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791 Sickle Cell Disease Model Mice Lacking 2,3-Dpg Show Reduced RBC Sickling and Improvements in Markers of Hemolytic Anemia

Program: Oral and Poster Abstracts
Session: 113. Hemoglobinopathies, Excluding Thalassemia—New Genetic Approaches to Sickle Cell Disease: Poster I
Hematology Disease Topics & Pathways:
sickle cell disease, Diseases, Hemoglobinopathies
Saturday, December 5, 2020, 7:00 AM-3:30 PM

Kelly M. Knee, PhD1, Amey Barakat, MS1*, Lindsay Tomlinson, DVM, DSc2*, Lila Ramaiah, DVM, PhD3*, Zane Wenzel, MS4*, Brendon Kapinos5*, Youngwook Ahn6*, Nathanael G. Lintner, PhD7*, S. Denise Field, PhD8*, Reema Jasuja, PhD1 and Betty A. Pettersen, PhD2*

1Rare Disease Research Unit, Pfizer Inc., Cambridge, MA
2Drug Safety Research and Development, Pfizer Inc, Cambridge, MA
3Drug Safety Research and Development, Pfizer Inc, Pearl River, NY
4Primary Pharmacology Group- Medicine Design, Pfizer Inc., Groton, CT
5Discovery Sciences, Pfizer Inc, Groton, CT
6Emerging Science and Innovation, Pfizer Inc, Cambridge, MA
7Medicine Design-Chemical Biology, Pfizer Inc, Cambridge, MA
8Medicine Design- Chemical Biology, Pfizer Inc, Cambridge, MA

Sickle cell disease (SCD) is a severe genetic disorder caused by a mutation in hemoglobin (b6Glu-Val), which allows the mutant hemoglobin to assemble into long polymers when deoxygenated. Over time, these polymers build up and deform red blood cells, leading to hemolytic anemia, vaso-occlusion, and end organ damage. A number of recent therapies for SCD have focused on modulating the mutant hemoglobin directly, however, reduction or elimination of 2,3-DPG to reduce Hb S polymerization and RBC sickling has recently been proposed as a therapeutic strategy for SCD. Current clinical studies focus on activation of pyruvate kinase to reduce 2,3-DPG, however, direct targeting of the enzyme which produces 2,3-DPG; Bisphosphoglycerate Mutase (BPGM) may also be possible.

In this study we evaluate the impact of elimination of 2,3-DPG on SCD pathology by complete knockout of BPGM in Townes model mice. Animals with complete knockout of BPGM (BPGM -/-) have no detectable 2,3-DPG, while animals that are heterozygous for BPGM (BPGM -/+) have 2,3-DPG levels comparable to Townes mice. Western Blot analysis confirms that BPGM -/- animals completely lack BPGM, while BPGM -/+ animals have BPGM levels that are nearly equivalent to Townes mice. As expected from the lack of 2,3-DPG, BPGM -/- animals have increased oxygen affinity, observed as a 39% decrease in p50 relative to Townes mice.

Complete elimination of 2,3-DPG has significant effects on markers of hemolytic anemia in BPGM -/- mice. Mice lacking 2,3-DPG have a 60% increase in hemoglobin (3.7 g/dL), a 53% increase in red blood cell count, and a 29% increase in hematocrit relative to Townes mice. The BPGM -/- mice also have a 57% decrease in reticulocytes, and a 61% decrease in spleen weight relative to Townes animals, consistent with decreased extramedullary hematopoiesis. Consistent with the reduction in hemolysis, BPGM -/- animals had a 59% reduction in red blood cell sickling under robust hypoxic conditions. BPGM -/+ animals had hemoglobin, RBC, and hematocrit levels that were similar to Townes animals, and a similar degree of RBC sickling to Townes mice. Liver phenotype was similar across all variants, with areas of random necrosis observed in BPGM -/-, BPGM -/+ and Townes mice. Higher percentages of microcytic and/or hyperchromic RBCs were observed in BPGM -/- animals relative to BPGM -/+ or Townes animals.

These results suggest that modulation of 2,3-DPG has a positive effect on RBC sickling and hemolytic anemia, which may have therapeutic benefits for SCD patients. However, the lack of improvement in organ damage suggests that modulation of 2,3-DPG alone may not be sufficient for complete elimination of SCD phenotypes, and further investigation of this therapeutic avenue may be necessary.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH