Selected Grantee Publications
Evolution of the Clinical-Stage Hyperactive TcBuster Transposase as a Platform for Robust Non-Viral Production of Adoptive Cellular Therapies
Skeate et al., Molecular Therapy. 2024.
https://pubmed.ncbi.nlm.nih.gov/38627969/
In this study, the authors report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure-guided and in vitro evolution approaches that achieve high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. This proof-of-principle TcB-M engineering of CAR-NK and CAR-T cells shows low integrated vector copy number, a safe insertion site profile, robust in vitro function, and improved survival in a Burkitt lymphoma xenograft model in vivo. Their work suggests that TcB-M is a versatile, safe, efficient, and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition. Supported by ORIP (F30OD030021), NCI, NHLBI, and NIAID.
AAV5 Delivery of CRISPR/Cas9 Mediates Genome Editing in the Lungs of Young Rhesus Monkeys
Liang et al., Human Gene Therapy. 2024.
https://pubmed.ncbi.nlm.nih.gov/38767512/
Genome editing in somatic cells and tissues has the potential to provide long-term expression of therapeutic proteins to treat a variety of genetic lung disorders. However, delivering genome-editing machinery to disease-relevant cell types in the lungs of primates has remained a challenge. Investigators of this article are participating in the NIH Somatic Cell Genome Editing Consortium. Herein, they demonstrate that intratracheal administration of a dual adeno-associated virus type 5 vector encoding CRISPR/Cas9 can mediate genome editing in rhesus (male and female) airways. Up to 8% editing was observed in lung lobes, including a housekeeping gene, GAPDH, and a disease-related gene, angiotensin-converting enzyme 2. Using single-nucleus RNA-sequencing, investigators systematically characterized cell types transduced by the vector. Supported by ORIP (P51OD01110, U42OD027094, S10OD028713), NCATS, NCI, and NHLBI.
Engineered IgM and IgG Cleaving Enzymes for Mitigating Antibody Neutralization and Complement Activation in AAV Gene Transfer
Smith et al., Molecular Therapy. 2024.
https://www.sciencedirect.com/science/article/pii/S1525001624003058?via%3Dihub=
Recombinant adeno-associated viral (AAV) vectors have emerged as the leading platform for therapeutic gene transfer, but systemic dosing of AAV vectors poses potential risk of adverse side effects, including complement activation triggered by anti-capsid immunity. In this study, investigators discovered an IgM cleaving enzyme (IceM) that degrades human IgM, a key trigger in the anti-AAV immune cascade. They engineered a fusion enzyme (IceMG) with dual proteolytic activity against human IgM and IgG. Antisera from animals treated with IceMG show decreased ability to neutralize AAV and activate complement. These studies have implications for improving the safety of AAV gene therapies and offer broader applications, including for organ transplantation and autoimmune diseases. Supported by ORIP (P51OD011107, U42OD027094), NHLBI, and NIAID.
Time of Sample Collection Is Critical for the Replicability of Microbiome Analyses
Allaband et al., Nature Metabolism. 2024.
https://pubmed.ncbi.nlm.nih.gov/38951660/
Lack of replicability remains a challenge in microbiome studies. As the microbiome field moves from descriptive and associative research to mechanistic and interventional studies, being able to account for all confounding variables in the experimental design will be critical. Researchers conducted a retrospective analysis of 16S amplicon sequencing studies in male mice. They report that sample collection time affects the conclusions drawn from microbiome studies. The lack of consistency in the time of sample collection could help explain poor cross-study replicability in microbiome research. The effect of diurnal rhythms on the outcomes and study designs of other fields is unknown but is likely significant. Supported by ORIP (T32OD017863), NCATS, NCI, NHLBI, NIAAA, NIAID, NIBIB, NIDDK, and NIGMS.
A Revamped Rat Reference Genome Improves the Discovery of Genetic Diversity in Laboratory Rats
de Jong, Cell Genomics. 2024.
https://www.cell.com/cell-genomics/fulltext/S2666-979X(24)00069-7
Rattus norvegicus has been used in many fields of study related to human disease; its genome was sequenced shortly after the genomes of Homo sapiens and Mus musculus. Investigators report extensive analyses of the improvements in mRatBN7.2, compared with the previous version. They conducted a broad analysis of a whole-genome sequencing data set of 163 samples from 120 inbred rat strains and substrains. Several additional resources have been created. This new assembly and its associated resources create a more solid platform for research on the many dimensions of physiology, behavior, and pathobiology of rats and can provide more reliable and meaningful translation of findings to human populations. Supported by ORIP (R24OD024617), NHGRI, NHLBI, and NIDA.
