Biology subjects

Pham, Q.

Publications and source records attributed to Pham, Q..

7 recordsLinked to original sources

bioRxiv2025

Modeling the Synergetic Dynamics of B cells and TFH cells in Germinal Center Reactions

B cells producing high-affinity antibodies arise through affinity maturation within germinal centers (GCs), where selection is driven by T follicular helper (TFH) cells. Recent studies have shown that, like GC B cells, TFH cells also undergo antigen-dependent selection, with competition among TFH clones dictated by their ability to recognize and stimulate B cells. This sensitivity-dependent selection process leads to dynamic remodeling of the TFH repertoire over time. Despite the essential role of TFH cells in B cell selection, the functional consequences of the time evolution of the TFH cell population remains poorly understood. To address this gap, we developed a population dynamics model that explicitly incorporates key TFH cell properties and dynamics. Our analysis predicts that dynamic feedback between B and TFH cell populations provides robust homeostatic regulation of their numbers in the GC, yielding a stable lymphocyte ratio that we verify experimentally. Moreover, our model predicts that TFH clone sensitivity dictates distinct evolutionary strategies during affinity maturation, with low-sensitivity TFH cells accelerating affinity gain at the expense of B cell diversity, while high-sensitivity TFH cells slow affinity maturation but preserve a broader B cell repertoire. These findings highlight the importance of co-regulation between TFH and B cells and suggest that reciprocal stimulation allows the immune system to tune the tradeoff between the speed of affinity gain and the breadth of B cell diversity--a principle that may extend to other adaptive systems. Significance StatementEffector B cells that secrete high-affinity antibodies and form immunological memory are essential for humoral immunity and arise from germinal center (GC) reactions. Within GCs, B cells undergo an accelerated version of Darwinian evolution to enhance antibody affinity. This process is orchestrated by T follicular helper (TFH) cells which provide stimulatory signals to selected B cells and undergo their own antigen-driven selection. To investigate this co-evolutionary process, we developed a tractable population-level model of the GC reaction. Our analysis reveals that the reciprocal stimulation of B and TFH cells provides a robust mechanism for regulating the B:TFH ratio and tuning the tradeoff between the speed of affinity maturation and the diversity of the antibody response.

immunology↗

bioRxiv2024

Efficient production of functional proaerolysin from E.coli

Proaerolysin is a bacterial toxin produced by Aeromonas hydrophila that specifically binds to GPI-anchored proteins on the plasma membrane, creating transmembrane pores that lead to cell death within a few hours. Leveraging this unique property, proaerolysin is widely used in diagnostic tests for paroxysmal nocturnal hemoglobinuria (PNH), a disease caused by somatic mutations in the PIGA gene, which is involved in the biosynthesis of GPI anchors. Additionally, proaerolysin serves as a counter-selection agent in genetic manipulations. Although bacterial expression and purification of proaerolysin have been previously reported, yields were low due to the absence of internal disulfide bonds, which are crucial for protein stability. Here, we demonstrate that using the Shuffle E. coli strain, which facilitates the formation of disulfide bonds in the cytoplasm, significantly improves the solubility and proper folding of proaerolysin. We achieved a high yield of proaerolysin, approximately 3 mg from a 50 ml bacterial culture, with a purity of over 99%. The functionality of recombinant proaerolysin was confirmed by testing in mouse embryonic stem cells (mESCs), demonstrating that this high-yield production method offers a reliable and cost-effective source of functional proaerolysin for a wide range of biotechnological applications.

biochemistry↗

bioRxiv2024

A facile chemical strategy to synthesize precise AAV-protein conjugates for targeted gene delivery

The efficacy of current gene therapy approaches using adeno associated virus (AAV) vectors is limited by the poor control over their tissue tropism. Untargeted AAV vectors require high doses to achieve therapeutic efficacy, which is associated with toxic off-target impacts and increased therapeutic costs. The ability to reprogram existing AAV vectors to selectively transduce target tissues is essential to develop next-generation human gene therapies that are safer, more efficacious, and less expensive. Using selective and high-affinity antibodies and antibody-like proteins to retarget existing AAV vectors to bind novel cell-surface receptors offers an attractive and modular approach to reprogram their tropism. However, attaching these proteins onto the complex and delicate AAV capsids remains challenging. Here, we report a versatile chemical strategy to covalently attach recombinant proteins onto the capsid of AAV, using a combination of genetic code expansion and bioorthogonal conjugation chemistry. This method is efficient, and allows precise control over the site and stoichiometry of protein attachment onto the AAV capsid, enabling systematic optimization of the resulting conjugate. Using this approach, we generated conjugates of AAV2 with an anti-HER2 nanobody and a full-length anti-HER2 IgG, which show highly efficient and selective gene delivery into HER2+ cancer cells. Remarkably, the optimized AAV2-nanobody conjugate facilitated efficient transduction of HER2+ tumor xenograft in mice with little off-target gene expression, including in the liver. Programmable synthesis of AAV-protein conjugates using this method offers a promising new strategy to rationally engineer next-generation gene therapy vectors.

