Amelioration of psoriasis by anti-TNF-alpha RNAi in the xenograft transplantation model

Abstract

Tumor necrosis factor-alpha (TNF-alpha) is upregulated in psoriatic skin and represents a prominent target in psoriasis treatment. The level of TNF-alpha-encoding mRNA, however, is not increased in psoriatic skin, and it remains unclear whether intervention strategies based on RNA interference (RNAi) are therapeutically relevant. To test this hypothesis the present study describes first the in vitro functional screening of a panel of short hairpin RNAs (shRNAs) targeting human TNF-alpha mRNA and, next, the transfer of the most potent TNF-alpha shRNA variant, as assessed in vitro, to human skin in the psoriasis xenograft transplantation model by the use of lentiviral vectors. TNF-alpha shRNA treatment leads to amelioration of the psoriasis phentotype in the model, as documented by reduced epidermal thickness, normalization of the skin morphology, and reduced levels of TNF-alpha mRNA as detected in skin biopsies 3 weeks after a single vector injection of lentiviral vectors encoding TNF-alpha shRNA. Our data show efficient lentiviral gene delivery to psoriatic skin and therapeutic applicability of anti-TNF-alpha shRNAs in human skin. These findings validate TNF-alpha mRNA as a target molecule for a potential persistent RNA-based treatment of psoriasis and establish the use of small RNA effectors as a novel platform for target validation in psoriasis and other skin disorders.

Figure 1

Functional screening of potential shRNA variants targeting human TNF-α. (a) Schematic representation of the TNF-α mRNA sequence and the distribution of shRNA target sequences. 5′ UTR and 3′ UTR indicate the 5′- and 3′-untranslated regions, respectively. ORF represents the open reading frame and P(A) the polyadenylation sequence. (b) Targeting the TNF-α sequence as detected by the dual-luciferase reporter assay. TNF-α cDNA was fused to the Renilla luciferase gene in the psiCHECK-2 vector and the plasmid co-transfected together with pBC.H1-shTNF-α1 through pBC.H1-shTNF-α7 and pBC.H1-shGFP (negative control) into HEK293 cells. Luciferase activities were determined 48 hours after transfection and the normalized values calculated relative to the negative shRNA control (pBC.H1-shGFP). P-values for the comparisons indicated by brackets were as follows: *P < 0.0001; **P = 0.0001; ***P = 0.0004. (c) Downregulation of transiently expressed TNF-α by transfection with lentiviral vector constructs encoding shRNAs. HEK293 cells were transfected with pSBT/CMV-TNF-α.EF1α-zeo and lentiviral vector constructs encoding shRNAs as indicated. TNF-α expression was determined by measuring TNF-α concentration in the medium by enzyme-linked immunosorbent assay. The asterisk indicates significant difference between the two groups indicated by the bracket (P = 0.0004). All experiments were performed in triplicates. Data are presented as mean ± SD. shRNA, short hairpin RNA; TNF-α, tumor necrosis factor-α.

Figure 2

In vitro knockdown of stable TNF-α expression by shTNF-α3. (a,b) Knockdown of stable TNF-α expression following transduction with lentiviral vector-encoded shTNF-α3. 293-TNF-α cells were transduced with LV/shTNF-α2, LV/shTNF-α3, LV/shTNF-α4, LV/shGFP, or LV/shIrr, as indicated below each column, at an estimated MOI of ~40. The column labeled “Vehicle” (a) represents cells that were transduced with LV/PGK-puro and therefore did not receive any shRNA. “TNF-α minus” refers to HEK293 cells that did not express TNF-α. The concentration of TNF-α in the medium of the transduced cells was determined (a) after 2 days or (b) after 2 and 4 days. Black and gray columns represent concentrations measured after 2 and 4 days, respectively. Relative TNF-α concentrations (normalized for cell number and amount of viral particles transferred to the cells) are presented in b. Significant differences between the most relevant groups (indicated by brackets) are indicated by asterisks representing the following P values; *P = 0.003; **P = 0.002; ***P = 0.0007. (c) Stable downregulation of TNF-α expression after vector treatment. 293-TNF-α cells were transduced with lentiviral vectors at an estimated MOI of ~2 followed by selection of transduced cells with puromycin. TNF-α expression was determined after 11 days by measuring TNF-α concentration in the medium by enzyme-linked immunosorbent assay. The relative amounts of TNF-α were determined by correlation of TNF-α concentration to the number of cells and to the amount of lentiviral vectors used for transduction (pg p24 Gag) (TNF-α concentration/cell number/pg p24 Gag). Significant difference (P = 0.0002) between cells treated with shIrr and shTNF-α3 is indicated by an asterisk. All experiments were done in triplicates. Data are presented as mean ± SD. (d) shTNF-α3 guide strand sequence complementarity with human and mouse TNF-α mRNA. Alignment at top shows the degree of sequence similiarity between the human (NM_000594) and mouse (NM_013693) TNF-α genes in the segment of the gene containing the shTNF-α3 target sequence. Numbers refer to distance from first nucleotide in start codon of the human TNF-α sequence. Nucleotides that differ between the two sequences are underlined in the mouse TNF-α sequence. Nucleotides that are not present in mouse sequence are indicated with hyphens. The two alignments below demonstrate that shTNF-α3 has perfect match with human TNF-α mRNA but matches only 9 out of 19 positions within this region of mouse TNF-α mRNA. TNF-α, tumor necrosis factor-α; shIrr, irrelevant shRNA; MOI, multiplicity of infection; shRNA, short hairpin RNA.

