Paired box 6 gene delivery preserves beta cells and improves islet transplantation efficacy

Abstract Loss of pancreatic beta cells is the central feature of all forms of diabetes. Current therapies fail to halt the declined beta cell mass. Thus, strategies to preserve beta cells are imperatively needed. In this study, we identified paired box 6 (PAX6) as a critical regulator of beta cell survival. Under diabetic conditions, the human beta cell line EndoC‐βH1, db/db mouse and human islets displayed dampened insulin and incretin signalings and reduced beta cell survival, which were alleviated by PAX6 overexpression. Adeno‐associated virus (AAV)‐mediated PAX6 overexpression in beta cells of streptozotocin‐induced diabetic mice and db/db mice led to a sustained maintenance of glucose homeostasis. AAV‐PAX6 transduction in human islets reduced islet graft loss and improved glycemic control after transplantation into immunodeficient diabetic mice. Our study highlights a previously unappreciated role for PAX6 in beta cell survival and raises the possibility that ex vivo PAX6 gene transfer into islets prior to transplantation might enhance islet graft function and transplantation outcome.

focusing more on T1D and T2D disease pathogenesis, rather than the background that led to the study of Pax6 vs. other transcription factors (e.g.Pax4).Moreover, the discussion does not include other potential therapeutic methods under development such as stem cell derived islet organoids, CRISPR corrected, or nanoparticle delivery systems.The authors do not discuss how Pax6, itself, enables improved beta cell function (as opposed to increased beta cell replication-which itself may not improve function).
Additional Suggestions: Figure 1: • The data from in vivo overexpression would be strengthened if the authors show similar effects of overexpression of Pax6 in EndoC cells • Explain rationale for why incretin hormones are used o Is proliferation being used as a measure of beta cell vitality?Why?A bit confusing given apparent lack of beta cell proliferation in adults o Insulin secretion data would strengthen the studies Figure 3: • T1D modeling via STZ usually requires multiple low doses; as presented the authors are using high dose to ablate β cells.This is not really a T1D model (more a model of insulin deficiency), and to present it as such is questionable.A NOD mouse model with Pax6 overexpression would strengthen this claim.
• The increase in beta cell proliferation and reduction in death should result in an increase in beta cell mass, but these data are not presented.
• Panel I quantifies Ins+ Ki67+ cells to measure beta cell proliferation; should panel J quantify cells that are both TUNEL+ and Ins+ to measure beta cell apoptosis?Figure 4: • The striking differences observed in the overexpression (A-H) does not appear to be reflected by the small differences observed in proliferation and death (I-J); again, presentation of beta cell mass would be helpful.Figure 5: • Glucose stimulated insulin secretion (GSIS) to show effect of overexpression would strengthen the study.The manuscript by So et al describes how Pax6 is important for beta-cell biology proliferation and survival.They present data that show that Pax6 overexpression may help treat diabetes both in mice models mimicking type 1 or type 2 diabetes.Moreover, they present data showing that Pax6 overexpression in human islets improve their function when transplanted in diabetic mice.The results are interesting and suggest that Pax6 overexpression may be part of new therapeutic strategies, at least in islet graft.Yet, they are several concerns about controls and experiments that are not presented.Thus, serious modifications of the present manuscript are required to improve the quality of the manuscript.
A main issue on the use of the AAV to overexpress Pax6 is to show that it actually targets beta cells and no other tissues.Controls of Pax6 expression in different tissues need to be shown.In addition, Pax6 overexpression in control normoglycemic mice, not just in diabetic mic, needs to be shown.In control mice, does Pax6 overexpression lead to a better glucose homeostasis?
Another main issue is the quantification of rare events.Proliferation and apoptosis rates were measured in alpha and beta cells in vivo, in STZ mice and Db mice.These events are rare, thus the positive cells (for example ki67+glucagon or TUNEL+insulin) are scarce.To obtain a precise measurement, it is required to count a minimum of these scarce events.For example, a bias can be easily made if a too low number of events is counted: 1 cells out of 1000 would suggest a rate of 0.1% while 2 cells out of 1000 would suggest a rate of 2% and a doubling of the rate with just one extra positive cell.The authors do not mention how many events where counted in each measurement and that is absolutely required.
On figure 1, it would be very logical to show Pax6 KD before showing the effects on the cells.It is a required control that have to be shown first to prove the efficiency of the KD.
Again, figure 1 needs to be re-organized: quantifications of WB need to be called as panels.Analysis of pAkt needs to display whether basal pAkt levels (not just insulin-stimulated) are different when Pax6 is KD.
Figure S1 may include the expression levels of more transcription factors, including Pax4, Tcfl2, hnf6 and hnf4alpha isl1 and neurod The experience with HGPA needs to be more documented: what are the glucose levels normally required to grow the cells?What are the glucose levels for HGPA?Glucose-stimulated insulin secretion should be measured on endobetaH1 cells in the different conditions.There is a need fora statistical analysis in between ctrl and HGPA cells.It is absolutely mandatory to describe properly the effect of HGPA.During HGPA, culture conditions may force cells to secrete more insulin thus the decreased ins content may reflect improved secretion.Therefore, insulin content must be related to GSIS.
Similarly to figure 1, the panels of figure 2 need to be re-organized.The control of Pax6 overexpression need to be shown first before the panels on proliferation and apoptosis.Also, the condition ctrl+pax6 overexpression is interesting and needs to be shown on figures.Please comment on how calculations for ratios of pAkt were done.Is there a normalization to total akt and to GAPDH and to control condition ?Please explain.It seems that pa6 overexpression reverts the effects of HGPA.Is it also true with Pax4 or pdx1 overexpression ?The question about the specificity of action of pax6 is central.STZ mice are not a T1D like phenotype.STZ mice are a model of diabetes to a lack of insulin (after chemical destruction of beta cells) but without auto-immune attack.Please correct.
The expression of pax 6 was measured on whole pancreas.Since stz kills beta cells, the number of beta cells is decreased in the stz+aav-ctrl group.thusnumber of pax6 expressing cells decreases, hence loss of pax6.where is the ctrl + pax6 aav group ?
In STZ mice and in Db/db mice, AAVPax6 treatment may modify beta-cell mass.Assessment of beta-cell mass needs to be performed in these mice.
A more precise description of the STZ model is needed.What is the dose of STZ ?Is it a one shot of STZ or many shots ?Is there a total or a partial destruction of beta cells ?
In STZ mice, pax6 is overexpressed in beta cells and stimulates proliferation but the stimulation of proliferation is not impressive but seems to be sufficient to restore beta cells and produce insulin.Please comment on this result.
TUNEL levels are very high in all groups while apoptosis in beta cells is supposed to be a very low level.Also, pictures of IF for TUNEL suggest that apoptotic cells are not beta cells but other cells.How was the co staining for TUNEL and insulin quantified ?
Was alpha cell mass measured in groups of mice with modified blood glucagon levels ?MafA IF should be nuclear but the picture show a weird staining in some cells.Please improve IF.
What is the phenotype of human normal islets + AAVPax6 ?Again, this group is missing and could provide interesting informations.
Line 262, the authors claim that "transplantation of T2D islets transduced with AAV-Ctrl (STZ/T2D-Ctrl) modestly normalized the blood glucose level".If glycose returns to a normal value, then it is called normalized, otherwise, it is decreased comparerd to control group.Modestly normalized is a non sense.
Insulin contents are compared in between STZ/Ctrl-L and STZ/L+Pax6 but it should also be compared to STZ/Ctrl-H.Referee #3 (Remarks for Author): The manuscript entitled "Pax6 gene delivery preserves beta cells and improves islets transplantation efficacy" by Wing Yan So, et al., describes the role of modulating Pax6 expression in pancreatic islets to preserve/restore beta cell function in different models of T2D, including preclinical (Db/Db, STZ treated mice) and human control and T2D islets, engrafted in STZ mice.This is an important study unravelling that modulation of Pax6 expression (i.e.increasing its levels) improves beta cell function.This is a well controlled and designed study, using pre-clinical relevant models, where functional tests are associated to molecular characterization of Pax6 over expression.The in vivo preclinical studies are clearly demonstrating that maintaining Pax6 levels may contribute to improve beta cell function/dedifferentiation during T2D.
Dear Dr. Lise Roth, Thanks for the message and the decision along with the comments from the reviewers.We have studied these comments and would like to discuss with you on the possibility of a reconsideraLon of the decision.
First, regarding the comments from the reviewers.We greatly appreciate the Lme and insights from all the reviewers, and would like to summarise the main points and our response/plans below: The main criLcism of Reviewer #1 is on the novelty of the study due to the extensive literature on PAX6 and its role in beta cell formaLon and funcLon.
Those extensive studies on PAX6 are mainly in mouse beta cells using mutant mice (PMID 15110714, 9224716, 26658466, 27941241, 23326594, 28377501).These studies show that PAX6 is physiologically essenLal for normal mouse islet development and funcLon using a lossof-funcLon approach.It is well documented that species differences do exist between human and rodent islets in terms of metabolism, cytoarchitecture and funcLonal implicaLons (PMID 21454710, 28422162, 16461897), which may influence the translatability of mouse studies to the human context.On top of using mouse models, we use human islets in our study to demonstrate the human relevance.Moreover, it is generally recognized that the capability of adult human beta cell replicaLon or regeneraLon is very limited when compared to rodent beta cells (PMID 22751699), and there is currently no clinical therapy proven to sLmulate beta cell proliferaLon or preserve beta cell mass once beta cell failure has ensued (PMID 27189025).Hence, strategies that can preserve beta cells, even in a modest manner, are valuable.With the use of cell line, mouse models and human islets, our results highlight previously unappreciated roles for PAX6 in maintaining human islet survival and the contribuLon to diabetes management.Although essenLal, understanding the physiological role of the criLcal beta cell regulators is just the first step.The ulLmate goal is to develop therapeuLc strategies by targeLng those regulators, which is not addressed by any of the aforemenLoned studies.In our study, we apply various in vitro and in vivo models to demonstrate the therapeuLc effects of AAV-mediated PAX6 gene delivery on beta cell preservaLon and diabetes management.In parLcular, we show that PAX6 gene delivery enhances both normal and T2D human islet potency, and thus might overcome two of the major unresolved barriers of clinical islet transplantaLon: post-transplant islet grad loss and scarcity of islet donors.By providing scienLfic basis to support targeLng PAX6 to overcome beta cell failure, our study hence shows clinical relevance which has not been demonstrated before.
For Reviewer #2, the first main issue is to include a group of AAV-PAX6 overexpression in normal mice and on the other hand show that the expression does not occur in other Lssues.We did not include the Ctrl + AAV-PAX6 group in our first drad as demonstraLng the therapeuLc potenLal of AAV-PAX6 in diseased mice would be more meaningful and in real pracLce, none of the medicaLons will be prescribed to normal subjects.We can provide those data in the revised version as requested.The second main issue is the quanLficaLon of Ki67 and TUNEL signals.For STZ mouse pancreas secLons, we quanLfy 8-10 pancreas secLons (each contains at least 3 islets) for each mouse and 6 mice for each group.For db/db mouse and human islet secLons, we quanLfy 4-5 secLons (each secLon contains at least 10 islets) for each batch of islets and 4-6 batches of islets for each group.For islet grad-bearing kidneys, we quanLfy 4-5 secLons for each mouse and 4-6 mice for each group.The number of mice/ batches of islets is indicated in the figure legend.The details can be added in the method secLon in the revised version.
For the addiLonal experiments suggested, we believe that they can be performed within a reasonable period of Lme.
We hope that these responses provide addiLonal evidence that our study indeed offers significant insights, and the requested experiments could be completed within a reasonable Lme period.Could we request that you reconsider your decision and allow a revised version to be submijed for consideraLon of publicaLon?
Best wishes, Weiping Dear Prof. Han, Thank you for your email and request to reconsider our decision on your manuscript.In order to make a formal appeal, I would suggest that you provide a detailed provisional point-by-point rebuttal letter in response to all referees' concerns.This would allow us to contact the original referees or an advisor and to make an informed decision.
Let me know if this is agreeable to you.

