By Jennifer Harbottle, PhD
Base editors, DNA double-strand break (DSB)-independent genome modification agents, have been recognized alongside prime editors as key technologies to watch this year,1 and advances to date have been encouraging. Although these technologies have yet to enter the clinic, the first candidate therapy received investigational new drug approval from the U.S. Food and Drug Administration in 2021—less than five years after the first base editor was described.2
Exciting preclinical data3 and research collaborations4 between eminent players in the field herald the important role base editing could play in advancing innovative cell and gene therapies, offering hope to patients with complex, debilitating diseases. Aside from their effective and highly efficient editing ability, there is growing impetus on the enhanced safety potential of base editing agents, particularly as concerns arise with the canonical CRISPR-Cas system.
A call for safer gene editing
The fields of cell and gene therapy and genome editing increasingly overlap, and results from published clinical trials that include CRISPR-based therapies show early evidence of feasibility and efficacy in humans. Both ex vivo and in vivo therapies have shown progress. To date, the ex vivo delivery of engineered cell therapies to address cancer and hemoglobinopathies has dominated this space, and it has provided promising proof-of-concept data. More recently, the first in vivo CRISPR-based gene therapy to treat hereditary transthyretin amyloidosis (ATTR) drew considerable attention when it was delivered in 2021.5
Advanced gene therapies not only show potential for curing rare, debilitating monogenic diseases, but they also offer hope for treating complex disorders, including cardiovascular disorders. The growing number of IND approvals for CRISPR-based drugs suggests that the transition to an exciting era of precision medicine is imminent.
However, this optimistic view comes with a caveat: With genome editing technologies, safety is as critical as efficacy.
Studies that highlight concerns surrounding DSBs are proliferating. For example, there are studies documenting how DSBs occur and persist through the use of genome modifying systems such as CRISPR-Cas, transcription activator-like effector nuclease (TALEN), and zinc finger nuclease (ZFN) systems.
DSB concerns were underscored in October 2021 when a clinical trial was halted following detection of a chromosomal abnormality in a lymphoma patient treated with a TALEN-engineered allogeneic chimeric antigen receptor (CAR) T-cell product.6 Although the DNA abnormality was not ascribed to the gene editing machinery in this instance, the potential for this to occur in the future remains, particularly as genome engineering applications for polygenic diseases become more complex and require simultaneous editing of multiple loci.
The FDA subsequently released a draft set of recommendations7 for preclinical safety assessment of new INDs; however, there is no standardized strategy for evaluation. The regulatory body suggests that a case-by-case approach should be employed, and that multiple orthogonal methods of detection and characterization are needed. These methods should incorporate the relevant human cell types from multiple donors and assays of “adequate sensitivity” to detect low-frequency events.
Ultimately, a growing body of research will help researchers elucidate the cell-specific endogenous DNA repair outcomes of cells treated with gene editing agents. There are concerted efforts to develop the most relevant and fit-for-purpose tools for assessing the scale and scope of potential DNA abnormalities.
Maintaining genomic integrity
Many of the deleterious, unintended off-target effects of CRISPR-Cas gene editing are underpinned by the system’s reliance on DSBs to edit target genes. Nuclease-induced formation of insertions and deletions (indels) at a guide RNA (gRNA)-specific target locus allows for genetic disruption and consequent functional protein knockout. This can provide an effective therapeutic strategy for some monogenic disorders, as was showcased in the first human in vivo gene therapy where liver-specific ablation of TTR transcription facilitated a decrease in pathogenic TTR protein accumulation in patients with hereditary ATTR.8
The phenotypic success of this CRISPR-Cas based gene therapy warrants acclaim; however, this therapy represents only the beginning of the in vivo gene editing journey. If the journey is to continue, safe and targeted means of delivery must be identified, and the biological consequences of triggering DSBs in the genome must be clarified.
Legitimate strategies to overcome gRNA-dependent off-target editing include rational gRNA design, the use of high-fidelity nuclease orthologues, the exercise of temporal control over editing activity, and the prevention of repeated nuclease cleavage.9 However, these strategies do not circumvent the risk of activating a cytotoxic and potentially oncogenic p53 response, nor do they eliminate the pernicious occurrence of “on target” structural variants.
