SHORT INTERFERING RNA TARGETING VARIANT C1858T OF GENE PTPN22
20210238607 · 2021-08-05
Assignee
Inventors
Cpc classification
C12N2320/32
CHEMISTRY; METALLURGY
A61K47/6913
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
A61K9/1271
HUMAN NECESSITIES
International classification
C12N15/113
CHEMISTRY; METALLURGY
A61K47/69
HUMAN NECESSITIES
A61K9/127
HUMAN NECESSITIES
Abstract
The present invention concerns a short Interfering RNA, which is variant C1858T PTPN22 short interfering RNA duplexes (siRNA), and uses thereof in medical field in the prevention and treatment of autoimmune diseases.
Claims
1) Short interfering RNA duplex targeting PTPN22 C1858T single nucleotide polymorphism, said short interfering RNA comprising or consisting of the sequence 5′-AUGAUUCAGGUGUCCAUAC-3′ (SEQ ID NO:2) and its complementary sequence 5′-GUAUGGACACCUGAAUCAU-3′ (SEQ ID NO:1).
2) Short interfering RNA duplex according to claim 1, wherein SEQ ID NO:1 and 2 have a dinucleotide at 3′ end, wherein said dinucleotide is chosen from the group consisting of dTdT, dAdA, dGdG and dCdC.
3) Short interfering RNA duplex according to claim 1, wherein said short interfering RNA is delivered by a carrier.
4) Short interfering RNA duplex according to claim 3, wherein the carrier is a liposome, such as a cationic liposome, nanocarrier, such as solid-lipid nanoparticles, or a PEGylated liposome.
5) Short interfering RNA duplex according to claim 4, wherein said cationic liposome comprises or consists of dimyristoyl-sn-glycero-phosphatidylcholine (DMPC) in combination with 2R,3S-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonium)-butane dibromide, or in combination with 2S,3S-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonium)-butane dibromide.
6) Short interfering RNA duplex according to claim 3, wherein said carrier is functionalized with monoclonal antibodies against T lymphocytes, such as anti-CD3 otelixizumab, teplizumab, antibodies against B lymphocytes, such as anti-CD20 antibody, such as a Fab anti-CD20, Rituximab, or antibodies anti LFA-1 lymphocyte function-associated antigen 1 (CD11a) or anti LFA-3 lymphocyte function-associated antigen 3.
7) Short interfering RNA duplex according to claim 3, wherein said carrier is functionalized with a sialoside analogue for Siglec-based cell targeting such as the sialic acid mimetic (SAM) PEG-lipid-F9 ligand for Siglec-10.
8) Pharmaceutical composition comprising or consisting of short interfering RNA duplex according to claim 1, in association with one or more excipients and/or adjuvants.
9) (canceled)
10) A method of preventing or treating an autoimmune disease, the method comprising administering to a subject in need thereof a short interfering RNA duplex according to claim 1 or a pharmaceutical composition comprising the short interfering RNA duplex, thereby preventing or treating the autoimmune disease.
11) The method of claim 10, wherein said subject carries a PTPN22 C1858T single nucleotide polymorphism.
12) The method of claim 10, wherein the autoimmune diseases are chosen from the group consisting of insulin-dependent diabetes mellitus (T1D), autoimmune thyroid disease (ATD), such as Graves' disease or Hashimoto's thyroiditis (HT), myasthenia gravis, multiple sclerosis, systemic lupus erythematosus (SLE), Wegener's granulomatosis, rheumatoid arthritis, juvenile idiopathic arthritis, celiac disease, vitiligo, Sjögren syndrome, primary adrenal insufficiency, alopecia areata, giant cell arteritis, polymyositis.
Description
[0030] The present invention is described by an illustrative, but not limitative way, according to preferred embodiments thereof, with particular reference to the enclosed drawings, wherein:
[0031]
[0032]
[0033]
[0034]
[0035]
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[0037]
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[0040]
[0041] Plasma membranes are stained with WGA, lipoplexes with PKH26 probe (positive dots, arrows) and nuclei with DAPI. Bars: 10 μm (A, C) and 5 μm (B, D).
[0042] For Figure in black and white, arrows in top right panels of A and C indicate PKH26 positive lipoplexes and bottom right panels their internalization in CD3+ and CD3− lymphocytes. Arrows in respective XY-Z orthogonal projections of confocal images (B, D) indicate lipoplexes internalization close to/beneath the cell membrane in CD3+ (B) and CD3− (D) cells.
