Description:

Field of the Invention:

The present invention relates to novel inhibitors for inhibiting the expression of Fos-related antigen-1 (Fra-1) or Fos-related antigen-2 (Fra-2) primarily in fibrogenic tissue cells and the use in the treatment of organ or tissue fibrosis, in particular liver fibrosis, pulmonary fibrosis or kidney fibrosis. The inhibitor is characterized in that it interferes with Fra-1 or Fra-2 expression, resulting in a reduction in the amount of Fra-1 or Fra-2 in said fibrogenic tissue cells to levels found in non- fibrotic healthy cells or tissues.

Background of the Invention:

Fibrotic tissue remodelling in liver fibrosis or pulmonary fibrosis often results in advanced (severe) organ malfunction and is associated with a high morbidity and mortality. Organ fibrosis, especially of the liver, lungs and kidneys, is the cause of almost 50% of worldwide morbidity and mortality due to chronic diseases (Friedmann SL et al., 2013; Flockey DC. et al., 2015; Distler JHW. et al., 2019). Currently no effective antifibrotic therapy is available in the clinic. Thus, there is a need for effective antifibrotic therapies for the treatment of liver fibrosis and pulmonary fibrosis.

The main causes of liver fibrosis and its end-stage, cirrhosis, worldwide include chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, alcohol abuse, biliary and autoimmune liver diseases (like autoimmune hepatitis, primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC), various congenital and genetic liver diseases, alcoholic and especially nonalcoholic steatohepatitis (NASH), whose prevalence has increased dramatically in parallel with overweight and type 2 diabetes (Schuppan D. et al., 2008; Younossi ZM. et a!., 2019;

Schuppan D. etal., 2018). The accumulation of excess scar tissue, subsequent to chronic injury, is due to overproduction of key molecules of the extracellular matrix, such as collagens and noncollagen proteins, and proteoglycans/glycosaminoglycans, in an attempt to repair the “wound that does not heal”. While in early stages of fibrosis organ integrity and function can be maintained, fibrosis progression to liver cirrhosis usually leads to organ failure, liver cancer, a high morbidity and finally premature death. Importantly, in liver diseases that can be treated causally in many cases, such as viral hepatitis B or C, fibrosis often still progresses despite effective antiviral treatment. Moreover, once advanced fibrosis or cirrhosis have developed, fibrosis regression with improved liver function rarely occurs. The excess synthesis and deposition of extracellular matrix proteins, synthesized mainly by activated hepatic stellate cells and myofibroblasts, replaces functional liver cells (mainly hepatocytes) by nonfunctional scar tissue and distorts the hepatic vasculature, which results in liver functional failure and/or extrahepatic complications like portal hypertension with e.g. bleeding esophageal or gastric varices, formation of ascites, coagulation disorders, hepatic encephalopathy and predisposition to septic complications. Moreover, cirrhosis increases the risk of developing a usually incurable primary hepatocellular carcinoma (HCC) by 100 to 200-fold, with a yearly incidence between 2 and 10% {Schuppan D. et al., 2008).

Similar molecular and cellular mechanisms, ECM molecules and ECM-producing cells, as they occur, for instance, in liver fibrosis also drive the development of pulmonary fibrosis and fibrosis of other mesenchymal-epithelial organs such as kidneys, skin, pancreas or the gastrointestinal tract {Friedman SL et a!., 2013; Rockey DC. eta!., 2016). Therefore, targeted antifibrotic therapies, i.e., therapies that interfere with fibrosis-specific mechanisms, cells or molecules, that are effective to inhibit fibrosis in liver or lung fibrosis, are also very likely to be effective in fibrosis of kidneys, skin, pancreas or the gastrointestinal tract, especially when they are directed towards these organs. That said, the cell biology, pathophysiology and the signaling pathways and molecules involved in liver, lung and kidney fibrosis share astonishing similarities. This means that most targeted antifibrotic therapies that work in one organ will likely be effective when target to another organ undergoing fibrosis.

Pulmonary fibrosis often develops in the context of environmental exposures such as certain toxins or allergens, or as an accompaniment of an (autoimmune) syndrome (Glass DS. eta!., 2020; Renzoni EA. et al., 2021). Common toxic and pro-inflammatory causes are exposure to asbestos, metal dusts or organic substances, radiation or medical drugs. In addition, severe microbial infections, hypoxia, hyperoxia or unknown causes (idiopathic pulmonary fibrosis) can lead to lung fibrosis. Similar to liver fibrosis, the disease is characterized by chronic inflammation and excess scar tissue (“collagen”) production and deposition with consequent distortion of lung vascular and airway structures including alveolar wall thickening, all leading to impaired pulmonary gas exchange, hypoxia, hypercapnia and pulmonary hypertension. Similar to liver fibrosis, pulmonary fibrosis dramatically increases the risk of lung cancer.

Kidney fibrosis results from autoimmune diseases, such as systemic lupus or IgA nephropathy, metabolic-vascular diseases such as in diabetes or nephrotoxins (Ruiz-Ortega M. et a!., 2020; Distler JHW. et al., 2019). Again, chronic inflammation and/or direct cellular damage (tubular or renal epithelial damage, similar to bile ductular damage in the liver or alveolar type 2 cell damage in the lungs). This leads to mesangial or interstitial fibroblast activation (similar to hepatic stellate cells and portal fibroblast activation in the liver, or to alveolar and interstitial lung fibroblast activation in the lungs, respectively). The result is mesangial (glomerular) or interstitial kidney fibrosis, with kidney failure as end stage condition.

There is an increasing interest in developing antifibrotic programs. However, efficacy of potentially promising therapeutic approaches has not yet been proven in humans, despite a vast potential market for effective antifibrotic agents (Friedmann SL. et a!., 2013; Schuppan D. et al., 2018). Moreover, current preclinical in vivo models of liver fibrosis are often not performed according to best standards and do often not replicate human chronic and fibrotic liver diseases which may result in disappointing phase 1-2 clinical trials (Popov Y. et al., 2009; Kim YO. etal., 2017; Farrell G. et al., 2019). Lack of clinical trials is due to the requirement of long follow-up studies and to the fact that liver biopsy, an invasive procedure, is still the gold-standard method for detecting changes in fibrosis. Notably, the requirement for long-term follow up in clinical phase 1-2 trials will likely not be necessary, since prediction of antifibrotic effects in liver but also in lung and kidney is now possible via the development of sensitive serum/plasma protein biomarkers of fibrosis and fibrogenesis (Karsdal MA. et al., 2020; Schuppan D. etal., 2021).

Authoritative and recent reviews have summarized the current state of the art of antifibrotic drug development for fibrosis of the liver (Friedmann SL. et al., 2013; Distler JHW. et al., 2019; Ruiz- Ortega M. et al., 2020; Schuppan D. et al., 2013; Schuppan D. etal., 2018), lungs (Friedmann SL. et al., 2013; Distler JHW. et al., 2019; Ruiz -Ortega M. et al., 2020; Schuppan D. et al., 2013; Schuppan D. etal., 2018) and kidneys (Friedmann SL. et al., 2013; Distler JHW. et al., 2019; Ruiz-Ortega M. et al., 2020; Schuppan D. etal., 2013; Schuppan D. et al., 2018).

Fos-related antigens-1 and -2 (Fra-1 and Fra-2) belong to the activator protein 1 (AP1 ) family of transcription factors and are involved in a broad variety of cellular processes, such as proliferation or differentiation. Fra-1 and Fra-2 form homodimers and heterodimers as part of a complex mode of transcriptional regulation, and are induced by a large variety of cellular signals. Mice that are transgenic for Fra-2 in all cells and overexpress Fra-2 constitutively develop a pulmonary and skin fibrosis, while mice overexpressing Fra-1 in all cells and constitutively, develop biliary fibrosis resembling human primary sclerosing cholangitis (Reich N. et al., 2010; Eferl PNAS, 2008).

Fra-2 is the most recently discovered and least described member of the AP-1 family of transcription factors. The Fra-2 gene consists of five regions homologous to other Fos proteins as members of the AP-1 family, including the basic leucine-zipper motif responsible for dimerization, but it differs from c-fos in lacking the strong transactivation domain J. Hess etal., 2004). Elevated Fra-2 expression has been described in several chronic lung diseases, such as chronic obstructive pulmonary disease, asthma and pulmonary fibrosis. Several stimuli can cause transcriptional upregulation of Fra-2, including phorbol esters (e.g. TPA), increased cAMP and Ca ²⁺ levels (Yoshida T. et al., 1993) and diverse growth factors such as PDGF-BB or TGFp (Biasin V. et al., 2014; Reich N. et al., 2010).

