Therapeutic macrophages

Abstract

The present invention relates to a macrophage genetically engineered to overexpress interleukin-10 (IL-10) or IL-10 in combination with matrix metallopeptidase 9 (MMP9). Such macrophages are useful for treating cirrhosis in a subject. In particular, the subject can be treated after having experienced a first liver decompensation event requiring hospitalization of the subject. The subject can have experienced a subsequent decompensation event prior to treatment.

Description

Technical Field

[0001] This invention relates to a method of treating cirrhosis in a subject by genetically engineering macrophages to express interleukin-10 (IL-10). Specifically, the subject may have experienced a first hepatic decompensation event requiring hospitalization, or may have refractory ascites. The subject may have experienced a subsequent decompensation event prior to treatment. Alternatively, the subject may have recovered from their first hepatic decompensation event and has not experienced a subsequent hepatic decompensation event since recovery. The invention also relates to populations of such macrophages and compositions comprising such macrophages for use in the method of the invention. Background Technology

[0002] Fibrosis is the common final pathway for chronic diseases of various etiologies, including toxic injury, viral infection, metabolic and genetic disorders, and autoimmune diseases. Acute, self-limiting fibrosis is likely to evolve into a reversible and protective response to injury. The balance between self-limiting and excessive fibrosis is finely regulated by multiple pathways and systems and is inherently dependent on the duration and recurrence of injury. The liver provides a biological paradigm for fibrosis formation and remodeling. Advanced chronic liver fibrosis, also known as cirrhosis, is a life-threatening condition. 1,2 Since the 1970s, liver disease has been the only leading cause of death in the UK with a steady annual increase, and it remains a major health burden worldwide. 3 Hepatic decompensation (HD) is defined as the acute development of one or more major complications of cirrhosis (i.e., ascites, encephalopathy, gastrointestinal variceal bleeding, and spontaneous bacterial peritonitis), and is a pathological progression in the clinical course of cirrhosis [Trebicka 2020]. 43 [Moreau 2013], and is the most common reason for hospitalization in patients with cirrhosis. 46 The risk of short-term death is high in HD patients [Moreau2013] 46 The first episode of hepatic decompensation (HD) (also referred to in this article as the first decompensated hepatic event) usually requires hospitalization and marks the transition from compensated to decompensated cirrhosis. Decompensated cirrhosis is characterized by recurrent episodes of HD. 42HD has two distinct clinical presentations, depending on the presence of other organ failure and the degree of systemic inflammation. The presence of multiple organ failure and high levels of systemic inflammation is hallmark of acute-on-chronic liver failure (ACLF), a syndrome associated with a very high 28-day mortality rate. HD with moderate systemic inflammation, without involvement of other organs, has a lower 28-day mortality rate (~2%, although this increases to 10% at 90 days, indicating heterogeneity in the clinical course of HD patients) [Trebicka 2020]. 43 However, this still foreshadowed disastrous results in the following years.

[0003] The only treatment for liver injury is removal of the harmful stimuli (e.g., administration of effective antiviral therapy) and liver transplantation. Treatment for liver failure caused by acute or chronic injury is limited to supportive care and / or transplantation, the latter requiring a donor. Currently, there is a lack of available organ donors, and surgery carries significant morbidity and mortality risks. Furthermore, patients will also be committed to lifelong immunosuppression. In addition, while etiological interventions (e.g., novel antiviral therapies for hepatitis C virus infection, abstinence from alcohol for alcoholic liver disease) have been relatively successful, many diseases (e.g., NASH) lack approved medical treatments, and patients often seek medical attention late, when cirrhosis and related complications have already occurred. [Starkey 2019] 44 Therefore, there is no specific treatment for cirrhosis, and thus, providing effective antifibrotic therapy is a major unmet clinical need for both chronic and acute liver injury. 4-6 .

[0004] Macrophages (Mɸ) play a crucial role in the inflammatory response of damaged liver. Two main populations of Mɸ exist in the liver: (i) resident macrophages (Kupffer cells, KC), and (ii) infiltrative macrophages. KCs perform patrolling functions in the hepatic sinusoids to engulf microbial debris that reaches the liver via the sinusoidal capillaries under homeostatic conditions. In the early stages of liver injury, KCs express chemokines such as CCL2 and CCL5, thereby facilitating the recruitment of monocytes from circulation. 2,7 The number of KCs decreases during fibrosis; then they refill the liver during the recovery phase of self-limiting fibrosis. 8Infiltrative monocyte-derived macrophages (MDMs) play a major role in the response to liver injury. Infiltrative MDMs are recruited via the CCR2 / CCL2 axis; once inside the liver parenchyma, they localize along the fibrotic septum 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 indicate a detrimental role for MDMs in progressive fibrosis. However, if MDMs are depleted at the onset of fibrotic remodeling, the remodeling process fails, and liver fibrosis persists. Macrophages are now generally considered to play a dual role in the formation and regression of fibrosis. 2,8-11 .

[0005] Macrophage therapy is considered beneficial for reducing chronic liver fibrosis due to the implied role of macrophages in fibrosis healing. It has been shown that bone marrow-derived macrophages (BMDM) reduce liver fibrosis when injected into a mouse model of chronic liver fibrosis. 12 Similar results were replicated in an immunodeficient mouse model of chronic liver fibrosis using human monocyte-derived macrophages (hMDM). 13 Furthermore, in the ongoing Phase II trial, a GMP-grade cell culture protocol is currently being used. 14 The production of hMDM for autologous transplantation in patients with cirrhosis (MATCH, Macrophage therapy for liver disease, ISRCTN10368050, EudraCT reference 2015-000963-15). Macrophages are also key to managing acute conditions. 38 .

[0006] However, to date, no therapy (whether macrophage therapy or others) has been proven to effectively treat patients with cirrhosis through four key mechanisms: recruitment of immune system cells to the liver (paracrine effect), conversion of recruited monocytes into pro-repair macrophages that are effective in spreading therapeutic effects (polarization), phagocytosis, and remodeling of fibrotic scars. In particular, to date, no therapy has been sufficiently effective through all these mechanisms for treating patients with decompensated cirrhosis and those who have experienced one or more hepatic decompensation events. Effective treatment for this clinically vulnerable population is needed. Summary of the Invention

[0007] Normally, human macrophages (hMDM) derived from monocytes in vitro will express IL-10, but... Figure 1The results presented show that the macrophages do not secrete large amounts of IL-10. In this paper, the inventors enhanced the antifibrotic and anti-inflammatory properties of macrophages by transiently transfecting and expressing and secreting IL-10 and MMP9, demonstrating the principle that additional expression of these genes can produce promising products to help resolve inflammatory conditions such as acute and chronic organ damage, resulting in macrophages that secrete IL-10 and active MMPs.

[0008] IL-10 is well-known for its anti-inflammatory properties, while some MMPs play a role in aiding fibrotic remodeling. However, the effects of additional combined expression of IL-10 and MMP9 are currently unclear. Furthermore, the inventors have demonstrated that macrophages engineered to overexpress IL-10 can be used in effective therapies, particularly for cirrhosis. Specifically, without being bound by theory or mechanism, the engineered macrophages described herein, particularly those expressing both IL-10 and MMP9, exhibit activity and / or improvement in each of the four mechanisms of action considered fundamental to the efficacy of therapeutic macrophages: 1) remodeling of fibrotic scars; 2) phagocytosis; 3) paracrine effects; and 4) polarization. Figure 32 This study demonstrates how these mechanisms of action contribute to the therapeutic efficacy of macrophages, and in particular, how the recruitment and polarization of endogenous monocytes can enhance the therapeutic effect of pro-repair macrophages (this enhancement is also known as a "virtuous cycle").

[0009] We demonstrate that, based on the expression of CD14, CD206 high (CD206high), 25F9, CD163, CCR2 low / neg (CCR2low / neg), CD86 low (CD86low), and HLA-Class II low (HLA-Class II low), human monocyte-derived macrophages (hMDM) transfected with mRNA expressing IL-10 or IL-10-p2A-MMP9 (referred to as IL-10+MMP9 in this paper) can polarize macrophages to a pro-repair phenotype. Furthermore, the inventors demonstrated in the embodiments that when IL-10 Trx and IL-10-MMP9 Trx hMDM were tested in a peripheral blood mononuclear cell (PBMC) migration assay, they recruited significantly more mononuclear cells compared to hMDM currently used in the MATCH clinical trials (trial registration numbers: ISRCTN10368050 and EudraCT; reference 2015-000963-15. Study protocol: multicenter, open-label, parallel-group, phase 2, randomized controlled trial of autologous macrophage therapy for cirrhosis). Recruitment can be tested in any suitable manner, such as Boyden chamber-based techniques. In the embodiments, the inventors tested immune cell migration in a transwell cell migration and invasion assay (Boyden chamber assay). Without being bound by theory or mechanism, IL-10-Trx macrophages are able to specifically recruit mononuclear cells while substantially not recruiting other immune cell types. Surprisingly, IL-10-MMP9 Trx hMDM exhibited a stronger ability to recruit monocytes compared to MMP9-Trx and IL-10-Trx. The two proteins appear to act synergistically in this process. In fact, only MMP9-transfected macrophages showed little or no ability to attract monocytes. Figure 12 ), while the activity of matrix metalloproteinases is typically increased in these cells (data not shown). Furthermore, IL-10 and MMP9Trx hMDM exhibit excellent anti-inflammatory properties and phagocytic capacity.

[0010] Finally, the inventors found that overexpression of IL-10 alone reduced total matrix metalloproteinase (MMP) activity and the level of MMP9 secreted in the supernatant of genetically engineered macrophages. The metalloproteinase assays used to determine activity levels were as described in the examples. The negative effect of IL-10 on MMP activity was consistent with findings in the literature (e.g., Roth et al., IL-10 Isan Autocrine Inhibitor of Human Placental Cytotrophoblast MMP-9 Production and Invasion, Developmental Biology, Vol. 205, No. 1, 1999, pp. 194-204; and Krishnamurthy et al., IL-10 inhibits inflammation and attenuates left ventricular remodeling after myocardial infarction via activation of STAT3 and suppression of HuR, Circ Res., 2009 Jan 30;104(2)).

[0011] Therefore, the inventors also provided macrophages engineered with a sequence encoding a specific MMP, namely MMP9. Interestingly, although MMP9 expression did not increase dramatically when comparing non-Trx and IL-10+MMP9 Trx hMDM, the total matrix metalloproteinase activity of IL-10+MMP9 Trx hMDM was significantly increased.

[0012] In summary, these data support the use of IL-10+MMP9 Trx hMDM as a therapeutic product for the treatment and / or prevention of inflammatory conditions (e.g., those related to organ damage) and predict its effectiveness in patients with severe disease, such as those with cirrhosis who have experienced one or more (e.g., multiple) hepatic decompensation events. While IL-10 is known to be an anti-inflammatory cytokine, this is somewhat surprising because previous data have shown that IL-4-stimulated macrophages (rather than IL-10-stimulated macrophages) are pro-repairing in animal models of acute liver disease. 15It is also conceivable that additional combinations of IL-10 and MMP9 expression (e.g., via transfection) could enhance the anti-fibrotic (by increasing matrix metalloproteinase activity) and pro-repair (by increasing monocyte migration) functions of macrophage-based therapeutic products. This recruitment of monocytes to sites of inflammation by macrophages expressing IL-10 and MMP9 is crucial, as the macrophages of the present invention recruit monocytes in situ in this manner to provide further therapeutic effects. The recruited monocytes can be converted into pro-repair macrophages, thereby enhancing the delivery of therapeutic macrophages. The pro-repair phenotype is described extensively herein.

[0013] Chronic inflammatory organ damage is often associated with fibrosis, such as in chronic liver disease. This fibrosis can lead to cirrhosis and, consequently, hepatic decompensation events, which can be severe enough to require hospitalization and / or result in death. Therefore, pro-repair therapeutic macrophages with anti-inflammatory and anti-fibrotic functions would be beneficial, especially for patients who have experienced one or more (e.g., multiple) hepatic decompensation events.

[0014] As described above, the inventors have found that macrophages engineered to overexpress IL-10 exhibit reduced total MMP activity. In contrast, macrophages engineered to additionally express MMP9, for example by engineering with nucleic acids encoding MMP9, exhibit enhanced total MMP activity, not just enhanced MMP9 activity. In this paper, through the combined expression of IL-10 and MMP9, the inventors, for the first time, have enhanced the total MMP activity of engineered macrophages to a greater extent than expected from data on macrophages transfected with IL-10 or MMP9 alone. Therefore, the inventors have demonstrated an unexpected synergistic effect of combined expression of IL-10 and MMP9 on total MMP activity. Therefore, macrophages are engineered to have additional / exogenous coding sequences for IL-10 and MMP9 in addition to those naturally present in the cells. Such combinations are not taught or suggested in the prior art. We show that macrophages transfected with a combined mRNA construct expressing IL-10 and MMP9 can be polarized into macrophages with a pro-repair phenotype, as described herein.

[0015] Furthermore, the inventors have demonstrated that IL-10-MMP9-Trx hMDM is located in the liver in a disease model of liver inflammation / fibrosis. The engineered macrophages have also demonstrated a significantly greater ability to recruit more monocytes compared to the unengineered hMDM currently used in MATCH. This recruitment study was conducted in vitro using the standard assays described herein. In summary, these data support the use of IL-10 and MMP9-Trx hMDM as an improved therapeutic product for treating and / or preventing inflammatory conditions such as those associated with organ damage. Indeed, the modified properties of the cells described herein suggest their potential to treat patients with particularly severe disease for whom liver transplantation was previously the only treatment option, such as those already hospitalized due to a first hepatic decompensation event, and those who subsequently experienced additional decompensation events.

[0016] As used herein, a “first hepatic decompensation event” or “first hepatic decompensation event” means a decompensation event that has not been preceded by an earlier decompensation event within the preceding six months. For example, the first decompensation event could be one that has not been preceded by an earlier decompensation event within the preceding 12 months. In some implementations, the first decompensation event has not been preceded by any earlier decompensation event in the subject, and in these implementations, the subject has never experienced an earlier decompensation event prior to the first decompensation event.

[0017] Matrix metalloproteases (MMPs), also known as matrix metalloproteinases or matrixins, are a family of zinc-dependent peptidases that collectively degrade various proteins in the extracellular matrix (ECM). MMPs play crucial roles in vivo, making them highly complex and difficult to understand. In the context of fibrosis, MMPs have been reported to contribute; however, they also play a key role in fibrotic regression through their matrix remodeling capabilities. Their dual role in fibrosis progression and regression can be understood to some extent through the complexity of their in vivo activities, ranging from cytokine and chemokine activation, immune cell recruitment and activation, to ECM degradation. Furthermore, the activation and subsequent activity of MMPs in vivo are highly regulated, primarily through tissue inhibitors of metalloproteases (TIMPs), which further complicates understanding their activity in fibrosis. Experimental models of pulmonary fibrosis have shown increased activity of MMP9 (a type IV collagenase targeting collagen), which is also associated with disruption of the alveolar epithelial cell membrane, suggesting a putative pro-fibrotic role for MMP9 in lung injury. However, in a bleomycin-induced pulmonary fibrosis model, although the lungs of MMP9-deficient mice exhibited limited alveolar bronchiectasis, MMP9-deficient mice developed pulmonary fibrosis similar to that of wild-type littermates. Overall, the exact role of MMPs in fibrosis is not fully understood. Furthermore, the MMP levels expressed by in vitro-derived hMDM are negligible; additionally, almost no activity was detected in the conditioned medium of these cells.

[0018] To date, there have been few reports in the field of MMP genetic engineering. MMP9 transfection in THP1 cells has been used as a research tool to understand the inflammatory response of macrophages in atherosclerosis. In therapeutics, overexpression of MMP9 in iPSCs via lentiviral transduction has been used to enhance the repair of damaged myocardium. MMP12 (an elastase targeting both soluble and insoluble elastin) has been overexpressed in endothelial progenitor cells for melanoma cell therapy. To the inventors' knowledge, previous work using MMP transfection of human macrophages has not produced macrophages with anti-fibrotic properties. Cabrera et al. demonstrated that overexpression of MMP9 in bleomycin-challenged transgenic mouse macrophages led to reduced fibrosis. 45 Differences in TIMP-1 levels were also observed between transgenic and wild-type mice. However, no differences in the expression levels or activities of other matrix metalloproteinases were reported.

[0019] MMPs possess complex biological properties, often being organ- and disease-specific. For example, MMP9 exhibits significant anti-fibrotic effects in models of chronic liver and chronic lung diseases. However, it appears to be detrimental to renal fibrosis.

[0020] WO2019 / 118888A1 (Therapeutic Use of Genetically Engineered Macrophages for the Treatment of Fibrosis) describes engineered macrophages for the treatment of fibrosis. For example, genetically engineered macrophages contain: recombinant extracellular matrix (ECM) targeting proteins; and / or recombinant proteases. The recombinant proteases can be matrix metalloproteinases (MMPs), such as one from a long list of possible MMPs including MMP9 and MMP12. However, no mention is made of IL-10 or cytokines associated with cell engineering.

[0021] WO2022 / 047119A1 (Modified Immune Cells for Fibrosis and Inflammation) describes modified immune cells comprising one or more nucleic acid sequences encoding: (i) at least one exogenous fibrinolytic agent, and / or (ii) at least one exogenous anti-inflammatory agent. The at least one exogenous fibrinolytic agent may include matrix metallopeptidases (MMPs), 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, cytokines, chemokines, or pentraxins, and the cytokines may include, for example, IL-10, IL-4, IL-13, and / or TGF-β. The modified immune cells may include macrophages. However, no specific combinations of these possible genes and cell types are disclosed, and furthermore, no combinations are illustrated in this application—only single genes are tested. Furthermore, the applicant has not attempted to determine, for example, the effect of IL-10 expression on the expression of other proteins, such as MMPs. Furthermore, data provided in application WO2022 / 047119A1 indicate that macrophages transfected with IL-10 produce at least one pro-inflammatory marker, CD-80, which is undesirable for treating inflammatory conditions.

[0022] WO2019 / 175595 describes the use of autologous, unengineered human macrophages in the treatment of liver diseases, including cirrhosis. However, the macrophages in this application are non-polarized and unengineered. Therefore, these cells are not very useful for treating inflammatory conditions and conditions with fibrotic components.

[0023] WO2012 / 062930 describes the use of a composition comprising macrophages overexpressing IL-10 from transfected mRNA as a medicine. However, that application does not disclose the combination of IL-10 and MMP9 or the treatment of cirrhosis. The inventors have demonstrated the advantageous role of macrophages engineered to express a specific combination of IL-10 and MMP9 in the treatment of, for example, inflammatory and fibrotic diseases.

[0024] Importantly, none of the cells described in the prior art have been shown to be effective in human patients with cirrhosis who have experienced hepatic decompensation events requiring hospitalization. The inventors have, for the first time, evaluated the safety and efficacy of macrophages overexpressing IL-10 and MMP9 in human subjects hospitalized for hepatic decompensation events. Specifically, the inventors evaluated a specific patient population who were clinically stable after a first hepatic decompensation event but subsequently experienced further decompensation events before administration of therapeutic engineered macrophages. It is understood that subjects who experienced subsequent decompensation events had a greater risk of morbidity and death than those who did not experience further decompensation events or those who stabilized after a single decompensation event.

[0025] As is evident from the above discussion of the prior art, to date, no therapy (whether macrophage therapy or any other) has been proven effective in treating this patient population. Data provided in the examples indicate that the macrophages described herein exert their effects through four key therapeutic mechanisms: recruitment of immune system cells to the liver (paracrine effect), conversion of recruited monocytes into pro-repair macrophages effective in spreading therapeutic effects (polarization), phagocytosis, and remodeling of fibrotic scars. Specifically, to date, there are no therapies capable of treating patients with decompensated cirrhosis who have experienced a first hepatic decompensation event requiring hospitalization and subsequent decompensation events. The macrophages described herein are expected to be effective in this particularly vulnerable patient population because the inventors have demonstrated that the macrophages can exert their effects through at least the four mechanisms described herein.

[0026] One or more aspects or embodiments of the claimed invention are designed to solve one or more of the aforementioned problems.

[0027] Macrophages used to treat diseases

[0028] This invention provides engineered macrophages for use in therapies for subjects in need, most notably cell therapy. The subject may suffer from a condition, disease, or symptom that would benefit from the administration of the macrophages of this invention. This condition, disease, or symptom may have inflammatory and / or fibrotic components. This condition, disease, or symptom may be acute or chronic, or slow-on-acute. This condition, disease, or symptom may cause organ damage. The organ may be any suitable organ, including the liver, lungs, or kidneys.

[0029] In some embodiments, the condition is a chronic inflammatory condition, such as, but not limited to, chronic liver disease / injury. In a preferred embodiment, the condition is not an acute inflammatory condition without cirrhosis. In other words, if the condition is an acute inflammatory condition, the subject preferably has cirrhosis. In some embodiments, the subject's condition, disease, or symptom is a chronic inflammatory condition with a fibrotic component. In some embodiments, the condition is chronic organ damage associated with chronic inflammation. In some embodiments, the condition is an acute inflammatory condition. In some embodiments, the condition is a subacute-on-chronic inflammatory condition.

[0030] In some implementations, the condition involves the kidneys, liver, or lungs. For example, the condition could be liver damage, kidney damage, or lung damage.

[0031] In some implementations, the condition may be acute-on-chronic liver failure (ACLF). ACLF is a syndrome characterized by acute decompensation of chronic liver disease and is associated with organ failure and high short-term mortality. Excessive systemic inflammatory response appears to play a crucial role in the development of ACLF.

[0032] In some embodiments, the condition may be liver injury. In a preferred embodiment, the liver injury is chronic liver injury, optionally inflammatory liver injury. In a preferred embodiment, the inflammatory liver injury has a fibrotic component. In a preferred embodiment, the condition is chronic inflammatory liver injury with a fibrotic component, preferably cirrhosis. Preferably, the liver injury is not acute liver injury. In particular, the liver injury is preferably not acute liver injury without underlying cirrhosis. In other words, if the condition is acute liver injury, the subject preferably has cirrhosis. In a preferred embodiment, the liver injury is not drug-induced liver injury (DILI).

[0033] Cirrhosis represents the end stage of chronic liver injury and progressive fibrosis (scarring), regardless of the underlying cause. It is characterized by severe liver fibrosis leading to structural damage, hepatocyte dysfunction, and portal hypertension. A variety of causes can lead to cirrhosis. Liver conditions with fibrotic components that can lead to fibrosis include, but are not limited to, non-alcoholic fatty liver disease (NAFL) (e.g., non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH)) or alcoholic liver disease (e.g., alcoholic fatty liver disease (AFLD) or alcoholic steatohepatitis (ASH)). To diagnose cirrhosis, subjects may be examined prior to treatment.

[0034] In a preferred embodiment, the present invention provides a method for treating cirrhosis in a subject, wherein the method includes administering engineered macrophages to the subject, the engineered macrophages being engineered to overexpress IL-10. The present invention also provides engineered macrophages for treating cirrhosis in a subject, wherein the method includes administering engineered macrophages to the subject, wherein the engineered macrophages are engineered to overexpress IL-10. The engineered macrophages may optionally be further engineered to overexpress MMP9.

[0035] Transient elastography (TE), also known as fibroscan, can be used to diagnose liver cirrhosis in subjects. Transient elastography uses ultrasound to provide a measurement of "liver stiffness," which can be measured in pressure units such as kilopascals (kPa). In some implementations, a subject can be diagnosed with liver cirrhosis based on a liver stiffness score greater than 15 kPa measured using transient elastography.

[0036] The diagnosis of cirrhosis can also be based on clinical and / or radiological features and / or laboratory values ​​associated with cirrhosis. Clinical features associated with cirrhosis may include, for example, jaundice, pruritus, ascites, edema, hepatic encephalopathy, hepatorenal syndrome / acute kidney injury (HRS / AKI), variceal bleeding, gastrointestinal bleeding, and spontaneous bacterial peritonitis (SBP). Radiological features detectable in the liver associated with cirrhosis may include hypertrophy of the caudate lobe and the lateral segment of the left lobe and / or atrophy of the posterior segment of the right lobe. Laboratory values ​​associated with cirrhosis may include elevated INR or PR, decreased platelet count (<150K x 10EE9 / L), elevated bilirubin, and / or decreased serum albumin.

[0037] The diagnosis of cirrhosis can also be made based on the histological characteristics of cirrhosis confirmed by liver biopsy. Cirrhotic tissue can be identified by the presence of fibrous tissue, for example, by staining for the presence of collagen.

[0038] In a preferred embodiment, the subject is diagnosed with cirrhosis based on the presence of at least one of the following conditions:

[0039] (a) Meeting the clinical or radiological characteristics of cirrhosis;

[0040] (b) Liver stiffness measurements (LSM) measured using transient elastography are greater than 15 kPa; and

[0041] (c) Histological features of cirrhosis confirmed by prior liver biopsy.

[0042] Causes of fibrosis can include, but are not limited to, steatotic liver disease (SLD), such as metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic-associated steatohepatitis (MASH), or Met-ALD. In some cases, the cause of steatotic liver disease may be unknown and may be termed cryptogenic SLD.

[0043] Metabolic steatohepatitis (MASLD) is a form of non-alcoholic fatty liver disease and is therefore also known as NAFLD. Metabolic steatohepatitis is a more severe form of MASLD and is also known as NASH (non-alcoholic steatohepatitis). "Met-ALD" refers to an individual with fatty liver disease who also drinks alcohol in moderation. In other words, an individual with a cause of non-alcoholic steatohepatitis may, in some cases, also have alcoholic liver disease (ALD) due to alcohol consumption. Subjects with NASH who also drink alcohol may also be considered to have a cause of NASH with some ALD. "Cryptogenic SLD" refers to SLD of unknown cause, such as individuals who do not carry any known metabolic risk factors for SLD.

[0044] In some embodiments, the condition is cirrhosis with NASH as a cause of NASH, with or without some ALD, or ALD. In some embodiments, the subject may have NASH with or without some ALD. In some embodiments, the subject may have ALD. In a preferred embodiment, when the subject has ALD, there has been no active alcohol abuse for at least 3 months prior to screening for treatment or starting treatment, and preferably no alcohol abuse. “Alcohol abuse” is defined as alcohol intake exceeding 3 units / day for women and 4 units / day for men, or binge drinking (>14 units / day). One unit equals 14 g of alcohol: half a pint (~240 mL) of beer, one glass (125 mL) of wine, or one serving (25 mL) of spirits. According to some embodiments, alcohol abuse may have different values, for example, if measured according to NIAID or UK values, where alcohol use >14 g / day for women or >21 g / day for men may be considered associated with ALD.

[0045] Fibrotic diseases, conditions, and illnesses may include mechanical trauma, biliary obstruction, autoimmune hepatitis, iron overload, hepatitis B (HBV) infection, and / or hepatitis C (HCV) infection. The engineered macrophages according to the invention are capable of treating cirrhosis regardless of the underlying cause. However, in a preferred embodiment, the subject's fibrosis is not caused by any viral hepatitis other than hepatitis C. In other words, if the cause of the subject's fibrosis or cirrhosis is viral, the causative virus is preferably hepatitis C virus (HCV). When the cause of the illness is viral, the viral infection has preferably been treated so that a sustained virological response can be observed in the subject. When the pathogen is HCV, the subject preferably has a sustained virological response to HCV and persistent cirrhosis. In some embodiments, HCV RNA is present in the subject's serum at a concentration of less than 25 IU / mL for at least 12 weeks from the start of HCV treatment. In a further preferred embodiment, the cause of the fibrosis or cirrhosis is not autoimmune or cholestasis. Cirrhosis with cholestatic etiology refers to cirrhosis caused by slowed bile flow through the biliary system, leading to inflammation in the liver. Cholestasis may be due to bile duct damage caused by an autoimmune response. Cholestatic causes of cirrhosis may include primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). Therefore, subjects preferably do not have or are known to have no autoimmune hepatitis, primary biliary cholangitis (PBC), and / or primary sclerosing cholangitis (PSC). In a preferred embodiment, the subject is identified as having cirrhosis and further has the following etiologies: pure metabolic dysfunction-associated steatotic liver disease (MASLD) or metabolic and alcohol-related / associated liver disease (Met-ALD) or HCV sustained virological response (HCV SVR) with persistent cirrhosis, or pure ALD (non-Met-ALD) only if alcohol abuse as described above is confirmed to be absent. According to some implementation schemes, subjects have a MELD 3.0 score of 12-20, as measured within two weeks of an eligible decompensated event.

[0046] Cirrhosis can be compensated or decompensated (also referred to herein as hepatic decompensation or HD). Decompensated cirrhosis is defined as an acute deterioration of liver function in a patient with cirrhosis, characterized by symptoms such as, 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 patient has compensated cirrhosis and has experienced at least one (or exactly one) decompensated cirrhosis event.

[0047] In some embodiments, the subject has experienced a first hepatic decompensation event or may have refractory ascites. In a preferred embodiment, refractory ascites begins within six months prior to macrophage administration. In a preferred embodiment, the subject has been hospitalized following or due to their first hepatic decompensation event. Hospitalization following a hepatic decompensation event is a measure of disease severity and provides a specific clinical context. Some symptoms of hepatic decompensation are similar to those of less severe cirrhosis, but when a patient is hospitalized after their first hepatic decompensation event, it indicates that their disease is severe enough to particularly benefit from the present invention.

[0048] Subjects may exhibit one or more clinical signs of hepatic decompensation selected from a list consisting of jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, variceal bleeding, and gastrointestinal bleeding, or have recovered from one or more of these clinical signs. In a particular implementation, a subject may exhibit, have recovered from, or have been admitted to the hospital due to ascites, hepatic encephalopathy, variceal bleeding (variceal bleeding), hepatorenal syndrome / acute kidney injury (HRS / AKI), and / or spontaneous bacterial peritonitis (SBP). Therefore, the treatment method may include steps for detecting clinical signs of hepatic decompensation in a subject, such as ascites, hepatic encephalopathy, variceal bleeding (variceal bleeding), hepatorenal syndrome / acute kidney injury (HRS / AKI), and / or spontaneous bacterial peritonitis (SBP).

[0049] In some implementations, the subject has been hospitalized for the first time due to a hepatic decompensation event, defined as the presence of the following signs: ascites, hepatic encephalopathy, variceal bleeding (variceal hemorrhage), hepatorenal syndrome / acute kidney injury (HRS / AKI), and / or spontaneous bacterial peritonitis (SBP). In some implementations, the subject is confirmed to be clinically stable after the first hepatic decompensation event requiring hospitalization and before the administration of engineered macrophages.

[0050] In a preferred embodiment, engineered macrophages are administered to subjects only if they are determined to be clinically stable after their first hepatic decompensation event, preferably within 6 weeks of their first visit (the visit in which their MELD score is first measured and informed consent is obtained), and assuming they have been clinically stable prior to administration. In a preferred embodiment, the patient has been clinically stable for at least 6 weeks prior to G-CSF mobilization.

[0051] As used in this article, “clinically stable” (also referred to as “recompensated” in this article) is defined as one or both of the following:

[0052] (i) A decrease in the Model 3.0 score for end-stage liver disease (e.g., a decrease of at least 1 point) in two independent MELD score assessments conducted within two weeks of each other; and

[0053] (ii) Subjects assessed by a physician, including a recorded MELD review, are sufficiently stable to be safely mobilized for cell mobilization (using G-CSF, such as Filgrastim), undergo apheresis, and / or be able to be treated with engineered macrophages without substantial risk to patient safety.

[0054] In a further preferred embodiment, after being determined to be clinically stable, a subject may experience subsequent additional decompensated events before the administration of engineered macrophages according to the invention. Thus, in some embodiments, a subject treated with the engineered macrophages of the invention may have experienced a second hepatic decompensated event after being determined to be clinically stable following a first hepatic decompensated event, and before the administration of engineered macrophages. For example, according to the preferred embodiment described above, a subject may experience a first hepatic decompensated event requiring hospitalization, become clinically stable, but subsequently experience at least one more hepatic decompensated event (after G-CSF mobilization, after apheresis, or after engineered macrophage creation), so the administration of engineered macrophages only occurs after the subject has become clinically stable after at least two hepatic decompensated events. In a preferred embodiment, the patient has been clinically stable for at least 6 weeks prior to G-CSF mobilization.

[0055] Therefore, in some embodiments, the subject to be treated has experienced more than one hepatic decompensation event before the administration of engineered macrophages according to the treatment according to the invention; preferably, the engineered macrophages are administered only after the subject has become clinically stable following more than one hepatic decompensation event; preferably, the subject has become clinically stable within 6 weeks after their first visit (the visit in which their MELD score is first measured and informed consent is screened). In a preferred embodiment, the patient has been clinically stable for at least 6 weeks prior to G-CSF mobilization.

[0056] The macrophages, compositions, and methods of the present invention are expected to be effective in treating patients who have been hospitalized following their first hepatic decompensation event, particularly those who have experienced additional, subsequent decompensation events. As is known in the art, once a subject has experienced more than one decompensation event, the severity and mortality of decompensated cirrhosis increase. 42Therefore, patients with more than one hepatic decompensation event are particularly at risk, regardless of theory or mechanism, and this population is preferably treated to minimize the number of additional decompensation events and increase the likelihood of transplant-free survival. Furthermore, the data provided in the examples indicate that the cells, compositions, and methods of the present invention are suitable for treating these specific patients with difficult-to-treat severe diseases.

[0057] Therefore, in a preferred embodiment, the subject experienced a subsequent additional hepatic decompensation event following the first decompensation event. As used herein, "additional" decompensation event refers to a decompensation event following the first hepatic decompensation event, also known as a "subsequent" decompensation event. In a preferred embodiment, the subject is treated with engineered macrophages after the subsequent decompensation event. In other words, the subject may have already experienced a further decompensation event prior to treatment. In some embodiments, the subject has not yet recovered from the subsequent decompensation event when engineered macrophages are administered, i.e., remains decompensated. Therefore, the subject may still exhibit one or more clinical signs of decompensation at the time of treatment, such as ascites, hepatic encephalopathy, variceal bleeding (variceal bleeding), hepatorenal syndrome / acute kidney injury (HRS / AKI), and / or spontaneous bacterial peritonitis (SBP). In other embodiments, the subject has recovered from the subsequent decompensation event and does not exhibit clinical signs of decompensation when macrophages are administered to the subject. In some implementations, the subject is determined to be clinically stable before the engineered macrophages are administered.

