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Syngeneic central nervous system transplantation of genetically transduced mature, adult astrocytes

Gene Therapy volume 9, pages 432–443 (2002)Cite this article

Abstract

Advances in the development of highly infectious, replication-deficient recombinant retroviruses provide an efficient means of stable transfer of gene expression. Coupled with ex vivo transduction, surrogate cell populations can be readily implanted into the brain, thus serving as vehicles for delivering selected gene products into the central nervous system (CNS). Here we report that rat astrocytes can be routinely and safely isolated from brain tissue of a living donor by use of short-term gelatin sponge implants. The mature, nontransformed astrocytes were easily expanded, maintained in long-term tissue cultures and were efficiently transduced with an amphotropic retrovirus harboring a heterologous, fused transgene. In vitro retroviral infection rendered the nontransformed cells essentially 100% viable after exposure. The level of efficiency of infection (30–50% effective genome integration of provirus and expression of transgene in target cell populations) and minimal cell toxicity obviated the need to harvest large numbers of target cells. Cultured transduced astrocytes were resilient and exhibited select peptide expression for up to 1 year. Subsequently, transduced astrocytes were used in a series of experiments in which cells were transplanted intracerebrally in syngeneic animals. Post-implantation, astrocytes seeded locally and either insinuated into the surrounding parenchyma in situ or exhibited a variable degree of migration, depending on the anatomic source of astrocytes and the targeted brain implantation site. Transduced astrocytes remained viable in excess of 8 months post-transplantation and exhibited sustained transgenic peptide expression of green fluorescent protein/neomycin phosphotransferase in vivo. The sequential isolation and culture of nontransformed, mature, adult astrocytes and recombinant retrovirus-mediated transduction in vitro followed by brain reimplantation represents a safe and effective means for transferring genetic expression to the CNS. This study lays the foundation for exploring the utility of using a human autologous transplantation system as a potential gene delivery approach to treat neurological disorders. Prepared and utilized in this manner, autologous astrocytes may serve as a vehicle to deliver gene therapy to the CNS.

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Acknowledgements

The authors wish to express their appreciation to Ms Grace Lombardo for expert secretarial support in the preparation of this manuscript. Atlas figures were reproduced from The Rat Brain with permission from Paxinos and Watson. This work was supported in part by grants from the Endowment for the Neurosciences and from the Roswell Park Alliance Foundation.

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Authors and Affiliations

  1. Department of Neurology, Laboratory of Neuroimmunology and Neurovirology, Roswell Park Cancer Institute, Buffalo, NY, USA

    SM Selkirk, SJ Greenberg & PO Spence

  2. Department of Experimental Pathology, Roswell Park Cancer Institute, Buffalo, NY, USA

    SJ Greenberg

  3. Department of Neurosurgery, Roswell Park Cancer Institute, Buffalo, NY, USA

    RJ Plunkett, TA Barone & A Lis

  4. Department of Neurology, State University of New York at Buffalo, Buffalo, NY, USA

    SJ Greenberg

  5. Department of Neurosurgery, State University of New York at Buffalo, Buffalo, NY, USA

    RJ Plunkett

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  1. SM Selkirk
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Selkirk, S., Greenberg, S., Plunkett, R. et al. Syngeneic central nervous system transplantation of genetically transduced mature, adult astrocytes. Gene Ther 9, 432–443 (2002). https://doi.org/10.1038/sj.gt.3301643

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