Recombinant human parvovirus B19 vectors: erythroid cell-specific delivery and expression of transduced genes

Abstract

A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562-566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111-1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited and acquired human diseases affecting cells of erythroid lineage.

FIG. 1

Schematic representation of the experimental strategy to generate recombinant B19-lacZ vectors. The recombinant plasmid pCMVp-lacZ, containing the lacZ gene under control of the CMV promoter inserted between the AAV ITRs, has been previously described (21, 23). Recombinant helper plasmids pSP-42 and pSP-46 contain the AAV rep genes under control of their authentic promoters, but the AAV cap genes have been replaced by the B19 cap genes (VP2 and VP1+VP2, respectively) under control of the CMV promoter and flanked by the adenovirus ITRs. The rest of the steps in generating the recombinant B19-lacZ vectors are described in Materials and Methods.

FIG. 2

Electron microscopic image of recombinant B19-lacZ particles. The recombinant virions were purified on CsCl gradients, as described in Materials and Methods. Samples were negatively stained with 3% phosphotungstic acid (pH 6.5), and the particles were visualized at a magnification of ×80,000 with a Philips 400 electron microscope (bar = 80 nm).

FIG. 3

Expression of the transduced lacZ gene mediated by recombinant AAV- and B19-lacZ vectors in human 293 and Epo-differentiated MB-02 cells. Approximately equivalent numbers of 293 cells (A, B, and C) and Epo-differentiated MB-02 cells (D, E, and F) were either mock infected or infected with 200 particles of AAV-lacZ (B and E) and B19-lacZ (C and F) recombinant vectors per cell under identical conditions. Forty-eight hours postinfection, the cells were fixed and stained for analysis of expression of the lacZ gene, as described in Materials and Methods.

FIG. 4

FACS analysis of lacZ gene expression in glycophorin A-positive primary human LDBM cells transduced with recombinant AAV- and B19-lacZ vectors. Glycophorin A-positive cells from human LDBM were either mock infected or infected with either 1 × 10⁵ particles of AAV-lacZ vector per cell or 2 × 10² particles of B19-lacZ vector per cell under identical conditions. Forty-eight hours postinfection, cells were harvested and processed for analysis of lacZ gene expression with fluorescent β-Gal product by using a Becton-Dickinson FACScan as described in Materials and Methods. The upper panels are FACS dot plots from a representative experiment indicating cells positive for lacZ (FL1-Height) and glycophorin A (FL2-Height). The results of dual positive cells from three separate experiments are plotted in the bar graph.

FIG. 5

FACS analysis of lacZ gene expression in erythroid and nonerythroid populations of primary human bone marrow-derived CD34⁺ cells transduced with recombinant AAV- and B19-lacZ vectors. CD34⁺ cells isolated from human LDBM were either mock infected or infected with either 1 × 10⁵ particles of AAV-lacZ vector per cell or 2 × 10² particles of B19-lacZ vector per cell under identical conditions. Forty-eight hours postinfection, cells were harvested and processed for analysis of lacZ gene expression in erythroid and nonerythroid cells by using fluorescent β-Gal product and PE-conjugated glycophorin A antibody, as described in Materials and Methods. The upper panels are FACS dot plots from a representative experiment indicating cells positive for lacZ (FL1-Height) and/or glycophorin A (FL2-Height). Nonerythroid cells expressing the lacZ gene are in the lower right quadrants, and erythroid cells expressing the lacZ gene are in the upper right quadrants. The results of dual positive cells from three separate experiments are plotted in the bar graph.

FIG. 6

FACS analysis of lacZ gene expression in erythroid- and myeloid-differentiated CD34⁺ primary human bone marrow cells transduced with recombinant AAV- and B19-lacZ vectors. Primary human bone marrow-derived CD34⁺ cells were allowed to undergo differentiation into erythroid or myeloid lineages with the use of respective cytokine combinations for 10 days in vitro, as described in Materials and Methods. Following differentiation, cells were either mock infected or infected with either 1 × 10⁵ particles of AAV-lacZ vector per cell or 2 × 10² particles of B19-lacZ vector per cell under identical conditions. Forty-eight hours postinfection, cells were harvested and stained with either PE-conjugated glycophorin A antibody (stippled bars) or PE-conjugated CD33 antibody (solid bars) and analyzed for lacZ gene expression in erythroid and myeloid populations by using fluorescent β-Gal product, as described in the legend to Fig. 4. Upper-row panels at the top are FACS dot plots from a representative experiment indicating cells positive for lacZ (FL1-H) and glycophorin A (FL2-H). Lower-row panels at the top represent cells expressing lacZ (FL1-H) and CD33 (FL2-H). The results of dual positive cells from three separate experiments are plotted in the bar graph.