Next Article in Journal
Generation of Induced Pluripotent Stem Cells and Neuroepithelial Stem Cells from a Family with the Pathogenic Variant p.Q337X in Progranulin
Previous Article in Journal
Transcriptional Profiling of Common Carp: A Microarray-Based Framework for Aquaculture Research
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 26
Issue 23
10.3390/ijms262311241
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Preclinical Assessment in Transgenic NOD Mice of a Novel Immunotherapy for Type 1 Diabetes: Lipoplexes Down-Modulate the Murine C1858T Ptpn22 Variant In Vitro

by
Irene Mezzani
1,
Antonella Accardo
2,
Emanuele Bellacchio
3,
Luca Fais
1,
Carlo Diaferia
2 and
Alessandra Fierabracci
1,*
1
Bambino Gesù Children’s Hospital, Scientific Institute for Research, Hospitalization and Healthcare (IRCCS), 00146 Rome, Italy
2
Research Centre on Bioactive Peptides (CIRPeB), Department of Pharmacy, University of Naples Federico II, 80134 Naples, Italy
3
Molecular Genetics and Functional Genomics, Bambino Gesù Children’s Hospital, Scientific Institute for Research, Hospitalization and Healthcare (IRCCS), 00146 Rome, Italy
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2025, 26(23), 11241; https://doi.org/10.3390/ijms262311241
Submission received: 7 October 2025 / Revised: 17 November 2025 / Accepted: 19 November 2025 / Published: 21 November 2025
(This article belongs to the Special Issue New Insights into the Pathogenesis of Type 1 Diabetes)
Browse Figures
Review Reports Versions Notes

Abstract

The C1858T PTPN22 (R620W) variant has been implicated in the pathogenesis of several autoimmune disorders and represents a promising immunotherapeutic target for Type 1 diabetes. We have been implementing a novel immunotherapeutic approach based on the use of lipoplexes that deliver siRNA duplexes. The efficacy and safety of lipoplexes was previously demonstrated in vitro in halting variant expression in the peripheral blood of patients. Preclinical safety and efficacy must be ascertained in vivo in appropriate animal models before clinical investigations can be undertaken, according to regulatory authorities in Europe. In the light of the foregoing, this study aims to verify that lipoplexes against the murine Ptpn22-R619W, equivalent to the human PTPN22-R620W, could be used for animal experimentation. The murine fibroblast cell line L929 was transfected with the PF62-pLentiPtpn22-R619W plasmid. We designed specific siRNA duplexes for the Ptpn22-R619W allele and formulated them into cationic lipoplexes in order to halt variant expression in the transfected L929 cell line. Transfection of fibroblasts expressing R619W using lipoplexes resulted in efficient silencing at 100 pmol siRNA after 48 h post-transfection, reaching higher significant knockdown after 72 h. Lipoplexes efficiently suppress pathogenic Ptpn22 variant expression in vitro, supporting the feasibility of a pre-clinical platform for testing of in vivo lipoplexes in CRISPR-engineered NOD/ShiLtJ mice carrying the R619W mutation.

1. Introduction

Type 1 diabetes mellitus (T1D) and autoimmune thyroid disease (ATD) frequently coexist as autoimmune polyglandular syndrome type 3 variant (APS3v), sharing a pathogenic basis in the destruction of endocrine cells by autoreactive T-cells [1,2]. The worldwide incidence of T1D is rising, especially in children <5 years, and is often accompanied by ATD, underscoring the need for therapies that preserve residual β-cells and thyrocytes [3]. The substitutive administration of the deficient hormones, namely insulin and levo-thyroxine (L-T4), is the standard treatment, although this does not halt the autoimmune process and does not rescue the residual hormone-producing cells [4]. Identification of innovative therapeutic interventions, especially those aimed at preserving the residual cells, is of crucial importance in improving the expectation of quality of life in pediatric patients [5].
Recently, the potential pathophysiological role played by the PTPN22 C1858T mutation in several autoimmune disorders, including T1D and APS3v, has been demonstrated [6,7,8,9,10]. PTPN22 encodes the protein tyrosine-protein phosphatase non-receptor type 22 (also referred to as lymphoid phosphatase, Lyp), which is a T-cell antigen receptor (TCR) signaling negative regulator, acting in concert with C-Src kinase. In Lyp, the C1858T variant produces the replacement of arginine 620 with a tryptophan residue (R620W), which leads to a gain of function with paradoxical reduced T-cell activation, affecting both innate and adaptive immune responses [11,12,13,14].
In light of this fact, it may be a valid drug target for the treatment of T1D and APS3v patients. We therefore implemented a novel immunotherapy based on the use of lipoplexes delivering siRNA that cause variant-allele-selective inhibition instead of complete gene knockdown [15,16,17,18,19]. We have already demonstrated the efficacy and tolerability of lipoplexes in peripheral blood mononuclear cells (PBMC) of T1D patients harboring the variant PTPN22. Furthermore, we developed strategies for functionalization of lipoplexes to improve selective delivery to specific immunocytes, in particular, and to allow the possibility of exposing Fab of monoclonal antibodies [19] to achieve specific silencing in B lymphocytes and cytotoxic T cells. An alternative functionalization strategy to target several immunocytes in the peripheral blood was exploited using a high-affinity Siglec 10 sialoside mimetic (SAM, PEG-lipid-F9) [19,20,21]. The Siglec family of sialic-acid-binding proteins is primarily expressed on cells of the immune system, which mediate innate and adaptive responses [21] functioning in the self and non-self discrimination. Thus, these endocytic receptors became attractive immunotherapeutic targets [21]. We proved the internalization and low toxicity of Lipo-siRNAR620W-PEGF9 (LiposiRNA-PEGF9) in PBMC and their efficacy in halting variant mRNA expression, as well as their functional efficacy by rescuing IL-2 secretion in PBMC of C1858T PTPN22 T1D patients. Lipoplexes were functionalized with sialoside mimetic PEGF9 and optimized for in vivo preclinical delivery studies [22].
In view of the prospective use of lipoplexes as immunotherapy of T1D and APS3v, a fundamental step forward is to conduct preclinical studies in animal models. For this purpose, according to international regulations, the biodistribution of lipoplexes and their functionalized compounds must be assessed by injection of fluorescently labeled compounds in murine strains such as C57BL/6 or BALB/c mice, commonly used for this kind of evaluation [23] as well as safety/efficacy in delaying or blocking the development of the disease. The NOD/ShiLtJ mouse (non-obese diabetic mice), a genetically predisposed strain for the development of autoimmune diabetes, closely mimicking the human model, appears to be ideal for preclinical studies to further investigate the influence of the R620W gene variant that causes the human disease, and to evaluate the efficacy of the novel pharmacological treatment [24,25,26]. In particular, phenotyping of the NOD model confirmed a 90% incidence of diabetic females by 30 weeks of age. The ideal NOD model carrying the R619W-Ptpn22 variant, equivalent to the human R620W, was generated using CRISPR-Cas9 technology [27]. This transgenic animal exhibits higher levels of anti-insulin antibodies, an earlier onset of the disease, and a higher prevalence in females.
In unraveling the feasibility of undertaking the proposed line of experimentation to ascertain the efficacy and safety of lipoplexes in the NOD transgenic mouse, we first aimed to effectively demonstrate the possibility of inhibiting the murine variant in vitro by using lipoplexes carrying the respective siRNA duplexes in the R619W-transfected L929 murine fibroblast cell line.

