Enhanced macrophages

Abstract

The present invention relates to macrophages engineered to express matrix metallopeptidase, and therapeutic uses thereof. Such macrophages may be used in the treatment of inflammatory conditions, including fibrotic components, in subjects, such as inflammatory organ damage. The inflammatory conditions may be acute or chronic and may involve fibrotic components.

Description

[Technical Field]

[0001] The present invention relates to macrophages that have been genetically engineered to express matrix metallopeptidase 9 (MMP9) and / or matrix metallopeptidase 12 (MMP12). The macrophages are preferably engineered with exogenous nucleic acids encoding MMP9 and / or MMP12. Such macrophages may be used to treat inflammatory conditions involving fibrotic elements in a subject, such as inflammatory organ damage. The inflammatory conditions may be acute or chronic and may involve fibrotic elements. The present invention further relates to populations of such macrophages, compositions comprising such macrophages or populations thereof, and methods of using such macrophages or populations thereof. The present invention further relates to methods of engineering such macrophages, including, for example, transient transfection with an MMP9 DNA or mRNA construct. The macrophages may be human macrophages. [Background technology]

[0002] Fibrosis is the final common pathway of chronic diseases with diverse etiologies, including toxic injury, viral infection, metabolic disorders, genetic disorders, and autoimmune diseases. Acute self-limiting fibrosis likely evolved as a reversible and protective response to injury. The balance between self-limiting and excessive fibrosis is finely regulated by multiple pathways and systems and essentially depends on the duration and repetition of injury. The liver provides a paradigm for the biology of fibrosis development and remodeling. End-stage chronic liver fibrosis, also known as cirrhosis, is a life-threatening condition. 1,2 Mortality attributable to liver disease is the single leading cause of death in the UK, where it has increased steadily every year since the 1970s, and remains a major health burden worldwide. 3 .

[0003] Hepatic decompensation (HD) is defined by the acute onset of one or more major complications of cirrhosis (i.e., ascites, encephalopathy, gastrointestinal variceal bleeding, and spontaneous bacterial peritonitis) and represents a progression of the disease during the clinical course of cirrhosis [Trebicka 2020]. 31 , the most common cause of hospitalization in patients with cirrhosis [Moreau 2013] 32 HD patients are at high risk for short-term mortality [Moreau 2013]. 32 The initial episode of HD (also referred to herein as the first liver decompensation event) often requires hospitalization and marks the transition from compensated to decompensated cirrhosis. Decompensated cirrhosis is characterized by recurrent episodes of HD. HD has two distinct clinical manifestations, depending on the presence or absence of other organ failure and the degree of systemic inflammation. The presence of multiple organ failure and severe systemic inflammation characterizes acute-on-chronic liver failure (ACLF), a syndrome with a very high 28-day mortality rate. HD with moderate systemic inflammation without additional organ involvement has a low 28-day mortality rate (approximately 2%, but increasing to 10% at 90 days, suggesting heterogeneity in the clinical course of HD patients) [Trebicka 2020]. 31 However, the prognosis remains poor for several years afterward.

[0004] The only treatments for liver injury are removal of the noxious stimulus (e.g., administration of effective antiviral therapy) and liver transplantation. Treatment of liver failure resulting from acute or chronic injury is limited to supportive care and / or transplantation, the latter of which requires a donor. Currently, there is a shortage of organ donors, and surgery carries significant morbidity and mortality risks. In addition, patients also commit to lifelong immunosuppression. Furthermore, despite the relative success of therapeutic interventions for specific etiologies (e.g., novel antiviral therapies for hepatitis C virus infection and abstinence for alcoholic liver disease), many diseases (e.g., NASH) lack approved treatments, and patients often delay seeking medical care when cirrhosis and related complications have already developed [Starkey 2019]. 33Therefore, there are no specific therapies for treating liver cirrhosis, and therefore there is a significant unmet clinical need to provide effective anti-fibrotic therapies for both chronic and acute liver injury. 4~6 .

[0005] Macrophages (MΦ) play a crucial role in the inflammatory response of injured livers. Two major populations of MΦ exist in the liver: (i) resident macrophages (Kupffer cells, KCs) and (ii) infiltrating macrophages. KCs perform a patrolling function within the hepatic sinusoids to phagocytose microbial debris that reaches the liver via the sinusoids under homeostatic conditions. In the early stages of liver injury, KCs express chemokines such as CCL2 and CCL5, thereby contributing to the recruitment of monocytes from the blood circulation. 2,7 The number of KCs is reduced during fibrosis and then repopulates within the liver during the recovery phase of self-limited fibrosis. 8 Infiltrating monocyte-derived macrophages (MDMs) play a key role in the response to liver injury. Once recruited via the CCR2 / CCL2 axis and entering the liver parenchyma, infiltrating MDMs locate along fibrous septa in the early stages of liver fibrosis and may promote fibrosis by releasing factors such as TGF-β, IL1, PDGF, and CCL2, which activate hepatic stellate cells and exacerbate inflammation. This may suggest a detrimental role for MDMs in progressive fibrosis. However, when MDMs are depleted at the onset of fibrotic remodeling, the remodeling process fails and liver fibrosis persists. It is now widely accepted that macrophages play a dual role in the establishment and resolution of fibrosis. 2,8~11 .

[0006] Given the suggested role of macrophages in the healing of fibrosis, macrophage cell therapy is considered a potential treatment for chronic liver fibrosis. Murine bone marrow-derived macrophages (BMDMs) have been shown to ameliorate liver fibrosis when injected into a mouse model of chronic liver fibrosis. 12Similar results have been reproduced using human monocyte-derived macrophages (hMDMs) in an immunodeficient mouse model of chronic liver fibrosis. 13 In addition, GMP-compliant cell culture protocols 14 is currently being used to generate hMDMs for autologous transplantation into patients with cirrhosis in an ongoing phase II trial (MATCH, Macrophage Therapy for Liver Disease, ISRCTN10368050).

[0007] hMDMs currently used in clinical practice do not have enhanced anti-fibrotic function. 14 , which may limit the scope and duration of their therapeutic effect. An ideal therapeutic macrophage for use in inflammatory organ injury containing a fibrotic component should possess anti-fibrotic properties.

[0008] Matrix metalloproteinases (MMPs), also known as matrix metalloproteinases or matrixins, are a family of zinc-dependent polypeptides that collectively degrade various proteins within the extracellular matrix (ECM). MMPs play many roles in vivo, making them extremely complex to understand. While MMPs have been reported to contribute to fibrosis, they are also crucial for the regression of fibrosis due to their ability to remodel the matrix. This dual role in the progression and regression of fibrosis can be understood in part through complex in vivo activities ranging from cytokine and chemokine activation, immune cell recruitment, and activation to extracellular matrix degradation. 15In addition, MMP activation and subsequent activity are highly regulated, primarily by tissue inhibitors of metalloproteinases (TIMPS), further complicating understanding MMP activity in fibrosis. Experimental models of pulmonary fibrosis have shown increased MMP9 (a type IV collagenase that targets collagen), which is also associated with destruction of the alveolar epithelial membrane, indicating a putative profibrotic role of MMP9 in lung injury. However, in a bleomycin-induced model of pulmonary fibrosis, MMP9-deficient mice developed similar pulmonary fibrosis to their wild-type littermates, although their lungs exhibited limited alveolar bronchiolosis. Overall, the precise role of MMPs in fibrosis is not fully understood. Furthermore, in vitro-derived hMDMs express only negligible levels of MMPs, and their activity is barely detectable in the conditioned medium of these cells.

[0009] Few studies have been reported in the field of MMP gene manipulation. MMP9 transfection in THP1 cells has been utilized as a research tool to understand the inflammatory response of macrophages in atherosclerosis. 16 Therapeutically, overexpression of MMP9 in iPSCs via lentiviral transduction has been utilized to enhance repair of damaged myocardium. 17 MMP12, an elastase that targets soluble and insoluble elastin, has been overexpressed in endothelial progenitor cells for use in melanoma cell therapy. 18 To the best of our knowledge, previous studies performed on MMP transfection of human macrophages have not yielded macrophages with antifibrotic properties. Cabrera et al. (2007) demonstrated that overexpression of MMP9 in macrophages from transgenic mice challenged with bleomycin reduced fibrosis. 21 Differences in the levels of TIMP-1 were also observed between transgenic and wild-type mice, but no differences in the expression levels or activities of other matrix metalloproteinases were reported. [Prior art documents] [Chartered documents]

[0010]

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Non-licensed literature

[0011] [Non-licensed document 1] Ramachandran, Gut. 2011 April; 60; A56 [Non-licensed document 2] Li YH et al. Cell Death Discov. 2021 Sep 13;7(1):239 [Non-licensed document 3] Ricardo Lamy et al. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4338

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[0012] Introduction to the Invention Typically, human macrophages (hMDMs), derived in vitro from monocytes, express MMPs under certain physiological conditions. As shown in Figures 1 and 4, in vitro-induced hMDMs express negligible levels of MMPs, with little activity detected in the conditioned medium of these cells (Figure 2). Here, we demonstrate that enhancing MMP expression, typically through transient transfection with sequences encoding MMP9 or MMP12, enhances the antifibrotic properties of macrophages. This provides proof-of-principle evidence for how increased expression of these genes delivers macrophages that secrete active MMPs, potentially contributing to fibrotic conditions such as the resolution of acute and chronic organ injury. Some MMPs play a role in supporting fibrosis remodeling. However, the impact of increased expression of MMP9 or MMP12 on overall MMP expression in macrophages is currently unknown.

[0013] We show that the most surprising feature of our MMP-transfected macrophages is their ability to co-upregulate other MMPs in addition to the selected MMP. In Figure 4B, we show that MMP9-transfected hMDMs strongly upregulate several other MMPs, including MMPs 1, 8, and 10, which may contribute significantly to the anti-fibrotic effects of macrophages, such as cell therapy. For example, MMP8 is normally contained in neutrophil granules and digests collagens I, II, and III, making it particularly interesting in the context of liver fibrosis, where most of the collagen in the fibrous septa is collagen I and III. 27,28 .

[0014] Thus, we also provide macrophages engineered with sequences encoding the specific MMPs MMP9 and / or MMP 12. Interestingly, the overall matrix metalloproteinase activity of hMDMs transfected with MMP9 and hMDMs transfected with MMP 12 is significantly increased, although this effect is not limited to the transfected proteases alone.

[0015] As demonstrated herein, MMP-transfected macrophages maintained cell surface expression of macrophage-specific markers, such as CD14, CD206, and 25F9 (Figure 3), demonstrating that transfection with MMP9 or MMP12 did not alter cell identity. Interestingly, however, MMP9-transfected cells were much better phagocytes than MMP12-transfected cells (Figure 5). This further demonstrates the versatility of the therapeutic approach described herein, as an appropriate MMP with the right combination of antifibrotic and pro-phagocytic properties can be selected depending on the disease target.

[0016] Collectively, these data support the use of MMP9TrxhMDM and / or MMP12TrxhMDM as therapeutic agents in the treatment and / or prevention of fibrotic conditions (e.g., those associated with inflammatory organ injury). Expression of MMP9 or MMP12 (e.g., by transfection) is expected to enhance the antifibrotic (antifibrotic via increased matrix metalloproteinase activity) function of macrophage-based therapeutics.

[0017] Organ damage due to chronic inflammation is often accompanied by fibrosis, such as in chronic liver disease, and therefore pro-restorative therapeutic macrophages with anti-inflammatory and anti-fibrotic functions would be beneficial.

[0018] For example, macrophages that have been engineered to additionally express MMP9 and / or MMP12 by engineering them with nucleic acids encoding MMP9 or MMP12 exhibit enhanced overall MMP activity, not just MMP9 / MMP12 activity.

[0019] Ramachandran et al. (2012) 8 and Ramachandran et al., Gut. 2011 April; 60; A56, evaluated macrophages in mice after liver injury and identified a subset of macrophages associated with fibrosis resolution. Cells associated with fibrosis resolution showed increased expression of various genes related to cellular debris clearance and anti-fibrotic pathways, and decreased expression of pro-fibrotic and pro-inflammatory genes. Among the genes with increased expression were MMP9 and MMP12. Moore JK et al. (2015) 13demonstrated that monocytes from patients with liver cirrhosis can differentiate into macrophages in vitro, and that these macrophages can express a series of genes related to anti-fibrotic pathways, such as MMPs 9 and 12. Li YH et al., Cell Death Discov. 2021 Sep 13;7(1):239, also demonstrated that upregulation of MMPs, such as MMPs 9 and 12, is an important mechanism by which anti-fibrotic macrophages resolve fibrosis, in addition to changes in the expression of other genes, such as downregulation of pro-inflammatory and fibrogenic cytokines. Ricardo Lamy et al., Invest. Ophthalmol. Vis. Sci. 2018;59(9):4338, demonstrated that MMP 12 has anti-fibrotic properties and that knockout of this MMP increases fibrosis. However, it has also been shown that MMP12 also regulates the expression of other genes, such as CCL2. Therefore, given the complex interactions that control gene expression, influencing MMP12 expression alone may produce unpredictable results. Wang M et al., Theranostics. 2020 Jan 1;10(1):36-49, demonstrates that MMP12 may exert anti-fibrotic effects by suppressing hepatic stellate cell activation and the release of pro-inflammatory cytokines.

[0020] However, none of these documents are directed to engineered cells. In fact, none of these documents exemplify or even suggest macrophages engineered to express MMP9 and / or MMP12, or that cells engineered to express only these genes (in the absence of other identified anti-fibrotic genes) would be therapeutically beneficial, given the complex nature of gene expression changes and the multiple contributing factors of fibrosis in vivo. Also, it is known that overexpression of MMPs can cause cells to upregulate tissue inhibitors of metalloproteinases (TIMPs); therefore, engineering cells to contain MMPs would be expected to upregulate TIMPs, negating any beneficial technical effects resulting from MMP expression.

[0021] WO 2019 / 118888A1 (Treatment of Fibrossis with Genetically Engineered Macrophages) describes engineered macrophages for the treatment of fibrosis. For example, genetically engineered macrophages contain a recombinant extracellular matrix (ECM)-targeting protein and / or a recombinant protease. The recombinant protease can be a matrix metalloproteinase (MMP), e.g., any one of a long list of possible MMPs. However, neither MMP9 nor MMP12 are exemplified, and therefore no effect on overall MMP activity levels has been observed. Furthermore, the engineered cells described therein have only been tested in models of pulmonary or cardiac fibrosis.

