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@#ObjectiveTo develop a national standard for genomic titer determination of recombinant type 5 adeno-associated virus(rAAV5).MethodsThe rAAV5-GFP stock solution prepared by the three-plasmid system was identified and verified for the appearance,pH,sterility,genomic titer,purity and infection titer according to the relevant requirements of Chinese Pharmacopoeia(Volume Ⅲ,2020 edition),which was diluted and subpackaged to prepare candidate standards according to the results;The stability of candidate standards was investigated by thermal acceleration test;Three laboratories were organized to collaboratively calibrate the candidate standards using droplet digital PCR(ddPCR).ResultsAll the detection indexes of the candidate standard and the stock solution met the relevant requirements;The genomic titer showed no significant decrease at 25,4,-20,-40,-80 ℃ for 1,3,4,6 months;Through collaborative calibration by three laboratories,the candidate standard was assigned a value of 2. 56 × 10(12)copies/mL,and the 95% confidence interval was 2. 48 ×10(12)copies/mL,and the 95% confidence interval was 2. 48 ×10(12)copies/mL ～ 2. 64 × 10(12)copies/mL ～ 2. 64 × 10(12)copies/mL.ConclusionThe developed national standard for the determination of rAAV5 genomic titer had good stability and might be used for the quality evaluation of rAAV5 related products.
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@#To improve the transduction efficiency of recombinant adeno-associated virus (rAAV) in NK92 cells, the number of cells, concentration of IL-2 in the medium, and serotype and dosage of rAAV were explored to optimize cell state and viral transfection conditions.Then, zinc chloride (ZnCl2), chloroquine, polyvinyl alcohol (PVA) and genistein with different concentration were added separately during transfection to further improve the viral transduction efficiency.The results showed that, at cell number of 5 × 105, the expression efficiency of enhanced green fluorescent protein (EGFP) was relatively high.When the IL-2 concentration was 1 000 IU/mL, NK92 cells were most suitable for virus transfection. The transduction efficiency of different serotypes of rAAV in NK92 cells was rAAV6, rAAV2 and rAAV9 in descending order.Pretreatment of NK92 cells with genistein could significantly increase the viral transduction efficiency, while the addition of other reagents had no significant effect.Through the optimization of the above conditions, the transduction efficiency of rAAV to NK92 cells could be significantly improved, which provided evidence for functional genetic modification of NK92 cells by rAAV.
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@#Adeno-associated virus (AAV) is a common viral vector used in gene therapy.Because of its high safety and its ability to target a variety of cells, it has been widely used in preclinical and clinical studies.However, during the design and production, AAV vectors have many key quality attributes that affect their safety and efficacy.The development and application of biological mass spectrometry technology provides a convenient platform for the research on biological macromolecules, especially in the aspects of protein sequence, structure and interaction.For AAV vectors, mass spectrometry can facilitate the determination or characterization of capsid protein ratio, post-translational modification, serotype, and empty capsid ratio, thus assisting in the quality control of AAV vectors.Compared with the existing methods, mass spectrometry has the advantages of smaller amount of sample size, faster and more sensitive analysis, being more suitable for the analysis of complete AAV vectors with higher mass resolution, and can distinguish empty capsids, full capsids and partial capsids.In the future, mass spectrometry technology is expected to play a more important role in the design and production of AAV vectors through the coupling of more efficient protein separation technology with mass spectrometry, the development of new information processing software platforms and new mass spectrometry detection techniques.
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Abstract Monogenic neuromuscular disorders are potentially treatable through gene therapy. Using viral vectors, a therapeutic transgene aims to restore normal levels of a protein not produced by the defective gene, or to silence a gene whose expression leads to toxic effects. Spinal Muscular Atrophy (SMA) is a good example of a monogenic disease that currently has an AAV9-based vector gene therapy as a therapeutic option. In this review, we intend to discuss the viral vectors and their mechanisms of action, in addition to reviewing the clinical trials that supported the approval of gene therapy (AVXS-101) for SMA as well as neuromuscular diseases that are potentially treatable with gene replacement therapy.