[0043]
[0044]
[0045]
[0046] Plasma membranes were stained with wheat germ agglutinin (WGA), lipoplexes with PKH26 probe (positive dots, arrows) and nuclei with DAPI. For Figure in black and white arrows in bottom panels indicate the internalization of lipoplexes in the indicated tissues.
[0047]
[0048]
EXAMPLE 1
Study of the Delivery of Short Interfering RNA of the Present Invention by Cationic Liposomes into PBMC of Type 1 Diabetes Patients and of Down-Regulation of Variant PTPN22 Gene in T Lymphocytes
[0049] Methods
[0050] Preparation and Characterization of Liposome Formulations
[0051] Gemini surfactant 2R,3S-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonium)-butane dibromide, 2, was prepared as previously reported (Bello 2006; Seebach 1977; Aleandri 2012, Perri 2017).
[0052] For the preparation of liposome and lipoplex formulations and siRNA stock solution, a freshly prepared buffer solution of 5 mM HEPES and 0.1 mM EDTA, at pH 7.4 (Sigma-Aldrich, Chemical Company (Co.), St Louis, Mo.) was used.
[0053] Liposomes composed of DM PC (purity>99%, Avanti Polar Lipids Inc. (Alabaster, Ala.)) and 2 at a 50/50 molar percentage were prepared following a previously described procedure (Hope 1992). A film of DMPC (3.0 μmol) and 2 (3.0 μmol) was prepared on the inside wall of a round-bottom flask by evaporation of a CHCl3 solution containing the proper amounts of the component. The film was then dried for 7 hours (hrs) under high vacuum, and 3.0 ml of buffer solution were added to have a final dispersion 1.0 mM in DMPC and 1.0 mM in 2 (Perri 2017). The solution was vortex-mixed, freezed-thawed six times from liquid nitrogen to 313 K, and finally extruded (10 times) through a 100 nm polycarbonate membrane (Whatman Nuclepore, Toronto, ON, Canada). Extrusions were carried out at 40° C., above the DMPC transition temperature (24.2° C.), on a 10 ml extruder (Lipex Biomembranes, Vancouver, Canada). For the preparation of lipoplexes (DMPC/2/siRNA or Lipo/siRNA), proper volumes of the siRNA stock solution (0.1 mM in buffer) were added to a diluted liposome solution to have the final concentrations: [siRNA]=1.3 μM, [DMPC]=50 μM, [2]=50 μM, corresponding to a charge ratio+/−=2.
[0054] Samples for confocal microscopy were prepared following the same procedure described above, adding a fluorescent probe (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)-ammonium salt, Avanti Polar Lipids) or PKH26 to the lipid solution, during the film preparation step. The fluorescent probe was used in a 0.1 mol percentage with respect to DMPC. Samples were analyzed by circular dichroism spectroscopy (CD) and dynamic light scattering (DLS) at different times after the preparation (9, 24, 48, 72 hrs) (Perri 2017).
[0055] Circular Dichroism Spectroscopy
[0056] CD spectra were recorded on a Jasco spectropolarimeter J-715 equipped with a Peltier device for the temperature control, using 0.5 cm path length quartz cuvettes. Measurements were run in the 330-220 nm spectral range at 25° C. CD spectra are the average of 16 scans obtained with an instrument scanning speed of 100 nm/min, response time of 1 second (s) and resolution of 1 nm (Perri 2017).
[0057] Dynamic Light Scattering
[0058] DLS measurements were obtained with a Brookhaven Instruments Corp. BI-200SM goniometer equipped with a BI-9000AT digital correlator using a solidstate laser (125 mW, λ=532 nm). Unless otherwise stated, measurements of scattered light were made at a scattering angle θ of 90°. Measurements were carried out at 25° C. (Perri 2017) and temperature was controlled with an accuracy of 0.1° C. Each experiment (duration in the range of 5-20 minutes) was repeated two or more times. CONTIN algorithm was used to fit the data.
[0059] siRNA Design
[0060] siRNA sequences were originally designed specifically for the target C1858T PTPN22 gene variant. These have been generated using a siRNA design algorithm licensed from Rosetta Inpharmatics (Sigma-Aldrich Chemical Co., http://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/siRNA/learning-center/mission-sup-reg0/siRNA-design-choosing.html).
