Abstract
INTRODUCTION : Despite advances in therapy, patients with relapsed AL amyloidosis die of resistant disease. New therapies are needed. siRNA directed at the constant regions of Ig light chains (LC) reduces LC mRNA and protein from patient cells, from human myeloma and AL cell lines, and in a flank plasmacytoma model with in vivo electroporation (Blood 2014;123:3440; Gene Ther 2016;23:727). To deliver siRNA in vivo, we first tested a series of biodegradable lipidoid nanoparticles (LPN) generated through Michael addition of aliphatic acrylates containing disulfide bonds responsive to intracellular glutathione that enhance siRNA transit from endosome to cytoplasm, and identified the 8B-3 LPN as safe and active in vitro. To provide an in vivo model, we tested RPMI8226, ALMC-1, NCI929 and JJN3 human myeloma reporter cell lines stably expressing FFL and GFP in NOD scid γ (NSG) mice using different routes of inoculation. We sought an optimal xenograft model that would provide reliable tumor-take, brief latency for circulating LC, rapid short-term increase in LC levels, measurable β2-microglobulin (β2M) levels and ease of administration of multiple injections of LPN. The NSG JJN3 intraperitoneal (IP) model met these standards. This model not only enables timely testing of this siRNA approach but also provides the significant challenge of rapid tumor growth. We now report the results in this model of delivery by the 8B-3 LPN of siIGKC targeting κ LC production.
MATERIALS AND METHODS: LPN/siRNA are formulated using a microfluid based mixer (NanoAssemblr, Precision Nanosystems, Inc), and are controlled for size by varying the relative flow rates of lipid and solvent. For in vivo delivery, cholesterol, DOPE and PEGylated co-lipids are used to form stable LPN with siNT or siIGKC (Dharmacon). Standard QA metrics are applied to each lot of 8B-3/siRNA and lot-to-lot checks for cell viability and in vitro FFL knockdown are performed. Coated loaded 8B-3 LPN are ~100nM in diameter and were tested in vitro at 8B-3:siRNA ratios of 10:1, 5:1 and 1.5:1. The 1.5:1 ratio was superior; cell viability was unaffected and κ LC reduction was 84%. NSG mice with JJN3 tumor implants (107 cells IP on day 1) are injected IP with 200μL 8B-3:siRNA (1.5:1 ratio) mixed with 400μL PBS once daily on days 5, 6 and 7. Luciferin imaging is obtained on day 5 and blood is obtained on day 5 (pre-injection) and on day 8 for ELISA for human κ LC and β2M.
RESULTS : Current JJN3 cells make only κ LC without IgA (Br J Haematol 1999;106:669) and in vitro at 48 hours after a single exposure to 8B-3/siIGKC we see reductions of 84% and 25% in κ LC and β2M secretion with no change in cell viability. Neither κ LC nor β2M reductions are seen with 8B3/siNT. The NSG JJN3 IP model has a 90% tumor-take and a 5-day LC latency. IP xenograft CD138+ cells are found in liver (subcapsular) and spleen. On day 5, the mice have median serum levels (Q1-Q3) of κ LC and β2M of 2.37μg/mL (1.68-3.32) and 1.56ρg/mL (0.58-5.38), values that strongly correlate (r=0.76, P<<0.01), as do the day 5 κ LC and FLUX values (r=0.88, P<<0.01). In 3 cohorts of 10 mice each, 5 siNT and 5 siIGKC per cohort, there were no differences in day 5 κ LC, FLUX and β2M, or in day 8 β2M, between the siNT and siIGKC groups (Table 1). On day 8 after 3 IP injections, the ratio of the medians of κ LCday 8/κ LCday 5 x 100% was lower in siIGKC mice (161% versus 264%) and trends towards significant reductions in κ LC with siIGKC were observed (Table 1). In a paired comparison of the means of the groups in the 3 cohorts the 33% reduction in κ LC with siIGKC was significant (Table 1). On day 8 there were no differences in the weights or behaviour of the mice.
CONCLUSIONS : We have previously shown that siIGKC, a pool of siRNA directed at consensus sequences in the κ LC constant region gene, can significantly reduce κ LC production in clonal plasma cells from patients, in human myeloma cell lines, and in vivo in a flank plasmacytoma xenograft model. In this work, we show that 8B-3 is a promising LPN for delivery of siRNA to human plasma cells and, when loaded with siIGKC, can with relative safety significantly reduce circulating κ LC in the NSG JJN3 IP model after 3 daily IP injections despite rapid tumor growth. We also show the utility of the NSG JJN3 IP model for the study of κ LC directed therapies. Extensive work lies ahead to identify and optimize a lead candidate for delivery of siRNA to human bone marrow plasma cells in vivo and to begin systematic pre-clinical safety studies.
Disclosures
Ma: Tufts Medical Center: Patents & Royalties: Patent: 9593332. Wang:Tufts University Medical School: Patents & Royalties: Patent: 9765022. Xu:Tufts University School of Medicine: Patents & Royalties: Patent: 9765022. Comenzo:Tufts Medical Center: Patents & Royalties: Patent: 9593332.
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