Intradermal delivery of lipophilic siRNAs enables prolonged skin retention and sustained gene silencing in a porcine model

Domain: neuroscience
Published: 2026-02-03
Editorial outcome: minor_revision

Abstract
Small interfering RNAs (siRNAs) offer significant therapeutic potential; however, extrahepatic applications, particularly to the skin, remain a challenge. Limited work has explored siRNA therapies for the skin, the largest organ in the human body, where dermatological conditions affect over one-third of the population worldwide. The skin’s external location makes it easily accessible for direct, local administration. Here, we present the in vivo intradermal delivery of therapeutic siRNAs into a porcine model whose skin structure most closely resembles that of human skin, demonstrating functional, and sustained gene silencing. We characterize two siRNA conjugates in human ex vivo and porcine in vivo skin models, showing that increased hydrophobicity significantly enhances skin retention and efficacy of siRNAs. Using a validated JAK1-targeting compound, we demonstrate that local delivery of siRNA enables accumulation across multiple cell types and suppression of JAK1-dependent inflammatory pathway in human skin ex vivo. In porcine models, intradermal injections result in prolonged skin siRNA retention for more than eight weeks, limited systemic tissue exposure, and sustained gene silencing for at least one month. These results underscore the importance of tailored siRNA conjugate design for achieving optimal skin biodistribution and therapeutic efficacy, providing a foundation for siRNA-based treatments for a broad range of dermatological conditions.

Introduction
RNA therapeutics are revolutionizing the way we treat diseases1. Small interfering RNAs (siRNA) represent a class of highly programmable molecules capable of silencing their complementary mRNA targets to prevent translation of disease-causing proteins2,3. Recent chemical advances in improving stability and in vivo delivery have enabled the translation of siRNAs into the clinic, with multiple FDA-approved drugs emerging in the past few years4. While this represents a significant leap forward for this modality, the clinical utility of siRNAs is currently limited to the liver2. As such, recent efforts in the field are focused on expanding the utility of siRNA drugs to extrahepatic organs (e.g., muscle5, lung6, CNS7) to treat previously “undruggable” diseases7,8.

One understudied organ of interest is the skin, where many diseases, such as cutaneous lupus erythematosus, psoriasis, vitiligo, alopecia areata, and atopic dermatitis, may benefit from treatment with siRNA9,10. According to the World Health Organization, skin diseases are among the most common human health conditions, impacting as many as one-third of the population worldwide, underscoring the demand for developing novel therapeutic approaches to address unmet medical needs11,12. As many of these conditions are primarily caused by the dysregulation of the immune system in the skin, delivering therapeutic modalities to the skin while avoiding systemic immunosuppression is critical13–15. While targeted delivery of siRNA to the skin following systemic administration remains a challenge, the inherent accessibility of skin makes local administration a clinically viable and straightforward approach9,10. Biologics, such as antibodies, are highly specific but are only efficient for targeting secreted or cell surface targets. Conventional small molecules require frequent dosing, which can be burdensome and limit patient compliance9,10,12,16,17. In contrast, siRNA therapeutics can overcome these challenges and offer sustained gene silencing when engineered for local skin administration, offering an ideal treatment option.

We have previously shown that the delivery properties of siRNAs can be altered by modifying conjugate chemistry, with lipophilic conjugates enhancing skin delivery and efficacy18–21. However, direct comparison of diverse siRNA conjugates for intradermal administration is lacking, and existing studies are largely confined to ex vivo tissues or rodent models21,22. These models present limitations in accurately assessing skin retention, systemic exposure, and therapeutic durability due to the inherent differences in rodent skin physiology and the limited viability of ex vivo human tissue15,23,24.

In this work, we investigate the pharmacokinetics and pharmacodynamics of lipophilic siRNAs in human and porcine skin models. We compare unconjugated siRNA (Unc-siRNA), amphiphilic dendritic siRNA (D-siRNA), and lipophilic docosanoic acid siRNA (DCA-siRNA). In human ex vivo skin, DCA-siRNA demonstrates enhanced cellular uptake and superior gene silencing in both the epidermis and dermis, suggesting that strong lipophilicity is ideal for siRNA skin efficacy. Targeting JAK1 expression with DCA-siRNA, a key immune regulator in skin diseases, leads to reduced downstream inflammatory chemokine release (CXCL9, 10, and 11)21,22.

Furthermore, we evaluate the pharmacokinetics and pharmacodynamic profile of DCA-siRNA in a porcine in vivo model. Intradermal injection resulted in prolonged skin retention (at least up to eight weeks), sustained JAK1 silencing (at least one month), and minimal systemic exposure. DCA-siRNA also exhibited an excellent safety profile, with no alterations to blood markers. These findings suggest that lipophilic DCA-siRNA is a promising therapeutic strategy for skin conditions with a defined target.

Results
Intradermal injection of lipophilic siRNAs into human skin leads to multiple cell type uptake and efficacy
Building on our previous findings that lipophilic conjugation enhances siRNA delivery to the skin18,19,21, we investigated how lipid conjugate hydrophobicity impacts cellular uptake and activity in human ex vivo skin after intradermal injection. From the library of conjugates previously assessed, docosanoic acid (DCA) demonstrated the highest skin retention and siRNA activity in mouse skin tissue, making it a compelling candidate for further evaluation18. Additionally, a recently developed amphiphilic dendrimer conjugate achieved effective skin delivery while being significantly less hydrophobic than DCA, warranting a direct comparison between the two19. For that, we assessed the cellular uptake of cyanine-3 (Cy3) labeled unconjugated (Unc-siRNA), the moderately lipophilic (dendritic, D-siRNA), and strongly lipophilic (docosanoic, DCA-siRNA) siRNAs, 24h post-injection into skin biopsies (Fig. 1a, b). Flow cytometry allowed for analysis of cell-type specific uptake analysis (Fig. 1c; gating strategy in Supplementary Fig. 1). While all tested siRNAs were internalized by all major skin cell populations, DCA-siRNA showed significantly increased uptake, particularly in melanocytes and immune cells (**p<0.01), compared to D-siRNA and Unc-siRNA. Overall, DCA-siRNA uptake was significantly higher than both Unc-siRNA (**p<0.01) and D-siRNA (***p<0.001).

To examine the influence of conjugate chemistry on siRNA efficacy, we employed our previously validated JAK1-targeting siRNA sequence (JAK13033)21, and intradermally administered Unc-JAK13033, DCA-JAK13033 and D-JAK13033 siRNAs into human skin biopsies. Four days post-injection, JAK1 mRNA levels were quantified using a QuantiGene 2.0 assay in both epidermal and dermal layers of the skin (Fig. 1d)21. All conjugates induced significant JAK1 mRNA silencing in the dermal layer. However, D-JAK13033, while showing a silencing trend, had considerable variability in the epidermal layer, failing to achieve statistical significance (p=0.07). This increased variability in the epidermis following an intradermal injection could be a result of the chemistry-dependent distribution away from the site of injection, leakage into the media, or issues with the separation of the various skin layers for processing (Fig. 1d).

Consistent with cellular uptake results, DCA-JAK13033 exhibited the most potent silencing, achieving approximately 50% and 75% reduction in JAK1 mRNA in the epidermis and dermis, respectively. In contrast, Unc-JAK13033 and D-JAK13033 yielded comparable silencing, with approximately 25% knockdown in the epidermis and 50% in the dermis (Fig. 1d). This indicates that increased hydrophobicity enhances siRNA-mediated JAK1 mRNA silencing in human skin following intradermal delivery. Furthermore, we observed dose-dependent silencing of DCA-JAK13033 in an escalating dose study (Supplementary Fig. 2).

JAK1 is a component of the IFN-γ signaling cascade; therefore, we sought to evaluate the impact of JAK1 silencing on downstream signaling molecules following IFN-γ stimulation21,25. Human skin biopsies were pre-treated with siRNAs for four days and subsequently stimulated with IFN-γ for 24h to induce chemokine expression (Fig. 1e). We then quantified mRNA levels of the IFN-γ-responsive chemokines CXCL9, CXCL10, and CXCL11. Consistent with JAK1 mRNA silencing, all siRNAs effectively suppressed chemokine expression, with DCA-JAK13033 exhibiting the most potent inhibition, achieving approximately 70% reduction across all three chemokines (Fig. 1e). D-JAK13033 and Unc-JAK13033 showed comparable levels of inhibition, approximately 50%, where D-JAK13033 demonstrated a slightly more robust inhibition than Unc-JAK3033. These findings demonstrate that siRNA-mediated JAK1 silencing not only reduces JAK1 mRNA expression but also effectively attenuates downstream chemokine signaling, which plays a pivotal role in the pathogenesis of numerous skin conditions26–28.

Intradermal administration of conjugated JAK13033 siRNAs in pigs demonstrates durable skin retention for at least 2 months, minimal systemic exposure, and a favorable safety profile
While JAK1 inhibitors have revolutionized inflammatory skin disease treatment, the potential for systemic toxicity can be minimized with localized delivery strategies14,15,29. To assess systemic distribution of intradermally administered JAK1-targeting siRNAs, we utilized a porcine model, chosen for its structural similarity to human skin17,30. Pigs received intradermal injections of D- or DCA-JAK13033 (50 µL of 200 µM siRNA/1 cm2 at an intradermal injection site; 10 nmol final dose/biopsy, a total of 80 injections per pig [40/side]; details in Supplementary Fig. 3). Skin and major organ biopsies (heart, spleen, liver, kidney) were collected at various time points, and siRNA accumulation was quantified using a peptide-nucleic acid hybridization assay (Fig. 2a)31. Notably, siRNAs exhibited prolonged skin retention for at least up to eight weeks, with 15 to 20-fold lower siRNA levels detected in systemic organs, indicating minimal systemic exposure (Fig. 2b). To evaluate potential immunogenicity, we measured 17 cytokines and interleukins in blood samples collected from days one to seven post-injection. No significant changes were observed for any analyte (Supplementary Fig. 4), confirming a favorable safety profile. Additionally, blood levels of platelets (thrombocytopenia), white blood cell count (inflammation), alanine aminotransferase (ALT, liver damage), and carbamide (kidney damage) had no significant changes (Fig. 2c). These findings, combined with sustained local skin retention and minimal systemic distribution, suggest that intradermal administration of hydrophobic siRNAs will have prolonged silencing activity in skin, with minimal toxicity and off-target effects.

Pharmacokinetic and pharmacodynamic assessment of DCA-siRNA in pigs
Building on DCA-siRNA’s superior potency in human skin ex vivo and its prolonged retention and safety in pig in vivo, we conducted a comprehensive pharmacokinetic/pharmacodynamic assessment in pigs to evaluate intradermal administration as a safe and effective therapeutic route for JAK1 skin silencing. We compared siRNA plasma circulation, tissue distribution, and skin retention following both intradermal and intravenous injections (Fig. 3a), utilizing a pig-reactive JAK1 siRNA sequence, JAK1883 (as our human-targeting JAK13033 is not cross-reactive). Intradermally injected DCA-JAK1883 demonstrated minimal systemic exposure compared to intravenous injection, showing an absolute bioavailability of 10.5% (AUClast) for DCA-JAK1883 (Fig. 3b), and significantly lower tissue accumulation across multiple organs seven days post-injection relative to the dose injected (Fig. 3c). Importantly, siRNA was only detected in the skin after intradermal injection, as intravenous administration failed to deliver sufficient siRNA to the skin (below the limit of detection by LC-MS/MS). We also observed sustained retention of DCA-JAK1883 in skin over time (Fig. 3d), with levels at approximately 300 µg/g at three hours post-injection, reducing to ~60 µg/g at 24h and maintaining this level to seven days post-intradermal injection. Finally, assessing siRNA activity by measuring JAK1 mRNA levels seven days post-intradermal injection (Fig. 3e), we found a ~50% reduction in both epidermal and dermal JAK1 mRNA levels (**p<0.01), confirming efficacious delivery of DCA-siRNA.

Furthermore, parallel studies with the human-targeting DCA-JAK13033 siRNA showed identical plasma, tissue, and skin accumulation profiles with an absolute bioavailability (AUClast) of 8.35% when injected intradermally (Supplementary Fig. 5), supporting the known principle that siRNA sequences minimally influence the biodistribution of this modality3. Hematological readouts from these studies were also unaltered, supporting the safety profile of DCA-siRNA, both intradermally and intravenously (Supplementary Fig. 5). We also confirmed that DCA-NTC does not impact JAK1 mRNA in ex vivo pig skin (Supplementary Fig. 6).

DCA-siRNA supports a minimum of one-month-long silencing in porcine skin following intradermal administration
Building on promising skin retention, a favorable safety profile, and one-week efficacy observed in pigs, we further investigated the durability and downstream signaling modulation of DCA-siRNA targeting JAK1 expression. We intradermally injected the pig-reactive DCA-JAK1883 (50 µL of 400 µM/1 cm2 site; 20 nmol final dose) and subsequently assessed JAK1 mRNA expression and downstream chemokines (CXCL9, 10, and 11 mRNA) in skin biopsies collected one-month post-injection following exposure to IFN-γ (Fig. 4a). DCA-JAK1883 effectively silenced JAK1 mRNA in both epidermal and dermal layers, achieving approximately 50% knockdown in the epidermis (***p<0.001) and 75% in the dermis (***p<0.001), compared to untreated skin, while intradermally administered PBS showed no silencing, as expected (Fig. 4b). Similarly, we observed significant reductions in CXCL9 (~50% in both epidermis and dermis, **p<0.01 in epidermis, ***p<0.001 in dermis) and CXCL10 (~50% in epidermis *p<0.05, ~40% in dermis p=0.28) mRNAs. Significant CXCL11 mRNA silencing was observed only in the dermis (~50% ****p<0.0001) (Fig. 4b). These results collectively demonstrate the potent and sustained JAK1 silencing and pathway modulation achievable with DCA-siRNA in a large animal model.

Methods
Ethics statement
All research performed complies with the relevant ethical regulations. De-identified surgically discarded fresh abdominal skin was obtained from the UMass Chan Biospecimen, Tissue, and Tumor Bank. The collection of these samples from patient donors was approved by the Institutional Review Board (IRB) at the University of Massachusetts Chan Medical School, and all participants gave written informed consent before surgical procedures. Minipig studies were conducted by SCANTOX (No. 79362) and Altasciences (No. 2181.04), under their approved IACUC protocols.

Oligonucleotide synthesis, deprotection and purification
Oligonucleotides were synthesized on a MerMade 6/12 synthesizer (Bioautomation) and AKTA Oligopilot 100 (GE Healthcare Life Sciences) following standard protocols. In brief, conjugated sense strands were synthesized at 5 to 20 μmol scales on custom-synthesized lipid-functionalized controlled pore glass (CPG) supports for DCA conjugate5,18,40. For the dendritic sense strand, synthesis was on a CPG functionalized with UnyLinker (ChemGenes) and commercially available amidites (Cy3, C6, C12, and symmetrical branching from ChemGenes) were used to build the dendritic moiety on the 5′-end as previously described41,42. All sense strands had a 2dT spacer between the strand and the conjugate. Antisense strands were synthesized on CPG functionalized with UnyLinker. All strands were cleaved and deprotected using 28% aqueous ammonium hydroxide solution for 16h at 50°C, followed by drying under vacuum at 45°C, and resuspended in Millipore H2O. The deprotected oligonucleotide solutions were then filtered to remove CPG residues. Oligonucleotides were purified using HPLC purification carried out on an Agilent 1290 Infinity II system as previously described18,19,21. Briefly, Sense strands were purified over a C18 column for lipid-conjugated sense strands and over anion-exchange column for antisense strands. Purified oligonucleotides were desalted by size-exclusion chromatography and characterized by liquid chromatography-mass spectrometry (LC/MS) analysis on an Agilent 6530 accurate-mass quadrupole time-of-flight (Q-TOF) LC/MS (Agilent Technologies).

In vivo oligonucleotide sequences
Oligonucleotide compounds and sequences used are listed in Table 1.

Testing siRNA efficacy in human ex-vivo skin
Freshly discarded post-panniculectomy abdomen skin was obtained from UMass Chan Biospecimen, Tissue, and Tumor Bank or was purchased from BIOIVT and ZENBIO. The human specimens were hydrated with PBS, and subcutaneous fat was excised prior to treatment. Then, the skin is submerged in an antifungal solution diluted in PBS to prevent any fungal infection or growth, followed by another PBS wash. Following that, siRNA is administered into the ex vivo skin and incubated in Iscove′s Modified Dulbecco′s Media (Sigma-Aldrich; #I3390) supplemented with 10% fetal bovine serum, 100 U/mL penicillin-streptomycin, and 0.05 μM 2-mercaptoethanol (termed complete media).

For intradermal testing, 8 mm skin biopsies were punched, and 10 nmols of siRNA was intradermally injected with a volume of 50 µL. The punches were cultured for 96h at 37°C in a 24-well plate with 2 mL of complete media per punch. To induce CXCL9, 10, and 11 expression, after 96h of siRNA treatment, skin biopsies were treated with new media containing 10 ng/mL recombinant human IFN-γ protein (R&D System, #285-IF-100) and 10 ng/mL recombinant human TNF protein (R&D System, #210-TA-020). mRNA expression was measured by the Quantigene 2.0 assay.

