Efficient Nuclease-free HR by Clade F AAV Requires High MOIs with High Quality Vectors
Authors: Saswati Chatterjee
Journal: Molecular Therapy (2019)

Two papers, 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar and Dudek and Porteus,2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar in the October issue of Molecular Therapy claim that homologous recombination (HR)-based genome editing by Clade F adeno-associated virus (AAV) is inefficient and virtually undetectable in the absence of double-stranded DNA breaks. These claims contradict our findings that Clade F AAVs mediate HR efficiently in primary cells and in vivo in the absence of double-stranded DNA breaks induced by exogenous nucleases.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 We showed that chromosomal insertion of donor sequences by hematopoietic stem cell-derived AAV (AAVHSC) vectors required flanking homology arms and was BRCA2-dependent. Since BRCA2 is an essential mediator of HR,4Prakash R. Zhang Y. Feng W. Jasin M. Homologous recombination and human health: the roles of BRCA1, BRCA2, and associated proteins.Cold Spring Harb. Perspect. Biol. 2015; 7: a016600Crossref PubMed Scopus (479) Google Scholar we concluded that Clade F AAV-mediated editing was accomplished by mediated HR. Importantly, we showed that high MOIs were required to observe augmented HR by Clade F AAV. AAVHSC-mediated HR-based genome editing was found to occur efficiently in vivo and was persistent. Neither 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar nor Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar actually replicated the studies described 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 There were significant discrepancies between our studies and those of Rogers at 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar and Dudek and Porteus.2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar These included the MOIs, AAV capsids, transduction, culture conditions, and vector quality. 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar and Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar used donor vectors encoding a promoter containing a GFP gene, in contrast to the promoterless editing vectors used in our studies. Both papers only tested in vitro editing with no actual sequence confirmation or in vivo evaluation. Due to these critical discrepancies between their studies and ours, we find their conclusions regarding the ability of Clade F AAV to mediate HR to be flawed. The most significant difference between 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar Dudek and Porteus,2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) 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 lies in the MOIs. 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 we clearly showed that efficient in vitro HR-based editing in the absence of nuclease-induced double-stranded DNA breaks was dependent upon the use of high MOIs. 150,000 vector genomes (vg)/cell or higher were required for efficient HR. Accordingly, our studies were performed at MOI: 150,000 or higher.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 In contrast, Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar used MOI: 5,000, 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar used an MOI: 10,000, 30-fold and 15-fold lower doses, respectively, than that required for efficient HR in absence of nucleases. Thus, it is not surprising that they failed to observe editing in the absence of nucleases at these MOIs. In fact, their studies simply replicated the low-dose data shown 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 which clearly showed that little to no HR was observed at these MOIs with any AAV tested. Curiously, both groups observed significant loss of viability at higher MOIs. 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar showed 66% toxicity at MOI: 50,000, with 50,000 vg/cell being their maximum tolerated dose for AAV6. Similarly, Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar were unable to use MOI >5,000 due to toxicity. This raises important concerns regarding the quality of their vectors and suggests the possible presence of toxic contaminants. Such high toxicities make it difficult to draw meaningful conclusions regarding the editing capacities of their vectors and raises serious questions regarding the validity of their conclusions. The toxicity observed 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar and Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar is in marked contrast to our data. As shown 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, we saw no difference in the viability of AAV-transduced and untransduced cells over a range of MOIs from 10,000 to >400,000 vg/cell. Interestingly, 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar also used a completely different method for packaging and purification of their AAV6 vectors as compared with their Clade F vectors, thus negating AAV6 as a true control. Although unaddressed in either 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar or Dudek and Porteus,2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar it is important for editing vectors to have intact inverted terminal repeats (ITRs) to mediate HR. These high G:C content palindromic termini of AAV genomes are important for recombination but are also prone to deletion and rearrangements.5Mroske C. Rivera H. Ul-Hasan T. Chatterjee S. Wong K.K. A capillary electrophoresis sequencing method for the identification of mutations in the inverted terminal repeats of adeno-associated virus.Hum. Gene Ther. Methods. 