In vitro assay for the efficacy assessment of AAV vectors expressing microdystrophin

Danilov, Kirill A.; Vassilieva, Svetlana G.; Polikarpova, Anna V.; Starikova, A.; Shmidt, A. A.; Galkin, Ivan I.; Tsitrina, Alexandra A.; Егорова, Т. В.; Orlov, Sergei N.; Kotelevtsev, Yuri

doi:10.1016/j.yexcr.2020.112033

Cited by 9 publications

(6 citation statements)

References 57 publications

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“…rAAV9 encoding µ Utrn and µ Dys were produced via the triple transfection method as previously described 42 . Briefly, adherent HEK293T (ATCC) cells were maintained in high-glucose DMEM (Gibco, USA) supplemented with 10% v/v fetal bovine serum (FBS, Biosera Europe, France) and Pen-Strep.…”

Section: Methodsmentioning

confidence: 99%

Therapeutic potential of highly functional codon-optimized microutrophin for muscle-specific expression

Starikova

Skopenkova

Polikarpova

et al. 2022

Sci Rep

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High expectations have been set on gene therapy with an AAV-delivered shortened version of dystrophin (µDys) for Duchenne muscular dystrophy (DMD), with several drug candidates currently undergoing clinical trials. Safety concerns with this therapeutic approach include the immune response to introduced dystrophin antigens observed in some DMD patients. Recent reports highlighted microutrophin (µUtrn) as a less immunogenic functional dystrophin substitute for gene therapy. In the current study, we created a human codon-optimized µUtrn which was subjected to side-by-side characterization with previously reported mouse and human µUtrn sequences after rAAV9 intramuscular injections in mdx mice. Long-term studies with systemic delivery of rAAV9-µUtrn demonstrated robust transgene expression in muscles, with localization to the sarcolemma, functional improvement of muscle performance, decreased creatine kinase levels, and lower immunogenicity as compared to µDys. An extensive toxicity study in wild-type rats did not reveal adverse changes associated with high-dose rAAV9 administration and human codon-optimized µUtrn overexpression. Furthermore, we verified that muscle-specific promoters MHCK7 and SPc5-12 drive a sufficient level of rAAV9-µUtrn expression to ameliorate the dystrophic phenotype in mdx mice. Our results provide ground for taking human codon-optimized µUtrn combined with muscle-specific promoters into clinical development as safe and efficient gene therapy for DMD.

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Section: Methodsmentioning

confidence: 99%

Therapeutic potential of highly functional codon-optimized microutrophin for muscle-specific expression

Starikova

Skopenkova

Polikarpova

et al. 2022

Sci Rep

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“…Therefore, it is attracting attention as a clinically applied vector for gene therapy in DMD. Dysfunctions in skeletal and cardiac muscles of DMD model mdx mice are significantly attenuated by partial restoration of dystrophin protein using the micro-dystrophin protein, which delivers a part of the cDNA copy of dystrophin to the affected tissues, based on internally deleted dystrophins [64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79]. However, there are some problems with the administration of AAV vectors into the skeletal muscle of canine X-linked muscular dystrophy model in Japan (CXMDj), which shows severe dystrophic phenotypes without immunosuppression, resulting in insufficient transgene expression with potent innate immune responses [80,81].…”

Section: Therapy Strategy Of Dmdmentioning

confidence: 99%

Therapeutic Application of Extracellular Vesicles-Capsulated Adeno-Associated Virus Vector via nSMase2/Smpd3, Satellite, and Immune Cells in Duchenne Muscular Dystrophy

Matsuzaka

Hirai

Hashido

et al. 2022

IJMS

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Duchenne muscular dystrophy (DMD) is caused by loss-of-function mutations in the dystrophin gene on chromosome Xp21. Disruption of the dystrophin–glycoprotein complex (DGC) on the cell membrane causes cytosolic Ca2+ influx, resulting in protease activation, mitochondrial dysfunction, and progressive myofiber degeneration, leading to muscle wasting and fragility. In addition to the function of dystrophin in the structural integrity of myofibers, a novel function of asymmetric cell division in muscular stem cells (satellite cells) has been reported. Therefore, it has been suggested that myofiber instability may not be the only cause of dystrophic degeneration, but rather that the phenotype might be caused by multiple factors, including stem cell and myofiber functions. Furthermore, it has been focused functional regulation of satellite cells by intracellular communication of extracellular vesicles (EVs) in DMD pathology. Recently, a novel molecular mechanism of DMD pathogenesis—circulating RNA molecules—has been revealed through the study of target pathways modulated by the Neutral sphingomyelinase2/Neutral sphingomyelinase3 (nSMase2/Smpd3) protein. In addition, adeno-associated virus (AAV) has been clinically applied for DMD therapy owing to the safety and long-term expression of transduction genes. Furthermore, the EV-capsulated AAV vector (EV-AAV) has been shown to be a useful tool for the intervention of DMD, because of the high efficacy of the transgene and avoidance of neutralizing antibodies. Thus, we review application of AAV and EV-AAV vectors for DMD as novel therapeutic strategy.

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“…In the initial report of AAV‐DJ, [ 8 ] CsCl density gradient centrifugation was used for purification, followed by later studies that used the same protocol [ 14,16,19,93 ] or iodixanol density gradient centrifugation. [ 11,15,94–97 ] Notably, AAV‐DJ contains the heparin‐binding domain from one of its parents, AAV2, which tempted Liu and Moon to try and purify AAV‐DJ via heparin affinity column chromatography. [ 13 ] Indeed, a relatively small, 5 mL heparin column in combination with FPLC was sufficient to enable the purification of 3 × 10 13 particles from 150 15 cm dishes of triple‐transfected HEK293 cells.…”

Section: Purification Of Aav‐djmentioning

confidence: 99%

“…Interestingly, it also turned out to be an excellent candidate for gene transfer into other cell types in vitro and in vivo, explaining why AAV‐DJ has been widely used to date for numerous applications and in various models of human gene therapy. [ 11,14–17 ] Moreover, its structure was resolved by cryo‐electron microscopy at 4.5 Å resolution, [ 18 ] or, more recently, even at 2.8 Å resolution in complex with a heparan sulfate analog. [ 19 ] Because of its interesting properties, its broad use and the advanced knowledge of its structure and biology, we will use AAV‐DJ as the prototype of a synthetic AAV capsid in this article that was created by forward‐directed molecular evolution.…”

Section: Introductionmentioning

confidence: 99%

Production, Processing, and Characterization of Synthetic AAV Gene Therapy Vectors

Andari

Grimm

2020

Biotechnology Journal

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Over the last two decades, gene therapy vectors based on wild‐type Adeno‐associated viruses (AAV) are safe and efficacious in numerous clinical trials and are translated into three approved gene therapy products. Concomitantly, a large body of preclinical work has illustrated the power and potential of engineered synthetic AAV capsids that often excel in terms of an organ or cell specificity, the efficiency of in vitro or in vivo gene transfer, and/or reactivity with anti‐AAV immune responses. In turn, this has created a demand for new, scalable, easy‐to‐implement, and plug‐and‐play platform processes that are compatible with the rapidly increasing range of AAV capsid variants. Here, the focus is on recent advances in methodologies for downstream processing and characterization of natural or synthetic AAV vectors, comprising different chromatography techniques and thermostability measurements. To illustrate the breadth of this portfolio, two chimeric capsids are used as representative examples that are derived through forward‐ or backwards‐directed molecular evolution, namely, AAV‐DJ and Anc80. Collectively, this ever‐expanding arsenal of technologies promises to facilitate the development of the next AAV vector generation derived from synthetic capsids and to accelerate their manufacturing, and to thus boost the field of human gene therapy.

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In vitro assay for the efficacy assessment of AAV vectors expressing microdystrophin

Cited by 9 publications

References 57 publications

Therapeutic potential of highly functional codon-optimized microutrophin for muscle-specific expression

Therapeutic potential of highly functional codon-optimized microutrophin for muscle-specific expression

Therapeutic Application of Extracellular Vesicles-Capsulated Adeno-Associated Virus Vector via nSMase2/Smpd3, Satellite, and Immune Cells in Duchenne Muscular Dystrophy

Production, Processing, and Characterization of Synthetic AAV Gene Therapy Vectors

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