In vivo efficacy and safety of systemically administered serinol nucleic acid-modified antisense oligonucleotides in mouse kidney

Abstract

Nucleic acid medicine encompassing antisense oligonucleotides (ASOs) has garnered interest as a potential avenue for next-generation therapeutics. However, their therapeutic application has been constrained by challenges such as instability, off-target effects, delivery issues, and immunogenic responses. Furthermore, their practical utility in treating kidney diseases remains unrealized. Recently, we developed a serinol nucleic acid-modified ASO (SNA-ASO) that exhibits significant nuclease resistance. In this study, we evaluated the in vivo efficacy of SNA-ASOs in mouse kidney. We subcutaneously administered various types of phosphorothioate-modified gapmer ASOs with SNA or 2'-O-methoxyethyl (2'-MOE) modifications (MOE-ASO) targeting sodium glucose cotransporter 2 (SGLT2) in mice. The subcutaneous administration of SGLT2-SNA-ASO led to a dose-dependent reduction in renal SGLT2 expression and subsequent glucosuria. The inhibitory effects of SGLT2-SNA-ASO were more potent and prolonged than those of ASOs without SNA. Moreover, SGLT2-SNA-ASO did not cause severe liver damage, unlike SGLT2-MOE-ASO. The administration of Cy5-labeled-ASOs demonstrated an early increase in renal uptake, particularly in the renal proximal tubules, when modified with SNA. In conclusion, systemic administration of SGLT2-ASO modified with the artificial nucleic acid SNA effectively suppressed renal SGLT2 expression and induced urinary glucose excretion. These results suggest that SNA-modified ASOs show potential for application in developing nucleic acid therapeutics.

Keywords: ASO; MT: Oligonucleotides: Therapies and Applications; SGLT2; SNA; SNA-ASO; antisense oligonucleotide; kidney; nucleic acid therapeutics; serinol nucleic acid; serinol nucleic acid-modified antisense oligonucleotide.

Graphical abstract

Figure 1

Effects of DNA-antisense oligonucleotides, serinol nucleic acid 2-ASO and 4-ASO, and 2′-methoxyethyl-ASO on sodium glucose cotransporter 2 -mRNA expression in the immortalized human proximal tubule epithelial cells (A) The chemical structure of serinol nucleic acid (SNA). In analogy to the 5′-3′ designation for DNA, the (S)-(R) designation for SNA based on the configuration at C2′ was used. (B) Sequences and structures of various antisense oligonucleotides (ASOs). Magenta letters indicate SNA, black letters indicate DNA, and blue letters indicate the 2′-methoxyethyl (2′-MOE) modification. ^mC, 5-methyl cytosine; subscript s, PS. (C) Human proximal tubule epithelial cells (HK-2) cells were transfected with control ASOs and sodium glucose cotransporter 2 (SGLT2)-ASOs for 24 h. Quantitative PCR (qPCR) analysis of SGLT2 mRNA expression (n = 6). β-Actin was used as the internal control. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (D) HK-2 cells were transfected with control ASOs and SGLT2-ASOs, and imaged every 4 h for up to 48 h (n = 3). The cell proliferation rate is plotted graphically, with 0 h as 100%. (E) Effect of modifications on ASO degradation in 50% fetal bovine serum (FBS). Each ASO was incubated with 50% FBS at 37°C. Aliquots taken at the indicated times of incubation were analyzed by 20% denaturing PAGE. Data represent means ± SEMs.

Figure 2

SGLT2-SNA-ASOs suppress renal SGLT2 expression in a dose-dependent manner SGLT2-ASOs and control-ASOs were subcutaneously (s.c.) administered to mice at doses of 1, 3, and 10 mg/kg/day thrice per week for 1 week. (A) Study design. (B–D) qPCR analysis of SGLT2 expression in the kidney (n = 4). β-Actin was used as the internal control. Data represent means ± SEMs. #p < 0.05; ##p < 0.01; ###p < 0.001 vs. respective control and PBS. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; N.S., not significant.

