in the central neural system (CNS) using rAAV2-retro remains undetermined. This study compared five commonly used promoters, including mouse phosphoglycerate kinase (mPGK), CMV early enhancer/chicken β-actin/short β-globulin intron (CAG), human cytomegalovirus (hCMV), chicken β-actin (CBA), and human synapsin (hSyn) promoters. The IM (unilateral gastrocnemius muscle) injection of rAAV2-retro vectors packaged with the reporter constructs containing each promoter was performed in the newborn C57BL/6J mice. The levels of gene expression and the types of cells were examined using the light-sheet illumination imaging technique and confocal microscopy. Our findings revealed that rAAV2-retro primarily targeted the brainstem and spinal cord within the CNS. Among the five promoters tested, CAG and hCMV showed the highest gene expression. Almost all the transduced cells were identified as LMNs. Additionally, gene expression driven by hCMV was found to be dependent of the inclusion of WPRE and β-globin intron elements. Importantly, none of the promoters induced hepatotoxicity, ensuring the safety of rAAV2-retro-mediated expression. This study provided valuable insights for optimizing the rAAV2-retro-mediated gene delivery system to LMNs in the brainstem and spinal cord, which might have potential implications for research on motor neuron-related diseases.
研究领域:
神经科学
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基因治疗
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分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Pharmaceuticals
·
2023
·Vol. 16
·期 4
·624
·
IF 4.6
Guoshuai Tian,Chunyu Cao,Shuyue Li,Wei Wang,Ye Zhang,Yafeng Lv
Krabbe disease is a rare neurodegenerative fatal disease. It is caused by deficiency of the lysosomal enzyme galactocerebrosidase (GALC), which results in progressive accumulation of galactolipid substrates in myelin-forming cells. However, there is still a lack of appropriate neural models and effective approaches for Krabbe disease. We generated induced pluripotent stem cells (iPSCs) from a Krabbe patient previously. Here, Krabbe patient-derived neural stem cells (K-NSCs) were induced from these iPSCs. By using nine kinds of recombinant adeno-associated virus (rAAV) vectors to infect K-NSCs, we found that the rAAV2 vector has high transduction efficiency for K-NSCs. Most importantly, rAAV2-GALC rescued GALC enzymatic activity in K-NSCs. Our findings not only establish a novel patient NSC model for Krabbe disease, but also firstly indicate the potential of rAAV2-mediated gene therapy for this devastating disease.Keywords:Krabbe disease;NSCs;rAAV vector;gene therapy;GALC;psychosine
研究领域:
神经科学
·
干细胞研究
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基因治疗
✓ 已验证
DOI
PubMed
高影响因子
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