Biology subjects

May, A. P.

Publications and source records attributed to May, A. P..

6 recordsLinked to original sources

bioRxiv2022

Ependymal cell maturation is heterogeneous and ongoing in the mouse spinal cord and dynamically regulated in response to injury

The spinal cord neural stem cell potential resides within the ependymal cells lining the central canal. These cells are, however, heterogeneous, and we know little about the biological diversity this represents. Here we use single-cell RNA-sequencing to profile adult mouse spinal cord ependymal cells. We uncover transcriptomes of known subtypes and a new mature ependymal cell state, that becomes more prominent with age. Comparison of ependymal cell transcriptomes from the brain and spinal cord revealed that ongoing cell maturation distinguishes spinal cord ependymal cells from their postmitotic brain counterparts. Using an ex vivo model of spinal cord injury, we show that ependymal cell maturation is reversible but also highly regulated. We revisit ependymal cell identities in adult human spinal cord and uncover evidence for their maturation and surprising ventralisation with age. This first in-depth characterisation of spinal cord ependymal cells paves the way to manipulation of distinct ependymal subtypes, provides insights into ependymal cell maturation dynamics and informs strategies for coaxing ependymal cell-driven spinal cord repair.

developmental biology↗

bioRxiv2020

Ageing compromises mouse thymus function and remodels epithelial cell differentiation

Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell death and compromised organ function. This is first observed in the thymus, the primary lymphoid organ that generates and selects T cells. However, the molecular and cellular mechanisms underpinning these ageing processes remain unclear. Here, we show that mouse ageing leads to less efficient T cell selection, decreased self-antigen representation and increased T cell receptor repertoire diversity. Using a combination of single-cell RNA-seq and lineage-tracing, we find that progenitor cells are the principal targets of ageing, whereas the function of mature thymic epithelial cells is compromised only modestly. Specifically, an early-life precursor cell population, retained in the mouse cortex postnatally, is virtually extinguished at puberty. Concomitantly, a medullary precursor cell quiesces, thereby impairing maintenance of the medullary epithelium. Thus, ageing disrupts thymic progenitor differentiation and impairs the core immunological functions of the thymus.

immunology↗

bioRxiv2019

Methods Matter -- Standard Production Platforms For Recombinant AAV Can Produce Chemically And Functionally Distinct Vectors

Different manufacturing approaches have been used in the production of recombinant adeno-associated virus (rAAV). The two leading approaches are transiently transfected human HEK293 cells and live baculovirus infection of Sf9 insect cells. Unexplained differences in vector performance have been seen clinically and preclinically. Thus, we performed for the first time a highly controlled comparative production analysis varying only the host cell species but keeping all other rAAV production parameters the same. We demonstrate that host cell species is critical for determining vector potency. Given these key findings, we then sought to deeply characterize differences in rAAVs when produced by these two manufacturing platforms with multiple analytical approaches including: proteomic profiling by mass spectrometry, isoelectric focusing, cryo-EM, denaturation assays, genomic and epigenomic sequencing of packaged genomes, human cytokine profiling, and comparative functional transduction assessments in vitro and in vivo, including in humanized liver mice. Using these tools weve made two major discoveries: 1) rAAV capsids have post-translational modifications (PTMs) including glycosylation, acetylation, phosphorylation, methylation and deamidation, and these PTMs differ between platforms; 2) rAAV genomes are methylated during production, and these methylation marks are also differentially deposited between platforms. In addition, our data also demonstrate that host cell protein impurities differ between platforms and can have their own PTMs including potentially immunogenic N-linked glycans. We show that human-produced rAAVs are more potent than baculovirus-Sf9 vectors in various cell types in vitro (P < 0.05-0.0001), in various mouse tissues in vivo (P < 0.03-0.0001), and in human liver in vivo (P < 0.005). Collectively, our findings were reproducible across vendors, including commercial manufacturers, academic core facilities, and individual laboratory preparations. These vector differences may have clinical implications for rAAV receptor binding, trafficking, expression kinetics, expression durability, vector immunogenicity as well as cost considerations.

molecular biology↗

bioRxiv2019

Nucleic Acid Cleavage with a Hyperthermophilic Cas9 from an Unculturable Ignavibacterium

