2 Common SCD Mouse Models Not Suited for Gene Editing Studies

Marisa Wexler, MS avatar

by Marisa Wexler, MS |

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Two mouse models commonly used to study sickle cell disease (SCD) are not suitable for testing certain gene editing treatment approaches, including those for fetal hemoglobin production, because of their specific genetic makeup, a study reported.

“This work provides a reminder for scientists to carefully consider the genetics of the mice that they are using to study human diseases and find the right mouse for the job,” Mitchell Weiss, MD, PhD, the study’s senior author at St. Jude Children’s Research Hospital, said in a press release.

The study, “Limitations of mouse models for sickle cell disease conferred by their human globin transgene configurations,” was published in the journal Disease Models & Mechanisms.

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Mouse models are an invaluable tool in scientific research, and they have been widely applied to study the biological mechanisms of SCD and to test potential treatments. In particular, two sickle cell mouse models commonly referred to as Berkeley and Townes are often used to study the condition.

SCD is caused by mutations in a gene coding for a subunit of hemoglobin, the protein that red blood cells use to carry oxygen. Specifically, the disease impacts the adult version of hemoglobin. An alternative form of the protein, called fetal hemoglobin, which is produced during fetal development but usually stops being made shortly after birth, is unaffected.

Unlike humans, mice do not have a fetal version of hemoglobin. In both the Berkeley and Townes models, animals are genetically engineered so that their genetic codes contain the human versions of hemoglobin genes, both adult and fetal, rather than the typical mouse genes.

Scientists at the Memphis research hospital attempted to use a gene editing technique called CRISPR/Cas-9 to reactivate fetal hemoglobin production in blood stem cells of Berkley mice. Restoring fetal hemoglobin production could compensate for the mutated adult hemoglobin that causes sickle cell, and it is being explored as a potential therapeutic strategy for SCD.

Unexpectedly, when scientists applied this treatment to Berkley mice, it was toxic — after just over four months, 70% of the animals died, and fetal hemoglobin was only produced by 3.1% of their blood stem cells. The team then conducted detailed analyses of the mice’s genetic code, hoping to understand what had happened.

“Despite the widespread use of [Berkeley and Townes] strains, the structure and regulation of their resident human [hemoglobin] genes is not fully defined or easily accessed through current databases or literature,” the researchers wrote. “This knowledge gap exists in part because the Berkeley and Townes strains were generated more than 25 and 15 years ago, respectively, when methods for genome manipulation and characterization were less advanced.”

Typically, a given cell has two copies of hemoglobin genes, one inherited from each biological parent. However, in the cells of Berkley mice, an average of nearly 22 copies of the mutated gene encoding adult hemoglobin and about 27 copies for fetal hemoglobin were found.

The gene editing technology CRISPR/Cas-9 works by first making a cut in a target DNA sequence of choice; the “editing” occurs as the cell repairs this cut. Further experiments revealed that, with so many copies of the target gene, the technology was effectively inducing widespread DNA damage in cells, ultimately causing toxicity.

Researchers also performed genetic characterizations of Townes mice, and results showed their cells had only one copy of the mutated gene for adult hemoglobin and the one for fetal hemoglobin. However, their genomes lacked certain elements that, while not part of the protein-coding hemoglobin genes, help to regulate their activity.

These genetic elements likely evolved to help control the switch from fetal to adult hemoglobin in humans, but they are absent in mice and were not introduced when Townes mice were first generated more than a decade ago. At that time, these regulatory elements were less well defined, the researchers noted.

When CRISPR/Cas-9 was used to reactivate fetal hemoglobin production in Townes mice, the animals survived and 57% of their blood stem cells produced the fetal protein. However, fetal hemoglobin levels were seven to 10 times lower in these mice cells than in human cells grown in the lab, and they were not high enough to alleviate the signs and symptoms of SCD. This was likely due to differences in the animals’ genetic regulatory elements.

“Our findings will help scientists using the Berkeley and Townes mice decide which to use to address their specific research question relating to sickle cell disease or hemoglobin,” Weiss said.