# Therapeutic potential of siRNA PMP22-SQ nanoparticles for Charcot-Marie-Tooth 1A neuropathy in rodents and non-human primates

Suzan Boutary, Guy Khalaf, Yosef Landesman, Mevidette El Madani, Didier Desmaële, Françoise Piguet, Rafael Alonso, Elena-Gaia Banchi, David Adams, Charbel Massaad, et al.

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## To cite this version:

Suzan Boutary, Guy Khalaf, Yosef Landesman, Mevidette El Madani, Didier Desmaële, et al. Therapeutic potential of siRNA PMP22-SQ nanoparticles for Charcot-Marie-Tooth 1A neuropathy in rodents and non-human primates. International Journal of Pharmaceutics, 2025, 671, 10.1016/j.ijpharm.2025.125234. hal-04919334

HAL Id: hal-04919334

https://hal.science/hal-04919334v1

Submitted on 29 Jan 2025

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# Therapeutic potential of siRNA PMP22-SQ nanoparticles for Charcot-Marie-Tooth 1A neuropathy in rodents and non-human primates

Suzan Boutary<sup>a,b</sup>, Guy Khalaf<sup>a</sup>, Yosef Landesman<sup>c</sup>, Mevidette el Madani<sup>a,d</sup>, Didier Desmaële<sup>e</sup>, Françoise Piguet<sup>f</sup>, Rafael Alonso<sup>f</sup>, Elena-Gaia Banchi<sup>f</sup>, David Adams<sup>a,g</sup>, Charbel Massaad<sup>b</sup>, Liliane Massaad-Massade<sup>a,\*</sup>

## ARTICLE INFO

### Keywords:

Small interfering RNA

Nanoparticles

Squalene

Charcot Marie Tooth 1A

Non-Human Primates

Biodistribution

Pharmacokinetics

Safety Profile

## ABSTRACT

Small interfering RNA (siRNA) has shown promising results for the treatment of Charcot-Marie-Tooth disease 1A (CMT1A) caused by overexpression of peripheral myelin protein (PMP22), leading to myelin dysfunction and axonal damage. Recently, we developed siRNA PMP22-squalene (SQ) nanoparticles (NPs) for intravenous use. Three consecutive injections of siRNA PMP22-SQ NPs at a cumulative dose of 1.5 mg/kg restored motor function in C61 transgenic mouse models. Pharmacokinetic studies showed a long half-life of antisense siRNA PMP22 in the sciatic nerve, and spinal cord, indicating targeted release potential. We further assessed the efficiency and safety of siRNA PMP22-SQ NPs in two healthy male non-human primates (Macaca fascicularis) after administering four escalating doses (0.1, 0.5, 2.5 and 4.5 mg/kg at one week interval). Interestingly, the siRNA PMP22-SQ NPs reduced PMP22 mRNA expression by approximately 70 % and probably induced an early-stage hereditary neuropathy with pressure palsies (HNPP)-like condition in two normal NHP. No preliminary toxicity was observed in organs or blood parameters of the two NHPs. Interestingly, the nerve conduction velocity decreased after the third injection of siRNA PMP22-SQ NPs. These results demonstrate the therapeutic potential of siRNA PMP22-SQ NPs, supporting advancement to further pre-clinical testing.

## 1. Introduction

Small interfering RNA (siRNA) has shown promising results for treating various diseases, including viral infections (Kang et al., 2023) and cancer (Zhang et al., 2023). Advances in bioconjugate chemistry and carrier technology have led to several siRNA-based drugs reaching treatments (Adams et al., 2018; Adams et al., 2021; Adams et al., 2023; Hu et al., 2020). Recently, we used the RNA interference approach for treating Charcot Marie tooth disease (CMT1A) (Boutary et al., 2021a). CMT1A is a hereditary motor and sensory neuropathy that leads to muscle weakness and sensory loss in the lower and upper limbs and affects more than 50 % of all patients with CMT (Saporta and Shy, 2013).

It is characterized by a 1.4 Mb tandem duplication on chromosome 17p11.2-p12, encompassing the peripheral myelin protein gene (PMP22) (Boutary et al., 2021b; Jerath and Shy, 2015; Lupski et al., 1991; Timmerman et al., 1992). This duplication results in 1.5-fold increased expression of PMP22, which is the primary underlying cause of the disease (Svaren et al., 2019; Vallat et al., 1996; Yoshikawa et al., 1994). Overexpression of PMP22 leads to repeated cycles of demyelination and remyelination, which leads to the formation of concentric layers of Schwann cell processes and collagen surrounding axons that are referred to as onion bulbs (Thomas, 1999). Supporting evidence for this mechanism comes from rodent models overexpressing PMP22, which exhibit a similar phenotype to patients with CMT1A (Fledrich

<footnote><sup>a</sup> Université Paris-Saclay, Inserm, Maladies et hormones du système nerveux, 94276 Le Kremlin-Bicêtre, France</footnote>

<footnote><sup>b</sup> Université Paris Cité, INSERM UMR_S 1124, 75006 Paris, France</footnote>

<footnote><sup>c</sup> NEXGEN BIOPHARMA SOLUTIONS INC, Brookline, Massachusetts, USA</footnote>

<footnote><sup>d</sup> National Research Centre, Cairo, Egypt</footnote>

<footnote><sup>e</sup> Institut Galien Paris-Sud, CNRS UMR 8612, Université Paris-Saclay, 17 avenue des Sciences, 91400 Orsay, France</footnote>

<footnote><sup>f</sup> Technological Innovation and Development Unit (TIDU) for Gene and Cell Therapy, TIDU GENOV, ICM, 47 boulevard de l'Hôpital, 75013 Paris, France</footnote>

<footnote><sup>g</sup> Neurology Department, AP-HP, Université Paris-Saclay and French Reference Center for Familial Amyloid Polyneuropathy and Other Rare Peripheral Neuropathies (CRMR-NNERF), Bicêtre University Hospital, Le Kremlin-Bicêtre, France</footnote>

<footnote>* Corresponding author at: Université Paris-Saclay, Inserm, Maladies et hormones du système nerveux, 94276 Le Kremlin-Bicêtre, France.
E-mail address: liliane.massade@inserm.fr (L. Massaad-Massade).</footnote>
et al., 2012; Huxley et al., 1996; Huxley et al., 1998; Sereda et al., 1996), including the C61-heterozygote CMT1A mouse model carrying four additional copies of the human PMP22 gene (Huxley et al., 1998).

Despite extensive research and numerous clinical studies, CMT1A remains an incurable neuropathy (Boutary et al., 2021b). Disease management primarily relies on rehabilitation therapy, corrective surgeries for deformities, and symptomatic medications (Pisciotta and Pareyson, 2023). The latest studies have shown a growing emphasis on silencing the PMP22 gene, particularly through the use of antisense oligonucleotides (Zhao et al., 2018), RNA interference (siRNA (Boutary et al., 2021a; Lee et al., 2017), shRNA (Gautier et al., 2021), miRNA (Stavrou et al., 2022)), and CRISPR/Cas9 editing (Lee et al., 2020). However, challenges remain in the systemic delivery of siRNA due to poor stability in circulation and unfavorable pharmacokinetics (PK) and biodistribution profiles.

To address these issues, we conjugated the siRNA PMP22 to squalene (SQ), a natural cholesterol precursor capable of forming nanoparticles (NPs) in water. Conjugation represents a promising methodology for delivering siRNA to tissues such as tumors (Ali et al., 2014b; Raouane et al., 2011; Urbinati et al., 2015) or sciatic nerve (Boutary et al., 2021a) without cytotoxic effects. A critical issue in developing siRNA nano-carriers is nanotoxicity, primarily due to their polycationic charges (Xue et al., 2014). We circumvented this problem by using squalene, a biocompatible and biodegradable lipid, conjugated to siRNA via copper-free click chemistry. The resulting NPs possess a slightly negative charge, with the potential to avoid toxicity. Additionally, we characterized the structure of siRNA-SQ NPs and their interactions with serum components to understand their assembly and fate after intravenous injection. The study revealed that siRNA-squalene nanoparticles self-assemble into stable 130-nm spherical structures and interact with serum components like albumin and low-density lipoprotein (LDL) (Caillaud et al., 2021).