Dissociation of epidermis and dermis skin biopsies
The skin biopsies were incubated in 0.5 mL of 30 mg/mL dispase II (Roche, #04942078001) in Dulbecco’s Modified Eagle’s Media (Corning Cellgro; #10-013CV) for 1h at 37°C. Epidermis and dermis were manually separated prior to being placed in RNAlater® for 24h as previously reported21. Epidermis and dermis were placed in 0.75 to 1 mL of homogenizing solution (Invitrogen; QS0517) containing 10 µL of 10 mg/mL proteinase K (Invitrogen, #AM3546) and were mechanically dissociated and homogenized. Specifically for the mechanical dissociation, the epidermis was dissociated using QIAGEN Tissue Lyser II as previously described following Quantigene protocols21, and the tougher dermis layer mechanical dissociation was carried out using manual dissociation probe or using the Bullet blender 5E Gold+ (Next Advance; #BB5EAUP) to ensure disruption of the dense extracellular matrix in skin tissue. Homogenates were then incubated at 55°C for 30 min, followed by centrifugation at 14,000 × g for 5 min. The clear supernatant was then collected for subsequent assays. Silencing of the target mRNA expression was measured through the Quantigene 2.0 assay.

mRNA quantification
The target mRNA levels were measured by using Quantigene 2.0 assays (Affymetrix). All QuantiGene detection probe sets were ordered from Thermo Fisher. For human skin, the following probes were used: human JAK1 (SA-50455), human CXCL9 (SA-12372), human CXCL10 (SA-50393), human CXCL11 (SA-50464), and human ACTB (SA-10008) as a housekeeping gene. For pig skin, the following probes were used: porcine JAK1 (SF-4295068), pig CXCL9 (SF-4296654), pig CXCL10 (SF-4295763), pig CXCL11 (SF-4299676), and pig ACTB (SF-4295627) as a housekeeping gene.

Human skin flow cytometry
Skin punch biopsies (4 mm) were injected with 50 µL of 100 µM Cy3-labeled siRNAs formulated in 1x PBS. Samples were then cultured for 24h in 2 mL/punch of Iscove’s Modified Dulbecco’s Medium (IMDM, Sigma; #I3390–500ML) supplemented with 10% fetal bovine serum (Gibco; #26140), 1x penicillin–streptomycin (Sigma; #P4333–100ML), and 50 µM of 2-mercaptoethanol (Sigma; #M3148). Skin biopsies were digested in IMDM media containing 1.6 mg/mL collagenase, type 4 (Worthington-biochem; #LS004186). Briefly, skin biopsies were diced and minced into small pieces and incubated at 37°C for 3h at 200 rpm in gentleMACS C tubes (Miltenyi Biotec, #130-093-237) on a MACS dissociator. The suspended cells were filtered through a 70 µm cell strainer (Fisher Scientific) and stained with the following dye and fluorescent antibodies: Human TruStain FcX (Biolegend, #422302), Sytox Blue (Thermo Fisher), BV605 anti-human CD45 (Biolegend), APC anti-human CD117 (c-kit) (Biolegend), PE-VIO770 anti-human CD140 (Biolegend) and BRIGHT-FTIC anti-Human CD49f (Biolegend). The samples were analyzed using BioRad ZE5 (BioRad), and data were processed using FlowJo 10 software. Gating of cell types were as follows: Immune cells, CD45+; Fibroblasts, CD45- CD140+; Endothelial cells, CD45- CD49f+; Keratinocytes, CD45- Ckit-; Melanocytes, Ckit+.

Peptide nucleic acid (PNA) hybridization assay
Tissue biodistribution levels of the siRNA were measured using a PNA hybridization assay as previously described. Briefly, the accumulation of the JAK13033 was quantified using a custom Cy3-labeled fluorescent PNA oligonucleotide probe (Alexa488-OO-GTACAAAGGAATCTGA-KK, PNABio) that is fully complementary to the antisense strand. Pre-weighted tissue punches were placed in 500 µL homogenizing solution (Invitrogen; QS0517) containing 0.2 mg/mL proteinase K (Invitrogen, #AM3546) in a QIAGEN collection microtube holding a 3-mm tungsten bead. The tissues were then homogenized for 10 min under 30 Hz frequency using a QIAGEN Tissue Lyser II. Homogenized tissues were incubated at 55°C for 30 min and centrifuged at 1000 × g for 10 min. Sodium dodecyl sulfate was precipitated from homogenates by using 3M potassium chloride, followed by a centrifugation for 15 min at 5000 × g. siRNAs in the supernatant were then collected and hybridized to the PNA probe at 95°C, followed by slow cooldown. Anion-exchanged chromatography was used to analyze the sample mixtures on an Agilent 1260 Infinity quad-pump HPLC with a 1260 FLD fluorescent detector, using a DNAPac PA100 column (Thermo Fisher) for peak separation. Gradient and buffers were used as previously described31,43. Cy3 fluorescence was monitored, and peaks were integrated. Final concentrations of oligos were determined by using a calibration curve generated by spiking known quantities of siRNA into tissue lysates from untreated animals.

Pig models for siRNA pharmacokinetics and pharmacodynamics
Pig experiments for in vivo intradermal and intravenous studies were performed by SCANTOX for the pharmacokinetics and accumulation of JAK13033, and by Altasciences for pharmacokinetics and pharmacodynamics (JAK13033 and JAK1883). Briefly, siRNAs were formulated in 1x PBS at 200 or 400 µM and administered intradermally or intravenously for the indicated doses. Intravenous dose was 10.76 mg/kg
For JAK13033 pharmacokinetics, a study was conducted in 10 male Göttingen SPF minipigs (n=5 per group, 4 months of age), where areas of treatment were tattooed prior to injection (50 µL of 200 µM siRNA/1 cm2 site; 10 nmol final dose/biopsy; 8 mm biopsy). Detailed biopsy location and collection are provided in Supplementary Fig. S3. Skin biopsies and blood/serum samples were collected over a 56-day period. Animals were euthanized at varying time periods over a 56-day period to obtain terminal heart, spleen, liver and kidney biopsies for systemic distribution. All biopsies were flash frozen and reconstituted in RNAlater® 24h prior to PNA hybridization analysis as described above. CBC and blood chemistry measurements were performed by SCANTOX.

For JAK1883 pharmacokinetics and pharmacodynamics, a study was conducted in 8 Göttingen SPF minipigs, n=2 pigs/sex for I.D. JAK13033 and JAK1883, a total of n=4 pigs per siRNA; similar setup for I.V. injection. Areas of treatment were tattooed prior to injection (50 µL of 400 µM siRNA/1 cm2 site; 20 nmol final dose/biopsy; 8 mm biopsy). No sex-dependent differences were observed; therefore, the data were based on combined female and male results. Skin biopsies and blood/plasma samples were collected over a one-week period. Animals were euthanized one week post administration, and tissues were collected to assess systemic biodistribution. All biopsies were flash frozen, and skin biopsies were reconstituted in RNAlater® 24h prior to mRNA quantification using Quantigene Assay 2.0. siRNA amounts in plasma, tissue, and skin were quantified by Altasciences via LC-MS/MS. JAK1 mRNA silencing in pigs for a one-month duration was conducted similarly, with the addition of the 24-h stimulation protocol to assess downstream chemokine measurements. For the one-month silencing study, a Göttingen SPF male minipig was injected intradermally (50 µL of 400 µM siRNA/1 cm2 site; 20 nmol final dose/biopsy; 8 mm biopsy; total of 4 biopsies per group), and samples were collected and processed as mentioned previously.

Pharmacokinetic parameters were determined using a non-compartmental approach within Phoenix® WinNonlin® software (version 8.0; Certara USA, Inc., Princeton, New Jersey), which is appropriate for both intravenous bolus and extravascular routes of administration. The linear trapezoidal method with linear interpolation was applied to estimate parameters from individual plasma and skin (biopsy) concentration-time data. Descriptive statistics for these parameters were calculated for males, females, followed by sexes combined, as no sex-dependent differences were observed. AUClast is defined as the area under the concentration versus time curve from the start of dose administration to the time after dosing at which the last quantifiable concentration was observed. Absolute bioavailability is defined as the percentage of the administered siRNA drug that reaches the systemic circulation intact.

Cytokine level measurement
Pigs were injected subcutaneously as described in Supplementary Fig. S3. Blood was collected pre-dosing, and on days one, two, and seven post-intradermal injection. Serum was separated and analyzed for cytokine concentration measurement using Porcine 17-plex Discovery Panel (CD31, GM-CSF, IFN-gamma, IL-1alpha, IL-1beta, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-18, MMP-1, PDGF-BB, SerpinE1, TNF, IL-12). Serum samples were analyzed as described in the manufacturer’s protocol via the UMass Metabolic Disease Research Center.

Statistics & reproducibility
The data was analyzed and graphed using the GraphPad Prism 9 software (GraphPad Software, Inc.). Details of methods for statistical analysis and sample sizes are included in each figure legend. P-values of less than 0.05 were considered significant for comparisons between groups. Most studies had at least 3 biologically independent replicates. No data points were excluded unless there was a failed assay measurement. No technical replicates were used, and all studies had at least one biological replicate. Randomization was followed. No blinding was necessary, hence not utilized in the experiments reported.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This a nicely conceived study demonstrating that intradermally administered siRNAS can provide proprolonged silencing of the Jak1gene which has downstream effects on inflammtory cytokines and chemokines. Overall this study has important implications for treating dermal inflammatory conditions like psoraiasis and eczema. The only shortcoming is that data feom scrambled or other control siRNAS seem to be missing.", "reviewer_2": "The manuscript presents a lipophilic siRNA conjugate strategy for treating skin diseases. The authors provide favorable safety and PK/ PD profile for administering siRNA in a localized manner without significant systemic drug exposure. The data presented across large animal model and clinically relevant human skin tissue is very encouraging. The overall study is well-planned and executed and presented in a clear and systematic manner, however, there are certain aspects of the work which need additional experimentation and clarification:\n1. Why did D-JAK13033 display considerable variability in the epidermal layer? Provide more explanation. Would increasing the sample size resolve the statistical significance?\n2. It is recommended to include non-targeting (scrambled) siRNA control in the in vivo porcine experiment to showcase differences in inflammatory markers. Since the siRNA is targeting a protein involved in an inflammatory cascade, there could be some unforeseen changes in the baseline levels which may not be accurately captured from untreated and PBS-treated study groups. This is evident from supplemental Figure S2, where PBS-treated human biopsy samples appear to have lower JAK1 mRNA levels compared to the non-targeting control group.\n3. What does the significance between PBS and on-target JAK1 siRNA look like in Figure S2?\n4. The authors report more than 8 weeks of siRNA retention in porcine tissue after intradermal injection. However, for the efficacy studies, they selected 1 month as the end-point. Why was this timeline chosen? Additionally, is there a siRNA threshold concentration needed at the target site to achieve therapeutic benefit?\n5. Make sure the graph axis is consistent across different figure panels for better visualization. For instance, in Fig. 3b, y-axis for CXCL levels in dermis vs. epidermis has different axis points.\n6. Given the translational potential of this technology and the mention of an on-going Phase 2a human clinical trial, the authors should add a discussion about other delivery systems (transdermal patches, ointments, and so on) that may be utilized as an alternative to painful intradermal injections where the intended treatment surface area is larger.", "reviewer_3": "This manuscript leverages a less commonly studied porcine model alongside a widely used ex vivo model, providing a valuable comparative perspective. The authors adeptly integrate lipid chemistry and RNA chemistry to enhance gene silencing efficacy and tissue retention in the skin, which is the body's largest organ. Furthermore, the analysis of unconjugated, dendrimer-conjugated, and their docosanoic siRNA conjugates allow for meaningful comparison and context within the field. Notably, the authors report increased silencing in JAK1 for their leading siRNA conjugate in both human skin and the porcine model.\nThis work makes important advances in intradermal delivery technology. The inclusion of comparative data across different siRNA conjugates, presented in a more human-compatible porcine model, provides novel insight and may inform future research directions.\nIn addition, the manuscript addresses a central challenge in the field: extrahepatic delivery. Building on previous research, the authors progress this area toward greater clinical relevance, as evidenced by the ongoing Phase 2a human clinical trial of the leading compound, DCA-JAK1. The manuscript is compelling in its broad inclusion of both efficacy outcomes and comprehensive cytotoxicity analyses, clearly oriented toward translatable research with potential positive clinical impact. I recommend this manuscript for publication in Nature Communications. However, I do have several comments/ questions that, if addressed, would further strengthen the work.\n1. Can authors offer any commentary on why they chose docosanoic acid as their lipophilic conjugate and their rationale as a whole regarding choosing their selected conjugates?\n2. The authors relay that the docosanoic conjugate is the most lipophilic conjugate tested. While looking at chemical structure one could come to the same conclusion, it would be nice to see an inclusion of some metric of lipophilicity of the selected conjugates showing this range, perhaps logP studies or some other analysis.\n3. Docosanoic acid has been implicated in anti-inflammatory processes in the body. The authors show that their DCA conjugated siRNA has an improved cytotoxic profile compared to other conjugates. Could the authors comment or provide any data regarding if the anti-inflammatory of DCA may be implicated in this study? Would its inclusion further increase skin retention due to its lipophilicity and some method of anti-inflammatory processes?"}, "author_response": "Remarks to the author\nReviewer #1\nThis is a nicely conceived study demonstrating that intradermally administered siRNAS\ncan provide proprolonged silencing of the Jak1gene which has downstream effects on\ninflammtory cytokines and chemokines. Overall this study has important implications for\ntreating dermal inflammatory conditions like psoraiasis and eczema. The only\nshortcoming is that data feom scrambled or other control siRNAS seem to be missing.\nWe thank the reviewer for pointing out the absence of a non-targeting control (NTC)\nsiRNA in the pig studies as a limitation. While we acknowledge this limitation, we wish\nto emphasize the data gathered from the human tissue samples in Figure 1. These\nsamples showed clear confirmation that NTC and PBS treatments are indistinguishable\nin their effect on JAK1 mRNA levels, which supports the specificity and on-target action\nof the therapeutic JAK1 siRNA.\nIn the minipig studies, three controls were included in parallel:\n• The solvent to control for procedural effects.\n• The human siRNA compound (non-cross-reactive) to control for\nchemistry-related effects, effectively serving as an NTC.\n• The minipig-specific compound to assess pharmacodynamic-related effects.\nWe selected the solvent as an additional control rather than a dedicated NTC for three\nreasons:\n1. The human compound provided a functional NTC in a non-cross-reactive\nspecies.\n2. This approach aligns with the 3Rs principles (Replacement, Reduction,\nRefinement) for large-animal studies.\n3. It reflects the clinical trial setting, where patients receive placebo (solvent) rather\nthan an NTC siRNA.\nThis design allowed differentiation of adverse events related to the procedure/solvent\nfrom those linked to siRNA chemistry or sequence. This design and data was formally\naccepted by the FDA as part of the IND-enabling package supporting Phase I/II trial\ninitiation.