Peer review of: Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
In this manuscript by G. Pavani et al., Authors performed targeted integration of a promoter less 
transgene cassette into the hemoglobin A genes (HBA) of hematopoietic stem/progenitor cells 
(HSPC) in order a to develop a robust protein delivery system based on stem cell–derived 
erythroid cells. This strategy was validated by showing increasing expression of different 
transgenes by the HBA promoter during in vitro erythroid differentiation of edited human cell lines 
and cord blood derived HSPC. Then, the Authors provided proof of concept for therapeutic 
application of this platform by showing secretion and preservation of enzymatic activity of a 
hyperactive factor IX (F9) and the lysosomal acid lipase (LAL), thus suggesting possible 
implementation as new treatment for hemophilia or lysosomal storage disorders. 
The reported experiments were accurately performed and the results appear convincing as they 
are often based on multiple lines of independent evidence. Despite, as fairly stated by the Authors, 
the use of erythroid cells as systemic source for transgene secretion was already reported by 
means of tissue-specific gene replacement vectors, the concept of exploiting the high 
transcriptional activity of HBA during erythroid differentiation is new and clever. Indeed, this 
strategy will allow to maximize transgene overexpression while avoiding the need for multiple 
vector integrations or strong artificial enhancer/promoters. 
Thus, the overall findings of this study can be considered of sufficient novelty and interest for the 
gene therapy field. However, the following points need to be addressed in order to improve the 
quality and general interest of this manuscript. 
Major points: 
Since NHEJ is the most common gene editing readout in HSPC, the choice of targeting a non-
translated region of the HBA genes will allow preserving their expression in a good fraction of the 
cells that does not contain homologous mediated integration. To verify this, the Authors performed 
quantitative PCR analyses on the HBA genes and found only low levels of genomic deletions 
between the two consecutive genes and showed a representative HPLC analysis that confirmed 
presence of the globin A subunit and HbA tetramer. However, since this is an important aspect of 
the proposed strategy, more experimental detail and scientific discussion have to be provided: 
- Genomic inversions that does not delete the HBA2 gene can occur when using a nuclease that 
cut two targets on the same chromosome. Since this event will result in functional KO of both the 
HBA genes involved, proper quantification of this rearrangement has to be reported (e.g. by ddPCR 
on the genomic junctions) 
- How many samples were analyzed by HPLC? Which is the fraction of edited cells in the analyzed 
population? 
- Targeted integration of the transgene expression cassette is designed to abrogate the expression 
of the edited allele. Despite this is not a concern, thanks to the genomic redundancy and the 
expected limited contribution of KI cells to the reconstituted hematopoiesis after transplant, these 
points have to be mentioned and discussed in the manuscript. 
- When performing homology driven integration into the HBA genes, up of 4 copies of the donor 
template could be knocked-in due to the high similarity of the homology sequences between the 
two loci (HBA1 and HBA2). The Authors should quantify these recombination events or comment 
on why they are not detected (e.g., in Fig.3d how are the integration measured?). 
The Authors reported a comprehensive off-target analysis for their selected gRNA, by combining in 
silico prediction and unbiased genome wide assays. However, better description of the results has 
to be reported to facilitate their correct interpretations: 
- Description of Suppl. Table 2 is currently unclear. The Authors claim in the text that “no indels 
were observed at any of the predicted off-target sites”, but from the table it rather appears that 
there is no difference in the levels of indels detected on HAB15 and control AAVS1 gRNAs. Is this 
the right interpretation of the table? What about OT16 that shows 2% of indels? 
- In Supl Fig.3b, it is not clear from the table why the Authors claim that no unique CLIS for 5’UTR 
(19 reported in the figure) were identified after correction for random integration (6 reported). 
Does this mean that more then one CLIS was found at the on-target site? 

- In the methods section it is reported that the levels of indels at the identified off-target sites was 
measured by TIDE analysis, which has a sensitivity of ~1%. Despite this threshold could be 
considered sufficient for a proof of concept study, this information has to be clearly stated in the 
main text to better guide future follow-up studies. 
Data on F9 and LAL secretion from the edited cells are an important proof of principle that 
thereported system allow production of proteins with therapeutic relevance and that at least these 
two enzymes are not toxic when overexpressed in differentiating HSPC. However, better 
description of the results and additional discussion points are to be included in the manuscript: 
- In Fig.3h, F9 activity should be indicated as aPPT and the scale line is probably mislabeled (are 
the reported data expressed as fold over untreated?). Data must also be reported as time 
(seconds) comparing edited and untreated cells. 
- Proteins ectopically expressed in differentiating erythrocytes would possibly contain complex 
post-translational modification, but since no direct and extended experimental proof are provided 
in the text (only indirect evidence of Glu6P could be argued), this claim have to be attenuated. 
- The Authors state that protein replacement therapy has several problems that could be overcame 
by their proposed strategy. However, the discussion should better address which are these 
advantages (e.g. single administration, possible induction of tolerance, …) and which could be 
possible disadvantage of the new proposed strategy. In particular, it will be important to mention 
possible problems related to dose control of the therapeutic enzyme after HSC transplantation 
(e.g. F9 Padua: low expression would result in limited efficacy while high engraftment could 
generate thrombophilia). 
- It could be of interest to add some speculations about possible effects of local over-concentration 
of some enzymes in the bone marrow, during erythrocytes differentiation, or in the erythrocyte 
degradation organ after red cell turnover. 
Minor points: 
- Is it not clear which of the gRNA tested in Fig.1a and Suppl. Fig.1 is used all over the manuscript 
(HBA15). 
- The data reported in Suppl. Fig 4b are not an indication of HSPC multipotency but rather of 
preservation of their clonogenic differentiation capacity. 
- Suppl. Fig. 3c is missing. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
In the article entitled “Ex vivo editing of human hematopoietic stem cells for erythroid expression 
of therapeutic proteins”, Pavani et al., take advantage of robust expression of the HBA loci 
encoding alpha-globin to develop a system for robust expression of transgenes specifically in 
erythrocytes. The authors take advantage of an evolutionary HBA tandem duplication (HBA2, HBA1 
genes) and tolerance of erythroid function to alpha-globin deficiency, even with the loss of three 
gene copies. The authors use this approach to express various proteins, which show potential for 
therapeutic activity in vitro and in vivo. 
 
Although the strategy to achieve reliable tissue-specific transgene expression through gene 
targeting has already been shown for other loci (such as albumin), reducing novelty of the overall 
approach, this report appears to be the first example of using HBA as an integration site. 
Moreover, erythrocytes derived from primary hematopoietic stem cells, or HSCs themselves, are 
good candidates for cellular therapy, and the possibility of ex vivo manipulation can improve safety 
by pre-screening. 
 
While the method has promise, the article in its current form fails to clearly demonstrate the 
aspect of safety (mentioned multiple times by the authors), nor does it establish a convincing 
standard for reproduction as a method. Specifically, the gRNAs, targeting vectors, and genotyping 
assays are poorly described or referenced in the main text. Fundamentally, the choice to target 
duplicated HBA2/1 sequences suggests the possibility of large deletions or rearrangements which 

are not explored with sufficiently with standard molecular assays. 
 
Specific Points: 
 
The authors select the 5’UTR gRNA as the standard for targeting, based on efficiency and 
expression. However, looking at UCSC Genome Data, this gRNA has perfect homology to both 
HBA2 and 1. It is well known that cleaving two adjacent loci with nucleases can result in deletions 
or inversions. The authors state that “All gRNA except 74 target both HBA1 and HBA2.” This 
suggests that deletions or rearrangements would be a prevalent outcome, yet they make little 
effort (beyond ddPCR) to screen for it. This is necessary data to validate the safety and usability of 
their system. To be convincing, genomic structural analysis is needed, such as long-range PCR, 
Southern blot, sequencing, or ddPCR with multiple probes. 
 
Along these lines, the ddPCR analysis of copy number (Fig. S1f) seems to show only a loss of one 
copy. Moreover, the data in Figure 3b is used to show correlation with GFP+ cells. However, HBA 
genes are both duplicated and autosomal (4 copies) what is the expected outcome of hetero and 
homozygous targeting? The authors completely ignore this possibility. Please clarify, and provide 
supporting data. 
 
The authors use GFP to enrich cells with vector integrations. What is the overall frequency of 
integration (ie. %GFP+). It seems the data is only shown for Factor IX integration. (Fig 3b Pre 
Sort). Please include this data to demonstrate the overall targeting efficiency. 
 
In the long run, is GFP a viable solution for therapeutic application? What is a reasonable 
alternative? Please discuss. 
 
The authors screen multiple gRNAs targeting the 5’UTR or introns, and select two sites as leads. 
However, the overall lack of detail on the homology arms, gRNAs and genotyping assays (apart 
from supplemental data) makes it difficult to understand the exact integration site chosen, or the 
rationale. Moreover, the IVS2 integrations site and trap vector should produce a truncated a-globin 
protein, which is not screened for (eg. Western blot) or even discussed by the authors. 
 
There are many instances of “data not shown”, against Nature policy. Also, colony screening data 
is not shown for Figure 3k, although HDR rates are reported (7/7). 
 
The screening process for gRNAs is not clear. In Figure 1a – does each grey bar represent a 
different gRNA? A map of the locus (found in Fig. S1a) or appropriate labelling is required in the 
main figure. 
 
Throughout the paper, the gRNA used for each experiment is not clear. 
Which guides were chosen from Supp2e/f to produce the data in Panel g? (HBB targeting) 
Moreover, the details of the target site are not clear. While the 5’UTR gRNA cuts upstream of the 
ATG, does the targeting vector correct the position of the insertion to be in-frame with the 
endogenous ATG, or does it insert directly at the CRISPR/Cas9 cut site? 
 
Genotyping data for insertion at HBB is not shown. 
 
“in line with TIDE-based indels analysis (8.5%±4.9, n=5)” 
The TIDE data is not shown? 
 
Remarkably, 5’UTR and IVS2 gRNA did not modify α-globin expression, measured as ratio between 
α- and β-like globin chains (Figure 1e and supplementary figure 1d). 
Was modification expected? Did the authors make an effort to map or avoid regulatory elements? 
What was the nature of the indels formed? The sequence analysis of indels is not made available. 
 

In the first set of results, the authors state “without affecting HSPC viability, differentiation 
potential and hemoglobin expression, thus representing ideal genomic sites for KI.”, However 
these data do not yet demonstrate transgene expression, and therefore do not support ideal sites 
for KI. Please re-write. 
 
What are the predicted PCR fragment sizes in S2c/d? Presumably the PCR fragments differ in sizes 
amongst the clones because of NHEJ insertion of the IDLV. Please explain. 
 
Do the two bars in Fig S1b represent two independent experiments, or two different gRNAs? 
 
Lanes are not labelled in Fig S2i, and there is no schematic for the PCR assay. 
 
“KI occurred mostly through HDR (>90% of CFC, n=40; supplementary fig 2j).” 
Fig S2j shows one sequencing result, not a summary of targeting efficiencies. 
 
The terms BFU-E, CFU-E, and CFU-GEMM are used in the text and need to be defined there. Is 
CFU-GM in Fig S2i the same as CFU-GEMM? The authors must be consistent. 
 
“PCR analysis of individual CFC showed integration in both red and white colonies (Supplementary 
figure 2i).” What is meant by red and white? 
 
Figure 2h, please use scale bars, not magnification. Also, why are only positive cells shown? 
Including examples of GFP negative cells would be more convincing. 
 
“no indels were observed at any of the predicted off-target sites (Supplementary table 2).” 
If that is the case, then what is the data in the table (column 4, last two columns)? 
 
“Off-target candidates were predicted in silico using two different software” 
Please state the software or provide a URL. 
 
“BFU-E derived erythroblasts were capable of secreting FIX (supplementary figure 3c).” 
Should the comment cite Fig. S4d?