siRNA conjugate with high albumin affinity and degradation resistance for delivery and treatment of arthritis in mice and guinea pigs

Abstract

Osteoarthritis and rheumatoid arthritis are debilitating joint diseases marked by pain, inflammation and cartilage destruction. Current osteoarthritis treatments only relieve symptoms, while rheumatoid arthritis therapies can cause immune suppression and provide variable efficacy. Here we developed an optimized small interfering RNA targeting matrix metalloproteinase 13 for preferential delivery to arthritic joints. Chemical modifications in a stabilizing 'zipper' pattern improved RNA resistance to degradation, and two independent linkers with 18 ethylene glycol repeats connecting to tandem C18 lipids enhanced albumin binding and targeted delivery to inflamed joints following intravenous administration. In preclinical models of post-traumatic osteoarthritis and rheumatoid arthritis, a single intravenous injection of the albumin-binding small interfering RNA achieved long-term joint retention, sustained gene silencing and reduced matrix metalloproteinase 13 activity over 30 days, resulting in decreased cartilage erosion and improved clinical outcomes, including reduced joint swelling and pressure sensitivity. This approach demonstrated superior efficacy over corticosteroids and small-molecule MMP inhibitors, highlighting the therapeutic promise of albumin 'hitchhiking' for targeted, systemic delivery of gene-silencing therapeutics to treat osteoarthritis and rheumatoid arthritis.

Fig. 1. Lipid conjugated siRNA constructs for albumin-mediated delivery to PTOA joints.

a–d, Left-knee mechanical loading was used to induce unilateral PTOA in mice, followed by i.v. delivery of 3 mg kg⁻¹ MSA-Cy5 (N = 5), 3 mg kg⁻¹ PEG-Cy5 (N = 3) and 200 µl of 2% w/v Evans blue (N = 4). Cy5 and Evans blue were measured by fluorescence imaging 24 h after delivery. Representative images are shown. a, Experimental timeline. b, MSA-Cy5 fluorescence in paired PTOA and healthy knees. c, Ratio of MSA-Cy5 and PEG-Cy5 fluorescence in PTOA and healthy knees for each treated mouse. d, Intravital Evans blue fluorescence in paired contralateral PTOA and contralateral non-loaded knees. e, PTOA and contralateral non-loaded knees were excised 24 h after Evans blue delivery. Evans blue was extracted and measured by densitometry (N = 3). Representative images of joints and resulting joint extracts are shown. f, Chemical modifications of siRNA ribose, backbone and terminus. g, Schematic of synthetic dicer-substrate siRNA and alternating 2′F and 2′OMe modified ‘zipper’ siRNA. h, siRNA stability in OA-derived synovial fluid: representative gel electrophoresis of dicer substrate and zipper siRNA sequences after incubation in synovial fluid collected from an untreated OA patient. i, Schematic representation of the si<(EG_XL)₂ series of zipper-modified siRNAs with divalent lipid end-modifications with variations in EG content. j, Left: FPLC chromatograph of elution of si<(EG_XL)₂ variants pre-incubated with human synovial fluid form normal joints. Right: percentage of total si<(EG_XL)₂ bound to albumin fractions. All error bars indicate s.d. of biological replicates. Knees labelled healthy indicate a non-loaded contralateral limb (right). Source data

Fig. 2. Accumulation of the albumin-binding siRNA–lipid conjugate, siRNA<(EG₁₈L)₂, in mouse PTOA knee joints following i.v. delivery.

a, Unilateral left knee mechanical loading, treatment (1 mg kg⁻¹ i.v.) and endpoint protocol used in b–d. b, Representative ex-vivo IVIS images and quantitation of Cy5-labelled siRNA<(EGxL)₂ accumulation in mouse knees 24 h after delivery. c, Left: representative intravital Cy5 fluorescence images taken 24 h after delivery of Cy5-labelled siRNA, siRNA-Chol or siRNA<(EG₁₈L)₂. Right: quantification of average Cy5 fluorescence in knees taken 24 h after mouse i.v. injection. Analysed using mixed effects analysis. d, DAPI-counterstained cryosections of knees from treated mice imaged and quantified for Cy5-labelled siRNA fluorescence in cartilage and synovial tissue. Dashed lines outline articular cartilage. e, In situ hybridization with an siMMP13 probe showing signal localization of siMMP13<(EG₁₈L)₂ in mice treated i.v. with 10 mg kg⁻¹. Representative images show siRNA presence in the femoral cartilage (F), meniscus (M), tibial cartilage (T) and synovium. Serial tissue sections incubated with a negative control scrambled probe showed no signal. f, Flow cytometry assessment of cell type-specific uptake of Cy5-siRNA<(EG₁₈L)₂ in synovium. Mice were treated i.v. with 10 mg kg⁻¹ following three bilateral mechanical loading sessions over a week. Cells isolated from synovia of 5 mice were pooled for analysis. Experimental and flow cytometry gating details are in Supplementary Fig. 11. All error bars indicate s.d. of biological replicates. Knees labelled healthy indicate a non-loaded contralateral limb (right). Source data

