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SARS-CoV-2, like SARS-CoV-1, uses the human cell surface protein angiotensin-converting enzyme 2 (ACE2) as its native receptor. Here, we design and characterize a mutant ACE2 that enables rapid affinity purification of a dimeric protein by altering the active site to prevent autoproteolytic digestion of a C-terminal His\n                      <jats:sub>10<\/jats:sub>\n                      epitope tag. In cultured cells, mutant ACE2 competitively inhibits lentiviral vectors pseudotyped with spikes from multiple SARS-CoV-2 variants and infectious SARS-CoV-2. Moreover, the protein can be nebulized and retains virus-binding properties. We developed a system for the delivery of aerosolized ACE2 to K18-hACE2 mice and demonstrated protection by our modified ACE2 when delivered as a prophylactic agent. These results show proof-of-concept for an aerosolized delivery method to evaluate anti-SARS-CoV-2 agents\n                      <jats:italic>in vivo<\/jats:italic>\n                      and suggest a new tool in the ongoing fight against SARS-CoV-2 and other ACE2-dependent viruses.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      The rapid evolution of SARS-CoV-2 variants poses a challenge for immune recognition and antibody therapies. However, the virus is constrained by the requirement that it recognizes a human host receptor protein. A recombinant ACE2 could protect against SARS-CoV-2 infection by functioning as a soluble decoy receptor. We designed a mutant version of ACE2 with impaired catalytic activity to enable the purification of the protein using a single affinity purification step. This protein can be nebulized and retains the ability to bind the relevant domains from SARS-CoV-1 and SARS-CoV-2. Moreover, this protein inhibits viral infection against a panel of coronaviruses in cells. Finally, we developed an aerosolized delivery system for animal studies and show the modified ACE2 offers protection in an animal model of COVID-19. These results show proof-of-concept for an aerosolized delivery method to evaluate anti-SARS-CoV-2 agents\n                      <jats:italic>in vivo<\/jats:italic>\n                      and suggest a new tool in the ongoing fight against SARS-CoV-2.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.00768-24","type":"journal-article","created":{"date-parts":[[2024,5,21]],"date-time":"2024-05-21T09:02:03Z","timestamp":1716282123000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":9,"title":["Development of a mutant aerosolized ACE2 that neutralizes SARS-CoV-2\n                    <i>in vivo<\/i>"],"prefix":"10.1128","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8657-9348","authenticated-orcid":true,"given":"Daniel L.","family":"Kober","sequence":"first","affiliation":[{"name":"Department of Biochemistry, The University of Texas Southwestern Medical Center, 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