Therapeutic macrophages

Abstract

The present invention relates to a macrophage that is genetically engineered to overexpress an interleukin-10 (IL-10) or an IL-10 in combination with a matrix metallopeptidase 9 (MMP9). Such macrophages may be used to treat inflammatory conditions, such as inflammatory organ injury, in a subject. The inflammatory condition may be acute or chronic, and may involve a fibrotic component.

Description

Technical Field

[0001] The present invention relates to macrophages that are genetically engineered to express a combination of interleukin-10 (IL-10) and matrix metallopeptidase 9 (MMP9). The macrophages are preferably engineered with exogenous nucleic acids encoding IL-10 and MMP9, and in some cases, they can be expressed from the same nucleic acid molecule. Such macrophages can be used to treat inflammatory conditions in a subject, such as inflammatory organ injury. The inflammatory condition can be acute or chronic and may involve a fibrotic component. The present invention also relates to populations of such macrophages or their populations, compositions comprising such macrophages or their populations, and methods of using such macrophages or their populations. The present invention also relates to methods of engineering such macrophages, for example, including transient transfection with a combined IL-10 and MMP9 mRNA construct. Background Art

[0002] Fibrosis is the final common pathway of chronic diseases of various etiologies, including toxic injury, viral infection, metabolic and genetic diseases, 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 essentially depends on the duration and recurrence of injury. The liver provides a biological paradigm for fibrogenesis and remodeling. Advanced chronic liver fibrosis, also known as cirrhosis, is a life-threatening condition 1,2 . Since the 1970s, deaths due to liver disease have been the only major cause of death that has steadily increased year by year in the UK and remain 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 morbid development in the clinical course of cirrhosis [Trebicka 2020 43 .], and is the most common cause of hospitalization in patients with cirrhosis [Moreau 2013 46 .]. Patients with HD are at high risk of short-term death [Moreau2013 46 .]. The first episode of HD (also referred to herein as the first hepatic decompensation event), which usually requires hospitalization, marks the transition from compensated cirrhosis to decompensated cirrhosis. Decompensated cirrhosis is characterized by recurrent episodes of HD 42HD has two different clinical presentations, depending on the presence of other organ failure and the degree of systemic inflammation. The presence of multiple organ failure and high systemic inflammation is characteristic of acute-on-chronic liver failure (ACLF), a syndrome associated with very high 28-day mortality. HD associated with moderate systemic inflammation without involvement of other organs has a lower 28-day mortality (∼2%, although this increases to 10% at 90 days, indicating a heterogeneous clinical course in HD patients) [Trebicka 2020 43 , but still portends poor outcomes in subsequent years.

[0003] The only treatment for liver injury is removal of the harmful stimulus (e.g., administration of an effective antiviral therapy) and liver transplantation. Treatment of liver failure caused by acute or chronic injury is limited to supportive care and / or transplantation, the latter requiring a donor. There is a current lack of available organ donors, and the surgery carries a significant risk of morbidity and mortality. In addition, patients will also be committed to lifelong immunosuppression. Moreover, although specific etiological treatment 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) do not yet have approved medical therapies, and patients typically present late when cirrhosis and related complications have already occurred [Starkey 2019 44 . Thus, there is no specific therapy for cirrhosis, and as such, providing effective antifibrotic therapies is a major unmet clinical need in chronic and acute liver injury 4-6 .

[0004] Macrophages (MΦ) play a key role in the inflammatory response of the damaged liver. There are two main MΦ populations in the liver: (i) resident macrophages (Kupffer cells, KC), and (ii) infiltrating macrophages. KC perform a patrolling function in the hepatic sinusoids to phagocytose microbial debris that reaches the liver via the sinusoidal capillaries under steady-state conditions. In the early stages of liver injury, KC express chemokines such as CCL2 and CCL5, thus contributing to the recruitment of monocytes from the circulation 2,7 . During fibrosis, the number of KC decreases; then they repopulate the liver during the recovery phase of self-limiting fibrosis 8。Infiltrating monocyte-derived MΦ (MDM) play a major role in the response to liver injury. Infiltrating MΦ are recruited via the CCR2 / CCL2 axis; once in the liver parenchyma, they localize along fibrotic septa in the early stages of liver fibrosis and may promote fibrosis by releasing factors such as TGF-β, IL1, PDGF, and CCL2, which activate hepatic stellate cells and exacerbate inflammation. This may suggest a detrimental role of MΦ in progressive fibrosis. However, if MΦ are depleted at the onset of fibrotic remodeling, the remodeling process fails and liver fibrosis persists. It is now generally accepted that macrophages play a dual role in the formation and resolution of fibrosis 2,8-11 。

