Use of cell-free retroviral vector preparations for transduction of cells from the marrow of chronic phase and blast crisis chronic myelogenous leukemia patients and from normal individuals.
Marrow cells were exposed to the LNL6 or G1N safety-modified variants of the N2 retrovirus, which contain the G418 bacterial resistance gene neo. The frequency of acquisition of the G418 resistance phenotype following exposure to LNL6 or G1N was compared among hematopoietic progenitor cells from the marrow of patients with chronic phase chronic myelogenous leukemia (CML), blast crisis CML, or from nonleukemic individuals. Under the conditions of our experiments, the myeloid committed progenitor cells from 3 of 6 nonleukemic individuals, 9 of 18 chronic-phase CML patients, and 2 of 4 blast crisis CML patients acquired resistance to at least 1 mg/ml G418 following incubation with cell-free supernatants from the PA317 LNL6 or PA317 G1N producer cell lines. Ten of the 32 colonies growing up in 0.8 mg/ml G418 from chronic-phase marrow exposed to LNL6 were shown to contain the neo gene by polymerase chain reaction (PCR) assay of DNA. These results were consistent with estimates of the transduction frequency based on acquisition of resistance to G418 as the number of colonies growing under G418 selection was always greater at 0.8 mg/ml G418 than at higher concentrations of G418 (1.0-1.4 mg/ml). The average transduction frequency at each G418 concentration (1.0, 1.2, and 1.4 mg/ml) in cells from blast crisis CML cells ranged from 2 to 14%, as measured by acquisition of G418 resistance. Chronic-phase CML showed slightly lower average frequencies of transduction (0.6-2.8% of the colonies are G418 resistant). The average transduction frequency of cells from nonleukemic marrow was as high as that seen from the marrow of chronic-phase CML individuals.(ABSTRACT TRUNCATED AT 250 WORDS)
Etkin
M
M
Department of Hematology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Filaccio
M
M
Ellerson
D
D
Suh
S P
SP
Claxton
D
D
Gaozza
E
E
Brenner
M
M
Moen
R
R
Belmont
J
J
Moore
K A
KA
D001483:Base Sequence; D001752:Blast Crisis / Q000235:genetics / Q000473:pathology*; D001853:Bone Marrow / Q000473:pathology*; D001854:Bone Marrow Cells; D002455:Cell Division; D002474:Cell-Free System; D004247:DNA; D004351:Drug Resistance / Q000235:genetics; D005822:Genetic Vectors; D005839:Gentamicins / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D015466:Leukemia, Myeloid, Chronic-Phase / Q000235:genetics / Q000473:pathology*; D008969:Molecular Sequence Data; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic
D005839:Gentamicins; D004247:DNA; C010680:antibiotic G 418