902. A Novel In Vivo, Lentivirus-Based Model of α-Spectrin Function
Authors: Sara Mootien, Nancy E. Siedel, David M. Bodine, Patrick G. Gallagher
Journal: Molecular Therapy (2004)

The study of α spectrin has yielded a better understanding of the structure and function of the membrane skeleton in erythroid and nonerythroid cells. The relationship between the primary, secondary, and tertiary structures of spectrin, a fundamental problem in hematology and biology, is complicated by the large size of spectrin, Mr 240kDa. A milestone in structure/function studies of spectrin is development of a gene delivery system capable of integrating the large (~7kb) α-spectrin cDNA in hematopoietic cells and cell lines. Two lentivirus vectors consisting of an HIV-1 vector backbone containing the ankyrin (Ank) erythroid promoter driving the human α-spectrin cDNA have been constructed. In one, pHR-Ank-αSP, the Ank promoter is in reverse orientation to the viral 5' LTR. In the other, pHR-Ank-αSP', the Ank promoter is in the same orientation and is adjacent to the viral 5' LTR. We chose the Ank promoter because it directed high-level, erythroid-specific expression with minimal position or enhancer dependence in TG mice, and, in lentiviral vectors, the Ank promoter directed erythroid-specific expression of target genes (Blood 98:2664, 2001). Lentivirus was produced by transient transfection of 293T cells with 3 plasmids: one of the lentiviral α-spectrin plasmids (pHR-Ank-alphaSP or pHR-Ank-alphaSP' an HIV-1 based packaging plasmid with the packaging signal deleted along with the LTR and envelope region, but including RRE-rev, gag/pol, and other elements, and a VSV-G envelope plasmid. Lentivirus containing supernatant was collected 48hrs after transfection. Analysis of lentivirus particles by slot blot analysis of RNA extracted from the supernatant revealed a titer of ~1x10e5. Lentivirus-conditioned supernatant was exposed to logarithmically expanding WT MEL cells in the presence of polybreen. PCR of genomic DNA from transduced cells using primers in the Ank promoter and exon 1 of the α-spectrin gene confirmed integration. The gross morphologic characteristics of the transduced MEL cells was similar to mock transduced cells. RNA extracted from the transduced cells was reverse transcribed using a human α-spectrin specific primer in exon 8. PCR of the RT product with primers corresponding to exons 7 and 8 and to the very 5' end of the human α-spectrin cDNA, exon 1 to exon 2, was positive. Immunoblot of cell lysates using a human-specific MoAB to human α-spectrin revealed a band at 240kDa in lysates from K562 cells (+ control), and both pHR-Ank-αSP and pHR-Ank-αSP' transduced cells. Similar to K562 cells, the 240kDa band was the only band seen in the transduced cells. Untransduced wild type MEL cells were negative. These experiments indicate that a very large membrane protein can be expressed using lentiviral vectors. There is also proof-of-principle for creation of in vivo model using lentiviral-mediated transfer of the α-spectrin gene into α-spectrin deficient MEL cells and HSC and progenitor cells from sph/sph mice, a murine model of α-spectrin deficient hemolytic anemia. Because sph/sph mice completely lack α-spectrin protein, this model could allow rapid assessment of many α-spectrin variants in vivo, without the need for laborious and time-consuming knock out/knock in experiments.