Date of Award
Doctor of Philosophy
Comparative and Experimental Medicine
Jonathan S. Wall
Daniel P. Kestler, Robert Donnell, Steven J. Kennel
A major contributor to mortality in patients with plasma cell dyscrasias (PCDs); i.e., multiple myeloma, light chain deposition disease and AL amyloidosis is the deposition as insoluble aggregates of monoclonal immunoglobulin light chain proteins (LC) in the kidneys and other organs. Currently anti-plasma cell chemotherapies are used to reduce LC synthesis, and slow deposition. While effective, these treatments are toxic, non-specific, expensive, and might not be appropriate in all cases, making the identification of an alternate means of reducing toxic LC species desirable. To this end, we have investigated whether RNA interference (RNAi) could achieve these goals.
Human (RPMI 8226, Bur) and transfected mouse myeloma (SP2/O-lambda 6) cells which produce measureable quantities of human LC protein were used as model systems for testing the efficacy of both synthetic small interfering RNAs (siRNAs) and short hairpin RNA (shRNA) expression vectors in reducing LC synthesis. Sequencing of LC genes provided the basis for design of siRNA duplexes targeting either the variable (V) or joining (J) regions of individual LCs, or the constant (C) region of either kappa or lambda LC isotypes. Myeloma lines were transfected with siRNAs using lipid-based transfection media. Cells receiving non-silencing siRNAs served as controls. Exposure of myeloma lines to siRNAs was well tolerated and no cytotoxicity was observed. LC mRNA expression was shown to be reduced ≥40% in 8226 and SP2/O- lambda 6 cell lines receiving siRNA treatment as compared with untreated controls. Exposure to siRNAs was also effective in significantly reducing both intracellular and secreted LC protein levels in cell lines tested as evidenced by flow cytometry or enzyme-linked immunosorbent assays (ELISAs).
Effective siRNA nucleotide sequences were used to generate shRNA cassettes which were ligated into lentiviral expression vectors under the control of the RNA polymerase III promoter, U6. These expression systems were used to generate replication incompetent lentiviral particles. Exposure of 8226 to lentiviral particles resulted in significant knockdown of LC mRNA and protein both in vitro and in xenograft tumor bearing immune compromised mice. These results provide positive evidence for the ability of RNAi based approaches to reduce LC secretion in models of PCD.
Phipps, Jonathan E, "Toward Personalized Medicine: The potential role of RNA interference in Plasma Cell Dyscrasia. " PhD diss., University of Tennessee, 2011.