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Increasing the size of rAAV-mediated expression cassettes in vivo by intermolecular joining of two complementary vectors

Nature Biotechnology volume 18, pages 527–532 (2000)Cite this article

Abstract

A major shortcoming to the use of adeno-associated virus (rAAV) vectors is their limited packaging size. To overcome this hurdle, we split an expression cassette and cloned it into two separate vectors. The vectors contained either a nuclear localizing Escherichia coli lacZ transgene (nlslacZ) with a splice acceptor, or the human elongation factor 1α ( EF1α) gene enhancer/promoter(s) (EF1αEP) with a splice donor. We co-injected a promoter-less nlslacZ vector with a vector containing either a single EF1αEP or a double copy of the EF1αEP in a head-to-head orientation, into the portal vein of mice. Gene expression, measured by both transduction efficiency and quantitation of the recombinant protein, was as much as 60–70% of that obtained from mice that received a single vector containing a complete EFαEP/nlslacZ expression cassette. This two-vector approach may allow development of gene therapy strategies that will carry exogenous DNA sequences with large therapeutic cDNAs and/or regulatory elements.

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Figure 1: Structures of the EF1αEP/nlslacZ expression cassette, nlslacZ plasmids, and rAAV vectors.
Figure 2: Transfection of 293 cells by the nlslacZ plasmids.
Figure 3: Xgal staining of the liver tissues from the rAAV-treated C57BL/6 rag1 mice.
Figure 4: RNA analyses of mouse livers transduced with AAV-Pless-nlsLacZ and/or EF1αEP rAAV vector.
Figure 5: Possible mechanisms of in vivo enhancement of transgene expression from AAV-Pless-nlsLacZ vector by AAV-EF1αEP or AAV-(EF1αEP) 2 vector.

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Acknowledgements

We thank Sally Fuess for technical assistance. This work was supported by NIH ROI HL53682.

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  1. Department of Pediatrics and Genetics, Program in Human Gene Therapy, Stanford University, Stanford, CA 94305

    Hiroyuki Nakai, Theresa A. Storm & Mark A. Kay

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  1. Hiroyuki Nakai
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Correspondence to Mark A. Kay.

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Nakai, H., Storm, T. & Kay, M. Increasing the size of rAAV-mediated expression cassettes in vivo by intermolecular joining of two complementary vectors. Nat Biotechnol 18, 527–532 (2000). https://doi.org/10.1038/75390

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