The presence of an AP-1 binding site in the promoter region of Fra-2 also suggests auto- regulatory mechanisms and cross regulation between different AP-1 family member subunits ( Yoshida T. et al., 1993; Sonobe M.H. etal., 1995). Indeed, Fra-2 expression can be induced by binding of the general AP-1 dimeric transcription factor c-jun/c-fos to the Fra-2 promoter. Once Fra-2 translation is activated, Fra-2 becomes highly abundant and the c-jun/c-fos dimers are replaced by a c-jun/Fra-2 complex, which is reported to have a lower transcriptional activity compared to c-fos/c-jun (Suzuki T. etal., 1991; Sonobe M.H. et al., 1995), suggesting an auto- regulatory negative feedback loop of Fra-2 expression. Fra-2 expression can also be influenced by epigenetic modifications, such as trimethylation of histone 3 (H3), which inhibits Fra-2 expression, whereas a lack of H3 trimethylation leads to increased Fra-2 expression (Kramer M. et al., 2013).

Fra-2 activity is influenced by TGF|3 (W. Tang, et al., 1998); J. Yue, et al., 2000) and by PDGF- BB (Reich N. etal. 2010), or by pro-inflammatory Th2 T cell cytokines such as IL-13 (Fichtner- Feigl S. et al., 2006). MAP kinases induce phosphorylation of Fra-2 in vitro to a similar extent and in a similar pattern as observed in vivo (Gruda M.C. etal., 1994). Phosphorylation of Fra-2 by MAP kinases increases its DNA binding activity (Gruda M.C. etal., 1994; Zoumpourlis V. et al., 2000).

Fra-2 has a role in the regulation of cell growth and differentiation, as well as in tissue homeostasis where it appears to integrate intra- and extracellular signals. Therefore, if aberrantly expressed, Fra-2 may be involved in the development of chronic diseases, especially in organs where it is normally expressed, such as the lung (Foletta V.C. et al., 1994). In healthy adults, lung immunostaining identified strong Fra-2 expression in some bronchial epithelial cells, vascular smooth muscle cells and in alveolar macrophages (Eferl R.et al., 2008; Biasin V. et al., 2014; BirnhuberA. et al., 2019; Ucero A.C. etal., 2019). Elevated levels of Fra-2 were reported in fibrotic lungs (and skin) of patients with systemic sclerosis (SSc) (Eferl R. et al., 2008; Reich N. et al., 2010; Maurer B. et al. 2009), interstitial lung disease and idiopathic pulmonary fibrosis (Eferl R. etal., 2008; Ucero A.C. et al. 2019), in the vasculature of patients with pulmonary hypertension (Biasin V. et al. 2014), and in pulmonary macrophages from patients with chronic obstructive pulmonary disease (Kent L et al. 2008).

A recent study showed an improved phenotype in bleomycin-induced pulmonary fibrosis, a model that is frequently used to model lung fibrosis, but that also shows limited resemblance to human pulmonary fibrosis, upon treatment with a general AP-1 family inhibitor and claimed that AP-1 inhibition should be considered as a therapeutic target for the treatment of pulmonary fibrosis Ucero A.C. et al. 2019). The AP-1 inhibitor T-5224 was used in order to investigate if AP-1 inhibition ameliorates fibrosis in the bleomcycin model and in Fra-2 ^Tg mice. However, it remains unclear how far this effect in the bleomycin lung fibrosis model was due to specific Fra-2 inhibition.

In the cause of fibrosis research and clinical translation, the expression of Fra-1 or Fra-2 has not been addressed by a specific- and organ-targeted pharmacological treatment. This is namely due to the lack of specific small molecule inhibitors and a lack of organ- and cell-specific targeting of of Fra-1 or Fra-2. Importantly, how far Fra-1 or Fra-2 are responsible for fibrosis in a specific organ with normal regulation of these transcription factors - as opposed to the constitutively active transgenes in all cells of the body, remains unclear. Moreover, it is likely that side effects will occur, especially when all AP-1 family members are therapeutically addressed, but also when an inhibitory drug is nor organ specific, necessitating target organ (and cell) specific delivery

EP1855524B1 describes a transgenic mouse that overexpresses the transcription factor Fos- related antigen 2 (Fra-2) in all cells of the body (Eferl PNAS, 2008). It was concluded that it may serve as a suitable model to study fibrotic lung and kidney diseases, since their transgenic mouse resulted in a phenotype resembling human pulmonary fibrosis and skin fibrosis resembling human scleroderma. However, it remained unclear how this reflects human pathology and what pathways were involved. Moreover, Fra-2 was overexpressed in all cells of the transgenic mouse and at a constant level, which cannot be expected to be the case in human fibrosis or in induced experimental fibrosis. Moreover, the cells that would overexpress Fra-2 in naturally occurring fibrotic diseases, or would primarily dive a fibrotic response dependent on Fra-2, remain ill- defined until today.

WO2014/178427A1 describes an agent for enhancing or suppressing the expression of semaphorin 3A. Serna 3A expression was significantly suppressed by suppressing the expression of GABPa, Sox 9, CREB, Jun B, Jun D, Fra-2. of the Invention:

Against this background it is the object of the present invention to provide alternative agents and pharmaceutical compositions that are suitable for the prevention or treatment of organ or tissue fibrosis, in particular for the treatment of lung fibrosis, liver fibrosis, pancreas fibrosis, or kidney fibrosis.

This object is solved by an inhibitor for Fra-1 or Fra-2 comprising the features of claim 1 . Preferred embodiments of the present invention are the subject-matter of the dependent claims.

The present invention identifies a novel in vivo target that is suitable for the treatment or prevention of organ fibrosis or tissue fibrosis, in particular for the treatment or prevention of an organ or tissue fibrosis that is kidney fibrosis, pancreas fibrosis, pulmonary fibrosis, vascular vessel fibrosis, skin fibrosis, bone marrow fibrosis, or liver fibrosis. As shown by the present invention, inhibition of Fra-1 and/or Fra-2 expression is a suitable target that can be utilized in the treatment or prevention of organ or tissue fibrosis in general. More specifically, the inhibitor of the invention is a molecule that interferes with Fra-1 or Fra-2 expression, resulting in a reduction in the amount of Fra-1 or Fra-2 in said fibrogenic cells to levels found in non-fibrotic healthy cells or tissues. Preferably, the inhibitor is a molecule that specifically and effectively interferes with Fra-1 or Fra-2 transcription, Fra-1 or Fra-2 post-transcriptional mRNA processing or Fra-1 or Fra-2 mRNA translation. As such the expression levels for Fra-1 or Fra-2 may refer to any stage of protein production, i.e. on transcription level, post-transcription level, translation level, or post post translation level.

Organ fibrosis as used in the context of the present invention is characterised by a loss of cellular homeostasis and disruption of the normal tissue architecture. Typical organ fibrosis is kidney fibrosis, pancreas fibrosis, pulmonary fibrosis, vascular vessel fibrosis, skin fibrosis, bone marrow fibrosis, or liver fibrosis.

Liver fibrosis as used in the context of the present invention refers to a condition in which a damaged liver tissue is transformed into a fibrotic tissue. The disease is associated with an excessive accumulation of extracellular matrix proteins (ECMs) including collagen that occurs in most types of chronic liver diseases.

Pulmonary fibrosis as used in the context of the present invention relates to a lung disease that occurs when lung tissue becomes damaged and scarred. Kidney fibrosis as used in the context of the present invention relates to a kidney disease that occurs when kidney tissue becomes damaged and scarred.

The present invention is based on the surprising finding that in fibrotic wild type mice, Fra-1 and Fra-2 is dominantly expressed in activated cholangiocytes, in macrophages and in activated myofibroblasts. Fra-1 or Fra-2 protein is equally overexpressed in affected fibrotic liver cells as compared to Fra-1 or Fra-2 protein found in normal, healthy cells. Similarly, the expression levels of Fra-1 or Fra-2 are significantly higher in affected fibrogenic cells than the respective expression levels in normal, healthy cells.

The inhibitors of the present invention are characterized in that they interfere with the expression apparatus of Fra-1 or Fra-2, i.e. Fra-1 or Fra-2 transcription, mRNA processing or mRNA translation, thereby reducing the amount of Fra-1 or Fra-2 protein in said fibrogenic tissue cells to levels comparable to the expression levels of Fra-1 or Fra-2 in non-fibrotic healthy cells.

