A Gene Therapy Study of Delayed Sickle Cell Disease in Mice

Sickle cell disease (SCD) is a debilitating disease that affects up to 40% of the population in some African countries. It is caused by mutations in the gene that makes hemoglobin, the protein that carries oxygen in red blood cells.

It may one day be possible to treat this disease using gene editing – by jump-starting the production of a healthy form of hemoglobin called fetal hemoglobin, which is usually only produced by the body when we are in the uterus.

But a new study testing this promising new treatment in mice has revealed that scientists still have a long way to go before they can try it in humans. The research has been published in Disease models and mechanisms.

Healthy red blood cells (RBCs) are donut-shaped, but with an indentation instead of a hole.

In sickle cell disease, abnormal hemoglobin distorts the shape of red blood cells when they are not carrying oxygen. Instead, sickle cells are C-shaped, like the agricultural tool called a “sickle”, and they become hard and sticky, and die sooner.

Sickle cell disease Scd
Human red blood cells with sickle cell disease. Credit: Image courtesy of Weiss Lab

Due to their shape, sickled red blood cells can get stuck and stop blood flow as they travel through small blood vessels. This causes patients to suffer episodes of excruciating pain, organ damage and reduced life expectancy.

Although current treatments have reduced complications and extended the life expectancy of affected children, most still die prematurely.

Red blood cells are made from hematopoietic stem cells from our bone marrow. These stem cells are able to grow into more than one cell type, in a process called hematopoiesis.

Researchers hope to modify the genes of these stem cells so that they produce red blood cells with fetal hemoglobin instead of the abnormal protein and can be reintroduced into the body to alleviate symptoms of SCD.

Unfortunately, they discovered that although two types of laboratory mice showed symptoms of sickle cell disease, their fetal hemoglobin gene and surrounding DNA were misconfigured, rendering stem cell treatment ineffective or even harmful.

These mice – called “Berkley” and “Townes” mice – have been genetically modified in different ways to carry several human hemoglobin genes (replacing mouse genes) so that scientists can study sickle cell disease in an animal model.

The researchers took stem cells from the mice and used CRISPIR-Cas9 to try to activate the gene for healthy fetal hemoglobin. They then put the reprogrammed stem cells back into the mice and monitored the animals for 18 weeks to find out how the treatment affected them.

Surprisingly, 70% of Berkley mice died from therapy, and fetal hemoglobin production was only activated in 3.1% of stem cells. In contrast, the treatment did not affect the survival of Townes mice and even activated the fetal hemoglobin gene in 57% of red blood cells.

Image of the nucleus of a berkley mouse stem cell, showing where the dna has been modified.
This shows the DNA-containing nucleus in a Berkeley mouse stem cell. The green and red dots show DNA that has been modified by this new stem cell gene therapy for sickle cell disease. These colored dots illustrate abnormal gain or loss of DNA segments after editing. Credit: Image courtesy of Weiss Lab

Even then, the levels of fetal hemoglobin produced were seven to 10 times lower than those seen when this approach was used in laboratory-grown human cells and were not high enough to reduce clinical signs of sickle cell disease.

“We realized that we didn’t know enough about the genetic configurations of these mice,” says lead author Dr Mitchell Weiss, head of the department of hematology at St Jude Children’s Research Hospital, USA.

The researchers sequenced the mice’s hemoglobin genes and surrounding DNA, and found that the Berkley mice – instead of having a single copy of the mutated human gene – had 22 randomly arranged broken copies of the mutated human gene. human sickle cell disease and 27 copies of human fetal hemoglobin.

This caused the lethal effects seen and means the mice cannot be used to test this treatment in the future.

“Our results will help scientists using Berkeley and Townes mice decide which to use to answer their specific sickle cell or hemoglobin research question,” Weiss concludes.

“Furthermore, this work reminds scientists to carefully examine the genetics of the mice they use to study human disease and find the right mouse for the job.”

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