| IntroductionRecombinant adeno-associated virus (rAAV) have been considered as the most promising tool for gene transfer, and have been frequently applied in clinical trials of gene therapy to treat many diseases. More importantly, Glybera, the first gene therapy product based on rAAV1, has been launched into the market at the end of 2012.However, there is still a certain distance to the final destination of curing diseases based on rAAV vectors. Due to the limited thansduction efficiency, high dosage of vectors needs to be employed, but high dosage of rAAV vectors can trigger the immune response, leading to the bad influence on health and the reduction in efficiency of follow-up treatment. Therefore, it has been a hot issue to improve the transduction efficiency.Since the life cycle of rAAV has been demonstrated gradually, many methods to improve the transduction efficiency of rAAV have been found, verified and applied, including selecting tissue specific tropism serotypes and variants, mutating capsid surface-exposed amino-acids, producing self-complementary AAV vectors(scAAV), modifying certain positions on the capsid surface with chemical group, biotin, or avidin, and utilizing proteasome inhibitors and glucocorticoids, et al.However, during the whole process of packaging viral vectors, infection and the gene expression post-infection, there are still several key steps being neglected, and a number of questions have not been clearly addressed. For example, whether ITR (cis-acting elements) and Rep (trans-acting factors) can infect the packaging efficiency and transduction efficiency? As proved by many researchers that increasing the multiplicity of infection (MOI) can improve the transduction efficiency, it still remains unknown that whether the transduction efficiency will increase or reduce when the cell density is increasing and MOI is fixed.Additionally, Traditional Chinese Medicine (TCM) has its own specific system of syndrome differentiation and treatment, furthermore, it contains enormous chemical compounds and can act in multiple-target way. Therefore, can we screen and confirm more components to enhance the transduction efficiency of rAAV vectors under the guidance of TCM theory?Focusing on the above mentioned questions, our research aim to find out new strategies to enhance the transduction efficiency of rAAV vectors, and the application of the new methods will also be investigated, in order to provide basis for expanding the application of these new methods.Methods1. The application of Rep3 and ITR3 in optimizing the packaging and transduction efficiency of rAAV3 vectorsWe constructed the plasmids made up of same capsid gene(Cap3) but different trans-acting factors (Rep2 or Rep3), pAAVR2C3, pAAVR3C3, and the plasmids with same target genes but different cis-acting elements (ITR2 or ITR 3), PITR2-CMVp-hrGFP,pITR3-CMVp-hrGFP,pITR2-CMVp-EGFP-Neo,pITR3-CMVp-EGF-Neo, followed by identification by endonucleases cutting and gene sequencing.These plasmids were transfected into HEK293 cells by the means of Polyetherimide (PEI), and transfection efficiency were evaluated by Western Blot or fluorescent microscope. Then these plasmids were transfected into HEK293 cells by the method of triple plasmid transfection, followed by qPCR to measure the packaging efficiency. The viral vectors of ssAAV3-ITR2-CMVp-EGFP-Neo and ssAAV3-ITR3-CMVp-EGFP-Neo, packaged by Rep2ITR2 and Rep3ITR3, were infected into human liver cancer cells, Huh7 and LH86, and fluorescent microscope was uesd to evaluate the transduction efficiency.2. The influence of cell density on transduction efficiency of rAAV vectorsThe cell density was increased by fixing the infection volume and increasing the cell number, or by fixing cell number and reducing the infection volume. K562 cells were infected by scAAV6-CBAp-EGFP or ssAAV6-CBAp-EGFP, with high or low cell density during or after infection. Fluorescent microscope and flow cytometry were utilized evaluate the transduction efficiency, and qPCR was applied to analyzed genome copy number of viral vectors. Moreover, K562 cells were infected with scAAV6-CBAp-EGFP at the high density with different MOIs, followed by fluorescent microscope and flow cytometry to evaluate the transduction efficiency.HEK293, K562 and Raji cells were infected with scAAV2-CBAp-EGFP at high or low cell density, followed by fluorescent microscope and flow cytometry to evaluate the transduction efficiency. The fluoresect antibodies of HSPG and FGFR1 and flow cytometry were used to analyze the expression level of receptor and co-receptor on the membrane