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Song L.,Central South University | Song L.,Florida College | Song L.,Shenzhen Institute of Xiangya Biomedicine | Kauss M.A.,Beckman Research Institute | And 24 more authors.
Cytotherapy | Year: 2013

Background aims: Although recombinant adeno-associated virus serotype 2 (AAV2) vectors have gained attention because of their safety and efficacy in numerous phase I/II clinical trials, their transduction efficiency in hematopoietic stem cells (HSCs) has been reported to be low. Only a few additional AAV serotype vectors have been evaluated, and comparative analyses of their transduction efficiency in HSCs from different species have not been performed. Methods: We evaluated the transduction efficiency of all available AAV serotype vectors (AAV1 through AAV10) in primary mouse, cynomolgus monkey and human HSCs. The transduction efficiency of the optimized AAV vectors was also evaluated in human HSCs in a murine xenograft model in vivo. Results: We observed that although there are only six amino acid differences between AAV1 and AAV6, AAV1, but not AAV6, transduced mouse HSCs well, whereas AAV6, but not AAV1, transduced human HSCs well. None of the 10 serotypes transduced cynomolgus monkey HSCs in vitro. We also evaluated the transduction efficiency of AAV6 vectors containing mutations in surface-exposed tyrosine residues. We observed that tyrosine (Y) to phenylalanine (F) point mutations in residues 445, 705 and 731 led to a significant increase in transgene expression in human HSCs in vitro and in a mouse xenograft model in vivo. Conclusions: These studies suggest that the tyrosine-mutant AAV6 serotype vectors are the most promising vectors for transducing human HSCs and that it is possible to increase further the transduction efficiency of these vectors for their potential use in HSC-based gene therapy in humans. © 2013 International Society for Cellular Therapy.

Song L.,Central South University | Song L.,Shenzhen Institute of Xiangya Biomedicine | Song L.,Florida College | Li X.,Florida College | And 14 more authors.
PLoS ONE | Year: 2013

We have observed that of the 10 AAV serotypes, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs), and that the transduction efficiency can be further increased by specifically mutating single surface-exposed tyrosine (Y) residues on AAV6 capsids. In the present studies, we combined the two mutations to generate a tyrosine double-mutant (Y705+731F) AAV6 vector, with which >70% of CD34+ cells could be transduced. With the long-term objective of developing recombinant AAV vectors for the potential gene therapy of human hemoglobinopathies, we generated the wild-type (WT) and tyrosine-mutant AAV6 vectors containing the following erythroid cell-specific promoters: β-globin promoter (βp) with the upstream hyper-sensitive site 2 (HS2) enhancer from the β-globin locus control region (HS2-βbp), and the human parvovirus B19 promoter at map unit 6 (B19p6). Transgene expression from the B19p6 was significantly higher than that from the HS2-βp, and increased up to 30-fold and up to 20-fold, respectively, following erythropoietin (Epo)-induced differentiation of CD34+ cells in vitro. Transgene expression from the B19p6 or the HS2-βp was also evaluated in an immuno-deficient xenograft mouse model in vivo. Whereas low levels of expression were detected from the B19p6 in the WT AAV6 capsid, and that from the HS2-βp in the Y705+731F AAV6 capsid, transgene expression from the B19p6 promoter in the Y705+731F AAV6 capsid was significantly higher than that from the HS2-βp, and was detectable up to 12 weeks post-transplantation in primary recipients, and up to 6 additional weeks in secondary transplanted animals. These data demonstrate the feasibility of the use of the novel Y705+731F AAV6-B19p6 vectors for high-efficiency transduction of HSCs as well as expression of the b-globin gene in erythroid progenitor cells for the potential gene therapy of human hemoglobinopathies such as β-thalassemia and sickle cell disease. © 2013 Song et al.

Aslanidi G.V.,Florida College | Rivers A.E.,Florida College | Rivers A.E.,University of Illinois at Chicago | Ortiz L.,Florida College | And 10 more authors.
PLoS ONE | Year: 2013

The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of AAV2 vectors, and phosphorylation of certain surface-exposed amino acid residues on the capsid provides the primary signal for ubiquitination. Removal of several critical tyrosine (Y) and serine (S) residues on the AAV2 capsid has been shown to significantly increase transduction efficiency compared with the wild-type (WT) vectors. In the present study, site-directed mutagenesis of each of the 17 surface-exposed threonine (T) residues was conducted, and the transduction efficiency of four of these mutants, T455V, T491V, T550V, and T659V, was observed to increase up to 4-fold in human HEK293 cells in vitro. The most critical Y, S, and T mutations were subsequently combined, and the quadruple-mutant (Y444+500+730F+T491V) AAV2 vector was identified as the most efficient. This vector increased the transduction efficiency ~24-fold over the WT AAV2 vector, and ~2-3-fold over the previously described triple-mutant (Y444+500+730F) vector in a murine hepatocyte cell line, H2.35, in vitro. Similar results were obtained in murine hepatocytes in vivo following tail vein injection of the Y444+500+730F+T491V scAAV2 vector, and whole-body bioluminescence imaging of C57BL/6 mice. The increase in the transduction efficiency of the Y-T quadruple-mutant over that of the Y triple-mutant correlated with an improved nuclear translocation of the vectors, which exceeded 90%. These observations suggest that further optimization of the AAV2 capsid by targeting amino acid residues involved in phosphorylation may not be possible. This study has thus led to the generation of a novel Y444+500+730F+T491V quadruple-mutant AAV2 vector with potential for use in liver-directed human gene therapy. © 2013 Aslanidi et al.