[0005] Due to the implied role of macrophages in fibrotic healing, macrophage therapy is considered beneficial for reducing chronic liver fibrosis. It has been shown that when injected into a murine model of chronic liver fibrosis, murine bone marrow-derived macrophages (BMDM) reduce liver fibrosis 12 。Similar results were reproduced using human monocyte-derived macrophages (hMDM) in an immunodeficient murine model of chronic liver fibrosis 13 。In addition, in an ongoing phase II trial, a GMP-grade cell culture protocol is currently used 14 to generate 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 resolving acute conditions 38 。However, to date, no therapy (macrophage therapy or otherwise) has been able to effectively treat patients with cirrhosis by acting through four key mechanisms: recruitment of cells of the immune system to the liver (paracrine effect), conversion of recruited monocytes into pro-repair macrophages effective in spreading the therapeutic effect (polarization), phagocytosis, and remodeling of fibrotic scars. In particular, to date, no therapy has been able to treat patients with decompensated cirrhosis and those who have experienced a liver decompensation event effectively enough through all of these mechanisms.

[0006] Introduction to the Invention

[0007] Generally, human macrophages (hMDM) derived in vitro from monocytes will express IL-10, but as Figure 1As shown by the results presented, the macrophage does not secrete a large amount of IL-10. In the present text, the inventors enhanced the anti-fibrotic and anti-inflammatory properties of macrophages by transient transfection to express and secrete IL-10 and MMP9, as a proof of principle of how the additional expression of these genes can produce promising products to help resolve inflammatory conditions such as acute and chronic organ injury, resulting in macrophages that secrete IL-10 and active MMP.

[0008] IL-10 is well-known for its anti-inflammatory properties, while some MMPs play a role in helping fibrotic remodeling. However, the effect of the additional expression of the combination of IL-10 and MMP9 is not currently clear. In addition, the inventors demonstrated that macrophages engineered to overexpress IL-10 can be used for effective treatment, particularly for liver 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 four mechanisms of action that are considered to be the basis of therapeutic macrophage efficacy, namely: 1) remodeling of fibrotic scars; 2) phagocytosis; 3) paracrine effects; and 4) polarization. Figure 32 It is shown how these mechanisms of action contribute to the therapeutic effect of macrophages, and in particular, the recruitment of endogenous monocytes and their polarization into pro-repair macrophages can enhance the therapeutic effect (this enhancement is also referred to as a "positive feedback loop").

[0009] We show that human monocyte-derived macrophages (hMDM) transfected (Trx) with mRNA expressing IL-10 or IL-10-p2A-MMP9 (referred to herein as IL-10+MMP9) can polarize macrophages into a pro-repair phenotype based on the expression of CD14, CD206 high (CD206high), 25F9, CD163, CCR2 low / negative (CCR2low / neg), CD86 low (CD86low), and HLA-Class II low. In addition, the inventors demonstrate in the Examples that when hMDM are tested in a peripheral blood monocyte (PBMC) migration assay, IL-10 Trx and IL-10-MMP9 Trx hMDM can recruit significantly more monocytes compared to hMDM currently used in the MATCH clinical trial (Trial registration numbers: ISRCTN10368050 and EudraCT reference 2015-000963-15. Study protocol: A 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 a Boyden chamber-based technique. In the Examples, the inventors tested immune cell migration in a transwell cell migration and invasion assay (Boyden chamber assay). Without wishing to be bound by theory or mechanism, IL-10-Trx macrophages are able to specifically recruit monocytes while essentially not recruiting other immune cell types. Surprisingly, IL-10-MMP9 Trx hMDM have a stronger ability to recruit monocytes compared to MMP9-Trx and IL-10-Trx. These two proteins appear to exhibit a synergistic effect in this process. In fact, only MMP9-transfected macrophages show little or no ability to attract monocytes ( Figure 12 ), while the activity of matrix metalloproteinases is usually increased in these cells (data not shown). In addition, IL-10 and MMP9 Trx hMDM show excellent anti-inflammatory characteristics and phagocytic ability.

