In Vitro Validation of a CRISPR-Mediated CFTR Correction Strategy for Preclinical Translation in Pigs

Lipiński

et al. 2020

Genes

Progress in genetic engineering over the past few decades has made it possible to develop methods that have led to the production of transgenic animals. The development of transgenesis has created new directions in research and possibilities for its practical application. Generating transgenic animal species is not only aimed towards accelerating traditional breeding programs and improving animal health and the quality of animal products for consumption but can also be used in biomedicine. Animal studies are conducted to develop models used in gene function and regulation research and the genetic determinants of certain human diseases. Another direction of research, described in this review, focuses on the use of transgenic animals as a source of high-quality biopharmaceuticals, such as recombinant proteins. The further aspect discussed is the use of genetically modified animals as a source of cells, tissues, and organs for transplantation into human recipients, i.e., xenotransplantation. Numerous studies have shown that the pig (Sus scrofa domestica) is the most suitable species both as a research model for human diseases and as an optimal organ donor for xenotransplantation. Short pregnancy, short generation interval, and high litter size make the production of transgenic pigs less time-consuming in comparison with other livestock species This review describes genetically modified pigs used for biomedical research and the future challenges and perspectives for the use of the swine animal models.

Section: Cystic Fibrosismentioning

confidence: 93%

Section: Cystic Fibrosismentioning

confidence: 99%

Application of Genetically Engineered Pigs in Biomedical Research

Lipiński

et al. 2020

Genes

Molecular Therapy - Methods & Clinical Development

“…Species differences between rodents and humans, as emphasized by gene therapy approaches to treat Duchenne muscular dystrophy (DMD) and cystic fibrosis (CF), 36,37 highlight that the safety and efficacy of CRISPR-Cas9-mediated in vivo gene therapy should be evaluated in a large animal model before clinical application. Therefore, in our present study, a novel FAH biallelic mutant pig model mimicking human HT1 was generated by intracytoplasmic microinjection combined with CRISPR-Cas9 technology.…”

Section: Discussionmentioning

confidence: 99%

Genetically blocking HPD via CRISPR-Cas9 protects against lethal liver injury in a pig model of tyrosinemia type I

Yang

Chen

et al. 2021

Hereditary tyrosinemia type I (HT1) results from the loss of fumarylacetoacetate hydrolase (FAH) activity and can lead to lethal liver injury (LLI). Therapeutic options for HT1 remain limited. The FAH À/À pig, a well-characterized animal model of HT1, represents a promising candidate for testing novel therapeutic approaches to treat this condition. Here, we report an improved single-step method to establish a biallelic (FAH À/À ) mutant porcine model using CRISPR-Cas9 and cytoplasmic microinjection. We also tested the feasibility of rescuing HT1 pigs through inactivating the 4-hydroxyphenylpyruvic acid dioxygenase (HPD) gene, which functions upstream of the pathogenic pathway, rather than by directly correcting the diseasecausing gene as occurs with traditional gene therapy. Direct intracytoplasmic delivery of CRISPR-Cas9 targeting HPD before intrauterine death reprogrammed the tyrosine metabolism pathway and protected pigs against FAH deficiency-induced LLI. Characterization of the F1 generation revealed consistent liver-protective features that were germline transmissible. Furthermore, HPD ablation ameliorated oxidative stress and inflammatory responses and restored the gene profile relating to liver metabolism homeostasis. Collectively, this study not only provided a novel large animal model for exploring the pathogenesis of HT1, but also demonstrated that CRISPR-Cas9-mediated HPD ablation alleviated LLI in HT1 pigs and represents a potential therapeutic option for the treatment of HT1.

“…31 These are supplemented by gene-editing approaches using CRISPR/Cas9. 32 Each of those approaches varies in the manner in which it delivers CFTR DNA or corrects the cellular DNA or protein, however, they all share the same challenges associated with delivery and measurement of success. The complexity of the lung anatomy and physiology, combined with the presence of thick sticky mucus and pathogens in the CF lung makes uniform deposition and uptake almost impossible to achieve, and hard to quantify.…”

Section: Cf Gene Therapymentioning

confidence: 99%

Will Airway Gene Therapy for Cystic Fibrosis Improve Lung Function? New Imaging Technologies Can Help Us Find Out

Parsons

Donnelley

2020

Human Gene Therapy

The promise of genetic therapies has turned into reality in recent years, with new first-line treatments for fatal diseases now available to patients. The development and testing of genetic therapies for respiratory diseases such as cystic fibrosis (CF) has also progressed. The addition of gene editing to the genetic agent toolbox, and its early success in other organ systems, suggests we will see rapid expansion of gene correction options for CF in the future. Although substantial progress has been made in creating techniques and genetic agents that can be highly effective for CF correction in vitro, physiologically relevant functional in vivo changes have been largely prevented by poor delivery efficiency within the lungs. Somewhat hidden from view, however, is the absence of reliable, accurate, detailed, and noninvasive outcome measures that can detect subtle disease and treatment effects in the lungs of humans or animal models. The ability to measure the fundamental function of the lung-ventilation, the effective transport of air throughout the lung-has been constrained by the available measurement technologies. Without sensitive measurement methods, it is difficult to quantify the effectiveness of genetic therapies for CF. The mainstays of lung health assessment are spirometry, which cannot provide adequate disease localization and is not sensitive enough to detect small early changes in disease; and computed tomography, which provides structural rather than functional information. Magnetic resonance imaging using hyperpolarized gases is increasingly useful for lung ventilation assessment, and it removes the radiation risk that accompanies X-ray methods. A new lung imaging technique, X-ray velocimetry, can now offer highly detailed regional lung ventilation information well suited to the diagnosis, treatment, and monitoring needs of CF lung disease, particularly after the application of genetic therapies. In this review, we discuss the options now available for imaging-based lung function measurement in the generation and use of genetic and other therapies for treating CF lung disease.