{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,16]],"date-time":"2026-10-16T03:12:54Z","timestamp":1792120374781,"version":"4.4.0"},"reference-count":119,"publisher":"MDPI AG","issue":"22","license":[{"start":{"date-parts":[[2023,11,20]],"date-time":"2023-11-20T00:00:00Z","timestamp":1700438400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100005633","name":"Finnish Foundation for Cardiovascular Research","doi-asserted-by":"publisher","award":["220051"],"award-info":[{"award-number":["220051"]}],"id":[{"id":"10.13039\/501100005633","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Adenoviral vectors are commonly used in clinical gene therapy. Apart from oncolytic adenoviruses, vector replication is highly undesired as it may pose a safety risk for the treated patient. Thus, careful monitoring for the formation of replication-competent adenoviruses (RCA) during vector manufacturing is required. To render adenoviruses replication deficient, their genomic E1 region is deleted. However, it has been known for a long time that during their propagation, some viruses will regain their replication capability by recombination in production cells, most commonly HEK293. Recently developed RCA assays have revealed that many clinical batches contain more RCA than previously assumed and allowed by regulatory authorities. The clinical significance of the higher RCA content has yet to be thoroughly evaluated. In this review, we summarize the biology of adenovirus vectors, their manufacturing methods, and the origins of RCA formed during HEK293-based vector production. Lastly, we share our experience using minimally RCA-positive serotype 5 adenoviral vectors based on observations from our clinical cardiovascular gene therapy studies.<\/jats:p>","DOI":"10.3390\/ijms242216519","type":"journal-article","created":{"date-parts":[[2023,11,20]],"date-time":"2023-11-20T10:32:32Z","timestamp":1700476352000},"page":"16519","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":38,"title":["Adenoviral Gene Therapy Vectors in Clinical Use\u2014Basic Aspects with a Special Reference to Replication-Competent Adenovirus Formation and Its Impact on Clinical Safety"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9590-0105","authenticated-orcid":false,"given":"Aleksi J.","family":"Leikas","sequence":"first","affiliation":[{"name":"Heart Center, Kuopio University Hospital, 70200 Kuopio, Finland"},{"name":"Gene Therapy Unit, Kuopio University Hospital, 70200 Kuopio, Finland"},{"name":"A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7593-2708","authenticated-orcid":false,"given":"Seppo","family":"Yl\u00e4-Herttuala","sequence":"additional","affiliation":[{"name":"Heart Center, Kuopio University Hospital, 70200 Kuopio, Finland"},{"name":"Gene Therapy Unit, Kuopio University Hospital, 70200 Kuopio, Finland"},{"name":"A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0847-107X","authenticated-orcid":false,"given":"Juha E. K.","family":"Hartikainen","sequence":"additional","affiliation":[{"name":"Heart Center, Kuopio University Hospital, 70200 Kuopio, Finland"},{"name":"Gene Therapy Unit, Kuopio University Hospital, 70200 Kuopio, Finland"},{"name":"School of Medicine, Faculty of Health Sciences, University of Eastern Finland, 70210 Kuopio, Finland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,11,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1056\/NEJMra1706910","article-title":"Gene Therapy","volume":"381","author":"High","year":"2019","journal-title":"N. Engl. J. Med."},{"key":"ref_2","unstructured":"(2022, September 22). Approved Cellular and Gene Therapy Products, Available online: https:\/\/www.fda.gov\/vaccines-blood-biologics\/cellular-gene-therapy-products\/approved-cellular-and-gene-therapy-products."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"567","DOI":"10.1111\/joim.13308","article-title":"Gene therapy for ischaemic heart disease and heart failure","volume":"290","author":"Korpela","year":"2021","journal-title":"J. Intern. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1038\/nrg2985","article-title":"Ex vivo gene transfer and correction for cell based therapies","volume":"12","author":"Naldini","year":"2011","journal-title":"Nat. Rev. Genet."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"464","DOI":"10.1016\/j.ymthe.2020.12.007","article-title":"Current Clinical Applications of In Vivo Gene Therapy with AAVs","volume":"29","author":"Mendell","year":"2021","journal-title":"Mol. Ther."},{"key":"ref_6","unstructured":"(2022, September 22). Gene Therapy Clinical Trials Worldwide\u2014Provided by the Journal of Gene Medicine. Available online: https:\/\/a873679.fmphost.com\/fmi\/webd\/GTCT."