synthetic biology↗

bioRxiv2024

Directed evolution of a bacterial leucyl tRNA in mammalian cells for enhanced noncanonical amino acid mutagenesis

The E. coli leucyl-tRNA synthetase (EcLeuRS)/tRNAEcLeu pair has been engineered to genetically encode a structurally diverse group of enabling noncanonical amino acids (ncAAs) in eukaryotes, including those with bioconjugation handles, environment-sensitive fluorophores, photocaged amino acids, and native post-translational modifications. However, the scope of this toolbox in mammalian cells is limited by the poor activity of tRNAEcLeu. Here, we overcome this limitation by evolving tRNAEcLeu directly in mammalian cells using a virus-assisted selection scheme. This directed evolution platform was optimized for higher throughput such that the entire acceptor stem of tRNAEcLeu could be simultaneously engineered, which resulted in the identification of several variants with remarkably improved efficiency for incorporating a wide range of ncAAs. The advantage of the evolved leucyl tRNAs was demonstrated by expressing ncAA mutants in mammalian cells that were challenging to express before using the wild-type tRNAEcLeu, by creating viral vectors that facilitated ncAA mutagenesis at a significantly lower dose, and by creating more efficient mammalian cell lines stably expressing the ncAA-incorporation machinery.

synthetic biology↗

bioRxiv2023

Precise manipulation of site and stoichiometry of capsid modification enables optimization of functional adeno-associated virus conjugates

The ability to engineer adeno-associated virus (AAV) vectors for targeted infection of specific cell types is critically important to fully harness its potential of human gene therapy. A promising approach to achieve this objective involves chemically attaching retargeting ligands onto the virus capsid. Site-specific incorporation of a bioorthogonal noncanonical amino acid (ncAA) into the AAV capsid proteins provides a particularly attractive strategy to introduce such modifications with exquisite precision. In this study, we show that using ncAA mutagenesis, it is possible to systematically alter the attachment site of a retargeting ligand (cyclic-RGD) on the AAV capsid to create diverse conjugate architectures, and that the site of attachment heavily impacts the retargeting efficiency. We further demonstrate that the performance of these AAV conjugates is highly sensitive to the stoichiometry of capsid labeling (labels per capsid), with an intermediate labeling density ([~]12 per capsid) providing optimal activity. Finally, we developed technology to precisely control the number of attachment sites per AAV capsid, by selectively incorporating a ncAA into the minor capsid proteins with high fidelity and efficiency, such that AAV-conjugates with varying stoichiometry can be synthesized in a homogeneous manner. Together, this platform provides unparalleled control over site and stoichiometry of capsid modification, which will enable the development of next-generation AAV vectors tailored with desirable attributes.

synthetic biology↗

bioRxiv2021

Topologically Associating Domain Boundaries are Commonly Required for Normal Genome Function

Topologically associating domain (TAD) boundaries are thought to partition the genome into distinct regulatory territories. Anecdotal evidence suggests that their disruption may interfere with normal gene expression and cause disease phenotype1-3, but the overall extent to which this occurs remains unknown. Here we show that TAD boundary deletions commonly disrupt normal genome function in vivo. We used CRISPR genome editing in mice to individually delete eight TAD boundaries (11-80kb in size) from the genome in mice. All deletions examined resulted in at least one detectable molecular or organismal phenotype, which included altered chromatin interactions or gene expression, reduced viability, and anatomical phenotypes. For 5 of 8 (62%) loci examined, boundary deletions were associated with increased embryonic lethality or other developmental phenotypes. For example, a TAD boundary deletion near Smad3/Smad6 caused complete embryonic lethality, while a deletion near Tbx5/Lhx5 resulted in a severe lung malformation. Our findings demonstrate the importance of TAD boundary sequences for in vivo genome function and suggest that noncoding deletions affecting TAD boundaries should be carefully considered for potential pathogenicity in clinical genetics screening.

genomics↗

bioRxiv2018

A cross-organism framework for supervised enhancer prediction with epigenetic pattern recognition and targeted validation

Enhancers are important noncoding elements, but they have been traditionally hard to characterize experimentally. Only a few mammalian enhancers have been validated, making it difficult to train statistical models for their identification properly. Instead, postulated patterns of genomic features have been used heuristically for identification. The development of massively parallel assays allows for the characterization of large numbers of enhancers for the first time. Here, we developed a framework that uses Drosophila STARR-seq data to create shape-matching filters based on enhancer-associated meta-profiles of epigenetic features. We combined these features with supervised machine learning algorithms (e.g., support vector machines) to predict enhancers. We demonstrated that our model could be applied to predict enhancers in mammalian species (i.e., mouse and human). We comprehensively validated the predictions using a combination of in vivo and in vitro approaches, involving transgenic assays in mouse and transduction-based reporter assays in human cell lines. Overall, the validations involved 153 enhancers in 6 mouse tissues and 4 human cell lines. The results confirmed that our model can accurately predict enhancers in different species without re-parameterization. Finally, we examined the transcription-factor binding patterns at predicted enhancers and promoters in human cell lines. We demonstrated that these patterns enable the construction of a secondary model effectively discriminating between enhancers and promoters.

bioinformatics↗