title: Development of targeted gene transfer into human primary T lymphocytes and macrophages using high-titer recombinant HIV vectors.
abstract: Primary human lymphocytes and macrophages are an important target cells for human immunodeficiency virus (HIV). For targeted gene transfer into CD4(+) lymphocytes and macrophages, we constructed HIV vectors with envelope glycoprotein (gp120) from the T-cell tropic BH10 strain and the macrophage tropic SF162, and developed an improved strategy for preparation of high-titer HIV vectors. Among several possible procedures, we found that ultrafiltration using CENTRIPREP columns was highly effective to concentrate HIV particles. The titer could be increased four orders of magnitudes. The total recovery was more than 80%. No replication-competent cytopathic HIV was detected in concentrated vector preparation. Using the high-titer HIV vector carrying the enhanced green fluorescent protein (EGFP) gene, we transduced human primary lymphocytes and macrophages. FACS analysis showed that the T-cell tropic vector could transduce 40-80% of CD4(+) T-cells stimulated with IL2 plus PHA and 20-50% of unstimulated cells. The macrophage tropic vector was shown to transduce approximately 20% of terminally differentiated macrophages. These results represent the initial report of targeted gene transfer into terminally differentiated macrophages. These results also indicate that these HIV vectors are useful for the manipulation of gene expression in HIV infectable cells and the development of gene therapy targeting lymphocytes and macrophages.
authors.affiliation: Department of Biochemistry and Molecular Biology, Division of Gene Therapy Research Center for Advanced Medical Technology, Nippon Medical School, Tokyo 113-8602, Japan. kmiyake@nms.ac.jp
authors.forename: Koichi
authors.initials: K
authors.lastname: Miyake
authors.forename: Noriko
authors.initials: N
authors.lastname: Miyake
authors.forename: Takashi
authors.initials: T
authors.lastname: Shimada
mesh_terms: D015496:CD4-Positive T-Lymphocytes; D004274:DNA, Recombinant / Q000235:genetics*; D005434:Flow Cytometry; D018390:Gene Targeting / Q000379:methods*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006678:HIV; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008264:Macrophages; D014161:Transduction, Genetic; D014462:Ultrafiltration
chemical_list: D004274:DNA, Recombinant; D015699:HIV Envelope Protein gp120; D049452:Green Fluorescent Proteins