Peer review of: Engineering monocyte/macrophage−specific glucocerebrosidase expression in human (2020)

Reviewers' comments: 

Reviewer #1 (CRISPR based therapy)(Remarks to the Author): 

Advances over the past ~2 decades have allowed clinic-scale, efficient targeted genetic 
engineering of hematopoietic stem and progenitor cells. One modality of such engineering is 
targeted integration into a genomic safe harbor locus of a transgene that would complement 
disease-causing loss-of- function mutations in a native gene. One potential limitation of this 
approach is potentially toxic, lineage-nonselective expression of the transgene, which in principle 
could be ameliorated via the use of a cell-type-specific promoter. In the submitted manuscript, 
Gomes-Ospina, Porteus, and colleagues investigate whether this approach can be used in the 
context of preclinical experiments for a human genetic disease (Gaucher’s), for which both there is 
both bone marrow transplant and enzyme replacement therapy evidence in support of such a 
strategy. This is a sensible set of studies, and the gene editing data (the area of this reviewer’s 
primary technical expertise) support the conclusions drawn. 

The authors choose the CCR5 safe harbor, which is appropriate, and use the CRISPR-Cas system 
in conjunction with an AAV-provided repair template, which is also appropriate since it’s clinic-
grade. The repair cassette contains a macrophage-specific promoter to limit expression of the 
transgene to the relevant lineage, which is a sensible approach. All this put together yields a 
robust efficiency of targeted integration as gauged by marker expression (Fig 1d) and edited allele 
quantitation (Fig 1e). The edited HSPCs produce macrophages that are marker-positive (Fig 2); 
the transgenic cassette using a lineage-specific promoter confines expression to macrophages (Fig 
3). Experiments transplanting the cells into immunodeficient mice show the edited cells engraft 
(Fig 4e-f), albeit at a reduced efficiency, and form macrophages (Fig 5c). 

Overall the data support the general conclusion (14-358) that the use of safe harbor transgenesis 
of a lineage-specific PTU is a relevant approach to pursue, including in the context of the 
development of an experimental therapeutic for GD. 

Comments 

One general comment: is there any evidence for AAV capture into potential off-target breaks 
induced by Cas9? 

Fig 2b: to complement the microscopy data in Fig 2c, would it make sense to also show the FACS 
data in Fig 2b for citrine-CD14 double-positive cells? (eg like in Fig 5c) 

7-149: should be “fluorescence” 

Fig 4c and 4d: the Results section mentions a ~10 fold difference in cell dose per mouse; would it 
be useful to color-code the data in these two figures according to the dose used (eg higher dose of 
cells per given mouse would be darker grey?) 

Fig 5d: it may be useful to comment on the 3 animals in the monocyte group that have robust 
targeted integration but are not citrine-positive. 

Discussion: it may be appropriate to mention the packaging limit of AAV as one factor to consider 
potential disease targets for this approach. 

16-409: a reference is missing 

Reviewed by: Fyodor Urnov 

Reviewer #2 (Gene therapy with stem cells)(Remarks to the Author): 

The manuscript by Scharenberg et al. characterizes a gene therapy approach to express the 
enzyme glucocerebrosidase in Gaucher Disease patients. The experimental approach allows 
expression of the GCase enzyme by Crispr/Cas9 editing and AAV6-mediated delivery of the gene in 
the CCR5 locus in human HSPCs. Several templates are tested, one of them allowing specific 
expression of the enzyme in the monocyte/macrophage lineage, which is then evaluated in vivo in 
immunodeficient humanized mice. The work is reasonably straightforward to follow, and the data 
are of high quality. This study provides the first example of transplantation of Crispr/Cas9-edited 
HSPCs expressing GCase and are potentially important for clinical applications in the field of gene 
therapy and more specifically Gaucher Disease. However, there are a number of issues that need 
to be addressed in order to improve the clarity and accuracy/quality of the paper. 
Major comments 
1- Figure 1: they authors should check CCR5 expression in edited versus non-edited cells. 

2- Figure 1C: Modifications of HSPCs with AAV+RNP using the SFFV construct show 2 populations 
of high and dim citrine+ cells but not in the CD68S construct. How do the authors explain this 
observation? 

3- Figure 1 D and E: In figure 1D, it seems that the SFFV promoter leads to a higher expression of 
Citrine relative to the CD68S promoter. However, in Figure 1E, allelic integration in enriched 
Citrine+ cells show a slightly better integration when using the CD68S-GCase-P2A-citrine construct 
in comparison with the SFFV promoter construct. Does the silencing of the CD68S promoter in the 
more primitive populations explain the lower citrine expression observed with bulk cells modified 
with CD68S promoter in Figure 1C? To support their hypothesis, the authors should clarify if these 
samples were also collected at 48 hours post-modification. This figure is confusing as it does not 
compare directly with Figure 1D. It would be good to add the data in non-enriched population in 
Figure 1E. 

