Targeting of human interleukin-12B by small hairpin RNAs in xenografted psoriatic skin

Abstract

BackgroundPsoriasis is a chronic inflammatory skin disorder that shows as erythematous and scaly lesions. The pathogenesis of psoriasis is driven by a dysregulation of the immune system which leads to an altered cytokine production. Proinflammatory cytokines that are up-regulated in psoriasis include tumor necrosis factor alpha (TNFα), interleukin-12 (IL-12), and IL-23 for which monoclonal antibodies have already been approved for clinical use. We have previously documented the therapeutic applicability of targeting TNFα mRNA for RNA interference-mediated down-regulation by anti-TNFα small hairpin RNAs (shRNAs) delivered by lentiviral vectors to xenografted psoriatic skin. The present report aims at targeting mRNA encoding the shared p40 subunit (IL-12B) of IL-12 and IL-23 by cellular transduction with lentiviral vectors encoding anti-IL12B shRNAs.MethodsEffective anti-IL12B shRNAs are identified among a panel of shRNAs by potency measurements in cultured cells. The efficiency and persistency of lentiviral gene delivery to xenografted human skin are investigated by bioluminescence analysis of skin treated with lentiviral vectors encoding the luciferase gene. shRNA-expressing lentiviral vectors are intradermally injected in xenografted psoriatic skin and the effects of the treatment evaluated by clinical psoriasis scoring, by measurements of epidermal thickness, and IL-12B mRNA levels.ResultsPotent and persistent transgene expression following a single intradermal injection of lentiviral vectors in xenografted human skin is reported. Stable IL-12B mRNA knockdown and reduced epidermal thickness are achieved three weeks after treatment of xenografted psoriatic skin with lentivirus-encoded anti-IL12B shRNAs. These findings mimick the results obtained with anti-TNFα shRNAs but, in contrast to anti-TNFα treatment, anti-IL12B shRNAs do not ameliorate the psoriatic phenotype as evaluated by semi-quantitative clinical scoring and by immunohistological examination.ConclusionsOur studies consolidate the properties of lentiviral vectors as a tool for potent gene delivery and for evaluation of mRNA targets for anti-inflammatory therapy. However, in contrast to local anti-TNFα treatment, the therapeutic potential of targeting IL-12B at the RNA level in psoriasis is questioned.

Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin

“…20,22 Briefly, xenotransplanted C.B-17 SCID mice were anesthetized by a subcutaneous injection of ketamine-xylazine or with 3.75% isoflurane (Forene; Abbott Scandinavia, Solna, Sweden) (2% isoflurane for anesthesia maintenance) and then injected intradermally (into the xenograft) with a total volume of 150 ll of viral vector suspension. rAAVs were injected at a dose of 5 · 10 10 VG/ mouse, whereas LV vectors were injected at a dose of 2 lg of p24 units per mouse.…”

“…20,22 Briefly, mice were injected intraperitoneally with the substrate d-luciferin potassium salt (Synchem OHG, Felsberg-Altenburg, Germany) at a dose of 150 lg/g of body weight and subsequently anesthetized with 3.75% isoflurane (Forene; Abbott Scandinavia). Anesthesia was maintained at 2% isoflurane during the bioluminescence scan in the IVIS Spectrum imaging system (Caliper Life Sciences, Hopkinton, MA).…”

“…20 In brief, the firefly luciferase gene was amplified by PCR from the commercially available pGL3-Control vector (Promega, Madison, WI) and cloned into the lentiviral vector pLV/PGK-puro, 22 using the BamHI/XhoI sites, thereby replacing the puromycin resistance gene. To generate the pLV/ CMV-FLuc vector, the CMV promoter sequence was amplified by PCR from the recombinant adenoassociated viral vector, rAAV-FLuc, purchased from the Gene Core Facility of the University of North Carolina (Chapel Hill, NC) and cloned into the pLV/PGK-FLuc vector, using the ClaI/BamHI sites, thereby replacing the phosphoglycerate kinase (PGK) promoter fragment.…”

“…20,21 We used such potency for treatment of an inflammatory skin disease model by small hairpin RNA (shRNA)-directed targeting of tumor necrosis factor-a. 22 In continuation of such observations, we wanted to explore the applicability of AAV-derived vectors for gene transfer to human skin.…”

Potent microRNA suppression by RNA Pol II-transcribed ‘Tough Decoy’ inhibitors

“…For Pol III-expressed inhibitors, we used the pCCL-PGK-Puro-H1-MCS plasmid, described in Bak et al (2011), in which the H1 promoter drives the expression of the miRNA inhibitors from an expression cassette located in the U3 region. We also generated a variant of this plasmid encoding eGFP (pCCL-PGK-eGFP-H1-MCS) by exchanging the XhoI/BamHIflanked puromycin resistance gene with the XhoI/BamHI-flanked eGFP from the lentiviral SIN-vector pCCL-PGK-eGFP (originally designated pCCL.WPS.PGK-eGFP.WHV; a kind gift from Dr. Patrick Aebischer, Swiss Federal Institute of Technology, EPFL, Lausanne, Switzerland).…”