Gene therapies are powerful research tools which deliver nucleic acids into diseased cells to directly treat illness – these are also being tested in human clinical trials for a range of applications. A thorough understanding of the currently available gene therapy methods is critical for successfully developing new gene therapy strategies and projects. Herein, we will discuss 4 gene therapy strategies: gene augmentation, gene silencing/inhibition, genome editing and gene suicide. Note that the last two options – genome editing and gene suicide – are both used to induce targeted death of diseased cells. Since gene suicide is mainly used to destroy tumor cells with an oncolytic virus, this article will not explore this topic in detail.
Gene augmentation therapy is used to treat diseases caused by loss-of-function mutations, which prevent the gene from producing a functional product. This gene therapy technique introduces DNA containing a functional version of the lost gene into the cell and aims to produce a functioning product at sufficient levels to replace the protein that was originally missing.
Gene augmentation therapy is the most common treatment option for spinal muscular atrophy (SMA). SMA is caused by a deficiency of a motor neuron protein called SMN1, so the basic concept of gene therapy treatment is to insert the normal SMN1 gene into the diseased cell. Importantly, vector AAV9 can deliver cDNA of SMN1 gene to the cells. The first gene therapy treatment for SMA was approved by the Food and Drug Administration (FDA) in 2019. Despite being an effective option to minimize the progression of SMA and improve survival, this treatment is costly. At present, the commonly used strategies of gene augmentation therapies include delivering of a new protein-coding gene, increasing the expression of growth factors and cytokines, as well as cellular cytokines and autophagy activation of the diseased protein.
If the mutation fragment length of the diseased gene is too large for the vector, one solution is to adopt alternative splicing. The alternative splicing method has achieved great success in Duchenne Muscular Dystrophy (DMD) treatment. Scientists have used antisense oligonucleotides (ASOs) to interfere with the translation of protein mRNA, preventing the mutant exons from being translated, and thereby avoiding the loss of protein function caused by disease-causing nonsense and frameshift mutations.
In cases where the addition of a functional gene does not resolve the disease phenotype, gene silencing therapy may be used to shut down (silence) the expression of an abnormal gene. For diseases caused by dominantly inherited disorders, just one abnormal allele can manifest the disease phenotype and related dysfunctionality of cells or organs. A common example is the constitutive expression of oncogene mutations in tumor cells, which requires gene therapy to inhibit the function and expression of pathogenic genes. RNA interference (RNAi) therapy has been applied in research across many polyglutamine (PolyQ)-related diseases, including Huntington’s disease (HD) and spinocerebellar ataxias (SCAs), with the aim to reduce the expression of toxic proteins. Although single-stranded ASOs can mediate gene silencing – small/short interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA) therapies typically provide stronger inhibitory function and durability.
In gene therapy applications, the use of gene editing technology has been closely tied to the development of CRISPR-Cas9 technology, which has made gene editing in organisms much easier and inexpensive. Importantly, CRISPR-Cas9 gene editing technology has become widely used in gene therapy and served as a breakthrough approach to many previous restrictions, such as the limitations by disease type (recessive or dominant disease), gene length, and in vitro or in vivo experimental model development. Those experimental limitations and others could be solved by gene editing (CRISPR-Cas9) technology. Below are four key gene editing strategies for gene therapy:
- (1) Directly inhibiting the expression of pathogenic genes, which is used for dominant diseases treatments;
- (2) Correcting the faulty gene via single base editing or direct knockouts (KOs);
- (3) Using homologous fragment repair strategy, which depends on the improvement of homologous recombination efficiency;
- (4) Introducing the normal gene by homologous recombination at the safe site, similar to gene augmentation.
Gene Editing Applications in Rare Disease Research
More than 80% of rare diseases are caused by genetic disorders. With the development of a gene therapy for a rare disease, it can provide hope of a one-time treatment for numerous rare diseases that currently have no specific therapeutic options.
At present, there are gene editing-based gene therapy R&D pipelines in progress for several rare diseases, including Duchenne muscular dystrophy (DMD), congenital immune deficiency, hepatitis B, hemophilia, and cystic fibrosis.
With Cyagen’s professional gene editing platform, we provide accurate genetic engineering disease models to help researchers explore key information on rare disease mechanisms and potential treatment approaches. Our model services may be customized to support drug development programs more efficiently transition from gene discovery and validation to pre-clinical safety and efficacy evaluations.
The research groups of Dr. Bin Zhou (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) and Dr. Hefeng Huang (International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University) co-published an article titled “In Vivo AAV-CRISPR/Cas9-mediated Gene Editing Ameliorates Atherosclerosis in Familial Hypercholesterolemia” in the journal Circulation.
