Construction of a high efficiency retroviral vector for gene therapy of Hunter's syndrome.
BACKGROUND
As an alternative method to the conventional therapies for Hunter's syndrome, which is a lethal lysosomal storage disorder, we have developed gene delivery vehicles using a series of retroviral vectors. The objective of this study was to develop a safe and efficient retroviral vector and to optimize conditions for efficient transduction of human bone marrow CD34+ stem cells using our vector.


METHODS
We constructed three types of MLV-based retroviral vectors expressing iduronate-2-sulfatase (IDS) which is deficient in patients suffering from Hunter's syndrome: MIN-IDS and MIM-IDS, which express IDS along with bacterial neo and human MDR genes, respectively, and MT-IDS lacking any selectable marker. Respective producer lines were derived from the packaging line, PG13, and compared for viral titer and levels of gene expression. After comparing, the retroviral vector, MT-IDS, was used to transduce human bone marrow CD34+ stem cells on fibronectin under various MOIs.


RESULTS
In comparison, MT-IDS not only produced the highest viral titer (close to 10(7) cfu/ml), but also showed the highest level of gene expression in various transduction assays and RNA analysis. When 1.5 x 10(5) human CD34+ bone marrow cells were transduced with MT-IDS under the most optimal MOIs, about 80% of total colony forming units were shown to contain the IDS cDNA.


CONCLUSIONS
Minimum-sized retroviral vector that contains no selective marker as well as a viral coding sequences could drive a high level of gene expression, be produced efficiently from the producer line, and enter hematopoietic cells at a high frequency. Our data suggest the great potential for using MT-based vector(s) in a gene therapy trial for Hunter's syndrome utilizing human CD34+ stem cells as target cells.
Hong
Youngtae
Y
Institute of Molecular Biology and Genetics, Seoul National University, Kwan-Ak-Gu, Seoul 151-742, Korea.
Yu
Seung Shin
SS
Kim
Jong-Mook
JM
Lee
Karim
K
Na
Young Soon
YS
Whitley
Chester B
CB
Sugimoto
Yoshikazu
Y
Kim
Sunyoung
S
D001854:Bone Marrow Cells / Q000378:metabolism; D002460:Cell Line; D019450:Genes, MDR; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D007066:Iduronate Sulfatase / Q000235:genetics / Q000378:metabolism; D016532:Mucopolysaccharidosis II / Q000628:therapy*; D012190:Retroviridae; D013234:Stem Cells / Q000378:metabolism; D014161:Transduction, Genetic
D007066:Iduronate Sulfatase