CELL AND GENE THERAPY

The newbies of nuclease-based gene editing

The role of base and prime editing in therapeutics
The new genetic engineering approaches of base editing
and prime editing promise the capacity for precise genemodifications, so could have a substantial impact on thedevelopment of therapeutics to treat rare disease and thenext generation of cell-based therapies. As newbies in
the fast-paced world of nuclease-based gene editing, baseand prime editing are being investigated intensely for
their advantages and limitations when it comes to clinicalapplication.
Since the discovery of DNA restriction enzymes in thelate 1960s enabled the manipulation of DNA, the idea ofaltering DNA for therapeutic gain has been with us. The
past 10 years has seen an unprecedented pace of change
in the rapidity and precision with which we can edit DNA.
Such advances have been driven by the use of nucleases,
such as zinc finger nucleases (ZFNs), transcription
activator-like effector nucleases (TALENs) and clusteredregularly interspaced short palindromic repeats-CRISPR
associated protein (CRISPR-Cas), but the precisionof these can be less than desired when contemplating
genetic changes in vivo that would be required to correct
monogenic diseases of childhood, such as Duchenne
muscular dystrophy, or making many simultaneous genetic
alterations to a cell ex vivo before placing it back into thepatient from whence it came.

The challenge of DNA double-strand breaks
The CRISPR-Cas9 system has been widely adopted as thego to method for gene editing, indeed this approach has
progressed impressively from its commercial debut in 2012
to its clinical trial debut in 2016. While ZFNs, TALENs andCRISPR-Cas are being used to drive gene and cell therapy
pipelines, they all share one substantial challenge in that
their mode of action generates DNA double-strand breaks.
These breaks are more often than not repaired in an error
prone fashion by the cell’s repair machinery and as suchcan cause unintended genetic changes within the wider
genome of engineered cells. Where these nuclease-based
gene editors are targeting just one single gene, there aremethods that can be used to make them error free.
However, it is becoming clear that for some cell therapies
to be successful, such as the use of chimeric antigen
receptor (CAR) T cells in the treatment of solid tumours,
multiple gene engineering events will be required toachieve efficacious therapies. There is a real risk that if one
employs standard gene-editing nucleases to make multipleedits, it could lead to genome-altering insertions, deletions
and/or chromosomal translocations. The impact of this ona patient could be that the gene or cell therapy is effective
but the off-target genetic changes lead to deleterious sideeffects, impacting patient recovery and potentially survival.
While base editing and prime editing make use of theCRISPR-Cas gene editing system, they use modified formsof these components that limit the introduction of DNA
double-strand breaks. This means that the off-target effects
8 June 2020 MedNousof these technologies in terms of insertions, deletions ortranslocations should be substantially reduced. However,
the true nature and extent of off-target effects arisingfrom these new gene editing approaches has yet to be fully
investigated.

Mechanics of base editing and prime editing
Base editing uses a deaminase enzyme to make a specificbase-pair change in the DNA. The base pair alteration is atransition (Figure 1), and can be A to G or C to T dependingon which deaminase is used. Prime editing uses a different
approach that involves a reverse transcriptase that enables
both transitions and transversions (Figure 1), as wellas multiple base-pair swaps, small insertions and smalldeletions.
Aside from its potential use to correct transitions in theDNA that cause human disease, base editing is well suitedfor introducing stop codon(s) into genes to knock out genetranscription from one or more genes. This is potentially
ideal for allogeneic CAR T cell therapy in patients withsolid tumours where it is expected that multiple genes
need to be silenced to provide a cloak of invisibility to thesecells such that they are not ‘seen’ as foreign by the treatedpatient’s immune system, as well as prolonging the survivalof the cells in the patient and enabling them to function
in an immune suppressive tumour microenvironment. Asbase editors do not cut both strands of the DNA in order tointroduce a base-pair transition, and have a relatively highgene engineering efficiency, it suggests that base editing
will have a starring role in the next generation of cell-based
therapies where multiple genetic changes are required.
Prime editing has the flexibility to correct nearly any
single nucleotide polymorphism within a target genome,and add in multiple base pairs using a template that is partof the prime editing machinery. Prime editing can introduce
insertions or deletions. These functionalities together
are ideal for gene therapies where corrections of smallstretches of DNA are required for establishing normal genefunction and a positive outcome for the patient. However,as the newest of the newbies on the block, the efficacy andefficiency of prime editing is still under investigation. Prime
editing can introduce unintended insertions and deletions
into the genome compared with base editing, but still atlevels below those evident when using CRISPR-Cas geneengineering.
One additional advantage of base editing and prime
editing is that they can modify the genome with precisionand efficacy in non-dividing cells. In the past, introducing
either single base changes or modifying small stretchesof DNA required a gene knock-in. Knocking stretches ofDNA into cells requires a process known as homologous
recombination, which is most efficient in dividing cells,
and is limited in primary cells, especially those that do
not divide. In a therapeutic setting where non-dividing
cells are often the target for gene therapy, establishedwww.mednous.com© 2020 Evernow Publishing Ltd