In our previous study, locomotor recovery and muscle strength were achieved at a cumulative dose of 1.5 mg/kg in JP18xJY13 mice (Boutary et al., 2021a). We validated this dose in the C61 CMT1A mouse model, which shows mild demyelination and intermediate nerve conduction velocity (Huxley et al., 1998), mimicking the human CMT1A phenotype thus making them a more clinically relevant model for testing siRNA PMP22-SQ NPs. The pharmacokinetic profile was determined by analyzing nanoparticle biodistribution over time using stem loop RT qPCR to track siRNA PMP22-SQ antisense strand concentration in various tissues. This comprehensive evaluation allowed us to determine both the immediate safety and the distribution patterns of the nanoparticles, providing critical insights into their therapeutic potential for CMT1A.

To estimate the safety and target organs of siRNA PMP22-SQ NPs, we conducted acute toxicity testing in C57Bl/6 mice, a wild type model used as a standard practice in preclinical research (Prior et al., 2020). WT Mice received a single dose injection of 1.5 mg/kg of siRNA PMP22-SQ NPs and no acute toxicity was observed. Additionally, we evaluated the efficiency, biodistribution and safety profile of siRNA PMP22-SQ NPs in two healthy male non-human primates (Macaca fascicularis). We administered four escalating doses of these nanoparticles, simulating a condition resembling an early-stage hereditary neuropathy with pressure palsies (HNPP), and observed no toxicity even at high concentrations. Our study underscores the therapeutic potential of siRNA PMP22-SQ NPs and paves the way for advancing to a phase I clinical trial.

# 2. Methods

## 2.1. Preparation of siRNA PMP22-SQ and siRNA Ct-SQ NPs

siRNA PMP22 used in this study is of common sequence between homo sapiens, macaca fascicularis, mus musculus and Rattus norvegicus. siRNA Ct is a scrambled sequence. siRNAs were purchased from

Eurogentec France as a single stranded RNA. Both siRNAs were conjugated to squalene by Cu-free click chemistry, and nano precipitated as previously described (Massaad-Massade et al., 2018). Briefly, dibenzocyclooctyne (DBCO)-sense siRNA strand was added to N₃-SQ at a ratio of 1:25 in a solution of acetone and DMSO then incubated for 12 h with stirring. The solvent was eliminated by an evaporation step using nitrogen flux followed by a lyophilization step overnight. Excess squalene was removed by high-performance liquid chromatography (HPLC) purification. For the formation of the doubled stranded siRNA-SQ bi-conjugates, equimolar quantities of sense siRNA-SQ and antisense RNA were added to a hybridization buffer containing 30 mM HEPES-KOH (pH 7.4), 2mM Mg acetate, and 100mM K acetate. The solution was incubated for 3-minutes (min) at 95 °C, then for 45 min at room temperature (RT). Finally, siRNA PMP22-SQ NPs and siRNA-Ct NPs were prepared by adding the siRNA-SQ by drops over an organic phase (acetone) with stirring in a 1:2 vol-to-volume ratio of siRNA-SQ:acetone. Acetone was removed by evaporation under nitrogen flux and size and concentration of the NPs was measured by dynamic light scattering (DLS) machine and nanodrop, respectively.

## 2.2. Mice studies

### 2.2.1. Mice models

For this study, C57BL/6 mice (WT) and C61 mice carrying 4 extra copies of the human PMP22 gene were used (Fledrich et al., 2012; Huxley et al., 1998). The C61 CMT1A model was chosen to validate the siRNA PMP22-SQ treatment since its phenotype is comparable to human CMT1A disease showing mild demyelination and intermediate nerve conduction velocity. The number of animals used per group, is in accordance with the 3R rule that aims to reduce the use of animals in preclinical research. Administration of treatments was performed under isoflurane anesthesia to minimize animal suffering and animals were sacrificed by cervical dislocation. All animals were housed in sterilized laminar flow caging system. Food, water, and bedding were sterilized before animals were placed in the cages. Food and water were given *ad libitum*.

### 2.2.2. Validation of siRNA PMP22-SQ NPs on C61 mouse model

Eight-week-old C61 heterozygote male and female mice were divided into 3 groups (n = 5 mice/group) and compared to wild-type C57BL/6J mice (n = 5). Group 1 received 5 % dextrose, group 2 received 1.5 mg/kg cumulative dose of siRNA Ct-SQ NPs, and group 3 received 1.5 mg/kg cumulative dose of siRNA PMP22-SQ NPs. The treatment of the siRNA Ct-SQ NPs and siRNA PMP22-SQ NPs was administered intravenously, which included three iv injections of 0.5 mg/kg dose separated by an interval of 3 days (Fig. 1A). Behavioral tests and Compound muscle action potential test (CMAP) were performed before and after treatment for all mice. Mice were sacrificed 1 week after the last injection and sciatic and brachial nerves were taken for molecular analysis. serum was collected to analyze toxicity of the siRNA-SQ NPs on blood biomarkers. Regarding the study of 2.5 mg/kg siRNA PMP22-SQ NPS, eight-week-old C61 heterozygote male and female mice were divided into 3 groups: group 1 received 5 % dextrose (n = 2), group 2 received 2.5 mg/kg cumulative dose of siRNA Ct-SQ NPs (n = 3), and group 3 received 2.5 mg/kg cumulative dose of siRNA PMP22-SQ NPs. The treatment was administered intravenously, which included five iv injections of 0.5 mg/kg dose separated by an interval of 3 days (Supplementary Fig. 1B). Mice were sacrificed after the last injection and Sciatic nerve was collected for RT-qPCR analysis.

#### a. Behavioral Tests:

### 2.2.3. Beam walking test

To determine the effect of siRNA PMP22-SQ NPs on the gait of the C61 mice groups and WT mice, the beam walking test was performed as previously described (Boutary et al., 2021a). Briefly, the mice were trained to cross a platform with a rod of 3 cm diameter, 70 cm length
<figure><img src="image_4.png" /><figcaption>**Fig. 1. Efficiency of siRNA PMP22-SQ NPs treatment at the dose of 1.5 mg/kg in C61 CMT1A mouse model.** A Treatment protocol design. B and C Time (sec) to perform the beam walking test and the Locotronic® test respectively where the mice treated by siRNA PMP22-SQ NPs showed a significant decrease in the time taken by the mice to do both tests compared to other C61 mice groups and compared to the same mice before treatment. D and E are the grip strength test analysis on both all limbs and forelimbs of the different mice groups. F CMAP analysis of treated C61 mice groups compared to WT mice group. G. PMP22 mRNA expression on brachial nerves of dextrose 5 %, siRNA Ct-SQ NPs and siRNA PMP22-SQ NPs mice groups. H. western blot image and analysis of PMP22 protein expression on sciatic nerves of dextrose 5 %, siRNA Ct-SQ NPs and siRNA PMP22-SQ NPs mice groups. Data presented as mean ± SD. \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.0001 using ANOVA analysis followed by Tukey's multiple comparison tests to compare two groups. CMAP, compound muscle action potential; NPs, nanoparticles.</figcaption></figure>

and around 30 cm above a flat surface from one side to the other then the test was performed three times on each mouse and recorded by a camera. The time to cross the platform was analyzed for each mouse and each trial. The test was performed at two time points before and after the end of the treatment. Data are presented as mean ± standard deviation (SD), (n = 5 in each group).

### 2.2.4. Locotronic Test

A locotronic apparatus (Intellibio innovations A-1805-00049) was used to test the locomotion of C61 mice groups and WT mice. The time required to cross the horizontal ladder of the apparatus was recorded for each mouse as previously described (Boutary et al., 2021a). The test was repeated three times for each mouse before and after treatment and the

average time was recorded. Data are presented as mean ± SD, (n = 5 in each group).

### 2.2.5. Grip strength test

An automated grip strength meter (BIOSEB Innovation Model: BIO-GS3) was used to assess the muscular activity of the C61 mice groups and WT mice at different time points as previously described (Boutary et al., 2021a). The maximum g-force of the forelimbs and total limbs was recorded for each mouse at designated time points during treatment. Data are presented as mean ± SD, (n = 5 in each group).

