High-Level Expression of Alkaline Phosphatase by Adeno-Associated Virus Vector Ameliorates Pathological Bone Structure in a Hypophosphatasia Mouse Model
Authors: Toshiki Tanase
Shinichi Abe
Takashi Okada
Masataka Kasahara
Yuko Nitahara-Kasahara
Yukihiko Hirai
Satoru Matsunaga
Seikou Shintani
Akira Yamaguchi
Aki Nakamura-Takahashi
Atsushi Watanabe
Journal: Calcified tissue international (2020)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Adeno-associated virus vector; Alkaline phosphatase; Bone structure; Enzyme replacement therapy; Hypophosphatasia.
Affiliations: Department of Pharmacology, Tokyo Dental College, 2-9-18, Kandamisaki-cho, Chiyoda-ku, Tokyo, 101-0061, Japan. ., Tokyo Dental College Research Branding Project, Tokyo Dental College, Tokyo, Japan. ., Department of Pediatric Dentistry, Tokyo Dental College, Tokyo, Japan., Tokyo Dental College Research Branding Project, Tokyo Dental College, Tokyo, Japan., Department of Anatomy, Tokyo Dental College, Tokyo, Japan., Department of Molecular Therapy, National Center of Neurology and Psychiatry, National Institute of Neuroscience, Tokyo, Japan., Division of Clinical Genetics, Kanazawa University Hospital, Ishikawa, Japan., Division of Molecular and Medical Genetics, The Institute of Medical Science, University of Tokyo, Tokyo, Japan., Oral Health Science Center, Tokyo Dental College, Tokyo, Japan.

Abstract:  Hypophosphatasia (HPP) is a systemic skeletal disease caused by mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNALP). We recently reported that survival of HPP model mice can be prolonged using an adeno-associated virus (AAV) vector expressing bone-targeted TNALP with deca-aspartate at the C terminus (TNALP-D10); however, abnormal bone structure and hypomineralization remained in the treated mice. Here, to develop a more effective and clinically applicable approach, we assessed whether transfection with TNALP-D10 expressing virus vector at a higher dose than previously used would ameliorate bone structure defects. We constructed a self-complementary AAV8 vector expressing TNALP driven by the chicken beta-actin (CBA) promoter (scAAV8-CB-TNALP-D10). The vector was injected into both quadriceps femoris muscles of newborn HPP mice at a dose of 4.5 × 1012 vector genome (v.g.)/body, resulting in 20 U/mL of serum ALP activity. The 4.5 × 1012 v.g./body-treated HPP mice grew normally and displayed improved bone structure at the knee joints in X-ray images. Micro-CT analysis showed normal trabecular bone structure and mineralization. The mechanical properties of the femur were also recovered. Histological analysis of the femurs demonstrated that ALP replacement levels were sufficient to promote normal, growth plate cartilage arrangement. These results suggest that AAV vector-mediated high-dose TNALP-D10 therapy is a promising option for improving the quality of life (QOL) of patients with the infantile form of HPP. 

