Peer review of: Frataxin gene editing rescues Friedreich’s ataxia pathology in dorsal root ganglia organoid- (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

In this manuscript by Mazzara et al., the authors developed a new system to model the sensory 
neurons affected in FRDA. In this work, the authors used patient derived iPSCs to generate dorsal 
root ganglia organoids (DRGOs). The first part of the manuscript is dedicated to characterizing the 
DRGOs. The patient derived DRGOs recapitulate neuronal impairment in survival, axonal 
pathogenic morphology and mitochondria dysregulation. The authors also established connectivity 
between DRGO sensory neurons and muscle fibers to recapitulate the proprioception sensory 
pathway in vitro. In the second part of the work, the authors report the excision of FXN intronic 
region by CRISPR/Cas9 in two different ways; 1- excision of the GAA tract only (PTL6-SD10) or 2- 
removal of the FXN intron-1 (PTL6-LD3). The authors then described the rescue of the previously 
identified phenotypes, which were only partial in the short deletion. An elegant approach used in 
this work is the addition of the microfluidic chamber device to evaluate mitochondrial health. The 
manuscript is well written and organized and the data is presented in a clear fashion. The overall 
story is intriguing and novel, and the subject is very relevant to the field. However, there are 
concerns that should be addressed before this manuscript can be accepted for publication. 

Major Issues: 

1. The authors used CRISPR/Cas9 targeted for deleting the GAA repeats in two different ways: one 
removing only the repeats (Short Deletion - SD), and a second removing FXN intron 1 (Long 
Deletion – LD). Even though the use of CRISPR/Cas9 is a pertinent approach, the restoration of 
the GAA repeat size to control levels in the “short deletion” iPSCs would be a more adequate 
control for this study. It should be used in the mitochondria, axon and cell death assays to 
compare the rescue of the phenotypes to the other 2 deletions. 

2. A main accomplishment of this work in the development of dorsal root ganglia organoids, 
however, the composition of the organoids was not completely described. Based on the results 
presented in Figure 2D, about 70% of total cells are neurons (BIII-Tub), which raises the question 
of what would the remaining 30% be. Single-cell PCR for not only neuronal, but also glial markers 
should help elucidate this issue. 

3. Finally, as described in this study “the removal of exclusively the expanded GAA tract was less 
effective in reverting the pathological hallmarks with respect to the larger deletion which ablated 
most of FXN intron-1”. The authors hypothesized that the inability to restore FXN expression in the 
removal of expended GAA tract cells was due to epigenetic mechanisms. There are 2 issues that 
should be addressed in the study of this hypothesis: 

a. The authors did not explore the possibility of the shorter deletion impacting the splicing of the 
intronic region. To exclude this possibility, a qRT-PCR should be performed to show that deletion of 
repeats alone does not disrupt the splicing of FXN. This should be carried out for all alleles 
(expanded, control, LD and SD). If negative, this should strengthen the author’s claim regarding 
the mechanism of FXN repression in the shorter deletion. 
b. It was not clear why the authors chose to investigate H3K9me3 specifically. Therefore, there is 
a need for an epigenetic screening as H3K9me3 may not be the only repressive factor involved in 
the FDRA pathogenesis. 

Minor Issues: 

1. Reference missing from sentence: “The length of the GAA expansion correlates proportionally 
with the severity of the disease and inversely with the age of onset which can occur from infancy 
to after 60 years of age.” 

2. The authors described electrophysiological measurements as well as synapsin and vGLUT 
analysis for characterization of the DRGOs, which raises the question: were there differences 
between affected and unaffected organoids? Especially, due to the axonal defect reported in figure 
6C and 6D. 

3. In the title: “FRDA deficits in Fe-S cluster protein biogenesis and antioxidant response are 
restored in PTL6-DL DRGOs”, please correct PTL6-DL to PTL6-LD 

4. In the legend of figure 5 E, please introduce the acronyms UC5’, UpSD, DwSD, UC3’. 

Reviewer #2 (Remarks to the Author): 

In their manuscript, Mazzara and colleagues report: 1) the generation of human induced 
pluripotent stem cells (iPSCs) from two patients with Friedreich’s ataxia (FRDA) and GAA repeat 
expansions of different lengths; 2) the generation of isogenic controls via the CRISPR-mediated 
excision of the GAA repeats or of most of FXN intron 1; the differentiation of these iPSCs into what 
they call dorsal root ganglia organoids (DRGOs), including the establishment of connections with 
co-cultured muscle spindles; excess cell death, loss of Fe-S proteins and abnormal antioxidant 
responses in FRDA-derived DRGOs. 
This is an interesting study with potential to provide relevant insight into FRDA pathogenesis. 
There are however, some concerns: 
1. No details are provided about the control iPSC line utilized. 
2. The morphological differences between DRGOs and the clusters formed in traditional 
differentiation protocols for SN should be better specified. In both cases cellular aggregates 
extending long neurites form. 
3. In FRDA, proprioceptive neurons are most vulnerable, showing developmental defects, while 
other SN neurons seem to be affected only later (e.g. Creigh P et al. Ann Clin Transl Neurol 2019; 
6:1718-1727), so it is important to understand the relative representation of different types of SN 
in the DRGOs. This is given only approximately. In particular, data suggest that a significant 
number of neurons are TrkB/TrkC double positives, i.e. their fate as proprioceptors or 
mechanoreceptors is not yet determined. This requires co-localization experiments of specific 
markers defining such subpopulation. IF images of better quality should be provided at different 
magnifications. Single-cell RNAseq may also be used. This is important also because most FXN in 
DRGs comes from proprioceptors (see Piguet F et al. Mol Ther. 2018; 26:1940-1952). 
4. The excess cell loss and abnormal morphology of FRDA DRGOs is an interesting observation, but 
it should be better specified. Is there a differentiation deficit? Does it specifically affect 
proprioceptors? If normally differentiated cells die prematurely, what are their identities, is there a 
specific vulnerability? 
5. Electrophysiology data confirm the neuronal phenotype of most analyzed cells, but again it is 
unclear what type of neurons they represent. Are there any corresponding, morphology data, i.e. 
after biocytin injection of clamped cells? 
6. In Figure 4 it is not specified if electrophysiology data are from FRDA, controls or isogenic 
control cells. These should be analyzed separately. 
7. The LD vs. SD observation is intriguing, but the only possible explanation is the persistence of 
inhibitory epigenetic marks in intron 1 after removal of GAA expansions. These may be histone 
PTMs or DNA methylation, which may last longer and has not been explored. There is some 
evidence that long repeats may be associated with more extensive DNA methylation in intron 1. 
The effect of continued culturing of iPSCs allowing more cell divisions should also be investigated. 
8. There are several imprecisions about the disease, including the statement that proprioceptive 
neurons and Purkinje cells are most vulnerable (page 6). This is incorrect, as the most vulnerable 
cells in the cerebellum are large projection neurons in the deep nuclei, while Purkinje cells are 

mostly preserved in number and morphology, except for their axonal endings in the deep nuclei 
undergoing grumose degeneration, possibly due to loss of target cells. 
9. The paper needs a revision for grammar and style, preferably by a native English speaker. 

Reviewer #3 (Remarks to the Author): 

Extremely interesting manuscript in which the authors developed an original and novel approach to 
generate organoid cultures of DRG sensory neurons from IPSC. This is the first example of a 
protocol for DRG organoids in the literature. Furthermore, the authors developed a very elegant 
experimental design to allow maturation of the DRGO in the presence of myogenic culture, and 
demonstrates the presence of muscle spindle. Overall, these novel experimental approaches could 
be important to develop human-derived models for sensory neuropathies. In the current 
manuscript, the approach is applied to IPSC cells derived from Friedreich’s Ataxia (FRDA) patients, 
a disease that primarily affects the proprioceptive neurons of the DRG. There is a current lack for 
good FRDA neuronal models, and the iPSC derived DRG organoids appears as a very powerful 
solution as it combines the complex genetic context of FRDA (large GAA repeat expansions) and 
the primarily affected cell type. 
Overall, this work represents an important advance in the field of stem cell research through the 
elaboration of a new DRG organoid differentiation protocol. There are however several weaknesses 
that temper the enthusiasm. 

Major comments: 
• Although not completely characterized, it is thought that there are multiple regulatory elements 
in intron 1 of the frataxin gene. Therefore, removing most of intron 1, as proposed in the 
manuscript, might have deleterious effect on the fine regulation of frataxin expression. In the 
current manuscript, the authors suggest that full frataxin recovery can only be achieved with the 
removal of a large portion of the intron 1 5figure 1F-G, text p6). However, other laboratories in 
several publications have demonstrated that removing the GAA expansion repeat restores the 
frataxin levels and prevents the heterochromatin epigenetic profile (Tremblay, Napierala, 
Gottesfeld laboratories). Furthermore, Lai et al. (2018) generated isogenic iPSC lines that different 
only in the length of the GAA repeat and demonstrated the restoration of frataxin expression. 
Could the authors elaborate on these differences and find a plausible explanation? The 
characterization of multiple clones for each deletion and each iPSC line would probably clarify this 
point. 
• The authors state that “PTL6 iPSCs showed the strongest frataxin reduction both at transcript 
and protein levels compared to PTS36”. However, the western blots and their quantification on 
Figure 1F-G are in disagreement with this statement. Indeed, frataxin levels appears to be down 
by approximately 60% in both lines, and if there is a difference, PTS36 appears to have less than 
PTL6. At the RNA level, there is indeed a clear difference. The authors should address this 
discrepancy. Again, analyzing several clones might help in clarifying. At the RNA level, PTS36 
appears to have 50% frataxin, levels equivalent to heterozygote carriers. At the protein level, both 
patients have approximately 40% frataxin, inconsistent with the 5-20% reported in the literature. 
Can the authors elaborate on this aspect? 
• The authors convincingly generated DRG organoids that are composed of all neuronal cell types. 
A technical question: what is the effect of the anti-mitotic treatment on glial cells, considering that 
these are extremely important for signaling and for myelination? 
The immunofluorescence images from 2B and 2C show very convincingly that the cultures are well 
organized and contains large sensory neurons that are positive for NF200. Considering that the 
RT-PCR is not quantitative, it is difficult to evaluate to which extent the culture is enriched for any 
of the 3 neuronal subtypes. The author should perform quantitative RT-PCR or Western Blot 
experiments to evaluate the enrichment. This statement holds for all the RT-PCR presented in the 
manuscript, quantitative would be better (Fig. 2, S2, S3). Furthermore, a better choice of specific 
antibodies or RT-PCR to determine the population of sensory neurons present in their culture 
would strengthen the manuscript. Indeed, the authors chose Runx3 and TrkC (NTRK3). Runx3 is 

specific of proprioceptive neurons during development and its expression stops when the neurons 
are mature (Marmigère and Ernfors, 2007). TrkC is expressed both by mechanoreceptive and 
proprioceptive neurons, as reported in Usoskin et al., 2015. Usoskin et al. present new 
proprioceptive specific genes such as CNTNAP2 and SPP1 that should be used. 
• While there is no doubt that the DRGO contain all subtypes of DRG sensory neurons, the authors 
should select images with a better staining pattern for the different markers (Figure 2G). Based on 
the images, it is difficult to understand how the authors manage to quantify properly the subtypes, 
considering the large amount of overlap in the staining. Furthermore, it is difficult to understand 
how the total percentage can be above 100% (Figure 2H – 114%), except if several neurons 
express two different receptors (as expected for mechanoreceptive neurons). The percentages 
given in the text (p. 7-8) do not correspond to the percentage shown on the graph Figure 2H. 
Indeed, the text states that the highest percentage is 44.5% corresponding to NTRK2, while Figure 
2H shows that NTRK3 has the highest percentage. Is there an inversion? 44.5% of neurons are 
positive for TrkC which is possible since it stains both proprioceptive and mechanoreceptive 
neurons. It is however incorrect to state that 44.5% are proprioceptive. The percentage of 
proprioceptive would be the TrkC positive part (44.5%) minus the TrkB positive part (exclusively 
mechanoreceptive) (35.8%), which would correspond to around 8-9%. Parvalbumin is specific for 
proprioceptive neurons in the DRG, and Figure 3E-F shows around 15% of Parvalbumin positive 
neurons, a percentage that is closer to the physiological number. Overall, the transcriptional profile 
demonstrates nicely that the DRGO present an expression profile similar to DRG albeit probably 
closest to developmental stages and immature. 
• The results obtained in figure 4 are clearly a highlight of the paper, showing the capacity to 
generate mature DRG organoids capable of muscle spindles forming and that are electrically 
active. These extremely impressive and innovative results could be strengthen by complementary 
experiments. Indeed, it would be interesting to identify the type of neurons that forms the muscle 
spindle, since in the physiological context, both motor and sensory neurons are found. This could 
be done using a Parvalbumin antibody to demonstrate the presence of proprioceptive neurons. 
With the current protocol, are muscle spindle sparse or are they found in a reasonable number? 
Although probably beyond the current manuscript, have the authors tried to myelinate the neurons 
to achieve better maturation and functionality? 
• Figures 5A to 5D show conflicting results with the mRNA and protein expression quantifications 
inversely correlated. The long repeat line (PTL6) shows lower protein level than the short repeat 
line (PTS36), as expected (although opposite to the result on the IPSC lines). Unexpectedly, the 
mRNA quantifications show the opposite. The authors should explain these conflicting results. The 
protein levels in the PTL6 line are this time in agreement with the levels described in the literature 
for FRDA patients. The effect of the SD or LD excision on the expression levels of frataxin appears 
not to be the same as for the iPSC lines, in particular for PTS36-SD23. Furthermore, high 
variability is seen – could this be a reflection of variation in the differentiation protocol with 
variable percentage of neurons from one organoid to another? The authors should discuss this 
issue in the manuscript. 
• In Figure 5F-G, the H3K9me3 pattern of PTL6 is different between the two figures. For example, 
in 5G, at the UC5’ site, the level of the mark is higher than any other site, although it is among the 
lowest in 5F. Could the authors comment these results? 
• It appears that the authors accidently present the exact same graphs in 6B as this is not in 
agreement with the rest (i.e. should the first graph be PTS36 and derivatives?). It would be worth 
for the authors to check. Overall Figure 6 shows a nice rescue of the phenotypes after the GAA 
repeats excision. However, there is a lack in tissue specificity, as the result show that every cell 
type is affected by FXN depletion, although proprioceptive neurons should be more sensitive than 
the rest (as explained earlier by the authors). Could the authors comment on this? Clearly the 
phenotype observed in the dish appears to agree with a developmental component of the disease 
as proposed more recently in the field. However, the authors also see an increase in death in FRDA 
DRGO compared to controls. In the text p 12, it is not always clear to which control the authors 
are referring to. This section should be re-written. 
• The authors explore the phenotype of the FRDA DRGO, with a particular focus on mitochondria, 
as it is known that mitochondria are particularly susceptible to FXN depletion. The authors have 

