In vitro and in vivo delivery of genes and proteins using the bacteriophage T4 DNA packaging machine

Abstract

The bacteriophage T4 DNA packaging machine consists of a molecular motor assembled at the portal vertex of an icosahedral head. The ATP-powered motor packages the 56-µm-long, 170-kb viral genome into 120 nm × 86 nm head to near crystalline density. We engineered this machine to deliver genes and proteins into mammalian cells. DNA molecules were translocated into emptied phage head and its outer surface was decorated with proteins fused to outer capsid proteins, highly antigenic outer capsid protein (Hoc) and small outer capsid protein (Soc). T4 nanoparticles carrying reporter genes, vaccine candidates, functional enzymes, and targeting ligands were efficiently delivered into cells or targeted to antigen-presenting dendritic cells, and the delivered genes were abundantly expressed in vitro and in vivo. Mice delivered with a single dose of F1-V plague vaccine containing both gene and protein in the T4 head elicited robust antibody and cellular immune responses. This "progene delivery" approach might lead to new types of vaccines and genetic therapies.

Fig. 1.

The bacteriophage T4 DNA packaging machine. Shown are various structural features of the T4 DNA packaging machine that are used in the design of progene delivery vehicle. (A) Structural model of the phage T4 DNA packaging machine showing the pentameric motor (cyan) assembled at the dodecameric portal vertex (red) of the head that can accommodate ∼170 kb DNA. (B) Ribbon models of portal and motor. (C) Capsomer showing the arrangement of the major capsid protein gp23* (green), Soc trimers (blue), and Hoc fiber (yellow). (D) Capsomer showing the ribbon models of gp23*, Soc, and Hoc. (E) Soc trimer showing the exposed N (green dots) and C (red dots) termini. (F) Hoc showing the exposed N terminus (green dot) at the tip of the fiber. The C-terminal Hoc domain 4 binds to capsid, but its structure has not yet been solved, hence shown as a yellow mass at the base of the fiber.

Fig. 2.

Experimental design for progene delivery. The bacteriophage T4 head is engineered to deliver genes and proteins into mammalian cells. The DNA packaging machine is assembled by binding of gp17 motor to the portal of empty Hoc⁻ Soc⁻ phage T4 head (the cut-out of the head shows both the exterior and the interior) (A). Using the energy from ATP hydrolysis the motor packages DNA molecules into the head (B). Soc-fused proteins (C) and Hoc-fused CPPs or targeting molecules (D) were displayed on the heads. The head particles bind to cells either nonspecifically (general delivery) (E) or through a host receptor (targeted delivery) (F and G) and are internalized (H). The surface displayed protein (e.g., β-galactosidase) (I) and encapsidated DNA (e.g., luciferase and GFP genes) (J) are released into the cytosol. The DNA enters the nucleus (K), priming transcription (L), and expression of luciferase and GFP proteins (M).

Fig. 3.

T4 heads efficiently deliver packaged DNA into mammalian cells. (A) Cryo electron micrograph of purified T4 heads. (B) Packaging of MluI-linearized pEGFP-C1 plasmid DNA (Upper) and BamHI-linearized psiCHECK2 plasmid DNA (Lower) into Hoc⁻ Soc⁻ T4 heads at increasing DNA-to-head ratios. The black arrow shows the packaged DNA. (C) Packaging of two plasmids (lane 1; psiCHECK2 plasmid DNA containing luciferase gene under the control of SV40 early promoter, blue arrow; pEGFP-C1 plasmid DNA containing eGFP gene under the control of the cytomegalovirus (CMV) promoter (green arrow), concatemerized luciferase DNA (lane 2, black arrow), and PCR-amplified luciferase expression cassette (lane 3, orange arrow). The red arrows show the 8-kb T4 DNA present in the heads. (D) In vitro assembly of CPPs on T4 heads. The red arrow shows bound Soc-T and Soc-P, and the blue arrow shows bound Hoc-T and Hoc-P; control lanes show purified heads. (E) Dose-dependent delivery of luciferase DNA into mammalian cells using CPP-decorated T4 heads. (F) Luciferase DNA delivery increases with increasing copy number of CPP. (G) Hoc-CPP decorated heads deliver genes more efficiently than Soc-CPP heads. (H) Gene delivery was at its highest when the heads were decorated with both Hoc-T and Soc-T CPPs. (I) eGFP DNA packaged heads decorated with (IV) or without (I) Hoc-CPPs are used for delivery. Fluorescence (III and VI) and phase contrast (II and V) micrographs of HEK293T cells in the presence (V and VI) or absence (II and III) of CPP. (J) Expression of packaged and delivered luciferase gene as plasmid, concatemer, or PCR product. (K) Comparison of the delivery efficiencies of T4, AAV-DJ, and lipofectamine. (In this experiment, due to the low capacity of AAV, the shorter pLuci plasmid was used instead of the psiCHECK2). Error bars represent SD; **P < 0.01; ***P < 0.001.

