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1.
Mol Ther Methods Clin Dev ; 32(2): 101268, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38911286

RESUMO

Excessive cytosolic calcium accumulation contributes to muscle degeneration in Duchenne muscular dystrophy (DMD). Sarco/endoplasmic reticulum calcium ATPase (SERCA) is a sarcoplasmic reticulum (SR) calcium pump that actively transports calcium from the cytosol into the SR. We previously showed that adeno-associated virus (AAV)-mediated SERCA2a therapy reduced cytosolic calcium overload and improved muscle and heart function in the murine DMD model. Here, we tested whether AAV SERCA2a therapy could ameliorate muscle disease in the canine DMD model. 7.83 × 1013 vector genome particles of the AAV vector were injected into the extensor carpi ulnaris (ECU) muscles of four juvenile affected dogs. Contralateral ECU muscles received excipient. Three months later, we observed widespread transgene expression and significantly increased SERCA2a levels in the AAV-injected muscles. Treatment improved SR calcium uptake, significantly reduced calpain activity, significantly improved contractile kinetics, and significantly enhanced resistance to eccentric contraction-induced force loss. Nonetheless, muscle histology was not improved. To evaluate the safety of AAV SERCA2a therapy, we delivered the vector to the ECU muscle of adult normal dogs. We achieved strong transgene expression without altering muscle histology and function. Our results suggest that AAV SERCA2a therapy has the potential to improve muscle performance in a dystrophic large mammal.

2.
PLoS One ; 19(1): e0295964, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38289946

RESUMO

Some acute exercise effects are influenced by postexercise (PEX) diet, and these diet-effects are attributed to differential glycogen resynthesis. However, this idea is challenging to test rigorously. Therefore, we devised a novel genetic model to modify muscle glycogen synthase 1 (GS1) expression in rat skeletal muscle with an adeno-associated virus (AAV) short hairpin RNA knockdown vector targeting GS1 (shRNA-GS1). Contralateral muscles were injected with scrambled shRNA (shRNA-Scr). Muscles from exercised (2-hour-swim) and time-matched sedentary (Sed) rats were collected immediately postexercise (IPEX), 5-hours-PEX (5hPEX), or 9-hours-PEX (9hPEX). Rats in 5hPEX and 9hPEX experiments were refed (RF) or not-refed (NRF) chow. Muscles were analyzed for glycogen, abundance of metabolic proteins (pyruvate dehydrogenase kinase 4, PDK4; peroxisome proliferator-activated receptor γ coactivator-1α, PGC1α; hexokinase II, HKII; glucose transporter 4, GLUT4), AMP-activated protein kinase phosphorylation (pAMPK), and glycogen metabolism-related enzymes (glycogen phosphorylase, PYGM; glycogen debranching enzyme, AGL; glycogen branching enzyme, GBE1). shRNA-GS1 versus paired shRNA-Scr muscles had markedly lower GS1 abundance. IPEX versus Sed rats had lower glycogen and greater pAMPK, and neither of these IPEX-values differed for shRNA-GS1 versus paired shRNA-Scr muscles. IPEX versus Sed groups did not differ for abundance of metabolic proteins, regardless of GS1 knockdown. Glycogen in RF-rats was lower for shRNA-GS1 versus paired shRNA-Scr muscles at both 5hPEX and 9hPEX. HKII protein abundance was greater for 5hPEX versus Sed groups, regardless of GS1 knockdown or diet, and despite differing glycogen levels. At 9hPEX, shRNA-GS1 versus paired shRNA-Scr muscles had greater PDK4 and PGC1α abundance within each diet group. However, the magnitude of PDK4 or PGC1α changes was similar in each diet group regardless of GS1 knockdown although glycogen differed between paired muscles only in RF-rats. In summary, we established a novel genetic approach to investigate the relationship between muscle glycogen and other exercise effects. Our results suggest that exercise-effects on abundance of several metabolic proteins did not uniformly correspond to differences in postexercise glycogen.


Assuntos
Glicogênio , Condicionamento Físico Animal , Ratos , Animais , Glicogênio/metabolismo , Glucose/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Modelos Genéticos , Músculo Esquelético/fisiologia , Condicionamento Físico Animal/fisiologia , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , RNA Interferente Pequeno/metabolismo , Insulina/metabolismo
3.
J Am Heart Assoc ; 12(3): e027480, 2023 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-36695318

RESUMO

Background Cardiomyopathy is a leading health threat in Duchenne muscular dystrophy (DMD). Cytosolic calcium upregulation is implicated in DMD cardiomyopathy. Calcium is primarily removed from the cytosol by the sarcoendoplasmic reticulum calcium ATPase (SERCA). SERCA activity is reduced in DMD. Improving SERCA function may treat DMD cardiomyopathy. Dwarf open reading frame (DWORF) is a recently discovered positive regulator for SERCA, hence, a potential therapeutic target. Methods and Results To study DWORF's involvement in DMD cardiomyopathy, we quantified DWORF expression in the heart of wild-type mice and the mdx model of DMD. To test DWORF gene therapy, we engineered and characterized an adeno-associated virus serotype 9-DWORF vector. To determine if this vector can mitigate DMD cardiomyopathy, we delivered it to 6-week-old mdx mice (6×1012 vector genome particles/mouse) via the tail vein. Exercise capacity, heart histology, and cardiac function were examined at 18 months of age. We found DWORF expression was significantly reduced at the transcript and protein levels in mdx mice. Adeno-associated virus serotype 9-DWORF vector significantly enhanced SERCA activity. Systemic adeno-associated virus serotype 9-DWORF therapy reduced myocardial fibrosis and improved treadmill running, electrocardiography, and heart hemodynamics. Conclusions Our data suggest that DWORF deficiency contributes to SERCA dysfunction in mdx mice and that DWORF gene therapy holds promise to treat DMD cardiomyopathy.


Assuntos
Cardiomiopatias , Distrofia Muscular de Duchenne , Camundongos , Animais , Distrofia Muscular de Duchenne/complicações , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Camundongos Endogâmicos mdx , Cálcio , Fases de Leitura Aberta , Cardiomiopatias/genética , Cardiomiopatias/terapia , Terapia Genética/métodos
4.
Hum Gene Ther ; 34(9-10): 449-458, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36515166

RESUMO

Adeno-associated virus (AAV)-mediated systemic micro-dystrophin (µDys) therapy is currently in clinical trials. The hope is to permanently improve the life quality of Duchenne muscular dystrophy (DMD) patients. Numerous preclinical studies have been conducted to support these trials. However, none examined whether a single therapy at a young age can lead to lifelong disease amelioration. To address this critical question, we injected 1 × 1013 vg particles/mouse of an AAV serotype-9 µDys vector to 3-month-old mdx mice through the tail vein. Therapeutic outcomes were evaluated at the age of 11 months (adulthood, 8 months postinjection) and 21 months (terminal age, 18 months postinjection). Immunostaining and Western blot showed saturated supraphysiological levels of µDys expression in skeletal muscle and heart till the end of the study. Treatment significantly improved grip force and treadmill running, and significantly reduced the serum creatine kinase level at both time points. Since cardiac death is a major threat in late-stage patients, we evaluated cardiac electrophysiology and hemodynamics by ECG and the closed-chest cardiac catheter assay, respectively. Significant improvements were observed in these assays. Importantly, many ECG and hemodynamic parameters (heart rate, PR interval, QRS duration, QTc interval, end-diastolic/systolic volume, dP/dt max and min, max pressure, and ejection fraction) were completely normalized at 21 months of age. Our results have provided direct evidence that a single systemic AAV µDys therapy has the potential to provide lifelong benefits in the murine DMD model.