#### b. Electrophysiological tests

For C61 mice, a compound muscle action potential test was performed using a standard EMG apparatus (Natus UltraPro S100 EMG) in
accordance with the guidelines of the American Association of Neuro-
muscular and Electrodiagnostic Medicine as previously described
(Boutary et al., 2021a). To measure the amplitude of the nerve signal,
the mouse was anesthetized using isoflurane and placed on its frontal
side on a heating pad to conserve its body temperature between 34 °C
and 36 °C. At the sciatic nerve notch level, the stimulator needle elec-
trode was positioned and the anode electrode was placed in the upper
base part of the tail, while at the gastrocnemius muscle level, the re-
ceptor needle was inserted in the medial part. Then, an electric signal (a
supramaximal square wave pulse) of 8 mA was delivered through the
stimulator needle and recorded by the receptor needle as an amplitude.
Data are recorded as mean ± SD.

c. mRNA extraction and real-time PCR (RT-qPCR)

Total RNA was extracted from total sciatic nerve and brachial nerves of C61 mice treated with siRNA Ct-SQ NPs and siRNA PMP22-SQ NPs using TRIzol/chloroform standard protocol. One µg of total RNA was used to generate the first-strand cDNA with M-MLV RT buffer pack (Invitrogen, Charbonnières-les-Bains, France). Then 50 ng of cDNA were used for RT-qPCR that was carried out using CFX96 BIORAD System with Maxima Syber Green Rox qPCR master Mix (Thermo Scientific, Villebon-sur-Yvette, France), according to the manufacturer's instructions. Human PMP22 gene expression was determined by 2⁻⁵ΔΔCt method and normalized to RPL13 mRNA levels. Relative mRNA PMP22 expressions of siRNA PMP22-SQ NPs treated C61 mice were compared to a siRNA Ct-SQ NPs treated mice and 5 % dextrose Ct mice.

d. Western Blot analysis

sciatic nerves from C61 mice treated groups with 5 % dextrose, siRNA CT-SQ NPs and siRNA PMP22-SQ NPs were used to obtain total protein using RIPA buffer (Sigma R0278) supplemented with a protease inhibitor cocktail (Roche, Neuilly sur Seine, France). Tissues were homogenized using Precellys 24® lysis and homogenizer (Bertin, Technologies, France) and then incubated for 2 h, under rotation at 4 °C for complete protein extraction. Samples were centrifuged at 13,000 rpm

for 20 min and supernatant containing total protein was stored at
-80 °C or quantified using BioRAD Assay according to the manufac-
turer's instructions.

5 µg of sciatic nerve proteins were loaded on a gradient poly-
acrylamide gel of 8–12 % (Invitrogen, Ref: NW04120) and transferred to
a nitrocellulose membrane using the iBlot Dry Blotting System (Invi-
trogen iBLOT 2, Cat# IB21001). Nitrocellulose Membranes were
blocked with 5 % BSA blocking solution for 1 h and incubated at 4 °C
overnight with the following primary antibodies: Rabbit Recombinant
Monoclonal PMP22 antibody (1:1000, abcam: ab270400) and mono-
clonal anti tubulin (1:10000, Cell signaling: mAb#3873). Blots were
then washed and incubated with the corresponding secondary anti-
rabbit and anti-mouse antibody conjugated to HRP (horseradish
peroxidase, 1:3000, Cell Signaling technology) for PMP22 and tubulin,
respectively. Bands were visualized by enhanced chemiluminescence
reagent (BioRAD, France) and quantified using image J software.

2.2.6. Acute toxicity study

Eight male and female C57Bl/6J mice, 2-months of age, received a single iv injection of 1.5 mg/kg of siRNA PMP22-SQ NPs. Body weights were recorded before treatment, 3 days after treatment and just prior to sacrifice (Fig. 2A). Mice were observed daily for any physical changes in appearance and sacrificed 7 days post-treatment. Blood was drawn directly from the heart of 4 injected mice followed by a heart perfusion using an antigen fix for fixation. The liver, kidney, spleen, lungs, sciatic nerve, heart and spinal cord were placed in an antigen fix solution for further histological analysis.

a. Serum biomarker analysis.

On the day of sacrifice, blood was collected directly from the heart of C57Bl/6J. Blood was allowed to rest at room temperature for 30 min and centrifuged at 2000 x g for 10 mins at 4 °C for serum collection. Serum samples were stored at -80 °C. Ten different biomarkers were quantified in the facilities of ENTEROSYS Prologue Biotech using Pentra 400

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Single iv injection of 1.5 mg/kg siRNA PMP22-SQ NPs shows no signs of acute toxicity in C57Bl/6 wt mice. A Protocol design. C57Bl/6 mice received a single iv dose of 1.5 mg/kg siRNA PMP22-SQ NPs and were compared to untreated C57Bl/6. Mice were sacrificed after 7-days. C Representative images of H&E staining of 5 µm sections of different organ including liver, kidney, heart, spleen, brain, spinal cord and sciatic nerve. Scale 200 µm.</figcaption></figure>
(HORIBA ABX, SAS). This included alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, HDL cholesterol, LDL cholesterol, total cholesterol, lactate dehydrogenase, albumin, bilirubin and creatinine.

b. Hematoxylin and eosin (H&E) staining.

At the time of sacrifice of the WT mice, kidney, liver, spleen, lungs and heart were harvested, fixed in antigen fix solution for 48 h, and then processed for paraffin embedding. Paraffin embedded tissues were shipped to EXCILONE (78990 Elancourt - France) for 5 µm sectioning and H&E staining of each tissue according to the standard protocol. Images were analyzed blindly by Pr. Clovis Adam, anatomo-cytopathologist at the Kremlin-Bicêtre Hospital.

## 2.3. Analysis of siPMP22 antisense biodistribution and pharmacokinetic using stem loop RT-qPCR (SL RT-qPCR)

Thirty-two age-matched male and female C61 mice were injected with a single iv dose of 1.5 mg/kg siRNA PMP22-SQ NPs and sacrificed at 0.083 h (5 min), 0.5 h (30 min), 1 h, 4 h, 6 h, 24 h, 48 h (n = 4/time point) and 72 h (n = 2/time point) post-injection (Supplementary Fig. 2A). Serum, kidney, liver, spleen, lungs, heart, sciatic nerve and spinal cord were collected. Biodistribution of siRNA PMP22 antisense was evaluated using the SL RT-qPCR method. Briefly, 50 mg of tissue sample was reconstituted in 1 ml of PBS containing 0.25 % Triton-X 100. Tissue suspension was homogenized using the Precellys Lysing Kit for Precellys homogenizer (cat # P000933-LYSK0-A). Homogenized samples were heated at 95 °C for 10 min then vortexed, and incubated on ice for 10 min. Samples were centrifuged at 20000 × g for 20 min at 4 °C and the supernatants were transferred to new vials. Next, SL RT reaction of siPMP22 antisense strand was performed using the TaqMan MicroRNA 200 kit (Cat # 4,366,596 Applied biosystems) followed by TaqMan qPCR as previously described (Chen et al., 2005; Landesman et al., 2010; Nair et al., 2017). The probes of SL RT and TaqMan qPCR primers/probes are listed in Table 1.

The same protocol described above was utilized for quantification of antisense siPMP22 in serum, samples following a 1:10 dilution in PBS containing 0.25 % Triton-X 100. Standard curves were plotted using a known concentration of siRNA PMP22-SQ NPs in serum (Supplementary Fig. 2B-C). The linear equation in slope-intercept form: y (CT) = -3,3758x + 15,739 was used to calculate the concentration in pg/mL in serum and pg/g in tissues.

The PK parameters were calculated by non-compartmental analysis methods from the serum and tissue concentration-time data via Phoenix WinNonlin (Version 8.3 or higher) by using guidelines listed in supplementary Table 1.

# 3. NHP studies

## 3.1. Non-human primate (NHP) model

*Macaca fascicularis* was used to study the efficacy and toxicity of siRNA PMP22-SQ NPs. *Macaca fascicularis* is a commonly used species for preclinical research that tolerates an administration protocol similar to a human clinical protocol (Ishigaki et al., 2018). siRNA PMP22-SQ NPs were administered weekly by intravenous (iv) injection under anesthesia (mixture of ketamine 3 mg/kg and dexdomitor 0.015 mg/kg). NHPs were housed in a temperature and humidity-controlled facility

with 12 h (h) light–dark cycle. Food and water were offered *ad libitum* except during the fasting experimental period. Two fruits per day and supplement of seeds or dry grapes were given. In addition, according to animal welfare, each NHP had access to enrichment in the cage, balls, scale, etc.