Fig. 4.

Targeted delivery of genes and proteins into DCs by T4 heads. (A) Display of DEC205mAb on T4 heads through GG domain. Hoc-GG, Soc-GG, and DEC205mAb bands are marked with green, blue, and red arrows, respectively. The top red arrow corresponds to heavy chain, and the bottom red arrow corresponds to light chain of IgG. (B) Targeted delivery of luciferase gene into DC2.4 cells but not into control Hep3B cells. (C) Fluorescence (II and IV) and phase contrast (I and III) micrographs of DC2.4 cells transduced with GFP heads in the presence (III and IV) or absence (I and II) of displayed DEC205mAb. (D) Display of tetrameric β-galactosidase on T4 heads at different ratios of β-galactosidase-Soc molecules to Soc binding sites. Red arrow shows bound β-galactosidase-Soc, and the 40:0 lane is the control lane showing no nonspecific binding of β-galactosidase-Soc in the absence of heads. (E) X-Gal cleavage activity of displayed β-galactosidase corresponding to lanes in D. (F and G) T4 heads displayed with β-galactosidase (blue) and CPP (I; red) (F) or DEC205mAb (V; yellow) (G) delivered functional β-galactosidase into DC2.4 cells. (H and I) T4 heads packaged inside with luciferase and GFP DNA and displayed outside with β-galactosidase (blue) and CPP (H; red) or DEC205mAb (I; yellow) are used for delivery. II and III show phase contrast and fluorescence micrographs, respectively, of DC2.4 cells transduced with control packaged heads containing no CPP or DEC205mAb; IV, stained for β-galactosidase activity with X-Gal; V, fluorescence of eGFP after staining with FITC-labeled antibody; VI, fluorescence of luciferase after staining with Rhodamine-labeled antibody; VII, merged fluorescence images of eGFP (V) and luciferase (VI).

Fig. 5.

In vivo delivery by T4. (A) Mice were injected intramuscularly with T4 heads packaged with pLuci plasmid and its outer surface decorated either with no ligand (panels 1 and 2) or with DEC205mAb (panels 3 and 4), CD40L (panels 5 and 6), or CPP (panels 7 and 8). (B) Luciferase expression cassettes used, without (pLuci) and with ITRs from AAV (pITR-luci), for packaging into T4 heads; in both the vectors, the luciferase gene is under the control of the CMV promoter. (C) Luciferase signal stays for a longer period if the expression cassette is flanked by ITRs. (D) Further analysis of targeted delivery into DCs. (1) The same amount of pITR-Luciferase was packaged into T4 heads decorated either with no ligand (lane I) or with CD40L (lane II); lane “M” shows molecular size standards. (2) Strong luciferase signal was seen at the site of injection with T4 heads containing no displayed ligand. (3) No signal was seen at the site of injection with T4 heads displayed with the DC-specific ligand, CD40L. (E) ELISA titration showed that both the no ligand group and the CD40L group induced the same level of anti-luciferase antibodies. y axis represents absorbance at 650 nm.

Fig. 6.

A single dose of T4-delivered plague vaccine induced robust humoral and cellular immune responses. (A) Five groups of mice were vaccinated by the intramuscular route (i.m.) with various formulations shown in the table. A mutant F1 gene from Y. pestis was fused to the N terminus of V gene and the F1-V fusion was then fused to the N terminus of Soc gene. The F1-V-Soc fusion protein was purified as a 66 kDa monomer. For the DNA group (#4), F1-V was cloned under the control of the CMV promoter. Spleens were harvested from three mice from each group at day 21 for Elispot assays, and sera were collected at day 28 for ELISAs. The ELISA (B) and Elispot (C) assays were performed according to the procedures described in Materials and Methods. SFC refers to spot-forming cells expressing IFN-γ. Error bars represent SD; **P < 0.01; ***P < 0.001.