The complement system is a pivotal player in multiple hematological conditions. Antibody blockade of the C5 component of complement has been approved as a treatment for both paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic-uremic syndrome (aHUS), validating C5 as an important therapeutic target.

Recently, we developed a robust RNAi therapeutics platform for the delivery of siRNAs to the liver using trivalent GalNAc conjugates, enabling silencing of hepatocyte-expressed genes following subcutaneous (SC) injection. The liver is a major source of C5 and other complement pathway components. The GalNAc conjugate technology allows rapid development of siRNAs targeting multiple members of the complement cascade and evaluation of their silencing in pre-clinical models.

To examine the utility of the siRNA approach for targeting complement pathway components we designed and synthesized GalNAc conjugated siRNAs targeting rodent, primate and human C5. Potent siRNA duplexes, showing greater than 95% silencing of C5 mRNA were selected using in vitro screening in human cell lines and mouse primary hepatocytes. C5 silencing and serum hemolytic activity inhibition were evaluated in rodents using single and multi-dose SC treatment regimens. A C5-targeting siRNA conjugate demonstrated a single dose ED50 of 0.625 mg/kg in the mouse with greater than 90% silencing of serum C5 achievable at higher doses. Serum C5 silencing was durable, with recovery starting two weeks after a single SC injection We went on to examine the efficacy of C5 silencing in the rat and observed robust lowering of serum C5 with 2.5 and 5 mg/kg multi-dose regimens, resulting in up to ∼90% inhibition of complement classical pathway hemolytic activity. Evaluation of the translation of this approach to higher species is in progress. Since PNH erythrocyte lysis is thought to be mediated by the activation of the alternative pathway of complement we initiated work on the development of siRNA conjugates targeting Factor B, an essential component of the alternative pathway C3 convertase. siRNAs targeting rodent, primate and human Factor B were identified by in vitro screening and demonstrate >90% silencing of Factor B mRNA in human cell lines and primary mouse hepatocytes. Evaluation of Factor B silencing in rodent models is ongoing.

siRNA-mediated silencing of liver-derived complement components is a promising novel therapeutic approach for inhibiting the activity of C5 and other complement pathway targets, with the potential to enable subcutaneous treatment for patients with PNH and related disorders.


Borodovsky:Alnylam: Employment. Yucius:Alnylam: Employment. Sprague:Alnylam: Employment. Butler:Alnylam: Employment. Fishman:Alnylam: Employment. Nguyen:Alnylam: Employment. Vaishnaw:Alnylam: Employment. Maier:Alnylam: Employment. Kallanthottathil:Alnylam: Employment. Kuchimanchi:Alnylam: Employment. Manoharan:Alnylam: Employment. Meyers:Alnylam: Employment. Fitzgerald:Alnylam: Employment.

Author notes


Asterisk with author names denotes non-ASH members.