Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci U S A

Institute for Human Gene Therapy and Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 10/2002; 99(18):11854-9. DOI: 10.1073/pnas.182412299
Source: PubMed

ABSTRACT Tissues from rhesus monkeys were screened by PCR for the presence of sequences homologous to known adeno-associated virus (AAV) serotypes 1-6. DNA spanning entire rep-cap ORFs from two novel AAVs, called AAV7 and AAV8, were isolated. Sequence comparisons among these and previously described AAVs revealed the greatest divergence in capsid proteins. AAV7 and AAV8 were not neutralized by heterologous antisera raised to the other serotypes. Neutralizing antibodies to AAV7 and AAV8 were rare in human serum and, when present, were low in activity. Vectors formed with capsids from AAV7 and AAV8 were generated by using rep and inverted terminal repeats (ITRs) from AAV2 and were compared with similarly constructed vectors made from capsids of AAV1, AAV2, and AAV5. Murine models of skeletal muscle and liver-directed gene transfer were used to evaluate relative vector performance. AAV7 vectors demonstrated efficiencies of transgene expression in skeletal muscle equivalent to that observed with AAV1, the most efficient known serotype for this application. In liver, transgene expression was 10- to 100-fold higher with AAV8 than observed with other serotypes. This improved efficiency correlated with increased persistence of vector DNA and higher number of transduced hepatocytes. The efficiency of AAV8 vector for liver-directed gene transfer of factor IX was not impacted by preimmunization with the other AAV serotypes. Vectors based on these novel, nonhuman primate AAVs should be considered for human gene therapy because of low reactivity to antibodies directed to human AAVs and because gene transfer efficiency in muscle was similar to that obtained with the best known serotype, whereas, in liver, gene transfer was substantially higher than previously described.

Download full-text


Available from: Mauricio R Alvira, Aug 27, 2015
  • Source
    • "d . Most of the work was focused on rAAV2 previously ( Peng et al . , 2000 ; Su et al . , 2000 ) and rAAV8 vectors more recently ( Hsu et al . , 2012 ) . However , rAAV2 vectors were generally inefficient through systemic injection ( Zincarelli et al . , 2008 ) and rAAV8 vectors have a broad tropism to normal tissues of mice other than the liver ( Gao et al . , 2002 ; Wang et al . , 2005 ; Zincarelli et al . , 2008 ) . In addition , most cocktail strategies to combine gene therapy and chemotherapy demonstrated only additive therapeutic effects . Only chemical drugs acting on specific molecular targets were utilized ( Liu et al . , 2014 ) . Our studies , on the other hand , report a synergistic effe"
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 severe 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.
    Human Gene Therapy 10/2014; 25(12). DOI:10.1089/hum.2014.099 · 3.62 Impact Factor
  • Source
    • "The rAAV8RSM is an rAAV vector serotype 8 (Gao et al., 2002) produced using a HEK293 transfection-based protocol. HEK293 cells were amplified from a cGMP master cell bank and transfected by the vector plasmid pTR-UF11 (Burger et al., 2004), the same plasmid used to generate the rAAV2RSM (Potter et al., 2008; Lock et al., 2010), and the pDP8 helper plasmid (see Materials and Methods section: Reference standard material manufacturing). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. Production of the rAAV8RSM was carried out using transient transfection and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to sixteen laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50x1011 pt/ml; CI, 4.26x1011 to 6.75x1011 pt/ml), vector genomes (mean, 5.75x1011 vg/ml; CI, 3.05x1011 to 1.09x1012 vg/ml), and infectious units (mean, 1.26x109 IU/ml; CI, 6.46x108 to 2.51x109 IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time where the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.
    Human Gene Therapy 10/2014; 25(11). DOI:10.1089/hum.2014.057 · 3.62 Impact Factor
  • Source
    • "Following, they were transferred into the previously described double stranded pdsAAV-LP1-EGFPmut AAV vector [10] using the restriction enzymes BglII and SalI. The plasmids encoding the miRNA constructs were cotransfected into HEK293T cells with the pDGΔVP helper plasmid [16] and a mutated p5E18-VD2/8 expression vector [17] encoding AAV2 rep and a mutated AAV8 cap protein (aa 589–592: QNTA to GNRQ). For virus production, HEK293T cells from six 80–90% confluent 15 cm plates were suspended in 1100 ml medium. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Disturbances in lipid homeostasis are hallmarks of severe metabolic disorders and their long-term complications, including obesity, diabetes, and atherosclerosis. Whereas elevation of triglyceride (TG)-rich very-low-density lipoproteins (VLDL) has been identified as a risk factor for cardiovascular complications, high-density lipoprotein (HDL)-associated cholesterol confers atheroprotection under obese and/or diabetic conditions. Here we show that hepatocyte-specific deficiency of transcription factor transforming growth factor β 1-stimulated clone (TSC) 22 D1 led to a substantial reduction in HDL levels in both wild-type and obese mice, mediated through the transcriptional down-regulation of the HDL formation pathway in liver. Indeed, overexpression of TSC22D1 promoted high levels of HDL cholesterol in healthy animals, and hepatic expression of TSC22D1 was found to be aberrantly regulated in disease models of opposing energy availability. The hepatic TSC22D1 transcription factor complex may thus represent an attractive target in HDL raising strategies in obesity/diabetes-related dyslipidemia and atheroprotection.
    04/2014; 3(2):155-66. DOI:10.1016/j.molmet.2013.12.007
Show more