[0058] In some implementations, when the engineered macrophages are autologous to the subject and generated from the subject's own monocytes harvested via apheresis, the subsequent decompensation event occurs after the monocytes are harvested from the subject. In other implementations, the subsequent decompensation event occurs after the engineered macrophages are generated. In other words, the apheresis and engineering steps can occur before the subsequent decompensation event.

[0059] Therefore, in some embodiments, the present invention provides engineered macrophages and compositions comprising them for use in a method of treating a patient exhibiting one or more clinical signs selected from a list consisting of jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, variceal bleeding, and gastrointestinal bleeding, wherein the one or more clinical signs require hospitalization.

[0060] In some embodiments, the present invention provides engineered macrophages and compositions comprising them for use in a method of treating a patient diagnosed with cirrhosis; optionally, said patient has the following etiologies of liver disease: (1) steatotic liver disease of pure metabolic dysfunction (MASLD) or metabolic and alcohol-related / associated liver disease (Met-ALD), or (2) sustained virological response to HCV with persistent cirrhosis (HCV SVR), or (3) alcohol-related liver disease (ALD or Met-ALD), only if the patient is confirmed to have consumed alcohol not exceeding the level of alcohol abuse and a value <25 mg / L in a PEth alcohol test; wherein said patient has:

[0061] (a) First hospitalization within six months due to a major hepatic decompensation event, defined as ascites, hepatic encephalopathy (HE), variceal bleeding, hepatorenal syndrome / acute kidney injury (HRS / AKI), spontaneous bacterial peritonitis (SBP), or a combination thereof; or

[0062] (b) Suffering from refractory ascites (also referred to herein as medically refractory ascites) that began within six months prior to treatment with engineered macrophages (second large volume paracentesis).

[0063] As used herein, the term "refractory ascites" or "medically refractory ascites" refers to ascites that cannot be mobilized or recurs to the point that it requires repeated (≥ 2) large-volume paracentesis (LVP) at least once every 8 weeks (i.e., therapeutic, non-diagnostic), in which case the date of onset of refractory ascites is defined as the date of the second LVP. As used herein, the term "refractory ascites" requires repeated (> 1) large-volume paracentesis (therapeutic, non-diagnostic), despite the best medical attempt to control the ascites using diuretics and dietary sodium restriction. As used herein, the term "recurrent ascites" refers to ascites that recurs (i.e., a second large-volume paracentesis) within a six-month period. In a preferred embodiment, the patient's refractory ascites began less than 6 months prior to treatment.

[0064] In some embodiments, the present invention provides engineered macrophages and compositions comprising them for treating a patient diagnosed with cirrhosis, wherein the patient has experienced a qualifying decompensated event; optionally, wherein the patient has the following etiologies of liver disease: (1) steatotic liver disease of pure metabolic dysfunction (MASLD) or metabolic and alcohol-related / associated liver disease (Met-ALD), or (2) persistent virological response to HCV with persistent cirrhosis (HCVSVR), or (3) alcohol-related liver disease (ALD or Met-ALD) only if the patient is confirmed to have consumed alcohol not exceeding the level of alcohol abuse and a value of <25 mg / L in a PEth alcohol test.

[0065] According to some embodiments, this document provides engineered macrophages of the present invention for treating cirrhosis in subjects who have experienced qualifying events. As used herein, a “qualifying event” in a subject (also referred to herein as a “qualifying decompensated event” or a “qualifying major decompensated event”) refers to a recent major liver decompensation event selected from the group consisting of:

[0066] (a) A recent major hepatic decompensation event requiring hospitalization of the subject, wherein the hospitalization is the first hospitalization within 6 months following any previous hospitalization for a decompensation event, and wherein the major hepatic decompensation event includes at least one of ascites (recurrent or medically refractory), hepatic encephalopathy, variceal bleeding, hepatorenal syndrome / acute kidney injury (HRS / AKI), and spontaneous bacterial peritonitis (SBP). Subjects experiencing such major hepatic decompensation events may also be referred to herein as hospitalized subjects or hospitalized treatment subjects; and

[0067] (b) Refractory ascites within the past 6 months. Subjects with refractory ascites may also be referred to as outpatient subjects in this article.

[0068] In some embodiments, the present invention provides engineered macrophages and compositions comprising them for a method of treating a patient exhibiting one or more clinical signs of hepatic decompensation selected from the group consisting of: ascites, hepatic encephalopathy, variceal bleeding, hepatorenal syndrome / acute kidney injury (HRS / AKI), spontaneous bacterial peritonitis (SBP), or a combination thereof, wherein the subject has previously been hospitalized for a single first decompensation event. The subject may exhibit ascites, hepatic encephalopathy, variceal bleeding (variceal bleeding), hepatorenal syndrome / acute kidney injury (HRS / AKI) and / or spontaneous bacterial peritonitis (SBP), or a combination thereof, as a first hepatic decompensation event requiring hospitalization. In other embodiments, the present invention provides engineered macrophages and compositions comprising them for a method of treating a patient exhibiting one or more clinical signs of liver decompensation selected from the group consisting of: ascites, hepatic encephalopathy, variceal bleeding, hepatorenal syndrome / acute kidney injury (HRS / AKI), spontaneous bacterial peritonitis (SBP), or a combination thereof, wherein the subject has previously been diagnosed with refractory ascites.

[0069] Hospitalization refers to admission to a hospital for treatment. Therefore, hospitalization typically requires a patient to stay in the hospital for at least 24 hours. Hospitalization is a measure of disease severity because symptoms cannot be controlled outside the hospital environment. In the context of cirrhosis, hospitalization is a recognized measure of disease severity and patient condition. 42 (See also, for example, Balcar et al., United European Gastroenterol J. 2021; 9(4): 427–437). In a preferred embodiment, the subject treated with the methods described herein and engineered macrophages has been hospitalized for the first time as an inpatient due to a major hepatic decompensation event, which may be referred to herein as an accredited event. A major hepatic decompensation event requiring hospitalization may be characterized by the presence of at least one of ascites, hepatic encephalopathy, variceal bleeding (variceal hemorrhage), hepatorenal syndrome / acute kidney injury (HRS / AKI), and / or spontaneous bacterial peritonitis (SBP).

[0070] In other embodiments, the engineered macrophages are used to treat the subject once the subject has recovered (recompensated) from their first hepatic decompensation event, optionally after the subject has been hospitalized following their first hepatic decompensation event. In some embodiments, the subject is treated with the cells, compositions, or cell populations of the present invention after discharge. According to some embodiments, recovery (recompensation) from a hepatic decompensation event is defined by a physician’s clinical assessment and / or by the absence of a significant increase in the MELD score between discharge and treatment. In some embodiments, the subject exhibits one or more clinical signs of hepatic decompensation and / or has recovered from one or more clinical signs of hepatic decompensation, selected from a list consisting of jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, variceal bleeding, and gastrointestinal bleeding.

[0071] Under some implementation schemes, subjects receive treatment after recovering from a first hepatic decompensation event that required hospitalization, but before experiencing additional hepatic decompensation events.

[0072] Further criteria can be used to select patients who may particularly benefit from the treatment. In a preferred embodiment, subjects exhibit any combination of the inclusion criteria listed in Example 25. In a preferred embodiment, subjects do not exhibit any of the exclusion criteria listed in Example 25.

[0073] In some implementations, the method includes assessing indicators that would be particularly beneficial to the subject, or that such indicators have been assessed in subjects to be treated. In a preferred implementation, the subject has one or more, or preferably all of the following characteristics: no known contraindications to treatment with G-CSF (especially filgrastim); no known contraindications to undergoing apheresis (especially leukapheresis); has been hospitalized for the first time as an inpatient for a liver decompensated event (eligible event) defined as ascites, hepatic encephalopathy, variceal bleeding (variceal bleeding), hepatorenal syndrome / acute kidney injury (HRS / AKI) and / or spontaneous bacterial peritonitis (SBP), preferably within six months prior to treatment or apheresis, or has been admitted as an outpatient for refractory ascites, preferably within six months prior to treatment or apheresis; a MELD 3.0 score of 12-20, measured within 2 weeks of the occurrence of an eligible event requiring hospitalization, or measured upon admission of an outpatient subject; and a Chronic Liver Failure Association Acute Decompensated Scale (CLIF-C) score. An AD score greater than 45 is required, and the event must be within 2 weeks of an eligible liver decompensation event requiring hospitalization; the following liver disease etiologies must be present: pure metabolic dysfunction-associated steatotic liver disease (MASLD), or metabolic and alcohol-related / associated liver disease (MetALD), or “pure” ALD without alcohol abuse (i.e., non-Met-ALD) with a negative PEth test prior to administration of engineered macrophages, or persistent HCV virological response (HCV SVR) with persistent cirrhosis; and a diagnosis of cirrhosis must be based on at least one of the following: (i) clinical and radiological features relevant to the diagnosis of cirrhosis, (ii) a liver stiffness score greater than 15 kPa as measured by transient elastography, or (iii) histological features of cirrhosis confirmed by previous liver biopsy. According to some implementation schemes, refractory ascites is considered an eligible event if the subject has refractory ascites.

[0074] Sustained virological response refers to the absence of virus in serum for an extended period after a treatment procedure. Therefore, sustained virological response can be determined by measuring viral load in blood or serum, for example, by quantifying viral RNA, at a time after treatment completion, such as 12 weeks. In some implementations, sustained virological response in a subject is defined as a viral load of less than 25 IU RNA per milliliter of blood over 12 weeks, or as measured at week 12 after treatment completion.

[0075] In some embodiments, the method includes assessing the subject for comorbidities and / or indicators of increased risk prior to treatment. In a preferred embodiment, the subject has no or is unaware of having and / or is suspected of having any combination of the following comorbidities or indicators of increased risk at the start of treatment: cirrhosis caused by any viral hepatitis other than HCV SVR; cirrhosis caused by autoimmune and cholestatic etiologies, including but not limited to primary biliary cholangitis and primary sclerosing cholangitis; any current organ failure requiring supportive care beyond outpatient care and unrelated to a hepatic decompensation event (a qualifying event); known splenomegaly, optionally wherein the subject's spleen has a maximum size greater than 16 cm; thrombocytopenia, optionally wherein thrombocytopenia is defined as less than 50 × 10⁻⁶ mmol / L. 9 Platelet count of 1 platelet / L; history of liver or other organ transplantation, such as having received at least one previous liver or organ transplant; acute-on-chronic liver failure (ACLF); positive microbial culture or recent sepsis as defined by a physician, unless stable and at least 5 weeks after completion of a full course of IV antibiotics; known infection with human immunodeficiency virus (HIV); pulmonary embolism; hepatocellular carcinoma (HCC) or active malignancy (excluding non-melanoma skin cancer, cervical carcinoma in situ, or superficial bladder cancer) within 5 years prior to the start of treatment; co-hepatic liver disease. The following comorbidities are contraindicated: hepatic pleural effusion (refractory to drug therapy) or portal vein thrombosis; chronic renal impairment in which the subject may require dialysis; unresolved acute kidney injury (AKI); acute heart failure; chronic heart failure; portal pulmonary hypertension; severe chronic lung disease, such as chronic obstructive pulmonary disease (COPD) or interstitial lung disease, in which forced expiratory volume in one second (FEV1) is less than 50% or 60%, and / or forced vital capacity (FVC) is less than 60%; hepatopulmonary syndrome; a history of chronic albumin therapy, or current chronic albumin therapy; and / or major untreated or unstable mental illness. In the preferred embodiment, the subject has none of the aforementioned comorbidities or indicators of increased risk. In other embodiments, the subject has no evidence of any life-threatening and / or clinically significant comorbidities that would contribute to the subject's cirrhosis.

[0076] In a preferred embodiment, the subject has none of the aforementioned comorbidities or indicators of increased risk, and the subject receives treatment after recovery or clinical stabilization (recompensation) following a first hepatic decompensation event requiring hospitalization, and wherein the subject has not experienced additional hepatic decompensation events after recovery from the first decompensation event (or after becoming clinically stable following the first decompensation event). In another preferred embodiment, the subject has none of the aforementioned comorbidities or indicators of increased risk, and the subject experienced a first hepatic decompensation event requiring hospitalization, experienced additional or subsequent hepatic decompensation events, and received treatment following the additional or subsequent decompensation events.

[0077] Forced expiratory volume (FEV1) refers to the volume of air an individual exhales during a forced exhalation, expressed as a percentage of a reference value. Forced vital capacity (FVC) refers to the maximum volume of air an individual exhales during a forced exhalation after a complete inspiration. Reference values ​​can be based on measurements of forced expiratory volume in a healthy population (i.e., individuals without restrictive lung disease). As used herein, FEV1 or FVC expressed as a percentage are relative to expected reference values ​​for healthy individuals.

[0078] Thrombocytopenia is defined as a low platelet count in a subject, such as a platelet count lower than that of a reference sample taken from a healthy individual. If a subject's platelet count is below a given reference point, such as below 150,000 platelets / mm³, this is considered thrombocytopenia. 3 Less than 100,000 platelets / mm 3 Or below 50,000 platelets / mm 3 The subject may be considered to have thrombocytopenia. In a specific implementation, if the subject's platelet count is less than 50,000 platelets / mm³, then the subject may be considered to have thrombocytopenia. 3 If the test result is negative, the subject can be considered to have thrombocytopenia.

[0079] In a preferred embodiment, the patient's MELD 3.0 score is between 10 and 20, or more preferably 10-16, 12-18, or 12-20. In the most preferred embodiment, the subject's MELD 3.0 score is 12-20.

[0080] MELD consists of three parts: bilirubin (BIL), creatinine (CRE), and international normalized ratio (INR), which are combined in the formula below to generate the MELD score.

[0081] The relevant scores that can be used according to the present invention include MELD, MELD 3.0, MELD 3.0 albumin-free and MELDNa, as discussed in Kim et al., Gastroenterology 2021;161:1887–1895, and are summarized below.

[0082] MELD = 9.57 x log e (creatinine) + 3.78 x log e (bilirubin) + 11.20 x log e (INR) +6.43, where creatinine (mg / dL), bilirubin (mg / dL) and INR values ​​below 1.0 are set to 1.0, and if serum creatinine ≥4 mg / dL or the patient has received 2 or more dialysis treatments in the previous week, the creatinine value is set to 4.0 mg / dL, rounded to the nearest integer.

[0083] MELD 3.0 = 1.33 (if female) + [4.56 × log e (bilirubin)] + [0.82 × (137 – Na)] – [0.24 × (137 – Na) × log e (bilirubin)] + [9.09 × log e (INR)] +[11.14 × log e [(creatinine)] + [1.85 × (3.5 – albumin)] – [1.83 × (3.5 – albumin) × log e (creatinine)] + 6, rounded to the nearest integer.

[0084] MELD 3.0 albumin-free = 1.40 (for women) + 4.85 * log e (bilirubin) + 0.88 *(137-Na) – 0.25 (137-Na) *log e (bilirubin) + 9.66 * log e (INR) + 10.47 * log e (creatinine) + 6.

[0085] MELDNa = MELD + 1.32*(137-Na) – [0.033*MELD*(137-Na)], where serum sodium concentration (Na) is limited to between 125 and 137 mmol / L, as defined by OPTN (Peng et al., Y, Khaled T, Liu J, et al. Data Request from the MELD Enhancement Subcommittee of the Liver and Intestinal Organ Transplantation Committee. Data Request ID: HR2011_02. June 7, 2011).

[0086] In some implementations, the subject's Chronic Liver Failure Association Acute Decompensation Score (CLIF-C AD) is greater than 45. CLIF-AD can be used to predict patients with acute decompensation but no chronic-on-acute liver failure and can be used to predict the survival of this group of patients. The CLIF-C AD score can be calculated as follows:

[0087] CLIF-C AD = 10 × [0.03 × age + 0.66 × Ln (creatinine mg / dL) + 1.71 × Ln(INR) + 0.88 × Ln (WBC 10 9 (cells / L) – 0.05 × (sodium mmol / L) + 8].

[0088] In some embodiments, the liver disease is a condition in which cirrhosis is caused by damage to hepatocytes, for example, a hepatocyte-derived disease such as those of viral origin (including treated (sustained viral response) hepatitis C (HCV), hepatitis B), damage caused by alcohol poisoning (alcohol-associated 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 α-1-antitrypsin deficiency. In some embodiments, the underlying cause has been eliminated (e.g., a patient who suffered damage due to alcohol poisoning no longer drinks alcohol, or a patient who suffered damage due to HCV no longer has HCV, etc.). In a preferred embodiment, when the cause of the disease is HCV, the subject exhibits a sustained virological response to HCV. In some embodiments, the patient with liver disease is at risk of end-stage renal disease.

[0089] In some embodiments, the condition is liver disease, specifically inflammatory liver disease. In some embodiments, the liver disease is steatotic liver disease (SLD). In some embodiments, the steatotic liver disease is metabolic dysfunction-associated steatotic liver disease (MASLD), which is Met-ALD or cryptogenic SLD. In some embodiments, the metabolic dysfunction-associated steatotic liver disease (MASLD) is metabolic-associated steatohepatitis (MASH). In some embodiments, the liver disease is non-alcoholic steatohepatitis, with or without some alcoholic liver disease (ALD). Preferably, when the liver disease is ALD, there is no active alcohol abuse, and there has been no alcohol abuse for at least 6 months prior to screening for treatment or starting treatment. In some implementations, the liver disease is pure metabolic dysfunction-associated steatotic liver disease (MASLD) or metabolic and alcohol-related / associated liver disease (Met-ALD), or HCV sustained virological response (HCV SVR) with persistent cirrhosis, or alcohol-related liver disease (ALD or Met-ALD), only if the patient is confirmed to have consumed alcohol at levels not exceeding alcohol abuse levels and a value < 25 mg / L in a PEth alcohol test.

[0090] Cirrhosis can lead to acute-on-chronic liver failure (ACLF). In some embodiments, the liver disease is ACLF. ACLF is a condition distinct from hepatic decompensation. Hepatic decompensation is characterized by ascites, hepatic encephalopathy, gastrointestinal bleeding, or any combination of these conditions in patients with cirrhosis. In contrast, ACLF is associated with organ failure and has a high short-term mortality rate exceeding 15% within 28 days. This syndrome has three main characteristics: ACLF occurs against a background of intense systemic inflammation; ACLF is often closely time-related to pro-inflammatory flare-ups such as infection or alcoholic hepatitis; and ACLF is associated with single-organ or multi-organ failure. In some embodiments, the engineered macrophages are used to treat cirrhosis in patients with ACLF.

[0091] Patients for treatment or use applicable to any aspect or embodiment of the invention may be patients suffering from the relevant disease and of the relevant severity.

[0092] In some implementations, macrophages engineered to express human IL-10 and human MMP9 can be used to treat ACLF.

[0093] The engineered macrophages can be autologous or allogeneic to the treated subject. In a preferred embodiment, the engineered macrophages are autologous to the subject. Autologous macrophages can be obtained by differentiating monocytes from the subject using apheresis. Prior to the apheresis step, the subject can be treated with G-CSF, preferably filgrastim. Treatment with G-CSF can be referred to as “mobilization.” Therefore, in some embodiments, the method of treating the subject’s inflammatory condition may include the following steps:

[0094] (a) With G-CSF, optionally filgrastim (such as but not limited to Neupogen) ® Subjects were treated, optionally at a dose of 10 μG / kg / day for 4 consecutive days;

[0095] (b) Obtaining monocytes from the subject by apheresis and then isolating the monocytes;

[0096] (c) Differentiating the monocytes into macrophages, optionally by incubating the monocytes in the presence of M-CSF;

[0097] (d) Engineered macrophages overexpressing IL-10 and / or MMP9; and

[0098] (e) Administer engineered macrophages to the subjects.

[0099] In some implementations, macrophages are cryopreserved and thawed before administration. According to some implementations, macrophages are reformulated before administration to a subject or before cryopreservation.

[0100] In some embodiments, subjects to be treated with the engineered macrophages of the present invention are pretreated with G-CSF (preferably recombinant G-CSF, preferably filgrastim), and preferably monocytes of the subjects are subsequently collected after treatment with G-CSF. In such embodiments, the collected monocytes can be differentiated to provide engineered macrophages for the treatment of the present invention.

[0101] In some embodiments, the present invention provides a method for treating cirrhosis in a subject using engineered macrophages as disclosed herein, wherein the method includes:

[0102] (a) With G-CSF, optionally filgrastim (such as but not limited to Neupogen) ® Subjects were treated, optionally at a dose of 10 μG / kg / day for 4 consecutive days;

[0103] (b) Obtaining monocytes from the subject by apheresis and then isolating the monocytes;

[0104] (c) Differentiating the monocytes into macrophages, optionally by incubating the monocytes in the presence of M-CSF;

[0105] (d) Engineered macrophages overexpressing IL-10 and / or MMP9; and

[0106] (e) Administer engineered macrophages to the subjects.

[0107] In some embodiments, the present invention provides engineered macrophages as disclosed herein and G-CSF (preferably recombinant G-CSF, optionally, filgrastim, such as but not limited to Neupogen) ® A method for treating cirrhosis in a subject, wherein the method includes:

[0108] (f) Subjects were treated with G-CSF, optionally at a dose of 10 μG / kg / day for 4 consecutive days;

[0109] (g) Obtaining monocytes from the subject by apheresis and then isolating the monocytes;

[0110] (h) Differentiating the monocytes into macrophages, optionally by incubating the monocytes in the presence of M-CSF;

[0111] (i) Engineered macrophages overexpressing IL-10 and / or MMP9; and

[0112] (j) Administer engineered macrophages to the subjects.

[0113] Before receiving G-CSF treatment, subjects may undergo screening to determine their suitability for treatment.

[0114] In embodiments where the macrophages are autologous to the subject and the subject has experienced subsequent or additional decompensation events following a first hepatic decompensation event requiring hospitalization, the step of obtaining monocytes from the subject via apheresis preferably occurs before the subsequent or additional decompensation event. In other words, the subsequent or additional decompensation event occurs after obtaining monocytes from the subject via apheresis. In some embodiments, one or more of the following steps occur before the subsequent or additional decompensation event: treatment with G-CSF, obtaining monocytes, differentiating the monocytes into macrophages, and engineering the macrophages to overexpress IL-10 and / or MMP9.

[0115] As described in detail below, macrophages derived from monocytes can be engineered prior to administration to a subject. In a preferred embodiment, the macrophages are engineered to overexpress IL-10 and MMP9.

[0116] According to the method of the invention, the engineered macrophages are administered to a subject. In a preferred embodiment, the engineered macrophages are administered to the subject after the subject has experienced an additional or subsequent decompensation event, wherein the subject has been or was previously hospitalized due to a previous, separate, first decompensation event.

[0117] In some embodiments, at least one dose of engineered macrophages is administered to the subject. In a preferred embodiment, up to four doses of engineered macrophages are administered to the subject. Suitably, one, two, three, or four doses may be administered to the subject. In a preferred embodiment, all doses of engineered macrophages are derived from mononuclear cells obtained from a single apheresis. Therapeutic macrophages may preferably be formulated into a dispersion for infusion, such as intravenous infusion. In some embodiments, doses may be administered at a concentration of 70 million to 130 million (i.e., 7 × 10⁻⁶). 7 Up to 1.3×10 8 Macrophages are administered in an amount of 1 × 10⁻⁶ macrophages. In a preferred embodiment, macrophages are administered in an amount of 1 × 10⁻⁶ macrophages. 8 Therapeutic macrophages are administered at doses of 1 × 10⁻⁶ macrophages. The engineered macrophages administered to the subject may be formulated in a pharmaceutical composition additionally comprising one or more pharmaceutically acceptable excipients. In embodiments where multiple doses are administered to the subject, the doses are preferably administered at 6-week intervals. In a preferred embodiment, the engineered macrophages or the pharmaceutical composition comprising said engineered macrophages are administered at doses of 1 × 10⁻⁶ macrophages. 8 Four doses of macrophages were administered to the subjects, with each dose spaced 6 weeks apart (i.e., a 6-week interval).

[0118] Suitably, the present invention may also provide pharmaceutical compositions that can be used in any of the methods of the present invention. The pharmaceutical composition may comprise engineered macrophages and one or more pharmaceutically acceptable excipients. The pharmaceutical composition may have the following excipient formulation: 33.13% saline, 0.17% human serum albumin, and 66.7% Cryostor-10, such that the final concentration of DMSO can be 6.7%. The pharmaceutical composition may be frozen.

[0119] engineered macrophages

[0120] According to an aspect of the invention, engineered macrophages are provided to overexpress IL-10. Engineered macrophages according to any embodiment described herein can be used according to any method for treating inflammatory diseases. In some embodiments, when in a 4x10... 6When cultured in vitro at a cell concentration of at least 10,000 pg / ml, the macrophages secrete IL-10 in the culture supernatant. As described herein, engineered macrophages that overexpress IL-10 alone have demonstrated a significant ability to recruit monocytes, exhibit anti-inflammatory secretomics, and convert unpolarized or pro-inflammatory macrophages into a pro-repair phenotype. These examples demonstrate that these macrophages can be used for therapeutic purposes, particularly for the treatment of cirrhosis.

[0121] According to another aspect of the invention, engineered macrophages overexpressing IL-10 and MMP9 are provided, such that the engineered macrophages express higher levels of IL-10 and MMP9 than untransfected macrophages. According to one aspect of the invention, engineered macrophages are provided, wherein the macrophages are engineered to express IL-10 and MMP9, preferably by providing additional or exogenous sequences encoding these proteins. Such IL-10 and MMP9 engineered macrophages can be used for treatment, for example, for treating inflammatory and / or fibrotic conditions in a subject.

[0122] In other aspects of the invention, engineered macrophages are provided that are engineered to overexpress IL-10 and IL-4; IL-10 and MMP12; IL-4, IL-13 and MMP9; or IL-4, IL-13 and MMP12.

[0123] As described herein, engineered macrophages according to any aspect of the invention may have specific structures and advantageous functional properties.

[0124] Exemplary structural and functional characteristics of the engineered macrophages, relative to the non-polarized, untransfected cells used in the MATCH study described in WO2019175595, are listed in Table 4. In some embodiments, the engineered macrophages of the present invention exhibit any combination of the characteristics described in Table 4.

[0125] Table 5 lists further structural and functional properties of the engineered macrophages. In some embodiments, the engineered macrophages of the present invention exhibit any combination of the properties described in Table 5. According to some embodiments, the engineered macrophages of the present invention exhibit at least all of the properties described in Table 5.

[0126] In a preferred embodiment, when exposed to unengineered macrophages, engineered macrophages polarize the unengineered macrophages to a pro-repair phenotype. When administered to a subject, the engineered macrophages can polarize host macrophages (such as macrophages derived from endogenous monocytes that have migrated to the liver) to a pro-repair phenotype. In some embodiments, the engineered macrophages can convert unpolarized host macrophages into pro-repair macrophages. In some embodiments, the engineered macrophages can convert pro-inflammatory host macrophages into pro-repair macrophages. In some embodiments, the conditioned medium derived from engineered macrophages can convert unengineered macrophages into a pro-repair phenotype. Therefore, the engineered macrophages of the present invention can convert unengineered macrophages into a pro-repair phenotype in vitro or in vivo. Conversion to a pro-repair phenotype may increase the expression of cell surface CD206 and CD163 and decrease the expression of CD86 and HLA-DR.

[0127] In some embodiments, the engineered macrophages exhibit at least a two-fold reduction in CD86 expression compared to unengineered, non-polarized cells. In some embodiments, the engineered macrophages exhibit at least a two-fold reduction in HLA-DR expression compared to unengineered, non-polarized cells. In some embodiments, the engineered macrophages exhibit at least a 1000-fold increase in IL-10 secretion compared to unengineered, non-polarized cells. In some embodiments, the engineered macrophages exhibit at least a 10-fold increase in MMP3 secretion compared to unengineered, non-polarized cells. In some embodiments, the engineered macrophages exhibit at least a 20-fold increase in MMP10 secretion compared to unengineered, non-polarized cells.

[0128] In some implementations, when using 4x10 6 When cultured in vitro at a cell concentration of 4 x 10 / ml, the macrophages of the present invention secrete IL-10 at a concentration of at least 10,000 pg / ml of culture supernatant. In some embodiments, when cultured at a concentration of 4 x 10 / ml... 6 When cultured in vitro at a cell concentration of 4 x 10⁶ / ml, the macrophages of the present invention secrete MMP9 at a concentration of at least 200 ng / ml of culture supernatant. In some embodiments, CD206 expression of the engineered macrophages is increased at least 5-fold compared to monocytes. In some embodiments, 25F9 expression of the engineered macrophages is increased at least 5-fold compared to monocytes. In some embodiments, CD80 expression of the engineered macrophages is decreased at least 10-fold compared to unengineered cells. In some embodiments, when cultured at a concentration of 4 x 10⁶ / ml, the macrophages secrete MMP9 at a concentration of at least 200 ng / ml of culture supernatant. 6When cultured in vitro at a cell concentration of 40 pg / ml, the macrophages of the present invention secrete TNF-α at a culture supernatant concentration of at least 40 pg / ml. In some embodiments, the engineered macrophages have the same phagocytic capacity as unengineered, non-polarized cells.

[0129] In one embodiment, the present invention provides engineered macrophages comprising one or more exogenous coding sequences for IL-10 and MMP. The exogenous coding sequences can be any suitable nucleic acid sequence. The exogenous coding sequences can exist as extrachromosomal nucleic acids in the cytoplasm or nucleus, or be integrated into the macrophage genome. The exogenous coding sequences can encode IL-10 and MMP9, or multiple exogenous coding sequences can each encode either IL-10 or MMP9.

[0130] In some implementations, the expression of endogenous IL-10 and / or MMP9 genes can be stimulated through genetic engineering. For example, gene editing technologies such as CRISPR can be used to turn endogenous genes encoding IL-10 and / or MMP9 on and off, producing engineered macrophages that express IL-10 and / or MMP9 under conditions where these proteins would not normally be expressed. This can be achieved, for example, by altering the promoter sequence.

[0131] Natural, unengineered macrophages can express IL-10 and / or MMP9 under relevant physiological conditions, but typically do not secrete significant levels of IL-10. However, this invention does not relate to these natural, unengineered macrophages, but rather to engineered macrophages in which, in particular, IL-10 expression levels have been elevated supraphysiologically, thereby improving the anti-inflammatory properties of therapeutic macrophages. As used herein, the inventors describe this as “overexpression” of IL-10. To overexpress IL-10, the macrophages can be engineered to have additional or exogenous IL-10 coding sequences. In some embodiments, the cells are transfected with mRNA encoding IL-10 / MMP9.

[0132] Cells overexpressing IL-10 and / or MMP9 contain coding sequences that express IL-10 and / or MMP9 at higher levels than unengineered cells. As mentioned above, overexpression can be achieved by introducing exogenous nucleic acids such as mRNA encoding IL-10 and / or MMP9, or by genetic modifications that stimulate the expression of IL-10 and / or MMP9 from endogenous coding sequences. Engineered macrophages overexpressing IL-10 and / or MMP9 do not necessarily secrete more IL-10 and / or MMP9 than unengineered macrophages.

[0133] In other embodiments, the macrophages may be engineered to activate endogenous genes encoding IL-10 and / or MMP9. In any case, the macrophages of this disclosure have been modified by altering the expression levels of IL-10 and / or MMP9 in any way, and are therefore referred to as engineered macrophages.

[0134] When IL-10 is overexpressed, the natural activity levels of MMPs, including MMP9, decrease.

[0135] Therefore, the engineered macrophages possess additional or exogenous MMP-coding sequences to maintain at least a "physiological" level of MMP9 expression, or only slightly above the physiological level. In some embodiments, MMP9 expression is modified to rescue, repair, or restore the macrophages to a comparable or increased level of MMP9 expression when compared to macrophages not transfected with IL-10. Thus, despite the fact that the engineered macrophages are engineered with additional / exogenous MMP9-coding sequences, the engineered macrophages exhibit MMP9 expression levels similar to or slightly higher than wild-type / naturally expressed levels. Slightly higher may mean an expression enhancement of 1.2 to 1.5 times compared to natural expression levels, such as 1.2, 1.3, 1.4, or 1.5 times the natural level.

[0136] The macrophages can be engineered to express both IL-10 and MMP9. In some embodiments, this expression can be driven by endogenous genes. In other embodiments, the macrophages are engineered to contain exogenous coding sequences for IL-10 and MMP9. Preferably, the macrophages overexpress IL-10. Preferably, the macrophages overexpress MMP9. Alternatively, the macrophages can be engineered to overexpress both IL-10 and MMP-9. The expression levels of IL-10 and / or MMP9 are increased compared to untransfected macrophages. The expression level of MMP9 is increased compared to macrophages transfected with IL-10 alone.

[0137] 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 will be discussed further below.

[0138] In some implementations, baseline macrophages (i.e., unengineered or natural) are referred to as unpolarized human monocyte-derived macrophages, also known as "resting" macrophages.

[0139] In some embodiments, the engineered macrophages are macrophages derived from iPSCs. Various methods for deriving macrophages from iPSCs are known in the art. By baseline, these will also be non-polarized or quiescent. Therefore, in some embodiments, the engineered macrophages are derived from pluripotent stem cells cultured in vitro. Preferably, such pluripotent stem cell-derived or iPSC-derived macrophages are low immunogenic due to the knockout or knockdown of at least one gene associated with any HLA class I and / or HLA class II cell surface molecules. For example, genes associated with HLA class I may be HLA-A, HLA-B, and HLA-C genes or the B2M gene. Genes associated with HLA class II may be HLA-DP, DM, DO, DQ, and DR or CIITA. This knockout or knockdown is evident in the macrophages, but because macrophages are differentiated from stem cells, they may also be introduced into any progenitor cells. In an exemplary method, the engineered macrophages of the present invention can be generated by: providing stem cells, such as induced pluripotent stem cells, optionally knocking out or down-expressing at least one sequence encoding a unit in the HLA I or HLA II complex; differentiating the stem cells into germ cells, optionally using BMP4, SCF, VEGF and / or a Rock inhibitor (Y-27632); differentiating the germ cells into macrophage progenitor cells, optionally using M-CSF and IL-3; and maturing the macrophage progenitor cells into functional macrophages, optionally using M-CSF.

[0140] As used in this article, the artificial expression of the genes involved in overexpression increases.

[0141] As used in the examples, protein expression levels were quantified between 16 and 24 hours post-transfection. The expression levels described herein are for a concentration of 4 x 10⁻⁶. 6 / ml (equivalent to per cm) 2 2x10 6 The results were given as a population of macrophages (cells). In this example, macrophages were transfected, separated by centrifugation, resuspended in TexMACs buffer supplemented with IL-3 and IL-14, and incubated at 37°C and 5% CO2. Those skilled in the art will recognize equivalent conditions suitable for determining the concentration of secreted proteins.