[0010] Finally, the inventors found that overexpression of IL-10 alone decreased total matrix metalloproteinase (MMP) activity and the level of MMP9 secreted in the supernatant of genetically engineered macrophages. The metalloproteinase assay for determining the activity level was 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 Is an 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] Accordingly, 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 un-Trx and IL-10 + MMP9 Trx hMDM, the total matrix metalloproteinase activity of IL-10 + MMP9 Trx hMDM increased significantly.

[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., associated with organ injury). Although IL-10 is known as an anti-inflammatory cytokine, this is somewhat surprising since previous data showed that in an animal model of acute liver disease, IL-4-stimulated macrophages (rather than IL-10-stimulated macrophages) were pro-reparative 15 . Combinations of additional IL-10 and MMP9 expression (e.g., by transfection) can also be envisaged to enhance the anti-fibrotic (by increasing matrix metalloproteinase activity) and pro-reparative (by increasing monocyte migration) functions of macrophage-based therapeutic products. The recruitment of monocytes to the site of inflammation by these macrophages expressing IL-10 and MMP9 is crucial since the macrophages of the present invention recruit monocytes in situ in this manner to provide a further therapeutic effect. The recruited monocytes can be converted into pro-reparative macrophages, thereby enhancing the effect of the therapeutic macrophages. The pro-reparative phenotype is described extensively herein.

[0013] Chronic inflammatory organ damage is usually associated with fibrosis, such as in chronic liver disease. Thus, reparative therapeutic macrophages with anti-inflammatory and anti-fibrotic functions would be beneficial.

[0014] As described above, the inventors have found that macrophages engineered to overexpress IL-10 exhibit a decrease in overall MMP activity. In contrast, macrophages engineered to additionally express MMP9, e.g., by engineering with a nucleic acid encoding MMP9, exhibit an enhancement in overall MMP activity, rather than just an enhancement in MMP9 activity. Herein, by the combined expression of IL-10 and MMP9, the inventors have for the first time enhanced the overall MMP activity of engineered macrophages to a greater extent than expected from the data of macrophages transfected with IL-10 or MMP9 alone. Thus, the inventors have demonstrated an unexpected synergistic effect of the combined expression of IL-10 and MMP9 on overall MMP activity. Accordingly, macrophages are engineered to have additional / exogenous coding sequences of them beyond those naturally present in the cell, IL-10 and MMP9. Such a combination is 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 reparative phenotype, as described herein.

[0015] Furthermore, the inventors have demonstrated that in a disease model of liver inflammation / fibrosis, IL-10-MMP9-Trx hMDMs localize to the liver. It has also been demonstrated that the engineered macrophages have the ability to recruit significantly more monocytes compared to non-genetically engineered hMDMs currently used in MATCH. Such recruitment studies were conducted in vitro using the standard assays described herein. Collectively, these data support the use of IL-10 and MMP9-Trx hMDMs as improved therapeutic products for the treatment and / or prevention of inflammatory conditions associated with, for example, organ damage.

[0016] Matrix metalloproteases (MMPs), also known as matrix metalloproteinases or matrixins, are a family of zinc-dependent polypeptide enzymes that collectively degrade various proteins in the extracellular matrix (ECM). MMPs have important roles in the body, making them very complex and difficult to understand. In the context of fibrosis, it has been reported that MMPs contribute; however, they also play a key role in fibrosis resolution through their matrix remodeling ability. Their dual role in fibrosis progression and regression can be understood to some extent by the complexity of their in vivo activities, from cytokine and chemokine activation, immune cell recruitment and activation to extracellular matrix degradation. In addition, the activation and subsequent activity of MMPs in vivo are highly regulated, mainly by tissue inhibitors of metalloproteinases (TIMPs), which adds to the difficulty of understanding their activity in fibrosis. Experimental models of pulmonary fibrosis have shown an increase in the activity of MMP9, a type IV collagenase that targets collagen, which is also associated with the disruption of the alveolar epithelial cell membrane, suggesting a putative profibrotic role of MMP9 in lung injury. However, in a bleomycin-induced model of pulmonary fibrosis, although the lungs of MMP9-deficient mice showed limited alveolar bronchiolization, MMP9-deficient mice developed similar pulmonary fibrosis to wild-type littermate mice. Overall, the exact role of MMPs in fibrosis is not fully understood. In addition, the levels of MMPs expressed by in vitro-derived hMDMs are negligible; furthermore, little if any activity was detected in the conditioned media of these cells.

