Research from this past year highlights the potential of CRISPR/Cas9 technology in modeling and treating rare disease
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How CRISPR-Cas9 Contributed to Rare Disease Research in 2021

Research from this past year highlights the potential of CRISPR-Cas9 technology in modeling and treating rare disease

Brydie Thomas-Moore, PhD
Dec 21, 2021 | 4 min read

CRISPR-Cas9 is a gene editing tool that has revolutionized modern biology, providing a relatively simple and cost-effective strategy to develop model systems and reveal disease mechanisms and target genes. And the simplicity, efficiency, and versatility that CRISPR-Cas9 technology offers in altering target genes has led to a growing interest in using this technology to treat disease.

As CRISPR-Cas9 targets specific DNA sequences, the technology works well for monogenic diseases—diseases caused by mutations in a single gene. With more than 80 percent of rare diseases caused by genetic mutations, could CRISPR-Cas9 help progress rare disease management? 

Rare diseases typically have few treatment options and diagnosis can be a lengthy process, if at all. Here, we explore research released in 2021 that highlights the potential of CRISPR-Cas9 technology in modeling and treating rare disease.

What is CRISPR-Cas9?

Clustered regularly interspaced short palindromic repeats—better known as CRISPR—and CRISPR-associated (Cas) proteins form an adaptive immune system found in many different types of microbial cells. As a microbial defense system, CRISPR-Cas9 degrades pathogenic DNA, such as from an invading virus. 

After infection with a virus, microbial cells can store short sections of viral DNA in their genetic material, known as “spacer” sequences. These spacer sequences are found as part of the CRISPR array, where different spacers are separated by repeating sequences. Consequently, the CRISPR loci encodes Cas proteins, precursor RNA or pre-crRNA (from the CRISPR array), and noncoding trans-activating crRNA or tracRNA.

The pre-crRNA is complementary to target DNA, such as DNA from invading viruses, and combines with tracRNA to form “guide RNA.” Once generated, the guide RNA forms a complex with Cas9 and directs Cas9 to the target DNA sequence. Cas9 works as a nuclease, cutting the target DNA by breaking the phosphodiester bonds that link adjacent nucleotides together.

How can CRISPR-Cas9 be used to treat disease?

By altering the guide RNA sequence, Cas9 can be directed to a different target DNA sequence—such as pathogenic gene variants in human disease. When Cas9 cuts the target DNA of a gene, the cell repairs the chopped DNA by adding or removing nucleotides to join the broken DNA strands back together. Repair can occur by:

Nonhomologous end-joining, where the edges of the broken DNA strands are joined back together, usually through random addition or deletion of nucleotides at the edges of the broken strands.

Homology-directed repair, where the broken strands are joined together by adding a DNA repair template.

Either way, the repair process can alter the genetic sequence, which can inactivate a faulty protein or generate a healthy protein.

Delivery of CRISPR-Cas9 for rare disease

CRISPR-Cas9 clinical applications depend on successful delivery of agents (CRISPR-Cas9 material) to the target cells. These agents are typically delivered to target cells through viral vectors, such as adeno-associated virus. But viral delivery systems can be immunogenic and have restrictions on the size or amount of agents that can be delivered. 

While non-viral systems, such as liposomes, may be able to offer safer delivery systems, they have been limited by factors such as poor efficiency and reproducibility

Recently, O’Keeffe et al. addressed some of these limitations, designing an efficient polymer-based vector to treat recessive dystrophic epidemolysis bullosa in vitro using human cells. 

Recessive dystrophic epidemolysis bullosa is a rare and severe skin disorder, where the skin is highly fragile from lack of collagen, causing blisters and wounds that can be fatal. Loss-of-function mutations in exon 80 of the type VII collagen gene (COL7A1) are linked to recessive dystrophic epidemolysis bullosis. 

O’Keeffee et al. used a positively-charged polymer—highly-branched poly(β-amino ester)—to deliver a guide RNA/Cas9 complex to skin cells, in vitro. The polymer vector was found to efficiently transfect cells, and the guide RNA/Cas9 complex deleted exon 80 by around 40 percent in treated cells, elevating levels of functional forms of type VII collagen.

CRISPR-Cas9 for modeling rare disease

With CRISPR-Cas9 able to alter specific genes, this technology can also be used to develop cell models for studying rare disease. Leoni et al. recently generated in vitro cell models for the rare bone disorder: osteogenesis imperfecta (OI) type XIV. 

OI type XIV is linked to abnormal type I collagen that can lead to clinical features such as bone fractures and growth deficiencies. OI type XIV has been linked to mutations in TMEM38B, which generates an ion channel (TRIC-B) that is involved in calcium ion homeostasis—but full details of how the disease develops is unclear.

Studying OI type XIV in cell models can be difficult as there is limited access to patient cell samples, partly due to the rarity of the disease and the complications associated with taking bone biopsies from affected individuals

Using CRISPR-Cas9, Leoni et al. were able to develop human fetal osteoblast cell lines, for the first time, to model OI type XIV. These cell models lacked TRIC-B by introducing loss-of-function mutations into TMEM38B using CRISPR-Cas9.

CRISPR-Cas9 for treating rare disease

The clinical potential of treating rare disease has recently been highlighted by initial results released by Gillmore et al. from a Phase 1 clinical trial (NCT04601051) treating transthyretin amyloidosis. Transthyretin amyloidosis is a rare and fatal disorder caused by misfolded forms of transthyretin protein accumulating in tissues, particularly in the nerves and heart.

Gillmore et al. designed a CIRSPR/Cas9 system that is delivered by lipid nanoparticles, termed “NTLA-2001.” With transthyretin primarily generated in the liver, Gillmore et al. used lipid nanoparticles that are taken up by liver cells. NTLA-2001 treatment reduced transthyretin levels by around 87 percent in sera from patients, with mild adverse effects in three of the six patients.

Gillmore et al. also address concerns over the ability of CRISPR-Cas9 to alter non-target DNA sequences, leading to off-target effects, and introducing pathogenic variants during DNA repair. These assessments identified only “low-risk” alterations—genetic changes that occurred in the transthyretin gene—but state that long-term follow is required to monitor safety of participants.

Advances in CRISPR-Cas9 systems have helped researches develop models and therapeutic strategies for treating rare diseases. With initial findings of a clinical trial study showing that CRISPR-Cas9 is capable of targeting proteins linked to transthyretin amyloidosis, CRISPR-Cas9 may soon offer a viable strategy for treating many types of rare disease.