2. Results

2.1. Transfection of the R619W-Ptpn22 Variant in the L929 Mouse Fibroblast Cell Line

The mouse L929 cell line (ATCC® CCL-1™, American Type Culture Collection, Manassas, VA, USA) was transfected with the PF62-pLentiPtpn22-R619W plasmid (Aurogene S.r.l., Rome, Italy).
The quantitative Real-Time PCR (qRT-PCR) results of transfection with the PF62-pLentiPtpn22-R619W plasmid versus empty vector (EV) are shown in the table below (Table 1).

2.2. siRNAs Are Delivered in the R619W-L929 Cell Line by a Commercial Transfection System and Efficiently Block Variant Ptpn22 Expression

In the experiment performed using the transfection kit to convey a siRNA sense/antisense (s/a) duplex with Lipofectamine (Lipofectamine™ RNAiMAX, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), which has a higher affinity for the target mRNA sequence, after 48 h of transfection, we observed a maximum inhibition of R619W mRNA in the range between 10 and 80 pmols of siRNA compared to untreated control cells. After 72 h from the beginning of the transfection, a higher degree of inhibition persisted at a 40 pmol concentration of siRNA duplex (Figure 1A,B).
These results highlight the specificity of the siRNA duplex, selected for the subsequent experiments of lipoplex variant inhibition reported below (vide infra).

2.3. Efficient Silencing of Variant Ptpn22 in R619W-L929 Cells by siRNA Delivery Using Lipoplexes

Lipoplexes efficiently block variant Ptpn22 expression in R619W-transfected L929 cells.
Within the range of siRNA doses between 60 and 100 pmols, the highest inhibition of variant mRNA was obtained with 100 pmol of siRNA after 48 h of transfection (Figure 2A). The efficiency increased after 72 h from the beginning of transfection and was shown even at the lower doses of 60 and 80 pmols of siRNA (Figure 2B).

