Enhancing cell-based therapies with synthetic gene circuits responsive to molecular stimuli.

De novo-designed proteins1-3 hold great promise as building blocks for synthetic circuits, and can complement the use of engineered variants of natural proteins4-7. One such designer protein-degronLOCKR, which is based on 'latching orthogonal cage-key proteins' (LOCKR) technology8-is a switch that degrades a protein of interest in vivo upon induction by a genetically encoded small peptide. Here we leverage the plug-and-play nature of degronLOCKR to implement feedback control of endogenous signalling pathways and synthetic gene circuits. We first generate synthetic negative and positive feedback in the yeast mating pathway by fusing degronLOCKR to endogenous signalling molecules, illustrating the ease with which this strategy can be used to rewire complex endogenous pathways. We next evaluate feedback control mediated by degronLOCKR on a synthetic gene circuit9, to quantify the feedback capabilities and operational range of the feedback control circuit. The designed nature of degronLOCKR proteins enables simple and rational modifications to tune feedback behaviour in both the synthetic circuit and the mating pathway. The ability to engineer feedback control into living cells represents an important milestone in achieving the full potential of synthetic biology10,11,12. More broadly, this work demonstrates the large and untapped potential of de novo design of proteins for generating tools that implement complex synthetic functionalities in cells for biotechnological and therapeutic applications.

Graves' disease is an autoimmune disorder that causes hyperthyroidism because of autoantibodies that bind to the thyroid-stimulating hormone receptor (TSHR) on the thyroid gland, triggering thyroid hormone release. The physiological control of thyroid hormone homeostasis by the feedback loops involving the hypothalamus-pituitary-thyroid axis is disrupted by these stimulating autoantibodies. To reset the endogenous thyrotrophic feedback control, we designed a synthetic mammalian gene circuit that maintains thyroid hormone homeostasis by monitoring thyroid hormone levels and coordinating the expression of a thyroid-stimulating hormone receptor antagonist (TSHAntag), which competitively inhibits the binding of thyroid-stimulating hormone or the human autoantibody to TSHR. This synthetic control device consists of a synthetic thyroid-sensing receptor (TSR), a yeast Gal4 protein/human thyroid receptor-α fusion, which reversibly triggers expression of the TSHAntag gene from TSR-dependent promoters. In hyperthyroid mice, this synthetic circuit sensed pathological thyroid hormone levels and restored the thyrotrophic feedback control of the hypothalamus-pituitary-thyroid axis to euthyroid hormone levels. Therapeutic plug and play gene circuits that restore physiological feedback control in metabolic disorders foster advanced gene- and cell-based therapies.

The ability to safely control transgene expression with simple synthetic gene switches is critical for effective gene- and cell-based therapies. In the present study, the signaling pathway controlled by human transient receptor potential (TRP) melastatin 8 (hTRPM8), a TRP channel family member1, is harnessed to control transgene expression. Human TRPM8 signaling is stimulated by menthol, an innocuous, natural, cooling compound, or by exposure to a cool environment (15-18 °C). By functionally linking hTRPM8-induced signaling to a synthetic promoter containing elements that bind nuclear factor of activated T cells, a synthetic gene circuit was designed that can be adjusted by exposure to either a cool environment or menthol. It was shown that this gene switch is functional in various cell types and human primary cells, as well as in mice implanted with engineered cells. In response to transdermal delivery of menthol, microencapsulated cell implants harboring this gene circuit, coupled to expression of either of two therapeutic proteins, insulin or a modified, activin type IIB, receptor ligand trap protein (mActRIIBECD-hFc), could alleviate hyperglycemia in alloxan-treated mice (a model of type 1 diabetes) or reverse muscle atrophy in dexamethasone-treated mice (a model of muscle wasting), respectively. This fully human-derived orthogonal transgene switch should be amenable to a wide range of clinical applications.

Cytomegalovirus (CMV) Reactivation within in the First Year after Chimeric Antigen Receptor (CAR) T Cell Therapy: Experience from the First Two Years at a Major Cancer Center

Precision medicine in oncology needs to enhance its capabilities to match diagnostic and therapeutic technologies to individual patients. Synthetic biology streamlines the design and construction of functionalized devices through standardization and rational engineering of basic biological elements decoupled from their natural context. Remarkable improvements have opened the prospects for the availability of synthetic devices of enhanced mechanism clarity, robustness, sensitivity, as well as scalability and portability, which might bring new capabilities in precision cancer medicine implementations. In this review, we begin by presenting a brief overview of some of the major advances in the engineering of synthetic genetic circuits aimed to the control of gene expression and operating at the transcriptional, post-transcriptional/translational, and post-translational levels. We then focus on engineering synthetic circuits as an enabling methodology for the successful establishment of precision technologies in oncology. We describe significant advancements in our capabilities to tailor synthetic genetic circuits to specific applications in tumor diagnosis, tumor cell- and gene-based therapy, and drug delivery.

Chimeric antigen receptor (CAR) T cell therapy has emerged as one of the major breakthroughs in cancer immunotherapy in the last decade. Outstanding results in hematological malignancies and encouraging pre-clinical anti-tumor activity against a wide range of solid tumors have made CAR T cells one of the most promising fields for cancer therapies. CAR T cell therapy is currently being investigated in solid tumors including glioblastoma (GBM), a tumor for which survival has only modestly improved over the past decades. CAR T cells targeting EGFRvIII, Her2, or IL-13Rα2 have been tested in GBM, but the first clinical trials have shown modest results, potentially due to GBM heterogeneity and to the presence of an immunosuppressive microenvironment. Until now, the use of autologous T cells to manufacture CAR products has been the norm, but this approach has several disadvantages regarding production time, cost, manufacturing delay and dependence on functional fitness of patient T cells, often reduced by the disease or previous therapies. Universal “off-the-shelf,” or allogeneic, CAR T cells is an alternative that can potentially overcome these issues, and allow for multiple modifications and CAR combinations to target multiple tumor antigens and avoid tumor escape. Advances in genome editing tools, especially via CRISPR/Cas9, might allow overcoming the two main limitations of allogeneic CAR T cells product, i.e., graft-vs.-host disease and host allorejection. Here, we will discuss how allogeneic CAR T cells could allow for multivalent approaches and alteration of the tumor microenvironment, potentially allowing the development of next generation therapies for the treatment of patients with GBM.

While chimeric antigen receptor (CAR) T cells have received FDA approvals in treating hematological malignancies, clinical responses of CAR T cells for solid tumors have been underwhelming. Intense investigation is underway to improve CAR T cells trafficking, persistence and efficacy. Interleukin-12 (IL-12) is a potent inflammatory cytokine that recruits and activates various endogenous and adoptively transferred immune cells, but development of its clinical applications is hindered by toxicity of systemic IL-12 signaling. In this study, we engineered and expressed membrane-bound IL-12 (mbIL12) in CAR T cells to overcome current limitations in CAR T cell and IL-12 therapies. We evaluated CAR T cell function using co-culture assays and preclinical murine models bearing human tumor xenografts. We modeled systemic metastasis of ovarian and breast cancer and assessed the impact of mbIL12 in CAR T cells on targeting regional and systemic diseases. Syngeneic mouse models were used to evaluate toxicity induced by mbIL12 in CAR T cells. We observed a greater number of CAR T cells in the peripheral blood of mice receiving locoregionally delivered CAR T cells engineered with mbIL12, suggesting that mbIL12 improves CAR T cell trafficking and persistence. Furthermore, mice bearing regional and systemic diseases experienced more potent and durable anti-tumor activity when treated with mbIL12 endowed CAR T cells. These results were mirrored in vitro where mbIL12 improved CAR T cells activation, proliferation and IFNg secretion when co-cultured with tumor cells, recapitulating our findings in vivo. Toxicities observed in immune-competent mice receiving systemic IL-12 delivery were not detected in those administered CAR T cells with mbIL12. We demonstrated that mbIL12 enhances anti-tumor function of CAR T cells for treatment of solid tumors. This study also showed that mbIL12 is safe, providing a translatable engineering strategy to improve CAR T cell therapy. IL-12 signaling is well known to modulate function of various immune cells, but the impact of CAR T cells with mbIL12 on immune tumor microenvironments remains to be elucidated. We are currently investigating changes in the immune landscape that CAR T cells with mbIL12 induce. Citation Format: Hee Jun Lee, Yuki Yamaguchi, Jackson Gibson, Cody Cullen, John P. Murad, Anthony K. Park, Isabel Monroy, Cari Young, Lea Christian, Lauren Adkins, Lawrence Stern, Wen-Chung Chang, Catalina Martinez, Stephen J. Forman, Saul J. Priceman. Regional administration of IL-12 endowed CAR T cells effectively targets systemic disease. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4090.

Objective: Stem cell therapy has emerged as a pioneering front in the biomedical domain, characterized by its rapid strides in innovation and progress. Such advancements hold profound implications for public health. Notably, Europe, the United States, Japan, and China have all crafted comprehensive regulatory frameworks to expedite the fruition of stem cell therapy. This article meticulously scrutinizes and evaluates the progression of clinical trials in cell therapy across major global jurisdictions. It delineates the trajectory of regulatory evolution in Europe, the United States, Japan, and China, and systematically collates the pivotal normative guidelines. This article is crafted with the intent to serve as a reference for regulatory agents, enterprises, and professionals in the field. Methods: Stem cell therapy, a paragon of innovative medical technology, heralds a new era of treatment possibilities for hitherto intractable diseases. It is more than just a benchmark of technological advancement; it represents a revolutionary shift in the pharmaceutical landscape. Since Dr.Thomas was awarded the Nobel Prize for his research on bone marrow transplantation in 1990, he has gradually moved from the laboratory to the clinical application stage, bringing new hope for treatment to patients. Stem cell therapy is a type of cell therapy. There are various types of cell therapy, which can be classified into stem cell therapy and immune cell therapy based on their cell sources. They can also be classified into autologous, allogeneic, and heterologous cells based on their donor sources. As a cutting-edge application technology in life science research, cell therapy has shown great therapeutic potential in various disease fields such as malignant tumors, genetic diseases, and chronic degenerative diseases. Multiple cell therapy products have been successfully launched and demonstrated excellent efficacy, among which stem cell therapy stands out due to its long application history and active research trend. Bone marrow/hematopoietic stem cell therapy is the earliest stem cell therapy, mainly used for bone marrow/hematopoietic stem cell transplantation to treat hematological malignancies such as leukemia. Human derived stem cells and their derived therapeutic products, as important regenerative medicine products, have great potential in cell replacement, tissue repair, disease treatment, and other fields. Nearly 70% of the stem cells used in clinical studies are hematopoietic stem cells and mesenchymal stem cells derived from bone marrow, peripheral blood, and umbilical cord. Technical methods include purification, in vitro culture and amplification, drug treatment, or gene modification. Immune cell therapy is a method of treating diseases using immune cells, which was originally mainly used to treat malignant tumors. According to the specificity of immune cell therapy, it can usually be divided into specific immune cell therapy and non-specific immune cell therapy. Specific immune cell therapy includes chimeric antigen receptor T-cell (CAR-T) therapy, T-cell receptor engineered T cell therapy, chimeric antigen receptor natural killer cell therapy (CAR-NK), and DC-CIK immunotherapy. Nonspecific immune cell therapy mainly includes lymphokine activated killer cell therapy and cytokine induced killer cell therapy. In addition, extracellular vesicles, as cell derivatives, have the advantage of non-living properties that are easier to characterize, store, package, and transport than living cells, and their research has shown explosive growth. Extracellular vesicle is a general term for lipid bilayer membrane-bound vesicles released by cells into the extracellular space, with a diameter range of 50-2,000 nanometers. Exosomes are extracellular vesicles with a diameter of 30-150 nanometers. At present, the Food and Drug Administration (FDA) has approved at least 8 extracellular vesicle products to enter phase II/III clinical trials. In recent years, the development of organoid technology, cell lineage tracing technology, single-cell spatial omics-sequencing technology, single-molecule technology, micro proteomics and proximity labeling technology has greatly promoted the progress of stem cell research and laid a good technical reserve for achieving functional organ reconstruction. Results: In the realm of emerging technologies, industry development often precedes regulatory frameworks. The swift progression of innovative stem cell therapy products not only propels medical innovation but also challenges existing regulatory technologies and institutions. Regulatory agencies must, therefore, continually innovate their strategies and technologies. Conclusion: Countries worldwide have crafted regulatory policies and technical evaluation systems tailored to their specific contexts. The variety of stem cell therapy types and applications defined by national regulations reflects this diversity. This article delves into the clinical research status of stem cell therapy through statistical analysis and dissects the regulatory frameworks governing it across nations, offering a valuable resource for stakeholders in this dynamic field.

