Reviewer's report
Title:Effective in vivo and ex vivo gene transfer to intestinal mucosa by
VSV-G-pseudotyped lentiviral vectors
Version: 2Date:17 August 2009
Reviewer: Kenneth Cornetta
Reviewer's report:
The manuscript by Matsumoto et al. is an interesting paper that seeks to obtain
gene transfer into colon tissue using lentiviral gene transfer. The paper is
succinct and well written. In general the data appears to be well presented and
solid, although the conclusions do not always follow the data (discussed below).
Overall comments:
This is a preliminary study to assess gene transfer. The paper would be
strengthened if there were evidence of a practical application in a relevant model.
Nevertheless, these types of preliminary studies are required before moving to a
disease model and the authors have done a careful study of gene transfer into
both mucosal and lymphoid tissues.
The authors found most gene transfer in the human explants was in cells of
hematopoietic origin. They present reasonable explanations for this in the
conclusions, but until these issues are settled we do not know if this will be a
viable method for gene transfer into the non-hematopoietic human colonic tissue.
Major Concerns:
1) Throughout the paper the authors claim the study demonstrates “safety” of this
method (Abstract conclusions, last 2 paragraph of the conclusions). This is an
overstatement; lack of adverse events in animals and some human explants
does not establish safety. No adverse events were seen in a limited number of
animals more precisely describes the data.
2) One explanation for the qPCR results in Fig. 3C is plasmid contamination of
the vector preparation. Vector made by transient transfection typically have
significant plasmid residual. Was the vector treated with DNAse prior to injection?
If not this possibility should be explained in the paper as a confounding issue, or
the samples should be retested for plasmid sequences (ex. ampicillin resistance
gene sequences) to assess for this possibility.
3) The “average copy number” per 300 ng of genomic DNA is presented, but the
authors should also estimate the number of integrations per cell so readers can
better estimate the clinical relevance of the gene transfer obtained.
4) The authors make several mentions that CD4 cells were particularly
susceptible to gene transfer. This is addressed briefly in the conclusions, but the
reason why CD4 and not CD8 is transduced is not explained, nor is there an
explanation of the clinical implications of this finding.
Minor Essential Concerns
1) In the second paragraph of the Background the authors claim that AAV vector
cannot be made at high titer. I do not believe this is an accurate comment and
should be referenced if they wish to make this point.
2) Fig 3C legend does not make sense.
Discretionary
Fig 1A could be simplified by removing all but the transgene diagram. Figure 1 B
is not needed. Figure 6 could be eliminated and the data put into the text.
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 am a founding member of Rimedion Inc. but there is no direct conflict to this
manuscript.
Reviewer's report
Title:Effective in vivo and ex vivo gene transfer to intestinal mucosa by
VSV-G-pseudotyped lentiviral vectors
Version: 2Date:17 August 2009
Reviewer: Kenneth Cornetta
Reviewer's report:
The manuscript by Matsumoto et al. is an interesting paper that seeks to obtain
gene transfer into colon tissue using lentiviral gene transfer. The paper is
succinct and well written. In general the data appears to be well presented and
solid, although the conclusions do not always follow the data (discussed below).
Overall comments:
This is a preliminary study to assess gene transfer. The paper would be
strengthened if there were evidence of a practical application in a relevant model.
Nevertheless, these types of preliminary studies are required before moving to a
disease model and the authors have done a careful study of gene transfer into
both mucosal and lymphoid tissues.
The authors found most gene transfer in the human explants was in cells of
hematopoietic origin. They present reasonable explanations for this in the
conclusions, but until these issues are settled we do not know if this will be a
viable method for gene transfer into the non-hematopoietic human colonic tissue.
Major Concerns:
1) Throughout the paper the authors claim the study demonstrates “safety” of this
method (Abstract conclusions, last 2 paragraph of the conclusions). This is an
overstatement; lack of adverse events in animals and some human explants
does not establish safety. No adverse events were seen in a limited number of
animals more precisely describes the data.
2) One explanation for the qPCR results in Fig. 3C is plasmid contamination of
the vector preparation. Vector made by transient transfection typically have
significant plasmid residual. Was the vector treated with DNAse prior to injection?
If not this possibility should be explained in the paper as a confounding issue, or
the samples should be retested for plasmid sequences (ex. ampicillin resistance
gene sequences) to assess for this possibility.
3) The “average copy number” per 300 ng of genomic DNA is presented, but the
authors should also estimate the number of integrations per cell so readers can
better estimate the clinical relevance of the gene transfer obtained.
4) The authors make several mentions that CD4 cells were particularly
susceptible to gene transfer. This is addressed briefly in the conclusions, but the
reason why CD4 and not CD8 is transduced is not explained, nor is there an
explanation of the clinical implications of this finding.
Minor Essential Concerns
1) In the second paragraph of the Background the authors claim that AAV vector
cannot be made at high titer. I do not believe this is an accurate comment and
should be referenced if they wish to make this point.
2) Fig 3C legend does not make sense.
Discretionary
Fig 1A could be simplified by removing all but the transgene diagram. Figure 1 B
is not needed. Figure 6 could be eliminated and the data put into the text.
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 am a founding member of Rimedion Inc. but there is no direct conflict to this
manuscript.
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 August 2009
Reviewer: Kenneth Cornetta
Reviewer's report:
The manuscript by Matsumoto et al. is an interesting paper that seeks to obtain
gene transfer into colon tissue using lentiviral gene transfer. The paper is
succinct and well written. In general the data appears to be well presented and
solid, although the conclusions do not always follow the data (discussed below).
Overall comments:
This is a preliminary study to assess gene transfer. The paper would be
strengthened if there were evidence of a practical application in a relevant model.
Nevertheless, these types of preliminary studies are required before moving to a
disease model and the authors have done a careful study of gene transfer into
both mucosal and lymphoid tissues.
The authors found most gene transfer in the human explants was in cells of
hematopoietic origin. They present reasonable explanations for this in the
conclusions, but until these issues are settled we do not know if this will be a
viable method for gene transfer into the non-hematopoietic human colonic tissue.
Major Concerns:
1) Throughout the paper the authors claim the study demonstrates “safety” of this
method (Abstract conclusions, last 2 paragraph of the conclusions). This is an
overstatement; lack of adverse events in animals and some human explants
does not establish safety. No adverse events were seen in a limited number of
animals more precisely describes the data.
2) One explanation for the qPCR results in Fig. 3C is plasmid contamination of
the vector preparation. Vector made by transient transfection typically have
significant plasmid residual. Was the vector treated with DNAse prior to injection?
If not this possibility should be explained in the paper as a confounding issue, or
the samples should be retested for plasmid sequences (ex. ampicillin resistance
gene sequences) to assess for this possibility.
3) The “average copy number” per 300 ng of genomic DNA is presented, but the
authors should also estimate the number of integrations per cell so readers can
better estimate the clinical relevance of the gene transfer obtained.
4) The authors make several mentions that CD4 cells were particularly
susceptible to gene transfer. This is addressed briefly in the conclusions, but the
reason why CD4 and not CD8 is transduced is not explained, nor is there an
explanation of the clinical implications of this finding.
Minor Essential Concerns
1) In the second paragraph of the Background the authors claim that AAV vector
cannot be made at high titer. I do not believe this is an accurate comment and
should be referenced if they wish to make this point.
2) Fig 3C legend does not make sense.
Discretionary
Fig 1A could be simplified by removing all but the transgene diagram. Figure 1 B
is not needed. Figure 6 could be eliminated and the data put into the text.
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 am a founding member of Rimedion Inc. but there is no direct conflict to this
manuscript.
assistant