[0061] From a list of siRNA sense/antisense (s/a) duplexes differing in mRNA target affinity (Table 1), generated without any backbone modification, the specific sequence with the higher affinity for the target was chosen for subsequent experiments, namely siRNA sequence (SNP_T sense 5′-GUAUGGACACCUGAAUCAU-3′ (SEQ ID NO: 1) with dTdT at 3′ end; SNP_T antisense 5′-AUGAUUCAGGUGUCCAUAC-3′ (SEQ ID NO: 2) with dTdT at 3′ end, Sigma Chemical Co.). dTdT was added in order to increase stability. Alternatives for any below cited sequence could be dAdA, dGdG and dCdC. Table 1 shows the design of duplex siRNAs SNP_T (sense and antisense) against the variant T1858 PTPN22 allele.
TABLE-US-00001 TABLE 1 siRNA Design Target Sense/ wherein each sequence Name Antisense has dTdT at 3′ end Start Target Sequence SNP_T s GUAUGGACACCUGAAUCAU 45 GTATGGACACCTGAATCAT (SEQ ID NO: 1) (SEQ ID NO: 11) SNP_T a AUGAUUCAGGUGUCCAUAC 45 ATGATTCAGGTGTCCATAC (SEQ ID NO: 2) (SEQ ID NO: 12) SNP_T s CUUCCUGUAUGGACACCUG 39 CTTCCTGTATGGACACCTG (SEQ ID NO: 3) (SEQ ID NO: 13) SNP_T a CAGGUGUCCAUACAGGAAG 39 CAGGTGTCCATACAGGAAG (SEQ ID NO: 4) (SEQ ID NO: 14) SNP_T s AUGGACACCUGAAUCAUUU 47 ATGGACACCTGAATCATTT (SEQ ID NO: 5) (SEQ ID NO: 15) SNP_T a AAAUGAUUCAGGUGUCCAU 47 AAATGATTCAGGTGTCCAT (SEQ ID NO: 6) (SEQ ID NO: 16) SNP_T s UGUAUGGACACCUGAAUCA 44 TGTATGGACACCTGAATCA (SEQ ID NO: 7) (SEQ ID NO: 17) SNP_T a UGAUUCAGGUGUCCAUACA 44 TGATTCAGGTGTCCATACA (SEQ ID NO: 8) (SEQ ID NO: 18) SNP_T s UAUGGACACCUGAAUCAUU 46 TATGGACACCTGAATCATT (SEQ ID NO: 9) (SEQ ID NO: 19) SNP_T a AAUGAUUCAGGUGUCCAUA 46 AATGATTCAGGTGTCCATA (SEQ ID NO: 10) (SEQ ID NO: 20)
[0062] C1858T PTPN22 Gene Variant Silencing in Human PBMC. Study Population
[0063] The study population was composed of 22 long-term T1D patients who were referred from the Department of Endocrinology at Bambino Gesu' Children's Hospital (OPBG). Of the total number of long-term patients, 16 were carriers of the C1858T PTPN22 polymorphism in heterozygosis, and 6 were non-carriers.
[0064] All enrolled patients were unrelated. All subjects entered the investigation after obtaining written informed consent. The study was approved by the local Institutional Review Board (IRB) of Bambino Gesu' Children's Hospital, regulating the use of human samples for experimental studies (N° 1385). The informed consent for children was obtained from the next of kin. Consent on behalf of children was written. Participant consent was recorded using a paper-based inventory system. The IRB approved the consent procedure.
[0065] Detection of the C1858T Variant in the PTPN22 Gene
[0066] Molecular analysis of the C1858T (R620W) polymorphism of the autoimmunity predisposing gene PTPN22 was evaluated in the DNA of patients and controls using a Xcml restriction fragment length polymorphism-PCR (polymerase chain reaction) method (Bottini 2004, Gianchecchi 2013).
[0067] Cell Preparation
[0068] Peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Hypaque (Histopaque, Sigma-Aldrich Chemical Co.) from sodium heparinized venous blood samples (5-10 ml) of recruited T1D patients. Subsequently, PBMC were frozen down in liquid nitrogen according to standard protocols (Gianchecchi 2014).
