Minding the Cs and Gs
Authors: Arthur M. Krieg
Journal: Molecular Therapy (2000)

Successful gene therapy requires the introduction of foreign DNA into host cells. Until recently, most investigators working in this field did not realize that the DNA sequences themselves contained immune stimulatory sequences, called CpG motifs, that activate immune responses as an unintended consequence of gene delivery. DNA containing CpG motifs activates both the innate and the acquired arms of immune responses (Fig. 1). CpG-induced innate immunity is exemplified by the rapid activation of NK cells, macrophages, and dendritic cells, with the concomitant production of proinflammatory cytokines. This response is a cause of the acute toxicity that has been observed in some gene therapy protocols (1Yew N.S. Zhao H. Wu I.-H. Song A. Tousingnant J.D. Przybylska M. Cheng S.H. Reduced inflammatory response to plasmid DNA vectors by elimination and inhibition of immunostimulatory CpG motifs.Mol. Ther. 2000; 1: 255-262Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Further, this activation of dendritic cells, as well as direct effects of CpG DNA on B cells (2Krieg A.K. Yi A.-K. Matson S. Waldschmidt T.J. Bishop G.A. Teasdale R. Koretzky G. Klinman D. CpG motifs in bacterial DNA trigger direct B-cell activation.Nature. 1995; 374: 546-549Crossref PubMed Scopus (3090) Google Scholar), initiates acquired immune responses, including the generation of antibody and cytotoxic T cell responses against delivered antigens (Fig. 1). Although bacterial DNA has been known to have immune stimulatory effects for many years (3Tokunaga T. Yamamoto H. Shimada S. Abe H. Fukuda T. Fujisawa Y. Furutani Y. Yano O. Kataoka T. Sudo T. Makiguchi N. Suganuma T. Antitumor activity of deoxyribonucleic acid fraction from mycobacterium bovis GCG. I. Isolation, physicochemical characterization, and antitumor activity.JNCI. 1984; 72: 955-962PubMed Google Scholar), it has only been a few years since the discovery that these immune stimulatory effects depend on the presence in bacterial DNA of unmethylated CpG dinucleotides in particular base contexts and that these effects can be reproduced by synthetic oligonucleotides containing these "CpG motifs" (2Krieg A.K. Yi A.-K. Matson S. Waldschmidt T.J. Bishop G.A. Teasdale R. Koretzky G. Klinman D. CpG motifs in bacterial DNA trigger direct B-cell activation.Nature. 1995; 374: 546-549Crossref PubMed Scopus (3090) Google Scholar). It is now generally accepted that immune activation by CpG motifs evolved as an immune defense against infection. CpG motifs are relatively rare and are heavily methylated in our own DNA. Thus, our DNA has a subtly different molecular pattern from prokaryotic DNAs, and because it lacks these unmethylated CpG "danger signals," it does not activate this defense. Because gene therapy vectors are unmethylated, they would normally "look" like pathogen DNA to this primitive innate immune defense and will, therefore, trigger it. Although the molecular mechanism of CpG-induced immune activation is still incompletely understood, it appears that the DNA must enter host cells and bind to an intracellular CpG pattern recognition receptor, which then leads to the activation of mitogen-activated protein kinases, NFkB, and other cell-signaling pathways (4Krieg A.M. Hartmann G. Yi A.-K. Mechanisms of action of CpG DNA.in: Wagner H. Current Topics in Microbiology and Immunology. Springer-Verlag, Heidelberg1999: 1-21Google Scholar). The immune effects of CpG dinucleotides depend critically on the flanking bases (Table 1). If the CG is preceded by a C and followed by a G, immune stimulation is almost abolished, and the activity of nearby stimulatory CpG motifs can be diminished (5Krieg A.M. Wu T. Weeratna R. Efler S.M. Love-Homan L. Zhang L. Yang L. Yi A.K. Short D. Davis H. Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs.Proc. Natl. Acad. Sci. USA. 1998; 95: 12631-12636Crossref PubMed Scopus (357) Google Scholar).TABLE 1Effects of Flanking Bases on Immune Stimulation by CpG ODNODNSequence (5'–3')IL-6 (pg/ml)aLevel of IL-6 ± SD of triplicate samples in supernatants of the murine B cell line WEHI-231 cultured for 24 h with the indicated ODN at 20 µM and measured by ELISA.ProliferationbB cell proliferation was measured using T-cell-depleted DBA/2 spleen cells by [3H]uridine incorporation; numbers are stimulation indices compared to untreated cells which had 2323 cpm.IgM (ng/ml)cMeasured by ELISA.NK LUdNatural killer lytic activity.512TCCATGTCGGTCCTGATGCT1300.8 ± 106.45.8 ± 0.33875 ± 4140.611637……C……………136.5 ± 27.41.7 ± 0.2166 ± 370.001615……G……………1201.8 ± 155.23.7 ± 0.32956 ± 4110.041614……A……………1533.7 ± 321.110.8 ± 0.66184 ± 2842.751636………A…………1181.6 ± 76.55.4 ± 0.43372 ± 2682.421634………C…………1049.6 ± 223.59.2 ± 0.95171 ± 11532.411619………T…………1555.7 ± 304.912.5 ± 1.07632 ± 6064.141618……A…T…………2109.4 ± 291.612.9 ± 0.7ND3.371639……AA…T…………1827.6 ± 83.211.5 ± 0.48068 ± 3992.221707……A…TC………ND4.0 ± 0.22267 ± 5620.521708……CA…TG………ND1.5 ± 0.1131 ± 802.14Note. Dots indicate identities; CpG dinucleotides are underlined; ND, not determined. Table reproduced with permission from R. S. Tuetken, A.