Fig. 3. Systemic delivery of siMMP13<(EG₁₈L)₂ decreases PTOA-induced hyperalgesia and joint histopathology.

a, Schematic timeline for knee loading and treatment. Bilateral knee loading (3× per week, 5 weeks) was used to induce severe PTOA for comparing therapeutic impact of treatment with i.v. siMMP13<(EG₁₈L)₂, i.a. Zilretta, i.p. CL-82198 and i.p. Marimastat. b, Left: mechanical hyperalgesia was measured via algometer through day 35. Data over time displayed as mean + s.e.m. Right: AUC of average Smalgo readings over time was assessed. N = 8. c, Left: total MMP activity in mouse knees was measured using MMPsense 750 Fast (N = 8). Middle: mAbCII binding in knee joints was measured by fluorescence imaging (N = 8). Right: C2C fragments in mouse serum was measured by ELISA (N = 3). d, Knee joints were stained with toluidine blue (top row, femoral condyles shown) and H&E (bottom row, synovium lining and meniscus shown). Asterisks: white, healthy articular surface; red, loss of articular surface; yellow, healthy synovial lining/meniscus; black, synovial thickening/meniscal expansion and calcification. e, Joint cartilage damage was quantitated with the OARSI osteoarthritis cartilage histopathology assessment system (top) and DJD score (bottom). NS, not significant. In all panels, error bars indicate s.d. of biological replicates, unless specified otherwise. Source data

Fig. 4. Intravenous delivery of siMMP13<(EG₁₈L)₂ achieves multijoint accumulation, MMP13 knockdown and diminished arthritis progression in a mouse RA model.

a,b, In a mouse inflammatory arthritis (K/BxN serum transfer) model, mice were treated with Cy5-siRNA<(EG₁₈L)₂, Cy5-siRNA-Chol and Cy5-siRNA (1 mg kg⁻¹ i.v.) 4 days after receiving K/BxN serum transfer. Joints of forepaw, hindpaw and knee were collected 24 h later and assessed by IVIS imaging (a) or fluorescence microscopy of hindpaw ankle joints (b). Representative images and quantitation of IVIS data are shown. N = 6. Dashed lines outline articular cartilage in fluorescence microscopy images of tissue cryosections. c, Relative Mmp13 mRNA in forepaws, knees and hindpaws of healthy untreated wild-type mice or in K/BxN serum recipients treated with siControl<(EG₁₈L)₂ or siMMP13<(EG₁₈L)₂ (10 mg kg⁻¹ i.v.) was measured by RT–qPCR. N = 6. Error bars represent s.d. of biological replicates. d–f, K/BxN serum recipients were treated with siMMP13<(EG₁₈L)₂, methylprednisolone or CL-82198 (10 mg kg⁻¹). d, Schematic timeline for K/BxN serum transfer and treatment. e, Photos of hindpaws were collected on treatment day 10, and representative images are shown. f, Ankle width change, clinical score, algometer ankle joint hyperalgesia and severity index were measured for 10 days after treatment. Error bars indicate s.e.m. (top) or s.d. (bottom) of biological replicates. Source data

Fig. 5. Intravenous siMMP13<(EG₁₈L)₂ treatment protects against cartilage/bone destruction and synovial inflammation in a mouse RA model.