},{"key":"ref_7","unstructured":"Curiel, D.T. (2016). Adenoviral Vectors for Gene Therapy, Academic Press. [2nd ed.]."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1038\/s41541-021-00356-x","article-title":"Adenoviral vector vaccine platforms in the SARS-CoV-2 pandemic","volume":"6","author":"Lorincz","year":"2021","journal-title":"NPJ Vaccines"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"421","DOI":"10.2174\/1566523213666131125095046","article-title":"Adenovirus vectors for gene therapy, vaccination and cancer gene therapy","volume":"13","author":"Wold","year":"2013","journal-title":"Curr. Gene Ther."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1016\/j.ymgme.2003.08.016","article-title":"Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer","volume":"80","author":"Raper","year":"2003","journal-title":"Mol. Genet. Metab."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1233","DOI":"10.1038\/sj.mt.6300175","article-title":"Current status of cardiovascular gene therapy","volume":"15","author":"Rissanen","year":"2007","journal-title":"Mol. Ther."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"709","DOI":"10.1038\/sj.gt.3300902","article-title":"Who\u2019s afraid of replication-competent adenoviruses?","volume":"6","author":"Fallaux","year":"1999","journal-title":"Gene Ther."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1485","DOI":"10.1089\/hum.1994.5.12-1485","article-title":"Emergence of early region 1-containing replication-competent adenovirus in stocks of replication-defective adenovirus recombinants (\u0394E1 + \u0394E3) during multiple passages in 293 cells","volume":"5","author":"Jani","year":"1994","journal-title":"Hum. Gene Ther."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1049","DOI":"10.1089\/hum.1997.8.9-1049","article-title":"Adenoviral vector-mediated gene expression in the nervous system of immunocompetent Wistar and T cell-deficient nude rats: Preferential survival of transduced astroglial cells in nude rats","volume":"8","author":"Hermens","year":"1997","journal-title":"Hum. Gene Ther."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"711","DOI":"10.1089\/hum.1995.6.6-711","article-title":"Trans-complementation of E1-deleted adenovirus: A new vector to reduce the possibility of codissemination of wild-type and recombinant adenoviruses","volume":"6","author":"Imler","year":"1995","journal-title":"Hum. Gene Ther."},{"key":"ref_16","unstructured":"U.S. Department of Health and Human Services Food and Drug Administration Center for Biologics Evaluation and Research (2022, October 24). Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy, Available online: https:\/\/www.fda.gov\/media\/72402\/download."},{"key":"ref_17","unstructured":"U.S. Department of Health and Human Services Food and Drug Administration Center for Biologics Evaluation and Research (2022, October 24). Guidance for Industry: Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs), Available online: https:\/\/www.fda.gov\/media\/113760\/download."},{"key":"ref_18","unstructured":"Peltonen, H., Mykk\u00e4nen, M., and Hassinen, M. (2019). Gene Therapy Vector Contamination Assay. (2019\/0292609 A1), U.S. Patent."},{"key":"ref_19","unstructured":"Fields, B.N., Knipe, D.M., and Griffin, D.E. (2007). Fields Virology, Wolters Kluwer Health\/Lippincott Williams & Wilkins. [5th ed.]."},{"key":"ref_20","unstructured":"Bennett, J.E., Dolin, R., and Blaser, J. (2015). Mandell, Douglas, and Bennett\u2019s Principles and Practice of Infectious Diseases, Elsevier Saunders. [8th ed.]."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1093\/oxfordjournals.aje.a120913","article-title":"The virus watch program: A continuing surveillance of viral infections in metropolitan New York families: Observations of adenovirus infections: Virus excretion patterns, antibody response, efficiency of surveillance, patterns of infection, and relation to illness","volume":"89","author":"Fox","year":"1969","journal-title":"Am. J. Epidemiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1016\/S1473-3099(09)70069-6","article-title":"Incubation periods of acute respiratory viral infections: A systematic review","volume":"9","author":"Lessler","year":"2009","journal-title":"Lancet Infect. Dis."