Sickle Cell Research to the Present and the Future

Three-dimensional rendering of sickle cell anemia blood cells. Credit: National Institutes of Health.

By Dr. Marcus Calkins, Part 3 of 3

Forty years after Linus Pauling and his lab demonstrated the molecular basis for Sickle cell disease and James Watson speculated that upregulation of fetal hemoglobin may protect from the disease, methods to control fetal hemoglobin specifically in red blood cells began to be developed. The molecular biology revolution of the late 20th century had produced extensive knowledge about the molecular systems that drive fetal hemoglobin production, but harnessing that intricate knowledge has taken another thirty years.

Hematopoietic Stem Cells (1990s)

Since the advent of radioactivity research, it has been well-established that red blood cells have a short lifespan of only about 115 days and are continually produced from precursors in the bone marrow. In order to replace defective blood cells in an individual, a protocol for whole-body irradiation and allogenic bone marrow transplant was pioneered by a group of doctors in Seattle in the 1970s, as a treatment for cancer patients.

However, the ability to specifically isolate and identify hematopoietic stem cells from patients was only developed in the 1990s. At that time, nuclear dye exclusion and flow cytometry characteristics were used to isolate the stem cells, but since then, a variety of cell surface markers have been identified, and protocols to isolate, expand and differentiate hematopoietic stem cells have become standardized. In addition, scientists have learned to modify the hematopoietic stem cells at a genetic level, creating the possibility that stem cells may be extracted, genetically modified ex vivo, and then used to reconstitute the bone marrow of patients with blood diseases.

For patients with Sickle cell disease, it may therefore be possible to extract hematopoietic stem cells and inactivate the BCL11A gene, which normally suppresses fetal hemoglobin. The red blood cell progeny of these altered stem cells would then produce fetal hemoglobin that could mask the effects of the disease-causing mutation in the β-globin gene. Afterward, the modified stem cells could be transplanted back into the same patients from which they were isolated, providing the person with a continual supply of red blood cells that expresses fetal hemoglobin and are resistant to sickling.

Gene Therapy and Genome Editors (2000s-2010s)

The final component of a therapy for Sickle cell disease has recently been realized, as it is now feasible to efficiently inactivate BCL11A in isolated hematopoietic stem cells. In the last two decades, several systems of modifying the genome (gene editors) have been developed. Although the first editors to be produced may still find clinical use, CRISPR has quickly overtaken previous technologies to become the most widely applied and well-known gene editing platform.

In the late 1990s, researchers invented two key methods of using proteins to make targeted edits to the genome. These early gene editor proteins are called TALENs and Zinc fingers, both of which are being tested in clinical trials today. Each of these editors is able to target highly specific DNA sequences and make an incision in the DNA helix at a predictable site. Once the DNA strand is incised, error-prone DNA repair processes are activated to fix the incision, often resulting in random base insertions, deletions and changes. In this way, the genetic code is disrupted in some cells, and these random disruptions often serve to inactivate the targeted gene. If those cells with an inactivated gene can be identified and expanded, whole populations of cells with the genetic alteration can be established.

The comparative difficulty of using TALENs and Zinc finger proteins instead of CRISPR is that targeting a particular site in the genome often requires a major technical effort. Since the proteins themselves target the DNA sequence of interest, each target sequence must have its own specialized editor. The introduction of CRISPR/Cas9 in the early 2010s allowed researchers to target various DNA sequences much more easily. This system uses a short guide RNA molecule for DNA targeting, so the same protein can be used to cut any genomic site. Since generating these guide RNAs is a relatively simple procedure, the amount of effort required to design and execute genome edits is greatly reduced.

Theoretically, TALENs, Zinc fingers and CRISPR could all be used to inactivate BCL11A in hematopoietic stem cells. However, design of an appropriate TALEN or Zinc finger might require relatively large investments of money and time.  On the other hand, CRISPR promises to be a more cost-effective and faster approach to editing the genome. In academic studies, CRISPR is already widely used and far more common than the other editing technologies for making genetic modifications to laboratory model organisms. However, with human patients, safety and efficacy greatly outweigh effort and cost. Time will tell which gene-editing platform proves to be most cost-effective, efficient and safest for clinical use.

A New Clinical Reality (2020s)

With these new tools at hand, Watson’s dream of increasing fetal hemoglobin in Sickle cell disease patients is finally within sight. At least two major collaborations to perform ex vivo gene therapy for Sickle cell disease have been initiated since 2018. Both use gene editors to inactivate the BCL11A gene and promote fetal hemoglobin expression in red blood cells.

One collaboration between Bioverativ and Sangamo is testing a protocol for gene editing with a Zinc finger. The estimated completion date for this trial is 2022. Another collaboration that has received a great deal of attention, and was recently published in the New England Journal of Medicine, involves CRISPR Therapeutics and Vertex Pharmaceuticals. This trial is among the first to attempt CRISPR in a clinical setting, and the results are highly anticipated by the research and medical communities, not only for their impact on Sickle cell disease, but also as a bellwether for the use of CRISPR in medical practice. So far the results of the trial are encouraging. As of December 2020, the first two patients to receive therapy were reportedly doing well and were free from symptoms more than one year after receiving the treatment. While this news is exciting, there is still much work to be done before the technique can be applied to a wider population.

It has taken many years and many twists, but the visions of the 1950s are finally beginning to be realized, bringing us to the cusp of an exciting new dawn in medicine. The slow march toward a cure for Sickle cell disease clearly demonstrates that through patience and continued investment in scientific discovery, we can continue to achieve the dreams of our predecessors and plant new seeds for future generations to reap the harvest.