K/BxN serum recipient mice were treated with siMMP13<(EG₁₈L)₂, methylprednisolone or CL-82198 (10 mg kg⁻¹). a–c, Histological analysis of toluidine blue-stained ankle cartilage sections (a, top) and H&E-stained hindpaw sections (a, bottom) were used for cartilage destruction scoring (b) and inflammation scoring (c). Asterisks: yellow, articular surface; red, articular surface damage; black, healthy synovium; white, synovial inflammation. d–g, MicroCT-based 3D renderings of hindpaw scans (d, representative images) were used to measure bone surface:volume ratio (e), bone mineral density (f) and bone fraction (g). HA, hydroxyapatite, White arrows in d show bone erosions. All error bars indicate s.d. of biological replicates. Source data

Fig. 6. MMP13 silencing by intravenous siMMP13<(EG₁₈L)₂ treatment in guinea pig ACL transection (ACLT) model of arthritis.

a, Study timeline for ACLT, treatment and sample collection in Dunkin Hartley guinea pigs. b–d, Cy5 fluorescence was measured ex vivo in healthy and ACLT knees (b) and organs (compared using multiple unpaired t-tests with no correction for multiple comparisons) (c) of guinea pigs by IVIS. Tissues were also assessed by fluorescence microscopy of sagittal ACLT knee cartilage/synovial cryosections (d) at 24 h after i.v. delivery of Cy5-siRNA or Cy5-siRNA<(EG₁₈L)₂ at 1 mg kg⁻¹. Representative images are shown. In d: blue, DAPI; red, Cy5 siRNA. e, Guinea pig Mmp13 mRNA expression (ACLT/healthy knee) as determined by RT–qPCR. f,g, MMP13 immunofluorescence in cartilage/synovial cryosections of ACLT knee joints that were untreated (left) or treated with siMMP13<(EG₁₈L)₂ (10 mg kg⁻¹ i.v.). Asterisks: white, synovium; magenta, femoral condyle cartilage. Representative images are shown with dashed lines outlining articular cartilage (f). MMP13 immunofluorescence intensity was quantitated using morphometric software (g). All error bars indicate s.d. of biological replicates. Knees labelled healthy indicate a contralateral limb (right) without ACL transection (non-surgical). Source data

Extended Data Fig. 1. Characterizing different routes of siRNA<(EG₁₈L)₂ delivery in PTOA mouse model.

a-c, Subcutaneous delivery of Cy5-siRNA<(EG₁₈L)₂ (2 mg/kg) was administered in a PTOA mouse model. Timeline for mechanical knee loading and treatment is shown (a). Intravital (b) and ex vivo (c) Cy5 fluorescence imaging was used to measure Cy5-siRNA<(EG₁₈L)₂ biodistribution to paired healthy and PTOA knees, along with organs, 24 hrs after subcutaneous treatment. N = 3. d, Quantification of subcutaneous vs. intravenous (i.v.) delivery route organ biodistribution 24 h after injection. Analyzed using multiple unpaired t-tests with no correction for multiple comparisons. e-g, Intra-articular delivery of Cy5-siRNA<(EG₁₈L)₂ (0.25 mg/kg) was characterized in a PTOA mouse model. Timeline for mechanical knee loading and treatment is shown (e). Ex vivo Cy5 fluorescence imaging was used to measure Cy5-siRNA<(EG₁₈L)₂ biodistribution to paired healthy and loaded knees, along with organs, 48 hrs after delivery (f). N = 3. Analyzed using multiple unpaired t-tests with no correction for multiple comparisons. (g) Cryohistology of the loaded knee joints 48 h after intra-articular injection with a focus on cartilage and synovial tissues. N = 3. Dashed lines outline articular cartilage. For all panels: Error bars indicate standard deviation of biological replicates. Radiant efficiency units are [photons/s] / [µW/cm²]. Knees labeled healthy indicate a non-loaded contralateral limb (right). Source data

Extended Data Fig. 2. MMP13 knockdown in PTOA joints by i.v. delivery of siMMP13<(EG₁₈L)₂.

a, Mmp13 mRNA was measured by qRT-PCR in healthy and PTOA knees of mice 5 days after i.v. delivery of siMMP13<(EG₁₈L)₂, siMMP13-Chol, or free siMMP13 (5 and 10 mg/kg, where indicated). Timeline for mechanical knee loading and treatment is shown in Extended Data Fig 1. b, Mmp13 mRNA was measured by qRT-PCR in PTOA mouse knees 5 days after i.v., i.a., or subcutaneous siMMP13<(EG₁₈L)₂ delivery at the doses indicated. N = 5-6. c, Mmp13 mRNA was measured by qRT-PCR in PTOA mouse knees 5 days after subcutaneous siMMP13<(EG₁₈L)₂ delivery at the doses indicated. N = 5-6. d-e, MMP13 IHC in PTOA knees (d), liver (e, top), and kidneys (e, bottom) 5 days after i.v. delivery (10 mg/kg) of siMMP13<(EG₁₈L)₂, siMMP13-Chol, free siMMP13, or no treatment. f, Relative Mmp13 mRNA expression in liver and kidneys of untreated mice (relative to PTOA knee Mmp13 levels). N = 3. g, Relative Mmp13 mRNA in kidneys, 5 days post-injection with 10 mg/kg siMMP13<(EG₁₈L)₂ or free siMMP13, vs. untreated controls. N = 3. For all panels: Error bars indicate standard deviation of biological replicates. Source data

