Human HPSE2 gene transfer ameliorates bladder pathophysiology in a mutant mouse model of urofacial syndrome

Reviewer #1 (Public Review):

Summary:

The authors try to use a gene therapy approach to cure urofacial symptoms in an HSPE2 mutant mouse model.

Strengths:

The authors have convincingly shown the expression of AAV9/HSPE2 in pelvic ganglion and liver tissues. They have also shown the defects in urethra relaxation and bladder muscle contraction in response to EFS in mutant mice, which were reversed in treated mice.

Weaknesses:

Some important and interesting data are missing. For example, whether the gene therapy can extend the life span of these mutants? The overall in vivo voiding function is missing. AAV9/HSPE2 expression in the bladder wall is not shown.

Reviewer #2 (Public Review):

In this study, Lopes and colleagues provide convincing evidence to support the potential for gene therapy to restore expression of heparanase-2 (Hpse2) in mice mutant for this gene, as occurs in urofacial syndrome. Beyond symptomatic relief for the consequences of outlet obstruction that results from Hpse2 mutation, no treatments exist. Building on prior studies describing the nature of urinary tract dysfunction in Hpse2 mutant mice, the authors applied a gene therapy approach to determine whether gene replacement could be achieved, and if so, whether restoration of HPSE2 expression could mitigate the urinary tract dysfunction and present a potential cure. Using an AAV9 viral vector encoding HPSE2, the authors performed gene replacement in neonatal wild-type or Hpse2 mutant mice and determined gene and protein expression as well as the impact on bladder outflow tract and bladder body physiology in juvenile mice. In addition to dose-dependent transduction of liver and pelvic ganglia (that innervate the bladder) with HPSE2, and demonstration of increased HPSE2 protein in Hpse2 mutant mice, the authors showed restoration of nerve-evoked outflow tract relaxation and bladder body contraction, both of which were deficient in mutant mice. They also showed that the viral vector-based approach was not deleterious to weight gain or to liver morphology. Based on these findings the authors concluded that AAV9-based HPSE2 replacement is feasible and safe, mitigates the physiological deficits in outflow tract and bladder tissue from Hpse2 mutant mice, and provides a foundation for gene replacement approaches for other genes implicated in lower urinary tract disorders.

Strengths include a rigorous experimental design, solid data in support of the conclusions, and a discussion of the limitations of the approach.

Weaknesses include a lack of discussion of the basis for differences in carbachol sensitivity in Hpse2 mutant mice, limited discussion of bladder tissue morphology in Hpse2 mutant mice, some questions over the variability of the functional data, and a need for clarification on the presentation of statistical significance of functional data

Reviewer #3 (Public Review):

Summary:

This is a really interesting study, looking at the efficacy of AAV-mediated delivery of wt HSPE2 gene into mouse mutants with the goal of rescuing lower urinary tract defects.

Strengths: Nice analysis of muscle physiology ex vivo, interesting approach.

Weaknesses: lack of rigor (see below). This is an awesome opportunity to learn much more about the disease, its affects on neurons, muscle, etc.

* Single-cell analysis of mutants versus control bladder, urethra including sphincter. This would be great also for the community.

* Detailed tables showing data from each mouse examined.

* Survival curves.

* Use of measurements that are done in vivo (spot assay for example). This sounds relatively simple.

* Assessment of viral integration in tissues besides the liver (could be done by QPCR).

* Discuss subtypes of neurons that are present and targeted in the context of mutants and controls.

Author Response

Our responses to the reviewers to go into the published pre-print. We thank the reviewers for their encouraging and thoughtful comments. These are good points that we would like to comment on as follows:

Reviewer 1:

Some important and interesting data are missing. For example, whether the gene therapy can extend the life span of these mutants? The overall in vivo voiding function is missing. AAV9/HSPE2 expression in the bladder wall is not shown.

A. Our study was not designed to determine whether gene therapy can improve life span of the Hpse2 mutant mice. We know that the mutant mice usually become ill after the first month of life and can die. However, we wanted to study the mice when they were generally well so that there would be no confounding effects on the bladder physiology caused by general ill health. Indeed, a recent study of Hpse2 inducible deletion in adult mice has shown evidence of exocrine pancreatic insufficiency (Kayal et al., PMID 37491420). We are currently exploring the status of the pancreas in our non-conditional juvenile Hpse2 mice, and whether gene transfer into the pancreas is possible.

B. We strongly agree that in vivo voiding studies will be important it the future, and suggest in vivo cystometry is the gold standard for this but is currently beyond the remit of this study.

C. It is correct that in this paper we have focussed on gene transduction into the pelvic ganglia, because the evidence is mounting that this is a neurogenic disease. Our ex vivo physiological studies show predominantly neurogenic defects that are corrected by the gene therapy. A detailed study of the bladder body is an interesting idea, in terms of possible transgene expression and detailed histology, and is something we will pursue in future studies.

Review 2:

Weaknesses include a lack of discussion of the basis for differences in carbachol sensitivity in Hpse2 mutant mice, limited discussion of bladder tissue morphology in Hpse2 mutant mice, some questions over the variability of the functional data, and a need for clarification on the presentation of statistical significance of functional data.

A. Yes, it is interesting that untreated male mutant mice have an increased bladder body contraction to carbachol compared with WT males. In a previous paper (Manak et al., 2020) we performed quantitative western blots for the M2 and M3 receptors and found levels were similar in mutants to the WTs, thus the increased sensitivity probably lies post-receptor.

B. A detailed study of the bladder body is an interesting idea, in terms of possible transgene expression and detailed histology, and is something we will pursue in future studies.

C. We have reported in our physiology graphs what we find. We do find some variability, particularly at lower frequencies, but our conclusions depend on analyses of the whole curve, which depend on multiple frequencies and show the expected overall pattern of frequency-dependent relaxation.

D. Thank you, the stats for Figure 8 will be corrected in the final version.

Reviewer 3:

Single-cell analysis of mutants versus control bladder, urethra including sphincter. This would be great also for the community.

A. Yes, in future we are very interested in using a single cell sequencing approach to look at the mutant, WT and rescued pelvic ganglia. In relation to this, there is a recent proof-of-principle paper pre-print in WT mouse pelvic ganglia, which suggests this may be feasible (Sivori et al., 2023).

Detailed tables showing data from each mouse examined.

B. In theory, it would be very interesting to correlate the strength of human gene transduction into the pelvic ganglia, with, for example, the effect on a physiological parameter. However, in general we used different sets of mice for these techniques so at the present we don’t have this information.

Use of measurements that are done in vivo (spot assay for example). This sounds relatively simple.

C. We strongly agree that in vivo voiding studies will be important it the future, and suggest in vivo cystometry is the gold standard for this but is currently beyond the remit of this study.

Assessment of viral integration in tissues besides the liver (could be done by QPCR).

D. This is an important point, and suggest the pancreas is a particularly interesting target for future studies. a recent study of Hpse2 inducible deletion in adult mice has shown evidence of exocrine pancreatic insufficiency (Kayal et al., PMID 37491420). We are currently exploring the status of the pancreas in our non-conditional juvenile Hpse2 mice, and whether gene transfer into the pancreas is possible.

Discuss subtypes of neurons that are present and targeted in the context of mutants and controls.

E. The make-up of the pelvic ganglia in Hpse2 mutant mice is a fascinating question. Future analysis using scRNA-Seq may be the most effective way to answer this question and is a molecular approach we are looking to pursue in the future.