Retracted: No

PubPeer comments:
 We have found two very similar data between this paper and different paper. Mol Ther Methods Clin Dev. 2016 Feb 3;3:15059. doi: 10.1038 mtm.2015.59. eCollection 2016. [https: www.ncbi.nlm.nih.gov pubmed ?term=10.1038%2Fmtm.2015.59] Five authors are in common among these two papers, but, each paper is described as a separate experiment. Actually, each paper has been approved by different Animal Research Ethics Committee. We tried to compare for check these images. (attached images) Please consider whether the data of these two papers are the same sample. ![file] ![file] ![file] ![file] ![file] ![file] ![file] , 
 Sorry for the late response due to COVID-19. 2016 paper samples and 2019 paper samples are different. These images are similar, because mice of the same age, and the imaging conditions are almost same. If you look closely, you can understand that the fine structure is different. We appreciate your letting us know about our data. Please be careful for a COVID-19. , 
 Thank you for your replay. I am glad that I could have a discussion with you. Thank you for your explanation about reason of similar images, but your explanation is not enough. Could you please explain about my questions with scientific evidence (e.g.: CT image, law data of X-ray images). I think that all images as described previously are quite similar still now. In these images, my major questions are following two. 1. ABOUT CT IMAGE. Q: Are they different slices of the same sample? Please watch the following image of femur bone (first image, one view). This is the merge CT-image of 2016 paper and 2020 paper. The outer shapes of the bone exactly match as shown in red line area. Bone shape of each individual is typically different, thus, the fact that shapes of two images from different samples continuously match has very low-probability. For this reason, I think that almost readers think u201cthese are different slices of the same sampleu201d. ![file] 2. ABOUT X-ray IMAGE OF TIBIA. Q: Why do two images including characteristic shapes match? Please watch the second image including four tibia bone views. The typical tibia bone shape is shown in image A in your 2020 paper. Then, the tibia bone shown in image-B has a characteristic shape shown in red arrow. Moreover, this characteristic shape is shown at both tibia bones in 2016 and 2020 papers. Next, please watch the image-C which is merged two images of 2016 and 2020 papers. Two kinds of tibia bone images including characteristic shapes exactly match. ![file] Fortunately, your research group has five authors in common among these two papers. Therefore, it is easy work for you that you upload all images of all samples. I am waiting for all images from you. , 
 I apologize for my delayed response, as I have been quite busy after returning from childcare leave. I understand your concern regarding sample similarity between the 2016 and 2020 papers. Please allow me to provide clarity to the situation by considering the information provided below. We stand firm in our statement that the samples used in the 2016 and 2020 papers are not the same and were obtained from different mice. Figure 1 shows the femur samples used in the 2016 paper (6 blue capped tubes) and those used in the 2020 paper (15 pink capped tubes). The corresponding femurs contained within each tube are shown and labeled accordingly in Figure 2. ![file] ![file] The specific femur, tibia, and fibula samples that you are concerned with regarding similarity are shown below in Figure 3 and 4. ![file] Considering this, our response to your questions are as follows: Question 1 You claimed that the femur samples used in the 2016 paper and those used in the 2020 paper are the same due to the matching red lines indicated in the merged CT-image. The control mice used in these two papers were the same age. In addition, you altered the total femur length in the merged image. Considering the above two points, small similarities are very likely to occur. Furthermore, a partial match does not indicate that the samples belong to the same individual. Such a conclusion can only be made when the outer shapes match exactly. The internal trabecular bone detection imagery differs depending on the utilized imaging device; however, the outer shape of the bone is unique foreach individual and cannot be changed. Please see Figure 5 below, followed by a description of the different listed sites. ![file] 1.tFemoral head: The two yellow triangles indicate unique features of the sample reported in the 2020 paper and the light blue triangle indicates unique features of sample reported in the 2016 paper. You will notice that the outer shape is completely different. 2.tGreater trochanter: The orange triangle indicates a unique feature of the sample reported in the 2020 paper. The outer shape is completely different compared to the 2016 sample. 3.tThe two green, red triangles and dark blue triangles indicate features that are unique to the sample used in the 2016 paper, while the three red triangles are indicative of features unique to the sample used in the 2020 paper. By comparing these features, a clear difference in width and outer shape exists for the two samples. 4.tFemoral head u2192 Trochanteric u2192 Greater trochanter (the black arrow): This area does not match completely, different from you pointed. The above information clearly indicates that the samples used in the 2016 paper and the 2020 paper are from different mice. Question 2 You suggest that the two samples are the same based on similarities between their characteristic shapes and features indicated by the black triangles. However, the characteristic shapes that you pointed out are, in fact, not characteristic shapes, but rather the anterior border ridge of the tibia, which is a normal anatomical structure. Please magnify Figure. 4, and you will be able to clearly see this for the TNALP-D10 and WT samples used in the 2016 paper, and the 1.5 u00d7 1011 sample used in the 2020 paper. Unfortunately, the TNALP sample used in the 2016 paper and the 1.5 u00d7 1011 sample used in the 2020 paper had low serum ALP activity, resulting in hypocalcification in the central part of the ridge. As such, they do not appear on the X-ray. Furthermore, we do not agree with your statement that the tibia bones match exactly. Please see Figure 6 below as well as the descriptive text that follows. ![file] 1.tThe anterior border ridge of the tibia: The yellow triangle indicates unique features of the sample used in the 2020 paper and the two purple triangles indicate unique features of the sample used in the 2016 paper. The outer shape is completely different. 2.tBased on the five black triangles indicated by you, it is unclear to us where you see an exact match. The three green triangles and the five light blue triangles represent unique features of the 2016 sample, while the two pink triangles represent unique features of the 2020 sample. Not only does this indicate differences in the outer shape of the two samples, but also in the width thereof. 3.tFibula: The five orange triangles represent unique features of the 2020 sample, and the dark blue triangles represent unique features of the 2016 sample. Since the fibula is a bone, it is impossible to change its outer shape. The above information indicate that the samples used in the 2016 paper are not the same as those used in the 2020 paper. In conclusion, unlike soft tissue, differences in the shape of hard tissue (such as the bone samples used in our prior work) cannot be associated with the same sample. , 
 #3 I never looked at x-ray slices of mice bones so I no authority here on expected similarities or differences. To a non-expert the differences do seem obvious, though. Did I match the right pairs? [ click to animate, Fig. 3d 2016 versus Fig. 2c 2020, taking the pairs suggested in #1 ] ![file] [ click to animate, Fig. 3d 2016 versus Fig. 2c 2020, taking the pairs suggested in #1 and scaling such that at least the right part matches a bit ] ![file] [ click to animate, Fig. 4a WT 2016 versus Fig. 3a Akp2+ +, pair as suggested in #1 ] ![file] , 
 Thank you for your comments on our manuscripts by providing easy-to-understand animation. You matched the right pairs. Your comments further demonstrate that the samples used in our 2016 and 2020 manuscripts are different one. I would like to express my heartfelt gratitude for your conscientious and kind comments. ,