Local application of engineered insulin-like growth factor I mRNA demonstrates regenerative therapeutic potential in vivo

Abstract

Insulin-like growth factor I (IGF-I) is a growth-promoting anabolic hormone that fosters cell growth and tissue homeostasis. IGF-I deficiency is associated with several diseases, including growth disorders and neurological and musculoskeletal diseases due to impaired regeneration. Despite the vast regenerative potential of IGF-I, its unfavorable pharmacokinetic profile has prevented it from being used therapeutically. In this study, we resolved these challenges by the local administration of IGF-I mRNA, which ensures desirable homeostatic kinetics and non-systemic, local dose-dependent expression of IGF-I protein. Furthermore, IGF-I mRNA constructs were sequence engineered with heterologous signal peptides, which improved in vitro protein secretion (2- to 6-fold) and accelerated in vivo functional regeneration (16-fold) over endogenous IGF-I mRNA. The regenerative potential of engineered IGF-I mRNA was validated in a mouse myotoxic muscle injury and rabbit spinal disc herniation models. Engineered IGF-I mRNA had a half-life of 17-25 h in muscle tissue and showed dose-dependent expression of IGF-I over 2-3 days. Animal models confirm that locally administered IGF-I mRNA remained at the site of injection, contributing to the safety profile of mRNA-based treatment in regenerative medicine. In summary, we demonstrate that engineered IGF-I mRNA holds therapeutic potential with high clinical translatability in different diseases.

Keywords: IGF-I; MT: RNA/DNA Editing; disc herniation; mRNA therapy; muscle injury; regeneration.

Graphical abstract

Figure 1

Acceleration of functional muscle regeneration by endogenous IGF-I mRNA (Cpd.1) in a mouse model of myotoxic injury (A) Baseline of TA muscle force in healthy mice. (B) Functional recovery of paralyzed TA muscle with vehicle or increasing dose of Cpd.1 mRNA treatment measured by muscle performance over the course of 28 days. (C) Area-under-the-curve (AUC) analysis of muscle performance data (∗p < 0.05, 30 μg versus vehicle; ∗∗p < 0.01, 10 μg versus vehicle). (D) Histological measurement of total TA muscle fiber count in legs of injured and contralateral (uninjured) animals treated with vehicle or increasing dose of Cpd.1 (∗∗p < 0.01, vehicle and 3 μg in injured versus uninjured). (E) Mean fiber cross-sectional area (CSA) in legs of injured and contralateral (uninjured) animals treated with vehicle or increasing dose of Cpd.1 (∗∗∗p < 0.001, vehicle and 3 μg in injured versus uninjured). (F) Frequency of TA muscle fiber types measured by antibodies against MyHC-I, MyHC-IIa, and MyHC-IIb in legs of injured and contralateral (uninjured) animals treated with vehicle or increasing dose of Cpd.1. (G) Frequency of slow muscle type I fibers in legs of injured and contralateral (uninjured) animals treated with vehicle or increasing dose of Cpd.1 (∗∗∗p < 0.001, vehicle and 3 μg in injured versus uninjured; ∗p < 0.05, 10 and 30 μg in injured versus uninjured; ∗p < 0.05, 10 and 30 μg uninjured). Data represent mean ± SEM of 7–10 mice per group.