well adapted a powerful technique long known to arrange neuronal axons in microgrooves, 
allowing to follow organelles movement and follow the direction of the movement, using 
microfluidic chips. The novelty here is that the authors have designed additional chambers 
compared to classic neuron adapted microfluidic chips, that permits the cultures of the DRGO. With 
this system, the authors were able to measure several parameters of mitochondrial morphology 
and dynamics. The authors demonstrated a decreased mitochondrial number (confirmed by 
mtDNA/gDNA quantifications in supplementary data), and overall decreased mitochondrial size. 
The graph and statistics for the circularity should be checked because there does not appear to be 
any difference between the 3 conditions. 
• In the last part of the manuscript, the authors investigated FRDA-related phenotypes such as 
iron-sulfur clusters (ISC) deficiency and oxidative stress. This is by far the weaker part of the 
manuscript and would benefit from being reinforced by a few complementary experiments. It is 
important to demonstrate whether there is an ISC cluster deficiency has this is the primary role of 
frataxin. The authors demonstrate a very strong decreased level of aconitase (around 90% in the 
PTL6 mutant), which seems unlikely as this protein is known to be the less sensitive to 
degradation when deprived of its ISC. There are numerous other proteins that can be tested by 
western blot to demonstrate indirectly a ISC deficiency, such as Complex I and II of the 
respiratory chain or Lipoic Acid bound to proteins. The activity of the enzyme (such as SDH or 
aconitase which are relatively standard measurements) would constitutes a more direct way to 
evaluate ISC loss. Increase in SOD1 and SOD2 mRNA are interesting results, but are not enough 
to conclude that there is the presence of an increase oxidative stress in the model. Staining with 
fluorescent probes specific to oxidative stress such as DHR123 or MitoSOX should be perform. 
Furthermore, using such probe could allow to investigate the phenotype specifically in the different 
subtypes of neurones. The tissue specificity is an important question in the FRDA field. 
Furthermore, the authors could also use a fluorescent probe that allows to investigate 
mitochondrial health status and activity such as TMRM, TMRE or JC-1. Additional information 
regarding iron metabolism would be much appreciated as it is part of the classical hallmarks of 
FRDA. 

Minor comments: 
1. The bibliography in the introduction is very sparse and an effort should be made to cite primary 
bibliography. 
2. The introduction is not completely updated with the latest development in the FRDA field that 
are important in the context of the current manuscript 
3. Cerebellar Purkinje cell are not extremely affected in FRDA, it is rather the dentate nucleus of 
the cerebellum that is affected. This should be corrected (p. 6) 
4. A throughout reading of the manuscript should be done to remove the multitude of typos or 
small annotation mistakes that can be found (some examples: legend figure 1B, “FRDA-DS and 
FRDA-DL” instead of FRDA-SD and FRDA-LD; legend figure 1, “organoids” instead of IPSC; Figure 
S1E, the oligos used are probably LF1+LR+SR and not LF2 as noted; ) 
5. Figure 3B – it is extremely difficult to discriminate between the different blues and green, and 
therefore it is difficult to discriminate between DRGO and IPSC. The colors should be changes 
6. The manuscript presents data on 6 different IPCS lines. For the graphs, it would be important to 
have a code which could differentiate between the different lines. This would considerably help. For 
example, PTL6 is grey in 5F and then red in 5G. Maybe using hatched bars for one of the lines 
would help, and color code could then stay. 

March 30th, 2020