With kind regards, Lise Roth
26th May 2023 Editor's correspondence Dear Dr. Roth, As promised, please find below our point-to-point response to the reviewers' comments.

Referee #1 (Comments on Novelty/Model System for Author):
The novelty of the studies is questionable.There is extensive literature on Pax6 and its role in beta cell formation and function, so I'm not sure how much this study adds.Delivery of a transcription factor as a therapeutic approach has been mentioned in the literature for years, but few approaches if any have materialized.Again, the novelty there is also marginal.
A: The referenced studies on PAX6 are mainly in mouse beta cells using mutant mice (PMID 15110714, 9224716, 26658466, 27941241, 23326594, 28377501).These studies show that PAX6 is physiologically essential for normal mouse islet development and function using a loss-of-function approach.It is well documented that species differences exist between human and rodent islets in terms of metabolism, cytoarchitecture and functional implications (PMID 21454710, 28422162, 16461897), which may influence the translatability of mouse studies to the human context.In contrast, we use human islets in our study to demonstrate the human relevance besides the experiments with mice.Moreover, it is generally recognized that the capability of adult human beta cell replication or regeneration is very limited when compared to rodent beta cells (PMID 22751699), and there is currently no clinical therapy proven to stimulate beta cell proliferation or preserve beta cell mass once beta cell failure has ensued (PMID 27189025).Hence, strategies that can preserve beta cells, even in a modest manner, are valuable.With the use of cell line, mouse models and human islets, our results highlight previously unappreciated roles for PAX6 in maintaining human islet survival and the contribution to diabetes management.While it is important to understand the physiological role of the critical beta cell regulators, the ultimate goal is to develop therapeutic strategies by targeting those regulators, which is not addressed by any of the aforementioned studies.In our study, we apply various in vitro and in vivo models to demonstrate the therapeutic effects of AAV-mediated PAX6 gene delivery on beta cell preservation and diabetes management.In particular, we show that PAX6 gene delivery enhances both normal and T2D human islet potency, and thus might overcome two of the major unresolved barriers of clinical islet transplantation: post-transplant islet graft loss and scarcity of islet donors.By providing scientific basis to support targeting PAX6 to overcome beta cell failure, our study hence shows clinical relevance which has not been demonstrated before.

Referee #1 (Remarks for Author):
Summary: In this study, the authors argue that Pax6 is a critical regulator of beta cell proliferation and cell death based on data using lentivirus and insulin promoted AAV to either knockdown or overexpress Pax6.Through numerous in vitro and in vivo model systems focusing on islets, the authors show that Pax6 ameliorates T1D (via single, high dose STZ although not adequately explained in methods) and T2D (via db/db or high glucose media, HGPA) disease systems.Moreover, they transplant T2D human islets treated with AAV-Pax6 8th Jun 2023 Appeal into STZ treated NSG mice to show functional glycemic control via increased insulin secretion.Bulk transcriptional analysis on control islets (unclear whether KO or WT Pax6 islets) versus AAV-Pax6 overexpressed islets show an increase in genes related to beta cell identity and pancreatic secretion.Islet transplantations in STZ treated NSG mice using islets of lower mass that have been exposed with AAV-Pax6 show similar improvement to the higher mass islets.Overall, the multiple experiments highlight that overexpression of Pax6 results in improved islet function.
Impact: Marginal, owing to the myriad other papers documenting the well-known role of Pax6.Moreover, this study does not account for AAV or lentiviral transfection efficiency issues that have been seen with other pancreatic genes.There may be marginal increase in knowledge depicting Pax6 as beneficial for low mass islets.
A: Our results have shown that the lentivirus and AAV approaches efficiently deliver PAX6 into cell line (fig.2D), mouse beta cells/ islets (fig.S2A, S2B, 4K) and human islets (fig.S5E) as shown by the significant increases in PAX6 protein levels.
Strengths: This paper has multiple in vitro and in vivo methods of testing knockout and overexpression capabilities for AAV mediated constructs.The authors effectively utilize these repetitive techniques throughout the paper to support their hypothesis.The paper is well written with easy flow and the figures as well as figure legends are easy to follow.For the most part, they cite papers that have addressed Pax6 within the field to a reasonable extent.
Criticisms: Although this paper has copious data, the implications are not entirely novel.The introduction is fairly generic, focusing more on T1D and T2D disease pathogenesis, rather than the background that led to the study of Pax6 vs. other transcription factors (e.g.Pax4).Moreover, the discussion does not include other potential therapeutic methods under development such as stem cell derived islet organoids, CRISPR corrected, or nanoparticle delivery systems.The authors do not discuss how Pax6, itself, enables improved beta cell function (as opposed to increased beta cell replication-which itself may not improve function).
A: PAX4 is a key regulator for beta cell development and maturation during embryogenesis.Although still detectable, PAX4 expression is low in adult beta cells (PMID 18234907).Acute overexpression of PAX4 has been found to protect beta cells against stress-induced apoptosis and stimulates proliferation.However, sustained PAX4 expression results in beta cell dedifferentiation (repression of MAFA and insulin), blunted glucose-stimulated insulin secretion and glucose intolerance (PMID 21521872).Besides, transcript levels of PAX4 are increased in human T2D islets (PMID 17989064), which also suggests that PAX4 upregulation triggers beta cell dedifferentiation in T2D and explains why PAX4 expression is normally suppressed in adult beta cells.These observations make PAX4 not a suitable therapeutic target.
PAX6 is found to be down-regulated in human T2D islets (PMID 22768844, 34193609) while PAX6 gene mutations are associated with diminished insulin secretion and glycemic perturbations in human (PMID 30151985, 15842522, 11756345).These observations suggest a strong correlation between PAX6 activity/ levels and human diabetes.Mechanistic studies are mainly done on mice, those studies suggest PAX6 critically regulates mouse beta cell function but its roles in human beta cells remain ambiguous.These justify the study of PAX6 in human islets.
Induced pluripotent stem cells (iPSC) are not yet suitable for large-scale production of patient-specific iPSC-beta cells for transplantation since individuals require several hundred thousand individual pancreatic islets.In addition, unlike whole pancreas or islet transplantation, iPSC-derived beta cells do not replace the lost function of alpha cells.Deficiency in glucagon production precipitates hypoglycemia unawareness.Until these challenges are addressed, transplantation of islets from cadaveric donors remains the preferred approach worldwide.Hence, strategies that enhance islet graft survival and reduce the islet mass for transplantation show clinical relevance and are valuable.
Both CRISPR and nanoparticle are other tools for gene delivery.CRISPR system is usually applied in stem-cell derived beta cells in which gene editing is first performed in stem cells followed by the differentiation into beta cells.There is one study demonstrating the use of CRISPR in human islet (PMID 33893274) and there is less study using nanoparticle to deliver genes into islets.Further studies and investigation are warranted to demonstrate their efficiency and safety in human islets.In contrast, AAV is a well-established gene delivery tool adopted in both mouse and human islets (PMID 29304344, 15858610, 31355778, 25676679, 25338552, 16567506, 18560422, 30400037), while multiple clinical studies have provided promising evidence of AAV-mediated long-term expression of therapeutic proteins without causing substantial adverse effects (PMID 23474247, 25409372, 25938638).Since our study did not aim to develop new gene delivery tool, but to demonstrate the therapeutic effects of PAX6 in diabetes management and islet transplant outcome, hence we chose AAV, the more well-established system as the gene delivery approach.These points can be discussed in the revised version.

Additional Suggestions:
Figure 1: • The data from in vivo overexpression would be strengthened if the authors show similar effects of overexpression of Pax6 in EndoC cells A: Overexpression of PAX6 in EndoC cells and the measurement of cell survival and function will be included in the revised version.

• Explain rationale for why incretin hormones are used
A: It is because previous studies have shown that PAX6 controls the GLP-1R and GIPR expression levels in rodent beta cells (PMID 22403172, 28377501, 27941241) and incretin signaling is known to critically regulate beta cell function and survival (PMID 16517403).These points will be emphasized in the revised version.
o Is proliferation being used as a measure of beta cell vitality?Why?A bit confusing given apparent lack of beta cell proliferation in adults A: We measure beta cell proliferation, cell death and insulin content, which are the indicators of beta cell survival and mass.The capability of cell proliferation in human beta cell is very limited.However, previous studies have shown that this can be induced (PMID 32051230, 30059217, 30581122).
o Insulin secretion data would strengthen the studies A: Glucose-stimulated insulin secretion will be included in the revised version.• T1D modeling via STZ usually requires multiple low doses; as presented the authors are using high dose to ablate β cells.This is not really a T1D model (more a model of insulin deficiency), and to present it as such is questionable.A NOD mouse model with Pax6 overexpression would strengthen this claim.
A: Both multiple low dose and single high dose of STZ are widely used in different mouse strains in different studies (PMID 21478271, 16755002) in order to ablate beta cells.
It is true that the STZ mouse model is not a T1D model.T1D is caused by the autoimmune destruction of beta cells while the STZ model is just to mimic the severe beta cell loss seen in T1D.Since we are not investigating the immunomodulation, we focus on the direct effect of PAX6 gene delivery on beta cell regeneration, the STZ model serves our purpose and hence is adopted in this study.
• The increase in beta cell proliferation and reduction in death should result in an increase in beta cell mass, but these data are not presented.
A: Measurement of beta cell mass will be included in the revised version.

• Panel I quantifies Ins+ Ki67+ cells to measure beta cell proliferation; should panel J quantify cells that are both TUNEL+ and Ins+ to measure beta cell apoptosis?
A: Figure 3J will be modified accordingly in the revised version.

Figure 4:
• The striking differences observed in the overexpression (A-H) does not appear to be reflected by the small differences observed in proliferation and death (I-J); again, presentation of beta cell mass would be helpful.
A: Measurement of beta cell mass will be included in the revised version.

Figure 5: • Glucose stimulated insulin secretion (GSIS) to show effect of overexpression would strengthen the study.
A: Results of GSIS on human islets will be included in the revised version.A: The control group is the AAV-Ctrl.The figure label will be modified in the revised version.

• Panel A-D: p-value adjustment for multiple hypothesis testing?/FDR?
A: This is Benjamini-Hochberg adjusted p-value.

• Panel G: are the listed pathways significantly enriched/overrepresented? Suggest showing GO analysis, statistics
A: Yes, the listed pathways are the top KEGG pathways enriched by the 843 CHIP-OE overlapping genes.

• Any cell cycle genes that are altered?
A: Yes, there are some genes involved in cell cycle altered which will be included in the revised version.

• Please show PCA plot to show variance between samples
A: PCA plot will be included in the revised version.

• Discuss why only 20% of genes are similar between CHIP and OE; what are the other genes doing in relation to beta cells
A: Genes bound by PAX6 with their expression unaltered by PAX6 overexpression suggest that PAX6 binds to these genes' promoters but do not regulate their expression.It is recognized that chromatin immunoprecipitation (ChIP) reveals the binding of transcription factors to thousands of genomic locations in the vicinity of both active and inactive regions.A number of weakly bound sites or the sites bound at low occupancy detected this way actually fail to drive gene transcription (PMID 24888900, 18271625, 23236164).
On the other hand, genes altered by PAX6 overexpression but not bound by PAX6 suggest that PAX6 indirectly regulates the expression of these genes without direct binding.This can be explained by the regulation of the expression/ activity of other transcription factors or master regulators such as MAFA, PDX1, NKX6.1, CREB and Akt by PAX6.These transcription factors/ regulators in turn regulate the expression of a bundle of genes.
These explain why only a portion of genes is shared by the ChIP and OE results.

Referee #2 (Remarks for Author):
The manuscript by So et al describes how Pax6 is important for beta-cell biology proliferation and survival.They present data that show that Pax6 overexpression may help treat diabetes both in mice models mimicking type 1 or type 2 diabetes.Moreover, they present data showing that Pax6 overexpression in human islets improve their function when transplanted in diabetic mice.The results are interesting and suggest that Pax6 overexpression may be part of new therapeutic strategies, at least in islet graft.Yet, they are several concerns about controls and experiments that are not presented.Thus, serious modifications of the present manuscript are required to improve the quality of the manuscript.

A main issue on the use of the AAV to overexpress Pax6 is to show that it actually targets beta cells and no other tissues. Controls of Pax6 expression in different tissues need to be shown. In addition, Pax6 overexpression in control normoglycemic mice, not just in diabetic mic, needs to be shown. In control mice, does Pax6 overexpression lead to a better glucose homeostasis?
A: AAV transduction is observed in beta cells (insulin positive cells) as shown in figure S2B.AAV transduction is minimally detected in other tissues (liver, kidney, muscle, spleen).The results will be included in the revised version.
We did not include the Ctrl + AAV-PAX6 group in our first draft as demonstrating the therapeutic potential of AAV-PAX6 in diseased mice would be more meaningful and in real practice, none of the medications will be prescribed to normal subjects.We can provide the data on normal mice in the revised version.
Another main issue is the quantification of rare events.Proliferation and apoptosis rates were measured in alpha and beta cells in vivo, in STZ mice and Db mice.These events are rare, thus the positive cells (for example ki67+glucagon or TUNEL+insulin) are scarce.To obtain a precise measurement, it is required to count a minimum of these scarce events.For example, a bias can be easily made if a too low number of events is counted: 1 cells out of 1000 would suggest a rate of 0.1% while 2 cells out of 1000 would suggest a rate of 2% and a doubling of the rate with just one extra positive cell.The authors do not mention how many events where counted in each measurement and that is absolutely required.
A: For STZ mouse pancreas sections, we quantify more than 10 pancreas sections (around 50 islets) for each mouse, 6 mice were included for each group.For db/db mouse and human islet sections, we quantify around 5 sections (over 50 islets) for each batch of islets and 4-6 batches of islets were included for each group.For islet graft-bearing kidneys, around 5 sections were quantified for each mouse and 4-6 mice were included for each group.The number of mice/ batches of islets is indicated in the figure legend.The details will be added in the method section in the revised version.

On figure 1, it would be very logical to show Pax6 KD before showing the effects on the cells. It is a required control that have to be shown first to prove the efficiency of the KD.
A: The graphs will be re-organized in the revised version.

Again, figure 1 needs to be re-organized: quantifications of WB need to be called as panels. Analysis of pAkt needs to display whether basal pAkt levels (not just insulin-stimulated) are different when Pax6 is KD.
A: Comparison of basal pAkt between Ctrl and PAX6 KD groups will be displayed in the revised version.

Figure S1 may include the expression levels of more transcription factors, including Pax4, Tcfl2, hnf6 and hnf4alpha isl1 and neurod
A: The measurement will be included in the revised version.

The experience with HGPA needs to be more documented: what are the glucose levels normally required to grow the cells? What are the glucose levels for HGPA? Glucosestimulated insulin secretion should be measured on endobetaH1 cells in the different conditions.
There is a need fora statistical analysis in between ctrl and HGPA cells.It is absolutely mandatory to describe properly the effect of HGPA.During HGPA, culture conditions may force cells to secrete more insulin thus the decreased ins content may reflect improved secretion.Therefore, insulin content must be related to GSIS.
A: Cells are maintained under normal condition (5.6 mmol/l glucose) or treated with HGPA (25 mmol/l D-glucose and 0.3 mmol/l palmitic acid) which has been stated in the "cell culture" section in materials and methods.Insulin content is significantly reduced under HGPA condition compared to normal condition as shown in figure 2C.Data on GSIS will be included in the revised version.
Similarly to figure 1, the panels of figure 2 need to be re-organized.The control of Pax6 overexpression need to be shown first before the panels on proliferation and apoptosis.Also, the condition ctrl+pax6 overexpression is interesting and needs to be shown on figures.
A: Overexpression of PAX6 in EndoC cells under normal condition and the measurement of cell survival and function will be included in the revised version.
Please comment on how calculations for ratios of pAkt were done.Is there a normalization to total akt and to GAPDH and to control condition ?Please explain.
A: As indicated in the quantification graph (figure 1E, right panel), pAkt is first normalized to total Akt.Then fold change is calculated by comparing all groups to the Ctrl+ vehicle group.

It seems that pa6 overexpression reverts the effects of HGPA. Is it also true with Pax4 or pdx1 overexpression ? The question about the specificity of action of pax6 is central.
A: PAX4 is a key regulator for beta cell development and maturation during embryogenesis.Although still detectable, PAX4 expression is low in adult beta cells (PMID 18234907).Acute overexpression of PAX4 has been found to protect beta cells against stress-induced apoptosis and stimulates proliferation.However, sustained PAX4 expression results in beta cell dedifferentiation (repression of MAFA and insulin), blunted glucose-stimulated insulin secretion and glucose intolerance (PMID 21521872).Besides, transcript levels of PAX4 are increased in human T2D islets (PMID 17989064), which also suggests that PAX4 upregulation triggers beta cell dedifferentiation in T2D and explains why PAX4 expression is normally suppressed in adult beta cells.These observations make PAX4 not a suitable therapeutic target.
It has been demonstrated that PAX6 regulates PDX1 expression in mouse beta cells (PMID 27941241) and our results show that PAX6 overexpression would enhance PDX1 expression in diabetic mouse and human islets (figure S4 and S5).In addition to PDX1, PAX6 also regulates the expression/ activity of other key regulators including MAFA and NKX6.1, suggesting PAX6 as a more comprehensive target.
A strong correlation between PAX6 activity/ levels and human diabetes is seen.While mechanistic studies are mainly done on mice, those studies suggest PAX6 critically regulates mouse beta cell function but its roles in human beta cells remain ambiguous.These also justify the study of PAX6 in human islets.

STZ mice are not a T1D like phenotype. STZ mice are a model of diabetes to a lack of insulin (after chemical destruction of beta cells) but without auto-immune attack. Please correct.
A: The sentence will be corrected accordingly.

The expression of pax 6 was measured on whole pancreas. Since stz kills beta cells, the number of beta cells is decreased in the stz+aav-ctrl group.thus number of pax6 expressing cells decreases, hence loss of pax6. where is the ctrl + pax6 aav group ?
A: Data on normal mice with AAV-PAX6 will be included in the revised version.
In STZ mice and in Db/db mice, AAVPax6 treatment may modify beta-cell mass.Assessment of beta-cell mass needs to be performed in these mice.
A: Measurement of beta cell mass will be included in the revised version.
A more precise description of the STZ model is needed.What is the dose of STZ ?Is it a one shot of STZ or many shots ?Is there a total or a partial destruction of beta cells ?
A: One single high dose of STZ is used in both C57BL/6J and NSG mice.For C57BL/6J (150mg/kg), mice with blood glucose levels exceeding 14 mmol/l for 2 consecutive times were used as recipients of AAV.Partial destruction of beta cells is required to allow AAV transduction in the remaining beta cells.For NSG (120mg/kg), mice with blood glucose levels exceeding 20 mmol/l for 2 consecutive times were used as recipients of islet transplant to mimic human context, as islet transplant is usually performed in patients with severe hyperglycemia.The details will be included in the revised version.
In STZ mice, pax6 is overexpressed in beta cells and stimulates proliferation but the stimulation of proliferation is not impressive but seems to be sufficient to restore beta cells and produce insulin.Please comment on this result.
A: Both stimulation of beta cell proliferation and inhibition of beta cell death account for the amelioration of hyperglycemia.Besides, relief on hyperglucagonemia also helps.