2012; 23: 128-136Crossref PubMed Scopus (8) Google Scholar Thus, stringent screening for intact ITRs is necessary for the production of high quality editing of AAV vectors.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 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar and Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar compared chromosomal insertion of AAV donor sequences by homology-dependent repair (HDR) after induction of double-stranded DNA breaks by either zinc finger nucleases (ZFNs)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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar or CRISPR/Cas9 (Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). Both groups used AAV6, AAV9, and mutagenized capsid variants of AAV9, while we used naturally occurring Clade F AAVs isolated from CD34+ cells6Smith 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 along with AAV2, AAV6, and AAV8 controls.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 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar erroneously claimed that AAVHSC13 is identical to AAVHSC17 and that AAV9 G505R represents both vectors. AAVHSC17, however, has an additional silent mutant at amino acid 60 (Smith et al.6Smith 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). Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar compared HDR by AAV9 A68V, AAV9 G505R, AAV9 S345A, AAV9 S345A G505R, AAV9, and AAV6. Of these, only a single capsid, AAV9G505R (AAVHSC13), is the same as one of the 14 AAV capsids tested 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 These include vectors that were not tested 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 and thus cannot be used to either substantiate or refute our data. Although Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar additionally list AAVHSC15 and AAVHSC17 among the AAV Clade F capsids tested, they do not show any data with these AAVs. Since AAVHSC15 and AAVHSC15 were used for many experiments 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 it would have been reasonable to use these vectors to replicate the studies 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 Both groups failed to acknowledge the presence of silent mutations. AAVHSC7 has a silent mutation at amino acid 308. It is well recognized that silent mutations are known to play important roles in protein folding, evolution, adaptation, and even disease pathogenesis7Brule C.E. Grayhack E.J. Synonymous Codons: Choose Wisely for Expression.Trends Genet. 2017; 33: 283-297Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 8Caspermeyer J. When silent mutations provide evolutionary advantage.Mol. Biol. Evol. 2016; 33: 1639Google Scholar, 9Sauna Z.E. Kimchi-Sarfaty C. Understanding the contribution of synonymous mutations to human disease.Nat. Rev. Genet. 2011; 12: 683-691Crossref PubMed Scopus (672) Google Scholar and therefore may not be inconsequential. Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar also discounted the possible role of the amino acid A68 in AAVHSC7 due to its location in the unresolved VP1 unique region.6Smith 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 They additionally argue that the internal location of T346A and the external location of G505R of AAVHSC17 (Smith et al.6Smith 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) do not suggest a role for these amino acids in HR. Since the role of capsid elements in HR remains unknown, there is no basis for such conclusions. Although the AAV vectors 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 were packaged with herpes simplex virus 1 (HSV1), we are aware of successful editing studies with AAV vectors manufactured using adenovirus helper genes and triple transfection.10Lobikin M. Ahmed S. Boyd M. Chittoda M. Eliya V. Ghazi I. Lehnert B. Mercaldi M. Seidel S. Kelly T. et al.Development of a Scalable Platform for GMP Production of High Quality, Novel Clade F rAAV Vectors Following Comparison of HEK293 Mammalian and the Sf9-Baculovirus Systems.Mol. Ther. 2019; 27: 160Google Scholar Our AAV vectors were purified through two rounds of cesium chloride density gradient centrifugation and were never heat inactivated. Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar raised the possibility of a role for HSV as a helper virus in elevating HR and of heat inactivation having biologic effects. Since all AAV vectors tested 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 were packaged and purified in an identical fashion, it is unlikely that HSV played a direct role in augmenting HR only in Clade F AAV. If that were the case, elevated levels of HR would have been seen across the board, not only for Clade F viruses. Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar additionally invoke the possibility of aberrant immune activation, which may lead to nuclease-free recombination or altered cellular responses to AAV in the context of the HSV1 helper in BRCA2−/− cells. However, we clearly showed that BRCA2 was absolutely required for AAVHSC HR.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 No recombination was observed in the absence of BRCA2, despite efficient transduction.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 Since no AAVHSC-mediated HR was observed in BRCA2−/− cells, this explanation offered by Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar does not apply. Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar raised additional concerns regarding the toxicity of potential residual HSV carried over into the final vector batches. Injection of purified AAV vectors packaged with HSV1 helper virus into immune-deficient NOD.CB17-Prkdc scid/NCrCrl mice did not result in toxicity or morbidity, as would be expected from HSV contamination.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,6Smith 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 Moreover, 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 documented a lack of toxicity in vitro over a range of MOIs from 10,000 to >400,000, indicating the absence of carryover of residual active HSV1, which might have confounded the results. 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 the editing assays were designed with a high degree of stringency. The editing vectors were designed to insert promoterless reporter genes into introns of target genes such that expression would be driven from the chromosomal promoter. A splice acceptor and T2A sequence were included to allow independent expression of the reporter genes. This ensured that expression from episomal AAV genomes did not confound the results, unlike what was shown 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar and Dudek and Porteus.2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar The absence of reporter expression in controls lacking homology arms indicated that possible minimal transcription from the ITRs did not contribute to reporter expression.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 In contrast, both 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar and Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar only used donor constructs with heterologous promoters driving reporter gene expression. Their donor vectors were designed such that expression could occur from both episomal vectors as well as edited chromosomes. Both studies then analyzed GFP expression in long-term cultures of AAV-transduced cells with the assumption that expression at 12–17 days implied editing, with no evidence offered to support this assumption. 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, we showed that the vector genome copy number quantitated in purified nuclei was higher in cells transduced with Clade F AAVs, followed by Clade A, then Clade E, and finally Clade B. Based on this observation, we suggested improved nuclear transport/entry as one possibility for augmentation of HR by Clade F AAV. In contrast, 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar evaluated whole cell-associated vector genome copy numbers, potentially including vector particles attached to the external cell surface and in the cytoplasm in addition to intranuclear copies. They correlated GFP expression on day 14 post-transduction, with vector genome copy number on day 2. It is unclear why a correlation between these values would be expected, since GFP and vector genomes copy number were assessed 12 days apart. Regardless, they showed that, with AAV6, they observed high levels of GFP expression and vector genome copies only in the presence of ZFN, and no GFP- or cell-associated vector genome copies were observed with AAV9 and AAVHSC13 (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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Since their AAV6 was packaged and purified by a completely different method than their AAV9 and AAVHSC13, these results could simply reflect the quality of their vector batches. Poor quality AAV9 and AAVHSC13 would likely not transduce or edit cells efficiently, even at high MOIs. Their observations of toxicity at MOIs >10,000 for AAV9 and AAVHSC13 versus MOI: 50,000 for AAV6 suggests that their AAV6 vector batches were of higher quality than their AAV9 and AAVHSC13 batches and supports this conclusion. The culture conditions for CD34+ cells used in both 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar and Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar were different from those 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 We have previously shown that AAV transduction of CD34+ cells is optimal for slowly dividing cells.11Paz H. Wong C.A. Li W. Forman S.J. Wong K.K. Chatterjee S. Quiescent subpopulations of human CD34-positive hematopoietic stem cells are preferred targets for stable recombinant adeno-associated virus type 2 transduction.Hum. Gene Ther. 2007; 18: 614-626Crossref PubMed Scopus (13) Google Scholar, 12Fisher-Adams G. Wong K.K. Podsakoff G.M. Forman S.J. Chatterjee S. Integration of adeno-associated virus vectors in CD34+ human hematopoietic progenitor cells after transduction.Blood. 