CRISPR Gene Therapies: Current Challenges and a Promising Future

January 11, 2024

Since its discovery, CRISPR and associated Cas proteins have been developed into a powerful gene-editing tool, revolutionizing biological research and offering exciting opportunities for the treatment of a range of genetic diseases. But despite the extensive applications of CRISPR in research and a plethora of clinical trial success stories, only one CRISPR gene-editing therapeutic has been approved for clinical use. Late in 2023, Vertex Pharmaceuticals received approval from both the UK and U.S. for their CRISPR-based treatment to treat sickle cell anaemia and beta thalassemia, by using CRISPR tools to “switch-on” the healthy infant hemoglobin gene in adults with the conditions.^1,2

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But while the CRISPR technology holds great promise, it also faces significant challenges and limitations that must be overcome to realize its full potential in clinical settings. In this article we will delve into the current challenges and limitations of CRISPR-based therapeutics and the efforts underway to overcome them and harness CRISPR for the treatment of human disease.

The CRISPR revolution

Unlike previous gene-editing technologies, such as Zinc Fingers and TALENS, CRISPR gene editing does not rely on engineered proteins but is RNA-based—a single guide RNA (sgRNA) directs the Cas endonuclease to the target site where a double-strand break (DSB) is introduced. The DSB then triggers endogenous DNA repair pathways, such as non-homologous end joining (NHEJ), an error-prone process that introduces an indel and subsequent frameshift mutation at the target site resulting in robust and efficient gene knockout. Alternatively, in the presence of an exogenous template, the DSB activates homologous repair resulting in desired mutations or sequence insertions. The flexibility, efficiency, and accuracy of CRISPR gene editing have seen it overtake both ZFNs and TALENs to become the primary gene-editing tool in use today.³

However, there remain significant challenges when looking to utilize CRISPR gene editing for clinical applications, such as the risk of off-target effects, in vivo stability, and the potential for host immunogenic responses—and addressing these are critical when looking to use CRISPR to treat disease.

Minimizing off-target activity

Arguably the greatest challenge to be overcome before the widespread clinical application of CRISPR-based gene editing is mitigating the risk of off-target effects—or unintended genetic alterations at sites other than the intended target.⁴ Researchers have made significant progress in improving the precision of CRISPR by developing more precise Cas9 variants and optimizing guide RNA designs. But the reliance on the introduction of a DSB can also cause significant safety issues, with CRISPR gene editing leading to cell death, or large base-deletions and chromosomal disorganization with the potential to cause malignant tumors.⁵ A key focus has been to develop CRISPR systems that do not introduce a DSB—systems such as base editing, prime editing, and the derivatives TWIN-PE and PASTE facilitate gene editing without a DSB, from single nucleotide changes to large-scale insertions of genetic material, with huge future potential for gene therapy.⁶

Challenges in delivery

Another significant hurdle in CRISPR-based therapeutics is the efficient and safe delivery of the editing machinery to target cells and tissues. For many genetic disorders, the editing components, including Cas9 and sgRNAs, need to be delivered to specific organs or tissues within the body. This can be particularly challenging when targeting internal organs or crossing the blood-brain barrier.

Viral vectors, such as adeno-associated viruses (AAV), are often used for CRISPR machinery delivery in vivo, however, they have a limited loading capacity and lack the ability to integrate into the genome. Other viral vectors such as lentiviruses are commonly used to overcome the limitation of AAV with a large loading capacity, but randomly integrate into the genome, which can be problematic if inserted into an oncogene. Non-viral delivery methods are also being explored including lipid-based nanocarriers, polymer nanoparticles, and exomes, which have all shown to be promising in the research setting.

Each approach has its advantages and limitations, and the choice of delivery method depends on the specific therapeutic application. Achieving efficient and targeted delivery while minimizing off-target effects and immune responses remains an ongoing challenge. But despite this, the continuous development and optimization shows promise for future clinical success. Researchers are hopeful that these challenges will lead to the development and application of tissue-specific vectors for gene-editing in vivo with enormous therapeutic potential.

Reducing immunogenicity

The immune system plays a crucial role in defending the body against foreign invaders, including viral vectors used for CRISPR delivery. The immunogenicity of gene therapy is a widely acknowledged risk following the death of 18-year-old Jesse Gelsinger in 1999 due to an inflammatory response induced by the adenovirus vector.⁷ But when CRISPR components are introduced into the body, they can also trigger an immune response, leading to the destruction of the edited cells or rendering the therapy ineffective. To mitigate this, scientists have attempted to develop Cas9 components that lack response-inducing exons. These Cas9 derivatives have been shown to successfully avoid eliciting humoral and cellular immune responses in mice models.⁸

Another method of mitigating the risk of immune responses to CRISPR gene therapies is the targeting of immune-privileged organs such as the eyes, brain, and testes.⁹ In a study of 179 patients with inherited retinal disease, scientists found that 76% of patients had a pathogenic allele that was a potential candidate for CRISPR gene-therapy repair, highlighting the applicability of CRISPR gene therapy in areas where no immunological response can occur.¹⁰

A promising future ahead for CRISPR gene editing

While there are still significant challenges to be overcome before CRISPR-based therapeutics become commonplace for the treatment of inherited and genetic disease, the recent approval of Vertex Pharmaceuticals CRISPR therapeutic offers a glimpse into the possible future. Continued effort to develop CRISPR systems with reduced off-target activity that avoid the introduction of a DSB, optimize CRISPR delivery, and reduce the risks associated with adverse immune responses mean that there are soon to be more proven CRISPR gene-editing therapeutic success stories in the coming years. The future of CRISPR-based therapies remains bright, offering hope for patients with genetic disorders and potentially transforming the landscape of medicine.

Key Takeaways
Despite its history of revolutionizing molecular and biological research and a variety of pre-/clinical trial success stories, only one CRISPR gene-editing therapeutic has currently been approved for clinical use.
CRISPR-based therapeutics hold immense promise for the treatment of genetic diseases, but they also face significant challenges and limitations. Addressing off-target effects, improving delivery methods, and mitigating immune responses, are critical for advancing the field.
The future is promising for the use of CRISPR gene editing to treat human disease thanks to the continued effort to improve delivery, as well as safety, stability and efficiency by omitting the DSB, and reducing immunogenicity.