CRISPR-Cas9 systems have been effectively harnessed to engineer the genomes of organisms from across the tree of life. Nearly all currently characterized Cas9 proteins are derived from mesophilic bacteria, and canonical Cas9 systems are challenged by applications requiring enhanced stability or elevated temperatures. We discovered IgnaviCas9, a Cas9 protein from a hyperthermophilic Ignavibacterium identified through mini-metagenomic sequencing of samples from a hot spring. IgnaviCas9 is active at temperatures up to 100 {degrees}C in vitro, which enables DNA cleavage beyond the 44 {degrees}C limit of Streptococcus pyogenes Cas9 (SpyCas9) and the 70 {degrees}C limit of both Geobacillus stearothermophilus Cas9 (GeoCas9) and Geobacillus thermodenitrificans T12 Cas9 (ThermoCas9). As a potential application of this enzyme, we demonstrated that IgnaviCas9 can be used in bacterial RNA-seq library preparation to remove unwanted cDNA from 16s ribosomal rRNA (rRNA) without increasing the number of steps, thus underscoring the benefits provided by its exceptional thermostability in improving molecular biology and genomic workflows. Taken together, IgnaviCas9 is an exciting addition to the CRISPR-Cas9 toolbox and expands its temperature range.

genomics↗

bioRxiv2018

Systematic characterization of genome editing in primary T cells reveals proximal genomic insertions and enables machine learning prediction of CRISPR-Cas9 DNA repair outcomes

The Streptococcus pyogenes Cas9 (SpCas9) nuclease has become a ubiquitous genome editing tool due to its ability to target almost any location in DNA and create a double-stranded break1,2. After DNA cleavage, the break is fixed with endogenous DNA repair machinery, either by non-templated mechanisms (e.g. non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ)), or homology directed repair (HDR) using a complementary template sequence3,4. Previous work has shown that the distribution of repair outcomes within a cell population is non-random and dependent on the targeted sequence, and only recent efforts have begun to investigate this further5-11. However, no systematic work to date has been validated in primary human cells5,7. Here, we report DNA repair outcomes from 1,521 unique genomic locations edited with SpCas9 ribonucleoprotein complexes (RNPs) in primary human CD4+ T cells isolated from multiple healthy blood donors. We used targeted deep sequencing to measure the frequency distribution of repair outcomes for each guide RNA and discovered distinct features that drive individual repair outcomes after SpCas9 cleavage. Predictive features were combined into a new machine learning model, CRISPR Repair OUTcome (SPROUT), that predicts the length and probability of nucleotide insertions and deletions with R2 greater than 0.5. Surprisingly, we also observed large insertions at more than 90% of targeted loci, albeit at a low frequency. The inserted sequences aligned to diverse regions in the genome, and are enriched for sequences that are physically proximal to the break site due to chromatin interactions. This suggests a new mechanism where sequences from three-dimensionally neighboring regions of the genome can be inserted during DNA repair after Cas9-induced DNA breaks. Together, these findings provide powerful new predictive tools for Cas9-dependent genome editing and reveal new outcomes that can result from genome editing in primary T cells.

cell biology↗

bioRxiv2017

Correction of autoimmune IL2RA mutations in primary human T cells using non-viral genome targeting

Human T cells are central to physiological immune homeostasis, which protects us from pathogens without collateral autoimmune inflammation. They are also the main effectors in most current cancer immunotherapy strategies1. Several decades of work have aimed to genetically reprogram T cells for therapeutic purposes2-5, but as human T cells are resistant to most standard methods of large DNA insertion these approaches have relied on recombinant viral vectors, which do not target transgenes to specific genomic sites6, 7. In addition, the need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells through homology-directed repair (HDR), but to date in human T cells this still requires viral transduction8, 9. Here, we developed a non-viral, CRISPR-Cas9 genome targeting system that permits the rapid and efficient insertion of individual or multiplexed large (>1 kilobase) DNA sequences at specific sites in the genomes of primary human T cells while preserving cell viability and function. We successfully tested the potential therapeutic use of this approach in two settings. First, we corrected a pathogenic IL2RA mutation in primary T cells from multiple family members with monogenic autoimmune disease and demonstrated enhanced signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR redirecting T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized the tumour antigen, with concomitant cytokine release and tumour cell killing. Taken together, these studies provide preclinical evidence that non-viral genome targeting will enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

genetics↗