## 3.2. Efficacy and toxicity evaluation of siRNA PMP22-SQ NPs in NHPs

Two healthy 3-year-old male NHPs received four escalating iv doses of siRNA PMP22-SQ NPs separated by a week interval (D0: 0.1 mg/kg; D7: 0.5 mg/Kg; D14: 2.5 mg/Kg; D21: 4.5 mg/Kg) then sacrificed 72 h after the last injection (Fig. 3A). Clinical observations were performed daily. Body weights were recorded weekly under anesthesia. Blood samples were collected from the femoral artery at baseline and 72 h after each injection for blood parameter analysis. Nerve conduction velocity measurements of the sciatic nerve were recorded at D3, D10, D17 and D24 for NHP1 and at baseline D17 and D24 for NHP2. On the day of sacrifice, NHPs were euthanized via iv delivery of euthasol (Vetcare) 140 mg/kg followed by perfusion of 1L PBS with heparin and 500 ml of 4 % PFA. The sciatic nerve, median nerve, radial nerve, kidney, spleen, liver, lungs, heart, testis, quadriceps, mesenteric lymph nodes, spinal cord, dorsal root ganglia, Meningis and Brain were collected for further study.

a. Electrophysiological tests for NHP

For the NHP studies, an electromyogram (EMG) was performed using the Mag2Health apparatus. The animals were tested once at baseline, D3, D10, D17 and D24 for NHP1 and at baseline D17 and D24 for NHP2. Basically, NHP were anesthetized under ketamine / dexmedetomidine by intramuscular injection. NHP were monitor for vital parameters (SPO2 and cardiac parameters all along the experiments. Electrode placement was determined on the feet and legs after shaving and alcohol disinfection. Up to 10 supramaximal stimuli were taken and the average was reported for the sciatic nerve for maximal amplitude. To calculate the conduction velocity (m/s), the distance (cm) from the recording electrode to the stimulating cathode was recorded and NCV was calculated by the software.

b. Hematoxylin and eosin (H&E) staining for NHP

For the NHP study, one sample of each organ including kidney, spleen, liver, lungs, heart, testis, quadriceps, mesenteric lymph nodes, spinal cord, dorsal root ganglia, meninges and brain was collected and fixed in 4 % PFA 48 h at 4 °C, washed in PBS for 48 h at 4 °C, placed in 70 % ethanol at room temperature, embedded in paraffin and cut in 5 µm thickness. Two sections of 5–10 µm thickness of each organ were stained with H&E according to standard protocol and scanned using Hamamatsu imaging system General anatomical review was conducted for reporting.

c. Analysis of siPMP22 antisense biodistribution using stem loop RT-qPCR (SL RT qPCR) in NHP

For the NHP biodistribution study, the same protocol as mentioned before for C61 mice biodistribution was used to measure the antisense siRNA PMP22 in the organs (sciatic nerve, median nerve, radial nerve, kidney, spleen, liver, lungs, heart, spinal cord and dorsal root ganglia) and fluids (serum, plasma and cerebrospinal fluids) collected after 72 h of the last injection of siRNA PMP22-SQ NPs.

d. Serum biomarker analysis

Blood samples from the femoral artery of NHPs were collected at the following time points: baseline, D3, D10, D17 and D24 after injection.

<caption>**Table 1**</caption>

Stem loop, and PCR primers as well as TaqMan probe used to amplify the siRNA PMP22 antisense strand.

<table><thead><tr><td>Name</td><td>Sequence</td></tr></thead><tbody><tr><td>Stem Loop Primer</td><td>GTCGTATCCAGTCAGGGTCCGAGGTATTCGCACTGGATACGACAAATACCAAC</td></tr><tr><td>Forward Primer</td><td>GCCCTAGTCCACACAGTTGG</td></tr><tr><td>Reverse Primer</td><td>GTGCAGGGTCCGAGGT</td></tr><tr><td>TaqMan probe</td><td>(6-FAM)TGGATACGACAAATACCAAC</td></tr></tbody></table>
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Effect of siRNA PMP22-SQ NPs injection in non-human primates. **A** Experimental design. Two age-matched male NHPs (3 years-old) received four escalating doses of siRNA PMP22-SQ NPs separated by a 1-week interval (Dose 1: 0.1 mg/kg, Dose 2: 0.5 mg/kg, Dose 3: 2.5 mg/kg; Dose 4: 4.5 mg/kg). Blood samples were taken at baseline, and directly 15 min after injection or 3 days post-injection. **B** Body weight of NHPs was recorded prior to injections and throughout the experiment. **C** Latency (ms) of the nerve signal in the sciatic nerve of treated NHPs at baseline and 3 days post-injection. A delay in the latency of the nerve signal was recorded directly after the first injection. **D** NCV measurement on the sciatic nerve at baseline and 3 days post-injection. **E** CMAP measurements at the distal or proximal part or pooled together of the sciatic nerve at 3 base lines and 3 days post injection. **F** mRNA expression in the sciatic nerve of one control NHP and the two treated NHPs normalized to B-actin. NHP, non-human primate; NCV, nerve conduction velocity; ms, millisecond; D, day; Ct, control.</figcaption></figure>

Blood was stored at RT up to 2 h after sampling until processing. The blood parameters studied were platelets, red blood cells, hemoglobin, hematocrit, WBC, neutrophils, lymphocytes, eosinophils, basophils, reticulocytes, Ionogram (sodium, potassium, calcium and chlore), total protein, uric acid, total bilirubin, creatinine, aspartate aminotransferase, alanine transaminase, gamma-glutamyl transferase, cholesterol, triglycerides, C-reactive protein, albumin, troponin T, calcium, alkaline phosphatase, prothrombin, LDL, HDL and glucose.

### e. mRNA extraction and real-time PCR (RT-qPCR)

Total RNA was extracted from 50 mg of sciatic nerve tissues of NHPs using TRIzol/chloroform standard method. One µg of total RNA was used to generate the first-strand cDNA with M-MLV RT buffer pack (Invitrogen, Charbonnières-les-Bains, France). Fifty ng of cDNA were used for RT-qPCR that was carried out using CFX96 BIORAD System with Maxima Syber Green Rox qPCR master Mix (Thermo Scientific, Villebon-sur-Yvette, France), according to the manufacturer's instructions. PMP22 gene expression was determined by 2⁻⁰⁻⁰ΔΔCt method and normalized to β-actin levels. Relative mRNA PMP22 expressions of siRNA PMP22 treated NHPs were compared to a non-treated NHPs nerves samples that were available from another study.

### f. Sciatic nerve semithin and ultra-thin sections

NHP sciatic nerves (middle area) were post fixed in 4 % Paraformaldehyde and 5 % glutaraldehyde solution overnight at 4 °C, then rinsed with 0.1 M Cocylate Buffer and contrasted with Osmium 1 % for around 1 h at RT, then washed and contrasted with Uranyl Acetate 5 % for 45 min at RT and washed. The nerves were then dehydrated using increasing percentage of ethanol bath and embedded in Epon Medium.

After that Nerves were cut to generate semithin sections, which were further stained with toluidine blue, and scanned using a Hamamatsu scanner. Ultrathin sections of 70 nm were contrasted with uranyl acetate solution and lead citrate for transmission electron microscopy analysis.

## 4. Statistics

Statistics were computed with GraphPad Prism 10 software. Outliers were identified using Grubbs' test. For the comparison between different groups before and after treatment, a two-way ANOVA followed by Tukey's multiple comparison test was performed. For the comparison between different groups, one-way ANOVA followed by Tukey's multiple comparison test was performed. The Mann-Whitney test was applied when comparing two groups. A value of p < 0.05 was considered significant. Data are presented as mean ± SD.

### 4.1. Study approval

Experiments on C61 heterozygote CMT1A mice were approved by the institutional Ethics Committee of Animal Experimentation and Research Council. This study was registered in the French Ministry of Higher Education and Research, Ministère de l'Enseignement Supérieur et de la Recherche; MESR, authorization N°: APAFIS#10131-2016112916404689 and was carried out according to French laws and regulations under the conditions established by the European Community (Directive 2010/63/UE). For the experiments in NHPs, all animal procedures and experiments were approved by the
local ethical committee (CE Darwin) under the number #40958 and the French Ministry of National Education, Higher Education and Research.

