{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,23]],"date-time":"2026-08-23T14:50:31Z","timestamp":1787496631551,"version":"build-2736575974"},"reference-count":53,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2022,12,24]],"date-time":"2022-12-24T00:00:00Z","timestamp":1671840000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Berlin Institute of Health (BIH)"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>The successful application of recombinant adeno-associated virus (rAAV) vectors for long-term transgene expression in clinical studies requires scalable production methods with genetically stable components. Due to their simple production scheme and the high viral titers achievable, first generation recombinant adenoviruses (rAdV) have long been taken into consideration as suitable tools for simultaneously providing both the helper functions and the AAV rep and cap genes for rAAV packaging. So far, however, such rAdV-rep\/cap vectors have been difficult to generate and often turned out to be genetically unstable. Through ablation of cis and trans inhibitory function in the AAV-2 genome we have succeeded in establishing separate and stable rAdVs for high-level AAV serotype 2 Rep and Cap expression. These allowed rAAV-2 production at high burst sizes by simple coinfection protocols after providing the AAV-ITR flanked transgene vector genome either as rAAV-2 particles at low input concentrations or in form of an additional rAdV. With characteristics such as the ease of producing the required components, the straightforward adaption to other transgenes and the possible extension to further serotypes or capsid variants, especially the rAdV-mediated rAAV amplification system presents a very promising candidate for up-scaling to clinical grade vector preparations.<\/jats:p>","DOI":"10.3390\/v15010064","type":"journal-article","created":{"date-parts":[[2022,12,27]],"date-time":"2022-12-27T07:02:12Z","timestamp":1672124532000},"page":"64","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["High-Level rAAV Vector Production by rAdV-Mediated Amplification of Small Amounts of Input Vector"],"prefix":"10.3390","volume":"15","author":[{"given":"Stefan","family":"Weger","sequence":"first","affiliation":[{"name":"Charit\u00e9\u2013Universit\u00e4tsmedizin Berlin, Corporate Member of Freie Universit\u00e4t Berlin and Humboldt-Universit\u00e4t zu Berlin, Clinic for Neurology with Experimental Neurology, Gene Therapy Group, Campus Benjamin Franklin, Hindenburgdamm27, 12203 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,12,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1038\/nrg2988","article-title":"Therapeutic in vivo gene transfer for genetic disease using AAV: Progress and challenges","volume":"12","author":"Mingozzi","year":"2011","journal-title":"Nat. Rev. Genet."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1994","DOI":"10.1056\/NEJMoa1407309","article-title":"Long-Term Safety and Efficacy of Factor IX Gene Therapy in Hemophilia B","volume":"371","author":"Nathwani","year":"2014","journal-title":"N. Engl. J. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1038\/gt.2012.43","article-title":"Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase deficiency: An open-label trial","volume":"20","author":"Gaudet","year":"2012","journal-title":"Gene Ther."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1597","DOI":"10.1016\/S0140-6736(09)61836-5","article-title":"Age-dependent effects of RPE65 gene therapy for Leber\u2019s congenital amaurosis: A phase 1 dose-escalation t trial","volume":"374","author":"High","year":"2009","journal-title":"Lancet"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1283","DOI":"10.1016\/j.ophtha.2012.11.048","article-title":"Three-year follow-up after unilateral subretinal delivery of adeno-associated virus in patients with Leber congenital Amaurosis type 2","volume":"120","author":"Testa","year":"2013","journal-title":"Ophthalmology"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"35ra42","DOI":"10.1126\/scitranslmed.3000830","article-title":"Systemic delivery of scAAV9 expressing SMN prolongs survival in a model of spinal muscular atrophy","volume":"2","author":"Valori","year":"2010","journal-title":"Sci. Transl. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"849","DOI":"10.1016\/S0140-6736(17)31868-8","article-title":"Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: A randomised, controlled, open-label, phase 3 trial","volume":"390","author":"Russell","year":"2017","journal-title":"Lancet"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1713","DOI":"10.1056\/NEJMoa1706198","article-title":"Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy","volume":"377","author":"Mendell","year":"2017","journal-title":"N. Engl. J. Med."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"16002","DOI":"10.1038\/mtm.2016.2","article-title":"Manufacturing of recombinant adeno-associated viral vectors for clinical trials","volume":"3","author":"Grieger","year":"2016","journal-title":"Mol. Ther. -Methods Clin. Dev."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1016\/S0378-1119(99)00347-9","article-title":"A helper virus-free packaging system for recombinant adeno-associated virus vectors","volume":"238","author":"Collaco","year":"1999","journal-title":"Gene"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2745","DOI":"10.1089\/hum.1998.9.18-2745","article-title":"Novel tools for production and purification of recombinant adenoassociated virus vectors","volume":"9","author":"Grimm","year":"1998","journal-title":"Hum. Gene Ther."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2224","DOI":"10.1128\/JVI.72.3.2224-2232.1998","article-title":"Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus","volume":"72","author":"Xiao","year":"1998","journal-title":"J. Virol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"287","DOI":"10.1038\/mt.2015.187","article-title":"Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector from the Culture Media for GMP FIX and FLT1 Clinical Vector","volume":"24","author":"Grieger","year":"2016","journal-title":"Mol. Ther."