Introduction: In an effort to develop a biomimetic vector for efficient and targeted gene transfer, we have engineered a multi-domain biomacromolecule. Using a novel technique, namely Function-based Combinatorial Vector Engineering, we have screened and identified the structure of a biomacromolecule where the functional propensity is scripted into the primary sequence structure to perform an array of self-guided functions. These include: a) efficient condensation of the plasmid DNA into deliverable nanoparticles by adenovirus mu peptide, b) delivery of the nanoparticles to model breast cancer cells using a combinatorially screened cyclic targeting peptide (TP) [1], c) endosomal disruption using a synthetic fusogenic peptide (FP) facilitating escape of the cargo into cytosol, and finally d) localization of the gene in the nucleus by HIV nuclear localization signal (NLS). By shuffling the order of the functional domains, a library of the DNA transporters was genetically engineered to screen the active candidate resulting in the optimum sequence shown in (Figure 1). Methods: Cloning and expression of the vectors: The genes encoding various motifs were synthesized and cloned into a pET21b expression vector. The expression vector was transformed into E. coli BL21(DE3) and induced with IPTG. The expressed vectors were purified to homogeneity on a Ni-NTA column. Transmission Electron Microscopy: The vector/pDNA nano-complexes were prepared, stained with uranyl acetate for 5 min, and visualized. Particle characterization: Vector/pDNA complexes were prepared at different N/P ratios by adding various amounts of the vector into 1 µg of pDNA (pEGFP). The mean hydrodynamic size of the complexes was measured by Photon Correlation Spectroscopy (PCS). Results are reported as Mean ± SEM. Hemolysis assay: Thoroughly washed sheep red blood cells were reconstituted to 3.2 x 108 cells/ml in phosphate buffered saline pH 7.4, 7.0 and 5.0 supplemented with various amounts of the vector. The lysis mixture was incubated at 37°C for 1h, centrifuged and the supernatant was read at 541 nm. Triton X-100 was used as positive control, whereas buffer only used as negative control. Cell culture and transfection: ZR-75-1 breast cancer and MCF-10A normal human mammary cells were seeded in 96-well tissue culture plates. Cells were approximately 70% confluent at the time of transfection. pEGFP was mixed with various ratios of the vector to form complexes followed by addition to the growth media. When used, chloroquine (100µM), bafilomycin (10nM), Nocodazole (10µM) were added to assist the particles escape into cytoplasm. The expression of GFP and the percent transfected cells was determined by flowcytometry. Inhibition assay: Cells were seeded at a density of 5 x 104 cells per well in a 96 well plate. Aliquots of targeting peptide in serum free media were added at concentrations of 0, 0.07, 0.7, 1.4, 2.8, and 5.5 nM to the wells. Aliquots of vector in complex with 1 µg pEGFP at N:P 10 were added to each of the wells. The control well with 0 µg/ml concentration received PBS. The measured percent transfected cells for the test groups (pre-treated with targeting peptide) are expressed as percent of the control defined as 100%. The data are shown as mean ± s.d., n=3. Results: As a result of the Function-Based Combinatorial Vector Engineering technique, the structure of a single chain biomacromolecule was identified that was able to: a) condense plasmid DNA into spherical and compact nanoparticles with 60 ± 5nm size, b) lyse the red blood cells efficiently at pH values less than 7.0, c) target ZR-75-1 breast cancer cells specifically with no significant effect on human normal mammary cells, d) disrupt endosome membranes efficiently, e) utilize microtubules to translocate the genetic material towards the nucleus, and f) mediate efficient gene transfer. Conclusion: This study demonstrates that by using Function-based Combinatorial Vector Engineering a multifunctional biomacromolecule can be engineered that is able to mimic virus characteristics and efficiently transfer genetic materials to the target cells. References: [1] Dane, K. Y. et al., J Immunol Methods 2006, 309, 120-9. Acknowledgements This work was funded by the Department of Defense Breast Cancer Concept Award W81XWH-06-BCRP-CA (BC062929).

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