David Baltimore

Programmed Delayed Splicing: A Mechanism for Timed Inflammatory Gene Expression 2267628

Inflammation involves timed gene expression, suggesting that the fine-tuned onset, amplitude, and termination of expression of hundreds of genes is of critical importance to organismal homeostasis. Recent study of post-transcriptional regulation of inflammatory gene expression led to the suggestion of a regulatory role for pre-mRNA splicing. Here, using a hybrid capture approach to purify incompletely spliced, chromatin-associated pre-mRNAs, we use deep sequencing to study pre-mRNA splicing of the NF- 𝜅 B transcriptome. By freezing transcription and examining subsequent splicing of complete transcripts, we find many introns splice tens to hundreds of times slower than average. Investigating the basis of these delays, we focused on evolutionarily conserved introns with suboptimal splice donor sequences and found that strengthening these donor sites by as few as two nucleotides in minigene reporter assays markedly increased gene expression for several targets. This suggests that such sites can act as timing elements that both delay mRNA production and limit expression amplitude. To broaden this mechanistic view, we applied deep learning sequence-to-function models with feature attribution to identify additional regulatory sequences—both intronic and exonic—that may contribute to delayed splicing through mechanisms independent of donor site strength. This integrated approach revealed non-canonical motifs enriched in slow-splicing introns, pointing to a broader repertoire of cis-elements that can fine-tune transcript maturation during inflammation. Together, these findings support a model in which the temporal regulation of pre-mRNA splicing serves as a layer of control in inflammatory gene expression, and raise the possibility that similar timing mechanisms operate in other rapid-response transcriptional programs.

Sequential transcriptional waves and NF-κB-driven chromatin remodeling direct drug-induced dedifferentiation in cancer

Hoffmann

NF-κB: master regulator of cellular responses in health and disease

Human cancer-targeted immunity via transgenic hematopoietic stem cell progeny

Adoptive transfer of genetically engineered T cells expressing a tumor-antigen-specific transgenic T cell receptor (TCR) can result in clinical responses in a variety of malignancies. However, these responses are frequently short-lived, and patients typically relapse within several months. This phenomenon is largely due to poor persistence of the transgenic T cells, as well as a progressive loss of their functionality and terminal differentiation in vivo. This underscores the need for cell therapy approaches able to sustain the initial antitumor efficacy and lead to long-term antitumor efficacy. Herein, we report the use of tandem cell therapies involving autologous T cells and hematopoietic stem cells engineered to express the NY-ESO-1 TCR for the treatment of solid tumors in a first-in-human phase I clinical trial (NCT03240861). This therapy is shown to be safe, feasible, and leads to initial tumor regression activity. T cell progeny from the HSC progenitors is shown to provide circulating transgenic NY-ESO-1 TCR-T cells, which display tumor-antigen-specific antitumor functionality, without any evidence of anergy or exhaustion. These results demonstrate the utility of transgenic HSCs to generate a self-renewing source of tumor-specific cellular immunotherapy in human participants. Clinicaltrials.gov: NCT NCT03240861.

Science, evidence, law, and justice

For nearly 25 y, the Committee on Science, Technology, and Law (CSTL), of the National Academies of Sciences, Engineering, and Medicine, has brought together distinguished members of the science and law communities to stimulate discussions that would lead to a better understanding of the role of science in legal decisions and government policies and to a better understanding of the legal and regulatory frameworks that govern the conduct of science. Under the leadership of recent CSTL co-chairs David Baltimore and David Tatel, and CSTL director Anne-Marie Mazza, the committee has overseen many interdisciplinary discussions and workshops, such as the international summits on human genome editing and the science of implicit bias, and has delivered advisory consensus reports focusing on topics of broad societal importance, such as dual use research in the life sciences, voting systems, and advances in neural science research using organoids and chimeras. One of the most influential CSTL activities concerns the use of forensic evidence by law enforcement and the courts, with emphasis on the scientific validity of forensic methods and the role of forensic testimony in bringing about justice. As coeditors of this Special Feature, CSTL alumni Tom Albright and Jennifer Mnookin have recruited articles at the intersection of science and law that reveal an emerging scientific revolution of forensic practice, which we hope will engage a broad community of scientists, legal scholars, and members of the public with interest in science-based legal policy and justice reform.

Non-viral precision T cell receptor replacement for personalized cell therapy

. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRβ). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

Multi-omic single-cell snapshots reveal multiple independent trajectories to drug tolerance in a melanoma cell line

The determination of individual cell trajectories through a high-dimensional cell-state space is an outstanding challenge for understanding biological changes ranging from cellular differentiation to epigenetic responses of diseased cells upon drugging. We integrate experiments and theory to determine the trajectories that single BRAF V600E mutant melanoma cancer cells take between drug-naive and drug-tolerant states. Although single-cell omics tools can yield snapshots of the cell-state landscape, the determination of individual cell trajectories through that space can be confounded by stochastic cell-state switching. We assayed for a panel of signaling, phenotypic, and metabolic regulators at points across 5 days of drug treatment to uncover a cell-state landscape with two paths connecting drug-naive and drug-tolerant states. The trajectory a given cell takes depends upon the drug-naive level of a lineage-restricted transcription factor. Each trajectory exhibits unique druggable susceptibilities, thus updating the paradigm of adaptive resistance development in an isogenic cell population.

Sequence-dependent dynamics of synthetic and endogenous RSSs in V(D)J recombination

Developing lymphocytes of jawed vertebrates cleave and combine distinct gene segments to assemble antigen-receptor genes. This process called V(D)J recombination that involves the RAG recombinase binding and cutting recombination signal sequences (RSSs) composed of conserved heptamer and nonamer sequences flanking less well-conserved 12- or 23-bp spacers. Little quantitative information is known about the contributions of individual RSS positions over the course of the RAG-RSS interaction. We employ a single-molecule method known as tethered particle motion to track the formation, lifetime and cleavage of individual RAG-12RSS-23RSS paired complexes (PCs) for numerous synthetic and endogenous 12RSSs. We reveal that single-bp changes, including in the 12RSS spacer, can significantly and selectively alter PC formation or the probability of RAG-mediated cleavage in the PC. We find that some rarely used endogenous gene segments can be mapped directly to poor RAG binding on their adjacent 12RSSs. Finally, we find that while abrogating RSS nicking with Ca2+ leads to substantially shorter PC lifetimes, analysis of the complete lifetime distributions of any 12RSS even on this reduced system reveals that the process of exiting the PC involves unidentified molecular details whose involvement in RAG-RSS dynamics are crucial to quantitatively capture kinetics in V(D)J recombination.

Myeloid cell–targeted miR-146a mimic inhibits NF-κB–driven inflammation and leukemia progression in vivo

NF-κB is a key regulator of inflammation and cancer progression, with an important role in leukemogenesis. Despite its therapeutic potential, targeting NF-κB using pharmacologic inhibitors has proven challenging. Here, we describe a myeloid cell-selective NF-κB inhibitor using an miR-146a mimic oligonucleotide conjugated to a scavenger receptor/Toll-like receptor 9 agonist (C-miR146a). Unlike an unconjugated miR146a, C-miR146a was rapidly internalized and delivered to the cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets (IRAK1 and TRAF6), thereby blocking activation of NF-κB in target cells. IV injections of C-miR146a mimic to miR-146a-deficient mice prevented excessive NF-κB activation in myeloid cells, and thus alleviated myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly, C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell-induced cytokine release syndrome. Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in a xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions, miR-146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using The Cancer Genome Atlas acute myeloid leukemia data set, we found an inverse correlation of miR-146a levels with NF-κB-related genes and with patient survival. Correspondingly, C-miR146a induced cytotoxic effects in human MDSL, HL-60, and MV4-11 leukemia cells in vitro. The repeated IV administration of C-miR146a inhibited expression of NF-κB target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results show the potential of using myeloid cell-targeted miR-146a mimics for the treatment of inflammatory and myeloproliferative disorders.

Alternative splicing coupled with transcript degradation modulates OAS1g antiviral activity

At the heart of an innate immune response lies a tightly regulated gene expression program. This precise regulation is crucial because small changes can shift the balance from protective to destructive immunity. Here we identify a frequently used alternative splice site in the gene oligoadenylate synthetase 1g ( Oas1g ), a key component of the 2–5A antiviral system. Usage of this splice site leads to the generation of a transcript subject to decay, and removal of the site leads to increased expression of Oas1g and an improved antiviral response. However, removal of the splice site also leads to an increase in apoptotic cell death, suggesting this splicing event exists as a compromise between the pathogen protective benefits and collateral damage associated with OAS1g activity. Across the innate immune response, we show that a multitude of alternative splicing events predicted to lead to decay exist, and thus have the potential to play a significant role in the regulation of gene expression in innate immunity.

Sequence-Dependent Dynamics of Synthetic and Endogenous RSSs in V(D)J Recombination

Developing lymphocytes in the immune system of jawed vertebrates assemble antigen-receptor genes by undergoing large-scale reorganization of spatially separated V, D, and J gene segments through a process known as V(D)J recombination. The RAG protein initiates this process by binding and cutting recombination signal sequences (RSSs) composed of conserved heptamer and nonamer sequences flanking less well-conserved 12- or 23-bp spacers. Little quantitative information is known about the contributions of individual RSS positions over the course of the RAG-RSS interaction. We employ a single-molecule method known as tethered particle motion to quantify the formation, stability, and cleavage of the RAG-12RSS-23RSS paired complex (PC) for numerous synthetic and endogenous 12RSSs. We thoroughly investigate the sequence space around a RSS by making 40 different single-bp changes and characterizing the reaction dynamics. We reveal that single-bp changes affect RAG function based on their position: loss of cleavage function (first three positions of the heptamer); reduced propensity for forming the PC (the nonamer and last four bp of the heptamer); or variable effects on PC formation (spacer). We find that the rare usage of some endogenous gene segments can be mapped directly to their adjacent 12RSSs to which RAG binds weakly. The 12RSS, however, cannot explain the high-frequency usage of other gene segments. Finally, we find that RSS nicking, while not required for PC formation, substantially stabilizes the PC. Our findings provide detailed insights into the contribution of individual RSS positions to steps of the RAG-RSS re-action that previously have been difficult to assess quantitatively. Summary V(D)J recombination is a genomic cut-and-paste process for generating diverse antigen-receptor repertoires. The RAG enzyme brings separate gene segments together by binding the neighboring sequences called RSSs, forming a paired complex (PC) before cutting the DNA. There are limited quantitative studies of the sequence-dependent dynamics of the crucial inter-mediate steps of PC formation and cleavage. Here, we quantify individual RAG-DNA dynamics for various RSSs. While RSSs of frequently-used segments do not comparatively enhance PC formation or cleavage, the rare use of some segments can be explained by their neighboring RSSs crippling PC formation and/or cleavage. Furthermore, PC lifetimes reveal DNA-nicking is not required for forming the PC, but PCs with nicks are more stable.

Trajectories from Snapshots: Integrated proteomic and metabolic single-cell assays reveal multiple independent adaptive responses to drug tolerance in a BRAF-mutant melanoma cell line

The determination of individual cell trajectories through a high-dimensional cell-state space is an outstanding challenge, with relevance towards understanding biological changes ranging from cellular differentiation to epigenetic (adaptive) responses of diseased cells to drugging. We report on a combined experimental and theoretic method for determining the trajectories that specific highly plastic BRAF V600E mutant patient-derived melanoma cancer cells take between drug-naïve and drug-tolerant states. Recent studies have implicated non-genetic, fast-acting resistance mechanisms are activated in these cells following BRAF inhibition. While single-cell highly multiplex omics tools can yield snapshots of the cell state space landscape sampled at any given time point, individual cell trajectories must be inferred from a kinetic series of snapshots, and that inference can be confounded by stochastic cell state switching. Using a microfludic-based single-cell integrated proteomic and metabolic assay, we assayed for a panel of signaling, phenotypic, and metabolic regulators at four time points during the first five days of drug treatment. Dimensional reduction of the resultant data set, coupled with information theoretic analysis, uncovered a complex cell state landscape and identified two distinct paths connecting drug-naïve and drug-tolerant states. Cells are shown to exclusively traverse one of the two pathways depending on the level of the lineage restricted transcription factor MITF in the drug-naïve cells. The two trajectories are associated with distinct signaling and metabolic susceptibilities, and are independently druggable. Our results update the paradigm of adaptive resistance development in an isogenic cell population and offer insight into the design of more effective combination therapies.

Sensitive Detection and Analysis of Neoantigen-Specific T Cell Populations from Tumors and Blood

Neoantigen-specific T cells are increasingly viewed as important immunotherapy effectors, but physically isolating these rare cell populations is challenging. Here, we describe a sensitive method for the enumeration and isolation of neoantigen-specific CD8+ T cells from small samples of patient tumor or blood. The method relies on magnetic nanoparticles that present neoantigen-loaded major histocompatibility complex (MHC) tetramers at high avidity by barcoded DNA linkers. The magnetic particles provide a convenient handle to isolate the desired cell populations, and the barcoded DNA enables multiplexed analysis. The method exhibits superior recovery of antigen-specific T cell populations relative to literature approaches. We applied the method to profile neoantigen-specific T cell populations in the tumor and blood of patients with metastatic melanoma over the course of anti-PD1 checkpoint inhibitor therapy. We show that the method has value for monitoring clinical responses to cancer immunotherapy and might help guide the development of personalized mutational neoantigen-specific T cell therapies and cancer vaccines.

Sequential transcriptional waves and NF-κB-driven chromatin remodeling direct drug-induced dedifferentiation in cancer

Drug-induced dedifferentiation towards a drug-tolerant persister state is a common mechanism cancer cells exploit to escape therapies, posing a significant obstacle to sustained therapeutic efficacy. The dynamic coordination of epigenomic and transcriptomic programs at the early-stage of drug exposure, which initiates and orchestrates these reversible dedifferentiation events, remains largely unexplored. Here we employ high-temporal-resolution multi-omics profiling, information-theoretic approaches, and dynamic system modeling to probe these processes in BRAF -mutant melanoma models and patient specimens. We uncover a hysteretic transition trajectory of melanoma cells in response to oncogene inhibition and subsequent release, driven by the sequential operation of two tightly coupled transcriptional waves, which orchestrate genome-scale chromatin state reconfiguration. Modeling of the transcriptional wave interactions predicts NF-κB/RelA-driven chromatin remodeling as the underlying mechanism of cell-state dedifferentiation, a finding we validate experimentally. Our results identify critical RelA-target genes that are epigenetically modulated to drive this process, establishing a quantitative epigenome gauge to measure cell-state plasticity in melanomas, which supports the potential use of drugs targeting epigenetic machineries to potentiate oncogene inhibition. Extending our investigation to other cancer models, we identify oxidative stress-mediated NF-κB/RelA activation as a common mechanism driving cellular transitions towards drug-tolerant persister states, revealing a novel and pivotal role for the NF-κB signaling axis in linking cellular oxidative stress to cancer progression.

MATE-Seq: Microfluidic Antigen-TCR Engagement Sequencing

Adaptive immunity is based on peptide antigen recognition. Our ability to harness the immune system for therapeutic gain relies on the discovery of the T cell receptor (TCR) genes that selectively target antigens from infections, mutated proteins, and foreign agents. Here we present a method that selectively labels peptide antigen-specific CD8+ T-cells in human blood using magnetic nanoparticles functionalized with peptide-MHC tetramers, isolates these specific cells within an integrated microfluidic device, and directly amplifies the TCR genes for sequencing. Critically, the identity of the peptide recognized by the TCR is preserved, providing the link between peptide and gene. The platform requires inputs on the order of just 100,000 CD8+ T cells, can be multiplexed for simultaneous analysis of multiple peptides, and performs sorting and isolation on chip. We demonstrate 1000-fold sensitivity enhancement of antigen-specific T-cell receptor detection and simultaneous capture of two virus antigen-specific T-cell receptors from samples of human blood.

Dual mechanisms of posttranscriptional regulation of Tet2 by Let-7 microRNA in macrophages

Significance Macrophages function as sentinel cells, constantly monitoring the host environment for bacterial infection. They are activated by bacterial components and then secrete proinflammatory cytokines, such as interleukin (IL)-6. This is one of the body’s most effective weapons for fighting infections but must be rigidly controlled to avoid pathologic overresponse. Many factors have been shown to act as feedback regulators of inflammatory processes. Using both knockout and myeloid cell-specific transgenic mouse models, we report here that a physiological role of the microRNA let-7adf cluster is to promote IL-6 secretion by lipopolysaccharide-activated macrophages through down-regulating Tet2. This microRNA cluster represses Tet2 using two different mechanisms: direct targeting and indirect enhancement of succinate accumulation, a compound that represses Tet2.

T Cell Receptor Immunotherapy Drives Human Immunodeficiency Virus Evolution in Humanized Mice

Summary T cell receptor mediated immunotherapy using engineered Hematopoietic Stem/Progenitor Cells leads to durable partial suppression of HIV in humanized mice. Sustained viral suppression is accompanied by viral evolution under selection pressure. This study highlights the potential for TCR immunotherapy and the need to target multiple epitopes. Abstract Effective CD8+ T cell responses targeted to the KK10 epitope of HIV presented by HLA-B*27:05, a protective HLA allele, correlate with the ability to control infection without antiretroviral therapy (ART). Here, we report an immunotherapy approach using two B*27:05-KK10-specific T Cell Receptors (TCRs) isolated from HIV controllers. Immunocompromised mice engrafted with human Hematopoietic Stem/Progenitor Cells (HSPCs) encoding for the TCRs showed differentiation into functionally active engineered T cells. Following infection with HIV, both TCRs showed sustained, albeit modest, viral suppression over 32 weeks, accompanied by a concomitant increase in CD4+ T cells. Sequencing of viral quasi-species from the plasma of infected mice demonstrated clear evidence for viral evolution under selection pressure from the TCRs. The most commonly observed mutation in the KK10 epitope was L6M, which preserved viral fitness but showed attenuated recognition by the TCRs. These studies show that TCR-immunotherapy was able to suppress HIV infection long-term while driving HIV evolution in humanized mice.

MATE-Seq: microfluidic antigen-TCR engagement sequencing

Adaptive immunity is based on peptide antigen recognition. Our ability to harness the immune system for therapeutic gain relies on the discovery of the T cell receptor (TCR) genes that selectively target antigens from infections, mutated proteins, and foreign agents. Here we present a method that selectively labels peptide antigen-specific CD8+ T cells using magnetic nanoparticles functionalized with peptide-MHC tetramers, isolates these specific cells within an integrated microfluidic device, and directly amplifies the TCR genes for sequencing. Critically, the identity of the peptide recognized by the TCR is preserved, providing the link between peptide and gene. The platform requires inputs on the order of just 100 000 CD8+ T cells, can be multiplexed for simultaneous analysis of multiple peptides, and performs sorting and isolation on chip. We demonstrate 1000-fold sensitivity enhancement of detecting antigen-specific TCRs relative to bulk analysis and simultaneous capture of two virus antigen-specific TCRs from a population of T cells.

Heterogeneous Responses of Hematopoietic Stem Cells to Inflammatory Stimuli Are Altered with Age

Long-term hematopoietic stem cells (LT-HSCs) maintain hematopoietic output throughout an animal's lifespan. However, with age, the balance is disrupted, and LT-HSCs produce a myeloid-biased output, resulting in poor immune responses to infectious challenge and the development of myeloid leukemias. Here, we show that young and aged LT-HSCs respond differently to inflammatory stress, such that aged LT-HSCs produce a cell-intrinsic, myeloid-biased expression program. Using single-cell RNA sequencing (scRNA-seq), we identify a myeloid-biased subset within the LT-HSC population (mLT-HSCs) that is prevalent among aged LT-HSCs. We identify CD61 as a marker of mLT-HSCs and show that CD61-high LT-HSCs are uniquely primed to respond to acute inflammatory challenge. We predict that several transcription factors regulate the mLT-HSCs gene program and show that Klf5, Ikzf1, and Stat3 play an important role in age-related inflammatory myeloid bias. We have therefore identified and isolated an LT-HSC subset that regulates myeloid versus lymphoid balance under inflammatory challenge and with age.

IND-Enabling Studies for a Clinical Trial to Genetically Program a Persistent Cancer-Targeted Immune System

Purpose: To improve persistence of adoptively transferred T-cell receptor (TCR)–engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application. Experimental Design: HSCs transduced with a lentiviral vector expressing NY-ESO-1 TCR and the PET reporter/suicide gene HSV1-sr39TK and T cells transduced with a retroviral vector expressing NY-ESO-1 TCR were coadministered to myelodepleted HLA-A2/Kb mice within a formal Good Laboratory Practice (GLP)–compliant study to demonstrate safety, persistence, and HSC differentiation into all blood lineages. Non-GLP experiments included assessment of transgene immunogenicity and in vitro viral insertion safety studies. Furthermore, Good Manufacturing Practice (GMP)–compliant cell production qualification runs were performed to establish the manufacturing protocols for clinical use. Results: TCR genetically modified and ex vivo–cultured HSCs differentiated into all blood subsets in vivo after HSC transplantation, and coadministration of TCR-transduced T cells did not result in increased toxicity. The expression of NY-ESO-1 TCR and sr39TK transgenes did not have a detrimental effect on gene-modified HSC's differentiation to all blood cell lineages. There was no evidence of genotoxicity induced by the lentiviral vector. GMP batches of clinical-grade transgenic cells produced during qualification runs had adequate stability and functionality. Conclusions: Coadministration of HSCs and T cells expressing an NY-ESO-1 TCR is safe in preclinical models. The results presented in this article led to the FDA approval of IND 17471.