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Although stable gene down-regulation in cells can be achieved with viral vectors expressing short hairpin RNAs (shRNAs), incorporating shRNAs into a microRNA (miR) backbone can improve both the efficacy and safety of RNAi by exploiting the endogenous miR-processing machinery. For gamma-retroviral vectors there are two main strategies, LTR (Pol II)- and U6 (Pol III)-driven expression of the miR-based shRNA, but both have limitations in viral titers generated, knock-down efficacy, and\/or protein\/reporter gene co-expression levels. Here, we sought to evaluate the effectiveness of inserting miR-30a-based shRNA within the psi\/packaging-containing intron of the murine stem cell virus (MSCV)-based splice-gag vector (MSGV) for which transcription is LTR-driven. Systematic comparison to previously described designs within the same viral backbone comprising a range of different shRNAs targets revealed maximal viral titer, knock-down and level of protein co-expression upon intronic insertion of miR-based shRNA. In addition, we demonstrated that the strategy can be extended to gene knock-down by wild-type miRs, as demonstrated for miR-30c1 and let-7a. Taken together, we have developed a gamma-retroviral vector design facilitating efficacious miR-mediated gene\u00a0knock-down and maintaining strong viral titers.<\/jats:p>","DOI":"10.1093\/narmme\/ugaf030","type":"journal-article","created":{"date-parts":[[2025,8,9]],"date-time":"2025-08-09T11:42:19Z","timestamp":1754739739000},"source":"Crossref","is-referenced-by-count":0,"title":["Gamma-retrovirus design for efficacious microRNA-mediated gene knock-down and protein co-expression without compromising titers"],"prefix":"10.1093","volume":"2","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6573-9752","authenticated-orcid":false,"given":"Romain","family":"Vuillefroy\u00a0de\u00a0Silly","sequence":"first","affiliation":[{"name":"Ludwig Institute for Cancer Research , University of Lausanne and Department of Oncology, Lausanne University Hospital (CHUV), CH-1011\u00a0 Lausanne ,","place":["Switzerland"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2479-6261","authenticated-orcid":false,"given":"Bili","family":"Seijo","sequence":"additional","affiliation":[{"name":"Ludwig Institute for Cancer Research , University of Lausanne and Department of Oncology, Lausanne University Hospital (CHUV), CH-1011\u00a0 Lausanne ,","place":["Switzerland"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-7471-7464","authenticated-orcid":false,"given":"Jimmy","family":"Maillard","sequence":"additional","affiliation":[{"name":"Ludwig Institute for Cancer Research , University of Lausanne and Department of Oncology, Lausanne University Hospital (CHUV), CH-1011\u00a0 Lausanne ,","place":["Switzerland"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Patrick","family":"Reichenbach","sequence":"additional","affiliation":[{"name":"Ludwig Institute for Cancer Research , University of Lausanne and Department of Oncology, Lausanne University Hospital (CHUV), CH-1011\u00a0 Lausanne ,","place":["Switzerland"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6849-7194","authenticated-orcid":false,"given":"Melita","family":"Irving","sequence":"additional","affiliation":[{"name":"Ludwig Institute for Cancer Research , University of Lausanne and Department of Oncology, Lausanne University Hospital (CHUV), CH-1011\u00a0 Lausanne ,","place":["Switzerland"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2025,8,12]]},"reference":[{"key":"2025121905382165600_B1","doi-asserted-by":"publisher","first-page":"4051","DOI":"10.1038\/35888","article-title":"Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans","volume":"391","author":"Fire","year":"1998","journal-title":"EMBO 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