[0069] Custom Liposome Transfection Protocol
[0070] T1D PBMC were thawed, washed in complete RPMI 1640 medium (EuroClone, Pero (Milan), Italy) supplemented with 10% fetal bovine serum (FBS, GE Healthcare Life Sciences, UT, USA) and L-glutamine (2 mM) (EuroClone). PBMC were then seeded at 1.5×10.sup.6 cells per well in 48-well plates (Falcon, Corning, NY, USA) in a final volume of 250 μl of FBS-free RPMI 1640 medium supplemented with L-glutamine (2 mM) and treated with different doses of Lipo/siRNA complexes (20, 60, 80, 100 pmols of siRNA). After an overnight (O/N) transfection, cells were washed by centrifugation at 1200 rpm for 5 minutes, seeded again in 48 wells flat bottom plates in complete RPMI medium at a final volume of 250 μl and incubated at 37° in a humidified atmosphere containing 5% CO.sub.2 for additional 24 and 48 hrs corresponding to a final transfection time of 48 and 72 hrs respectively.
[0071] RNA Extraction and Quantitative Real Time-PCR Analysis
[0072] Total RNA from untreated or treated PBMC was isolated with TRIzol™ Reagent (Invitrogen, Life Technologies Corporation, Carlsbad, Calif., USA) following the manufacturer's instructions. After in vitro reverse transcription (500 ng) with the High-Capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, Calif.), quantitative Real-Time PCR (rtq-PCR) was performed using 7900HT Fast Real-Time PCR System (Applied Biosystems) and Power SYBR Green PCR Master Mix (Applied Biosystems) with the following primers: [0073] (i) GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (human)
TABLE-US-00002 forward (fwd): (SEQ ID NO: 21) 5′-CGACCACTTTGTCAAGCTCA-3′ reverse (rev): (SEQ ID NO: 22) 5′-AGGGGTCTACATGGCAACTG-3′ [0074] (ii) PTPN22 (human)
TABLE-US-00003 fwd: (SEQ ID NO: 23); 5′-GCTGTACTAGCAACTGCTCC-3′ rev: (SEQ ID NO: 24) 5′-CCAGCTTCCTCAACCACAAT-3′ [0075] (iii)PTPN22T.sup.1868 (human)
TABLE-US-00004 fwd: (SEQ ID NO: 25) 5′-CAGCTGTACTAGCAACT-3′ rev: (SEQ ID NO: 26) 5′-AGGTGTCCATACAGGAA-3′
For the analysis, the mRNA levels, normalized to GAPDH, were calculated as follows:
2.sup.−[ΔCt(Lipo/siRNA)−ΔCt(RPMI)]=2−ΔΔCt,
where ΔCt=C.sub.t (PTPN22 or PTPN22.sup.T1868)−C.sub.t (GAPDH).
[0076] Rtq-PCR products were purified by means of a Gel and PCR clean up kit (Qiagen, Hilden, Germany) following the manufacturer's instructions and subsequently analyzed using the Genetic Analyzer 3500 (Applied Biosystems).
[0077] Confocal Microscopy Analysis
[0078] T1D PBMC from both wild-type and heterozygous patients were seeded at 1.5×10.sup.6 cells per well in 48-well plates (Falcon) in a final volume of 250 μl of FBS-free RPMI 1640 medium (EuroClone) supplemented with L-glutamine (2 mM) (EuroClone) and treated with Lipo/siRNA complexes marked with rhodamine (100 pmols of siRNA) for 4 and a half hrs. At the end of the incubation period, cells were harvested, washed in PBS, and fixed with 4% paraformaldehyde (Sigma-Aldrich Chemical Co.). Fixed cell suspensions were distributed drop wise onto positive charged microscope slides (Super Frost plus, Menzel-Glaser, Germany) and dried at 37° C. After rehydration in PBS, cell permeabilization was obtained incubating microscope slides with 0.1% PBS-Triton X-100 (Sigma-Aldrich Co.) for 5 minutes. Subsequently, 30 minutes blocking with 5% BSA (Bovine Serum Albumin, Sigma-Aldrich Co.) was performed and cells were then stained with primary mouse anti-human CD3 (Clone UCHT1 BD Biosciences, San Jose, Calif., 1:30, incubated for 1 hour at room temperature (RT)) followed by secondary antibody (Ab) goat anti-mouse Cy-5 conjugate (Invitrogen, 1:100, incubated for 1 hour at RT). Finally, to counterstain plasma membrane and nuclei, WGA conjugated to Oregon Green1488 (Invitrogen, 1:200) and Hoechst 33342 (Invitrogen, 1 μg/ml) or DAPI (4′,6-diamidino-2-phenylindole) were used respectively. Confocal imaging was performed on an Olympus Fluoview FV1000 confocal microscope equipped with FV10-ASW version 2.0 software, Multi Ar (458±488 and 515 nm), 2× He/Ne (543 and 633 nm), and 405-nm diode lasers, using a 60× (1.40 NA oil) objective. Optical single sections were acquired with a scanning mode format of 1024×1024 pixels, sampling speed of 40 ms/pixel (pixel size of 0.2 mm), and Z-reconstructions of serial single optical sections were carried out with an electronic zoom at 2.5. Fluorochromes unmixing was performed by acquisition of automated-sequential collection of multi-channel images, in order to reduce spectral crosstalk between channels (Perri 2017).