4- Figure 2B: according to the title of the paragraph (line 124) it seems that the cell phenotype 
was analyzed in edited human HSPCs but it would be more rigorous to compare this phenotype 
with non-edited cells as well. 

5- Supplementary Figure 4: If the authors have looked into T cell compartment as well it would be 
informative to add these data. 

6- Figure 4E: It is unclear what do pre- and post- mean on the x-axis. Probably pre- and post-
transplant but please clarify in the figure legend or in the results section. Additionally, it is unclear 
in which cell population was the targeted alleles frequency determined. Indeed, this would help a 
more accurate evaluation of the extent of editing in this in vivo experiment. The authors should 
explain whether it was from the PBMCs fraction including mouse and human CD45+ cells or 
isolated human CD45+ cells or myeloid cells or else. 

7- Figure 5B: why are only 5 mice represented when 8 mice received a secondary transplant? 

8- Figure 5: It is unclear if these data are from the primary or secondary transplant (except for 
figure 5B). There is persistence of edited human cells and citrine expression at 16 weeks (or 32 
weeks?) post editing, but it is important to confirm GCase expression. The authors should analyze 
expression and function of GCase at 32 weeks. The authors could also use the sorted cells from 
myeloid and B cells lineages to further confirm the specificity of CD68S promoter in vivo as well 
and show GCase activity from these sorted cells relative to other lineages. 

9- Figure 5C: it is confusing if these data were obtained from primary or secondary transplanted 
mice? If these are from primary transplant, then the same should be shown using mice from the 

secondary transplants to demonstrate long term persistence of protein expression from edited cells. 
It is important to show expression and function of GCase as well in both primary and secondary 
transplanted mice. 

10- Supplementary figure 5A and B: How do the authors explain the drop in modified engrafted 
cells relative to the level of editing pre-transplant? The authors should comment on this 
observation in the results section. 
Minor comments 
1- Please be specific as to which in vivo model is used in the study 

2- Figure 1D: the authors should clarify in the figure legend if these data were obtained by flow 
cytometry? 

3- Figure 1E: the authors should describe how were the citrine+ versus citrine- cells enriched. 

4- Figure 3A: how many days post-modifications were the cells sorted? 

5- Figure 3D: how long after the sort was the GCase activity assessed? The authors should provide 
some statistical analyses for these data to determine if these differences are statistically significant. 

6- Figure 4B: it would be informative to know how many days post-modifications were the citrine+ 
/ citrine- cells sorted so we have a better understanding how early in the GM differentiation phase 
can the CD68S promoter allow citrine expression and support the hypothesis of an expression in 
granulocyte/monocyte-primed progenitors. 

7- From lines 239 to 243, it is written “The median percentage of myeloid cells and B-cells in the 
bone marrow was 24.9% and 63.6%, respectively, for the mice transplanted with CD68S-GCase-
targeted HSPCs, and 18% and 66.8%, respectively, for the mice transplanted with CD68S-GCase-
P2A-Citrine-targeted HSPCs.” However, in the myeloid cells panel (Figure 5A) the % is higher for 
CD68S-GCase-P2A-Citrine than CD68S-GCase whereas the numbers indicate the opposite (18% 
versus 24.9%). Does Figure 5A represent the bone marrow data or else or are the myeloid 
numbers inverted for CD68S-GCase and CD68S-GCase-P2A-Citrine? 

8- Figure 5A: The authors should clarify in the figure legend if these cell samples were collected 
from the bone marrow at necropsy? Were these samples collected at 16 weeks too? Are these 
mice from the primary transplant? 

9- Supplementary figure 5A and B: on the x-axis, citrine is written “cirine”, please correct. 

10- Supplementary figure 6: It is unclear why the authors didn’t use the same panel as in primary 
transplant to monitor the level of human cells engraftment and differentiation? 

11- In the discussion (lines 346 to 353) it would be informative to discuss the study from Dunbar 
et al. who attempted transplant of transduced CD34+ cells in GD patients without success 
(reference 18) and why is it expected that the authors strategy would be more efficient. 

12- In the Methods section, line 371, the origin if the CD68S sequence is missing. Similarly, line 
409 the reference for 2′-O-methyl-3’-phosphorothioate is missing and line 520 the reference for 
the GCAse activity assay is missing. Please clarify.