[0142] In some embodiments, the macrophages are engineered to overexpress IL-10, wherein the secreted IL-10 protein level is greater than about 300 pg / ml. Suitably, the secreted IL-10 protein level is greater than about: 300 pg / ml or 400 pg / ml or 500 pg / ml or 600 pg / ml or 700 pg / ml or 800 pg / ml or 900 pg / ml or 1,000 pg / ml or 2,000 pg / ml or 3,000 pg / ml or 4,000 pg / ml or 5,000 pg / ml or 6,000 pg / ml or 7,000 pg / ml or 8,000 pg / ml or 9,000 pg / ml or 10,000 pg / ml or 11,000 pg / ml. Suitablely, these IL-10 protein levels can be measured by culturing macrophages as described above, wherein the concentration of macrophages in the culture medium is 4 x 10⁻⁶. 6 cells / ml, equivalent to 2 x 10 6 cells / cm 2 And measure the protein concentration in the culture medium. In a preferred embodiment, when using 4x10... 6 When cultured in vitro at a cell concentration of 19,000 pg / ml, the macrophages secrete IL-10 at a culture supernatant concentration of at least 10,000 pg / ml, 15,000 pg / ml, or 20,000 pg / ml. In a particularly preferred embodiment, IL-10 is secreted at a culture supernatant concentration of 49,000 pg / ml or higher. In some embodiments, the engineered macrophages may secrete IL-10 at levels up to 1000 times higher than unengineered, non-polarized hMDM. Unengineered, non-polarized hMDM is described in the art, for example, in WO2019175595.

[0143] In some embodiments, the macrophages are engineered to express MMP9, wherein the secreted MMP9 protein level is greater than about 200 ng / ml. Suitably, the secreted MMP9 protein level is greater than about: 300 ng / ml, 400 ng / ml, 500 ng / ml, 600 ng / ml, 700 ng / ml, 800 ng / ml, 900 ng / ml, or 1000 ng / ml. Suitably, the secreted MMP9 protein level is between about 200 ng / ml and 2000 ng / ml. In a preferred embodiment, the secreted MMP9 protein level is greater than 200 ng / ml. In one embodiment, the secreted MMP9 protein level is greater than 1500 ng / ml. Suitably, the engineered macrophages (containing IL-10 and MMP9) secrete MMP9 protein levels higher than the average level of MMP9 protein secreted by macrophages engineered with IL-10 alone. In some embodiments, the engineered macrophages (containing IL-10 and MMP9) secrete MMP9 protein levels at least equal to the average level of MMP9 protein secreted by unpolarized, untransfected macrophages. Suitably, the total MMP activity of the engineered macrophages of the present invention is also higher than that of macrophages engineered with IL-10 alone. In other embodiments, the total MMP activity of the engineered macrophages is higher than that of untransfected macrophages. In a preferred embodiment, the MMP activity of the engineered macrophages is at least 1.5 times higher than that of untransfected macrophages. Suitably, these IL-10 and MMP9 protein levels can be measured by culturing macrophages as described above, wherein the concentration of macrophages in the culture medium is 4 x 10⁻⁶. 6 cells / ml, equivalent to 2 x 10 6 cells / cm 2 The concentration of proteins in the culture medium is measured. In some embodiments, the engineered macrophages may secrete greater amounts of other matrix metalloproteinases. In a particular embodiment, the engineered macrophages may secrete up to 10 times more matrix metalloproteinase-3 (MMP3) than unengineered, non-polarized cells. In a particular embodiment, the engineered macrophages may secrete up to 10 times more matrix metalloproteinase-10 (MMP10) than unengineered, non-polarized cells.

[0144] In some embodiments, the macrophages have a pro-repair phenotype and are anti-inflammatory and anti-fibrotic. This phenotype is further defined herein.

[0145] Macrophages engineered to express IL-10 and MMP9 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 technology. Therefore, in some embodiments, the macrophages can be engineered with one or more exogenous coding sequences. These exogenous coding sequences may encode IL-10 and / or MMP9 and / or they may encode gene-editing proteins, such as CRISPR, nickases, etc., which can alter the cell's genome to increase the expression of endogenous IL-10 and / or endogenous MMP9. This can be achieved, for example, by editing promoter or enhancer sequences.

[0146] Known viral vectors used for transfecting macrophages include lentiviruses, adenoviruses, and adeno-associated viruses (AAVs).

[0147] As used herein, “coding” refers to the ability of a sequence of nucleotides (such as genes, cDNA, or mRNA) to serve as a template for the synthesis of macromolecules (such as defined amino acid sequences) in biological processes. Therefore, a coding sequence can be any suitable nucleic acid sequence that provides instructions for the synthesis of the relevant entity (e.g., IL-10 or MMP9). The coding sequence can be included in the same vector / construct or in different vectors / constructs.

[0148] As used herein, “exogenous” means any substance introduced from or produced outside a specific cell, particularly genetic material. As used herein, in some embodiments, the exogenous coding sequence or engineered macrophages encodes / expresses IL-10 or MMP9. In some embodiments, the IL-10 and / or MMP9 are human. Those skilled in the art will understand that sequence variations of these genes / coding sequences are also included in this invention. Ideally, the gene / coding sequence is human. The gene / coding sequence may be codon-optimized. The gene / coding sequence can be modified, and it should be understood that if the reference sequence is RNA, then different nucleotides may be present in the DNA vector.

[0149] Preferably, the provided IL-10 coding sequence is at least 80% similar to or homologous to the NCBI reference sequence: NM_000572.3 (human IL10 mRNA sequence), and more preferably at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, or 99% similar to the natural sequence.

[0150] Preferably, the provided MMP9 coding sequence is at least 80% similar to or homologous to the NCBI reference sequence: NM_004994.3 (Homo sapiens matrix metallopeptidase 9 (MMP9), mRNA), and preferably at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, or 99% similar to the natural sequence.

[0151] Preferably, the expressed IL-10 protein is at least 80% similar to or homologous to the sequence shown in SEQ ID No. 4, and more preferably at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, or 99% similar to SEQ ID No. 4.

[0152] Preferably, the expressed MMP9 protein is at least 80% similar to or homologous to the sequence shown in SEQ ID No. 6, and more preferably at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, or 99% similar to SEQ ID No. 6.

[0153] In some embodiments, the macrophages are non-virally engineered and engineered using a nucleic acid vector. In some embodiments, macrophages are transfected using a DNA vector, such as a naked DNA vector. Suitably, the DNA vector may encode both IL-10 and MMP9 on a single vector. In some embodiments, the nucleic acid vector is not derived from a viral genome. In some embodiments, macrophages are transfected using one or more cell-free nucleic acids or vectors. In some embodiments, macrophages are transfected using an RNA vector. In some embodiments, macrophages are transfected using mRNA. Suitably, macrophages are transfected using a single mRNA construct expressing IL-10 and MMP9.

[0154] In some embodiments, IL-10 and MMP9 are provided to macrophages as mRNA. Thus, the engineered cells are provided with exogenous mRNA molecules. MMP9 and IL-10 may be on the same mRNA molecule or on different mRNA molecules. Preferably, MMP9 and IL-10 are on the same mRNA molecule. More preferably, the mRNA is a bicistronic vector encoding both MMP9 and IL-10. Suitably, the mRNA molecule comprises a sequence as described in any one of SEQ ID Nos. 1 to 3, 10, 13, or 14.

[0155] In some embodiments, the mRNA comprises a sequence encoding IL-10. In some embodiments, the mRNA comprises at least 80% of a sequence identical to that of SEQ ID NO: 13. In some embodiments, the mRNA comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a sequence identical to that of SEQ ID NO: 13. In a preferred embodiment, the mRNA comprises the sequence described in SEQ ID NO: 13.

[0156] In some embodiments, the mRNA comprises a sequence encoding MMP9. In some embodiments, the mRNA comprises at least 80% of the sequence identical to SEQ ID NO: 14. In some embodiments, the mRNA comprises at least 85%, at least 90%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence identical to SEQ ID NO: 14. In a preferred embodiment, the mRNA comprises the sequence described in SEQ ID NO: 14.

[0157] In some embodiments, the macrophages are engineered with mRNA encoding both IL-10 and MMP9. In some embodiments, the macrophages are engineered with mRNA comprising both the sequences described in SEQ ID NO: 13 and SEQ ID NO: 14. In some embodiments, the sequences described in SEQ ID NO: 13 and 14 are located on the same mRNA molecule. In some embodiments, the sequences described in SEQ ID NO: 13 and 14 are located on different molecules. In a preferred embodiment, the mRNA comprises the sequences described in SEQ ID NO: 13 and 14 on the same mRNA molecule, separated by an mRNA encoding a self-cleaving adapter. In a preferred embodiment, the self-cleaving adapter has the amino acid sequence described in SEQ ID NO: 9. In a preferred embodiment, the mRNA encoding the self-cleaving adapter has the sequence described in SEQ ID NO: 15.

[0158] In some embodiments, the mRNA construct is expressed via a cleavable linker fused to IL-10 in MMP9. In some embodiments, the linker is p2A. p2A may have the amino acid sequence described in SEQ ID No. 9, which may be encoded by the mRNA sequence described in SEQ ID No. 8 or 15. In a preferred embodiment, p2A is encoded by the mRNA sequence described in SEQ ID NO: 15. p2A is part of the 2A self-cleaving peptide family or 2A peptides. These peptides are 18-22 amino acid long and can induce ribosome skipping during intracellular protein translation. These peptides share the core sequence motif of DxExNPGP. This allows IL-10 and MMP9 to be expressed as separate proteins, even though they exist in the same mRNA molecule.

[0159] In a preferred embodiment, both IL-10 and MMP9 are expressed from an mRNA molecule. In some embodiments, the mRNA molecule contains at least 80% of the sequence identical to SEQ ID NO: 10. In some embodiments, the mRNA molecule contains at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence identical to SEQ ID NO: 10. In a preferred embodiment, the mRNA containing the sequence described in SEQ ID NO: 10 further contains a polyA tail, optionally 65 to 250 residues long, preferably 90 to 120 residues long, preferably about 90 residues long, and / or a 5' cap. In a particularly preferred embodiment, the mRNA containing the sequence described in SEQ ID NO: 10 further contains a polyA tail, optionally 65 to 250 residues long, preferably 90 to 120 residues long, such as 90 residues long, and a 5' cap. In a preferred embodiment, the exogenous mRNA sequence comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence listed in SEQ ID NO: 16. In a particularly preferred embodiment, the exogenous mRNA sequence comprises the sequence listed in SEQ ID NO: 16, and preferably also comprises a 5' cap.

[0160] In some embodiments, the exogenous mRNA sequence comprises a 5' untranslated region (5'UTR) sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence listed in SEQ ID NO: 19. In a preferred embodiment, the exogenous mRNA sequence comprises a 5' untranslated region (5'UTR) sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence listed in SEQ ID NO: 17. In a preferred embodiment, the exogenous mRNA sequence comprises a 5' UTR sequence as listed in SEQ ID NO: 17.

[0161] In some embodiments, the exogenous mRNA sequence comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical 3' untranslated region (3'UTR) to the sequence listed in SEQ ID NO: 20. In a preferred embodiment, the exogenous mRNA sequence comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical 3' untranslated region (3'UTR) to the sequence listed in SEQ ID NO: 18. In a preferred embodiment, the exogenous mRNA sequence comprises a 3' UTR sequence as listed in SEQ ID NO: 18. In a preferred embodiment, the exogenous mRNA comprises both a 5' UTR sequence as listed in SEQ ID NO: 17 and a 3' UTR sequence as listed in SEQ ID NO: 18.

[0162] Alternatively or additionally, IRES sites can be inserted between the coding sequences, which technicians will understand will interrupt expression to form two separate proteins.

[0163] In some embodiments, the mRNA molecule contains a poly-A tail. A poly-A tail is a long chain of adenine nucleotides added to the mRNA molecule during RNA processing to increase molecular stability. A suitable poly-A tail is 65 to 250 residues long, preferably 90 to 120 residues long. The poly-A tail makes the RNA molecule more stable and prevents its degradation.

[0164] In some embodiments, the mRNA molecule contains at least one cap. The 5'-cap is a hallmark of eukaryotic mRNA. Chemically, the 5'-cap consists of an inverted 7-methylguanosine, linked to the remainder of the eukaryotic mRNA via a 5'-5' triphosphate bridge. This so-called "cap0" is particularly helpful for the stability of eukaryotic mRNA, the initiation of translation, and mRNA decay. Artificial cap structures have been designed to improve the success rate of in vitro translation. Synthesized anti-reverse cap analogs include additional methylation and the trinucleotide CleanCap. ® AG, methylated at the first adenosine. At least one cap can be a synthetic cap analogue, preferably CleanCap. ® .

[0165] In some embodiments, the mRNA is modified RNA. In some embodiments, the mRNA modification includes chemical modification of uridine and / or chemical modification of cytidine. In some embodiments, the mRNA modification includes pseudouridine, N1-methylpseudouridine, 5-methoxy-uridine, 5-methyl-cytidine, preferably 5-methoxy-uridine.

[0166] In one embodiment, the mRNA is modified with 5-methoxy-uridine and includes at least one CleanCap. ® .

[0167] In some implementations, the mRNA construct contains the sequence of SEQ ID NO. 2 or a sequence that is at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, or 99% similar to or homologous to SEQ ID No. 2.

[0168] In some embodiments, the macrophages are engineered using a nucleic acid vector. In some embodiments, macrophages are transfected via electroporation. Other suitable transfection methods include nuclear transfection.

[0169] In some embodiments, the mRNA is delivered to macrophages via nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles, which may be described, for example, in U.S. Patent Nos. 8,058,069, 8,492,359, 8,822,668, 9,364,435, 9,504,651, and 11,141,378.

[0170] In some embodiments, the macrophages are autologous or allogeneic to the subject. In some embodiments, the macrophages are derived from progenitor cells, such as iPSCs, hematopoietic stem cells, or monocytes.

[0171] In some embodiments, the macrophages are derived from monocytes by culturing monocytes under suitable conditions. Suitably, these conditions may include:

[0172] An in vitro method for generating macrophages, comprising:

[0173] (b) Culture monocytes in a culture medium for 3-5 days to produce macrophages, wherein the culture medium contains one or more growth factors that stimulate macrophage production;

[0174] Step (a) is performed entirely in the same culture medium.

[0175] Suitably, the culture medium is suitable for generating macrophages from monocytes. Suitably, the culture medium is a T cell culture medium. Suitably, the culture medium may be selected from: X-Vivo 10, X-Vivo 15, TexMACS, AIMv, RPMI, DMEM, and DMEM / F12. Suitably, the culture medium is TexMACS (Miltenyi).

[0176] Suitably, the culture medium is serum-free. Suitably, the culture medium is free of foreign proteins. Suitably, the culture medium conforms to GMP standards.

[0177] Suitably, the culture medium may contain one or more factors. Suitable factors include growth factors, polysaccharides, cytokines, and chemokines. Suitable factors may include MCSF and GM-CSF. Suitably, therefore, the factors are growth factors. Suitably, the one or more factors comply with GMP. In one embodiment, the culture medium may contain one or more growth factors, which may include MCSF or GM-CSF. Monocytes are most commonly cultured with MCSF or GM-CSF. Cultured monocytes with GM-CSF bias them toward an "inflammatory" phenotype, while cultured monocytes with MCSF bias them toward a "pro-repair" phenotype. In other embodiments, the one or more growth factors do not include a combination of MCSF and GM-CSF. Therefore, if MCSF is used as a growth factor to generate macrophages in any method of the present invention, it is preferable that GM-CSF is not used to generate macrophages from monocytes. This applies to the step of culturing monocytes until macrophages are generated.

[0178] According to another aspect of the invention, a population of engineered macrophages as described herein is provided. This population is applicable to the therapies described above.

[0179] According to another aspect of the invention, a composition comprising engineered macrophages is provided. In some embodiments, the engineered macrophage composition is used in the therapy described above, or a population of engineered macrophages is used in the therapy described above. The composition may also contain other pharmaceutically acceptable components, such as suitable cell culture media, excipients, etc.

[0180] According to any aspect of the invention, suitably, the use includes administering an effective amount of engineered macrophages to a subject.

[0181] According to any aspect of the invention, the use may include delivering engineered macrophages to a subject via systemic administration, suitably via systemic injection. In some embodiments, administration is via local injection, for example, for the kidneys and lungs. In some embodiments, administration is via a nebulizer to the lungs. For example, peripheral intravenous injection is advantageous for liver conditions to avoid invasive procedures in patients with cirrhosis. Local injection, such as via the renal artery for kidney conditions, may be better tolerated.

[0182] Preferably, the macrophages used in the therapy are engineered in vitro and delivered to the patient.

[0183] However, in some embodiments, the macrophages can be engineered in vivo. In some embodiments, the macrophages are engineered in vivo by administering to a subject a formulation containing exogenous coding sequences of IL-10 and MMP9 suitable for transfecting macrophages. The formulation may include any exogenous coding sequences discussed herein. Suitable delivery vehicles for in vivo engineering may include macrophage-targeting molecules due to their cell surface markers. If in vivo transfection of macrophages is envisioned, local application of the formulation may be more effective, such as local injection of the formulation into the liver or kidney, or nebulization into the lungs. Such treatment can be formulated as nanoparticles to assist macrophage targeting.

[0184] According to a fourth aspect of the invention, a method for improving the migration of monocytes to sites of inflammation is provided, comprising using engineered macrophages according to a first aspect of the invention, a population of engineered macrophages according to a second aspect of the invention, or a composition according to a third aspect of the invention.

[0185] In some embodiments, the method polarizes host monocytes / macrophages to a pro-repair phenotype. In some embodiments, the method polarizes unpolarized host macrophages to an anti-inflammatory and / or pro-repair phenotype. In some embodiments, the method polarizes inflammatory host macrophages to an anti-inflammatory and / or pro-repair phenotype.

[0186] In some embodiments, one or more of the following biomarkers may be used to describe a pro-repair phenotype: increased CD206 and / or CD163, and decreased inflammatory biomarkers such as CD86 and / or type II (HLA-DR). These increases / decreases are compared to non-polarized (resting) or pro-inflammatory macrophages. In terms of secretory profile, these macrophages are expected not to express TNFα, IFNg, and IL1β, which are typically associated with pro-inflammatory and pro-fibrotic profiles. In a preferred embodiment, the engineered macrophages express CD206 at a level 5-fold higher than those in unengineered, non-polarized cells, such as those described in WO2019 / 17559.

[0187] In some embodiments, the engineered macrophages do not secrete TNFα, IFNg, IL1β, IL-12p70, and / or IL-2. In a preferred embodiment, the engineered macrophages secrete less than 40 pg / ml of TNFα. In some embodiments, the engineered macrophages secrete the same or similar levels of TNFα, IFNg, IL1β, IL-12p70, and / or IL-2 as unengineered, non-polarized cells.

[0188] The engineered macrophages secrete IL-10 and / or MMP9. In a preferred embodiment, the engineered macrophages secrete at least 10,000 pg / ml of IL-10. In a preferred embodiment, the engineered macrophages secrete at least 200 ng / ml of MMP9. In some embodiments, the engineered macrophages exhibit enhanced MMP activity. In a preferred embodiment, the engineered macrophages exhibit 1.5 times higher MMP activity than unengineered, non-polarized cells. In some embodiments, the engineered macrophages secrete IL-6 and / or CXCL8.

[0189] According to another aspect of the invention, a method for generating engineered macrophages expressing IL-10 and MMP9 is provided. In some embodiments, the method includes introducing a nucleic acid comprising at least one sequence encoding IL-10 and at least one sequence encoding MMP9 into macrophages or cells of origin thereof.

[0190] In certain embodiments, the method includes transiently transfecting macrophages with an mRNA construct comprising at least one sequence encoding IL-10 and at least one sequence encoding MMP9. In another embodiment, a method is provided for generating engineered macrophages expressing IL-10, such that the engineered macrophages secrete at least 10,000 pg / mL of IL-10, the method comprising introducing a nucleic acid encoding IL-10 into macrophages or cells of origin thereof. In some embodiments, the method further includes exposing the macrophages to an anti-inflammatory treatment, such as IL-4 and IL-13, after transfection. In some embodiments, the method includes exposing the macrophages to IL-4 and IL-13. In some embodiments, the macrophages are incubated with IL-4, IL-13, and M-CSF.

[0191] In some embodiments, the method includes electroporation. In some embodiments, after electroporation, the macrophages are contacted with anti-inflammatory treatment.

[0192] In some embodiments, the present invention provides:

[0193] 1. An engineered macrophage comprising an exogenous coding sequence for IL-10 and an exogenous coding sequence for MMP9.

[0194] 2. The engineered macrophages according to embodiment 1, wherein the expression of the exogenous coding sequence has a synergistic effect in restoring MMP activity compared to engineered macrophages containing a single exogenous IL-10 sequence, and / or a synergistic effect in monocyte recruitment of macrophages.

[0195] 3. The engineered macrophages according to embodiment 1 or embodiment 2, wherein the macrophages and / or coding sequences are human.

[0196] 4. An engineered macrophage according to any one of embodiments 1 to 3, wherein the exogenous coding sequence is present on one or more nucleic acid molecules or integrated into the genome of the macrophage.

[0197] 5. The engineered macrophage according to embodiment 4, wherein the nucleic acid molecule is a DNA or RNA molecule, preferably an mRNA molecule.

[0198] 6. The engineered macrophage according to embodiment 5, wherein the mRNA molecule contains chemically modified residues, preferably modified uracil residues, and optionally at least one synthetic cap.

[0199] 7. The engineered macrophage according to any of the foregoing embodiments, wherein the exogenous coding sequence of IL-10 and the exogenous coding sequence of MMP9 are on the same nucleic acid.

[0200] 8. Engineered macrophages according to any of the foregoing embodiments, wherein the macrophages are engineered to overexpress IL-10, preferably, wherein in a 4x10 6 At a cell concentration of 100 μg / ml, the secreted IL-10 protein level is greater than approximately 300 pg / ml.

[0201] 9. The engineered macrophage according to any of the foregoing embodiments, wherein the metalloproteinase activity level is at least 1.5 times that of the metalloproteinase activity of non-engineered macrophages.

[0202] 10. Engineered macrophages according to any one of embodiments 1 to 7, wherein the metalloproteinase activity is restored relative to the reduced metalloproteinase activity in macrophages engineered solely with the IL-10 coding sequence.

[0203] 11. Engineered macrophages according to any of the foregoing embodiments, wherein the macrophages are transiently transfected, optionally via electroporation.

[0204] 12. Engineered macrophages according to embodiment 10, wherein the transfection is non-viral.

[0205] 13. The engineered macrophages according to any of the foregoing embodiments, wherein the macrophages have a repair-promoting phenotype.

[0206] 14. A population of engineered macrophages according to any of the foregoing embodiments.

[0207] 15. A therapeutic composition comprising a population of macrophages as described in embodiment 14 and a pharmaceutically acceptable medium.

[0208] 16. Engineered macrophages according to any one of embodiments 1 to 13, a population of macrophages according to embodiment 14, or a composition according to embodiment 15, used in a therapy.

[0209] 17. Engineered macrophages, populations, or compositions according to embodiment 16, wherein the therapy is administered to a subject in need.

[0210] 18. Engineered macrophages according to any one of embodiments 1 to 13, a population of macrophages according to embodiment 14, or a composition according to embodiment 15, for treating an inflammatory condition in a subject.

[0211] 19. Engineered macrophages, populations, or compositions according to embodiment 17 or embodiment 18, wherein the macrophages are autologous or allogeneic to the subject.

[0212] 20. Engineered macrophages, populations, or compositions according to embodiment 18 or embodiment 19, wherein the condition is a chronic inflammatory condition with fibrotic components, optionally wherein the condition is organ damage associated with chronic inflammation.

[0213] 21. Engineered macrophages, populations, or compositions according to embodiment 18 or embodiment 19, wherein the condition is acute-on-chronic liver failure (ACLF).

[0214] 22. A method for improving the migration of monocytes to sites of inflammation, comprising using engineered macrophages, populations of engineered macrophages, or compositions thereof according to any one of the foregoing embodiments.

[0215] 23. The method according to embodiment 22, wherein the method polarizes host monocytes / macrophages to a pro-repair phenotype and / or away from a pro-inflammatory phenotype.

[0216] 24. A method for generating engineered macrophages according to any one of embodiments 1 to 13, comprising transiently transfecting macrophages with mRNA molecules encoding IL-10 and / or MMP9.

[0217] 25. The method according to embodiment 24, which includes contacting macrophages with IL-4 and IL-13 before, during, or after transfection.

[0218] 26. The method according to embodiment 24 or 25, wherein a bicistronic vector linked by a p2A adapter sequence is used to co-transfect mRNA molecules encoding IL-10 and MMP9.

[0219] 27. Engineered macrophages according to any one of embodiments 1 to 13, wherein the macrophages are engineered with an mRNA construct encoding human IL-10 fused to the human MMP9 protein via a cleavable adapter.

[0220] 28. A method for treating inflammation and / or fibrosis, comprising administering to a subject in need a therapeutically effective amount of engineered macrophages according to any one of embodiments 1-13.

[0221] The present invention also provides the following embodiments, which can be combined with any other embodiments:

[0222] 1. An engineered macrophage, said engineered macrophage being engineered to overexpress IL-10.

[0223] 2. Engineered macrophages according to implementation scheme 1, wherein when 4x10 6When cultured in vitro at a cell concentration of at least 10,000 pg / ml, the macrophages secrete IL-10 at a culture supernatant concentration of at least 10,000 pg / ml.

[0224] 3. Engineered macrophages according to embodiment 1 or embodiment 2, wherein the macrophages are additionally engineered to overexpress MMP9.

[0225] 4. The engineered macrophages according to embodiment 3, wherein the engineered macrophages comprise an exogenous coding sequence for IL-10 and an exogenous coding sequence for MMP9.

[0226] 5. The engineered macrophages according to embodiment 4, wherein the expression of the exogenous coding sequence has a synergistic effect in restoring MMP activity compared to engineered macrophages containing a single exogenous IL-10 sequence, and / or a synergistic effect in monocyte recruitment of macrophages.

[0227] 6. The engineered macrophage according to any one of embodiments 1-5, wherein the macrophage and / or coding sequence is human.

[0228] 7. The engineered macrophage according to any one of embodiments 4-6, wherein the exogenous coding sequence of IL-10 encodes a protein having an amino acid sequence that is at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% identical to that of SEQ ID NO: 4, optionally wherein the IL-10 protein comprises an amino acid sequence identical to that of SEQ ID NO: 4.

[0229] 8. The engineered macrophage according to any one of embodiments 4-7, wherein the exogenous coding sequence of MMP9 encodes a protein having at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% of the same amino acid sequence as SEQ ID NO: 6, optionally wherein the IL-10 protein comprises the same amino acid sequence as SEQ ID NO: 6.

[0230] 9. An engineered macrophage according to any one of embodiments 4, 5, 7 or 8, wherein the exogenous coding sequence is present on one or more nucleic acid molecules or integrated into the genome of the macrophage.

[0231] 10. The engineered macrophage according to embodiment 9, wherein the nucleic acid molecule is a DNA or RNA molecule, preferably an mRNA molecule, optionally wherein IL-10 and MMP9 are expressed by the same mRNA molecule, further optionally wherein the mRNA molecule encodes IL-10 and MMP9 linked by a adapter sequence, further optionally wherein the adapter is a self-cleaving 2A adapter, further optionally wherein the adapter is p2A.

[0232] 11. The engineered macrophage according to embodiment 10, wherein the nucleic acid molecule is an mRNA molecule containing at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same sequence as SEQ ID NO: 13, optionally wherein the nucleic acid contains SEQ ID NO: 13.

[0233] 12. The engineered macrophage according to embodiment 10 or 11, wherein the nucleic acid molecule is an mRNA molecule containing at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same sequence as SEQ ID NO: 14, optionally wherein the nucleic acid contains SEQ ID NO: 14.

[0234] 13. The engineered macrophages according to embodiments 10-12, wherein the nucleic acid molecule is an mRNA molecule encoding IL-10 and MMP9 linked by a linker sequence, and wherein the linker sequence encodes a protein comprising the amino acid sequence described in SEQ ID NO: 7, optionally wherein the protein encoded by the linker sequence comprises the amino acid sequence described in SEQ ID NO: 9.

[0235] 14. The engineered macrophages according to embodiments 10-13, wherein the nucleic acid molecule is an mRNA molecule encoding IL-10 and MMP9 linked by a adapter sequence, and wherein the adapter sequence comprises an mRNA having the sequence described in SEQ ID NO: 15.

[0236] 15. The engineered macrophages according to embodiments 10-14, wherein the nucleic acid molecule is an mRNA molecule comprising at least 80% identical, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 10, optionally wherein the mRNA further comprises a polyA tail of 65 to 250 residues long, preferably 90 to 120 residues long, and / or a 5' cap.

[0237] 16. The engineered macrophage according to embodiments 10-15, wherein the nucleic acid molecule is an mRNA molecule containing at least 80% identical, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 16, and optionally, wherein the mRNA further comprises a 5' cap.

[0238] 17. The engineered macrophage according to embodiment 16, wherein the mRNA molecule contains chemically modified residues, preferably modified uracil residues, and optionally at least one synthetic cap.

[0239] 18. Engineered macrophages according to any one of embodiments 4 to 17, wherein the exogenous coding sequence of IL-10 and the exogenous coding sequence of MMP9 are on the same nucleic acid.

[0240] 19. The engineered macrophage according to any one of the foregoing embodiments, wherein the macrophage is engineered by editing the endogenous promoter of the IL-10 gene and / or the MMP9 gene, or wherein the macrophage is engineered by regulating the expression of an endogenous silencing RNA or by introducing an exogenous silencing RNA sequence, optionally wherein the silencing RNA is a miRNA.

[0241] 20. The engineered macrophage according to any of the foregoing embodiments, wherein the metalloproteinase activity level is at least 1.5 times that of the metalloproteinase activity of non-engineered macrophages.

[0242] 21. The engineered macrophage according to any one of the foregoing embodiments, wherein the CD86 expression of the engineered macrophage is reduced by at least two-fold compared with that of non-engineered, non-polarized cells.

[0243] 22. The engineered macrophage according to any one of the foregoing embodiments, wherein the HLA-DR expression of the engineered macrophage is reduced by at least two-fold compared with that of non-engineered, non-polarized cells.

[0244] 23. The engineered macrophage according to any one of the foregoing embodiments, wherein the engineered macrophage secretes at least 1000 times more IL-10 than non-engineered, non-polarized cells.

[0245] 24. The engineered macrophage according to any one of the foregoing embodiments, wherein the engineered macrophage secretes MMP3 at least 10 times more than non-engineered, non-polarized cells.

[0246] 25. The engineered macrophage according to any one of the foregoing embodiments, wherein the engineered macrophage secretes at least 20-fold more MMP10 than non-engineered, non-polarized cells.

[0247] 26. Engineered macrophages according to any one of the foregoing embodiments, wherein when 4 x 10 6 When cultured in vitro at a cell concentration of at least 10,000 pg / ml, the macrophages secrete IL-10 at a culture supernatant concentration of at least 10,000 pg / ml.

[0248] 27. Engineered macrophages according to any one of the foregoing embodiments, wherein when 4 x 10 6 When cultured in vitro at a cell concentration of 200 ng / ml, the macrophages secrete MMP9 at a concentration of at least 200 ng / ml of culture supernatant.

[0249] 28. The engineered macrophage according to any one of the foregoing embodiments, wherein the CD206 expression of the engineered macrophage is increased by at least 5-fold compared with that of monocytes.

[0250] 29. The engineered macrophage according to any one of the foregoing embodiments, wherein the 25F9 expression of the engineered macrophage is increased by at least 5-fold compared with monocytes.

[0251] 30. The engineered macrophage according to any one of the foregoing embodiments, wherein the CD80 expression of the engineered macrophage is reduced by at least 10 percent compared with that of non-engineered, non-polarized cells.

[0252] 31. Engineered macrophages according to any one of the foregoing embodiments, wherein when 4 x 10 6 When cultured in vitro at a cell concentration of 40 pg / ml, the macrophages secreted TNF-α at a culture supernatant concentration of up to 40 pg / ml.

[0253] 32. The engineered macrophage according to any one of the foregoing embodiments, wherein the engineered macrophage has at least the same phagocytic capacity as unengineered, non-polarized cells.

[0254] 33. Engineered macrophages according to any one of embodiments 1-32, wherein the metalloproteinase activity is restored relative to the reduced metalloproteinase activity in macrophages engineered solely with the IL-10 coding sequence.

[0255] 34. Engineered macrophages according to any of the foregoing embodiments, wherein the macrophages are transiently transfected, optionally via electroporation.

[0256] 35. Engineered macrophages according to embodiment 34, wherein the transfection is non-viral.

[0257] 36. The engineered macrophages according to any of the foregoing embodiments, wherein the macrophages have a repair-promoting phenotype.

[0258] 37. A population of engineered macrophages according to any of the foregoing embodiments.

[0259] 38. A therapeutic composition comprising a population of macrophages according to embodiment 37 and a pharmaceutically acceptable medium.

[0260] 39. Engineered macrophages according to any one of embodiments 1 to 36, a population of macrophages according to embodiment 37, or a composition according to embodiment 38, used in a therapy.

[0261] 40. Engineered macrophages, populations, or compositions according to embodiment 39, wherein the therapy is administered to a subject in need.

[0262] 41. Engineered macrophages according to any one of embodiments 1 to 36, a population of macrophages according to embodiment 37, or a composition according to embodiment 38, for treating an inflammatory condition in a subject.

[0263] 42. Engineered macrophages, populations, or compositions according to embodiments 40 or 41, wherein the macrophages are autologous or allogeneic to the subject.

[0264] 43. Engineered macrophages, populations, or compositions according to embodiment 41, wherein the inflammatory condition is liver injury, optionally chronic liver injury.

[0265] 44. Engineered macrophages, populations, or compositions according to embodiments 41 or 43, wherein the condition is a chronic inflammatory condition with fibrotic components, optionally wherein the condition is organ damage associated with chronic inflammation.

[0266] 45. Engineered macrophages, populations, or compositions according to any one of embodiments 1, 43, or 44, wherein the condition is fibrosis, and wherein the fibrosis is located in or affects an organ selected from the group consisting of: liver, lung, heart, kidney, pancreas, skin, gastrointestinal tract, bone marrow, hematopoietic tissue, nervous system, eye, and combinations thereof.