Assuntos
Distrofia Muscular de Duchenne , Camundongos , Animais , Distrofia Muscular de Duchenne/genética , Distrofina/genética , Dependovirus/genética , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Terapia Genética/métodos
5.
Hum Gene Ther ; 34(9-10): 459-470, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36310439

RESUMO

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by dystrophin deficiency. Dystrophin consists of the amino terminus, central rod domain with 24 spectrin-like repeats and four hinges (H), cysteine-rich domain, and carboxyl terminus. Several highly abbreviated micro-dystrophins (µDys) are currently in clinical trials. They all carry H1 and H4. In this study, we investigated whether these two hinges are essential for µDy function in murine DMD models. Three otherwise identical µDys were engineered to contain H1 and/or H4 and were named H1/H4 (with both H1 and H4), ΔH1 (without H1), and ΔH4 (without H4). These constructs were packaged in adeno-associated virus serotype-9 and delivered to the tibialis anterior muscle of 3-month-old male mdx4cv mice (1E12 vector genome particles/muscle). Three months later, we detected equivalent µDys expression in total muscle lysate. However, only H1/H4 and ΔH1 showed correct sarcolemmal localization. ΔH4 mainly existed as sarcoplasmic aggregates. H1/H4 and ΔH1, but not ΔH4, fully restored the dystrophin-associated protein complex and significantly improved the specific muscle force. Eccentric contraction-induced force decline was best protected by H1/H4, followed by ΔH1, but not by ΔH4. Next, we compared H1/H4 and ΔH1 in 6-week-old male mdx mice by intravenous injection (1E13 vector genome particles/mouse). Four months postinjection, H1/H4 significantly outperformed ΔH1 in extensor digitorum longus muscle force measurements but two constructs yielded comparable electrocardiography improvements. We conclude that H4 is essential for µDys function and H1 facilitates force production. Our findings will help develop next-generation µDys gene therapy.


Assuntos
Distrofia Muscular de Duchenne , Masculino , Camundongos , Animais , Distrofia Muscular de Duchenne/genética , Distrofina/genética , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Terapia Genética
7.
Hum Gene Ther ; 33(9-10): 518-528, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35350865

RESUMO

Adeno-associated virus (AAV)-mediated clustered regularly interspaced short palindromic repeats (CRISPR) editing holds promise to restore missing dystrophin in Duchenne muscular dystrophy (DMD). Intramuscular coinjection of CRISPR-associated protein 9 (Cas9) and guide RNA (gRNA) vectors resulted in robust dystrophin restoration in short-term studies in the mdx mouse model of DMD. Intriguingly, this strategy failed to yield efficient dystrophin rescue in muscle in a long-term (18-month) systemic injection study. In-depth analyses revealed a selective loss of the gRNA vector after long-term systemic, but not short-term local injection. To determine whether preferential gRNA vector depletion is due to the mode of delivery (local vs. systemic) or the duration of the study (short term vs. long term), we conducted a short-term systemic injection study. The gRNA (4e12 vg/mouse in the 1:1 group or 1.2e13 vg/mouse in the 3:1 group) and Cas9 (4e12 vg/mouse) vectors were coinjected intravenously into 4-week-old mdx mice. The ratio of the gRNA to Cas9 vector genome copy dropped from 1:1 and 3:1 at injection to 0.4:1 and 1:1 at harvest 3 months later, suggesting that the route of administration, rather than the experimental duration, determines preferential gRNA vector loss. Consistent with our long-term systemic injection study, the vector ratio did not influence Cas9 expression. However, the 3:1 group showed significantly higher dystrophin expression and genome editing, better myofiber size distribution, and a more pronounced improvement in muscle function and electrocardiography. Our data suggest that the gRNA vector dose determines the outcome of systemic AAV CRISPR therapy for DMD.