\nWe did conduct however an ex vivo pig study, similar to the human ex vivo study, where\npig skin was injected with PBS, DCA-NTC and DCA-JAK1883 and showed no significant\ndifference in expression between NTC and PBS. The figure has been added to the\nsupplemental information (Supplementary Figure 6), as well as the paragraph below\ndiscussing the limitation of the pig study, in the discussion section:\nnoisserpxE\nANRm\n1KAJ\n)SBP\nfo\n%(\n2\n1\n1\n0\n5\n0\n5\n0\n0\n0\n0\n0\ns i R N\nn s\n2\nn\nA\ns\n0\nn\nn\ns\n1\nd\n✱\ns\n0\n✱\no\n5\ns\n2\ne\n. 5\n(\n2\nn\n0\nm o l )\nP\nD\nD\nB\nC\nC\nS\nA\nA\n-\n-\nN\nJ A\nT C\nK 1\n8 8 3\n“Another limitation is the absence of a non-targeting control (NTC) siRNA group in the\npig study. Due to the significant cost constraints of large animal studies, and the\ninclusion of these data into the Investigational New Drug (IND)-enabling toxicity\npackage, PBS (the placebo used in clinical trial), was selected as the control. However,\nthe impact of the siRNA chemistry in the form of an NTC control is mitigated by several\nlines of evidence: human biopsy data showed that NTC-treated and PBS-treated tissues\nwere identical in their inability to affect JAK1 mRNA levels; additionally, the\nhuman-targeting JAK13033 compound—which acts as an NTC in pigs since it is not\ncross-reactive—demonstrated no alterations in CBC, blood chemistry, or inflammatory\nmarkers at various timepoints and in two different conjugates. These findings support\nthe on-target specificity of the JAK1 siRNA and confirm that the siRNA chemistry itself\ndoes not cause non-specific inflammation”\nReviewer #2\nThe manuscript presents a lipophilic siRNA conjugate strategy for treating skin\ndiseases. The authors provide favorable safety and PK/ PD profile for administering\nsiRNA in a localized manner without significant systemic drug exposure. The data\npresented across large animal model and clinically relevant human skin tissue is very\nencouraging. The overall study is well-planned and executed and presented in a clear\nand systematic manner, however, there are certain aspects of the work which need\nadditional experimentation and clarification:\n1. Why did D-JAK13033 display considerable variability in the epidermal layer? Provide\nmore explanation. Would increasing the sample size resolve the statistical significance?\nWe thank the reviewer for their comment. There are two potential explanations for\nvariability in the epidermal layer: 1) the albumin-binding, amphiphilic nature of the\ndendrimer conjugate that can leak out of the epidermal layer into the solution more\nreadily, decreasing its retention and efficacy; and 2) the variability coming from the\nseparation process of epidermal layer, as it is a very thin layer being peeled.\nWhile the statistics show no significant difference in the epidermis for dendrimer\n(p=0.07) which might be improved with a larger sample size, it ultimately does not affect\nthe conclusion of the stronger potency observed in the dermis and epidermis with\nDCA-siRNA which is the lead chemistry. We have adjusted the main text to discuss this,\nand reproduced below:\n“However, D-JAK13033 while showing a silencing trend, it had considerable variability in\nthe epidermal layer, failing to achieve statistical significance (p=0.07). This increased\nvariability in the epidermis following an intradermal injection could be a result of the\nchemistry-dependent distribution away from the site of injection, leakage into the media,\nor issues with the separation of the various skin layers for processing.”\n2. It is recommended to include non-targeting (scrambled) siRNA control in the in vivo\nporcine experiment to showcase differences in inflammatory markers. Since the siRNA\nis targeting a protein involved in an inflammatory cascade, there could be some\nunforeseen changes in the baseline levels which may not be accurately captured from\nuntreated and PBS-treated study groups. This is evident from supplemental Figure S2,\nwhere PBS-treated human biopsy samples appear to have lower JAK1 mRNA levels\ncompared to the non-targeting control group.\nWe thank the reviewer for their critical feedback. We have elaborated on this general\nlimitation in our response to Reviewer 1 (above) and addressed it within the manuscript\ndiscussion. Particularly for the impact on inflammatory markers, no changes were\nobserved at any of the 17 measured inflammatory markers (Figure S4) from pigs\ninjected with D-JAK13033 and DCA-JAK3033 and no alteration to pathology readouts from\nWBC and blood chemistry counts (Figure 2C). Here the siRNAs are JAK13033 which\ntarget only the human transcript and not the pig, essentially acting as NTC controls in\npigs. We have also added WBC and blood chemistry data from the JAK1883 PK and\nefficacy study (Figure 3) which also show no changes to the levels pre-dosing in either\nintravenous or intradermal dosing of DCA-JAK1833 siRNAs. The panel is reproduced\nbelow, and added to supplemental figure S5 – E):\neulaV\n1 08642 00000\n211\n00000\n05050\np\nI n\nre -in\nt\nje\nr\nc\na\ntio\nv\nn\ne n o\nD\nu\na y\ns\nWAB\n9 la te le ts (1 0\nL T (μ k a t/L )\nU N (m g /d L )\n/L )\neulaV\n1 08642 00000\n211\n00000\n05050\np re\nI n\n-in\nt\nje\nr a\nc tio\nd\nn\ne r m\nD a\na\ny\nl\nWAB\n9 la te le ts (1 0\nL T (μ k a t/L )\nU N (m g /d L )\n/L )\nSpecifically concerning Figure S2, we confirm there is no statistically significant\ndifference between the NTC and PBS human biopsy groups in either Figure 1D or the\nrevised Figure S2 (where statistics against both controls have now been added). The\nslight, non-significant trend observed is limited to the epidermal layer, which we believe\nis a result of normal experimental variation. Since this variation is not replicated in\nFigure 1D and did not reach statistical significance in any comparison, we are confident\nit does not impact our overall conclusions. Lastly, in our in vivo comparisons to\nuntreated and PBS, which in Figure S2 trend to have slightly lower JAK1 expression\nthan the NTC, this leads to a more conservative measure of apparent potency of the\nsiRNA.\n3. What does the significance between PBS and on-target JAK1 siRNA look like in\nFigure S2?\nWe have added the significance to PBS to Figure S2, where slight statistic changes\nwere observed to the epidermal layer, but no changes were observed in the dermal\nlayer which is the site of injection.\n4. The authors report more than 8 weeks of siRNA retention in porcine tissue after\nintradermal injection. However, for the efficacy studies, they selected 1 month as the\nend-point. Why was this timeline chosen? Additionally, is there a siRNA threshold\nconcentration needed at the target site to achieve therapeutic benefit?\nWe appreciate the reviewer's comment regarding the duration of the efficacy study.\nWhile we do have supporting siRNA retention data in skin up to 8 weeks (Figure 2B),\nthis specific time point was limited in sample size (single biopsy at 8 weeks compared to\nn=2-3 at 4 weeks) and utilized the more potent JAK13033 siRNA sequence rather than\nthe pig-reactive JAK1883 sequence used in the efficacy study. Given these constraints,\nand the high cost associated with large animal studies, we chose the 4-week endpoint\nto ensure robust knockdown. The ongoing clinical investigation aims to assess clinical\nefficacy 4 months after the last injection day.\nBy combining many of our experiments where pharmacokinetic (PK) and\npharmacodynamic (PD) assessments were performed with different concentrations of\nsiRNA and different application modes (transepidermal, topical, or intradermal),\nwe\nwere able to estimate the minimum required level for silencing. In this approach, we\nonly included biopsies from time points where samples were collected at least 7 days\nafter the last application or injection to ensure that silencing had time to establish and\nthat non-functional, recently applied siRNA does not blur the PK accumulation without\npharmacodynamic activity. it allowed us to estimate that to achieve 50% silencing, a\nthreshold of 0.5 µg/g of JAK1883 is required in the dermis. However, many limitations\nexist to this approach (among them, for example: two different biopsies are used for PK\nand PD; different experiments or pigs are combined in the same graph; different modes\nof application, different timepoints from last application (7 to 28 days) etc.), which is why\nwe did not include this into a publication.