High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Chen, Ling; Lü, Yuan; Cheng, Baohua; McGoogan, Katherine E.; Gee, Samantha W.Y.; Ma, Wenqin; Li, Baozheng; Aslanidi, George; Srivastava, Arun

doi:10.3791/2538

Cited by 24 publications

(23 citation statements)

References 14 publications

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“…1E, indicated that a large amount of rAAV8 vector genomes were present in the liver, whereas the numbers of rAAV3 vector genomes were minimal, corroborating our previous observations. These studies, together with our previous publications (Glushakova et al, 2009;Ling et al, 2010Ling et al, , 2011Cheng et al, 2012), provide a clear rationale to further pursue rAAV3 vector-mediated potential gene therapy of human HCC.…”

Section: Raav3 Vectors Selectively Target Human Liver Tumors In a Murmentioning

confidence: 69%

“…Among all commonly used rAAV serotypes, rAAV3, which fails to efficiently transduce any normal murine tissue in vivo (Zincarelli et al, 2008;Ling et al, 2010;Markakis et al, 2010), was shown in our previous studies to transduce human HCC cells highly efficiently both in vitro (Ling et al, 2011) and in vivo (Cheng et al, 2012). Although rAAV3 vectors also transduce primary human hepatocytes in vitro (Glushakova et al, 2009) and in humanized mice in vivo (Lisowski et al, 2014), the transgene expression could be restricted to malignant cells by using an HCCspecific promoter, alpha-fetoprotein promoter (AFPp).…”

Section: Introductionmentioning

confidence: 95%

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SelectiveIn VivoTargeting of Human Liver Tumors by Optimized AAV3 Vectors in a Murine Xenograft Model

et al. 2014

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Current challenges for recombinant adeno-associated virus (rAAV) vector-based cancer treatment include the low efficiency and the lack of specificity in vivo. rAAV serotype 3 (rAAV3) vectors have previously been shown to be ineffective in normal mouse tissues following systemic administration. In the present study, we report that rAAV3 vectors can efficiently target and transduce various human liver cancer cells in vivo. Elimination of specific surface-exposed serine and threonine residues on rAAV3 capsids results in further augmentation in the transduction efficiency of these vectors, without any change in the viral tropism and cellular receptor interactions. In addition, we have identified a potential chemotherapy drug, shikonin, as a multifunctional compound to inhibit liver tumor growth as well as to significantly enhance the efficacy of rAAV vector-based gene therapy in vivo. Furthermore, we also document that suppression of tumorigenesis in a human liver cancer xenograft model can be achieved through systemic administration of the optimized rAAV3 vectors carrying a therapeutic gene, and shikonin at a dose that does not lead to liver damage. Our research provides a novel means to achieve not only targeted delivery but also the potential for gene therapy of human liver cancer.

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Section: Raav3 Vectors Selectively Target Human Liver Tumors In a Murmentioning

confidence: 69%

Section: Introductionmentioning

confidence: 95%

SelectiveIn VivoTargeting of Human Liver Tumors by Optimized AAV3 Vectors in a Murine Xenograft Model

et al. 2014

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“…Highly purified stocks of self-complementary (sc) AAV2-WT or 26 capsid mutants of AAV2 vectors or AAV8-WT vector carrying the enhanced green fluorescent protein (EGFP) gene driven by the chicken b-actin promoter were generated by polyethyleneimine-based triple transfection of AAV-293 cells (Ling et al, 2011). Briefly, forty 150-mm 2 dishes 80% confluent with AAV-293 cells were transfected with AAV2 rep/cap (p.ACG2), transgene (dsAAV2-EGFP), and AAV-helper free (p.helper) plasmids.…”

Section: Generation Of Recombinant Vectorsmentioning

confidence: 99%

Bioengineering of AAV2 Capsid at Specific Serine, Threonine, or Lysine Residues Improves Its Transduction Efficiencyin Vitroandin Vivo

Gabriel

Hareendran

Sen

et al. 2013

Human Gene Therapy Methods

107

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We hypothesized that the AAV2 vector is targeted for destruction in the cytoplasm by the host cellular kinase/ ubiquitination/proteasomal machinery and that modification of their targets on AAV2 capsid may improve its transduction efficiency. In vitro analysis with pharmacological inhibitors of cellular serine/threonine kinases (protein kinase A, protein kinase C, casein kinase II) showed an increase (20-90%) on AAV2-mediated gene expression. The three-dimensional structure of AAV2 capsid was then analyzed to predict the sites of ubiquitination and phosphorylation. Three phosphodegrons, which are the phosphorylation sites recognized as degradation signals by ubiquitin ligases, were identified. Mutation targets comprising eight serine (S) or seven threonine (T) or nine lysine (K) residues were selected in and around phosphodegrons on the basis of their solvent accessibility, overlap with the receptor binding regions, overlap with interaction interfaces of capsid proteins, and their evolutionary conservation across AAV serotypes. AAV2-EGFP vectors with the wild-type (WT) capsid or mutant capsids (15 S/T/alanine [A] or 9 K/arginine [R] single mutant or 2 double K/R mutants) were then evaluated in vitro. The transduction efficiencies of 11 S/T/A and 7 K/R vectors were significantly higher (*63-90%) than the AAV2-WT vectors (*30-40%). Further, hepatic gene transfer of these mutant vectors in vivo resulted in higher vector copy numbers (up to 4.9-fold) and transgene expression (up to 14-fold) than observed from the AAV2-WT vector. One of the mutant vectors, S489A, generated *8-fold fewer antibodies that could be cross-neutralized by AAV2-WT. This study thus demonstrates the feasibility of the use of these novel AAV2 capsid mutant vectors in hepatic gene therapy.

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“…We have reported that Huh7 cells, a well-known human hepatocellular carcinoma cell line (Nakabayashi et al, 1982), can be efficiently transduced by AAV3 vectors (Glushakova et al, 2009;Ling et al, 2010Ling et al, , 2011Cheng et al, 2012), and that the transduction efficiency of these vectors can be further augmented by site-directed mutagenesis of surface-exposed tyrosine residues (Cheng et al, 2012). To further examine whether phosphorylation of surface-exposed serine and/or threonine residues also affects the transduction efficiency of AAV3 vectors, Huh7 cells were either mock-treated or treated with two different serine/threonine kinase inhibitors, c-Jun N-terminal kinase ( JNK) inhibitor or p38 mitogenactivated protein kinase (MAPK) inhibitor (SB), followed by infection with scAAV3-CBAp-EGFP vectors, depicted schematically in Fig.…”

Section: Resultsmentioning

confidence: 99%

Limitations of Encapsidation of Recombinant Self-Complementary Adeno-Associated Viral Genomes in Different Serotype Capsids and Their Quantitation

Wang

Chen

Song

et al. 2012

Human Gene Therapy Methods

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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.

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High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Cited by 24 publications

References 14 publications

SelectiveIn VivoTargeting of Human Liver Tumors by Optimized AAV3 Vectors in a Murine Xenograft Model

SelectiveIn VivoTargeting of Human Liver Tumors by Optimized AAV3 Vectors in a Murine Xenograft Model

Bioengineering of AAV2 Capsid at Specific Serine, Threonine, or Lysine Residues Improves Its Transduction Efficiencyin Vitroandin Vivo

Limitations of Encapsidation of Recombinant Self-Complementary Adeno-Associated Viral Genomes in Different Serotype Capsids and Their Quantitation

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