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"950","DOI":"10.1016\/j.vaccine.2009.10.145","article-title":"International epidemiology of human pre-existing adenovirus (Ad) type-5, type-6, type-26 and type-36 neutralizing antibodies: Correlates of high Ad5 titers and implications for potential HIV vaccine trials","volume":"28","author":"Mast","year":"2010","journal-title":"Vaccine"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1002\/rmv.589","article-title":"Clinical features and treatment of adenovirus infections","volume":"18","author":"Lenaerts","year":"2008","journal-title":"Rev. Med. Virol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2000","DOI":"10.1128\/JVI.80.4.2000-2012.2006","article-title":"Genetic identification of adenovirus type 5 genes that influence viral spread","volume":"80","author":"Subramanian","year":"2006","journal-title":"J. Virol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3549","DOI":"10.1128\/JVI.80.7.3549-3558.2006","article-title":"Replication properties of human adenovirus in vivo and in cultures of primary cells from different animal species","volume":"80","author":"Jogler","year":"2006","journal-title":"J. Virol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"819","DOI":"10.1038\/sj.cgt.7700765","article-title":"Experimental infections of humans with wild-type adenoviruses and with replication-competent adenovirus vectors: Replication, safety, and transmission","volume":"11","author":"Lichtenstein","year":"2004","journal-title":"Cancer Gene Ther."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"6141","DOI":"10.1128\/JVI.73.7.6141-6146.1999","article-title":"Frequency and stability of chromosomal integration of adenovirus vectors","volume":"73","author":"Harui","year":"1999","journal-title":"J. Virol."},{"key":"ref_29","first-page":"917","article-title":"Transformation of rodent cells by adenovirus 19 and other group D adenoviruses","volume":"43","author":"McAllister","year":"1969","journal-title":"J. Natl. Cancer Inst."},{"key":"ref_30","first-page":"1293","article-title":"Adenovirus isolates from urine of patients with acquired immunodeficiency syndrome","volume":"1","author":"Valderrama","year":"1983","journal-title":"Lancet"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"921","DOI":"10.3201\/eid1206.050245","article-title":"Co-infections of adenovirus species in previously vaccinated patients","volume":"12","author":"Vora","year":"2006","journal-title":"Emerg. Infect. Dis."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1835","DOI":"10.1016\/S0161-6420(93)31389-8","article-title":"Prolonged recovery of desiccated adenoviral serotypes 5, 8, and 19 from plastic and metal surfaces in vitro","volume":"100","author":"Gordon","year":"1993","journal-title":"Ophthalmology"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1419","DOI":"10.1128\/AAC.50.4.1419-1424.2006","article-title":"Efficacy of hospital germicides against adenovirus 8, a common cause of epidemic keratoconjunctivitis in health care facilities","volume":"50","author":"Rutala","year":"2006","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1093\/milmed\/160.6.300","article-title":"Adenovirus vaccines in the U.S. military","volume":"160","author":"Gaydos","year":"1995","journal-title":"Mil. Med."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1093\/oxfordjournals.aje.a120866","article-title":"Characteristics of vaccine-induced and natural infection with adenovirus type 4 in naval recruits","volume":"88","author":"Rosenbaum","year":"1968","journal-title":"Am. J. Epidemiol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1006\/smvy.1997.0139","article-title":"Control of apoptosis by human adenovirus genes","volume":"5","author":"Chinnadurai","year":"1998","journal-title":"Semin. Virol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1038\/sj.onc.1201540","article-title":"The large E1B protein together with the E4orf6 protein target p53 for active degradation in adenovirus infected cells","volume":"16","author":"Steegenga","year":"1998","journal-title":"Oncogene"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1080\/08830180490265556","article-title":"Functions and mechanisms of action of the adenovirus E3 proteins","volume":"23","author":"Lichtenstein","year":"2004","journal-title":"Int. Rev. Immunol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2296","DOI":"10.1128\/jvi.70.4.2296-2306.1996","article-title":"The adenovirus death protein (E3-11.6K) is required at very late stages of infection for efficient cell lysis and release of adenovirus from infected cells","volume":"70","author":"Tollefson","year":"1996","journal-title":"J. Virol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1356","DOI":"10.1073\/pnas.84.5.1356","article-title":"\u201cE3\/19K\u201d protein of adenovirus type 2 inhibits lysis of cytolytic T lymphocytes by blocking cell-surface expression of histocompatibility class I