The gene dosage compensation hypothesis presents a mechanism through which the expression of certain genes is modulated to compensate for differences in the dose of genes when additional chromosomes are present. It is one of the means through which cancer cells actively cope with the potential damaging effects of aneuploidy, a hallmark of most cancers. Dosage compensation arises through several processes, including downregulation or overexpression of specific genes and the relocation of dosage-sensitive genes. In cancer, a majority of compensated genes are generally thought to be regulated at the translational or post-translational level, and include the basic components of a compensation loop, including sensors of gene dosage and modulators of gene expression. Post-translational regulation is mostly undertaken by a general degradation or aggregation of remaining protein subunits of macromolecular complexes. An increasingly important role has also been observed for transcriptional level regulation. This article reviews the process of targeted gene dosage compensation in cancer and other biological conditions, along with the mechanisms by which cells regulate specific genes to restore cellular homeostasis. These mechanisms represent potential targets for the inhibition of dosage compensation of specific genes in aneuploid cancers. This article critically examines the process of targeted gene dosage compensation in cancer and other biological contexts, alongside the criteria for identifying genes subject to dosage compensation and the intricate mechanisms by which cells orchestrate the regulation of specific genes to reinstate cellular homeostasis. Ultimately, our aim is to gain a comprehensive understanding of the intricate nature of a systems-level property. This property hinges upon the kinetic parameters of regulatory motifs, which we have termed "gene dosage sensor loops." These loops have the potential to operate at both the transcriptional and translational levels, thus emerging as promising candidates for the inhibition of dosage compensation in specific genes. Additionally, they represent novel and highly specific therapeutic targets in the context of aneuploid cancer.

The hepatic transport systems mediating bile acid uptake and excretion undergo independent, stage-specific expression during development in the rat. In this study, the mechanisms underlying ontogenic regulation of both the Na(+)-dependent basolateral bile acid transporter and canalicular bile acid transporter/ecto-ATPase were examined. Steady state mRNA levels for the basolateral transporter were less than 20% of adult values prior to birth, increased to 35% on the first postnatal day, and reached adult levels by 1 week of age. This was paralleled by transcription rates, which were low prior to birth, reached 47% by day 1, and were maximal by 1 week of age. Steady state mRNA levels for ecto-ATPase were 12% of adult values prior to birth and showed a 2-fold increase by the first day of life. Thereafter, there was a gradual increase in mRNA for this transporter, with adult levels being reached at 4 weeks of age. Transcription rates paralleled this increment, although adult levels were reached earlier. Surprisingly, for both transporters, the full complement of protein was present well before adult levels of mRNA were reached. The basolateral protein was expressed at 82% of adult levels on the first day of life but was of lower apparent molecular mass (39 kDa), a difference that persisted until 4 weeks of age. N-Glycanase digestion suggested that this difference could be fully accounted for by N-linked glycosylation. The ecto-ATPase protein was present at 33% of adult levels prior to birth, 77% by 1 day, and 84% of adult levels by 1 week of age. Unlike the basolateral transporter, the apparent molecular weight of this protein did not change during development. In summary, the ontogeny of bile acid transporters on the plasma membrane of the hepatocyte is complex and appears to be regulated at transcriptional, translational, and post-translational levels.

BackgroundSporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, is a fatal neurodegenerative disease with currently no treatment options. Stem cell therapy for neurodegenerative diseases is emerging as a possible treatment option. However, while there are a few clinical trials for other neurodegenerative disorders such as Parkinson’s disease, prion disease cell therapy research has so far been confined to animal models.MethodsHere, we use a novel approach to study cell therapies in sCJD using a human cerebral organoid model. Cerebral organoids can be infected with sCJD prions allowing us to assess how neural precursor cell (NPC) therapy impacts the progression of sCJD. After 90 days of sCJD or mock infection, organoids were either seeded with NPCs or left unseeded and monitored for cellular composition changes, prion infection parameters and neuroelectrophysiological function at 180 days post-infection.ResultsOur results showed NPCs integrated into organoids leading to an increase in neuronal markers and changes in cell signaling irrespective of sCJD infection. Although a small, but significant, decrease in protease-resistant PrP deposition was observed in the CJD-infected organoids that received the NPCs, other disease-associated parameters showed minimal changes. However, the NPCs had a beneficial impact on organoid function following infection. sCJD infection caused reduction in neuronal spike rate and mean burst spike rate, indicative of reduced action potentials. NPC seeding restored these electrophysiological parameters to the uninfected control level.ConclusionsTogether with the previous animal studies, our results support that cell therapy may have some functional benefit for the treatment of human prion diseases.

Human neurological disorders such as Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, multiple sclerosis (MS), stroke, and spinal cord injury are caused by a loss of neurons and glial cells in the brain or spinal cord. Cell replacement therapy and gene transfer to the diseased or injured brain have provided the basis for the development of potentially powerful new therapeutic strategies for a broad spectrum of human neurological diseases. However, the paucity of suitable cell types for cell replacement therapy in patients suffering from neurological disorders has hampered the development of this promising therapeutic approach. In recent years, neurons and glial cells have successfully been generated from stem cells such as embryonic stem cells, mesenchymal stem cells, and neural stem cells, and extensive efforts by investigators to develop stem cell-based brain transplantation therapies have been carried out. We review here notable experimental and preclinical studies previously published involving stem cell-based cell and gene therapies for Parkinson's disease, Huntington's disease, ALS, Alzheimer's disease, MS, stroke, spinal cord injury, brain tumor, and lysosomal storage diseases and discuss the future prospects for stem cell therapy of neurological disorders in the clinical setting. There are still many obstacles to be overcome before clinical application of cell therapy in neurological disease patients is adopted: 1) it is still uncertain what kind of stem cells would be an ideal source for cellular grafts, and 2) the mechanism by which transplantation of stem cells leads to an enhanced functional recovery and structural reorganization must to be better understood. Steady and solid progress in stem cell research in both basic and preclinical settings should support the hope for development of stem cell-based cell therapies for neurological diseases.

CTGCT, Centre of Excellence for the Technologies of Gene and Cell Therapy: Collaborative Translation of Scientific Discoveries into Advanced Treatments for Neurological Rare Genetic Diseases and Cancer

