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Previously, we showed that expression of the p14 fusion-associated small transmembrane (FAST) fusogenic protein could enhance oncolytic HAdV efficacy and reduce tumor growth rate in a human xenograft mouse model of cancer. We now explore whether co-expression of the adenovirus death protein (ADP) with p14 FAST protein could synergize to further enhance oncolytic vector efficacy. ADP is naturally encoded within the early region 3 (E3) of HAdV, a region which is frequently removed from HAdV-based vectors, and functions to enhance cell lysis and progeny release. We evaluated a variety of approaches to achieve optimal expression of the two proteins, the most efficient method being insertion of an expression cassette within the E3 deletion, consisting of the coding sequences for p14 FAST protein and ADP separated by a self-cleaving peptide derived from the porcine teschovirus-1 (P2A). However, the quantities of p14 FAST protein and ADP produced from this vector were reduced approximately 10-fold compared to a similar vector-expressing only p14 FAST protein and wildtype HAdV, respectively. Compared to our original oncolytic vector-expressing p14 FAST protein alone, reduced expression of p14 FAST protein and ADP from the P2A construct reduced cell-cell fusion, vector spread, and cell-killing activity in human A549 adenocarcinoma cells in culture. These studies show that a self-cleaving peptide can be used to express two different transgenes in an armed oncolytic HAdV vector, but also highlight the challenges in maintaining adequate transgene expression when modifying vector design.<\/jats:p>","DOI":"10.3390\/ijms252212451","type":"journal-article","created":{"date-parts":[[2024,11,20]],"date-time":"2024-11-20T03:57:04Z","timestamp":1732075024000},"page":"12451","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Development and Characterization of an Oncolytic Human Adenovirus-Based Vector Co-Expressing the Adenovirus Death Protein and p14 Fusion-Associated Small Transmembrane Fusogenic Protein"],"prefix":"10.3390","volume":"25","author":[{"given":"Kathy L.","family":"Poulin","sequence":"first","affiliation":[{"name":"Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8420-8903","authenticated-orcid":false,"given":"Ryan G.","family":"Clarkin","sequence":"additional","affiliation":[{"name":"Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada"},{"name":"Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1N 6N5, Canada"},{"name":"Eric Poulin Centre for Neuromuscular Disease, University of Ottawa, Ottawa, ON K1N 6N5, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2391-1010","authenticated-orcid":false,"given":"Joshua","family":"Del Papa","sequence":"additional","affiliation":[{"name":"Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada"},{"name":"Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1N 6N5, Canada"},{"name":"Eric Poulin Centre for Neuromuscular Disease, University of Ottawa, Ottawa, ON K1N 6N5, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Robin J.","family":"Parks","sequence":"additional","affiliation":[{"name":"Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada"},{"name":"Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1N 6N5, Canada"},{"name":"Eric Poulin Centre for Neuromuscular Disease, University of Ottawa, Ottawa, ON K1N 6N5, Canada"},{"name":"Department of Medicine, University of Ottawa, Ottawa, ON K1N 6N5, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,11,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"209","DOI":"10.3322\/caac.21660","article-title":"Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries","volume":"71","author":"Sung","year":"2021","journal-title":"CA Cancer J. 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