antigens","volume":"84","author":"Burgert","year":"1987","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"4114","DOI":"10.1128\/jvi.65.8.4114-4123.1991","article-title":"The 10,400- and 14,500-dalton proteins encoded by region E3 of adenovirus function together to protect many but not all mouse cell lines against lysis by tumor necrosis factor","volume":"65","author":"Gooding","year":"1991","journal-title":"J. Virol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"3270","DOI":"10.1074\/jbc.M008218200","article-title":"Three adenovirus E3 proteins cooperate to evade apoptosis by tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2","volume":"276","author":"Benedict","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"5815","DOI":"10.1074\/jbc.273.10.5815","article-title":"Interaction of the adenovirus 14.7kDa protein with FLICE inhibits Fas ligand-induced apoptosis","volume":"273","author":"Chen","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"4904","DOI":"10.1128\/jvi.70.8.4904-4913.1996","article-title":"The adenovirus E3-14.7K protein and the E3-10.4K\/14.5K complex of proteins, which independently inhibit tumor necrosis factor (TNF)-induced apoptosis, also independently inhibit TNF-induced release of arachidonic acid","volume":"70","author":"Krajcsi","year":"1996","journal-title":"J. Virol."},{"key":"ref_45","first-page":"177","article-title":"Regulation of adenovirus E2 transcription unit","volume":"199","author":"Swaminathan","year":"1995","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"2605","DOI":"10.1128\/jvi.63.6.2605-2615.1989","article-title":"Adenovirus early region 4 encodes two gene products with redundant effects in lytic infection","volume":"63","author":"Huang","year":"1989","journal-title":"J. Virol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"631","DOI":"10.1128\/jvi.63.2.631-638.1989","article-title":"Redundant control of adenovirus late gene expression by early region 4","volume":"63","author":"Bridge","year":"1989","journal-title":"J. Virol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"2022","DOI":"10.1128\/JVI.72.3.2022-2032.1998","article-title":"In vitro and in vivo biology of recombinant adenovirus vectors with E1, E1\/E2A, or E1\/E4 deleted","volume":"72","author":"Lusky","year":"1998","journal-title":"J. Virol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1587","DOI":"10.1089\/hum.1998.9.11-1587","article-title":"Toxicological comparison of E2a-deleted and first-generation adenoviral vectors expressing alpha1-antitrypsin after systemic delivery","volume":"9","author":"Zhou","year":"1998","journal-title":"Hum. Gene. Ther."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1452","DOI":"10.1161\/01.ATV.20.6.1452","article-title":"Second-generation adenoviral vectors do not prevent rapid loss of transgene expression and vector DNA from the arterial wall","volume":"20","author":"Wen","year":"2000","journal-title":"Arterioscler. Thromb. Vasc. Biol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1080\/10623320109051563","article-title":"Effects of first generation E1E3-deleted and second generation E1E3E4-deleted\/modified adenovirus vectors on human endothelial cell death","volume":"8","author":"Jornot","year":"2001","journal-title":"Endothelium"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1681","DOI":"10.3390\/v2081681","article-title":"Adenoviral producer cells","volume":"2","author":"Kovesdi","year":"2010","journal-title":"Viruses"},{"key":"ref_53","unstructured":"Curiel, D.T. (2016). Adenoviral Vectors for Gene Therapy, Academic Press. [2nd ed.]."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"222","DOI":"10.2174\/156652308785160647","article-title":"Advances in helper-dependent adenoviral vector research","volume":"8","author":"Segura","year":"2008","journal-title":"Curr. Gene Ther."},{"key":"ref_55","first-page":"247","article-title":"Helper-dependent adenovirus vectors devoid of all viral genes cause less myocardial inflammation compared with first-generation adenovirus vectors","volume":"99","author":"Fleury","year":"2004","journal-title":"Basic Res. Cardiol."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"761","DOI":"10.1089\/hum.2013.071","article-title":"Transgene expression up to 7 years in nonhuman primates following hepatic transduction with helper-dependent adenoviral vectors","volume":"24","author":"Ng","year":"2013","journal-title":"Hum. Gene. Ther."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"743","DOI":"10.1038\/gt.2016.53","article-title":"Long-term, high-level hepatic secretion of acid \u03b1-glucosidase for Pompe disease achieved in non-human primates using helper-dependent adenovirus","volume":"23","author":"Rastall","year":"2016","journal-title":"Gene Ther."