Such variants could pose a threat to genomic integrity or cell function. Also, the loose ends of DSBs that occur at edited target loci, gRNA-dependent off-target sites, or less predictable sites in the wider genome may fuse to form chromosomal translocations—the rate and propensity of which are exacerbated in a multigene editing context.7
Other potentially deleterious effects include large and complex genomic rearrangements, segmental duplications, and terminal chromosomal truncations. In addition, CRISPR-Cas can induce the formation of micronuclei and chromosome bridges as well as low levels of chromothripsis.10
Although the functional and clinical implications of these events remain unclear and have yet to be characterized in a cell-, genomic-, and disease-specific context, they do highlight the need to carefully monitor their occurrence in any preclinical development of a genetically engineered therapeutic product. However, avoiding DSBs altogether offers a favorable and promising alternative to accelerate the progression of advanced cell and gene therapies.
Breaking boundaries, not DNA
Leveraging the targeting power of the traditional CRISPR-Cas system, base editors recruit a deaminase enzyme to the target nucleotide, enabling a chemical modification that facilitates a C–G > T–A (cytidine base editors, CBEs)2 or A–G > T–C transition (adenine base editors, ABEs).11 The capacity to induce a single-base conversion can be harnessed to correct a pathogenic point mutation, silence a disease-causing gene, skip a disease-causing exon, activate a specific gene, or fine-tune engineered immune cell therapies, providing multiple strategies to address a wide range of diseases.
Although the deleterious outcomes of triggering DSBs are mitigated using base editing, researchers continue to “pressure test” the system to comprehensively understand the mechanisms of action and evaluate the safety profile of this next-generation gene editing tool to ensure a smooth transition to the clinic. Concerns initially surrounded unwanted, widespread gRNA-independent single nucleotide variants (SNVs) in the transcriptome and genome of base-editor-treated cells. However, these concerns have been largely abated through the use of split-engineered base editors12 or transient, therapeutically relevant reagents alongside the incorporation of deaminase and/or nuclease variants within the base editor architecture that have less RNA and DNA off-target activity.
The occurrence of bystander editing, that is, base conversion beyond that of the target nucleotide within the editing window, has also called into question the precision of base editors, particularly in a gene therapy context aiming to correct a pathogenic SNV with single-base granularity. To clear the bystander hurdle and facilitate precise DNA correction at single-base resolution, researchers may consider alternative strategies. These include the use of modified or evolved deaminase variants with a narrower editing window or sequence-specific activity, the use of imperfect gRNA,13 and switching from DSB-dependent platforms to prime editing, a complementary DSB-independent platform.
Innovation for cell and gene therapies
Building upon the foundations and learnings of CRISPR-based therapeutics, the emergence of base editing has been accompanied by the construction of a fast-tracked path toward the clinic. Base editing offers a distinct advantage over its predecessors: It facilitates precise, viable, multigene editing in a wide range of therapeutically relevant cell types.
The rapid progress and promising outcomes to date make base editing a strong contender in the development of innovative cell and gene therapies. It is with much anticipation that researchers and clinicians worldwide await evidence of its efficacy in humans.
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3. Musunuru K, Chadwick AC, Mizoguchi T, et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature 2021; 593(7859): 429–434. DOI: 10.1038/s41586-021-03534-y.
4. Pfizer and Beam Enter Exclusive Multi-Target Research Collaboration to Advance Novel In Vivo Base Editing Programs for a Range of Rare Diseases [Press Release]. Beam Therapeutics. Posted January 10, 2022. Accessed June 2, 2022.
5. Gillmore JD, Gane E, Taubel J, et al. CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis. N. Engl. J. Med. 2021; 385: 493-502. DOI: 10.1056/NEJMoa2107454.
6. Allogene Therapeutics Reports FDA Clinical Hold of AlloCAR T Trials Based on a Single Patient Case in ALPHA2 Trial [Press Release]. Allogene Therapeutics. Posted October 7, 2021. Accessed June 2, 2022.
7. Human Gene Therapy Products Incorporating Human Genome Editing [Document No. FDA-2021-D-0398]. Center for Biologics Evaluation and Research. U.S. Food & Drug Administration. Posted March 2022. Accessed June 2, 2022.
8. Intellia and Regeneron Announce Updated Phase 1 Data Demonstrating a Single Dose of NTLA-2001, an Investigational CRISPR Therapy for Transthyretin (ATTR) Amyloidosis, Resulted in Rapid, Deep and Sustained Reduction in Disease-Causing Protein [Press Release]. Intellia Therapeutics. Posted February 28, 2022. Accessed April 19, 2022.
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10. Leibowitz ML, Papathanasiou S, Doerfler PA, et al. Chromothripsis as an on-target consequence of CRISPR–Cas9 genome editing. Nat. Genet. 2021; 53(6): 895–905. DOI:10.1038/s41588-021-00838-7.
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