CRISPR-based ex vivo gene editing of donor organs

Abstract

Donor organs are frequently discarded because of concerns about quality or pathogen risk, challenges that could be mitigated through ex vivo gene editing or silencing during machine perfusion. Here, we discuss the development of CRISPR-based approaches for ex vivo gene silencing in human donor organs, from proof of concept in kidney biopsies to the challenges of organ-scale translation.

Organ transplantation is constrained not only by the scarcity of donor organs but also by the limited ability to repair donated organs; for example, many donated kidneys must be discarded because of concerns about quality or expected graft function^1,2. In particular, early graft injury and pathogen-related complications remain major barriers to the long-term clinical success of organ transplantation. Transplant surgeons thus routinely face decisions about whether to accept or decline marginal donor organs, knowing that patients on the waitlist are running out of time. Even organs that are successfully transplanted carry risks from latent viruses, such as cytomegalovirus and human polyomavirus 1, which can reactivate under the immunosuppression regime required to prevent rejection and cause serious complications years after transplantation. Therefore, eliminating these viruses ex vivo would reduce immunogenicity and extend graft longevity. In addition, ischaemia–reperfusion injury, the tissue damage that occurs when blood flow is restored after a period of oxygen deprivation, can compromise the quality of donor organs.

Machine perfusion can preserve donor organs ex vivo by restoring circulation, controlling temperature, enabling gas exchange and delivering pharmacological agents^3–6. Importantly, machine perfusion provides direct access to the donor organ ex vivo in a controlled environment, thereby offering a window to alter organ biology using gene editing tools, such as CRISPR–Cas. Such tools could be applied for targeted DNA editing or RNA silencing to suppress ischaemia–reperfusion injury pathways and reduce viral burden, ultimately preventing early graft injury and improving organ quality prior to transplantation (Fig. 1).

Fig. 1 |. Ex vivo gene silencing in donor organs using CRISPR–Cas during machine perfusion.

A donor organ connected to a machine perfusion circuit is treated with CRISPR–Cas, which is administered in the circulating perfusate, during the ex vivo window (37 °C) prior to transplantation. This could allow RNA knockdown to eliminate viral contamination and modification of ischaemia injury-related pathways.

We founded CasNx in 2024 to develop CRISPR-based therapies for ex vivo organ treatment during machine perfusion.

ψDNA-guided Cas12a for organ treatment

As one instance of this approach, at the University of Florida, we developed a pseudo-guide DNA (ψDNA)-guided Cas12a platform, in which the guide RNA is replaced with a short DNA oligonucleotide to direct sequence-specific RNA silencing. Such DNA-guided Cas12a can achieve programmable RNA knockdown in cultured cells. Importantly, DNA guides benefit from their chemical stability, resistance to nuclease degradation in biological fluids and lower costs to synthesize at scale when compared with guide RNA⁷.

These properties are especially relevant in ex vivo organ treatment. During machine perfusion, the platform must remain active at physiological temperature over prolonged exposure periods, function in the biochemical environment of perfusate and reach target cells within intact tissue architecture. Moreover, the platform must reproducibly function across donor organs that differ in quality, ischaemic history and perfusion behavior^8,9.

Proof of concept in human organs

To test the CRISPR platform in human organs, we established a collaboration with an organ procurement organization to obtain biopsy samples from human kidneys and livers that had been declined for transplantation. CRISPR performs efficiently at a temperature of 37 °C; however, sustaining tissue viability at that temperature for the extended period needed for meaningful silencing proved challenging. Accordingly, initial experiments failed because tissue integrity deteriorated before a measurable effect could be detected. Therefore, we iterated through multiple perfusate formulations to support viability during the treatment window. In addition, because we did not have access to a clinical perfusion device, we created a bench-top peristaltic pump using basic lab tools.

With these adjustments, we achieved substantial target RNA knockdown in human kidney biopsies ex vivo, demonstrating that the platform can drive a measurable molecular effect in human organ tissue. However, to translate results from biopsies to whole human organs, the formulation and delivery conditions must be further optimized.