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1128\/jvi.37.1.171-180.1981","article-title":"Cloning of a DNA fragment from the left-hand terminus of the adenovirus type 2 genome and its use in site-directed mutagenesis","volume":"37","author":"Stow","year":"1981","journal-title":"J. Virol."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1016\/S0169-409X(01)00215-0","article-title":"Approaches for generating recombinant adenovirus vectors","volume":"52","author":"Mizuguchi","year":"2001","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_60","unstructured":"Curiel, D.T. (2016). Adenoviral Vectors for Gene Therapy, Academic Press. [2nd ed.]."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"2577","DOI":"10.1089\/hum.1998.9.17-2577","article-title":"Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method","volume":"9","author":"Mizuguchi","year":"1998","journal-title":"Hum. Gene Ther."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"502","DOI":"10.2144\/99263rr01","article-title":"Novel cloning method for recombinant adenovirus construction in Escherichia coli","volume":"26","author":"Souza","year":"1999","journal-title":"Biotechniques"},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"80","DOI":"10.1038\/sj.gt.3301047","article-title":"New tools for the generation of E1- and\/or E3-substituted adenoviral vectors","volume":"7","author":"Danthinne","year":"2000","journal-title":"Gene Ther."},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1186\/1743-422X-6-18","article-title":"A simplified in vitro ligation approach to clone an E1B55k-deleted double-targeted conditionally-replicative adenovirus","volume":"6","author":"Haviv","year":"2009","journal-title":"Virol. J."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1099\/0022-1317-36-1-59","article-title":"Characteristics of a human cell line transformed by DNA from human adenovirus type 5","volume":"36","author":"Graham","year":"1977","journal-title":"J. Gen. Virol."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"8802","DOI":"10.1073\/pnas.91.19.8802","article-title":"An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3","volume":"91","author":"Bett","year":"1994","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"614","DOI":"10.1016\/0042-6822(88)90302-9","article-title":"A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5","volume":"163","author":"McGrory","year":"1988","journal-title":"Virology"},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"4805","DOI":"10.1128\/jvi.70.7.4805-4810.1996","article-title":"Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli","volume":"70","author":"Chartier","year":"1996","journal-title":"J. Virol."},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"1236","DOI":"10.1038\/nprot.2007.135","article-title":"A protocol for rapid generation of recombinant adenoviruses using the AdEasy system","volume":"2","author":"Luo","year":"2007","journal-title":"Nat. Protoc."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"6186","DOI":"10.1073\/pnas.91.13.6186","article-title":"Efficient manipulation of the human adenovirus genome as an infectious yeast artificial chromosome clone","volume":"91","author":"Ketner","year":"1994","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1089\/104303403321208934","article-title":"Hybrid yeast-bacteria cloning system used to capture and modify adenoviral and nonviral genomes","volume":"14","author":"Hokanson","year":"2003","journal-title":"Hum. Gene Ther."},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"2667","DOI":"10.1089\/10430349950016708","article-title":"A high-efficiency Cre\/loxP-based system for construction of adenoviral vectors","volume":"10","author":"Ng","year":"1999","journal-title":"Hum. Gene Ther."},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"13565","DOI":"10.1073\/pnas.93.24.13565","article-title":"A helper-dependent adenovirus vector system: Removal of helper virus by Cre-mediated excision of the viral packaging signal","volume":"93","author":"Parks","year":"1996","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"S18","DOI":"10.1038\/sj.gt.3302612","article-title":"Gutless adenovirus: Last-generation adenovirus for gene therapy","volume":"12","author":"Alba","year":"2005","journal-title":"Gene Ther."},{"key":"ref_75","first-page":"223","article-title":"Preparation and titration of CsCl-banded adenovirus stocks","volume":"130","author":"Tollefson","year":"2007","journal-title":"Methods Mol. Med."