The use of base editing in stem cell-based therapies

Kevin Hemphill, R&D Manager at PerkinElmer’s Horizon Discovery explores how base editing has emerged as an attractive gene editing option for researchers wanting to develop stem cell-based therapies.  

Increasing interest in CRISPR-Cas gene editing systems over recent years has delivered innovative developments in precision genome engineering technologies, holding enormous potential for applications across the healthcare sector. In cell therapy, regenerative and adoptive immunotherapies continue to bridge the gap between genetic engineering strategies and their clinical application. Initial methods relied heavily on engineering primary cells from patient samples; however, widespread adoption requires the development of scalable production methods capable of supporting the demand for allogeneic cell therapy products.  

Pluripotent stem cells (PSCs) and their inherent ability to differentiate into any cell of the body, represent an attractive option in the manufacture of novel cell-based therapeutics. A second generation CRISPR-Cas gene editing system, base editing, reduces the risk of potentially deleterious effects associated with other gene editing technologies, presenting a safe and effective means of editing cells for therapeutic applications. The combined potential of base editing and PSCs holds great promise for the future of personalised medicine, offering an unlimited supply of universal donor cells that can be tailored to meet the needs of each individual patient. 

Induced pluripotent stem cells 

Initially obtained from surplus early-stage embryos generated for in vitro fertilisation, embryonic stem cells (ESCs) were the first example of pluripotent stem cells to offer potential for cell therapies within the clinical space1; although, the therapeutic impact was constrained by a limited supply of donor material as well as important ethical considerations. Being able to differentiate into any cell within the human body, PSCs hold a huge amount of promise within the cell-based therapeutics industry. Pioneering work in the mid-2000s, led by the Yamanaka group at Kyoto University, further advanced the field of PSCs. Adult fibroblasts were cultured in a cocktail of transcription factors to reactivate embryonic gene expression patterns2. This demonstrated, for the first time, that differentiated adult cells could be ‘induced’ back into a pluripotent state, thus creating the first induced pluripotent stem cells (iPSCs) (Figure 1). 

Figure 1: Derivation and differentiation of iPSCs. Using a select cocktail of transcription factors, fully differentiated adult cells such as fibroblasts and lymphocytes can be induced back to a pluripotent state known as an iPSC. These self-renewing cells can subsequently be expanded and differentiated into any cell within the human body under select conditions, presenting a host of therapeutic options. (Image by Dr Andrea Frapporti, PerkinElmer’s Horizon Discovery)

Like ESCs, iPSCs can be stably propagated in culture, are amenable to transgenesis, gene editing, and clonal expansion, and can be banked for future use. This circumvents several technical challenges inherent to the genetic manipulation of differentiated cells in ex vivo culture or intact tissues. Further innovative research in the fields of developmental biology and bioengineering have since advanced and refined our understanding of iPSCs, allowing researchers intricate control over iPSC differentiation with the potential to repair and replace diseased and damaged tissues. These cells present a highly scalable platform, capable of manufacturing advanced cellular therapeutics. Several clinical trials are underway to evaluate the therapeutic potential of iPSCs across a wide range of applications3 prompting a global shift towards the uptake of iPSCs as a therapeutic option.  

Addressing histocompatibility 

Despite iPSCs being a relatively new research platform, results have shown that patient-derived (autologous) cells present far fewer risks than donor-derived (allogeneic) cells because autologous cells avoid alloreactivity (immune cell rejection) which can lead to attack of donor cells by host immune cells (graft-versus-host disease), and vice versa 4-7. 

To avoid these histocompatibility challenges, early clinical trials often opt for an autologous graft-based approach 8,9 to subvert the need for prolonged immunosuppressive treatment and streamline the grafting process. However, the added cost of providing autologous grafts may outweigh the medical costs associated with providing continued immunosuppressive treatment. Additionally, the requirement for donor cells to be obtained from a patient, rather than prior collection from a third-party donor, adds an additional time requirement for autologous treatments that may not fit within disease progression.  

Due to the cost and time constraints associated with autologous-derived iPSCs, efforts have turned to the manufacture and bank storage of allogeneic iPSCs derived from healthy donors. Although many human leukocyte antigen (HLA) variants exist across the population, a large majority are extremely rare. Allelic distribution modelling of the Japanese population, with its low diversity has suggested that a bank of only 170 cell lines would be capable of providing either full or beneficial matching at the three most influential HLA markers for 80% of the Japanese population10. Estimates in the UK suggest that around 150 randomly selected iPSC donors could provide full or beneficial matching for 20% and 38% of the population, respectively. Furthermore, models suggest that only 10 perfect homozygous donors could provide full matches for 38% of the population and beneficial matches for 67% 11. Despite the distinct benefits to having at least partial HLA matching, the risk of graft rejection remains, and the added need for immunosuppressants brings with it a prolonged risk to the patient’s health and a significant impact to their quality of life. In addition, iPSC lines, despite their pluripotency, have skewed suitability towards certain cell types 12,13. Taken together, this helps strengthen the case for either fully HLA-matched, autologous iPSC therapies in as many instances as possible, or to develop precision gene editing approaches capable of generating allogeneic iPSCs adept at avoiding immune rejection. 