[0267] 46. ​​Engineered macrophages, populations, or compositions according to any one of embodiments 43 or 44, wherein the condition is cirrhosis.

[0268] 47. The engineered macrophages, populations, or compositions according to embodiment 46, wherein the cirrhosis is caused by 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, biliary obstruction, autoimmune hepatitis, iron overload, hepatitis B infection (HBV), and hepatitis C infection (HCV).

[0269] 48. Engineered macrophages, populations, or compositions according to embodiment 46, wherein the cirrhosis is caused by steatotic liver disease (SLD), optionally wherein the steatotic liver disease is metabolic dysfunction-related steatotic liver disease, metabolic-associated steatohepatitis, Met-ALD, or cryptogenic SLD.

[0270] 49. Engineered macrophages, populations, or compositions according to any one of embodiments 46-48, wherein the cirrhosis is selected from compensated cirrhosis and decompensated cirrhosis.

[0271] 50. Engineered macrophages, populations, or compositions according to any one of embodiments 41-49, wherein the condition is acute-on-chronic liver failure (ACLF).

[0272] 51. Engineered macrophages, populations, or compositions according to embodiments 46-49 for treating a subject who has recovered (recompensated) from a first hepatic decompensation event, optionally wherein the first hepatic decompensation event required hospitalization of the subject, preferably wherein the subject has not experienced additional hepatic decompensation events after recovering from the first hepatic decompensation event.

[0273] 52. Engineered macrophages, populations, or compositions according to embodiments 46-49 and 51, wherein the subject exhibits one or more clinical signs of hepatic decompensation, or has recovered from one or more clinical signs of hepatic decompensation, the one or more clinical signs being selected from a list consisting of jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, variceal bleeding, and gastrointestinal bleeding.

[0274] 53. An engineered macrophage, population or composition according to any one of embodiments 1-52, wherein the macrophage is derived from human monocyte-derived macrophages (hMDM) or stem cells, optionally wherein the stem cells are induced pluripotent stem cells (iPSCs).

[0275] 54. The engineered macrophages, populations, or compositions according to embodiment 53, wherein the macrophages are derived from iPSCs and the iPSCs substantially lack functional HLA I and II complexes on their surface.

[0276] 55. A method for improving the migration of monocytes to sites of inflammation, comprising using engineered macrophages, populations of engineered macrophages, or compositions thereof according to any one of the foregoing embodiments.

[0277] 56. The method according to embodiment 55, wherein the method polarizes host monocytes / macrophages to a pro-repair phenotype and / or away from a pro-inflammatory phenotype.

[0278] 57. A method for generating engineered macrophages according to any one of embodiments 1 to 36, comprising transiently transfecting macrophages with mRNA molecules encoding IL-10 and / or MMP9.

[0279] 58. The method according to embodiment 57, which includes contacting macrophages with IL-4, IL-13 and M-CSF before, during or after transfection.

[0280] 59. The method according to embodiment 57 or 58, wherein a bicistronic vector linked via a p2A adapter sequence is used to co-transfect mRNA molecules encoding IL-10 and MMP9.

[0281] 60. Engineered macrophages according to any one of embodiments 1 to 36, wherein the macrophages are engineered using an mRNA construct encoding human IL-10 fused to the human MMP9 protein via a cleavable adapter.

[0282] 61. A method for treating inflammation and / or fibrosis, comprising administering to a subject in need a therapeutically effective amount of engineered macrophages according to any one of embodiments 1-36.

[0283] 62. A method for polarizing macrophages to a pro-repair phenotype, wherein polarized macrophages have increased expression of CD163 and CD206 and decreased expression of HLA DR and CD86 compared to cells not polarized to a pro-repair phenotype, wherein the method comprises engineering the macrophages to express IL-10 and MMP9 at levels above endogenous levels.

[0284] 63. The method according to embodiment 62, wherein the macrophages are engineered to express IL-10 and MMP9 by introducing exogenous nucleic acids containing nucleotide sequences encoding IL-10 and MMP9.

[0285] 64. The method according to embodiment 63, wherein the nucleotide sequences encoding IL-10 and MMP9 are present on the same nucleic acid molecule.

[0286] 65. The method according to embodiment 63, wherein the nucleotide sequences encoding IL-10 and MMP9 are present on different nucleic acid molecules.

[0287] 66. The method according to any one of embodiments 63-65, wherein the nucleic acid is mRNA.

[0288] 67. A method for polarizing macrophages to a pro-repair phenotype, wherein polarized macrophages have increased expression of CD163 and CD206 and decreased expression of HLA DR and CD86 compared to cells not polarized to a pro-repair phenotype, wherein the method comprises engineering the macrophages to overexpress IL-10, optionally, wherein when at 4x10 6 When cultured in vitro at a cell concentration of at least 10,000 pg / ml, the macrophages secrete IL-10 at a culture supernatant concentration of at least 10,000 pg / ml.

[0289] 68. The method according to embodiment 67, wherein the macrophages are engineered to express IL-10 by introducing an exogenous nucleic acid containing a nucleotide sequence encoding IL-10.

[0290] 69. The method according to embodiment 67 or 68, wherein the nucleic acid is mRNA.

[0291] 70. A method for improving the hypothermic resistance of macrophages, comprising incubating macrophages in a culture medium containing IL-4, IL-13 and M-CSF.

[0292] 71. A method for cryopreserving macrophages, comprising incubating macrophages in a culture medium containing IL-4, IL-13 and M-CSF prior to cryopreservation.

[0293] 72. The method according to embodiment 70 or 71, wherein the concentrations of IL-4 and IL-13 in the culture medium are 20 ng / ml, the concentration of M-CSF is 100 ng / ml, and the concentration of macrophages is 4 x 10⁻⁶. 6 Cells / ml.

[0294] 73. The method according to embodiments 70-72, wherein the cells are incubated overnight in a culture medium containing IL-4, IL-13 and M-CSF.

[0295] 74. Cryopreserved macrophages obtained by the method according to any one of embodiments 70-73.

[0296] According to a preferred embodiment, the present invention provides an engineered macrophage, engineered to overexpress IL-10 and optionally further engineered to overexpress MMP9, for the treatment of cirrhosis in a subject, wherein the method includes administering the engineered macrophage to the subject, wherein the subject has been hospitalized following a first hepatic decompensation event and, prior to administering the engineered macrophage to the subject, experienced a subsequent additional decompensation event following the first decompensation event.

[0297] According to a preferred embodiment, the present invention provides an engineered macrophage, engineered to overexpress IL-10 and optionally further engineered to overexpress MMP9, for use in treating cirrhosis in a subject, wherein the method includes administering the engineered macrophage to the subject, wherein the subject suffers from refractory ascites.

[0298] According to some implementation schemes, “cirrhosis” in subjects refers to cirrhosis caused by a liver disease etiology selected from at least one of the following: (1) steatotic liver disease associated with pure metabolic dysfunction (MASLD), or (2) metabolic and alcohol-related / associated liver disease (Met-ALD), or (3) HCV sustained virological response with persistent cirrhosis (HCV SVR), or (4) alcohol-related liver disease (ALD or Met-ALD) only if the patient is confirmed to have consumed alcohol within the Met-ALD limit (i.e., no alcohol abuse) and has a value of <25 mg / L in the PEth alcohol test.In a particularly preferred embodiment, the present invention provides an engineered macrophage, engineered to overexpress IL-10 and optionally further engineered to overexpress MMP9, for use in treating cirrhosis in a subject, wherein the method includes administering the engineered macrophage to the subject, wherein the subject has been hospitalized following a first hepatic decompensation event, and prior to administering the engineered macrophage to the subject, the subject has experienced a subsequent additional decompensation event following the first decompensation event, and wherein the subject has all of the following characteristics indicating benefit from treatment. Possibility of benefit: No known inconsistencies with G-CSF or leukapheresis procedures; age between 18 and 17 years; diagnosis of cirrhosis based on at least one of the following: a. clinical and radiological features relevant to the diagnosis of cirrhosis; liver stiffness score >15 kPa measured by transient elastography (TE; Fibroscan); and c. histological features of cirrhosis confirmed by previous liver biopsy; etiology of the following liver diseases: a. steatotic liver disease of pure metabolic dysfunction (MASLD) or metabolic and alcohol-related / associated liver disease (Met-ALD) or persistent virological response to HCV with persistent cirrhosis (HCV). a. SVR); b. Alcohol-related liver disease (ALD or Met-ALD), only if the subject is confirmed to have consumed alcohol within the Met-ALD limit, defined as an intake of >14 units / week, or more than 2 units / day for women and more than 3 units / day for men (1 unit is equivalent to 8 g of alcohol); c. Alcohol-related liver disease (ALD or Met-ALD), only if the subject's PEth test value is <25 mg / L; MELD 3.0 score of 12-20, within two weeks of an "eligible" decompensated event, or if the participant is eligible for outpatient status, their MELD at screening (Visit 1). A score of 3.0 must be between 12 and 20; hospitalization for a major hepatic decompensation event, including ascites, hepatic encephalopathy, variceal bleeding, hepatorenal syndrome / acute kidney injury (HRS / AKI) or spontaneous bacterial peritonitis (SBP), which is the only hospitalization for a hepatic decompensation event in the past 6 months, or refractory ascites, defined as ascites that cannot be mobilized or recurs within 8 weeks after LVP (the date of onset of refractory ascites is defined as the time of the second LVP).

[0299] In some implementations, subjects do not possess the following characteristics that indicate contraindication to treatment: cirrhosis caused by any viral hepatitis other than hepatitis C virus (HCV SVR); cirrhosis caused by autoimmune and cholestatic etiologies, including but not limited to primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC); acute liver disease without underlying cirrhosis, including but not limited to drug-induced liver injury (DILI); any current organ failure requiring supportive care beyond outpatient care and unrelated to the participant's liver decompensation event; known splenomegaly ≥16 cm; thrombocytopenia < 50 × 10⁻⁶. 9 / L; any of the following comorbidities are present or suspected: history of liver or other organ transplantation, ACLF, sepsis (positive microbial culture), unless stable and at least 4 weeks after completion of a full course of IV antibiotics, human immunodeficiency virus (HIV), known human immunodeficiency virus, human T-lymphotropic virus 1, pulmonary embolism (PE), hepatocellular carcinoma (HCC) or active malignancy within the past 5 years (excluding non-melanoma skin cancer, cervical carcinoma in situ, superficial bladder cancer, benign polyps, etc.), liver comorbidities such as portal vein thrombosis, hepatic pleural effusion unless it is a small amount of pleural effusion that is clinically inconspicuous and is incidentally detected by radiological evaluation without clinical intervention, chronic renal impairment (on dialysis) or unresolved acute kidney injury (AKI), acute or chronic heart failure (New York Heart Association Class III / IV), portal pulmonary hypertension, severe chronic lung disease such as chronic obstructive pulmonary disease (COPD) or interstitial lung disease in which FEV1 is less than 50% and / or FEV1 / FVC less than 60%, hepatopulmonary syndrome, history of or current chronic albumin therapy, major untreated / unstable mental illness, TIPS within the past 6 months; evidence of any life-threatening comorbidities; current or planned use of immunomodulatory or immunosuppressive drugs (low-dose corticosteroids are permitted up to 10 mg / kg / day of prednisone or equivalent, or inhaled steroids to control asthma); prior to any gene or cell therapy; participants with known hypersensitivity to dimethyl sulfoxide (DMSO); female patients only – pregnant or breastfeeding or planning to become pregnant in the following year, or of childbearing potential and unwilling to comply with contraception requirements; current / recent alcohol abuse in the three months prior to treatment, defined as alcohol intake greater than 3 units / day for women and greater than 4 units / day for men, or binge drinking (>14 units / day); intake of non-medically supervised abused substances.

[0300] In another preferred embodiment, the present invention provides engineered macrophages engineered to overexpress IL-10 and optionally further engineered to overexpress MMP9 for use in a method of treating cirrhosis in a subject, wherein the method comprises administering the engineered macrophages to the subject, wherein the subject has been hospitalized following a first hepatic decompensation event and, prior to administering the engineered macrophages to the subject, experienced a subsequent additional decompensation event following the first decompensation event, and the subject, at the start of treatment, has no or is unaware of having and / or is suspected of having any of the following comorbidities or indicators of increased risk: cirrhosis caused by any viral hepatitis other than HCV SVR; cirrhosis caused by autoimmune and cholestatic etiologies, including but not limited to primary biliary cholangitis and primary sclerosing cholangitis; any current organ failure requiring supportive care beyond outpatient care and unrelated to a hepatic decompensation event (a qualifying event); known splenomegaly, optionally wherein the subject's spleen has a maximum size greater than 16 cm. cm; thrombocytopenia, optionally, wherein thrombocytopenia is defined as less than 50 × 10⁻⁶. 9 A platelet count of 1 platelet / L; a history of liver or other organ transplantation, such as having received at least one previous liver or organ transplant; acute-on-chronic liver failure (ACLF); a positive microbial culture or recent sepsis as defined by a physician, unless stable and at least 5 weeks after completion of a full course of IV antibiotics; known infection with human immunodeficiency virus (HIV); pulmonary embolism; hepatocellular carcinoma (HCC) or active malignancy (excluding non-melanoma skin cancer, cervical carcinoma in situ, or superficial bladder cancer) within 5 years prior to the start of treatment; liver complications, such as hepatic pleural effusion (responding to drug treatment). (Therapeutic refractory) or portal vein thrombosis; chronic renal impairment in which the subject may require dialysis; unresolved acute kidney injury (AKI); acute heart failure; chronic heart failure; portal pulmonary hypertension; severe chronic lung disease, such as chronic obstructive pulmonary disease (COPD) or interstitial lung disease, in which forced expiratory volume in one second (FEV1) is less than 50% or 60%, and / or forced vital capacity (FVC) is less than 60%; hepatopulmonary syndrome; history of chronic albumin therapy, or current chronic albumin therapy; and / or major untreated or unstable mental illness.

[0301] In a particularly preferred embodiment, the present invention provides an engineered macrophage, engineered to overexpress IL-10 and optionally further engineered to overexpress MMP9, for the treatment of cirrhosis in a subject, wherein the method comprises administering the engineered macrophage to the subject, wherein the subject has been hospitalized following a first hepatic decompensation event, wherein the subject experienced a subsequent additional decompensation event prior to the administration of the engineered macrophage to the subject, and wherein the subject has none of the aforementioned comorbidities or indicators of increased risk and exhibits all of the aforementioned characteristics indicating that they may benefit from treatment.

[0302] According to a preferred embodiment, the present invention provides an engineered macrophage, engineered to overexpress IL-10 and optionally further engineered to overexpress MMP9, for use in treating cirrhosis in a subject, wherein the method comprises administering the engineered macrophage to the subject, wherein the subject has been hospitalized following their first hepatic decompensation event, wherein the subject has recovered from their first hepatic decompensation event, wherein the subject has not experienced subsequent or additional decompensation events prior to treatment, and wherein the subject possesses all of the following characteristics that indicate a likelihood of benefiting from treatment: the absence of known inconsistencies with G-CSF or apheresis procedures; and age. Age between 17 and 18 years; diagnosed with cirrhosis based on at least one of the following: a. clinical and radiological features associated with the diagnosis of cirrhosis; liver stiffness score >15 kPa measured by transient elastography (TE; Fibroscan); and c. histological features of cirrhosis confirmed by previous liver biopsy; and the following etiologies of liver disease: a. steatotic liver disease of pure metabolic dysfunction (MASLD) or metabolic and alcohol-related / associated liver disease (Met-ALD) or HCV persistent virological response (HCVSVR) with persistent cirrhosis; b. alcohol-related liver disease (ALD or Met-ALD) only if the subject is confirmed to have consumed alcohol not exceeding the Met-ALD limit, defined as an intake > 14 units / week, or more than 2 units / day for women and more than 3 units / day for men (1 unit is equivalent to 8 g of alcohol); c. Alcohol-related liver disease (ALD or Met-ALD), only if the subject's PEth test value is <25 mg / L; MELD 3.0 score of 12-20, within two weeks of an "eligible" decompensated event, or if the participant is eligible for outpatient treatment, their MELD 3.0 score at screening (Visit 1) must be 12-20; hospitalization as an inpatient due to a major hepatic decompensated event, including ascites, hepatic encephalopathy, variceal bleeding, hepatorenal syndrome / acute kidney injury (HRS / AKI) or spontaneous bacterial peritonitis (SBP), which is the only hospitalization for a hepatic decompensated event in the past 6 months, or refractory ascites, defined as ascites that cannot be mobilized or recurs within 8 weeks after LVP (the date of onset of refractory ascites is defined as the time of the second LVP).

[0303] In some implementations, subjects do not possess the following characteristics that indicate contraindication to treatment: cirrhosis caused by any viral hepatitis other than hepatitis C virus (HCV SVR); cirrhosis caused by autoimmune and cholestatic etiologies, including but not limited to primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC); acute liver disease without underlying cirrhosis, including but not limited to drug-induced liver injury (DILI); any current organ failure requiring supportive care beyond outpatient care and unrelated to the participant's liver decompensation event; known splenomegaly ≥16 cm; thrombocytopenia < 50 × 10⁻⁶. 9 / L; any of the following comorbidities are present or suspected: history of liver or other organ transplantation, ACLF, sepsis (positive microbial culture), unless stable and at least 4 weeks after completion of a full course of IV antibiotics, human immunodeficiency virus (HIV), known human immunodeficiency virus, human T-lymphotropic virus 1, pulmonary embolism (PE), hepatocellular carcinoma (HCC) or active malignancy within the past 5 years (excluding non-melanoma skin cancer, cervical carcinoma in situ, superficial bladder cancer, benign polyps, etc.), liver comorbidities such as portal vein thrombosis, hepatic pleural effusion unless it is a small amount of pleural effusion that is clinically inconspicuous and is incidentally detected by radiological evaluation without clinical intervention, chronic renal impairment (on dialysis) or unresolved acute kidney injury (AKI), acute or chronic heart failure (New York Heart Association Class III / IV), portal pulmonary hypertension, severe chronic lung disease such as chronic obstructive pulmonary disease (COPD) or interstitial lung disease in which FEV1 is less than 50% and / or FEV1 / FVC less than 60%, hepatopulmonary syndrome, history of or current chronic albumin therapy, major untreated / unstable mental illness, TIPS within the past 6 months; evidence of any life-threatening comorbidities; current or planned use of immunomodulatory or immunosuppressive drugs (low-dose corticosteroids are permitted up to 10 mg / kg / day of prednisone or equivalent, or inhaled steroids to control asthma); prior to any gene or cell therapy; participants with known hypersensitivity to dimethyl sulfoxide (DMSO); female patients only – pregnant or breastfeeding or planning to become pregnant in the following year, or of childbearing potential and unwilling to comply with contraception requirements; current / recent alcohol abuse in the three months prior to treatment, defined as alcohol intake greater than 3 units / day for women and greater than 4 units / day for men, or binge drinking (>14 units / day); intake of non-medically supervised abused substances.

[0304] According to another preferred embodiment, the present invention provides engineered macrophages engineered to overexpress IL-10 and optionally further engineered to overexpress MMP9 for use in treating cirrhosis in a subject, wherein the method comprises administering the engineered macrophages to the subject, wherein the subject has been hospitalized following a first hepatic decompensation event, wherein the subject has recovered from the first hepatic decompensation event, wherein the subject has not experienced subsequent or additional decompensation events prior to treatment, and wherein the subject has no or is unaware of any of the following comorbidities or indicators of increased risk at the start of treatment: cirrhosis caused by any viral hepatitis other than HCV SVR; cirrhosis caused by autoimmune and cholestatic etiologies, including but not limited to primary biliary cholangitis and primary sclerosing cholangitis; any current organ failure requiring supportive care beyond outpatient care and unrelated to the hepatic decompensation event (a qualifying event); known splenomegaly, optionally wherein the subject's spleen has a maximum size greater than 16 cm; thrombocytopenia, optionally wherein thrombocytopenia is defined as less than 50 × 10⁻⁶ mmol / L. 9 A platelet count of 1 platelet / L; a history of liver or other organ transplantation, such as having received at least one previous liver or organ transplant; acute-on-chronic liver failure (ACLF); a positive microbial culture or recent sepsis as defined by a physician, unless stable and at least 5 weeks after completion of a full course of IV antibiotics; known infection with human immunodeficiency virus (HIV); pulmonary embolism; hepatocellular carcinoma (HCC) or active malignancy (excluding non-melanoma skin cancer, cervical carcinoma in situ, or superficial bladder cancer) within 5 years prior to the start of treatment; liver complications, such as hepatic pleural effusion (responding to drug treatment). (Therapeutic refractory) or portal vein thrombosis; chronic renal impairment in which the subject may require dialysis; unresolved acute kidney injury (AKI); acute heart failure; chronic heart failure; portal pulmonary hypertension; severe chronic lung disease, such as chronic obstructive pulmonary disease (COPD) or interstitial lung disease, in which forced expiratory volume in one second (FEV1) is less than 50% or 60%, and / or forced vital capacity (FVC) is less than 60%; hepatopulmonary syndrome; history of chronic albumin therapy, or current chronic albumin therapy; and / or major untreated or unstable mental illness.

[0305] According to another preferred embodiment, the present invention provides an engineered macrophage, engineered to overexpress IL-10 and optionally further engineered to overexpress MMP9, for the treatment of cirrhosis in a subject, wherein the method comprises administering the engineered macrophage to the subject, wherein the subject has been hospitalized following a first hepatic decompensation event, wherein the subject has recovered from the first hepatic decompensation event (or is clinically stable after the first hepatic decompensation event) prior to the administration of the engineered macrophage, wherein the subject has not experienced subsequent or additional decompensation events prior to treatment, and the subject has no of the aforementioned comorbidities or indicators of increased risk, and exhibits all of the aforementioned characteristics indicating that they may benefit from the treatment.

[0306] In a particularly preferred embodiment, the present invention provides an engineered macrophage, engineered to overexpress IL-10 and optionally further engineered to overexpress MMP9, for the treatment of cirrhosis in a subject, wherein the method comprises administering the engineered macrophage to the subject, wherein the subject suffers from refractory ascites, and wherein the subject has all of the following characteristics that indicate a potential benefit from treatment: no known inconsistencies with G-CSF or leukapheresis procedures; age between 18 and 17 years; a diagnosis of cirrhosis based on at least one of the following: a. clinical and radiological features associated with the diagnosis of cirrhosis; a liver stiffness score >15 kPa measured by transient elastography (TE; Fibroscan); and c. histological features of cirrhosis confirmed by previous liver biopsy; and the following etiologies of liver disease: a. steatotic liver disease of pure metabolic dysfunction (MASLD) or metabolic and alcohol-related / associated liver disease (Met-ALD) or persistent virological response to HCV with persistent cirrhosis (HCV). a. SVR); b. Alcohol-related liver disease (ALD or Met-ALD), only if the subject is confirmed to have consumed alcohol within the Met-ALD limit, defined as an intake of >14 units / week, or more than 2 units / day for women and more than 3 units / day for men (1 unit is equivalent to 8 g of alcohol); c. Alcohol-related liver disease (ALD or Met-ALD), only if the subject's PEth test value is <25 mg / L; MELD 3.0 score of 12-20, within two weeks of an "eligible" decompensated event, or if the participant is eligible for outpatient status, their MELD at screening (Visit 1). A score of 3.0 must be between 12 and 20; hospitalization for a major hepatic decompensation event, including ascites, hepatic encephalopathy, variceal bleeding, hepatorenal syndrome / acute kidney injury (HRS / AKI) or spontaneous bacterial peritonitis (SBP), which is the only hospitalization for a hepatic decompensation event in the past 6 months, or refractory ascites, defined as ascites that cannot be mobilized or recurs within 8 weeks after LVP (the date of onset of refractory ascites is defined as the time of the second LVP).

[0307] In some implementations, subjects do not possess the following characteristics that indicate contraindication to treatment: cirrhosis caused by any viral hepatitis other than hepatitis C virus (HCV SVR); cirrhosis caused by autoimmune and cholestatic etiologies, including but not limited to primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC); acute liver disease without underlying cirrhosis, including but not limited to drug-induced liver injury (DILI); any current organ failure requiring supportive care beyond outpatient care and unrelated to the participant's liver decompensation event; known splenomegaly ≥16 cm; thrombocytopenia < 50 × 10⁻⁶.9 / L; any of the following comorbidities are present or suspected: history of liver or other organ transplantation, ACLF, sepsis (positive microbial culture), unless stable and at least 4 weeks after completion of a full course of IV antibiotics, human immunodeficiency virus (HIV), known human immunodeficiency virus, human T-lymphotropic virus 1, pulmonary embolism (PE), hepatocellular carcinoma (HCC) or active malignancy within the past 5 years (excluding non-melanoma skin cancer, cervical carcinoma in situ, superficial bladder cancer, benign polyps, etc.), liver comorbidities such as portal vein thrombosis, hepatic pleural effusion unless it is a small amount of pleural effusion that is clinically inconspicuous and is incidentally detected by radiological evaluation without clinical intervention, chronic renal impairment (on dialysis) or unresolved acute kidney injury (AKI), acute or chronic heart failure (New York Heart Association Class III / IV), portal pulmonary hypertension, severe chronic lung disease such as chronic obstructive pulmonary disease (COPD) or interstitial lung disease in which FEV1 is less than 50% and / or FEV1 / FVC less than 60%, hepatopulmonary syndrome, history of or current chronic albumin therapy, major untreated / unstable mental illness, TIPS within the past 6 months; evidence of any life-threatening comorbidities; current or planned use of immunomodulatory or immunosuppressive drugs (low-dose corticosteroids are permitted up to 10 mg / kg / day of prednisone or equivalent, or inhaled steroids to control asthma); prior to any gene or cell therapy; participants with known hypersensitivity to dimethyl sulfoxide (DMSO); female patients only – pregnant or breastfeeding or planning to become pregnant in the following year, or of childbearing potential and unwilling to comply with contraception requirements; current / recent alcohol abuse in the three months prior to treatment, defined as alcohol intake greater than 3 units / day for women and greater than 4 units / day for men, or binge drinking (>14 units / day); intake of non-medically supervised abused substances.

[0308] In another preferred embodiment, the present invention provides engineered macrophages engineered to overexpress IL-10 and optionally further engineered to overexpress MMP9 for use in a method of treating cirrhosis in a subject, wherein the method comprises administering the engineered macrophages to the subject, wherein the subject has refractory ascites and the subject, at the start of treatment, has no or is unaware of having and / or is suspected of having any of the following comorbidities or indicators of increased risk: cirrhosis caused by any viral hepatitis other than HCV SVR; cirrhosis caused by autoimmune and cholestatic etiologies, including but not limited to primary biliary cholangitis and primary sclerosing cholangitis; any current organ failure requiring supportive care beyond outpatient care and unrelated to a liver decompensation event (a qualifying event); known splenomegaly, optionally wherein the subject's spleen has a maximum size greater than 16 cm; thrombocytopenia, optionally wherein thrombocytopenia is defined as less than 50 × 10⁻⁶ mmol / L. 9 A platelet count of 1 platelet / L; a history of liver or other organ transplantation, such as having received at least one previous liver or organ transplant; acute-on-chronic liver failure (ACLF); a positive microbial culture or recent sepsis as defined by a physician, unless stable and at least 5 weeks after completion of a full course of IV antibiotics; known infection with human immunodeficiency virus (HIV); pulmonary embolism; hepatocellular carcinoma (HCC) or active malignancy (excluding non-melanoma skin cancer, cervical carcinoma in situ, or superficial bladder cancer) within 5 years prior to the start of treatment; liver complications, such as hepatic pleural effusion (responding to drug treatment). (Therapeutic refractory) or portal vein thrombosis; chronic renal impairment in which the subject may require dialysis; unresolved acute kidney injury (AKI); acute heart failure; chronic heart failure; portal pulmonary hypertension; severe chronic lung disease, such as chronic obstructive pulmonary disease (COPD) or interstitial lung disease, in which forced expiratory volume in one second (FEV1) is less than 50% or 60%, and / or forced vital capacity (FVC) is less than 60%; hepatopulmonary syndrome; history of chronic albumin therapy, or current chronic albumin therapy; and / or major untreated or unstable mental illness.

[0309] In a particularly preferred embodiment, the present invention provides an engineered macrophage, engineered to overexpress IL-10 and optionally further engineered to overexpress MMP9, for use in treating a subject with cirrhosis, wherein the method comprises administering the engineered macrophage to the subject, wherein the subject has refractory ascites, and wherein the subject has none of the aforementioned comorbidities or indicators of increased risk, and exhibits all of the aforementioned characteristics indicating that they may benefit from the treatment.

[0310] According to a preferred embodiment, the present invention provides engineered macrophages engineered to overexpress IL-10 for the treatment of cirrhosis in a subject hospitalized after a first hepatic decompensation event. In a preferred embodiment, the present invention provides engineered macrophages engineered to overexpress IL-10 for the treatment of cirrhosis in a subject hospitalized after a first hepatic decompensation event, wherein the subject is treated with the engineered macrophages once the subject has recovered from their first hepatic decompensation event and before experiencing an additional hepatic decompensation event. In a particularly preferred embodiment, the present invention provides engineered macrophages engineered to overexpress IL-10 and MMP9 for the treatment of cirrhosis in a subject hospitalized after a first hepatic decompensation event, wherein the subject is treated with the engineered macrophages once the subject has recovered from their first hepatic decompensation event and before experiencing an additional hepatic decompensation event.

[0311] According to a further preferred embodiment, the present invention provides engineered macrophages derived from iPSCs and engineered to overexpress IL-10. Preferably, the engineered iPSC-derived macrophages are used to treat cirrhosis, particularly in subjects hospitalized after a first hepatic decompensation event. In a particularly preferred embodiment, the engineered iPSC-derived macrophages are used to treat cirrhosis, particularly in subjects hospitalized after a first hepatic decompensation event, wherein the subject is treated with the engineered macrophages once the subject has recovered from their first hepatic decompensation event and before experiencing an additional hepatic decompensation event.

[0312] According to a further preferred embodiment, the present invention provides engineered macrophages engineered to overexpress IL-10 and MMP9, wherein the macrophages comprise exogenous mRNA encoding IL-10 and MMP9, optionally separated by a cleavable adapter. Preferably, the exogenous mRNA comprises the sequence shown in SEQ ID NO: 10, a polyA tail optionally 65-250 residues long, preferably 90-120 residues long, and a 5' cap. In another preferred embodiment, the exogenous mRNA sequence comprises the sequence shown in SEQ ID NO: 16 and a 5' cap. Preferably, the engineered macrophages are used to treat cirrhosis, particularly cirrhosis in subjects hospitalized after a first hepatic decompensation event. In a particularly preferred embodiment, the engineered macrophages are used to treat cirrhosis, particularly cirrhosis in subjects hospitalized after a first hepatic decompensation event, wherein the subject is treated with the engineered macrophages once the subject has recovered from their first hepatic decompensation event and before experiencing an additional hepatic decompensation event.

[0313] According to a further preferred embodiment, the present invention provides engineered macrophages derived from iPSCs and engineered to overexpress IL-10 and MMP9, wherein the macrophages contain exogenous mRNA encoding IL-10 and MMP9, optionally separated by a cleavable adapter. Preferably, the exogenous mRNA contains the sequence shown in SEQ ID NO: 10, optionally a polyA tail of 65-250 residues long, preferably 90-120 residues long, preferably about 90 residues long, and / or a 5' cap. In another preferred embodiment, the exogenous mRNA sequence contains the sequence shown in SEQ ID NO: 16 and a 5' cap. Preferably, the iPSC-derived engineered macrophages are used to treat cirrhosis, particularly cirrhosis in subjects hospitalized after a first hepatic decompensation event. In a particularly preferred embodiment, the iPSC-derived engineered macrophages are used to treat cirrhosis, particularly cirrhosis in subjects who have been hospitalized following a first hepatic decompensation event, wherein the subject is treated with the engineered macrophages once the subject has recovered from their first hepatic decompensation event and before experiencing an additional hepatic decompensation event.

[0314] According to a further preferred embodiment, the present invention provides engineered macrophages that are engineered to overexpress IL-10, wherein when expressed at a concentration of 4 x 102... 6 When cultured in vitro at a cell concentration of 4 x 10⁶ pg / ml, the macrophages secrete IL-10 at a culture supernatant concentration of at least 10,000 pg / ml, in a method for treating cirrhosis, particularly in subjects hospitalized after a first hepatic decompensation event. In a particularly preferred embodiment, the invention provides engineered macrophages engineered to overexpress IL-10, wherein when cultured at a ... 6 When cultured in vitro at a cell concentration of at least 10,000 pg / ml, the macrophages secrete IL-10 at a culture supernatant concentration of at least 10,000 pg / ml. This is used in a method of treating cirrhosis, particularly in subjects hospitalized after a first hepatic decompensation event, wherein the engineered macrophages are used to treat the subject once the subject has recovered from their first hepatic decompensation event and before experiencing an additional hepatic decompensation event.

[0315] According to a further preferred embodiment, the present invention provides an engineered macrophage, which is engineered to overexpress IL-10 and MMP9, wherein the engineered macrophage: has a metalloproteinase activity level at least 1.5 times that of unengineered macrophages; CD86 expression is at least two-fold lower than that of unengineered, non-polarized cells; HLA-DR expression is at least two-fold lower than that of unengineered, non-polarized cells; IL-10 secretion is at least 1000-fold higher than that of unengineered, non-polarized cells; MMP3 secretion is at least 10-fold higher than that of unengineered, non-polarized cells; MMP10 secretion is at least 20-fold higher than that of unengineered, non-polarized cells; CD206 expression is at least 5-fold higher than that of monocytes; 25F9 expression is at least 5-fold higher than that of monocytes; CD80 expression is at least 10 percent lower than that of unengineered, non-polarized cells; when expressed at 4 x 10-10... 6 When cultured in vitro at a cell concentration of 1000 pg / ml, IL-10 is secreted at a concentration of at least 10,000 pg / ml of culture supernatant; when cultured at a concentration of 4 x 10⁶ pg / ml, IL-10 is secreted. 6 When cultured in vitro at a cell concentration of / ml, MMP9 is secreted at a concentration of at least 200 ng / ml of culture supernatant; and when cultured at 4 x 10 6 When cultured in vitro at a cell concentration of / ml, TNF-α is secreted at a culture supernatant concentration of up to 40 pg / ml. In a particularly preferred embodiment, the engineered macrophages are derived from iPSCs, and the macrophages contain exogenous mRNA encoding IL-10 and MMP9, optionally separated by a cleavable adapter. Preferably, the iPSC-derived engineered macrophages are used to treat cirrhosis, particularly cirrhosis in subjects hospitalized following a first hepatic decompensation event. The invention will now be further described with reference to the following heading sections. Any feature under any section may be combined with any aspect or embodiment of the invention in any feasible order.