[0017] 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. Therapeutically, overexpression of MMP9 in iPSCs by lentiviral transduction has been used to enhance the repair of damaged myocardium. MMP12, an elastase that targets soluble and insoluble elastin, has been overexpressed in endothelial progenitor cells for melanoma cell therapy. To the knowledge of the present inventors, no work has ever been done to transfect human macrophages with MMPs to produce macrophages with antifibrotic properties. Cabrera et al. demonstrated that overexpression of MMP9 in transgenic mouse macrophages challenged with bleomycin led to attenuated 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 metalloproteases were reported.

[0018] MMPs have complex biological properties, which are generally organ- and disease-specific. For example, MMP9 has a significant antifibrotic effect in models of chronic liver disease and chronic lung disease. However, it appears to be detrimental to renal fibrosis.

[0019] WO2019 / 118888A1 (Treatment of fibrosis with genetically engineered macrophages) describes macrophages engineered for the treatment of fibrosis. For example, genetically engineered macrophages that comprise: a recombinant extracellular matrix (ECM) targeting protein; and / or a recombinant protease. The recombinant protease can be a matrix metalloproteinase (MMP), such as one from a long list of possible MMPs including MMP9 and MMP12. However, IL-10 or cytokines related to cell engineering are not mentioned.

[0020] 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 can include matrix metallopeptidase (MMP), such as one or more from a long list of possible MMPs including MMP9 and MMP12. The at least one exogenous anti-inflammatory agent can include, for example, cytokines, chemokines or pentraxins, and the cytokines can include, for example, IL-10, IL-4, IL-13 and / or TGF-β. The modified immune cells can include macrophages. However, specific combinations of these possible genes and cell types are not disclosed, and furthermore, no combinations are exemplified in this application - only single genes are tested. In addition, this applicant did not attempt to determine, for example, the effect of IL-10 expression on the expression of other proteins such as MMPs. Furthermore, the data provided in the WO2022 / 047119A1 application shows that macrophages transfected with IL-10 produce at least one pro-inflammatory marker CD-80 on the macrophages, which is not desirable for the treatment of inflammatory conditions.

[0021] WO2019 / 175595 describes the treatment of liver diseases, including cirrhosis, using autologous isolated non-engineered human macrophages. However, the macrophages in this application are non-polarized and non-engineered. Therefore, such cells are of little use for the treatment of inflammatory conditions and conditions with a fibrotic component.

[0022] WO2012 / 062930 describes the use of a composition comprising macrophages overexpressing IL-10 from transfected mRNA as a medicament. However, this application does not disclose the combination of IL-10 and MMP9 or the treatment of cirrhosis.

[0023] The present inventors have for the first time demonstrated the beneficial effect of macrophages engineered to express a specific combination of IL-10 and MMP9 in the treatment of, for example, inflammatory and fibrotic diseases.

[0024] One or more aspects or embodiments of the claimed invention are directed to solving one or more of the above problems. Summary of the Invention

[0025] According to a first aspect of the present invention, there is provided an engineered macrophage engineered to overexpress IL-10. In certain embodiments, when cultured in vitro at a cell concentration of 4x10 6 / ml, the macrophages secrete IL-10 at a culture supernatant concentration of at least 10,000 pg / ml. As described herein, engineered macrophages that overexpress IL-10 alone exhibit a significant ability to recruit monocytes, have an anti-inflammatory secretome, and convert unpolarized or pro-inflammatory macrophages into a pro-repair phenotype. These examples demonstrate that such macrophages can be used for treatment, particularly for the treatment of liver cirrhosis.