\n5. Make sure the graph axis is consistent across different figure panels for better\nvisualization. For instance, in Fig. 3b, y-axis for CXCL levels in dermis vs. epidermis has\ndifferent axis points.\nFigure 4B y-axis have been adjusted and are now consistent\n6. Given the translational potential of this technology and the mention of an on-going\nPhase 2a human clinical trial, the authors should add a discussion about other delivery\nsystems (transdermal patches, ointments, and so on) that may be utilized as an\nalternative to painful intradermal injections where the intended treatment surface area is\nlarger.\nWe have now added a discussion on alternative delivery strategies beyond intradermal\ninjections, reproduced below:\n“Beyond intradermal injections, there are currently multiple advanced delivery systems\nbeing explored to administer therapeutics, to the skin.35 These non-invasive strategies\ncan potentially serve as alternatives for treating diseases that require coverage of much\nlarger skin surface areas. Key examples include microneedle patches (MNs) 36,37, which\ncreate transient microchannels for enhanced drug uptake, ointments and other topical\nformulations, and novel delivery enhancers utilizing materials such as ionic liquids38 and\nstar particles 39. These strategies can potentially be utilized as alternatives to deliver\ntherapeutic siRNAs to much larger skin surface areas for diseases that require bigger\ncoverage.”\nReviewer #3\nThis manuscript leverages a less commonly studied porcine model alongside a widely\nused ex vivo model, providing a valuable comparative perspective. The authors adeptly\nintegrate lipid chemistry and RNA chemistry to enhance gene silencing efficacy and\ntissue retention in the skin, which is the body's largest organ. Furthermore, the analysis\nof unconjugated, dendrimer-conjugated, and their docosanoic siRNA conjugates allow\nfor meaningful comparison and context within the field. Notably, the authors report\nincreased silencing in JAK1 for their leading siRNA conjugate in both human skin and\nthe porcine model. This work makes important advances in intradermal delivery\ntechnology. The inclusion of comparative data across different siRNA conjugates,\npresented in a more human-compatible porcine model, provides novel insight and may\ninform future research directions. In addition, the manuscript addresses a central\nchallenge in the field: extrahepatic delivery. Building on previous research, the authors\nprogress this area toward greater clinical relevance, as evidenced by the ongoing\nPhase 2a human clinical trial of the leading compound, DCA-JAK1. The manuscript is\ncompelling in its broad inclusion of both efficacy outcomes and comprehensive\ncytotoxicity analyses, clearly oriented toward translatable research with potential\npositive clinical impact. I recommend this manuscript for publication in Nature\nCommunications. However, I do have several comments/ questions that, if addressed,\nwould further strengthen the work.\nWe thank the reviewer for their comments and feedback\n1. Can authors offer any commentary on why they chose docosanoic acid as their\nlipophilic conjugate and their rationale as a whole regarding choosing their selected\nconjugates?\nWe thank the reviewer for their comment. DCA was chosen based on our previous work\nwhere we compared a much larger range of hydrophobic conjugates (Biscans et al\nNucleic Acid Research 2019) and DCA showed the highest retention in the skin, which\nwas then further confirmed to provide the strongest siRNA efficacy in skin (Tang et al\nMolecular Therapy 2022). Similarly, the recently developed Dendrimer conjugate (Fakih\net al Moleculary Therapy Nucleic Acid 2023) showed promising skin retention and\nactivity, while having an amphiphilic character and lower hydrophobicity than DCA,\nwhich warranted us to compare it to DCA in human skin tissue and larger animal models\nin the current study. We have elaborated the results section discussing the conjugate\nrationale to capture these reasons:\n“From the library of conjugates previously assessed, docosanoic acid (DCA)\ndemonstrated the highest skin retention and siRNA activity in mouse skin tissue, making\nit a compelling candidate for further evaluation.18,25 Additionally, a recently developed\namphiphilic dendrimer conjugate achieved effective skin delivery while being\nsignificantly less hydrophobic than DCA, warranting a direct comparison between the\ntwo.19”\n2. The authors relay that the docosanoic conjugate is the most lipophilic conjugate\ntested. While looking at chemical structure one could come to the same conclusion, it\nwould be nice to see an inclusion of some metric of lipophilicity of the selected\nconjugates showing this range, perhaps logP studies or some other analysis.\nWe thank the reviewer for the excellent suggestion. We have previously compared the\nhydrophobicity of the conjugates used in the form of retention time on reverse phase\nHPLC analysis, which clearly shows the hydrophobic nature of the tested compounds\nand providing a nice range of hydrophobicity tested. That work was published (Fakih et\nal Molecule Therapy Nucleic Acids 2 023) and the figure (Figure 1A bottom) has been\nreproduced below. We have also edited the main text to highlight this as mentioned in\nresponse to the first comment.\n[Figure Redacted]\n3. Docosanoic acid has been implicated in anti-inflammatory processes in the body. The\nauthors show that their DCA conjugated siRNA has an improved cytotoxic profile\ncompared to other conjugates. Could the authors comment or provide any data\nregarding if the anti-inflammatory of DCA may be implicated in this study? Would its\ninclusion further increase skin retention due to its lipophilicity and some method of\nanti-inflammatory processes?\nWe thank the reviewer for this comment. While Docosahexaenoic Acid (DHA), the\npoly-unsaturated version of Docosanoic acid (DCA), plays an important role in\nanti-inflammatory processes, we did not find any evidence of DCA’s role in inflammatory\nprocesses. This is due to DHA being a polyunsaturated fatty acid (22 carbon single\nbonds, 6 double bonds) which are precursors of biologically active lipid mediators.\nHowever, DCA is fully saturated (22 carbon single bonds,0 double bonds) lipid and does\nnot play an anti-inflammatory role. Therefore, we do not believe that the DCA conjugate\nin DCA-siRNA is playing an anti-inflammatory role, an assumption that is further\nsupported by our DCA conjugated non-targeting control which shows similar JAK1\nexpression to PBS (Figure 1D)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "This is an important study demonstrating that dermal delivry of ASOs targeting the Jak0Stat pathway can be used to ammeliorate inflammatory skin conditions. The concerns of the previous reviews have been adequately addressed.", "reviewer_2": "The authors have sufficiently addressed my comments, provided clarification where needed, and modified the main text accordingly. Overall, the study demonstrates compelling pre-clinical data, and presents a clear path for clinical trials to investigate their lead siRNA conjugate. I recommend the manuscript for publication in Nature Communications.", "reviewer_3": "Authors have effectively answered and responded to comments made by myself and other reviewers. This manuscript is even stronger. I recommended this manuscript for acceptance.\nVery minor detail: on line 95 \" 95 For that, We assessed the cellular uptake of cyanine-\" should be...\"For that, we assessed the cellular uptake of cyanine-\" lowercase we. Again very minor."}, "author_response": "We thank the reviewer for their feedback and we are glad we were able to address the concerns.\nWe thank the reviewer for their feedback and we are glad we were able to address the comments.\nWe thank the reviewer for their feedback and we are glad we were able to address the comments.\nWe have fixed this typo, we thank the reviewer for their keen eye."}]

Decision letter