[0022] WO 2022 / 047119A1 (MODIFIED IMMUNE CELLS FOR FIBROSIS AND INFLAMMATION) describes modified immune cells containing one or more nucleic acid sequences encoding (i) at least one exogenous fibrolytic agent and / or (ii) at least one exogenous anti-inflammatory agent. The at least one exogenous fibrolytic agent may include a matrix metallopeptidase (MMP), such as one or more from a long list of possible MMPs, including MMP9 and MMP12. The at least one exogenous anti-inflammatory agent may include, for example, a cytokine, chemokine, or pentraxin, and the cytokine may include, for example, IL-10, IL-4, IL-13, and / or TGF-beta. The modified immune cells may include macrophages. However, similarly, neither MMP9 nor MMP12 is exemplified, and therefore no effect on overall MMP activity levels has been observed.

[0023] The present inventors are the first to demonstrate the unexpected beneficial effects of macrophages engineered to express MMP9 or MMP12 in the treatment of, for example, fibrotic and / or inflammatory diseases / conditions. [Means for solving the problem]

[0024] The present invention relates to genetically engineered macrophages with enhanced antifibrotic function by transfection with exogenous sequences encoding MMP9 and / or MMP12. Such macrophages are suitable for use in treating inflammatory conditions, preferably those with a fibrotic component. The genetically engineered macrophages described herein exhibit multiple beneficial properties, including (1) the ability to secrete active MMP9 and / or MMP12; (2) upregulation of functional MMP9 and / or MMP12 without upregulation of TIMPs; and, most surprisingly, (3) co-upregulation of other MMPs in addition to the transfected MMP. This surprising effect demonstrates the versatility of the present invention, as it allows for the selection of an appropriate exogenous MMP (MMP9 or MMP12) with the appropriate combination of antifibrotic and pro-phagocytic properties depending on the disease target. The present invention further relates to genetically engineered macrophage populations, methods for generating the macrophages, and compositions for use therewith.

[0025] The macrophages of the present invention may suitably be human macrophages.

[0026] According to a first aspect of the present invention, there is provided an engineered macrophage, which has been engineered to express MMP9 or MMP12, preferably by the provision of additional or exogenous sequences encoding these proteins. Such engineered macrophages may have therapeutic utility, for example, for use in treating inflammatory and / or fibrotic conditions in a subject.

[0027] In one embodiment, the present invention provides an engineered macrophage comprising one or more exogenous coding sequences for MMP9 and / or MMP12. The exogenous coding sequences may be any suitable nucleic acid sequence. The exogenous coding sequences may be present in the cytoplasm or nucleus as extrachromosomal nucleic acid, or may be integrated into the macrophage genome.

[0028] In some embodiments, expression of endogenous MMP9 and / or MMP12 genes can be stimulated by genetic engineering. For example, gene editing techniques such as CRISPR can be used to turn on and off the endogenous genes encoding these MMPs, generating engineered macrophages that express MMP12 and / or MMP9 under conditions that would not otherwise express these proteins. This can be done, for example, by modifying the promoter sequence.

[0029] In some embodiments, the macrophages are engineered by introducing an exogenous nucleic acid encoding MMP9. In some embodiments, the macrophages are engineered by gene editing, optionally using a CRISPR-Cas9 system or a genetic modification to increase MMP9 expression. In some specific embodiments, the engineering may include editing a promoter sequence to increase MMP9 expression, preferably the MMP9 promoter is edited. In other specific embodiments, the engineering includes gene editing or genetic modification to increase expression of a transcription factor that upregulates MMP9 expression, optionally the transcription factor upregulates MMP9 expression by interacting with or binding to the MMP9 promoter. In other specific embodiments, the gene editing or genetic modification causes downregulation of or inhibits the activity of an miRNA that suppresses MMP9 expression, optionally the gene editing or genetic modification includes deleting a sequence encoding the miRNA.

[0030] Natural, unengineered macrophages are capable of expressing MMP9 and / or MMP12 under relevant physiological conditions. The present invention does not relate to these natural, unengineered macrophages, but rather to engineered macrophages that have elevated, improved, or enhanced levels of MMP activity compared to native cells. Thus, in one aspect of the present invention, engineered macrophages are provided, which have been engineered to express MMP9 at levels higher than the levels endogenously expressed in unengineered macrophages.

[0031] In other embodiments, macrophages can be engineered to activate endogenous genes encoding MMP9 and / or MMP 12. In either case, the macrophages of the present disclosure have been modified by altering expression levels by any means, and are therefore referred to as engineered macrophages.

[0032] As described, engineered macrophages are provided with additional or exogenous MMP9 or MMP12 coding sequences to enhance the overall activity of the MMPs, preferably by 1.2 to 1.5 times the wild-type activity, e.g., 1.2, 1.3, 1.4, or 1.5 times the natural level / wild-type.

[0033] The macrophages are engineered to express MMP9 or MMP12. In some embodiments, this expression can be induced from an endogenous gene. In other embodiments, the macrophages are engineered to contain an exogenous coding sequence for MMP9 or MMP12. It may be preferable for the macrophages to overexpress MMP9. It may be preferable for the macrophages to overexpress MMP12. The expression levels of MMP9 and / or MMP12 are increased when compared to non-transfected macrophages.

[0034] In some embodiments, the engineered macrophages are human monocyte-derived macrophages. In some embodiments, the macrophages are derived from monocytes by culturing in the presence of MCSF. Suitable culture conditions are discussed further below.

[0035] In some embodiments, baseline macrophages (i.e., pre-engineered or natural) are referred to as non-polarized human monocyte-derived macrophages, also called "resting" macrophages.

[0036] In some embodiments, the engineered macrophages are iPSC-derived macrophages. Various methods for deriving macrophages from iPSCs are known in the art. At baseline, these will also be unpolarized or quiescent.

[0037] The macrophages are isolated, and therefore the cells can be described as ex vivo.

[0038] Advantageously, said macrophages are suitable for use in the treatment of an inflammatory condition, preferably an inflammatory condition with a fibrotic component.

[0039] Where used in the examples, protein expression levels were quantified 16-24 hours after transfection. The expression levels listed herein are within the range of 4 x 10 6 pieces / ml(1cm 2 2 x 10 6 Secreted protein concentrations are obtained for macrophage populations at a concentration of 1000 μg / mL (equivalent to 1000 μg / mL of cells). In the examples, macrophages were transfected, isolated by centrifugation, resuspended in TexMACs buffer supplemented with IL-3 and IL-14, and incubated at 37°C under 5% CO2. Those skilled in the art will be aware of equivalent conditions suitable for determining secreted protein concentrations.

[0040] In some embodiments, macrophages are engineered to overexpress MMP9, and the secreted MMP9 protein level exceeds about 200 ng / ml. Preferably, the secreted MMP9 protein exceeds about 300 ng / ml, 400 ng / ml, 500 ng / ml, 600 ng / ml, 700 ng / ml, 800 ng / ml, 900 ng / ml, or 1,000 ng / ml. Preferably, the secreted MMP9 protein level is between about 200 ng / ml and 2,000 ng / ml. Preferably, the engineered macrophages have a higher level of secreted MMP9 protein than the average level of secreted MMP9 protein in non-engineered macrophages. Preferably, the total MMP activity of the engineered macrophages of the present invention is also higher than that of non-engineered macrophages. Preferably, these protein levels are measured by culturing the macrophages described above, wherein the concentration of the macrophages in the culture medium is 4×10 6 cells / ml (2 x 10 6 cells / cm 2 Thus, in some embodiments, the level of secreted MMP9 can be measured by culturing a sample of 1000 cells / ml of MMP9 at a concentration of 4×10 or less, which is equal to 4×10 6 When cultured in vivo at a cell concentration of 1000 ng / ml, the concentration exceeds 200 ng / ml.

[0041] In some embodiments, macrophages are engineered to express MMP12, and the level of secreted MMP12 protein exceeds about 200 ng / ml. Preferably, the secreted MMP12 protein exceeds about 300 ng / ml, 400 ng / ml, 500 ng / ml, 600 ng / ml, 700 ng / ml, 800 ng / ml, 900 ng / ml, or 1,000 ng / ml. Preferably, the level of secreted MMP12 protein is between about 200 ng / ml and 2,000 ng / ml. Preferably, the engineered macrophages have a higher level of secreted MMP12 protein than the average level of secreted MMP12 protein in non-engineered macrophages. Preferably, the total MMP activity of the engineered macrophages of the present invention is also higher than the total MMP activity of non-engineered macrophages. Preferably, these MMP12 protein levels are measured by culturing macrophages as described above, with the concentration of macrophages in the culture medium being 4×10 6 cells / ml (2 x 10 6 cells / cm 2 The concentration of the protein in the culture medium can be measured by culturing the protein (corresponding to

[0042] Known viral vectors for transfecting macrophages include lentiviruses, adenoviruses, and adeno-associated viruses (AAV).

[0043] As used herein, "encoding" refers to the ability of a sequence of nucleotides, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of a macromolecule in a biological process, such as a defined amino acid sequence. Thus, a coding sequence can be any suitable nucleic acid sequence that provides instructions for synthesizing the relevant entity (e.g., MMP9 or MMP12).

[0044] As used herein, "exogenous" refers to any material (especially genetic material) introduced from or produced outside of a particular cell. As used herein, in some embodiments, the exogenous coding sequence or engineered macrophage encodes / expresses MMP9 or MMP12. In some embodiments, the MMP9 or MMP12 is human. One of skill in the art will understand that variations in the sequence of these genes / coding sequences are also encompassed by the present invention. Ideally, the gene / coding sequence is human. The gene / coding sequence may be codon-optimized. The gene / coding sequence may be adjusted, and if the reference sequence is RNA, different nucleotides may be present in the DNA vector.

[0045] Preferably, the sequence of the MMP9 coding sequence provided is at least 80% similar or homologous to SEQ ID NO: 1 (human matrix metallopeptidase 9 (MMP9), mRNA), preferably at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% similar to SEQ ID NO: 1.

[0046] Preferably, the expressed MMP9 protein is preferably at least 80% similar or homologous to the sequence presented as SEQ ID NO:2, preferably at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% homologous to SEQ ID NO:2.

[0047] Preferably, the sequence of the MMP12 coding sequence provided is at least 80% similar or homologous to SEQ ID NO: 3 (Homo sapiens matrix metallopeptidase 12 (MMP12), mRNA), preferably at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% homologous to SEQ ID NO: 3.

[0048] Preferably, the expressed MMP12 protein is preferably at least 80% similar or homologous to the sequence presented as SEQ ID NO:4, preferably at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% similar to SEQ ID NO:4.

[0049] In preferred embodiments of the aspects described in the preceding paragraph, the coding sequence and protein are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprise, or consist of, SEQ ID NO: 1, 2, 3, or 4.

[0050] Preferably, the sequence of the MMP9 coding sequence provided is at least 80% similar or homologous to SEQ ID NO: 5, preferably at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, or 99% similar to SEQ ID NO: 5. In preferred embodiments, the MMP9 coding sequence provided is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprises, or consists of, SEQ ID NO: 5. Preferably, the sequence optionally further comprises a poly-A tail of 65 to 250 residues in length, preferably 90 to 120 residues in length, preferably about 90 residues in length, and / or a 5' cap.

[0051] In some embodiments, the macrophages are non-virally engineered and are engineered with a nucleic acid vector. In some embodiments, the macrophages are transfected with a DNA vector (e.g., a naked DNA vector or a plasmid). In some embodiments, the nucleic acid vector is not derived from a viral genome. In some embodiments, the macrophages are transfected with one or more free nucleic acids or vectors. In some embodiments, the macrophages are transfected with an RNA vector. In some embodiments, the macrophages are transfected with mRNA.

[0052] In some embodiments, MMP9 or MMP12 is provided to the macrophages as mRNA, such that the engineered cells are provided with an exogenous mRNA molecule.

[0053] In some embodiments, the mRNA molecule comprises a poly-A tail, which is a long chain of adenine nucleotides added to an mRNA molecule during RNA processing to increase the stability of the molecule. Suitable poly-A tails are 65-250 residues in length, preferably 90-120 residues in length. The poly-A tail makes the RNA molecule more stable and prevents its degradation.

[0054] In some embodiments, an mRNA molecule contains at least one cap. The 5' cap is characteristic of eukaryotic mRNA. Chemically, the 5' cap consists of an inverted 7-methylguanosine connected to the rest of the eukaryotic mRNA via a 5'-5' triphosphate bridge. This so-called "cap0" contributes, among other things, to eukaryotic mRNA stabilization, translation initiation, and mRNA degradation. Artificial cap structures have been designed to enhance the success of in vitro translation. Synthetic anti-inverted cap analogs include the trinucleotide CleanCap® AG, which is methylated at the first adenosine with additional methylation. At least one cap can be a synthetic cap analog, preferably CleanCap®.

[0055] In some embodiments, the mRNA is modified RNA. In some embodiments, the mRNA modification comprises a chemical modification of uridine and / or a chemical modification of cytidine. In some embodiments, the mRNA modification comprises pseudouridine, N1-methylpseudouridine, 5-methoxy-uridine, 5-methyl-cytidine, preferably 5-methoxy-uridine.

[0056] In certain embodiments, the mRNA is modified with 5-methoxyuridine and contains at least one CleanCap®.

[0057] In some embodiments, macrophages are engineered using nucleic acid vectors. In some embodiments, macrophages are transfected via electroporation. Other suitable transfection methods include nucleofection.

[0058] In some embodiments, the nucleic acid vector is delivered to macrophages by a nanoparticle, which in some embodiments is a lipid nanoparticle, such as those described in U.S. Patent Nos. 8,058,069, 8,492,359, 8,822,668, 9,364,435, 9,504,651, and 11,141,378.

[0059] In some embodiments, the macrophages are autologous or allogeneic to the subject, hi some embodiments, the macrophages are derived from progenitor cells such as iPSCs, hematopoietic stem cells, or monocytes.

[0060] In some embodiments, macrophages are derived from monocytes by culturing the monocytes under suitable conditions. Such conditions may suitably include: 1. An in vitro method for producing macrophages, comprising: (a) culturing monocytes in a medium for 3 to 5 days to produce macrophages, the medium containing one or more growth factors to stimulate macrophage production; A method in which step (a) is all carried out in the same medium.