Preferably, the fibrogenic tissue cells are selected from the group consisting of liver cells, lung cells, kidney cells and skin cells. In a preferred embodiment, the inhibitor perturbs Fra-1 or Fra-2 expression by gene silencing or protein silencing mediated by siRNA-LNPs of the present invention.

It has been found that inhibition of Fra-1 and/or Fra-2 transcription factor in target cells that overexpress Fra-1 and/or Fra-2 is associated with antifibrotic activity. As demonstrated herein, the application of an inhibitor of the present invention in vivo resulted in a down-regulation of the expression of Fra-1 or Fra-2 in the affected fibrotic cells, as exemplified in hepatic, pulmonary and renal tissue. Target cells that previously overexpressed Fra-1 or Fra-2 showed expression levels that did not significantly differ from the expression levels of Fra-1 or Fra-2 found in normal, healthy cells. Most importantly, treatments with Fra-1 and/or Fra-2 inhibitors in form of siRNA- LNPs (siRNA lipid nanoparticles) did not show any unwanted (e.g. pro-inflammatory or toxic) side effect post transfection.

A preferred inhibitor of Fra-1 or Fra-2 expression therefore comprises small interfering RNA (siRNA) operating within the RNA interference (RNAi) pathway. In alternative embodiments, the invention also comprises small molecules that inhibit Fra-2 activity.

Alternatively, also miRNAs can be used which are derived from regions of RNA transcripts that fold back on themselves to form short hairpins. They are usually selected from shorter regions of dsRNA and mediate silencing of genes by repression of translation. In alternative embodiments, the inhibitor is microRNA (miRNA) comprising a nucleotide sequence that is at least partially complementary to a nucleotide sequence of Fra-1 or Fra-2 mRNA, or parts thereof.

In a further alternative embodiment, the inhibitor is an antisense oligonucleotide (ASO) comprising a nucleotide sequence that is at least partially complementary to a nucleotide sequence encoding Fra-1 or Fra-2, or parts thereof. Preferred ASOs are single-stranded antisense oligonucleotides that inhibit the expression of Fra-1 and/or Fra-2. In preferred embodiments, said ASO molecule comprises one or more modifications, including, but not limited to at least one locked nucleic acid (LNA) molecule, 2’-sugar modification, modified internucleotide linkage or a combination thereof.

The invention also comprises Fra-2-specific ASOs that do not require lipid or other nanoparticle for efficient delivery, especially to the liver, but also other organs like lungs or kidneys, and here preferably to non-epithelial cells. In preferred embodiments, the administration of such pharmacologically active gents such as ASOs is conducted via parenteral administration, preferably subcutaneous, intraperitoneal or intravenous administration. In humans, subcutaneous administration is preferred, whereas in animals the agents are preferably administered intraperitoneally or intravenously in order to produce a comparable knockdown and antifibrotic effect, as demonstrated by the present invention. Importantly, in preferred embodiments, the ASOs as used in the present invention can be produced in a standardized fashion, and that do not have solid method IP character. Importantly, the ASO therapy can be transferred to human phase 1 -2 studies using biomarker-assisted efficacy readouts. This therapeutic approach is very safe and does not rely on a lipid component.

A preferred siRNA inhibitor according to the present invention comprises a nucleotide sequence that is at least partially complementary to a nucleotide sequence of Fra-1 or Fra-2 mRNA, or parts thereof. siRNA comprising such complementary nucleotide sequences interferes with the expression of Fra-1 or Fra-2 by degrading their mRNA after transcription, thereby preventing Fra- 1 or Fra-2 translation. A preferred siRNA of the present invention is a double-stranded RNA (dsRNA) having a length of preferably between 15 to 30 bp, preferably between 20 to 25 bp. In a preferred embodiment, siRNAs of the invention can be equipped with optional overhanging nucleotides and can be modified by any method known in the art to, e.g., in order to increase stability.

By using siRNA that is complementary to the Fra-1 or Fra-2 nucleotide sequence, the expression of Fra-1 or Fra-2 can be silenced in an efficient way such that the affected cells no longer overexpress Fra-1 or Fra-2 transcription factor. Preferably, the siRNAs of the present invention are synthetic siRNAs that induce RNA interference in the fibrosis inducing liver, lung, kidney or cells.

The inhibitors of the present invention may also mediate siRNA-induced post-transcriptional gene silencing by cleaving mRNA molecules encoding for Fra-1 or Fra-2 using the RNA-induced silencing complex (RISC) pathway.

Alternatively, siRNA can be expressed by using an expression vector, in order to obtain a durable knockdown or gene silencing in the affected fibrotic liver or lung cells.

In a preferred embodiment, the siRNA inhibitor for inhibiting Fra-1 or Fra-2 expression according to the invention comprises a nucleotide sequence which comprises the following nucleic acid sequences or parts thereof. The invention also covers mutants, variants or derivatives of these sequences. Variants my be shortened, elongated or chemically modified.

Sense: 5’-GCUCACCGCAGAAGCAGUAUU-3’ (SEQ ID NO: 1 )

Antisense: 5 -UACUGCUUCUGCGGUGAGCUU-3’ (SEQ ID NO: 2)

Preferred siRNA inhibitors for inhibiting Fra-1 or Fra-2 expression contain additional modifications, such as 2’-O-methyl groups in every 2 ^nd C and/or phosphorothioate at the 3’ ends. Additional substitutions, such as 2’F-pyrimidines, or locked nucleotides can be introduced to enhance siRNA stability. The invention also covers a fragment of a nucleic acid as defined in SEQ ID NO: 1 or SEQ ID NO: 2 that retains the ability to inhibit Fra-2 expression. The invention also covers mutants of these nucleic acid sequences in which one or more nucleic acids, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleic acids, are substituted, chemically modified or deleted in the nucleic acid sequences of SEQ ID NO: 1 or SEQ ID NO: 2. The invention also covers siRNA which comprises nucleic acids that are extended versions of nucleic acid sequences of SEQ ID NO: 1 or SEQ ID NO: 2.

In another aspect of the invention, direct derivatization of siRNA (or ASOs) is possible with covalently coupled sugars that can direct the inhibitor to surface receptors of specific cells. Examples include but are not limited to mannose derivatization for targeting the mannose receptors on macrophages, or mannose-6-phosphate for targeting fibroblasts/myofibroblast/stellate cells and endothelial cells. In order to specifically direct siRNA that is directed against Fra-1 or Fra-2 to the affected fibrosisinducing organ or tissue cells, the siRNA is preferably complexed with a lipid-mediated nucleic acid carrier. Preferably, the lipid-mediated nucleic acid carrier is composed of lipid nanoparticles (LNPs) formulated as Lipoplex. Lipoplexes are nano-structured complexes that have been shown to be useful vehicles in the therapeutic context of the present invention in particular for the treatment or prevention of organ fibrosis, such as liver fibrosis, pulmonary fibrosis or kidney fibrosis.

Modulating the ratio of cationic lipids and nucleic acids affects the binding of the vectors with negatively charged cell surfaces. Lipoplex suspensions are known to be unstable in aqueous suspension for long-term storage, especially with respect to hydrolysis and size stability. In a preferred embodiment, the Lipoplex is therefore composed of lipidoid, cholesterol, 1 ,2-dioleoyl- sn-glycero-3-phosphatidylcholine (DSPC), 1 ,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG) and siRNA.

Preferably, the lipidoid used in the lipoplex of the present invention comprises the following structure: wherein Ox is selected from the group consisting of C12H25 (dodecyl), C11H23 (undecyl), C13H27 (tridecyl), or C14H29 (tetradecyl).

In alternative embodiments, higher delivery efficiency, correct particle size and zeta potential can be achieved by variations in a molecular group of said lipidoid. Examples include but are not limited to lipid nanoparticle curvature and thus size, and low in vivo toxicity or unspecific immune stimulation.

The inventive lipidoid nanoparticles mediate potent gene knockdown of Fra-1 or Fra-2 transcription factor in fibrosis-inducing (e.g. liver, lung and kidney) cells upon administration to a subject that is in need thereof.

The inventors found that siRNA-LNP mediated protein silencing in vivo can be significantly increased by altering the structural composition of the nanoparticles. Usually, the same efficiency obtained in vitro cannot be obtained in vivo. In a preferred embodiment, potent lipidoids for use in conjunction with siRNAs of the present invention are synthesized from acyl-amines with three or more substitution sites.

