Particle assembly incorporating a VP22–BH3 fusion protein, facilitating intracellular delivery, regulated release, and apoptosis  N.D. Brewis, A. Phelan, N. Normand, E. Choolun, P. O'Hare  Molecular Therapy  Volume 7, Issue 2, Pages 262-270 (February 2003) DOI: 10.1016/S1525-0016(02)00054-0 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Expression and purification of VP22.C1, BH3-22, and BH3m-22. (A) Schematic of the BH3-22 expression construct. A fusion protein comprising the BH3 domain of Bak (B) and amino acids 159–301 of VP22 was expressed under the control of a T7 promoter. (B) Primary sequence of the BH3 domain of human Bak. The mutation BH3A78, termed BH3m, has a single amino acid substitution (L to A) that abolishes the proapoptic activity of Bak BH3. (C) The proteins were purified from the bacterial extracts using Ni–NTA affinity chromatography as described under Materials and Methods. The eluates (10 μg protein per lane) were analyzed on a 10–20% gradient SDS–PAGE gel (Invitrogen, Carlsbad, CA, USA) stained with Coomassie brilliant blue R-250. Lanes: 1, molecular weight standards; 2, VP22.C1; 3, BH3-22; 4, BH3m-22. Molecular Therapy 2003 7, 262-270DOI: (10.1016/S1525-0016(02)00054-0) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Uptake into cells and light-induced redistribution of vectosome complexes formed with BH3-22 protein and fluorescent ODN. Cos-1 cells were incubated with vectosomes prepared using VP22.C1 (a, b), BH3-22 (c, d), and BH3m-22 (e, f) protein (as described under Materials and Methods). Free ODN alone (g, h) was used as a control. The cells were washed with PBS and live cells then examined using confocal microscopy before (a,c,e,g) and 2 min after (b, d, f, h) epifluorescence illumination (10 s). After light activation particles were observed to disrupt, resulting in rapid release of the F-ODN into the cytoplasm and nucleus. A video (Vectosome.mov) showing light-induced disruption of BH3-22 vectosomes can be downloaded and viewed with the article on ScienceDirect (www.sciencedirect.com). Molecular Therapy 2003 7, 262-270DOI: (10.1016/S1525-0016(02)00054-0) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Light-triggered release of BH3-22 vectosomes induces cell death. (A) Cos-1 cells were incubated overnight with vectosomes formed using VP22 (a, b), BH3-22 (c, d), or BH3m-22 (e, f). The medium was changed as for Fig. 2, and the cells in b, d, and e were illuminated by epifluorescence illumination at low magnification (3 min). Fresh medium was added and the cells were assayed for cell viability using a fluorescence-based live/dead assay (Molecular Probes). Images were taken with a ×10 objective. As a control, cells were incubated with a synthetic BH3 peptide at 0.9 μM (g). Cytotoxicity was observed only for the BH3-22 particles and only after light-induced disruption. (B) Localized activation of BH3-22 vectosomes. Cos-1 cells were incubated with BH3-22 vectosomes as above. Areas of the monolayer were illuminated for 10 s using a high-magnification objective creating a series of three circles (outlined). Fresh medium was then added and the cells were assayed after 24 h incubation using the live/dead assay and viewed with a ×10 objective. Dead cells were observed almost exclusively in the area subject to illumination and not in the remainder of the monolayer. Molecular Therapy 2003 7, 262-270DOI: (10.1016/S1525-0016(02)00054-0) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Light-activated BH3-22 vectosomes induce apoptosis. Cos-1 cells were incubated overnight with BH3-22 vectosomes (c, d, e, f) as described above. Control monolayers received a similar amount of PBS buffer (a, b). Cell in e and f were then illuminated in suspension (2 min) while cells in a, b, c, and d were processed in parallel but with no illumination. The cells were replated with fresh medium for 16 h before analysis using flow cytometry for DNA content using propidium iodide stain (a, c, e) or for cell surface phosphatidylserine using a GFP–annexin V stain (b, d, f). Annexin V fluorescence is displayed on a logarithmic scale. Cells containing the BH3-22 vectosomes and subject to light activation showed significant amounts of sub-G1 DNA and surface-exposed annexin V staining. Molecular Therapy 2003 7, 262-270DOI: (10.1016/S1525-0016(02)00054-0) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Time course of BH3-22 vectosome-induced cell death. (A) Cos-1 cells and (B) skeletal muscle cells were incubated overnight with VP22 vectosomes or BH3-22 vectosome as indicated. The medium was replaced with PBS and an area of cells was subjected to epifluorescence illumination. The cells were then incubated in fresh medium and assayed for viability at different times after light activation using the live/dead stain as above. Cytotoxicity could be observed within 4 h after vectosome redistribution. Molecular Therapy 2003 7, 262-270DOI: (10.1016/S1525-0016(02)00054-0) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 6 The effect of light-activated BH3-22 vectosomes on CT26 cells. (A) CT26 cells were incubated overnight with VP22 (a, b) or BH3-22 (c, d) vectosomes. Cells were illuminated (3 min, b and d) and assayed (24 h later) using the live/dead stain as described above. (B) CT26 cells were incubated overnight with VP22 or BH3-22 vectosomes, detached, and counted and 5×104 were injected subcutaneously into each of 10 Balb/C mice for each test sample. After 24 h, the injected area was illuminated using a fiber optic cold light source (as described under Materials and Methods). Tumor progression, plotted as average tumor size was monitored over 40 days. Molecular Therapy 2003 7, 262-270DOI: (10.1016/S1525-0016(02)00054-0) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions