Reviewer's report
Title:Effective in vivo and ex vivo gene transfer to intestinal mucosa by
VSV-G-pseudotyped lentiviral vectors
Version: 2Date:17 September 2009
Reviewer: Steven Polyak
Reviewer's report:
Dear Authors,
The manuscript entitled “Effective in vivo and ex vivo gene transfer to intestinal
mucosa by VSV-G-pseudotyped lentiviral vectors” fills an important void in LV
mediated gastrointestinal gene transfer. The research is exploratory in nature
with sound science and interesting results. The findings would be interesting to
the readers of BMC Gastroenterology. Overall, several corrections and
clarifications are required to provide a manuscript that is appropriate for
publication. The introduction is appropriate but would be nice to know what cell
types the authors are interested in targeting or if this is purely an exploratory
study where the investigators seek to determine the tropism of VSV-G LV. The
methods are appropriate for a gastroenterology journal without detailed emphasis
on vector biology/production; however, to benefit the readers, more detail is
required. In the results, discussion and conclusion sections, a few errors, holes
and some unsubstantiated conclusions need to be corrected. Below the
comments are organized into Major and Minor Revisions for each section of the
manuscript. New experiments are not requested therefore I believe you can
provide the appropriate changes to allow for publication.
Major compulsory revisions:
Abstract
1) Authors mention histologic evaluation was done to assess for inflammation,
but this is not provided in the manuscript.
Methods
1) The readers of a GI journal will likely not have knowledge about the need and
the function of polybrene in LV transduction. Please provide a brief sentence
describing why polybrene is used.
2) In the in vivo studies section if the mice are divided into untreated control,
vehicle solution control and LV groups. Somewhere in the methods the
composition of the vehicle solution must be mentioned.
3) A 50% ETOH enema pretreatment was used. In the methods the purpose for
using this pretreatment should be reported, i.e. mucosal disruption. In the
discussion (see below) why this pretreatment was necessary and the
consequences and potential influence of this pretreatment on mucosal infection
or vector translocation should be discussed.
4) In the abstract and results authors report that histologic assessment for
inflammation was performed. If it was done, please report the scoring system or
evaluation technique used in the methods. (also see comment # 3 in the Results
section below)
5) Fig 1B is superfluous and should be removed. This same figure is available in
the appropriately referenced manuscript [30].
6) There is a discrepancy in the IF staining of the explants tissues. Paragraph 1
on page 9 states that tissues were fixed in formaldehyde, embedded in Paraffin
and sectioned for IF staining. However the second paragraph states that explants
cryosections were used in IF staining. Please clarify.
7) The manuscript states that AF488 was used for all antibodies. Please correct
anti-b-gal to a red fluorophore, which was reported in the results.
Results & Discussion
1) The Caco2 transduction efficiency of 13.7% differs from Seppen et al results
(76% dividing & 24% polarized). Could you please comment why?
2) Please report how many mice were used in the in vivo experiments in each
group, missing in second paragraph of page 11 or in the corresponding methods
section. Was it 8 in each group as reported in the bioluminescence
examinations?
3) The authors report that no significant differences were seen in histopathologic
assessments in the murine studies, but there is no description of what histologic
scoring method was used. This is important especially at day 2. This either needs
to be added to the methods or removed from the results.
4) Include discussion as to why ETOH enemas were used as a pretreatment and
its consequences.
5) Transduced cells did not show “robust” amplification. 231 copy numbers is not
considered robust. Marginal at best. Please correct in manuscript.
6) Please shorten fig 3 legend, much is repeated from the text. In the legend a
supplemental fig 1 is reported, but this is not part of the viewable text, please
correct or remove.
7) There is a discrepancy in the fig 3C legend and the text: sensitivity is either 50
or 20 copies. Please correct.
8) The authors do not provide the number of mice used to obtain the x-gal
positive cells in the in vivo experiments. Please add this to results text or figure 4
legend.
9) The authors do not show significance marks in x-gal positive cells between
groups in figure 4. I suspect this is just missing from the figure. If it is please
correct. If not then report in the text.
10) Immunofluroescence is very challenging in the intestine. Nonspecific green
staining is commonly seen. Negative controls are vital and necessary. Please
provide a supplemental figure showing the negative control stains for each image
in figure 5.
11) Please report the correct fluorophores used in the methods section (also see
comment #7 in the methods section).
12) The representative images in fig 5 are difficult to correlate with the reported
numbers. There are no observed b-gal positive only cells in 5B, C&F.
Additionally, 5D appears to have more colocalizing cells than 5C. Either new
representative images are required or this must be explained in the text or at
least the strong conclusions cannot be drawn.
13) Figure 6. To remain consistent label the control as NC or Mock to remain
consistent no 0ng/p24.
14) Figure 6 legend. Report number of explants used to determine number.
15) Figure 7. Similar to figure 6 a negative control for each image should be
shown as a supplemental figure.
16) More positive cells in the LP of the explants is not a surprising finding given
that the vector has access to the LP ex vivo and the samples were treated with
polybrene.
17) There are significant differences between the in vivo and ex vivo data. These
are not surprising but needs to be emphasized and explained. Experimental
confounders also need to be reported for example use of pretreatment ETOH
enemas in vivo, the use of polybrene ex vivo and 2-14 days vs 24 hrs.
Conclusion
1) Please make the conclusion section more succinct. Much of this is discussion.
This section should remain more focused in the conclusions drawn from the
experimental findings.
2) Paragraph 2 page 16 needs to be removed. The investigators do not show
data to support or refute this conclusion and so does not belong in this
manuscript.
3) Paragraph 2 page 18: Be careful drawing conclusions when comparing
tropism between the different experiments especially when comparing to other
studies using different viral vectors which clearly have different cellular and tissue
tropisms.
4) Paragraph 3 page 18: authors conclude that LV exhibited a remarkable ability
to transduce up to 50% of CD4 positive cell population. The conclusion should
not be so strong since the immunofluorescence images do not support such a
strong conclusion. If the authors would like to be certain I recommend isolation of
LPMCs and conducting flow experiments.
5) Paragraph 1 page 19: Please remove the hydrostatic pressure in the rectal
lumen comments as this was analyzed thus is speculative. Plus pipette
instillation of vector solution would likely not create enough pressure, unless
there is methodology that is missing from the manuscript. The ETOH
pretreatment more likely contributed to increased mucosal penetration into the
epithelium. It would be interesting to compare with and without ETOH
pretreatment.
6) Paragraph 2 page 17 and Paragraph 2 page 19: One cannot draw conclusions
on species tropism differences from these experiments given that murine explant
experiments and human rectal instillation experiments were not done. Cannot
compare the result from two different experiments to draw a conclusion.
Minor essential revisions:
Abstract
1) A second conclusion was that you demonstrated the differences between
model systems (in vivo vs ex vivo) in evaluating vector gene transfer.
Background
1) Last sentence first paragraph is same as in abstract and with same grammar
problem.
2) You provide references for non-viral vector, retroviral vector and adenoviral
vector history in GI transduction, but you do not reference AAV history in GI tract.
It would be appropriate to do so.
3) “limited by low packaging capacity” is misleading. I think you mean the limited
size of transgene insert?
Methods
1) It is customary to provide the names and source company for experimental
kits or chemicals if appropriate or mention that the items were made in the lab.
For example:
a. p24 ELISA kit or lab made?
b. Polybrene
c. All the antibodies in the IMF staining studies.
2) How was seepage prevented after enema?
3) What was the fixative in the murine tissues used in x-gal staining?
4) Are the anti-B-gal and anti-keratin & CD antibodies directly conjugated to
AF488 or was there a secondary involved? Describe the IMF staining technique.
Did you use antigen retrievals etc?
5) What microscope and software did you use to do the quantitative
morphometry and confocal analysis?
Results & Discussion
1) The manual counting of cells for the determination of transduction efficiency in
vector gene transfer has high inter-observer variability. This is an important issue
when it comes to reporting transduction efficiencies in an evolving field with
relatively low efficiencies. Computerized pixel counting or flow is best to
determine cell line transduction efficiency. Given the possibility of nonspecific
staining in intestinal cells a clear negative control needs to be shown or reported.
This should also be mentioned as a limitation.
2) Please report how this statement is determined: “The highest dose tested was
10ng p24 equivalent units of LV, which corresponds to a biological infectious titer
of 10^6 transducing units (TU) on a standardized cell line such as 293T”. Did you
determine this or is this accepted from a reference?
3) Was the luminescence (transgene expression) limited to the rectum because
the ETOH enema was limited to the rectum or the application of the vector was
limited to this area?
4) Given that LP immune cells are the major cell types that are transduced by LV,
it is important to report presence or absence of vector genome in mesenteric
lymph nodes. Please report or acknowledge that this information is not available.
5) Figure 7. If DAPI staining is present in all images appear modified as the blue
cannot be seen.
Conclusion
1) Paragraph 1 page 16: without local or systemic toxicity in vivo needs to be
changes to without macroscopic or systemic toxicity in vivo. Cytokines were not
measured and so drawing strong conclusions is difficulty.
2) Paragraph 3 page 16: The correlation between bioluminescence signal and
transgene expressing cell quantification is interesting. In review of ref #40
Hiraoka et al I could not see how this was achieved. Could you please expound
in the manuscript.
Level of interest: An article of importance in its field
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.
Reviewer's report
Title:Effective in vivo and ex vivo gene transfer to intestinal mucosa by
VSV-G-pseudotyped lentiviral vectors
Version: 2Date:17 September 2009
Reviewer: Steven Polyak
Reviewer's report:
Dear Authors,
The manuscript entitled “Effective in vivo and ex vivo gene transfer to intestinal
mucosa by VSV-G-pseudotyped lentiviral vectors” fills an important void in LV
mediated gastrointestinal gene transfer. The research is exploratory in nature
with sound science and interesting results. The findings would be interesting to
the readers of BMC Gastroenterology. Overall, several corrections and
clarifications are required to provide a manuscript that is appropriate for
publication. The introduction is appropriate but would be nice to know what cell
types the authors are interested in targeting or if this is purely an exploratory
study where the investigators seek to determine the tropism of VSV-G LV. The
methods are appropriate for a gastroenterology journal without detailed emphasis
on vector biology/production; however, to benefit the readers, more detail is
required. In the results, discussion and conclusion sections, a few errors, holes
and some unsubstantiated conclusions need to be corrected. Below the
comments are organized into Major and Minor Revisions for each section of the
manuscript. New experiments are not requested therefore I believe you can
provide the appropriate changes to allow for publication.
Major compulsory revisions:
Abstract
1) Authors mention histologic evaluation was done to assess for inflammation,
but this is not provided in the manuscript.
Methods
1) The readers of a GI journal will likely not have knowledge about the need and
the function of polybrene in LV transduction. Please provide a brief sentence
describing why polybrene is used.
2) In the in vivo studies section if the mice are divided into untreated control,
vehicle solution control and LV groups. Somewhere in the methods the
composition of the vehicle solution must be mentioned.
3) A 50% ETOH enema pretreatment was used. In the methods the purpose for
using this pretreatment should be reported, i.e. mucosal disruption. In the
discussion (see below) why this pretreatment was necessary and the
consequences and potential influence of this pretreatment on mucosal infection
or vector translocation should be discussed.
4) In the abstract and results authors report that histologic assessment for
inflammation was performed. If it was done, please report the scoring system or
evaluation technique used in the methods. (also see comment # 3 in the Results
section below)
5) Fig 1B is superfluous and should be removed. This same figure is available in
the appropriately referenced manuscript [30].
6) There is a discrepancy in the IF staining of the explants tissues. Paragraph 1
on page 9 states that tissues were fixed in formaldehyde, embedded in Paraffin
and sectioned for IF staining. However the second paragraph states that explants
cryosections were used in IF staining. Please clarify.
7) The manuscript states that AF488 was used for all antibodies. Please correct
anti-b-gal to a red fluorophore, which was reported in the results.
Results & Discussion
1) The Caco2 transduction efficiency of 13.7% differs from Seppen et al results
(76% dividing & 24% polarized). Could you please comment why?
2) Please report how many mice were used in the in vivo experiments in each
group, missing in second paragraph of page 11 or in the corresponding methods
section. Was it 8 in each group as reported in the bioluminescence
examinations?
3) The authors report that no significant differences were seen in histopathologic
assessments in the murine studies, but there is no description of what histologic
scoring method was used. This is important especially at day 2. This either needs
to be added to the methods or removed from the results.
4) Include discussion as to why ETOH enemas were used as a pretreatment and
its consequences.
5) Transduced cells did not show “robust” amplification. 231 copy numbers is not
considered robust. Marginal at best. Please correct in manuscript.
6) Please shorten fig 3 legend, much is repeated from the text. In the legend a
supplemental fig 1 is reported, but this is not part of the viewable text, please
correct or remove.
7) There is a discrepancy in the fig 3C legend and the text: sensitivity is either 50
or 20 copies. Please correct.
8) The authors do not provide the number of mice used to obtain the x-gal
positive cells in the in vivo experiments. Please add this to results text or figure 4
legend.
9) The authors do not show significance marks in x-gal positive cells between
groups in figure 4. I suspect this is just missing from the figure. If it is please
correct. If not then report in the text.
10) Immunofluroescence is very challenging in the intestine. Nonspecific green
staining is commonly seen. Negative controls are vital and necessary. Please
provide a supplemental figure showing the negative control stains for each image
in figure 5.
11) Please report the correct fluorophores used in the methods section (also see
comment #7 in the methods section).
12) The representative images in fig 5 are difficult to correlate with the reported
numbers. There are no observed b-gal positive only cells in 5B, C&F.
Additionally, 5D appears to have more colocalizing cells than 5C. Either new
representative images are required or this must be explained in the text or at
least the strong conclusions cannot be drawn.
13) Figure 6. To remain consistent label the control as NC or Mock to remain
consistent no 0ng/p24.
14) Figure 6 legend. Report number of explants used to determine number.
15) Figure 7. Similar to figure 6 a negative control for each image should be
shown as a supplemental figure.
16) More positive cells in the LP of the explants is not a surprising finding given
that the vector has access to the LP ex vivo and the samples were treated with
polybrene.
17) There are significant differences between the in vivo and ex vivo data. These
are not surprising but needs to be emphasized and explained. Experimental
confounders also need to be reported for example use of pretreatment ETOH
enemas in vivo, the use of polybrene ex vivo and 2-14 days vs 24 hrs.
Conclusion
1) Please make the conclusion section more succinct. Much of this is discussion.
This section should remain more focused in the conclusions drawn from the
experimental findings.
2) Paragraph 2 page 16 needs to be removed. The investigators do not show
data to support or refute this conclusion and so does not belong in this
manuscript.
3) Paragraph 2 page 18: Be careful drawing conclusions when comparing
tropism between the different experiments especially when comparing to other
studies using different viral vectors which clearly have different cellular and tissue
tropisms.
4) Paragraph 3 page 18: authors conclude that LV exhibited a remarkable ability
to transduce up to 50% of CD4 positive cell population. The conclusion should
not be so strong since the immunofluorescence images do not support such a
strong conclusion. If the authors would like to be certain I recommend isolation of
LPMCs and conducting flow experiments.
5) Paragraph 1 page 19: Please remove the hydrostatic pressure in the rectal
lumen comments as this was analyzed thus is speculative. Plus pipette
instillation of vector solution would likely not create enough pressure, unless
there is methodology that is missing from the manuscript. The ETOH
pretreatment more likely contributed to increased mucosal penetration into the
epithelium. It would be interesting to compare with and without ETOH
pretreatment.
6) Paragraph 2 page 17 and Paragraph 2 page 19: One cannot draw conclusions
on species tropism differences from these experiments given that murine explant
experiments and human rectal instillation experiments were not done. Cannot
compare the result from two different experiments to draw a conclusion.
