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Non-immunostimulatory nonviral vectors

FENG LIU^*,1, LISA M SHOLLENBERGER and LEAF HUANG^*,1

^* The Center for Pharmacogenetics, School of Pharmacy, and
School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

¹Correspondence: The center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA. E-mail: fliu@pitt.edu or leafh{at}pitt.edu

SPECIFIC AIMS

Proinflammatory cytokines induced by nonviral vectors have limited their applications in gene therapy. The specific aim of this study is to develop a non-immunostimulatory vector, which has been named "safeplex." The safeplex could efficiently deliver a gene to the target cells and inhibit the cytokines.

PRINCIPAL FINDINGS

1. Inhibition of cytokines by the safeplex
Tumor necrosis factor (TNF-) is one of the major cytokines induced by CpG motif DNA. It has been shown that intraperitoneal injection of dexamethasone (DEX) suppressed cationic lipid vector-mediated TNF- induction. Therefore, TNF- was used as an index and DEX was selected as the suppressor to evaluate the effect of the safeplex on inhibiting cytokines. It is known that high levels of gene transfer into the lung requires a high charge ratio of cationic lipid to DNA, the higher the charge ratio the higher efficiency of gene transfer. However, data in Fig. 1 a revealed that an increase in the charge ratio resulted in increased TNF- production. The data showed that injection of plasmid DNA alone did not induce TNF-, and a low level of TNF- was observed after injection of lipoplexes or safeplexes with a low charge ratio (±0.5). TNF- levels in blood climbed as high as 2000 pg/mL when the charge ratio of the lipoplexes was increased to 12:1. However, when plasmid DNA was delivered by the safeplexes, the levels of TNF- were significantly decreased, even at the highest charge ratio of 12:1 (P<0.01). It is known that the major organs involved in the production of cytokines are the spleen, followed by the liver and lung. Theoretically, inflammatory suppressors delivered by the safeplex should accomplish their desired functions in these organs. The data in Fig. 1b confirmed that TNF- levels in these organs were greatly decreased by the safeplex. In addition, the levels of other cytokines, IL-12 and IFN-, decreased significantly (Fig. 1c ). These data suggest that the safeplex can serve as a non-immunostimulatory gene vector to inhibit the inflammatory toxicity induced by CpG motif pDNA.

View larger version (15K): [in this window] [in a new window]   Figure 1. Comparison of TNF- levels in blood, organs (a, b) and IL-12 and IFN- (c) after injection of the lipoplexes and safeplexes. The charge ratio of DOTAP to DNA (±) varied from 0 to 12 (a) and was fixed at 6 to 1 (b, c). The levels of TNF- were detected 2 or 6 h after injection (c) of the lipoplexes and safeplexes containing 25 µg pDNA. **P <0.001 (3 mice in each group).

2. Effect of other candidate suppressors on inhibition of cytokines
Glucocorticoids—for example, DEX and prednisone—are widely used for their anti-inflammatory and immunosuppressive properties, which antagonize the activation of the NF-B pathway by direct and indirect mechanisms. Other pharmacologic agents have been described to inhibit NF-B activity. These agents include nonsteroidal anti-inflammatory drugs (NSAIDs) and natural compounds. The following experiment examined whether the safeplex can carry, in addition to DEX, other inflammatory inhibitors such as prednisone (another glucocorticoid drug), indomethacin (NSAID), tetrandrine (herbal medicine), and gliotoxin (fungal metabolite). Tetrandrine is a natural compound extracted from a Chinese herbal remedy known as Hanfngji. Tetrandrine has been shown to inhibit NF-B activation by suppressing signal-induced degradation of IB (a cytoplasmic inhibitor of NF-B transcription factor). Gliotoxin, used as an inhibitor of NF-B, exhibited profound immunosuppressive activity in vivo. Previous study showed that sublethally irradiated mice with an intraperitoneal injection of gliotoxin showed a significant delay in the recovery of immune cells. As shown in Fig. 2 , all of the above inflammatory inhibitors showed an inhibitory effect on TNF- production when they were carried by the safeplexes with a ratio of 1/6 (inhibitor/DOTAP, mol/mol). However, the NSAID and natural suppressors did not inhibit TNF- production as efficiently as DEX.

View larger version (13K): [in this window] [in a new window]   Figure 2. Inhibition of TNF- production by other suppressors. Levels of TNF- in the blood were analyzed 2 h after the injection of lipoplexes and safeplexes with a charge ratio of DNA to DOTAP of 1/6 (mol/mol). **P <0.001 (n=3).

CONCLUSIONS AND SIGNIFICANCE

In this study, we designed a new nonviral vector, which is non-immunostimulatory, to decrease the inflammatory toxicity. The rationale for designing this vector, as shown in Fig. 3 , is encapsulation of an inflammatory suppressor into the cationic liposome and formation of a new type of complex (cationic liposome/inflammatory suppressor/DNA). The DNA and suppressor will be codelivered to an individual immune cell where the cytokines are produced. The inflammatory suppressor will be released from the vector in the cytoplasm and interrupt the cytokine production by inhibiting NF-B at one or multiple activation steps of the signaling pathway. We named the non-immunostimulatory vector safeplex. We reported for the first time the development of the nonimmunostimulatory nonviral vector for systemic delivery of genes.

View larger version (35K): [in this window] [in a new window]   Figure 3. Schematic depiction summarizing the function of inflammatory suppressors that interrupt the cytokine production by inhibiting NF-B at one or multiple activation steps of the signaling pathway. Modified from ref 16 of full-text article.

An inflammatory response is invariably associated with administration of gene transfer complexes (lipoplexes) composed of cationic lipids and plasmid DNA (pDNA). In addition to causing inflammation, the unmethylated CpG in pDNA has been shown to induce apoptosis of lung endothelia cells when delivered systemically. Another concern is the potential for augmenting an antibody response to the transgene product or to the pDNA itself. The T help-1 response to the immunostimulatory CpG motifs makes pDNA an excellent adjuvant for immunization to a variety of protein antigens. This property is useful and indeed has been exploited for genetic vaccines, but is deleterious for the treatment of genetic disorders that require repeated delivery of lipoplexes. Bacterial DNA has also been shown to be capable of inducing the production of anti-DNA antibodies in mice. This finding suggests the possibility of developing an autoimmune response in patients as a result of treatment. Such adverse responses are clearly undesirable and development of strategies to abate this inflammatory response is warranted. Therefore, our goal in this study is to develop a non-immunostimulatory vector to increase the safety profile and hence the therapeutic index of nonviral vectors for gene therapy.

In summary, our data demonstrate that many inflammatory suppressors were successfully delivered by the safeplex, such as glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), NF-B inhibitor, and a natural compound from an herbal medicine. As a result, the gene vector of safeplex dramatically decreased the proinflammatory cytokines induced by plasmid DNA. The vector has a great potential for gene therapy in clinical settings. Acting at the transcriptional and translational levels, potential enhancers can be codelivered into target cells to improve the efficiency of gene transfer by the nonviral vector. Moreover, the unique features of this vector, simultaneously inducing the inflammation and carrying inflammatory suppressors into the same type of cells, will provide an easy and powerful tool to screen anti-inflammatory drugs in vivo. Using this vector, a candidate drug from synthesis or herbal medicines can be tested quickly to reveal the anti-inflammatory effect.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2187fje;

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