-K. Yi, and A. M. Krieg (1998). The immune effects of bacterial DNA and their possible role in the pathogenesis of lupus. In Lupus: Molecular and Cellular Pathogenesis (G. C. Tsokos and G. Kammer, Eds.), Humana Press.a Level of IL-6 ± SD of triplicate samples in supernatants of the murine B cell line WEHI-231 cultured for 24 h with the indicated ODN at 20 µM and measured by ELISA.b B cell proliferation was measured using T-cell-depleted DBA/2 spleen cells by [3H]uridine incorporation; numbers are stimulation indices compared to untreated cells which had 2323 cpm.c Measured by ELISA.d Natural killer lytic activity. Open table in a new tab Note. Dots indicate identities; CpG dinucleotides are underlined; ND, not determined. Table reproduced with permission from R. S. Tuetken, A.-K. Yi, and A. M. Krieg (1998). The immune effects of bacterial DNA and their possible role in the pathogenesis of lupus. In Lupus: Molecular and Cellular Pathogenesis (G. C. Tsokos and G. Kammer, Eds.), Humana Press. Because viral or nonviral gene therapy vectors contain hundreds of CpG motifs, which are responsible for at least some of the observed immunotoxicity, counter measures are urgently needed if the clinical development of gene therapy is to realize its full promise. Unfortunately, so simple a measure as methylation of these CpGs within a gene therapy vector is not likely to be fruitful, because of the resulting decreased expression of the encoded genes. In this issue of Molecular Therapy, Yew et al. describe two approaches to at least partially reverse the immune effects of CpG motifs (1Yew N.S. Zhao H. Wu I.-H. Song A. Tousingnant J.D. Przybylska M. Cheng S.H. Reduced inflammatory response to plasmid DNA vectors by elimination and inhibition of immunostimulatory CpG motifs.Mol. Ther. 2000; 1: 255-262Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). First, the authors show that the heroic approach of cloning out more than half of the 526 CpG motifs from a gene therapy vector works well, reducing the level of cytokine induction by 50% or more. Second, the authors show that antimalarial compounds, which previously have been reported to block the signaling pathways induced by CpG DNA in vitro, also significantly reduce cytokine responses to gene therapy vectors in vivo. Since these antimalarial compounds are widely used in humans, and have proven to be safe and well tolerated, this approach should not require a great deal of further developmental work before evaluation in humans. Although the results of this study are clear-cut and convincing, unanswered questions remain. Only a human clinical trial using a CpG-depleted gene therapy vector can show whether such a partial reduction in CpG content will reduce immune activation to a clinically insignificant level. As well, there may be practical limitations to the number of CpG motifs that can be removed, since many of these may be in essential binding sites for transcription factors or other regulatory regions that are not amenable to mutagenesis. Presumably, the combination of antimalarial drugs and CpG-depleted vectors should further reduce the immune effect. One disappointing aspect in the results of Yew et al. is that the addition of previously described CpG-neutralizing sequences (5Krieg A.M. Wu T. Weeratna R. Efler S.M. Love-Homan L. Zhang L. Yang L. Yi A.K. Short D. Davis H. Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs.Proc. Natl. Acad. Sci. USA. 1998; 95: 12631-12636Crossref PubMed Scopus (357) Google Scholar) to a gene therapy vector failed to diminish the immune stimulation. The existence of immune-neutralizing sequences in vertebrate genomic DNA can be inferred from the fact that even when the DNA is demethylated, it fails to induce cytokine expression (6Sun S. Beard C. Jaenisch R. Jones P. Sprent J. Mitogenicity of DNA from different organisms for murine B cells.J. Immunol. 1997; 159: 3119-3125PubMed Google Scholar). That is, the mere presence of unmethylated CpG motifs is not sufficient to make a DNA fragment immune stimulatory. A major challenge to investigators in the field will be to uncover the structural features of DNA that determine its immune effects. It is worth pointing out that the immune stimulatory effects of CpG DNA are not simply a thorn in the side of the genetic engineer. In fact, synthetic oligodeoxy-nucleotides containing CpG motifs have proven to be safe and effective vaccine adjuvants and are currently in human clinical trials (7Davis H.L. Use of CpG DNA for enhancing specific immune responses.in: Wagner H. Current Topics in Microbiology and Immunology. Springer-Verlag, Heidelberg2000: 171-183Crossref Google Scholar). CpG DNA has also shown efficacy for the immunotherapy of cancer and allergic diseases in rodent models and is currently entering human clinical trials. In performing gene therapy for cancer immunotherapy, CpG motifs can even contribute to a therapeutic effect (8Dow S.W. Frandkin L.G. Liggitt D.H. Willson A.P. Heath T.D. Potter T.A. Lipid–DNA complexes induce potent activation of innate immune responses and antitumor activity when administered intravenously.J. Immunol. 1999; 163: 1552-1561PubMed Google Scholar). For most gene therapy applications, however, the immune stimulatory effects of CpG motifs are quite unwelcome. The approaches demonstrated by Yew et al. should be useful in reducing the risk of giving the immune system a "danger signal," when one really desires to introduce a gene. It is anticipated that this challenge will be the subject of many further studies.