[0061] Preferably, the medium is suitable for generating macrophages from monocytes. Preferably, the medium is a T cell medium. Preferably, the medium can be selected from X-Vivo 10, X-Vivo 15, TexMACS, AIMv, RPMI, DMEM, and DMEM / F12. Preferably, the medium is TexMACS (Miltenyi).

[0062] Preferably, the medium is serum-free. Preferably, the medium is xenoprotein-free. Preferably, the medium is GMP compliant.

[0063] Preferably, the medium may contain one or more factors. Suitable factors include growth factors, polysaccharides, cytokines, and chemokines. Suitable factors may include MCSF and GM-CSF. Thus, preferably, the factors are growth factors. Preferably, the one or more factors are GMP-compliant. In one embodiment, the medium may contain one or more growth factors, which may include MCSF or GM-CSF. Monocytes are most commonly cultured with either MCSF or GM-CSF. Culturing monocytes with GM-CSF biases them toward an "inflammatory" phenotype, while culturing monocytes with MCSF biases them toward a "pro-restorative" phenotype. In other embodiments, the one or more growth factors do not include a combination of MCSF and GM-CSF. Thus, in any of the methods of the present invention, if M-CSF is used as a growth factor to generate macrophages, it may be preferable not to also use GM-CSF to generate macrophages from monocytes. This is true for culturing monocytes until macrophages are generated.

[0064] According to a second aspect of the present invention there is provided an engineered macrophage, preferably engineered to express MMP9 by providing an additional or exogenous sequence encoding MMP9, for use in the treatment of inflammatory conditions with a fibrotic component in the lung or liver.

[0065] According to a third aspect of the present invention there is provided an engineered macrophage, preferably engineered to express MMP12 by providing an additional or exogenous sequence encoding MMP12, for use in the treatment of inflammatory conditions with a fibrotic component in the liver.

[0066] According to any aspect of the present invention, the inflammatory condition with a fibrotic component may preferably be cirrhosis of the liver.

[0067] According to any aspect of the invention, the use may include delivering the engineered macrophages to a subject by systemic administration, preferably by systemic injection. In some embodiments, administration is by local injection, for example, to the kidneys and lungs. In some embodiments, administration is to the lungs by nebulizer. Peripheral intravenous injection is preferred for liver conditions to avoid invasive procedures, for example, in patients with cirrhosis. Local injection, such as into the renal artery, for kidney conditions may be better tolerated.

[0068] Preferably, the macrophages used in therapy are engineered ex vivo and delivered to the patient.

[0069] However, in some embodiments, macrophages may be engineered in vivo. In some embodiments, macrophages are engineered in vivo by administering to a subject a preparation of an exogenous coding sequence for MMP9 or MMP12 suitable for transfection of the macrophages. The preparation may include any of the exogenous coding sequences discussed herein. Delivery vehicles suitable for in vivo manipulation may include targeting molecules for macrophages that utilize cell surface markers of macrophages. When in vivo transfection of macrophages is envisioned, local injection of the preparation into the liver or kidney, or local application of the preparation, such as spraying into the lungs, may be more effective. Such treatments may be prepared as nanoparticles to assist in macrophage targeting.

[0070] According to a fourth aspect of the present invention there is provided a population comprising engineered macrophages for use in accordance with any aspect of the present invention.

[0071] According to a fifth aspect of the present invention there is provided a composition comprising an engineered macrophage for use in accordance with the first, second or third aspect of the invention, or a population of engineered macrophages for use in accordance with the fourth aspect of the invention.

[0072] According to a sixth aspect of the present invention there is provided a method of improving the resolution of fibrosis in a chronic condition, comprising the use of an engineered macrophage according to the first, third or third aspect of the invention, a population of engineered macrophages according to the fourth aspect of the invention or a composition according to the fifth aspect of the invention, in other words a method of treating such a condition.

[0073] According to any aspect of the invention, the engineered macrophages are ex vivo macrophages, optionally human ex vivo macrophages.

[0074] The engineered macrophages may be derived or isolated from any suitable source. The engineered macrophages may be isolated from humans. The engineered macrophages may be derived from any suitable progenitor cells, including hematopoietic cells. The engineered macrophages may be derived from human monocytes. It is preferred that the engineered macrophages are derived from human monocytes. Thus, the use of human monocyte-derived macrophages (hMDMs) may be particularly preferred.

[0075] Suitably, the matrix metalloproteinase may be selected from MMP9 or MMP 12. Preferably, the matrix metalloproteinase is one or more of MMP9 and MMP12.

[0076] According to all aspects of the present invention, macrophages are engineered with an exogenous nucleic acid comprising a coding sequence encoding an MMP (either MMP9 or MMP12). This coding sequence can be a gene encoding MMP9 or MMP12. Thus, macrophages are capable of expressing said MMP after transfection. In the case of a DNA vector, the coding sequence is preferably operably linked to a promoter allowing expression of said MMP. It may be preferable for the promoter to be constitutive for certain applications or inducible for other applications, thereby allowing control of MMP expression.

[0077] The exogenous nucleic acid may be any suitable nucleic acid. The exogenous nucleic acid may be present in the cytoplasm or nucleus as an extrachromosomal nucleic acid, or may be integrated into the macrophage genome. The exogenous nucleic acid comprising a coding sequence may encode only MMP9 or MMP12, may encode both MMP9 and MMP12, may encode an additional matrix metalloproteinase, or may encode a coding sequence for a non-MMP protein. When multiple coding sequences are encoded, they may be on the same exogenous nucleic acid or on different exogenous nucleic acids.

[0078] Natural, unengineered macrophages are capable of expressing MMP9 and / or MMP12 under relevant physiological conditions. However, the present invention does not relate to these natural, unengineered macrophages, but instead relates to macrophages in which the expression levels of MMP9 or MMP12 are elevated above physiological levels, thereby improving the antifibrotic properties of the therapeutic macrophages. As used herein, we refer to this as "overexpression" of MMP9 or MMP12. To overexpress these matrix metalloproteinases, macrophages can be engineered to carry additional or exogenous coding sequences for MMP9 and / or MMP12.

[0079] Macrophages may be transfected or engineered with DNA molecules, RNA molecules, or non-viral vectors. Preferably, engineered macrophages may be transfected with a DNA vector. Preferably, the vector may not be derived from a viral genome. Preferably, macrophages may be transfected with an mRNA vector. It may be preferable to engineer macrophages without viral transfection, as this may polarize or activate the macrophages.

[0080] Macrophages may be transfected by any suitable means. Engineered macrophages may be transfected via electroporation. Alternatively, macrophages may be transfected by nucleofection. Any suitable means of transfection may be utilized.

[0081] Macrophages can be suitably manipulated by modifying their genomes through gene editing. Gene editing allows for the permanent insertion of exogenous coding sequences. Numerous gene editing techniques are known, including those that require the use of nucleases. A variety of nucleases are known that can be used to modify genomes, ranging from base editing technology, prime editing technology, to gene editing. Many nucleases are known, including zinc finger, TALEN, and guided nucleases. These can be induced by RNA ("RNA"-guided nucleases), and can be induced by enzymes involved in CRISPR, including, but not limited to, Cas9, Cas12a, Cas13, Mad7, etc. Macrophages themselves can be the subject of gene editing, or progenitor cells can be gene-edited before being converted into macrophages.

[0082] As used herein, "encoding" refers to the ability of a sequence of nucleotides, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of a macromolecule in a biological process, such as a defined amino acid sequence. As such, a coding sequence can be any suitable nucleic acid sequence that provides instructions for synthesizing the relevant entity (herein, MMP9 and / or MMP12). When multiple coding sequences are introduced, the coding sequences can be contained within the same vector / construct or can be contained in different vectors / constructs.

[0083] As used herein, "exogenous" refers to any material (especially genetic material) that is introduced into or produced outside of a particular cell.

[0084] As used herein, an exogenous nucleic acid encodes MMP9 or MMP12, and the engineered macrophages express the coding sequence of MMP9 or MMP12 from the exogenous nucleic acid. Those skilled in the art will understand that variations in the sequences of these genes / coding sequences are also encompassed by the present invention. Ideally, the gene / coding sequence is human. The gene / coding sequence may be codon-optimized. The gene / coding sequence may be tailored. Preferably, the coding sequence for MMP9 is at least 80% similar to NM_004994.3, and preferably at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, or 99% similar to NM_004994.3. Preferably, the sequence of MMP12 is at least 80% similar to NM_002426.6, preferably at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% similar to NM_002426.6.

[0085] In some embodiments, the macrophages are non-virally engineered and contacted with a nucleic acid vector. In some embodiments, the macrophages are transfected with a DNA vector (e.g., a naked DNA vector). Preferably, the DNA vector may encode MMP9 or MMP12. In some embodiments, the nucleic acid vector is not derived from a viral genome. In some embodiments, the macrophages are transfected with one or more free nucleic acids or vectors. In some embodiments, the macrophages are transfected with an RNA vector. In some embodiments, the macrophages are transfected with mRNA. Preferably, the macrophages are transfected with a single mRNA construct expressing MMP9 or MMP12.

[0086] In some embodiments, MMP9 and / or MMP12 are provided to macrophages as mRNA, thus providing the engineered cells with exogenous mRNA molecules.

[0087] In some embodiments, the mRNA is delivered to macrophages via nanoparticles, which in some embodiments are lipid nanoparticles, such as those described in U.S. Patent Nos. 8,058,069, 8,492,359, 8,822,668, 9,364,435, 9,504,651, and 11,141,378.

[0088] The engineered macrophages of the present invention may have a transient or permanent suppression of the STING pathway. Suppression of the STING pathway may be achieved by any suitable means. The STING pathway of the engineered macrophages of the present invention may be suppressed by directly inhibiting one or more components of the STING pathway. The engineered macrophages may be contacted with one or more STING pathway inhibitors. The engineered macrophages may be contacted with one or more STING pathway inhibitors after transfection. Alternatively, the engineered macrophages may be contacted with one or more STING pathway inhibitors before or during transfection. The engineered macrophages may be contacted with one or more STING pathway inhibitors, including IL-10, IL-4, and IL-13.

[0089] It may be preferable that the engineered macrophages can exhibit a pro-restorative / pro-regenerative phenotype. Such phenotypes are further defined herein. The engineered macrophages can exhibit an M2 or M2-like phenotype and can be anti-inflammatory and anti-fibrotic. An M2 or M2-like phenotype is pro-restorative or pro-regenerative. In some embodiments, the pro-restorative phenotype can be described using one or more of the following markers: increased CD206 and / or CD163, decreased inflammatory markers such as CD86 and / or MHC class II (HLA-DR). These increases / decreases are relative to non-polarized (resting) or pro-inflammatory macrophages. In terms of secretory profile, these macrophages are expected not to express TNFα, IFNg, and IL1b, which are typically associated with pro-inflammatory and pro-fibrotic profiles.

[0090] In some embodiments, the method comprises contacting the macrophages with an anti-inflammatory treatment (e.g., IL-4 and IL-13) after transfection. In some embodiments, the method comprises contacting the macrophages with IL-4 and IL-13. In some embodiments, the method comprises electroporation. In some embodiments, the macrophages are contacted with an anti-inflammatory treatment, e.g., IL-4 and IL-13, after electroporation.

[0091] The engineered macrophages can be autologous or allogeneic to the subject. In some embodiments, the engineered macrophages can be isolated from a human, either the subject or another human. In some embodiments, the macrophages are derived from progenitor cells, such as hematopoietic stem cells, or monocytes. In some embodiments, the macrophages are derived from monocytes by culturing the monocytes under appropriate conditions as discussed herein.

[0092] The inflammatory condition may be any suitable inflammatory condition in any tissue or organ of the body. Preferably, the inflammatory condition may be present in the tissues of the liver, lungs, kidneys, heart, gastrointestinal tract, brain, pancreas, thyroid, bone, or uterus. The inflammatory condition may be acute or chronic. It may be preferable for the condition to be chronic. Chronic inflammation refers to mild, long-term inflammation that persists for an extended period of time, such as months to years. Whether the inflammatory condition is acute or chronic, it may be resistant to conventional treatment.

[0093] Inflammatory conditions can have a fibrotic component. Fibrosis, or scarring, is defined by the accumulation of excess extracellular matrix components.

[0094] If the engineered macrophages are engineered with an exogenous nucleic acid comprising an MMP9 coding sequence, the inflammatory condition can be, for example, in the lung or liver, preferably in the liver, more preferably the inflammatory condition comprises cirrhosis of the liver. If the inflammatory condition is in the lung, the condition can comprise pulmonary fibrosis.

[0095] Where the engineered macrophages are engineered with an exogenous nucleic acid comprising an MMP12 coding sequence, the inflammatory condition can be, for example, in the liver, and preferably the inflammatory condition comprises cirrhosis of the liver.

[0096] Cirrhosis represents the final stage of chronic liver injury and progressive fibrosis (scarring), regardless of the underlying etiology. Cirrhosis is characterized by severe liver fibrosis, which causes the collapse of liver structure, hepatocyte dysfunction, and portal hypertension. Various etiologies can lead to cirrhosis. Liver disorders with fibrotic components that can lead to fibrosis include, but are not limited to, nonalcoholic fatty liver disease (NAFL) (e.g., nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH)), or alcoholic liver disease (e.g., alcoholic fatty liver disease (AFLD) or alcoholic steatohepatitis (ASH)). Fibrotic diseases, disorders, and conditions can include mechanical trauma, biliary obstruction, autoimmune hepatitis, iron overload, hepatitis B infection (HBV), and / or hepatitis C infection (HCV). Liver disorders with fibrotic components may also include, but are not limited to, fatty liver disease (SLD), which in turn may include metabolic dysfunction-associated fatty liver disease (MASLD), metabolic-associated steatohepatitis (MASH), Met-ALD, or idiopathic SLD.

[0097] Fatty liver disease can also be known as fatty liver.Metabolic dysfunction-related fatty liver disease refers to non-alcoholic fatty liver disease, and therefore can also be known as NAFLD.Metabolic-related steatohepatitis refers to a more severe form of MASLD, and can also be known as NASH."Met-ALD" refers to individuals who suffer from fatty liver disease and also drink alcohol."Idiopathic SLD" refers to SLD of unknown cause, such as individuals who do not have any known metabolic risk factors for SLD.

