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CRISPR Tackles Diverse Single-Gene Conditions

The end-of-year FDA approval of the first CRISPR-based therapy, for sickle cell disease, came a mere dozen years after Jennifer Doudna and Emmanuelle Charpentier introduced the technology. They shared the Nobel Prize in Chemistry in 2020.

CRISPR is one of the better abbreviations in genetics. It’s certainly more memorable than RFLPs, GWAS, and even SNPs, so euphonious that few reports – technical or otherwise – actually use the term “clustered regularly interspaced short palindromic repeats.” CRISPRs are short DNA sequences, peppered with repeats, that latch onto DNA-cutting enzymes, commandeering and directing them to snip certain parts of a chromosome.

The genomes of certain bacteria naturally harbor CRISPR sequences. The microbes deploy them to dismantle the genetic material of infecting viruses, a little like an immune response.

Unlike gene therapy as originally-envisioned since the 1950s, which adds a functioning copy of a gene, CRISPR can add, remove, or replace a DNA sequence. So it’s termed gene editing if targeted to a single gene, or genome editing if more than one. My book The Forever Fix: Gene Therapy and the Boy Who Saved It, describes early gene therapy attempts, beginning with the first clinical trial in 1990.

Just as the 1990s saw gene therapies for several single-gene conditions inch towards and enter clinical trials, the next few years are certain to see gene and genome editing emerge as a more refined tool. The eclectic applications are being tailored to the specific natures of the mutations that drive specific diseases. The first FDA approval of CRISPR gene editing targets the mutation in the beta globin gene that causes the excruciating blocked circulation of sickle cell disease, correcting a single DNA base substitution that otherwise bends red blood cells into curved, spiky shapes.

Sickle Cell Disease

The CRISPR-based treatment for sickle cell disease is Casgevy, from CRISPR Therapeutics and Vertex Pharmaceuticals.

Casgevy alters stem cells from a patient’s blood so that they can carry more oxygen. The altered cells are reinfused into the patient, where they home to the bone marrow, attach, multiply, and release into the circulation cells that ramp up production of fetal hemoglobin.

Specifically, Casgevy turns on a silenced gene (BCL11A) that normally directs manufacture of a form of hemoglobin in the fetus that can carry more oxygen than the “adult” form. With more fetal hemoglobin, more oxygen reaches narrow places where the bendy red blood cells can deform, preventing the sickling that causes the excruciating pain crises.

The therapy is “ex vivo and, autologous”: it’s done outside the body using the patient’s cells.

What will the next CRISPR-based treatment for a single-gene disease be? Perhaps one of these.

Phenylketonuria (PKU)

A few drops of blood from the heel of newborns in many nations is used to screen for several dozen genetic metabolic diseases. The first, phenylketonuria (PKU), affects about 1 in 15,000 people. A classic “inborn error of metabolism,” PKU causes buildup of the amino acid phenylalanine. Severe brain damage, causing seizures and intellectual disability, follows unless an extremely rigid “medical food” diet is adhered to for many years.

Researchers at the Perelman School of Medicine at the University of Pennsylvania are using CRISPR on the affected gene, which encodes the enzyme phenylalanine hydroxylase, in a mouse model and in liver cells from patients with PKU. The intervention corrects the genetic glitch.

Duchenne Muscular Dystrophy

Gene-based treatment of DMD has faced hurdles: the huge size of the gene and the deletion that causes most cases, and the necessity to alter many muscle cells to alleviate symptoms. So far clinical trials have had mostly discouraging results.

In late September 2023, a multicenter team reported the death of a patient in a clinical trial of a high-dose gene therapy using CRISPR. A safer approach might be to alter muscle stem cells from patients outside the body, and then implant them, like the sickle cell strategy. A different approach is described in Stem Cell Reports. It uses “multi-exon skipping” to correct the mutation by snipping out portions of the gene.