3. Discussion

According to regulatory authorities in Europe (EMA, European Medicines Agency) and the US (FDA, Food and Drug Administration), the interpretation of results from animal studies is necessary to support the future clinical application of novel therapeutic modalities. Indeed, preclinical safety and efficacy assessment of new medicinal products ensures that benefits will outweigh the risks associated with their use [23].
Nevertheless, the main challenge in developing a proof of concept for advancing to a clinical trial is, in most circumstances, the unavailability of appropriate animal models with similar functional characteristics to the intended human disorder.
A current line of investigation in our laboratory is to exploit the feasibility of the novel immunotherapy for T1D and APS3v based on the use of lipoplexes targeting the C1858T PTPN22 variant [16,17,18,19]. In light of the arguments raised (vide supra), preclinical assessment is requested at this stage of the investigation to ascertain the safety and efficacy of lipoplexes in the ideal NOD mice model made transgenic for the variant R619W-Ptpn22 through CRISPR-Cas9 technology [27]. For this purpose, it was necessary to have confirmation in preliminary settings of the possibility of blocking the variant R619W of Ptpn22 mRNA in the transgenic L929 fibroblast cell line using lipoplexes carrying the appropriate siRNA duplex. The murine line was transfected with the PF62-pLentiPtpn22-R619W plasmid vector (which is commercially available). Lipoplexes carrying siRNA duplexes against the variant efficiently reduced variant R619W-Ptpn22 expression, as revealed using qRT-PCR, although with less efficacy than with the standard Lipofectamine system used as a control. Indeed, lipoplexes produced a significant inhibitory effect on variant mRNA expression at higher doses (80–100 pmols of siRNA concentration) and with a longer time interval for internalization than with the Lipofectamine transfection control. The lower silencing efficiency observed with lipoplexes compared to Lipofectamine can be determined by several factors, such as cellular uptake and intracellular processing mechanisms. Lipofectamine-based systems are optimized commercial reagents that promote efficient siRNA complexation, membrane fusion, and endosomal escape, resulting in higher cytoplasmic release of siRNA molecules. In contrast, lipoplex formulations may exhibit lower stability and less efficient endosomal release. Moreover, differences in lipid composition and charge ratio can influence the kinetics of complex disassembly and siRNA release within the cytoplasm [28].
These results encourage us to proceed with assessment through in vivo animal experimentation on lipoplexes, the results of which will possibly allow future advancement to Phase I/II clinical trials of T1D immunotherapy in adults, which are necessary before a pediatric investigation plan can be implemented.

4. Materials and Methods

4.1. siRNA Design

Authentic siRNA sequences were designed (Design ID: ABHSP52) to specifically target the C1858T Ptpn22 murine gene variant (Life Technologies, Thermo Fisher, Milan, Italy).

4.2. Liposome/Lipoplex Preparation

Phospholipids [1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), N-[1-(2,3-Dioleoyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTAP), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000-Maleimide)] were acquired from Avanti Polar Lipids (Alabaster, Alabama). Liposomal solutions were assembled in 100 mM phosphate buffer (PBS, Euroclone, Milan, Italy) at a pH of 7.4. DOTAP/DOPE/DSPEPEG2000-Maleimeide (47.5/47.5/5 molar ratio) liposomes were formulated using the lipid film method as previously described [18]. Briefly, phospholipids previously dissolved in chloroform were mixed, and the organic solvent was evaporated under a nitrogen stream to obtain a homogeneous film. The film was subsequently hydrated with 1.0 mL of 100 mM PBS (pH = 7.4) to achieve a final concentration of 800 μM. The suspension was sonicated for 30 min (min) and successively extruded 10 times at room temperature (RT), using a thermo barrel extruder system (Northern Lipids Inc., Vancouver, BC, Canada) under nitrogen through a polycarbonate membrane (Nucleopore Track Membrane, 25 mm, Whatman, Brentford, UK) with a pore size of 0.1 μm. Then, 500 μL of a siRNA solution (5.2 μM in water) was mixed with an equivalent volume of liposomes, and the resulting solution was stirred at RT for 3 h.
Lipoplexes were generated by mixing an equal volume of the liposomal solution and 2.6 μM siRNA at RT for 3 h. The resulting solution contains lipoplexes in which both the liposomal and siRNA concentrations are halved. The mean diameter of lipoplexes (150 nm) did not show differences with respect to the formulation previously prepared by us using the same liposomal composition and the human variant of the siRNA [19] (Figure 3).

4.3. Generation of R619W-Transfected L929 Murine Fibroblast Cell Line

The L929 mouse line was transfected with the R619W mouse variant using the PF62-pLentiPtpn22-R619W plasmid available from Aurogene S.r.l. (Rome, Italy).

4.4. L929 Cell Culture

R619W-Ptpn22-transfected L929 cells (ATCC® CCL-1™, American Type Culture Collection, Manassas, VA, USA) were cultured at a density of 2 × 105 cells/mL in T75 flasks (Falcon Labware Becton Dickinson (BD) Biosciences, Oxnard, CA, USA). Cells were cultured in complete Dulbecco’s Modified Eagle Medium high-glucose medium (DMEM HG, Euroclone, Milan, Italy) supplemented with 10% fetal bovine serum (FBS) (Hyclone, South Logan, UT, USA), L-glutamine (2 mM, Euroclone, Milan, Italy), and 1% penicillin/streptomycin (pen/strep) (Euroclone), incubated at 37 °C in a 5% CO2 humidified atmosphere, and sub-cultured twice per week at the same seeding density.