Resumo Doenças neuromusculares monogênicas são potencialmente tratáveis através de terapia gênica. Utilizando-se de vetores virais, um transgene terapêutico objetiva repor os níveis normais de uma proteina não produzida pelo gene defeituoso ou silenciar um gene cuja expressão leva a efeitos tóxicos. A Atrofia Muscular Espinhal (AME) é um bom exemplo de doença monogenica que atualmente tem uma terapia gênica com vetor viral AAV9 como opção terapêutica. Nesta revisão, pretendemos discutir os vetores virais e macanismos de ação utilizados, além de revisar os ensaios clínicos que embasaram a aprovação da terapia gênica (AVXS-101) para AME, assim como doenças neuromusculares potencialmente tratáveis com terapia de reposição gênica.
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Amyotrophic lateral sclerosis is a neurodegenerative disease caused by the loss of motor neurons in the brain and spinal cord. There is currently no effective cure. The emergence of gene therapy brings hope to treatment, which can be achieved by delivering transgenes to replace or correct defective genes, as well as the expression of neurotrophic factors. The vectors of gene therapy can be viral vectors and non-viral vectors. Lentiviral vectors can be used to deliver therapeutic sequences to motor neurons in the central nervous system. Adeno-associated viruses can effectively mediate gene expression and delivery of neurotrophic factors. Gene editing and antisense oligonucleotides therapy are also perspective treatment options. This article summarizes gene therapy for amyotrophic lateral sclerosis from basic experiments and clinical trials.
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Objective:To investigate the role of integrin-linked kinase (ILK)-small interfering RNA (siRNA)-adeno-associated virus (AAV) in scar formation after glaucoma filtering surgery in rat eyes.Methods:Forty-eight Sprague Dawley rats of SPF grade, aged 8 to 9 weeks old, were selected and divided into blank control group, ILK-siRNA-AAV group, NC-siRNA-AAV group and mitomycin C (MMC) group by random number table method, with 12 rats in each group.Left eyes of the rats were taken as experimental eyes, and no intervention was administered to fellow eyes.The bulbar conjunctival filtering bleb after glaucoma filtration surgery in rats was established by anterior chamber drainage tube implantation.One day after operation, phosphate buffer solution, ILK-siRNA-AAV, and NC-siRNA-AAV were injected into the filtering bleb of blank control group, ILK-siRNA-AAV group and NC-siRNA-AAV group, 5 μl each group, respectively.Cotton tablets containing 0.4 mg/ml MMC were placed under conjunctival flap for 5 minutes during operation in MMC group.Intraocular pressure (IOP) was measured with a handheld tonometer before surgery and at 1, 2, 3, 7, 14, 21, 28 days after surgery.Formation of filtering blebs in rats was observed with a surgical microscope at 1, 2, 3, 7, 14, 21, 28 days after operation, and the bleb survival time was calculated by Kaplan-Meier survival analysis.The mRNA and protein expression levels of ILK in conjunctival and subconjunctival tissues at the surgical sites were detected by reverse transcription PCR and Western blot, respectively, on the 28th day after operation.Silencing of ILK gene was identified.Effect of ILK gene silencing on the morphology of drainage pathway was observed by hematoxylin-eosin staining.Effect of ILK gene silencing on collagen fiber deposition in the bulbar conjunctiva at filtration area was examined by Masson staining, and the percentage of positive area of collagen fiber staining in the total tissue visual field was calculated.The use and care of the animals complied with the ARVO Statement.This research protocol was approved by an Ethics Committee of Xi'an Jiaotong University (No.2013-772). Results:There were statistically significant differences in IOP at different time points between before and after surgery among four groups ( Fgroup=76.84, P<0.001; Ftime=114.49, P<0.001). The IOP of ILK-siRNA-AAV group on the 1st, 7th, 14th and 28th day after operation and the IOP of MMC group on the 2nd, 3rd, 7th, 14th and 28th day after operation were lower than those of blank control group, and the differences were statistically significant (all at P<0.05). The IOP of ILK-siRNA-AAV group was lower on 7th, 14th, 21st and 28th day after operation