Lentiviral vectors escape innate sensing but trigger p53 in human hematopoietic stem and progenitor cells

Abstract Clinical application of lentiviral vector (LV)‐based hematopoietic stem and progenitor cells (HSPC) gene therapy is rapidly becoming a reality. Nevertheless, LV‐mediated signaling and its potential functional consequences on HSPC biology remain poorly understood. We unravel here a remarkably limited impact of LV on the HSPC transcriptional landscape. LV escaped innate immune sensing that instead led to robust IFN responses upon transduction with a gamma‐retroviral vector. However, reverse‐transcribed LV DNA did trigger p53 signaling, activated also by non‐integrating Adeno‐associated vector, ultimately leading to lower cell recovery ex vivo and engraftment in vivo. These effects were more pronounced in the short‐term repopulating cells while long‐term HSC frequencies remained unaffected. Blocking LV‐induced signaling partially rescued both apoptosis and engraftment, highlighting a novel strategy to further dampen the impact of ex vivo gene transfer on HSPC. Overall, our results shed light on viral vector sensing in HSPC and provide critical insight for the development of more stealth gene therapy strategies.

transduction of human hematopoietic progenitor cells had not yet been done. It is a significant advance in describing the biology underlining gene therapy based on viral vectors • Potential to increase early stages of hematopoietic reconstitution following LV mediated GT is a little but overemphasized since : a) neutropenia related death as mentioned p19, following SCTs is no longer as high as reported in the cited 2009 paper b) it is not excluded that p53 inhibition or ATM inhibition might exert unwanted effects not seen in the experimental setting. I therefore suggest to temper the presentation of the medical interest of this approach • P3, DAI (NLR family) could also be cited as a cytosolic receptor of DNA • All over the manuscript, the term "HSC" is used to define cord blood or bone marrow CD34+ cells. This is not fully accurate since only small fractions are bonafide HSC. The term of hematopoietic progenitor cells would thus be more appropriate. We thank the reviewers for retaining our work technically well performed, novel and of significant advance in understanding the biology underlying gene therapy based on viral vectors. We have modified the manuscript based on the reviewers' insightful criticisms and suggestions and believe to have properly addressed most of his/her concerns and to have significantly improved the overall quality of this work.
Briefly, we have now modified the manuscript to address the concerns specifically raised by the Reviewer as follows: Referee #1 (Remarks): • Potential to increase early stages of hematopoietic reconstitution following LV mediated GT is a little but overemphasized since : a) neutropenia related death as mentioned p19, following SCTs is no longer as high as reported in the cited 2009 paper Regarding this specific point, we thank the Reviewer for this observation and have now modified the discussion on neutropenia-related mortality in hematopoietic stem cell transplantation (HSCT) accordingly and have included more recent references, page 18 of the revised manuscript, as suggested by the Reviewer. b) it is not excluded that p53 inhibition or ATM inhibition might exert unwanted effects not seen in the experimental setting. I therefore suggest to temper the presentation of the medical interest of this approach As the Reviewer correctly suggests, there are certainly some safety concerns associated with the potential application of transient ATM inhibition during ex vivo HSPC gene therapy to be taken into account and carefully addressed before any clinical implementation can be foreseen. Based on this useful comment, we have now tempered the discussion regarding the applicability of ATM inhibition, page 19 of the revised manuscript, acknowledging these relevant safety concerns, as rightly pointed out by the Reviewer.
• P3, DAI (NLR family) could also be cited as a cytosolic receptor of DNA As the Reviewer rightly points out, also other cytosolic nucleic acid sensor may be involved in vector sensing in HSPC. In particular, we have now cited also the DNA-dependent activator of interferon-regulatory factors (DAI) as a potential sensor of vector nucleic acids in the introduction, page 3 of the revised manuscript, as suggested by the Reviewer • All over the manuscript, the term "HSC" is used to define cord blood or bone marrow CD34+ cells. This is not fully accurate since only small fractions are bonafide HSC. The term of hematopoietic progenitor cells would thus be more appropriate.
We fully agree with the Reviewer that including the progenitor compartment in our definition of CD34+ population is more appropriate. Accordingly, we have modified the term "HSC" to "HSPC" throughout the revised version of manuscript in order to refer more correctly to the heterogeneous population of CD34 + hematopoietic stem and progenitor cells, as kindly suggested by the Reviewer.
Finally, we have also modified our manuscript to comply to the EMBO Molecular Medicine editorial requirements as follows:

