Genome Editing in Induced Pluripotent Stem Cells: Facilitating Therapeutic Development

For many vexing diseases, such as heart failure, advanced diabetes, hemophilia, myeloma, advanced cirrhosis, etc., there is no complete cure for these diseases. The most effective method is allogeneic transplantation. Patient-specific induced pluripotent stem cells (iPSCs) are considered a multifunctional resource in the field of biomedicine. Since iPSCs are generated on an individual basis, it eliminates the risk of immune rejection and has the potential for continuous self-renewal and multidirectional differentiation. Based on their properties, iPSCs may be the optimal cellular material to use for disease modeling, drug discovery, and the development of patient-specific cellular therapies. To gain an in-depth understanding of human pathologies, patient-specific iPSCs have been used to model human diseases with some iPSC-derived cells recapitulating pathological phenotypes in vitro.

The use of genome editing has extended the potential of iPSCs, generating new models for a number of disorders, such as Alzheimer's and Parkinson's Disease. In the past decade, researchers have made great progress in genome editing techniques, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPRs/Cas9).

â» Using ZFNsr
So far, ZFNs have been widely used in many organisms and cell lines, and have also been utilized for a variety of therapeutic purposes, including a Phase I clinical trial using ZFN-modified autologous CD4 T cells with a mutated CCR5 gene to treat HIV infection. However, it is technically challenging to design or assemble multiple modular zinc finger motifs to achieve the high efficiency of genome editing.

â» Using TALENsr
Similar to ZFNs, TALENs are a fusion protein consisting of a nonspecific FokI endonuclease and an adaptable TALE DNA-binding domain. TALEs were discovered in the plant pathogenic bacteria genus Xanthomonas. These bacterial TALEs are secreted proteins that invade plant cell nuclei to activate gene transcription through binding to target gene promoters, thereby contributing to the establishment of bacterial infections.

Compared with ZFNs, the single base recognizing feature of TALE motif offers greater design flexibility. This also leads to an increased technical challenge for the TALE arrays cloning due to extensive repeat sequences.

â» Using CRISPRs/Cas9
The CRISPR/Cas9 is a novel genome editing tool that is rapidly gaining popularity. This state-of-the-art gene editing method uses a single non-sequence specific protein combined with a small guiding RNA molecule to achieve efficient and accurate gene modification. By doing so, this technique enables the generation of isogenic controls or cell lines with isogenic mutations to focus on the pathologies caused by a specific mutation.

In particular, the use of CRISPR/Cas9 technology has extended the potential of iPSCs, allowing scientists to acquire deep insight into the molecular bases of many diseases and develop pharmacological research. Recently, researchers from the Abramson Cancer Center of the University of Pennsylvania demonstrated that CRISPR-edited immune cells can persist, thrive, and function after a cancer patient receives these cells for months. These new findings will open the door to later-stage studies to investigate and extend this approach to a broader field beyond cancer.

“This is the first confirmation of the ability of CRISPR/Cas9 technology to target multiple genes at the same time in humans and illustrates the potential of this technology to treat many diseases that were previously not able to be treated or cured,” said Carl June, the Professor at the Perelman School of Medicine at the University of Pennsylvania.

The use of iPSC genome editing to model human diseases is rapidly becoming a standard strategy in biomedical research. Combined with iPSCs and genome-wide studies, it will dramatically expand researchers’ understanding of the pathophysiology of metabolic disorders and is expected to develop new therapies in the foreseeable future. However, these technologies also face many challenges, which must be overcome to better achieve personalized stem cell therapy for genetic diseases. In particular, it is necessary to improve reprogramming mechanism efficiency and to set up standard protocols to avoid incomplete reprogramming and the onset of de novo mutations. In addition, there is the need to evaluate the safety aspects for human trials, both for the application of genome editing techniques and iPSCs.

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