Wang Y.,Shanghai University of Traditional Chinese Medicine | Wang Y.,Florida College | Ling C.,Florida College | Song L.,Florida College | And 10 more authors.
Human Gene Therapy Methods | Year: 2012

We previously reported that self-complementary adeno-associated virus (scAAV) type 2 genomes of up to 3.3 kb can be successfully encapsidated into AAV2 serotype capsids. Here we report that such oversized AAV2 genomes fail to undergo packaging in other AAV serotype capsids, such as AAV1, AAV3, AAV6, and AAV8, as determined by Southern blot analyses of the vector genomes, although hybridization signals on quantitative DNA slot-blots could still be obtained. Recently, it has been reported that quantitative real-time PCR assays may result in substantial differences in determining titers of scAAV vectors depending on the distance between the primer sets and the terminal hairpin structure in the scAAV genomes. We also observed that the vector titers determined by the standard DNA slot-blot assays were highly dependent on the specific probe being used, with probes hybridizing to the ends of viral genomes being significantly overrepresented compared with the probes hybridizing close to the middle of the viral genomes. These differences among various probes were not observed using Southern blot assays. This overestimation of titer is a systemic error during scAAV genome quantification, regardless of viral genome sequences and capsid serotypes. Furthermore, different serotypes capsid and modification of capsid sequence may affect the ability of packaging intact, full-length AAV genomes. Although the discrepancy is modest with wild-type serotype capsid and short viral genomes, the measured titer could be as much as fivefold different with capsid mutant vectors and large genomes. Thus, based on our data, we suggest that Southern blot analyses should be performed routinely to more accurately determine the titers of recombinant AAV vectors. At the very least, the use of probes/primers hybridizing close to the mutant inverted terminal repeat in scAAV genomes is recommended to avoid possible overestimation of vector titers. © 2012 Mary Ann Liebert, Inc.

Li B.-N.,Experimental Hematology Laboratory | Li B.-N.,Shenzhen Institute of Xiangya Biomedicine | Li Z.-Y.,Experimental Hematology Laboratory | Li Z.-Y.,Shenzhen Institute of Xiangya Biomedicine | And 6 more authors.
Progress in Biochemistry and Biophysics | Year: 2011

The adeno-associated virus (AAV) has many safety features that favor its use in the treatment of arteriosclerosis; however, the conventional, adeno-associated virus (AAV) mediated single-gene delivery is inefficient for arteriosclerosis. This has been attributed that the incidence of atherosclerosis is caused by a variety of genetic defects but not a particular gene. To overcome this, double-gene delivery was evaluated for the treatment of atherosclerosis. Four experimental groups were administered the following AAV vector constructs: rAAV-apoAI-IRES-SR-BI, rAAV-apoAI-GFP, rAAV-IRES-GFP, and PBS. ApoAIand SR-BIgene expression was detected using RT-PCR. The apoAIand SR-BIprotein expression was determined by Western blotting and ELISA. Diet-induced hypercholesterolemia and atherosclerosis in rats was adopted and rAAV was administered through the tail vein injection. HepG2 cells were cultured and infected with the three viral vectors. The apoAIand SR-BIsecreted from HepG2 cells in the AAV- apoAI/SR-BIgroup enhanced cholesterol efflux and resulted in a stronger RCT ability, respectively. In the rats' model with diet-induced hypercholesterolemia and atherosclerosis, GFP expression could be detected at 8 weeks post-injection. The rAAV vector had superior gene expressing activity. Eight weeks after gene transfer, plasma total cholesterol and LDL-cholesterol concentrations were significantly reduced (P < 0.05) compared to control for rAAV-IRES-GFP (AAV-GFP) treated group. No effect on HDL-cholesterol concentrations occurred. Ultrasound determined intima-media thickness also has been significantly reduced compared to control. Serum hs-CRP and SOD levels increased significantly (P< 0.01). Serum MDA levels decreased significantly. Gene mRNA expression was detected in atherosclerosis rats' model. The results show that rAAV-hapoAI-IRES-hSR-BIvector can anti-inflammatory, reduce atherosclerotic macrophage content and increases lesion stability of pre-existing plaques through quenching of NF- κB activity and reducing plasma cholesterol. Simultaneous over-expression of apoAIand SR-BIby AAV-mediated gene transfer may have a favorable effect on diet-induced hypercholesterolemia and arteriosclerosis in rats. These results may provide a new method for gene therapy of arteriosclerosis.

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