Scientists have worked tirelessly to develop ever more precise and efficient CRISPR-Cas systems to reach the ultimate goal: safe and effective CRISPR-Cas-based medical treatments. Over the years, scientists have found and created Cas variants, developed vast libraries of guide RNAs (gRNAs), and invented new editing approaches. These advancements have been instrumental in bringing CRISPR into the clinic.
At the end of 2023, there were 84 ongoing or completed clinical trials around the world involving CRISPR-Cas systems. Also, the first CRISPR-based gene editing therapy gained approval from the U.S. Food and Drug Administration. This therapy, Casgevy, was developed by Vertex Pharmaceuticals and CRISPR Therapeutics, and it was approved for the treatment of sickle cell disease and transfusion-dependent b-thalassemia.
A more sophisticated CRISPR-Cas9 system
The success of Casgevy highlights the tremendous potential of CRISPR-Cas9 systems by achieving gene knockouts with high efficiency and high specificity. However, most genetic disorders are the result of a combination of mutations, deletions, and duplications, requiring more sophisticated editing strategies, such as correction and insertion-based editing. These methods currently don’t have the same level of efficiency and specificity, but
with time they will, suggests Christof Fellmann, PhD, head of CRISPR-X, CRISPR Therapeutics.
CRISPR Therapeutics uses a combination of knockout and insertion editing to engineer allogenic chimeric antigen receptor (CAR) T cells with an enhanced potency. Two investigational product candidates are in clinical testing for the treatment of certain cancers, and two additional clinical trials are expected to be initiated in the first half of 2024 targeting systemic lupus erythematosus and hematologic malignancies.
CRISPR Therapeutics is also advancing clinical trials for in vivo CRISPR-Cas9 therapies, which offer the possibility for one-time treatments for a variety of diseases. This includes two investigational treatments using lipid nanoparticles to deliver a CRISPR-Cas9 system to the liver, where it disables a specific gene associated with cardiovascular disease. After preclinical data for both treatments demonstrated durable reductions of the key proteins, they are now in clinical testing.
The CRISPR-X team is pushing the boundaries even further and is now focused on developing methods for in vivo gene correction and insertion in the liver and other organs. “CRISPR-X is exploring and developing new editing modalities that we believe will enable us to address new potential disease indications across various target tissues,” Fellmann says. “We believe the possibilities are vast. Over time, we will see the impact of CRISPR across all disease areas, both common and rare.”
A detailed, real-time CRISPR quality control platform
Developing a high-performance CRISPR gene editing system is largely a matter of choosing the right system components, that is, the right guide RNAs (gRNAs) and Cas proteins. Doing so, however, typically involves the use of laborious, time-consuming, and expensive methods that incorporate cell-based assays and sequencing-based assessments of on/off-target editing. Unfortunately, these methods may not permit detailed assessments of complex formation or target binding.
An alternative approach is available from CRISPR QC. The company has an exclusive license to the Cardea Bio CRISPR-Chip, a real-time analytics platform that can capture data on performance metrics such as binding efficiency, binding strength, and cleavage rate. Unlike other CRISPR analyzers, CRISPR-Chip works quickly and doesn’t require technical expertise. Most importantly, it allows scientists to do more research by cutting costs.
“Our core platform can help reduce the number of formations that need to be tested because it can identify the formations that are less likely to work,” says Kiana Aran, PhD, co-founder and board member at CRISPR QC. “It’s really allowing us to not only scale up the gene editing process and reduce time, but also to gain a better understanding on how CRISPR works, which will ultimately allow us to design better formulations.”
CRISPR QC has been standardizing gene editing procedures in collaboration with the National Institute of Standards and Technology’s Genome Editing Consortium. According to CRISPR QC, details about this work will be published later in the year.
CRISPR-Chip can theoretically be used to test any CRISPR system. At this point, it hasn’t been used to test any next-generation gene editing systems, such as base or prime editing systems, but it will soon, according to CRISPR QC.
Establishing clinical safety
The advent of base editing in 2016 gave scientists a precise way to edit single nucleotides without making double-strand breaks in the DNA. This meant that CRISPR could be safer. It could avoid the double-strand breaks that result in off-target editing, translocations, and deletions, and that cause cells to shut down or die.
