Enhancement of retroviral gene transduction on a dish coated with a cocktail of two different polypeptides: one exhibiting binding activity toward target cells, and the other toward retroviral vectors.
CH-296, a recombinant fragment of human fibronectin (FN) composed of the cell-binding domain (C-domain), heparin-binding domain II (H-domain), and CS1 site, enhances the retrovirus-mediated gene transduction (GT) of hematopoietic stem cells. The RGD sequence in the C-domain is recognized by a variety of cell types through integrin VLA-5, and the LDV sequence in the CS1-site is recognized by integrin VLA-4. Retrovirus particles were also found to bind to the H-domain. Consequently, the CH-296 fragment can enhance GT through binding to both retrovirus particles and target cells that express integrins VLA-5 and/or VLA-4. In this study, we found that the GT efficiency can be maintained at levels comparable to that of CH-271, a FN fragment similar to CH-296 but lacking the CS1 site, when a cocktail of separated functional domains of CH-271 is used. When a dish was coated with a mixture of the C-domain and H-domain (molar ratio, 1:10), the GT efficiency of NIH3T3 cells reached the same level as that of the mother fragment, CH-271. The H-domain in the cocktail can be replaced with other virus-binding components, polylysine, FGF, and the insulin-binding domain of ColV, without the loss of GT efficiency. With other than FN fragments, a cocktail of erythropoietin and polylysine caused higher GT efficiency of Epo-receptor expressing TF-1 cells than in the case of each component alone.
Asada
K
K
Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Otsu, Shiga 520-2193, Japan.
Uemori
T
T
Ueno
T
T
Hashino
K
K
Koyama
N
N
Kawamura
A
A
Kato
I
I
D000818:Animals; D002460:Cell Line; D005353:Fibronectins; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D006801:Humans; D051379:Mice; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012190:Retroviridae
D005353:Fibronectins; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins