Recombinant polymer-protein fusion: a promising approach towards efficient and targeted gene delivery.
BACKGROUND
Synthetic vectors such as polymers have the potential to reduce the safety problems associated with viral vectors; however, their low transfection efficiency limits their clinical utility. To study the critical steps involved in an efficient transgene expression, there is a need for creative approaches that allow a systematic correlation between gene carrier structure and properties necessary for successful gene transfer. Using recombinant techniques a prototype vector comprised of tandem repeating units fused to a targeting moiety was biosynthesized to mediate gene transfer in mammalian cell lines. The carrier was designed to have the structure of (KHKHKHKHKK)6-FGF2 where lysine (K) residues would allow complexation with plasmid DNA, basic fibroblast growth factor (FGF2) to target cells over-expressing FGF2 receptors (FGFR), and histidine (H) residues to facilitate escape from the endosomal compartments.


METHODS
The gene carrier was biosynthesized in E. coli, purified using a Ni-NTA column, characterized, complexed with pDNA, and the complexes were used to transfect NIH 3T3, T-47D and COS-1 mammalian cell types known to express FGFR.


RESULTS
Results demonstrate the successful cloning and expression of the gene carrier with over 95% purity. The molecular weight of the gene carrier was determined by MALDI-TOF to be 27 402. Amino acid content analysis and Western blot confirmed the expression of the gene carrier in E. coli. The vector was able to condense pDNA, induce cell proliferation in NIH 3T3 fibroblasts, and mediate transgene expression in NIH 3T3, T-47D and COS-1 mammalian cell types.


CONCLUSIONS
Genetic engineering techniques show promise for systematic investigation of structure-activity relationships of non-viral gene delivery vectors.
Hatefi
A
A
Department of Pharmaceutical Sciences and Center for Nanomedicine and Cellular Delivery, University of Maryland, Baltimore, Baltimore, MD 21201-1075, USA.
Megeed
Z
Z
Ghandehari
H
H
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D049109:Cell Proliferation; D002522:Chlorocebus aethiops; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011108:Polymers / Q000737:chemistry*; D013329:Structure-Activity Relationship
D011108:Polymers; D016222:Fibroblast Growth Factor 2; D004247:DNA