CRISPR Therapy Progress and Prospects

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins are core components of fast-evolving therapeutic gene editing tools. Scientists have used CRISPR-Cas9 technology to develop therapies for genetic disorders relatively easily and cost-effectively. In this article, explore the milestones in CRISPR therapy development, from designing carriers for delivering CRISPR therapeutics, to understanding important underlying mechanisms, applications, and improvement strategies.

What Is CRISPR Therapy?

CRISPR therapy is a promising gene editing approach that uses genome-targeting tools such as the CRISPR-Cas9 system to treat diseases by correcting mutations or otherwise modifying disease-causing genes with high precision and efficiency.¹

Prominent milestones in the CRISPR odyssey

In 1987, molecular biologist Yoshizumi Ishino and his team at Osaka University first discovered CRISPR in the DNA sequence of Escherichia coli.² Technological advances enabled researchers to determine CRISPR’s biological function as part of an adaptive prokaryotic immune system and identify genes associated with CRISPR sequences that encode DNA repair proteins, known as cas genes.^3,4 CRISPR guide RNAs (gRNAs) help Cas proteins cleave DNA or RNA sequences at specific target sites, ultimately enabling researchers to develop CRISPR technologies for gene editing.⁵

In 2016, scientists conducted the first CRISPR-related clinical trial involving human recipients and demonstrated the possibility and safety of its clinical application.¹ In this clinical trial, researchers edited T cells to reduce programmed cell death protein-1 (PD-1) expression and injected the CRISPR-Cas9 -edited T cells into patients who had advanced non-small-cell lung cancer. These cells survived for several weeks without causing substantial adverse effects.

Since the first clinical trial, scientists have continued to investigate many therapeutic applications for CRISPR gene editing. In 2023, the US Food and Drug Administration (FDA) authorized Casgevy^® (exagamglogene autotemcel), the first and only approved CRISPR-Cas9-based cell therapy, for treating sickle cell disease (SCD) in patients aged twelve years and above.

How CRISPR therapies work

CRISPR therapies typically use the CRISPR-Cas system to cut a specific target site in a gene, which the cell’s endogenous DNA repair pathways mend, correcting aberrant expression or mutations in the process.⁶

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Genetics

CRISPR Gene Editing: Cas9 and Beyond

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In the case of SCD, a common monogenic blood disorder caused by a single point mutation in the hemoglobin subunit beta (HBB) gene, the CRISPR-Cas9 system allows scientists to replace a disease-causing variant with a functional one. Using a gRNA complementary to the HBB gene sequence, scientists employ Cas9 and a donor strand of DNA carrying the corrected DNA sequence to accurately edit the SCD mutation.

For cancer treatment, scientists aim to inactivate oncogenes, such as MYC, that play a vital role in cancer development.⁷ For instance, researchers have designed CRISPR gRNAs that specifically detect and bind to an oncogene to prevent its transcription. This strategy has effectively reduced tumor growth in animal models of lymphoma. Scientists are also exploring ways to use the CRISPR-Cas9 system to repair mutations that increase the risk of cancer, such as errors in the BRCA1 and BRCA2 genes.

Delivering CRISPR Therapies

Delivering CRISPR-Cas system components to their target site is crucial for efficient gene editing. Scientists can deliver CRISPR therapies via in vivo or ex vivo approaches.¹⁰ For in vivo delivery, they typically directly inject packaged plasmid DNA or mRNA encoding the CRISPR components into the bloodstream or target tissues. Ex vivo methods most commonly involve extracting cells from a donor, modifying them with CRISPR in the laboratory, and reintroducing the engineered cells into a living system, such as a model organism or patient.

For both in vivo and ex vivo approaches, researchers must use appropriate carriers or packaging materials to protect the CRISPR components, ensuring they reach the target location and function optimally. The key requirements for efficient CRISPR tool delivery include the following.¹

• The carrier must be stable in the bloodstream, preventing immune clearance or degradation.
• The carrier must accumulate in target tissues and induce cellular endocytosis.
• The CRISPR system must evade the lysosome to regulate gene expression or perform genome editing.

To overcome limitations associated with CRISPR delivery such as susceptibility to degradation and immune clearance, scientists have developed various protective carriers that improve delivery efficiency. Common carriers for CRISPR delivery include the following.¹

• Viral vectors: Nonpathogenic viruses such as adeno-associated virus (AAV), lentivirus, and baculovirus can deliver the CRISPR system into target cells, often without generating long-term genetic alterations.
• Polymers: Polymer-based nanoparticles or micelles can encapsulate CRISPR components and result in low immunogenicity and high cellular uptake upon delivery. For example, polycaprolactone (PCL) is a synthetic, biodegradable polymer that exhibits the potential for targeted in vivo CRISPR delivery in animal experiments.
• Nanocarriers: Lipid nanoparticles (LPN) exhibit high biocompatibility and low immunogenicity and can efficiently encapsulate CRISPR components for targeted delivery and effective gene editing. Scientists have encapsulated gold nanoparticles (AuNP) in LPN to develop AuNP-Cas9-sgPlk-1 plasmid (LACP) for melanoma treatment. The inherent anti-inflammatory and antibacterial properties of AuNPs could further support tumor treatment.
• Exosomes: Exosomes are small extracellular vesicles that can directly package plasmids, mRNA, gRNAs, and Cas proteins and deliver them to the target cells, effectively reducing off-target side effects during transport.

