{"doi":"10.1002/ctm2.1561","title":"Intravital imaging of muscle damage and response to therapy in a model of Pompe disease","abstract":"Dear Editor, Pompe disease (acid alpha-glucosidase [GAA] deficiency) is a severe multisystem lysosomal glycogen storage disorder that is primarily marked by progressive deterioration of muscle tissues.1 The limited efficacy of enzyme replacement therapy (ERT) – the only available treatment option – spearheaded efforts to develop gene therapy approaches and new drugs with improved muscle targeting for ERT.1 Over the years, our studies using a mouse model (knockout [KO]) provided a large body of evidence indicating that: 1) the dysfunction of glycogen-laden lysosomes leads to a major secondary abnormality in the diseased muscle – impaired autophagy and massive autophagic buildup; 2) the buildup resolution upon treatments occurs only when glycogen levels and lysosomal pool return to normal; and 3) the elimination of autophagic buildup is a reliable indicator of therapeutic efficacy.2-4 We have recently reported that a newly developed recombinant adeno-associated virus (rAAV)-based systemic gene therapy resulted in a fast and long-lasting reversal of pathology in multiple tissues of KO mice, including the limb muscle (gastrocnemius).2 Preclinical testing of new therapies is a lengthy undertaking that requires a large number of animals and involves an array of techniques to analyze samples ex vivo. High-resolution intravital microscopy (IVM), a powerful technique with a wide range of research applications, offers the possibility to visualize and quantify the effectiveness of new treatments in live animals within the natural tissue context. In this study, we revisited the same gene therapy approach2 and applied IVM to peer inside living muscle cells in a reporter KO model expressing green fluorescent protein (GFP) fused to autophagosomal marker LC3. The IVM imaging of gastrocnemius muscle was performed seven weeks after a single intravenous administration of an AAV9 vector (2.5 × 1013 vg/kg) expressing human GAA transgene. The animals were 5–7 months of age at the start of therapy. (The mouse strain, referred to as GFP-LC3:KO, and the vector are described in Supporting Information.) As expected, autophagic accumulation spanning along the longitudinal fibre axis can be seen in virtually all myofibers (94.6 ± 5.8%; n = 122 from three animals) of untreated GFP-LC3:KO (Figure 1A–C, GFP-LC3 middle panels). Consistent with our previous data,2 gene therapy reversed the pathology, as indicated by near complete elimination of autophagic buildup – 93.1 ±6.3 % fibres (n = 112 from four animals) were buildup-free (Figure 1A–C, GFP-LC3 bottom panels; Videos S1–S3). However, unlike in the previous study, the reversal can be directly observed in live GFP-LC3:KO without laborious biochemical and molecular biology techniques and with a much-reduced number of animals. Furthermore, we took advantage of the fact that the GFP signal can be collected together with the NAD(P)H fluorescence signal; endogenous NAD(P)H can be excited by two-photon (2P) microscopy and used to measure mitochondrial function and metabolic activity in live animals at subcellular resolution.5-7 The observed NAD(P)H fluorescence intensity in the buildup areas in the muscle of GFP-LC3:KO appeared weaker than in the neighbouring buildup-free regions (Figure 1C). Quantitative measurements (Supporting Information) confirmed visual observations – NAD(P)H levels were significantly reduced in the buildup areas (Figure 1D), in agreement with the disruption of mitochondrial morphology/function previously reported by us and others.8, 9 Next, we explored the possibility of evaluating therapeutic efficacy by using non-invasive imaging of the tongue muscle as a substitute for skeletal muscle (tongue involvement in Pompe patients is described in Supporting Information). This would allow for extended follow-up to monitor the disease progression and the effect of therapies. In preliminary experiments, we used a conventional ex vivo method – confocal microscopy of fixed single fibres isolated from","journal":"Clinical and Translational Medicine","year":2024,"id":467467,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9668,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":317884,"name":"Yeap Ng","orcid":"0000-0002-5702-8296","position":1,"is_corresponding":false},{"id":274359,"name":"Davide Randazzo","orcid":"0000-0002-2000-3060","position":2,"is_corresponding":false},{"id":317885,"name":"Roberto Weigert","orcid":"0000-0003-0740-4465","position":3,"is_corresponding":false},{"id":263646,"name":"Rosa Puertollano","orcid":"0000-0002-1106-5489","position":4,"is_corresponding":false},{"id":275026,"name":"Nina Raben","orcid":"0000-0001-9519-3535","position":5,"is_corresponding":false},{"id":275025,"name":"Naresh Kumar Meena","orcid":"0000-0001-5450-5184","position":0,"is_corresponding":true}],"reference_count":10,"raw_metadata":null,"created_at":"2026-07-19T02:05:15.025255Z","pmid":"38445455","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}