H3K27M-mutant diffuse midline gliomas (DMG) are aggressive pediatric brain tumors with dismal prognoses and no effective treatments. GD2, a disialoganglioside overexpressed on DMG cells, is a promising target for chimeric antigen receptor (CAR) T cell therapy. In Arm A of the Phase I clinical trial (NCT04196413), 12 patients (11 on study) received GD2-CAR T cells through an initial intravenous (IV) infusion, and if patients had clinical or radiographic improvement, they subsequently received intracerebroventricular (ICV) administrations. This regimen achieved clinical benefit in ten patients, with >50% tumor volumetric reductions in 5 patients and a durable ongoing complete response in one patient (Majzner, Ramakrishna et al. Nature 2022; Monje et al. Nature 2024). However, disease progression ultimately occurred in 11 patients underscoring challenges in therapeutic optimization. To elucidate mechanisms underlying treatment responses and resistance, we performed a comprehensive multi-omic analysis, integrating single-cell RNA and TCR sequencing, mass cytometry, and clinical and molecular datasets. Sequencing 1, 062, 398 cells from patient apheresis, CAR T products, and cerebrospinal fluid (CSF) samples collected longitudinally at pre-defined trigger events, we reconstructed the dynamic landscape of immune and tumor cell populations in 12 DMG patients. Our analysis revealed profound CAR T cell infiltration into the CSF after treatment, accompanied by increases in endogenous T cell, mast cell, and monocyte populations and a relative decrease in microglia evident in CSF. This works sought to deeply characterize the lymphocyte profiles in these samples. Inflammatory and proliferation pathways were elevated in CAR T cells at early post-ICV time points, transitioning into an NK-like state and TGF-β receptor signaling at later stages. TCR clonal tracking identified persistent CAR T clones, alongside endogenous T cell activation and expansion. Moreover, non-responders to the therapy showed elevated Treg signatures in the CSF. Using CellChat-based cell-cell communication analysis, we uncovered differential cytokine-receptor interactions in responders versus non-responders, offering novel insights into immune-tumor interactions in the context of CAR T cell therapy. By contextualizing single-cell data within multi-omic frameworks and clinical outcomes, this study advances our understanding of the GD2-CAR T cell therapy, the tumor microenvironment, and biomarkers of response and resistance. These findings provide critical insights for optimizing CAR T cell therapies in CNS malignancies and inform the development of next-generation immunotherapies. Citation Format: Yiyun Chen, Moksha H. Desai, Zachary J. Ehlinger, Kun Wei Song, Skyler Rietberg, Hossein Daghagh, Mark P. Hamilton, Snehit Prabhu, Sonia Partap, Jasia Mahdi, Elena Sotillo, Bita Sahaf, Sneha Ramakrishna, Zinaida Good, Michelle Monje, Crystal Mackall. Unravelling temporal dynamics of GD2-CAR T cell therapy in H3K27M-mutant diffuse midline glioma through comprehensive single-cell and multi-omic profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7123.

Synthetic biology has significantly advanced the design of synthetic control devices, gene circuits and networks that can reprogram mammalian cells in a trigger-inducible manner. Prokaryotic helix-turn-helix motifs have become the standard resource to design synthetic mammalian transcription factors that tune chimeric promoters in a small molecule-responsive manner. We have identified a family of Actinomycetes transcriptional repressor proteins showing a tandem TetR-family signature and have used a synthetic biology-inspired approach to reveal the potential control dynamics of these bi-partite regulators. Daisy-chain assembly of well-characterized prokaryotic repressor proteins such as TetR, ScbR, TtgR or VanR and fusion to either the Herpes simplex transactivation domain VP16 or the Krueppel-associated box domain (KRAB) of the human kox-1 gene resulted in synthetic bi- and even tri-partite mammalian transcription factors that could reversibly program their individual chimeric or hybrid promoters for trigger-adjustable transgene expression using tetracycline (TET), γ-butyrolactones, phloretin and vanillic acid. Detailed characterization of the bi-partite ScbR-TetR-VP16 (ST-TA) transcription factor revealed independent control of TET- and γ-butyrolactone-responsive promoters at high and double-pole double-throw (DPDT) relay switch qualities at low intracellular concentrations. Similar to electromagnetically operated mechanical DPDT relay switches that control two electric circuits by a fully isolated low-power signal, TET programs ST-TA to progressively switch from TetR-specific promoter-driven expression of transgene one to ScbR-specific promoter-driven transcription of transgene two while ST-TA flips back to exclusive transgene 1 expression in the absence of the trigger antibiotic. We suggest that natural repressors and activators with tandem TetR-family signatures may also provide independent as well as DPDT-mediated control of two sets of transgenes in bacteria, and that their synthetic transcription-factor analogs may enable the design of compact therapeutic gene circuits for gene and cell-based therapies.