Rett Syndrome

A mutation in a gene called MECP2, on the X chromosome, causes Rett syndrome. It affects 1 in every 10,000-15,000 girls. The gene is a transcription factor, which means that it controls many other genes.

In Rett syndrome, a mutation blocks some neuron-neuron signaling in the brain, slowing development as a child reaches preschool age. She may have difficulty walking and develop seizures, autism and/or intellectual disability. A telltale sign is a distinctive holding and wringing of the hands. Many die young.

Investigators at the University of Siena in Italy are evaluating gene editing as a therapeutic approach for Rett syndrome in cells from patients who have any of four mutations in the gene, and in mice.

LCA10 Retinal Blindness 

Researchers at Editas Medicine are evaluating efficacy of a CRISPR-based gene editing treatment to provide vision in patients with specific mutations in the CEP290 gene, which cause LCA10, a form of retinal blindness.

The treatment is a single administration beneath the retina. One of the first FDA-approved gene therapies was to treat LCA2, another form of retinal blindness. It restores vision in just days.


Some other applications of CRISPR on single-gene diseases are earlier in development. That’s the case for the rare infantile cystinosis, in which an amino acid, cystine, builds up and damages cells in the kidneys and eyes. Kidney problems typically begin from 6 to 12 months of age. Treatment includes drugs to lower cystine, feeding tubes, dialysis, and kidney transplant.

Researchers at the University at Buffalo envision a stem-cell-based CRISPR treatment for infantile cystinosis. They compared stem cells from a healthy person and from an individual with infantile cystinosis. They added the precise mixture of insulin, growth factors, and other proteins required to coax the cells to divide and specialize as the proximal renal tubule, the part of a kidney’s nephron subunit that the disease affects. Mimicking the proximal tubule in a lab dish has been elusive for a long time – some body parts are challenging to replicate in a dish.

The next step would bring in CRISPR to edit the mutation behind cystinosis.

“The normal gene can be introduced in the genome of cystinotic hiPSCs (human induced pluripotent stem cells), which can then be injected in the kidney to replace the defective proximal tubules of individuals with infantile cystinosis,” said Mary Taub, senior author of the group’s paper in The International Journal of Molecular Sciences. Only the proximal tubules need be replaced, not the entire kidney, and because the stem cells originate from the patient, tissue rejection shouldn’t happen, she added.    

Spinal Muscular Atrophy

The precision of CRISPR-based approaches can improve upon “classical” gene therapy in several ways. Consider spinal muscular atrophy (SMA). It is the second most common single-gene disease of childhood following cystic fibrosis. As motor neurons in the spinal cord degenerate, skeletal (voluntary) muscle loses function, producing weakness and impairing mobility. Also called “floppy baby syndrome,”

SMA results from mutation in the “survival motor neuron” gene (SMN1). A peculiarity of SMA is that a second gene, SMN2, lies near the first on chromosome 5. Normally, SMN2 is dormant because of a mutation. The two FDA-approved, gene-based approaches for SMA (Spinraza and Zolgensma) reawaken SMN2 – but not in all cells. Spinraza does so only in the spinal cord, while Zolgensma uses a gene therapy vector that becomes diluted over time. Although some children have had remarkable improvements, others have only achieved tiny advances, like being able to hold the head up a few seconds longer. With Spinraza costing $805,000 for the first year of therapy and $380,000 per year thereafter, and Zolgensma’s one time-treatment topping $2 million, a new approach is needed.

A multi-institution team in the US is using a variation of CRISPR called base editing to permanently refine SMN2 to have a lasting effect on restoring production of the encoded protein needed to sustain the motor neurons in the spinal cord. The research was recently published in Nature Biomedical Engineering.

What’s Next?

CRISPR has the potential to tackle conditions other than those caused by mutations in a single gene. A future DNA Science post will consider applications of CRISPR in the more common cancers, metabolic disorders, infectious diseases, and in agriculture, probiotic development, and bringing back extinct species.  

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