1996; 88: 492-504Crossref PubMed Google Scholar, 13Santat L. Paz H. Wong C. Li L. Macer J. Forman S. Wong K.K. Chatterjee S. Recombinant AAV2 transduction of primitive human hematopoietic stem cells capable of serial engraftment in immune-deficient mice.Proc. Natl. Acad. Sci. USA. 2005; 102: 11053-11058Crossref PubMed Scopus (25) Google Scholar For retrovirus and lentivirus transduction, CD34+ cells are often prestimulated and placed in culture at high cytokine concentrations, since rapid cycling promotes better transduction. However, for AAV transduction, we have reported that low cytokine concentrations is better,11Paz H. Wong C.A. Li W. Forman S.J. Wong K.K. Chatterjee S. Quiescent subpopulations of human CD34-positive hematopoietic stem cells are preferred targets for stable recombinant adeno-associated virus type 2 transduction.Hum. Gene Ther. 2007; 18: 614-626Crossref PubMed Scopus (13) Google Scholar, 12Fisher-Adams G. Wong K.K. Podsakoff G.M. Forman S.J. Chatterjee S. Integration of adeno-associated virus vectors in CD34+ human hematopoietic progenitor cells after transduction.Blood. 1996; 88: 492-504Crossref PubMed Google Scholar, 13Santat L. Paz H. Wong C. Li L. Macer J. Forman S. Wong K.K. Chatterjee S. Recombinant AAV2 transduction of primitive human hematopoietic stem cells capable of serial engraftment in immune-deficient mice.Proc. Natl. Acad. Sci. USA. 2005; 102: 11053-11058Crossref PubMed Scopus (25) Google Scholar similar to that 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 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar prestimulated their CD34+ cells, and both studies used 5–10-fold higher cytokine concentrations than were 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 Neither study provided convincing molecular proof of editing. The lack of sequence confirmation of editing in both studies is troubling. Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar provided no molecular evidence of editing, while 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar only performed a PCR-based targeted integration assay without sequence confirmation. 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar additionally lacked important controls for the specificity of their targeted integration PCRs. Sequence confirmation is critical for any conclusions regarding successful editing. Both studies made the assumption that GFP expression at 12–17 days post-transduction indicated successful editing. However, it is well known that infection with single-stranded AAV initiates a cellular DNA damage response, checkpoint activation, and cell cycle arrest.14Fragkos M. Jurvansuu J. Beard P. H2AX is required for cell cycle arrest via the p53/p21 pathway.Mol. Cell. Biol. 2009; 29: 2828-2840Crossref PubMed Scopus (121) Google Scholar,15Jurvansuu J. Fragkos M. Ingemarsdotter C. Beard P. Chk1 instability is coupled to mitotic cell death of p53-deficient cells in response to virus-induced DNA damage signaling.J. Mol. Biol. 2007; 372: 397-406Crossref PubMed Scopus (30) Google Scholar AAV-induced cell-cycle arrest is associated with activation of checkpoint proteins ATR, Chk1, and H2AX.14Fragkos M. Jurvansuu J. Beard P. H2AX is required for cell cycle arrest via the p53/p21 pathway.Mol. Cell. Biol. 2009; 29: 2828-2840Crossref PubMed Scopus (121) Google Scholar,15Jurvansuu J. Fragkos M. Ingemarsdotter C. Beard P. Chk1 instability is coupled to mitotic cell death of p53-deficient cells in response to virus-induced DNA damage signaling.J. Mol. Biol. 2007; 372: 397-406Crossref PubMed Scopus (30) Google Scholar In 12–17-day culture experiments performed 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar and Dudek and Porteus,2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar transduced cells containing single-stranded AAV genomes that would preferentially undergo mitotic arrest, while untransduced cells would proliferate normally. Therefore, over time in culture, a reduction of the proportion of AAV-transduced cells would be expected and is actually observed.