# 5. Results

## 5.1. Efficacy of cumulative dosing of 1.5 mg/kg of siRNA PMP22-SQ in heterozygote C61 CMT1A transgenic mice

In our previous study, we administered a total of 2.5 mg/kg siRNA PMP22-SQ NPs to JP18/JY13 transgenic mice to evaluate its long-lasting therapeutic effect. Motor activity was completely restored starting from the third injection (cumulative dose: 1.5 mg/kg). Building on these findings, we tested the same cumulative dose (1.5 mg/kg via three injections of 0.5 mg/kg on days 0, 4, and 7) in C61 mice to assess if similar results could be achieved. Heterozygous C61 mice were divided into three groups: 5 % dextrose (vehicle, n = 5), siRNA Ct-SQ NPs (control, n = 5), siRNA PMP22-SQ NPs (n = 5), and wild-type (WT, n = 5) mice (Fig. 1A). By day 10, mice treated with siRNA PMP22-SQ NPs showed significant improvements in locomotor activity (beam walking and Locotronic® tests) and normalized muscular activity, supported by increased compound muscle action potential (CMAP) (Fig. 1, from B to F). This was accompanied by a reduction in human PMP22 mRNA expression in brachial nerves (Fig. 1G) and human PMP22 protein levels in sciatic nerves (Fig. 1H) compared to the siRNA Ct-SQ NPs group, indicating specificity to the siRNA PMP22-SQ NPs.

Therefore, the same protocol used in the JP18/JY13 mice (2.5 mg/kg via five injections separated by three days) was applied to C61 mice. Herein, a significant reduction in PMP22 mRNA expression was observed in siRNA PMP22-SQ NP-treated mice, compared to those treated with dextrose or with siRNA CT-SQ NPs (Supplementary Fig. 1C), confirming the findings from the JP18/JY13 model and supporting the efficacy of this therapeutic approach.

Next, we investigated whether there were any signs of systemic toxicity following treatment with siRNA PMP22-SQ NPs. No significant alterations in liver (alanine transaminase [ALT] or aspartate aminotransferase [AST]) or kidney biomarkers (albumin and creatinine) were detected (Table 2). Given the prediction of siRNA-SQ NP are transported in blood by LDL (Boutary et al., 2021a; Caillaud et al., 2021), it was important to also measure the blood lipid profile including total cholesterol, LDL and HDL. As such, a significant decrease in serum total cholesterol and HDL levels was identified in the siRNA-SQ treatment groups (Table 2).

## 5.2. siRNA PMP22 antisense concentration in serum and tissues of heterozygote C61 CMT1A mice

Serum concentrations of siRNA PMP22 antisense after a single dose injection of 1.5 mg/kg of siRNA PMP22-SQ NPs in C61 mice are

provided in Table 3. Individual serum and tissue concentrations of siRNA PMP22 antisense are listed in Supplementary Tables 2-3, respectively. Two of the four mice had an initial rapid drop after 0.083 h in serum concentrations of antisense siRNA PMP22 followed by a slow elimination. The other two mice showed an initial rise in serum concentrations to a peak at 0.5 h followed by a rapid decline then a slow terminal elimination. All tissues evaluated had measurable concentrations of antisense siRNA PMP22. In all tissues, the highest concentrations observed were measured 5 min after PMP22-SQ NPs injection (0.083 h), which then began to fall in a biphasic manner. There was a rapid distribution of antisense siRNA PMP22 to tissues followed by a slow elimination showing retention of siRNA PMP22 in tissues. The tissue with the highest concentrations was heart followed by liver and kidney. The remaining examined tissues, including sciatic nerve and spinal cord, were exposed to siRNA PMP22 but at lower concentrations.

## 5.3. siRNA PMP22 antisense serum and tissue pharmacokinetic parameters in heterozygote C61 CMT1A mice

Mean PK parameters for antisense siRNA PMP22 in serum and tissue following a single 1.5 mg/kg injection of siRNA PMP22-SQ NPs are shown in Table 3. Individual PK serum and tissue parameters for siRNA PMP22 antisense are shown in Supplementary Tables 4 and 5, respectively. The mean serum $C_{max}$, $AUC_{last}$ and $AUC_{inf}$ values for antisense siRNA PMP22 were 1242 pg/mL, 932 pg\*h/mL, and 934 pg\*h/mL, respectively. The mean $C_{max}$ occurred at a median $T_{max}$ of 0.291 h. As mentioned above two animals had $T_{max}$ values at the earliest sampling time of 0.083 h with the other two mice had $T_{max}$ values of 0.5 h. Following an initial rapid drop in concentrations the terminal half-life of siRNA PMP22 was long at 13.1 h. The clearance of siRNA PMP22 was rapid at 4112 L/h/kg with a high $V_{dss}$ of 11,344 L/kg.

The distribution of antisense siRNA PMP22 to tissue was rapid as peak concentrations were observed in all tissues at the first sampling time of 0.083 h. The highest siRNA PMP22 $C_{max}$ value was observed in heart followed by liver and kidney. Overall, siRNA PMP22 tissue have a rapid fall, which appears to decline slowly thereafter. The half-life of antisense siRNA PMP22 in sciatic nerve, and spinal cord was long ranging from 54.2 and 71.6 h respectively, which suggests siRNA was being retained in the central and peripheral nervous system. Moreover, siRNA PMP22 was also retained in highly perfused tissues with half-lives of 21.2, 61.8, 28.2, and 45.1 h in heart, kidney, liver, and lung, respectively. Although the terminal half-life estimates had some imprecision due to the limitation in the time points taken, as $R^2$ adjusted values were < 0.8 or the $AUC_{extrap}$ values > 25 %, measurable levels of siRNA PMP22 were observed out to at least 48 h supporting the retention of siRNA PMP22 in the examined tissues (Supplementary Table 5).

<table><caption>**Table 2**
Serum biomarker analyses of mice after 1.5 mg/kg siRNA PMP22-SQ NPs cumulative dose.</caption><thead><tr><th>Treatment Groups</th><th>ALP (U/L)</th><th>ALT (U/L)</th><th>AST (U/L)</th><th>LDH (U/L)</th><th>Albumin (µmol/L)</th><th>Bilirubin (µmol/L)</th><th>Total cholesterol (mmol/l)</th><th>HDL (mmol/L)</th><th>LDL (mmol/L)</th><th>Creatinine (µmol/L)</th></tr></thead><tbody><tr><td>WT (n = 6)</td><td>86.9 ± 19.9</td><td>28.8 ± 6.7</td><td>146.5 ± 34.2</td><td>994.2 ± 288.1</td><td>433.7 ± 26.3</td><td>4.4 ± 0.7</td><td>2.04 ± 0.06</td><td>1.5 ± 0.1</td><td>0.2 ± 0.1</td><td>12.5 ± 4.5</td></tr><tr><td>5 % Dextrose (n = 5)</td><td>87.3 ± 23.8</td><td>26.6 ± 3.2</td><td>106.2 ± 25.3</td><td>948.9 ± 346.4</td><td>409.8 ± 38.6</td><td>3.9 ± 0.5</td><td>2.45 ± 0.24**</td><td>1.8 ± 0.1*</td><td>0.2 ± 0.1</td><td>13.2 ± 3.0</td></tr><tr><td>siRNA CT-SQ NPs(n = 5)</td><td>85.7 ± 26.9</td><td>25.0 ± 3.4</td><td>104.4 ± 18.5</td><td>866.0 ± 392.9</td><td>419.2 ± 38.3</td><td>3.9 ± 0.3</td><td>2.02 ± 0.18##</td><td>1.5 ± 0.2</td><td>0.2 ± 0.1</td><td>14.4 ± 3.0</td></tr><tr><td>siRNA PMP22-SQ NPs (n = 5)</td><td>84.5 ± 26.6</td><td>26.4 ± 8.9</td><td>131.4 ± 33.8</td><td>859.0 ± 321.9</td><td>412.5 ± 32.1</td><td>4.2 ± 0.4</td><td>1.9 ± 0.12##</td><td>1.3 ± 0.1##</td><td>0.2 ± 0.1</td><td>13.8 ± 5.0</td></tr></tbody></table>

ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase; HDL, high-density lipoprotein; LDL, low-density lipoprotein; WT, wild type; NPs, nanoparticles; SQ, squalene.

* P < 0.05 wt and 5 % Dextrose group.

# P < 0.05 5 % dextrose group and siRNA Ct-SQ NP and siRNA PMP22-SQ NPs.
<caption>**Table 3**</caption>

Serum (pg/ml) and tissue (pg/g) concentrations for siRNA PMP22 antisense following a single iv injection of 1.5 mg/kg siRNA PMP22-SQ NPs.

