AAV6 Is Superior to Clade F AAVs in Stimulating Homologous Recombination-Based Genome Editing in Human HSPCs
Authors: Amanda M. Dudek, Matthew H. Porteus
Journal: Molecular Therapy (2019)

Rogers et al.1Rogers G.L. Chen H.-Y. Morales H. Cannon P.M. Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break.Mol. Ther. 2019; 27 (this issue): 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar provide a detailed study of a novel class of clade F adeno-associated virus (AAV) capsids (AAV hematopoietic stem cells [AAVHSCs])2Smith L.J. Ul-Hasan T. Carvaines S.K. Van Vliet K. Yang E. Wong Jr., K.K. Agbandje-McKenna M. Chatterjee S. Gene transfer properties and structural modeling of human stem cell-derived AAV.Mol. Ther. 2014; 22: 1625-1634Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar to facilitate homologous recombination in the presence or absence of site-specific DNA cleavage in human CD34+ hematopoietic stem and progenitor cells (HSPCs). The recent publication that clade F AAVs can facilitate high-efficiency homologous recombination in the absence of a targeted DNA break3Smith L.J. Wright J. Clark G. Ul-Hasan T. Jin X. Fong A. Chandra M. St Martin T. Rubin H. Knowlton D. et al.Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing.Proc. Natl. Acad. Sci. USA. 2018; 115: E7379-E7388Crossref PubMed Scopus (23) Google Scholar was surprising due to the high sequence homology of AAVHSC variants to the relatively inefficient AAV9 and the long-standing knowledge in the field that AAV-based viral vectors do not undergo high frequency-targeted integration (1% or less).4Russell D.W. Hirata R.K. Human gene targeting by viral vectors.Nat. Genet. 1998; 18: 325-330Crossref PubMed Scopus (266) Google Scholar, 5Miller D.G. Trobridge G.D. Petek L.M. Jacobs M.A. Kaul R. Russell D.W. Large-Scale Analysis of Adeno-Associated Virus Vector Integration Sites in Normal Human Cells.J Virol. 2005; 79: 11434-11442Crossref PubMed Scopus (124) Google Scholar, 6Porteus M.H. Mammalian gene targeting with designed zinc finger nucleases.Mol Ther. 2006; 13: 438-446Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar Rogers et al.1Rogers G.L. Chen H.-Y. Morales H. Cannon P.M. Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break.Mol. Ther. 2019; 27 (this issue): 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar provide a direct comparison with AAV6, the serotype most commonly utilized for this method and cell type and demonstrate that long-lived (>14 days) expression of a GFP-reporter encoding donor template is only observed after specific induction of a DNA break using zinc-finger nucleases (ZFNs). They additionally report that the highest efficiency of editing is observed using AAV6. Here we provide additional independently generated confirmation of the findings that AAV6 is a superior vector for homologous recombination-based genome editing and discuss potential technical differences in the presented studies that may influence the data interpretation and conflicting interpretation of these studies. As gene therapy and gene editing become more widely applied therapeutics, transparency and reproducibility is critical for the successful development and implementation as these therapies transition into the clinic. Genome editing-based gene therapies are being developed as a class of molecular therapeutics that have the potential to cure previously untreatable diseases through permanent correction of a disease-causing genetic mutation.7Porteus M.H. A New Class of Medicines through DNA Editing.N. Engl. J. Med. 2019; 380: 947-959Crossref PubMed Scopus (121) Google Scholar The development of ZFNs,8Durai S. Mani M. Kandavelou K. Wu J. Porteus M.H. Chandrasegaran S. Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells.Nucleic Acids Res. 2005; 33: 5978-5990Crossref PubMed Scopus (353) Google Scholar, 9Porteus M.H. Carroll D. Gene targeting using zinc finger nucleases.Nat. Biotechnol. 2005; 23: 967-973Crossref PubMed Scopus (495) Google Scholar transcription activator-like effector nuclease (TALENs),10Christian M. Cermak T. Doyle E.L. Schmidt C. Zhang F. Hummel A. Bogdanove A.J. Voytas D.F. Targeting DNA double-strand breaks with TAL effector nucleases.Genetics. 2010; 186: 757-761Crossref PubMed Scopus (1316) Google Scholar, 11Li T. Huang S. Jiang W.Z. Wright D. Spalding M.H. Weeks D.P. Yang B. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain.Nucleic Acids Res. 2011; 39: 359-372Crossref PubMed Scopus (401) Google Scholar, 12Miller J.C. Tan S. Qiao G. Barlow K.A. Wang J. Xia D.F. Meng X. Paschon D.E. Leung E. Hinkley S.J. et al.A TALE nuclease architecture for efficient genome editing.Nat. Biotechnol. 2011; 29: 143-148Crossref PubMed Scopus (1566) Google Scholar and Cas913Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (9324) Google Scholar, 14Cho S.W. Kim S. Kim J.M. Kim J.-S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease.Nat. Biotechnol. 2013; 31: 230-232Crossref PubMed Scopus (1375) Google Scholar-based gene editing methodologies have allowed specific manipulation of a DNA sequence of interest by targeted insertion, deletion, or single base editing with high efficiency. Recombinant AAV is an ideal delivery vehicle to provide DNA templates for gene correction through homology-directed repair after induction of a specific DNA break because it is a non-integrating virus and thus has the key property for a "hit and run" process in dividing cells. The combination of a nuclease-induced break with transduction of AAV6 to deliver the donor has been efficient in a wide variety of primary cell types, including HSPCs and T cells, and genetic loci.15Dever D.P. Bak R.O. Reinisch A. Camarena J. Washington G. Nicolas C.E. Pavel-Dinu M. Saxena N. Wilkens A.B. Mantri S. et al.CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells.Nature. 2016; 539: 384-389Crossref PubMed Scopus (511) Google Scholar, 16Pavel-Dinu M. Wiebking V. Dejene B.T. Srifa W. Mantri S. Nicolas C.E. Lee C. Bao G. Kildebeck E.J. Punjya N. et al.Gene correction for SCID-X1 in long-term hematopoietic stem cells.Nat. Commun. 2019; 10: 1634Crossref PubMed Scopus (97) Google Scholar, 17Eyquem J. Mansilla-Soto J. Giavridis T. van der Stegen S.J.C. Hamieh M. Cunanan K.M. Odak A. Gönen M. Sadelain M. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection.Nature. 2017; 543: 113-117Crossref PubMed Scopus (942) Google Scholar, 18Wang J. Exline C.M. DeClercq J.J. Llewellyn G.N. Hayward S.B. Li P.W.-L. Shivak D.A. Surosky R.T. Gregory P.D. Holmes M.C. Cannon P.M. Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors.Nat. Biotechnol. 2015; 33: 1256-1263Crossref PubMed Scopus (205) Google Scholar, 19Chen S. Sun S. Moonen D. Lee C. Lee A.Y.-F. Schaffer D.V. He L. CRISPR-READI: Efficient Generation of Knockin Mice by CRISPR RNP Electroporation and AAV Donor Infection.Cell Rep. 2019; 27: 3780-3789.e4Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 20De Ravin S.S. Reik A. Liu P.-Q. Li L. Wu X. Su L. Raley C. Theobald N. Choi U. Song A.H. et al.Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease.Nat. Biotechnol. 2016; 34: 424-429Crossref PubMed Scopus (142) Google Scholar Smith et al.3Smith L.J. Wright J. Clark G. Ul-Hasan T. Jin X. Fong A. Chandra M. St Martin T. Rubin H. Knowlton D. et al.Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing.Proc. Natl. Acad. Sci. USA. 2018; 115: E7379-E7388Crossref PubMed Scopus (23) Google Scholar claimed that a novel set of AAV capsid variants, which they called AAVHSC, were able to mediate high frequencies of targeted integration into CD34+ human HSPCs without the need for a double-strand break (DSB). These clade F AAVHSC variants are a highly related subgroup of AAV capsids that are most closely related to AAV9, most of which differ by only one or two single amino acid substitutions in the capsid. Since 1998, when Russell and Hirata4Russell D.W. Hirata R.K. Human gene targeting by viral vectors.Nat. Genet. 1998; 18: 325-330Crossref PubMed Scopus (266) Google Scholar published their seminal paper showing that AAV could integrate in a targeted fashion at much higher frequencies than naked plasmid DNA, AAV has been used in this manner for research purposes. The frequency without selection, however, has been 1% or lower.21Hiramoto T. Li L.B. Funk S.E. Hirata R.K. Russell D.W. Nuclease-free Adeno-Associated Virus-Mediated Il2rg Gene Editing in X-SCID Mice.Mol. Ther. 2018; 26: 1255-1265Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 22Barzel A. Paulk N.K. Shi Y. Huang Y. Chu K. Zhang F. Valdmanis P.N. Spector L.P. Porteus M.H. Gaensler K.M. Kay M.A. Promoterless gene targeting without nucleases ameliorates haemophilia B in mice.Nature. 2015; 517: 360-364Crossref PubMed Scopus (177) Google Scholar, 23Hirata R.K. Russell D.W. Design and packaging of adeno-associated virus gene targeting vectors.J. Virol. 