Figure 3

In vivo transduction of human psoriasis skin with eGFP-encoding lentiviral vectors. Psoriatic plaque skin biopsies were transplanted onto severe combined immunodeficiency mice in the psoriasis xenograft transplantation model. Following healing, eGFP-encoding lentiviral vectors were injected intradermally into the human psoriasis skin grafts in an amount corresponding to 5.3 µg p24 Gag/mouse. Three days after transduction skin biopsies were taken, fixed, embedded in paraffin, and stained with anti-eGFP antibody. eGFP expression was analyzed by fluorescence microscopy. (a) Negative control (untreated transplanted grafts) stained with Hoechst to visualize cell nuclei. (b) Negative control stained with anti-GFP antibody. (c) Skin biopsy treated with lentiviral-mediated transfer of eGFP (LV/PGK-eGFP) and stained with Hoechst. (d) Skin biopsy treated with LV/PGK-eGFP and stained with anti-GFP antibody. Bar = 100 µm. eGFP, enhanced GFP.

Figure 4

Comparison of control treatments of psoriatic plaques in the xenograft transplantation model. (a) Semiquantitative clinical psoriasis scores were given twice weekly for 3 weeks to mice treated with the irrelevant monoclonal antibody (filled circles, n = 10) or LV/shIrr (filled squares, n = 7). The two control treatments showed no significant difference in semiquantitative clinical psoriasis scores. (b) Epidermal thickness of skin biopsies after treatment with the irrelevant monoclonal antibody and LV/shIrr. Upon sacrifice 3 weeks after treatment biopsies were taken, fixed and paraffin-embedded, and stained. Epidermal thickness was measured in irrelevant antibody-treated (black column, n = 10) or LV/shIrr-treated (gray column, n = 7) mice. The two control treatments showed no significant difference in the epidermal thickness. Data are presented as mean + SEM. shIrr, irrelevant shRNA.

Figure 5

In vivo knockdown of TNF-α in human psoriasis skin by shTNF-α3. (a) The schedule for treatment of psoriasis plaques in vivo. Psoriatic plaque skin biopsies were transplanted onto SCID mice in the psoriasis xenograft transplantation model. All mice were allowed to heal for 10 days before onset of treatment. Grafts were treated either by a single injection with LV/shTNF-α3 or LV/shIrr, or five times weekly with CsA injections (positive control) and killed 3 weeks after treatment. Arrows indicate injection time points. (b) Semiquantitative clinical psoriasis scores were given twice weekly for 3 weeks to mice treated with negative control (filled squares, n = 17), LV/shTNF-α3 (open squares, n = 10), or CsA (positive control) (filled triangles, n = 12). shTNF-α3 significantly decreased the semiquantitative clinical psoriasis score (*P = 0.03). Intradermal injections were performed at day 0. (c) LV/shTNF-α3 treatment decreased the epidermal thickness in psoriasis skin grafts. Mice were killed after treatment and biopsies were taken, fixed, paraffin-embedded and stained with H&E. Epidermal thickness was measured in negative control-treated (black column, n = 17), LV/shTNF-α3-treated (dark gray column, n = 10), and CsA-treated (light gray column, n = 10) grafts. shTNF-α3 significantly decreased the epidermal thickness as compared to negative control (*P = 0.03). A measurement of nonlesional skin (n = 12) is included for comparison (white column). (d) Reduction of TNF-α gene expression in psoriasis skin in LV/shTNF-α3-treated skin. Biopsies were taken 3 weeks after treatment and TNF-α mRNA levels were measured by qPCR in negative control-treated (black column, n = 17) and in LV/shTNF-α3-treated (dark gray column, n = 9) grafts (P = 0.10). The black/white-graduated column (n = 7) represents the mean level of TNF-α mRNA detected in psoriatic plaque skin biopsies obtained from seven skin donors and which were not grafted onto mice. (e) The level of mouse TNF-α mRNA in the spleen is unaffected by shTNF-α3 treatment. Quantitative reverse transcription-PCR was performed on total RNA derived from spleens of mice treated with LV/shIrr (black column; n = 7) and LV/shTNF-α3 (dark gray column; n = 10), respectively (P = 0.77). (f) Histological assessment of grafted skin samples. Representative tissue samples of each treatment group are shown. Bar = 100 µm. All data are presented as mean + SEM. TNF-α, tumor necrosis factor-α; SCID, severe combined immunodeficiency; CsA, cyclosporin A; shIrr, irrelevant shRNA; H&E, hematoxylin and eosin.