CLINICAL RESEARCH Hepatic Lactate Dehydrogenase A: An RNA Interference Target for the Treatment of All Known Types of Primary Hyperoxaluria Gema Ariceta1,2, Kelly Barrios3, Bob D. Brown3, Bernd Hoppe3,4, Ralf Rosskamp3 and Craig B. Langman5,6 1 Division of Pediatric Nephrology, Hospital Universitari Vall d’Hebron, Universitat Autonoma de Barcelona, Barcelona, Spain; 2 Nefrología Pediátrica, Hospital Infantil, Hospital Universitari Vall d’Hebron, Passeig de la Vall d’Hebron, Barcelona, Spain; 3 Dicerna Pharmaceuticals, Inc., Lexington, Massachusetts, USA; 4German Hyperoxaluria Center Cologne/Bonn, Bonn, Ger- many; 5Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA; and 6Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, Illinois, USA Introduction: Primary hyperoxaluria (PH) is a family of 3 rare genetic disorders of hepatic glyoxylate metabolism that lead to overproduction and increased renal excretion of oxalate resulting in progressive renal damage. LDHA inhibition of glyoxylate-to-oxalate conversion by RNA interference (RNAi) has emerged as a potential therapeutic option for all types of PH. LDHA is mainly expressed in the liver and muscles. Methods: Nonclinical data in mice and nonhuman primates show that LDHA inhibition by RNAi reduces urinary oxalate excretion and that its effects are liver-specific without an impact on off-target tissues, such as the muscles. To confirm the lack of unintended effects in humans, we analyzed data from the phase I randomized controlled trial of single-dose nedosiran, an RNAi therapy targeting hepatic LDHA. We con- ducted a review of the literature on LDHA deficiency in humans, which we used as a baseline to assess the effect of hepatic LDHA inhibition. Results: Based on a literature review of human LDHA deficiency, we defined the phenotype as mainly muscle-related with no liver manifestations. Healthy volunteers treated with nedosiran experienced no drug- related musculoskeletal adverse events. There were no significant alterations in plasma lactate, pyruvate, or creatine kinase levels in the nedosiran group compared with the placebo group, signaling the uninterrupted interconversion of lactate and pyruvate and normal muscle function. Conclusion: Phase I clinical data on nedosiran and published nonclinical data together provide substantial evidence that LDHA inhibition is a safe therapeutic mechanism for the treatment of all known types of PH. Kidney Int Rep (2021) 6, 1088–1098; https://doi.org/10.1016/j.ekir.2021.01.029 KEYWORDS: lactate dehydrogenase A; nedosiran; primary hyperoxaluria; RNA interference; small interfering RNA ª 2021 International Society of Nephrology. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). rimary hyperoxaluria (PH) is a family of rare, Glyoxylate is normally converted to either glycine (by P autosomal recessive genetic disorders of glyoxylate metabolism resulting in overproduction of oxalate in alanine:glyoxylate aminotransferase), glycolate (by glyoxylate reductase/hydroxypyruvate reductase), or the liver.1,2 Three genetic types of PH (PH1, PH2, and oxalate (by lactate dehydrogenase [LDH]) in the liver. In PH3) have been defined, each caused by mutations that PH1 and PH2, reduced alanine:glyoxylate aminotrans- result in a deficiency, mislocalization, or loss-of- ferase or glyoxylate reductase/hydroxypyruvate reduc- function of key enzymes (Figure 1) involved in tase activity leads to an increased buildup of glyoxylate glyoxylate metabolism.3–5 and a resultant overproduction of oxalate by LDH. In Common to all PH types is the accumulation of PH3, 2 mechanisms for oxalate overproduction have glyoxylate, which is metabolized to oxalate in the liver. been proposed, both of which ultimately lead to the accumulation of cytosolic glyoxylate and its subsequent conversion to oxalate by LDH.2,6 Oxalate cannot be Correspondence: Craig B. Langman, Ann and Robert H. Lurie metabolized further and is almost exclusively excreted Children’s Hospital of Chicago, Box MS37, 225 E Chicago Ave, Chicago IL 60611, USA. E-mail: c-langman@northwestern.edu by the kidneys, where it exerts its hazardous effects Received 5 January 2021; accepted 18 January 2021; published because of precipitation of its calcium salt (calcium ox- online 3 February 2021 alate or CaOx).2,7 1088 Kidney International Reports (2021) 6, 1088–1098 Ariceta et al.: LDHA is an RNA Interference Target for PH CLINICAL RESEARCH Figure 1. Glyoxylate metabolism in primary hyperoxaluria. Lactate dehydrogenase (LDH)–mediated conversion of glyoxylate to oxalate in the liver is the ultimate step resulting in oxalate