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"796","DOI":"10.1089\/hum.2009.094","article-title":"Large-scale adeno-associated viral vector production using a herpesvirus-based system enables manufacturing for clinical studies","volume":"20","author":"Knop","year":"2009","journal-title":"Hum. Gene Ther."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"986","DOI":"10.1038\/sj.gt.3300937","article-title":"High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap","volume":"6","author":"Conway","year":"1999","journal-title":"Gene Ther."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"861","DOI":"10.1089\/hum.2009.004","article-title":"Scalable recombinant adeno-associated virus production using recombinant herpes simplex virus type 1 coinfection of suspension-adapted mammalian cells","volume":"20","author":"Thomas","year":"2009","journal-title":"Hum. Gene Ther."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1021","DOI":"10.1089\/hum.2010.250","article-title":"Reproducible high yields of recombinant adeno-associated virus produced using invertebrate cells in 0.02-to 200-liter cultures","volume":"22","author":"Cecchini","year":"2011","journal-title":"Hum. Gene Ther."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1935","DOI":"10.1089\/10430340260355347","article-title":"Insect cells as a factory to produce adeno-associated virus type 2 vectors","volume":"13","author":"Urabe","year":"2002","journal-title":"Hum. Gene Ther."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"212","DOI":"10.1089\/hum.2013.184","article-title":"OneBac: Platform for scalable and high-titer production of adeno-associated virus serotype 1\u201312 vectors for gene therapy","volume":"25","author":"Mietzsch","year":"2014","journal-title":"Hum. Gene Ther."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"754","DOI":"10.1126\/science.149.3685.754","article-title":"Adenovirus-Associated Defective Virus Particles","volume":"149","author":"Atchison","year":"1965","journal-title":"Science"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"704","DOI":"10.1038\/sj.gt.3301454","article-title":"Recombinant adenovirus expressing adeno-associated virus cap and rep proteins supports production of high-titer recombinant adeno-associated virus","volume":"8","author":"Zhang","year":"2001","journal-title":"Gene Ther."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"922","DOI":"10.1089\/hum.2009.125","article-title":"Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production","volume":"20","author":"Zhang","year":"2009","journal-title":"Hum. Gene Ther."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1006\/mthe.2002.0634","article-title":"An adenoviral expression system for AAV rep78 using homologous recombination","volume":"6","author":"Carlson","year":"2002","journal-title":"Mol. Ther."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2615","DOI":"10.1073\/pnas.96.6.2615","article-title":"Site-specific integration mediated by a hybrid adenovirus\/adeno-associated virus vector","volume":"96","author":"Recchia","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"660","DOI":"10.1016\/j.ymthe.2004.07.003","article-title":"Site-specific integration of functional transgenes into the human genome by adeno\/AAV hybrid vectors","volume":"10","author":"Recchia","year":"2004","journal-title":"Mol. Ther."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"11699","DOI":"10.1128\/JVI.00779-06","article-title":"A helper-dependent capsid-modified adenovirus vector expressing adeno-associated virus rep78 mediates site-specific integration of a 27-kilobase transgene cassette","volume":"80","author":"Wang","year":"2006","journal-title":"J. Virol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"14294","DOI":"10.1073\/pnas.1102883108","article-title":"Computationally designed adeno-associated virus (AAV) Rep 78 is efficiently maintained within an adenovirus vector","volume":"108","author":"Sitaraman","year":"2011","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"3981","DOI":"10.1128\/JVI.03120-15","article-title":"A Regulatory Element Near the 3\u2032 End of the Adeno-Associated Virus rep Gene Inhibits Adenovirus Replication in cis by Means of p40 Promoter-Associated Short Transcripts","volume":"90","author":"Weger","year":"2016","journal-title":"J. Virol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"14126","DOI":"10.1128\/JVI.02350-14","article-title":"Differential contribution of adeno-associated virus type 2 Rep protein expression and nucleic acid elements to inhibition of adenoviral replication in cis and in trans","volume":"88","author":"Weger","year":"2014","journal-title":"J. Virol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"4646","DOI":"10.1128\/jvi.70.7.4646-4654.1996","article-title":"A \u201chumanized\u201d green fluorescent protein cDNA adapted for high-level expression in mammalian cells","volume":"70","author":"Zolotukhin","year":"1996","journal-title":"J. Virol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"5099","DOI":"10.1128\/JVI.06991-11","article-title":"Roles of E4orf6 and VA I RNA in adenovirus-mediated stimulation of human parvovirus B19 DNA replication and structural gene expression","volume":"86","author":"Winter","year":"2012","journal-title":"J. Virol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2509","DOI":"10.1073\/pnas.95.5.2509","article-title":"A simplified system for generating recombinant adenoviruses","volume":"95","author":"He","year":"1998","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_33","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_34","doi-asserted-by":"crossref","first-page":"7807","DOI":"10.1128\/JVI.00198-06","article-title":"Effects of adeno-associated virus on adenovirus replication and gene expression during coinfection","volume":"80","author":"Timpe","year":"2006","journal-title":"J. Virol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1716","DOI":"10.1093\/emboj\/cdg153","article-title":"PKA\/PrKX activity is a modulator of AAV\/adenovirus interaction","volume":"22","author":"Chiorini","year":"2003","journal-title":"EMBO J."