High levels of AAV vector integration into CRISPR-induced DNA breaks
Authors: Miko≈Çaj P Zaborowski
Jeffrey S Chamberlain
Sara P Garcia
J Keith Joung
Adrienn Volak
Bence Gy√∂rgy
Martin Ingelsson
Alissa Muller
Casey A Maguire
Alexander A Sousa
David P Corey
Killian S Hanlon
Benjamin P Kleinstiver
Stefan E Spirig
Niclas E Bengtsson
Xandra O Breakefield
Shengdar Q Tsai
Camilla L√∂√∂v
Martin J Aryee
Journal: Nature communications (2019)
Discipline: Multidisciplinary
Keywords: Adeno-associated virus; AAV vectors; CRISPR-Cas nucleases; integration; double-strand breaks
Affiliations: Department of Neurobiology, Harvard Medical School, Boston, MA, 02115, USA., Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital, Charlestown, MA, 02129, USA., Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA., Department of Pathology, Massachusetts General Hospital, Boston, MA, USA., Department of Pathology, Harvard Medical School, Boston, MA, USA., Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, 02129, USA., Center for Cancer Research and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA., Program in Neuroscience, Harvard Medical School, Boston, MA, 02115, USA., Department of Gynecology, Obstetrics and Gynecologic Oncology, Division of Gynecologic Oncology, Pozna≈Ñ University of Medical Sciences, 60-535, Pozna≈Ñ, Poland., Institute of Molecular and Clinical Ophthalmology Basel, 4031, Basel, Switzerland., Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA., Department of Neurology, University of Washington, Seattle, WA, 98195, USA., Uppsala University, Department of Public Health and Caring Sciences, Geriatrics, Uppsala, Sweden., Department of Biostatistics, Harvard T. H. Chan School of Public Health, Boston, MA, USA., Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital, Charlestown, MA, 02129, USA. ., Program in Neuroscience, Harvard Medical School, Boston, MA, 02115, USA. ., Department of Neurobiology, Harvard Medical School, Boston, MA, 02115, USA. ., Institute of Molecular and Clinical Ophthalmology Basel, 4031, Basel, Switzerland. .

Abstract:  Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-Œª465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465Œª in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing. 

Retracted: No

PubPeer comments:
 [My pre-print on this data was rejected. ] . 15 days later they publish - but dont mention Cas9 integration, as that would stop all their funding, etc. [You basically dont fight this kind of financial juggernaut.]  , 
 Their statement ** hepatocellular carcinoma induction has not been associated with AAV vectors in NHPs and humans** is grossly incorrect. They have failed to cite previous work - 1.JC Nault, et al 2015 u2013 u201cRecurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomasu201d 2. JC. Nault et al 2016 u201cWild-type AAV Insertions in Hepatocellular Carcinoma Do Not Inform Debate Over Genotoxicity Risk of Vectorized AAVu201d And then there is a new study from the same group (2019) showing the same: https: pubpeer.com publications BA5F5E8274602FCD6D74AB40EC8028#1 Where is the peer-reviewing? , 
 This is another paper that should have been cited. [Severe Toxicity in Nonhuman Primates and Piglets Following High-Dose Intravenous Administration of an Adeno-Associated Virus Vector Expressing Human SMN ] , 
 This is now a pre-print in [F1000] ... , 
 And as I said above  Their statement  hepatocellular carcinoma induction has not been associated with AAV vectors in NHPs and humans is grossly incorrect. [ Solid Biosciencesu2019 DMD gene therapy trial failure u2013 is there any other plausible reason than AAV integration? ] Imagine the problems with Cas9 integration - how will any therapy work? , 
 [Virus used in gene therapies may pose cancer risk, dog study hints]  After ending the experiment and studying the livers of six dogs, her team found that in every one of them, AAV-ferried DNAu2014the factor VIII gene or, more often, fragments of regulatory sequencesu2014had integrated in many spots across the genome in the dogsu2019 liver cells, sometimes near genes affecting cell growth. Some of those cells had divided more than other cells, forming pockets of multiple cells or u201cclonesu201d in some animals. Sabatinou2019s team suspectsu2014although it canu2019t proveu2014that the insertions activated growth genes, explaining both the clones and the rise in blood levels of factor VIII in the two dogs. (Sabatino declined to discuss the results because they have not yet been published.) Imagine when the report Cas9 expressing dogs, which is surely happening. , 
 Here s some more data from the long term dog study - compare this with the table I provided in the 1st post above (as usual, they *dont* check for Cas9 integration - that would close the whole project).  ,