Generating Antibodies by Genetic Immunization
Intravenous Gene Transfer

Hydrodynamic Tail Vein Delivery & Pathway IV ™ Delivery to Skeletal Muscle

Mirus has developed proprietary methods for bloodstream-mediated delivery of nucleic acids to target tissues and organs outside the blood stream. The most widely used methods are based upon the principle of transiently increasing blood vessel permeability. In rodents, a preferred protocol involves the rapid injection into a tail vein of a sufficient volume of nucleic acid solution to elevate the intravascular pressure and enhance the vessel’s permeability. This protocol has been commonly referred to as “hydrodynamic” tail vein (HTV) delivery. It results in particularly high gene transfer to the liver (10-40% of all hepatocytes). This technique has been described in the literature and is increasingly being used for gene therapy and other biological studies. The true utility of this methodology lies in its inherent simplicity and low toxicity. The procedure takes only minutes to perform and 100% of the animals survive. The side-effects from the procedure are few, transient and mild. As a result, the Mirus invention has become the method-of-choice for in vivo gene delivery to the liver.

We have compared our delivery techniques with the classic method for GI, the direct injection of pDNA into skeletal muscle (Fig. 1). The results clearly demonstrate that hydrodynamic delivery of naked pDNA resulted in much higher titers and more rapid induction of anti-luciferase antibodies than conventional injection into skeletal muscle.

Figure 1. Anti-luciferase antibody titers in mice genetically immunized with 10µg pCMV-luc+ (vector expressing luciferase under control of the human cytomegalovirus promoter) using different immunization routes (black squares, hydrodynamic delivery of naked pDNA; red circles, intramuscular delivery of naked DNA). Mice were primed on day 0 and boosted on days 14, 21,28 and 35. Sera were assayed by ELISA, and compared to a commercially available anti-luciferase antibody. Data points are the averaged values of 5 mice in each group.

Selection of the promoter driving antigen expression impacts the immune response. In many genetic immunization experiments, plasmid DNA vectors with the CMV promoter are employed. It is well known that CMV-driven expression in the liver reaches maximum levels between 10 and 20 hours after delivery, but diminishes very rapidly afterwards. Therefore, a single delivery of a CMV vector does not result in a very robust antibody response; high antibody titers are only obtained after 1 or more boosts (Fig. 2). By using expression vectors that result in sustained expression, e.g., ubiquitin C, high antibody titers can be induced by a single tail vein delivery (Fig. 2). Boosting further increases titers, yet its main advantage is the shorter amount of time that is required to reach very high specific antibody titers. This time span is shorter than is observed with other genetic immunization methods.

Figure 2. Anti-luciferase antibody titers in genetically immunized mice. The mice were immunized with 10 µg plasmid DNA vectors expressing luciferase under transcriptional control of either the CMV or the ubiquitin C promoter. Some groups of mice were boosted with the same pDNA on days 14, 21 and 28. Blood samples were assayed for anti-luciferase antibody analysis as described above.

Hydrodynamic Intravenous Gene Delivery to Muscle

Nucleic acids can also be delivered very efficiently to limb skeletal muscle following intravenous injection. With blood flow in a limb temporarily occluded by a tourniquet, a pDNA solution is rapidly injected IV. This elevates the pressure within the occlusion zone, transiently disrupting the endothelial cell wall and allowing the pDNA to migrate into the adjoining muscle cells. Blood flow is then restored to normal within a few minutes, with no adverse affects to the vasculature or muscle tissue. This hydrodynamic limb vein (HLV) protocol, trademarked Pathway IV ™, is simple, effective, and can be used in small rodents (mice, rats, guinea pig), rabbits and larger animals (goats, primates), as well as in humans. Figure 3 (left panel) shows a comparison of antibody titers obtained by Mirus’ two intravenous gene delivery routes in mice: hydrodynamic tail vein delivery to liver and saphenous vein delivery to hind limb skeletal muscle. From this experiment it is clear that the kinetics and levels of antibody induction for the two delivery routes are very similar. The right panel in Fig. 3 demonstrates antibody titers obtained after intravenous gene transfer via the saphenous vein in rabbits. The rabbit data indicate that high levels of antigen-specific antibodies can be induced in this much larger species.

