How Next-Generation Editing Technologies are Transforming Cell and Gene Therapies

Genomics and genome editing have gone through a revolution in the past decade, brought on by the discovery of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems.¹ These RNA-guided DNA endonucleases have made gene editing in prokaryotic and eukaryotic cells simple and accessible, empowering pre-clinical and clinical researchers to apply them in various areas, including genomics, imaging, diagnostics, and cell and gene therapeutics.^2-5

Therapeutic applications have received the most attention, given the potential for developing one-dose treatments for genetic diseases. Promising results from Intellia’s ongoing, first-in-human trials in patients with hereditary angioedema (HAE) and transthyretin (ATTR) amyloidosis have heightened excitement about the potential of curing other rare genetic diseases.^6,7 Accordingly, several other companies are targeting rare diseases, such as sickle cell diseases, beta-thalassemia, and inherited retinal disorders.^8-10 CRISPR-Cas systems are also being investigated in ex vivo therapeutic strategies, where engineered T-cell receptors are introduced into immune cells isolated from patients, then transferred back into patients to induce anti-tumor immunity.^11,12

CRISPR 2.0: New Tools, New Therapeutic Applications

Thus far, the CRISPR-Cas systems that have made inroads into the clinic use traditional genome editing approaches, relying on the generation of double-stranded breaks (DSB) in host genomic DNA. 

While powerful, this strategic approach can lead to increased safety concerns and decreased efficacy. It also limits the number of genetic diseases that drug developers can target. 

CRISPR-mediated genetic engineering has evolved and changed since the landmark Science paper by Jinek et al. to address some of these shortcomings.¹³ This next generation of new CRISPR-Cas techniques has expanded the genetic engineer’s toolkit, increasing the scope of diseases that drug developers could cure. 

The General Concept of Gene Editing

While there are many old and new tools for gene editing, the concept is the same: A guide RNA targets a particular site so that an enzyme, such as a DNA endonuclease, can generate a downstream change in the target sequence.¹⁴‍

In the case of CRISPR-Cas nucleases, DNA sequences are targeted by a complementary, target-specific, single guide RNA (sgRNA) flanked by a short protospacer adjacent motif (PAM).^15,16 Several different CRISPR-Cas nucleases and fusion proteins enable researchers to employ their desired gene editing strategy.

Non-Homologous End Joining and Homology-Directed Repair

The most widely used CRISPR-Cas system, CRISPR-Cas9, creates double-stranded breaks (DSBs) in genomes.^15,16 A cell’s endogenous DNA repair pathways then take over to repair the break. 

There are two distinct repair pathways, non-homologous end joining (NHEJ) and homology-directed repair (HDR), which introduce different types of mutations.^15,16‍

NHEJ is an imperfect process that can generate small insertion or deletion mutations, collectively called indels.¹⁷ These indels can mutate a gene by introducing a sense mutation or knock-out a gene by introducing a frameshift or stop codon mutation. Intellia’s ATTR clinical candidate uses this approach, inducing NHEJ that introduces frameshift mutations into the mutated TTR gene associated with disease pathology.

HDR, by contrast, is accomplished by inducing a CRISPR-mediated DSB in a target sequence, which then gets repaired with an exogenous piece of DNA that has homology to the target DNA sequence.¹⁸ Through homologous recombination, this DNA is used as a template to repair the DSB, thus creating a gene knock-in. Short, single-stranded DNA can be used, but double-stranded DNA is required for larger insertions. Researchers can use this to introduce a new gene into the genome or a functional version of a defective gene.¹⁹ Most CRISPR-based therapeutics being clinically investigated use this genome editing technique.

There is a vast selection of CRISPR-Cas systems for NHEJ and HDR gene editing strategies. Some introduce DSBs, but others are nickases, which cleave only a single strand of the target DNA. In some experimental situations, using a nickase can be more robust and result in less off-target gene editing.^13,20,21

Correcting Certain Point Mutations with Base Editing

Base editing is a newer form of genome editing that can alter specific point nucleotides without inducing DSBs. CRISPR-Cas base editing systems were designed and developed by using a nicking or cleavage-deficient version of Cas9 (called dCas9), fused to a:

• Escherichia coli–derived uracil DNA N-glycosylase or rat APOBEC1, which can induce C→G transversions,^22,23
• cytidine deaminase, which can induce C→T transitions,²⁴ or 
• adenine deaminase, which can induce A→G transitions.²⁵