4.5. Mutant Ptpn22-Gene Silencing in a Transfected L929 Fibroblast Line (R619W-L929)

4.5.1. siRNA Transfection Using a Commercial Transfection System

Selected siRNA sense/antisense (s/a) duplexes (R619W-Forward: 5′-TCCCCTCCGAATAGTGCTGA-3′-R619W-Reverse: 5′-CATTCAGGGAGTGGCGG-3′) with R619W mRNA target affinity were first validated by measuring their capability to silence L929 cells using a commercial transfection kit (Lipofectamine™ RNAiMAX, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and observing the resulting decrease in mRNA levels through qRT-PCR.
Briefly, on the first day, L929 cells were rescued from T75 culture flasks using a trypsin/EDTA solution (Euroclone), washed in PBS (Euroclone) and seeded at 1.5 × 105 per well in 12-well plates (Falcon, Corning Incorporated, Corning, NY, USA) in 1 mL of DMEM HG (Euroclone) supplemented only with 1% pen/strep and L-glutamine (2 mM) and then maintained for 24 h at 37 °C in a 5% CO2 humidified atmosphere.
On day 2, cells were washed with PBS (Euroclone) and then treated with different concentrations of R619W-Ptpn22 siRNA duplex (10, 40, 80 pmols of siRNA) in transfection medium (Gibco, Thermo Fisher Scientific) and incubated overnight (O/N) at 37 °C in a 5% CO2 humidified atmosphere. To evaluate siRNA internalization efficiency, cells were incubated using the fluorescent (Alexa Fluor 555-conjugated) siRNA provided by the manufacturer.
On day 3, complete DMEM HG medium (Euroclone) supplemented with 20% FBS and 2% pen/strep was added to stop the O/N incubation. The control group was treated with the fluorescent siRNA and subsequently analyzed by flow cytometry using a FACS Canto II instrument (Becton and Dickinson, Sunnyvale, CA, USA) and PC FACS Diva software (BD Biosciences, San Jose, CA, USA).
On day 4 and day 5, after 48 and 72 h, respectively, from the beginning of each transfection with different concentrations of R619W-Ptpn22 siRNA duplex, cells were harvested, washed with PBS, pelleted at 500× g for 5 min, and then lysed in RLT buffer (Qiagen, Hilden, Germany) for subsequent RNA extraction. Each experimental condition was assessed in two independent replicates.

4.5.2. Custom Liposome Transfection Protocol of R619W-L929 Fibroblast Line

On the first day, L929 cells were harvested from T75 culture flasks using a trypsin/EDTA solution (Euroclone) and cultured at 0.7 × 105 cells per well in 24-well plates (Falcon) in 500 μL of DMEM HG (Euroclone) enriched with 10% FBS, 1% pen/strep and L-glutamine (2 mM) for 24 h at 37 °C in a 5% CO2 humidified atmosphere.
On day 2, cells were first rinsed with PBS (Euroclone) and then cultured using 500 μL of the same FBS and pen/strep-free medium, containing different amounts (60, 80, 100 pmols) of siRNA duplex in relation to the Ptpn22 variant (vide supra), assembled as lipoplexes. Cells were incubated O/N with the complexes at 37 °C in a 5% CO2 humidified atmosphere. Appropriate controls were set up by growing cells using complete DMEM HG (Euroclone) medium alone. Each experimental condition included at least three independent determinations (triplicates). At the end of the incubation period, cells were harvested from the cell culture plates, washed in PBS by centrifugation at 500× g for 5 min, and cultured in complete DMEM HG (Euroclone) at a final volume of 1 mL.
On day 4 and on day 5, after 48 and 72 h, respectively, from the beginning of each transfection, cells were washed in PBS and then lysed in RLT buffer (Qiagen) for subsequent RNA extraction.

4.5.3. qRT-PCR

Relative gene expression levels were assessed by quantitative Real-Time PCR (qRT-PCR). Cells were lysed in RLT-Buffer at 48 and 72 h post-treatment. After RNA extraction (RNeasy Plus Mini Kit, Qiagen), quantification was performed as instructed by the manufacturer by spectrophotometry (Nanodrop 2000, Thermo Fisher Scientific, Waltham, MA, USA), followed by reverse transcription (SuperScript™ IV First-Strand Synthesis System, Thermo Fisher Scientific) and qRT-PCR amplification (PowerUp™ SYBR™ Green Master Mix, Thermo Fisher Scientific) employing the primers listed below to amplify the murine R619W-Ptpn22 variant:
  • R619W forward (R619W-forward): 5′-TCCCCTCCGAATAGTGCTGA-3′;
  • R619W reverse (R619W-reverse): 5′-CATTCAGGGAGTGGCGG-3′.
Murine GAPDH was used as the internal housekeeping control. The following primers were used:
  • GAPDH forward (GAPDH-FW): 5′-ATTCAACGGCACAGTCAAGG-3′;
  • GAPDH reverse (GAPDH-RV): 5′-ATCATAAACATGGGGGCATC-3′.
Relative expression levels were calculated using the 2–ΔΔCt method.

4.6. Statistical Analysis

Statistical analyses were performed using GraphPad Prism 10.0 (GraphPad Software, San Diego, CA, USA). One-way analysis of variance (ANOVA), followed by Šidák’s post hoc test for multiple comparisons, was used to identify significant differences among groups. Statistical significance was defined as a * p ≤ 0.05. Results are expressed as means ± standard deviation (SD) in Supplementary Table S1 and % of inhibition in Supplementary Figure S1.