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"3721","DOI":"10.1128\/JVI.02693-06","article-title":"Adeno-associated virus type 2 p5 promoter: A rep-regulated DNA switch element functioning in transcription, replication, and site-specific integration","volume":"81","author":"Murphy","year":"2007","journal-title":"J. Virol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"144","DOI":"10.1016\/j.virol.2003.11.024","article-title":"Rep68 protein of adeno-associated virus type 2 interacts with 14-3-3 proteins depending on phosphorylation at serine 535","volume":"320","author":"Han","year":"2004","journal-title":"Virology"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"559","DOI":"10.1016\/0092-8674(85)90113-8","article-title":"Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro","volume":"42","author":"Jones","year":"1985","journal-title":"Cell"},{"key":"ref_39","first-page":"559","article-title":"Functional relationships between transcriptional control signals of the thymidine kinase gene of herpes simplex virus","volume":"42","author":"McKnight","year":"1985","journal-title":"Cell"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1016\/0092-8674(84)90321-0","article-title":"The distal transcription signals of the herpesvirus tk gene share a common hexanucleotide control sequence","volume":"37","author":"McKnight","year":"1984","journal-title":"Cell"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"4300","DOI":"10.1128\/jvi.71.6.4300-4309.1997","article-title":"The adeno-associated virus type 2 p40 promoter requires a proximal Sp1 interaction and a p19 CArG-like element to facilitate Rep transactivation","volume":"71","author":"Pereira","year":"1997","journal-title":"J. Virol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1747","DOI":"10.1128\/jvi.71.3.1747-1756.1997","article-title":"The cellular transcription factor SP1 and an unknown cellular protein are required to mediate Rep protein activation of the adeno-associated virus p19 promoter","volume":"71","author":"Pereira","year":"1997","journal-title":"J. Virol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1182\/blood-2013-01-306647","article-title":"Immune responses to AAV vectors: Overcoming barriers to successful gene therapy","volume":"122","author":"Mingozzi","year":"2013","journal-title":"Blood"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1016\/j.jviromet.2006.11.019","article-title":"Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography","volume":"140","author":"Qu","year":"2007","journal-title":"J. Virol. Methods"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"194ra92","DOI":"10.1126\/scitranslmed.3005795","article-title":"Overcoming preexisting humoral immunity to AAV using capsid decoys","volume":"5","author":"Mingozzi","year":"2013","journal-title":"Sci. Transl. Med."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1089\/hum.2010.118","article-title":"Preclinical evaluation of a recombinant adeno-associated virus vector expressing human alpha-1 antitrypsin made using a recombinant herpes simplex virus production method","volume":"22","author":"Chulay","year":"2011","journal-title":"Hum. Gene Ther."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"5059","DOI":"10.1073\/pnas.0810614106","article-title":"An inducible system for highly efficient production of recombinant adeno-associated virus (rAAV) vectors in insect Sf9 cells","volume":"106","author":"Aslanidi","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"688","DOI":"10.1089\/hum.2015.050","article-title":"OneBac 2.0: Sf9 Cell Lines for Production of AAV5 Vectors with Enhanced Infectivity and Minimal Encapsidation of Foreign DNA","volume":"26","author":"Mietzsch","year":"2015","journal-title":"Hum. Gene Ther."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1217","DOI":"10.1016\/j.ymthe.2005.08.018","article-title":"Successful production of pseudotyped rAAV vectors using a modified baculovirus expression system","volume":"12","author":"Kohlbrenner","year":"2005","journal-title":"Mol. Ther."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1016\/j.omtm.2020.05.018","article-title":"Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors","volume":"18","author":"Rumachik","year":"2020","journal-title":"Mol. Ther. -Methods Clin. Dev."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"1182","DOI":"10.1038\/s41467-022-28738-2","article-title":"Self-attenuating adenovirus enables production of recombinant adeno-associated virus for high manufacturing yield without contamination","volume":"13","author":"Su","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1157","DOI":"10.1089\/hum.2004.15.1157","article-title":"The innate immune response to adenovirus vectors","volume":"15","author":"Muruve","year":"2004","journal-title":"Hum. Gene Ther."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"3199","DOI":"10.1002\/bit.27472","article-title":"Tustian, Moving from the bench towards a large scale, industrial platform process for adeno-associated viral vector purification","volume":"117","author":"Adams","year":"2020","journal-title":"Biotechnol. Bioeng."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/15\/1\/64\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:50:32Z","timestamp":1760147432000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/15\/1\/64"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,12,24]]},"references-count":53,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2023,1]]}},"alternative-id":["v15010064"],"URL":"https:\/\/doi.org\/10.3390\/v15010064","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,12,24]]}}}