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We have included five keywords, page 1 of the revised manuscript; -- We have moved the methods and related references of Viral Vectors Colony-forming unit (CFU) assay and Transplantation of human HSPC in NSG mice, Statistical analysis, Limiting Dilution Assay and Homing Assay from the supplementary materials to the main manuscript as requested, pages 21-24; -- We have updated the references to comply the EMBO Molecular Medicine guidelines; -- We have updated all figure legends to comply to the Author Guidelines regarding statistical testing, pages 32-36; -- We have filled-in the complete Author Checklist; -- We have included source data for all the Western blots shown in the main and supplementary figures of the manuscript; -- We have included a statement in the Materials and Methods section of the revised manuscript identifying the institutional and/or licensing committee approving the experiments, page 22; -- We have included a Synopsis of our findings and prepared a visual abstract accompanying it; -- We have included the ORCID ID for the corresponding author.
2nd Editorial Decision 02 June 2017 Thank you for the submission of your revised manuscript to EMBO Molecular Medicine.
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No prior studies were used to estimate the statistical power of the study The animal sample size was chosen in agreement with available published work in which similar experiments were performed (Zonari et al., Stem Cell reports, 2017;Nucera et al, Cancer Cell, 2016, Giustacchini et al. , Cell stem cell, 2012 All the mice used in this study were female NSG, transplanted between 8--10 weeks of age. In the homing and LDA assay both pre--established criteria and stringent internal controls (not transplanted mice) were used to establish exlusion/inclusion of the mice in the analysis. For example, in the LDA experiment a mouse was considered as engrafted if the percentages of human CD45+ within the BM was above 0.10 (basal level of noise signal at flow cytometer observed in the negative controls), and if both human myeloid and lymphoid cells were observed in the mouse (pre--established criteria based on the expected stem cell repopulation capacity). In the primary transplant experiment (Fig 3A--B) we excluded all the mice of one experiment due to technical issues during the transplant procedure that lead to unaccettably low and random levels of engraftment among the groups (also in untreated controls). Yes, the in vitro experiments were mostly performed with pools of CD34+ donors to decrease the impact of interindividual biological variability.
The animal study was randomized.

Yes
Data were analyzed with Graph Pad Prism version 5.0a and expressed as the mean ± standard error mean (SEM) if not otherwise stated. Non--parametric Wilcoxon Signed Rank test was used to assess the different level of expression of specific genes respect to internal control set as 1. Non-parametric (Kruskall--Wallis) was used for unpaired dataset, while the non--parametric (Friedman test) was used for matched paired observations. In both cases Dunn's adjustment was used for No estimation of variation was performed as all tests were non--parametric. For further details, please refer to Material and Methods of the manuscript NA 6. To show that antibodies were profiled for use in the system under study (assay and species), provide a citation, catalog number and/or clone number, supplementary information or reference to an antibody validation profile. e.g., Antibodypedia (see link list at top right), 1DegreeBio (see link list at top right). 7. Identify the source of cell lines and report if they were recently authenticated (e.g., by STR profiling) and tested for mycoplasma contamination. 11. Identify the committee(s) approving the study protocol.
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E--Human Subjects
All the mice used in this study werefemale NSG, purchased from Jackson lab, transplanted between 8--10 weeks of age. Animals were housed at the Ospedale San Raffaele animal facility in sterility conditions complying with the immunosuppressed phenotype of the NSG mice. Transplantions, bleeding and sacrifice were performed within thwe animal facility BSL2 room, according to National and European regulatory standards.
All animal experiments were performed according to National and European regulation in the context of a protocol approved by the Ospedale San Raffaele Ethical Committee and the Italian Ministry of Health (IACUC 611).
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