In a preferred embodiment, the siRNA-lipoplexes contain helper lipids such as cholesterol, DSPC and PEG2000-DMG. The choice of helper lipids did not affect the relative efficiency of the lipidoid compounds of the present invention. Lipoplex nanoparticles composed of lipidoid, cholesterol, DSPC, PEG200-DMG and siRNA against Fra-1 or Fra-2 were optimized with four aliphatic side chains and further optimized in size and zeta potential after loading with siRNAs using microfluidic or dual centrifugation technology. As further efficiency criteria, the particle size of the LNPs comprise a diameter that ranges preferably from 50 to 200 nm, preferably from 50 to 120 nm, which makes them suitable to reach the target cells in the affected liver, lung or kidney tissue. The invention also encompasses particles having a desired value within this range, or may be produced within any range within the given range of 50 to 200 nm. If the diameter of the nanoparticles should be too large, efficient delivery to a fibrotic lung, kidney tissue or liver tissue is negatively affected. Furthermore, it should be considered that lipidoids are expected to be degraded in the presence of cellular or tissue enzymes, including precursor enzymes in the blood, particularly as they raise when these LNPs are transfected in vivo.

Preferably, the siRNA-LNPs of the present invention were formulated at a lipidoid:cholesterol:DSPC:PEG molar ratio of 50:38,5:10:1 ,5.

In a preferred embodiment, the siRNA-LNPs of the present invention are formulated using the following ingredients and concentrations:

50% lipidoid 10% DSPC 38,5% cholesterol 1 ,5% DMG-PEG2000

The surface pKa of the LNPs of the invention also plays an important role in increasing in vivo efficiency. A critical pKa value averages at 5,5. For values less than 5,5, average efficiency decreased monotonically with pKa. Therefore, it is preferred that the pKa is greater or equal to 5,5. To increase efficiency, the LNPs of the present invention preferably contain one or more tertiary amines, 013 tail, or more than two tails with a pKa > 5,4 in order to achieve more than 80 % protein silencing. Preferably, the zeta potential of the inventive LNPs ranges from 0 to 100 mV, preferably > 0 to 50 mV. All intermediate values or ranges within the range of 0 to 50 mV are comprised by the present invention.

As shown herein, the siRNA loaded LNPs of the present invention result in a knockdown of Fra-1 or Fra-2 expression in the target cells when intravenously injected into fibrotic mice, thereby preventing the development of or significantly reducing liver fibrosis in vivo in models of CCI4- induced parenchymal fibrosis and in spontaneously developing secondary biliary fibrosis (Mdr2KO mice). In both models, delayed Fra-2 lipoplex treatment reduced Fra-2 transcript levels by at least 80 %, and liver fibrosis was reduced by more than 50 %.

The present invention also relates to pharmaceutical compositions, comprising an agent for inhibiting the expression of Fos-related antigen-1 (Fra-1) or Fos-related antigen-2 (Fra-2) in fibrogenic tissue for use in the treatment of organ or tissue fibrosis as described by the present invention. In a preferred aspect of the invention, the pharmaceutical composition of the present invention is suitable for the prevention or treatment of kidney fibrosis, pancreas fibrosis, pulmonary fibrosis, vascular vessel fibrosis, skin fibrosis, bone marrow fibrosis, or liver fibrosis.

The pharmaceutical composition of the present invention can include carriers, diluents, excipients or mixtures thereof commonly used in biological preparations. The pharmaceutically acceptable carrier can be any carrier that is able to deliver the composition of the present invention in the living body without limitation such as saline, sterilized water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture thereof. If necessary, a general additive such as antioxidant, buffer, and bacteriostatic agent can be additionally added.

When formulating the pharmaceutical composition according to the present invention, generally used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants can be added.

The composition of the present invention can be formulated as an oral or parenteral preparation. Oral preparations can include solid formulations and liquid formulations. In one aspect, the solid formulation can be tablets, pills, powders, granules, capsules or troches. Such solid formulation can be prepared by adding at least one excipient to the composition. The excipient can be starch, calcium carbonate, sucrose, lactose, gelatine, or a mixture thereof. In addition, the solid preparation can contain lubricants such as magnesium stearate and talc. In preferred embodiments, the liquid formulation can be suspensions, solutions, emulsions or syrups. In this case, the liquid formulation can contain excipients such as wetting agents, sweetening agents, fragrances, and preservatives. In preferred embodiments, the parenteral preparation can include injections, suppositories, powders for respiratory inhalation, spray aerosols, powders and creams. The injection can include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, and the like. Non-aqueous solvent or suspension, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, or injectable esters such as ethyl oleate can be used.

The composition of the present invention can be administered orally or parenterally according to a desired method. Parenteral administration can include intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection.

The invention also relates to methods of treating or preventing a disease associated with organ or tissue fibrosis in a subject comprising administering to the subject a pharmaceutical composition as defined herein. In preferred embodiments, the fibrogenic organ or tissue cells are selected from the group consisting of liver cells, lung cells, kidney cells, pancreas cells, or skin cells. In further preferred embodiments, the organ or tissue fibrosis is selected from the group consisting of kidney fibrosis, pancreas fibrosis, pulmonary fibrosis, vascular vessel fibrosis, skin fibrosis, bone marrow fibrosis, or liver fibrosis.

In preferred embodiments, the method comprises the administration of ASOs comprising a nucleotide sequence that is at least partially complementary to a nucleotide sequence encoding Fra-1 or Fra-2, or parts thereof. Preferred ASOs in the composition are singlestranded anti-sense oligonucleotides ASOs that inhibit the expression of Fra-1 and/or Fra-2.

In preferred embodiments in the compositions or methods of the invention, Fra-2-specific ASOs that do not require lipid or other nanoparticle for efficient delivery are used. This allows delivery to non-epithelial cells, in particular non-epithelial of the organs (e.g. liver) to be treated. In preferred embodiments, the administration of ASOs is conducted via parenteral administration, preferably subcutaneous, intraperitoneal or intravenous administration.

The pharmaceutical composition can be administered by the pharmaceutically effective amount to the subject to be treated, i.e. a human or animal. The effective amount can be determined according to the type of disease, the severity, the activity of the drug, the patient's sensitivity to the drug, the time of administration, the route of administration, the duration of treatment, the drugs being used simultaneously, and the like. The composition of the present invention can be administered alone or in combination with other therapeutic agents. In combination administration, the administration can be sequential or simultaneous. In a preferred embodiment, the method of treatment includes the step of monitoring the progress of treatment using one or more serum/plasma protein biomarkers of fibrosis and fibrogenesis.

The invention is explained in more detail in the following examples.

In the following examples, murine 3T3 fibroblasts, cholangiocytes and macrophages were transfected with increasing doses of different Fra-2 or control-luciferase siRNAs. Major fibrosis- and inflammation-related transcripts (coll a1 , acta2, timpl ) were determined at different time points post transfection. BALB/c mice were treated in vivo with escalating doses of CCL4 for four weeks to induce advanced liver fibrosis. One group received optimized Cy5-labeled Fra-2 siRNA- lipoplexes four times intravenously from week 3 to 4, and two control groups received luciferase- siRNA-lipoplexes or PBS. The same procedure was applied to Mdr2KO mice that develop spontaneous biliary fibrosis from age 8 to 10 weeks. Liver fibrosis was assessed by Sirius Red morphometry and hydroxyproline quantification. The expression of fibrosis and inflammation- related genes was assessed by qPCR.

Compared to mock siRNA-treated controls, the expression of coll al , acta2 and timpl in TGFbl - stimulated fibroblasts was downregulated 2-fold, in correlation with efficient Fra-2 knockdown. In vivo, using near infrared whole-body imaging, the Fra-2 siRNA-lipoplexes were entirely located in the liver two hours after injection. Fra2-siRNA lipoplex treatment, although being started in a later phase of fibrogenesis, resulted in significant inhibition of liver fibrosis, usually 50 %, with collagen levels close to normal controls in CCI4-induced parenchymal fibrosis, and with no further fibrosis progression in Mdr2KO mice. This was accompanied by an up to 4-fold downregulation of Coll al and other fibrosis related transcripts. No signs of toxicity were observed. Comparable results can be obtained with Fra-1 siRNA nanoparticles.

Moreover, the present invention confirms results using an alternative knockdown strategy, i.e., the use of mRNA neutralizing antisense oligonucleotides (ASOs) that can be employed without a carrier lipid (parenterally) and that mainly target the liver, and here prominently nonparenchymal (non-hepatocyte) cells, such as macrophages, (myo-) fibroblasts, but also activated fibrogenic stem cells/cholangiocytes (Weng SY et al., 2018).