[0098] However, the engineered macrophages of the present invention are capable of treating cirrhosis regardless of the underlying etiology.

[0099] Cirrhosis can be either compensated cirrhosis or decompensated cirrhosis (also referred to herein as liver decompensation or HD). Decompensated cirrhosis is defined as a rapid decline in liver function in cirrhotic patients, and is characterized by symptoms including, but not limited to, jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, variceal bleeding, gastrointestinal bleeding, and any combination thereof. In other embodiments, the liver disease is decompensated cirrhosis. In some embodiments, the liver disease is compensated cirrhosis. In some embodiments, the subject has compensated cirrhosis. In some embodiments, the subject shows one or more clinical signs of decompensated cirrhosis selected from the list consisting of jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, variceal bleeding, and gastrointestinal bleeding.

[0100] In some embodiments, the liver disease is one in which cirrhosis is caused by hepatocyte damage, such as diseases of viral origin (including treated (sustained viral response) hepatitis C (HCV), hepatitis B), alcoholism damage (alcohol-related liver disease (ALD)), or non-alcoholic fatty liver disease (NAFLD) (including non-alcoholic steatohepatitis (NASH) (including NASH caused by diabetes or obesity), cryptogenic cirrhosis, hemochromatosis, or alpha-1-antitrypsin deficiency). In some embodiments, the underlying etiology has been eliminated (e.g., a patient suffering from alcoholism damage is no longer drinking, or a patient suffering from HCV damage no longer has HCV, etc.). In some embodiments, the patient with liver disease is at risk of end-stage renal disease.

[0101] In some embodiments, the liver disease is fatty liver disease (SLD). In some embodiments, the fatty liver disease is metabolic dysfunction-associated fatty liver disease (MASLD), Met-ALD, or idiopathic SLD. In some embodiments, the metabolic dysfunction-associated fatty liver disease is metabolic-associated steatohepatitis (MASH).

[0102] Cirrhosis can lead to acute-on-chronic liver failure (ACLF). In some embodiments, the liver disease is ACLF. ACLF is a condition distinct from liver decompensation, which is characterized by the development of ascites, hepatic encephalopathy, gastrointestinal bleeding, or any combination of these conditions in patients with cirrhosis. In contrast, ACLF is associated with organ failure and is associated with a high short-term mortality rate of over 15% at 28 days. Three main characteristics characterize this syndrome: ACLF occurs in the setting of intense systemic inflammation; ACLF frequently occurs in close temporal association with an inflammation-induced exacerbating event (e.g., infection or alcoholic hepatitis); and ACLF is associated with single or multiple organ failure. In some embodiments, engineered macrophages are used in the treatment of cirrhosis in subjects with ACLF.

[0103] Affected subjects suitable for treatment or use according to any aspect or embodiment of the present invention may be subjects with the relevant disease and severity.

[0104] The engineered macrophages of the present invention may be used for therapy. The macrophages of the present invention may be used for treating an inflammatory condition and / or a fibrotic condition in a subject. As defined above, treating herein may refer to the prevention, reduction, or elimination of inflammation / fibrosis / organ damage. For example, the engineered macrophages may be administered to a subject in an acute inflammatory stage for the purpose of preventing a chronic inflammatory condition. The engineered macrophages may also be administered to a subject in a chronic inflammatory stage for the purpose of preventing / reducing chronic fibrosis. The engineered macrophages may also be administered to a subject experiencing an acute exacerbation of a chronic inflammatory stage, such as acute exacerbation of chronic liver failure (ACLF).

[0105] In some embodiments, the condition may be acute-on-chronic liver failure (ACLF). ACLF is a syndrome characterized by acute decompensation of chronic liver disease accompanied by organ failure and high short-term mortality. Excessive systemic inflammatory response is thought to play an important role in the development of ACLF.

[0106] In some embodiments, macrophages that have been engineered to express human MMP9 or MMP12 may be used in the treatment of liver cirrhosis.

[0107] In some embodiments, macrophages that have been engineered to express human MMP9 or MMP12 may be used in the treatment of ACLF.

[0108] Further optional features are set out in the dependent claims. Any feature under any section may be combined with any of the aspects or embodiments of the invention, in any practicable order.

[0109] In certain embodiments, the present invention provides: 1. Engineered macrophages for use in the treatment of inflammatory conditions, preferably inflammatory conditions with a fibrotic component, wherein the macrophages are engineered with an exogenous nucleic acid encoding MMP9 or MMP12.

[0110] 2. Engineered macrophages as described in Example 1, which are derived from progenitor cells.

[0111] 3. The engineered macrophage of embodiment 1 or 2, wherein the matrix metalloproteinase is MMP9.

[0112] 4. The engineered macrophage of embodiment 3, wherein the inflammatory condition is in the lung or liver, preferably in the liver, more preferably the inflammatory condition comprises cirrhosis or ACLF.

[0113] 5. The engineered macrophage of embodiment 1 or 2, wherein the matrix metalloproteinase is MMP12.

[0114] 6. The engineered macrophage of embodiment 5, wherein the inflammatory condition is in the liver, more preferably, the inflammatory condition comprises cirrhosis or ALCF.

[0115] 7. The engineered macrophage of any one of embodiments 1 to 6, which overexpresses the coding sequence encoding MMP9 or MMP12.

[0116] 8. The engineered macrophage of any one of embodiments 1 to 7, wherein the exogenous nucleic acid comprises a DNA molecule, an RNA molecule, or a non-viral vector.

[0117] 9. The engineered macrophage of any one of embodiments 1 to 8, wherein the exogenous nucleic acid is transfected into the macrophage, preferably via electroporation.

[0118] 10. The engineered macrophage of any one of embodiments 1 to 9, wherein the STING pathway is inhibited or the macrophage is treated with an anti-inflammatory agent.

[0119] 11. The engineered macrophage of any one of embodiments 1 to 10, which exhibits a pro-restorative phenotype.

[0120] 12. The engineered macrophage of any one of embodiments 1 to 11, which is autologous or allogeneic to the subject.

[0121] 13. The engineered macrophage of any one of embodiments 1 to 12, wherein the inflammatory condition is chronic.

[0122] 14. A population of engineered macrophages comprising engineered macrophages for use according to any one of embodiments 1 to 13.

[0123] 15. A composition comprising an engineered macrophage or a population of engineered macrophages for use according to any one of embodiments 1 to 14.

[0124] 16. A method for improving the resolution of fibrosis in a chronic condition, comprising the use of an engineered macrophage, a population of engineered macrophages, or a composition described in any one of embodiments 1 to 15.

[0125] 17. A method for treating an inflammatory condition, preferably an inflammatory condition with a fibrotic component, comprising the use of macrophages that have been engineered with an exogenous sequence encoding MMP9 or MMP12.

[0126] The present invention also provides the following embodiments, which may be combined with any other embodiment: 1. An engineered macrophage for use in the treatment of an inflammatory condition with a fibrotic component, wherein the macrophage is engineered with an exogenous nucleic acid encoding MMP9.

[0127] 2. The engineered macrophage of embodiment 1, which is derived from a progenitor cell.

[0128] 3. The engineered macrophage of embodiment 1 or 2, wherein the inflammatory condition is in the lung or liver, preferably in the liver, more preferably the inflammatory condition comprises cirrhosis or ACLF.

[0129] 4. The engineered macrophage of embodiment 3, wherein the inflammatory condition comprises liver cirrhosis.

[0130] 5. The engineered macrophage of embodiment 4, wherein the cirrhosis results from at least one disease or condition selected from the group consisting of non-alcoholic fatty liver disease (NAFL) (e.g., non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH)), alcoholic liver disease (e.g., alcoholic fatty liver disease (AFLD) or alcoholic steatohepatitis (ASH)), mechanical trauma to the liver, bile duct obstruction, autoimmune hepatitis, iron overload, hepatitis B infection (HBV), and hepatitis C infection (HCV).

[0131] 6. The engineered macrophage of embodiment 4, wherein the cirrhosis is caused by fatty liver disease (SLD), and optionally the fatty liver disease is metabolic dysfunction-associated fatty liver disease (MASLD), metabolic-associated steatohepatitis (MASH), Met-ALD, or idiopathic SLD.

[0132] 7. The engineered macrophage of any one of embodiments 4 to 6, wherein the cirrhosis is selected from compensated cirrhosis and decompensated cirrhosis.

[0133] 8. The engineered macrophage of embodiment 7, wherein the cirrhosis is decompensated cirrhosis, and the subject being treated exhibits one or more clinical signs of decompensated cirrhosis selected from the list consisting of jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, variceal bleeding, and gastrointestinal bleeding.

[0134] 9. An engineered macrophage according to any one of embodiments 1 to 8, which overexpresses the coding sequence encoding MMP9.

[0135] 10. The engineered macrophage of any one of claims 1 to 9, wherein the exogenous nucleic acid comprises a DNA molecule, an RNA molecule, or a non-viral vector.

[0136] 11. The engineered macrophage of any one of embodiments 1 to 10, wherein the exogenous nucleic acid is transfected into the macrophage, preferably via electroporation.

[0137] 12. The engineered macrophage of any one of embodiments 1 to 11, wherein the STING pathway is inhibited or the macrophage is treated with an anti-inflammatory agent.

[0138] 13. The engineered macrophage of any one of embodiments 1 to 12, which exhibits a pro-restorative phenotype.

[0139] 14. The engineered macrophage of any one of embodiments 1-13, wherein the macrophage is autologous or allogeneic to the subject, and optionally, the macrophage is derived from a human monocyte or stem cell, and further optionally, the stem cell is an induced pluripotent stem cell.

[0140] 15. The engineered macrophage of any one of embodiments 1 to 14, wherein the inflammatory condition is chronic.

[0141] 16. Engineered macrophages secrete MMP9 and induce 4 × 10 6 16. The engineered macrophage of any one of embodiments 1 to 15, wherein the level of secreted MMP9 protein exceeds 200 ng / ml when cultured in vitro at a cell concentration of 100 ng / ml.

[0142] 17. A population of engineered macrophages comprising engineered macrophages for use according to any one of embodiments 1 to 16.

[0143] 18. A composition comprising an engineered macrophage or a population of engineered macrophages for use according to any one of embodiments 1 to 17.

[0144] 19. A method for improving the resolution of fibrosis in a chronic condition, comprising the use of an engineered macrophage, a population of engineered macrophages, or a composition described in any one of embodiments 1 to 18.

[0145] 20. A method for treating an inflammatory condition with a fibrotic component, comprising the use of macrophages that have been engineered with an exogenous sequence encoding MMP9.

[0146] 21. An engineered macrophage, wherein the macrophage is engineered to express MMP9.

[0147] 22. The engineered macrophage of embodiment 21, wherein the engineered macrophage comprises one or more exogenous coding sequences for MMP9.

[0148] 23. The engineered macrophage of embodiment 22, wherein the exogenous nucleic acid is transfected into the macrophage, optionally via electroporation.

[0149] 24. The engineered macrophage of embodiment 22 or 23, which is transfected with a DNA vector, an RNA vector, optionally mRNA, a vector not derived from a viral genome, or one or more free nucleic acids.

[0150] 25. The engineered macrophage of any one of embodiments 21 to 24, wherein the expressed MMP9 protein is at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% homologous or similar to the sequence shown as SEQ ID NO: 2.

[0151] 26. The engineered macrophage of any one of embodiments 21 to 25, wherein the sequence of the provided MMP9 coding sequence is at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% homologous or similar to SEQ ID NO: 1.

[0152] 27. The engineered macrophage of any one of embodiments 21 to 25, wherein the sequence of the provided MMP9 coding sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:5.

[0153] 28. The engineered macrophage of embodiment 21, which has been engineered to turn on or upregulate an endogenous gene encoding MMP9.

[0154] 29. An engineered macrophage according to embodiment 21 or 28, which has been engineered to block or downregulate an endogenous gene that downregulates MMP9 expression.

[0155] 30. An engineered macrophage described in any one of embodiments 21 to 29, which has been engineered to overexpress MMP9.

[0156] 31. The engineered macrophage according to any one of embodiments 21 to 30, which is derived from human monocytes or stem cells, optionally derived from induced pluripotent stem cells.

[0157] 32. The engineered macrophage of any one of embodiments 21 to 31, wherein the STING pathway is inhibited or the macrophage is treated with an anti-inflammatory agent.

[0158] 33. The engineered macrophage of any one of embodiments 21-32, which exhibits a pro-restorative phenotype.

[0159] 34. Engineered macrophages secrete MMP9 and 4 × 10 6 34. The engineered macrophage of any one of embodiments 21 to 33, wherein the level of secreted MMP9 protein exceeds 200 ng / ml when cultured in vitro at a cell concentration of 100 ng / ml.

[0160] 35. A population of engineered macrophages comprising the engineered macrophages of any one of embodiments 21 to 34.

[0161] 36. A composition comprising a population of engineered macrophages according to any one of embodiments 21 to 34 or engineered macrophages according to embodiment 35.

[0162] 37. The engineered macrophage, population of engineered macrophages, or composition according to any one of embodiments 21 to 36, for use in the treatment of an inflammatory condition, preferably an inflammatory condition with a fibrotic component, preferably cirrhosis of the liver.

[0163] 38. A method for improving the resolution of fibrosis in a chronic condition, comprising the use of an engineered macrophage, a population of engineered macrophages, or a composition described in any one of embodiments 21 to 36.

[0164] Further advantages are described below.

[0165] The invention will be further explained below with reference to exemplary embodiments and the accompanying drawings. [Brief explanation of the drawings]

[0166] [Figure 1] (A,B): Expression levels of MMP9 (A) and MMP12 (B) in macrophages transfected with plasmids encoding MMP9 or MMP12, respectively, as determined by qPCR of mRNA levels normalized to PIK3C2A; (C,D): Expression levels of MMP9 (C) and MMP12 (D) proteins in cell culture supernatants were also determined using ELISA. Expression is measured 24 hours after transfection. NT = non-transfected; NT + STINGi = non-transfected + STING inhibitor cocktail (rIL4 + rIL13). MMPx Trx = transfected with MMPx. [Figure 2] FIG. 1 shows total MMP activity measured in cell culture supernatants of different populations of hMDMs by fluorescence emission. [Figure 3A]Figure 1 shows heatmap representation of flow cytometry analysis of cell surface marker expression ((A) CD14) in non-transfected (NT), NT cultured in the presence of a STING inhibitor cocktail (STINGi), MMP9-transfected (MMP9 Trx), and MMP12-transfected (MMP12 Trx) hMDMs 24 hours post-transfection. Each row represents a different donor. Mean fluorescence intensity (MFI) values ​​were used to generate the heatmap (darker = lower MFI, lighter = higher MFI). [Figure 3B] Heatmap representation of flow cytometry analysis of cell surface marker expression ((B)CD206) in non-transfected (NT), NT cultured in the presence of a STING inhibitor cocktail (STINGi), MMP9-transfected (MMP9 Trx), and MMP12-transfected (MMP12 Trx) hMDMs 24 hours post-transfection. Each row represents a different donor. Mean fluorescence intensity (MFI) values ​​were used to generate the heatmap (darker = lower MFI, lighter = higher MFI). [Figure 3C] Heatmap representation of flow cytometry analysis of cell surface marker expression ((C)25F9) in non-transfected (NT), NT cultured in the presence of a STING inhibitor cocktail (STINGi), MMP9-transfected (MMP9 Trx), and MMP12-transfected (MMP12 Trx) hMDMs 24 hours post-transfection. Each row represents a different donor. Mean fluorescence intensity (MFI) values ​​were used to generate the heatmap (darker = lower MFI, lighter = higher MFI). [Figure 4](A) Doses of multiple cytokines in cell culture supernatants of non-transfected (NT), NT + Sting inhibitor cocktail (NT + STINGi), and CCR2-, MMP9-, and MMP12-transfected (Trx) hMDMs. (B) Doses of multiple MMPs in cell culture supernatants of NT, NT + STINGi, and MMP9-Trx hMDMs. (A-B) Values ​​used to generate heat maps are averages of at least four donors / groups. Black indicates the highest expression level for each analyte, and light gray indicates the lowest expression level for each analyte. [Figure 5] FIG. 1 shows the percentage of phagocytosed hMDMs measured by flow cytometry after 1 hour of incubation with pH-sensitive beads coated with E. coli. DETAILED DESCRIPTION OF THE INVENTION

[0167] The present invention relates to engineered macrophages that have been engineered with the coding sequence of at least one matrix metalloproteinase (MMP), preferably MMP9 or MMP12. The resulting engineered macrophages have desirable anti-fibrotic properties, making them suitable for use in the treatment of inflammatory conditions that may have a fibrotic component.

[0168] "Macrophage," as used herein, refers to a phagocyte involved in the detection, engulfment, and destruction of pathogens and apoptotic cells. Macrophages may be generated by differentiation of any suitable progenitor cell, including monocytes. "Engineered macrophages" of the present invention refer to macrophages that have been engineered to overexpress one or more matrix metalloproteinases (MMPs).

[0169] matrix metalloproteinases Matrix metalloproteinases (MMPs), also known as matrix metalloproteinases or matrixins, are a family of peptidases that can collectively cleave all components of the extracellular matrix (ECM). MMPs can also process bioactive mediators such as growth factors, cytokines, chemokines, and cell surface receptors. Twenty-five mammalian MMPs have been identified (Table 1), which play various roles in ECM maintenance and tissue repair processes, and play both inhibitory and stimulatory roles in fibrosis.

[0170] [Table 1]

[0171] MMP9 (matrix metallopeptidase 9) is a type IV collagenase, a matrix metalloproteinase. MMP9 is also known as 92 kDa type IV collagenase, 92 kDa gelatinase, or gelatinase B (GELB).

[0172] In some embodiments, the MMP9 is human MMP9, as set forth in GenBank Accession No. NM_004994.3. In some embodiments, the human MMP9 comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, this amino acid sequence is encoded by an mRNA comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 5.

[0173] MMP12 (matrix metallopeptidase 12) is a matrix metalloproteinase that degrades soluble and insoluble elastin. MMP12 is also known as macrophage metalloelastase (MME) or macrophage elastase (ME).

[0174] In some embodiments, the MMP12 is human MMP12 as set forth in GenBank Accession No. NM_002426.6. In some embodiments, the human MMP12 comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the amino acid sequence is encoded by an mRNA comprising the sequence of SEQ ID NO: 3.

[0175] Macrophages can be transfected with an exogenous nucleic acid comprising a coding sequence encoding MMP9 or MMP12 for use in treating inflammatory conditions with a fibrotic component.

[0176] Engineered macrophages Macrophages may be transfected with one or more exogenous nucleic acid constructs, such as one or more coding sequences encoding an MMP (MMP9 or MMP12). The construct may be any suitable construct, but is preferably a DNA or RNA construct.

[0177] Macrophages engineered to express MMP9 or MMP12 can be genetically engineered in any suitable manner, for example, using viral or non-viral vectors, DNA or RNA constructs, or gene editing using any suitable technique. Thus, in some embodiments, macrophages can be engineered with one or more exogenous coding sequences. These exogenous coding sequences can encode MMP9 and / or MMP12 and / or gene editing proteins, such as CRISPR or nickase, that can modify the cell's genome to increase the expression of endogenous MMP9 and / or MMP12. This can be achieved, for example, by editing promoter or enhancer sequences.

[0178] The present inventors have surprisingly discovered that transfecting macrophages with an MMP9 or MMP12 expression construct results in the upregulation and secretion of active MMPs without the upregulation of TIMPs. The present inventors have also discovered that transfecting macrophages with an MMP expression construct results in the co-upregulation of other MMPs in addition to the specific transfected MMP (see Examples and Figure 4B). This latter result was particularly surprising. Both of these properties of the macrophages of the present invention would be expected to be beneficial in antifibrotic treatments. Therefore, engineering macrophages with either MMP9 or MMP12 increases the activity of multiple MMPs.

[0179] The results herein demonstrate that transfection of macrophages with a plasmid encoding MMP9 significantly increased MMP9 transcription within 24 hours compared to untransfected macrophages (Figure 1A). Furthermore, the level of secreted MMP9 protein was also significantly increased compared to untransfected controls (Figure 1C). This evidence suggests that the electroporation method employed efficiently transfects macrophages with the MMP9-encoding plasmid, resulting in a rapid, robust, and consistent increase in MMP9 at both the transcript and protein levels.

[0180] The results herein also show that transfection of macrophages with an MMP12-encoding construct significantly increased MMP12 transcription within 24 hours compared to untransfected macrophages (Figure 1B). Furthermore, the level of secreted MMP12 protein was also significantly increased compared to untransfected controls (Figure 1D). This evidence suggests that the electroporation method employed efficiently transfects macrophages with the MMP12-encoding plasmid, resulting in a rapid, robust, and consistent increase in MMP12 at both the transcript and protein levels.

[0181] Preferably, the engineered macrophages can be engineered non-virally. Preferably, the macrophages can be transfected with a DNA vector. Preferably, the DNA vector is a naked DNA vector, such that it is free of proteins and / or lipids. Preferably, the DNA vector is not derived from a viral genome. The DNA vector can be an episomal vector, such that it can function without being integrated into the macrophage chromosome.

[0182] The DNA vector may comprise at least one sequence encoding MMP9 and / or MMP12.

[0183] The DNA vector may comprise at least one sequence encoding MMP9 and / or MMP12 operably linked to a promoter.

[0184] A "promoter" is a nucleotide sequence that initiates and controls the transcription of a polynucleotide. Promoters can include inducible promoters (expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. The terms "promoter" or "regulatory element" are intended to include full-length promoter regions and functional segments of these regions (such as segments that control transcription or translation).

[0185] "Operably linked" refers to an arrangement of elements in which the components so described are configured to perform their normal functions. Thus, a given promoter operably linked to a nucleic acid sequence can affect the expression of that sequence when the appropriate enzymes are present. The promoter need not be contiguous with the sequence, so long as it functions to drive expression of that sequence. Thus, for example, a promoter sequence can still be considered "operably linked" to a coding sequence even if untranslated but transcribed sequences exist between the promoter sequence and the nucleic acid sequence. Thus, the term "operably linked" is intended to encompass any spacing or orientation between the promoter element and the DNA sequence of interest that allows initiation of transcription of the DNA sequence of interest upon recognition of the promoter element by the transcription complex. In one embodiment, the DNA vector can include one or more liver-specific or cirrhosis-specific promoters. In some embodiments, the DNA vector can include the CX3CR1 promoter, insulin-like growth factor 1 (IGF1), or CD1 IB promoter.

[0186] Preferably, the macrophages are genetically engineered macrophages that contain extrachromosomal / episomal constructs that overexpress MMP9 and / or MMP12.

[0187] Preferably, macrophages may be transfected with exogenous RNA (preferably exogenous mRNA) encoding MMP9 and / or MMP12.

[0188] The engineered macrophages can be transfected with any suitable nucleic acid via electroporation. Other suitable transfection methods include nucleofection.

[0189] The engineered macrophages may be transfected with one or more free nucleic acids or vectors.

[0190] The engineered macrophages may be engineered to overexpress MMP9 and / or MMP12.

[0191] Characterization of engineered macrophages The present inventors have discovered that transfecting macrophages with constructs encoding MMP9 or MMP12 achieves upregulation and secretion of multiple active MMPs without upregulating TIMPs. The present inventors have also discovered that transfected macrophages exhibit coexpression of MMPs other than the exogenously encoded MMP (see Examples and Figure 4B). This is a desirable characteristic for engineered macrophages for use in treating inflammatory conditions with fibrotic components.

[0192] TIMPS When cells overexpress MMPs, they may upregulate their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), which render the MMPs non-functional.

[0193] We have demonstrated that MMP-transfected macrophages have sustained MMP activity, suggesting that functional MMPs are upregulated without upregulating TIMPs.

[0194] Preferably, the transfected engineered macrophages of the present invention are capable of overexpressing the transfected MMP with minimal or no upregulation of TIMPs.

[0195] Co-regulation of other MMPs Most surprisingly, the present inventors have discovered that engineered macrophages transfected with MMPs can co-regulate other MMPs in addition to the MMP introduced by transfection. Figure 4B shows that macrophages transfected with MMP9 strongly up-regulate several other MMPs, such as MMP1, MMP8, and MMP10. Thus, multiple other MMPs are affected. The co-upregulation of these additional MMPs may significantly contribute to the anti-fibrotic effects of the macrophage cell therapy of the present invention. For example, MMP8 is normally contained in neutrophil granules and digests collagens I, II, and III, making it particularly interesting for liver fibrosis, where most of the collagen in the fibrous septum is collagen I and III. 27,28 .

[0196] Advantageously, the transfected engineered macrophages of the present invention are capable of upregulating the expression of multiple other MMPs in addition to the MMP introduced by transfection.

[0197] Engineered macrophages for use in treating inflammatory conditions - Patent Application 20070122999 Preferably, the present invention relates to a cell therapy product for inflammatory organ injury based on macrophages that have been genetically modified with exogenous nucleic acids to enhance their anti-fibrotic capacity. Preferably, the engineered macrophages have a pro-restorative phenotype and are anti-inflammatory and anti-fibrotic.

[0198] In some embodiments, the pro-restorative phenotype may be described using one or more of the following markers: increased CD206 and / or CD163, decreased inflammatory markers such as CD86 and / or MHC class II (HLA-DR). These increases / decreases are relative to non-polarized (resting) or pro-inflammatory macrophages. In terms of secretory profile, these macrophages are expected to not express TNFα, IFNγ, and IL1b, which are typically associated with pro-inflammatory and pro-fibrotic profiles.

[0199] "Treatment," as used herein, refers to an intervention in a physiological condition that prevents, reduces, or eliminates clinical symptoms associated with a given physiological condition in a subject.

[0200] "Subject," or "individual," or "animal," or "patient," means any subject (particularly a mammalian subject) for whom diagnosis, prognosis, or treatment is desired, except where the subject is defined as a "healthy subject." Mammalian subjects include humans; domestic animals; farm animals; such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and dairy cows.

[0201] Preferably, the subject may be in need of treatment. Thus, preferably, the subject may be suffering from or at risk of developing a disease. Preferably, the subject may exhibit one or more symptoms of a disease.

[0202] The engineered macrophages of the present invention are for use in treating inflammatory conditions, preferably inflammatory conditions that have or may develop fibrotic components in a subject. As defined above, treating herein can refer to preventing, reducing, or eliminating inflammation and / or fibrosis. For example, engineered macrophages may be administered to a subject in an acute inflammatory stage to prevent a chronic inflammatory condition with fibrotic components. Engineered macrophages may also be administered to a subject in a chronic inflammatory stage to prevent / reduce chronic fibrosis. In a preferred embodiment, the chronic inflammatory liver injury with fibrotic components is cirrhosis. In some embodiments, the liver disease is decompensated cirrhosis. In some embodiments, the liver disease is compensated cirrhosis.

[0203] In some embodiments, the condition is decompensated cirrhosis and the subject exhibits one or more clinical signs of liver decompensation selected from the list consisting of jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, variceal bleeding, and gastrointestinal bleeding.

[0204] In some embodiments, the liver disease is one in which cirrhosis is caused by hepatocyte damage, such as diseases of viral origin (including treated (sustained viral response) hepatitis C (HCV), hepatitis B), alcoholism damage (alcohol-related liver disease (ALD)), or non-alcoholic fatty liver disease (NAFLD) (including non-alcoholic steatohepatitis (NASH) (including NASH caused by diabetes or obesity), spontaneous cirrhosis, hemochromatosis, or alpha-1-antitrypsin deficiency). In some embodiments, the underlying etiology has been eliminated (e.g., a patient suffering from alcoholism damage is no longer drinking, or a patient suffering from HCV damage no longer has HCV, etc.). In some embodiments, the liver disease patient is at risk of end-stage renal disease.

[0205] In some embodiments, the liver disease is fatty liver disease (SLD). In some embodiments, the fatty liver disease is metabolic dysfunction-associated fatty liver disease (MASLD), Met-ALD, or idiopathic SLD. In some embodiments, the metabolic dysfunction-associated fatty liver disease (MASLD) is metabolic-associated steatohepatitis (MASH).

[0206] Metabolic dysfunction-related fatty liver disease refers to non-alcoholic fatty liver disease, and therefore may also be known as NAFLD.Metabolic-related steatohepatitis refers to a more severe form of MASLD, and may also be known as NASH."Met-ALD" refers to individuals who suffer from fatty liver disease and also drink alcohol."Idiopathic SLD" refers to SLD of unknown cause, such as individuals who do not have any known metabolic risk factors for SLD.