[0079] Toxicity Assay
[0080] Toxicity evaluation of lipoplexes was assessed by monitoring cell morphology, viability, quantity and quality of cell pellets and quantification of protein extract concentration at the end of the experimental procedure. T1D PBMC were seeded at 1.5×10.sup.6 cells per well in 48-well plates (Falcon) in a final volume of 250 μl of FBS-free RPMI 1640 medium (EuroClone) supplemented with L-glutamine (2 mM) (EuroClone) and treated with different doses of Lipo/siRNA complexes marked with rhodamine (20, 60, 80, 100 pmols of siRNA) for 4 and a half hrs. Subsequently the cells were harvested with complete medium, centrifuged 1200 rpm for 5 minutes, washed once in PBS and resuspended in PBS 2% FBS. To detect and quantify dead cells the blue fluorescent cell impermeant dye DAPI (4′,6-diamidine-2′-phenylindole dihydrochloride, Invitrogen) was added at a final concentration of 0.2 μM, 5 minutes prior to analysis of cells by flow-cytometer BD LSR Fortessa X-20 (BD, Sunnyvale, Calif.). DAPI specifically enters only dead cells when used on live cells. 20,000 events were acquired and data analyzed by BD FACSDiva software 8.0 (BD Biosciences). The evaluation was carried out on triplicate biological determinations.
[0081] Functional Assay
[0082] A functional assay was implemented to verify the effect of lipoplexes on T cell activation by evaluating interleukin 2 (IL-2) concentration in supernatants of patients PBMC transfected O/N with different doses of Lipo/siRNA complexes (Lipo/siRNA complexes 60, 80 and 100 pmols of siRNA) then treated with Dynabeads Human T-activator CD3/CD28 beads (Invitrogen). After transfection, cells were washed by centrifugation, seeded 2.5×10.sup.5 per well in 96 wells flat bottom plates in complete RPMI medium, then activated with the indicated anti-CD3/CD28 beads at different bead-to-cell ratios. Cells were subsequently incubated at 37° in a humidified atmosphere containing 5% CO.sub.2 for 20 hrs. In an additional experimental condition to specifically address immunomodulation, cells were stimulated with a suboptimal bead-to-cell ratio 1:50 carried out for 5 days (Perri 2017). At the end of the incubation period, supernatants and cells were collected and separated by centrifugation at 1200 rpm for 5 minutes. The concentration of IL-2 in supernatants was estimated by means of the human IL-2 ELISA development kit (Mabtech, Nacka strand, Sweden) following the manufacturer's guideline. Plates were then read at 405 nm by Bench-mark Plus microplate spectrophotometer (Bio-Rad, CA). The evaluation was carried out with at least triplicate biological determinations.
[0083] Statistical Analysis
[0084] Differences between siRNA+CD3+ and siRNA+CD3− cells, among wild-type and heterozygous T1D PBMC, representing the population's specific transfection efficiency, were statistically evaluated using the unpaired t test. The analysis was performed scoring a total of 600 cells by two independent observers (MP, SP). A resulting P value<0.05 was considered statistically significant. For the Lipo/siRNA efficacy experiments, evaluated in rtq-PCR and IL-2 ELISA assays, differences between each test condition and the control condition were assessed for statistical significance with one-way ANOVA analysis of variance and Bonferroni multiple comparison test. To analyze the difference of IL-2 production in functional assays between wild-type and heterozygous C1858T PTPN22 stimulated T1D PBMC unpaired t test was used. The statistical study was performed analyzing multiple biological determinations with Graph Pad Prism software version number 5 (San Diego, Calif.).