As shown herein, 6 out of 7 tested Fra-2 neutralizing ASOs reduced Fra-2 transcripts by up to 70% in fibroblasts, macrophages and cholangiocytes (the key cell types involved in liver fibrogenesis), which was accompanied by markedly decreased profibrotic and increased antifibrotic transcripts, by up to 80%, such as for the key fibrogenic effectors coll a1 , Timpl or Mmp9 in these cells, with variable modulation according to the used ASO, the cell type studies, and also with a highly significant modulation of certain inflammation-related genes. Most notable was a dramatic increase in macrophage genes, such as iNos (up to more than 100-fold) that is implicated in (antifibrotic) M1-type polarization, but also modulation of II4 and Timpl (down) or Tnfa, Mmp9, 1110 (up). No in vitro cellular toxicity was observed up to 200 pg/ml.

The effect of ASO7 was also investigated in the MDR2-/- model of spontaneous biliary fibrosis, resembling the human, currently untreatable, progressive biliary liver disease primary sclerosing cholangitis. 24 male 8-week-old MDR2-/- mice were divided into three groups of eight animals each. To determine the degree of fibrosis prior to treatment initiation, at eight weeks of age, the first group was sacrificed untreated. The two remaining groups were treated with either ASO7 or PBS. The amount administered was 50 mg/kg. Administration was performed twice weekly by i.p. Injection for a period of four weeks in total. After 12 weeks, both groups were sacrificed and samples were preserved for further processing.

As for the CCL4-induced fibrosis, serum chemistry for in vivo liver toxicity only showed a slight increase of AST in untreated and ASO7 treated mice. ALT, ALP, creatinine and bilirubin remained normal compared to 8-week-old untreated Mdr2KO mice, indicating lack of liver and renal toxicity. Hepatic hydroxyproline (total collagen) content, the key quantitative biochemical parameter of fibrous tissue accumulation, was highly significantly reduced by factor of two compared to the PBS-treated controls. Similar results were obtained by morphometric quantification of collagen by Sirius Red morphometry (factor 2.5 reduction).

To further confirm the therapeutic efficacy of Fra-2 knockdown by ASO technology, fibrosis relevant hepatic transcript levels were determined. Quantitative RT-PCR analysis confirmed a significant reduction of profibrogenic hepatic transcripts in ASO7 treated Mdr2KO mice, such as asma (acta2), coll a1 , timpl , and a strong upregulation of putatively fibrolytic mmp9. Immunohistochemistry showed a significant reduction of CD68 positive (M2-type, profibrogenic) macrophages.

Finally quantitative high-end proteomic analysis was performed on age matched healthy (wildtype, non-fibrotic) FVB strain controls compared to Mdr2KO mice treated with Fra-2 ASO7 or PBS (control). This label-free proteomic technology allows detection and quantification of >2,500 proteins in complex tissues like liver (Kaps et aL, 2022). 3 samples of each group were analyzed and each sample was tested for reproducibility in 4 replicates. The results indicate a near normalization of the most up- and downregulated proteins in untreated fibrotic Mdr2KO mice, which further supports the unique antifibrotic effect of suppressing Fra-2 via ASO- and siRNA- mediated knockdown in vivo, and likely also by specific small molecules to be developed.

It therefore can be concluded that Fra-2 or Fra-1 is an attractive target for antifibrotic therapy wherein optimized Fra-1 or Fra-2-siRNA-loaded LNPs show a strong antifibrotic activity when injected in advanced phases of hepatic fibrogenesis.

Description of the Figures:

Fig. 1 Lipoplex formulation and characterization.

A. Lipoplex composition: a) Proprietary Lipidoid: dodecyl 3-[3-[3-[bis(3-dodecoxy-3- oxo-propyl)amino]propyl-methyl-amino]propyl-(3-dodecoxy-3-ox o- propyl)amino]propanoate (formula C67H131 N3O8, M.W. [g/mol]: 1106,8); b-d) standard lipoplex components. B. Microfluidic technology and setup. C. CryoTEM characterization of siRNA-lipoplex. D. Dynamic light scattering analysis of lipoplex. E. zeta potential distribution (mV).

Fig. 2 Cellular uptake and lack of toxicity of proprietary LNP-siRNA-lipoplexes in vitro. A. (proprietary) LNP-sFra2 uptake in 3T3 fibroblasts. Blue fluorescence: nuclei stained with DAPI; red fluorescence: LNP-Cy5-siFra2; green fluorescence: cytoplasmatic membranes stained by lipophilic carbocyanine dye DiO. B. Cell viability in murine 3T3 fibroblasts, 603B cholangiocytes and bone marrow derived macrophages (BMDM, M2 polarized). Controls are the non-transfected cells, the cells treated with naked 25 or 50 nM of Luc-control siRNA, and the cells treated with DMSO for 10 min (dead cells). siRNA transfection was performed with 25nM or 50nM siRNA using EndoFectin or proprietary lipoplex.

Fig. 3A-B LNP-siFra knockdown in 3T3 fibroblast (A) and 603B cholangiocytes (B). Relative gene expression normalized to GAPDH transcript levels. Naive: untreated cells. Negative controls: cells transfected with either EndoFectin-siLuc or LNP-siLuc. Positive control: cells transfected with EndoFectin-siFra2. Comparisons with LNP- siFra2 transfection. Means ±SEM; n=3-5 per test. siFra2 transfection using either Endofectin or LNP-siFra2 reduce transcript levels of fra2, collal and acta2 (encoding a-sma) in activated fibroblasts by 50-60%, and expression of fra2, but not of tgfbl , by 60-70% in activated cholangiocytes. ****p <0.0001 ; *p <0.05; **p <0.01 (all naive vs. LNP-siFra2). Fig. 3C-D LNP-siFra2 induced knockdown in MO (C) and M2 (D) polarized macrophages reduces M2-type and increases M1 -type macrophage transcripts. Relative gene expression normalized to GAPDH transcript levels. Naive: untreated cells. Negative controls: cells transfected with either EndoFectin-siLuc or LNP-siLuc. Positive control: cells transfected with EndoFectin-siFra2. Comparisons with LNP-siFra2 transfection. MO: naive bone marrow derived macrophages (BMDM); M2: M2 polarized BMDM using IL-4 and IL-13. Means ±SEM; n=3-5 per test. C. fra2, mrc1 , il6, ill 0: ***p <0.001 (naive vs. LNP-siFra2). arg1 , inos: ****p <0.0001 (naive vs. LNP- siFra2). D. fra2, mrc1 , arg 1 , inos: ****p <0.0001 (naive vs. LNP-siFra2); ill 0 ***p <0.001 (naive vs. LNP- siFra2); tgfbl : no significance.

Fig. 3E Fra2 knockdown does not affect Fra1 expression.

Relative fra1 mRNA expression in fibroblasts, macrophages and cholangiocytes after LNP-siFra2 KO. Fra1 mRNA remains unaffected.

Fig. 4 Biodistribution of LNP-siRNAs in CCI4-f ibrotic mice.

A. Near infrared (NIR) fluorescence emission after 0.5, 2 and 24 h post intravenous injection of LNP-siLuc, LNP-siFra2 and PBS (mice #1 -2 injected with LNP-siLuc, mice #3-4 injected with LNP-siFra2 and mouse #5 injected with PBS). B. NIR fluorescence in key organs explanted 48 h after injection. C. Quantitative assessment of organ specific uptake from explanted organs shows major accumulation of the RNA-loaded LNPs in liver, followed by the kidneys.

Fig. 5 Treatment of mice with intravenous LNP-siFra2 highly significantly reduces CCI4- induced advanced liver fibrosis. A. Experimental scheme. Gavage with escalating doses of CCI4 gavage for 4 weeks in Balb/c mice; 4 intravenous injections of LNP- siLuc and LNP-siFra2 vs PBS in late-stage fibrosis from weeks 5-6 (n=8 per group).

