Preclinical development of siRNA therapeutics for AL amyloidosis

Abstract

Amyloid light chain (AL) amyloidosis is a rare hematologic disorder characterized by the accumulation of a misfolded monoclonal immunoglobulin (Ig) light chain (LC) as fibrillar protein deposits. Current treatments, including cytotoxic chemotherapy and immunomodulatory therapy, are directed at killing the plasma cells that produce the LCs, but have significant toxicity for other cell types. We have designed small interfering RNAs (siRNAs) targeting the amyloidogenic LC messenger RNA (mRNA) in order to reduce expression of the amyloid precursor protein. Using nanomolar concentrations of siRNAs, we have inhibited synthesis of LC in transfected cells in vitro in a dose-dependent fashion. Furthermore, in an in vivo plasmacytoma mouse model of AL amyloidosis, we have demonstrated that these siRNAs can significantly reduce local production and circulating levels of LC. This model system highlights the therapeutic potential of siRNA for AL amyloidosis.

Figure 1. siRNA target sites on a prototype κ1 LC

Diagram of siRNA target sites on patient amyloidogenic κ1 immunoglobulin LC, AL-009-κ1. Two siRNAs were designed to target different regions of the light chain variable (VL) domain and one to target the light chain constant (CL) domain. One siRNA, GG1.1, was designed as a control directed at the κ1 germline gene, but not the AL-009-κ1 sequence.

Figure 2. In vitro κ1 mRNA and protein expression after siRNA treatment

(A) NIH-3T3 cells stably expressing AL-009-κ1 were treated for 48 hours with 20nM of either VK1.1, VK1.2, or CK1.2 siRNAs; mean relative mRNA levels are depicted (* indicates p < 0.5). Reduction in cellular LC protein at 48 hours with increasing concentrations of siRNA are plotted in (B); representative immunoblots are shown in (C). Time course of reduction of protein levels cells following treatment with 20 nM of VK1.1, VK1.2 or CK1.2 siRNA are plotted in (D); representative immunoblots are shown (E).

Figure 3. Effect of siRNA, delivered by in vivo electroporation, on plasmacytoma LC mRNA and protein levels

Plasmacytomas were formed over 2–3 weeks in mice by subcutaneous injection of SP2/0 cells transfected with human amyloidogenic LC. The plasmacytomas were then injected with 12 μg of control or experimental (VK1.2) siRNA and in vivo electroporation was performed. 48 hours later, the mice were sacrificed for analysis and plasmacytoma tissue was collected. (A) Plasmacytoma κ1 LC mRNA expression levels relative to Blimp1, a plasma cell specific marker, are depicted (** indicates p = 0.0016, n = 10 per group). (B) Plasmacytoma lysates immunoblotted for human κ1 LC; each lane represents an individual plasmacytoma, control treated samples underlined and in italics. Data depicted as the mean for control siRNA vs. VK1.2 siRNA treated plasmacytoma LC protein levels (** indicates p = 0.0051, n = 10 per group). (C) Representative sections of plasmacytomas treated with control or experimental siRNA, brown staining for human κ1 LC.

Figure 4. Effect of siRNA, delivered by in vivo electroporation, on circulating LC protein levels

(A) Comparison by immunoblot of κ1 LC levels in sera taken from the same mouse pre- and post-treatment (48 hours). Control-treated samples are underlined and italicized. (B) Graph depicting the ratio of post-treatment to pre-treatment circulating κ1 Ig LC levels (post/pre) quantitated from the associated immunoblot for control and experimental siRNA treatment (*** p = 0.0003, n = 10 per group).