Examples of CRISPR Therapy Applications

CRISPR systems hold immense therapeutic potential for many diseases, from monogenic conditions to complex disorders.¹¹

For example, scientists currently use ex vivo CRISPR-Cas9 strategies to correct pathogenic mutations that cause heritable diseases such as SCD, cystic fibrosis, or Duchenne muscular dystrophy, or silence aberrantly expressed genes in tumor cells. In vitro or ex vivo CRISPR-based screens also help researchers identify genes with therapeutic potential, such as viable targets for anticancer immunotherapy.

CRISPR gene editing tools can also augment existing therapies, including photothermal therapy for malignant tumor treatment.⁷ Photothermal therapy often fails to effectively kill tumor cells because of their tolerance to high temperatures. Scientists have constructed a hypoxia-responsive nanoparticle based on gold nanorods to overcome this limitation and deliver CRISPR specifically into hypoxic cancer cells.¹² This nanocarrier packages a CRISPR-Cas9 system that targets heat shock protein 90α (HSP90α), causing the cancer cells to lose their thermotolerance. Subsequently, scientists can use mild heat from near-infrared light to ablate the tumor without damaging paracancerous tissue.

Additionally, researchers are investigating CRISPR therapies in pre-clinical and clinical trials for diseases beyond cancer and monogenic conditions. For example, a pre-clinical experiment demonstrated that the CRISPR-dCas9-VP64 system encapsulated in exosomes can activate the transcriptional hepatocyte differentiation regulator hepatocyte nuclear factor 4α (HNF4α) to alleviate liver fibrosis.¹³

Scientists are also actively investigating CRISPR technology in clinical trials for diseases such as transthyretin (TTR) amyloidosis, Leber congenital amaurosis type 10 (LCA10), cardiovascular disease, bone regeneration, Alzheimer’s disease, and obesity.¹⁴ These trials evaluate the safety and efficacy of editing specific genes associated with these conditions.

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From CRISPR to Prime Editing: The Evolution of the Genome Editing Revolution

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Challenges and Research Innovations

CRISPR therapies, despite their immense potential, involve several challenges, including narrow genome editing ranges, off-target effects, and ethical considerations.

Target sequence limitations

CRISPR-Cas9 can theoretically edit any position in the genome, but it is limited to sequences that are three to four nucleotides upstream of a protospacer adjacent motif (PAM).¹ If the conventional Cas9 PAM sequence, NGG, where N can be any nucleotide, is not located downstream of the target sequence, Cas9 cannot effectively bind the target for editing.

To overcome this limitation, scientists have developed multiple Cas9 variants by adding modified structural domains or mutating the Cas9 PAM-recognition site. For example, researchers developed xCas9 by phage-assisted continuous evolution, creating a Cas9 variant that recognizes many PAMs including GAA, NG, and GAT.¹⁵ The broader targeting capacity of xCas9 could facilitate new CRISPR gene therapy development for many genetic diseases, including thalassemia and inherited retinal diseases.

Off-target effects

In conventional CRISPR-Cas9 systems, off-target effects can occur when Cas9 acts on untargeted genomic sequences, potentially leading to adverse outcomes. A mismatch between the gRNAs and the DNA sequence, tolerated by Cas9, results in genomic cleavage at nontargeted sites. In addition, genomic regions that are almost identical to target sequences are more likely to become off-target sites.

Scientists have created Cas9 variants to improve the accuracy of CRISPR tools and therapies. For instance, the nuclease dead Cas9 variant (dCas9) has two amino acid substitutions, H840A and D10A, that prevent dCas9 from cleaving the DNA that it binds.^1,16 Researchers have incorporated transcriptional activators or repressors into CRISPR-dCas9 systems, creating tools that can activate (CRISPRa) or inhibit (CRISPRi) target gene transcription without editing the DNA.¹⁷ One key feature of CRISPRa and CRISPRi, which could be the next major focus in developing novel gene therapies, is their ability to modulate gene expression without causing permanent DNA changes.¹⁸

Researchers have further developed CRISPR fusion technologies such as base editing and prime editing that use a Cas9 variant called a nickase, which bears only nuclease domain substitution and creates single-stranded DNA breaks instead of double-stranded breaks. Scientists have attached the Cas9 nickase to cytosine deaminase to create cytidine base editors (CBEs), adenosine deaminase to create adenine base editors (ABEs), and a reverse transcriptase domain to create prime editors. Nickase-based tools reduce off-target effects by introducing cuts that favor a less error-prone DNA repair pathway, homology directed repair, which opens the door for safer CRISPR therapies.

Base editing systems also minimize unintended genomic alterations by directly modifying a single base type within a specific DNA sequence, which prevents random base pair insertions or deletions.¹⁹ Base editors are currently being explored widely in gene therapy and biotechnology. ABEs have demonstrated higher efficiency and precision than conventional Cas9 in correcting the SCD mutation ex vivo and in animal models.²⁰

Scientists have also developed CRISPR systems to edit RNA, including the REPAIR system that enables A-to-I (G) replacement and the RESCUE system for C-to-U replacement.¹¹ Unlike conventional CRISPR tools, RNA-targeting CRISPR systems operate without requiring a specific PAM sequence, and because they edit transient RNA transcripts rather than the heritable genome, they help reduce the possible consequences of permanent off-target edits. RNA editing is a promising therapeutic approach for diseases like Huntington's disease, where scientists aim to silence mutated HTT mRNA to reduce pathological protein aggregates.²¹

Ethical consideration

While research innovation in therapeutic CRISPR-Cas systems is expanding, scientists remain wary of the significant bioethical implications, particularly the long-term consequences of unintended changes in the genome or misuse of germline editing.²² Scientists address these issues via a multifaceted approach involving strong regulatory frameworks, increased public engagement to address misinformation, and collaborations between scientists, policymakers, ethicists, and the public to ensure responsible and ethical CRISPR technology applications.