overproduction in primary hyperoxaluria types 1, 2, and 3. Nedosiran is an investigational RNA interference therapy designed to inhibit hepatic LDH (encoded by LDHA). AGT, alanine:glyoxylate aminotransferase; GO, glycolate oxidase; GRHPR, glyoxylate reductase/hydroxypyruvate reductase; HOG, 4-hydroxy-2-oxoglutarate; HOGA, 4-hydroxy-2-oxoglutarate aldolase. The earliest and most common clinical manifesta- known for its function in the interconversion of py- tions of all PH types are nephrolithiasis and progressive ruvate and lactate via the Cori cycle.12–14 In PH, LDH nephrocalcinosis resulting from the deposition of plays a critical role in hepatic oxalate production. The 2 highly insoluble CaOx crystals throughout the kid- most common subunits of LDH are LDHA (or M sub- ney.2,6 This may lead to progressive renal damage and unit) and LDHB (or H subunit), which are encoded by chronic kidney disease, which often culminates in renal the LDHA and LDHB genes, respectively. These 2 failure.2,8 PH1 is the most severe of all PH types. It is subunits can assemble into 5 different tetrameric estimated that 20% to 50% of patients with PH (mainly combinations or isozymes (LDH1–LDH5). The LDHA in PH1) have advanced renal disease, and 27% have subunit is predominantly expressed in the muscle and renal failure at the time of or before PH diagnosis.6,9 As liver, whereas LDHB is the predominant form in the kidney injury progresses, a second phase of insult oc- heart, spleen, kidney, brain, and erythrocytes.15 LDH curs when the kidneys are unable to excrete the load of plays a central role in oxalate metabolism in the liver oxalate because of the declining filtration rate. This (Figure 1) because it is the key enzyme responsible for causes oxalate buildup in the plasma and deposition of the conversion of glyoxylate to oxalate,16–19 thereby CaOx crystals in virtually all tissues in a process called controlling the ultimate step of oxalate production and systemic oxalosis, which can be life-threatening.2,6 its subsequent urinary excretion. Liver-specific Most current PH therapies are conservative measures knockdown of LDHA and the resultant reduction of and are poorly effective. A liver-kidney transplant is LDHA, therefore, offers the potential to reduce hepatic currently the only option to prevent systemic oxalosis oxalate production and urinary oxalate excretion in all in patients progressing to renal failure in PH1.10 In genetically defined types of PH.20 PH2, isolated kidney transplantation is the method of Nedosiran (formerly DCR-PHXC) is an investiga- choice, although a combined liver-kidney transplant tional ribonucleic acid interference (RNAi) therapy21 may be required in some severe cases.11 being developed as a treatment for PH. Nedosiran is a The lack of approved pharmacologic therapies for synthetic, double-stranded RNA oligonucleotide (i.e., PH1 poses a major unmet medical need. Therapies tar- small interfering RNA [siRNA]) designed to target the geted at glyoxylate metabolism to reduce hepatic oxa- mRNA encoding LDHA. It is being evaluated in clin- late burden have gained interest in the PH arena. One ical trials for the treatment of all 3 genetically defined such target is LDH, a tetrameric enzyme primarily types of PH (PH1, PH2, and PH3). The 2-part phase I trial, PHYOX1 (ClinicalTrials.gov identifier NCT03392896) has been completed. The first part of the 1 Lumasiran, an RNAi therapy targeting hepatic GO, was recently trial (group A; N ¼ 25) was a placebo-controlled, sin- approved as a treatment for PH1. gle-blind trial in healthy volunteers (HVs), while the Kidney International Reports (2021) 6, 1088–1098 1089 CLINICAL RESEARCH Ariceta et al.: LDHA is an RNA Interference Target for PH second part (group B; N ¼ 18) was an open-label trial in articles were excluded from further review: articles patients with genetically confirmed PH (PH1 and PH2). describing other subtype deficiencies (e.g., LDHB/H) Liver-specific targeting of nedosiran is achieved by and articles reporting nonclinical data. conjugating the RNA oligonucleotide to N-acetyl galac- tosamine (GalNAc) aminosugar residues. This GalNAc PHYOX1 Study Design conjugation enables specific binding to the asialoglyco- PHYOX1 (EudraCT No: 2017-003534-89; Clinicaltrials. protein receptors (ASGPRs), which are predominantly gov identifier NCT03392896) is a 2-part, phase I, expressed on the surface of hepatocytes.22–25 Once single-ascending dose study of subcutaneous nedosiran preferentially taken up by the liver, nedosiran engages in HVs (group A) and adult patients with PH1 or PH2 the