},{"key":"ref_76","doi-asserted-by":"crossref","first-page":"318","DOI":"10.1089\/hum.2014.007","article-title":"Challenges in manufacturing adenoviral vectors for global vaccine product deployment","volume":"25","author":"Vellinga","year":"2014","journal-title":"Hum. Gene Ther."},{"key":"ref_77","doi-asserted-by":"crossref","first-page":"560","DOI":"10.1089\/hum.2015.081","article-title":"Process Development of Adenoviral Vector Production in Fixed Bed Bioreactor: From Bench to Commercial Scale","volume":"26","author":"Lesch","year":"2015","journal-title":"Hum. Gene Ther."},{"key":"ref_78","doi-asserted-by":"crossref","first-page":"376","DOI":"10.1089\/hum.2019.247","article-title":"Benchmarking of Scale-X Bioreactor System in Lentiviral and Adenoviral Vector Production","volume":"31","author":"Leinonen","year":"2020","journal-title":"Hum. Gene Ther."},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"8459","DOI":"10.1128\/jvi.70.12.8459-8467.1996","article-title":"Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence","volume":"70","author":"Hehir","year":"1996","journal-title":"J. Virol."},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"1909","DOI":"10.1089\/hum.1998.9.13-1909","article-title":"New helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses","volume":"9","author":"Fallaux","year":"1998","journal-title":"Hum. Gene Ther."},{"key":"ref_81","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1089\/10430349950019246","article-title":"Characterization of replication-competent adenovirus isolates from large-scale production of a recombinant adenoviral vector","volume":"10","author":"Zhu","year":"1999","journal-title":"Hum. Gene Ther."},{"key":"ref_82","doi-asserted-by":"crossref","first-page":"1341","DOI":"10.1089\/hum.1994.5.11-1341","article-title":"Trans complementation of an E1A-deleted adenovirus with codelivered E1A sequences to make recombinant adenoviral producer cells","volume":"5","author":"Goldsmith","year":"1994","journal-title":"Hum. Gene Ther."},{"key":"ref_83","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1016\/S0378-4347(00)00557-0","article-title":"Characterization and quality control of recombinant adenovirus vectors for gene therapy","volume":"752","author":"Roitsch","year":"2001","journal-title":"J. Chromatogr. B. Biomed. Sci. Appl."},{"key":"ref_84","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1016\/j.jviromet.2007.05.010","article-title":"A rapid and sensitive assay for detection of replication-competent adenoviruses by a combination of microcarrier cell culture and quantitative PCR","volume":"145","author":"Schalk","year":"2007","journal-title":"J. Virol. Methods"},{"key":"ref_85","doi-asserted-by":"crossref","first-page":"1009","DOI":"10.1016\/j.ymthe.2003.09.001","article-title":"Detection of replication-competent adenoviruses spiked into recombinant adenovirus vector products by infectivity PCR","volume":"8","author":"Uchida","year":"2003","journal-title":"Mol. Ther."},{"key":"ref_86","doi-asserted-by":"crossref","unstructured":"Gao, M., Yngve, E., Yu, D., and Jin, C. (2022). A qPCR-Based Method for Quantification of RCA Contaminants in Oncolytic Adenovirus Products. Front. Mol. Biosci., 9.","DOI":"10.3389\/fmolb.2022.883249"},{"key":"ref_87","doi-asserted-by":"crossref","first-page":"909","DOI":"10.1089\/10430340252939023","article-title":"A single short stretch of homology between adenoviral vector and packaging cell line can give rise to cytopathic effect-inducing, helper-dependent E1-positive particles","volume":"13","author":"Murakami","year":"2002","journal-title":"Hum. Gene Ther."},{"key":"ref_88","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1038\/emm.2001.25","article-title":"Development of a packaging cell line for propagation of replication-deficient adenovirus vector","volume":"33","author":"Kim","year":"2001","journal-title":"Exp. Mol. Med."},{"key":"ref_89","doi-asserted-by":"crossref","first-page":"2105","DOI":"10.1089\/104303400750001417","article-title":"Efficient transformation of primary human amniocytes by E1 functions of Ad5: Generation of new cell lines for adenoviral vector production","volume":"11","author":"Schiedner","year":"2000","journal-title":"Hum. Gene Ther."},{"key":"ref_90","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1007\/s10616-006-9023-6","article-title":"A new complementing cell line for replication-incompetent E1-deleted adenovirus propagation","volume":"51","author":"Xu","year":"2006","journal-title":"Cytotechnology"},{"key":"ref_91","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1089\/10430340360535797","article-title":"Optimization of the generation and propagation of gutless adenoviral vectors","volume":"14","author":"Sakhuja","year":"2003","journal-title":"Hum. Gene Ther."