Reviewer's report
Title:Hepatitis B Virus Inhibition in Mice by Lentiviral Vector Mediated Small
Interference RNA
Version: 1Date:25 May 2009
Reviewer: Anja Ehrhardt
Reviewer's report:
The manuscript by Deng et al. describes lentivirus-based siRNA delivery against
hepatitis B virus (HBV) infection. The authors explored two siRNAs against HBV
expressed from a lentiviral vector in a transient mouse model for HBV infection.
In order to establish the system Deng et al. used hydrodynamic injection for
co-delivery of siRNA expressing lentiviruses and a plasmid containing the HBV
genome. They detected a significant reduction of parameters specific for HBV
infection. Reduction of viral transcripts, viral DNA and viral antigens are shown.
They conclude that lentiviruses may be a valuable therapeutic alternative for
delivery of siRNAs against HBV.
The designed experiments are straight forward and clear to the reader. However,
for publication the authors need to address the following issues.
Major Compulsory Revisions:
• What was the rational for using hydrodynamic delivery of the siRNA expressing
lentiviruses? Also, I was wondering whether there is any previous literature on
performance of lentiviral vectors after hydrodynamic delivery. In general, the
purpose of using viruses for liver-directed gene transfer is to minimize damage of
the target organ. Therefore, I was wondering, whether the authors have tried to
sequentially inject the plasmid pTHBV2 and the lentiviral vector? The authors
may inject the plasmid first and a few hours later or the next day the siRNA
expressing lentivirus. This is a more relevant experiment because the virus would
be injected in a therapeutically relevant manner. For instance, have the authors
investigated transduction efficiencies in mouse liver (percentage of transduced
hepatocytes) and duration of transgene expression after high pressure tail vein
injection of a lentiviral vector? Has anyone analyzed integration efficiencies of
lentiviral vectors after hydrodynamic delivery? Moreover, have the authors
considered quantifying and analyzing the molecular status of lentiviral vector
genomes after hydrodynamic delivery? This could be accomplished by
quantitative PCR and/or Southern blot analysis. Any of this information would
make the manuscript much stronger.
• The authors should provide a Northern blot showing siRNA expression from the
lentiviral vectors on days 1, 4, and 7 post-injection. Levels of siRNA expression
at various time points post-injection will provide important insights into this
therapeutic strategy.
• What are the actual levels expressed in ng per ml of serum HBsAg after
injection of 12 µg of the plasmid pTHBV2? The authors should provide more
information about the calculation of the S/N value. What is the cut off of this
measurement?
• Were the siRNA sequences already used in previous studies and were they
tested in vitro? What was the rational for choosing these two siRNA sequences?
• The authors should add a figure which schematically shows DNA sequences
contained in all lentiviral vectors used in this study. This could be a subfigure of
figure 1.
• There are some results the authors decided not to show. However, for instance
siRNA-mediated HBV DNA reduction should be depicted in an appropriate figure.
This is important information which needs to be included in the revised version of
the manuscript.
Minor Essential Revisions:
• The introduction section should provide a brief summary of alternatively used
transfer vehicles for in vivo delivery of siRNAs against HBV infection. Besides
non-viral approaches AAV vectors and adenoviral vectors were also explored for
delivery of siRNA against HBV replication.
• The last sentence in the introduction section needs to be rewritten.
• In general, I would suggest that the manuscript should be proof-read by a native
English speaking person.
• The discussion section should also discuss limitations of this approach. For
example potential insertional mutagenesis may occur using an integrating viral
vector or potential liver damage due to vector-related toxic side effects may
occur.
• Materials and methods section, “Lentivirus production and transduction”: the
authors state that the method used for determination of viral titers was described
previously. However, no reference was provided.
• Materials and methods section, “Animal experiments”: the paragraph in which
the authors describe how much virus and plasmid was used for hydrodynamic
delivery needs to be rewritten. This is an essential method for this study and
should be clear to the reader.
• Materials and methods section, “Quantitative real-time PCR assay”: instead of
providing an additional table for primer sequences the authors should include this
information directly into the materials and methods section.
• It is confusing for the reader that the naming of the lentiviral vectors starts with
the letter “p” (e.g pWPT-GFP-siRNA-1). Is this a plasmid (“p”) or the actual virus?
• The authors should carefully go through all their references. The numbering of
various references in the text is not consistent with listed references at the end of
the manuscript. Here are just two examples: reference 8 does not show that HBV
infection is reduced by RNA interference. Moreover, in the materials and
methods section the manuscript by Yang et al. is listed as reference 43 and not
46.
Level of interest: An article of importance in its field
Quality of written English: Needs some language corrections before being
published
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests:
I declare that I have no competing interests.
