Base Editing: Brightening the Future for Stem Cell-Based Therapy

Since the first stem cell transplant more than 60 years ago, stem cells have been revered for their unique ability to self-renew and differentiate into many cell types to treat disease. The advent of gene-editing technologies, such as CRISPR-Cas9, has further advanced this space; bespoke stem cell therapies are becoming a reality, enabling the treatment of previously untreatable diseases. As engineered stem cells will persist throughout a lifetime in the patient, it is essential that gene-editing technologies are safe, with minimal risk of introducing unintended genomic modification.

CRISPR-Cas9 and base editing

CRISPR-Cas9 gene engineering has been performed on multiple cell types for more than a decade and successful clinical trials are ongoing. However, its reliance on genotoxic DNA double-strand breaks (DSBs) poses significant safety concerns and limits its usefulness in cells sensitive to DNA damage response such as stem cells. Moreover, gene editing using conventional CRISPR-Cas9 is restricted to the generation of protein “knockouts” by the generation of indels at the cut site, creating a heterogenous population of edited cells; or relies on the inefficient homology directed repair (HDR) mechanism to introduce a corrected sequence.

Base editing is a second-generation CRISPR-Cas9 technology capable of generating homogenous single nucleotide edits at precise locations with high efficiency without relying on DNA DSBs, making it a far safer and more efficient alternative to conventional CRISPR-Cas9. Base editors rely on deaminase enzymes to introduce point mutations via chemical modification of nucleobases. Currently, two main types of base editors exist, cytidine base editors and adenine base editors, which convert C to T, and A to G, respectively.¹ In both cases, a CRISPR-Cas9 module, defective in its ability to introduce DSBs, is used to recruit the deaminase to the target site. Upon binding of the CRISPR-Cas9 module to DNA, a short stretch of single-stranded DNA is exposed and serves as the substrate of the deaminase. Because the technology relies on a defective Cas enzyme, the risk of unwanted genotoxic effects is mitigated, especially in cases where multiple simultaneous edits are required.

Hematopoietic stem and progenitor cells

With their ability to repopulate all blood components, hematopoietic stem and progenitor cells (HSPCs) from healthy donors have been used as stem cell therapies for many years. Allogeneic transplantations that rely on finding a suitable compatible donor have revolutionized medicine but are associated with a substantial risk of graft-versus-host-disease in treated patients.

More recent autologous stem cell therapies utilize the patient’s own cells, either engineered to express a healthy copy of the mutated gene by viral gene transfer, or with the disease-causing mutation corrected by gene-editing technologies such as CRISPR-Cas9, and then transfused back into the patient to offer a life-long cure to diseases previously considered untreatable. The semirandom integration of the viral genome, with the possibility of disrupting other genetic elements such as tumor suppressor genes, and the genotoxic risk associated with DSBs formed by CRISPR-Cas9, makes these approaches both heterogenous and risky.

Base editing offers a more precise and versatile alternative since single base modifications can be introduced at specific loci to directly correct a point mutation and repair defective genes. Although there are some limitations to which nucleotide substitutions can be introduced, the preclinical and clinical data demonstrating gene correction in HSPCs is mounting. Multiple proof of concept data has been generated to prove that base editors can efficiently correct β-thalassemia- and sickle cell disease (SCD)-causing mutations.² Additionally, in 2021, the U.S. Food and Drug Administration (FDA) approved an ex vivo base editing product, which introduces a single nucleotide change into HSPCs to reactivate HbF production for the treatment of SCD and β-thalassemia, as the first Investigational New Drug (IND) based on base editing technology.³ This pioneering clinical trial paves the way for development of similar therapies for the treatment of other genetic diseases.

Induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs), another promising candidate for stem cell therapy, are generated by culturing adult differentiated cells in a defined cocktail of transcription factors. Once pluripotency is established, they can self-renew, be engineered to fix defective genes, then differentiate into a multitude of therapeutically useful cell types useful for regenerative medicine or adoptive cell therapy.⁴ iPSCs engineered with other gene-editing technologies are already in clinical trials for the treatment of various diseases that affect the eye, nervous system, heart, and cancer. However, the hypersensitivity of these cells to the DNA damage response limits the therapeutic scope of DSB-dependent technologies for creating highly engineered iPSCs as the future of regenerative medicine. With the superior preclinical safety profile of base editors, and their ability to generate a homogenous population of edited cells with high efficiency without the activation of the DNA damage response, it is only a matter of time before scientists look to base editing of iPSCs as a potential therapy.

Future prospects

Although base editing is still in its infancy, preclinical and early clinical data show enormous potential for its use for the generation of advanced stem cell therapies. Compared to CRISPR-Cas9, base editors offer an improved safety profile and increased efficiency for the introduction of single nucleotide changes. Notably, base editing has also opened the door to potentially new therapeutic strategies, such as epitope masking by single nucleotide change, to make transplanted HSPCs resistant to cancer targeting drugs while retaining full cell functionality. Although not yet fully evaluated in the clinic, with convincing preclinical data, base editors are a promising candidate for creating the next generation of gene and cell therapies, especially in sensitive cell types such as stem cells.

References

1. Rees and Liu. Base editing: precision chemistry on the genome and transcriptome of living cells. Nature Reviews Genetics. 2018, 19:770. 

2. Antoniou et al. Base and prime editing technologies for blood disorders. Frontiers in Genome Editing. 2021, 3. 

3. Beam Therapeutics. BEAM-101 IND Cleared by FDA for Evaluation as a Treatment for Sickle Cell Disease. 2021

4. Blassberg. Genome editing of pluripotent stem cells for adoptive and regenerative cell therapies. GEN Biotechnology. 2022, 1(1):77.