Figure 3. Left panel – mice were immunized with 10 µg pDNA (UbC- luc+ expression vector) on days 0, 14 and 21. DNA was delivered via the tail vein (HTV delivery) or the saphenous vein (HLV delivery). Anti-luciferase antibodies were measured at different time points after genetic immunization (the average value of 5 animals in each group is plotted). Right panel – two New Zealand White rabbits were immunized via the saphenous vein on days 0, 14, 21, and 28. Anti-luciferase antibody titers for each individual rabbit are shown.

Generation of monoclonal antibodies against post-translationally modified antigens

Genetic immunization is eminently suited to generate antibodies against post-translationally modified antigens. Figure 4 shows the results from a representative project using Ki67, a nuclear protein expressed exclusively in proliferating cells. Its localization within the interphase nucleus and on condensed mitotic chromosomes during mitosis is regulated by cell cycle-specific kinases and phosphatases. Figure 4A shows staining of HeLa cells with a commercially available Ki-67 monoclonal antibody (mAb). Panels B–F show results using five of the mAbs generated through the HTV genetic immunization method. The differences in the staining patterns are due to the antibodies reacting with specific phosphorylation-dependent Ki-67 epitopes present only at certain stages of the cell cycle. For example, the mAb shown in panel D stains the perinucleolar region in interphase cells, but cannot recognize Ki-67 when it is hyperphosphorylated during mitosis (lack of staining on the condensed mitotic chromosomes). The mAb shown in panel E reacts strongly with the mitotic form of the protein and stains the nucleolar region of interphase cells very weakly. In contrast, the mAb shown in panel C yields very intense staining with all forms of Ki-67. There were several other mAbs that were able to distinguish even the various stages of mitosis, staining the chromosomes in pro- and metaphase, but not in anaphase or telophase and vice versa (data not shown). Immunization with bacterially produced, non-phosphorylated recombinant proteins would not have yielded such an extensive palette of mAbs that enables the study of the dynamic changes of this protein during the cell cycle. The project was successful in numerical terms, as well: of 960 wells of fused hybridomas, 164 passed the initial ELISA screen for reactivity with the expressed Ki-67 fragment. In a more stringent screen based on immunocytochemistry and Western blotting, 46 of these positive hybridoma lines gave characteristic Ki-67 staining patterns. None of these 46 mAbs cross-reacted with the mouse or hamster Ki-67 orthologs (44% identity to the human protein), only two cross-reacted with the rat ortholog (45% identity), while almost all could recognize the monkey ortholog (determined by immunohistochemistry).

Figure 4. Immunocytochemical detection of Ki-67 with monoclonal antibodies generated after genetic immunization by hydrodynamic tail vein delivery of plasmid DNA. Mice were genetically immunized by HTV delivery of pCI-Ki67 on day 0, 14, 21, 28 and 35. The mice were boosted again on day 104, sacrificed on day 108 and splenocytes obtained and fused with myeloma cells. Supernatants of 164 of 960 wells containing fused cells were reactive with the expressed Ki-67 antigen. Of these, 46 showed immunocytochemistry staining anticipated for Ki-67 on HeLa cells. Five different monoclonal antibodies are shown in panels B-F and compared to a commercially available anti-hKi67 mAb used at a concentration of 1 µg/ml (panel A). All supernatants were diluted 1:5 and detected with Cy3-labeled anti-mouse IgG (red). Cells were counterstained with TO-PRO®-3 DNA stain (blue) and Alexa488-Phalloidin actin stain (yellow). Each panel shows an area of 97.5 x 97.5 µm.

Summary

In summary, Mirus’ delivery technology is a robust platform for the induction of antibodies in animals. Its key advantages over alternative delivery modalities include:

• Higher antibody titers can be elicited than with IM pDNA injection
  • Up to 100 fold greater immune response can be generated in the same time span
• Antibody induction is more rapid than with IM injection
• Antibodies can be raised against immunogens whether expressed intracellularly, membrane bound or secreted
• Repeat or boost immunizations can further elevate humoral response
• Protocols are simple to use
  • No special equipment need be purchased or maintained
• Procedure is well tolerated
• Effective in rabbits and even larger animals

Bibliography for Mirus’ Nucleic Acid Delivery Procedures

Genetic Immunization using Hydrodynamic Plasmid DNA Delivery:

Bates MK, Zhang G, Sebestyén MG, Neal ZC, Wolff JA and Herweijer H. 2006. Genetic immunization for antibody generation in research animals by intravenous delivery of plasmid DNA. BioTechniques 40:199-208.