[0316] illustrate

[0317] The following definitions are provided.

[0318] As used herein, a “payload” is at least one nucleic acid encoding a therapeutically targeted gene (such as, but not limited to, DNA or RNA) that is introduced into macrophages, optionally, via transfection.

[0319] As used in this article, “non-polarized macrophage” refers to a mature macrophage that has not received any further stimulation to induce specific functional capabilities. Non-polarized macrophage can also refer to naïve or unactivated macrophages.

[0320] "Mature macrophages" refer to macrophages that express mature cell surface markers, preferably CCR2-, CD14+, and 25F9+.

[0321] Macrophages can acquire various states, known as “polarization,” which are generally, but simply, divided into two main extremes: “pro-inflammatory” (or classically activated, “M1”, “M1-like”) and “pro-regenerative” (or “pro-repair”, alternatingly activated, “M2”, “M2-like”, anti-inflammatory or anti-fibrotic). However, macrophages may adopt a state between these two extremes, which may be “unpolarized,” quiescent or primitive (M0), or more inclined to an anti-inflammatory or pro-inflammatory state.

[0322] M1 macrophages are generally considered pro-inflammatory, while M2 macrophages are responsible for 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 employ different phenotypes and even switch between them in response to a multitude of stimuli they are exposed to. These in vivo macrophage phenotypes cannot be precisely reproduced in tissue culture models, highlighting the importance of characterizing macrophages on a functional basis. Macrophages acquire a "pro-regenerative" state under the combined action of various factors, including macrophage colony-stimulating factor (M-CSF), IL-4, IL-13, IL-10, and TGF-β. These macrophages primarily mediate wound healing and tissue regeneration.

[0323] Due to the complexity of macrophage biology, the classification of "M1" and "M2" can be considered overly simplistic. For example, "M2" macrophages are actually considered to be a range dependent on their environment. Therefore, attempts have been made to further subdivide the regenerative M2-like states, such as the following subclassification proposed by Gharavi, AT et al., "The role of macrophage subtypes and exosomes in immunomodulation", Cell Mol Biol Lett 27, 83 (2022). In this subclassification, for example:

[0324] “M2a” cells are considered to be anti-inflammatory, pro-fibrotic, and play a role in allergies and wound healing. These cells are classified by the expression of IL-10, IL-1R, IL-27a, CCL1, CCL17, CCL18, CCL22, CD11b, CD45, CD206, YM1, RELMa, IGF1, DCIR, stable protein 1, factor XIII-A, Ly6C, TREM-2, and DC-SIGN. The M2a state may be acquired under the influence of factors such as IL-4, IL-13, IL-10, and PPARγ.

[0325] “M2b” cells are believed to be involved in the activation of helper T cell type 2 (Th2) responses, immune regulation, and the promotion of tumor progression. These cells are classified by the expression of IL-6, TNF-α, CD86, and SPHK1. M2b status may be acquired under the influence of IL-1β or through exposure to LPS.

[0326] “M2c” cells are involved in immunosuppression, phagocytosis, tissue repair, and extracellular matrix remodeling. These cells are classified by the expression of IL-10, CXCL13, CD163, CD206, CXCR4, TGF-β, and MerTK. M2c status may be acquired under the stimulation of IL-10, glucocorticoids, IL-6, IL-10, TNF-α, and TLR.

[0327] “M2d” cells are involved in tumor progression, angiogenesis, and clearance of apoptotic tissue. These cells are classified by the expression of IL-10, VEGF, and TGF-β. The M2d state may be acquired upon exposure to LPS.

[0328] Among the biomarkers associated with each of these pro-repair states, the engineered macrophages of this invention typically express IL-10, CCL22, CD11b, CD45, CD206, CD86, CD163, and CXCR4. The engineered macrophages of this invention also typically express MHC II, which is more generally associated with pro-inflammatory macrophages. Therefore, it can be seen that the engineered macrophages of this invention differ from the pro-repair, anti-inflammatory macrophages described in the literature because they express biomarkers associated with multiple subtypes, such as both M2a and M2c-like cells. However, in all aspects of this invention, the engineered macrophages of this invention express biomarkers consistent with those of functional human monocyte-derived macrophages, such as CD45, CD14, CD206, CCR2, CD163, CD169, and 25F9.

[0329] The engineered macrophages of this invention (containing IL-10 and MMP9) can function as “pro-repair” macrophages. For example, as described in Ramachandran et al, Proc Natl Acad Sci USA. 2012 Nov 13; 109(46):E3186–E3195, pro-repair macrophages exhibit loss of pro-inflammatory gene expression, increased expression of matrix-degrading enzymes, and enrichment of phagocytosis-related genes. Furthermore, the phenotype of pro-repair macrophages does not belong to the M1 / M2 paradigm, highlighting the limitations of this classification in the in vivo environment. Pro-repair macrophages play an important role in tissue remodeling, such as fibrosis regression.

[0330] Ramachandran et al. reported that macrophages possess high MMP activity. Furthermore, in Figure 5 In this study, the authors show several genes regulated in pro-repair macrophages. Notably, they observe upregulation of Mrc1 (now CD206), which is also expressed in the macrophages of the examples described herein. Furthermore, they exhibit low levels of TNFα and IL1β, which is also confirmed in the examples given herein. Therefore, this could be a marker of a pro-repair phenotype. However, these macrophages described herein naturally express low levels of IL-10.

[0331] As used herein, “GMP compliant” means that the method described conforms to good manufacturing practice principles and is interchangeable with “GMP compatible” and “GMP grade”. For example, GMP compliant culture media must be free of serum, antibiotics, animal materials, and foreign proteins. The WHO provides guidance on good manufacturing practice requirements: “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 (Second Update). WHO Press. pp. 17–18. ISBN 9789241547086.

[0332] As used herein, “UT,” “NTRx,” or “UT N / T” refers to untransfected macrophages that differentiate from monocytes in the same process used to differentiate transfected macrophages, but without any further incubation and / or transfection with additional factors. These macrophages are similar to the non-polarized, untransfected cells used in the MATCH study described in WO2019175595 (therefore, UT can also be described as MATCH-like cells in this application); however, the macrophages in the MATCH study matured for 7 days, while the UT macrophages in this application matured for 5 days.

[0333] As used herein, “UT + TR,” “NTRx + Tr,” or “UT T” refers to untransfected cells (as described above) that have undergone treatment similar to that of transfected cells (i.e., incubation with IL-4+IL-13+M-CSF after mock transfection). Incubation with IL-4+IL-13+M-CSF is described in more detail in the examples herein.

[0334] This article also describes hMDM cells transfected with constructs encoding specific products, such as MMP9 and / or IL-10, which were not treated with IL-4+IL-13+M-CSF as described herein. These cells are referred to as “TRx”, for example, cells transfected with IL-10 but not incubated with IL-4+IL-13+M-CSF are denoted as “IL-10 TRx”.

[0335] This article also describes hMDM transfected with a construct encoding a specific product, and said hMDM was further treated with IL-4+IL-13+M-CSF. Such cells are denoted as “TRx+TR”, such as “IL-10 TRx+TR”. This article also describes hMDM transfected with an exogenous bicistronic mRNA construct expressing IL-10 and MMP9. Such cells are denoted as “RTX001”. Without being bound by theory or mechanism, overexpression of IL-10 and MMP9 implies that RTX001 cells can self-polarize to an anti-inflammatory and / or pro-repair phenotype.

[0336] It should be noted that the term "a" or "an" entity refers to one or more of the entities in question.

[0337] “Approximately” means + / - 10%, + / - 9%, + / - 8%, + / - 7%, + / - 6%, + / - 5%, + / - 4%, + / - 3%, + / - 2%, + / - 1% of a given value, unless otherwise stated.

[0338] Engineered macrophages used in cell therapy

[0339] In a preferred embodiment, the present invention relates to an engineered macrophage, wherein the macrophage comprises one or more exogenous coding sequences of IL-10 and MMP9.

[0340] This invention relates to an engineered macrophage, wherein the macrophage is engineered to express IL-10 and MMP9. The macrophage can be engineered to express these proteins by including exogenous coding sequences. The exogenous coding sequences can be extrachromosomal or integrated into the cell's genome. The engineered macrophage expresses exogenous coding sequences for IL-10 and MMP9.

[0341] In some embodiments, the engineered macrophages carry one or more exogenous sequences that can activate endogenous expression of IL-10 and / or MMP9. Any macrophage genetically modified in any way by an exogenous sequence (i.e., a sequence that is not part of the natural macrophage genome) is an engineered macrophage according to the present invention.

[0342] The engineered macrophages can be used in therapies, such as to treat inflammatory and / or fibrotic conditions in subjects.

[0343] Preferably, the administration of engineered macrophages to the subject is not related to the inflammatory response. In particular, the administration of engineered macrophages to the subject is preferably not related to an increase in the concentration of inflammatory cytokines such as IL-1β and / or TNF-α in plasma.

[0344] As used herein, a "macrophage" refers to a phagocytic cell that is responsible for detecting, engulfing, and destroying pathogens and apoptotic cells and is produced through the differentiation of monocytes. The "engineered macrophage" of this invention is a macrophage engineered to express IL-10 and / or MMP9. Specifically, expression is higher than endogenous levels, such that the engineered macrophage expresses IL-10 and / or MMP9 at higher levels than unengineered cells. The macrophage can be engineered to express these proteins by including exogenous coding sequences. In some embodiments, when expressed at 4x10... 6 When cultured in vitro at a cell concentration of 4 x 10 / ml, the macrophages secrete IL-10 at a concentration of at least 10,000 pg / ml of culture supernatant. In some embodiments, when cultured at 4 x 10 / ml... 6 When cultured in vitro at a cell concentration of 200 ng / ml, the macrophages secrete MMP9 at a concentration of at least 200 ng / ml of culture supernatant.

[0345] In some embodiments, the IL-10 amino acid sequence is encoded by mRNA containing the sequence SEQ ID NO. 1. The native mRNA sequence of IL-10 is provided at: NCBI Reference Sequence: NM_000572.3. Variants and homologs of this sequence are also included.

[0346] MMP9 (matrix metallopeptidase 9) is a matrix metalloproteinase, a type IV collagenase. MMP9 is also known as 92 kDa type IV collagenase, 92 kDa gelatinase, or gelatinase B (GELB). Matrix metalloproteinases (MMPs), also called matrix metalloproteinases or matrixins, are a family of peptidases that collectively cleave all components of the extracellular matrix (ECM). MMPs also process biologically active mediators such as growth factors, cytokines, chemokines, and cell surface receptors. Twenty-five mammalian MMPs have been identified, each playing a different role in the maintenance of the extracellular matrix and tissue repair, and exhibiting both inhibitory and stimulatory effects in fibrosis.

[0347] In some embodiments, the MMP9 coding sequence is human MMP9, as described in NCBI reference sequence: NM_004994.3. In some embodiments, the amino acid sequence is encoded by mRNA containing the sequence of SEQ ID NO. 3 or a variant that is at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, or 99% similar to SEQ ID No. 3.

[0348] In some embodiments, the mRNA is modified. In some embodiments, the mRNA modification is selected from chemical modifications of uridine and / or cytidine. Pseudouridine. In some embodiments, the mRNA modification includes pseudouridine, N1-methylpseudouridine, 5-methoxy-uridine, 5-methyl-cytidine, preferably 5-methoxy-uridine. In a preferred embodiment, all endogenous uridine is replaced by 5-methoxy-uridine.

[0349] In some embodiments, the mRNA includes a poly-A tail at its 3' end. A suitable poly-A tail is 65 to 250 residues long, preferably 90 to 120 residues long. In a preferred embodiment, the 3' UTR and 5' UTR are modified relative to the endogenous 3' UTR and 5' UTR. In some embodiments, the poly-A tail is 120 residues long. In a preferred embodiment, the poly-A tail is longer than the endogenous poly-A tail. In a particularly preferred embodiment, the poly-A tail is 90 residues long. In a particularly preferred embodiment, the mRNA comprises the sequence of SEQ ID No. 10 or a variant similar to SEQ ID NO: 10 by at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In another preferred embodiment, the exogenous mRNA sequence comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence listed in SEQ ID NO: 16. In a particularly preferred embodiment, the exogenous mRNA sequence comprises the sequence listed in SEQ ID NO: 16. As described herein, the endogenous uridine in SEQ ID NO: 10 and 16 is replaced with 5-methoxy-uridine. In a preferred embodiment, the mRNA comprises the sequence of SEQ ID NO: 10, a 90-residue polyA tail, and a 5' cap. In a preferred embodiment, the mRNA comprises the sequence of SEQ ID NO: 16 and a 5' cap.

[0350] In some embodiments, the mRNA is resistant to degradation. In some embodiments, the mRNA is non-immunogenic.

[0351] In some embodiments, the IL-10 and MMP9 proteins are expressed by different exogenous coding sequences or nucleic acid molecules. In some embodiments, the IL-10 and MMP9 proteins are expressed as fusion proteins encoded by a single mRNA molecule linked by a cleavable linker.

[0352] In some embodiments, the mRNA comprises the sequence of SEQ ID NO. 2 or a variant that is at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, or 99% similar to SEQ ID NO. 2.

[0353] In some embodiments, one or both of the 5' and 3' ends of the mRNA have been modified by any means to improve stability and / or the ability to express the protein encoding.

[0354] In some embodiments, one or more ribonucleotides of the mRNA molecule have been modified. In some embodiments, one or more uracil bases have been modified, preferably with 5-methoxyuridine.

[0355] In some embodiments, one or both caps of the mRNA molecule are synthetic, preferably CleanCap. ® .

[0356] Overexpression is understood as “excessive” or higher levels of gene expression, such as that caused by an increased frequency of gene transcription. Therefore, it can also be considered as expression above wild-type or normal levels. Overexpression can be defined by the amount of protein produced by a cell population, or by a fold increase in expression levels compared to wild-type or normal levels. Expression levels can be described as the amount of protein secreted per unit volume of cell culture. However, increased transcription does not necessarily lead to an increased amount of secreted protein.

[0357] Cells overexpressing IL-10 and / or MMP9 contain coding sequences that express IL-10 and / or MMP9 at higher levels than unengineered cells. As mentioned above, overexpression can be achieved by introducing exogenous nucleic acids such as mRNA encoding IL-10 and / or MMP9, or by genetic modifications that stimulate the expression of IL-10 and / or MMP9 from endogenous coding sequences. Engineered macrophages overexpressing IL-10 and / or MMP9 do not necessarily secrete more IL-10 and / or MMP9 than unengineered macrophages.

[0358] As used herein, overexpression involves artificially expressing the gene at an increased amount relative to the expression level of the unmodified gene (which may be referred to herein as wild-type or native macrophages). As used in the examples, expression levels were quantified between 16 and 24 hours post-transfection. The expression levels described herein are for a concentration of 4 x 10⁻⁶. 6 / ml (equivalent to per cm) 2 2x10 6 The expression is given by a population of macrophages (cells). In a preferred embodiment, overexpression is achieved by introducing exogenous mRNA into macrophages. In an example, macrophages are transfected, separated by centrifugation, resuspended in TexMACs buffer supplemented with IL-3 and IL-14, and incubated at 37°C and 5% CO2. Those skilled in the art will know the equivalent conditions suitable for determining the concentration of secreted proteins. Under these conditions, the following levels were experimentally determined:

[0359] In the implementation, IL-10 overexpression means that the secreted IL-10 protein level is greater than about 300 pg / ml. Suitably, the IL-10 expression level is greater than about: 300 pg / ml, 400 pg / ml, 500 pg / ml, 600 pg / ml, 700 pg / ml, 800 pg / ml, 900 pg / ml, 1,000 pg / ml, 2,000 pg / ml, 3,000 pg / ml, 4,000 pg / ml, 5,000 pg / ml, 6,000 pg / ml, 7,000 pg / ml, 8,000 pg / ml, 9,000 pg / ml, 10,000 pg / ml, or 11,000 pg / ml. In a preferred embodiment, the secreted IL-10 protein level is greater than 10,000 pg / ml.

[0360] In the implementation scheme, “relative overexpression” of IL-10 in IL-10 engineered macrophage cultures means an increase in the level of secreted IL-10 protein in the culture of approximately 100-300 pg / ml, or greater than approximately 300 pg / ml, relative to the average wild-type protein secretion of wild-type macrophage cultures cultured under the same conditions. Suitably, the increase in IL-10 expression level is greater than about: 300 pg / ml or 400 pg / ml or 500 pg / ml or 600 pg / ml or 700 pg / ml or 800 pg / ml or 900 pg / ml or 1,000 pg / ml or 2,000 pg / ml or 3,000 pg / ml or 4,000 pg / ml or 5,000 pg / ml or 6,000 pg / ml or 7,000 pg / ml or 8,000 pg / ml or 9,000 pg / ml or 10,000 pg / ml or 11,000 pg / ml. In a preferred embodiment, the secreted IL-10 protein level is greater than 10,000 pg / ml. Suitably, these IL-10 protein levels can be measured by culturing macrophages as described above, wherein the concentration of macrophages in the culture medium is 4 x 10⁻⁶. 6 cells / ml, equivalent to 2 x 10 6 cells / cm 2 And measure the protein concentration in the culture medium. Therefore, when using 4x10 6 When cultured in vitro at a cell concentration of at least 10,000 pg / ml, the macrophages secrete IL-10 at a culture supernatant concentration of at least 10,000 pg / ml. In some embodiments, the engineered macrophages may secrete IL-10 at levels up to 1000 times higher than unengineered, non-polarized hMDM. Unengineered, non-polarized hMDM is described in the art, for example, in WO2019175595.

[0361] In the embodiments, MMP9 expression means that the level of secreted MMP9 protein is between about 200 ng / ml and 2000 ng / ml. Suitably, the level of secreted MMP9 protein is greater than about: 300 ng / ml or 400 ng / ml or 500 ng / ml or 600 ng / ml or 700 ng / ml or 800 ng / ml or 900 ng / ml or 1000 ng / ml. Suitably, the level of secreted MMP9 protein is between about 200 ng / ml and 2000 ng / ml. In a preferred embodiment, the level of secreted MMP9 is greater than 200 ng / ml. Suitably, the level of MMP9 protein secreted by the engineered macrophages (containing IL-10 and MMP9) is higher than the average level of MMP9 protein secreted by macrophages engineered with IL-10 alone. Suitably, the total MMP activity of the engineered macrophages of the present invention is also higher than the total MMP activity of macrophages engineered with IL-10 alone. In a preferred embodiment, the total MMP activity of the engineered macrophages is at least 1.5 times higher than that of untransfected macrophages.

[0362] In the implementation scheme, "relative overexpression of MMP9" in MMP9-engineered macrophage cultures, relative to the average wild-type protein expression of wild-type macrophage cultures cultured under the same conditions, means an increase in the secreted MMP9 protein level in the culture of approximately 200 ng / ml to 2000 ng / ml. Suitably, the increase in the secreted MMP9 protein level relative to the average wild-type protein expression of wild-type macrophage cultures cultured under the same conditions is greater than approximately: 300 ng / ml, 400 ng / ml, 500 ng / ml, 600 ng / ml, 700 ng / ml, 800 ng / ml, 900 ng / ml, or 1000 ng / ml. Suitably, the increase in the secreted MMP9 protein level in the culture of the culture of approximately 200 ng / ml to 2000 ng / ml, relative to the average wild-type protein expression of wild-type macrophage cultures cultured under the same conditions. Suitably, these MMP9 protein levels can be measured by culturing macrophages as described above, wherein the concentration of macrophages in the culture medium is 4 x 10⁻⁶. 6 cells / ml, equivalent to 2 x 10 6 cells / cm 2 And measure the protein concentration in the culture medium. Therefore, when using 4x10 6When cultured in vitro at a cell concentration of at least 200 ng / ml, the macrophages secrete MMP9 at a culture supernatant concentration of at least 200 ng / ml. In a preferred embodiment, the secreted MMP9 level is greater than 200 ng / ml. Suitably, the increase in secreted MMP9 levels in IL-10-MMP9 engineered macrophage cultures (i.e., containing IL-10 and MMP9) relative to the average wild-type protein expression of wild-type macrophage cultures cultured under the same conditions is greater than the average increase in MMP9 protein secreted in cultures of macrophages engineered with MMP9 alone. Suitably, the total MMP activity of the engineered macrophages of the present invention is also higher than the total MMP activity of macrophages engineered with IL-10 alone. In some embodiments, these increases are synergistic. In a preferred embodiment, the total MMP activity of the engineered macrophages is at least 1.5 times higher than that of untransfected macrophages.

[0363] In the implementation scheme, "relatively low expression of MMP9" in IL-10 engineered macrophage cultures, relative to the average wild-type protein expression of wild-type macrophage cultures cultured under the same conditions, means a reduction in secreted MMP9 protein levels of approximately 50 ng / ml to 100 ng / ml, approximately 100 ng / ml to 200 ng / ml, or approximately 200 ng / ml to 2000 ng / ml in the culture. Suitably, the reduction in secreted MMP9 protein levels relative to the average wild-type protein expression of wild-type macrophage cultures cultured under the same conditions is greater than approximately: 100 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 600 ng / ml, 700 ng / ml, 800 ng / ml, 900 ng / ml, or 1000 ng / ml. Appropriately, the level of secreted MMP9 protein in the culture was reduced by approximately 200 ng / ml to 500 ng / ml compared to the average wild-type protein expression in wild-type macrophage cultures cultured under the same conditions.

[0364] The engineered macrophages of this invention (containing IL-10 and MMP9) secrete higher levels of MMP9 than macrophages engineered with IL-10 alone (see: Figure 1 (Comparison of IL-10-MMP9 Trx and IL-10 Trx). In one embodiment, the macrophages are engineered with at least one DNA vector encoding IL-10 and / or MMP9. It should be understood that the DNA vector may require one or more auxiliary sequences, such as promoters, terminators, poly(A) signaling sequences, etc.

[0365] A promoter is a nucleotide sequence that initiates and regulates polynucleotide transcription. Promoters can include inducible promoters (where the expression of a polynucleotide sequence operatively linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressive promoters (where the expression of a polynucleotide sequence operatively linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. The term "promoter" or "control element" is intended to include both the full-length promoter region and the functional (e.g., controlling transcription or translation) segments of these regions.

[0366] In one embodiment, the DNA vector may include one or more liver-specific promoters or cirrhosis-specific promoters. In some embodiments, the DNA vector may include the CX3CR1 promoter, insulin-like growth factor 1 (IGF1), or CD1 IB promoter.

[0367] "Operationally linked" refers to an arrangement of elements in which the components, as described so far, are configured to perform their usual functions. Thus, a given promoter operably linked to a nucleic acid sequence can influence the expression of that sequence when a suitable enzyme is present. The promoter need not be adjacent to the sequence, as long as it directs its expression. Therefore, for example, an inserted, untranslated but transcribed sequence can exist between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered "operationally linked" to the coding sequence. Therefore, the term "operationally linked" is intended to include any spacing or location between the promoter element and the DNA sequence of interest, which allows transcription of the DNA sequence of interest to be initiated when the transcription complex recognizes the promoter element.

[0368] In this invention, “treatment” refers to an intervention in a physiological condition that prevents, alleviates, or eliminates clinical symptoms associated with a given physiological condition of the subject.

[0369] The "therapeutic effective amount" of macrophages described in this specification is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from disease onset or promotes disease remission, demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods, or prevention of damage or disability caused by disease suffering. The ability of a therapeutic agent to promote disease remission can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predicting human efficacy, or by determining the activity of the therapeutic agent in an in vitro assay. Therapeutic effective amounts and dosage regimens can be empirically determined by testing in known in vitro or in vivo (e.g., animal model) systems.

[0370] "Subject," "individual," "animal," or "patient" means any subject who requires diagnosis, prognosis, or treatment, especially a mammalian subject, unless the subject is defined as a "healthy subject." Mammal subjects include humans; livestock; farm animals; such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, dairy cows, etc.

[0371] Suitablely, the subject may require treatment. Therefore, suitablely, the subject may have a disease, condition, or symptom, or be at risk of developing a disease, condition, or symptom. Suitablely, the subject may exhibit one or more symptoms of a disease, condition, or symptom.

[0372] The engineered macrophages of this invention can be used in therapy. The macrophages of this invention can be used to treat inflammatory and / or fibrotic conditions in subjects. As described above, treatment here can refer to the prevention, reduction, or elimination of inflammation / fibrosis / organ damage. For example, engineered macrophages can be administered to subjects during the acute inflammatory phase to prevent chronic inflammatory conditions. Engineered macrophages can also be administered to subjects in the chronic inflammatory phase to prevent / reduce chronic fibrosis. Engineered macrophages can also be administered to subjects experiencing a slow-on-acute inflammatory state, such as slow-on-acute liver failure (ACLF).

[0373] Suitablely, acute illness or injury can be classified as illness or injury with an onset time of less than 24 weeks. Suitablely, chronic illness can be classified as illness or injury lasting more than 6 months. Suitablely, subacute-on-chronic illness can be classified as illness or injury with an onset time of less than 24 weeks from the cause in patients with a chronic illness lasting more than 6 months. Suitablely, the engineered macrophages of the present invention can be administered to subjects with acute exacerbations to prevent or increase the transition to chronic inflammation and fibrosis.

[0374] Fibrosis refers to the deposition of extracellular matrix and connective tissue following tissue injury, which may lead to replacement of the solid tissue and, if excessive, eventually result in scarring. In some implementations, the condition is fibrosis.

[0375] In some implementations, the condition is present in or affects organs selected from the group consisting of: liver, lungs, heart, kidneys, pancreas, skin, gastrointestinal tract, bone marrow, hematopoietic tissue, nervous system, eyes, or combinations thereof.

[0376] Suitablely, the condition is chronic organ damage associated with chronic inflammation. Suitablely, the condition involves the kidneys, liver, or lungs. For example, the condition could be inflammatory liver injury, inflammatory kidney injury, or inflammatory lung injury.

[0377] Suitablely, the present invention relates to cell therapy products for inflammatory organ injury, which are based on monocyte-derived macrophages genetically modified with a payload to induce a repair-promoting phenotype.

[0378] Suitablely, the engineered macrophages have a pro-repair phenotype and are anti-inflammatory and anti-fibrotic.

[0379] The M2-like phenotype is pro-repair, while the M1-like phenotype is pro-inflammatory.

[0380] The engineered macrophages include exogenous coding sequences for IL-10 and MMP9. These exogenous coding sequences can be provided in any suitable manner. This can be in the form of a nucleic acid vector, delivered as is, or genetically modified. For example, using viral or non-viral vectors, DNA or RNA constructs, or through gene editing using any suitable technology.

[0381] Macrophages engineered to express IL-10 and MMP9 can be genetically engineered in any suitable manner. For example, using viral or non-viral vectors, DNA or RNA constructs, or through gene editing using any suitable technology.

[0382] The macrophages can be virally engineered. In this genetic engineering approach, a virus, such as a lentivirus, adenovirus, or AAV, is used to introduce a foreign coding sequence / payload into the macrophages. The virus can provide the foreign coding sequence / payload as an "extrachromosomal" construct, or the gene can be integrated into the macrophage genome. As described in the art, viral engineering of macrophages requires careful techniques to prevent the macrophages from phagocytosing the virus.

[0383] Suitablely, the engineered macrophages are non-viral engineered, for example, using a nucleic acid vector comprising a foreign coding sequence. The nucleic acid can be any suitable nucleic acid, including DNA and RNA. Suitablely, the macrophages are transfected with a DNA vector. Suitablely, the DNA vector is a naked DNA vector, such that it does not bind to proteins and / or lipids. Suitablely, the DNA vector is not derived from a viral genome. Optionally, the DNA vector is a non-integrating vector, so that it can function without integrating into the chromosome of the macrophage. Alternatively, the nucleic acid vector is a messenger RNA (mRNA) molecule.

[0384] Suitable, the DNA vector contains at least one sequence encoding IL-10 and / or MMP9, which is operatively linked to a promoter.

[0385] As used in this article, “encoding” refers to the ability of a specific nucleotide sequence (such as a gene or mRNA) to serve as a template for the synthesis of macromolecules (such as proteins) in a cell. If the transcription and translation of the mRNA corresponding to a gene produces a protein in the cell, then that gene encodes a protein.

[0386] Suitablely, the engineered macrophage is a genetically engineered macrophage comprising a nucleic acid construct that overexpresses IL-10 and MMP9.

[0387] Suitablely, the mRNA molecule expresses IL-10 and / or MMP9. In other words, the mRNA includes coding sequences for IL-10 and MMP9, which are exogenous to macrophages. The mRNA may be chemically modified. The chemical modification can be any suitable modification, most notably improving the half-life of the mRNA in the cell. Suitable modifications are discussed extensively here.

[0388] Nucleic acid vectors can be introduced into cells using any suitable transfection method, such as, but not limited to, cationic liposome-mediated transfection, lipid transfection, polymer encapsulation, peptide-mediated transfection, or bioballistic particle delivery systems, such as a "gene gun." Suitablely, the macrophages are transfected with nucleic acids via electroporation. Other suitable transfection methods include nuclear transfection.

[0389] Macrophages can be appropriately engineered by altering their genome through gene editing. Gene editing allows for the permanent insertion of exogenous coding sequences. Many gene editing techniques are known, including those that require the use of nucleases. From base editing and lead editing to gene editing, various nucleases are known to be used to modify the genome. Many nucleases are known—such as zinc finger nucleases, TALENs, and guide nucleases. These can be RNA-guided (“RNA-guided nucleases”)—such as the enzymes involved in CRISPR, including but not limited to Cas9, Cas12a, Cas13, Mad7, etc. The macrophages themselves can be gene-edited, or progenitor cells can be gene-edited before transforming into macrophages.

[0390] Appropriately, macrophages may be transfected with one or more free nucleic acids or vectors.

[0391] Suitable, macrophages were transfected with one or more exogenous coding sequences of IL-10 and MMP9.

[0392] Suitablely, the macrophage has at least one exogenous coding sequence for IL-10 and at least one exogenous coding sequence for MMP9.

[0393] Suitable, the macrophages are engineered to overexpress IL-10 and MMP9.

[0394] Suitable, the macrophages are autologous or allogeneic to the subject.

[0395] This invention also relates to populations of engineered macrophages as described herein. These populations can be used for cell therapy.

[0396] Suitablely, the use includes administering an effective amount of engineered macrophages to a subject.

[0397] The present invention also relates to compositions comprising engineered macrophages or populations thereof of the present invention. Suitably, the compositions are pharmaceutical compositions.

[0398] Suitably, the engineered macrophages can be formulated into a pharmaceutical composition. Suitably, the composition is suitable for administration to a subject. Suitably, the composition is a liquid. Suitably, the composition is an infusionable liquid.

[0399] Suitably, the engineered macrophages are used to administer to a subject via any route. Delivery to the subject can be by local or systemic administration. In some embodiments, administration is by, for example, local injection, nebulizer, or systemic injection. Suitably, the engineered macrophages are used to administer to a subject by infusion. Suitably, the engineered macrophages are used to administer to a subject parenterally, and suitably intravenously. Suitably, the engineered macrophages are used to administer to a subject by injection or infusion. Suitably, the engineered macrophages are used to administer to a subject intravenously by infusion.

[0400] The present invention also relates to a method for improving the migration of monocytes to inflammation, comprising using engineered macrophages according to a first aspect of the invention, a population of macrophages according to a second aspect of the invention, or a composition according to a third aspect of the invention.

[0401] Suitablely, the engineered macrophages, engineered macrophages used in therapy, or engineered macrophages used in methods to improve the migration of monocytes to inflammation have a chemical inducing effect on monocytes. The engineered macrophages may have a chemical inducing effect on monocytes in vitro or in vivo. Recruiting host monocytes at sites of inflammation / damage to organs is beneficial for treating inflammatory conditions. In some embodiments, the engineered macrophages have a specific chemical inducing effect on monocytes but not on other immune cell types. In some embodiments, when administered to a subject with fibrosis, such as a subject with a chronic inflammatory condition containing fibrotic components, the engineered macrophages localize to the fibrotic site and / or recruit monocytes to the fibrotic site. As described in the embodiments herein, the presence of engineered macrophages or monocytes at the fibrotic site can be determined by flow cytometry. In some embodiments, the fibrotic site may be the lung or liver.

[0402] Generate engineered macrophages expressing IL-10 and MMP9

[0403] The present invention also relates to a method for generating engineered macrophages expressing IL-10 and MMP9, comprising transiently transfecting macrophages with a combined mRNA construct containing at least one sequence encoding IL-10 and at least one sequence encoding MMP9. In some embodiments, the macrophages are contacted with an anti-inflammatory treatment after transfection. In some embodiments, the macrophages are contacted with IL-4 and IL-13. In other embodiments, the macrophages are contacted with IL-4, IL-13, and M-CSF. Suitably, the engineered macrophages overexpress IL-10 and restore MMP activity due to co-expression with MMP9 (otherwise MMP activity is inhibited by IL-10). Suitably, the engineered macrophages are pro-repair. Suitably, the generated engineered macrophages can be used for cell therapy.

[0404] Notably, engineered macrophages overexpressing IL-10, or IL-10 and MMP9, exhibit a pro-regenerative phenotype and require no further anti-inflammatory treatment after transfection. Suitablely, the resulting engineered macrophages are manufactured according to GMP-compliant standards. Therefore, suitablely, the engineered macrophages and their populations conform to GMP standards.

[0405] In other embodiments, methods for generating engineered macrophages include introducing sequences encoding IL-10 and / or MMP9 into the genome of macrophages.

[0406] In a preferred embodiment, at least 80% of the engineered macrophages produced by this method secrete IL-10.

[0407] This document also provides a method for improving the cryoresilience of macrophages, comprising incubating macrophages in a medium containing IL-4, IL-13, and M-CSF. According to some embodiments, incubation is performed with IL-4, IL-13, and M-CSF prior to cryopreservation. According to some embodiments, IL-4, IL-13, and M-CSF are removed from the medium prior to cryopreservation, such that the cryopreservation medium is free of IL-4, IL-13, and M-CSF. According to some embodiments, this document provides a method for cryopreserving macrophages, comprising incubating macrophages in a medium containing IL-4, IL-13, and M-CSF prior to cryopreservation. According to some embodiments, the method for improving cryoresilience or for cryopreserving macrophages further includes removing IL-4, IL-13, and M-CSF from the medium prior to cryopreservation. As used herein, the term "cryoresilience" (also referred to as "recovery") refers to the survival rate of macrophages after cryopreservation, optionally measured by the percentage of cryopreserved macrophages that survived freezing. In some embodiments, the concentrations of IL-4 and IL-13 in the culture medium are 20 ng / ml, the concentration of M-CSF is 100 ng / ml, and the concentration of the macrophages is 4 x 10⁻⁶. 6 Cells / ml. In some embodiments, the cells are incubated overnight in a medium containing IL-4, IL-13, and M-CSF.

