Immunomodulation of the donor lung with CRISPR-mediated activation of IL-10 expression

Abstract

Background: Inflammatory injury in the donor lung remains a persistent challenge in lung transplantation that limits donor organ usage and post-transplant outcomes. Inducing immunomodulatory capacity in donor organs could address this unsolved clinical problem. We sought to apply clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) technologies to the donor lung to fine-tune immunomodulatory gene expression, exploring for the first time the therapeutic use of CRISPR-mediated transcriptional activation in the whole donor lung.

Methods: We explored the feasibility of CRISPR-mediated transcriptional upregulation of interleukin 10 (IL-10), a key immunomodulatory cytokine, in vitro and in vivo. We first evaluated the potency, titratability, and multiplexibility of the gene activation in rat and human cell lines. Next, in vivo CRISPR-mediated IL-10 activation was characterized in rat lungs. Finally, the IL-10-activated donor lungs were transplanted into recipient rats to assess the feasibility in a transplant setting.

Results: The targeted transcriptional activation induced robust and titrable IL-10 upregulation in vitro. The combination of guide RNAs also facilitated multiplex gene modulation, that is, simultaneous activation of IL-10 and IL1 receptor antagonist. In vivo profiling demonstrated that adenoviral delivery of Cas9-based activators to the lung was feasible with the use of immunosuppression, which is routinely applied to organ transplant recipients. The transcriptionally modulated donor lungs retained IL-10 upregulation in isogeneic and allogeneic recipients.

Conclusions: Our findings highlight the potential of CRISPR epigenome editing to improve lung transplant outcomes by creating a more favorable immunomodulatory environment in the donor organ, a paradigm that may be extendable to other organ transplants.

Keywords: CRISPR; IL-10; dSaCas9-VPR; gene activation; lung transplant.