Assuntos
Distrofina , Distrofia Muscular de Duchenne , Animais , Sistemas CRISPR-Cas/genética , Dependovirus/genética , Dependovirus/metabolismo , Distrofina/genética , Distrofina/metabolismo , Edição de Genes/métodos , Terapia Genética/métodos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo
8.
Gene Ther ; 29(6): 333-345, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-34611321

RESUMO

Inverted terminal repeats (ITRs) are the only wild-type components retained in the genome of adeno-associated virus (AAV) vectors. To determine whether ITR modification is a viable approach for AAV vector engineering, we rationally deleted all CpG motifs in the ITR and examined whether CpG elimination compromises AAV-vector production and transduction. Modified ITRs were stable in the plasmid and maintained the CpG-free nature in purified vectors. Replacing the wild-type ITR with the CpG-free ITR did not affect vector genome encapsidation. However, the vector yield was decreased by approximately 3-fold due to reduced vector genome replication. To study the biological potency, we made micro-dystrophin (µDys) AAV vectors carrying either the wild-type ITR or the CpG-free ITR. We delivered the CpG-free µDys vector to one side of the tibialis anterior muscle of dystrophin-null mdx mice and the wild-type µDys vector to the contralateral side. Evaluation at four months after injection showed no difference in the vector genome copy number, microdystrophin expression, and muscle histology and force. Our results suggest that the complete elimination of the CpG motif in the ITR does not affect the biological activity of the AAV vector. CpG-free ITRs could be useful in engineering therapeutic AAV vectors.


Assuntos
Dependovirus , Vetores Genéticos , Animais , Dependovirus/genética , Distrofina/genética , Terapia Genética , Vetores Genéticos/genética , Camundongos , Camundongos Endogâmicos mdx
9.
Nat Commun ; 12(1): 6769, 2021 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-34819506

RESUMO

Adeno-associated virus (AAV)-mediated CRISPR-Cas9 editing holds promise to treat many diseases. The immune response to bacterial-derived Cas9 has been speculated as a hurdle for AAV-CRISPR therapy. However, immunological consequences of AAV-mediated Cas9 expression have thus far not been thoroughly investigated in large mammals. We evaluate Cas9-specific immune responses in canine models of Duchenne muscular dystrophy (DMD) following intramuscular and intravenous AAV-CRISPR therapy. Treatment results initially in robust dystrophin restoration in affected dogs but also induces muscle inflammation, and Cas9-specific humoral and cytotoxic T-lymphocyte (CTL) responses that are not prevented by the muscle-specific promoter and transient prednisolone immune suppression. In normal dogs, AAV-mediated Cas9 expression induces similar, though milder, immune responses. In contrast, other therapeutic (micro-dystrophin and SERCA2a) and reporter (alkaline phosphatase, AP) vectors result in persistent expression without inducing muscle inflammation. Our results suggest Cas9 immunity may represent a critical barrier for AAV-CRISPR therapy in large mammals.


Assuntos
Sistemas CRISPR-Cas/imunologia , Terapia Genética/efeitos adversos , Vetores Genéticos/imunologia , Músculo Esquelético/imunologia , Distrofia Muscular de Duchenne/terapia , Animais , Sistemas CRISPR-Cas/genética , Dependovirus/genética , Modelos Animais de Doenças , Cães , Distrofina/genética , Distrofina/imunologia , Edição de Genes/métodos , Genes Reporter/genética , Genes Reporter/imunologia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Humanos , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/imunologia , Distrofia Muscular de Duchenne/patologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/imunologia
10.
Hum Mol Genet ; 30(14): 1321-1336, 2021 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-33949649

RESUMO

ΔR4-R23/ΔCT micro-dystrophin (µDys) is a miniaturized version of dystrophin currently evaluated in a Duchenne muscular dystrophy (DMD) gene therapy trial to treat skeletal and cardiac muscle disease. In pre-clinical studies, µDys efficiently rescues cardiac histopathology, but only partially normalizes cardiac function. To gain insights into factors that may impact the cardiac therapeutic efficacy of µDys, we compared by mass spectrometry the composition of purified dystrophin and µDys protein complexes in the mouse heart. We report that compared to dystrophin, µDys has altered associations with α1- and ß2-syntrophins, as well as cavins, a group of caveolae-associated signaling proteins. In particular, we found that membrane localization of cavin-1 and cavin-4 in cardiomyocytes requires dystrophin and is profoundly disrupted in the heart of mdx5cv mice, a model of DMD. Following cardiac stress/damage, membrane-associated cavin-4 recruits the signaling molecule ERK to caveolae, which activates key cardio-protective responses. Evaluation of ERK signaling revealed a profound inhibition, below physiological baseline, in the mdx5cv mouse heart. Expression of µDys in mdx5cv mice prevented the development of cardiac histopathology but did not rescue membrane localization of cavins nor did it normalize ERK signaling. Our study provides the first comparative analysis of purified protein complexes assembled in vivo by full-length dystrophin and a therapeutic micro-dystrophin construct. This has revealed disruptions in cavins and ERK signaling that may contribute to DMD cardiomyopathy. This new knowledge is important for ongoing efforts to prevent and treat heart disease in DMD patients.