Regenerative and adoptive therapies 

A large portion of the research efforts within the iPSC field has focussed on regenerative and adoptive therapies, with the intention to use iPSCs to repair or replace damaged or diseased cells and tissues, including that of the immune system. 

The first in-person iPSC-derived regenerative therapy trial took place at Kyoto University, Japan. The trial saw positive results from grafting a sheet of retinal pigment epithelial cells differentiated from iPSCs into the eye of a patient with macular degeneration8. The results were promising, with further clinical trials since being initiated for macular degeneration as well as retinal pigmentosa across Japan and the USA9,14,15. 

Regenerative iPSC-derived therapies are not just limited to ophthalmic diseases. A recent US and South Korean pharmaceutical partnership unveiled plans to trial a diabetes treatment using iPSC-derived pancreatic beta cells16. Other clinical trials have also been initiated to evaluate the safety and efficacy of transplanting iPSC- and ESC-derived dopaminergic neural progenitors into the brain of Parkinson’s disease patients 17. 

Adoptive therapies using iPSC-derived cells is another area that has, so far, shown great promise, particularly in chimeric antigen receptor (CAR) T cell therapy. Transgenic expression of an engineered CAR is an approach used to direct immune cells to therapeutically relevant target cells expressing a specific antigen that bypasses the requirement to collect them from the site of disease. Studies have shown that CAR-engineered T, natural killer and macrophage cells have the potential to treat circulating and solid tumour cancer cells18-21. A major obstacle to overcome is the preparation of sufficient quantities of cells to provide patient treatment. Many trials have successfully modified iPSCs with the CAR transgene, providing a highly scalable bank of cells that can easily be expanded and differentiated into therapeutic products (22). 

Base editing — unlocking the full potential of iPSC therapeutics 

With the development of novel precision base editing approaches, the potential to ‘customise’ universal allogeneic iPSC stocks has become a more likely prospect than ever before. Such stocks have already been created using conventional CRISPR-Cas9 approaches; however, cells edited with CRISPR-Cas9 have an increased risk of random insertions and deletions (indels) which are introduced during a cell’s intrinsic DNA repair process following a DNA double strand break (DSB) from Cas9. In the engineering of more complex therapeutic solutions, multiple gene editing events are often required, raising the risk of additional unexpected cytotoxic/oncogenic effects through both indel formation, as well as chromosomal translocation events arising from multiple DSBs.  

Enter, base editing. 2016/17 saw the emergence of this novel CRISPR-Cas9-based approach. Base editing uses a guide RNA in partnership with a nickase version of Cas9, mutated to cut only a single strand of DNA, along with a deaminase enzyme to enable alteration of a single nucleotide 23,24. Cytosine base editors (CBEs) and adenine base editors (ABEs) are the two main classes of base editing enzymes mediating the base pair conversion from C–G to T–A and A–T to G–C, respectively. This precision editing approach allows for a host of potential therapeutic applications, including gene silencing, either via splice site disruption or the addition of premature stop codons, as well as gene correction in single nucleotide variant diseases. Development and use of DNA-free delivery of base editing systems can enable genome engineering in both dividing and terminally differentiated cells. 

The success of base editing has led to rapid advances toward commercialisation and clinical application. The Pin-point base editing system utilises a Cas component with an independent deaminase that is recruited to a DNA target site via a guide RNA with a tethered aptameric sequence25. This system offers unique advantages in the ability to flexibly connect and sequester the Cas and deaminase components through targeted use of the guide RNA with and without an aptamer recruiting scaffold, thus enabling additional modular control of the deaminase for a wider array of base editing along with other genome engineering applications.  

Although gene editing approaches often focus on gene knockout and other harder to achieve gene correction applications, as the technology continues to develop, new technologies such as base editing can be applied to more readily enhance or modulate protein function by installing known polymorphisms or rationally designed modifications into the genes encoding them in a multiplex fashion. For editing of stem cells, this raises the possibility of designing iPSC-derived cell therapies where gene regulatory networks can be altered through the silencing or activation of specific genes and pathways. Such advances would allow a highly customisable, context-dependent iPSC solution that would have scope to be used as a therapeutic to address unmet medical needs. 

On the horizon 

Advancements within the fields of precision gene editing and cell therapy continue to progress rapidly and the potential future of these two areas of research is largely interwoven. From a precision base editing approach through to the development of novel iPSC technologies, researchers can affect precise adjustments to help cells subvert immune detection and to silence or reactivate genes and pathways. With the current interest and continued development in the field, gene editing strategies tailored to an individual could offer therapeutic potential to a wide range of diseases. 

About the author: 

Kevin Hemphill is an R&D Manager at PerkinElmer’s Horizon Discovery. Since joining Horizon in 2017, he has supported the development of new products and technologies in the gene editing and modulation space and has contributed towards the technology development and commercialisation of Horizon’s Pin-point base editing platform. Previously, Kevin worked as a scientist at GE Healthcare’s Dharmacon site after obtaining degrees in Biochemistry from New Mexico State University and Molecular Biology from the University of Colorado Boulder. 


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