2000; 74: 4612-4620Crossref PubMed Scopus (108) Google Scholar We demonstrated that AAV6 mediates targeted integration without a break in HSCs, for example, at a frequency of ∼0.5%.24Bak R.O. Porteus M.H. CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors.Cell Rep. 2017; 20: 750-756Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar The frequency of targeted integration without a break by AAV is dose dependent.23Hirata R.K. Russell D.W. Design and packaging of adeno-associated virus gene targeting vectors.J. Virol. 2000; 74: 4612-4620Crossref PubMed Scopus (108) Google Scholar, 25Porteus M.H. Cathomen T. Weitzman M.D. Baltimore D. Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks.Mol. Cell. Biol. 2003; 23: 3558-3565Crossref PubMed Scopus (134) Google Scholar Rogers et al.1Rogers G.L. Chen H.-Y. Morales H. Cannon P.M. Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break.Mol. Ther. 2019; 27 (this issue): 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar directly compared one variant, AAV9-G505R (AAVHSC13), to AAV9 and AAV6 in transducing and serving as a template for HDR genome editing. They demonstrate that, although AAV6, AAV9, and AAV9-G505R can all transduce HSPCs as shown by GFP expression from an exogenous promoter (phosphoglycerate kinase promoter [PGK]), high efficiency targeted integration at multiple loci (CCR5 and AAVS1) can only occur in the presence of a site-specific ZFN-induced DNA break. This was demonstrated using flow cytometry to demonstrate sustained high-level expression of GFP after 14 days as well as by molecular characterization of genomic integration by digital droplet PCR (ddPCR) primers spanning the targeted integration site. Importantly, GFP expression observed from HSPC transduction by any of the three tested serotypes encapsidating the CCR5-PGK-GFP construct at day 2 was lost by day 14 in the absence of a site-specific break. These data suggest that AAVs, including the novel class of clade F AAVHSCs, are unable to facilitate long-lasting genomic integration in HSPCs without a DNA DSB. We have independently confirmed that AAV6 is a superior vector for homology-directed genome editing in HSPCs as compared to AAV9-G505R as well as a panel of other AAVHSC viral vectors by using an additional method for generating DNA DSBs (CRISPR/Cas9 nuclease) and an additional therapeutically relevant genomic locus (HBB). Using site-directed mutagenesis, we generated a small panel of AAVHSC variants (Figure 1A) that were previously characterized to have high-level AAV production as single-stranded AAV (ssAAV) and high transduction in HSPCs.2Smith L.J. Ul-Hasan T. Carvaines S.K. Van Vliet K. Yang E. Wong Jr., K.K. Agbandje-McKenna M. Chatterjee S. Gene transfer properties and structural modeling of human stem cell-derived AAV.Mol. Ther. 2014; 22: 1625-1634Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar We characterized the ability of these capsids to facilitate homology-directed repair (HDR) in the presence and absence of a site-specific Cas9/ribonucleoprotein (RNP) complex in comparison to their parental capsid AAV9 as a control as well as the commonly used AAV6 and highly related AAV1. This panel of capsids was used to encapsidate a homology donor construct containing GFP driven from ubiquitin C (UBC) promoter with homology arms compatible with the HBB gene (Figure 1B).26Vakulskas C.A. Dever D.P. Rettig G.R. Turk R. Jacobi A.M. Collingwood M.A. Bode N.M. McNeill M.S. Yan S. Camarena J. et al.A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells.Nat. Med. 2018; 24: 1216-1224Crossref PubMed Scopus (366) Google Scholar Episomal expression of this construct demonstrates low intensity fluorescence, which is lost over time as the AAV genome is diluted through cell division, or high intensity fluorescence after site-specific genomic integration following HDR.27Charlesworth C.T. Camarena J. Cromer M.K. Vaidyanathan S. Bak R.O. Carte J.M. Potter J. Dever D.P. Porteus M.H. Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting.Mol. Ther. Nucleic Acids. 2018; 12: 89-104Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar The reported capsid sequence of each of these variants was reported to differ by only one (AAVHSC7, HSC13) or two (AAVHSC7) amino acids relative to AAV9 (Figure 1A). The location of AAV9-A68 cannot be visualized in the AAV9 crystal structure28DiMattia M.A. Nam H.J. Van Vliet K. Mitchell M. Bennett A. Gurda B.L. McKenna R. Olson N.H. Sinkovits R.S. Potter M. et al.Structural insight into the unique properties of adeno-associated virus serotype 9.J. Virol. 