than those of NC-siRNA-AAV group, with statistically significant differences (all at P<0.05). The bleb survival time of blank control group, NC-siRNA-AAV group, ILK-siRNA-AAV group and MMC group was (3.50±1.51), (5.00±3.41), (31.50±3.15) and (31.33±2.46) days, respectively, with a significant difference among them ( F=395.83, P<0.05). The bleb survival time of ILK-siRNA-AAV group and MMC group was higher than that of blank control group and NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). There were statistically significant differences in the relative expression levels of ILK mRNA and protein among four groups ( F=222.32, 752.69; both at P<0.05), and the relative expression levels of ILK mRNA and protein were significantly lower in ILK-siRNA-AAV group than blank control group and NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). Proliferative fibrous connective tissue and a large number of cells at surgical sites were found in blank control group and NC-siRNA-AAV group, and the fibroblasts were of a high density and grew in clumps.In ILK-siRNA-AAV group, the bulbar conjunctiva was thin, and the arrangement of fibrous connective tissue was loose, and a few proliferative fibroblasts were found.In MMC group, the conjunctival fibrous layer was loose and thin, forming cavities, and scarce cells were found.There was statistically significant difference in the percentage of collagen fiber positive staining area among four groups ( F=741.66, P<0.05). The positive staining percentage of ILK-siRNA-AAV group and MMC group was significantly lower than that of blank control group, among which there was lower positive staining percentage in ILK-siRNA-AAV group than NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). Conclusions:Silencing of ILK can inhibit the scar formation after glaucoma filtering surgery and maintain low IOP in rats.
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The use of small interfering RNAs (siRNAs) has been under investigation for the treatment of several unmet medical needs, including acute lung injury/acute respiratory distress syndrome (ALI/ARDS) wherein siRNA may be implemented to modify the expression of pro-inflammatory cytokines and chemokines at the mRNA level. The properties such as clear anatomy, accessibility, and relatively low enzyme activity make the lung a good target for local siRNA therapy. However, the translation of siRNA is restricted by the inefficient delivery of siRNA therapeutics to the target cells due to the properties of naked siRNA. Thus, this review will focus on the various delivery systems that can be used and the different barriers that need to be surmounted for the development of stable inhalable siRNA formulations for human use before siRNA therapeutics for ALI/ARDS become available in the clinic.
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OBJECTIVE@#To express and purify the antigenic peptide of adeno-associated virus (AAV) capsid conserved regions in prokaryotic cells and prepare its rabbit polyclonal antibody.@*METHODS@#The DNA sequence encoding the conserved regions of AAV capsid protein was synthesized and cloned into the vector pET30a to obtain the plasmid pET30a-AAV-CR for prokaryotic expression and purification of the conserved peptides. Coomassie blue staining and Western blotting were used to identify the AAV conserved peptides. Japanese big ear white rabbits were immunized with AAV conserved region protein to prepare polyclonal antibody, with the rabbits injected with PBS as the control group. The antibody titer was determined with ELISA, and the performance of the antibody for recognizing capsid protein sequences of AAV1-AAV10 was assessed with Western blotting and immunofluorescence assay.@*RESULTS@#The plasmid pET30a-AAV-CR was successfully constructed, and a recombinant protein with a relative molecular mass of 17000 was obtained. The purified protein induced the production of antibodies against the conserved regions of AAV capsid in rabbits, and the titer of the purified antibodies reached 1:320 000. The antibodies were capable of recognizing a wide range of capsid protein sequences of AAV1-AAV10.@*CONCLUSION@#We successfully obtained the polyclonal antibodies against AAV capsid conserved region protein from rabbits, which facilitate future studies of AAV vector development and the biological functions of AAV.
