Selected Grantee Publications
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- HIV/AIDS
- Somatic Cell Genome Editing
- CRISPR
Amphiphilic Shuttle Peptide Delivers Base Editor Ribonucleoprotein to Correct the CFTR R553X Mutation in Well-Differentiated Airway Epithelial Cells
Kulhankova et al., Nucleic Acids Research. 2024.
https://academic.oup.com/nar/article/52/19/11911/7771564?login=true
Effective translational delivery strategies for base editing applications in pulmonary diseases remain a challenge because of epithelial cells lining the intrapulmonary airways. The researchers demonstrated that the endosomal leakage domain (ELD) plays a crucial role in gene editing ribonucleoprotein (RNP) delivery activity. A novel shuttle peptide, S237, was created by flanking the ELD with poly glycine-serine stretches. Primary airway epithelia with the cystic fibrosis transmembrane conductance regulator (CFTR) R533X mutation demonstrated restored CFTR function when treated with S237-dependent ABE8e-Cas9-NG RNP. S237 outperformed the S10 shuttle peptide at Cas9 RNP delivery in vitro and in vivo using primary human bronchial epithelial cells and transgenic green fluorescent protein neonatal pigs. This study highlights the efficacy of S237 peptide–mediated RNP delivery and its potential as a therapeutic tool for the treatment of cystic fibrosis. Supported by ORIP (U42OD027090, U42OD026635), NCATS, NHGRI, NHLBI, NIAID, NIDDK, and NIGMS.
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.
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.
Preclinical Safety and Biodistribution of CRISPR Targeting SIV in Non-Human Primates
Burdo et al., Gene Therapy. 2024.
https://pmc.ncbi.nlm.nih.gov/articles/PMC11090835/
Nonhuman primates have served as a valuable resource for evaluating novel eradication and cure strategies for HIV infection. Using a male rhesus macaque model, researchers demonstrated the safety and utility of CRISPR gene-editing technology for targeting integrated simian immunodeficiency virus (SIV). Their work suggests that a single intravenous inoculation for HIV gene editing can be utilized to reach viral reservoirs throughout the body. Additionally, no off-target effects or abnormal pathology were observed. Together, these findings support the continued development of HIV eradicative cure strategies using CRISPR technology in humans. Supported by ORIP (P40OD012217, U42OD021458).
Focused Ultrasound–Mediated Brain Genome Editing
Lao et al., PNAS. 2023.
https://www.pnas.org/doi/epdf/10.1073/pnas.2302910120
Gene editing in the brain has been challenging because of the restricted transport imposed by the blood–brain barrier (BBB). In this study, investigators described a safe and effective gene‑editing technique by using focused ultrasound (FUS) to transiently open the BBB for the transport of intravenously delivered CRISPR machinery to the brain in mice. By combining FUS with adeno-associated virus–mediated gene delivery, researchers can achieve more than 25% editing efficiency of particular cell types. This method has the potential to expand toolkit options for CRISPR delivery and opens opportunities for treating diseases of the brain, such as neurodegenerative disorders, with somatic genome editing. Supported by ORIP (U42OD026635) and NINDS.
AAV5 Delivery of CRISPR-Cas9 Supports Effective Genome Editing in Mouse Lung Airway
Liang et al., Molecular Therapy. 2022.
https://www.cell.com/molecular-therapy-family/molecular-therapy/fulltext/S1525-0016(21)00530-X
Genome editing in the lung has the potential to provide long-term expression of therapeutic protein to treat lung genetic diseases. The authors illustrated that AAV5 can efficiently deliver CRISPR-Cas9 to mouse lung airways and was the first to achieve ∼20% editing efficiency in those airways. Results were confirmed through independent experiments at two different institutes. This highly efficient dual AAV platform will facilitate the study of genome editing in the lung and other tissue types. Supported by ORIP (U42OD026645).
Thresholds for Post-Rebound SHIV Control after CCR5 Gene-Edited Autologous Hematopoietic Cell Transplantation
Cardozo-Ojeda et al., eLife. 2021.
https://elifesciences.org/articles/57646
Investigators developed a mathematical model to project the minimum threshold of C-C chemokine receptor type 5 (CCR5) gene-edited cells necessary for a functional cure from HIV. This was based on blood T cell reconstitution and plasma simian-HIV (SHIV) dynamics from SHIV-1157ipd3N4-infected juvenile pig-tailed macaques that underwent autologous transplantation with CCR5 gene editing. The model predicts that viral control can be obtained following analytical treatment interruption (ATI) when: (1) transplanted hematopoietic stem and progenitor cells (HSPCs) are at least fivefold higher than residual endogenous HSPCs after total body irradiation and (2) the fraction of protected HSPCs in the transplant achieves a threshold (76–94%) sufficient to overcome transplantation-dependent loss of SHIV immunity. Under these conditions, if ATI is withheld until transplanted gene-modified cells engraft and reconstitute to a steady state, spontaneous viral control is projected to occur. Supported by ORIP (P51OD010425), NCATS and NIAID.