5. Conclusions

Allele-specific inhibition via siRNA-containing lipoplexes represents a promising strategy for selectively silencing the PTPN22 genetic mutation C1858T in immunocytes implicated in the pathogenesis of APS3v/T1D.
In vitro results shown in this manuscript confirm the possibility of targeting the pathogenic (R619W-Ptpn22) allele in CRISPR-modified NOD/ShiLtJ mice, which harbors the variant, paving the way for preclinical translation. Experimental design for safety and efficacy studies will include the use of this knocking mice model (https://www.jax.org/strain/036948 (accessed on 30 September 2025) available from Jackson Laboratories (Bar Arbor, ME, USA) [27]. Heterozygous transgenic mice, generated from two couples breeding, will be treated with lipoplexes by intravenous injection in the lateral tail vein. Animals will be monitored daily for health status assessment, weighed twice a week for the overall 7-month time course of the experimental procedure. Once a week, a blood sample will be taken from the mandibular plexus [29] to measure blood glucose. Glycosuria will be tested once a week until diabetes onset, then once a month toward the end of the experimental procedure. Lipoplexes functionalized with Fab anti-murine CD3 will also be developed and tested in the animal model in parallel experiments [18]. Biodistribution of lipoplexes will also be estimated in the Balb/c mice strain by injection, through the tail lateral vein, of lipoplexes and anti-CD3Fab functionalized lipoplexes to specifically target T lymphocytes, both labeled with ATTO647NDOPE (ATTOTEC, Siegen, Germany). Mice will be sacrificed after 48 h post-injection, and PBMC will be taken and cryopreserved. Lymphoid and non-lymphoid organs, including lymph nodes, thymus, spleen, pancreas, liver, and heart, will be snap-frozen and embedded in OCT (Optimal cutting temperature) compound. At different times within 48 h post-injection, IVIS (in vivo imaging system) will be applied to monitor lipoplexes’ biodistribution. These findings provide a solid basis for future biodistribution, pharmacokinetic, and efficacy studies as required by regulatory authorities in Europe before clinical investigations can be undertaken.

6. Patents

Italian Patent 102018000005182 issued on 26.05.2020; PCT/IT2019/050095 filed on 8.05.2019, extended in Europe, USA, and Hong Kong, issued in USA on 9 September 2025. Inventor: Alessandra Fierabracci, MD, PhD.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms262311241/s1.

Author Contributions

Conceptualization, A.F.; methodology, A.F., I.M., A.A., and C.D.; software, I.M. and E.B.; validation, A.A. and A.F.; formal analysis, I.M., E.B., and A.F.; investigation, I.M., A.A., L.F., C.D., and A.F.; resources, A.A.; data curation, I.M., L.F., and A.A.; writing—original draft preparation, I.M., A.A., and A.F.; writing—review and editing, I.M., C.D., and A.F.; visualization, E.B.; supervision, A.F.; project administration, A.F.; funding acquisition, A.F. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Italian Ministry of Health through applied research (Ricerca Finalizzata) funding under Grant RF-2019-12369889 assigned to Alessandra Fierabracci, and current research funds.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Acknowledgments

We acknowledge Anna Lo Russo and Alessia Palma for technical assistance in cell culture and molecular biology experiments. Carlo Diaferia acknowledges grant CN00000041 from the National Center for Gene Therapy and Drugs based on RNA Technology (concession number 1035 of 17 June 2022-PNRR MUR-M4C2—Investment 1.4 Call “National Centers”, financed by EU—NextGenerationEU), project code MUR:CN00000041–CUP UNINA: E63C22000940007.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
Ptpn22Protein tyrosine phosphatase N22
LypLymphoid phosphatase
NODNon-Obese Diabetic mice
siRNASmall interfering RNA
T1DType 1 Diabetes
ATDAutoimmune Thyroid Disease
CtCycle Threshold
APS3vAutoimmune Polyglandular Syndrome type 3 variant
L-T4Levothyroxine
ArgArginine
TrpTryptophan
SAMSiglec 10 sialoside mimetic
CRISPR-Cas9Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated protein 9 (Cas9)
RISCRNA-Induced Silencing Complex
s/asense/antisense
EMAEuropean Medicines Agency
FDAFood and Drug Administration
DOPEPhospholipids [1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine
DOTAPN-[1-(2,3-Dioleoyloxy) propyl]-N, N, N-trimethylammonium chloride
DSPE-PEG2000-Maleimide1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide (polyethylene glycol)-2000] (ammonium salt)
Min.Minutes
ATCCAmerican Type Culture Collection
DMEM HGDulbecco’s Modified Eagle Medium High Glucose
%Percentage
OCTOptimal cutting temperature
IVISIn vivo imaging system

