Development of a competitive PCR method for in vitro and in vivo quantification of herpes simplex virus thymidine kinase and neomycin resistance-expressing cells used in a clinical trial
S. Maddens et al., Development of a competitive PCR method for in vitro and in vivo quantification of herpes simplex virus thymidine kinase and neomycin resistance-expressing cells used in a clinical trial, J HEMATH ST, 9(2), 2000, pp. 225-236
Citations number
33
Categorie Soggetti
Hematology,"Medical Research Diagnosis & Treatment
The aim of this study was to set up a sensitive and specific method to quan
tify the number of gene-modified cells in a gene therapy clinical trial cur
rently underway at our institution. This trial involves the use of retrovir
ally transduced allogeneic T cells expressing the herpes simplex-1 thymidin
e kinase (HSV-TK) and neomycin-phosphotransferase (NeoR) resistance gene. Q
uantification by competitive PCR was performed, with two homologous interna
l standards (Delta TK, Delta NeoR), 30 bp shorter than the target sequences
(TK, NeoR), coupled to fluorescent laser-based detection. Assessment of th
e amplification systems procedures was carried out for each sequence. The 3
0-bp deletion did not affect the amplification efficiency significantly. De
termination of the plateau phase of both amplified sequences demonstrated t
hat each sample must be quantified during the predetermined exponential pha
se. Finally, a blinded study of a transduced cell dilutions panel validated
the overall methodology. The competitive PCR was applied to quantification
of the retroviral transduction process by quantifying the NeoR gene in tra
nsduced PBMC samples (prior to G418 selection) from 18 donors in our clinic
al trial. A mean transduction efficiency of 9.78% +/- 1.37% was observed. W
e also quantified TK-expressing donor transgenic T cells in a murine GVHD m
odel. Results demonstrated on initial expansion of donor HSV-TK- expression
T cells as well as a significant ganciclovir (GCV)-induced decrease correl
ated with the number of circulating gene-modified T cells. Therefore, we ha
ve developed an efficient gene quantification tool that should be useful fo
r in vivo monitoring of gene-modified cells.