Extended Data Fig. 3. Systemic (i.v.) siMMP13<(EG₁₈L)₂ (10 mg/kg) treatment achieves long-term retention and MMP13 silencing in PTOA knee joints.

a, Timeline for mouse left knee mechanical loading (3 times / week) and single treatment (day 0; 7 days after initiation of mechanical loading) with Cy5-siRNA<(EG₁₈L)₂ or siMMP13<(EG₁₈L)₂. b, Cryosection confocal microscopy imaging of Cy5-siRNA<(EG₁₈L)₂ in joint tissues taken down on day 1 post-treatment. c-e, Cy5-siRNA<(EG₁₈L)₂ was visualized on day 30 post-treatment by confocal microscopy of knee joint cryosections (c, representative images with dashed lines outlining articular cartilage), and at time points from 0-30 days post-treatment by longitudinal intravital Cy5 imaging (d). Semiquantitative analysis of the AUC (Cy5 fluorescence x days) was calculated (e) (N = 4). f-g, Knee joint Mmp13 was measured at the indicated time points by qRT-PCR (e) and IHC (f) in healthy or PTOA knee tissues collected at the indicated time points from mice treated with or without siMMP13<(EG₁₈L)₂. N = 5-6. For all panels: Error bars indicate standard deviation of biological replicates. Knees labeled healthy indicate a non-loaded contralateral limb (right). Source data

Extended Data Fig. 4. Healthy and PTOA knee joint retention after intra-articular delivery over a 30-day time course.

a, Representative IVIS images of healthy and PTOA mouse knee joints over 30 days after a single 1 mg/kg intra-articular (i.a.) injection of Cy5-siRNA<(EG₁₈L)₂ or Cy5-siRNA. b, Semiquantitative analysis of time-course fluorescent radiant efficiency within healthy and OA mouse knee joints over 30 days (N = 3). AUC was calculated based on the fluorescence intensity profiles. Radiant efficiency over time plotted as mean + SEM. AUC plot error bars indicate standard deviation of biological replicates. c, Semiquantitative analysis of time-course fraction of retention within healthy and OA mouse knee joints over 30 days (N = 3). AUC was calculated based on the fraction of retention profiles. Radiant efficiency over time plotted as mean + SEM. AUC plot error bars indicate standard deviation of biological replicates. d, Representative confocal microscopy of knee joint cryosections at the 30-day endpoint showing i.a. Cy5-siRNA<(EG₁₈L)₂, and i.a. Cy5-siRNA in loaded, PTOA, knees with a specific focus on synovial and cartilage/meniscus tissues. Dashed lines outline articular cartilage. Knees labeled healthy indicate a non-loaded contralateral limb (right). Source data

Extended Data Fig. 5. Intravenous albumin-hitchhiking siMMP13<(EG₁₈L)₂ treatment provides whole knee joint protection by reducing osteophyte formation and pathologic mineralization and is associated with a reduction in MMP13 protein and collagen degradation fragments.

a, MicroCT-based 3-dimensional (3D) renderings of meniscal/ectopic mineralization and osteophyte growth in healthy and PTOA mice treated with siMMP13<(EG₁₈L)₂ or siControl<(EG₁₈L)₂. Red asterisk = osteophyte outgrowth. b, Measurements of femoral and tibial osteophyte size at largest outgrowth from normal cortical bone structure. N = 8. c, Ectopic mineralization (measured as mg of hydroxyapatite) in menisci and in the form of osteophytes was quantified. N = 8. d-f, MMP13 IHC analyses of PTOA joint cartilage (d), meniscus (e), and synovium (f) show reduced MMP13 protein levels in animals treated with siMMP13<(EG₁₈L)₂. g, IHC on C1,2 C collagen 2 degradation fragments. Samples here are from the PTOA therapeutic study in Main Fig. 3. For panels b-c: Error bars indicate standard deviation of biological replicates. Source data

Extended Data Fig. 6. Systemic siMMP13<(EG₁₈L)₂ delivery alters inflammatory gene expression profiles in mechanically-loaded PTOA joints.