References

  1. Redondo, M.J.; Morgan, N.G. Heterogeneity and endotypes in type 1 diabetes mellitus. Nat. Rev. Endocrinol. 2023, 9, 542–554. [Google Scholar] [CrossRef]
  2. Dittmar, M.; Kahaly, G.J. Genetics of the autoimmune polyglandular syndrome type 3 variant. Thyroid 2010, 20, 737–743. [Google Scholar] [CrossRef] [PubMed]
  3. Norris, J.M.; Johnson, R.K.; Stene, L.C. Type 1 diabetes-early life origins and changing epidemiology. Lancet Diabetes Endocrinol. 2020, 8, 226–238. [Google Scholar] [CrossRef] [PubMed]
  4. Rapini, N.; Schiaffini, R.; Fierabracci, A. Immunotherapeutic Strategies for the Prevention and Treatment of Distinct Stages of Type 1 Diabetes: An Overview. Int. J. Mol. Sci. 2020, 21, 2103. [Google Scholar] [CrossRef]
  5. Woittiez, N.J.C.; Roep, B.O. Impact of disease heterogeneity on treatment efficacy of immunotherapy in Type 1 diabetes: Different shades of gray. Immunotherapy 2015, 7, 163–174. [Google Scholar] [CrossRef] [PubMed]
  6. Gianchecchi, E.; Palombi, M.; Fierabracci, A. The putative role of the C1858T polymorphism of protein tyrosine phosphatase PTPN22 gene in autoimmunity. Autoimmun. Rev. 2013, 12, 717–725. [Google Scholar] [CrossRef]
  7. Su, Y.; Li, X.; Wu, P.-D.; Zhang, Y.-L.; Fang, P.-F.; Wu, F.-F.; He, X.-F. The Association between PTPN22 SNPs and Susceptibility to Type 1 Diabetes: An Updated Meta-Analysis. PLoS ONE 2025, 20, e0321624. [Google Scholar] [CrossRef]
  8. Žak, R.; Navasardyan, L.; Hunák, J.; Martinů, J.; Heneberg, P. PTPN22 intron polymorphism rs1310182 (c.2054-852T>C) is associated with type 1 diabetes mellitus in patients of Armenian descent. PLoS ONE 2023, 18, e0286743. [Google Scholar] [CrossRef]
  9. Fichna, M.; Małecki, P.P.; Żurawek, M.; Furman, K.; Gębarski, B.; Fichna, P.; Ruchała, M. Genetic variants and risk of endocrine autoimmunity in relatives of patients with Addison’s disease. Endocr. Connect. 2023, 12, e230008. [Google Scholar] [CrossRef]
  10. Pasha, U.; Nisar, H.; Nisar, H.; Abid, R.; Ashraf, N.M.; Sadaf, S. Molecular dynamic simulations unravel the underlying impact of missense mutation in autoimmunity gene PTPN22 on predisposition to rheumatoid arthritis. J. Interferon Cytokine Res. 2023, 43, 121–132. [Google Scholar] [CrossRef]
  11. Vang, T.; Congia, M.; Macis, M.D.; Musumeci, L.; Orrú, V.; Zavattari, P.; Nika, K.; Tautz, L.; Taskén, K.; Cucca, F.; et al. Autoimmune-associated lymphoid tyrosine phosphatase is a gain-of-function variant. Nat. Genet. 2005, 37, 1317–1319. [Google Scholar] [CrossRef]
  12. Anderson, W.; Barahmand-Pour-Whitman, F.; Linsley, P.S.; Cerosaletti, K.; Buckner, J.H.; Rawlings, D.J. PTPN22 R620W gene editing in T cells enhances low-avidity TCR responses. eLife 2023, 12, e81577. [Google Scholar] [CrossRef]
  13. Belcastro, E.; Cudini, A.; Mezzani, I.; Petrini, S.; D’Oria, V.; Schiaffini, R.; Scarsella, M.; Russo, A.L.; Fierabracci, A. Effect of the autoimmune-associated genetic variant PTPN22 R620W on neutrophil activation and function in patients with insulin-dependent diabetes mellitus. Front. Immunol. 2025, 16, 1554570. [Google Scholar] [CrossRef]
  14. Orozco, R.C.; Marquardt, K.; Pratumchai, I.; Shaikh, A.F.; Mowen, K.; Domissy, A.; Sherman, L.A. Autoimmunity-associated allele of tyrosine phosphatase gene PTPN22 enhances anti-viral immunity. PLoS Pathog. 2024, 20, e1012095. [Google Scholar] [CrossRef]
  15. Bottini, N.; Musumeci, L.; Alonso, A.; Rahmouni, S.; Nika, K.; Rostamkhani, M.; MacMurray, J.; Meloni, G.F.; Lucarelli, P.; Pellacchia, M.; et al. A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat. Genet. 2004, 36, 337–338. [Google Scholar] [CrossRef]
  16. Perri, V.; Pellegrino, M.; Ceccacci, F.; Scipioni, A.; Petrini, S.; Gianchecchi, E.; Lo Russo, A.; De Santis, S.; Mancini, G.; Fierabracci, A. Use of short interfering RNA delivered by cationic liposomes to enable efficient down-regulation of PTPN22 gene in human T lymphocytes. PLoS ONE 2017, 12, e0175784. [Google Scholar] [CrossRef] [PubMed]
  17. Pellegrino, M.; Ceccacci, F.; Petrini, S.; Scipioni, A.; De Santis, S.; Cappa, M.; Mancini, G.; Fierabracci, A. Exploiting novel tailored immunotherapies of type 1 diabetes: Short interfering RNA delivered by cationic liposomes enables efficient down-regulation of variant PTPN22 gene in T lymphocytes. Nanomedicine 2019, 18, 371–379. [Google Scholar] [CrossRef] [PubMed]
  18. Arena, A.; Belcastro, E.; Accardo, A.; Sandomenico, A.; Pagliarosi, O.; Rosa, E.; Petrini, S.; Conti, L.A.; Giorda, E.; Corsetti, T.; et al. Preparation and In Vitro Evaluation of RITUXfab-Decorated Lipoplexes to Improve Delivery of siRNA Targeting C1858T PTPN22 Variant in B Lymphocytes. Int. J. Mol. Sci. 2021, 23, 408. [Google Scholar] [CrossRef] [PubMed]
  19. Arena, A.; Belcastro, E.; Ceccacci, F.; Petrini, S.; Conti, L.A.; Pagliarosi, O.; Giorda, E.; Sennato, S.; Schiaffini, R.; Wang, P.; et al. Improvement of Lipoplexes With a Sialic Acid Mimetic to Target the C1858T PTPN22 Variant for Immunotherapy in Endocrine Autoimmunity. Front. Immunol. 2022, 13, 838331. [Google Scholar] [CrossRef]
  20. Büll, C.; Heise, T.; Adema, G.J.; Boltje, T.J. Sialic Acid Mimetics to Target the Sialic Acid-Siglec Axis. Trends Biochem. Sci. 2016, 41, 519–531. [Google Scholar] [CrossRef]
  21. Rillahan, C.D.; Schwartz, E.; McBride, R.; Fokin, V.V.; Paulson, J.C. Click and pick: Identification of sialoside analogues for siglec-based cell targeting. Angew. Chem. Int. Ed. Engl. 2012, 51, 11014–11018. [Google Scholar] [CrossRef]
  22. Sennato, S.; Paldino, G.; Bombelli, C.; Mezzani, I.; Petrini, S.; Delfino, D.V.; Fiorentino, F.; Marianecci, C.; Ciogli, A.; Rotili, D.; et al. Optimization of Lipoplexes Functionalized with a Sialic Acid Mimetic (F9-PEG) to Target the C1858T PTPN22 Variant for Preclinical Assessment of a Novel Immunotherapy in Endocrine Autoimmunity. Pharmaceutics 2025, 17, 710. [Google Scholar] [CrossRef] [PubMed]
  23. Cavagnaro, J.; Silva Lima, B. Regulatory acceptance of animal models of disease to support clinical trials of medicines and advanced therapy medicinal products. Eur. J. Pharmacol. 2015, 759, 51–62. [Google Scholar] [CrossRef] [PubMed]
  24. Bernard, N.F.; Ertug, F.; Margolese, H. High incidence of thyroiditis and anti-thyroid autoantibodies in NOD mice. Diabetes 1992, 41, 40–46. [Google Scholar] [CrossRef]