In this study, researchers find that adeno-associated virus (AAV) delivers CRISPR/Cas9 to achieve Ldlr gene correction that can partially rescue LDLR expression and effectively ameliorate atherosclerosis phenotypes in Ldlr mutant mice generated by Cyagen. The nonsense point mutation mouse line, LdlrE208X, is based on a gene mutation relevant to familial hypercholesterolemia - providing a potential therapeutic approach for the treatment of patients with the rare disease.
The Research Map
- Custom Rodent Models: TurboKnockout® gene targeting mice, Rosa26 Transgenic, Knockout (KO), conditional KO (cKO/floxed), Large-fragment Knockin (LFKI), point mutation, and much more
- Cell line generation: knockout (KO), knockin, point mutation, overexpression
- Cell products: research-use stem cells, media, differentiation kits, cell culture supplements and reagents
- DNA vector construction and BAC modification (Recombineering) services
- Lentivirus packaging services
About Cyagen – The Custom Animal Model Experts
In under 15 years since its founding, Cyagen has become a leading provider of custom mouse and rat models – delivering over 78,400 models to researchers worldwide and receiving over 4,750 peer-reviewed citations. Cyagen prides itself on its premium customer service: including price-matching, client access to complimentary technical consultations, full confidentiality, and a 100% money-back service guarantee.
Why Choose Cyagen?
- Complete research solutions – from custom rodent model generation to therapeutic viral packaging and injection.
- Both in vivo (animal) or in vitro (cell) experimental models available
- Expert scientific team provides in-depth technical support and assistance in formulating research model strategies.
Cyagen Expert Consultation
If you are engaged with or interested in gene therapy research, Cyagen’s experts can provide you with in-depth technical support and discuss your project plans. We also provide customize model generation strategies, helping develop your gene therapy research and publish high-impact articles.
Cyagen Knockout (KO) Catalog Models Repository
- Validated genome, guaranteed models (Reducing risk)
- 100% pure C57BL/6 background (Not mixed)
- Delivered in as few as 3 months (Fast turnaround)
Animal Models for Drug Development
Animal models of disease are essential tools for studying the mechanisms of human disease occurrence and development, drug efficacy screenings, and therapeutic assessment (TA). Excellent animal models created for a particular disease pathology can accelerate the development of new drugs. With the aim of helping researchers more easily obtain high-quality disease animal models from countries in Europe and the United States, Cyagen has formed strong partnerships with top animal model providers overseas to actively introduce animal models of superior quality. Simultaneously, Cyagen has invested heavily in research and development, recruited high-end talents, continued optimizing our suite of gene editing technologies, and has expanded our genetically engineered mouse model repository. Additionally, Cyagen has established a drug development and animal screening platform to serve research in cancer, immunity, endocrinology, cardiovascular disease (CVD), neurology, and infectious diseases. In short, Cyagen hopes to provide domestic researchers, CRO institutions, and new drug development institutions with world-class animal model services that meet every anticipated need to accelerate research progress worldwide.
Tumor Models and Drug Efficacy Evaluations
Cancers and malignant tumors are among the major pathologies that seriously endanger human health. Animal models of tumor are essential tools for studying the mechanisms of human tumor occurrence and development, drug efficacy screenings, and therapeutic assessment (TA). In accordance with your needs, Cyagen’s innovative animal model and drug development platform can provide various effective tumor models - such as immunodeficient nude mice, NOD SCID, C-NKG, and BRGSF, immune checkpoint (ICP) humanized mice, syngeneic models, human tumor tissue xenograft models (CDX) – in addition to genetically modified models and phenotype analysis services. Furthermore, we can construct various subcutaneous, in situ, or metastatic tumor models to deliver highly customized in vivo pharmacodynamic services for your specific model requirements.
Animal Model Supporting Services
In addition to animal model generation, Cyagen has established a range of supporting services, such as animal breeding, embryo/sperm cryopreservation, histology and transcriptome profiling. Outsourcing these services to Cyagen is a more cost-effective way of conducting your research. All these services are performed by experienced specialists following standardized procedures, so we guarantee to deliver high quality services and data with unbeatable price and turnaround.
Cyagen’s Germ-Free Animal Platform
Cyagen has introduced a complete range of germ-free (GF) animal husbandry and testing programs from our partner and leading provider of research models, Taconic Biosciences. Together, we have assembled an excellent germ-free (GF) animal technical team and established a standardized sterile animal feeding and breeding detection technology system. We can provide customers with germ-free (GF) mouse technical services that meet a wide variety of SPF standards as well as after-sales and training services.
Custom Cell Line Services
Cyagen can provide various types of custom cell line services such as vector construction, virus packaging and over expression, interference, gene knockout cell line, point mutation cell line and gene knockin cell line. Leverage our experts and optimized CRISPR/Cas9 cell line modeling service platform CRISPR-Pro for a hassle-free research. CRISPR-Pro enables large fragments excision or correct mutations in various cell lines.