CELL AND GENE THERAPY
methods for gene knock-ins
are too inefficient to have
utility. As base editing andprime editing are not reliant onthese established methods for
introducing one or more basepair changes, they might prove
to be the systems of choice foraltering DNA in primary, nonproliferative cells. Early datasuggest that base editing has
a higher level of gene editing
efficiency compared with primeediting, which could offer baseediting a clear advantage inmodifying primary cells fortherapeutic use1.

What don’t we know yet?

Figure 1. Mechanics of base editing and prime editing
ONH2NNNNHAGTransversions

In summary
With any new technology, success is always dependent
on the details and ‘real world’ data. If over the next year
peer-reviewed data show that base editing has a highNHguanine

Transitions

adenineNH2Npurines

The concern with theestablished methods ofCRISPR, ZFNS and TALENs
for making multiple genetic
changes in a cell is theirNH2
off-target effects. The offC
target effects of base editing
Nand prime editing are stillN
under investigation. WhileOH
their reduced capacity forcytosine
introducing changes in theDNA likely to bring aboutsubstantial genetic effect suchas translocations and large
deletions is evident, the off-target effects of both systems
are still largely unknown. Similarly, factors influencing
the editing efficiency of either technology require moreinvestigation.
Similar to CRISPR-Cas gene editing, the design of theguide RNA will be crucial to the efficacy and precision ofbase editing and prime editing. Fortunately, optimisationof guide RNA design has been necessary for the CRISPRCas gene engineering revolution and key learnings from thepast decade should be applicable to both base editing andprime editing.
The off-target activities of the reverse transcriptase anddeaminase for prime editing and base editing, respectively,
also need investigation. It is well known among virologiststhat reverse transcriptases are error prone and the levelof error that might occur with prime editing is currently
unclear. It is possible that prime editing might be able toaccurately change one or two bases per edit but beyond
that the error rate increases. Similarly, for base editing theoff-target deamination activity of the deaminase is being
investigated and will need to be carefully controlled toensure that the deaminase does not randomly deaminatebase pairs from the target site bound by the guide RNA.

© 2020 Evernow Publishing LtdNHNTransversions

OTTransitions

NHpyrimidinesNOH
thyminegene engineering efficiency with limited off-target effects
compared with the established methods of CRISPR, ZFNs
and TALENs, then it will potentially be the gene editor ofchoice for cell-based therapies where multiple knockouts
are required. Similarly, if prime editing proves to be precise
and efficacious at transition, transversion and introducing
short stretches of DNA to correct a gene of interest, thenthis might become the method of choice for gene therapy.
In the fast-moving field of research that is gene editing
today, we should expect much greater understanding of
the capabilities and limitations of base editing and prime
editing and their applications to gene and cell therapy
within the next 12 months.
References:
1. Anzalone AV, Randolph PB, Davis JR, et al. Search-and-replace
genome editing without double-strand breaks or donor DNA.
Nature. 2019;576(7785):149-157. doi:10.1038/s41586-019-1711-4Figure 1: https://commons.wikimedia.org/w/index.
php?curid=19884125

This article was prepared by Jonathan Frampton, PhD,
Corporate Development Partner at Horizon Discovery ofCambridge, UK

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