<table><thead><tr><th>Time (h)</th><th>Serum (mean ± SD)</th><th>Liver (mean ± SD)</th><th>Kidney (mean ± SD)</th><th>Heart (mean ± SD)</th><th>Lung (mean ± SD)</th><th>Spleen (mean ± SD)</th><th>Spinal Cord (mean ± SD)</th><th>Sciatic Nerve (mean ± SD)</th></tr></thead><tbody><tr><td>0.083</td><td>753.25 ± 128.42</td><td>484.15 ± 598.00</td><td>221.13 ± 62.77</td><td>7699.51 ± 6799.97</td><td>85.06 ± 87.77</td><td>3.44 ± 3.59</td><td>110.15 ± 132.75</td><td>149.84 ± 79.08</td></tr><tr><td>0.5</td><td>849.33 ± 910.30</td><td>57.30 ± 35.01</td><td>13.12 ± 11.82</td><td>69.63 ± 39.70</td><td>26.76 ± 20.69</td><td>1.95 ± 1.31</td><td>4.97 ± 1.73</td><td>6.19 ± 4.73</td></tr><tr><td>1</td><td>255.86 ± 308.61</td><td>45.75 ± 42.49</td><td>27.34 ± 30.83</td><td>67.34 ± 68.83</td><td>4.48 ± 1.75</td><td>3.05 ± 1.04</td><td>7.56 ± 6.75</td><td>9.80 ± 9.30</td></tr><tr><td>4</td><td>11.87 ± 12.67</td><td>4.78 ± 3.67</td><td>3.19 ± 2.63</td><td>5.39 ± 4.62</td><td>3.24 ± 3.06</td><td>2.95 ± 0.57</td><td>2.06 ± 1.01</td><td>8.34 ± 8.86</td></tr><tr><td>6</td><td>6.98 ± 7.94</td><td>5.12 ± 4.03</td><td>2.72 ± 1.92</td><td>8.34 ± 8.11</td><td>4.82 ± 5.25</td><td>2.51 ± 0.58</td><td>2.94 ± 1.28</td><td>3.06 ± 1.50</td></tr><tr><td>24</td><td>1.37 ± 1.09</td><td>4.29 ± 3.72</td><td>2.21 ± 1.30</td><td>2.45 ± 1.94</td><td>1.46 ± 1.08</td><td>2.72 ± 0.53</td><td>1.98 ± 1.08</td><td>1.73 ± 0.45</td></tr><tr><td>48</td><td>1.05 ± 1.10</td><td>3.17 ± 2.48</td><td>1.99 ± 1.05</td><td>5.34 ± 4.59</td><td>1.21 ± 0.52</td><td>2.54 ± 0.90</td><td>2.66 ± 2.01</td><td>1.59 ± 0.43</td></tr><tr><td>72</td><td>0.18 ± 0.06</td><td>1.21 ± 0.51</td><td>0.89 ± 0.07</td><td>1.60 ± 0.59</td><td>1.03 ± 0.38</td><td>4.27 ± 1.49</td><td>0.00 ± 0.00</td><td>2.56 ± 1.78</td></tr></tbody></table>

## 5.4. Assessment of siRNA PMP22-SQ NP acute toxicity in C57Bl/6 mice

Given the rapid distribution of siRNA PMP22-SQ NPs to different organs, it was necessary to do a rapid screening to confirm the safety of the test dose (1.5 mg/kg) and to identify toxicity of target organs. For this purpose, a single iv injection of 1.5 mg/kg of siRNA PMP22-SQ NPs was administered to C57Bl/6 wt mice. Mice were observed daily for any change in their physical appearance or body weight for 7 days prior to sacrifice (Fig. 2A). No changes in physical appearance or fluctuations in body weight were detected (Table 5A). Additionally, no significant changes in any serum biomarkers measured were detected between treated mice compared to control mice, with the exception of a slight increase in the albumin levels (Table 5B). Moreover, no signs of inflammation or toxicity was observed in histological H&E-stained sections for all organs studied (Fig. 2B).

## 5.5. Effect of siRNA PMP22-SQ NPs in healthy non-human primates (NHP)

Next, we expanded our pre-clinical studies by evaluating the effect of siRNA PMP22-SQ NPs in healthy NHP. Two male NHPs received four escalating iv doses of siRNA PMP22-SQ NPs at one-week intervals (Fig. 3A). As shown in Fig. 3B, we did not observe any significant change in body weight of NHPs and also in their behavior at baseline and after treatment. However, a delay in the latency of the sciatic nerve signal was recorded (Fig. 3C) as well as a decrease in the nerve conduction velocity (NCV) that started to appear on day 17, three days following the third siRNA PMP22-SQ NPs injection (Fig. 3D). Regarding the CMAP analysis a decrease in the proximal CMAP levels were measured in both NHPs starting from D17 of the treatment. However this drop in CMAP level was only confirmed in the distal region of NHP 1 (Fig. 3E). Moreover, mRNA expression of PMP22 in the sciatic nerve was around 70% lower than that in control, untreated NHP (Fig. 3F). Furthermore, toluidine

blue staining of the sciatic nerve revealed improper folding of the myelin sheath of the NHPs treated with siRNA PMP22-SQ NPs as well as structural fragility (Fig. 4, A-B). This event was confirmed by transmission electron microscopy images showing the ultra-structure of the abnormal myelinated axons in both treated NHPs (Fig. 4C). Quantification of the percent of abnormal myelinated axons in sciatic nerves of PMP22-SQ NPs injected NHP demonstrated a 20% and 10% increase of abnormal myelinated axons for NHP1 and NHP2, respectively (Fig. 4D). G-Ratio analysis on TEM images showed alteration in the myelin sheath of one of the NHPs showing demyelination (Fig. 4E). Collectively, these results demonstrate the efficiency of siRNA PMP22-SQ NPs in NHP however a larger cohort study is required using different doses and period of treatment to confirm the positive effects of siRNA PMP22-SQ NPS.

## 5.6. siRNA PMP22 antisense concentration in fluids and tissues of healthy NHPs

Next, the biodistribution profile of siRNA PMP22-SQ NPs in serum, plasma and different organs of treated NHPs was evaluated. Antisense siRNA PMP22 was detected in high quantities in the serum samples directly taken after injection (D7, D14 and D21) and in low quantities 72 h later (Table 6), which suggests siRNA PMP22-SQ NP stability within serum and efficient distribution to different organ systems. Of note, antisense siRNA PMP22 was also detected in the plasma and cerebrospinal fluid samples taken 72 h after each injection (Table 6). The concentration of antisense siRNA PMP22 was also assessed in different organs of the NHPs 72 h after the last injection (Table 7). Interestingly, the antisense siRNA PMP22 was detected in all the organs tested but it was found to be more concentrated in the peripheral nerves (sciatic, median and radial nerves), spinal cord and dorsal root ganglia (Table 7). We also plotted PMP22 levels in respect to the amount of the antisens siRNA PMP22 found in sciatic nerve of each NHP. We showed that the

<table><caption>**Table 4**
Mean pharmacokinetic parameters for siRNA PMP22 in serum and tissues following a single iv injection of 1.5 mg/kg siRNA PMP22-SQ NPs.</caption><thead><tr><th>Tissue</th><th>Half-life (hour)</th><th>T<sub>max</sub>(hour)</th><th>C<sub>max</sub>(pg/mL or pg/g)</th><th>AUC<sub>last</sub>(pg*h/mL or pg*h/g)</th><th>AUC<sub>inf</sub>(pg*h/mL or pg*h/g)</th><th>Cl(L/h/kg)</th><th>Vss(L/kg)</th><th>T/S</th></tr></thead><tbody><tr><td>Serum</td><td>13.1</td><td>0.291</td><td>1242</td><td>932</td><td>934</td><td>4112</td><td>11,344</td><td>NA</td></tr><tr><td>Heart</td><td>21.2</td><td>0.083</td><td>7699</td><td>1424</td><td>1471</td><td>NA</td><td>NA</td><td>1.53</td></tr><tr><td>Kidney</td><td>61.8</td><td>0.083</td><td>221</td><td>202</td><td>292</td><td>NA</td><td>NA</td><td>0.217</td></tr><tr><td>Liver</td><td>28.2</td><td>0.083</td><td>488</td><td>390</td><td>439</td><td>NA</td><td>NA</td><td>0.418</td></tr><tr><td>Lung</td><td>45.1</td><td>0.083</td><td>85.1</td><td>141</td><td>206</td><td>NA</td><td>NA</td><td>0.151</td></tr><tr><td>Sciatic Nerve</td><td>71.6</td><td>0.083</td><td>150</td><td>161</td><td>345</td><td>NA</td><td>NA</td><td>0.173</td></tr><tr><td>Spinal Cord</td><td>54.2</td><td>0.083</td><td>110</td><td>139</td><td>440</td><td>NA</td><td>NA</td><td>0.150</td></tr><tr><td>Spleen</td><td>NC</td><td></td><td>4.49</td><td>163</td><td>NC</td><td>NA</td><td>NA</td><td>0.175</td></tr></tbody></table>

PK Parameters: AUClast: Area under plasma concentration-time curve from time 0 to last quantifiable concentration, AUCinf: Area under plasma concentration-time curve from time 0 to infinity, Cmax: Maximum observed concentration, Tmax: Time corresponding to occurrence of Cmax, Cl: Total body clearance, Vss: Volume of distribution at steady state, t1/2: Terminal half-life.