natural endogenous RNAi machinery to selectively (group B) conducted between November 2017 and reduce LDHA mRNA levels, thereby reducing the pro- November 2019. The primary objective of the group A duction of LDHA protein and LDH enzyme in hepato- portion of the study was to evaluate the safety and cytes. Ultimately, the final step responsible for the tolerability of nedosiran. Clinical laboratory data and overproduction of hepatic oxalate is inhibited. The musculoskeletal AE data from group A will be dis- proof-of-concept that LDHA-targeted siRNAs can cussed in this manuscript, and methods applying only reduce oxalate production has been demonstrated using to that portion of the trial will be presented here. The animal models of PH1 and PH2.20,26 remainder of the PHYOX1 clinical trial will be While selective inhibition of hepatic LDH enzyme described in a separate article. promises to be an elegant therapeutic target for all Group A was designed as a placebo-controlled, genetically defined types of PH, it is prudent to prove participant-blind, single ascending-dose study that inhibition of the hepatic isozyme does not produce wherein participants were randomized into 5 sequen- unintended consequences in off-target tissues, such as tial dose cohorts (0.3, 1.5, 3.0, 6.0, and 12.0 mg/kg muscles. Nonclinical testing of siRNAs that target he- nedosiran or placebo) with 5 participants (3 active, 2 patic LDHA20,26 has provided evidence to support the placebo) in each cohort (Supplementary Figure S1). hypothesis that liver-specific LDHA inhibition using Male or female HVs between 18 and 55 years of age GalNAc-conjugated siRNAs, such as nedosiran, pro- (inclusive) with a body mass index of 19.0 to 32.0 kg/ duces no apparent adverse effects in off-target (non- m2 (inclusive) were eligible to take part in the study if hepatic) tissues. Herein, we provide clinical data to they met all eligibility criteria set forth in the study corroborate the safety of nedosiran-mediated hepatic protocol (Supplementary Table S1). Participants were LDHA inhibition by 1) defining the human phenotype enrolled on day 0 and randomized on day 1 to receive a of congenital LDHA deficiency (LDHAD) based on a single dose of nedosiran or placebo. Participants were comprehensive review of cases published in the litera- discharged from the study site on day 3 and returned ture and 2) correlating the LDHAD phenotype with to the site at specified time points through day 29 (end clinical data in the form of biomarker levels and muscle- of study). The study protocol and amendments were related adverse events (AEs) in HVs (group A) ran- approved by an independent ethics committee, and the domized to receive single-dose nedosiran or placebo in study was conducted according to the International the phase I trial. People with congenital LDHAD lack Conference on Harmonization guidelines and relevant LDHA expression in all tissues. They therefore repre- country-specific laws and regulations. sent the most extreme scenario, which we can use as a baseline to assess the effect of hepatic LDHA inhibition. PHYOX1 Safety Analyses Safety analyses were performed at scheduled time points throughout the study and included physical METHODS examination, vital signs, electrocardiograms, standard Literature Review clinical laboratory testing, concomitant medications, A comprehensive review of literature was performed and AEs (coded using MedDRA version 21.0). Of these, on PubMed with no time limits on publication date AEs classified under the “musculoskeletal and con- using the following search terms: L-lactate dehydro- nective tissue disorders” system organ class (SOC) were genase/deficiency, L-lactate dehydrogenase contains: discussed herein. Blood samples were collected from dehydrogenase, L-lactate, L lactate dehydrogenase, participants on days 0 (baseline), 2, 8, 15, 22, and 29 lactate dehydrogenase, dehydrogenase, lactate. English (end of study) for lactate, pyruvate, and creatine kinase language articles that reported human LDHAD cases (CK) analyses. Fasted samples were obtained at the were included. Foreign language articles were included clinical site at approximately the same time of the day if their abstracts were in English and provided relevant on days 0, 2, 15, and 29. Samples on days 8 and 22 were patient information on LDHAD. The following types of outpatient samples, so fasting was variable and not 1090 Kidney International Reports (2021) 6, 1088–1098 Ariceta et al.: LDHA is an RNA Interference Target for PH CLINICAL RESEARCH Figure 2. Patients with lactate dehydrogenase A (LDHA) deficiency: symptoms reported in the published literature. The symptoms of LDHA deficiency reported for the 14 patients were primarily muscle-related (green bars). Multiple symptoms were reported in a single patient in some cases. always as long as for samples obtained at the clinical on the same patient(s) with LDHAD. To reduce dupli- site. For lactate analysis, blood was collected into a 1.2- cation of patient numbers, unique cases were identified mL fluoride/ethylenediamine tetraacetic acid monov- and matched based on cross-referencing of citations ette and sent for analysis within 15 minutes of sample and family pedigree charts (to the extent possible in a collection. For pyruvate, blood was collected in a 1.2- literature review setting). Based on this process, 14 mL lithium heparin monovette and transported on unique patients (6 female and 8 male) with congenital wet ice for immediate analysis. For CK, blood was LDHAD were identified: 11 cases in 7 families in Japan, collected in a 4.5-mL plain monovette. Serum was 1 case in an Italian family, and 2 cases in 2 U.S. families. separated by centrifuging at 4  C for 10 minutes using Figure 2 categorizes the symptoms reported in pa- centrifugal force equal to 2000 g, transferred into a 2.5- tients with LDHAD identified in the literature. Of the mL false bottom propylene tube, and stored refriger- 23 symptoms reported, 74% (17/23) were related to ated at approximately 4  C pending analysis. Lactate, some form of muscular manifestation and the remaining pyruvate, and CK levels in plasma/serum samples were 26% (6/23) were skin-related. There were no liver assessed by enzymatic assays using cobas c analyzers manifestations reported among any patients. Exertional (Roche Diagnostics, Indianapolis, Indiana, USA) ac- muscle pain was the most common symptom followed cording to the manufacturer’s instructions and by skin lesions and exertional pigmenturia (myoglo- methods reported in the literature.27 binuria and hemoglobinuria). Three of the 6 female patients (50%) reported uterine stiffness during preg- Statistical Analyses nancy. Chest pain was reported in 1 patient but not Graphs were prepared using Prism software (version directly linked to LDHAD by the authors.42 In 3 pa- 8.4.3; GraphPad Software, San Diego, CA). All statisti- tients, no symptoms were reported in these article(s). cal analyses were performed using SAS software (version 9.4; IBM Corp., Chicago, IL). The Wilcoxon Literature Review: Alterations in Lactate, rank sum test was used to compare biomarker data Pyruvate, and CK Levels in Patients With LDHAD between nedosiran and placebo groups. Biomarker Symptoms were typically more common in patients changes from days 0 to 29 within each group were with LDHAD when they were engaged in various types compared using the Wilcoxon signed rank test. The of exercise (e.g., judo wrestling, pole climbing, 100- criterion for statistical significance was P < 0.05. meter dash). In general, these patients did not report symptoms under nonexercise conditions (Table 1).30,31 RESULTS At rest, plasma levels of both lactate and pyruvate in Literature Review: Patients With Congenital patients with LDHAD were found to be similar to those LDHAD Do not Show Signs of Liver in non-LDHAD control subjects. When subjected to Manifestations ischemic exercise testing of the forearm, plasma pyru- The literature search yielded 137 articles (see Methods vate was increased significantly in these patients for search criteria), of which 20 articles were relevant compared with control subjects. The expected increase to human LDHAD cases.28–47 Multiple articles reported in lactate levels was reduced compared with control Kidney International Reports (2021) 6, 1088–1098 1091 CLINICAL RESEARCH Ariceta et al.: LDHA is an RNA Interference Target for PH Table 1. Summary of lactate dehydrogenase A deficiency CK. No significant changes were observed from day 0 to phenotype defined by the literature day 29 for any of the 3 biomarkers within the nedo- Key takeaways from the literature review of lactate dehydrogenase A deficiency siran group (Figures 3–5). There were also no apparent  No liver manifestations biomarker trends based on nedosiran dose levels  Patients largely asymptomatic except under exercise conditions (Figures 3–5, insets). A low sample size (n ¼ 3 per  Normal lactate levels at rest (range 0.4--0.6 mM)a group) precludes meaningful statistical analysis at the  Normal pyruvate levels at rest (range 0.07--0.09 mM)a,b  Elevations in creatine kinase levels at rest noted in some cases dose level. Both the nedosiran and placebo groups (range 48--26,290 U/L)c showed similar lactate, pyruvate, and CK profiles dur-  Marked elevation of plasma pyruvate (range 0.68--0.88 mM)d and creatine kinase ing the course of the study as seen from measurements under exercise challenge at timepoints between days 0 and 29 (Figure 6a–c).  Blunted elevation of plasma lactate (range 3.4--4.0 mM)d under exercise challenge a Mean biomarker values in either group did not exceed Based on a single article33 reporting numerical values of plasma lactate and pyruvate on 4 patients. the upper limit of normal during the study timeframe b Significant pyruvate elevations were noted in a single patient during her third trimester (upper limits of normal: lactate, 2.2 mmol/L; pyruvate, of pregnancy; her lactate levels were “almost within normal range” as described by the authors.39 0.13 mmol/L; CK, 469.5 U/L). c Elevation in creatine kinase levels at rest were noted in 3 articles.30,32,43 d Based on a single article33 reporting numerical values of plasma lactate and pyruvate on 4 patients subjected to incremental exercise on a bicycle ergometer; creatine kinase values were not reported. PHYOX1: No Drug-Related Musculoskeletal AEs Were Observed in the Single-Dose Nedosiran subjects.28,30,31,33 As further evidence of predominant Group skeletal muscle involvement, a marked increase in Seven participants in group A (3 in placebo; 4 in plasma CK relative to control subjects was also nedosiran) experienced $1 AE, all of which were of observed when challenged under ischemic exercise mild or moderate severity. We were interested in conditions.28 assessing the effect of nedosiran on off-target tissues, specifically the muscles. Therefore, AEs classified un- der musculoskeletal and connective tissue disorders PHYOX1: HVs Treated With Single-Dose SOC were considered relevant (other AEs will be dis- Nedosiran Showed no Alterations in Plasma cussed in a separate article on the entire PHYOX1 trial). Lactate, Pyruvate, or CK Levels Musculoskeletal AEs were experienced by 2 partici- The first part (group A) of the PHYOX1 trial was a pants in the nedosiran group (Table 4), both of which placebo-controlled, participant-blind, single ascending (back pain and myalgia) were considered by the dose study. Group A enrolled 25 HVs at a single site, all investigator to be unrelated to the study drug. Both of whom completed the study. Five cohorts were dosed events resolved within 3 days of onset and appeared to at 0.3, 1.5, 3.0, 6.0, or 12.0 mg/kg of nedosiran or have no clinical sequelae. placebo (3:2 randomization; Table 2). The mean age of all participants was 33.2 years (range, 19–55 years). The mean body mass index of all participants DISCUSSION was 25.4 kg/m2. PH is a family of rare, autosomal recessive genetic Among HVs treated with a single dose of nedosiran disorders of glyoxylate metabolism that lead to hepatic (0.3–12.0 mg/kg), there were no significant changes in overproduction of oxalate and progressive renal dam- plasma lactate, pyruvate, or CK levels compared with age, which may culminate in renal failure and systemic placebo at the end of study (day 29; Table 3). Within oxalosis.2,6 LDH is the final common enzyme respon- the placebo group, changes from day 0 (baseline) to day sible for the conversion of glyoxylate to oxalate in all 29 were significant for lactate and pyruvate, but not for genetically defined types of PH, which makes it an Table 2. Demographic characteristics of PHYOX1 group A (healthy volunteers) Nedosiran, mg/kg Characteristic Placebo (N [ 10) 0.3 (n [ 3) 1.5 (n [ 3) 3.0 (n [ 3) 6.0 (n [ 3) 12.0 (n [ 3) Overall (n [ 15) All participants (N [ 25) Age (y) Mean (SD) 35.5 (9.9) 28.7 (5.7) 28.0 (7.2) 37.3 (17.5) 35.3 (10.5) 29.3 (6.4) 31.7 (9.6) 33.2 (9.7) Sex, n (%) Male 8 (80.0) 2 (66.7) 1 (33.3) 1 (33.3) 2 (66.7) 0 6 (40.0) 14 (56.0) Female 2 (20.0) 1 (33.3) 2 (66.7) 2 (66.7) 1 (33.3) 3 (100) 9 (60.0) 11 (44.0) BMI (kg/m2) Mean (SD) 26.47 (3.28) 26.37 (4.27) 24.36 (3.31) 26.93 (3.14) 21.96 (1.41) 23.81 (2.53) 24.69 (3.20) 25.40 (3.29) BMI, body mass index; SD, standard deviation. 