},{"key":"ref_92","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1002\/jgm.844","article-title":"A system for efficient generation of adenovirus protein IX-producing helper cell lines","volume":"8","author":"Vellinga","year":"2006","journal-title":"J. Gene Med."},{"key":"ref_93","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.ymthe.2004.09.018","article-title":"Adenovirus protein IX: A new look at an old protein","volume":"11","author":"Parks","year":"2005","journal-title":"Mol. Ther."},{"key":"ref_94","doi-asserted-by":"crossref","first-page":"1575","DOI":"10.1089\/hum.1995.6.12-1575","article-title":"Development of cell lines capable of complementing E1, E4, and protein IX defective adenovirus type 5 mutants","volume":"6","author":"Krougliak","year":"1995","journal-title":"Hum. Gene Ther."},{"key":"ref_95","doi-asserted-by":"crossref","first-page":"714","DOI":"10.1038\/nm0696-714","article-title":"Second-generation adenovirus vectors","volume":"2","author":"Wang","year":"1996","journal-title":"Nat. Med."},{"key":"ref_96","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s12276-019-0299-y","article-title":"No more helper adenovirus: Production of gutless adenovirus (GLAd) free of adenovirus and replication-competent adenovirus (RCA) contaminants","volume":"51","author":"Lee","year":"2019","journal-title":"Exp. Mol. Med."},{"key":"ref_97","doi-asserted-by":"crossref","first-page":"1954","DOI":"10.1038\/sj.gt.3301321","article-title":"Trans-complementation of vector replication versus Coxsackie-adenovirus-receptor overexpression to improve transgene expression in poorly permissive cancer cells","volume":"7","author":"Fechner","year":"2000","journal-title":"Gene Ther."},{"key":"ref_98","doi-asserted-by":"crossref","first-page":"e01782-16","DOI":"10.1128\/JVI.01782-16","article-title":"Efficient Transformation of Primary Human Mesenchymal Stromal Cells by Adenovirus Early Region 1 Oncogenes","volume":"91","author":"Speiseder","year":"2016","journal-title":"J. Virol."},{"key":"ref_99","doi-asserted-by":"crossref","first-page":"3823","DOI":"10.1073\/pnas.86.10.3823","article-title":"Role of early region 3 (E3) in pathogenesis of adenovirus disease","volume":"86","author":"Ginsberg","year":"1989","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_100","doi-asserted-by":"crossref","first-page":"2547","DOI":"10.1093\/eurheartj\/ehx352","article-title":"Adenoviral intramyocardial VEGF-D\u0394N\u0394C gene transfer increases myocardial perfusion reserve in refractory angina patients: A phase I\/IIa study with 1-year follow-up","volume":"38","author":"Hartikainen","year":"2017","journal-title":"Eur. Heart J."},{"key":"ref_101","doi-asserted-by":"crossref","first-page":"761","DOI":"10.1089\/hum.2020.299","article-title":"Efficacy and Safety of Clinical-Grade Human Vascular Endothelial Growth Factor-D\u0394N\u0394C Gene Therapy Containing Residual Replication-Competent Adenoviruses","volume":"32","author":"Leikas","year":"2021","journal-title":"Hum. Gene Ther."},{"key":"ref_102","doi-asserted-by":"crossref","first-page":"32127","DOI":"10.1074\/jbc.274.45.32127","article-title":"Biosynthesis of vascular endothelial growth factor-D involves proteolytic processing which generates non-covalent homodimers","volume":"274","author":"Stacker","year":"1999","journal-title":"J. Biol. Chem."},{"key":"ref_103","doi-asserted-by":"crossref","first-page":"1015","DOI":"10.1016\/j.jacc.2006.09.053","article-title":"Vascular endothelial growth factors: Biology and current status of clinical applications in cardiovascular medicine","volume":"49","author":"Rissanen","year":"2007","journal-title":"J. Am. Coll. Cardiol."},{"key":"ref_104","doi-asserted-by":"crossref","first-page":"1029","DOI":"10.1161\/01.CIR.0000115519.03688.A2","article-title":"Adenoviral catheter-mediated intramyocardial gene transfer using the mature form of vascular endothelial growth factor-D induces transmural angiogenesis in porcine heart","volume":"109","author":"Rutanen","year":"2004","journal-title":"Circulation"},{"key":"ref_105","doi-asserted-by":"crossref","first-page":"1716","DOI":"10.1136\/heartjnl-2016-309373","article-title":"AdVEGF-B186 and AdVEGF-D\u0394N\u0394C induce angiogenesis and increase perfusion in porcine myocardium","volume":"102","author":"Nurro","year":"2016","journal-title":"Heart"},{"key":"ref_106","doi-asserted-by":"crossref","unstructured":"Pajula, J., L\u00e4hteenvuo, J., L\u00e4hteenvuo, M., Honkonen, K., Halonen, P., H\u00e4tinen, O.P., Kuivanen, A., Heikkil\u00e4, M., Nurro, J., and Hartikainen, J. (2022). Adenoviral VEGF-D\u0394N \u0394C gene therapy for myocardial ischemia. Front. Bioeng. Biotechnol., 10.","DOI":"10.3389\/fbioe.2022.999226"},{"key":"ref_107","doi-asserted-by":"crossref","first-page":"1098","DOI":"10.1161\/01.RES.0000073584.46059.E3","article-title":"VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses","volume":"92","author":"Rissanen","year":"2003","journal-title":"Circ. Res."