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Wilken, M. S.

Publications and source records attributed to Wilken, M. S..

5 recordsLinked to original sources

X-Cell: Scaling Causal Perturbation Prediction Across Diverse Cellular Contexts via Diffusion Language Models

Causal models of cellular systems hold the promise to empower broad biological discovery, including the systematic identification of novel targets for drug discovery. Predicting how genetic and pathway perturbations reshape gene expression across diverse cellular contexts is a prerequisite for building generalizable cellular foundation models. However, current methods typically fail to extrapolate beyond their training distributions because they rely predominantly on observational expression atlases rather than interventional perturbation data. We present X-Atlas/Pisces, the largest genome-wide CRISPRi Perturb-seq compendium to date, comprising 25.6 million perturbed single-cell transcriptomes across 16 biologically diverse contexts, including widely used cell lines, induced pluripotent stem cells (iPSCs), resting and CD3/CD28 activated Jurkat T lymphoma cells, and multi-lineage differentiating iPSCs. Leveraging this resource, we develop X-Cell, a diffusion language model that predicts perturbation responses by iteratively refining control-to-perturbed state transitions through cross-attention to multi-modal biological priors derived from natural language, protein language models, interaction networks, genetic dependency maps, and morphological profiles. X-Cell outperforms existing state-of-the-art models by up to five-fold on key metrics such as Pearson{Delta} (correlation between predicted and observed perturbation-induced log-fold changes), and demonstrates zero-shot prediction of T cell inactivating perturbations in stimulated Jurkat cells. We scale X-Cell to 4.9 billion parameters (X-Cell-Ultra), the largest causal perturbation model to date. We demonstrate for the first time that perturbation prediction follows power-law scaling with an exponent matching large language models. X-Cell-Ultra demonstrates zero-shot generalization to novel biological contexts, including unseen iPSC-derived melanocyte progenitors and primary human CD4+ T cells from multiple donors, and outperforms all baselines after self-supervised test-time adaptation. These results demonstrate that coordinated scaling of causal perturbation data and model capacity yields foundation models capable of generalizable perturbation prediction across cellular contexts, with potential applications for improving computational target identification, validation, and context-specific therapeutic prioritization.

systems biology↗

Large-scale discovery of potent, compact and lineage specific enhancers for gene therapy vectors

Regulation of gene expression during cell development and differentiation is chiefly orchestrated by distal noncoding regulatory elements that precisely modulate cell selective gene activity. Gene therapy vectors rely on the cellular and context specificity of regulatory DNA elements to express therapeutic transgenes in the correct location and time. Here, we develop a straight-forward, one-shot approach to screen putative regulatory sequences identified in large-scale epigenomics profiling experiments for precise and programmable control of transgenes encoded within gene therapy viral vectors. We designed a library of 15,000 short sequences ([~]200bp) derived from a set of developmentally active DHS elements during human ex vivo erythropoiesis and cloned them into a GFP reporter lentiviral vector. In an erythroid progenitor cell line, these elements display a gradient of transcriptional enhancer activity, with some demonstrating equivalent activity to the canonical {beta}-globin LCR despite a 9-fold smaller size. We show that these elements are both highly cell type restricted and developmental stage specific both in vitro and in vivo. Finally, we replace the LCR element with one of the novel short enhancers in a {beta}-thalassemia lentiviral therapeutic vector and efficiently correct the thalassemic phenotype in patient-derived HSPCs. More broadly, our approach provides further insights into enhancer biology with wider implications into the development of highly cell type specific and efficacious viral vectors for human gene therapy.

genomics↗

Promoter keyholes enable specific and persistent multi-gene expression programs in primary T cells without genome modification

Human genes are regulated quantitatively, yet the ability to specify the expression level of a native gene accurately and specifically using a defined reagent has remained elusive. Here we show that precise targeting of KRAB repressive domain within regulatory DNA unlocks an endogenous quantitative dial that can be engaged at nucleotide resolution to program expression levels across a wide physiologic range, with single-gene specificity and high reproducibly in primary cells.

synthetic biology↗

Cis-Regulatory Accessibility Directs Muller Glial Development and Regenerative Capacity

Diseases and damage to the retina lead to losses in retinal neurons and eventual visual impairment. Although the mammalian retina has no inherent regenerative capabilities, fish have robust regeneration from Muller glia (MG). Recently, we have shown that driving expression of Ascl1 in adult mouse MG stimulates neurogenesis similar to fish regeneration. The regeneration observed in the mouse is limited in the variety of neurons that can be derived from MG; Ascl1-expressing MG primarily generate bipolar cells. To better understand the limits of MG-based regeneration in mouse retinas, we used ATAC- and RNA-seq to compare newborn progenitors with MG. Our analysis demonstrated striking similarities between MG and progenitors, with losses in regulatory motifs for neurogenesis genes. Young MG were found to have intermediate expression profiles and accessible DNA, which is mirrored in the ability of Ascl1 to direct bipolar neurogenesis in young MG. When comparing what makes bipolar and photoreceptor cells distinct from glial cells, we find that bipolar-specific accessible regions are more frequently linked to bHLH motifs and Ascl1 binding, indicating that Ascl1 preferentially binds to bipolar regions. Overall, our analysis indicates a loss of neurogenic gene expression and motif accessibility during glial maturation that may prevent efficient reprogramming.

developmental biology↗

STAT pathway activation limits the Ascl1-mediated chromatin remodeling required for neural regeneration from Muller glia in adult mouse retina.

Muller glia can serve as a source for retinal regeneration in some non-mammalian vertebrates. Recently we found that this process can be induced in mouse Muller glia after injury, by combining transgenic expression of the proneural transcription factor Ascl1 and the HDAC inhibitor TSA. However, new neurons are only generated from a subset of Muller glia in this model, and identifying factors that limit Ascl1-mediated MG reprogramming could potentially make this process more efficient, and potentially useful clinically. One factor that limits neurogenesis in some non-mammalian vertebrates is the STAT pathway activation that occurs in Muller glia in response to injury. In this report, we tested whether injury induced STAT activation hampers the ability of Ascl1 to reprogram Muller glia into retinal neurons. Using a STAT inhibitor, in combination with our previously described reprogramming paradigm, we found a large increase in the ability of Muller glia to generate neurons, similar to those we described previously. Single-cell RNA-seq showed that the progenitor-like cells derived from Ascl1-expressing Muller glia have a higher level of STAT signaling than those that become neurons. Using Ascl1 ChIP-seq and DNase-seq, we found that developmentally inappropriate Ascl1 binding sites (that were unique to the overexpression context) had enrichment for the STAT binding motif. This study provides evidence that STAT pathway activation reduces the efficiency of Ascl1-mediated reprogramming in Muller glia, potentially by directing Ascl1 to inappropriate targets.

developmental biology↗