[0085] Preliminar Biodistribution Studies
[0086] Evaluation of Internalization of PKH26-Labelled Lipo/siRNA in Human PBMC.
[0087] Following the same procedure reported for rhodamine marked Lipo/siRNA lipoplexes (vide supra) PBMC were treated with Lipo/siRNA complexes marked with PKH26 (0.5-10 with 80-100 pmols of siRNA) for 4 and a half hrs. Internalization was evaluated by confocal microscopy analysis as above described.
[0088] Evaluation of Toxicity of PKH26-Labelled Lipo/siRNA for In Vivo Studies in Mice.
[0089] Lipo/siRNA complexes marked with PKH26 (0.5-10 with 80, 100 pmols of siRNA) were administered for 4 and a half hrs. Cell morphology was assessed as above described.
[0090] Preliminar Biodistribution Studies of PKH26-Labelled Lipo/siRNA in Mice.
[0091] PKH26-labelled PEGylated lipoplexes with 50 μg of siRNA were injected intravenously in a C56BL6 8 weeks old male mice via lateral tail vein in 80 μl volume of Hepes 5 mM/EDTA 0.1 mM, pH 7.4. 24 hrs atter injection, the animal was sacrificed by cervical dislocation. For histological analysis under confocal microscopy lymphoid organs spleen, bone marrow, thymus and nonlymphoid organs liver, heart and lung were removed and snap frozen with Optical Cutting Temperature (OCT) method. Peripheral blood samples were also taken in EDTA.
[0092] Histological Analysis
[0093] Serial 10 μM thick cryostatic sections were cut from frozen tissue blocks. Cytospun slides were prepared from mice PBMC, isolated under Ficoll-Hypaque procedure (Histopaque, Sigma-Aldrich Chemical: St Louis, Mo., USA); cells were fixed in 4% paraphormaldehyde, then dropped onto microscopic slides. For microscopic examination plasma membrane and nuclei were stained with WGA conjugated to Oregon Green1488 (Invitrogen, 1:200) and Hoechst 33342 (Invitrogen, 1 μg/ml) respectively. Confocal microscopy analysis was carried out as above described. Cells identified by PKH26-lipoplexes were counted by two independent observers under Nikon Eclipse E600 Optical microscope by scoring at least 1000 elements onto two consecutive sections at 60× magnification under oil immersion.
[0094] Results
[0095] Evaluation of Size and Polydispersion of Liposomes and Lipoplexes by DLS Measurements
[0096] DLS experiments on the liposome formulation of DMPC/2 and on lipoplexes (DMPC/2/siRNA or Lipo/siRNA) were performed as previously reported (Perri 2017). The investigations on the liposomes formulation confirmed what it was already described: liposome formulation of DMPC/2 shows a narrow single population centered at about 40 nm after 9 hrs from the extrusion, whereas, after 72 hrs from the preparation, DMPC/2 liposomes increase significantly in dimensions (Perri 2017).
[0097] The lipoplexes composed of the siRNA against the variant PTPN22 with the liposomes of DMPC/2 show a behavior similar to that observed for lipoplexes of wild type siRNA (Perri 2017). In fact, also in this case lipoplexes dimensions do not seem to be heavily affected by the presence of siRNA, the dimensions being around 70 nm in diameter. In addition, the lipoplexes DMPC/2/siRNA do not change considerably with time, increasing slightly up to ˜90 nm, thus suggesting that lipoplexes are rather stable.
[0098] CD Investigations into the Conformational Stability of siRNA in Lipoplexes
[0099] CD investigations of the lipoplexes composed of the siRNA against the variant PTPN22 with the liposomes of DMPC/2 were performed following the same approach previously described for wild type siRNA (Perri 2017). Also in this case, the CD spectrum of siRNA in lipoplexes (DMPC/2/siRNA or Lipo/siRNA), measured at different times after their preparation, resembles that of free siRNA in buffer solution, the bands of lipoplexes being less intense than those of free siRNA. These observations indicate that the association between liposomes and siRNA does not affect significantly the conformational stability of siRNA designed against the variant PTPN22. In addition, the absence of marked variations in the CD spectrum over a 72 hrs period is an indirect confirmation of lipoplexes stability.
