R-Spondin Mimetic, SZN-043, Induced Proliferation and Wnt Activity, Two Features Deficient in Human Alcohol-Associated Liver Disease

Reviewer #1 (Public review):

Summary:

The work by Fisher et al describes the role of novel RSPO mimetics in the activation of WNT signaling and hepatocyte regeneration. However, the results of the experiments and weaknesses of the methods used do not support the conclusions of the authors that the new therapy can promote liver regeneration in alcohol-induced liver cirrhosis.

Strengths:

Similarly to its precursor, aASGR1-RSPO2-RA-IgG, SZN-043 can upregulate Wnt target genes and promote hepatocyte proliferation in the liver.

Weaknesses:

(1) The authors rely on the expression of a single gene, CYP1A1, as a readout of Wnt/ß-catenin target gene expression. A more systemic evaluation of Wnt/ß-catenin activity should be performed.

(2) The lack of the mRNA upregulation of cell cycle genes is not sufficient to draw a conclusion of the impaired regeneration in cirrhotic livers.

(3) The authors present single-dose pharmacokinetic (PK) profile of SZN-04. It is not clear how that compares to its precursor, to justify better pharmacokinetic properties.

(4) The specificity of Wnt/ß-catenin activation should be evaluated in ß-catenin KO mice to show no target gene induction in the absence of ß-catenin.

(5) The authors demonstrated that the drug promoted hepatocyte proliferation. How it affects liver functional parameters in alcohol-fed mice, hepatocyte differentiation markers, albumin production, and coagulation factor synthesis is not clear.

(6) Female mice only were used for alcohol studies; the effect on the male mice needs to be evaluated as well.

(7) Alcohol feeding did not reduce Wnt/ß-catenin target gene expression in mice suggesting that it is a bad model to study the efficacy of the SZN-043 in alcohol-induced liver cirrhosis.

(8) The authors used CCl4-induced fibrosis as a model of ALD fibrosis. However, this is not a suitable fibrosis model for ALD studies. Adding alcohol to CCl4 treatment could potentially address this issue. Alternatively, the authors should use an ALD model that produces significant fibrosis.

(9) Sex for the CCl4-treated mice is not indicated.

(10) Histology and fibrosis assessment data for alcohol-fed mice should be presented.

(11) The rationale for using 13.5-month-old aging mice for alcohol studies and immunodeficient mice only for CCl4 studies is not clear.

Reviewer #2 (Public review):

Summary:

The study by Fisher et al investigates a therapeutic role for SZN-043, a hepatocyte-targeted R-spondin mimetic, for its potential role in restoring Wnt signaling and promoting liver regeneration in alcohol-associated liver disease (ALD). Using multiple preclinical models, the compound was shown to promote hepatocyte proliferation and reduce fibrosis. This study highlights the efficacy of promoting liver regeneration while maintaining controlled signaling. Limitations include a need for further exploration of off-target effects and fibrosis mechanisms. The findings support SZN-043 as a promising candidate for ALD therapy, warranting further clinical evaluation. This is a well-designed study with thorough investigation using multiple disease models.

Strengths:

(1) Well-written manuscript with clear design, robust methods, and discussion.

(2) Using multiple models strengthens the findings and expands beyond ALD.

(3) Identification of SZN-043 as a novel potent drug for liver regeneration.

Weaknesses:

(1) The introduction needs to be re-structured with an emphasis on liver regeneration. It seems that the entire manuscript is focused on liver regeneration, however, only the last two sentences or so describe liver regeneration. The frequency of liver transplants owing to a reduced ability for liver regeneration in AH patients needs to be highlighted.

(2) In Figure 4, it appears that the humanized mice liver was injected with the SZN-043. Is it possible that using a partial hepatectomy model will be beneficial for assessing the effects of SZN-043 rather than using them in mice without any hepatocyte damage?

(3) Figure 4B. Panel 3 has 10mpk merged inside the figure. Please correct this.

(4) Figure 4B. DAPI staining will be vital to show the Ki67 staining specific to hepatocytes (at least visually we can do co-localization with a double nucleus in each cell). The current image shows some cells show Ki67 staining which shows some cells which are not binuclear.

(5) The alcohol feeding was performed for 8 weeks and is described as NIAAA model in the methods section. NIAAA model is 11 days of alcohol+ one binge. Please correct this or clarify it in the methods section, as this is not reflected. ASGR1 may be also expressed by macrophages so it's important to show the specificity.

(6) Is it possible that the SZN-043 also has effect on macrophages promoting an anti-inflammatory state? This should be discussed.

(7) Potential off-target effects of SZN-043, particularly in stellate cell activation in the context of fibrosis should be discussed.

(8) Discuss the limitations of current models and how they might influence the interpretation of the results.

(9) Clearly explain how SZN-043 overcomes limitations of prior RSPO-based therapies.