[0026] According to another aspect of the present invention, there is provided an engineered macrophage that overexpresses IL-10 and MMP9, such that the engineered macrophage expresses higher levels of IL-10 and MMP9 than untransfected macrophages. According to one aspect of the present invention, there is provided an engineered macrophage, wherein the macrophage is 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 conditions and / or fibrotic conditions in a subject.

[0027] In other aspects of the present invention, there are provided engineered macrophages 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.

[0028] As described herein, engineered macrophages according to any aspect of the present invention can have specific structures and advantageous functional properties.

[0029] Exemplary structural and functional properties of the engineered macrophages relative to non-polarized, non-transfected 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 properties described in Table 4.

[0030] 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.

[0031] In a preferred embodiment, when exposed to non-engineered macrophages, the engineered macrophages polarize the non-engineered macrophages into a pro-repair phenotype. When administered to a subject, the engineered macrophages can polarize host macrophages (such as endogenous monocyte-derived macrophages that migrate to the liver) into 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 from the engineered macrophages can convert non-engineered macrophages into a pro-repair phenotype. Thus, the engineered macrophages of the present invention can convert non-engineered 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.

[0032] In some embodiments, the CD86 expression of the engineered macrophages is reduced by at least two-fold compared to non-engineered, non-polarized cells. In some embodiments, the HLA-DR expression of the engineered macrophages is reduced by at least two-fold compared to non-engineered, non-polarized cells. In some embodiments, the IL-10 secretion of the engineered macrophages is increased by at least 1000-fold compared to non-engineered, non-polarized cells. In some embodiments, the MMP3 secretion of the engineered macrophages is increased by at least 10-fold compared to non-engineered, non-polarized cells. In some embodiments, the MMP10 secretion of the engineered macrophages is increased by at least 20-fold compared to non-engineered, non-polarized cells.

[0033] In some embodiments, when cultured in vitro at a cell concentration of 4x10 6 / ml, the macrophages of the present invention secrete IL-10 at a concentration of at least 10,000 pg / ml in the culture supernatant. In some embodiments, when cultured in vitro at a cell concentration of 4x10 6 / ml, the macrophages of the present invention secrete MMP9 at a concentration of at least 200 ng / ml in the culture supernatant. In some embodiments, the CD206 expression of the engineered macrophages is increased by at least 5-fold compared to monocytes. In some embodiments, the 25F9 expression of the engineered macrophages is increased by at least 5-fold compared to monocytes. In some embodiments, the CD80 expression of the engineered macrophages is reduced by at least ten-fold compared to non-engineered cells. In some embodiments, when at 4x10 6When cultured in vitro at a cell concentration of / ml, the macrophages of the present invention secrete TNF-a at a concentration of at least 40 pg / ml in the culture supernatant. In some embodiments, the engineered macrophages have the same phagocytic ability as non-engineered, non-polarized cells.

[0034] In one embodiment, the present invention provides engineered macrophages comprising one or more exogenous coding sequences for IL-10 and MMP. The exogenous coding sequence can be any suitable nucleic acid sequence. The exogenous coding sequence can exist as an extrachromosomal nucleic acid in the cytoplasm or nucleus, or be integrated into the macrophage genome. The exogenous coding sequence can encode IL-10 and MMP9, or multiple exogenous coding sequences can each encode IL-10 or MMP9.

[0035] In some embodiments, the expression of endogenous IL-10 and / or MMP9 genes can be stimulated by genetic engineering. For example, gene editing techniques such as CRISPR can be used to turn on and off endogenous genes encoding IL-10 and / or MMP9, generating engineered macrophages that express IL-10 and / or MMP9 under conditions where these proteins would not otherwise be expressed. For example, this can be achieved by altering the promoter sequence.

[0036] Natural, non-engineered macrophages are capable of expressing IL-10 and / or MMP9 under relevant physiological conditions, but typically natural macrophages do not secrete significant levels of IL-10. However, the present invention does not relate to these natural, non-engineered macrophages, but rather to engineered macrophages in which the expression level of IL-10 in particular has been increased to supra-physiological levels, thereby improving the anti-inflammatory properties of the 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 an additional or exogenous IL-10 coding sequence. In some embodiments, the cells are transfected with mRNA encoding IL-10 / MMP9.