},{"key":"ref_108","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1038\/s41434-021-00295-1","article-title":"Long-term safety and efficacy of intramyocardial adenovirus-mediated VEGF-D\u0394N\u0394C gene therapy eight-year follow-up of phase I KAT301 study","volume":"29","author":"Leikas","year":"2022","journal-title":"Gene Ther."},{"key":"ref_109","doi-asserted-by":"crossref","first-page":"e3437","DOI":"10.1002\/jgm.3437","article-title":"Intramyocardial adenoviral vascular endothelial growth factor-D\u2206N\u2206C gene therapy does not induce ventricular arrhythmias","volume":"24","author":"Leikas","year":"2022","journal-title":"J. Gene Med."},{"key":"ref_110","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1006\/mthe.2002.0638","article-title":"Increased vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: A randomized, placebo-controlled, double-blinded phase II study","volume":"6","author":"Manninen","year":"2002","journal-title":"Mol. Ther."},{"key":"ref_111","doi-asserted-by":"crossref","first-page":"2677","DOI":"10.1161\/01.CIR.0000070540.80780.92","article-title":"Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: Phase II results of the Kuopio Angiogenesis Trial (KAT)","volume":"107","author":"Hedman","year":"2003","journal-title":"Circulation"},{"key":"ref_112","doi-asserted-by":"crossref","first-page":"629","DOI":"10.1038\/gt.2009.4","article-title":"Eight-year safety follow-up of coronary artery disease patients after local intracoronary VEGF gene transfer","volume":"16","author":"Hedman","year":"2009","journal-title":"Gene Ther."},{"key":"ref_113","doi-asserted-by":"crossref","first-page":"253","DOI":"10.2174\/157488606777934440","article-title":"Safety profile of plasmid\/liposomes and virus vectors in clinical gene therapy","volume":"1","author":"Wirth","year":"2006","journal-title":"Curr. Drug. Saf."},{"key":"ref_114","doi-asserted-by":"crossref","first-page":"823","DOI":"10.1016\/S1470-2045(13)70274-2","article-title":"Adenovirus-mediated gene therapy with sitimagene ceradenovec followed by intravenous ganciclovir for patients with operable high-grade glioma (ASPECT): A randomised, open-label, phase 3 trial","volume":"14","author":"Westphal","year":"2013","journal-title":"Lancet Oncol."},{"key":"ref_115","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1038\/nm0697-639","article-title":"ONYX-015, an E1B gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents","volume":"3","author":"Heise","year":"1997","journal-title":"Nat. Med."},{"key":"ref_116","doi-asserted-by":"crossref","first-page":"12483","DOI":"10.1128\/JVI.76.24.12483-12490.2002","article-title":"Evidence that replication of the antitumor adenovirus ONYX-015 is not controlled by the p53 and p14(ARF) tumor suppressor genes","volume":"76","author":"Edwards","year":"2002","journal-title":"J. Virol."},{"key":"ref_117","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1038\/mt.2009.266","article-title":"Current issues and future directions of oncolytic adenoviruses","volume":"18","author":"Yamamoto","year":"2010","journal-title":"Mol. Ther."},{"key":"ref_118","doi-asserted-by":"crossref","first-page":"910","DOI":"10.1002\/jgm.1096","article-title":"An inventory of shedding data from clinical gene therapy trials","volume":"9","author":"Wagemaker","year":"2007","journal-title":"J. Gene Med."},{"key":"ref_119","doi-asserted-by":"crossref","first-page":"1211","DOI":"10.1002\/1097-0142(195611\/12)9:6<1211::AID-CNCR2820090624>3.0.CO;2-7","article-title":"Studies on the use of viruses in the treatment of carcinoma of the cervix","volume":"9","author":"Huebner","year":"1956","journal-title":"Cancer"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/22\/16519\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T21:26:03Z","timestamp":1760131563000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/22\/16519"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,11,20]]},"references-count":119,"journal-issue":{"issue":"22","published-online":{"date-parts":[[2023,11]]}},"alternative-id":["ijms242216519"],"URL":"https:\/\/doi.org\/10.3390\/ijms242216519","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,11,20]]}}}