NA = not applicable; NC = not calculated as terminal phase could not be estimated.

Serum is reported as pg/mL and tissues as pg/g.

Tmax is reported as median.

T/S is tissue/serum ratio.
<table><caption>**Table 5**
Single iv injection of siPMP22-SQ NPs at 1.5 mg/kg in C57Bl/6 wt mice.</caption><thead><tr><th>A Days</th><th colspan="4">1</th><th colspan="2">3</th><th colspan="2">7</th></tr><tr><th>Body weight (g)</th><td colspan="4">22.22 ± 4.04</td><td colspan="2">22.42 ± 4.49</td><td colspan="2">22.87 ± 4.26</td></tr><tr><th>B Parameters</th><th>ALP<br>U/L</th><th>ALT<br>(U/<br>L)</th><th>AST<br>(U/<br>L)</th><th>LDH<br>(U/L)</th><th>Albumin<br>(µmol/L)</th><th>Bilirubin<br>(µmol/L)</th><th>Total<br>Cholesterol<br>(mmol/L)</th><th>HDL<br>(mmol/<br>L)</th><th>LDL<br>(mmol/<br>L)</th><th>Creatinine<br>(µmol/L)</th></tr></thead><tbody><tr><td>Untreated<br>(n=7)</td><td>76.6<br>± 23</td><td>36 ±<br>19</td><td>160<br>± 76</td><td>696±<br>148</td><td>351.5 ±<br>17.5</td><td>1.3± 0.8</td><td>1.63 ± 0.2</td><td>1.05 ±<br>0.15</td><td>0.11 ±<br>0.06</td><td>21 ± 5.5</td></tr><tr><td>siRNA PMP22-<br>SQ NPs (n =<br>4)</td><td>97.5<br>± 20</td><td>38 ±<br>12</td><td>152<br>± 57</td><td>582<br>±<br>113</td><td>391 ±<br>25.5*</td><td>2.1 ± 0.8</td><td>1.71 ± 0.3</td><td>1.13 ±<br>0.31</td><td>0.06 ±<br>0.03</td><td>23.8 ± 4.1</td></tr></tbody></table>

A. Body weights of mice recorded on days 1, 3 and 7.

B. Blood serum biomarkers. Data represent mean ± SD.

*p < 0.05 vs. untreated.

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Sciatic nerve structure of siRNA PMP22-SQ NPs treated NHPs. **A** and **B**. Representative images of toluidine blue sciatic nerve semi-thin sections of untreated NHP and two siRNA PMP22-SQ NPs treated NHP (NHP1 and NHP2). Scale bar 100 µm and 25 µm. Yellow arrows indicate tomaculas. **C**. Transmission electron microscopy (TEM) images of ultrathin sciatic nerve sections of NHPs. Scale bar 2 µm. **D** analysis of the abnormal myelinated axon in sciatic nerve done on ultra-thin sections showing a statistical increase between the NHP injected by escalating doses of siRNA PMP22-SQ NPs (20 % for NHP1 and 10 % for NHP2). **n** = the number of axons analyzed in each NHP. **G**. g-Ratio analysis of myelin thickness of the sciatic nerves ultra thin sections of NHP1, 2 and Control.</figcaption></figure>

ratio are quasi-identical in both NHP (0.04 for NHP1 and 0.05 for NHP2) Supplementary Fig. 3. Moreover, it is important to note that the anti-sense siRNA does not accumulate in the liver, heart or lungs but is retained in kidneys, its potential organ of elimination (Table 7).

## 5.7. Safety profile of antisense siRNA PMP22 in healthy NHPs

Next, we evaluated the preliminary safety profile of the siRNA PMP22-SQ NPs in serum and plasma samples collected 72 h after each injection. No changes were observed in the total blood cell counts of both NHPs, except for a reduction in red blood cells and hematocrit, which is likely due to the frequency of the 15 ml blood collections from

each NHP per week (Supplementary Fig. 4). The blood ionogram analysis did not show any variations between the baseline (before start of treatment) and D24 (day of sacrifice, Supplementary Fig. 5). Histological analysis by H&E staining of sections from different organs indicated an overall tolerance in all the organs evaluated, with no abnormal findings detected in either NHP (Fig. 5).

## 6. Discussion

The RNA interference concept was first introduced in 1998 (Fire et al., 1998) and has since garnered support as an innovative therapeutic approach for many disease states (Friedrich and Aigner, 2022). This led
<table><caption>**Table 6**
Concentration of antisense siRNA PMP22 NHPs in fluids after iv injection at escalating doses of siRNA PMP22-SQ NPs (0.1, 0.5, 2.5 and 4.5 mg/kg).</caption><thead><tr><th rowspan="2">Time-point</th><th colspan="3">NHP1 (pg/mL)</th><th colspan="3">NHP2 (pg/mL)</th></tr><tr><th>Serum</th><th>Plasma</th><th>CSF</th><th>Serum</th><th>Plasma</th><th>CSF</th></tr></thead><tbody><tr><td>Day 3: 3 days after injection at 0.1 mg/kg</td><td>1.63</td><td>1.7</td><td>5.02</td><td>2.78</td><td>3.67</td><td>11.18</td></tr><tr><td>Day 7: 15 min after injection at 0.5 mg/kg</td><td>2430</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>Day 10: 3 days after injection at 0.5 mg/kg</td><td>1.31</td><td>3.46</td><td>55.01</td><td>2.44</td><td>5.88</td><td>4.78</td></tr><tr><td>Day 14: 15 min after injection at 2.5 mg/kg</td><td>6968</td><td>NA</td><td>NA</td><td>127,171</td><td>NA</td><td>NA</td></tr><tr><td>Day 17: 3 days after injection at 2.5 mg/kg</td><td>1.02</td><td>1.77</td><td>NA</td><td>1.63</td><td>2.69</td><td>7.00</td></tr><tr><td>Day 21: 15 min after injection at 4.5 mg/kg</td><td>111,434</td><td>NA</td><td>NA</td><td>98,305</td><td>NA</td><td>NA</td></tr><tr><td>Day 24: 3 days after injection at 4.5 mg/kg</td><td>0.92</td><td>0.79</td><td>5.12</td><td>1.53</td><td>7.1</td><td>5.48</td></tr></tbody></table>

Using stem loop RT qPCR, anti-sense siRNA PMP22 concentration was measured in serum collected at 15 min and 72 h after each injection. For Plasma and cerebrospinal fluids, siRNA PMP22 was measured 72hr after each injection. The calculations of the quantity in pg/ml are based on a standard curve of known concentrations of siRNA PMP22-SQ NPs.
CSF: cerebrospinal fluid; NA: no blood sampling done.