While this certainly set base editors apart from conventional CRISPR-Cas systems, unwanted edits at the target site and elsewhere remain a key challenge for both editing methods. “The most important challenge of base editing tools is unintended edits,” affirms Priya Chockalingam, PhD, vice president and head of clinical bioanalytics and translational sciences, Beam Therapeutics.
Monitoring and assessing these changes is a critical part of clinical testing. But even before a proposed genome editing therapeutic can enter the clinical testing phase, the drug’s sponsor must complete a rigorous preclinical assessment to identify and explore the potential for unwanted edits and evaluate the associated biological consequences.
“Preclinical studies are conducted to assess and characterize the risks of gene edited products,” Chockalingam says. “Workflow for the evaluation of off-target editing includes in silico prediction and experimental detection, nomination and validation of off-target edits, and finally clinical monitoring of the validated off-target edits.”
Beam Therapeutics currently has two products in clinical testing, one for the treatment of sickle cell disease and the other to produce donor-derived CAR T cells. The company recently received clearance to initiate another clinical study in the United Kingdom for an in vivo therapy for patients with a1-antitrypsin deficiency, an inherited genetic condition that can cause serious lung and liver disease.
Prime editing goes big
The creator of base editing, David R. Liu, PhD, of Harvard University and the Broad Institute, went on to develop prime editing in 2019. Instead of introducing changes to single nucleotides, prime editing can search the genome, find a precise location, and replace faulty DNA with a corrected copy, again without causing a double-strand break in the DNA. This led to a significantly improved safety profile.
“Prime editing occurs with high specificity, and no detectable double-strand breakage or detectable bystander edits have been observed in our lead programs,” says Jeremy Duffield MD, PhD, FRCP, chief scientific officer, Prime Medicine.
Prime Medicine is working to improve the editing capabilities of two systems that are currently in the discovery phase. Their dual-flap or twin prime editing system can make a broader range of edits over a larger DNA sequence, lending itself to the development of treatments for repeat expansion diseases such as Huntington’s disease and amyotrophic lateral sclerosis. In addition, the company’s PASSIGE system allows for a gene-sized piece of DNA to be precisely added to the genome. Whether used alone or in combination, these technologies can deliver great improvements to prime editing systems.
“The range and scope of prime editing technology continues to expand,” Duffield notes. “We estimate that more than 90% of all disease-causing mutations can be addressed by this technology.”
However, one limitation of prime editing systems is their larger size, which can complicate delivery if delivery vehicles have limited capacity. Prime Medicine is actively addressing this by developing smaller prime editors and finding ways around size constraints of delivery methods. One option is to use two adeno-associated virus (AAV) vectors.
A more diverse toolbox
To address delivery constraints and other limitations,
Metagenomi is developing a comprehensive CRISPR toolbox. “Metagenomi’s toolbox contains thousands of CRISPR nucleases with diverse abilities to target different parts of the genome,” says Chris Brown, PhD, vice president of discovery, Metagenomi. “Given the measured targeting density of our toolbox, we believe that essentially any codon in the human genome could be addressed with our gene editing systems.”
One of Metagenomi’s applications is an in vivo therapy for hemophilia A. In this therapy, the company’s CRISPR editing system inserts a functional FVIII gene into the genome to restore normal blood clotting. Two system components—the company’s propietary novel nuclease, MG29-1, and a small, engineered gRNA—are packed within a lipid nanoparticle that targets the liver. A functional FVIII gene is delivered by an AAV vector.
In an ongoing nonhuman primate study, Metagenomi demonstrated integration of a FVIII cassette and observed therapeutically relevant levels of protein over nearly five months following treatment.
Metagenomi’s toolbox is also designed to be compatible with viral and nonviral delivery technologies, particularly through the use of smaller components. For example, the company has developed an ultrasmall system called SMART. It is roughly three times smaller than SpCas9, which allows the company’s editors to be packaged in one AAV and which expands potential target tissues beyond just the liver.
“As the genetic medicine field continues to rapidly evolve, our platform positions us to be at the forefront of unlocking the full potential of genome editing through the continuous discovery of new editing systems and the development of the next wave of genetic medicines,” Brown declares.