[0408] Macrophages for engineered

[0409] Suitablely, the macrophages used for engineering have been generated from any suitable progenitor cells. Suitablely, the macrophages have been generated in vitro.

[0410] Suitablely, the macrophages used for engineering are monocyte-derived. Suitablely, they are human monocyte-derived macrophages (hMDM). Monocyte-derived refers to macrophages differentiated from monocytes. Monocytes are the natural precursors of macrophages and dendritic cells; they are present in the blood and bone marrow. Suitablely, the macrophages are derived from peripheral blood monocytes; suitablely, the macrophages are peripheral blood monocyte-derived macrophages. Suitablely, the macrophages are human peripheral blood monocyte-derived macrophages. Suitablely, the monocytes are isolated from human subjects.

[0411] Suitablely, the macrophages are derived from monocytes by culturing monocytes (suitably in vitro). Suitablely, the macrophages are derived from monocytes by any suitable culture method.

[0412] Suitably, the macrophages are generated in vitro from monocytes by a culture method lasting 3 to 8 days, optionally 4 to 8 days. Suitably, the macrophages are generated in vitro from monocytes by a culture method lasting 3 to 7 days, particularly 4 to 7 days, or 5 to 7 days. In one embodiment, the macrophages are generated in vitro from monocytes by a culture method lasting 3-5 days, 4 or 5 days, or 7 days, referred to as the Day 5 method or the Day 7 method, respectively. An example of an in vitro method for generating macrophages from monocytes is described in WO2019 / 175595. The "Day 5" method is described in application number PCT / GB2021 / 051294 (the contents of which are incorporated herein by reference).

[0413] Suitablely, the macrophages are produced via a "day 5" method, the method comprising:

[0414] (a) Mononuclear cells are cultured in a culture medium for 3-5 or 4-5 days to produce macrophages, wherein the culture medium contains one or more growth factors that stimulate macrophage production;

[0415] Step (a) is performed entirely in the same culture medium.

[0416] Suitably, the culture medium contains one or more growth factors selected from the CSF family, preferably M-CSF. Suitably, the culture medium contains M-CSF at a concentration between 25-150 ng / mL. Suitably, the culture medium contains recombinant human macrophage colony-stimulating factor 1 (rhM-CSF-1; also known as "rh (recombinant human) CSF-1") at a level of 100 ng / mL LMP.

[0417] In other embodiments, the macrophages have been derived from stem cells, such as pluripotent or multipotent stem cells. In a particular embodiment, the macrophages have been derived from induced pluripotent stem cells (iPSCs).

[0418] Anti-inflammatory treatment

[0419] In one embodiment, a method for generating engineered macrophages engineered with a combination of IL-10 and MMP9 includes transiently transfecting macrophages with exogenous coding sequences for IL-10 and MMP9. These coding sequences can be provided by transfection with nucleic acids, such as one or more mRNA molecules.

[0420] The macrophages have at least one exogenous coding sequence for IL-10 and at least one exogenous coding sequence for MMP9.

[0421] In some embodiments, after transfection with an exogenous coding sequence, the macrophages are contacted with an anti-inflammatory treatment. Suitably, the anti-inflammatory treatment comprises anti-inflammatory cytokines. In some embodiments, the macrophages are contacted with an anti-inflammatory treatment comprising IL-4 and IL-13.

[0422] In some implementations, anti-inflammatory therapy can be incorporated into the method of transfecting macrophages.

[0423] Transfected macrophages can be exposed to these anti-inflammatory cytokines (IL4 and IL13) for a period of approximately 2 to approximately 48 hours, preferably 4 to 40 hours, preferably 12 to 24 hours, and optionally approximately 16 hours.

[0424] Transfected macrophages can be exposed to these anti-inflammatory cytokines (IL4 and IL13) at concentrations between 2 ng / mL and 200 ng / mL, preferably between 5 ng / mL and 150 ng / mL, preferably between 10 ng / mL and 100 ng / mL, preferably between 15 ng / mL and 75 ng / mL, preferably between 20 ng / mL and 50 ng / mL.

[0425] Suitablely, the anti-inflammatory treatment is used as a solution.

[0426] For the step of contacting macrophages with IL4+IL13, the following plating method is suitable: 2 x 102 cells. 6 hMDM / cm 2 , with 4x10 6 / mL.

[0427] The invention will now be described with reference to the following figures. Attached Figure Description

[0428] Figure 1 - A graph depicting the experimental results of transfecting macrophages with IL-10 and IL-10+MMP9 - Macrophages secrete high levels of IL-10. A: hMDM transfected with IL-10 shows decreased MMP9 expression levels. B: hMDM transfected with IL-10+MMP9 shows increased MMP9 levels. The dashed line represents the minimum level required for the product.

[0429] Figure 2 - A graph depicting the experimental results of transfecting macrophages with multiple constructs. It shows the percentage (%) of IL-10-secreting cells over a 2-hour timeframe using flow cytometry capture assay. Both constructs produced a high percentage of secretory cells. Each symbol represents an individual donor.

[0430] Figure 3- A graph depicting experimental results of macrophage transfection with various constructs regarding cell surface markers. Flow cytometry analysis of hMDM for recognizing cell surface markers. The black solid lines represent the normalized levels of expression on unengineered cells. Each graph represents a different macrophage surface marker: A: CD45, B: CD14, C: CD206; D: CCR2, E: CD163, F: CD169, and G: 25F9.

[0431] Figure 4 - A graph depicting experimental results of macrophage transfection with various constructs regarding pro-inflammatory markers. Pro-inflammatory markers CD86 (A) and HLA-DR (B), as measured by flow cytometry, were significantly downregulated in engineered cells. Other inflammatory markers remained largely unchanged compared to non-Trx hMDM. CD80 expression was ideally no more than 20% or 10-15% higher than in non-NTx (NTx) cells. Solid black lines represent levels in non-Trx cells. Dashed red lines represent maximum expected levels. Each symbol represents an independent donor.

[0432] Figure 5 - A graph depicting experimental results of macrophage transfection with various constructs in terms of phagocytic capacity. Phagocytosis was measured using pH-sensitive (pHrodo) beads coated with *E. coli*. The percentage of phagocytic macrophages was measured by flow cytometry. Each symbol represents an individual donor. The dashed line represents the minimum expected percentage of phagocytic macrophages.

[0433] Figure 6 - A graph depicting experimental results of macrophages transfected with multiple constructs for M1 (A and B) and M2 markers (C and D). M1 and M2 markers were used as indicators of pro-inflammatory and anti-inflammatory phenotypes in macrophages produced in vitro. Flow cytometry analysis of M1 and M2 cell surface markers in macrophages treated overnight with supernatants from untreated, untreated with +Trx, IL-10 Trx, and IL-10+MMP9 Trx hMDM. M2 = positive control: macrophages from the same donor were polarized with high levels of IL-10 (dashed line). Figure A shows the results for CD86, Figure B shows the results for HLA DR. Figure C shows the results for CD206, and Figure D shows the results for CD163.

[0434] Figure 7 - A graph depicting the experimental results of transfecting macrophages with various constructs on the migration of other cells. PBMC migration assay results were measured by flow cytometry. Only monocytes showed significant migration. Data were normalized to Ntrx cells (black line), with minimum expected increase represented by dashed lines.

[0435] Figure 8 - A graph depicting experimental results of MMP expression in macrophages transfected with various constructs. MMP activity assays are shown in the supernatants of tested NTrx, NTrx+ treatment, IL-10 Trx, and IL-10+MMP9 Trx hMDM. Dashed lines represent 1.5-fold increases in activity levels measured in NTrx hMDM supernatant. Each symbol represents an independent donor.

[0436] Figure 9 - A graph depicting experimental results regarding the localization of macrophages transfected with various constructs at sites of injury in vivo. The percentage (%) of live (7AAD-) human macrophages measured using flow cytometry in digests from fibrotic livers is shown. Each point represents a different mouse. Data are reported as mean ± standard deviation (SD). Figure A shows localization in the lungs, and Figure B shows localization in the liver.

[0437] Figure 10 - A graph depicting experimental results regarding the in vivo expression of IL-10 and MMP9 by transfecting macrophages with various constructs. Human IL-10 and human MMP9 in circulation in mice with chronic liver fibrosis are shown by ELISA at different time points following cell injection. Each point represents a different mouse. Data are reported as mean ± SD. Figure A shows IL-10 in plasma, and Figure B shows MMP9 in plasma.

[0438] Figure 11 - A graph depicting the experimental results of macrophages transfected with various constructs after injection into a mouse model. Inflammatory cytokines IL-1β and TNF-α in mouse plasma were measured by ELISA to validate inflammation (if present) induced by injection of engineered and unengineered cells. Measurements were taken at different time points after cell injection. The black dashed line represents the maximum tolerated level. Each point represents a different mouse. Data are reported as mean ± SD. Figure A depicts the results for mIL-1β, and Figure B depicts the results for mTNF-α.

[0439] Figure 12 – As shown, human monocytes were recruited from conditioned media derived from unengineered hMDM or hMDM transfected with various genes. Assays were performed according to the instructions in Materials and Methods, and results were analyzed by flow cytometry. Each symbol represents conditioned media from an independent donor. Data are reported as single donor dispersion, mean, and standard deviation.

[0440] Figure 13- Regulated medium from hMDM transfected with IL-10 specifically recruited monocytes. The data shown are based on data using... Figure 12 The same protocol was used, but migration of individual cell types is shown. Each symbol represents conditioned medium from an independent donor. Data are reported as single-donor dispersion, mean, and standard deviation.

[0441] Figure 14 - Conditioned medium derived from IL-10-transfected macrophages polarized unpolarized macrophages to a pro-repair phenotype. Assays were performed as described in section (A)—macrophages cultured for 5 days were treated with conditioned medium derived from unengineered cells treated with IL-4, IL-13, and M-CSF, cells engineered with a payload of IL-10 or MERTK, or polarization media containing 50 ng / ml MexMACS and IL-10. The expression of HLA-DR (B), CD86 (C), 25F9 (D), CD206 (E), and CD163 (F) was assessed using flow cytometry as described in the examples.

[0442] Figure 15 - Conditioned culture medium derived from IL-10-transfected macrophages rescued pro-inflammatory macrophages and promoted a pro-repair phenotype. Assays were performed as described in section (A)—macrophages cultured for 5 days were treated with conditioned medium derived from unengineered cells treated with IL-4, IL-13, and M-CSF, cells engineered with IL-10 or MERTK payloads, or polarization media containing 50 ng / ml MexMACS and IL-10. The expression of HLA-DR (B), CD86 (C), 25F9 (D), CD206 (E), and CD163 (F) was assessed using flow cytometry as described in the examples.

[0443] Figure 16 Macrophages transfected with IL-10 alone had a secretosome comparable to that of unengineered, non-polarized macrophages. Cells were treated as shown in the figure: untransfected cells, cells post-transfection treated with IL-4, IL-13, and M-CSF (UT TR) or without post-transfection treatment (UT), or cells transfected with specific payloads (IL-10, MERTK, MMP9, or MMP12, as shown on the x-axis). The concentrations of IL-10 (A), IL-6 (B), CXCL8 (C), IL-12p70 (D), IL-2 (E), IL-1β (F), TNF-α (G), and IFN-γ (H) in the culture supernatant were measured, wherein the cells were cultured at a concentration of 4 x 10⁻⁶ cells. 6Cells / ml were cultured at concentrations as described in this article.

[0444] Figure 17 - After administration to mice with a liver fibrosis model, macrophages transfected with IL-10 alone localized to the liver. Similar to that described in Example 8, IL-10-transfected cells or PBS were administered intravenously to mice with a CCl4-induced fibrotic liver injury model.

[0445] Figure 18 Macrophages transfected with MMP9 showed increased MMP activity. Total MMP activity was measured in untransfected macrophages with or without interferon gene stimulating factor inhibitor (iSTING) or in macrophages transfected with a specific payload, as described in the examples. Data are presented as mean ± SD. *** p ≤ 0.005.

[0446] Figure 19 MMP9 transfection of macrophages increased the expression of other matrix metalloproteinases. The expression of MMP1, 3, 7, 8, and 10 was measured in untransfected (NT), macrophages treated with or without the STING inhibitor (STINGi), or macrophages transfected with MMP9 alone. Deeper chroma indicates stronger expression.

[0447] Figure 20 -RTX 001 macrophages recruit monocytes in vitro. RTX001 macrophages are generated by transfection with a bicistronic construct having the sequence shown in SEQ ID NO: 16. PBMC migration assays were performed as described in the examples, and the results were measured by flow cytometry. Results were normalized to untransfected (NTrx) cells.

[0448] Figure 21 -RTX 001 macrophages recruit monocytes in vivo. The recruitment of total myeloid cells was assessed by flow cytometry to detect the proportion of CD11b+ Tim4- cells to total CD45+ cells (left panel). Monocytes were identified as Ly6Chi CD64- CD45+ cells by flow cytometry (right panel). Recruitment was measured in a mouse CCl4-induced liver fibrosis model. RTX001-transfected macrophages, untransfected macrophages, or PBS-mediated macrophages were injected into mice, and recruitment was assessed 24 hours after administration.

[0449] Figure 22 - RTX001 macrophages promote an anti-inflammatory environment in vivo. 24 hours after administration of RTX001 macrophages, untransfected macrophages, or a PBS control to a CCL4-induced mouse model of liver fibrosis, mouse IL-10 in liver homogenate was measured.

[0450] Figure 23Macrophages engineered to express IL-10 alone, as well as those expressing IL-10 and MMP9, exhibited similar pro-repair secretion profiles. The levels of secreted IL-2 (A), IL-12p70 (B), IFNg (C), TNF-α (D), and IL1B (E) were measured in the supernatant of the culture medium, with the engineered cells cultured at a concentration of 4 x 10⁶ cells / ml.

[0451] Figure 24 - Administration of RTX001 macrophages reduced scar-forming cells in vivo. RTX001 macrophages, untransfected macrophages, or PBS-mediated liver fibrosis were administered to a CCL4-induced liver fibrosis mouse model. Tissue sections were taken from the mice one week after administration and stained for α-SMA (left panel). The percentage area of ​​α-SMA-positive regions was calculated, and the results are shown in the right panel.

[0452] Figure 25 RTX001 macrophages with CM significantly reduced LX-2 αSMA expression. (A) Fold change in mean fluorescence intensity (MFI) of αSMA relative to the TGF-β-stimulated control group (without CM). (B) Graph comparing the percentage of cells expressing αSMA. Data were averaged from three replicates and are expressed as mean ± SD (n=1). ** p ≤ 0.01.

[0453] Figure 26 - Optimized bicistronic mRNAs resulted in improved pro-repair properties. Cells were transfected with either unoptimized mRNA (SEQ ID NO:2, which also contains a 120-residue polyA tail) or optimized mRNA (SEQ ID NO: 16), both of which are bicistronic mRNAs encoding both IL-10 and MMP9. The amounts of secreted MMP9 (left panel) and IL-10 (right panel) in the culture supernatant were measured, and MMP activity was measured as described herein. MMP activity was normalized to untransfected cells.

[0454] Figure 27-RTX001 is stable in an inflammatory environment. Changes in CD80 and CD86 (inflammatory markers) and CD206 (pro-repair markers and general macrophage recognition markers) were measured in engineered macrophages exposed to specific concentrations of IFN-γ. No significant changes were observed in pro-inflammatory markers (CD80 and CD86) or the macrophage recognition marker CD206 (A). MATCH-like (Ntrx) and RTX0001 (Trx+tr) cells were incubated with IFN-γ (1.05, 10.5, or 105 ng / mL) for 24 h. The MFI of pro-inflammatory markers HLA-DR and CD80 was assessed using flow cytometry. Data shown are fold changes relative to Ntrx cells cultured without IFN-γ (B and C).

[0455] Figure 28 A table showing the expression of macrophage markers or cytokine secretion in specific macrophage products. Macrophage products consist of: untransfected and untreated (UT) or treated with IL-4, IL-13, and M-CSF (UT + TR); transfected with IL-10 and MMP9 and untreated (IL-10 MMP9 TRx) or transfected and treated with IL-4, IL-13, and M-CSF (IL-10 MMP9 TRx + TR). Mean fluorescence intensity was measured by flow cytometry as described herein.

[0456] Figure 29 - Post-transfection treatment did not lead to further polarization of engineered macrophages. Expression of cD86 (left) and MHC II (right) in macrophage products was detected by flow cytometry. The macrophage products tested consisted of: untransfected macrophages, untreated (NTx) or treated with IL-4, IL-13, and M-CSF (NTx+TR); macrophages transfected with both MMP9 and IL-10, untreated (IL-10-MMP9) or further treated with IL-4, IL-13, and M-CSF after transfection (IL-10-MMP9+TR).

[0457] Figure 30Treatment with IL-4, IL-13, and M-CSF improved cryogenic resistance. Viability of the macrophage product population was determined after cryopreservation. The macrophage products consisted of: untreated macrophages (NTx) or macrophages further treated with IL-4, IL-13, and M-CSF (NTrx + Tr); and macrophages transfected with both MMP9 and IL-10 (Trx) or macrophages further treated with IL-4, IL-13, and M-CSF after transfection (Trx + Tr). Viability was assessed by measuring the proportion of cells surviving after cryopreservation relative to the total number of surviving cells before cryopreservation.

[0458] Figure 31 - Summary of protocols used to measure the efficacy of the mouse-on-mouse model in Example 25.

[0459] Figure 32 - A schematic diagram outlining the mechanism that is believed to underlie the therapeutic effects of engineered macrophages.

[0460] The data shown in these figures demonstrate that the inventors have demonstrated a surprising and effective combination of expression of two specific genes in tandem (IL-10 and MMP9) that yields unforeseen and surprising benefits to the phenotype of engineered macrophages. Surprisingly, expression of MMP9 alone can rescue the repressed MMP expression / activity observed in IL-10-transfected macrophages. As shown above, this effect appears to be synergistic. Furthermore, the excellent recruitment of monocytes by combined overexpression was not predicted, as entities such as cytokines are routinely indicated to be involved in the recruitment process.

[0461] Figure 33 A schematic diagram summarizing the EMERALD clinical trial design.

[0462] Figure 34 This describes the timeline of the mobilization, mononuclear cell harvesting, and pre-treatment patient assessment phases in the EMERALD clinical trial.

[0463] Figure 35 This document outlines the timeline for the engineered macrophage manufacturing and therapeutic phases in the EMERALD clinical trial.

[0464] Figure 36 This describes the timeline for treatment allocation and the phases of treatment with engineered macrophages (RTX001) in the EMERALD clinical trial. Detailed Implementation

[0465] Genetic engineering of macrophages allows for the combined overexpression of IL-10 and MMP9, providing many of the features and functions required for cell therapy, which can be used in inflammatory conditions such as organ damage regeneration. The inventors have demonstrated that the combination of IL-10 and MMP9 produces:

[0466] - Reliable macrophage properties, unaffected by engineering processes.

[0467] - Strong anti-inflammatory phenotype.

[0468] - The ability to pattern primitive macrophages into a pro-repair phenotype.

[0469] - Excellent ability to absorb prey.

[0470] - Reassuring safety and biodistribution characteristics, including penetration in damaged organs and rapid clearance / loss in other organs.

[0471] The above-mentioned features are shared with macrophages engineered solely with IL-10 previously studied by the inventors. However, the combination of IL-10 and MMP9 provides some specific and surprising features that are key to delivering the desired therapeutic effects, such as:

[0472] - The powerful ability to attract monocytes, which is then patterned into a pro-repair phenotype.

[0473] - The ability to restore MMP activity (a substitute for fibrosis / extracellular matrix (ECM) remodeling) that was eliminated by IL-10 alone.

[0474] Therefore, the inventors believe that engineered macrophages using a combination of IL-10 and MMP9 will provide an effective product capable of providing anti-inflammatory and anti-fibrotic functions in therapeutic applications, such as for several organ injury conditions, both acute and chronic. In cases of acute injury, remodeling of ECM components is crucial to ensure tissue restoration to its original state and proper regeneration.

[0475] equivalent

[0476] Those skilled in the art will recognize or be able to determine equivalents of the specific embodiments described herein using only conventional experimentation. These equivalents are intended to be included by the appended claims. Any combination of embodiments disclosed in any of the dependent claims or embodiments is considered to be within the scope of this disclosure.

[0477] By incorporating via reference

[0478] All references in this article, as well as the contents of every patent, patent application publication, and scientific publication, are incorporated herein by reference in their entirety, including the contents of the accompanying figures.

[0479] sequence list

[0480] Italics – Encoding Proteins

[0481] Bold – Connector

[0482] In all mRNA sequences, uridine is transcribed into thymine.

[0483] SEQ ID No. 1

[0484] 1. Single hIL10 mRNA

[0485]

[0486] SEQ ID No. 2

[0487] 2. Bicistronic hIL10-P2A-hMMP9

[0488]

[0489] SEQ ID No. 3

[0490] 3.MMP9

[0491]

[0492] SEQ ID No. 4

[0493] Protein sequence of human IL10 (bold = open reading frame)

[0494]

[0495] SEQ ID No. 5

[0496] Protein sequence IL10–p2a-MMP9 (bold = open reading frame, underline = adapter)

[0497]

[0498] SEQ ID No. 6

[0499] Human MMP9 protein sequence (bold = open reading frame)

[0500]

[0501] SEQ ID No. 7

[0502] p2A motif

[0503] DXEXNPGP

[0504] SEQ ID No. 8

[0505] Unoptimized p2A mRNA sequence

[0506] GGAAGCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCT

[0507] SEQ ID No. 9

[0508] p2A amino acid sequence

[0509] GSGATNFSLLKQAGDVEENPGP

[0510] SEQ ID No. 10

[0511] Optimized bicistronic mRNA coding sequence (adapter sequence underlined). All uridines (transcribed here as thymine) are 5-methoxyuridines.

[0512]

[0513] SEQ ID No. 11

[0514] The amino acid sequence encoded by the optimized bicistronic mRNA sequence (IL-10-p2A-MMP9 fusion protein) (adaptor sequence is underlined).

[0515] MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN GSGATNFSLLKQAGDVEENPGPMSLWQPLVLVLLVLGCCFAAPRQRQSTLVLFPGDLRTNLTDRQLAEEYLYRYGYTRVAEMRGESKSLGPALLLLQKQLSLPETGELDSATLKAMRTPRCGVPDLGRFQTFEGDLKWHHHNITYWIQNYSEDLPRAVIDDAFARAFALWSAVTPLTFTRVYSRDADIVIQFGVAEHGDGYPFDGKDGLLAHAFPPGPGIQGDAHFDDDELWSLGKGVVVPTRFGNADGAACHFPFIFEGRSYSACTTDGRSDGLPWCSTTANYDTDDRFGFCPSERLYTQDGNADGKPCQFPFIFQGQSYSACTTDGRSDGYRWCATTANYDRDKLFGFCPTRADSTVMGGNSAGELCVFPFTFLGKEYSTCTSEGRGDGRLWCATTSNFDSDKKWGFCPDQGYSLFLVAAHEFGHALGLDHSSVPEALMYPMYRFTEGPPLHKDDVNGIRHLYGPRPEPEPRPPTTTTPQPTAPPTVCPTGPPTVHPSERPTAGPTGPPSAGPTGPPTAGPSTATTVPLSPVDDACNVNIFDAIAEIGNQLYLFKDGKYWRFSEGRGSRPQGPFLIADKWPALPRKLDSVFEERLSKKLFFFSGRQVWVYTGASVLGPRRLDKLGLGADVAQVTGALRSGRGKMLLFSGRRLWRFDVKAQMVDPRSASEVDRMFPGVPLDTHDVFQYREKAYFCQDRFYWRVSSRSELNQVDQVGYVTYDILQCPED-

[0516] SEQ ID No. 12

[0517] Mouse mRNA-based transcript sequence introduced into mRTX001.

[0518]

[0519] SEQ ID NO: 13

[0520] Optimized sequence of IL-10 mRNA. All uridines (transcribed here as thymine) are 5-methoxyuridines.

[0521] ATGCACAGCTCCGCCCTGCTGTGCTGCCTGGTGCTGCTGACCGGCGTGCGGGCCAGCCCCGGCCAGGGCACACAGTCCGAGAACAGCTGCACCCACTTCCCAGGCAATCTCCCCAACATGCTGAGAGACCTGA GGGACGCCTTCTCCCGCGTGAAGACATTCTTCCAGATGAAGGACCAGCTGGACAATCTCCTGCTGAAGGAGAGCCTGCTGGAGGACTTCAAGGGCTACCTGGGCTGCCAGGCCCTGTCCGAGATGATCCAGTTC TACCTGGAGGAGGTGATGCCACAGGCCGAGAACCAGGACCCCGACATCAAGGCCCACGTGAACAGCCTGGGCGAGAACCTGAAGACCCTGCGGCTGAGACTGAGGCGCTGCCACCGGTTCCTGCCATGCGAGA ACAAGTCCAAGGCCGTGGAACAAGTGAAGAACGCCTTCAACAAGCTGCAGGAGAAGGGCATCTACAAGGCCATGAGCGAGTTCGACATCTTCATCAACTACATCGAGGCCTACATGACAATGAAGATCAGAAAC

[0522] SEQ ID NO: 14

[0523] Optimized sequence of MMP9 mRNA. All uridines (transcribed here as thymine) are 5-methoxyuridines.

[0524]

[0525] SEQ ID NO: 15

[0526] Optimized adapter mRNA sequence. All uridines (transcribed here as thymine) are 5-methoxyuridines.

[0527] GGCTCCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCA

[0528] SEQ ID NO: 16

[0529] Optimized bicistronic mRNA (including 5'UTR and 3'UTR). All uridines (transcribed here as thymine) are 5-methoxyuridines.

[0530]

[0531] SEQ ID NO: 17 - 5' UTR of the optimized bicistronic mRNA construct (all uridines, transcribed here as thymine, are 5-methoxy-uridines).

[0532] AGGCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATTCACAACCAGGCCTCCACAACC

[0533] SEQ ID NO: 18 - 3' UTR of the optimized bicistronic mRNA construct (all uridines, transcribed here as thymine, are 5-methoxyuridines).

[0534] ACTCGAGTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTATCCCAGTGTATATTGTCGAC

[0535] SEQ ID NO: 19 - 5' UTR of unoptimized bicistronic mRNA construct

[0536] AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC

[0537] SEQ ID NO: 20 - 3' UTR of unoptimized bicistronic mRNA construct

[0538] GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG

[0539] References

[0540] 1. Williams, R. et al. Addressing liver disease in the UK: ablueprint for attaining excellence in health care and reducing prematuremortality from lifestyle issues of excess consumption of alcohol, obesity,and viral hepatitis. Lancet 384, 1953-1997, doi:10.1016 / S0140-6736(14)61838-9(2014).

[0541] 2. Pellicoro, A., Ramachandran, P., Iredale, J. P. & Fallowfield, J.A. Liver fibrosis and repair: immune regulation of wound healing in a solidorgan. Nat Rev Immunol 14, 181-194, doi:10.1038 / nri3623 (2014).

[0542] 3. Fallowfield, J. A. Future mechanistic strategies for tacklingfibrosis--an unmet need in liver disease. Clin Med (Lond) 15 Suppl 6, s83-87,doi:10.7861 / clinmedicine.15-6-s83 (2015).

[0543] 4. Forbes, S. J. & Newsome, P. N. Liver regeneration - mechanisms andmodels to clinical application. Nat Rev Gastroenterol Hepatol, doi:10.1038 / nrgastro.2016.97 (2016).

[0544] 5. Thomas, J. A., Ramachandran, P. & Forbes, S. J. Studies ofmacrophage therapy for cirrhosis - From mice to men. J Hepatol 68, 1090-1091,doi:10.1016 / j.jhep.2017.11.043 (2018).

[0545] 6. Forbes, S. J., Gupta, S. & Dhawan, A. Cell therapy for liverdisease: From liver transplantation to cell factory. J Hepatol 62, S157-169,doi:10.1016 / j.jhep.2015.02.040 (2015).

[0546] 7. Falasca, L., Bergamini, A., Serafino, A., Balabaud, C. & Dini, L.Human Kupffer cell recognition and phagocytosis of apoptotic peripheral bloodlymphocytes. Exp Cell Res 224, 152-162, doi:10.1006 / excr.1996.0123 (1996).

[0547] 8. Ramachandran, P. et al. Differential Ly-6C expression identifiesthe recruited macrophage phenotype, which orchestrates the regression ofmurine liver fibrosis. Proc Natl Acad Sci U S A 109, E3186-3195, doi:10.1073 / pnas.1119964109 (2012).

[0548] 9. Wynn, T. A. & Barron, L. Macrophages: master regulators ofinflammation and fibrosis. Semin Liver Dis 30, 245-257, doi:10.1055 / s-0030-1255354 (2010).

[0549] 10. Zigmond, E. et al. Infiltrating monocyte-derived macrophages andresident kupffer cells display different ontogeny and functions in acuteliver injury. J Immunol 193, 344-353, doi:10.4049 / jimmunol.1400574 (2014).

[0550] 11. Campana, L., Esser, H., Huch, M. & Forbes, S. Liver regenerationand inflammation: from fundamental science to clinical applications. Nat RevMol Cell Biol 22, 608-624, doi:10.1038 / s41580-021-00373-7 (2021).

[0551] 12. Thomas, J. A. et al. Macrophage therapy for murine liver fibrosisrecruits host effector cells improving fibrosis, regeneration, and function.Hepatology 53, 2003-2015, doi:10.1002 / hep.24315 (2011).

[0552] 13. Moore, J. K. et al. Phenotypic and functional characterization ofmacrophages with therapeutic potential generated from human cirrhoticmonocytes in a cohort study. Cytotherapy 17, 1604-1616, doi:10.1016 / j.jcyt.2015.07.016 (2015).

[0553] 14. Fraser, A. R. et al. Development, functional characterization andvalidation of methodology for GMP-compliant manufacture of phagocyticmacrophages: A novel cellular therapeutic for liver cirrhosis. Cytotherapy19, 1113-1124, doi:10.1016 / j.jcyt.2017.05.009 (2017).

[0554] 15. Starkey Lewis, P. J., Moroni, F. & Forbes, S. J. Macrophages as aCell-Based Therapy for Liver Disease. Semin Liver Dis 39, 442-451, doi:10.1055 / s-0039-1688502 (2019).

[0555] 16. Moroni, F. et al. Safety profile of autologous macrophage therapyfor liver cirrhosis. Nat Med 25, 1560-1565, doi:10.1038 / s41591-019-0599-8(2019).

[0556] 17. Das, S. T. et al. Monomeric and dimeric CXCL8 are both essentialfor in vivo neutrophil recruitment. PLoS One 5, e11754, doi:10.1371 / journal.pone.0011754 (2010).

[0557] 18. Wilgus, T. A., Roy, S. & McDaniel, J. C. Neutrophils and WoundRepair: Positive Actions and Negative Reactions. Adv Wound Care (NewRochelle) 2, 379-388, doi:10.1089 / wound.2012.0383 (2013).

[0558] 19. Lin, Z. Q., Kondo, T., Ishida, Y., Takayasu, T. & Mukaida, N.Essential involvement of IL-6 in the skin wound-healing process as evidencedby delayed wound healing in IL-6-deficient mice. J Leukoc Biol 73, 713-721,doi:10.1189 / jlb.0802397 (2003).

[0559] 20. Wynn, T. A. & Vannella, K. M. Macrophages in Tissue Repair,Regeneration, and Fibrosis. Immunity 44, 450-462, doi:10.1016 / j.immuni.2016.02.015 (2016).

[0560] 21. Mantovani, A. et al. The chemokine system in diverse forms ofmacrophage activation and polarization. Trends Immunol 25, 677-686, doi:10.1016 / j.it.2004.09.015 (2004).

[0561] 22. Mosser, D. M. & Edwards, J. P. Exploring the full spectrum ofmacrophage activation. Nat Rev Immunol 8, 958-969, doi:10.1038 / nri2448(2008).

[0562] 23. Acharya, D., Li, X. R. L., Heineman, R. E. & Harrison, R. E.Complement Receptor-Mediated Phagocytosis Induces Proinflammatory CytokineProduction in Murine Macrophages. Front Immunol 10, 3049, doi:10.3389 / fimmu.2019.03049 (2019).

[0563] 24. Dreschers, S. et al. Metalloproteinases TACE and MMP-9Differentially Regulate Death Factors on Adult and Neonatal Monocytes AfterInfection with Escherichia coli. Int J Mol Sci 20, doi:10.3390 / ijms20061399(2019).

[0564] 25. Mosser, D. M. & Zhang, X. Interleukin-10: new perspectives on anold cytokine. Immunol Rev 226, 205-218, doi:10.1111 / j.1600-065X.2008.00706.x(2008).

[0565] 26. Riley, J. K., Takeda, K., Akira, S. & Schreiber, R. D.Interleukin-10 receptor signaling through the JAK-STAT pathway. Requirementfor two distinct receptor-derived signals for anti-inflammatory action. JBiol Chem 274, 16513-16521, doi:10.1074 / jbc.274.23.16513 (1999).

[0566] 27. Campana, L. et al. The STAT3-IL-10-IL-6 Pathway Is a NovelRegulator of Macrophage Efferocytosis and Phenotypic Conversion in SterileLiver Injury. J Immunol 200, 1169-1187, doi:10.4049 / jimmunol.1701247 (2018).

[0567] 28. Braat, H., Peppelenbosch, M. P. & Hommes, D. W. Interleukin-10-based therapy for inflammatory bowel disease. Expert Opin Biol Ther 3, 725-731, doi:10.1517 / 14712598.3.5.725 (2003).

[0568] 29. Nelson, D. R., Lauwers, G. Y., Lau, J. Y. & Davis, G. L.Interleukin 10 treatment reduces fibrosis in patients with chronic hepatitisC: a pilot trial of interferon nonresponders. Gastroenterology 118, 655-660,doi:10.1016 / s0016-5085(00)70134-x (2000).

[0569] 30. Marlow, G. J., van Gent, D. & Ferguson, L. R. Why interleukin-10supplementation does not work in Crohn's disease patients. World JGastroenterol 19, 3931-3941, doi:10.3748 / wjg.v19.i25.3931 (2013).