<table><caption>**Table 7**
Concentrations of antisense siRNA PMP22 NHPs in tissues 72 h after iv injection at escalating doses of siRNA PMP22-SQ NPs (0.1, 0.5, 2.5 and 4.5 mg/kg).</caption><thead><tr><th>Organs</th><th>NHP 1 (pg/g)</th><th>NHP 2 (pg/g)</th></tr></thead><tbody><tr><td>Liver</td><td>5.13</td><td>3.18</td></tr><tr><td>Kidney</td><td>21.3</td><td>79.2</td></tr><tr><td>Heart</td><td>7.80</td><td>2.21</td></tr><tr><td>Lung</td><td>2.92</td><td>1.24</td></tr><tr><td>Spleen</td><td>2.94</td><td>4.16</td></tr><tr><td>Sciatic nerve</td><td>32.1</td><td>21.3</td></tr><tr><td>Median nerve</td><td>86.7</td><td>3.40</td></tr><tr><td>Radial nerve</td><td>15.1</td><td>5.39</td></tr><tr><td><strong>Spinal cord</strong></td><td></td><td></td></tr><tr><td>- cervical</td><td>4.14</td><td>10.8</td></tr><tr><td>- thoracic</td><td>26.5</td><td>8.31</td></tr><tr><td>- lumbar</td><td>8.03</td><td>9.00</td></tr><tr><td><strong>Dorsal root ganglia (DRG)</strong></td><td></td><td></td></tr><tr><td>- cervical</td><td>9.67</td><td>7.98</td></tr><tr><td>- thoracic</td><td>6.21</td><td>8.22</td></tr><tr><td>- lumbar</td><td>12.12</td><td>7.46</td></tr></tbody></table>

Using stem loop RT qPCR, anti-sense siRNA PMP22 concentration was measured in organs, at the time of sacrifice, 72 h after last injection of siRNA PMP22-SQ NPs. The calculations of the quantity in pg/g are based on a standard curve of known concentrations of siRNA PMP22-SQ NPs.

the US Food and Drug Administration and the European Medicines Agency to approve drugs based on siRNA for clinical use (Patisiran (Adams et al., 2018; Hoy, 2018) and Givosiran (Scott, 2020)). In our laboratory, we focus on delivering the siRNA to cancer tissues (Ali et al., 2014a; Urbinati et al., 2015; Urbinati et al., 2016) and to the peripheral nervous system (Boutary et al., 2021a) in the form of squalenoyl nanoparticles. Previously, we provided evidence that treatment with squalenoyl siRNA PMP22-SQ NPs led to a phenotypic improvement in two CMT1A mouse models. More specifically, siRNA PMP22-SQ NPs normalized the PMP22 protein expression and ameliorated both motor

and sensory activity of CMT1A mice (Boutary et al., 2021a). Here, we expanded our initial findings and examined the efficacy, pharmacokinetic profile and safety of the siRNA PMP22-SQ NPs in the heterozygote C61 mouse model of CMT1A at the active dose 1.5 mg/kg and in ascending doses in non-human primates.

Regarding the efficacy of siRNA PMP22-SQ NP treatment, our findings revealed that intravenous administration of siRNA PMP22-SQ NPs effectively reversed the pathological phenotype in heterozygote C61 mice. The outcomes from administering a cumulative 1.5 mg/kg dose to two-month age C61 mice are consistent with our previous findings in JP18/JY13 mice, where siRNA PMP22-SQ NPs restored functional activity after three doses of 0.5 mg/kg, demonstrating preclinical benefits at the same cumulative dose of 1.5 mg/kg (Boutary et al., 2021a).

We acknowledge, however, that the rapid timing of recovery observed in both studies may appear surprising, given the complexity of the cellular and molecular processes involved, including siRNA expression, silencing of PMP22, Schwann cell regulation, remyelination, and axonal repair. Interestingly, similar rapid initiation of recovery has been observed by Perea et al., where overexpression of PMP22 was halted by the administration of tetracycline, leading to prompt remyelination in JP18/JY13 mice (Perea et al., 2001). This supports the notion that interventions targeting PMP22 overexpression can rapidly trigger recovery processes. Nevertheless, further studies are required to fully elucidate the precise mechanisms underlying this rapid response and to validate these findings.

Efficacy studies in NHPs demonstrated an inhibition of 70 % in the PMP22 mRNA expression as well as an increase in the percentage of the abnormal axons in the NHP treated with siPMP22-SQ NPs thus likely inducing a phenotype akin to an early stage HNPP disease in healthy NHPs.

With respect to the biodistribution and pharmacokinetic parameters of siRNA PMP22, we provide evidence that the antisense siRNA PMP22 is distributed to different organs, including the spinal cord and sciatic nerve of both mice and NHPs. The lipid-rich composition of myelin (Jackman et al., 2009; Poitelon et al., 2020), notably high in cholesterol (Saher et al., 2009), likely facilitates the uptake of siRNA-SQ NPs by the myelin sheath of peripheral and central nervous system. This holds significance for the potential therapeutic application of siRNA PMP22-SQ NPs for neurodegenerative diseases. Moreover, even though the antisense siRNA PMP22 was found in the heart and liver of both mice and NHPs, there was no drug accumulation after 72 h or toxicity confirmed by bioanalytical and histological analysis. Furthermore, the likely efficient renal clearance of siRNA PMP22-SQ NPs did not impact creatinine and albumin levels.

In addition, we provide robust evidence supporting the safety profile of siRNA PMP22-SQ NPs with no indications of organ or blood toxicity observed in either mice or NHPs, even at the injection site. Moving forward, the siRNA component and squalene as the carrier are two crucial factors that must be taken into consideration when considering future clinical studies. While numerous studies have demonstrated the safety of siRNA (Adams et al., 2021; Adams et al., 2023; Pirillo and Catapano, 2022; Ray et al., 2023), the potential toxicity of squalene when conjugated to siRNA remains to be elucidated. The conjugation process utilizing copper (CU)-free click chemistry resulted in NPs carrying a slightly negative charge, a pivotal characteristic anticipated to mitigate potential toxicity concerns. As such, positively charged NPs have been shown to induce hepatotoxicity, inflammation, and immune responses in mice when compared to neutral or negatively charged NPs (Kedmi et al., 2010). This is an accordance with our findings and suggest that the utilization of our slightly negatively charged squalene-conjugated siRNA PMP22-SQ NPs have a safer profile. Furthermore, our current data expands upon our previous findings that siRNA-SQ NPs interact with serum LDL and albumin (Caillaud et al., 2021) and then is distributed to organs of interest. To our knowledge, no clinically tested SQ-NPs have been approved or reported. Alternative injection methods, such as intrathecal administration, could potentially reduce systemic
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Safety profile of sirna pmp22-sq nps in nhps. representative images of H&E staining on 5 µm sections of different organs of treated NHPs. Scale bar 500 µm or 250 µm depending on the organ size.</figcaption></figure>

exposure but are more invasive and less practical in most contexts.

Compared to approved siRNA therapies such as patisiran (Adams et al., 2018), which uses lipid nanoparticles (LNPs) for delivery, SQ-NPs may offer advantages in tissue specificity through LDL receptor-mediated uptake. Additionally, the efficacy of SQ-NPs has been demonstrated at doses comparable to or lower than those required for some LNP-based therapies. This highlights the potential of SQ-NPs for achieving targeted delivery with reduced systemic exposure.

## 7. Conclusion

The present study constitutes the first step in the preclinical development of siRNA PMP22-SQ NPs as a treatment for CMT1A disease. We demonstrate the efficacy, safety profile, and biodistribution of siRNA PMP22-SQ NPs in the treatment of CMT1A disease in rodent and likely in large animals. Future studies on NHPs will aim to determine the minimal effective dose by testing lower doses at different time points. These studies will provide critical data on the optimal dosing regimen, which is essential before moving forward to clinical studies. Collectively, our findings provide the foundation for a new approach to using squalenoyl siRNA NPs as a vehicle for the treatment for hereditary diseases of the nervous system.

## Funding source

This work was supported by the "SATT Paris Saclay", program

Maturation siCMT and by Fondation NRJ-Institut de France.

## CRediT authorship contribution statement

**Suzan Boutary:** Writing – original draft, Methodology, Investigation, Formal analysis, Conceptualization. **Guy Khalaf:** Writing – review & editing, Methodology, Investigation. **Yosef Landesman:** Writing – review & editing, Formal analysis. **Mevidette el Madani:** Writing – review & editing, Formal analysis, Data curation. **Didier Desmaële:** Writing – review & editing, Supervision, Investigation. **Françoise Piguet:** Writing – review & editing, Validation, Methodology, Formal analysis, Conceptualization. **Rafael Alonso:** Writing – review & editing, Methodology. **Elena-Gaia Banchi:** Writing – review & editing, Methodology. **David Adams:** Writing – review & editing, Supervision, Conceptualization. **Charbel Massaad:** Writing – review & editing, Supervision, Conceptualization. **Liliane Massaad-Massade:** Writing – review & editing, Validation, Supervision, Investigation, Funding acquisition.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Liliane Massade reports was provided by U1195 INSERM. Liliane Massade reports a relationship with Diseases and Hormones of the Nervous System that includes:. If there are other authors, they declare
that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Authors thank the team of the animal core facility of BioMedTech Facilities INSERM US36 | CNRS UMS2009 | Université Paris Cité. JetPub Scientific Communications LLC assisted the authors in the preparation of this manuscript.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijpharm.2025.125234.

Data availability

Data will be made available on request.

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