Assuntos
Cardiomiopatias , Distrofia Muscular de Duchenne , Animais , Cardiomiopatias/genética , Distrofina/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/metabolismo , Miócitos Cardíacos/metabolismo , Proteômica
11.
Mol Ther Methods Clin Dev ; 18: 856-868, 2020 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32953935

RESUMO

Adeno-associated virus (AAV) is one of the most important gene delivery vehicles for in vivo gene therapy. Intramuscular (i.m.) and intravascular (i.v.) injection are commonly used for AAV gene transfer. Unfortunately, the fate of AAV vectors following administration remains unclear at the histological level. Taking advantage of RNAscope, a recently developed in situ hybridization technique that can reveal high-resolution viral DNA localization information, in this study, we evaluated body-wide distribution of an AAV9 vector in the context of the cell and tissue microenvironments. We observed distinctive kinetics of cell and nuclear entry of the AAV DNA in striated muscle and liver following i.m. and i.v. injection. We also found characteristic distribution patterns of the AAV DNA in various histological structures in internal organs, including gonads and lymph nodes, following i.v. injection. Finally, we showed significantly body-wide spreading of the AAV DNA following i.m. injection. These results add a new dimension to our understanding of AAV transduction biology and provide a basis for assessing the full impact of AAV gene therapy.

12.
Mol Ther Methods Clin Dev ; 18: 664-678, 2020 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32775499

RESUMO

Vector production scale-up is a major barrier in systemic adeno-associated virus (AAV) gene therapy. Many scalable manufacturing methods have been developed. However, the potency of the vectors generated by these methods has rarely been compared with vectors made by transient transfection (TT), the most commonly used method in preclinical studies. In this study, we blindly compared therapeutic efficacy of an AAV9 micro-dystrophin vector generated by the TT method and scalable herpes simplex virus (HSV) system in a Duchenne muscular dystrophy mouse model. AAV was injected intravenously at 5 × 1014 (high), 5 × 1013 (medium), or 5 × 1012 (low) viral genomes (vg)/kg. Comparable levels of micro-dystrophin expression were observed at each dose in a dose-dependent manner irrespective of the manufacturing method. Vector biodistribution was similar in mice injected with either the TT or the HSV method AAV. Evaluation of muscle degeneration/regeneration showed equivalent protection by vectors made by either method in a dose-dependent manner. Muscle function was similarly improved in a dose-dependent manner irrespective of the vector production method. No apparent toxicity was observed in any mouse. Collectively, our results suggest that the biological potency of the AAV micro-dystrophin vector made by the scalable HSV method is comparable to that made by the TT method.

13.
Elife ; 92020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32648542

RESUMO

Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging.


Assuntos
Envelhecimento/efeitos dos fármacos , Cardiopatias/tratamento farmacológico , Mitocôndrias/fisiologia , Oligopeptídeos/administração & dosagem , Estresse Oxidativo , Animais , Metabolismo Energético , Feminino , Cardiopatias/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução
14.
Mol Ther ; 28(3): 845-854, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-31981493