1092 Kidney International Reports (2021) 6, 1088–1098 Ariceta et al.: LDHA is an RNA Interference Target for PH CLINICAL RESEARCH Table 3. Biomarker values in healthy volunteers Placebo (N [ 10) Nedosiran (N [ 15) Biomarker and timeline Mean (SD) Median (min, max) Mean (SD) Median (min, max) P value Plasma lactate (mmol/L) Baseline (day 0) 1.32 (0.66) 1.05 (0.60, 2.60) 0.87 (0.27) 0.90 (0.40, 1.50) .821a End of study (day 29) 0.78 (0.32) 0.70 (0.40, 1.40) 0.75 (0.20) 0.70 (0.50, 1.30) .189b .027c Plasma pyruvate (mmol/L) Baseline (day 0) 0.018 (0.012) 0.014 (0.005, 0.046) 0.013 (0.007) 0.012 (0.000, 0.029) .622a End of study (day 29) 0.010 (0.009) 0.006 (0.001, 0.026) 0.012 (0.010) 0.008 (0.000, 0.031) .616b .023c Plasma creatine kinase (U/L) Baseline (day 0) 164.10 (121.81) 125.00 (65.00, 405.00) 95.20 (41.00) 91.00 (24.00, 172.00) .398a End of study (day 29) 172.60 (147.90) 103.00 (57.00, 510.00) 95.33 (33.38) 101.00 (38.00, 150.00) .689b 1.000c SD, standard deviation. a Day 29 nedosiran versus day 29 placebo. b Day 0 nedosiran versus day 29 nedosiran. c Day 0 placebo versus day 29 placebo. attractive therapeutic target for fulfilling an unmet tissues, specifically the muscle (where LDHA is also need in all known types of PH.20 expressed, along with the liver). Animal studies by Lai Nedosiran is an investigational RNAi therapy et al.20 and Wood et al.26 have demonstrated the lack of (administered monthly via subcutaneous injections) such off-target effects; both studies found that GalNAc- designed to inhibit hepatic LDHA. It is intended to conjugated siRNAs achieved liver-specific knockdown reduce the expression of LDHA and the resultant LDH of LDHA in mice with no significant alterations to its activity in the liver by harnessing the natural RNAi expression in other tissues, such as the muscle, skin, pathway. GalNAc conjugation achieves liver-specific kidneys, heart, or uterus. Lai et al.20 also noted liver- targeting of nedosiran by binding to ASGPRs. specific knockdown of LDHA in nonhuman primates Although ASGPRs have been detected in nonhepatic and chimeric mice with humanized livers, which pro- tissues, their expression on hepatocytes far exceeds vides an additional level of evidence for liver-specific that in other locations, and ASGPRs have no known RNAi delivery across species. role elsewhere in the body.22–25 Therefore, in theory To further explore the lack of off-target effects with and by design, it would be unlikely to see extrahepatic siRNAs, we reviewed the data from animal studies delivery of these siRNAs. Nevertheless, it is imperative against the human phenotype of congenital LDHAD, to demonstrate the lack of RNAi effects in off-target which represents the most extreme scenario of loss of Figure 3. Plasma lactate distribution in healthy volunteers. Scatter plot showing plasma lactate levels in healthy volunteers at baseline (day 0) and at the end of the study (day 29) in placebo and nedosiran groups. Inset graph (in gray) shows plasma lactate distribution by nedosiran dose. SD, standard deviation Kidney International Reports (2021) 6, 1088–1098 1093 CLINICAL RESEARCH Ariceta et al.: LDHA is an RNA Interference Target for PH Figure 4. Plasma pyruvate distribution in healthy volunteers. Scatter plot showing plasma pyruvate levels in healthy volunteers at baseline (day 0) and at the end of the study (day 29) in placebo and nedosiran groups. Inset graph (in gray) shows plasma pyruvate distribution by nedosiran dose. SD, standard deviation. LDHA expression. The human LDHAD phenotype was the human counterparts with LDHAD. Emerging data defined based on a comprehensive review of medical from repeat dose toxicity studies in nonhuman pri- literature spanning >35 years, which identified a total mates (monthly dosing of LDHA siRNA for #9 months) of 14 such patients worldwide. This deficiency presents also show no adverse skeletal muscle effects (unpub- with a predominantly muscle-related phenotype with lished data). In keeping with the lack of liver mani- some reports of skin lesions. Interestingly, none of the festations noted in the human phenotype, animal data patients with LDHAD had liver manifestations. We also show no liver histopathology or acute liver therefore deduced that evidence of any off-target ef- toxicity with siRNA-LDHA inhibition. No changes in fects would manifest in the skeletal muscles. Lai et al.20 body composition or weight loss were noted, rein- ruled out such manifestations as they observed normal forcing the overall safety of this approach.20,26 muscle performance when Ldha-knockdown mice were Another aspect of LDHA inhibition is its potential subjected to exercise in a treadmill endurance test. No effect on the Cori cycle and related metabolic path- signs of exercise-induced myopathy were noted, unlike ways, such as gluconeogenesis. Patients with LDHAD Figure 5. Plasma creatine kinase (CK) distribution in healthy volunteers. Scatter plot showing plasma CK levels in healthy volunteers at baseline (day 0) and at the end of the study (day 29) in placebo and nedosiran groups. Inset graph (in gray) shows plasma CK distribution by nedosiran dose. SD, standard deviation. 1094 Kidney International Reports (2021) 6, 1088–1098 Ariceta et al.: LDHA is an RNA Interference Target for PH CLINICAL RESEARCH Figure 6. Superimposed lactate, pyruvate, and creatine kinase (CK) distribution profiles in healthy volunteers. Superimposed scatter plots showing (a) lactate, (b) pyruvate, and (c) CK profiles in the nedosiran (N ¼ 15) and placebo (N ¼ 10) groups at timepoints between days 0 and 29 (days 2, 8, 15, and 22). One participant had a missing lactate value, and another participant had a missing pyruvate value, both on day 2 within the placebo group. SD, standard deviation. showed a marked elevation of plasma pyruvate and CK signs of lactic acidosis, as suggested by the absence of because of impaired glycolysis under exercise challenge plasma lactate elevations in mice. Similarly, liver- conditions. Wood et al.26 noted a significant rise in specific LDHA inhibition did not result in an eleva- both liver and plasma pyruvate levels under non- tion of lactate in nonhuman primates. In addition, mice exercise conditions in a PH1 mouse model treated with showed normal muscle function during exercise, liver-specific siRNA targeted at LDHA. In contrast to implying an uninterrupted glucose resupply from liver these findings, Lai et al.20 noted that liver-specific to muscles, indicating a properly functioning hepatic siRNA-LDHA inhibition in mice did not result in sig- gluconeogenesis pathway.20 The lack of significant in- nificant elevations of plasma pyruvate under non- creases in plasma lactate and pyruvate after hepatic exercise conditions and during exercise. There were no LDHA inhibition was also corroborated by clinical data Kidney International Reports (2021) 6, 1088–1098 1095 CLINICAL RESEARCH Ariceta et al.: LDHA is an RNA Interference Target for PH Table 4. Musculoskeletal adverse events in healthy volunteers Nedosiran, mg/kg System organ class and preferred term Placebo (N [ 10) 0.3 (n [ 3) 1. 5 (n [ 3) 3.0 (n [ 3) 6.0 (n [ 3) 12.0 (n [ 3) Overall (n [ 15) Participants with $1 TEAE, n (%), no. of TEAEs 3 (30.0), 4 0 0 1 (33.3), 1 2 (66.7), 3 1 (33.3), 2 4 (26.7), 6 Musculoskeletal and connective tissue disorders 0 0 0 0 1 (33.3), 1 1 (33.3), 1 2 (13.3), 2 Back pain 0 0 0 0 1 (33.3), 1 0 1 (6.7), 1 Myalgia 0 0 0 0 0 1 (33.3), 1 1 (6.7), 1 TEAE, treatment-related adverse event. Adverse events were coded using MedDRA version 21.0. TEAEs are events that occurred or worsened on or after the first dose of study drug. Participants are counted once for each system organ class and once for each preferred term. obtained from group A of the PHYOX1 trial. HVs biochemical effects of LDHA inhibition. It is also treated with up to 12 mg/kg of nedosiran showed no important to note that participants were able to significant changes in plasma lactate, pyruvate, or CK perform activities of daily living, without any drug- levels compared with placebo during the clinical trial related AEs after the administration of nedosiran. The observation period. There were no significant changes ongoing pivotal (PHYOX2)48 and open-label extension in lactate, pyruvate, or CK within the nedosiran (PHYOX3)49 trials are multidose trials evaluating group between baseline and end of study (day 29), nedosiran. They are designed to capture safety, further confirming the lack of any systemic effects of including muscle-related AEs and CK elevations, and hepatic LDHA inhibition in humans (especially its efficacy assessments following chronic nedosiran role in the Cori cycle and other interconnected dosing. Thus, they are prospectively monitoring the metabolic pathways). The statistically significant chronic effects of LDHA inhibition. changes in lactate and pyruvate within the placebo In summary, an analysis of the published nonclinical group could be attributed to the variability in the and recent clinical evidence supports RNAi-mediated baseline values within the group. These changes are LDHA inhibition as a potentially safe therapeutic not thought to be clinically meaningful, which is mechanism for all genetic variants of PH. corroborated by the fact that the participants in the placebo group were asymptomatic and did not experience any adverse events that would be DISCLOSURE attributable to such a reduction in either pyruvate or CBL is a consultant for Dicerna Pharmaceuticals, Inc. lactate. Overall, an analysis of the existing nonclin- (Lexington, MA) and Allena Pharmaceuticals, Inc. ical data and the current phase I clinical trial data (Newton, MA). GA reports personal fees and nonfinancial show that RNAi-mediated LDHA inhibition in the support from Alexion Pharmaceuticals, personal fees and liver appears to be safe for disease targeting. Pre- nonfinancial support from Recordati Rare Diseases, sumably, this inhibition has a predominant impact Advicenne, nonfinancial support from Kyowa Kirim, on oxalate formation as opposed to the interconver- personal fees from Dicerna Pharmaceuticals, personal sion of lactate and pyruvate. None of the HVs fees and other from Chiesi, outside the submitted work. experienced any drug-related musculoskeletal AEs KB, BDB, BH, and RR are employees of Dicerna after treatment with nedosiran, which also supports Pharmaceuticals, Inc. BDB reports patents issued/pending the specificity of RNAi action and the lack of unin- relevant to the submitted work. tended consequences in off-target tissues. We acknowledge that the biochemical findings and ACKNOWLEDGMENTS lack of drug-related musculoskeletal AEs presented We thank all PHYOX1 participants and their caregivers. here are based on a single dose of nedosiran in HVs We thank Annelize Koch, who was the principal investi- alone. The AE data on patients with PH are beyond the gator for group A of the trial. The PHYOX1 trial was scope of this article and will be discussed in a follow-on funded by Dicerna Pharmaceuticals, Inc. Dicerna was article reporting the totality of results from the involved in the design and conduct of the study and PHYOX1 study. The biomarker data presented here provided logistical support during the trial. BH was an were collected under clinical trial conditions that were investigator and CBL was the chair of the safety review not intended to replicate exercise challenge conditions. committee of the trial. GA is an investigator in the The transient nature of the pyruvate and lactate alter- ongoing PHYOX2 and PHYOX3 trials. Jing Zhou ations after exercise made it impractical to capture in a (Dicerna) conducted the statistical analysis reported in clinical setting. Animal models are an alternative, on this manuscript. Aparna Raghavan (Dicerna) provided which we based our conclusions for exercise-related medical writing support. Dicerna was permitted to 1096 Kidney International Reports (2021) 6, 1088–1098 Ariceta et al.: LDHA is an RNA Interference Target for PH CLINICAL RESEARCH review the manuscript and suggest changes. The authors 12. Doherty J, Cleveland J. Targeting lactate metabolism for had full editorial control of the paper and provided their cancer therapeutics. J Clin Invest. 2013;123(9):3685–3692. final approval of all content. The participant data that 13. Valvona CJ, Fillmore HL, Nunn PB, et al. The regulation and support the conclusions of this manuscript are included function of lactate dehydrogenase A: therapeutic potential in brain tumor. Brain Pathol. 2015;26(1):3–17. within the manuscript and its supplemental materials. A written request may be submitted to the corresponding 14. Adeva-Andany M, López-Ojén M, Funcasta-Calderón R, et al. author for data from this trial for the sole purpose of Comprehensive review on lactate metabolism in human health. Mitochondrion. 2014;17:76–100. replicating procedures and results presented in the article, provided that necessary approvals are obtained 15. Granchi C, Paterni I, Rani R, et al. Small-molecule inhibitors of human LDH5. Future Med Chem. 2013;5(16):1967–1991. from the data owner in accordance with all US legisla- tion on the general data protection regulation. Provision 16. Poore RE, Hurst CH, Assimos DG, et al. Pathways of hepatic of data may depend upon applicable current, pending, or oxalate synthesis and their regulation. Am J Physiol. 1997;272(1):C289–C294. future regulatory authority submissions. For additional 17. Mdluli K, Booth M, Brady R, et al. A preliminary account of the trial details, visit https://clinicaltrials.gov/ct2/show/NCT033 properties of recombinant human glyoxylate reductase 92896. (GRHPR), LDHA and LDHB with glyoxylate, and their potential roles in its metabolism. Biochim Biophys Acta. 2005;1753(2): SUPPLEMENTARY MATERIAL 209–216. Supplementary Material (PDF) 18. Williams H, Smith L. Hyperoxaluria in L-glyceric aciduria: Figure S1. PHYOX1-group A study design schematic. possible pathogenic mechanism. Science. 1971;171(3969): Table S1. Inclusion and exclusion criteria for PHYOX1- 390–391. group A participants. 19. Smith L, Bauer R, Craig J, et al. Inhibition of oxalate synthe- sis: In vitro studies using analogues of oxalate and glycolate. CONSORT statement. 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