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C. Russell Y. Cruz, MD, PhD
Senior Editor, ISCT Telegraft
Associate Professor, Children's National Hospital
United States

Cell and gene therapies have slowly forced a shift from our understanding of pharmacology. These therapies are no longer passive molecules, but complex, dynamic systems with no straightforward relationship to dose, pharmacokinetics, route of administration, and drug compartments. Recent studies may make us rethink one other element: time.

Time, it seems, is an active variable too. Biology, after all, is strongly rhythmic.

Recent studies continue to make us rethink our age-old concepts of pharmacology, including how they apply to biological therapies like those we investigate in our field. Leukocyte trafficking,¹ antigen presentation,² and cytokine secretion³ all vary according to the time of day; and known circadian-influenced systems like glucocorticoids,⁴ catecholamines,⁵ and body temperature⁶ play a role in immune regulation.  It is thus not surprising how chronobiology can be an important parameter when determining the efficacy of cell and gene therapies. Perhaps it’s time to rethink the value of, well, time, in cell and gene therapy.

Current evidence, for example, underscores the importance of timing in CAR T-cell therapy. Work by Lyons et al showed that morning infusions of CAR T cells lead to better survival and lower toxicity. Each hour earlier in the day boosts outcomes and reduces severe ICANs. This effect is most evident in women and those treated with CD19 CAR T cells.⁷ This dependence may involve circadian control of immune activity or daytime variability in tumor accessibility.

Indeed, other forms of immunotherapy seem to follow the same relationship with infusion time. A retrospective study by Huang et al⁸ also hints that giving immunochemotherapy earlier in the day may enhance its effectiveness. The randomized phase 3 LungTIME-C01 trial enrolled 210 patients with advanced non-small cell lung cancer who had not received prior treatment and did not have driver mutations. After a median follow-up of nearly 29 months, patients treated earlier in the day had a median PFS of 11.3 months compared to 5.7 months for those treated later, with a significant reduction in the risk of disease progression. Median OS was also longer for the early group (28.0 months versus 16.8 months). Of note, in the early treatment group, levels of circulating CD8+ T cells rose, while in the late group, they declined. The ratio of activated to exhausted CD8+ T cells was also higher in the early group.⁸ A similar earlier study of time of infusion of checkpoint blockers showed that giving these inhibitors earlier in the day in patients with metastatic renal cell carcinoma was linked to possibly better response and survival.⁹

Because DNA repair pathways can also be potentially influenced by diurnal cycles, gene therapies may also benefit from optimizing time of infusion. Double stranded break repair appears to be regulated by circadian rhythms,¹⁰ and researchers are beginning to explore how manipulating circadian-regulated DNA repair processes could optimize therapeutic strategies, particularly where timing of the intervention may influence the precision of genomic edits by CRISPR for example.¹⁰

Beyond time of infusion, we can also potentially use circadian clocks as a means to further engineer cells. Using promoters that respond to circadian cues, for example, will help us design cells whose actions are orchestrated in time. For example, we can restrict CAR signaling to specific windows of the day (to occur during maximal immune activation), or use programmed cytokine release to coincide with the natural cytokine rhythm to efficiently minimize exhaustion and toxicity.

If our understanding of how time influences biological therapies continues to mature, we may need to start thinking of restructuring clinical operations. Infusion centers, apheresis units, and manufacturing facilities are, necessarily, modeled to follow current work schedules, and seldom take biological rhythms into account. Infusions are scheduled during business hours when staff are there. But if evidence continues to support the value of time-optimized treatment schedules, infusion services will need to be restructured to enable dosing during “biologic prime time.” Practically, this may require more in-patient procedures or outpatient services that open in the early morning. Prescriptions need to accommodate time of infusion as part of the instructions. Additionally, educating both patients and clinicians about the importance of timing will be crucial for ensuring adherence and acceptance. Ultimately, this is not just a matter of tweaking procedures, but a systems-level redesign challenge for cell and gene therapy-centric hospitals.

To get to this level of understanding, unfortunately, is challenging. So far, most trials (and even preclinical work) do not consider time as a variable. Most protocols neither randomize nor stratify by time of infusion, fail to collect serial immune and cytokine measurements dense enough to map biological rhythms, and do not record light exposure, sleep patterns, or shift work status—all factors that can alter circadian rhythms. Achieving a deeper understanding of time’s role in cell and gene therapy is hindered by this lack of information. A practical roadmap for the field would need to incorporate such studies in ongoing trials. We also need to develop preclinical models that simulate circadian cues. The added complexity should hopefully be worthwhile in the end.

Reimagining time as a critical pharmacologic element may transform our current cell and gene therapy approaches. There is a possible future where we specify circadian phase as part of a therapy alongside dose. We have successfully incorporated spatial data into analysis of the efficacy of our cellular products (with spatial transcriptomics etc). The next generation of biologics may need to incorporate when they are expected to work optimally.

Ultimately, embracing time as an active variable may be the key to unlocking the full potential of living therapeutics.

references

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7. Kafka MS, Benedito MA, Roth RH, Steele LK, Wolfe WW, Catravas GN. Circadian rhythms in catecholamine metabolites and cyclic nucleotide production. Chronobiol Int. 1986;3(2):101-15. doi: 10.3109/07420528609066354. PMID: 2824067.
8. Patrick G Lyons, Emily Gill, Prisha Kumar, Melissa Beasly, Brenna Park-Egan, Zulfiqar Lokhandwala, Brandon Hayes-Lattin, Catherine L Hough, Nathan Singh, Guy Hazan, Huram Mok, Janice Huss, Colleen A McEvoy, Jeffrey Haspel. Diurnal rhythms in chimeric antigen receptor T cell performance: an observational study of 670 patients. medRxiv 2025.05.15.25327612; doi: https://doi.org/10.1101/2025.05.15.25327612
9. Huang Z, Zeng L, Ruan Z, Zeng Q, Yan H, Jiang W, Xiong Y, Zhou C, Yang H, Liu L, Dai J, Zou N, Xu S, Wang Y, Wang Z, Deng J, Chen X, Wang J, Xiang H, Li X, Duchemann B, Chen G, Xia Y, Mok T, Scheiermann C, Lévi F, Yang N, Zhang Y. Time-of-day immunochemotherapy in non-small cell lung cancer: a randomized phase 3 trial. Nat Med. 2026 Feb 2. doi: 10.1038/s41591-025-04181-w. Epub ahead of print. PMID: 41629425.
10. Dizman N, Govindarajan A, Zengin ZB, Meza L, Tripathi N, Sayegh N, Castro DV, Chan EH, Lee KO, Prajapati S, Feng M, Loo V, Pace M, O'Brien S, Bailey E, Barragan-Carrillo R, Chehrazi-Raffle A, Hsu J, Li X, Agarwal N, Pal SK. Association Between Time-of-Day of Immune Checkpoint Blockade Administration and Outcomes in Metastatic Renal Cell Carcinoma. Clin Genitourin Cancer. 2023 Oct;21(5):530-536. doi: 10.1016/j.clgc.2023.06.004. Epub 2023 Jun 25. PMID: 37495481.

11. Chen Y, Xu X, Chen Z, Chen L, Jiang Y, Mao Z. Circadian factors CLOCK and BMAL1 promote nonhomologous end joining and antagonize cellular senescence. Life Med. 2024 Feb 4;3(2):lnae006. doi: 10.1093/lifemedi/lnae006. PMID: 39872667; PMCID: PMC11749561.