Leveraging Rational Protein Engineering to Improve mRNA Therapeutics.

Messenger RNA (mRNA) is a promising new class of therapeutics that has potential for treatment of diseases in fields such as immunology, oncology, vaccines, and inborn errors of metabolism. mRNA therapy has several advantages over DNA-based gene therapy, including the lack of the need for nuclear import and transcription, as well as limited possibility of genomic integration. One drawback of mRNA therapy, especially in cases such as metabolic disorders where repeated dosing will be necessary, is the relatively short in vivo half-life of mRNA (∼6-12 h). We hypothesize that protein engineering designed to improve translation, yielding longer-lasting protein, or modifications that would increase enzymatic activity would be helpful in alleviating this issue. In this study, we present two examples where sequence engineering improved the expression and duration, as well as enzymatic activity of target proteins in vitro. We then confirmed these findings in wild-type mice. This work shows that rational engineering of proteins can lead to improved therapies in vivo.

Plasmacytoid dendritic cells, interferon signaling, and FcγR contribute to pathogenesis and therapeutic response in childhood immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder of childhood characterized by immune-mediated destruction of platelets. The mechanisms underlying the pathogenesis of ITP and the therapeutic efficacy of intravenous immunoglobulins (IVIG) in this disorder remain unclear. We show that monocytes from patients with ITP have a distinct gene expression profile, with increased expression of type I interferon response (IR) genes. Plasma from ITP patients had increased levels of several cytokines indicative of immune activation, including an increase in interferon-α. ITP patients also had an increase in plasmacytoid dendritic cells (pDCs) compared to healthy donors. Therapy-induced remission of ITP was associated with abrogation of the IR gene signature in monocytes without reduction in the levels of circulating interferon-α itself. IVIG altered the ratio of activating/inhibitory Fcγ receptors (FcγRs) in vivo primarily by reducing FcγRIII (CD16). The engagement of activating FcγRs was required for IVIG-mediated abrogation of monocyte response to exogenous interferon-α in culture. Moreover, plasma from ITP patients led to activation of monocytes and myeloid DCs in culture with an increase in T cell stimulatory capacity; this activation depended on the engagement of activating FcγRs and interferon-α receptor (IFNAR) and was inhibited by antibody-mediated blockade of these pathways. These data point to a central role of type I interferon in the pathogenesis of ITP and suggest targeting pDCs and blockade of IR as potential therapeutic approaches in this disorder. They also provide evidence for the capacity of IVIG to extinguish IR in vivo, which may contribute to its effects in autoimmunity.