B. Sirius red staining and morphometry for hepatic collagen deposition staining shows an 80% reduction of stainable collagen in liver sections; ****p <0.0001 (LNP-siLuc vs. LNP- siFra2 or PBS). C. Quantitative RT-PCR: highly significant reduction of relative gene expression of fibrosis and M2-type macrophage related gene expression, and increase of M1 -type macrophage related gene expression in livers after treatment with LNP-siFra2 vs controls. Fra2, coll a1 , acta2/asma, timpl , arg1 , il6; all ****p <0.0001 ; inos: <0.01. D. Hydroxyproline (biochemical collagen) content: **p <0.01 (LNP-siLuc vs LNP-siFra2), ***p <0.001 (PBS vs LNP-siFra2). Fig. 6 Histological expression of Fra2 and macrophage markers in CCI4-fibrotic mice with and without LNP-mediated Fra2 knockdown. A. Fra2 is prominently expressed in parenchymal liver cells compatible with macrophages. Fra2, CD68 (activated macrophages) and YM1 (M2-type macrophages) were all reduced in LNP-siFra2 vs control treated livers. B. Quantitative morphometry for CD68 and YM1 was performed on 10 random liver sections (40x magnification) per mouse and data are the means ± SD from 8 mice per group. ***p <0.001 .

Fig. 7 Serum ALT and liver to body weights are reduced by LNP-siFra2 vs control treatment in CCI4-fibrotic mice. A-B. No change in body weight with LNP-siFra2 treatment, while the liver/body weight ratio is significantly decreased in LNP-siFra2 treated CCI4- fibrotic mice. C. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are decreased, and of alkaline phosphatase (ALP) increased in LNP-siFra2 vs control treated mice (means ±SD; n=8 per group). ****p <0.0001 .

Fig. 8 Treatment of mice with intravenous LNP-siFra2 highly significantly reduces progressive biliary fibrosis in Mdr2-/- mice. A. Experimental scheme. 5 intravenous injections of LNP-siLuc and LNP-siFra2 in spontaneously progressive biliary fibrosis from week 5-7 of age (n=8 per group). B. Sirius red staining and morphometry for hepatic collagen deposition staining shows a 35% reduction of stainable (portal tract) collagen in liver sections; **p <0.01 (LNP-siLuc vs. LNP-siFra2). C. Quantitative RT- PCR: (highly) significant reduction of relative gene expression of fibrosis and M2-type macrophage related gene expression, and increase of M1-type macrophage related gene expression in livers after treatment with LNP-siFra2 vs the LNP-siLuc control. D. Hydroxyproline (biochemical collagen) content. *p <0.05; **p <0.01 ; ****p<0.0001 .

Fig. 9 Histological expression of macrophage markers CD86 and YM1 and levels of liver enzymes in Mdr2-/- mice treated with LNP-siFra2 vs LNP-siLuc.

A. Immunohistochemistry for YM1 expressing M2-type macrophages. B. Quantitative morphometry for CD68 and YM1 performed on 10 random liver sections (40x magnification) per mouse; data are the means ± SD from 8 mice per group. C. Serum levels of ALT and ALP are decreased, in LNP-siFra2 vs control treated mice (means ±SD; n=8 per group). For comparison: serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are decreased, and of alkaline phosphatase (ALP) increased in LNP-siFra2 vs control treated mice (means +SD; n=8 per group). **p <0.01 ; ***p <0.001 ; ****p <0.0001 (not shown). Fig. 10 Expression of Fra-2 transcripts in normal and fibrotic mouse livers. Groups of 8 mice were analyzed by quantitative RT-PCR. Hepatic Fra-2 mRNA levels are highly significantly increased in active parenchymal (CCL-4 induced) and biliary (Mdr2KO) fibrosis. Levels normalize quickly when CCL4 application is discontinued leading to slow fibrosis regression within 1 -4 weeks.

Fig. 11 In vitro validation of Fra-2 antisense oligonucleotides, (a) 3T3 murine fibroblasts were incubated with 7 different Fra-2 ASOs (kindly produced by lonis pharmaceuticals) and a control ASO. All except ASO n°1 could significantly reduce Fra-2 expression; (b) ASO5 significantly reduced II4 and increased Tnfa, Mmp9 and iNos mRNA expression in RAW 264.7 murine macrophages. ASO7 additionally increased 1110 mRNA; (c) After treatment with ASO5, 603B cells produced significantly lower levels of Col1 a1 and Mmp13, but higher levels of Col3a1 transcripts. Treatment with ASO7 reduced Col1a1 more than ASO5. In addition, Tgfbl was decreased and Mmp13 increased; (d) The effect on fibroblasts was likewise antifibrotic. ASO5 reduced Col1a1 and Col3a1 . ASO7 was also able to reduce the expression of Tgfbl and increase Mmp9 and Tnfa expression; (e) MTT assay showed no signs of cytotoxicity of ASO7 in fibroblasts, macrophages and cholangiocytes up to a concentration of 200 pg/mL. (*p < 0.05, **p < 0.001 , ***p < 0.0001 , means ± SD, n = 3).

Fig.12 Experimental scheme of the in vivo testing of ASO7 in MDR2KO mice. 8 weeks old mice were injected twice weekly either with PBS or ASO7 for 4 weeks

Fig. 13 Collagen quantification in MDR2KO mice. Sirius Red staining of 8 weeks (a), 12 weeks and PBS injected (b) and 12 weeks and ASO7-treated (c) animals. Together with the data from the hydroxyproline (HYP) analysis (d) and the quantitative analysis of the Sirius Red staining, the antifibrotic effect of ASO7 is evident. The treated mice showed an approximately 50% lower liver collagen content than the same-aged experimental animals administered PBS alone. (*p < 0.05, **p < 0.001 , ***p < 0.0001 , 12 weeks + PBS vs. 12 weeks + ASO 7; ± SD, n = 8).

Fig. 14 Fibrosis related and serum safety parameters of MDR2KO mice with or without ASO treatment, (a, b, c, g) Immunohistochemical staining for CD68+ cells revealed a significant difference in macrophage occurrence in the treated group. The number of macrophages in 8- and 12-weeks old mice were significantly higher than in Fra-2 ASO7-treated animals; (d, f) Serum chemistry showed no sign of acute liver toxicity due to treatment with ASO7; (e) Treatment with ASO7 vs PBS (control) significantly decreased profibrogenic asma, collal and Timpl transcripts, and increased (fibrolytic) Mmp9 transcripts. (*p < 0.05, **p < 0.001 , ***p < 0.0001 , 12 weeks + PBS vs. 12 weeks + ASO 7; ± SD, n = 8)

Fig. 15 High-end quantitative proteomics indicates near normalization of the biliary fibrotic liver proteome by Fra2-ASO7. Label-free high-end quantitative proteomics of healthy age-matched FVB wildtype livers vs biliary fibrotic livers from Mdr2KO mice treated with Fra-2 ASO7 vs PBS alone. Near normalization of the advanced fibrotic (and precancerous) microenvironment. Analysis also reveals numerous biologically plausible down- or upregulated targets after ASO7 therapy (no shown).

Material and Methods:

Lipidoid synthesis and lipid compounds

The lipidoid dodecyl 3-[3-[3-[bis(3-dodecoxy-3-oxo-propyl)amino]propyl-methyl-ami no]propyl- (3-dodecoxy-3-oxo- propyl)amino]propanoate (formula C67H131 N3O8, M.W. [g/mol]: 1106,8), was synthesized by ChiroBlock (Wolfen, Germany) based on a screen of in silico candidates for effective siRNA delivery (33). 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and 1 ,2-dimyristoyl-rac -glycero-3-methoxypolyethylene glycol-2000, (DMG-PEG 2000) were purchased from Avanti Polar Lipids (City, Alabama, USA).

Formulation of lipid nanoparticles

The recipe for the lipoplex (LNP) included the lipidoid, cholesterol, DSPC and DMG-PEG 2000. The cationic lipid, cholesterol, DSPC, and DMG-PEG2000 were combined and solubilized in 90% ethanol respectively at molar ratio of cationic lipid: DSPC: cholesterol: DMG-PEG2000 of 50 : 10 : 38,5 : 1 ,5. The polynucleotide (PN) was dissolved at a concentration 0.4 mg/mL in 10 mM citrate, pH 3.0. To prepare LNPs, the PN (here siRNA) solution, the lipid solution and PBS buffer were injected into a microfluidic device (interdigital and caterpillar micro mixers, Fraunhofer IMM) at relative volumetric flow rates of PN: lipids: buffer of 1 :1 :2 using three disposable syringes, (Braun Melsungen, Germany) that were controlled by two syringe pumps (Harvard elite 1 1 (Fig. 1 D). The two solutions (PN and lipids) were simultaneously injected into the first microfluidic channel using two syringes that were controlled by the same syringe pump with a flow rate of 5mL/min. The mixed solution was then directly injected into the second microfluidic channel, simultaneously with PBS buffer from the third syringe, using a second syringe pump with a flow rate of 10mL/min. The thus freshly prepared LNPs were dialyzed against PBS buffer using membranes or dialysis tubes (Slide-A-Lyzer® MINI Dialysis 3.5K MWCO, ThermoFisher Scientific, Germany) to remove ethanol, exchange buffer and uncomplexed siRNAs. Dynamic light scattering measurement

Effective diameter, which describes the intensity-averaged hydrodynamic diameter of the LNPs, was determined using dynamic light scattering and used as a convenient measure of relative particle sizes. The effective diameters were calculated with NANO-flex® (microtrac, particle Metrix GmbH).

