Development of Substrate Degradation Enzyme Therapy for Mucopolysaccharidosis IVA Murine Model
Round 1
Reviewer 1 Report
Thank you very much for this work. Substrate Degradation Enzyme Therapy that you propose seems to be a promising theraputic option not only to MPS IVA but to all mucopolysaccharidoses and is worth further investigation.
Author Response
Response to Reviewer 1 Comments:
Point 1: Thank you very much for this work. Substrate Degradation Enzyme Therapy that you propose seems to be a promising therapeutic option not only to MPS IVA but to all mucopolysaccharidoses and is worth further investigation.
Response 1: Thank you very much your positive comment. SDET using thermostable keratanase has the potential for not only MPS IVA but MPS IVB since both diseases accumulate KS. Other bacterial GAG endoglycosidases such as chondroitinase ABC, chondroitinase B, or heparinase would be applicable to treat other types of MPS. We described this point in Discussion Section.
Reviewer 2 Report
The manuscript: “Development of Substrate Degradation Enzyme Therapy for Mucopolysaccharidosis IVA Murine Model” by Kazuki Sawamoto and Shunji Tomatsu describes that systemic treatment of the mouse model of Mucopolysaccharidosis IVA (MPS IVA) with bacterial keratanase, endo-β-N-acetylglucosaminidase. The enzyme can efficiently digest circulating keratan sulfate polymer reducing its levels for up to 4 weeks after a single injection. After 3 consequent administrations (1/month starting from neonatal age) authors could detect a substantial improvement of bone pathology.
Overall this is an interesting novel finding which may lead to development of a new approach to treat the bone disease in MPS IV patients not responding to the currently available enzyme replacement therapy with recombinant human N-acetylgalactosamine-6-sulfate sulfatase. At the same time ,several points need to be addressed before the paper becomes acceptable for publication.
Can the authors provide a measure for statistical significance of changes in vacuolization of chondrocytes (Table 1)? They show the reduction in % of severely affected group and an increase in % of animals with moderate and small lesions but it would be important to know how the size of lesions/cell is reduced on average and if it is significant. They also should describe how exactly the histopathological scores were determined?
The major concern with treatments involving bacterial enzymes is an adverse immune reaction, resulting in rapid elimination of the replacement enzyme and causing potentially harmful side-effects. The authors mention this possibility in the discussion and suggest that modification of the bacterial enzyme with a polymer such as PEGylation can prevent the immune response. Previous studies have demonstrated however that such approach can reduce but not prevent the immune response (see for example PMID: 30366815). This must be discussed in details together with potential applicability of other immunosuppressing procedures. Also if the authors have already studied the immune response in treated mice to TSK it would be important to present the data.
The method part contains description of the experiments which results are not shown or described, such as toxicology study. Either results need to be shown (or added as supplementary data) or the description of these experiments needs to be removed.
Two types of histochemical staining (H&E and toluidine blue) has been performed. Which of them is shown in Fig. 3C? Add this information either to results or to the figure legend.
Did authors measure circulating cytokines including (TNF-alpha) in plasma of treated and untreated mice? Providing these data would bring more depth to discussion of the potential effect of TSK treatment on inflammation.
Author Response
Response to Reviewer 2 Comments:
Thank you very much for your comments.
Point 1: Can the authors provide a measure for statistical significance of changes in vacuolization of chondrocytes (Table 1)? They show the reduction in % of severely affected group and an increase in % of animals with moderate and small lesions but it would be important to know how the size of lesions/cell is reduced on average and if it is significant. They also should describe how exactly the histopathological scores were determined?
Response 1: Veterinary pathologist supported us to make pathological slides and evaluate pathological scores in MPS IVA mice. After repeated administration of 2U/kg thermostable keratanase, pathological scores of vacuolization and column structure showed the tendency of improvement; however, there was not significant difference in pathological scores and chondrocyte cell size. Further studies are required under consideration of the following factors; i) dose-dependent administration and ii) frequency and duration of administration, iii) the number of mice to confirm therapeutic efficacy of thermostable keratanase on bone and cartilage lesions. We have already described these factors in Discussion Section.