Reviewer's report
Title:Hepatitis B Virus Inhibition in Mice by Lentiviral Vector Mediated Small
Interference RNA
Version: 1Date:25 May 2009
Reviewer: Anja Ehrhardt
Reviewer's report:
The manuscript by Deng et al. describes lentivirus-based siRNA delivery against
hepatitis B virus (HBV) infection. The authors explored two siRNAs against HBV
expressed from a lentiviral vector in a transient mouse model for HBV infection.
In order to establish the system Deng et al. used hydrodynamic injection for
co-delivery of siRNA expressing lentiviruses and a plasmid containing the HBV
genome. They detected a significant reduction of parameters specific for HBV
infection. Reduction of viral transcripts, viral DNA and viral antigens are shown.
They conclude that lentiviruses may be a valuable therapeutic alternative for
delivery of siRNAs against HBV.
The designed experiments are straight forward and clear to the reader. However,
for publication the authors need to address the following issues.
Major Compulsory Revisions:
• What was the rational for using hydrodynamic delivery of the siRNA expressing
lentiviruses? Also, I was wondering whether there is any previous literature on
performance of lentiviral vectors after hydrodynamic delivery. In general, the
purpose of using viruses for liver-directed gene transfer is to minimize damage of
the target organ. Therefore, I was wondering, whether the authors have tried to
sequentially inject the plasmid pTHBV2 and the lentiviral vector? The authors
may inject the plasmid first and a few hours later or the next day the siRNA
expressing lentivirus. This is a more relevant experiment because the virus would
be injected in a therapeutically relevant manner. For instance, have the authors
investigated transduction efficiencies in mouse liver (percentage of transduced
hepatocytes) and duration of transgene expression after high pressure tail vein
injection of a lentiviral vector? Has anyone analyzed integration efficiencies of
lentiviral vectors after hydrodynamic delivery? Moreover, have the authors
considered quantifying and analyzing the molecular status of lentiviral vector
genomes after hydrodynamic delivery? This could be accomplished by
quantitative PCR and/or Southern blot analysis. Any of this information would
make the manuscript much stronger.
• The authors should provide a Northern blot showing siRNA expression from the
lentiviral vectors on days 1, 4, and 7 post-injection. Levels of siRNA expression
at various time points post-injection will provide important insights into this
therapeutic strategy.
• What are the actual levels expressed in ng per ml of serum HBsAg after
injection of 12 µg of the plasmid pTHBV2? The authors should provide more
information about the calculation of the S/N value. What is the cut off of this
measurement?
• Were the siRNA sequences already used in previous studies and were they
tested in vitro? What was the rational for choosing these two siRNA sequences?
• The authors should add a figure which schematically shows DNA sequences
contained in all lentiviral vectors used in this study. This could be a subfigure of
figure 1.
• There are some results the authors decided not to show. However, for instance
siRNA-mediated HBV DNA reduction should be depicted in an appropriate figure.
This is important information which needs to be included in the revised version of
the manuscript.
Minor Essential Revisions:
• The introduction section should provide a brief summary of alternatively used
transfer vehicles for in vivo delivery of siRNAs against HBV infection. Besides
non-viral approaches AAV vectors and adenoviral vectors were also explored for
delivery of siRNA against HBV replication.
• The last sentence in the introduction section needs to be rewritten.
• In general, I would suggest that the manuscript should be proof-read by a native
English speaking person.
• The discussion section should also discuss limitations of this approach. For
example potential insertional mutagenesis may occur using an integrating viral
vector or potential liver damage due to vector-related toxic side effects may
occur.
• Materials and methods section, “Lentivirus production and transduction”: the
authors state that the method used for determination of viral titers was described
previously. However, no reference was provided.
• Materials and methods section, “Animal experiments”: the paragraph in which
the authors describe how much virus and plasmid was used for hydrodynamic
delivery needs to be rewritten. This is an essential method for this study and
should be clear to the reader.
• Materials and methods section, “Quantitative real-time PCR assay”: instead of
providing an additional table for primer sequences the authors should include this
information directly into the materials and methods section.
• It is confusing for the reader that the naming of the lentiviral vectors starts with
the letter “p” (e.g pWPT-GFP-siRNA-1). Is this a plasmid (“p”) or the actual virus?
• The authors should carefully go through all their references. The numbering of
various references in the text is not consistent with listed references at the end of
the manuscript. Here are just two examples: reference 8 does not show that HBV
infection is reduced by RNA interference. Moreover, in the materials and
methods section the manuscript by Yang et al. is listed as reference 43 and not
46.
Level of interest: An article of importance in its field
Quality of written English: Needs some language corrections before being
published
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests:
I declare that I have no competing interests.