  25. Beales, P.E.; Castri, F.; Valiant, A.; Rosignoli, G.; Buckley, L.; Pozzilli, P. Adrenalitis in the non-obese diabetic mouse. Autoimmunity 2002, 35, 329–333. [Google Scholar] [CrossRef] [PubMed]
  26. Chen, D.; Thayer, T.C.; Wen, L.; Wong, F.S. Mouse Models of Autoimmune Diabetes: The Nonobese Diabetic (NOD) Mouse. Methods Mol. Biol. 2020, 2128, 87–92. [Google Scholar] [CrossRef]
  27. Lin, X.; Pelletier, S.; Gingras, S.; Rigaud, S.; Maine, C.J.; Marquardt, K.; Dai, Y.D.; Sauer, K.; Rodriguez, A.R.; Martin, G.; et al. CRISPR-Cas9-Mediated Modification of the NOD Mouse Genome with Ptpn22R619W Mutation Increases Autoimmune Diabetes. Diabetes 2016, 65, 2134–2138. [Google Scholar] [CrossRef]
  28. Chernousova, S.; Epple, M. Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent. Gene Ther. 2017, 24, 282–289. [Google Scholar] [CrossRef]
  29. Golde, W.; Gollobin, P.; Rodriguez, L. A rapid, simple, and humane method for submandibular bleeding of mice using a lancet. Lab Anim. 2005, 34, 39–43. [Google Scholar] [CrossRef]
Ijms 26 11241 g001
Figure 1. Lipofectamine-siRNA-mediated silencing of Ptpn22 mRNA expression. (A) qRT-PCR analysis performed 48 h post-transfection with Lipofectamine showed a dose-dependent reduction in Ptpn22 transcript levels, with maximum silencing at 80 pmol siRNA (0.0660 ± 0.0186; 93.4% of inhibition), compared to untreated controls. (B) At 72 h post-transfection with Lipofectamine, Ptpn22 mRNA expression remained reduced across all siRNA concentrations, with the strongest silencing effect detected at 40 pmol (0.1822 ± 0.0928, 81.78% of inhibition) siRNA. Data are expressed as mean ± SEM of two replicates in Supplementary Table S1 (** p < 0.01, *** p < 0.001) and as percentage (%) of inhibition in Supplementary Figure S1.
Figure 1. Lipofectamine-siRNA-mediated silencing of Ptpn22 mRNA expression. (A) qRT-PCR analysis performed 48 h post-transfection with Lipofectamine showed a dose-dependent reduction in Ptpn22 transcript levels, with maximum silencing at 80 pmol siRNA (0.0660 ± 0.0186; 93.4% of inhibition), compared to untreated controls. (B) At 72 h post-transfection with Lipofectamine, Ptpn22 mRNA expression remained reduced across all siRNA concentrations, with the strongest silencing effect detected at 40 pmol (0.1822 ± 0.0928, 81.78% of inhibition) siRNA. Data are expressed as mean ± SEM of two replicates in Supplementary Table S1 (** p < 0.01, *** p < 0.001) and as percentage (%) of inhibition in Supplementary Figure S1.
Ijms 26 11241 g001
Ijms 26 11241 g002
Figure 2. LiposiRNA-mediated silencing of Ptpn22 mRNA expression. (A) qRT-PCR analysis performed 48 h post-transfection with LiposiRNA complexes showed a dose-dependent reduction in Ptpn22 transcript levels, with maximum silencing at 100 pmol siRNA (0.3853 ± 0.0226, 61.47% of inhibition), compared to untreated controls. (B) At 72 h post-transfection with LiposiRNA complexes, Ptpn22 mRNA expression remained reduced across all siRNA concentrations, with the strongest knockdown confirmed at 100 pmol (0.0788 ± 0.0150, 92.12% of inhibition) siRNA. Data are expressed as mean ± SEM of two replicates in Supplementary Table S1 (ns = not significant, * p < 0.05, ** p < 0.01) and % of inhibition in Supplementary Figure S1.
Figure 2. LiposiRNA-mediated silencing of Ptpn22 mRNA expression. (A) qRT-PCR analysis performed 48 h post-transfection with LiposiRNA complexes showed a dose-dependent reduction in Ptpn22 transcript levels, with maximum silencing at 100 pmol siRNA (0.3853 ± 0.0226, 61.47% of inhibition), compared to untreated controls. (B) At 72 h post-transfection with LiposiRNA complexes, Ptpn22 mRNA expression remained reduced across all siRNA concentrations, with the strongest knockdown confirmed at 100 pmol (0.0788 ± 0.0150, 92.12% of inhibition) siRNA. Data are expressed as mean ± SEM of two replicates in Supplementary Table S1 (ns = not significant, * p < 0.05, ** p < 0.01) and % of inhibition in Supplementary Figure S1.
Ijms 26 11241 g002
Ijms 26 11241 g003
Figure 3. Schematic representation of the siRNA-based therapeutic strategy targeting the pathogenic R619W mutation in the mouse Ptpn22 gene. The illustration depicts the application of a 21-nucleotide antisense siRNA strand designed to perfectly complement a segment within exon 14 of murine Ptpn22 mRNA, specifically encompassing the mutated codon (highlighted in green). The mouse model used in this study (NOD) harbors the R619W mutation, corresponding to the pathogenic human mutation R620W, along with three additional nucleotide variants located proximally within the same exon (indicated by yellow bars on the mRNA sequence).
Figure 3. Schematic representation of the siRNA-based therapeutic strategy targeting the pathogenic R619W mutation in the mouse Ptpn22 gene. The illustration depicts the application of a 21-nucleotide antisense siRNA strand designed to perfectly complement a segment within exon 14 of murine Ptpn22 mRNA, specifically encompassing the mutated codon (highlighted in green). The mouse model used in this study (NOD) harbors the R619W mutation, corresponding to the pathogenic human mutation R620W, along with three additional nucleotide variants located proximally within the same exon (indicated by yellow bars on the mRNA sequence).
Ijms 26 11241 g003
Table 1. qRT-PCR analysis. Results of L929 cells transduced with the PF62-pLentiPtpn22-R619W plasmid and with the empty vector (EV) are shown. Ct, Cycle threshold.
Table 1. qRT-PCR analysis. Results of L929 cells transduced with the PF62-pLentiPtpn22-R619W plasmid and with the empty vector (EV) are shown. Ct, Cycle threshold.
Sample IDPtpn22 Ctβ-Actin CtΔCtΔΔCtFold Change (2−ΔΔCt)
L929-EV36.8616.99−19.600.001.00
L929-EV36.9317.09
L929-EV36.9817.35
L929-Ptpn22-R619W26.4317.10−10.46−10.461409.15
L929-Ptpn22-R619W26.2117.14
L929-Ptpn22-R619W26.2417.21
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Mezzani, I.; Accardo, A.; Bellacchio, E.; Fais, L.; Diaferia, C.; Fierabracci, A. Preclinical Assessment in Transgenic NOD Mice of a Novel Immunotherapy for Type 1 Diabetes: Lipoplexes Down-Modulate the Murine C1858T Ptpn22 Variant In Vitro. Int. J. Mol. Sci. 2025, 26, 11241. https://doi.org/10.3390/ijms262311241

