It is no surprise that technical progress in altering genes precisely, permanently, and safely is driving advances in biotechnology research. Genome editing is the key to delivering on the promise of the human genome project (HGP). Once scientists deduce relationships between genotypes and phenotypes and convert sequencing data into meaningful and actionable biological and clinical insights, genome editing holds the potential to alter errant genetic codes to treat diseases that were once deemed incurable.
Initial breakthroughs in genome editing were enabled by zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)—powerful, precise and programmable chimeric nucleases with sequence-specific DNA binding modules and sequence-agnostic scissors. ZFNs and TALENs enable a range of genetic modifications but early versions of the technology were more complex than what’s available today, limiting their use.
The CRISPR-Cas system, and subsequent innovations in base and prime editing, offer simplicity in the face of such technical hurdles. Clustered, regularly interspaced, short, palindromic repeats (CRISPR) found in bacterial genomes integrate snippets of viral code, providing key immune intelligence against future viral attacks. These repeats can be expressed individually as a short guide RNAs that bind to and target Cas enzymes to cut its matching genomic sequence, circumventing the need to design a new protein for every edit.
Compared to standard CRISPR-Cas systems, base editing is now recognized as a more precise, and potentially safer option. It introduces single-nucleotide changes while minimizing the risk of double-stranded DNA cuts by tethering an editing deaminase to a mutated Cas that makes a single-stranded cut at the target. Yet, it is limited by potential off-target edits that are hard to predict, and the ability to only introduce four of twelve possible single-nucleotide changes. Prime editing, although currently less efficient than base editing, employs a more complex prime editing guide RNA (pegRNA) that can introduce any single-nucleotide change, in addition to insertions and deletions of hundreds of base pairs.
Impactful advances in genome editing
Talking to GEN in an exclusive interview, Jason Potter, director of cell biology at Thermo Fisher Scientific, shares his perspective on biopharma’s ability to harness CRISPR’s evolving toolkit for precision editing applications. He sees strong potential in leveraging CRISPR-based drug screening and disease modeling for therapeutic development and discusses how his company is focused on growing its portfolio of precise editing solutions.
Potter believes several innovations will have an impact on the biotech industry in the decade ahead, including delivery of CRISPR machinery, editing techniques that work without making double-strand DNA breaks, allogeneic workflows where cells are edited, characterized, and expanded to eliminate off-target and GvHD (graft versus host disease) issues, and the use of CRISPR-based recombinases for targeted insertions.
Getting a grip on targeted edits without viral delivery
The production of CAR-T cells that involves precise, ex vivo genome editing sets the standard in genome editing-based therapeutics. “To advance the field, targeted insertion at a site where we know what’s going on, [such as] how many copies are inserted, is going to be more desirable than random approaches,” says Potter.
Although adeno-associated viruses (AAVs) are used to deliver CRISPR tools in CAR-T cell production, Potter says, “There’s a desire to move away from viral-based donors from a safety and cost perspective. But naked donors, that is just DNA, still have significantly lower performance than AAV systems.” With better protection and improved nuclear delivery, Potter says, “non-viral delivery will be preferred over viral delivery.”
Lipid nanoparticles (LNPs) are increasingly being used as a vehicle for ex vivo and in vivo delivery in animal model studies. “There’s potential in LNPs for both targeted and systemic therapies, but they’re not good enough yet,” said Potter. “You need at least 15% of the cells to have the edits you want for therapeutic efficacy in most diseases. We can hit that range but if you’re dealing with an autologous cell population, such as for CAR-T cells, you need as many cells as possible to carry the edits. If you can only edit 15%, cost increases, efficacy decreases, and you’ll need more cells to get the same result. This can be a non-starter, as often you can’t get more cells from patients.”
When it is not possible to edit a larger fraction of the initial cell population, a viable option would be to expand the cells that have been successfully edited. Innovations in cell expansion technologies, not just for suspension cells cultures such as those involved in T cell-based products but also for adherent cultures, have seen rapid growth. For instance, this Learning Lab discusses a new cell expansion technology that cultures adherent cells within hollow microfibers to reduce development costs and production timelines for cell therapy products.