TUNEL levels are very high in all groups while apoptosis in beta cells is supposed to be a very low level. Also, pictures of IF for TUNEL suggest that apoptotic cells are not beta cells but other cells. How was the co staining for TUNEL and insulin quantified ?
A: TUNEL level is low in normal islets but high in diabetic subjects (fig.3J, 4J, 5D).STZ targets beta cells and causes beta cell death and more apoptotic cells are detected in STZ mice.Co-staining is quantified by counting the TUNEL and insulin double positive cells and divided by the total number of insulin positive cells.

Was alpha cell mass measured in groups of mice with modified blood glucagon levels ?
A: Alpha cell mass was not quantified, the pancreatic alpha-to-beta cell ratio was quantified instead.

MafA IF should be nuclear but the picture show a weird staining in some cells. Please improve IF.
A: The image will be improved accordingly.

What is the phenotype of human normal islets + AAVPax6 ? Again, this group is missing and could provide interesting informations.
A: Figure 7, S6C to S6F demonstrates the effects of AAV-PAX6 on normal human islets with the post-transplant islet graft function and survival evaluated.
Line 262, the authors claim that "transplantation of T2D islets transduced with AAV-Ctrl (STZ/T2D-Ctrl) modestly normalized the blood glucose level".If glycose returns to a normal value, then it is called normalized, otherwise, it is decreased comparerd to control group.Modestly normalized is a non sense.
A: The sentence will be corrected accordingly.

Insulin contents are compared in between STZ/Ctrl-L and STZ/L+Pax6 but it should also be compared to STZ/Ctrl-H.
A: Comparison between STZ/Ctrl-H and STZ/Ctrl-L will be included in the revised version.

Referee #3 (Remarks for Author):
The manuscript entitled "Pax6 gene delivery preserves beta cells and improves islets transplantation efficacy" by Wing Yan So, et al., describes the role of modulating Pax6 expression in pancreatic islets to preserve/restore beta cell function in different models of T2D, including preclinical (Db/Db, STZ treated mice) and human control and T2D islets, engrafted in STZ mice.This is an important study unravelling that modulation of Pax6 expression (i.e.increasing its levels) improves beta cell function.This is a well controlled and designed study, using pre-clinical relevant models, where functional tests are associated to molecular characterization of Pax6 over expression.The in vivo preclinical studies are clearly demonstrating that maintaining Pax6 levels may contribute to improve beta cell function/dedifferentiation during T2D.
I have some comments concerning the study.
Concerning the AAV8 approach, the authors do not describe why they have used this serotype, and whether the AAVs infect both human and mouse pancreatic islets.Is the infection specific to pancreatic islets or are other tissues also affected after AAVs injection?How efficient is the transaction of AAVs in the middle of pancreatic islets?
A: AAV8 has been demonstrated to stably transduce the pancreas, including beta cell in islets.The tissue specificity would be greatly enhanced when combining with a tissue specific promoter.Insulin promoter is used in the present study and other studies for beta cell transduction (PMID 31355778, 25676679, 25338552, 16567506, 18560422, 30400037).AAV8 is also able to efficiently transduce human islets as shown in this study (figure 5E) and other studies (PMID 29304344, 15858610).Details will be added in the revised version.
AAV transduction is minimally detected in other tissues (liver, kidney, muscle, spleen).The results will be included in the revised version.Transduction in the middle of islets is observed as shown in figure S2B; while quantification of protein expression (figure S2A, 4K and 5E) also shows the significant increase in PAX6 expression.
It is interesting to observe that Pax6 over expression modulates proliferation and/or apoptosis, in response (or not) to Insulin or Glp1.In the RNA-seq/ChIP-seq integrated analysis, none of the proliferative nor apoptosis pathways were found to be regulated by Pax6, although this seems to be the major functional effect (and, consequently, subsequent insulin secretion effect).The authors should, at least, discuss this point.
A: The RNA-seq/ChIP-seq integrated analysis only displays the pathways/ genes that are directly regulated by PAX6 (genes bound by PAX6 and are also altered by PAX6 overexpression).However, PAX6 also regulates the expression/ activity of other transcription factors or master regulators such as MAFA, PDX1, NKX6.1, CREB and Akt.These transcription factors/ regulators in turn regulate the expression of a bundle of genes.These genes/ pathways are not directly regulated by PAX6 (no direct binding) and hence are not displayed in the ChIP/ OE overlapping result.This point can be discussed in the revised version.Please note that based on the initial reports and on the amount of work needed to address the referees' concerns, we cannot guarantee at this stage that the eventual outcome will be favorable.Addressing the reviewers' concerns in full will be necessary for further considering the manuscript in our journal, and a revised manuscript will once again be subject to review.
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Yours sincerely, Lise Roth
Lise Roth, PhD Senior Editor EMBO Molecular Medicine Dear Dr. Roth, Thank you for your decision letter on 12 June 2023.We appreciate the insightful comments from all 3 reviewers and have performed a number of new experiments based on their suggestions to strengthen the study and address their concerns.We have also made changes to the text in the current revised version as highlighted for your kind consideration of publication in EMBO Molecular Medicine.Please find below our point-to-point response to the reviewers' comments.

Referee #1 (Comments on Novelty/Model System for Author):
The novelty of the studies is questionable.There is extensive literature on Pax6 and its role in beta cell formation and function, so I'm not sure how much this study adds.Delivery of a transcription factor as a therapeutic approach has been mentioned in the literature for years, but few approaches if any have materialized.Again, the novelty there is also marginal.
A: The referenced studies on PAX6 are mainly in mouse beta cells using mutant mice (PMID 15110714, 9224716, 26658466, 27941241, 23326594, 28377501).These studies show that PAX6 is physiologically essential for normal mouse islet development and function using a loss-of-function approach.It is well documented that species differences exist between human and rodent islets in terms of metabolism, cytoarchitecture and functional implications (PMID 21454710, 28422162, 16461897), which may influence the translatability of mouse studies to the human context.In contrast, we use human islets in our study to demonstrate the human relevance besides the experiments with mice.Moreover, it is generally recognized that the capability of adult human beta cell replication or regeneration is very limited when compared to rodent beta cells (PMID 22751699), and there is currently no clinical therapy proven to stimulate beta cell proliferation or preserve beta cell mass once beta cell failure has started (PMID 27189025).Hence, strategies that can preserve beta cells, even in a modest manner, are valuable.With the use of cell line, mouse models and human islets, our results highlight previously unappreciated roles for PAX6 in maintaining human islet survival and the potential contribution to diabetes management.While it is important to understand the physiological role of the critical beta cell regulators, the ultimate goal is to develop therapeutic strategies by targeting those regulators, which is not addressed by any of the aforementioned studies.In our study, we apply various in vitro and in vivo models to demonstrate the therapeutic effects of AAV-mediated PAX6 gene delivery on beta cell preservation and diabetes management.In particular, we show that PAX6 gene delivery enhances both normal and T2D human islet potency, and thus might overcome two of the major unresolved barriers of clinical islet transplantation: post-transplant islet graft loss and scarcity of healthy islets from donors.By investigating scientific basis to support targeting PAX6 to overcome beta cell failure, our study hence provides the first evidence for the potential clinical relevance of PAX6 in diabetes management.

Referee #1 (Remarks for Author):
Summary: In this study, the authors argue that Pax6 is a critical regulator of beta cell proliferation and cell death based on data using lentivirus and insulin promoted AAV to 12th Sep 2023 2nd Authors' Response to Reviewers either knockdown or overexpress Pax6.Through numerous in vitro and in vivo model systems focusing on islets, the authors show that Pax6 ameliorates T1D (via single, high dose STZ although not adequately explained in methods) and T2D (via db/db or high glucose media, HGPA) disease systems.Moreover, they transplant T2D human islets treated with AAV-Pax6 into STZ treated NSG mice to show functional glycemic control via increased insulin secretion.Bulk transcriptional analysis on control islets (unclear whether KO or WT Pax6 islets) versus AAV-Pax6 overexpressed islets show an increase in genes related to beta cell identity and pancreatic secretion.Islet transplantations in STZ treated NSG mice using islets of lower mass that have been exposed with AAV-Pax6 show similar improvement to the higher mass islets.Overall, the multiple experiments highlight that overexpression of Pax6 results in improved islet function.
A: A single high dose of STZ was intraperitoneally injected into C57BL/6J (150 mg/kg) or NSG (120 mg/kg) mice to render hyperglycemia.The details have been added in the Materials and Methods section (lines 571-573).

Impact: Marginal, owing to the myriad other papers documenting the well-known role of Pax6. Moreover, this study does not account for AAV or lentiviral transfection efficiency issues that have been seen with other pancreatic genes. There may be marginal increase in knowledge depicting Pax6 as beneficial for low mass islets.
A: With all due respect, we disagree on the reviewer's suggestion that our study provides only marginal increase in knowledge.The novelty and the significance of the current study have been elaborated above in response to the first question.Regarding the transfection efficiency, our results have shown that the lentivirus efficiently causes PAX6 knockdown in the EndoC-bH1 cell line (Appendix fig.S1A).Moreover, we also show that our lentivirus and AAV approaches efficiently deliver PAX6 into cell line (Appendix fig.S1C and S1D), mouse beta cells/ islets (Appendix fig.S2A, S2B, S3A, S3B and fig.4K) and human islets (fig.5E) as shown by the significant increases in PAX6 protein levels.

Strengths: This paper has multiple in vitro and in vivo methods of testing knockout and overexpression capabilities for AAV mediated constructs. The authors effectively utilize these repetitive techniques throughout the paper to support their hypothesis. The paper is well written with easy flow and the figures as well as figure legends are easy to follow. For the most part, they cite papers that have addressed Pax6 within the field to a reasonable extent.
Criticisms: Although this paper has copious data, the implications are not entirely novel.The introduction is fairly generic, focusing more on T1D and T2D disease pathogenesis, rather than the background that led to the study of Pax6 vs. other transcription factors (e.g.Pax4).

Moreover, the discussion does not include other potential therapeutic methods under development such as stem cell derived islet organoids, CRISPR corrected, or nanoparticle delivery systems. The authors do not discuss how Pax6, itself, enables improved beta cell function (as opposed to increased beta cell replication-which itself may not improve function).
A: PAX4 is a key regulator for beta cell development and maturation during embryogenesis.Although still detectable, PAX4 expression is low in adult beta cells (PMID 18234907).Acute overexpression of PAX4 has been found to protect beta cells against stress-induced apoptosis and stimulates proliferation.However, sustained PAX4 expression results in beta cell dedifferentiation (repression of MAFA and insulin), blunted glucose-stimulated insulin secretion and glucose intolerance (PMID 21521872).Besides, transcript levels of PAX4 are increased in human T2D islets (PMID 17989064), which also suggests that PAX4 upregulation triggers beta cell dedifferentiation in T2D and explains why PAX4 expression is normally suppressed in adult beta cells.These observations strongly argue against PAX4 as a suitable therapeutic target.
PAX6 is found to be down-regulated in human T2D islets (PMID 22768844, 34193609) while PAX6 gene mutations are associated with diminished insulin secretion and glycemic perturbations in human (PMID 30151985, 15842522, 11756345).These observations suggest a strong correlation between PAX6 activity / levels and human diabetes.While mechanistic studies are mainly done on mice, those studies suggest PAX6 critically regulates mouse beta cell function but its roles in human beta cells remain ambiguous.These justify the study of PAX6 in human islets.The background of PAX4 and PAX6 has been included in the Introduction section (lines 99-112).
Induced pluripotent stem cells (iPSC) are not yet suitable for large-scale production of patient-specific iPSC-beta cells for transplantation since individuals require a large number of individual pancreatic islets.In addition, unlike whole pancreas or islet transplantation, iPSCderived beta cells do not replace the lost function of alpha cells.Deficiency in glucagon production precipitates hypoglycemia unawareness.Until these challenges are addressed, transplantation of islets from cadaveric donors remains the preferred approach worldwide.Hence, it is clinically relevant to develop strategies that enhance islet graft survival and reduce the islet mass for transplantation.These points have been discussed in the Discussion section (lines 517-525).
CRISPR and nanoparticle are additional tools for gene delivery.The CRISPR system is usually applied in stem-cell derived beta cells.There is only one study demonstrating the use of CRISPR in human islets, which involves extensive manipulations including islet dispersion and reaggregation into pseudoislets (PMID 33893274).There are also limited number of studies using nanoparticles to deliver genes into islets (PMID 36706177, 26530912).Further investigation is warranted to demonstrate their efficiency and effects on islet physiology of these approaches.In contrast, AAV is the leading platform for gene delivery for the treatment of various human diseases as it provides long-term gene modification without causing substantial adverse effects (PMID 30710128).AAV is also widely adopted in both mouse and human islets (PMID 29304344, 15858610, 31355778, 25676679, 25338552, 16567506, 18560422, 30400037).Since our study was not aimed at developing a new gene delivery tool, but demonstrating the therapeutic effects of PAX6 delivery in diabetes management and islet transplant outcome, hence we chose AAV, the well-established system as the gene delivery approach.These points have been discussed in the Discussion section (lines 415-427).
The effects of PAX6 manipulations on glucose-stimulated insulin secretion (GSIS) have been examined as suggested and shown in fig.1C, 2C, S1G, S6A and S8B.Our study demonstrated PAX6 as a critical regulator of beta cell function, survival and identity, which orchestrate to enhance the potency of human beta cells and islets.

• The data from in vivo overexpression would be strengthened if the authors show similar effects of overexpression of Pax6 in EndoC cells
A: Overexpression of PAX6 in EndoC-bH1 cells has been performed as suggested .It was found that PAX6 overexpression (Appendix fig.S1D) increased cell proliferation stimulated by insulin, exendin-4, GIP and IGF1 (Appendix fig.S1E).PAX6 overexpression also inhibited cell apoptosis (Appendix fig.S1F), increased GSIS (Appendix fig.S1G) and insulin content (Appendix fig.S1H).Moreover, PAX6 overexpression also enhanced insulin and incretin signalings (Appendix fig.S1I to S1L).

• Explain rationale for why incretin hormones are used
A: Hormones or factors that are well-known to stimulate beta cell proliferation including insulin, exendin-4 (GLP-1 analog), glucose-dependent insulinotropic polypeptide (GIP) and insulin-like growth factor 1 (IGF1) were used to demonstrate the effects of PAX6 manipulations on beta cell proliferation.Besides, previous ChIP-sequencing data reveal that PAX6 binds to genes related to the insulin signaling (PMID 27941241) and on the other hand PAX6 was found to control the GLP-1R and GIPR expression levels in rodent beta cells (PMID 22403172, 28377501, 27941241).Both insulin and incretin signalings are known to critically regulate beta cell function and survival (PMID 18481923, 16517403) but whether PAX6 modulates these signaling events in human beta cells is yet to be confirmed.These points have been included in the Result and Discussion sections (lines 143-146 and lines 445-450).

o Is proliferation being used as a measure of beta cell vitality? Why? A bit confusing given apparent lack of beta cell proliferation in adults
A: We measure beta cell proliferation, cell death and insulin content, which are the indicators of beta cell survival and mass.EndoC-bH1 cell line is an immortalized cell line capable of proliferation as shown in our data and previous publication (PMID 21865645).On the other hand, the capability of cell proliferation in primary human beta cell is very limited.However, previous studies have shown that this can be induced (PMID 32051230, 30059217, 30581122).
o Insulin secretion data would strengthen the studies A: The effects of PAX6 manipulations on GSIS in EndoC-bH1 cells have been examined as suggested.As shown in fig.1C, PAX6 knockdown significantly reduces GSIS while in fig.2C and S1G, PAX6 overexpression enhances GSIS in both normal and HGPA conditions.