2012; 86: 6947-6958Crossref PubMed Scopus (132) Google Scholar due to its location in the VP1 unique region, which is unresolved in AAV structures: the physical locations of G505 (Figure 1C, top) and T346 (Figure 1C, bottom) are highlighted in magenta mapped on the AAV9 crystal structure, with individual VP3 monomers highlighted in white, gray, and black. G505 is a surface-exposed residue located at the interface of two adjacent monomers, while T346 is located on the inner surface of the capsid on individual monomers. The highly distinct locations of these two different point mutations does not suggest an obvious underlying mechanism by which the two amino acid variants may be specifically influencing the homologous recombination (HR) activity of these capsids. Quantification of the total number of GFP-positive or highly GFP expressing cells was used as a surrogate readout for HDR, with no virus and no RNP conditions as the gating controls (Figure 1D). Transgene expression levels and the amount of targeted integration using this donor has been previously characterized with AAV6 and is consistent with our observations.15Dever D.P. Bak R.O. Reinisch A. Camarena J. Washington G. Nicolas C.E. Pavel-Dinu M. Saxena N. Wilkens A.B. Mantri S. et al.CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells.Nature. 2016; 539: 384-389Crossref PubMed Scopus (511) Google Scholar While some low intensity GFP expression can be seen after transduction with each vector, an appreciable number of cells demonstrating high intensity GFP expression after HDR is only observed in the AAV6+RNP condition (Figure 1D). These data are consistent with previous reports that AAV6 is more efficient at transducing HSPCs than other AAV serotypes.29Ellis B.L. Hirsch M.L. Barker J.C. Connelly J.P. Steininger 3rd, R.J. Porteus M.H. A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1-9) and one engineered adeno-associated virus serotype.Virol. J. 2013; 10: 74Crossref PubMed Scopus (133) Google Scholar, 30Veldwijk M.R. Sellner L. Stiefelhagen M. Kleinschmidt J.A. Laufs S. Topaly J. Fruehauf S. Zeller W.J. Wenz F. Pseudotyped recombinant adeno-associated viral vectors mediate efficient gene transfer into primary human CD34(+) peripheral blood progenitor cells.Cytotherapy. 2010; 12: 107-112Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar Long-term GFP expression was quantified at 12 to 14 days post-transduction in three independent experiments using cord blood-derived CD34+ HSPCs from three different individual donors (Figure 1D). The percentage of cells with episomal or HDR GFP expression is presented on a log scale to highlight the large differences in efficacy of these vectors. Our data are in agreement with Rogers et al.,1Rogers G.L. Chen H.-Y. Morales H. Cannon P.M. Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break.Mol. Ther. 2019; 27 (this issue): 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar which highlights AAV6 as the superior vector for delivery of HDR donors and the requirement of a site-specific nuclease for this reaction to occur at high efficiency and maintain long-term expression. While the results presented here and in Rogers et al.1Rogers G.L. Chen H.-Y. Morales H. Cannon P.M. Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break.Mol. Ther. 2019; 27 (this issue): 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar are in conflict with the previous reports of high efficiency HSPC gene editing by clade F AAVs, Rogers et al.1Rogers G.L. Chen H.-Y. Morales H. Cannon P.M. Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break.Mol. Ther. 2019; 27 (this issue): 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar point out that there are potential technical differences that exist between the studies that may have influenced the conflicting results or interpretation of data. Here we discuss the technical differences between the studies in hopes that further clarification of the methodology will reconcile these conflicting results. The sequences between flanking inverted terminal repeats (ITRs) are the same in Rogers et al.1Rogers G.L. Chen H.-Y. Morales H. Cannon P.M. Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break.Mol. Ther. 2019; 27 (this issue): 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar and Smith et al.:3Smith L.J. Wright J. Clark G. Ul-Hasan T. Jin X. Fong A. Chandra M. St Martin T. Rubin H. Knowlton D. et al.Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing.Proc. Natl. Acad. Sci. USA. 2018; 115: E7379-E7388Crossref PubMed Scopus (23) Google Scholar the backbone containing the ITR sequences is from different plasmid constructs (pAAV-MCS or pSaiLuc AAV2, respectively) and the potential for sequence differences in the ITR may alter transgene expression. Sequences downstream of the right homology arm may influence episomal GFP expression, as a minimal enhancer-promoter element has been described in this region,31Logan G.J. Dane A.P. Hallwirth C.V. Smyth C.M. Wilkie E.E. Amaya A.K. Zhu E. Khandekar N. Ginn S.L. Liao S.H.Y. et al.Identification of liver-specific enhancer-promoter activity in the 3′ untranslated region of the wild-type AAV2 genome.Nat. Genet. 2017; 49: 1267-1273Crossref PubMed Scopus (63) Google Scholar although it is unclear whether this sequence would be functional in HSPCs. Conversely, other studies suggest that ITR sequences may have a negative impact on transgene expression.32Yan Z. Sun X. Feng Z. Li G. Fisher J.T. Stewart Z.A. Engelhardt J.F. Optimization of Recombinant Adeno-Associated Virus-Mediated Expression for Large Transgenes, Using a Synthetic Promoter and Tandem Array Enhancers.Hum. Gene Ther. 2015; 26: 334-346Crossref PubMed Scopus (39) Google Scholar AAV production protocols differ between the studies, as we and Rogers et al.1Rogers G.L. Chen H.-Y. Morales H. Cannon P.M. Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break.Mol. Ther. 2019; 27 (this issue): 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar used a helper virus-free system in which helper functions are provided by co-transfection of a helper plasmid,33Xiao X. Li J. Samulski R.J. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.J. Virol. 1998; 72: 2224-2232Crossref PubMed Google Scholar whereas vector used by Smith et al.3Smith L.J. Wright J. Clark G. Ul-Hasan T. Jin X. Fong A. Chandra M. St Martin T. Rubin H. Knowlton D. et al.Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing.Proc. Natl. Acad. Sci. USA. 2018; 115: E7379-E7388Crossref PubMed Scopus (23) Google Scholar produced AAV using replication-competent herpes simplex virus type 1 (HSV1) as a helper.34Chatterjee S. Wong J.K.K. Adeno-Associated Viral Vectors for the Delivery of Antisense RNA.Methods. 1993; 5: 51-59Crossref Scopus (14) Google Scholar Although the described purification method cites either heat inactivation or cesium chloride purification for inactivation of HSV1, there is still the potential for contamination of live helper virus or incomplete heat inactivation, which may boost the signal observed from AAV transduction experiments. Interestingly, inactivated HSV has been shown to cause induction of expression of endogenous retroviruses (ERVs),35Brudek T. Lühdorf P. Christensen T. Hansen H.J. Møller-Larsen A. Activation of endogenous retrovirus reverse transcriptase in multiple sclerosis patient lymphocytes by inactivated HSV-1, HHV-6 and VZV.J Neuroimmunol. 2007; 187: 147-155Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar which are observed at the AAVS1 locus (USC Genome Browser), and could therefore lead to stalled replication forks, inducing double-stranded breaks, and subsequent integration being detected at this locus. Of note, the target loci used in Smith et al.3Smith L.J. Wright J. Clark G. Ul-Hasan T. Jin X. Fong A. Chandra M. St Martin T. Rubin H. Knowlton D. et al.Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing.Proc. Natl. Acad. Sci. USA. 2018; 115: E7379-E7388Crossref PubMed Scopus (23) Google Scholar have been shown to undergo nuclease-free recombination,36Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain.Nat. Genet. 1999; 21: 70-71Crossref PubMed Scopus (4153) Google Scholar which may occur at higher frequency in the presence of heat inactivated HSV1. Additionally, BRCA2−/− cells have been shown to have aberrant immune activation,37Reisländer T. Lombardi E.P. Groelly F.J. Miar A. Porru M. Di Vito S. Wright B. Lockstone H. Biroccio A. Harris A. et al.BRCA2 abrogation triggers innate immune responses potentiated by treatment with PARP inhibitors.Nat. Commun. 