The cytidine base editors (CBEs) and adenine base editors (ABEs) expand the therapeutic potential of CRISPR-Cas systems by enabling cell and gene therapy developers to efficiently correct many of the disease-associated single nucleotide polymorphisms (SNPs) without the risk of introducing DSBs.^24,25 In addition, CBEs and ABEs have very low rates of introducing unwanted, off-target edits (<0.1%). The recent development of optimized C-to-G base editors further expands the scope of SNPs that can be introduced or corrected.^22,23‍

Thus far, proof-of-concept base editing in animal models has yielded promising outcomes. ABEs have been used to correct a mutant dystrophin gene in a Duchenne muscular dystrophy mouse model.^26. Base editing can correct other loss-of-function mutations, such as phenylketonuria, or silence the expression of disease-associated genes by introducing premature stop codons within a gene’s coding region.²⁷ 

Changing Any DNA Base to Another Using Prime Editing

Prime editing is conceptually similar to base editing but expands its scope to all four possible transition and eight transversion mutations.²⁸ This capability allows genome editing to correct up to 89% of disease-associated SNPs.²⁸

The system uses a Cas9 nickase fused to the reverse transcriptase (RT) enzyme. Instead of a sgRNA, the enzyme uses a prime editing guide RNA (pegRNA), which contains a complementary targeting sequence and the desired edit.²⁸ After nicking the target strand, the Cas9-RT fusion uses the pegRNA as a template for reverse transcription, incorporating the desired mutation directly into the nicked strand. The mismatch in the unedited strand is then corrected by a distinct Cas9 nickase, sgRNA, and endogenous DNA repair systems that use the edited strand as a template for repair.²⁶‍

This method can also insert DNA sequences of up to 50 nucleotides and deletions of up to 80 nucleotides.²⁶‍

The therapeutic application of prime editing is in its early days, but there is broad therapeutic potential given the scope of mutations that can be corrected. Companies such as Prime Medicine hope to capitalize upon this potential. 

Beyond Genome Editing: Targeting RNA

The discovery of the Cas13 enzyme, a programmable RNase, opened up the possibility of targeting disease-associated RNAs.²⁹ Because Cas13 lacks any DNase activity, it removes the risk of off-target DNA cleavage or inducing a DNA damage response. RNA editing is also reversible: Once Cas13 is eliminated from a cell, edited RNA would eventually be degraded, allowing the transcriptome to return to homeostasis. 

These advantages make it an attractive editing technique for knocking down disease-associated transcripts and modulating alternative splicing events. In addition, researchers have fused adenosine deaminases to dCas9 to direct RNA editing (both A to I and C to U conversions) to specific transcripts.^30,31 Like base and prime editing, proof-of-concept experiments are underway, and first-in-human studies are on the horizon.

CRISPR-Driven Innovations in Cell and Gene Therapies

Thanks to the development of the new gene editing techniques described above, cell and gene therapies have made incredible progress. In particular, gene therapy, which suffered significant safety setbacks in the late 1990s, is only getting safer with the possibility of CRISPR-based editing that doesn’t require DSBs.³²‍

For that reason, base and prime editing are racing toward the clinic: In early 2022, Beam Therapeutics entered into a research collaboration with Pfizer to target several rare diseases using base editing.³³‍

Yet, careful pre-clinical vetting of off-target edits and overall editing precision is still essential for drug developers looking to take these next-generation technologies into humans. Regulatory agencies are keeping a careful watch on this rapidly evolving field and have published detailed guidelines for standardizing the pre-clinical development of gene editing technologies.³⁴ 

As a part of the emerging pre-clinical and clinical framework for evaluating the safety and efficacy of gene editing technologies, computational tools, including AI and ML algorithms, are sure to be an integral piece of overcoming cell and gene therapy commercialization challenges.

Recommended Reading

Want to learn more about how computational tools can help shape the pre-clinical development of cell and gene therapies? Check out our white paper on CRISPRank, a novel computational tool that offers an end-to-end solution for designing genome editing experiments and selecting the most appropriate strategy for your cell and gene therapy.

Conclusion

New CRISPR-Cas systems are maturing, driving a new research and drug development era. The next-generation editing technologies discussed above solve many ongoing concerns and risks initially raised as CRISPR-based gene therapies entered the clinic. As investment floods into the growing bioeconomy, a robust biomanufacturing infrastructure is forming, enabling the continued refinement, development, and commercialization of novel biologics and the possibility of safe and effective cures for a range of diseases.