Knockdown of Pathologic Immuoglobulin Light Chain Protein by Small Interfering RNA Molecules.
Authors: Phipps Jonathan E , Foster James S , Kestler Daniel P , Donnell Robert , Solomon Alan , Wall Jonathan S 
Published: October 2019

Abstract
Abstract 4407

Introduction
A major contributing factor to the morbidity and mortality of patients with plasma cell dyscrasias (i.e., light chain deposition disease, light chain amyloidosis (AL), etc.) is the overproduction and deposition of immunoglobulin light chain (LC) protein. Accumulation of insoluble fibrillar or amorphous aggregates within vital organs, particularly the kidneys, contributes to progressive loss of function and failure. Recent reports have shown that reducing levels of precursor LC proteins by 50% correlated with improved prognosis in patients with AL. Current therapies to suppress the production of monoclonal free LC rely on conventional or high dose myeloablative chemotherapeutic intervention. These therapies carry inherent risk and may not be appropriate in every situation, thus there is a need to employ alternative or adjuvant therapeutic approaches. To this end, we have investigated the ability of small interfering RNA molecules to decrease production of LC mRNA and protein secretion in both a synthetic and naturally occurring myeloma cell line. We report here that exposure of these lines to small interfering RNA (siRNA) oligonucleotides inhibited LC production in a specific and non-cytotoxic manner.

Methods
SP2/O mouse myeloma cells were stably transfected with a construct encoding a human λ6 LC protein (Wil) and designated SP2/O-1 for this study. The human λ2 producing cell line RPMI 8226 was purchased from ATCC. For both lines, LC mRNA was sequenced and used to design siRNA molecules targeting the V-, J-, or C-region of the LC. Cells were cultured for 1-3 d with sequence-specific siRNA, sham siRNA, or with media alone after which time LC mRNA levels were assessed by real-time PCR, and cellular or secreted LC protein concentrations were measured using flow cytometry or ELISA, respectively.

Results
Treatment with siRNA was well tolerated and no significant reduction in cell viability was observed in either the SP2/O-1 or RPMI 8226 cells at any of the timepoints assayed. Exposure of either SP2/O-1 or 8226 cells to siRNAs targeting the LC resulted in reductions of mRNA as compared to sham siRNA-treated controls. Flow cytometric analysis of 8226 cells immunostained with anti-LC antibodies at 48 h post transfection, indicated that ∼63% and 83% of the total cell number contained reduced amounts of cytoplasmic LC following treatment with V-, or C- region-specific siRNA, respectively. The 8226 LC protein was also significantly reduced by 20-60% in culture supernatants over the 72h period in cells exposed to experimental siRNAs as compared with sham-treated cells. Similarly, treating the SP2/O-1 cells with siRNAs led to significant (20-52%; P < 0.05) reductions in secreted LC Wil over 72 h as compared to sham-siRNA treatments.

Conclusions
We demonstrate here that siRNA provides a viable option for preventing the synthesis and thus secretion of pathologic LC protein using two cell lines, and that this reduction is carried out in a specific and non-cytotoxic manner. These results provide support for the potential use of siRNA to decrease the secretion of pathologic LC proteins in patients diagnosed with plasma cell dyscrasias. Our future goal will be to evaluate the action of siRNAs on LC production in vivo using a murine model of the disease.

Disclosures:
No relevant conflicts of interest to declare.