of HEK293, K562 and Raji cells. Hematopoetic stem cells (HSC) were infected with scAAV2-CBAp-EGFP, scAAV6-CBAp-EGFP, scAAV2-4M-CBAp-EGFP or scAAV6-3M-CBAp-EGFP. In high or low cenn density. Fluorescent microscope and flow cytometry were utilized evaluate the transduction efficiency.3. The effect and machanism of Bufalin to enhance the transduction efficiency of rAAV vectorsHEK293 and HeLa cells were infected with ssAAV2-CBAp-EGFP, scAAV2-CMVp-hrGFP and scAAV2-4M-CBAp-EGFP. During infection, cells were treated with DMSO, Bufalin, MG132 or the combination of Bufalin and MG132. Fluorescent microscope and flow cytometry were utilized evaluate the transduction efficiency. Western blot was followed to evaluate the content of ubiquitination.C57 mice were injected with Bufalin or DMSO intraperitoneally from Day 1 to Day 10, and ssAAV8-CBAp-fluc-EYFP (1×109 vgs) vectors were administrated on Day 6. Two weeks post-injection of the viral vectors, in vivo imaging system was applied to evaluate the expression of reporter gene.4. The effect and machanism of Shikonin to improve the transduction efficiency of rAAV vectors in hematopoetic cellsK562 cells were infected with scAAV6-CBAp-EGFP, and cells were treated with DMSO, Shikonin, Bufalin, MG132, NAC, the combination of NAC and Shikonin, et al. during infection. Fluorescent microscope and flow cytometry were utilized evaluate the transduction efficiency. K562 and HEK293 cells were infected with scAAV6-CBAp-EGFP and scAAV2-CBAp-EGFP, and during infection, cells were treated with proteasome inhibitors and protease inhibitors. Fluorescent microscope and flow cytometry were utilized evaluate the transduction efficiency. DHE staining was applied to analyze the content of Reactive Oxygen Species (ROS), followed by qPCR to investigate the DNA copy number of rAAV. Western Blot was used to evaluate the level of ubiquitination.HSC were infected with scAAV6-CBAp-EGFP, and during infection, cells were treated with DMSO, Shikonin, MG132. Fluorescent microscope and flow cytometry were utilized evaluate the transduction efficiency.5. The influence of Glycyrrhizic acid ammonium salt and Schizandrin on gene expression efficiency of rAAV vectors post-stable transductionC57 mice were administrated with ssAAV2-CBAp-Fluc-EYFP (5×108 vgs), ssAAV8-CBAp-Fluc-EYFP (5×109 vgs), and scAAV2-CBAp-Gluc (5×108 vgs) through tail vein injection.2 months post-transduction, DMSO, Glycyrrhizic acid ammonium salt and Schizandrin et al. were treated to the mice intraperitoneally. In vivo imaging system and Gaussia luciferase analysis kit were applied to evaluate the expression of reporter gene before and after the treatment.HEK293 cells were infected with scAAV2-CBAp-EGFP, and during infection, cells were treated with DMSO, Glycyrrhizic acid ammonium salt and Schizandrin. Fluorescent microscope and flow cytometry were utilized evaluate the transduction efficiency.6. StatisticsAll results were expressed as mean ±standard deviation. Statistical analysis was performed by SPSS 16.0. Analysis of variance (ANOVA) was used to evaluate the effect of Bufalin, Shikonin, Glycyrrhizic acid ammonium salt or Schizandrin under different concentrations. For other difference between two groups, Student’s t test was used when data have homogeneity of variance while Mann-Whitney U test was used when data lack of homogeneity of variance. P<0.05 was considered to be statistically significant.Results1. The application of Rep3 and ITR3 in optimizing the packaging and transduction efficiency of rAAV3 vectors1.1 The construction and of plasmids and the analysis of transfection effciencyResults showed that the expression of Rep2 was close to Rep3, there was no significant difference in the green florescence mediated by ITR2 and ITR3(P>0.05, P>0.05).1.2 The effect of trans-acting factors on the replication efficiency of target genes containing cis-acting elementsWith the help of Rep 2, there was no significant difference of DNA copy number, whereas with the help of Rep 3, there was no significant difference of DNA copy number in pITR2-CMVp-hrGFP and pITR3-CMVp-hrGFP, but the DNA copy number of pITR2-CMVp-EGF-Neo was significantly lower than pITR3-CMVp-EGF-Neo (P<0.01).1.3 The influence of trans-acting factors and containing cis-acting elements on the packaging efficiencyCompared with Rep2ITR2, the vector number of Rep2ITR3 was similar (P>0.05), Rep3ITR2 was significantly lower (P<0.05), and Rep3ITR3 was 4.5 folder higher (P<0.01).1.4 The influence of trans-acting factor and containing cis-acting elements on the transduction efficiency of the packaged vectorsGreen