CRISPR/Cas genome editing is a simple, cost effective, and highly specific technique for introducing genetic variations. In mammalian cells, CRISPR/Cas can facilitate non-homologous end joining, homology- directed repair, and single-base exchanges. Cas9/Cas12a nuclease, dCas9 transcriptional regulators, base editors, PRIME editors and RNA editing tools are widely used in basic research. Currently, a variety of CRISPR/Cas-based therapeutics are being investigated in clinical trials. Among many new findings that have advanced the field, we highlight a few recent advances that are relevant to CRISPR/Cas-based gene therapies for monogenic human genetic diseases.

Advances in CRISPR/Cas-based Gene Therapy in Human Genetic Diseases.

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.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/biot.202000025

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

Muscular dystrophies (MDs) encompass a wide variety of inherited disorders that are characterized by loss of muscle tissue associated with a progressive reduction in muscle function. With a cure lacking for MDs, preclinical developments of therapeutic approaches depend on well-characterized animal models that recapitulate the specific pathology in patients. The mouse is the most widely and extensively used model for MDs, and it has played a key role in our understanding of the molecular mechanisms underlying MD pathogenesis. This has enabled the development of therapeutic strategies. Owing to advancements in genetic engineering, a wide variety of mouse models are available for the majority of MDs. Here, we summarize the characteristics of the most commonly used mouse models for a subset of highly studied MDs, collated into a table. Together with references to key publications describing these models, this brief but detailed overview would be useful for those interested in, or working with, mouse models of MD.

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Mouse models for muscular dystrophies: an overview.

Introduction: Duchenne muscular dystrophy (DMD) is an X-linked handicapping disease due to the loss of an essential muscle protein dystrophin. Dystrophin-null animals have been extensively used to study disease mechanisms and to develop experimental therapeutics. Despite decades of research, however, treatment options for DMD remain very limited.Areas covered: High-throughput high-content screening and precision medicine offer exciting new opportunities. Here, the authors review animal models that are suitable for these studies.Expert opinion: Nonmammalian models (worm, fruit fly, and zebrafish) are particularly attractive for cost-effective large-scale drug screening. Several promising lead compounds have been discovered using these models. Precision medicine for DMD aims at developing mutation-specific therapies such as exon-skipping and genome editing. To meet these needs, models with patient-like mutations have been established in different species. Models that harbor hotspot mutations are very attractive because the drugs developed in these models can bring mutation-specific therapies to a large population of patients. Humanized hDMD mice carry the entire human dystrophin gene in the mouse genome. Reagents developed in the hDMD mouse-based models are directly translatable to human patients.

Duchenne muscular dystrophy animal models for high-throughput drug discovery and precision medicine.

CRISPR Technologies for the Treatment of Duchenne Muscular Dystrophy

Exon skipping is a promising strategy for Duchenne muscular dystrophy (DMD) disease-modifying therapy. To make this approach safe, ensuring that excluding one or more exons will restore the reading frame and that the resulting protein will retain critical functions of the full-length dystrophin protein is necessary. However, in vivo testing of the consequences of skipping exons that encode the N-terminal actin-binding domain (ABD) has been confounded by the absence of a relevant animal model. We created a mouse model of the disease recapitulating a novel human mutation, a large de novo deletion of exons 8-34 of the DMD gene, found in a Russian DMD patient. This mutation was achieved by deleting exons 8-34 of the X-linked mouse Dmd gene using CRISPR/Cas9 genome editing, which led to a reading frame shift and the absence of functional dystrophin production. Male mice carrying this deletion display several important signs of muscular dystrophy, including a gradual age-dependent decrease in muscle strength, increased creatine kinase, muscle fibrosis and central nucleation. The degrees of these changes are comparable to those observed in mdx mice, a standard laboratory model of DMD. This new model of DMD will be useful for validating therapies based on skipping exons that encode the N-terminal ABD and for improving our understanding of the role of the N-terminal domain and central rod domain in the biological function of dystrophin. Simultaneous skipping of exons 6 and 7 should restore the gene reading frame and lead to the production of a protein that might retain functionality despite the partial deletion of the ABD.

CRISPR/Cas9-generated mouse model of Duchenne muscular dystrophy recapitulating a newly identified large 430 kb deletion in the human DMD gene

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

Many genetic diseases that are responsible for muscular disorders have been described to date. Gene replacement therapy is a state-of-the-art strategy used to treat such diseases. In this approach, the functional copy of a gene is delivered to the affected tissues using viral vectors. There is an urgent need for the design of short, regulatory sequences that would drive a high and robust expression of a therapeutic transgene in skeletal muscles, the diaphragm, and the heart, while exhibiting limited activity in non-target tissues. This review focuses on the development and improvement of muscle-specific promoters based on skeletal muscle α-actin, muscle creatine kinase, and desmin genes, as well as other genes expressed in muscles. The current approaches used to engineer synthetic muscle-specific promoters are described. Other elements of the viral vectors that contribute to tissue-specific expression are also discussed. A special feature of this review is the presence of up-to-date information on the clinical and preclinical trials of gene therapy drug candidates that utilize muscle-specific promoters.

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Muscle-Specific Promoters for Gene Therapy

Duchenne muscular dystrophy is a complex and severe orphan disease. It develops when the organism lacks the expression of dystrophin - a large structural protein. Dystrophin is transcribed from the largest gene in the human genome. At the moment, there is no cure available. Dozens of groups all over the world search for cure. Animal models are an important component of both the fundamental research and therapy development. Many animal models reproducing the features of disease were created and actively used since the late 80’s until present. The species diversity spans from invertebrates to primates and the genetic diversity of these models spans from single mutations to full gene deletions. The models are often non-interchangeable; while one model may be used for particular drug design it may be useless for another. Here we describe existing models, discuss their advantages and disadvantages and potential applications for research and therapy development.

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Duchenne Muscular Dystrophy Animal Models

The experiments were carried out on 5 groups of cage housed broiler chickens of the Ross 308 cross aged from 1 to 35 days. It was found that dried chicory root, oregano, and common nettle are valuable additives in compound poultry feed. The addition of dried chicory root, oregano, and common nettle in amounts of 2 kg/t; 0.5 kg/t, and 2.0 kg/t, respectively, to broiler diets allows obtaining good livability (100%) and live bodyweight, as well as low feed conversion. In the experimental groups the dried medicinal plants were added to the feed without the use of in-feed antibiotics. It was statistically proven that the live bodyweight in broilers at the end of growing in the experimental groups was significantly in compare to control by 7.08-8.04%. This increase was accompanied by a decrease in feed conversion ratio by 10.33-10.45% due to improved digestibility and retention of dietary nutrients. With the introduction of dried medicinal plants into broiler feed, an increase in the protein content in the pectoral muscles by 1.31-1.56% and a decrease in fat content by 2.23-2.97% were found. The sensory quality of the roasted meat was good.

/pdf/the-use-of-medicinal-plants-in-the-compound-poultry-feed-t9rihhizbn.pdf

Tatiana V. Egorova

Papers

CRISPR/Cas9-generated mouse model of Duchenne muscular dystrophy recapitulating a newly identified large 430 kb deletion in the human DMD gene

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

Muscle-Specific Promoters for Gene Therapy

Duchenne Muscular Dystrophy Animal Models

The use of medicinal plants in the compound poultry feed