[0037] Cells overexpressing IL-10 and / or MMP9 contain coding sequences that express IL-10 and / or MMP9 at higher levels than non-engineered cells. As described above, overexpression can be achieved by introducing exogenous nucleic acids such as mRNA encoding IL-10 and / or MMP9, or by genetic modification that stimulates the expression of IL-10 and / or MMP9 from endogenous coding sequences. Engineered macrophages overexpressing IL-10 and / or MMP9 do not necessarily secrete a greater amount of IL-10 and / or MMP9 than non-engineered macrophages.

[0038] In other embodiments, the macrophages can be engineered to turn on endogenous genes encoding IL-10 and / or MMP9. In any case, the macrophages of the present disclosure have been modified by altering the IL-10 and / or MMP9 expression levels in any way and are thus referred to as engineered macrophages.

[0039] When IL-10 is overexpressed, the native activity levels of MMPs, including MMP9, decrease.

[0040] Accordingly, the engineered macrophages have an additional or exogenous MMP-encoding sequence to maintain at least a "physiological" level of MMP9 expression, or a level just above the physiological level. In some embodiments, MMP9 expression is modified to rescue, repair, or restore the macrophages to a comparable or increased MMP9 expression level when compared to macrophages not transfected with IL-10. Thus, despite the fact that the engineered macrophages are engineered with an additional / exogenous MMP9-encoding sequence, the engineered macrophages exhibit an MMP9 expression level similar to or slightly higher than the wild-type / native expression. Slightly higher may mean an expression enhanced 1.2 to 1.5 times the native expression level, such as 1.2, 1.3, 1.4, or 1.5 times the native level.

[0041] 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. It may be preferred that the macrophages overexpress IL-10. It may be preferred that the macrophages overexpress MMP9. Alternatively, the macrophages can be engineered such that both IL-10 and MMP-9 are overexpressed. When compared to untransfected macrophages, the expression levels of IL-10 and / or MMP9 are increased. When compared to macrophages transfected with IL-10 alone, the expression level of MMP9 is increased.

[0042] 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.

[0043] In some embodiments, the baseline macrophages (i.e., pre-engineered or native) are referred to as unpolarized human monocyte-derived macrophages, also known as "resting" macrophages.

[0044] In some embodiments, the engineered macrophages are macrophages derived from iPSCs. There are various methods known in the art for deriving macrophages from iPSCs. Baseline-wise, these will also be non-polarized or resting. Thus, in certain embodiments, the engineered macrophages are derived from pluripotent stem cells cultured in vitro. Preferably, due to the knockout or knockdown of at least one gene associated with any HLA class I and / or HLA class II cell surface molecule, such pluripotent stem cell-derived or iPSC-derived macrophages are hypoimmunogenic. For example, the genes associated with HLA class I can be HLA-A, HLA-B, and HLA-C genes or the B2M gene. The genes associated with HLA class II can be HLA-DP, DM, DO, DQ, and DR or CIITA. Such knockout or knockdown is evident in macrophages, but since macrophages are differentiated from stem cells, it may also be introduced into any progenitor cells. In an exemplary method, the engineered macrophages of the present invention can be produced by: providing stem cells, such as induced pluripotent stem cells, optionally knocking out or knocking down the expression of at least one sequence encoding a unit in the HLA I or HLA II complex; differentiating the stem cells into embryoid bodies, optionally using BMP4, SCF, VEGF, and / or a Rock inhibitor (Y-27632); differentiating the embryoid bodies into macrophage progenitor cells, optionally using M-CSF and IL-3; and maturing the macrophage progenitor cells into functional macrophages, optionally using M-CSF.

[0045] As used herein, overexpression involves an increase in the artificial expression amount of a gene.

[0046] As used in the examples, the expression level of the protein was quantified between 16 and 24 hours after transfection. The expression levels described herein are for a macrophage population at a concentration of 4x10 6 / ml (equivalent to 2x10 2 cells per cm 6 ). In the examples, 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. Equivalent conditions suitable for determining the concentration of secreted proteins will be known to those skilled in the art.

