PCRX-201, a novel IL-1Ra gene therapy treatment approach for low back pain resulting from intervertebral disc degeneration

Abstract

Low back pain is the leading cause of global disability with intervertebral disc (IVD) degeneration a major cause. However, no current treatments target the underlying pathophysiological causes. PCRX-201 presents a novel gene therapy approach that addresses this issue. PCRX-201 codes for interleukin-1 receptor antagonist, the signalling inhibitor of the pro-inflammatory cytokine interleukin-1, which orchestrates the catabolic degeneration of the IVD. Here, the ability of PCRX-201 to transduce human nucleus pulposus cells to increase IL-1Ra production was assessed together with effects on catabolic pathways. When transduced with PCRX-201, the production and release of IL-1Ra was increased in degenerate human nucleus pulposus cells and tissue. Whereas, the production of downstream proteins, including IL-1β, IL-6, MMP3, ADAMTS4 and VEGF were decreased in both cells and tissue, indicating a reduction in IL-1-induced catabolic signalling. Here, a novel gene therapy vector, PCRX-201, was shown to transduce degenerate NP cells and tissue, increasing the production of IL-1Ra. The increased IL-1Ra resulted in decreased production of catabolic cytokines, enzymes and angiogenic factors, whilst also increasing aggrecan expression. This demonstrates PCRX-201 enables the inhibition of IL-1-driven IVD degeneration. The ability of PCRX-201 to elicit anti-catabolic responses is promising and warrants further development to determine the efficacy of this exciting, novel gene therapy.

Fig. 1. Metabolic activity and IL-1Ra production in monolayer-cultured, PCRX-201-transduced, degenerate human NP cells.

a Metabolic activity of human NP cells 48 h after transduction with PCRX-201 at different multiplicities of infection (MOI), normalised to untreated controls. IL-1Ra production in cell culture supernatant after (b) 2 d PCRX-201 transduction ±1 d 10 ng/mL IL-1β stimulation, c 5 d PCRX-201 transduction ±4 d 10 ng/mL IL-1β stimulation. Horizontal lines represent median values. Significant differences determined by Kruskal–Wallis with Dunn’s multiple comparison tests *p ≤ 0.05.

Fig. 2. Long-term production of IL-Ra following initial PCRX-201 transduction in 3D alginate bead-cultured human NP cells.

IL-1Ra release into cell culture supernatant (a) 2 d, (b) 1w, (c) 2w, (d) 3w, (e) 4w, (f) 6w, (g) 8w and (h) 10w after initial transduction with PCRX-201 at a multiplicity of infection (MOI) of 3000, compared to non-infected control cells (CTR). i Time course of IL-1Ra release from 2 d to 10w in PCRX-201 transduced human NP cells (MOI:3000). Horizontal lines represent median values. Significant differences determined by (a–h) Mann–Whitney or (i) Kruskal–Wallis with Dunn’s multiple comparison tests *p ≤ 0.05.

Fig. 3. Viability and downstream protein production in 3D cultured human NP cells following PCRX-201 transduction.

Degenerate human NP cells (n = 6 patients in triplicate) were transduced with PCRX-201 at a multiplicity of infection (MOI) of 0, 750 or 3000, resuspended in alginate beads and cultured for 3 weeks, +/−100 pg/mL IL-1β for the last week of culture. Following culture (a) total DNA/alginate bead was measured to determine effects of culture on NP cell viability. ELISA was used to determine the downstream production of (b) IL-1Ra, (c) IL-1β, (d) IL-6, (e) IL-8, (f) MMP3, (g) ADAMTS4, and (h) VEGF within cell culture supernatant after 3w culture. Horizontal lines represent median values. Significant differences determined by Kruskal–Wallis with Dunn’s multiple comparison tests *p ≤ 0.05.

Fig. 4. Paracrine effect on downstream protein production in 3D alginate bead-cultured human NP cells when exposed to PCRX-201-conditioned media.

Degenerate human NP cells (n = 6 patients in triplicate) encapsulated in alginate beads were treated for 3 days with conditioned media from NP cells transduced with PCRX-201 (MOI:3000) for 1 week previously. Cells treated with media from previously non-transduced cells served as controls (CTR). ELISA was used to determine the effects on downstream production of (a) IL-1β, (b) IL-6, (c) MMP3, (d) ADAMTS4 and (e) VEGF within cell culture supernatant after 3 days of culture. Horizontal lines represent median values. Significant differences determined by Mann-Whitney tests *p ≤ 0.05.

Fig. 5. Modulation of IL-1Ra and downstream protein production in ex vivo human NP tissue explants following direct PCRX-201 injection.

Human NP explants (n = 5) (Table 1) were injected with PCRX-201 at a multiplicity of infection (MOI) of 3000 and cultured for 2 weeks within a semi-constrained plastic ring to limit tissue swelling. Non-transduced tissue explants served as controls (CTR). Following culture, the production of (a) IL-1Ra, (b) IL-1β, (c) IL-6, (d) MMP3, (e) ADAMTS4, (f) aggrecan, and (g) VEGF within cell culture supernatant was determined using ELISA. Horizontal lines represent median values. Significant differences determined by Mann–Whitney tests *p ≤ 0.05.

Fig. 6. Expression of IL-1Ra and catabolic proteins in ex vivo human NP tissue explants following direct PCRX-201 injection.

Expression of (a) IL-1Ra, (b) IL-1β, (c) VEGF and (d) NGF in native human nucleus pulposus (NP) cells, determined by immunohistochemistry, following 2-week ex vivo culture of human NP tissue explants (n = 5). Immunopositivity (brown staining) was determined by counting 200 NP cells per sample and expressing the percentage of cells positively stained for proteins of interest. Cell nuclei counterstained blue with Mayer’s haematoxylin. CTR non-transduced control tissue, PCRX-201 tissue transduced with PCRX-201 at a multiplicity of infection (MOI) of 3000. Horizontal lines represent median values. Significant differences determined by Mann–Whitney tests *p ≤ 0.05.

Fig. 7. Expression of extracellular matrix proteins and matrix degrading enzymes in ex vivo human NP tissue explants following direct PCRX-201 injection.

Expression of (a) collagen type II, (b) aggrecan, (c) MMP3 and (d) ADAMTS4 in native human nucleus pulposus (NP) cells, determined by immunohistochemistry, following 2-week ex vivo culture of human NP tissue explants (n = 5). Immunopositivity (brown staining) was determined by counting 200 NP cells per sample and expressing the percentage of cells positively stained for proteins of interest. Cell nuclei counterstained blue with Mayer’s haematoxylin. CTR non-transduced control tissue, PCRX-201 tissue transduced with PCRX-201 at a multiplicity of infection (MOI) of 3000. Horizontal lines represent median values. Significant differences determined by Mann–Whitney tests *p ≤ 0.05.