Obesity Causes Mitochondrial Fragmentation and Dysfunction in White Adipocytes Due to RalA Activation
Xia et al., Nature Metabolism. 2024.
https://pubmed.ncbi.nlm.nih.gov/38286821/
This study presents a molecular mechanism for mitochondrial dysfunction as a characteristic trait of obesity. Chronic activation of the small GTPase RalA in inguinal white adipocytes (iWAT), in male mice fed a high-fat diet (HFD) represses energy expenditure by shifting mitochondrial dynamics toward excessive fission, contributing to weight gain and metabolic dysfunction. Targeted deletion of RalA in iWAT attenuated HFD-induced obesity due to increased energy expenditure and mitochondrial oxidative phosphorylation. Mechanistically, RalA dephosphorylates inhibitory Serine637 on fission protein Drp1, leading to excessive fission in adipocytes and mitochondrial fragmentation. Expression of a human homolog of Drp1—DNM1L—in adipose tissue is positively correlated with obesity and insulin resistance. These findings open avenues to investigate RalA-Drp1 axis in energy homeostasis. Supported by ORIP (S10OD023527), NCI, NHLBI, and NIDDK.
Macrophages Derived From Human Induced Pluripotent Stem Cells (iPSCs) Serve As a High-Fidelity Cellular Model for Investigating HIV-1, Dengue, and Influenza viruses
Yang et al., Journal of Virology. 2024.
https://pubmed.ncbi.nlm.nih.gov/38323811/
Macrophages can be weaponized by viruses to host viral reproduction and support long-term persistence. The most common way of studying these cells is by isolating their precursors from donor blood and differentiating the isolated cells into macrophages. This method is costly and technically challenging, and it produces varying results. In this study, researchers confirmed that macrophages derived from iPSC cell lines—a model that is inexpensive, consistent, and modifiable by genome editing—are a suitable model for experiments involving HIV and other viruses. Macrophages derived from iPSCs are as susceptible to infection as macrophages derived from blood, with similar infection kinetics and phenotypes. This new model offers researchers an unlimited source of cells for studying viral biology. Supported by ORIP (R01OD034046, S10OD021601), NIAID, NIDA, NIGMS, and NHLBI.
De Novo Variants in FRYL Are Associated With Developmental Delay, Intellectual Disability, and Dysmorphic Features
Pan et al., The American Journal of Human Genetics. 2024.
https://www.cell.com/ajhg/fulltext/S0002-9297(24)00039-9
FRY-like transcription coactivator (FRYL) belongs to a Furry protein family that is evolutionarily conserved from yeast to humans, and its functions in mammals are largely unknown. Investigators report 13 individuals who have de novo heterozygous variants in FRYL and one individual with a heterozygous FRYL variant that is not confirmed to be de novo. The individuals present with developmental delay; intellectual disability; dysmorphic features; and other congenital anomalies in cardiovascular, skeletal, gastrointestinal, renal, and urogenital systems. Using fruit flies, investigators provide evidence that haploinsufficiency in FRYL likely underlies a disorder in humans with developmental and neurological symptoms. Supported by ORIP (U54OD030165), NHLBI, NICHD, and NCATS.
Newly Identified Roles for PIEZO1 Mechanosensor in Controlling Normal Megakaryocyte Development and in Primary Myelofibrosis
Abbonante et al., American Journal of Hematology. 2024.
https://pubmed.ncbi.nlm.nih.gov/38165047/
Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix to promote lineage differentiation are only partially understood. The authors report that PIEZO1, a mechanosensitive cation channel, is expressed in mouse and human Mks, and activation of PIEZO1 increased the number of immature Mks in mice. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Together, these data suggest that PIEZO1 places a brake on Mk maturation and platelet formation in physiology, and its upregulation might contribute to aggravating disease. Supported by ORIP (K01OD025290), NHGRI, NHLBI, and NCATS.
Stable HIV Decoy Receptor Expression After In Vivo HSC Transduction in Mice and NHPs: Safety and Efficacy in Protection From SHIV
Li, Molecular Therapy. 2023.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10124088/
Autologous hematopoietic stem cell (HSC) gene therapy offers a promising HIV treatment strategy, but cost, complexity, and toxicity remain significant challenges. Using female mice and female nonhuman primates (NHPs) (i.e., rhesus macaques), researchers developed an approach based on the stable expression of eCD4-Ig, a secreted decoy protein for HIV and simian–human immunodeficiency virus (SHIV) receptors. Their goals were to (1) assess the kinetics and serum level of eCD4-Ig, (2) evaluate the safety of HSC transduction with helper-dependent adenovirus–eCD4-Ig, and (3) test whether eCD4-Ig expression has a protective effect against viral challenge. They found that stable expression of the decoy receptor was achieved at therapeutically relevant levels. These data will guide future in vivo studies. Supported by ORIP (P51OD010425) and NHLBI.