[0100] Lipo/siRNA SNP_T Lipoplexes are Effectively Internalized in T1D PBMC
[0101] Internalization of rhodamine-conjugated Lipo/siRNA complexes (100 pmols of siRNA) was visualized in T1D PBMC following 4 and a half hrs of incubation (
[0102] The internalization was confirmed specifically in both CD3+ (white) and CD3− T lymphocytes (
[0103] Lipo/siRNA SNP_T Lipoplexes are not Toxic to T1D PBMC
[0104] PBMC treated with rhodamine-marked lipoplexes (20, 60, 80 and 100 pmols of siRNA) did not show signs of toxicity during the culture period as assessed by quality and quantity of cell pellet and quantification of protein extract concentration at the end of the experimental procedure.
[0105] T1D PBMC treated with different doses of rhodamine-conjugated lipoplexes for 4 and a half hrs retained proper morphology both of the cell membrane (green) and of the nuclei (blue) as revealed by confocal microscopy (
[0106] In Flow-cytometry analysis, T1D PBMC revealed high percentage of rhodamine+ cells implying relevant transfection efficacy and internalization and, at the same time, showed low percentage of dead cells (Rhodamine+DAPI+ cells) (
[0107] Lipo/siRNA SNP_T Lipoplexes Treatment Downregulates PTPN22 mRNA
[0108] The mRNA obtained from PBMC derived from 16 heterozygous C1858T PTPN22 patients and 6 wild-type PTPN22 patients was analyzed by rtq-PCR after treating cells with different doses of lipoplexes (20, 60, 80 and 100 pmols of siRNA) for 48 and 72 hrs. Either time point of the lipoplexes treatment led to a decrease in the target PTPN22 mRNA levels in 13 out of 16 heterozygous patients (
[0109] To ascertain lipoplexes variant specificity, a second set of primers aimed to detect T1858 variant mRNA solely was designed. These primers were first validated by performing the rtq-PCR on PBMC derived from wild-type PTPN22 T1D patients using both set of primers, the new specific one and the first one able to recognize all target gene mRNA. The result of this validation showed the inability of the specific set to detect wild-type PTPN22 mRNA where the T1858 SNP is not present (
[0110] Lipo/siRNA SNP_T Lipoplexes Efficacy Toward Lyp Biological Activity
[0111] Autoimmune disease associated R620W Lyp variant is a gain-of-function form of the enzyme (Nang 2005; Lin 2016), meaning that a more potent phosphatase activity of the protein is indeed present. Data from literature (Nang 2005) showing a decreased secretion of IL-2 by heterozygous C1858T PTPN22 PBMC in comparison to wild-type PTPN22 PBMC after stimulation with anti-CD3/CD28 beads were confirmed in T1D patients (
[0112] After TCR engagement, an increased concentration of IL-2 upon lipoplexes treatment in respect to untreated cells (RPMI) was observed in heterozygous C1858T PTPN22 T1D PBMC in comparison to wild-type PTPN22 T1D PBMC (
[0113] Evaluation of Lipo/siRNA for In Vivo Studies in Mice.
[0114] Internalization of PKH26-labelled Lipo/siRNA complexes (100 pmols of siRNA) was visualized in HD PBMC following 4 and a half hrs of incubation (
[0115] HD PBMC treated with PKH26-conjugated lipoplexes for 4 and a half hrs retained proper morphology both of the cell membrane (WGA stain) and of the nuclei (DAPI stain) as revealed by confocal microscopy (
[0116] In Flow-cytometry analysis, HD PBMC revealed high percentage of PKH26+ cells implying relevant transfection efficacy and internalization and, at the same time, showed low percentage of dead cells (PKH26+DAPI+ cells) (
[0117] Preliminar Evaluation of Biodistribution of Lipo/siRNA in Representative Mice.
[0118] After 24 hours from injection PKH26-labelled lipoplexes were not identified in the peripheral blood while these identified different percentages of cells within the organs under investigation (
[0119] Preliminary Evaluation of Incorporation and Toxicity on Human PBMC of Lipoplexes Functionalized with PEG-lipid-F9 (PEG-F9 Lipoplexes)
[0120] Preliminary experiments where conducted by confocal microscopy and Flow cytometry (data not shown) demonstrating that PEG-lipid-F9 lipoplexes marked with PKH26 are incorporated in PBMC after 4 and a half hrs of incubation and are not toxic to treated cells.
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