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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR–Cas9 ribonucleoprotein

Lipid nanoparticle (LNP) delivery of clustered regularly interspaced short palindromic repeat (CRISPR) ribonucleoproteins (RNPs) could enable high-efficiency, low-toxicity and scalable in vivo genome editing if efficacious RNP–LNP complexes can be reliably produced. Here we engineer a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) to generate iGeoCas9 variants capable of >100× more genome editing of cells and organs compared with the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP–LNP complexes edit a variety of cell types and induce homology-directed repair in cells receiving codelivered single-stranded DNA templates. Using tissue-selective LNP formulations, we observe genome-editing levels of 16-37% in the liver and lungs of reporter mice that receive single intravenous injections of iGeoCas9 RNP–LNPs. In addition, iGeoCas9 RNPs complexed to biodegradable LNPs edit the disease-causing SFTPC gene in lung tissue with 19% average efficiency, representing a major improvement over genome-editing levels observed previously using viral or nonviral delivery strategies. These results show that thermostable Cas9 RNP–LNP complexes can expand the therapeutic potential of genome editing.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A Murine Model of Radionuclide Lung Contamination for the Evaluation of Americium Decorporation Treatments

The hydroxypyridinone ligand 3,4,3-LI(1,2-HOPO) (HOPO), has been previously characterized as a promising chelating agent for in vivo decorporation of actinides, with decorporation being the removal of internally deposited contaminants from the body after exposure. The large majority of relevant literature reports have detailed the efficacy profile of HOPO as a decorporation agent in rodent models, where controlled radionuclide contamination is conducted via intravenous injection. However, this method of contamination does not necessarily reflect an accurate predictive model of the most probable biodistribution of free metal in the body. In the event of a radiological dispersal device or nuclear power plant accident scenario, it is most likely that first responders, military personnel, and victims of the event will be contaminated via air and water transmission. Therefore, research into the efficacy of chelating agents to treat lung-contaminated in vivo models needs to be carried out. Here, we establish a murine model with controlled, reproducible lung contamination using two different radionuclides, 89Zr and 241Am, for orthogonal biodistribution validation by positron emission tomography and ex vivo radioanalysis, respectively. In addition, we report effective chelation treatment of 241Am-contaminated lungs using HOPO, which improves decorporation by up to 40% compared to Ca-DTPA, the current standard of care.

Arino, Trevor↗
DOE OSTI2024

Evaluation of two inoculation routes of an adenovirus-mediated viral protein inhibitor in a Crimean-Congo hemorrhagic fever mouse model

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne nairovirus with a wide geographic spread that can cause severe and lethal disease. No specific medical countermeasures are approved to combat this illness. The CCHFV L protein contains an ovarian tumor (OTU) domain with a cysteine protease thought to modulate cellular immune responses by removing ubiquitin and ISG15 post-translational modifications from host and viral proteins. Viral deubiquitinases like CCHFV OTU are attractive drug targets, as blocking their activity may enhance cellular immune responses to infection, and potentially inhibit viral replication itself. We previously demonstrated that the engineered ubiquitin variant CC4 is a potent inhibitor of CCHFV replication in vitro. A major challenge of the therapeutic use of small protein inhibitors such as CC4 is their requirement for intracellular delivery, e.g., by viral vectors. In this study, we examined the feasibility of in vivo CC4 delivery by a replication-deficient recombinant adenovirus (Ad-CC4) in a lethal CCHFV mouse model. Since the liver is a primary target of CCHFV infection, we aimed to optimize delivery to this organ by comparing intravenous (tail vein) and intraperitoneal injection of Ad-CC4. While tail vein injection is a traditional route for adenovirus delivery, in our hands intraperitoneal injection resulted in higher and more widespread levels of adenovirus genome in tissues, including, as intended, the liver. However, despite promising in vitro results, neither route of in vivo CC4 treatment resulted in protection from a lethal CCHFV infection.

59 BASIC BIOLOGICAL SCIENCES↗