| AL (Amyloid Light Chain) amyloidosis is a rare, sporadic human hematologic disorder characterized by the accumulation of misfolded monoclonal immunoglobulin light chains as fibrillar protein deposits. The goal of this project was to test innovative therapeutics and develop better models for AL amyloidosis. In the first aim, we hypothesized that siRNA could be used to downregulate the expression of amyloidogenic light chains in in vitro and in vivo models of AL amyloidosis as a novel treatment for this disorder. In vitro, five of eight siRNAs targeting amyloidogenic light chains kappa1-AL-009 or lambda6-AL-080 were effective in reducing protein levels. In kinetic and dose response studies, siRNAs reduced protein levels by ≥75% for up to five or more days. These were tested using in vivo electroporation in a mouse model based upon transplantation of light chain-secreting plasmacytoma cells, and found to reduce plasma cell expression mRNA and protein expression as well as serum levels of light chains. These experiments provide proof-of-concept of this approach. For the second aim, we have engineered two novel constructs that will drive light chain expression in terminally differentiating plasma cells and better recapitulate the human disorder. The constructs encodes kappa promoter/enhancer sequences and human c-Myc, but utilizes a stop codon which prevents translation of Myc until the B cell is fully differentiated and undergoes somatic hypermutation (SHM). We have added sequences encoding amyloidogenic light chains 3' to c-Myc, linked by a self-cleaving peptide sequence, to produce clonally expanded transformed plasma cells that will secrete human amyloidogenic light chains. 125 C57BL/6 oocytes have been microinjected with the transgene construct and transferred into pseudo-pregnant mice. Sixteen potential Vk*Myc-F2A-lambda6 founders were born, three of which were shown to be transgene positive. Ten Vk*Myc-F2A-kappa1 founders have been born. |
