The purpose of the present study was to explore the use of siRNAs as a potential treatment for AL amyloidosis in vitro
and in vivo
. It has been shown that a reduction in serum LC levels leads to organ function improvement in patients with AL amyloidosis24, 25
. Thus, it is plausible that reducing LC levels through an siRNA-mediated mechanism, alone or in combination with other therapies, could halt AL disease progression and enable patients to recover organ function.
We designed siRNAs that were directed at either the variable (VL) or the constant (CL) regions of the LC in order to reduce amyloidogenic precursor protein expression. The basis of our strategy for targeting the unique VL domain was to develop sequence- and patient-specific siRNAs for treatment directed specifically at the amyloidogenic LC; one aim of this approach was to spare other LCs, i.e. non-amyloidogenic proteins from same LC family that might contribute to immune competence in a patient. Alternatively, by targeting shared sequences in the CL domain, siRNAs might target many populations of LC proteins and could be effective for many patients. All three siRNAs, designed against the amyloidogenic AL-009-κ1 protein, caused a significant reduction in LC levels compared to controls in vitro. The most potent siRNAs had IC50s between 1 and 10 nM, and were effective at reducing LC levels by 75–80% at concentrations of 20 nM. In kinetic studies, LC reduction in vitro was maximal at 48–96 hours and persisted for at least 7 days, suggesting a weekly dosing regimen might be feasible.
In addition to our in vitro
studies, we sought to provide proof of concept in vivo
. For these studies, an innovative electroporation technique was used for delivery of siRNA to plasmacytomas secreting human amyloidogenic LC. In this model, we were able to reduce mRNA and protein levels in the plasmacytomas and also in the circulation with a single dose of siRNA. In these short-term experiments limited by plasmacytoma growth kinetics, we were not able to study the effects of siRNA treatment on amorphous LC deposition in the kidney or on the LC-induced bradycardia. Nonetheless, these results provide a proof-of-concept for this approach of reducing LC message and protein levels. Another recent publication has made use of siRNA against AL LC in vitro 26
, and antisense RNA against LC has also been explored using multiple myeloma cell lines 27
Advantages of the siRNA approach include high specificity and low toxicity. There is minimal sequence homology between immunoglobulin LC and other genes; therefore, there should be few off target effects on other mRNAs and proteins. A consequence of LC knockdown could be a reduction in the humoral immune repertoire. By testing siRNA targeting both the variable and constant domains of the LC, we explored the balance between a sequence-specific, patient-specific therapy and subfamily therapy. VL-targeted sequences would affect only the pathologic LC sequence, but each siRNA would be different and would have to be designed and tested for each patient. Development of CL-directed siRNA sequences would have broader specificity, but still should not significantly impair immune responses, as anti-pathogen antibody responses are not confined to particular subfamilies. This is an advantage over current chemotherapy and non-specific immunomodulatory therapy, which is broadly myelo- and immuno-suppressive.
While our studies indicate that a LC-directed siRNA approach to treatment for AL amyloidosis is feasible, several issues are yet to be addressed in translating this technology to a clinical application. Patients will need to have their amyloidogenic LC isotype, subfamily, and possibly even specific sequence determined to select an appropriate siRNA. In our database of LC sequences, AL-Base, 26% (124/477) of the AL light chain sequences are κ and of those 81% (100/124) are κ1 light chain with 40% (47/124) derived from the κ1 018 germline gene donor19
. Thus, a single effective κ1 siRNA targeted toward the framework regions of the variable domain could be used for almost half of κ patients. Patients with λ light chain AL amyloidosis have more genetic diversity. Thus, the specific light chain subfamily would have to be targeted, unless an siRNA could be identified that targeted all λ V regions. This seems an almost impossible requirement due to the sequence dissimilarity of the framework regions. In the lambda family, λ1, λ2, λ3, and λ6 are found at similar frequencies (19.4%, 15.7%, 19.9%, and 17.6% respectively) in our AL clinic population19
. Though not been synthesized or validated, several siRNAs have been designed in silico
to target the λ constant domains from λ1, λ2, λ3, and λ6, the highest represented AL λ light chain families. These siRNAs have 100% identity in the seed region and minor mismatches in the remainder of the oligonucleotides28
, and can be tested in future experiments in vitro
. Additionally, of the lambda sequences, λ6 is of particular interest because it is rarely found in non-AL settings and represents <2% of sequences from patients with multiple myeloma or from polyclonal B cells. Preliminary results from in vitro
studies indicate that, like those siRNAs targeting κ1 LC, λ6 targeting siRNAs are effective in reducing LC mRNA and protein levels (data not shown).
Further preclinical studies aimed at optimizing the pharmacokinetics and improving the delivery of LC-directed siRNA are necessary prior to testing in patients with AL amyloidosis. We have shown in vitro
that siRNA knockdown can persist for up to 7 days. This suggests that intermittent therapy is possible in vivo
. Nonetheless, it is likely that siRNA oligonucleotides will need to be altered to extend their duration in the circulation as unmodified oligonucleotides typically have a serum half-life of minutes to hours. By modifying the oligonucleotides, half-life can be extended to days29
. Approaches include encapsulating the oligonucleotides in liposomes, nanoparticles, or other substances that are non-toxic and non-immunogenic30
Another requirement in the development of siRNA as an AL therapeutic is for a delivery system that specifically targets plasma cells in the bone marrow, since amyloidogenic plasmacytomas are rare in patients. Possible technologies to direct siRNA delivery include conjugation with aptamers, lipophilic substances, peptides, or antibodies31
. For example, antibodies against CD138 could be employed for targeted delivery to plasma cells, although CD138 (syndecan-1), a heparan sulfate-bearing proteoglycan, is also expressed on epithelial cells32
. There would presumably be no effect of targeting LC siRNA to epithelial cells; however, these cells would provide a “sink” for the injected siRNA.
In summary, siRNA directed against amyloidogenic light chain can reduce light chain synthesis and secretion in cells in culture and in vivo. With the development of improved delivery and targeting techniques, siRNA therapeutics hold promise as an effective and less toxic approach for treatment of AL amyloidosis and possibly as a therapy in other protein misfolding and deposition diseases.