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 With AAV transductions performed at MOI: 5,000–10,000 vg/cell, as 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar and Dudek and Porteus,2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar an excess of intracellular single-stranded AAV genomes is expected even after HR or HDR-mediated insertion of one or two copies into the genome, leading to activation of checkpoint proteins and mitotic arrest. It is precisely for this reason that long-term in vitro experiments with AAV vectors do not yield useful information. For these reasons, the evaluation of AAV vectors is best addressed in vivo. Unfortunately, neither 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar nor Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar performed in vivo evaluation of editing. In contrast, 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, in addition to in vitro studies, we showed long-term stable in vivo editing by AAVHSC of the murine Rosa26 locus by serial bioluminescent assays and Southern blot and sequence analyses. Notably, we never questioned the ability of AAV6 to transduce CD34+ cells or K562 cells. In fact, we have previously published on AAV6 transduction of human CD34+ hematopoietic stem progenitor cells (HSPCs) in vitro and, importantly, in vivo.16Song L. Kauss M.A. Kopin E. Chandra M. Ul-Hasan T. Miller E. Jayandharan G.R. Rivers A.E. Aslanidi G.V. Ling C. et al.Optimizing the transduction efficiency of capsid-modified AAV6 serotype vectors in primary human hematopoietic stem cells in vitro and in a xenograft mouse model in vivo.Cytotherapy. 2013; 15: 986-998Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar In this collaborative study, we showed robust, stable long-term transduction of xenografted CD34+ HSPCs up to 6 months post-transplantation.16Song L. Kauss M.A. Kopin E. Chandra M. Ul-Hasan T. Miller E. Jayandharan G.R. Rivers A.E. Aslanidi G.V. Ling C. et al.Optimizing the transduction efficiency of capsid-modified AAV6 serotype vectors in primary human hematopoietic stem cells in vitro and in a xenograft mouse model in vivo.Cytotherapy. 2013; 15: 986-998Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar Thus, we have no doubt that AAV6 is a good vector for the delivery of donor constructs for HDR-mediated insertion at sites of double-stranded DNA breaks induced by ZFNs or CRISPR/Cas9. In the presence of intranuclear single-stranded AAV donor templates, nuclease-mediated double-stranded DNA breaks are often repaired by the insertion of the AAV donor. In contrast, the BRCA2-dependant editing by AAV described 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 does not require the presence of prior double-stranded DNA breaks. Single-stranded AAV has long been known to mediate HR in the absence of DNA breaks.17Khan I.F. Hirata R.K. Russell D.W. AAV-mediated gene targeting methods for human cells.Nat. Protoc. 2011; 6: 482-501Crossref PubMed Scopus (138) Google Scholar,18Miller D.G. Wang P.R. Petek L.M. Hirata R.K. Sands M.S. Russell D.W. Gene targeting in vivo by adeno-associated virus vectors.Nat. Biotechnol. 2006; 24: 1022-1026Crossref PubMed Scopus (94) Google Scholar 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 we simply reported that Clade F AAVs mediate BRCA2-dependent HR at higher efficiencies. We showed editing by 14 different AAVs in primary cells in short-term cultures in vitro and by AAVHSC15 and AAV8 long-term in vivo. In each case, we found editing by Clade F AAVs to be more efficient.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 In closing, while there are numerous differences among 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 Roger 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar and Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, we believe that their use of very low MOIs is the primary reason for their inability to observe augmented HR by Clade F AAVs. Moreover, their use of AAV vectors that were toxic, even at low doses, further impeded their ability to observe augmented HR by Clade F AAV. Finally, in order to successfully observe HR-based editing by Clade F AAV, it is critical to (1) ensure the presence of intact and complete inverted terminal repeats; (2) use highly purified batches of AAV vectors; (3) use AAV vectors with single-stranded genomes; and (4) target cells that are not cycling too fast to be efficiently transduced by AAV. Augmented HR by Clade F AAVs requires high MOIs. This in turn requires the use of high-titered, high quality, non-toxic AAV vectors. The inability of 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: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar and Dudek and Porteus2Dudek A.M. Porteus M.H. AAV6 is superior to Clade F AAVs in stimulating homologous recombination-based genome editing in human HSPCs.Mol. Ther. 2019; 27: 1701-1705Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar to transduce cells at MOI: 150,000 or higher in the absence of accompanying toxicity or to accurately replicate our studies clearly impacted their ability to make meaningful conclusions regarding the HR capacities of Clade F AAVs. Meanwhile, we remain committed to advancing this technology to better comprehend the underlying mechanisms and explore its potential for use in correcting genetic diseases. S.C. is co-founder of and has an equity stake in Homology Medicines Inc.