Reply to “Efficient Nuclease-free HR by Clade F AAV Requires High MOIs with High Quality Vectors”
Authors: Amanda M. Dudek, Matthew H. Porteus
Journal: Molecular Therapy (2019)

We appreciate the interest and comments on our recent report comparing the ability of different adeno-associated virus (AAV) serotypes to mediate targeted genome integration into human CD34+ hematopoietic stem and progenitor cells (HSPCs).1Dudek 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 Here we provide further concise clarification on four points: (1) our nomenclature for describing different AAV serotypes; (2) agreement on the need to establish quality control standards for AAV preps; (3) the issue of different MOIs; and (4) the importance of transparency and independent validation of reproducibility in studies that may one day impact the lives of patients. We generated clade F AAV9 variants (hematopoietic stem cell-derived AAV [AAVHSC] capsids) as peer-reviewed and publicly described by Smith et al.2Smith 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 engineered the amino acid variants that Smith et al.2Smith 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,3Smith 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 claimed as being most relevant to the frequency of targeted integration. By re-engineering these variants, we could study them in an unencumbered manner. We apologize for any confusion in nomenclature in our letter. We used the AAV6 capsid numbering system in the legend to Figure 1E but used the AAV9 numbering system throughout the rest of the paper. This change in nomenclature may have created some confusion, but we did recreate the previously reported clade F variants. We will deposit these clade F variants into Addgene for independent study and free access to the community. The issue of quality control of AAV preps is critically important as we increasingly recognize the cellular response of cells to AAV transduction. We agree that vector quality is of utmost importance both when developing potential clinical therapeutics and for studies using preclinical vectors for the initial design of AAV-based gene therapies in vivo and in vitro. Although the studies by Dudek and Porteus1Dudek 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 Rogers et al.4Rogers 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. Cold Spring Harbor Laboratory, 2019Abstract Full Text Full Text PDF Scopus (16) Google Scholar made use of a variety of different purification methods, none of these studies provided information on vector purity and quality. We suggest that the AAV community establish a set of quality control standards for publication of pre-clinical studies using AAV vectors. A key difference, as we identified and discussed in our original letter, among the Smith et al. studies,2Smith 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,3Smith 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 our study,1Dudek 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 Rogers et al.4Rogers 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. Cold Spring Harbor Laboratory, 2019Abstract Full Text Full Text PDF Scopus (16) Google Scholar is the issue of MOI. Clearly, at low MOI’s, AAV6 is superior at serving as a template for homologous recombination following a nuclease-induced double-strand break compared to clade F variants. We believe, however, that the issue of what occurs at significantly higher MOI’s with and without a double-strand break, where effects of the method of AAV titering and how cells respond to AAV transduction, needs further independent study. We hope one outcome of this exchange of letters is to prompt such experiments. Finally, we concluded our original letter and conclude this one by reiterting that it is critical for the therapeutic translation of targeted integration strategies to have transparent methodologies allowing independent validation of results and reproducibility. That independent groups have achieved different results and arrived at different conclusions reflects a robust scientific process. We look forward to the genome editing community working through these differences moving forward so that we can better develop innovative therapies that are safe and effective for patients who currently have large unmet medical needs. M.H.P. serves on the SAB and has equity in CRISPR Tx and Allogene Tx.