Subject(s)
Animals , Rabbits , Antibodies , Capsid , Capsid Proteins/genetics , Dependovirus/genetics , Prokaryotic Cells , Recombinant Proteins/geneticsABSTRACT
Hemophilia is a congenital hemorrhagic disease caused by genetic abnormalities in coagulation factor Ⅷ or factor Ⅸ. Current conventional therapy to prevent bleeding requires frequent intravenous injections of coagulation factor concentrates from early childhood. Hemophilia provides an attractive target for gene therapy studies, due to the monogenic nature of these disorders and easily measurable endpoints (factor levels and bleed rates). All successful, pre-clinical and clinical studies to date have utilized recombinant adeno-associated virus(AAV) vectors for factor Ⅷ or Ⅸ hepatocyte transduction. Although this is likely to alter the paradigm of hemophilia care in the near future, it will be important to overcome immune responses against AAV. This review focuses on current successes of clinical trials, and the future direction of hemophilia gene therapy.
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Leber hereditary optic neuropathy (LHON) is a matrilineal hereditary optic neuropathy in which mitochondrial DNA mutations lead to retinal ganglion cell degeneration. At present, the treatment for LHON is limited. Early symptomatic treatment and medical treatment may improve the vision of patients. In recent years, rapid progress has been made in gene therapy. Many clinical studies have confirmed its safety and efficacy. Monocular gene therapy is helpful to improve the visual function of LHON patients, and it can also improve the visual acuity of uninjected eyes. Patients do not have serious eye or systemic adverse events during the treatment period, showing good safety and tolerance. Studies with larger sample size and longer follow-up time are needed to further verify the efficacy and safety of gene therapy in the future. Gene therapy is expected to become a safe and effective treatment, bringing hope to LHON patients.
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Objective:To evaluate the safety and clinical effect of gene therapy for Leber hereditary optic neuropathy (LHON).Methods:A multi-center prospective non-randomized controlled trial was conducted.Eighty eyes of 40 LHON patients with mitochondrial DNA 11778 mutation were enrolled in Taihe Hospital from December 2017 to February 2018.Intravitreal injection of recombinant adeno associated virus 2-NADH dehydrogenase 4 (rAAV2- ND4) was carried out in the unilateral eye with worse visual acuity or the right eye (if the visual acuity of both eyes was equal) of each subject as the treated group and the fellow eyes as the untreated group.The best corrected visual acuity (BCVA) was detected using a standard logarithmic chart and intraocular pressure (IOP) was measured with a non-contact tonometer before treatment and 1, 3, 6, 12 months after treatment.The manifestations of the ocular anterior segment and fundus were examined by slit lamp microscopy and color photography.The changes of visual acuity and IOP before and after gene therapy were compared, and complications were evaluated between the treated group and the untreated group.The effective rate defined as visual acuity improved ≥0.3 LogMAR at the end of follow-up was assessed.This study adhered to the Declaration of Helsinki and the study protocol was approved by an Ethics Committee of Taihe Hospital (No.201807). Written informed consent was obtained from each subject prior to any medical examination and treatment. Results:The visual acuity improved 6 eyes in the treated group and 4 eyes in the untreated group, and 13 patients showed bilateral improvement.The visual acuity improvement ≥0.3 LogMAR in 23 patients with the effective rate 57.5%.The BCVA was (1.51±0.62) LogMAR and (1.62±0.58) LogMAR at the end of following-up in the untreated group and treated group, respectively, which were significantly higher than (1.75±0.46) LogMAR and (1.83±0.47) LogMAR before treatment (both at P<0.01), and no significant difference was found in BCVA between the two groups ( Fgroup=0.084, P=0.772). There was no significant difference in IOP between the two groups before and after treatment ( Fgroup=0.557, P=0.575; Ftime=2.314, P=0.106). No serious complications were found in all subjects during following-up. Conclusions:rAAV2- ND4 gene therapy is safe and effective for LHON, and binocular vision can be improved by monocular intravitreal injection of rAAV2- ND4 gene.
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Objective:To investigate the protective effect of adeno-associated virus (AAV) vector-mediated heme oxygenase-1 ( HO-1) gene overexpression on retinitis pigmentosa (RP) models in rats. Methods:Eighty healthy male SD rats were selected and randomized into the blank control group, RP model group, AAV-GFP group and AAV-HO-1-GFP group, with 20 rats in each group.The RP model was established via tail vein injection of 1% sodium iodate solution at the dose of 50 mg/kg.Rats in the RP model group, AAV-GFP group and AAV-HO-1-GFP group were subretinally injected with normal saline, AAV-GFP virus and AAV-HO-1-GFP virus according to grouping.Rats in the blank control group were administrated with subretinal injection of equal volume of normal saline.The eyeballs of rats were enucleated on 14th day after molding.The retinal structure and thickness were detected by hematoxylin-eosin staining; apoptosis in retinal tissue was detected by TUNEL assay; the expression levels of cysteinyl aspartate specific proteinase 3 (caspase 3), B-lymphoma-2 (bcl-2) and HO-1 protein in rats retina was identified by Western blot.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology (ARVO). The study protocol was approved by an Ethics Committee of The Second Affiliated Hospital of Fujian Medical University (No.202188).Results:Hematoxylin-eosin staining results showed normal retinal structure in the blank control group, obvious retinal structure damage with thinned and wavy outer nuclear layer in the RP model group and AAV-GFP group, and mild retinal structure damage with slightly thinned outer nuclear layer in the AAV-HO-1-GFP group.Compared with the blank control group, the outer nuclear layer was significantly thinner in the RP model group and AAV-GFP group (both at P<0.05). Relative expression level of HO-1 protein in the RP model group, AAV-GFP group, and AAV-HO-1-GFP group was 0.76±0.21, 0.76±0.16, 0.92±0.05, respectively, which were all significantly higher than 0.48±0.25 in the blank control group (all at P<0.05). The relative expression level of HO-1 protein in the AAV-HO-1-GFP group was significantly higher than that in the RP model group and the AAV-GFP group (both at P<0.05). There were significant differences in apoptosis rate, the relative expression levels of bcl-2 and caspase 3 protein among the four groups ( F=1 596.333, 1 043.806, 364.331; all at P<0.01). The apoptosis rate and the relative expression level of caspase 3 were significantly higher, and the relative expression level of bcl-2 was lower in the RP model group and AAV-GFP group than those in the AAV-HO-1-GFP group (all at P<0.05). Conclusions:AAV-mediated overexpression of HO-1 gene can protect the retina of RP rats.
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Acetaminophen (APAP) overdose is the leading cause of drug-induced liver injury, and its prognosis depends on the balance between hepatocyte death and regeneration. Sirtuin 6 (SIRT6) has been reported to protect against oxidative stress-associated DNA damage. But whether SIRT6 regulates APAP-induced hepatotoxicity remains unclear. In this study, the protein expression of nuclear and total SIRT6 was up-regulated in mice liver at 6 and 48 h following APAP treatment, respectively.
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The 2020 Nobel Prize in Chemistry recognized CRISPR-Cas9, a super-selective and precise gene editing tool. CRISPR-Cas9 has an obvious advantage in editing multiple genes in the same cell, and presents great potential in disease treatment and animal model construction. In recent years, CRISPR-Cas9 has been used to establish a series of rat models of drug metabolism and pharmacokinetics (DMPK), such as
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Whether direct manipulation of Parkinson's disease (PD) risk genes in the adult monkey brain can elicit a Parkinsonian phenotype remains an unsolved issue. Here, we used an adeno-associated virus serotype 9 (AAV9)-delivered CRISPR/Cas9 system to directly co-edit PINK1 and DJ-1 genes in the substantia nigras (SNs) of two monkey groups: an old group and a middle-aged group. After the operation, the old group exhibited all the classic PD symptoms, including bradykinesia, tremor, and postural instability, accompanied by key pathological hallmarks of PD, such as severe nigral dopaminergic neuron loss (>64%) and evident α-synuclein pathology in the gene-edited SN. In contrast, the phenotype of their middle-aged counterparts, which also showed clear PD symptoms and pathological hallmarks, were less severe. In addition to the higher final total PD scores and more severe pathological changes, the old group were also more susceptible to gene editing by showing a faster process of PD progression. These results suggested that both genetic and aging factors played important roles in the development of PD in the monkeys. Taken together, this system can effectively develop a large number of genetically-edited PD monkeys in a short time (6-10 months), and thus provides a practical transgenic monkey model for future PD studies.
Subject(s)
Animals , Brain , CRISPR-Cas Systems/genetics , Dependovirus/genetics , Haplorhini , Phenotype , Protein Kinases/geneticsABSTRACT
OBJECTIVE@#Plant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells.@*METHODS@#A miR122 target (122T) sequence was incorporated into the 3' untranslated region of the tcs cDNA sequence. The firefly luciferase (fluc) transgene was used as an appropriate control. Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells. The effects of miR122 overexpression on cell growth, transgene expression, and rAAV production were determined.@*RESULTS@#The presence of 122T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line (in vitro), fresh human hepatocytes (ex vivo), and mouse liver (in vivo). Also, the normal liver physiology was unaffected by delivery of 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells.@*CONCLUSION@#HEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.
Subject(s)
Animals , Humans , Mice , Dependovirus/genetics , Genetic Therapy , Genetic Vectors/genetics , HEK293 Cells , MicroRNAs/genetics , TrichosanthinABSTRACT
Whether direct manipulation of Parkinson’s disease (PD) risk genes in the adult monkey brain can elicit a Parkinsonian phenotype remains an unsolved issue. Here, we used an adeno-associated virus serotype 9 (AAV9)-delivered CRISPR/Cas9 system to directly co-edit PINK1 and DJ-1 genes in the substantia nigras (SNs) of two monkey groups: an old group and a middle-aged group. After the operation, the old group exhibited all the classic PD symptoms, including bradykinesia, tremor, and postural instability, accompanied by key pathological hallmarks of PD, such as severe nigral dopaminergic neuron loss (>64%) and evident α-synuclein pathology in the gene-edited SN. In contrast, the phenotype of their middle-aged counterparts, which also showed clear PD symptoms and pathological hallmarks, were less severe. In addition to the higher final total PD scores and more severe pathological changes, the old group were also more susceptible to gene editing by showing a faster process of PD progression. These results suggested that both genetic and aging factors played important roles in the development of PD in the monkeys. Taken together, this system can effectively develop a large number of genetically-edited PD monkeys in a short time (6–10 months), and thus provides a practical transgenic monkey model for future PD studies.
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Objective: To investigate the expression of LacZ gene mediated by intravenous injection of the single-stranded adeno-associated virus serotype 9 (ssAAV9) containing hypoxia-responsive element (HRE) promoter in the cerebral ischemic area, and further identify the types of brain cells that can be transfected by the vector. Methods: A mouse model of permanent left distal middle cerebral artery occlusion (dMCAO) was established. The expression of hypoxia-inducible factor-1 (HIF-1) in cerebral ischemic area was detected at 1 and 5 days after ischemia. The ssAAV vector containing HRE-regulated LacZ gene was packaged into the capsid of AAV9 virus (AAV9-H9LacZ), and AAV9-H9LacZ was injected into mice through jugular vein 1 h after the establishment of dMCAO model. Five days after injection of AAV9-H9LacZ, X-gal staining was used to detect the expression of β-galactosidase (β-gal) encoded by the LacZ gene in the ischemic area and liver tissue. Immunofluorescence double staining was used to detect the expression of β-gal in astrocyte, neurons and vascular endothelial cells. Results: The expression of HIF-1 was increased 1 and 5 days after ischemia. β-gal was mainly expressed in ischemic penumbra of mice injected with AAV9-H9LacZ. There was no positive expression of β-gal in the liver tissue of mice. β-gal was mainly co-expressed with glial fibrillary acidic protein as an astrocyte-specific marker, and a little of β-gal was co-expressed with neuron-specific nuclear protein. Conclusion: After cerebral ischemia, intravenous injection of AAV9-H9LacZ can effectively mediate gene expression in astrocyte in the cerebral ischemia area. HRE can effectively control the expression of the LacZ gene in cerebral ischemia.
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Objective@#To evaluate the effects of adeno-associated virus (AAV) carrying hFⅧ by serotype 8 (AAV8/hFⅧ) on hemophilia A (HA) mice by gene therapy strategy.@*Methods@#pAAV-CB-EGFP, pH22 (serotype 2) and pfΔ6 (adenovirus helper) were used to package AAV into HEK-293 cells in different conditions (ratios of cells to plasmids). The efficiency of transfection and infection were evaluated using immunofluorescence microscope to seek an optimized package condition. pAAV-TTR-hFⅧ, pH 28 (serotype 8) and pfΔ6 were applied to package AAV8/hFⅧ in HEK-293 cells using the optimized package condition. The purified AAV8/hFⅧ were intravenously injected into HA mice and the effects of gene therapy were estimated.@*Results@#The efficiency of package was evaluated according to the amount and intensity of enhanced green fluorescent protein (EGFP) under immunofluorescence microscope. Four package conditions including 10 cm-dish to transfect 10 μg plasmids, 20 cm-dish to 20 μg, 30 μg and 40 μg plasmids were employed, and the condition of 20 cm-dish to transfect 20 μg plasmids reached the highest transfection efficiency at 24 h, 48 h and 72 h after transfection. The small scale AAV-EGFP was packaged using the optimized condition and an AAV crude extract was harvested by a freeze-thaw method. HEK-293 and 16095 cells were infected by the AAV crude extract, and the preferential infection efficiency was recognized in 16095 cells under immunofluorescence microscope. Then, AAV8/hFⅧ was packaged and purified based on the optimized transfection condition, and the high purity of AAV8/hFⅧ was detected by Western blot. Fractions of AAV8/hFⅧ at the dose of 8×1012 vg/kg were injected into HA mice through tail vein, an eye-bleeding was performed at every two weeks, and the activity of FⅧ was measured by aPTT assay. Results showed that the activity of FⅧ maintained at the therapeutic level and lasted up to 12 weeks after injection.@*Conclusion@#The purified AAV8/hFⅧ based on the optimized package condition could play a role in HA mice gene therapy, and the long-term therapeutic effects of AAV8/hFⅧ were observed in vivo.
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Peroxisome proliferator-activated receptor (PPAR) is a transcriptional coactivator that binds to a diverse range of transcription factors. PPAR coactivator 1 (PGC-1) coactivators possess an extensive range of biological effects in different tissues, and play a key part in the regulation of the oxidative metabolism, consequently modulating the production of reactive oxygen species, autophagy, and mitochondrial biogenesis. Owing to these findings, a large body of studies, aiming to establish the role of PGC-1 in the neuromuscular system, has shown that PGC-1 could be a promising target for therapies targeting neuromuscular diseases. Among these, some evidence has shown that various signaling pathways linked to PGC-1 are deregulated in muscular dystrophy, leading to a reduced capacity for mitochondrial oxidative phosphorylation and increased reactive oxygen species (ROS) production. In the light of these results, any intervention aimed at activating PGC-1 could contribute towards ameliorating the progression of muscular dystrophies. PGC-1 is influenced by different patho-physiological/pharmacological stimuli. Natural products have been reported to display modulatory effects on PPAR activation with fewer side effects in comparison to synthetic drugs. Taken together, this review summarizes the current knowledge on Duchenne muscular dystrophy, focusing on the potential effects of natural compounds, acting as regulators of PGC-1.