Investigational RNAi Mediated Oxalate Reduction TherapyAuthors: John Knight1, Abigail Liebow2, Kyle Wood1, Sonia Fargue1, W. Todd Lowther3, David Erbe2, Ross Holmes1

Affiliations: 1University of Alabama at Birmingham, Birmingham AL, 2Alnylam Pharmaceuticals, Cambridge MA, 3Wake Forest School of Medicine, Wake Forest NC

Primary Hyperoxaluria Type 1 is an autosomal recessive disorder of glyoxylate metabolism from loss of function of alanine-glyoxylate aminotransferase in hepatocyte peroxisomes of affected individuals, resulting in profound oxalate overproduction. End stage renal disease is common at an early age with necessary treatment via dual liver-kidney transplantation. siRNA-GalNAc conjugate technology has provided a way to specifically target this defective pathway in hepatocyte peroxisomes by silencing the mRNA for the upstream enzyme, glycolate oxidase, and starving the substrate for oxalate production. Evaluation of such an approach is ongoing in an early stage clinical trial in patients with the investigational RNAi therapeutic ALN-GO1.

To expand the potential application of substrate reduction therapy to other diseases of oxalate excess, the current study targeted the downstream enzyme in oxalate synthesis – lactate dehydrogenase (LDH). LDH was shown as early as 1973 to be the key enzyme in oxidizing glyoxylate to oxalate in human livers. Thus, its inhibition could have the potential to treat the full array of patients with primary hyperoxaluria (types, 1, 2, 3, and idiopathic) and even common stone forming diseases. Of course, LDH and its metabolites are also well established regulators of energetic flux in liver cells (e.g., Cori Cylcle, Krebs Cycle) and the safety of its inhibition must be explored carefully to understand any impact on hepatic and whole body metabolism.

Figure 1. Oxalate Synthesis in Hepatocytes

Figure 2. Hydroxyproline Catabolism and Oxalate Synthesis in Primary Hyperoxaluria Patients

Figure 3. LDHA-GalNAc siRNA Dose Response

WT mice, single dose subQ, day 10 sacrifice

Experimental Design and Methods• 13-15 week old male mice (wild type and Agxt Ko),

n=6• Mice dosed weekly with 10 mg/Kg siRNA or PBS

for 4 weeks; Ultra-low oxalate diet• 24-hour urine collections in Nalgene metabolic cages• Animals fasted 6 hours prior to necropsy• Tissue and plasma rapidly frozen in liquid nitrogen• Anion chromatography/mass spectrometry to

measure urinary glycolate and oxalate, liver and plasma lactate and glycolate

• High Pressure Liquid Chromatography to measure liver glyoxylate and liver pyruvate

Figure 4. Specific Liver LDH Activity

• No change seen in LDH activity in thigh muscle or heart of RNAi treated mice

• No effect on body weight, no overt changes in behavior or activity

Figure 5. 24-hr Oxalate and Glycolate Excretion

Figure 6. Plasma levels of lactate and glycolate

Figure 7. Liver Levels of Lactate and Pyruvate

Figure 8. Liver Levels of Glyoxylate and Glycolate

Table I. Liver Anion Metabolome

Ion chromatography coupled to mass spectrometry

Summary1. In mice, liver-specific knockdown of LDH resulted

in profound oxalate lowering in both healthy and diseased animals, indicating the exciting potential for this approach in decreasing oxalate synthesis in disease.

2. However, additional findings contradicted expectations - instead of the predicted increase in glycolate levels that was anticipated, treated mice showed profound whole-body decreases in glycolate levels – in their urine, plasma, and livers.

3. Additionally, substantial changes were seen in levels of lactate, pyruvate and TCA Cycle organic acids in the livers of treated animals, consistent with the known role of LDH in carbohydrate metabolism.

4. The importance of these changes will need to be investigated fully, including in chronic toxicology studies, to help define the potential of LDH-based therapy in hyperoxalurias while ensuring the safety of this approach.