RESUMO

Loss of dystrophin leads to Duchenne muscular dystrophy (DMD). A pathogenic feature of DMD is the significant elevation of cytosolic calcium. Supraphysiological calcium triggers protein degradation, membrane damage, and eventually muscle death and dysfunction. Sarcoplasmic/endoplasmic reticulum (SR) calcium ATPase (SERCA) is a calcium pump that transports cytosolic calcium to the SR during excitation-contraction coupling. We hypothesize that a single systemic delivery of SERCA2a with adeno-associated virus (AAV) may improve calcium recycling and provide long-lasting benefits in DMD. To test this, we injected an AAV9 human SERCA2a vector (6 × 1012 viral genome particles/mouse) intravenously to 3-month-old mdx mice, the most commonly used DMD model. Immunostaining and western blot showed robust human SERCA2a expression in the heart and skeletal muscle for 18 months. Concomitantly, SR calcium uptake was significantly improved in these tissues. SERCA2a therapy significantly enhanced grip force and treadmill performance, completely prevented myocardial fibrosis, and normalized electrocardiograms (ECGs). Cardiac catheterization showed normalization of multiple systolic and diastolic hemodynamic parameters in treated mice. Importantly, chamber dilation was completely prevented, and ejection fraction was restored to the wild-type level. Our results suggest that a single systemic AAV9 SERCA2a therapy has the potential to provide long-lasting benefits for DMD.


Assuntos
Cardiomiopatia Dilatada/etiologia , Cardiomiopatia Dilatada/terapia , Expressão Gênica , Terapia Genética , Distrofia Muscular de Duchenne/complicações , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Administração Intravenosa , Animais , Dependovirus/genética , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Retículo Sarcoplasmático/metabolismo , Fatores de Tempo , Transdução Genética
15.
Mol Ther ; 27(9): 1568-1585, 2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31327755

RESUMO

CRISPR editing of muscle stem cells (MuSCs) with adeno-associated virus serotype-9 (AAV9) holds promise for sustained gene repair therapy for muscular dystrophies. However, conflicting evidence exists on whether AAV9 transduces MuSCs. To rigorously address this question, we used a muscle graft model. The grafted muscle underwent complete necrosis before regenerating from its MuSCs. We injected AAV9.Cre into Ai14 mice. These mice express tdTomato upon Cre-mediated removal of a floxed stop codon. About 28%-47% and 24%-89% of Pax7+ MuSCs expressed tdTomato in pre-grafts and regenerated grafts (p > 0.05), respectively, suggesting AAV9 efficiently transduced MuSCs, and AAV9-edited MuSCs renewed successfully. Robust MuSC transduction was further confirmed by delivering AAV9.Cre to Pax7-ZsGreen-Ai14 mice in which Pax7+ MuSCs are genetically labeled by ZsGreen. Next, we co-injected AAV9.Cas9 and AAV9.gRNA to dystrophic mdx mice to repair the mutated dystrophin gene. CRISPR-treated and untreated muscles were grafted to immune-deficient, dystrophin-null NSG.mdx4cv mice. Grafts regenerated from CRISPR-treated muscle contained the edited genome and yielded 2.7-fold more dystrophin+ cells (p = 0.015). Importantly, increased dystrophin expression was not due to enhanced formation of revertant fibers or de novo transduction by residual CRISPR vectors in the graft. We conclude that AAV9 effectively transduces MuSCs. AAV9 CRISPR editing of MuSCs may provide enduring therapy.


Assuntos
Dependovirus/genética , Distrofina/genética , Edição de Genes , Vetores Genéticos/genética , Mioblastos/metabolismo , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Modelos Animais de Doenças , Distrofina/química , Expressão Gênica , Técnicas de Transferência de Genes , Genes Reporter , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , RNA Guia de Cinetoplastídeos/genética , Regeneração , Células Satélites de Músculo Esquelético/metabolismo , Transdução Genética
16.
Mol Med ; 25(1): 31, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266455