Tail Vein Procedure in Rodents:

Liu F, Song Y, Liu D. 1999. Hydrodynamics-based Transfection in Animals by Systemic Administration of Plasmid DNA. Gene Therapy 6:1258-1266.

Maruyama H, et al. 2004. Rat liver-targeted naked plasmid DNA transfer by tail vein injection. Mol Biotechnol. 26:165-72.

Zhang G, Budker V and Wolff JA. 1999. High Levels of Foreign Gene Expression in Hepatocytes after Tail Vein Injections of Naked Plasmid DNA. Human Gene Therapy 10:1735-1737.

Alternative Hydrodynamic Procedures in Larger Animals:

Eastman SJ, et al. 2002. Development of catheter-based procedures for transducing the isolated rabbit liver with plasmid DNA. Human Gene Therapy. 13:2065-77.

Zhang, G., D. Vargo, et al. (1997). Expression of naked plasmid DNA injected into the afferent and efferent vessels of rodent and dog livers. Human Gene Therapy 8:1763-72.

Intravenous Gene Delivery to skeletal muscle:

Hagstrom et al., 2004. A Facile Non-Viral Method for Delivering Genes and siRNAs to Skeletal Muscle of Mammalian Limbs. Mol. Ther. 10:386-392..

Toumi H, Hegge J, Subbotin V, Noble M, Herweijer H, Best TM and Hagstrom JE. 2006. Rapid intravascular injection into limb skeletal muscle: a damage assessment study. Mol. Ther. 13:229-236.

Expression of Secreted and Other Proteins in Animal Models:

Alino SF, Crespo A, Dasi F.2003.Long-term therapeutic levels of human alpha-1 antitrypsin in plasma after hydrodynamic injection of nonviral DNA. Gene Ther.10:1672-9.

Dagnaes-Hansen F,Holst HU, Sondergaard M, Vorup-Jensen T, Flyvbjerg A, Jensen UB, Jensen TG. 2002.Physiological effects of human growth hormone produced after hydrodynamic gene transfer of a plasmid vector containing the human ubiquitin promotor. J Mol Med. 80:665-70.

Dai C, Yang J, Liu Y. 2002. Single injection of naked plasmid encoding hepatocyte growth factor prevents cell death and ameliorates acute renal failure in mice. J Am Soc Nephrol. 13:411-22.

Danko I et al. 2004. Nonviral gene transfer into liver and muscle for treatment of hyperbilirubinemia in the gunn rat. Hum Gene Ther 15:1279-1286.

Hodges BL and Scheule RK 2003. Hydrodynamic delivery of DNA. Expert Opin Biol Ther 3:911-918.

Jiang J, et al. 2001. Intravenous delivery of naked plasmid DNA for in vivo cytokine expression. Biochem Biophys Res Commun. 289:1088-92.

Knapp JE and Liu D 2004. Hydrodynamic delivery of DNA. Methods Mol Biol 245:245-250.

Lui VW, et al. 2001. Systemic production of IL-12 by naked DNA mediated gene transfer: toxicity and attenuation of transgene expression in vivo. J Gene Med. 3:384-93.

Maruyama H et al. 2004. Rat liver-targeted naked plasmid DNA transfer by tail vein injection. Mol Biotechnol 26:165-172.

Miao CH, et al. 2001. Long-term and therapeutic-level hepatic gene expression of human factor IX after naked plasmid transfer in vivo. Mol Ther. 3:947-57.

Miao CH 2005. A novel gene expression system: non-viral gene transfer for hemophilia as model systems. Adv Genet 54:143-177.

Ortaldo JR. 2005. In vivo hydrodynamic delivery of cDNA encoding IL-2: rapid, sustained redistribution, activation of mouse NK cells, and therapeutic potential in the absence of NKT cells. J Immunol 175:693-699.

Smyth MJ et al. 2005. Sequential activation of NKT cells and NK cells provides effective innate immunotherapy of cancer. J Exp Med 201:1973-1985.

Zhang X et al. 2004. Regional hydrodynamic gene delivery to the rat liver with physiological volumes of DNA solution. J Gene Med 6:693-703.