St. Jude Sickle Cell Research Team Warns Geneticists to Choose ‘the Right Mouse for the Job’

By Brittany Wade 

July 12, 2022 | In the 1970s, sickle cell disease (SCD)–characterized by prolonged bouts of extreme pain, edema, immunodeficiency, and the presence of the eponymous sickle-shaped red blood cells–became the first disease identified as a genetic disorder. Once considered on the cutting edge of biomedical research, it inspired many therapeutic interventions. Nevertheless, a one-time permanent cure has eluded scientists for decades.  

Now, over fifty years later, Mitchell Weiss, Chairman of Hematology at St. Jude Children’s Research Hospital, and fellow researchers are devising autologous hematopoietic stem cell (HPC) gene-editing techniques with hopes of developing a long-standing treatment.  

Their work comes after the groundbreaking study from the Sarah Cannon Research Institute following the progression of a 34-year-old woman whose genes were modified to alleviate SCD symptoms. The patient is showing tremendous improvement, and the work opened the door to more accessible treatments. Unfortunately, the standard treatment, bone marrow transplants, can be hard to achieve thanks to a shortage of eligible donors and potentially deadly side effects. 

SCD is caused by a single base-pair mutation in the Hbb gene, which codes for the protein beta-hemoglobin. Alpha- and beta-hemoglobin are substituents of the larger tetrameric protein hemoglobin that resides in red blood cells and shuttles oxygen to tissues and organs. 

Hbb’s point mutation converts the nucleic acid base adenine to thymine, remodeling beta-hemoglobin and, by default, hemoglobin. As a result, multiple hemoglobin molecules adhere to one another, leading to abnormal red blood cells mimicking crescents or sickles. Symptoms include severe anemia, vascular occlusion, multi-organ failure, and even death. With over 300,000 newborns diagnosed annually, a permanent cure would be a welcomed reprieve. 

Of Mice and Men 

On the hunt to revolutionize SCD therapies, the St. Jude team used CRISPR-based gene editing in mice HPCs to increase fetal hemoglobin expression (DOI: 10.1242/dmm.049463, Disease Models & Mechanisms). Fetal hemoglobin–a naturally-occurring protein during human gestation that diminishes immediately preceding birth–mitigates SCD symptoms.  

The team’s pre-clinical study followed two immunodeficient mouse groups: “Berkeley” and “Townes.” Berkeley mice share histopathological symptoms with human SCD patients, and Townes mice possess several human hemoglobin genes useful for SCD study. The scientific community has relied heavily on these two strains for over 15 years, giving Weiss’ team confidence in their use for future research. 

What seemed like the makings of a promising treatment ultimately ended in catastrophe. The majority of the Berkeley mice perished post gene editing. Though over half the Townes mice expressed fetal hemoglobin, levels were insufficient to alleviate clinical signs of the disease. 

Taking a Closer Look 

Such discouraging outcomes highlight the need to delineate a study design error from a therapeutic failure. Of course, not all disappointing results point directly to faulty treatments. 

After taking a closer look, the team learned a valuable lesson: an experiment is only as good as its mice. “We realized that we did not know enough about the genetic configurations of these mice,” said Weiss in a press release. Though they have been widely used for years, their resident human globin gene structure and regulatory requirements are largely unknown. Furthermore, what little documentation existed was not easily accessible.  

Such limited information is due, in part, to the less-advanced manipulation techniques that existed when the strains were created nearly two decades ago. Thus, the team sequenced each group’s hemoglobin genes to gather information aligned with modern genetic standards. 

With the Berkeley mice containing anywhere from four to 22 human transgenes, gene editing produced an overwhelming amount of double-strand DNA breaks, causing death. Adding base or prime editors to lower the number of double-strand breaks may reduce mortality rates in future pre-clinical studies. Even though widespread death was not observed in Townes mice, low therapeutic gene expression levels could indicate a lack of critical DNA regulatory elements.  

The team outlined their findings in great detail in the Disease Models & Mechanisms article, hoping the information would help others with SCD research. They underscore the importance of thoroughly understanding a subject’s genomic structure, recognizing the limitations of a particular study design, and acknowledging the critical nature of distal DNA elements in gene therapy.  

“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. Additionally, 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,” advises Weiss.  

The team also urges scientists to consider the possibility of varying transgene expression levels and high gene conversion rates in successive generations. Perhaps with these new considerations, that ever-elusive cure will be a reality in no time.