[0570] 31. Iyer, S. S. & Cheng, G. Role of interleukin 10 transcriptionalregulation in inflammation and autoimmune disease. Crit Rev Immunol 32, 23-63, doi:10.1615 / critrevimmunol.v32.i1.30 (2012).

[0571] 32. Hung, K. S. et al. Interleukin-10 gene therapy reversesthioacetamide-induced liver fibrosis in mice. Biochem Biophys Res Commun 336,324-331, doi:10.1016 / j.bbrc.2005.08.085 (2005).

[0572] 33. Chou, W. Y. et al. Electroporative interleukin-10 gene transferameliorates carbon tetrachloride-induced murine liver fibrosis by MMP andTIMP modulation. Acta Pharmacol Sin 27, 469-476, doi:10.1111 / j.1745-7254.2006.00304.x (2006).

[0573] 34. Huang, Y. H. et al. Therapeutic effect of interleukin-10 on CCl4-induced hepatic fibrosis in rats. World J Gastroenterol 12, 1386-1391, doi:10.3748 / wjg.v12.i9.1386 (2006).

[0574] 35. Zhang, L. J., Zheng, W. D., Shi, M. N. & Wang, X. Z. Effects ofinterleukin-10 on activation and apoptosis of hepatic stellate cells infibrotic rat liver. World J Gastroenterol 12, 1918-1923, doi:10.3748 / wjg.v12.i12.1918 (2006).

[0575] 36. Cypel, M. et al. Functional repair of human donor lungs by IL-10gene therapy. Sci Transl Med 1, 4ra9, doi:10.1126 / scitranslmed.3000266(2009).

[0576] 37. Cua, D. J., Hutchins, B., LaFace, D. M., Stohlman, S. A. &Coffman, R. L. Central nervous system expression of IL-10 inhibits autoimmuneencephalomyelitis. J Immunol 166, 602-608, doi:10.4049 / jimmunol.166.1.602(2001).

[0577] 38. Starkey Lewis, P. et al. Alternatively activated macrophagespromote resolution of necrosis following acute liver injury. J Hepatol 73,349-360, doi:10.1016 / j.jhep.2020.02.031 (2020).

[0578] 39. Ma, P. F. et al. Cytotherapy with M1-polarized macrophagesameliorates liver fibrosis by modulating immune microenvironment in mice. JHepatol 67, 770-779, doi:10.1016 / j.jhep.2017.05.022 (2017).

[0579] 40. Wilson, H. M. et al. Bone-marrow-derived macrophages geneticallymodified to produce IL-10 reduce injury in experimental glomerulonephritis.Mol Ther 6, 710-717, doi:10.1006 / mthe.2002.0802 (2002).

[0580] 41. Boehler, R. M. et al. Lentivirus Delivery of IL-10 to Promote andSustain Macrophage Polarization Towards an Anti-Inflammatory Phenotype.Biotechnol Bioeng. 2014 June; 111(6): 1210–1221. doi:10.1002 / bit.25175(2014).

[0581] 42. D’Amico et al, Further decompensation as a new prognostic stagein cirrhosis. Results of a large multicenter cohort study supporting BavenoVII statements. Journal of Hepatology 78(S1) S105-106. (2023)

[0582] 43. Trebicka J, et al. International Variceal Bleeding ObservationalStudy Group and Baveno Cooperation. Rebleeding and mortality risk areincreased by ACLF but reduced by pre-emptive TIPS. J Hepatol; 73(5):1082-1091. (2020)

[0583] 44. Starkey Lewis PJ, Moroni F, Forbes SJ. Macrophages as a Cell-Based Therapy for Liver Disease. Semin Liver Dis., 39(4):442-451.(2019)

[0584] 45. Cabrera S, Gaxiola M, Arreola JL, Ramírez R, Jara P, D'ArmientoJ, Richards T, Selman M, Pardo A. Overexpression of MMP9 in macrophagesattenuates pulmonary fibrosis induced by bleomycin. Int J Biochem Cell Biol;39(12):2324-38. (2007)

[0585] 46. Moreau R, et al. CANONIC Study Investigators of the EASL–CLIFConsortium. Acute-on-chronic liver failure is a distinct syndrome thatdevelops in patients with acute decompensation of cirrhosis.Gastroenterology, 144(7):1426-37 1437.e1-9. (2013)

[0586] 47. Fan, Y. Y., Ding, W., Zhang, C., Fu, L., Xu, D. X., & Chen, X.Obeticholic acid prevents carbon tetrachloride-induced liver fibrosis throughinteraction between farnesoid X receptor and Smad3. Internationalimmunopharmacology, 77, 105911 (2019)

[0587] 48. Younossi, Z. M., Ratziu, V., Loomba, R., Rinella, M., Anstee, Q.M., Goodman, Z., Bedossa, P., Geier, A., Beckebaum, S., Newsome, P. N.,Sheridan, D., Sheikh, M. Y., Trotter, J., Knapple, W., Lawitz, E.,Abdelmalek, M. F., Kowdley, K. V., Montano-Loza, A. J., Boursier, J.,Mathurin, P., … REGENERATE Study Investigators. Obeticholic acid for thetreatment of non-alcoholic steatohepatitis: interim analysis from amulticentre, randomised, placebo-controlled phase 3 trial. Lancet (London,England), 394(10215), 2184–2196. (2019)

[0588] 49. Wettstein, G., Luccarini, J. M., Poekes, L., Faye, P., Kupkowski,F., Adarbes, V., Defrêne, E., Estivalet, C., Gawronski, X., Jantzen, I.,Philippot, A., Tessier, J., Tuyaa-Boustugue, P., Oakley, F., Mann, D. A.,Leclercq, I., Francque, S., Konstantinova, I., Broqua, P., & Junien, J. L.The new-generation pan-peroxisome proliferator-activated receptor agonistIVA337 protects the liver from metabolic disorders and fibrosis. Hepatologycommunications, 1(6), 524–537 (2017)

[0589] 50. Francque, S. M., Bedossa, P., Ratziu, V., Anstee, Q. M.,Bugianesi, E., Sanyal, A. J., Loomba, R., Harrison, S. A., Balabanska, R.,Mateva, L., Lanthier, N., Alkhouri, N., Moreno, C., Schattenberg, J. M.,Stefanova-Petrova, D., Vonghia, L., Rouzier, R., Guillaume, M., Hodge, A.,Romero-Gómez, M., … NATIVE Study Group. A Randomized, Controlled Trial ofthe Pan-PPAR Agonist Lanifibranor in NASH. The New England journal ofmedicine, 385(17), 1547–1558 (2021)

[0590] Examples

[0591] Materials and methods

[0592] The following scheme is used to generate the data described in the following embodiments.

[0593] Macrophage culture

[0594] We isolated monocytes from the erythrocyte sedimentation rate (ESR) amber layer from healthy volunteers from the Scottish National Blood Transfusion Service (SNBTS) using a Ficoll gradient (GE Healthcare) followed by magnetic column selection with CliniMACS CD14 reagent (Miltenyi Biotec). Monocytes were then matured in culture for 1 to 7 days 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, Biotechne). hMDM was used at 2 x 10-1 6 / cm 2 The density was incubated in 6-well multi-well plates (Corning Costar) for 5 days. hMDM was counted using an automated counter (TC20, BioRad).

[0595] Macrophage engineering

[0596] Small to medium scale transfection of mRNA

[0597] Mature macrophages were precipitated at 300 μg for 5 minutes, then the supernatant was removed, and the cells were resuspended in replenished buffer P3 (Lonza) at a density of 50 × 10⁶ or 100 × 10⁶ cells / ml. IL-10 (798 NT) mRNA was added at a concentration of 2 μg / 10⁶ cells or IL-10-MMP9 (2985 NT) mRNA at a concentration of 8 μg / 10⁶ cells, and thoroughly mixed by pipetting. The cell suspension was transferred to an electroporation cuvette / cassette (100 μl / 1 ml). Cells were transfected on Lonza Nucleofectors using pulse code CM-137. Cells were collected in sterile Falcon tubes. Wash the electroporation cuvette / cassette with 100 μl / 1 ml TexMACS medium supplemented with M-CSF (100 ng / ml), IL4 (20 ng / ml), and IL13 (20 ng / ml), and add the cells to the cell suspension. Incubate the cells in a TC incubator for 20 minutes. Count the cells and adjust the cell density to 4 × 10⁶ / ml. Continue incubation at a density of 2 × 10⁶ / cm². 2 Inoculate the cells and place them in a cell culture incubator.

[0598] Massive fluidic transfection of mRNA

[0599] Precipitate mature macrophages at 300 g for 5 minutes. Remove the supernatant and resuspend the cells at a density of 200 x 10^6 cells / ml in replenished buffer P3 (Lonza). Transfer mRNA containing IL10 (798 NT) at a concentration of 2 μg / 10^6 cells or IL10-MMP9 (2985 NT) at a concentration of 8 μg / 10^6 cells to sterile Falcon tubes and top with replenished buffer P3 to the same volume as the cell suspension. Set up Lonza Nucleofector LV transfection. Transfer the cell suspension and mRNA to a 4D-Nucleofector LV reservoir as needed. Connect cell culture bags pre-filled with TexMACS medium supplemented with M-CSF (100 ng / ml), IL4 (20 ng / ml), and IL13 (20 ng / ml) to the tubing of the electroporation chamber to elute the cells. Cells were transfected into Lonza Nucleofector using pulse code CM-137. The bags containing the cells were transferred to a TC incubator for 20 minutes. Cell counting was performed and the cell density was adjusted to 4 × 10⁶ / ml. The density was then increased to 2 × 10⁶ / cm³. 2 Inoculate the cells and place them in a cell culture incubator.

[0600] Small-scale transfection – pDNA

[0601] As used in this article, a “payload” is one or more genes with therapeutic purposes that are introduced via transfection to test their effects on macrophages.

[0602] On day 5, human monocyte-derived macrophages (hMDM) were resuspended in CliniMACS at a density of 75*10^6 or 150*10^6 cells / ml. ® In electroporation buffer (Miltenyi Biotec, #170-076-625), transfer 100-300 μl of suspension to electroporation cuvettes with a gap size of 0.2 cm. Add 5 μg of plasmid DNA per 5*10^6 cells directly to the cuvette and mix the plasmid DNA with the cells by gently shaking the cuvette. Use CliniMACS Prodigy ® Controlled CliniMACS ® Cells were transfected using an electroporator. The electroporation parameters are listed in Table 1 below (as further described in patent application PCT / GB2021 / 051300 (published as WO2021240167)):

[0603] Table 1 shows the electroporation transfection parameters of the IL10 expression plasmid in hMDM.

[0604]

[0605] After transfection, cells were retrieved from the cuvette into sterile TexMACS using an 18G sterile needle attached to a 1 mL syringe or similar tool. ™ Cells were cultured in GMP medium (Miltenyi Biotec, #170-076-306). Cell counting was performed using a TC-20 automated cell counter (Bio-Rad). Cells were rotated at 300*g for 5 minutes at room temperature. The supernatant was aspirated, and the cells were resuspended at a concentration of 4*10^6 cells / mL 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 density of 2*10^6 cells / cm³. 2 decking.

[0606] Used to generate Figure 1-11 Macrophages, as shown in data 20-30, were transfected with an RNA construct. This was used to produce... Figure 12-16 Macrophages from data shown in 18 and 19 were transfected with plasmid DNA constructs.

[0607] Low temperature stress resistance treatment

[0608] To improve the low-temperature resistance of transfected engineered macrophages, macrophages were incubated overnight in TexMACS serum-free medium (Miltenyi Biotec), which contained 100 ng / mL M-CSF (BioTechne) and 20 ng / mL IL-4 and IL-13 (BioTechne), with a cell concentration of 4 x 10⁻⁶ cells / mL. 6 cells / mL and 2x10 6 / cm 2 .

[0609] Phagocytosis assay

[0610] In summary, macrophages for imaging analysis were prepared by seeding them at a density of 150,000 cells / well in 96-well clear-bottom imaging plates (Grenier). The supernatant was removed, and the cells were stained for 30 minutes at 37°C and 5% CO2 with 100 µL PBS + NucBlue (ThermoFisher) + 5 µg / ml Cellmask Deep Red Plasma Membrane Stain (Invitrogen). The cells were washed three times with 100 µL PBS. 50 µL PBS was added to the cells for T0 analysis using the OperaPhenix high-content screening system. After T0, 50 µL of 0.2 mg / ml pHrodo Red Yeast Glycan Beads (Life Technologies) were added to the cells. A series of images were acquired over a 96-minute timeframe to monitor phagocytosis. The images were analyzed using Columbus data imaging software and the Tibco Spotfire data analysis system. Graphs were plotted using GraphPad Prism 9.2.0.

[0611] By dividing the cells into 2x10 6Cells were resuspended in PBS + 0.5 mM EDTA (Life Technologies) at a concentration of / ml to prepare cells for flow cytometry analysis. 50 µL of cell suspension was dispensed into low-adhesion round-bottom 96-well plates. 50 µL of resuspended pHrodo beads (prepared according to manufacturer's instructions) was added to each test well. Cells were incubated at 37°C and 5% CO2 for 2 hours. At the end of the 1-hour incubation, the plate was rotated at 300 x g at 4°C for 5 minutes to remove the supernatant, and the pellet was resuspended in 100 μL of 1:100 FcR blocking PEA solution per well. The cells were then incubated in the dark at 4°C for 15 minutes, followed by the addition of antibody (see Table 3) to the appropriate test wells and incubation at 4°C for 20 minutes. Cells were washed with PBS + 0.5 mM EDTA and rotated at 300 g for 5 minutes. The supernatant was discarded, and the cells were resuspended in PBS + 0.5 mM EDTA + 1:1000 DRAQ7. Incubate at 4°C for 5 minutes. Wash as described above, then resuspend in 100 μL of PBS + 0.5 mM EDTA + 0.1% human serum. Collect 50 μL of cells using a Novocyte 3000 or Novocyte Quanteon (Agilent). Perform flow cytometry analysis on NovoExpress software, using the following gating strategies to identify actively phagocytic macrophages: "Cell gate" to exclude debris, "single-line gate" to exclude double cells, "live gate" to exclude dead cells, "CD14+ gate" to identify iMACS, and "phRodo+ve gate" to measure the percentage of phagocytic macrophages.

[0612] IL-10 capture assay

[0613] For each condition, 1x10 6Cells were resuspended in 80 μL of cold TexMACS and 20 μL of IL-10 capture reagent was added. The cells were incubated on ice for 5 minutes. Then, 10 mL of warm TexMACS was added to the payload-transfected cells (test group), and 10 mL of cold TexMACS was added to another payload-transfected cell line as a negative control. The test group was continuously incubated at 37 °C with rotation for 1.5 hours. The negative control was kept on ice. After incubation, the cells were filled to 15 mL with cold buffer and rotated slowly at 4 °C. The cells were then washed again with 10 mL of cold buffer and rotated slowly at 4 °C. The cells were resuspended in 80 μL of cold buffer, 20 μL of detection antibody and 5 μL of CD14 VioBlue were added, and the cells were incubated on ice for 10 minutes. The cells were washed with 5 mL of cold buffer and rotated slowly at 4 °C. The cells were then resuspended in cold buffer with 1 mL of 1:1000 Draq7 and 100 μL of sample was transferred to a 96-well plate. Rotate the plate at a reduced speed, resuspend it in 100 μL of cold buffer, and collect the samples on a flow cytometer.

[0614] Flow cytometry labeling

[0615] Macrophages were 1x10 6 Resuspend cells at a concentration of / ml in PBS + 0.5mM EDTA (Life Technologies) + FcR Block 1:100 (Miltenyi). Aliquot 100µl of cells into low-adhesion round-bottom 96-well plates. Incubate cells for 5 minutes, then add appropriate antibody to the appropriate test wells (see Table 2) and incubate at 4°C for 20 minutes. Wash cells with PBS + 0.5mM EDTA and rotate at 300g for 5 minutes. Discard the supernatant and resuspend cells in PBS + 0.5mM EDTA + 1:1000 DRAQ7. Incubate at 4°C for 5 minutes. Wash as described above, then resuspend in 100µl of PBS + 0.5mM EDTA + 0.1% human serum. Collect 50µl of cells using a Novocyte3000 or Novocyte Quanteon (Agilent).

[0616] Table 2 - Flow cytometry-labeled antibodies for hMDM.

[0617]

[0618] MSD V-plex cytokine dosage

[0619] Cytokines in cell culture supernatant were analyzed using the V-PLEX Human Biomarker 10-Plex Kit on a MESO Quickplex SQ 120, following the manufacturer's instructions (Meso Scale Discovery). 10 μL of supernatant was tested. Results are expressed in pg / mL. Values ​​were adjusted for dilution at the time of 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 alone (TexMACS) from the amount of cytokines detected in the cell culture supernatant.

[0620] PBMC attraction / migration assay

[0621] ESR (erythrocyte sedimentation rate) amber layer was donated from SNBTS at a sample treatment level of 20–17. Peripheral blood mononuclear cells were isolated from the ESR amber layer using standard methods and cultured in CryoStor CS10 (STEMCELL) at 50 x 10⁻⁶ cells / mL. 6 Frozen at a density of / mL and stored until needed. To establish a migration assay, PBMCs were thawed and stored at 4.6 x 10⁻⁶. 6 Resuspend cells in TexMACS (Miltenyi). Add 75 μL of cells from TexMACS to the top chamber of a 5 μm 96-well transwell (Corning). Add 150 μL of freeze-thawed conditioned medium to the bottom chamber. Place the transwell plate in an incubator (37°C, 5% CO2). After 3 hours, remove the top chamber and stain the migrating cells in the bottom chamber with CD45-PerCP, CD14-VioBlue, CD15-Pevio770, CD16-BV605, CD56-PE, CD3-FITC, and CD19-APC using a standard flow cytometry staining procedure. Collect the staining with Novocyte3000 or Novocyte Quanteon (Agilent).

[0622] Polarization assay – nonpolarized macrophages

[0623] Mature macrophages on day 5 were processed at a rate of 2 x 10 6 / cm 2Cells were seeded at a density of 100 ng / ml in TexMACS + 100 ng / ml MCSF (R&D) in 96-well plates (Corning). 50 ng / ml IL-10 (R&D) was added to the control wells (M2 polarization medium). After cell attachment (5 h), the medium was removed and replaced with conditioned medium. Cells and conditioned medium were incubated in an incubator (37°C, 5% CO2) for 18 h, followed by standard flow cytometry staining for CD14-VioBlue, CD45-PerCP, CD206-BV711, 25F9-eF660, HLA-DR-PeCy7, CD86-PE, and CD163-FITC. DRAQ7 was used to stain dead cells. Cells were then harvested using Novocyte3000 or Novocyte Quanteon (Agilent).

[0624] MMP activity assay

[0625] 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; when intact, it does not fluoresce. The cleaved peptide no longer quenches the fluorescent signal, thus resulting in fluorescence emission, which was measured in relative fluorescence units (RFU). The assay was performed according to the manufacturer's instructions (https: / / www.abcam.com / ps / products / 112 / ab112146 / documents / ab112146%20MMP%20Activity%20Assay%20Kit%20Fluorometric%20-%20Green%20v4b%20(website).pdf - ab112146 MMP ActivityAssay Kit Fluorometric - Green v4b), testing 25 μL of cell culture supernatant. All data shown represent the activity of secreted MMP over a 24-hour period. Results are plotted as RFU minus the background fluorescence of the culture medium alone (TexMACS).

[0626] statistics

[0627] Each reported point represents a different donor. Unless otherwise stated, at least three donors are analyzed for each condition. Where appropriate, data are presented as mean ± SD. Where appropriate, a two-tailed t-test is performed on paired data.

[0628] result

[0629] Example 1: Efficient transfection of IL-10 and IL-10+MMP9 in hMDM

[0630] To generate transfected (Trx) human monocyte-derived macrophages (hMDM) for cell therapy, a significant increase in the expression and secretion levels of the desired payload is required. Based on internal and published results, we set minimum thresholds for our selected payloads IL-10 and MMP9. The levels of secreted proteins in the cell culture supernatant 24 hours post-transfection were measured by ELISA. Interestingly, both IL-10 and IL-1-0+MMP9 (bicistronic vector) transfection resulted in a significant increase in IL-10 secretion. Figure 1 Flow cytometry based on IL-10 capture assays also confirmed increased IL-10 secretion in engineered macrophages. This assay monitored IL-10 secretion in macrophages over a 2-hour period. Figure 2 However, MMP9 secretion did not show a remarkable increase: only IL-10 transfection resulted in a slight decrease in MMP9 secretion, which was at least partially salvaged by co-transfection of IL-10 and MMP9. Figure 1 This indicates that MMP9 has sufficient transfection efficiency. In all experiments presented in this paper, we always analyzed untransfected (NTrx), NTx + recovery treatment (IL4 + IL13), Trx cells with only IL-10, and IL-10 + MMP9Trx. All Trx cells received recovery treatment. IL-10 and MMP9 were co-transfected using a bicistronic vector ligated with a p2A adapter sequence.

[0631] Example 2: hMDM transfected with IL-10 and IL-10+MMP9 showed retention of macrophage recognition markers.

[0632] The key to obtaining safe and effective macrophage therapy is maintaining macrophage surface recognition markers. This indicates that the genetic engineering procedure did not fundamentally alter the cell characteristics. The panleukocyte marker CD45 was present in all cell types. In IL-10+MMP9, the expression intensity of the bone marrow marker CD14 was slightly decreased (MFI fold change), but the percentage of positive cells remained unchanged, so we are satisfied that the cells maintained their bone marrow characteristics. The mature macrophage marker 25F9 was slightly increased in engineered cells, a positive marker of strong macrophage characteristics. Compared with unengineered cells, all other markers analyzed in engineered cells (CD206, CD163, CCR2, CD169) were unchanged or slightly increased. These data support the view that our engineering method is safe and does not interfere with cell characteristics. Data as follows Figure 3 As shown.

[0633] Example 3: hMDM transfected with IL-10 and IL-10+MMP9 exhibits significant anti-inflammatory properties.

[0634] Obtaining highly anti-inflammatory macrophages is important for treating acute and chronic inflammatory conditions, such as those associated with organ damage. Surprisingly, not only did the transfected macrophages retain all the recognition markers ( Figure 3 Furthermore, they also showed reductions in several pro-inflammatory markers, such as CD86 and HLA-DR ( Figure 4 This highlights the autocrine-paracrine effect of engineered IL-10 in inducing a strong anti-inflammatory phenotype in engineered macrophages. The slight increase in CD80 levels observed with IL-10+MMP9 is unlikely to be significant in terms of biological effects, as it is within 10% of the desired level (red dashed line).

[0635] Example 4: hMDM transfected with IL-10 and IL-10+MMP9 exhibits excellent phagocytic ability.

[0636] In acute and chronic inflammatory conditions, such as those associated with organ injury, another key aspect of effective macrophage therapy is its efficient phagocytic capacity. Here, we report that both engineered and unengineered macrophages effectively phagocytosed, exceeding the minimum required level (dashed line). Figure 5 ).

[0637] Example 5: hMDM transfected with IL-10 and IL-10+MMP9 polarized primitive macrophages to a pro-repair phenotype.

[0638] During inflammatory organ injury, local macrophages require a pro-repair phenotype to support fibrotic remodeling and / or tissue regeneration. In this assay, we evaluated the ability of cell culture supernatants from NTrx, NTrx+-treated, IL-10 Trx, and IL-10+MMP9 Trx macrophages 24 hours post-transfection to polarize macrophages from unrelated donors. M2 macrophages polarized from the same donor using recombinant IL-10 served as a positive control (red dashed line). The expected outcome was a decrease in M1 markers CD86 and HLA-DR, and similar or increased M2 markers CD206 and CD163, in macrophages treated with supernatants from engineered hMDM compared to unengineered hMDM. Supernatants from IL-10 Trx and IL-10+MMP9 Trx hMDM were both effective in promoting the conversion of unrelated donor macrophages to a pro-repair phenotype (decreased CD86 and HLA-DR, increased CD206, and similar levels of CD163). Figure 6 ).

[0639] Example 6: Compared with hMDM transfected with IL-10, hMDM transfected with IL-10+MMP9 showed better monocyte counts. Fundraising capacity

[0640] One of the important functions of macrophages is to recruit new monocytes in situ, thus enabling them to model themselves as pro-repair phenotypes. Figure 6Evidence was provided regarding the ability of IL-10 Trx and IL-10+MMP9 Trx hMDM to induce this phenotype in nonpolar macrophages. Figure 7 The data shown surprisingly support the view that only the supernatant from IL-10+MMP9 Trx hMDM induced significant monocyte migration when tested in PBMC migration assays.

[0641] Example 7: In terms of metalloproteinase activity, macrophages transfected with IL-10+MMP9 were superior to those transfected with IL-10. macrophages

[0642] A final key feature of macrophage therapies designed to induce tissue remodeling is their ability to digest extracellular matrix (ECM) components. This assay measures the ability of the total library of MMPs in the cell culture supernatant to digest ECM components using a fluorescent probe. Surprisingly, IL-10 Trx significantly reduced (-50%) the total MMP activity measured in the supernatant compared to NTrx hMDM. Notably, co-transfection with MMP9 alone was sufficient to restore this ability in the supernatant, making it even higher than in NTrx hMDM. The MMP activity of hMDM co-expressing IL-10 and MMP9 was 1.5 times that of NTrx hMDM, indicating that MMP9 and IL-10 synergistically increase the MMP activity of hMDM. This may also highlight the ability of MMP9 transfection to increase the activation of other MMPs. Data as follows Figure 8 As shown.

[0643] Example 8: In a chronic liver disease model, hMDM transfected with IL-10 and IL-10+MMP9 showed significant differences in efficacy 24 hours post-injection and It was located in the liver within 72 hours and was rapidly cleared from the lungs.

[0644] For macrophage therapy to be effective, it needs to be localized to the site of injury after injection. In this experiment, chronic liver disease was induced in immunodeficient mice (NSG strain) by injection of a toxin (CCl4). Four weeks after fibrosis induction, hMDM was injected via the tail vein, and the liver was retrieved at different time points for enzymatic digestion to recover the non-parenchymal portion. The results showed that both IL-10 Trx and IL-10+MMP9 Trx hMDM initially localized to the lungs and liver, but only persisted in the liver for 72 hours. As expected, the genetically engineered cells were cleared one week after injection. Figure 9 Therefore, they exhibit pharmacokinetics and distribution consistent with efficacy and safety.

[0645] Example 9: In a chronic liver disease model, hMDM transfected with IL-10 and IL-10+MMP9 maintained [their condition] 24 hours after injection. Expression of effective payload

[0646] In the same experiment described above, circulating human IL-10 and MMP9 were measured in mouse plasma at different time points following cell therapy injection. Interestingly, although macrophages were concentrated in the liver and lungs, IL-10 and MMP9 were detectable systemically (in blood circulation) in the expected mouse group. Figure 10 Appropriate controls should be implemented to ensure reliable detection of human proteins without cross-reactivity with their mouse counterparts.

[0647] Example 10: IL-10 and IL-10+MMP9 have good safety profiles in a chronic liver disease model.

[0648] Finally, for success in a clinical setting, cell therapy needs to be safe, both at the time of injection and at subsequent time points. In particular, it is crucial that the cell therapy exhibits no off-target effects in undamaged organs and is cleared within a safe timeframe. Data support the safety profile of both IL-10 Trx and IL-10+MMP9 Trx hMDM. Indeed, no embolism was detected at the time of injection, and no systemic inflammation was detected systemically in mice with chronic liver disease at different time points after injection (Table 3 and...). Figure 11 These data, plus Figure 10 The rapid clearance observed in the cells negates the possibility that these cells are tumorigenic or have any long-term toxicity.

[0649] Table 3 - Summary of safety features and results required to meet safety requirements.

[0650]

[0651] Example 11: Engineered macrophages overexpressing IL-10 specifically recruit monocytes. As described in this article, the measurement response is as follows Figure 12 PBMCs from hMDM-conditioned medium migrated as described in the treatments. "D6 UT" cells were removed after 6 days of culture, untransfected and without IL-4 or IL-13 transfection and treatment (as described in the recovery treatment). "D6 UT + TR" cells were removed after 6 days of culture, untransfected but treated with IL-4 / IL-13 (as described in the recovery treatment). The remaining treatment groups were transfected with the genes shown. Transfection with IL-10 alone induced the largest monocyte recruitment, and transfection with CCR2 was the only other treatment that produced macrophages capable of significantly recruiting monocytes. Figure 12 Only the recruitment of monocytes identified by flow cytometry is shown. Figure 13 Showing the use of Figure 12 The subset of macrophages treated under the conditions shown (UT and UT TR correspond to) Figure 12The recruitment of D6 UT and D6 UT TR cells (described in the text) in other cell types, such as those identified using flow cytometry (B cells, T cells, NK cells, neutrophils, and monocytes). Figure 13 The study demonstrated that IL-10 transfection specifically recruited monocytes, but not other cell types. Conditioned media from untransfected macrophages and macrophages overexpressing MMP9 had no effect on monocyte recruitment.

[0652] Example 12: Engineered macrophages overexpressing IL-10 transform both nonpolarized and pro-inflammatory macrophages. To promote phenotype repair. To test the ability of IL-10-overexpressing macrophages to convert monocyte-derived macrophages into pro-repair macrophages, the inventors tested the effect of CMs derived from IL-10-overexpressing macrophages on the phenotypes of M0 and M1 macrophages. When M0 macrophages (derived from monocytes incubated for 5 days in the presence of 100 ng / ml recombinant human macrophage colony-stimulating factor rhM-CSF, essentially as described in WO 2021 / 240162) were co-incubated with CMs derived from IL-10-overexpressing cells, they exhibited a spectrum of markers associated with pro-repair M2 macrophages (i.e., downregulation of HLA-DR & CD86 and upregulation of 25F9, CD206, and CD163). This effect was equivalent to incubating M0 macrophages with a medium containing M2 polarization mediators (TexsMACS + 50 ng / ml IL-10). Figure 14 Flow cytometry and measurement of mean fluorescence intensity (MFI) were used to evaluate surface markers.

[0653] Similarly, CM incubated with macrophages overexpressing IL-10 was able to "rescue" M1 macrophages and convert their phenotype to the pro-repair M2 phenotype. For testing, the CM was co-incubated with macrophages pre-polarized to the M1 phenotype using 100 ng / ml LPS + 50 ng / ml IFN-γ. This rescue capability is similar to that achieved using M2 polarization mediators (…). Figure 15 ).

[0654] Example 13: Engineered macrophages overexpressing IL-10 possess an anti-inflammatory secretomic profile. The secretion levels of pro-inflammatory cytokines were measured in macrophages overexpressing IL-10 alone, macrophages overexpressing MMP9 alone, macrophages overexpressing both IL-10 and MMP9, and untransfected non-polarized macrophages. Macrophages overexpressing IL-10 exhibited an anti-inflammatory secretomic profile. Specifically, when macrophages overexpressed IL-10, no secretion of pro-inflammatory factors such as TNF-α and IFN-γ was observed. Figure 16 Surprisingly, although macrophages that overexpress MMP9 alone showed increased secretion of pro-inflammatory cytokines such as IL2, IL12p70, IFNg, TNFα, and IL1β, Figure 16 However, macrophages overexpressing MMP9 and IL-10 showed similar secretion levels to macrophages overexpressing IL-10 alone. Figure 23 Similarly, the role of macrophages in systemic inflammation was assessed in vivo. Briefly, NSG mice were subjected to 4–5 weeks of CCl4 poisoning. In SD 23, mice were randomly assigned to receive either macrophages overexpressing IL-10 alone (IL-10 Trx hMDM) or macrophages overexpressing both IL-10 and MMP9 (IL-10-MMP Trx hMDM). Mice injected with phosphate-buffered saline (PBS) served as vehicle controls. IL1β and TNFα levels in mouse liver homogenates were measured using an MSD assay. Surprisingly, despite in vitro measurements of the pro-inflammatory secretomic profile of macrophages expressing MMP9 alone, neither macrophages overexpressing IL-10 alone nor those overexpressing both IL-10 and MMP9 induced an increase in inflammatory cytokines compared to the vehicle control. Figure 11 ).

[0655] Example 14: Engineered macrophages can be delivered to the liver and persist therein. Furthermore, intravenous injection of IL-10-overexpressing human macrophages into mice with modeled localized liver fibrosis, and their persistence for at least 72 hours post-administration, suggests that they can be delivered to the treatment site in patients with liver fibrosis. Figure 17 ).

[0656] Example 15: Engineered macrophages exhibit MMP and scar remodeling activity. To salvage the scar remodeling capacity of macrophages overexpressing IL-10, the inventors considered introducing matrix metalloproteinases (MMPs), as MMPs are known to play an important role in degrading scar tissue in inflamed liver (e.g., Campana et al, Nature Reviews Molecular Cell Biology, Vol. 22, pp. 608–624 (2021)). Since macrophages overexpressing either MMP9 or MMP12 exhibit the ability to maintain some phagocytic capacity, the inventors tested the scar remodeling capacity of macrophages expressing either MMP. Using a FRET-based fluorophore method to measure total MMP activity, both MMP9 and MMP12 induced an increase in total MMP activity, with MMP9 inducing a greater increase. Figure 1 ).

[0657] Example 16 - Recruiting macrophages engineered to overexpress both MMP9 and IL-10 in vitro and in vivo Nuclear cells. RTX001 macrophages were generated by transfecting hMDM with a single bicistronic mRNA containing sequences encoding both MMP9 and IL-10. Figure 20Results of a PBMC migration assay performed as described in Materials and Methods are shown, comparing NTRx cells and RTX001 macrophages in vitro. The ability of macrophages overexpressing both MMP9 and IL10 to recruit monocytes was also confirmed in vivo in a mouse model of liver fibrosis (liver injury induced by 4–5 weeks of CCl4 poisoning) 24 hours after administration. These assay results are as follows... Figure 21 As shown. NSG mice underwent 4–5 weeks of CCl4 poisoning. RTX001 and NTrx cells were administered intravenously to SD 23 mice, and readings were collected 24 hours after cell administration. Mice injected with phosphate-buffered saline (PBS) served as a mediator control. Flow cytometry counts were performed to determine the percentage of bone marrow cells (defined as CD45+, SiglecF-, Ly6G-, Tim4-, CD11b+) and classical monocytes (defined as CD45+, SiglecF-, Ly6G-, Tim4-, CD11b+, CD64-, Ly6Chi) recruited to mouse CD45 leukocytes. In vivo migration was defined as cell recruitment to the liver compared to the mediator control. Figure 21 As can be seen, compared with mice treated with untransfected cells (NTRx) or PBS, mice receiving cells overexpressing IL-10 and MMP9 (RTX001) showed an increased percentage of monocytes (right panel) and bone marrow cells recruited from total white blood cells (left panel).