RESUMO

BACKGROUND: Delocalization of neuronal nitric oxide synthase (nNOS) from the sarcolemma leads to functional muscle ischemia. This contributes to the pathogenesis in cachexia, aging and muscular dystrophy. Mutations in the gene encoding dystrophin result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). In many BMD patients and DMD patients that have been converted to BMD by gene therapy, sarcolemmal nNOS is missing due to the lack of dystrophin nNOS-binding domain. METHODS: Dystrophin spectrin-like repeats 16 and 17 (R16/17) is the sarcolemmal nNOS localization domain. Here we explored whether R16/17 protein therapy can restore nNOS to the sarcolemma and prevent functional ischemia in transgenic mice which expressed an R16/17-deleted human micro-dystrophin gene in the dystrophic muscle. The palmitoylated R16/17.GFP fusion protein was conjugated to various cell-penetrating peptides and produced in the baculovirus-insect cell system. The best fusion protein was delivered to the transgenic mice and functional muscle ischemia was quantified. RESULTS: Among five candidate cell-penetrating peptides, the mutant HIV trans-acting activator of transcription (TAT) protein transduction domain (mTAT) was the best in transferring the R16/17.GFP protein to the muscle. Systemic delivery of the mTAT.R16/17.GFP protein to micro-dystrophin transgenic mice successfully restored sarcolemmal nNOS without inducing T cell infiltration. More importantly, R16/17 protein therapy effectively prevented treadmill challenge-induced force loss and improved muscle perfusion during contraction. CONCLUSIONS: Our results suggest that R16/17 protein delivery is a highly promising therapy for muscle diseases involving sarcolemmal nNOS delocalizaton.


Assuntos
Músculo Esquelético/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Sarcolema/metabolismo , Utrofina/metabolismo , Animais , Humanos , Camundongos , Camundongos Transgênicos , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Mutação/genética , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/farmacologia , Ligação Proteica/genética , Sarcolema/genética , Sarcolema/patologia , Utrofina/genética
17.
Hum Gene Ther ; 30(9): 1039-1051, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31184217

RESUMO

Krabbe disease is an inherited neurodegenerative disease caused by mutations in the galactosylceramidase gene. In the infantile form, patients die before 3 years of age. Systemic adeno-associated virus serotype 9 (AAV9) gene therapy was recently shown to reverse the disease course in human patients in another lethal infantile neurodegenerative disease. To explore AAV9 therapy for Krabbe disease, we engineered a codon-optimized AAV9 galactosylceramidase vector. We further incorporated features to allow AAV9-derived galactosylceramidase to more efficiently cross the blood-brain barrier and be secreted from transduced cells. We tested the optimized vector by a single systemic injection in the twitcher mouse, an authentic Krabbe disease model. Untreated twitcher mice showed characteristic neuropathology and motion defects. They died prematurely with a median life span of 41 days. Intravenous injection in 2-day-old twitcher mice reduced central and peripheral neuropathology and significantly improved the gait pattern and body weight. Noticeably, the median life span was extended to 150 days. Intraperitoneal injection in 6- to 12-day-old twitcher mice also significantly improved the motor function, body weight, and median life span (to 104 days). Our results far exceed the ≤70 days median life span seen in all reported stand-alone systemic AAV therapies. Our study highlights the importance of vector engineering for Krabbe disease gene therapy. The engineered vector warrants further development.


Assuntos
Dependovirus/genética , Galactosilceramidase/genética , Terapia Genética , Vetores Genéticos/genética , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/terapia , Transgenes , Animais , Modelos Animais de Doenças , Ativação Enzimática , Galactosilceramidase/metabolismo , Expressão Gênica , Ordem dos Genes , Técnicas de Transferência de Genes , Engenharia Genética , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/isolamento & purificação , Leucodistrofia de Células Globoides/metabolismo , Leucodistrofia de Células Globoides/fisiopatologia , Camundongos , Fenótipo , Transdução Genética , Resultado do Tratamento
18.
Methods Mol Biol ; 1937: 281-294, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30706404

RESUMO

Many diseases affect multiple tissues and/or organ systems, or affect tissues that are broadly distributed. For these diseases, an effective gene therapy will require systemic delivery of the therapeutic vector to all affected locations. Adeno-associated virus (AAV) has been used as a gene therapy vector for decades in preclinical studies and human trials. These studies have shown outstanding safety and efficacy of the AAV vector for gene therapy. Recent studies have revealed yet another unique feature of the AAV vector. Specifically, AAV can lead to bodywide gene transfer following a single intravascular injection. Here we describe the protocols for effective systemic delivery of AAV in both neonatal and adult mice and dogs. We also share lessons we learned from systemic gene therapy in the murine and canine models of Duchenne muscular dystrophy.