From biopsy to whole-organ treatment

To achieve whole-organ treatment, delivery, reproducibility, safety and manufacturing must be considered. The relevant cellular targets vary with the intended intervention. For antiviral applications, CRISPR would need to reach the specific donor-organ cells that contain intracellular viral RNA; for example, in kidney grafts, renal tubular epithelial and urothelial compartments are typically infected by human polyomavirus 1, whereas cytomegalovirus primarily infects endothelial, epithelial and stromal cells. To reduce graft immunogenicity or ischaemia–reperfusion injury, the most important targets are probably vascular endothelial cells, which form the immunological interface between the graft and recipient circulation, together with parenchymal cells that propagate inflammatory, stress and injury pathways after transplantation.

The CRISPR platform can be delivered in the perfusate, which flows through the vasculature; however, relevant target cells might lie underneath the endothelial interface. Therefore, we need to assess whether CRISPR–Cas12a complexes can distribute broadly through intact organ architecture to achieve meaningful and uniform silencing. Thus, an essential translational question is whether the CRISPR complexes can distribute from the perfusate into intact organ tissue sufficiently to silence viral, inflammatory or injury-related transcripts in the cellular compartments that drive post-transplant complications. This challenge is compounded by donor-organ heterogeneity, including ischaemia time, donor age, organ quality and local perfusion dynamics, which may influence tissue penetration and reproducibility. Here, heterogeneity matters because different regions of the organ may receive different levels of perfusate exposure owing to variation in blood flow, tissue damage and swelling, leading to uneven CRISPR delivery and variable silencing. This can be tackled by optimizing perfusion conditions and measuring delivery and knockdown across multiple regions of the organ.

Importantly, transient RNA knockdown must persist through transplantation and the early post-operative period. In addition, longitudinal organ-level off-target assessment is required, as results cannot be extrapolated from cell culture experiments. As the organs are genetically modulated ex vivo, systemic recipient exposure might be less of a concern, compared with in vivo gene editing approaches; however, residual Cas12a protein and ψDNA in the graft need to be characterized and controlled. Furthermore, an ex vivo therapeutic product requires scalable, consistent manufacturing of good manufacturing practice (GMP)-grade Cas12a protein, synthetic ψDNA guides and delivery agents. We are primarily exploring lipid nanoparticles. In addition, engineered virus-like particles and viral vectors may be used depending on organ and target cell.

Clinical path and market entry

If organ-scale knockdown can be demonstrated, ex vivo CRISPR treatment may have an important translational advantage as it can be integrated into transplant workflows. The likely early use case is improvement of organ quality and usability in perfusion workflows, for example, by suppressing injury–response pathways in marginal organs. One example is transient silencing of p53, a pro-apoptotic mediator, to reduce renal ischaemia–reperfusion injury¹⁰.

The US regulatory pathway will depend on the final product configuration and therapeutic claims, probably involving the Center for Biologics Evaluation and Research. Ex vivo CRISPR cell therapies, such as CASGEVY, provide only partial precedents, because donor-organ treatment during perfusion raises distinct questions around graft assessment, release criteria, residual reagent characterization and demonstration of clinical benefit. From a reimbursement perspective, adoption will depend on whether transplant centres view the added step as justified by improvements in organ quality or clinical outcomes.

Lessons from building the platform

Several lessons have shaped our approach. First, scientific novelty is necessary but not sufficient. Technologies can be mechanistically sound but fail if the development path is impractical. In transplantation, the key question is not whether an intervention works in principle, but whether it can be integrated into the constraints of procurement timelines, organ handling and clinical decision-making.

Second, access shapes innovation. We started with discarded human organs and improvised perfusion hardware because those were the resources available to us. At early stages, having access to an adequate biological system can matter more than having ideal infrastructure.

Third, platform companies often move on multiple timescales. While ex vivo organ treatment is a long-horizon translational effort, we are simultaneously pursuing rapid CRISPR–Cas-based pathogen diagnostics that can progress more quickly. Working across both efforts has influenced how we think about iteration, validation and company survival.

Finally, ex vivo organ engineering is inherently interdisciplinary. Molecular engineering, perfusion practice and transplant surgery usually sit in different professional worlds. Progress depends on integrating those perspectives early rather than sequentially. That convergence created CasNx, founded by a transplant surgeon, a CRISPR researcher, and a chemical engineer.

Related links

CASGEVY: https://www.fda.gov/news-events/press-announcements/fda-approves-first-gene-therapies-treat-patients-sickle-cell-disease