Minor essential revisions:
Abstract
1) A second conclusion was that you demonstrated the differences between
model systems (in vivo vs ex vivo) in evaluating vector gene transfer.
Background
1) Last sentence first paragraph is same as in abstract and with same grammar
problem.
2) You provide references for non-viral vector, retroviral vector and adenoviral
vector history in GI transduction, but you do not reference AAV history in GI tract.
It would be appropriate to do so.
3) “limited by low packaging capacity” is misleading. I think you mean the limited
size of transgene insert?
Methods
1) It is customary to provide the names and source company for experimental
kits or chemicals if appropriate or mention that the items were made in the lab.
For example:
a. p24 ELISA kit or lab made?
b. Polybrene
c. All the antibodies in the IMF staining studies.
2) How was seepage prevented after enema?
3) What was the fixative in the murine tissues used in x-gal staining?
4) Are the anti-B-gal and anti-keratin & CD antibodies directly conjugated to
AF488 or was there a secondary involved? Describe the IMF staining technique.
Did you use antigen retrievals etc?
5) What microscope and software did you use to do the quantitative
morphometry and confocal analysis?
Results & Discussion
1) The manual counting of cells for the determination of transduction efficiency in
vector gene transfer has high inter-observer variability. This is an important issue
when it comes to reporting transduction efficiencies in an evolving field with
relatively low efficiencies. Computerized pixel counting or flow is best to
determine cell line transduction efficiency. Given the possibility of nonspecific
staining in intestinal cells a clear negative control needs to be shown or reported.
This should also be mentioned as a limitation.
2) Please report how this statement is determined: “The highest dose tested was
10ng p24 equivalent units of LV, which corresponds to a biological infectious titer
of 10^6 transducing units (TU) on a standardized cell line such as 293T”. Did you
determine this or is this accepted from a reference?
3) Was the luminescence (transgene expression) limited to the rectum because
the ETOH enema was limited to the rectum or the application of the vector was
limited to this area?
4) Given that LP immune cells are the major cell types that are transduced by LV,
it is important to report presence or absence of vector genome in mesenteric
lymph nodes. Please report or acknowledge that this information is not available.
5) Figure 7. If DAPI staining is present in all images appear modified as the blue
cannot be seen.
Conclusion
1) Paragraph 1 page 16: without local or systemic toxicity in vivo needs to be
changes to without macroscopic or systemic toxicity in vivo. Cytokines were not
measured and so drawing strong conclusions is difficulty.
2) Paragraph 3 page 16: The correlation between bioluminescence signal and
transgene expressing cell quantification is interesting. In review of ref #40
Hiraoka et al I could not see how this was achieved. Could you please expound
in the manuscript.
Level of interest: An article of importance in its field
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.
Please review this paper.
user
Reviewer's report
Title:Effective in vivo and ex vivo gene transfer to intestinal mucosa by
VSV-G-pseudotyped lentiviral vectors
Version: 2Date:17 September 2009
Reviewer: Steven Polyak
Reviewer's report:
Dear Authors,
The manuscript entitled “Effective in vivo and ex vivo gene transfer to intestinal
mucosa by VSV-G-pseudotyped lentiviral vectors” fills an important void in LV
mediated gastrointestinal gene transfer. The research is exploratory in nature
with sound science and interesting results. The findings would be interesting to
the readers of BMC Gastroenterology. Overall, several corrections and
clarifications are required to provide a manuscript that is appropriate for
publication. The introduction is appropriate but would be nice to know what cell
types the authors are interested in targeting or if this is purely an exploratory
study where the investigators seek to determine the tropism of VSV-G LV. The
methods are appropriate for a gastroenterology journal without detailed emphasis
on vector biology/production; however, to benefit the readers, more detail is
required. In the results, discussion and conclusion sections, a few errors, holes
and some unsubstantiated conclusions need to be corrected. Below the
comments are organized into Major and Minor Revisions for each section of the
manuscript. New experiments are not requested therefore I believe you can
provide the appropriate changes to allow for publication.
Major compulsory revisions:
Abstract
1) Authors mention histologic evaluation was done to assess for inflammation,
but this is not provided in the manuscript.
Methods
1) The readers of a GI journal will likely not have knowledge about the need and
the function of polybrene in LV transduction. Please provide a brief sentence
describing why polybrene is used.
2) In the in vivo studies section if the mice are divided into untreated control,
vehicle solution control and LV groups. Somewhere in the methods the
composition of the vehicle solution must be mentioned.
3) A 50% ETOH enema pretreatment was used. In the methods the purpose for
using this pretreatment should be reported, i.e. mucosal disruption. In the
discussion (see below) why this pretreatment was necessary and the
consequences and potential influence of this pretreatment on mucosal infection
or vector translocation should be discussed.
4) In the abstract and results authors report that histologic assessment for
inflammation was performed. If it was done, please report the scoring system or
evaluation technique used in the methods. (also see comment # 3 in the Results
section below)
5) Fig 1B is superfluous and should be removed. This same figure is available in
the appropriately referenced manuscript [30].
6) There is a discrepancy in the IF staining of the explants tissues. Paragraph 1
on page 9 states that tissues were fixed in formaldehyde, embedded in Paraffin
and sectioned for IF staining. However the second paragraph states that explants
cryosections were used in IF staining. Please clarify.
7) The manuscript states that AF488 was used for all antibodies. Please correct
anti-b-gal to a red fluorophore, which was reported in the results.
Results & Discussion
1) The Caco2 transduction efficiency of 13.7% differs from Seppen et al results
(76% dividing & 24% polarized). Could you please comment why?
2) Please report how many mice were used in the in vivo experiments in each
group, missing in second paragraph of page 11 or in the corresponding methods
section. Was it 8 in each group as reported in the bioluminescence
examinations?
3) The authors report that no significant differences were seen in histopathologic
assessments in the murine studies, but there is no description of what histologic
scoring method was used. This is important especially at day 2. This either needs
to be added to the methods or removed from the results.
4) Include discussion as to why ETOH enemas were used as a pretreatment and
its consequences.
5) Transduced cells did not show “robust” amplification. 231 copy numbers is not
considered robust. Marginal at best. Please correct in manuscript.
6) Please shorten fig 3 legend, much is repeated from the text. In the legend a
supplemental fig 1 is reported, but this is not part of the viewable text, please
correct or remove.
7) There is a discrepancy in the fig 3C legend and the text: sensitivity is either 50
or 20 copies. Please correct.
8) The authors do not provide the number of mice used to obtain the x-gal
positive cells in the in vivo experiments. Please add this to results text or figure 4
legend.
9) The authors do not show significance marks in x-gal positive cells between
groups in figure 4. I suspect this is just missing from the figure. If it is please
correct. If not then report in the text.
10) Immunofluroescence is very challenging in the intestine. Nonspecific green
staining is commonly seen. Negative controls are vital and necessary. Please
provide a supplemental figure showing the negative control stains for each image
in figure 5.
11) Please report the correct fluorophores used in the methods section (also see
comment #7 in the methods section).
12) The representative images in fig 5 are difficult to correlate with the reported
numbers. There are no observed b-gal positive only cells in 5B, C&F.
Additionally, 5D appears to have more colocalizing cells than 5C. Either new
representative images are required or this must be explained in the text or at
least the strong conclusions cannot be drawn.
13) Figure 6. To remain consistent label the control as NC or Mock to remain
consistent no 0ng/p24.
14) Figure 6 legend. Report number of explants used to determine number.
15) Figure 7. Similar to figure 6 a negative control for each image should be
shown as a supplemental figure.
16) More positive cells in the LP of the explants is not a surprising finding given
that the vector has access to the LP ex vivo and the samples were treated with
polybrene.
17) There are significant differences between the in vivo and ex vivo data. These
are not surprising but needs to be emphasized and explained. Experimental
confounders also need to be reported for example use of pretreatment ETOH
enemas in vivo, the use of polybrene ex vivo and 2-14 days vs 24 hrs.
Conclusion
1) Please make the conclusion section more succinct. Much of this is discussion.
This section should remain more focused in the conclusions drawn from the
experimental findings.
2) Paragraph 2 page 16 needs to be removed. The investigators do not show
data to support or refute this conclusion and so does not belong in this
manuscript.
3) Paragraph 2 page 18: Be careful drawing conclusions when comparing
tropism between the different experiments especially when comparing to other
studies using different viral vectors which clearly have different cellular and tissue
tropisms.
4) Paragraph 3 page 18: authors conclude that LV exhibited a remarkable ability
to transduce up to 50% of CD4 positive cell population. The conclusion should
not be so strong since the immunofluorescence images do not support such a
strong conclusion. If the authors would like to be certain I recommend isolation of
LPMCs and conducting flow experiments.
5) Paragraph 1 page 19: Please remove the hydrostatic pressure in the rectal
lumen comments as this was analyzed thus is speculative. Plus pipette
instillation of vector solution would likely not create enough pressure, unless
there is methodology that is missing from the manuscript. The ETOH
pretreatment more likely contributed to increased mucosal penetration into the
epithelium. It would be interesting to compare with and without ETOH
pretreatment.
6) Paragraph 2 page 17 and Paragraph 2 page 19: One cannot draw conclusions
on species tropism differences from these experiments given that murine explant
experiments and human rectal instillation experiments were not done. Cannot
compare the result from two different experiments to draw a conclusion.
Minor essential revisions:
Abstract
1) A second conclusion was that you demonstrated the differences between
model systems (in vivo vs ex vivo) in evaluating vector gene transfer.
Background
1) Last sentence first paragraph is same as in abstract and with same grammar
problem.
2) You provide references for non-viral vector, retroviral vector and adenoviral
vector history in GI transduction, but you do not reference AAV history in GI tract.
It would be appropriate to do so.
3) “limited by low packaging capacity” is misleading. I think you mean the limited
size of transgene insert?
Methods
1) It is customary to provide the names and source company for experimental
kits or chemicals if appropriate or mention that the items were made in the lab.
For example:
a. p24 ELISA kit or lab made?
b. Polybrene
c. All the antibodies in the IMF staining studies.
2) How was seepage prevented after enema?
3) What was the fixative in the murine tissues used in x-gal staining?
4) Are the anti-B-gal and anti-keratin & CD antibodies directly conjugated to
AF488 or was there a secondary involved? Describe the IMF staining technique.
Did you use antigen retrievals etc?
5) What microscope and software did you use to do the quantitative
morphometry and confocal analysis?
Results & Discussion
1) The manual counting of cells for the determination of transduction efficiency in
vector gene transfer has high inter-observer variability. This is an important issue
when it comes to reporting transduction efficiencies in an evolving field with
relatively low efficiencies. Computerized pixel counting or flow is best to
determine cell line transduction efficiency. Given the possibility of nonspecific
staining in intestinal cells a clear negative control needs to be shown or reported.
This should also be mentioned as a limitation.
2) Please report how this statement is determined: “The highest dose tested was
10ng p24 equivalent units of LV, which corresponds to a biological infectious titer
of 10^6 transducing units (TU) on a standardized cell line such as 293T”. Did you
determine this or is this accepted from a reference?
3) Was the luminescence (transgene expression) limited to the rectum because
the ETOH enema was limited to the rectum or the application of the vector was
limited to this area?
4) Given that LP immune cells are the major cell types that are transduced by LV,
it is important to report presence or absence of vector genome in mesenteric
lymph nodes. Please report or acknowledge that this information is not available.
5) Figure 7. If DAPI staining is present in all images appear modified as the blue
cannot be seen.
Conclusion
1) Paragraph 1 page 16: without local or systemic toxicity in vivo needs to be
changes to without macroscopic or systemic toxicity in vivo. Cytokines were not
measured and so drawing strong conclusions is difficulty.
2) Paragraph 3 page 16: The correlation between bioluminescence signal and
transgene expressing cell quantification is interesting. In review of ref #40
Hiraoka et al I could not see how this was achieved. Could you please expound
in the manuscript.
Level of interest: An article of importance in its field
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.
assistant