Figure 3: • T1D modeling via STZ usually requires multiple low doses; as presented the authors are using high dose to ablate β cells. This is not really a T1D model (more a model of insulin deficiency), and to present it as such is questionable. A NOD mouse model with Pax6 overexpression would strengthen this claim.
A: Both multiple low dose and single high dose STZ are widely used in different mouse strains in different studies (PMID 21478271, 16755002) in order to ablate beta cells.
We agree that the STZ mouse model is not ideal as a T1D model.T1D is caused by the autoimmune destruction of beta cells while the STZ model is just to mimic the severe beta cell loss seen in T1D.Since we are not investigating the immunomodulation, we focus on the direct effect of PAX6 gene delivery on beta cell regeneration, the beta cell deficit feature in the STZ model serves our purpose and hence is adopted in this study.
• The increase in beta cell proliferation and reduction in death should result in an increase in beta cell mass, but these data are not presented.
A: Measurement of beta cell mass in STZ mice has been performed as suggested and shown in Appendix fig.S2D.It was found that beta cell mass was significantly reduced by STZ, but increased by PAX6 delivery.

• Panel I quantifies Ins+ Ki67+ cells to measure beta cell proliferation; should panel J quantify cells that are both TUNEL+ and Ins+ to measure beta cell apoptosis?
A: Figure 3J has been modified accordingly.

Figure 4:
• The striking differences observed in the overexpression (A-H) does not appear to be reflected by the small differences observed in proliferation and death (I-J); again, presentation of beta cell mass would be helpful.
A: Measurement of beta cell mass in db/db mice has been performed as suggested and shown in Appendix fig.S4A.It was found that PAX6 gene delivery increases beta cell mass in db/db mice.On top of promoting beta cell survival, our results demonstrate that PAX6 gene delivery also increases insulin content and insulin secretion, maintains beta cell identity while causing a complementary reduction in glucagon levels.These modulations orchestrate to relieve glycemic perturbation.A: The control group is the AAV-Ctrl.The figure labels and Result section have been modified accordingly (lines 343-345).

• Panel A-D: p-value adjustment for multiple hypothesis testing?/FDR?
A: Panel A is Benjamini-Hochberg adjusted p-value while panels B-D are FDR adjusted pvalues.These are stated in the figure legend (lines 1125 and 1127).
• Panel G: are the listed pathways significantly enriched/overrepresented? Suggest showing GO analysis, statistics A: Yes, the listed pathways are the top and significantly enriched KEGG pathways enriched by the 843 CHIP-OE overlapping genes adjusted p-value < 0.05; cutoff log 2 FC > 0.5).This is stated in the figure legend (lines 1129-1132).The GO pathways were not included because there are many redundant terms, but relevant pathways such as hormone/insulin secretion or transport, glucose homeostasis, positive regulation of MAPK cascade, are consistently overrepresented.

• Any cell cycle genes that are altered?
A: Yes, there is a list of genes involved in the regulation of DNA replication and cell proliferation upregulated in PAX6-overexpressing islets, which has been included in Appendix fig.S8E.

• Please show PCA plot to show variance between samples
A: The PCA plot has been included in Appendix fig.S8D as suggested.

• Discuss why only 20% of genes are similar between CHIP and OE; what are the other genes doing in relation to beta cells
A: That expression of genes bound by PAX6 was unaltered by PAX6 overexpression suggests that PAX6 binds to these genes' promoters but do not regulate their expression.It is recognized that chromatin immunoprecipitation (ChIP) reveals the binding of transcription factors to thousands of genomic locations in the vicinity of both active and inactive regions.A number of weakly bound sites or the sites bound at low occupancy detected this way actually fail to drive gene transcription (PMID 24888900, 18271625, 23236164).
On the other hand, that expression of genes that were not bound by PAX6 was altered by PAX6 overexpression suggests that PAX6 indirectly regulates the expression of these genes.This can be explained by the regulation of the expression / activity of other transcription factors or master regulators such as MAFA, PDX1, NKX6.1, CREB and Akt by PAX6.These factors in turn regulate the expression of a bundle of effector genes.These points have been highlighted in the Result section (lines 364-373).

Referee #2 (Remarks for Author):
The manuscript by So et al describes how Pax6 is important for beta-cell biology proliferation and survival.They present data that show that Pax6 overexpression may help treat diabetes both in mice models mimicking type 1 or type 2 diabetes.Moreover, they present data showing that Pax6 overexpression in human islets improve their function when transplanted in diabetic mice.The results are interesting and suggest that Pax6 overexpression may be part of new therapeutic strategies, at least in islet graft.Yet, they are several concerns about controls and experiments that are not presented.Thus, serious modifications of the present manuscript are required to improve the quality of the manuscript.
A main issue on the use of the AAV to overexpress Pax6 is to show that it actually targets beta cells and no other tissues.Controls of Pax6 expression in different tissues need to be shown.In addition, Pax6 overexpression in control normoglycemic mice, not just in diabetic mic, needs to be shown.In control mice, does Pax6 overexpression lead to a better glucose homeostasis?
A: AAV-mediated exogenous PAX6 expression is observed in beta cells (insulin positive cells) as shown in Appendix fig.S2B and S3B, while exogenous PAX6 is minimally detected in other tissues (liver, kidney, muscle, spleen) as shown in Appendix fig.S2C.
The effects of AAV-PAX6 in normal mice have been investigated and included in Appendix fig.S3 and the Results section (lines 222-235).Unlike the marked effects in STZ mice, PAX6 gene delivery in normal mouse beta cells (Appendix fig.S3A and S3B) resulted in a mild reduction in blood glucose (Appendix fig.S3C) and slight improvement in glucose tolerance (Appendix fig.S3D and S3E).Serum insulin level and pancreatic insulin content were modestly increased by AAV-PAX6 administration (Appendix fig.S3F and S3G) while serum and pancreatic glucagon levels were not significantly altered (Appendix fig.S3H and S3I).Immunofluorescence staining showed that the islet alpha-to-beta cell ratio was not altered by PAX6 overexpression (Appendix fig.S3J).Moreover, the anti-apoptotic effect of AAV-PAX6 was not evident in normal mouse islets as both AAV-Ctrl-and AAV-PAX6-treated mice displayed a low amount of TUNEL-positive apoptotic cells in islets (Appendix fig.S3K).Notably, similar to the effects in STZ-treated mice, AAV-PAX6 increased beta cell proliferation (Appendix fig.S3L) and beta cell mass (Appendix fig.S3M) in normal mice whereas alpha cell mass was not significantly altered (Appendix fig.S3M).Taken together, AAV-PAX6 stimulates beta cell proliferation but improves glucose homeostasis only mildly in normal mice.
Another main issue is the quantification of rare events.Proliferation and apoptosis rates were measured in alpha and beta cells in vivo, in STZ mice and Db mice.These events are rare, thus the positive cells (for example ki67+glucagon or TUNEL+insulin) are scarce.To obtain a precise measurement, it is required to count a minimum of these scarce events.For example, a bias can be easily made if a too low number of events is counted: 1 cells out of 1000 would suggest a rate of 0.1% while 2 cells out of 1000 would suggest a rate of 2% and a doubling of the rate with just one extra positive cell.The authors do not mention how many events where counted in each measurement and that is absolutely required.
A: For normal and STZ-treated mouse pancreas sections, more than 10 pancreas sections containing over 50 islets were quantified for each mouse and 5 to 6 mice were included for each group.For db/db mouse and human islet sections, more than 5 sections containing over 50 islets were quantified for each batch of islets and 5 to 6 batches of islets were included for each group.More than 4000 beta cells per mouse or batch of islets were counted.For islet graft-bearing kidney sections, more than 5 sections were quantified for each mouse and 4 to 6 mice were included for each group.The number of mice or batches of islets is indicated in the figure legend.The details have been added in the Materials and Methods section (lines 683-690).
On figure 1, it would be very logical to show Pax6 KD before showing the effects on the cells.It is a required control that have to be shown first to prove the efficiency of the KD.
A: Figure 1 has been re-organized with the quantification of PAX6 protein level shown first (Appendix fig.S1A).
Again, figure 1 needs to be re-organized: quantifications of WB need to be called as panels.Analysis of pAkt needs to display whether basal pAkt levels (not just insulin-stimulated) are different when Pax6 is KD.The experience with HGPA needs to be more documented: what are the glucose levels normally required to grow the cells?What are the glucose levels for HGPA?Glucosestimulated insulin secretion should be measured on endobetaH1 cells in the different conditions.
There is a need fora statistical analysis in between ctrl and HGPA cells.It is absolutely mandatory to describe properly the effect of HGPA.During HGPA, culture conditions may force cells to secrete more insulin thus the decreased ins content may reflect improved secretion.Therefore, insulin content must be related to GSIS.
A: The EndoC-bH1 cells are normally maintained in medium containing 5.6 mmol/l glucose, according to the standard protocol (PMID 21865645).HGPA medium contains 25 mmol/l Dglucose and 0.3 mmol/l palmitic acid.These are stated in the Materials and Methods section (lines 551-554).
GSIS in EndoC-bH1 cells under HGPA condition with PAX6 overexpression has been included as suggested (fig.2C) and it was found that HGPA significantly suppressed GSIS but PAX6 overexpression could rescue the impaired GSIS to some extent.
Statistical comparison between Ctrl and HGPA has been displayed in the relevant graphs (fig.2C and 2D).
It is recognized that only a few percent of insulin within a beta cell is released during a 1 hour glucose stimulation (PMID 17919179, 21865645), so the subtle reduction in insulin content caused by insulin secretion is not evident and might not be reflected.Conversely, it has been revealed that insulin content positively correlated with the pulse mass of insulin secretion and first and second-phase insulin secretory response in human islets (PMID 12574208, 14764798, 12865318) while reduction of insulin content in T2D patients (PMID3538738, 28887444, 21104225) associates with a concomitant decline in insulin secretion (PMID 10950818, 21104225).In line with these observations, our results demonstrated that HGPA significantly reduced insulin secretion and insulin content while PAX6 overexpression reversed these (fig.2C and 2D).
Similarly to figure 1, the panels of figure 2 need to be re-organized.The control of Pax6 overexpression need to be shown first before the panels on proliferation and apoptosis.Also, the condition ctrl+pax6 overexpression is interesting and needs to be shown on figures.
A: Figure 2 has been re-organized with the quantification of PAX6 protein level shown first (Appendix fig.S1C).
Please comment on how calculations for ratios of pAkt were done.Is there a normalization to total akt and to GAPDH and to control condition ?Please explain.
A: As indicated in the quantification panel (fig.1F and 2F, right panels), pAkt is normalized to total Akt and hence pAkt/ Akt is displayed.Then the pAkt/Akt ratio of all groups is compared to the pAkt/Akt ratio of the Ctrl + vehicle group and thus the pAkt/Akt ratio of the Ctrl + vehicle group is set as 1.
It seems that pa6 overexpression reverts the effects of HGPA.Is it also true with Pax4 or pdx1 overexpression ?The question about the specificity of action of pax6 is central.
A: PAX4 is a key regulator for beta cell development and maturation during embryogenesis.Although still detectable, PAX4 expression is low in adult beta cells (PMID 18234907).Acute overexpression of PAX4 has been found to protect beta cells against stress-induced apoptosis and stimulates proliferation.However, sustained PAX4 expression results in beta cell dedifferentiation (repression of MAFA and insulin), blunted glucose-stimulated insulin secretion and glucose intolerance (PMID 21521872).Besides, transcript levels of PAX4 are increased in human T2D islets (PMID 17989064), which also suggests that PAX4 upregulation triggers beta cell dedifferentiation in T2D and explains why PAX4 expression is normally suppressed in adult beta cells.These observations argue against PAX4 as a suitable therapeutic target.
PAX6 is found to be down-regulated in human T2D islets (PMID 22768844, 34193609) while PAX6 gene mutations are associated with diminished insulin secretion and glycemic perturbations in human (PMID 30151985, 15842522, 11756345).These observations suggest a strong correlation between PAX6 activity / levels and human diabetes.While mechanistic studies are mainly done on mice, those studies suggest PAX6 critically regulates mouse beta cell function but its roles in human beta cells remain ambiguous.These justify the study of PAX6 in human islets.The background of PAX4 and PAX6 has been included in the Introduction section (lines 99-112).
It has been demonstrated that PAX6 regulates PDX1 expression in mouse beta cells (PMID 27941241) and our results show that PAX6 overexpression would enhance PDX1 expression in diabetic mouse and human islets (figure S5A, S5B, S6B and S6C).In addition to PDX1, PAX6 also regulates the expression / activity of other key regulators including MAFA and NKX6.1, suggesting PAX6 as a more suitable target.
STZ mice are not a T1D like phenotype.STZ mice are a model of diabetes to a lack of insulin (after chemical destruction of beta cells) but without auto-immune attack.Please correct.
The expression of pax 6 was measured on whole pancreas.Since stz kills beta cells, the number of beta cells is decreased in the stz+aav-ctrl group.thusnumber of pax6 expressing cells decreases, hence loss of pax6.where is the ctrl + pax6 aav group ?
A: The effects of AAV-PAX6 in normal mice have been investigated and included in Appendix fig.S3 and lines 222-235.
In STZ mice and in Db/db mice, AAVPax6 treatment may modify beta-cell mass.Assessment of beta-cell mass needs to be performed in these mice.
A: Measurement of beta cell mass in STZ mice and db/db has been included as suggested and shown in Appendix fig.S2D and S4A.Results show that AAV-PAX6 increased beta cell mass in both STZ-treated and db/db mice.
A more precise description of the STZ model is needed.What is the dose of STZ ?Is it a one shot of STZ or many shots ?Is there a total or a partial destruction of beta cells ?
A: One single high dose of STZ is used in both C57BL/6J and NSG mice.For C57BL/6J mice (150 mg/kg), mice with blood glucose levels exceeding 14 mmol/l for 2 consecutive times were used as recipients of AAV.Partial destruction of beta cells is required to allow AAV transduction in the remaining beta cells.For NSG (120 mg/kg), mice with blood glucose levels exceeding 20 mmol/l for 2 consecutive times were used as recipients of islet transplant to mimic human context, as islet transplant is usually performed in patients with severe hyperglycemia.These details have been included in the Materials and Methods section (lines 571-573, 578-579, 633-634).
In STZ mice, pax6 is overexpressed in beta cells and stimulates proliferation but the stimulation of proliferation is not impressive but seems to be sufficient to restore beta cells and produce insulin.Please comment on this result.
A: Apart from stimulation of beta cell proliferation, inhibition of beta cell death and increase in insulin content account for the increase in insulin levels and amelioration of hyperglycemia.Besides, relief on hyperglucagonemia also helps relieve glycemic disturbance.

TUNEL levels are very high in all groups while apoptosis in beta cells is supposed to be a very low level. Also, pictures of IF for TUNEL suggest that apoptotic cells are not beta cells but other cells. How was the co staining for TUNEL and insulin quantified ?
A: TUNEL level is low in normal islets but high in diabetic subjects (fig.3J, 4J, 5D).Costaining is quantified by dividing the TUNEL and insulin double positive cell number by the total insulin positive cell number.

Was alpha cell mass measured in groups of mice with modified blood glucagon levels ?
A: Alpha cell mass has been quantified.For STZ-treated mice, STZ caused a dramatic increase in alpha cell mass which was reduced by PAX6 overexpression (Appendix fig.S2G).
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Figure 5: • Glucose stimulated insulin secretion (GSIS) to show effect of overexpression would strengthen the study.A: GSIS in human islets has been performed as suggested.As shown in Appendix fig.S6Aand S8B, AAV-PAX6 transduction promotes GSIS in both normal and T2D human islets.

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