Cryo transmission electron microscopy (CryoTEM)

Sample preparation: Before grid preparation, samples were vortexed for 30s. Grids were hydrophilized by oxygen plasma (negative surface charge). Each sample was preserved in vitrified ice supported by holey carbon films on 200-mesh copper grids (QuantiFoil® R2/1 ). Each sample was prepared by applying a 6 pL drop of sample suspension to a cleaned grid, removing buffer carefully with filter paper, and immediately proceeding with vitrification in liquid ethane at -180°C with a Leica EM GP. Grids were stored under liquid Nitrogen until being transferred to the electron microscope for imaging.

Measurement: Cryogenic TEM imaging was performed by means of a Zeiss Libra® 120 under liquid N2 cryoconditions on holey carbon-coated copper grids after freezing the solution. The microscope was used at 120 kV acceleration voltage and the images were taken with a Gatan UltraScan® ccd camera. Vitreous ice grids were transferred into the electron microscope using a cryostage that maintains the grids at a temperature below -170 °C.

Determination of zeta potential

Zeta potential measurements were acquired on a Zetasizer Nano ZS (Malvern, Westborough, MA), and reported values were the average of 10-25 runs.

Fra-2 antisense oligonucleotides (ASOs)

Fra-2 ASOs were synthesized by lonis Pharmaceuticals, Carlsbad, CA, USA, in a scientific collaboration using standard advanced technology employing modified nucleosides and/or ribose moieties containing a 10 base pair double stranded DNA sequence complementary to Fra-2 mRNA (gapmers) flanked at both termini by stabilizing sequences of 3 double stranded base pairs (Weng et al., 2018, Seth et al., 2008).

Cell culture

Murine 3T3 fibroblasts and murine 603B cholangiocytes were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin, 1% L- glutamine, and 1% streptomycin (Sigma Aldrich) in 5% CO2 at 37 °C. The medium was changed every 2 days and the cells were separated by trypsinization using trypsin-EDTA (0.05%; LifeTechnologies, =Darmstadt, Germany) before reaching confluency. Bone marrow cells were aseptically isolated from femur and tibia bones of 6-8 weeks old C57BL/6 mice. Using a 21 G needle and 1 ml syringe the marrow was flushed out into IMDM (Iscove's Modified Dulbecco's Medium) plus 10% heat inactivated FBS. The cells were passed through a 100 pm cell strainer to remove cell clumps, bone, hair and tissue, and spun down at 500 x g for 5 min at 4 °C. Red blood cells were removed by adding 3-5ml of Red Blood Cell lysis buffer, incubated at RT for 10 min, cells spun down at 500xg for 5 min at 4 °C and resuspended in a petri dish containing bone marrow derived macrophage (BMDM) growth medium (3x10 ⁶ cells/10ml; Invivogen, CA). On day 7, formation of mature BMDM was assessed by viewing under the microscope. For M0 polarization of macrophages, only BMDM medium was used. For M2 polarization, cells were incubated in BMDM medium containing 20 ng/ml IL-4 and 20 ng/ml IL-13. Incubation ties was for 48h.

In vitro gene knockdown using siRNA lipoplexes

Four different siRNA sequences selected and tested for efficient Fra2 knockdown in cells using EndoFectin as in vitro transfectant (not shown). The most efficient siRNA was labeled with Cy5- near infrared (NIR) dye and complexed into the optimized lipoplex formulation for in vivo knockdown.

Cy5-labeled siFra2 (anti-Fra2 siRNA), based on the sequences:

Sense: 5’-Cyanine 649 -GCUCACCGCAGAAGCAGUAUU-3’ (SEQ ID NO: 3) Antisense: 5’P-UACUGCUUCUGCGGUGAGCUU-3’ (SEQ ID NO: 4)

HPLC-purified Cy5- siRNA was purchased from Dharmacon (Cambridge, UK).

Cy5-labeled siLuc (anti-Luciferase siRNA, Biospring, Frankfurt, Germany) served as negative control:

Antisense: 5’-P-UCGAAGuACUcAGCGuAAGdTsdT-3’ (SEQ ID NO: 5)

Sense: 5’-Cy5-cuuAcGcuGAGuAcuucGAdTsdT-3’ (SEQ ID NO: 6)

N = unmodified nucleotide, n = 2’O-methyl-modified nucleotide, s = phosphorothioate, P = phosphate

To assess knockdown efficiency, collagen producing murine 3T3 fibroblasts, murine cholangiocytes 603B, and M2-polarized or unpolarized BMDM were seeded in 12-well culture plates at a density of 250,000 cells per well and allowed to adhere overnight. 24 h before the knockdown 3T3 and 603B cells were preincubated with supplemented DMEM containing 5 ng/mL TGF[31 (R&D,, Minneapolis, USA). M0 and M2 were incubated with BMDM media or 20 ng/ml IL-4 and 20 ng/ml IL-13 for 48h respectively. Afterwards the cells were incubated with siFra2 or siLuc (complexed by LNP) at a final concentration of 50 nM for 24 h at 37 °C. siFra2 and siLuc mixed with EndoFectin transfection reagent (Gene Copoeia, USA) served as positive and negative in vitro transfection controls. All experiments were performed in triplicates

In vitro gene knockdown using ASOs

RAW macrophages, TGF[31 -activated 603b cholangiocytes, and TGF[31 -activated 3T3 fibroblasts were incubated with selected ASOs at 20 or 40 pg/ml for 24 h followed by analysis of fibrosis-related gene expression.

Quantitative RT-PCR

After indicated incubation times of 3T3 fibroblasts, 603B and BMDM with LNP or EndoFectin -siFra2 or -siLuc and after isolation of cells from homogenized harvested livers by Tissue Lyser II (Qiagen, Venlo, Netherlands), RNA was extracted, and 1 pg of total RNA was reverse-transcribed into cDNA using the qScript cDNA SuperMix (Quantas, Beverly, USA). The following TaqMan primers and probes for Acta2, Col1a1, Gapdh, Timpl, Tgfbl transcripts were used (Applied Biosystems, Darmstadt, Germany):

Acta2 forward 5'-ACAGCCCTCGCACCCA-3' (SEQ ID NO: 7) reverse 5'-CAAGATCATTGCCCCTCCAGAACGC-3' (SEQ ID NO: 8) probe 5’-GCCACCGATCCAGACAGAGT-3’ (SEQ ID NO: 9)

Col1a1 forward 5'-ACGCATGGCCAAGAAGACA-3' (SEQ ID NO: 10) reverse 5'-AAGCATACCTCGGGTTTCCAC-3' (SEQ ID NO: 11 ) probe 5’-AGCTGCATACACAATGGCCTAAGGGTCC-3’ (SEQ ID NO: 12)

Gapdh forward 5’- CCTGCCAAGTATGATGACATCAAGA-3’ (SEQ ID NO: 13) reverse 5’- GTAGCCCAGGATGCCCTTTAGT-3’ (SEQ ID NO: 14) probe 5’-TGGTGAAGCAGGCGGCCGAG-3’ (SEQ ID NO: 15)

Timpl forward 5'- TCCTCTTGTTGCTATCACTGATAGCTT-3' (SEQ ID NO: 16) reverse 5'- CGCTGGTATAAGGTGGTCTCGTT-3' (SEQ ID NO: 17) probe 5’- TTCTGCAACTCGGACCTGGTCATAAGG-3’ (SEQ ID NO: 18) Tgfbl forward 5’-AGAGGTCACCCGCGTGCTAA-3’ (SEQ ID NO: 19) reverse 5'-TCCCGAATGTCTGACGTATTGA-3’ (SEQ ID NO: 20) probe 5’-ACCGCAACAACGCCATCTATGAGAAAACCA-3’ (SEQ ID NO: 21 )

The following primer sequences were used for the SYBR Green methodology (Metabion,

Planegg, Germany):

Fra1 forward 5’-GAGACGCGAGCGGAACAAG-3’ (SEQ ID NO: 22) reverse 5’-CTTCCAGCACCAGCTCAAGG-3’ (SEQ ID NO: 23)