Please review this paper.
user
Reviewer's report
Title:Hepatitis B Virus Inhibition in Mice by Lentiviral Vector Mediated Small
Interference RNA
Version: 1Date:25 May 2009
Reviewer: Anja Ehrhardt
Reviewer's report:
The manuscript by Deng et al. describes lentivirus-based siRNA delivery against
hepatitis B virus (HBV) infection. The authors explored two siRNAs against HBV
expressed from a lentiviral vector in a transient mouse model for HBV infection.
In order to establish the system Deng et al. used hydrodynamic injection for
co-delivery of siRNA expressing lentiviruses and a plasmid containing the HBV
genome. They detected a significant reduction of parameters specific for HBV
infection. Reduction of viral transcripts, viral DNA and viral antigens are shown.
They conclude that lentiviruses may be a valuable therapeutic alternative for
delivery of siRNAs against HBV.
The designed experiments are straight forward and clear to the reader. However,
for publication the authors need to address the following issues.
Major Compulsory Revisions:
• What was the rational for using hydrodynamic delivery of the siRNA expressing
lentiviruses? Also, I was wondering whether there is any previous literature on
performance of lentiviral vectors after hydrodynamic delivery. In general, the
purpose of using viruses for liver-directed gene transfer is to minimize damage of
the target organ. Therefore, I was wondering, whether the authors have tried to
sequentially inject the plasmid pTHBV2 and the lentiviral vector? The authors
may inject the plasmid first and a few hours later or the next day the siRNA
expressing lentivirus. This is a more relevant experiment because the virus would
be injected in a therapeutically relevant manner. For instance, have the authors
investigated transduction efficiencies in mouse liver (percentage of transduced
hepatocytes) and duration of transgene expression after high pressure tail vein
injection of a lentiviral vector? Has anyone analyzed integration efficiencies of
lentiviral vectors after hydrodynamic delivery? Moreover, have the authors
considered quantifying and analyzing the molecular status of lentiviral vector
genomes after hydrodynamic delivery? This could be accomplished by
quantitative PCR and/or Southern blot analysis. Any of this information would
make the manuscript much stronger.
• The authors should provide a Northern blot showing siRNA expression from the
lentiviral vectors on days 1, 4, and 7 post-injection. Levels of siRNA expression
at various time points post-injection will provide important insights into this
therapeutic strategy.
• What are the actual levels expressed in ng per ml of serum HBsAg after
injection of 12 µg of the plasmid pTHBV2? The authors should provide more
information about the calculation of the S/N value. What is the cut off of this
measurement?
• Were the siRNA sequences already used in previous studies and were they
tested in vitro? What was the rational for choosing these two siRNA sequences?
• The authors should add a figure which schematically shows DNA sequences
contained in all lentiviral vectors used in this study. This could be a subfigure of
figure 1.
• There are some results the authors decided not to show. However, for instance
siRNA-mediated HBV DNA reduction should be depicted in an appropriate figure.
This is important information which needs to be included in the revised version of
the manuscript.
Minor Essential Revisions:
• The introduction section should provide a brief summary of alternatively used
transfer vehicles for in vivo delivery of siRNAs against HBV infection. Besides
non-viral approaches AAV vectors and adenoviral vectors were also explored for
delivery of siRNA against HBV replication.
• The last sentence in the introduction section needs to be rewritten.
• In general, I would suggest that the manuscript should be proof-read by a native
English speaking person.
• The discussion section should also discuss limitations of this approach. For
example potential insertional mutagenesis may occur using an integrating viral
vector or potential liver damage due to vector-related toxic side effects may
occur.
• Materials and methods section, “Lentivirus production and transduction”: the
authors state that the method used for determination of viral titers was described
previously. However, no reference was provided.
• Materials and methods section, “Animal experiments”: the paragraph in which
the authors describe how much virus and plasmid was used for hydrodynamic
delivery needs to be rewritten. This is an essential method for this study and
should be clear to the reader.
• Materials and methods section, “Quantitative real-time PCR assay”: instead of
providing an additional table for primer sequences the authors should include this
information directly into the materials and methods section.
• It is confusing for the reader that the naming of the lentiviral vectors starts with
the letter “p” (e.g pWPT-GFP-siRNA-1). Is this a plasmid (“p”) or the actual virus?
• The authors should carefully go through all their references. The numbering of
various references in the text is not consistent with listed references at the end of
the manuscript. Here are just two examples: reference 8 does not show that HBV
infection is reduced by RNA interference. Moreover, in the materials and
methods section the manuscript by Yang et al. is listed as reference 43 and not
46.
Level of interest: An article of importance in its field
Quality of written English: Needs some language corrections before being
published
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests:
I declare that I have no competing interests.
assistant