2019; 10: 3143Crossref PubMed Scopus (95) Google Scholar which may lead to nuclease-free recombination or altered cellular responses to AAV, especially in the context of an HSV1 helper. When considered in aggregate, it is clear that more mechanistic characterization of the recombination induced by this novel class of AAVs will lead to a better understanding of their utility as vectors for gene editing. An additional important consideration when comparing different capsids is the method of titration and dilution buffer used for titration. Both groups used TaqMan qPCR for AAV genome titration, either using GFP-, AAVS1-, or Luc-specific primer-probes (Smith et al.)3Smith L.J. Wright J. Clark G. Ul-Hasan T. Jin X. Fong A. Chandra M. St Martin T. Rubin H. Knowlton D. et al.Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing.Proc. Natl. Acad. Sci. USA. 2018; 115: E7379-E7388Crossref PubMed Scopus (23) Google Scholar or ITR-specific primer-probe with AAV2 reference standard material from American Type Culture Collection (ATCC).1Rogers G.L. Chen H.-Y. Morales H. Cannon P.M. Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break.Mol. Ther. 2019; 27 (this issue): 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar Neither group mentions whether surfactant is used in vector dilutions for qPCR titration, which is an important consideration when comparing different capsids, as the lack of surfactant can increase capsid adherence to plastic and cause under-titration by up to 10-fold for some capsids.38Sanmiguel J. Gao G. Vandenberghe L.H. Quantitative and Digital Droplet-Based AAV Genome Titration. Springer New York, 2019: 51-83Google Scholar The reported genome dose used by Rogers et al.1Rogers G.L. Chen H.-Y. Morales H. Cannon P.M. Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break.Mol. Ther. 2019; 27 (this issue): 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar is 104 vector genomes (vg)/cell, while Smith et al.3Smith L.J. Wright J. Clark G. Ul-Hasan T. Jin X. Fong A. Chandra M. St Martin T. Rubin H. Knowlton D. et al.Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing.Proc. Natl. Acad. Sci. USA. 2018; 115: E7379-E7388Crossref PubMed Scopus (23) Google Scholar report using up to 4 × 105 vg/cell. We and others have observed a cytotoxic effect from high vg/cell transduction of extremely efficient AAV capsids and, as such, the high dose of AAV may be causing apoptosis in this sensitive cell population. This would be consistent with previous reports that high levels of AAV cause p53-induced apoptosis in other stem cells such as human embryonic stem cells (hESCs)39Hirsch M.L. Fagan B.M. Dumitru R. Bower J.J. Yadav S. Porteus M.H. Pevny L.H. Samulski R.J. Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.PLoS ONE. 2011; 6: e27520Crossref PubMed Scopus (33) Google Scholar and relates back to the method of genome titration for the different capsids. For a more in-depth discussion of AAV-mediated toxicity in stem cells, we refer readers to a review by Brown et al.40Brown N. Song L. Kollu N.R. Hirsch M.L. Adeno-Associated Virus Vectors and Stem Cells: Friends or Foes?.Hum. Gene Ther. 2017; 28: 450-463Crossref PubMed Scopus (20) Google Scholar The recent publication of high efficiency genome editing induced by AAVHSC variants in the absence of a DNA break and the conflicting report presented by Rogers et al.1Rogers G.L. Chen H.-Y. Morales H. Cannon P.M. Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break.Mol. Ther. 2019; 27 (this issue): 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar highlight the importance of a better mechanistic understanding of how HDR occurs in the context of these gene editing and gene delivery technologies. A thorough examination of HDR induced by AAV9-G505R suggests that the previously reported DSB independent mechanism of site-specific recombination facilitated by capsid may need revision or at least further exploration of technical differences leading to such drastically conflicted studies. We have discussed potential technical differences that may influence experimental outcomes presented by these studies with the goal of aiding in the transparency and reproducibility of novel gene therapy methodologies as an effort to consolidate our understanding of how to develop gene editing-based therapeutics. Download .pdf (.09 MB) Help with pdf files Document S1. Supplemental Methods