Potter is convinced, better methods to expand edited cells could offer a potent solution to achieve high editing efficiencies. “If we could figure out methods that kept the edited cells and expand them out for several days, increasing the number of edited cells from the initial population—that’s also a significant path. There’s research into that space but I am not aware of a method that addresses edited cell expansion well enough yet,” says Potter.
Editing without double stranded DNA breaks
The problem with an editing system that generates double-strand-breaks in DNA is that it can have effects other than the intended edit. Potter says, “Where indels occur randomly you’re going to disrupt your gene in different ways. You might make a gene where the indel causes a long frame shift making a peptide or a protein that you don’t want. But the bigger issue is chromosomal rearrangements.”
With base editing, the chances of double-strand breaks are minimal as it only nicks one strand, resulting in improved cellular viability and less off target cutting. “However, in base editing there’s still the problem with random deamination due to the added cytosine or adenosine deaminase domain to some degree. The other restriction is the need to have a PAM at the right distance from the desired base change position. It has made great progress but it’s still a little limiting,” says Potter.
Prime editing, developed by Andrew Anzalone in David Liu’s group and first published in Nature in 2019, uses a pegRNA that identifies the target sequence and a nickase fused to a reverse transcriptase. This allows the introduction of small insertions and deletions in addition to a full repertoire of single-base changes.
Potter says, “Here you’re either getting the edit you want or you’re getting wild type. That’s better than getting random indels because you can still go through another round to edit the wildtype population.”
Twin prime editing (twinPE), a development from David Liu’s group first reported in 2021, further refines prime editing. As the name suggests, the method uses two pegRNAs that form the template for the synthesis of complementary DNA flaps on opposing strands to introduce targeted recombinase recognition sites separated by considerable distances. These twin sites then enable recombinase-mediated deletions, replacements, inversions, or insertions of large, gene-sized DNA sequences.
Allogeneic workflows: off target and graft-vs-host issues
Potter sees a shift occurring from often expensive autologous therapies to more scalable off-the shelf allogeneic therapies. However, allogeneic therapies can provoke graft-versus-host disease (GVHD), an immune response resulting from mismatched donor and recipient immune signatures.
“Stripping cells of MHC (major histocompatibility complex) receptors so that they can be used in multiple patients will allow us to both stockpile cells and bring therapies to patients at a much lower cost,” says Potter.
He looks forward to developments in using progenitor or induced pluripotent stem cells (iPSCs) as starting material for gene editing and characterization required for allogeneic therapies. Unlike autologous pipelines, allogeneic workflows offer the ability to screen the results, pick out intended clones, differentiate, and expand them to quantities that can treat many patients.
“The difficulty is demonstrating you can differentiate them in the lab so that they have the same immune potential and maturation profile as naturally differentiated cells,” says Potter. He remains optimistic about the therapeutic potential of allogeneic workflows.
Systemic delivery of gene editing platforms
Despite rapid advances in fine-tuning the molecular machinery that edits genes, delivering the gene-editing machinery or edited cells to functional niches in the body remains a challenge. Jennifer Doudna, who won the 2020 Nobel Prize in chemistry for her work on CRISPR, summed it up in a CNBC interview saying, “We have the editors; we just don’t know how to get them where they need to go.”
Intellia Therapeutics, a company co-founded by Doudna, has developed a biodegradable and well-tolerated LNP-based delivery platform that has successfully transported the CRISPR gene-editing apparatus to durably cut down on transthyretin in patients where the misfolded protein builds up on nerves and results in systemic problems.
Potter says, “That’s the first example that I’m aware of in humans. This was a knockout, but it opens the pathway for new discoveries. Before that, most of this work has been ex vivo, where we’re taking the cells out and modifying them before putting them back in.”
LNP-mediated delivery can be targeted specifically either by modulating LNP formulation or by including receptor-specific ligands. Effective delivery of the gene editing apparatus in vivo to targeted cells will also reduce off-target risks.
“Delivery is the real challenge! We need a zip code system for human tissues,” said Francis Collins, MD, PhD, former NIH director and currently President Biden’s acting scientific advisor, while speaking at the 2022 American Society of Gene and Cell Therapy (ASGCT) annual conference, in Washington, DC.
Toward precision editing
Precision of CRISPR editing tools have been heightened at many fronts with increased fidelity of Cas enzymes, unique gRNA designing tools, and chemical modifications of gRNAs to increase specificity.
To address the need for more precise genome editing products, Thermo Fisher has recently released a TrueCut HiFi Cas9 that cuts like the wild type enzyme but is engineered for high on-target activity with low off-target potential. In addition, the company has developed a design software (Invitrogen TrueDesign Genome Editor) that enables scientists to design, select and order reagents for their genome editing experiments.
“The software has a scoring algorithm that tells you the potential of alternative sites in the genome that have a similar gRNA sequence. The less unique your gRNA is, the more likely it is that off-targets can occur,” said Potter. Together, a high-fidelity enzyme and a unique gRNA can significantly minimize off-target edits.
To confirm off target risks with a particular gRNA and Cas9 combination, Potter uses techniques like GUIDE-Seq (Genome-wide, Unbiased Identification of DSBs Enabled by Sequencing) to detect off-target events to an extent where they cannot be detected by sensitive next-generation sequencing (NGS). GUIDE-Seq allows unbiased, in vitro detection of off-target edits in living cells. In a 2018 paper in Biotechniques, Potter’s team presented TEG-Seq—an improvement over the GUIDE-Seq protocol developed by Keith Joung’s group. TEG-Seq uses an Ion Torrent NGS platform to detect genome-wide off-target edits. By reducing nonspecific amplification, TEG-Seq increases sensitivity for off-target detection.
“In silico tools like TrueDesign can identify potential off-target edit sites by similarity to the gRNA sequence, but there are biological reasons why, even if it has the potential, nothing’s going to happen at that site. So, in conjunction with in silico analysis, tools like TEG-Seq can experimentally confirm whether or not your off-target sites are really going to be a problem for you. And if they are, you can consider whether you need a high-fidelity Cas or a modified gRNA to mitigate that, or if you need to change the guide to something else that has a lower off-target potential,” says Potter.
In addition to refining the CRISPR toolkit, Thermo Fisher is also investigating the understudied potential of TAL effectors. “There does seem to be a lot of potential there both for specificity and the ability to cut. They seem to have the same ability as CRISPR. The workflows for TAL effectors have been improved on and can be competitive. It’s just that the field has been focused on CRISPR,” says Potter. “For TAL effectors the big challenge was getting your TAL made. Now TALs are much cheaper and can be delivered in the same timeframe as you can get a gRNA.”
Thermo Fisher’s R&D team is working on improving TAL effectors. “We screen rigorously for specific, on-target binding. That can take more work but those can be highly specific enzymes, potentially more specific than CRISPR currently,” says Potter. To bolster specificity of TAL-based editing, Potter’s team often uses protein or mRNA systems that are transient so that the payload sticks around in target cells for only short periods. “We only need to make one cut per cell, so we don’t need it to be around for a long time,” says Potter. Using a transient toolkit improves fidelity and safety in gene editing strategies in vivo.
Although most of the genome editing field is currently focused on the CRISPR-Cas systems, which have a reputation for being simpler, faster and more cost-effective, Potter believes probing into the untapped potential of other nucleases is worthwhile.
“The only big disadvantage of CRISPR is that it needs a PAM site, so you’re limited on where you can use it,” says Potter. Updated versions of TAL-nucleases do not have sequence specificity and can potentially be used to cut at every base in the genome. These new TALs include a non-specific DNA-cleaving nuclease fused to a sequence-specific DNA-binding domain that can be targeted to any sequence.
The success of the HGP has snowballed into the identification of nearly 7,000 human genetic disorders. While gene therapies based on ex vivo gene editing, such as CAR-T, have had a profound impact, most patients with genetic diseases need in vivo solutions, which require clinically precise editing tools, and safe and specific delivery systems that can be scaled to benefit the entire patient population.