Fra2 forward 5’-CACTCCCGGCACTTCAAAC-3’ (SEQ ID NO: 24) reverse 5'-GAGTCTGATGACTGGTCCCC-3’ (SEQ ID NO: 25)

Mrc1 forward 5’-AAGGCTATCCTGGTGGAAGAA-3’ (SEQ ID NO: 26) reverse 5’-AGGGAAGGGTCAGTCTGTGTT-3’ (SEQ ID NO: 27) forward 5’-CTCCAAGCCAAAGTCCTTAGAG-G (SEQ ID NO: 28) reverse 5’-AGGAGCTGTCATTAGGGACATC (SEQ ID NO: 29) inos forward 5’-CTATCTCCATTCTACTACTACTACCAGATCGA-3’ (SEQ ID NO: 30) reverse 5’- CCTGGGCCTCAGCTTCTCAT-3’ (SEQ ID NO: 31 )

116 forward 5’-ACCAGAGGAAATTTTCAATAGGC-3’ (SEQ ID NO: 32) reverse 5’-TGATGCACTTGCAGAAAACA-3’ (SEQ ID NO: 33)

1110 forward 5'-GCTCTTACTGACTGGCATGAG-3’ (SEQ ID NO: 34) reverse 5’-CGCAGCTCTAGGAGCATGTG-3’ (SEQ ID NO: 35)

TaqMan and SYBR Green reaction mixtures were from ThermoFisher Scientific, Darmstadt, Germany).

Gapdh was used to normalize data (ratio of target gene to Gapdh) and to control for RNA integrity. The TaqMan and SYBR Green reactions were performed using a Step One Plus sequence amplification system (Applied Biosystems, Foster City, CA).

Cell uptake

Lipoplex uptake was monitored in vitro in 3T3 murine fibroblasts. The cells were cultured as described above, fixed in 4% paraformaldehyde for 30 mins 24 h after transfection and labeled with muclear dye (5pl/mL, ThermoFisher, Germany) and NeuroDiO (PK-CA707-30021 , PromoCell, Heidelberg, Germany) staining cytoplasmic membrane and intracellular membrane structures.

Cell viability

3T3 cells were transfected for 48h using EndoFectin or lipoplex complexed with siRNAs, followed by incubation with Fixable Viability Dye eFluor780 (eBioscience) to assess viability by fluorescence-activated cell sorting (FACS Canto II, BD Bioscience, Mississauga, Canada). 10,000-50,000 cells were measured per staining, with OneComp eBeads for standardization (eBioscience) using BD FACS Diva software version 7.0. Further data analysis was carried out using open source Flowing Software 2.5.0 (Perttu Terho, Turku, Finland).

Fibrosis models

All animal studies were approved by the local ethics committee on animal care (G 17-1 -030, Government of Rhineland Palatinate, Germany). 6 weeks old female Balb/c mice were purchased from Janvier Labs (Germany) and kept under 12 h light-dark cycles at 25 °C and 40-60% humidity. Mice had access to regular chow and water ad libitum. Carbon tetrachloride (Sigma-Aldrich, St. Louis, US) diluted in mineral oil (Sigma-Aldrich, St. Louis, US) was given by oral gavage 3 times a week in an escalating dose protocol (first dose 0.875 mL/kg; 1.75 mL/kg week 1-2; 2.5 mL/kg week 3-4) for 4 weeks. Mdr2 knockout mice were 5 weeks old before treatment. At predetermined time point mice were sacrificed by cervical dislocation, blood and organs were collected for analysis.

In vivo gene knockdown

During the third and fourth week of fibrosis induction with CCL, mice (n=8 per group) were anesthetized with isoflurane gas and injected intravenously 4 times (two times per week) with 1 mg/kg LNP-siFra2 control LNP-siLuc (10:1 weight-to-weight ratio LNP: siRNA; volume 50 pL), or 50 pL PBS. Five weeks old Mdr2-/- mice were injected intravenously 5 times (twice weekly in week 1 -2 and once per in week 3). 48h after the last injection, organs and blood were harvested.

Clinical chemistry

Serum was analysed for ALP (alkaline phosphatase), AST (aspartate aminotransferase), ALT (alanine aminotransferase) and creatinin by standardized assays by the clinical chemical laboratory of Mainz University Medical Center standard laboratory test. All creatinine values were <0.10 mg/dl, below the level of detection.

Hydroxyproline determination

Liver collagen content was determined colorimetrically as hydroxyproline. Briefly, snap frozen liver specimens from the left and middle lobes, totalling200-300 mg, were combined, homogenized in 3 mL 6N HOI, and hydrolyzed for 16 h at 110 °C. Triplicates of 5pL were placed in a transparent 96 well-plate (Greiner bio-one, Kremsmunster, Austria), mixed with 150 pL 0.1 M citrate buffer, pH 6.0, and 100 pL containing 150 mg/mL chloramine T. After 30 min incubation at RT 100 pL of Ehrlich's reagent (1.25 g dimethyl-benzaldehyde dissolved in 100 mL distilled water) was added and incubated at 65 °C for 30 min. Absorbance was measured at 550 nm in an Infinite M200Pro spectrophotometer (TECAN, Austria). Total (pg/liver) and relative (pg/g liver) hydroxyproline content were calculated).

Collagen (Sirius red) staining and morphometry

Formalin fixed liver sections were stained 5% Piero-Sirius red (Sigma-Aldrich, Germany) at RT for 1 h and washed in distilled water and 0.5% acetic acid. 10 randomly selected fields (x40) were photographed using a Zeiss Scope A.1 microscope and an AxioCam MRC Zeiss camera (Jena, Germany). The percentage of the Sirius red-stained area was measured by Imaged software with an adjusted threshold setting. Sections were subject to morphometry with and without portal areas, the latter representing quantitatively less but functionally more relevant collagen deposition. The means of 10 Sirius red stained areas in 10 random sections (40x magnification), 5 each from the two major liver lobes were assessed per mouse and data are the means ± SD from 8 mice per group.

Immunohistochemistry & immunofluorescence

4 pm thick formalin fixed liver sections were boiler-treated with citrate buffer, pH 6.0, for 30 min, preincubated with 3% hydrogen peroxide for 10 min and blocked with 5% normal goat serum (Invitrogen) for 60 min at RT, followed by rat monoclonal anti mouse Fra2 (clone REY146C, Merck, MABS1261 ) 1/100 for 60 min at RT, followed by biotinylated goat anti-rat IgG 4°C overnight. Rabbit polyclonal anti-mouse CD68 (Abeam, ab125212) and rabbit anti- mouse Ym1 (StemCell #60130) 1/400 were followed by biotinylated goat anti-rabbit IgG 1/500). Colour was developed with Avidin-Biotin-enzyme Complex (ABC) at RT for 30 min and the DAB (3, 3’-diaminobenzidine) solution (all from Vector Labs, Burlingame, USA) for 1 min. Nuclei were counterstained with Hematoxylin (Sigma-Aldrich) for 10 seconds, followed by dehydration. Pictures were taking using a Scope A.1 microscope and an AxioCam MRC camera (both from Carl Zeiss). Morphometry was performed as outlined for Sirius red staining.

In vivo imaging of near infrared (NIR) labeled siRNA-lipoplexes

In vivo NIR fluorescence imaging of Cy5-dye labeled LNP-siFra2 and LNP-siLuc was performed with the IVIS Spectrum Imaging system (Caliper LifeSciences, Hopkinton, US). After injection at predetermined time points, 5 random mice (mouse 1 -2 treated with LNP- siLuc, mouse 3-4 treated with LNP-siFra2, and mouse 5 injected only with PBS as control) were transferred into the machine's image chamber and anesthetized temporarily with isoflurane. A picture integration time of 4 s was set for the fluorescence source. Filters were adjusted with excitation at 640 nm and emission at 700 nm to visualize Cy5-dye labeled LNP- siFra2 and LNP-siLuc.

Ex vivo imaging of organs

48 h after the last injection of LNP-siFra2 or LNP-siLuc the mice were sacrificed and liver, spleen, lungs, heart and kidneys were immediately transferred into the imaging chamber of the IVIS Spectrum Imaging system. Image acquisition was performed with the same settings as described above. The organs of two mice (mouse 1 -mouse 3) and control (only PBS) were checked.

Statistics

Statistical significances of differences were evaluated by one-way ANOVA using GraphPad Prism version 5.0 (San Diego, USA). For statistical differences between two groups, unpaired Student’s t-test was used. Data are expressed as means ± SD

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