Point 2: The major concern with treatments involving bacterial enzymes is an adverse immune reaction, resulting in rapid elimination of the replacement enzyme and causing potentially harmful side-effects. The authors mention this possibility in the discussion and suggest that modification of the bacterial enzyme with a polymer such as PEGylation can prevent the immune response. Previous studies have demonstrated however that such approach can reduce but not prevent the immune response (see for example PMID: 30366815). This must be discussed in details together with potential applicability of other immunosuppressing procedures. Also if the authors have already studied the immune response in treated mice to TSK it would be important to present the data.
Response 2: We agree with the reviewer’s comments. We have already described that the use of a non-mammalian enzyme may evoke a serious immune response. Development of immunosuppression strategies will be required, such as biodegradable biomaterials (like PEGylation) encapsulating the enzyme for clinical use. Pegvaliase, which is bacterial derived enzyme conjugated with PEG, is already approved for phenylketonuria. However, PEGylated drugs sometimes induce a clinically relevant anti-PEG antibody response (Verhoef et al.2013, Hershfield et al. 2014). Pegvaliase also produces anti-PEG IgG and anti-PEG IgM (Gupta et al. 2018). Therefore, co-administration regimen with immunosuppressants should be required in clinical practice. We added this discussion in our manuscript.
As we described in toxicity study, serious adverse effects were not shown after repeated injection of 250 U/kg thermostable keratanase in wild-type mice. We are developing the assay method for antibody titers against keratanase. We will publish the method once available.
Point 3: The method part contains description of the experiments which results are not shown or described, such as toxicology study. Either results need to be shown (or added as supplementary data) or the description of these experiments needs to be removed.
Response 3: We removed a part of the method section about toxicity study since it is underway in parallel of the establishment of antibody assay.
Point 4: Two types of histochemical staining (H&E and toluidine blue) has been performed. Which of them is shown in Fig. 3C? Add this information either to results or to the figure legend.
Response 4: Fig.3C shows representative pathological pictures by toluidine blue staining. We added this information in the figure legend.
Point 5: Did authors measure circulating cytokines including (TNF-alpha) in plasma of treated and untreated mice? Providing these data would bring more depth to discussion of the potential effect of TSK treatment on inflammation.
Response 5: Thank you very much for the critical suggestion. Recent studies indicate that inflammation is associated with progression of symptoms in MPS (we described it in discussion). We understand blood TNF-αlevel is a useful biomarker for treatment efficacy in MPS patients. Polgreen et al. indicated that TNF-α was associated with pain and physical disability in patients with MPS I, II, and VI (Polgreen et al. 2016). Our recent study also showed blood TNF-α, IL-6, and IL-1b levels were elevated in MPS IVA patients compared to in controls (Fujitsuka et al. 2019). We will assay serum cytokine with immune response study, which is under development.
Reviewer 3 Report
The manuscript by Sawamoto and Tomatsu describes a new treatment approach for MPS IVA in mice. MPS IVA patients suffer from accumulation of keratan sulfate and chondroitin-6-sulfate in many tissues, especially in cartilage and ECM leading to skeletal dysplasia and associated clinical features.
The authors discuss the current available ERT and hematopoietic stem cell treatments forMPS IVA and explain disadvantages of both treatment options, e.g. minimal impact on bone lesions.
The authors propose a novel therapy that utilizes a keratan sulfate degrading enzyme from Bacillus circulans, which they named “thermostable keratanase” and which can degrade keratan sulfate in blood circulation and which may prevent accumulation of KS in tissues and ECM.
Thermostable keratanase was tested in 3D chondrocyte cultures and in neonatal as well as adult MPS IVA knockout mice. Upon treatment, serum and tissue KS levels were decreased and remained low for at least 4 weeks. Furthermore, the pathological score in the treated mice was lower than in untreated mice. In a small cohort toxicity study, no adverse effects were noted.
The therapeutic efficacy of thermostable keratanase seems promising. The authors discuss the need to consider immune responses, because of using a non-mammalian enzyme.
The paper is written in a clear way. Results are presented in logical order.
Minor comment:
Chapter 2.2 and Figure 1:
o how long were the chondrocyte cells treated with keratanase?
o Change “treatement of” into “treatment with”
Author Response
Response to Reviewer 3 Comments:
Thank you very much for your comments.
Point 1: (Chapter 2.2 ) how long were the chondrocyte cells treated with keratanase?
Response 1: Incubation time was 72hrs. We described it in Material and Methods.
Point 2: (Figure 1) Change “treatement of” into “treatment with”
Response 2: We fixed it.