a, qRT-PCR of IL-1B, TNFalpha, NGF, and P16INK4a (Cdkn2a) at days 5, 10, 20, and 30 after a single i.v. injection of 10 mg/kg siMMP13<(EG₁₈L)₂. Analyzed using a mixed effects analysis. N = 3-7. Error bars indicate standard deviation. b, Unsupervised sorting of treatment groups as quantified by nanoString at day 10 after treatment of PTOA knees with 10 mg/kg siMMP13<(EG₁₈L)₂. Gene expression is shown as high- (green) or low-expression (red) sorted vertically by differences between treatment groups. c, Gene cluster expression changes at day 10 post-treatment in PTOA knees. Source data

Extended Data Fig. 7. Characterizing albumin-based drug delivery in the K/BxN serum transfer arthritis model.

a-c, Evans Blue or Cy5-MSA was delivered i.v. to wild type (healthy) and K/BxN serum recipient mice 4 days after serum transfer. Joints of forepaws and hindpaws were used for Evans Blue extraction (a, representative images), measuring Evans Blue absorbance in extracts (a, right). Evans Blue was also visualized by fluorescence microscopy in joint cryosections (b). MSA-Cy5 delivery to joints was visualized by intravital Cy5 fluorescence imaging (c). d, qRT-PCR was used to measure relative gene expression of albumin transport-associated genes (Sparc and Caveolin-1 [Cav1]) in healthy and K/BxN arthritis hind paws. N = 7 for Sparc; N = 6 for Cav1. e-g, Cy5-siRNA<(EG₁₈L)₂ (1 mg/kg, i.v.) was delivered to healthy and K/BxN serum recipient mice. Cy5 fluorescence in organs (e) and limb joints (f), analyzed using multiple unpaired t-tests with no correction for multiple comparisons, were measured ex vivo at 24 hrs. Cy5 fluorescence was assessed in knee and hindpaw cryosections by fluorescence microscopy (g). For panel e, N = 3 for heart, lungs, liver, and spleen; N = 6 for kidney, forepaw, knee, and hindpaw. For all panels: Error bars indicate standard deviation of biological replicates. Source data

Extended Data Fig. 8. Intravenous siMMP13<(EG₁₈L)₂ delivery silences MMP13 and protects cartilage in the joints of mice given K/BxN serum transfer.

K/BxN STA mice were treated with or without siMMP13<(EG₁₈L)₂ (10 mg/kg, i.v.), then assessed at day 10. a, MMP13 IHC analysis of cartilage and synovium in multiple joints was used to assess silencing of MMP13 expression. b, Fluorescent mAbCII delivered to mice was detected by ex vivo fluorescence imaging to measure relative cartilage damage in healthy and K/BxN STA mice undergoing treatment (representative images shown). N = 14. c, Pan-MMP activity was measured via delivery of MMPsense 750 Fast to mice, followed by detection using ex vivo fluorescence imaging. N = 14. For all panels: Error bars indicate standard deviation of biological replicates. Source data

Extended Data Fig. 9. Intravenous delivery of siMMP13<(EG₁₈L)₂ protects cartilage and bone while reducing inflammation in multiple joints of the K/BxN STA model.

a-b, Toluidine Blue stained histological knee and forepaw sections (a) were scored using the Cartilage Destruction Score (b). Hindpaw data is shown in Fig. 5. c-d, H&E-stained histological knee and forepaw sections (c) were scored using the Inflammation Score (d). Hindpaw data is shown in Fig. 5. e, Histological bone destruction score quantification of hindpaw, knee, and forepaw joint tissues. For panels b, d, and e: N = 7. Error bars indicate standard deviation of biological replicates. Source data

Extended Data Fig. 10. Intravenous siMMP13<(EG₁₈L)₂ treatment reduces presence of cartilage degradation fragments in serum and diminishes the inflammatory gene expression profile in the joints of the K/BxN STA model.

a, Relative serum C2C collagen 2 degradation fragment levels in serum were measured on day 10 following treatment by ELISA. Error bars indicate standard deviation of biological replicates. N = 6-7. b, Presence and localization of C1,2C collagen 2 degradation fragments were assessed by IHC on day 10 post-treatment. c-d, RNA harvested from K/BxN recipient mouse joints was assessed by the nanoString nCounter Mouse Inflammatory 294 Gene Expression panel. Clustering of gene expression changes [high- (green) or low- (red)] sorted vertically by differences between treatment groups (c). Analysis of relevant gene clusters from tissues that were harvested on day 10 post-treatment (d). N = 4. Source data