florescence mediated by ssAAV3-ITR3-CMVp-EGFP-Neo was significantly higher than ssAAV3-ITR2-CMVp-EGFP-Neo in Huh7 and LH86 cells (P<0.01, P<0.01).2. The influence of cell density on transduction efficiency of rAAV vectors2.1 The influence of cell density on transduction efficiency of rAAV6 vectrors in K562 cellsWith the increase of cell number, the percentage of fluorescence positive cells and mean fluorescence were increased in a limited range.Results showed that the percentage of fluorescence positive cells and mean fluorescence of high density group were significantly increased than those of low density followed by mix to high density (P<0.01, P<0.01), and the percentage of fluorescence positive cells and mean fluorescence in the group of high density followed by dilution to low density were significantly higher than those of low density followed by mix to high density (P<0.01, P<0.01).The percentage of fluorescence positive cells and mean fluorescence in high density group were significantly higher than those in low density group (P<0.01, P<0.05). qPCR was applied to analyzed genome copy number of viral vectors, and results showed that the copy number in high density group was 4.5 fold higher than that in low density group (P<0.01).The percentage of fluorescence positive cells and mean fluorescence were reduced with the increase of infection volume, and the copy number in the group of 50μL and 500μL was significantly lower than that in the group of lmL.With high cell density, rAAV6 vectors can be successfully transduced into K562 cells though MOI was reduced into 300 or 30 vgs/cell.2.2 The influence of cell density on transduction efficiency of rAAV2 vectrors in HEK293, K562 and Raji cellsUnder the same MOI and cell density, the percentage of fluorescence positive cells and mean fluorescence were reduced in the following order, HEK293, K562 and Raji. Additioally, with the same MOI, the percentage of fluorescence positive cells and mean fluorescence of HEK293 cells were not increased with the increase of cell density, and the percentage of fluorescence positive cells and mean fluorescence of K562 and Raji cells were increased (P<0.01, P<0.01).Among these 3 cell lines, HEK293 expressed the highest level of receptor and co-receptor, followed by K562 and Raji cells, and the level of FGFR1 in K562 cells was similar to that of Raji cells.2.3 The influence of cell density on transduction efficiency of rAAV2 and rAAV6 vectrors in hematopoetic stem cellsResults showed that with same MOI, the percentage of fluorescence positive cells and mean fluorescence in high cell density group were higher than those in low cell density, and with same number of viral vectors, the percentage of fluorescence positive cells and mean fluorescence in high cell density group were also higher than those in low cell density. Moreover, the percentage of fluorescence positive cells and mean fluorescence in the groups of mutated rAAV vectors (scAAV2-4M-CBAp-EGFP and scAAV6-3M-CBAp-EGFP) were higher than those in the groups of wtAAV vectors (scAAV2-CBAp-EGFP and scAAV6-CBAp-EGFP).3. The effect and machanism of Bufalin to enhance the transduction efficiency of rAAV vectors3.1 The effect of Bufalin on the transduction efficiency of rAAV vectors in vitroResults showed that the green florescence pixels mediated rAAV2 including single-stranded, double-stranded, wide type and mutated vectors was significantly enhanced by Bufalin.3.2 The mechanism for Bufalin to enhance the transduction efficiency of rAAV vectors in vitroResults showed that the green florescence pixels in the groups of Bufalin and MG132 were significantly increased by contrast to DMSO (P<0.05, P<0.01), accompanied with the increase in the percentage of fluorescence positive cells and mean fluorescence. Furthermore, the green florescence pixels in the combination group was significantly higher than that in both Bufalin and MG132 groups (P<0.05, P<0.05).Western blot results demonstrated that ubiquitination was significantly accumulated in the group of MG132, whereas the ubiquitination mediated by Bufalin was similar to that of DMSO.3.3 The effect of Bufalin on the transduction efficiency of rAAV vectors in vivo Compared to DMSO, the firefly luciferase was not significantly increased by Bufalin.4. The effect and machanism of Shikonin to improve the transduction efficiency of rAAV vectors in hematopoetic cells4.1 The effect of Shikonin on the transduction efficiency of rAAV6 vector in K562 cellsShikonin possessed the most promising activity to enhance the percentage of fluorescence positive cells and mean