Figure 2

Improvement of IGF-I secretion through signal peptide optimization (A) HEK293 cells were transfected Cpd.1 and Cpd.2 IGF-I mRNA using lipofection, and IGF-I protein was analyzed in the supernatant after 24 h using ELISA. Statistical analysis was carried out using Student’s t test (∗∗∗p < 0.001). (B) Dose-response study on induction of IGF-I secretion in HEK293T cells after mRNA transfection with Cpd.1 or Cpd.2. Cells were transfected with Cpd.1 or Cpd.2 at different concentrations (0, 0.02, 0.06, 0.2, 0.6, or 2 μg), and secreted IGF-I was measured after 24 h in the cell culture supernatant using a specific ELISA. Cpd.2 induced IGF-I secretion significantly more potent (EC₅₀ = 0.13 μg) than Cpd.1 (EC₅₀ = 0.89 μg). Data represent mean ± SEM of 2 replicates. Significance (∗∗∗p < 0.001) was assessed using two-way ANOVA of the two curves. (C) Time course study of Cpd.2 IGF-I mRNA and IGF-I protein levels in HEK293 cells. The time points at which RNA and IGF-I quantified include 5, 17, 29, 41, 53, and 65 h. The mRNA levels are measured by relative gene expression method (2-ΔΔCt) where untransfected samples were used as control group and the expression level set to 1. Expression was normalized using human PPIA as a reference gene. Secreted IGF-I was measured in the cell culture supernatant using ELISA with respective time point. (D) IGF-I secretion from mouse skeletal muscle cells (C2C12) by mRNA transfection with Cpd.1-Cpd.2. C2C12 cells were transfected with each 2 μg Cpd.1-Cpd.2, and secreted IGF-I was measured after 24 h in the cell culture supernatant using a specific ELISA. Cpd.2 induced IGF-I secretion significantly higher than Cpd.1 (6.1-fold). Data represent mean ± SEM of 4 replicates. Significance (∗∗∗p < 0.001) was assessed using Student’s t test. (E) Induction of IGF-I secretion from human primary skeletal muscle cells (HSkMCs) by mRNA transfection with Cpd.1 and Cpd.2. HSkMCs were transfected with each 2 μg Cpd.1 or Cpd.2, and secreted IGF-I was measured after 24 h in the cell culture supernatant using a specific ELISA. Cpd.2 induced IGF-I secretion significantly higher than Cpd.1 (3.1-fold). Data represent mean ± SEM of 3 replicates. Significance (∗∗p < 0.01) was assessed using Student’s t test. F) Proliferation assay with MCF-7 cells with increasing concentration of rhIGF-I (INCRELEX) or IGF-I derived from HEK293 cells supernatant transfected with Cpd.2 mRNA. Dose-response curve of MCF-7 cells (3,000 cells/well) after 96 h with treatments was plotted with four parameter non-linear fitted curve. No statistical difference between Cpd.2 and INCRELEX group was observed.

Figure 3

Comparison of endogenous and optimized IVT IGF-I mRNA in mouse myotoxic injury model (A) Muscle function of non-injured TA muscles. TA muscles from non-injured animals (n = 10) were assessed for muscle function on days 7, 14, and 28 each, and data were sampled. Data are from a total of n = 30 measurements. (B) Functional recovery of TA muscle after notexin injury. After notexin injury via intramuscular injection (day 0), IGF-I mRNA treatments (Cpd.1, 10 μg; Cpd.2, 1 μg) were applied by intramuscular injection on days 1 and 4 (see arrowheads). The control group received vehicle solution. Muscle function was assessed on days 1, 4, 7, 10, 14, 21, and 28 post-injury. Data represent mean ± SEM of 5 mice per group and time point. A 2-way ANOVA revealed highly significant difference between both Cpd.1 and Cpd.2 curves compared with vehicle (∗∗∗p < 0.001). (C) Area-under-the-curve (AUC) calculation of functional recovery after myotoxic injury. Animals were subjected to notexin injury on day 0, and Cpd.1 or Cpd.2 at indicated doses or vehicle was injected i.m. on days 1 and 4. TA muscle function was assessed on days 4, 7, 10, 14, 17, 21, and 28 from the same mice, and AUC was calculated from each animal. Data are represented as mean ± SEM, with 4 or 5 animals per group. Statistical analysis was done using a one-way ANOVA followed by Dunnett’s multiple-comparison test. ∗∗p < 0.01 vs. vehicle. (D) Dose-response curves (0.05–30 μg) from AUC of functional recovery after myotoxic injury. Animals were subjected to notexin injury on day 0, and Cpd.2 or Cpd.1 at indicated doses or vehicle injected i.m. on days 1 and 4. TA muscle function was assessed on days 4, 7, 10, 14, 17, 21, and 28 from the same mice, and AUC for maximum force was calculated from each animal. From the XY plots, EC₅₀ values were calculated to 0.4 μg for Cpd.2 and 6.6 μg for VMB-Cpd.1 by a non-linear Hill fit. Data are represented as mean ± SEM, with 4–14 animals per group.

