Advances with RNAi-Based Therapy for Hepatitis B Virus Infection

Round 1

Reviewer 1 Report

line 10   current HBV estimate from Polaris WHO is 292 M people who are HBsAg+

line 14 HBV needs to be treated also to reduce infectivity 

lin 16 is there any peer reviewed publication that shows that any patient infected with HBV ever clears completely cccDNA ? I am not able to find any document of this being cleared even with acute HBV (Protzer 1996)

also line 86

Line 253 discuss proposed GTJ and recombinants and how that may effect iRNA targeting

expand on GT A1 and GT A2 very different viruses with different interferon responses

line 359 need to discuss melittin toxicity in primates and the end of the ARC 520 development expand on the dual deliver system used by Arrowhead including using the cholesterol receptor

need to include ARC 521 data and the new ARO program and the transition of the 521 project to Janssen, as well as expand on the data on combination therapy including the large phase IIb trial with triple combination therapy that is ongoing

the development of iRNA helped researchers reach profound insights into the activity of integrated HBV DNA producing truncated HBsAg mRNA transcripts and that integrants were producing major amounts of HBsAg

VIR took over the Alnylam project and is entering phase II

Arbutus has had iRNA data published, inclusion here would be useful

the authors really need to emphasize that combination therapy to reach a sterlizing cure with removal of all integrants is key

Author Response

Revised manuscript submitted to Viruses (Ms. Ref. No.: Viruses-868620): Advances with RNAi-based therapy for hepatitis B virus infection, by Fiona van den Berg et al

Thank you for the feedback on our manuscript which is being considered for publication in Viruses. In response to the helpful comments of the two reviewers, we have made revisions to the submission. Changes in the revised manuscript are tracked and the ‘point by point’ responses are provided below. 

Reviewer 1

Comment 1: line 10   current HBV estimate from Polaris WHO is 292 M people who are HBsAg+

Authors’ Response: We have updated the current estimate of HBV prevalence. (Because this appears in the Abstract, the information is not cited with a reference.)

Comment 2: line 14 HBV needs to be treated also to reduce infectivity

Authors’ Response: We have added that current drug treatments also “reduce infectivity”.

Comment 3: lin 16 is there any peer reviewed publication that shows that any patient infected with HBV ever clears completely cccDNA ? I am not able to find any document of this being cleared even with acute HBV (Protzer 1996)

also line 86

Authors’ Response: This is a good comment and we have adjusted the text to reflect the concern. Importantly, exact assessment of elimination of cccDNA from HBV-infected individuals is difficult to carry out. As discussed later in the manuscript, assays for cccDNA are not yet reliable and accurately quantitative. It has been thought that most acutely infected individuals eliminate cccDNA when all markers of the virus become negative. However there may well be residual presence of the cccDNA minichromosome in individuals who have apparently eliminated the infection. Another point is that performing a liver biopsy on recovered HBV-infected individuals is considered unethical. The procedure is invasive and carries risks of complications.

Along similar lines the text of line 107, which describes the action of interferon alpha, has also been modified to indicate that the treatment has been associated with HBsAg seroconversion rather than elimination of cccDNA, which as the reviewer indicates has not been established conclusively.

Comment 4: Line 253 discuss proposed GTJ and recombinants and how that may effect iRNA targeting

expand on GT A1 and GT A2 very different viruses with different interferon responses

Authors’ Response: As discussed in this section, “sequence variation among genotypes and subgenotypes could influence silencing efficacy. Given the sensitivity of gene silencers to target sequence changes, designing siRNAs, shRNAs and artificial micro RNAs (amiRNAs) against the conserved sites is essential to ensure activity across multiple genotypes”. As RNAi is sequence-specific, this statement applies to all genotypes and subgenotypes, including the potential genotype J and subgenotypes A1 and A2, irrespective of different clinical manifestations. This statement has been moved to the start of the section for emphasis. We have clarified that “subgenotype A1 predominates in Africa, whereas subgenotype A2 is mainly found in Europe.”

Comment 5: line 359 need to discuss melittin toxicity in primates and the end of the ARC 520 development

expand on the dual deliver system used by Arrowhead including using the cholesterol receptor

Authors’ Response: The following has been added at this point in the text:

“A similar two-vial formulation was used in the well-known clinical trial candidate ARC-520 (Arrowhead Pharmaceuticals), in which two distinct cholesterol-conjugated siRNAs were mixed with NAG-MLP prior to injection. ARC-520 initially showed promising preclinical and clinical results [100,101], but lethal toxicity of the EX1 dynamic polyconjugate (DPC) delivery vehicle, a version of NAG-MLP, in a related safety study in non-human primates led to the discontinuation of the ARC-520 clinical trial (Table 1).”

Comment 6: need to include ARC 521 data and the new ARO program and the transition of the 521 project to Janssen, as well as expand on the data on combination therapy including the large phase IIb trial with triple combination therapy that is ongoing

the development of iRNA helped researchers reach profound insights into the activity of integrated HBV DNA producing truncated HBsAg mRNA transcripts and that integrants were producing major amounts of HBsAg

VIR took over the Alnylam project and is entering phase II

Arbutus has had iRNA data published, inclusion here would be useful

Authors’ Response: These are good comments that we have addressed by updating and expanding information presented in Section 7.3 on clinical trials. The focus has however been on dealing with published data. Table 1 has been edited to explain information better. Specifics are indicated below.

1. Details on ARC520, ARC-521 and ARO-HBV have been expanded, including the collaboration with Janssen Pharmaceuticals. The clinical trials involving ARO-HBV combinations have also been included.
2. The contribution of HBsAg from integrated HBV copies has been emphasised and details from the relevant references (Woodell, et al., 2017) (yuen, et al., 2019) have been included.

• “These studies showed the previously underappreciated contribution of HBsAg expression from integrated copies of the HBV genome, as opposed to cccDNA, and highlighted the need for additional siRNAs capable of targeting all HBV transcripts, regardless of origin.”

2218. The Vir Biotechnology / Alnylam Pharmaceuticals collaboration has been indicated for VIR-2218.
2219. The preliminary Arbutus data has been included.

Comment 7: the authors really need to emphasize that combination therapy to reach a sterlizing cure with removal of all integrants is key

Authors’ Response: This valid point has now been emphasized in section 3 (lines 88-90)

Reviewer 2 Report

In this review, the author summarized different RNAi-based approaches to target HBV.

The manuscript is well written. Below are few comments:

• There are some issues with the labelling of the figures. Indeed, in page 2, it is indicated several times to go to Figure 1 but Figure 1 is not the HBV life cycle. Page 5, figures xB and xC et cD are mentioned but not displayed in the manuscript…
• I would rearrange the different part of the manuscript to present everything on HBV first (parts 2, 3, beginning of 5 and 6), then RNAi principle and delivery to finally talk about RNAi and HBV.
• The part on HBV and RNAi, that is actually the purpose of the review, is small compared to the others. Descriptions of the different clinicals trials is very short and very limited in vitro data have been included.
• Some tables or figures to summarize the different biochemical modifications of the siRNA as well as delivery strategies would help the reader.
• IFN alpha or TGF beta are not correctly written (probably due to informatic issues).
• Lines 149, an additional “re” is present.

Author Response

Revised manuscript submitted to Viruses (Ms. Ref. No.: Viruses-868620): Advances with RNAi-based therapy for hepatitis B virus infection, by Fiona van den Berg et al

Thank you for the feedback on our manuscript which is being considered for publication in Viruses. In response to the helpful comments of the two reviewers, we have made revisions to the submission. Changes in the revised manuscript are tracked and the ‘point by point’ responses are provided below. 

Reviewer 2

In this review, the author summarized different RNAi-based approaches to target HBV.

The manuscript is well written. Below are few comments:

Comment 1: There are some issues with the labelling of the figures. Indeed, in page 2, it is indicated several times to go to Figure 1 but Figure 1 is not the HBV life cycle. Page 5, figures xB and xC et cD are mentioned but not displayed in the manuscript

Authors’ Response: We apologize for the error and the order and labelling of Figures 1 and 2 in the text has been corrected.

Comment 2: I would rearrange the different part of the manuscript to present everything on HBV first (parts 2, 3, beginning of 5 and 6), then RNAi principle and delivery to finally talk about RNAi and HBV. The part on HBV and RNAi, that is actually the purpose of the review, is small compared to the others.

Authors’ Response: We feel that each section of the manuscript is written in the context of RNAi treatments for HBV. Our aim has been to interweave discussion of these two topics in the manuscript to provide adequate substance to each of the themes. We feel that re-arranging the sections would disrupt the clarity and context that has been provided in earlier sections of the current format.

Comment 3: Descriptions of the different clinicals trials is very short and very limited in vitro data have been included.

Authors’ Response: Section 7.3 on clinical trials has been updated and expanded to include additional details, but with a focus on published data. This point is similar to that of reviewer 1 (Comment 6).

Comment 4: Some tables or figures to summarize the different biochemical modifications of the siRNA as well as delivery strategies would help the reader.

Authors’ Response: Including additional Table and Figure is probably not necessary as it would duplicate what appears in the text. Also these topics have been dealt with comprehensively in specialised reviews on delivery and chemical modifications of siRNAs.

Comment 5: IFN alpha or TGF beta are not correctly written (probably due to informatic issues).

Authors’ Response: This has been corrected on our side but should be checked again in the final proof.

Comment 6: Lines 149, an additional “re” is present.

Authors’ Response: This has been corrected to “are”.

We thank the reviewers for their critiques. Responding to the points has improved the manuscript and we hope that the revised version is acceptable for publication in Viruses.

Round 2

Reviewer 1 Report

full response to the previous review appreciated