AMA Style

Mezzani I, Accardo A, Bellacchio E, Fais L, Diaferia C, Fierabracci A. Preclinical Assessment in Transgenic NOD Mice of a Novel Immunotherapy for Type 1 Diabetes: Lipoplexes Down-Modulate the Murine C1858T Ptpn22 Variant In Vitro. International Journal of Molecular Sciences. 2025; 26(23):11241. https://doi.org/10.3390/ijms262311241

Chicago/Turabian Style

Mezzani, Irene, Antonella Accardo, Emanuele Bellacchio, Luca Fais, Carlo Diaferia, and Alessandra Fierabracci. 2025. "Preclinical Assessment in Transgenic NOD Mice of a Novel Immunotherapy for Type 1 Diabetes: Lipoplexes Down-Modulate the Murine C1858T Ptpn22 Variant In Vitro" International Journal of Molecular Sciences 26, no. 23: 11241. https://doi.org/10.3390/ijms262311241

APA Style

Mezzani, I., Accardo, A., Bellacchio, E., Fais, L., Diaferia, C., & Fierabracci, A. (2025). Preclinical Assessment in Transgenic NOD Mice of a Novel Immunotherapy for Type 1 Diabetes: Lipoplexes Down-Modulate the Murine C1858T Ptpn22 Variant In Vitro. International Journal of Molecular Sciences, 26(23), 11241. https://doi.org/10.3390/ijms262311241

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop