Search

Mantle cell lymphoma is an aggressive subtype of non-Hodgkin’s lymphoma that claims the lives of tens of thousands of people every year. Although combination chemotherapy treatments such as CHOP have yielded promising outcomes in the clinic, the development of chemoresistance in patients has limited their long-term success. The lack of in vitro chemoresistance models has limited our ability to understand the mechanisms by which cells develop resistance, and thus our ability to develop novel therapeutics to overcome this issue. Here, we describe the development of a clinically relevant chemoresistant mantle cell lymphoma model using the JeKo-1 cell line. This was achieved through a stepwise treatment selection strategy using gradually increasing concentrations of CHOP. We show that resistant JeKo-1 cells display strong recovery and fast proliferation after treatment with an IC(50) dose of CHOP. We also found that resistant JeKo-1 cells overexpress three oncogenes implicated in the development of mantle cell lymphoma—Cyclin D1, Mcl-1, and Bcl-2—compared to normal JeKo-1 cells. We anticipate that in vitro models such as this one will enable the discovery of new therapeutic strategies for overcoming chemoresistance and improve clinical outcomes in mantle cell lymphoma patients. IMPACT STATEMENT: Mantle cell lymphoma remains one of the deadliest subtypes of non-Hodgkin’s lymphoma, in large part because patients become resistant to frontline chemotherapy. The development of strategies to treat advanced disease will be contingent upon testing in appropriate models. Most in vitro models of resistant mantle cell lymphoma are laboratory grade models that do not recapitulate the low level of chemoresistance typically observed in patients, limiting their utility. This study develops a clinically relevant in vitro model that can be used to establish the mechanisms of resistance and test new therapeutics intended to treat recurrent disease.

In recent years, messenger RNA (mRNA) has come into the spotlight as a versatile therapeutic with the potential to prevent and treat a staggering array of diseases. Billions of dollars have been invested into the commercial development of mRNA drugs, with ongoing clinical trials focused on vaccines (for example, influenza and Zika viruses) and cancer immunotherapy (for example, myeloma, leukemia, and glioblastoma). Although significant progress has been made in the design of in vitro transcribed mRNA that retains potency while minimizing unwanted immune responses, the widespread use of mRNA drugs requires the development of safe and effective drug delivery vehicles. Unfortunately, delivery vehicle development has been stymied by an inadequate understanding of how the molecular properties of a vehicle confer efficacy. Recently, it was found that a class of lipid-like materials, known as lipidoids, can be formulated into lipid nanoparticles (LNPs) that potently deliver a variety of nucleic acids in vitro and in vivo. Here, we investigate the use of these materials for the delivery of messenger RNA both in a variety of cell types and in mice. By synthesizing a library of 400 lipidoids, we show that the material chemistry has a large impact on the mRNA delivery efficacy in vitro. Using a smaller library of 11 lipidoids, we show that LNPs formulated from these materials potently deliver mRNA to a variety of organs in mice. The two most potent materials from the library, 306O10 and 306Oi10, have 10-carbon tails and identical molecular weights, and vary only in that the 306O10 tail is straight and the 306Oi10 tail has a one-carbon branch. Remarkably, this small difference in structure conferred a 10-fold improvement in 306Oi10 efficacy. The enhanced potency of this branched-tail lipidoid was attributed to its strong surface ionization upon entry into the endosomal compartment of target cells. We also show that delivery of the top LNP, 306Oi10, enabled higher levels of protein expression than two gold standard lipids, C12-200 and DLin-MC3-DMA in mice. We also demonstrate that this material is sufficiently potent to encapsulate and deliver three mRNAs of varying lengths within the same formulation. Furthermore, 306Oi10 co-delivered Cas9 mRNA and single guide RNAs (sgRNAs), facilitating CRISPR-mediated gene editing in the livers of mice. Intravenous delivery of this material also did not significantly increase serum cytokine or IgG levels, nor did it cause liver toxicity, as determined by histology. Finally, we explore the impact of mRNA nucleoside modifications on resultant protein expression in mice. Specifically, we probe the influence of the delivery vehicle on the efficacy of mRNA modifications by delivering a variety of modified mRNAs using four LNPs which target either the liver, spleen, or lungs. We demonstrate that the delivery vehicle has a large impact on the efficacy of modified mRNAs. We also show that the majority of protein expression enhancement occurs in the spleen due to enhanced transfection as well as increased translation in cells in this organ.