Figure 3

Adverse effects of SGLT2-ASOs SGLT2-ASOs were subcutaneously administered to mice at doses of 1, 3, 10, and 30 mg/kg/day thrice per week for 1 week. (A) Serum aspartate aminotransferase (AST) levels (n = 4). (B) Serum alanine aminotransferase (ALT) levels (n = 4). (C) Total bilirubin levels (n = 4). (D) qPCR analysis of SGLT1 expression in the kidneys (n = 4). β-Actin was used as the internal control. Data are presented as the means ± SEMs. #p < 0.05; ##p < 0.01; ###p < 0.001 vs. respective 1 mg/kg group; ∗∗∗p < 0.001.

Figure 4

Administration of various SGLT2-ASOs for 3 weeks in mice SGLT2-ASOs were subcutaneously administered to mice at doses of 10 mg/kg/day thrice per week for 3 weeks. (A) Study design. (B) qPCR analysis of SGLT2 expression in kidneys (n = 4–8). (C) Western blotting analysis of SGLT2 expression in the kidneys (n = 3). β-Actin was used as the internal control. (D) Serum AST levels (n = 4–8). (E) Serum ALT levels (n = 4–8). (F) Serum alkaline phosphatase (ALP) levels (n = 4–8). Data are presented as the means ± SEMs. #p < 0.05; ##p < 0.01; ###p < 0.001 vs. PBS; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 5

SGLT2-SNA2-ASO suppressed renal SGLT2 expression without severe liver damage SGLT2-SNA2-ASO, SNA-modified control-ASO (cSNA-ASO) or SGLT2-MOE-ASO were subcutaneously administered to mice at doses of 10 mg/kg/day thrice per week for 3 weeks (A) qPCR analysis of SGLT2 expression in the kidneys (n = 8). (B) Serum AST levels (n = 4–8). (C) Serum ALT levels (n = 4–8). (D–G) qPCR analysis for detecting TNF-α (D), TGF-β1 (E), F4/80 (F), and MCP-1 (G) expression in the liver (n = 8). We used 18S as the internal control. (H) Urinary NGAL levels (n = 8). (I) Quantification of CD45 positive area in kidneys (n = 4). (J and K) Representative images and quantification of KIM-1⁺ area in kidneys (n = 4). Kidney specimens from renal ischemia-reperfusion injury (IRI) were used as a positive control for KIM-1 staining. Scale bar, 500 μm. (L) Serum IL-6 levels (n = 4). Data are presented as the means ± SEMs. #p < 0.05; ##p < 0.01; ###p < 0.001 vs. PBS; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; †p < 0.05 by post hoc Tukey’s multiple comparisons after one-way ANOVA, with the exception of the MOE-ASO group.

Figure 6

Evaluation of the in vivo stability of systemically administered SNA-ASOs SGLT2-DNA-ASO, SGLT2-SNA2-ASO, and SGLT2-SNA4-ASO were subcutaneously administered to the mice twice at a dose of 10 mg/kg/day (A) Study design. (B) qPCR analysis of SGLT2 expression in the kidney on days 4, 11, and 18 (n = 8). (C) Western blot analysis of SGLT2 protein in kidney samples on day 18 (n = 6). β-Actin was used as the internal control. Data are presented as the means ± SEMs. #p < 0.05; ###p < 0.001 vs. PBS; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; N.S., not significant.

Figure 7

Evaluation of the in vivo distribution of systemically administered various ASOs Cy-5-labeled SGLT2-DNA-ASO, SGLT2-SNA2-ASO, SGLT2-SNA4-ASO, SGLT2-MOE-ASO, or PBS (vehicle control) were subcutaneously administered to mice as a single dose of 3 mg/kg (A) Study design. (B) Representative fluorescence microscopy images of kidneys from mice 24 h after the administration of Cy-5 labeled SNA2-ASO or PBS. Scale bar, 100 μm. (C) Representative images of fluorescence intensity in each organ, including the kidney (K), liver (Li), brain (B), lung (Lu), heart (H), intestine (I), eye (E), spleen (S), and epididymal fat (F), on days 1, 8, and 15 after ASO or PBS administration. Right: layout of each tissue. (D and E) Fluorescence intensities in the kidney (D) and liver (E) on days 1, 8, and 15 after ASO administration (n = 4). (F) qPCR analysis of SGLT2 expression in the kidney on days 1, 8, and 15 (n = 4). β-Actin was used as the internal control. Data are presented as the means ± SEMs. #p < 0.05; ###p < 0.001 vs. respective PBS; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.