[0658] Example 17 – Engineered macrophages transform liver cells into a pro-repair phenotype in vivo. NSG mice were subjected to CCl4 poisoning for 4–5 weeks. RTX001 and NTrx cells were administered intravenously to SD 23 mice, and readings were collected 24 hours after cell administration. Mice injected with phosphate-buffered saline (PBS) served as a mediator control. Mouse cytokine levels, including IL-10 in liver homogenate, were assessed using an MSD assay. Figure 22 As can be seen, administration of human cells (RTX001) overexpressing IL-10 and MMP9 induced an increase in IL-10 in mice (as measured in liver homogenate). This indicates that human IL-10 induces an anti-inflammatory phenotype, and given the well-known polarization characteristics of IL-10 (notably, untransfected, non-polarized cells did not induce any increase in IL-10 levels in mice), this suggests that mouse cells are transitioning to a pro-repair phenotype.

[0659] Example 18 - RTX 001 macrophages reduced α-SMA expression in vitro and in vivo.The beneficial effects of macrophage overexpression of IL-10 and MMP9 have been demonstrated in vivo in a mouse model of liver fibrosis (mice 4-5 weeks after CCl4 poisoning) one week after administration. When macrophages were intravenously injected into mice, reduced activation of scar-forming cells (i.e., activated hepatic stellate cells, HSCs) was observed in scar tissue compared with untransfected (NTrx) macrophages and PBS controls (as seen by staining for α-SMA, a marker of HSC activation, which was quantified in the results shown in the right figure). Figure 24 ).

[0660] To further demonstrate the role of IL-10 and MMP9-expressing macrophages (RTX001) in liver fibrosis, an in vitro system was developed using the LX-2 cell line (Sigma Aldrich, SCC064). The LX-2 cell line is a human hepatic stellate cell line that has been extensively characterized and shown to retain key features of hepatic stellate cells, thus serving as a suitable model for human liver fibrosis. In this system, LX-2 cells were activated by treating them with 50 ng / ml TGF-β in DMEM medium for 24 hours, a process that occurs during liver fibrosis. The cells were then incubated for another 24 hours in DMEM conditioned medium (CM), where the following macrophages grew for approximately 18 hours:

[0661] 1. Untreated / untransfected / UT human macrophages (non-Trx CM).

[0662] 2. Human macrophages expressing constructs encoding IL-10 and MMP9 (RTX001 CM).

[0663] 3. Human macrophages expressing a construct encoding IL-10 alone (control CM).

[0664] For each of these groups, conditioned medium from macrophages from six different donors was used as a biological replicate. After incubation with conditioned medium, we measured the number of LX-2 cells expressing α-SMA (Figure B) and the expression level of α-SMA in the cells (Figure B). Figure 25 A). Since α-SMA is a marker of activation of human hepatic stellate cells, a decrease in its level indicates reduced activation. Because hepatic stellate cells are involved in the formation of liver scarring, a decrease in their activation predicts a reduction in liver fibrosis. Figure 25 As shown in Figure B, LX-2 cells treated with RTX001 showed a decrease in the number of α-SMA-expressing cells and lower α-SMA expression, indicating the inhibitory effect of RTX001 on hepatic stellate cell activation.

[0665] Example 19 - Optimized bicistronic mRNA leads to improved IL-10 secretion. like Figure 26As shown in the right figure, transfection with optimized bicistronic mRNA resulted in significantly higher IL-10 secretion. Surprisingly, despite IL-10's inhibitory effect on MMP9 secretion, such as... Figure 1 As shown in B, compared with untransfected macrophages (NTRx), the optimized sequence resulted in significantly higher MMP9 secretion and significantly higher MMP activity. Figure 26 (See the left and bottom images). In these experiments, the conditions and mRNA concentrations were the same for both optimized and unoptimized mRNAs.

[0666] Example 20 – Engineered macrophages are stable in inflammatory environments. In the livers of patients with advanced chronic liver disease, there is an accumulation of pro-inflammatory macrophages that secrete pro-inflammatory cytokines and enhance the inflammatory response (see Campana et al, 2021).

[0667] To demonstrate that macrophages expressing IL-10+MMP9 do not revert to a pro-inflammatory phenotype once administered to patients and exposed to an inflammatory environment in the liver, we conducted a stability study. In this study, we demonstrated the stability of the pro-repair phenotype of macrophages expressing IL-10+MMP9 in an inflammatory environment (by modeling the liver environment of patients with end-stage chronic liver disease using hIFN-γ levels in patients).

[0668] To test the stability of the phenotype, we used the following experimental procedure to test the phenotype of cells after co-incubation with hIFN-γ:

[0669] To obtain macrophages, PBMCs were isolated from steady-state leukapheresis or active blood samples. CD14+ cells were isolated from PBMCs and plated in TexMacs + M-CSF for 5 days to differentiate into macrophages. On day 5 of culture, macrophages were harvested and transfected with IL-10 + MMP9 or left untransfected. After transfection, macrophages were incubated overnight (~16 hours) at 37°C and 5% CO2 in TexMacs + 100 ng / mL M-CSF, IL-4 (20 ng / mL), and IL-13 (20 ng / mL). After the resting period, macrophages were harvested and plated at a density of 2 × 10⁵ cells / well in TexMacs with or without stimulation (hIFN-γ) in U-bottom ultra-low adhesion sterile culture plates (Corning Cat #7007). The concentration of hIFN-γ was calculated using the following information: Total IFN-γ in human liver was calculated using the total IFN-γ concentration in the liver of damaged mice, which was determined through previous in vivo pharmacological experiments and scaled up to the weight of human liver. The total IFN-γ level was then divided by the planned treatment cell dose to obtain the IFN-γ level per cell. This was used to establish a concentration gradient to account for the possibility of patients experiencing IFN-γ levels higher or lower than the calculated concentration. All groups were plated to provide three technical replicates. Macrophages were then cultured for 24 hours, followed by centrifugation at 300xG for 5 minutes. The culture supernatant was collected and frozen at -20°C, and the cells were used for flow cytometry to assess the levels of CD80, CD86, MHCII, 25F9, CD14, CD206, and CD163 present on the cell surface. After staining, cells were analyzed on Novocyte Quanteon, and data were analyzed using NovoExpress and GraphPad Prism software. There were no significant changes in pro-inflammatory markers (HLA-DR, CD80, and CD86) or macrophage recognition marker CD206.

[0670] from Figure 27 It can be seen that the phenotype of cells expressing MMP9 and IL10 is stable in an inflammatory environment.

[0671] Example 21 - The expression of macrophage markers varies among macrophage products. Macrophage products consisted of: untransfected and untreated (UT) or treated with IL-4, IL-13, and M-CSF (UT + TR); transfected with IL-10 and MMP9 and untreated (IL-10 MMP9 TRx) or transfected and treated with IL-4, IL-13, and M-CSF (IL-10 MMP9 TRx + TR). Mean fluorescence intensity was measured by flow cytometry as described herein, and the results are as follows: Figure 28 As shown.

[0672] Example 22 - Post-transfection treatment did not lead to further polarization of engineered macrophages, but it did improve low... Temperature resistance. In cells transfected with bicistronic constructs encoding MMP9 and IL-10, the expression of CD86 (A) and MHC II (B) was measured, either without treatment after transfection (IL-10-MMP9) or further treated with IL-4, IL-13, and M-CSF (IL-10-MMP9+ TR). No difference in CD86 and MHC II expression was detected between the IL-10-MMP9 and IL-10-MMP9+ TR groups. Figure 29 Therefore, due to IL-10 secretion, transfected cells are completely self-polarized (e.g., the reduction in HLA-DR and CD86 is solely due to IL-10 secretion by transfected cells). However, as... Figure 30 As shown, untransfected (NTRx) or transfected (TRx) macrophages incubated with IL-4+IL-13 exhibited higher resistance to low temperatures, as measured by the percentage of cells that remained viable after cryopreservation.

[0673] Example 23 - Measuring the anti-fibrotic properties of engineered macrophages in an immunocompromised mouse model of liver fibrosis able

[0674] The macrophages described in this article are of human origin. To understand their behavior in mammalian systems, immunocompromised mice that allow for transient implantation of human material in vivo are required. Otherwise, immune-active wild-type recipient strains would lead to rapid elimination of the administered human cells via xenogeneic acute rejection mechanisms. The highly immunocompromised “NSG” strain is genetically modified to eliminate the activity of host T cells, B cells, and NK cells. Not wishing to be bound by theories or mechanisms, the mode of action (MoA) of macrophages depends in part on the recruitment of host innate immune effector cells, including monocytes (Thomas et al. 2011. Ma et al. 2017). While NSG mice can accept transfer of human cells, they lack several host functional immune cells that provide a secondary immune response following macrophage treatment. Therefore, the full pharmacological effects, as expected in humans, cannot be modeled in immunocompromised strains. Furthermore, due to species differences in receptor binding and downstream signaling pathways, some human proteins in the macrophage secretome may not function in mice. Therefore, the limitations of immunocompromised strains, in the best-case scenario, would underestimate or completely exclude the absolute efficacy evidence of macrophages described in this article (evidence of a statistically significant reduction in liver fibrosis). However, the in vivo experimental model used to test efficacy is described below.

[0675] The macrophages used in this embodiment were primary human monocyte-derived macrophages (hMDM) that had been phenotypically modified via transient transfection to provide bicistronic mRNA transcripts encoding IL-10 and MMP-9 with a P2A self-cleaving peptide. To demonstrate the antifibrotic properties of the tested cell therapy, experimental liver fibrosis was modeled in an immunocompromised mouse strain. NSG mice (full name: NOD.Cg-PrkdcSCID Il2rgtm1Wjl / SzJ, from Charles River Laboratories) lacked T cells, B cells, and NK cells, resulting in immunodeficiency that allowed for transient in vivo implantation of human cells. Liver fibrosis was induced in NSG mice by twice-weekly administration of carbon tetrachloride (CCl4, intraperitoneal injection, 0.4 μL / g body weight, diluted in olive oil) for 12 weeks. The CCl4 fibrosis model is a well-established and easily manageable rodent fibrosis model, and its clinical predictability with obeticholic acid (Fan et al., 201947; Younossi et al., 201948) and rennetine (Wettstein et al., 201749; Francque et al., 202150) has been demonstrated. When establishing liver fibrosis, cells (1 × 10⁻⁶) are delivered intravenously 8 weeks after CCl4 administration. 6 To test the antifibrotic properties of the tested macrophages, PBS-treated damaged mice were used as a control. Additionally, untransfected human monocyte-derived macrophages (NTrx hMDM, 1x10⁻¹) were used. 6Damaged mice (intravenously) served as a control group for the engineered step. Twenty-four hours after the 17th dose of CCl4, cells or the medium alone (100 μL, intravenously, in PBS) were administered. All mice continued to receive CCl4 for an additional 4 weeks. All mice were humanely euthanized by exsanguination 24 hours after the 24th and final doses of CCl4, under terminal anesthesia and prior to cervical dislocation. Whole blood was collected via cardiac puncture and processed to release plasma or serum for assessment of liver chemobiomarkers. Liver, spleen, lung, heart, and kidney were harvested and fixed for histological analysis. To determine the antifibrotic properties of macrophages, picrosirius red (PSR) staining was used in histological liver sections to visualize collagen fibers and quantify liver fibrosis. PSR-stained sections were digitized using a microscopic slide scanner (Zeiss Axioscan, Zeiss AG) and liver fibrosis was quantified using image analysis software (Zen, Zeiss AG). The antifibrotic properties of macrophages were evaluated by comparing the percentage of PSR in mice treated with macrophages with those treated with the mediator alone or NTrx-hMDM.

[0676] Example 24 - Measuring the anti-inflammatory effects of engineered macrophages in an immunocompromised mouse model of fibrotic fatty liver disease Fiberization properties

[0677] To demonstrate the antifibrotic properties of the engineered macrophages of Example 23, experimental liver fibrosis was modeled in an immunocompromised mouse strain with a background of fatty liver disease. NSG mice (full name: NOD.Cg-PrkdcSCIDIl2rgtm1Wjl / SzJ, from Charles River Laboratories) lacked T cells, B cells, and NK cells, resulting in immunodeficiency that allowed for transient in vivo implantation of human cells. Liver fibrosis was induced in mice by feeding them a choline-deficient, amino acid-restricted high-fat diet (CDAA HFD) with restricted methionine content. Random feeding of CDAA HFD induced hepatic steatosis and inflammation, leading to a pronounced fibrotic histological pattern, but no complete weight loss was observed in the animals. The CDAA HFD model is valuable because, unlike other fibrosis models, CDAA HFD-induced fibrosis regresses slowly and exhibits stable fibrosis for at least two weeks after cessation of dietary damage. Therefore, the characteristics of this model allow for the evaluation of the test article in the absence of ongoing damage. Here, NSG mice were given CDAA-HFD (random) for 12 weeks to induce liver fibrosis in the context of fatty liver disease. After 12 weeks, the mice were switched back to a standard diet. The anti-fibrotic properties of engineered macrophages were tested after intravenous administration following restoration to a normal diet. Damaged mice receiving PBS alone served as a mediator control. Additionally, untransfected human monocyte-derived macrophages (NTrx hMDM, 1x10⁻¹) were also used. 6 Mice with liver fibrosis (intravenous injection) were used as a control group for the engineered steps. Twenty-four hours after resuming a standard (RM3) diet, cells or a single medium (100 μL, intravenous, in PBS) were administered. All mice continued on the standard diet for an additional four weeks. At the end of the study, all mice were humanely euthanized by exsanguination under terminal anesthesia prior to cervical dislocation. Whole blood was collected via cardiac puncture and processed to release plasma or serum for assessment of liver chemobiomarkers. Liver, spleen, lung, heart, and kidney were harvested and fixed for histological analysis. To measure liver fibrosis, histological liver sections were stained with Sirius red (PSR) to visualize collagen fibers. PSR-stained sections were digitized using a microscopic slide scanner (ZeissAxioscan, Zeiss AG), and liver fibrosis was quantified using image analysis software (QuPath image analysis open-source software). The antifibrotic properties of engineered macrophages were evaluated by comparing the percentage of PSR-positive staining in macrophage-treated mouse samples with those from mice treated with either the macrophage alone or NTrx-hMDM.

[0678] Table 4. Exemplary structural and functional characteristics of engineered macrophages

[0679]

[0680] Table 5. Further exemplary structural and functional characteristics of engineered macrophages

[0681]

[0682] in conclusion

[0683] In summary, the genetic engineering of IL-10 in combination with MMP9 provides many of the features and functions required for cell therapy, such as for the prevention / treatment of inflammatory conditions and / or organ damage regeneration. The combination of IL-10 and MMP9 provides:

[0684] - Reliable macrophage properties, unaffected by engineering processes.

[0685] - Strong anti-inflammatory phenotype.

[0686] - The ability to pattern primitive macrophages into a pro-repair phenotype.

[0687] - Excellent ability to absorb prey.

[0688] - Reassuring safety and biodistribution characteristics, including penetration in damaged organs and rapid clearance / loss in other organs.

[0689] The aforementioned characteristics are shared with macrophages engineered using IL-10 alone. However, the combination of IL-10 and MMP9 provides some specific and surprising characteristics that are key to delivering the desired therapeutic effects, such as:

[0690] - The powerful ability to attract monocytes, which is then patterned into a pro-repair phenotype.

[0691] - The ability to restore MMP activity (a substitute for fibrosis / ECM remodeling) that was eliminated by IL-10 alone.

[0692] Therefore, we believe that engineered macrophages with a combination of IL-10 and MMP9 will provide an effective product with anti-inflammatory and anti-fibrotic functions in several organ injury settings, both acute and chronic (remodeling of ECM components is also crucial in acute injury settings to ensure tissue restoration to its original state and proper regeneration).

[0693] Example 25: Trial of Autologous Mononuclear Cell-Derived Macrophage Therapy in Humans

[0694] To evaluate the safety, tolerability, and efficacy of engineered autologous monocyte-derived macrophages (RTX001, as described above) overexpressing IL-10 and MMP9, an open-label phase 1 / 2 multicenter clinical trial (EMERALD) was conducted in participants with decompensated cirrhosis, particularly those with cirrhosis who were hospitalized for the first time due to hepatic decompensation. Details of the study are described below.

[0695] Qualification Standards

[0696] The safety and efficacy of autologous monocyte-derived macrophage products will be evaluated in subjects who become clinically stable after experiencing an qualifying event. Subjects will also be assessed for the following inclusion criteria and will only be included in the trial if they also meet all of the following inclusion criteria (and have no exclusion criteria):

[0697] Inclusion criteria

[0698] i. Participants must be between 18 and 75 years old.

[0699] ii. A diagnosis of cirrhosis is made based on at least one of the following:

[0700] a. Clinical and radiological features relevant to the diagnosis of cirrhosis;

[0701] b. Liver stiffness score >15 kPa as measured by transient elastography (TE; Fibroscan); and

[0702] c. Previous liver biopsy confirmed the histological features of cirrhosis.

[0703] iii. The following are causes of liver disease:

[0704] a. Pure metabolic dysfunction-related steatotic liver disease (MASLD) or metabolic and alcohol-related / associated liver disease (Met-ALD) or HCV sustained virological response (HCV SVR) with persistent cirrhosis.

[0705] b. Alcohol-related liver disease (ALD or Met-ALD), only if the subject is confirmed to have consumed alcohol within the Met-ALD limit, defined as an intake of >14 units / week, or more than 2 units / day for women and more than 3 units / day for men. One unit equals 8g of alcohol: half a pint (~240 mL) of beer, one glass (125 mL) of wine, or one serving (25 mL) of spirits.

[0706] c. Alcohol-related liver disease (ALD or Met-ALD), only if the subject's PEth test value is <25 mg / L.

[0707] iv. The Model for End-Stage Liver Disease (MELD) score of 12–20 was required within two weeks of an “eligible” decompensation event. If participants were eligible for outpatient status, their MELD 3.0 score had to be 12–20 at screening (Visit 1).

[0708] v. There are no known contradictions with G-CSF or apheresis procedures.

[0709] vi. Inpatient treatment as a hospitalized patient due to a major hepatic decompensation event, including ascites, hepatic encephalopathy, variceal bleeding, hepatorenal syndrome / acute kidney injury (HRS / AKI) or spontaneous bacterial peritonitis (SBP), which is the only hospitalization for hepatic decompensation event in the past 6 months.

[0710] vii. Outpatients: Refractory ascites (only): defined as ascites that cannot be mobilized or recurs within 8 weeks after LVP. The date of onset of refractory ascites is defined as the time of the second LVP.

[0711] Participants were also evaluated according to the following exclusion criteria, and were excluded if they met any of the following criteria:

[0712] Exclusion criteria

[0713] i. Cirrhosis due to the following reasons:

[0714] a. Any viral hepatitis other than hepatitis C virus (HCV SVR), or

[0715] b. Autoimmune and cholestatic causes, including but not limited to primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC).

[0716] ii. Acute liver disease without underlying cirrhosis, including but not limited to drug-induced liver injury (DILI).

[0717] iii. Any current organ failure requiring supportive care beyond that of an outpatient and unrelated to the participant's liver decompensation event.

[0718] iv. Splenomegaly ≥16 cm

[0719] v. Thrombocytopenia < 50 × 10 9 / L

[0720] vi. Any of the following comorbidities are present or suspected:

[0721] a. History of liver transplant or other organ transplant

[0722] b.ACLF

[0723] c. Sepsis (positive microbial culture), or, according to the PI's definition, unless stable and at least 4 weeks after completion of the full course of IV antibiotics, human immunodeficiency virus (HIV).

[0724] d. Known human immunodeficiency viruses

[0725] e. Human T-lymphotropic virus 1

[0726] f. Pulmonary embolism (PE)

[0727] g. Hepatocellular carcinoma (HCC) or active malignant disease within the past 5 years (excluding non-melanoma skin cancer, cervical carcinoma in situ, superficial bladder cancer, benign polyps, etc.)

[0728] h. Liver complications, such as portal vein thrombosis

[0729] i. Exclude patients with hepatic pleural effusion unless it is a small amount of pleural effusion that is not clinically apparent and is incidentally detected by radiological evaluation without clinical intervention.

[0730] j. Chronic renal impairment (on dialysis) or unresolved acute kidney injury (AKI).

[0731] k. Acute or chronic heart failure (New York Heart Association Class III / IV)

[0732] 1. Portal pulmonary hypertension

[0733] m. Severe chronic lung disease, such as chronic obstructive pulmonary disease (COPD) or interstitial lung disease, where FEV1 is less than 50% and / or FEV1 / FVC is less than 60%.

[0734] n. Hepatopulmonary syndrome

[0735] o. History of chronic albumin therapy or current treatment

[0736] p. Major untreated / unstable mental illness

[0737] q. Tips from the past 6 months

[0738] vii. At the researcher's discretion, any evidence of life-threatening or clinically significant comorbidities may limit adherence to research procedures.

[0739] viii. Currently using or planning to use immunomodulatory or immunosuppressive drugs; Note: Low doses of corticosteroids, up to 10 mg / kg / day of prednisone or equivalent, or inhaled steroids are permitted to control asthma.

[0740] ix. Has received gene or cell therapy at any time.

[0741] x. Participants with known hypersensitivity to dimethyl sulfoxide (DMSO)

[0742] xi. If a participant may not comply with research procedures, limitations, and requirements, the researcher shall make the judgment that the participant should not participate in the research.

[0743] xii. For female participants only—those who are pregnant or breastfeeding, plan to become pregnant in the following year, or are likely to have children and do not wish to comply with contraception requirements.

[0744] xiii. Current / recent alcohol abuse in the three months prior to screening, defined as alcohol intake greater than 3 units / day for women and greater than 4 units / day for men, or binge drinking (>14 units / day), as determined by the investigator. Note: One unit equals 14 g of alcohol: half a pint (~240 mL) of beer, one glass (125 mL) of wine, ...

Claims

1. A method for treating cirrhosis in a subject, wherein the method comprises administering engineered macrophages to the subject to overexpress IL-10.

2. A method for treating cirrhosis in a subject by engineering macrophages that overexpress IL-10, wherein the method includes administering the engineered macrophages to the subject.

3. The method of claim 1 or the engineered macrophages of claim 2, wherein the subject (a) experienced a first hepatic decompensation event requiring hospitalization; or (b) suffered from refractory ascites, optionally wherein the refractory ascites began within six months prior to administration of the macrophages.

4. The method or engineered macrophages according to any one of claims 1-3, wherein the subject exhibits one or more clinical signs of hepatic decompensation, or has recovered from one or more clinical signs of hepatic decompensation, or has become clinically stable after one or more clinical signs of hepatic decompensation, wherein the one or more clinical signs of hepatic decompensation are optionally selected from ascites, hepatic encephalopathy, variceal bleeding (variceal bleeding), hepatorenal syndrome / acute kidney injury (HRS / AKI) and / or spontaneous bacterial peritonitis (SBP).

5. The method or engineered macrophages according to any one of claims 1-4, wherein the macrophages are autologous or allogeneic to the subject.

6. The method or engineered macrophages of claim 5, wherein the macrophages are autologous to the subject, and wherein the macrophages are differentiated from monocytes, the monocytes being obtained from the subject by apheresis followed by separation of the monocytes, optionally by incubating the monocytes in the presence of M-CSF.

7. The method of claim 6, wherein the subject is treated with G-CSF before obtaining mononuclear cells from the subject via apheresis; or the engineered macrophages of claim 6, wherein the subject has previously been treated with G-CSF and then mononuclear cells are subsequently obtained from the subject via apheresis; optionally, wherein the G-CSF is recombinant G-CSF, and optionally, wherein the recombinant G-CSF is filgrastim.

8. The method or engineered macrophages according to any one of claims 3-7, wherein the subject is confirmed to be clinically stable after the first hepatic decompensation event requiring hospitalization and before the administration of the engineered macrophages.

9. The method or engineered macrophages of claim 7 or 8, wherein the subject is confirmed to be clinically stable after the first hepatic decompensation event requiring hospitalization and before treatment with G-CSF, optionally wherein the subject was clinically stable for at least 6 weeks prior to treatment with G-CSF.

10. The method or engineered macrophages according to any one of claims 3-7, wherein the subject experienced a subsequent additional liver decompensation event after the first liver decompensation event requiring hospitalization and before the administration of the engineered macrophages to the subject, optionally wherein the subject was confirmed to be clinically stable after the subsequent additional liver decompensation event and before the administration of the engineered macrophages to the subject.

11. The method or engineered macrophages of any one of claims 10, wherein the engineered macrophages are autologous to the subject, wherein the subject experienced a subsequent additional liver decompensation event after the first decompensation event and after obtaining mononuclear cells from the subject via apheresis to manufacture engineered macrophages.

12. The method or engineered macrophages of claim 10 or 11, wherein the subject is confirmed to be clinically stable after a subsequent additional hepatic decompensation event and before the engineered macrophages are administered to the subject.

13. The method or engineered macrophages according to any one of claims 1-9, wherein the subject has recovered from (or has become clinically stable) their first hepatic decompensation event, and wherein the subject has not experienced any additional hepatic decompensation events since recovering from (or becoming clinically stable after) the first decompensation event.

14. The method or engineered macrophages according to any one of claims 1-13, wherein the subject is screened for suitability for treatment prior to administration of the engineered macrophages.

15. The method or engineered macrophages according to any one of claims 1-14, wherein the cause of the cirrhosis is: (a) Malabsorption-associated steatosis liver disease (MASLD), (b) Metabolic and alcohol-related / associated liver disease (Met-ALD), (c) HCV sustained virological response (HCV SVR) with persistent cirrhosis, or (d) Alcohol-related liver disease (ALD or Met-ALD), provided that the patient’s alcohol consumption does not exceed the Met-ALD limit and the value in the PEth alcohol test is <25 mg / l.

16. The method or engineered macrophages according to any one of claims 1-15, wherein the subject has no known contraindications to G-CSF or apheresis.

17. The method or engineered macrophages according to any one of claims 1-16, wherein the subject has a Chronic Liver Failure Association acute decompensation score greater than 45.

18. The method or engineered macrophages according to any one of claims 1-17, wherein the subject's end-stage liver disease model 3.0 (MELD 3.0) score is 12-20.

19. The method or engineered macrophages according to any one of claims 1-17, wherein the diagnosis of cirrhosis is made based on the presence of at least one of the following: (a) Meeting the clinical or radiological characteristics of cirrhosis; (b) Liver stiffness measurements (LSM) measured using transient elastography (TE) are greater than 15 kPa; and (c) Histological features of cirrhosis confirmed by prior liver biopsy.

20. The method or engineered macrophages of claim 18, wherein the diagnosis of cirrhosis is made based on the presence of all of (a)-(c).

21. The method or engineered macrophages according to any one of claims 1-20, wherein the macrophages are additionally engineered to overexpress MMP9.

22. The method or engineered macrophage according to any one of claims 1-21, wherein the engineered macrophage comprises an exogenous coding sequence for IL-10 and an exogenous coding sequence for MMP9.

23. The method or engineered macrophages according to any one of claims 1-22, wherein the expression of the exogenous coding sequence has a synergistic effect in restoring MMP activity compared to engineered macrophages containing a single exogenous IL-10 sequence, and / or a synergistic effect in monocyte recruitment of macrophages.

24. The method or engineered macrophage of any one of claims 22 or 23, wherein the macrophage and / or coding sequence is human.

25. The method or engineered macrophage according to any one of claims 22-24, wherein the exogenous coding sequence of IL-10 encodes a protein having at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO: 4, optionally wherein the IL-10 protein comprises the same amino acid sequence as SEQ ID NO:

4.

26. The method or engineered macrophage according to any one of claims 22-25, wherein the exogenous coding sequence of MMP9 encodes a protein having at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO: 6, optionally wherein the IL-10 protein comprises the same amino acid sequence as SEQ ID NO:

6.

27. The method or engineered macrophage according to any one of claims 22-26, wherein the exogenous coding sequence is present on one or more nucleic acid molecules or integrated into the genome of the macrophage.

28. The method or engineered macrophage of claim 27, wherein the nucleic acid molecule is a DNA or RNA molecule, preferably an mRNA molecule, optionally wherein IL-10 and MMP9 are expressed by the same mRNA molecule, further optionally wherein the mRNA molecule encodes IL-10 and MMP9 linked by a adapter sequence, further optionally wherein the adapter is a self-cleaving 2A adapter, further optionally wherein the adapter is p2A.

29. The method or engineered macrophage of claim 28, wherein the nucleic acid molecule is an mRNA molecule comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same sequence as SEQ ID NO: 13, optionally wherein the nucleic acid comprises SEQ ID NO:

13.

30. The method or engineered macrophage according to claim 28 or 29, wherein the nucleic acid molecule is an mRNA molecule comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same sequence as SEQ ID NO: 14, optionally wherein the nucleic acid comprises SEQ ID NO:

14.

31. The method or engineered macrophage according to any one of claims 28-30, wherein the nucleic acid molecule is an mRNA molecule encoding IL-10 and MMP9 linked by a adapter sequence, and wherein the adapter sequence encodes a protein comprising the amino acid sequence described in SEQ ID NO: 7, optionally wherein the protein encoded by the adapter sequence comprises the amino acid sequence described in SEQ ID NO:

9.

32. The method or engineered macrophage according to any one of claims 28-31, wherein the nucleic acid molecule is an mRNA molecule encoding IL-10 and MMP9 linked by a adapter sequence, and wherein the adapter sequence comprises an mRNA having the sequence described in SEQ ID NO:

15.

33. The method or engineered macrophage according to any one of claims 28-32, wherein the nucleic acid molecule is an mRNA molecule comprising at least 80% identical, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 10, optionally wherein the mRNA further comprises a polyA tail of 65 to 250 residues long, preferably 90 to 120 residues long, preferably about 1 / 2', and / or a 5' cap.

34. The method or engineered macrophage according to any one of claims 28-33, wherein the nucleic acid molecule is an mRNA molecule comprising at least 80% identical, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 16, optionally wherein the mRNA further comprises a 5' cap.

35. The method or engineered macrophage of claim 34, wherein the mRNA molecule contains chemically modified residues, preferably modified uracil residues, and optionally at least one synthetic cap.

36. The method or engineered macrophages according to any one of claims 22-35, wherein the exogenous coding sequence of IL-10 and the exogenous coding sequence of MMP9 are on the same nucleic acid.

37. The method or engineered macrophage according to any one of the preceding claims, wherein the macrophage is engineered by editing the endogenous promoter of the IL-10 gene and / or the MMP9 gene, or wherein the macrophage is engineered by regulating the expression of an endogenous silencing RNA or by introducing an exogenous silencing RNA sequence, optionally wherein the silencing RNA is a miRNA.

38. The method or engineered macrophage according to any one of the preceding claims, wherein the metalloproteinase activity level of the engineered macrophage is at least 1.5 times that of the metalloproteinase activity of the non-engineered macrophage.

39. The method or engineered macrophages according to any one of the preceding claims, wherein the CD86 expression of the engineered macrophages is reduced by at least two-fold compared to non-engineered, non-polarized cells.

40. The method or engineered macrophages according to any one of the preceding claims, wherein the HLA-DR expression of the engineered macrophages is reduced by at least two-fold compared to non-engineered, non-polarized cells.

41. The method or engineered macrophage according to any one of the preceding claims, wherein the engineered macrophage secretes at least 1000 times more IL-10 than non-engineered, non-polarized cells.

42. The method or engineered macrophage according to any one of the preceding claims, wherein the engineered macrophage secretes MMP3 at least 10-fold more than non-engineered, non-polarized cells.

43. The method or engineered macrophage according to any one of the preceding claims, wherein the engineered macrophage secretes at least 20-fold more MMP10 than non-engineered, non-polarized cells.

44. The method or engineered macrophages according to any one of the preceding claims, wherein when 4 x 10 6 When cultured in vitro at a cell concentration of at least 10,000 pg / ml, the engineered macrophages secrete IL-10 at a culture supernatant concentration of at least 10,000 pg / ml.

45. The method or engineered macrophages according to any one of the preceding claims, wherein when 4 x 10 6 When cultured in vitro at a cell concentration of at least 200 ng / ml, the engineered macrophages secrete MMP9 at a culture supernatant concentration of at least 200 ng / ml.

46. ​​The method or engineered macrophage according to any one of the preceding claims, wherein the CD206 expression of the engineered macrophage is increased by at least 5-fold compared with that of monocytes.

47. The method or engineered macrophages according to any one of the preceding claims, wherein the 25F9 expression of the engineered macrophages is increased by at least 5-fold compared to monocytes.

48. The method or engineered macrophages according to any one of the preceding claims, wherein the CD80 expression of the engineered macrophages is reduced by at least 10 percent compared with that of unengineered, non-polarized cells.

49. The method or engineered macrophages according to any one of the preceding claims, wherein when 4 x 10 6 When cultured in vitro at a cell concentration of 40 pg / ml, the engineered macrophages secreted TNF-α at a culture supernatant concentration of up to 40 pg / ml.

50. The method or engineered macrophage according to any one of the preceding claims, wherein the engineered macrophage has at least the same phagocytic capacity as unengineered, non-polarized cells.

51. The method or engineered macrophages according to any one of the preceding claims, wherein the metalloproteinase activity of the engineered macrophages is restored relative to the reduced metalloproteinase activity in macrophages engineered solely with the IL-10 coding sequence.

52. The method or engineered macrophages according to any one of the preceding claims, wherein the macrophages are transiently transfected, optionally via electroporation.

53. The method or engineered macrophages of claim 51, wherein the transfection is non-viral.

54. The method or engineered macrophage according to any one of the preceding claims, wherein the macrophage has a pro-repair phenotype.

55. The method or engineered macrophages according to any one of the preceding claims, wherein one or more doses of the engineered macrophages are administered to the subject.

56. The method or engineered macrophages of claim 55, wherein the subject is administered a dose of the engineered macrophages greater than one dose, and the dose is administered to the subject at 6-week intervals.

57. The method or engineered macrophages according to any one of the preceding claims, wherein the engineered macrophages are produced at a concentration of 1 × 10⁻⁶. 8 A dose of one macrophage was administered to the subject.

58. The method or engineered macrophages according to any one of the preceding claims, wherein the subject is administered 4 doses of the engineered macrophages.

Images