fluorescence, in a dose-dependent manner.4.2 The effect of proteasome inhibitors and protease inhibitors on the transduction efficiency of rAAV6 vector in K562 cellsMG132 was able to significantly increse the green fluorescence pixels in rAAV2 or rAAV6 transduced HEK 293 cells, rather than K562 cells. Among all proteasome inhibitors and protease inhibitors, only Carfilzomib can significantly increase the mean fluorescence of rAAV6 treated K562 cells (P<0.01), without any influence on the percentage of fluorescence positive cells.4.3 The mechanism for Shikinon to enhance the transduction efficiency of rAAV6 vector in K562 cellsNAC had no effect on the percentage of fluorescence positive cells and mean fluorescence (P>0.05, P>0.05), but can significantly reduce the increased percentage of fluorescence positive cells and mean fluorescence by Shikonin (P<0.01, P<0.01). Shikonin can increase ROS, however, the increased ROS was reduced to normal after NAC treatment. DNA copy number of rAAV was not increased by Shikonin, NAC or their comnbination. Western Blot results showed that Carfilzomib and MG132 resulted in accumulation of ubiquitination, and the amount of ubiquitination were much higher than Shikonin.4.4 The effect of Shikonin on the transduction efficiency of rAAV6 vector in HSCsShikonin can increase the mean fluorescence, and its effect was higher than MG132.5. The influence of Glycyrrhizic acid ammonium salt and Schizandrin on gene expression efficiency after rAAV vectors post-stable transduction5.1 Screening of chemical compound from Chinese herbs to enhance the gene expression efficiency post-transduction in vivoCompared with DMSO, the level of firefly luciferase in the group of Glycyrrhizic acid ammonium salt and Schizandrin was significantly higher than pre-injection (P<0.01, P<0.01).5.2 The effect of Glycyrrhizic acid ammonium salt and Schizandrin on the geneexpression efficiency post-transduction in vivo Glycyrrhizic acid ammonium salt and Schizandrin can increase the level of firefly luciferase and Gaussia luciferase in relatively low doage rAAV transduced mice, but they were inert in relatively high doage rAAV transduced mice.5.3 The effect of Glycyrrhizic acid ammonium salt and Schizandrin on the geneexpression efficiency post-transduction in vitro Glycyrrhizic acid ammonium salt and Schizandrin had no influence the percentage of fluorescence positive cells and mean fluorescence.ConclusionIn order to enhance the transduction efficiency of rAAV vectors, we applized TCM with its advantages of enormous components and multiple targeting efficiency, focused on the key points which are less invsetigated in self-optimization of rAAV vectors. In the present research, we had found out positive results, which deserved further investigation and application.Compared with the commonly applied elements in packaging rAAV vectors, Rep2 and ITR2, Rep 3 and ITR3 can achieve better package efficiency of rAAV3. accompanied with higher transduction efficiency. During infection, the increased cell density was proven to induce increased intracellular DNA copy number of rAAV and higher transduction efficiency, which can be applied to multiple floating cell lines (K562 and Raji), several serotypes (rAAV2 and rAAV6), and different type of packaged genome (sc and ss). Furthermore, high density can trigger higher transduction efficiency in HSCs.By comparing with Celastrol, Pristimerin and Shikonin, which had been isolated from TCM medicine and identified to improve the transduction efficiency, we screened new TCM components and found out that Bufalin can strikingly increase the transduction efficiency in vitro, dependent on other mechanism rather than the widely reported proteasome inhibition, however Bufalin was not effective in vivo. Moreover, dependent on increasing intracellular ROS, Shikonin was effective in enhancing the transduction efficiency in hematopoetic cells, especially in HSCs.Since few researches have been aimed to find new strategies to enhance the gene expression post-transduction of rAAV vectors, under the guidence of tCM theory, we screened numbers of chemical compoud, which were major ingrendients of the TCM herbs targeting liver, a major targeting organ of rAAV. Results showed that though Glycyrrhizic acid ammonium salt and Schizandrin had no influence on the gene expression post-transduction of rAAV vectors in vitro, they can significantly increase the gene expression post-transduction of rAAV vectors in mice, and without limitation by serotype, reporter gene, genome type (sc and ss). |
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