Figure 4

PK/PD of IGF-I mRNA in rat TA muscle (A) Scheme of TA muscle processing and generation of tissue pieces. The red circle indicates the site of the punch injury and 1L and 1R where Cpd.2 injections had been located. Segments 1L–4L were processed for qPCR analysis of Cpd.2 and downstream biomarkers, segments 1R–4R were processed for protein analysis of IGF-I and pAKT. (B) Time course of Cpd.2 mRNA exposure in TA muscle of 1L segment at time points 0–72 h after i.m. injection by qRT-PCR. (C) AUC analysis (0–72 h) to measure mRNA exposure from 1L to 4L at different doses (1, 3, or 10 μg) and vehicle treatment. (D) Half-life of Cpd.2 exposure in 1L and 2L segment, assessed for the 3 and 10 μg doses. (E) IGF-I protein expression after i.m. injection of 3 or 10 μg Cpd.2, at time points 0–72 h. 1R muscle samples were taken and analyzed for IGF-I protein by IQELISA. (F) Tissue phosphorylation of AKT as downstream signal of IGF-I at time points 0–72 h after i.m. injection of Cpd.2 in TA muscle. 2R muscle samples were taken and analyzed for phosphorylation of AKT at Ser473 by a phosphor-specific and total AKT ELISA kit. Data are expressed as area under the curve (AUC) and represent mean ± SEM of 8 animals per group.

Figure 5

Changes in downstream biomarkers expression in rat injury model (A) Illustration of skeletal muscle regenerative process within 7 days after injury. Early processes within the first three days include processes of inflammation and satellite cell activation, proliferation, and fusion, but also early myogenic processes including embryonic myosin isoforms to be expressed, whereas adult myosins are only expressed after that time period. Pax7, MYH3, and MYH8 were investigated for their time course over three days after injury and the potential change due to Cpd.2 treatment (adapted from Ciciliot and Schiaffino³⁰). (B–D) qPCR analysis of Pax7, MYH3, and MYH8 mRNA levels after muscle injury. Punch injuries were applied to TA muscle in rats, and vehicle or Cpd.2 applied i.m. at different doses (1, 3, or 10 μg) directly after injury as described. At times indicated, tissues were harvested and analyzed for Pax7, MYH3, and MYH8 using qRT-PCR. Relative changes were calculated by normalizing individual values to the respective control of each treatment group. Dose dependence and time course of these 3 markers were investigated in 1L and 2L segment. Arrows point to changes occurring 3 days after injury. Data are expressed as mean ± SEM of 5 (vehicle) and 8 (Cpd.2 dose groups) animals per data point. Gray dashed line indicates the control level of 1.

Figure 6

Prevention of functional deterioration in rabbit spinal disc herniation model (A) In vitro assessment of Cpd.1 and Cpd.3 mRNA (300 ng/well) based expression and secretion of IGF-I in HEK293 cells (12,000 cells/well). (B) In vitro assessment of Cpd.1 and Cpd.3 mRNA (300 ng/well) based expression and secretion of IGF-I in HepG2 cells (20,000 cells/well). (C) In vitro assessment of Cpd.1 and Cpd.3 mRNA (300 ng/well) based expression and secretion of IGF-I in primary rat motoneurons (10,000 cells/well). All three cell types were transfected with Cpd.1 and Cpd.3 IGF-I mRNA using Lipofectamine 2000, and IGF-I protein was analyzed in the supernatant after 24 h using ELISA. Statistical analysis was carried out using a Student’s t test (∗∗∗p < 0.001). (D) Schematic presentation of group allocation for treatments and each rabbit (n = 8) serving as its own control by receiving vehicle solution into the L2/L3 intervertebral space (vehicle), a sham procedure without injury in the L3/L4 disc (sham), and 20 μg Cpd.3 into the L4/L5 intervertebral space (Cpd.3). (E) Optical control of the puncture injury process using a stab. (F) X-ray picture to indicate L2/L3, L3/L4, and L4/L5 discs. (G) Body weight evolution of animals during the study over period of 90 days. Each line corresponds to one animal, and all eight-animal data are included with the measurement day. (H) Changes in the intervertebral disc height index (%DHI) in function of treatment. (H′) Calculation of disc height. Data are expressed as mean ± SEM of 8 rabbits. Statistical comparison was done using 2-way ANOVA and Dunnett’s multiple-comparison test against saline group.

Figure 7

Prevention of pathological progression in rabbit spinal disc herniation model (A) Representative histological pictures of the vehicle-treated, sham-treated, and Cpd.3-treated discs stained with toluidine blue and Masson’s trichrome. (B) Histological grading score derived from vehicle-treated, sham-treated, and Cpd.3-treated discs. Data are expressed as mean ± 95% CI of 8 rabbits. Statistical comparison was performed using the non-parametric Mann-Whitney test between all groups.