[0207] Suitably, an acute disease or injury may be classified as a disease or injury that occurs less than 24 weeks from the cause. Suitably, a chronic disease may be classified as a disease or injury that has persisted for more than 6 months. Suitably, an acute exacerbation of a chronic disease may be classified as a disease or injury that occurs less than 24 weeks from the cause in a patient already suffering from a chronic disease that has persisted for more than 6 months. Suitably, the engineered macrophages of the present invention may be administered to a subject with an acute disease to prevent the transition to or increase in chronic inflammation and fibrosis.

[0208] In some embodiments, the condition is in or affects an organ selected from the group consisting of the liver, lung, heart, kidney, pancreas, skin, gastrointestinal, bone marrow, hematopoietic tissue, nervous system, eye, or a combination thereof.

[0209] In some embodiments, the condition can be acute exacerbation of chronic liver failure (ACLF). ACLF is a syndrome characterized by acute decompensation of chronic liver disease accompanied by organ failure and high short-term mortality. An excessive systemic inflammatory response is thought to play an important role in the development of ACLF. Preferably, the condition is chronic organ damage associated with chronic inflammation. Preferably, the condition relates to the kidney, liver, or lung. For example, the condition can be inflammatory liver injury, inflammatory kidney injury, or inflammatory lung injury.

[0210] Preferably, the macrophages are autologous or allogeneic to the subject.

[0211] The present invention also relates to engineered macrophage populations for use as cell therapy.

[0212] Preferably, the use comprises administering to a subject an effective amount of the engineered macrophages.

[0213] The present invention also relates to a composition comprising the engineered macrophages or populations thereof of the present invention. Preferably, the composition is a pharmaceutical composition.

[0214] Preferably, the engineered macrophages can be formulated into a pharmaceutical composition. Preferably, the composition is suitable for administration to a subject. Preferably, the composition is a liquid. Preferably, the composition is an insoluble liquid.

[0215] Preferably, delivery to the subject is by systemic administration, preferably by systemic injection.

[0216] Preferably, the engineered macrophages are administered to the subject by any route. Preferably, the engineered macrophages are administered to the subject by injection. Preferably, the engineered macrophages are administered to the subject parenterally (preferably intravenously). Preferably, the engineered macrophages are administered to the subject by injection or infusion. Preferably, the engineered macrophages are administered to the subject intravenously by infusion.

[0217] Preferably, administration may be by local injection, for example to the kidney or lung. Preferably, administration may be by nebulizer, for example to the lung.

[0218] Preferably, macrophages for therapeutic use are engineered ex vivo and delivered to a patient.

[0219] However, in some embodiments, macrophages may be engineered in vivo. In some embodiments, macrophages are engineered in vivo by administering to a subject a preparation of an exogenous coding sequence for MMP9 or MMP12 suitable for transfection of the macrophages. The preparation may include any of the exogenous coding sequences discussed herein. Delivery vehicles suitable for in vivo manipulation may include targeting molecules for macrophages that utilize cell surface markers of macrophages. When in vivo transfection of macrophages is envisioned, local injection of the preparation into the liver or kidney, or local application of the preparation, such as spraying into the lungs, may be more effective.

[0220] Advantageously, the present invention relates to a cell therapy product for inflammatory organ injury based on monocyte-derived macrophages genetically modified with a payload that induces a pro-restorative phenotype.

[0221] The M2-like phenotype is pro-restorative and the M1-like phenotype is pro-inflammatory.

[0222] Generation of engineered macrophages that overexpress matrix metalloproteinases The present invention also relates to methods for producing engineered macrophages that overexpress one or more MMPs. The present invention also relates to methods for producing engineered macrophages that overexpress MMP9 and / or MMP12, comprising transfecting macrophages with an exogenous nucleic acid comprising at least one sequence encoding MMP9 and / or MMP12, and optionally contacting the macrophages with one or more STING inhibitors / anti-inflammatory treatments. Preferably, the produced engineered macrophages may be used in cell therapy.

[0223] Preferably, the engineered macrophages produced are manufactured in accordance with GMP standards, and therefore preferably the engineered macrophages and populations thereof are GMP compliant.

[0224] Macrophages for transfection Macrophages for transfection can be isolated from a human, preferably a subject. Macrophages can be produced from any suitable progenitor cells. Suitable progenitor cells include hematopoietic cells. Preferably, the progenitor cells are monocytes. Preferably, the macrophages are produced in vitro.

[0225] The macrophages for transfection may be derived from monocytes. The macrophages for transfection may be human monocyte-derived macrophages (hMDMs). Monocyte-derived refers to macrophages differentiated from monocytes. Monocytes are the natural precursors of macrophages and dendritic cells and are found in blood and bone marrow. Preferably, the macrophages are derived from peripheral blood monocytes, and preferably, the macrophages are peripheral blood monocyte-derived macrophages. Preferably, the macrophages are human peripheral blood monocyte-derived macrophages. Preferably, the monocytes are isolated from a human subject.

[0226] Macrophages may be derived from monocytes by culturing the monocytes, preferably in vitro. Macrophages may be derived from monocytes using any suitable culture method.

[0227] Macrophages may be produced in vitro from monocytes by a culture method lasting 3-8 days, optionally lasting 4-8 days. Preferably, macrophages are produced in vitro from monocytes by a culture method lasting 3-7 days, particularly 4-7 days or 5-7 days. In one embodiment, macrophages are produced in vitro from monocytes by a culture method lasting 3-5 days, 4 or 5 days, or 7 days, known as the 5-day method or the 7-day method, respectively. An example of an in vitro method for producing macrophages from monocytes is described in WO 2019 / 175595. The "5-day" method is described in WO 2021 / 240167, the contents of which are incorporated herein by reference.

[0228] Macrophages are "day 5" method, (a) culturing monocytes in a medium for 3-5 days or 4-5 days to produce macrophages, wherein the medium contains one or more growth factors to stimulate macrophage production; The method may be carried out by a method in which all steps (a) are carried out in the same medium.

[0229] The medium may contain one or more growth factors selected from the CSF family, preferably M-CSF. The medium may contain M-CSF at a concentration of 25 to 150 ng / mL. The medium may contain 100 ng / mL GMP-grade recombinant human macrophage colony-stimulating factor 1 (rhM-CSF-1, also known as "rh(recombinant human) CSF-1").

[0230] Transfection Macrophages may be transfected with exogenous nucleic acid and, at a suitable point in the transfection protocol, may further be contacted with one or more STING inhibitors, or indeed anti-inflammatory treatments.

[0231] A suitable exogenous nucleic acid may be a DNA vector, preferably a non-viral DNA vector. Suitable vectors include DNA constructs, for example, episomal constructs such as plasmids. Preferably, the exogenous nucleic acid encodes one or more MMPs and a promoter. Preferably, the exogenous nucleic acid encodes all sequences necessary and sufficient for transient overexpression of an MMP by macrophages. Preferably, the encoded MMP protein can be secreted by macrophages. Preferably, the promoter is a constitutive promoter. Preferably, the MMP is either or both of MMP9 and MMP12.

[0232] The transfection method can be nucleofection of macrophages, where the nucleic acid enters the nucleus of the cell, and can include transfecting lipid-nucleic acid complexes into the cells via electroporation.

[0233] Transfection methods can include electroporation. Typically, electroporation involves applying an electric current to a cell using electrodes. Typically, a cell is placed between two electrodes, and then a current pulse is generated across the cell. The electric pulse induces the formation of transient pores in the cell membrane, allowing nucleic acids to pass through the pores and enter the cell by passive diffusion or, if the nucleic acid is charged, by active electrophoretic movement induced by an electric field.

[0234] The electroporation step can be performed using any electroporator device by programming the desired electroporation conditions into the electroporator. The electroporation step can be performed using a cliniMACS electroporator (Miltenyi) or other suitable electroporator. The electroporation step can be performed using a Lonza GMP Amaxa.

[0235] Prior to electroporation, macrophages may be contacted with nucleic acids (NAs). Macrophages may be contacted with NAs in a solution. Preferably, the solution is conductive. The solution may be an electroporation solution. The electroporation solution may be a buffer suitable for use with the selected electroporator.

[0236] Preferably, the steps of seeding cells before and after electroporation are performed at 4×10 6 2 x 10 cells / mL 6 hMDM / cm 2 The method includes a step of sowing the above.

[0237] At the time of electroporation, the cell density was 50 x 10 6 cells / mL ~ 150 x 10 6 At the time of electroporation, macrophages should be at least 5 x 10 cells / mL. 7 The cells may be present in solution at a cell density of 100 cells / mL.

[0238] Suitable transfection methods for producing macrophages that express a protein of interest, including suitable electroporation methods, are described in WO 2021 / 240167. Other suitable electroporation methods may be used.

[0239] The transfection method according to the present invention comprises: (a) contacting macrophages with a DNA vector comprising at least one sequence encoding an MMP; (b) electroporating the macrophages with a first pulse phase, the first pulse phase comprising a burst of unipolar pulses or rectangular pulses, each pulse being 750-1000V, and the first pulse phase lasting for a total duration of 20-500 μs; (c) electroporating the macrophages with a second pulse phase, the second pulse phase comprising a burst of pulses or rectangular pulses, each pulse being between 50 and 225 V, and the second pulse phase lasting for a total duration between 2,000 and 50,000 μs. may include:

[0240] The MMP can be MMP9 and / or MMP12.

[0241] The method for producing engineered macrophages that overexpress MMPs includes: (a) contacting macrophages with a DNA vector comprising at least one sequence encoding an MMP; (b) electroporating the macrophages; and (c) contacting the macrophages with one or more STING inhibitors may include:

[0242] The MMP can be MMP9 and / or MMP12.

[0243] Preferably, the method for producing engineered macrophages that overexpress MMPs comprises: (a) contacting macrophages with a DNA vector comprising at least one sequence encoding an MMP; (b) electroporating the macrophages with a first pulse phase, the first pulse phase comprising a burst of unipolar pulses or rectangular pulses, each pulse being 750-1000V, and the first pulse phase lasting for a total duration of 20-500 μs; (c) electroporating the macrophages with a second pulse phase, the second pulse phase comprising a burst of pulses or rectangular pulses, each pulse being between 50 and 225 V, and the second pulse phase lasting for a total duration between 2,000 and 50,000 μs; and (d) contacting the macrophages with one or more STING inhibitors may include:

[0244] The MMP can be MMP9 and / or MMP12.

[0245] STING inhibitor / anti-inflammatory treatment Methods of producing engineered macrophages according to the present invention include transfecting macrophages with an exogenous nucleic acid (optionally a nucleic acid vector, such as a DNA vector or an RNA vector) comprising at least one sequence encoding an MMP of interest, and contacting the macrophages with one or more stimulator of interferon genes (STING) inhibitors or anti-inflammatory treatments.

[0246] The STING inhibitor is a STING pathway inhibitor. Preferably, the STING pathway inhibitor suppresses the STING pathway. Preferably, the suppression of the STING pathway prevents the activation of interferon secretion. Some STING inhibitors are also anti-inflammatory.

[0247] The STING pathway inhibitor may be added during the process of transfecting macrophages.

[0248] The STING pathway inhibitor may be added after the electroporation step. Thus, the method may comprise contacting the macrophage with the STING pathway inhibitor, preferably contacting the transfected macrophage with the STING pathway inhibitor.

[0249] The method is: (a) contacting the engineered macrophages with a DNA vector comprising at least one sequence encoding an MMP (MMP9 or MMP12); (b) electroporating the macrophages; and (c) contacting the macrophages with one or more STING inhibitors may include:

[0250] The MMP can be MMP9 and / or MMP12.

[0251] Inhibition or suppression of the STING pathway can be achieved by contacting transfected macrophages with anti-inflammatory cytokines. In particular, transfected macrophages can be contacted with IL10, IL4, and IL13 (IL4 and IL13). Preferably, the STING inhibitor cocktail includes IL4 and IL13. This provides a good balance between suppressing the STING pathway and enabling effective overexpression of one or more MMPs in the transfected macrophages.

[0252] The transfected macrophages may be contacted with these anti-inflammatory cytokines (IL10 and / or IL4 / IL13) for a period of about 2 hours to about 48 hours, preferably for a period of 4 hours to 40 hours, preferably for a period of 12 to 24 hours, optionally for about 16 hours.

[0253] The transfected macrophages may be contacted with these anti-inflammatory cytokines (IL4 / IL13) at a concentration of 2 ng / mL to 200 ng / mL, preferably at a concentration of 5 ng / mL to 150 ng / mL, preferably at a concentration of 10 ng / mL to 100 ng / mL, preferably at a concentration of 15 ng / mL to 75 ng / mL, preferably at a concentration of 20 ng / mL to 50 ng / mL.

[0254] Alternatively, the STING pathway inhibitor may be a small molecule. Preferably, the STING inhibitor may be selected from, for example, BX-795, H-151, Amlexanox, and MRT67307.

[0255] Preferably, the STING pathway inhibitor is used in solution.

[0256] In contacting macrophages with one or more STING inhibitors, cells may be seeded as follows: 4×10 6 2 x 10 cells / mL 6 macrophages / cm 2 .

[0257] Inhibition of the STING pathway may be achieved by inhibiting or suppressing one or more elements of the STING pathway and / or one or more elements that regulate the STING pathway. Such inhibition or suppression of the STING pathway may include inhibiting or blocking activation of the STING pathway, and thus suitably, inhibition or suppression of the STING pathway may include deactivating the STING pathway.

[0258] Preferably, the STING pathway may be considered suppressed or inhibited. Preferably, the STING pathway is suppressed or inhibited compared to transfected macrophages not contacted with or not containing a heterologous nucleic acid encoding a STING pathway inhibitor. The STING pathway may be suppressed by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% compared to transfected macrophages not contacted with or not containing a heterologous nucleic acid encoding a STING pathway inhibitor.

[0259] The following definitions are provided:

[0260] "Payload," as used herein, refers to a gene of therapeutic interest that is introduced by transfection and tested for its effect on macrophages.

[0261] "Non-polarized macrophages," as used herein, refer to mature macrophages that have not received any further stimulation to elicit a specific functional capability; non-polarized macrophages may also refer to naive, resting, or non-activated macrophages.

[0262] "Mature macrophages" refer to macrophages that express mature cell surface markers (preferably CCR2-, CD14+, and 25F9+).

[0263] Macrophages can adopt a variety of states termed "polarization," which is usually simplistically divided into two main extremes: "pro-inflammatory" (or classically activated, "M1," "M1-like") and "pro-regenerative" (or "pro-resolving," alternatively activated, "M2," "M2-like," anti-inflammatory, or anti-fibrotic). However, macrophages can adopt states between these extremes; states can be "non-polarized," resting, or naive (M0), or can approach anti-inflammatory or pro-inflammatory states.

[0264] It is generally assumed that M1 macrophages are pro-inflammatory, while M2 macrophages are involved in immune regulation and wound healing responses. However, it is becoming increasingly clear that this binary classification does not address the more complex heterogeneity in vivo, where macrophages adopt distinct phenotypes and even switch phenotypes in response to various stimuli. These in vivo macrophage phenotypes cannot be accurately reproduced in tissue culture models, highlighting the importance of functional characterization of macrophages.

[0265] Macrophages acquire a "pro-regenerative" state under the action of a combination of various factors, such as macrophage colony-stimulating factor (M-CSF), IL-4, IL-13, IL-10, and TGF-β. Macrophages primarily mediate wound healing and tissue regeneration.

[0266] The engineered macrophages of the present invention can function as "pro-restorative" macrophages. As described, for example, in Ramachandran et al., Proc Natl Acad Sci USA. 2012 Nov. 13; 109(46): E3186-E3195, pro-restorative macrophages exhibit a loss of pro-inflammatory gene expression, an increase in expression of matrix-degrading enzymes, and an enrichment of phagocytosis-related genes. Furthermore, the phenotype of pro-restorative macrophages falls outside the M1 / M2 paradigm, highlighting the limitations of this classification in an in vivo setting. Pro-restorative macrophages play an important role in tissue remodeling (e.g., resolution of fibrosis). The engineered macrophages can be subjected to anti-inflammatory treatment.

[0267] The macrophages reported in Ramachandran et al. have high MMP activity. Furthermore, in Figure 5, the authors show several genes that are regulated in pro-restorative macrophages. Notably, there is upregulation of Mrc1 (now CD206), which is also expressed in the macrophages of the examples presented here. In addition, low levels of TNFα and IL1b are shown. This may therefore be indicative of a pro-restorative phenotype.

[0268] "GMP-compliant," as used herein, means that a method complies with the principles of good manufacturing practice and may be used interchangeably with "GMP-compatible" and "GMP-grade." For example, GMP-compliant media must be serum-free, antibiotic-free, animal-substance-free, and xenoprotein-free. The WHO provides the following guidance on what is required for good manufacturing practice: "Chapter 1: WHO good manufacturing practices: Main principles for pharmaceutical products." Quality Assurance of Pharmaceuticals: A compendium of guidelines and related materials - Good manufacturing practices and inspection. 2 (2nd updated ed.). WHO Press. pp. 17-18. ISBN 9789241547086.

[0269] It should be noted that the term "a" or "an" entity refers to one or more of that entity.

[0270] "About" means + / -10%, + / -9%, + / -8%, + / -7%, + / -6%, + / -5%, + / -4%, + / -3%, + / -2%, + / -1% of the stated value, unless otherwise defined.

[0271] A "therapeutically effective amount" of macrophages, as described herein, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from developing disease or promotes disease regression as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of functional impairment or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to the skilled practitioner, for example, in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in an in vitro assay. Therapeutically effective amounts and administration regimens can be determined empirically by testing in known in vitro or in vivo (e.g., animal model) systems.

[0272] Preferably, the subject may be in need of treatment. Thus, preferably, the subject may be suffering from a disease, condition, or disorder, or may be at risk of developing a disease, condition, or disorder. Preferably, the subject may exhibit one or more symptoms of a disease, condition, or disorder.

[0273] equivalent Those skilled in the art will recognize, or be able to recognize using no more than routine experimentation, equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the scope of the following claims. Any combination of the embodiments disclosed in any multiple dependent claims or examples is contemplated as being within the scope of the present disclosure.

[0274] Incorporation by Reference The disclosures of any and all patents, patent application publications, and scientific publications mentioned herein, as well as the contents of any drawings thereof, are specifically incorporated herein by reference in their entirety.

[0275] Sequence Listing Italics - protein coding

[0276] SEQ ID NO: 1 Human MMP9 mRNA coding sequence NCBI reference: NM_004994.3 (uridine is transcribed here as thymine) [ka]

[0277] SEQ ID NO: 2 Protein sequence human MMP9 (bold = open reading frame) [ka]

[0278] SEQ ID NO: 3 MMP12 mRNA coding sequence Human: NCBI Reference Sequence: NM_002426.6 (Uridine is transcribed here as thymine) [ka] [ka]

[0279] SEQ ID NO: 4: MMP12 preprotein sequence Human: NP_002417.2 [ka]

[0280] SEQ ID NO: 5. Optimized mRNA sequence of MMP9. Uridines are transcribed here as thymines, and all uridines are 5-methoxy-uridines. [ka]

[0281] (References) TIFF2025533510000009.tif229157TIFF2025533510000010.tif234157TIFF2025533510000011.tif229157TIFF2025533510000012.tif47157 [Example]

[0282] Materials and Methods hMDM cell culture Monocytes were isolated from buffy coat products from healthy volunteers provided by the Scottish National Blood Transfusion Service (SNBTS) using a Ficoll gradient (GE Healthcare) followed by magnetic column selection using CliniMACS CD14 Reagent (Miltenyi Biotec). Monocytes were matured for 1–7 days in culture in phenol red-free TexMACS (Miltenyi Biotec) in the presence of 100 ng / mL GMP-grade recombinant human macrophage colony-stimulating factor (rhM-CSF) (R&D System, Biotech). hMDMs were cultured at 2 × 10 for 5 days. 6 pieces / cm 2 hMDMs were cultured in 6-well multiwell plates (Corning Costar) at a density of 1000 x 1000. hMDMs were counted using an automatic counter (TC20, BioRad).

[0283] Payload transfection Day 5 human monocyte-derived macrophages (hMDMs) were resuspended in CliniMACS® El electroporation buffer (Miltenyi Biotec, #170-076-625) at a density of 75 x 10^6 or 150 x 10^6 cells / ml, and 100–300 μl of the suspension was transferred to an electroporation cuvette with a 0.2 cm gap. 5 μg of plasmid DNA per 5 x 10^6 cells was added directly to the cuvette, and the cuvette was gently tapped to mix with the cells. Cells were transfected using a CliniMACS® electroporator controlled by a CliniMACS Prodigy®. Electroporation parameters are outlined in Table 2 below (Patent No. PCT / GB2021 / 051300).

[0284] [Table 2]

[0285] After transfection, cells were harvested from the cuvette into sterile TexMACS™ GMP medium (Miltenyi Biotec, #170-076-306) using an 18G sterile needle attached to a 1 mL syringe or similar device. Cell counts were performed using a TC-20 automated cell counter (Bio-Rad). Cells were spun down at 300 × g for 5 minutes at room temperature. The supernatant was aspirated and the cells were resuspended in sterile TexMACS™ GMP medium (Miltenyi Biotec, #170-076-306) supplemented with 100 ng / mL rhM-CSF (R&D systems, #AFL216), 20 ng / mL rhIL4 (R&D systems, #AFL204), and 20 ng / mL rhIL13 (R&D systems, #213-ILB / CF) at a concentration of 4 x 10 cells / mL and 2 x 10 cells / cm. 2 were sown at a density of

[0286] Flow cytometry labeling Macrophages were cultured at 1 × 10 in PBS + 0.5 mM EDTA (Life Technologies) + FcR block 1:100 (Miltenyi). 6 Cells were resuspended at a concentration of 100 μL / ml. 100 μL of cells were dispensed into a low-adhesion round-bottom 96-well plate. Cells were incubated for 5 minutes, then the appropriate antibody (see Table 3) was added to the appropriate test well and left at 4°C for 20 minutes. Cells were washed with PBS + 0.5 mM EDTA and spun down at 300 g for 5 minutes. The supernatant was removed and cells were resuspended in PBS + 0.5 mM EDTA + 1:1000 DRAQ7. Incubated at 4°C for 5 minutes. Washed as before and then resuspended in 100 μL of PBS + 0.5 mM EDTA + 0.1% human serum. 50 μL of cells were dispensed into a Novocyte 3000 or Novocyte Quanteon (Agilent).

[0287] [Table 3]

[0288] MSD V-plex cytokine dosage Cytokines in cell culture supernatants were analyzed using the V-PLEX Human Biomarker 10-Plex kit on a MESO Quickplex SQ 120 according to the manufacturer's instructions (Meso Scale Discovery). 10 μL of supernatant was tested. Results are shown in pg / mL. Values ​​have been adjusted to account for the dilution during testing. All data shown represent secretion over a 24-hour period. Reported data are net concentrations calculated by subtracting the amount of a given cytokine in the culture medium (TexMACS) alone from the amount of cytokine detected in the cell culture supernatant.

[0289] RNA extraction and qPCR RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. One-step real-time qPCR was performed using the extracted RNA with the QuantiTect SYBR Green RT PCR Kit and QuantiTech Primers (Qiagen). Briefly, a master mix was prepared, RNA was diluted to 5 ng / μL, and 1 μL of master mix was added per test. One-step qPCR was performed in a 384-well Quant Studio 5 (ThermoFisher) with a program of 50°C for 10 minutes, then 95°C for 15 minutes for reverse transcription and cDNA synthesis, followed by 40 cycles of 94°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. PIK3C2A and HPRT1 were two housekeeping genes used for normalization, and RNA expression was measured using 2% RI. -ΔCt The calculation was performed using the method (ΔCt = Ct (gene of interest) - Ct (geometric mean of two housekeeping genes)).

[0290] MMP activity assay MMP activity was confirmed by successful cleavage of a standard MMP peptide. The standard MMP peptide is flanked by a quencher and a fluorescent signal and does not fluoresce when intact. The cleaved peptide no longer quenches the fluorescent signal, thus releasing fluorescence, measured as relative fluorescence units (RFU). The assay was performed according to the manufacturer's instructions: http: / / www.abcam.com / ps / products / 112 / ab112146 / documents / ab112146%20MMP%20Activity%20Assay%20Kit%20Fluorometric%20-%20Green%20v4b%20(website).pdf (ab112146 MMP Activity Assay Kit Fluorometric - Green v4b (website).pdf (abcam.com)), testing 25 μl of cell culture supernatant. All data shown represent the activity of secreted MMPs over a 24-hour period. Results are plotted as RFU minus the background fluorescence of the culture medium (TexMACS) alone.

[0291] MSD R-plex MMP dosage MMPs in cell culture supernatants were analyzed using the R-PLEX Human Biomarker 7-Plex kit on a MESO Quickplex SQ 120 according to the manufacturer's instructions (Meso Scale Discovery). 15uL of supernatant was tested. Results are shown in pg / mL. Values ​​are adjusted to account for the dilution used. All data shown represent secretion within 24 hours. Data reported are net concentrations calculated by subtracting the amount of a given cytokine in culture medium alone (TexMACS) from the amount of cytokine detected in the cell culture supernatant.

[0292] Phagocytosis - Live Imaging hMDMs were seeded at a density of 250,000 cells / well in a 96-well clear-bottom imaging plate (Grenier) and allowed to attach for 24 hours at 37°C and 5% CO2 in 100 μl of TexMACS + MCSF (non-transfected hMDMs) or TexMACS + MCSF + IL4 / IL13 (STING inhibitor cocktail, transfected hMDMs). The supernatant was removed, and cells were stained with 100 μl of PBS + NucBlue (ThermoFisher) and 5 μg / ml Cellmask Deep red Plasma membrane stain (Invitrogen) for 30 minutes at 37°C and 5% CO2. Cells were washed three times with 100 μl of PBS. For TO analysis using the Opera Phenix High Content Screening System, 50 μl of PBS was added to the cells. After TO, 50 μl of 0.2 mg / ml pHrodo Red Zymosan beads (Life Technologies) were added to the cells. A series of images were acquired over a 96-minute period to monitor phagocytosis. Images were analyzed using Columbus data imaging software and a Tibco Spotfire data analysis system. Graphs were plotted using GraphPad Prism 9.2.0.

[0293] statistics All dots reported represent separate donors. At least three donors were analyzed for each condition unless otherwise specified. Data are presented as mean ± SD where appropriate. Two-tailed t-tests were performed for paired data where appropriate.

[0294] result hMDMs can be efficiently transfected with plasmids encoding MMPs Ideal therapeutic macrophages used in the treatment of inflammatory organ injury with a fibrotic component must possess antifibrotic properties. Untransfected, nonpolarized macrophages express little or no MMPs. Therefore, hMDMs were transfected with either MMP9 or MMP12. Expression of MMP9 and MMP12 was examined 24 h posttransfection. Significant increases in MMP9 and MMP12 mRNA levels were observed in transfected hMDMs compared with untransfected hMDMs (Figure 1A-B). Furthermore, MMP9 and MMP12 protein expression was also significantly increased compared with untransfected hMDMs (Figure 1C-D).

[0295] MMP-transfected hMDMs secrete active and functional MMPs in the extracellular space. When cells overexpress MMPs, they may upregulate their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). Upregulation of TIMPs renders MMPs nonfunctional. An enzymatic assay was developed to verify the functionality of MMPs after secretion into the cell culture medium of non-transfected (NT), non-transfected treated with a STING inhibitor cocktail (NT+STINGi), MMP9-transfected (MMP9 Trx), MMP12-transfected (MMP12 Trx), and CCR2-transfected (CCR2 Trx) hMDMs. The results showed that MMP9-transfected hMDMs had significantly higher MMP activity in their cell culture supernatants, and transfection with MMP12 tended to upregulate MMP activity (Figure 2). The lack of a significant increase in MMP activity in cell culture supernatants of CCR2-transfected hMDMs confirms that the increased activity is a specific effect of the transfected payload and not a general side effect of transfection.

[0296] The increase in MMP activity in MMP-transfected macrophages is significant, and in many cases, when MMPs are upregulated, tissue inhibitors of MMPs (TIMPs) are also upregulated, thereby limiting the effectiveness of the extracellular activity of MMPs. 15 Therefore, the results suggest that the transfection method used achieves increased secretion of active MMPs without significant upregulation of TIMPs.

[0297] Taken together, these data support the use of MMP-transfected hMDMs in inflammatory diseases with a fibrotic component. MMPs can be transfected alone or in combination.

[0298] MMP-transfected hMDMs maintain the expression of macrophage-specific markers To demonstrate that MMP-transfected hMDMs have a favorable safety profile, we measured the expression of markers associated with macrophage lineage and function. A complete report of the mean MFI detected in non-transfected hMDMs (NT), NT treated with a STING inhibitor cocktail (STINGi), MMP9-transfected (MMP9 Trx), and MMP12 Trx hMDMs can be found in Table 4. Figure 3 is a graph representing the data in Table 4.

[0299] [Table 4]

[0300] Figure 3 shows that some markers, such as CD206, are modulated while others, such as 25F9, remain stable. Overall, expression of macrophage lineage and functional markers remains detectable by flow cytometry, confirming that transfection does not significantly affect cell identity.

[0301] MMP9 transfection alters the proinflammatory and antifibrotic secretory profile of hMDMs Other important features of antifibrotic cell therapy are its proinflammatory profile and its ability to secrete MMPs other than those specifically overexpressed. 8 .

[0302] Both properties were investigated using a multi-protein delivery system. To assess the inflammatory profile, CCR2 Trx hMDMs were used as a control to distinguish between payload-specific effects and those of the transfection procedure. MMP9-Trx and MMP12-Trx hMDMs up-regulate pro-inflammatory cytokines such as TNFα, IL12p70, and IL8 (Figure 4A and Table 5). This was only semi-surprising, as pro-inflammatory macrophages stimulated with IFNγ and LPS have previously been reported to up-regulate MMP9. 16 However, previous studies have also found upregulation of MMP9 in human pro-restorative macrophages after in vitro stimulation with IL-10. 17 Furthermore, studies performed using mouse models of chronic liver injury have identified a population with a hybrid pro-inflammatory / pro-restorative phenotype that is involved in liver regeneration and overexpresses MMP9 and MMP12 but not pro-inflammatory mediators such as TNFα or IL12p70. 8 Finally, in THP-1 cells, a direct correlation between MMP9 overexpression and a proinflammatory phenotype has been described. 18 .

[0303] To assess MMP secretion profiles, we used a multi-protein administration method that allows for simultaneous evaluation of MMPs 1, 3, 7, 8, and 10. Conditioned media from non-transfected (NT), non-transfected + STING inhibitor cocktail (NT + STINGi), and MMP-transfected hMDMs were analyzed. IL-4 has previously been demonstrated to suppress MMP expression. 19IL4, along with IL13, is part of the STING inhibitor cocktail. Therefore, the use of STINGi could potentially suppress the expression of one or more MMPs, including MMP9 itself. Surprisingly, MMP Trx hMDMs strongly upregulated all administered MMPs in addition to MMP9 itself (Figure 4B). Therefore, the genetic engineering method presented here is suitable for creating cell therapies with antifibrotic properties.

[0304] [Table 5]

[0305] MMP9-transfected hMDMs have similar phagocytosis to untransfected macrophages, whereas MMP12-transfected hMDMs do not. The final step in characterizing MMP-transfected macrophages was measuring their phagocytic activity. MMP9 Trx, MMP12, non-transfected (NT) hMDMs, and NTs treated with a STING inhibitor cocktail (STINGi) were loaded with pH-sensitive beads coated with Escherichia coli (E. coli) for 1.5 hours. Phagocytosis was measured by live imaging.

[0306] The data in Figure 5 show that MMP12-transfected hMDMs have reduced phagocytic activity compared to NT and NT+STINGi hMDMs, whereas MMP9-transfected hMDMs exhibit similar phagocytosis as NT and NT+STINGi hMDMs.

[0307] These data suggest that careful consideration is needed when selecting appropriate MMPs to be used as a therapeutic strategy. Furthermore, these data support the use of different MMPs in combination to maximize target diversity in the fibrous extracellular matrix while preserving other functions such as phagocytosis.

[0308] Conclusions and Discussion MMPs have complex biology, which is often organ and disease specific. 20 For example, MMP9 has significant anti-fibrotic effects in models of chronic liver disease and chronic lung disease. 21~23 However, this appears to be detrimental in renal fibrosis. 24 The results presented here support the feasibility of genetically modifying macrophages using DNA vectors expressing specific MMPs of choice, allowing for tailoring antifibrotic cell therapy to the organ and condition of interest.

[0309] In pathophysiology, macrophages are among the largest expressers of MMPs. However, as shown in Figures 1 and 4, in vitro derived hMDMs express only minimal levels of MMPs, and furthermore, activity is barely detectable in the conditioned medium of these cells (Figure 2). In models of chronic liver disease, it has been demonstrated that macrophages with hybrid phenotypes but pro-restorative functions exist, characterized by expression of MMP9 and MMP128. 8 Although it is very difficult to recapitulate this phenotype in vitro, in Figure 4B we show that the IL4+IL13 STING inhibitory cocktail promotes the expression of a diverse set of MMPs compared to untreated, untransfected macrophages. However, both cell types also have negligible MMP activity detected in their supernatants (Figure 2), making them unsuitable as good antifibrotic agents. MMPs are typically upregulated during infection. 25 , or is upregulated upon in vitro stimulation with LPS 16Stimulating hMDMs with LPS is not a viable option for cell therapy, and therefore, alternative strategies for upregulating MMPs must be explored. In this study, specific MMPs of interest were introduced into macrophages by transfection. As a proof-of-principle, day 5 hMDMs were transfected with plasmids expressing MMP9 (type IV collagenase) and MMP12 (elastase), two MMPs with distinct target proteins in the extracellular matrix. As shown in Figure 1, this method achieved significant overexpression of both MMP9 and MMP12. Furthermore, a significant increase in MMP activity was detected in the conditioned medium from transfected hMDMs compared with untransfected hMDM medium (Figure 2). A CCR2-expressing plasmid was used as a control to demonstrate that the upregulation of MMP activity in the conditioned medium was payload-specific and not simply a result of the transfection procedure. The increase in MMP activity in transfected cells is not trivial for two reasons: (i) RNA expression of MMPs does not necessarily lead to secretion of active enzymes into the extracellular space; 20,23,26 However, the approach outlined here achieves the ability to secrete active MMPs. (ii) It is known that when MMPs are upregulated, their tissue inhibitory factors (TIMPs) are also upregulated in a negative feed loop. 15, 23 Thus, the method described herein achieves a sustained increase in MMP activity, demonstrating that this technique surprisingly achieves upregulation of functional MMPs without upregulating TIMPs.

[0310] The most surprising feature of the MMP-transfected macrophages of the present invention is their ability to co-upregulate other MMPs in addition to the exogenously encoded MMP. Figure 4B shows that MMP9 Trx hMDM strongly upregulates several other MMPs, such as MMPs 1, 8, and 10, which may contribute significantly to the anti-fibrotic effects of the macrophage cell therapy of the present invention. For example, MMP8 is normally contained in neutrophil granules and digests collagens I, II, and III, and is therefore of particular interest in the context of liver fibrosis, where most of the collagen in the fibrous septa is collagen I and III. 27,28 .

[0311] MMP-transfected macrophages maintained cell surface expression of macrophage-specific markers such as CD14, CD206, and 25F9 (Figure 3), demonstrating that transfection with MMPs did not alter cell identity. Interestingly, however, MMP9-transfected cells were significantly better phagocytes than MMP12-transfected cells (Figure 5). This further demonstrates the versatility of the therapeutic approach described herein, as appropriate MMPs with the right combination of antifibrotic and pro-phagocytic properties can be selected depending on the disease target.

[0312] MMP-transfected macrophages exhibit a more proinflammatory profile compared with non-transfected hMDMs and CCR2-transfected hMDMs. Therefore, when antifibrotic therapy is used in the context of chronic inflammatory diseases, additional interventions to suppress inflammation in MMP-transfected hMDMs may be required. Suitable anti-inflammatory treatments may include treatment with IL-4 / 11-13, etc.

[0313] Therefore, the present invention relates to a novel approach to anti-fibrotic cell therapy by transfecting macrophages to overexpress one or more metalloproteinases selected from MMP9 and MMP12. The type and combination of metalloproteinases can be tailored depending on the fibrotic disease being targeted.

Claims

1. An engineered macrophage for use in the treatment of an inflammatory condition with a fibrotic component, the macrophage being engineered with an exogenous nucleic acid encoding MMP9.

2. 10. The engineered macrophage of claim 1, which is derived from a progenitor cell.

3. 3. The engineered macrophage of claim 1 or 2, wherein the inflammatory condition is in the lung or liver, preferably in the liver, more preferably the inflammatory condition comprises cirrhosis or ACLF.

4. 4. The engineered macrophage of claim 3, wherein the inflammatory condition comprises liver cirrhosis.

5. 5. The engineered macrophage of claim 4, wherein the cirrhosis results from at least one disease or condition selected from the group consisting of non-alcoholic fatty liver disease (NAFL) (e.g., non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH)), alcoholic liver disease (e.g., alcoholic fatty liver disease (AFLD) or alcoholic steatohepatitis (ASH)), mechanical trauma to the liver, bile duct obstruction, autoimmune hepatitis, iron overload, hepatitis B infection (HBV), and hepatitis C infection (HCV).

6. 5. The engineered macrophage of claim 4, wherein the cirrhosis results from fatty liver disease (SLD), and optionally the fatty liver disease is metabolic dysfunction-associated fatty liver disease (MASLD), metabolic-associated steatohepatitis (MASH), Met-ALD, or idiopathic SLD.

7. 7. The engineered macrophage of any one of claims 4 to 6, wherein the cirrhosis is selected from compensated cirrhosis or decompensated cirrhosis.

8. 8. The engineered macrophage of claim 7, wherein the cirrhosis is decompensated cirrhosis and the subject being treated exhibits one or more clinical signs of decompensated cirrhosis selected from the list consisting of jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, variceal bleeding, and gastrointestinal bleeding.

9. 9. The engineered macrophage of any one of claims 1 to 8, which overexpresses the coding sequence encoding MMP9.

10. 10. The engineered macrophage of claim 1, wherein the exogenous nucleic acid comprises a DNA molecule, an RNA molecule, or a non-viral vector.

11. 11. The engineered macrophage of any one of claims 1 to 10, wherein the exogenous nucleic acid is transfected into the macrophage, preferably via electroporation.

12. 12. The engineered macrophage of any one of claims 1 to 11, wherein the STING pathway is inhibited or the macrophage is treated with an anti-inflammatory agent.

13. 13. The engineered macrophage of any one of claims 1 to 12, which exhibits a pro-restorative phenotype.

14. 14. The engineered macrophage of any one of claims 1 to 13, wherein the macrophage is autologous or allogeneic to the subject, optionally wherein the macrophage is derived from a human monocyte or stem cell, further optionally wherein the stem cell is an induced pluripotent stem cell.

15. 15. The engineered macrophage of any one of claims 1 to 14, wherein the inflammatory condition is chronic.

16. The engineered macrophages secreted MMP9 and stimulated 4 × 10 6 16. The engineered macrophage of any one of claims 1 to 15, wherein the level of secreted MMP protein exceeds 200 ng / ml when cultured in vitro at a cell concentration of 100 ng / ml.

17. 17. A population of engineered macrophages comprising engineered macrophages for use according to any one of claims 1 to 16.

18. 18. A composition comprising an engineered macrophage or a population of engineered macrophages for use according to any one of claims 1 to 17.

19. 20. A method of improving the resolution of fibrosis in a chronic condition, comprising the use of an engineered macrophage, population of engineered macrophages, or composition of any one of claims 1 to 18.

20. A method for treating an inflammatory condition with a fibrotic component, comprising the use of macrophages that have been engineered with an exogenous sequence encoding MMP9.

21. Macrophages that have been engineered to express MMP9.

22. 22. The engineered macrophage of claim 21, comprising one or more exogenous coding sequences for MMP9.

23. 23. The engineered macrophage of claim 22, wherein the exogenous nucleic acid is transfected into the macrophage, optionally via electroporation.

24. 24. The engineered macrophage of claim 22 or 23, transfected with a DNA vector, an RNA vector, optionally mRNA, a vector not derived from a viral genome, or one or more free nucleic acids.

25. 25. The engineered macrophage of any one of claims 21 to 24, wherein the expressed MMP protein is at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% homologous or similar to the sequence presented as SEQ ID NO:

2.

26. 26. The engineered macrophage of any one of claims 21 to 25, wherein the sequence of the provided MMP9 coding sequence is at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% homologous or similar to SEQ ID NO:

1.

27. 26. The engineered macrophage of any one of claims 21 to 25, wherein the sequence of the provided MMP9 coding sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:

5.

28. 22. The engineered macrophage of claim 21, engineered to turn on or upregulate an endogenous gene encoding MMP9.

29. 30. The engineered macrophage of claim 21 or 28, engineered to block or downregulate an endogenous gene that downregulates MMP9 expression.

30. 30. The engineered macrophage of any one of claims 21 to 29, which has been engineered to overexpress MMP9.

31. 31. The engineered macrophage of any one of claims 21 to 30, derived from human monocytes or stem cells, optionally derived from induced pluripotent stem cells.

32. 32. The engineered macrophage of any one of claims 21 to 31, wherein the STING pathway is inhibited or the macrophage is treated with an anti-inflammatory agent.

33. 33. The engineered macrophage of any one of claims 21 to 32, which exhibits a pro-restorative phenotype.

34. The engineered macrophages secreted MMP9 and 4 × 10 6 34. The engineered macrophage of any one of claims 21 to 33, wherein the level of secreted MMP protein exceeds 200 ng / ml when cultured in vitro at a cell concentration of 100 ng / ml.

35. 35. A population of engineered macrophages comprising the engineered macrophage of any one of claims 21 to 34.

36. 36. A composition comprising the engineered macrophage of any one of claims 21 to 34 or the population of engineered macrophages of claim 35.

37. 37. The engineered macrophage, population of engineered macrophages, or composition of any one of claims 21 to 36 for use in the treatment of an inflammatory condition, preferably an inflammatory condition with a fibrotic component, preferably cirrhosis of the liver.

38. 37. A method of improving the resolution of fibrosis in a chronic condition, comprising the use of an engineered macrophage, population of engineered macrophages, or composition of any one of claims 21 to 36.

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