Assuntos
Dependovirus/genética , Vetores Genéticos/administração & dosagem , Transdução Genética/métodos , Administração Intravenosa , Animais , Animais Recém-Nascidos , Cães , Terapia Genética , Humanos , Camundongos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia
19.
JCI Insight ; 3(23)2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30518686

RESUMO

Adeno-associated virus-mediated (AAV-mediated) CRISPR editing is a revolutionary approach for treating inherited diseases. Sustained, often life-long mutation correction is required for treating these diseases. Unfortunately, this has never been demonstrated with AAV CRISPR therapy. We addressed this question in the mdx model of Duchenne muscular dystrophy (DMD). DMD is caused by dystrophin gene mutation. Dystrophin deficiency leads to ambulation loss and cardiomyopathy. We treated 6-week-old mice intravenously and evaluated disease rescue at 18 months. Surprisingly, nominal dystrophin was restored in skeletal muscle. Cardiac dystrophin was restored, but histology and hemodynamics were not improved. To determine the underlying mechanism, we evaluated components of the CRISPR-editing machinery. Intriguingly, we found disproportional guide RNA (gRNA) vector depletion. To test whether this is responsible for the poor outcome, we increased the gRNA vector dose and repeated the study. This strategy significantly increased dystrophin restoration and reduced fibrosis in all striated muscles at 18 months. Importantly, skeletal muscle function and cardiac hemodynamics were significantly enhanced. Interestingly, we did not see selective depletion of the gRNA vector after intramuscular injection. Our results suggest that gRNA vector loss is a unique barrier for systemic AAV CRISPR therapy. This can be circumvented by vector dose optimization.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Distrofina/genética , Edição de Genes , Distrofia Muscular de Duchenne/genética , Animais , Dependovirus , Modelos Animais de Doenças , Feminino , Fibrose , Terapia Genética , Vetores Genéticos , Masculino , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/patologia , Mutação , Miocárdio/metabolismo , Miocárdio/patologia , Doenças Neuromusculares , RNA Guia de Cinetoplastídeos
20.
Skelet Muscle ; 8(1): 36, 2018 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-30466494

RESUMO

BACKGROUND: Loss of sarcolemmal nNOSµ is a common manifestation in a wide variety of muscle diseases and contributes to the dysregulation of multiple muscle activities. Given the critical role sarcolemmal nNOSµ plays in muscle, restoration of sarcolemmal nNOSµ should be considered as an important therapeutic goal. METHODS: nNOSµ is anchored to the sarcolemma by dystrophin spectrin-like repeats 16 and 17 (R16/17) and the syntrophin PDZ domain (Syn PDZ). To develop a strategy that can independently restore sarcolemmal nNOSµ, we engineered an R16/17-Syn PDZ fusion construct and tested whether this construct alone is sufficient to anchor nNOSµ to the sarcolemma in three different mouse models of Duchenne muscular dystrophy (DMD). RESULTS: Membrane-associated nNOSµ is completely lost in DMD. Adeno-associated virus (AAV)-mediated delivery of the R16/17-Syn PDZ fusion construct successfully restored sarcolemmal nNOSµ in all three models. Further, nNOS restoration was independent of the dystrophin-associated protein complex. CONCLUSIONS: Our results suggest that the R16/17-Syn PDZ fusion construct is sufficient to restore sarcolemmal nNOSµ in the dystrophin-null muscle.


Assuntos
Proteínas Associadas à Distrofina/metabolismo , Distrofina/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Sarcolema/metabolismo , Animais , Distrofina/genética , Proteínas Associadas à Distrofina/química , Proteínas Associadas à Distrofina/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos DBA , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Domínios PDZ , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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