HHSN261201200095C (NIH NCI) and SBIR grant 2R44GM088894. (NIH NIGMS) to G.W.J.. Discussion / Conclusion. The data presen
Topical DNA aptamers to the antimicrobial peptide LL37 for the potential treatment of psoriasis
Department of Dermatology
and Venereology George W. (Bill) Jackson†, Michel Gilliet҂ † Base Pair Biotechnologies, Houston, TX, ҂ Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland
Abstract Psoriasis is a chronic inflammatory autoimmune skin disorder which affects a significant number of people worldwide. Although this disease is not lifethreatening, it can lead to a significantly poor quality of life and negatively impact the mental health of the patients affected. For more severe psoriasis, both topical and systemic prescription medications exist. Unfortunately, all of these treatments carry significant risks that deter patient adoption, such as general immunosuppression, the development of cancer, increased bacterial and fungal infections, damage to the kidney, lungs, or liver, and the development of blood abnormalities. We are currently developing and testing DNA and modified DNA aptamers to target an extracellular, upstream mediator of psoriasis, the so called, “LL37” peptide. Promising candidate molecules are already in hand which show biological inhibitory activity. Using a patented multiplex approach to aptamer selection, we are developing a number of new candidate aptamers with a modified DNA chemistry. This novel aptamer chemistry has the potential to greatly enhance transport through the skin and will be incorporated during selection of the LL37 antagonists.
LL37 antimicrobial peptide as a target Extracellular – avoids issues with intracellular delivery Plasmacytoid dendritic cells (pDC’s) specifically involved. pDC’s only sense nucleic acids with TLR7 and TLR9 PROVEN modulator of psoriatic inflammatory cascade [1-3] LL37 is only expressed in injured skin or in psoriasis
Advantages of Aptamers vs. Antibodies Synthetic – chemically defined; no lot-to-lot variation Easier to produce at GMP grade Enhanced transport properties incorporated during selection More stable, ready topical formulation
Future Work
Selection of tighter LL37 binders (“Apt_222” is 60 nM) Incorporation of non-natural nucleotides Skin transport studies with modified DNA polymers Additional IFN-α measurements for aptamer candidates in pDC cell culture Test most promising in vitro candidates in xenoplant mouse model
Discussion / Conclusion
SKIN
Figure 2. LL37-specific aptamer #222 but not #223 blocks ability of LL37 to form immunogenic complexes with DNA. IFN-α secreted by pDC after overnight stimulation at the indicated conditions. Control experiments are indicated by red star and orange arrows . CONTROLS: Red Star = aptamer alone does not induce anti-viral or inflammatory response via TLR9. Green Arrows = a known CpG oligo agonist of TLR9 shows viability of cells and receptor .
Modified DNA is likely to have improved transport properties through the skin [4] A
Figure 1. Role of LL37 in psoriasis development. Inflamed and injured tissue results in cell death, where LL37 (red cylinders) subsequently binds to extracellular human genomic DNA (hgDNA). The LL37+hgDNA complex activates endosomal TLR9 receptors in plasmacytoid dendritic cells (pDCs), leading to a production of IFN-α in response. This cytokine causes local recruitment of macrophages and T-cells, which produce further cytokines and induces an inflammation response in the region. These molecular events cause an autoimmune inflammatory cascade that is self-propagating.
Figure 4. Fluorescence imaging cross-section of modified DNA 76-mer with C5-propyne deoxy C substitution (chemistry B from Fig. 3). The DNA is located well into the dermis.
B
C
Figure 3. Modified nucleotides with unsaturation for improved transport across skin. (A) Amino-allyl deoxyUTP, (B) 5-Propynyl-2'-deoxycytidine-5'Triphosphate (C) C8-alkyne-dCTP. All 3 of these triphosphates have been successfully incorporated during amplification of our aptamer library and transport efficiency through murine skin has been confirmed.
The data presented here demonstrate that it is possible to select a DNA aptamer with greater affinity for LL37 than its natural binding partner, genomic DNA. Furthermore “Aptamer_222” completely abrogates IFN-α production in pDC cell culture and thus shows great promise as a potential lead compound for psoriasis treatment. In future experiments discussed above, we plan to generate additional, aptamerbased lead compounds and characterize them in human xenograft mouse models.
References 1. Lande R, Gregorio J, Facchinetti V, Chatterjee B, Wang Y-H, Homey B, Cao W, Wang YH, Su B, Nestle FO, Zal T, Mellman I, Schroder J-M, Liu Y-J, Gilliet M: Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature 2007, 449:564–569. 2. Gilliet M, Cao W, Liu Y-J: Plasmacytoid dendritic cells: sensing nucleic acids in viral infection and autoimmune diseases. Nat. Rev. Immunol 2008, 8:594–606. 3. Gregorio J, Meller S, Conrad C, Di Nardo A, Homey B, Lauerma A, Arai N, Gallo RL, Digiovanni J, Gilliet M: Plasmacytoid dendritic cells sense skin injury and promote wound healing through type I interferons. J. Exp. Med 2010, 207:2921–2930. 4. White et al. C-5 propyne-modified oligonucleotides penetrate the epidermis in psoriatic and not normal human skin after topical application. J. Invest. Dermatol 2002, 118:1003–1007.
Acknowledgements This work was supported in part by an SBIR Contract HHSN261201200095C (NIH NCI) and SBIR grant 2R44GM088894 (NIH NIGMS) to G.W.J.