[0047] 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 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. Suitably, these IL-10 protein levels can be measured by culturing the macrophages as described above, wherein the concentration of macrophages in the culture medium is 4x 10 6 cells / ml, equivalent to 2x10 6 cells / cm 2 , and measuring the concentration of the protein in the culture medium. In a preferred embodiment, when cultured in vitro at a cell concentration of 4x10 6 / 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 can secrete IL-10 at a level 1000-fold higher than non-engineered, non-polarized hMDMs. Non-engineered, non-polarized hMDMs are described in the art, for example in WO2019175595.

[0048] 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 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 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 MMP9 protein level secreted by the engineered macrophages (comprising IL-10 and MMP9) is higher than the average level of MMP9 protein secreted by macrophages engineered with IL-10 alone. In some embodiments, the MMP9 protein level secreted by the engineered macrophages (comprising IL-10 and MMP9) is at least equal to the average level of MMP9 protein secreted in unpolarized, untransfected macrophages. Suitably, the overall MMP activity of the engineered macrophages of the present invention is also higher than the overall MMP activity of macrophages engineered with IL-10 alone. In other embodiments, the overall 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 the MMP activity 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 medium is 4x10 6 cells / ml, equivalent to 2x10 6 cells / cm 2 , and measuring the concentration of the protein in the medium. In some embodiments, the engineered macrophages may secrete a greater amount of other matrix metalloproteinases. In a specific embodiment, the amount of matrix metalloproteinase-3 (MMP3) secreted by the engineered macrophages can be 10 times higher than that of non-engineered, non-polarized cells. In a specific embodiment, the amount of matrix metalloproteinase-10 (MMP10) secreted by the engineered macrophages can be 10 times higher than that of non-engineered, non-polarized cells.

[0049] The macrophages of the present invention can be used in therapies for subjects in need thereof, most notably cell therapy. The subject may have a condition, disease or disorder that would benefit from the administration of the macrophages of the present invention. Such a condition, disease or disorder may have an inflammatory and / or fibrotic component. Such a condition, disease or disorder can be acute or chronic, or slow plus acute. Such a condition, disease or disorder may result in organ damage. The organ can be any suitable organ, including the liver, lung or kidney.

[0050] In some embodiments, the subject's condition, disease, or disorder 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 slow plus acute inflammatory condition.

[0051] In some embodiments, the condition involves the kidney, liver, or lung. For example, the condition can be liver injury, kidney injury, or lung injury.

[0052] In some embodiments, the condition can be acute-on-chronic liver failure (ACLF). ACLF is a syndrome characterized by the 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.

[0053] In some embodiments, the condition can 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.

[0054] Cirrhosis represents the end stage of chronic liver injury and progressive fibrosis (scarring) and is independent of the underlying cause. It is characterized by severe liver fibrosis, leading to structural disruption, hepatocyte dysfunction, and portal hypertension. Various etiologies can cause cirrhosis. Liver conditions with a fibrotic component 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)).

[0055] Etiologies that can lead to fibrosis can include, but are not limited to, steatohepatitis liver disease (SLD), such as metabolic dysfunction-associated steatohepatitis (MASLD), metabolic-associated steatohepatitis (MASH), or Met-ALD. In some cases, the cause of fatty liver disease may be unknown and may be referred to as cryptogenic SLD.

[0056] Metabolic dysfunction-associated steatohepatitis refers to a type of non-alcoholic fatty liver disease and can thus also be referred to as NAFLD. Metabolic-associated steatohepatitis refers to a more severe form of MASLD and can also be referred to as NASH. "Met-ALD" refers to an individual with steatohepatitis liver disease who also consumes alcohol. "Cryptogenic SLD" refers to SLD of unknown cause, such as an individual who does not carry any known metabolic risk factors for SLD.

[0057] Fibrotic diseases, disorders, and conditions can include mechanical trauma, biliary obstruction, autoimmune hepatitis, iron overload, hepatitis B infection (HBV) and / or hepatitis C infection (HCV). However, the engineered macrophages according to the present invention are capable of treating cirrhosis regardless of the underlying cause.

[0058] Cirrhosis can be compensated or decompensated cirrhosis (also referred to herein as hepatic decompensation or HD). Decompensated cirrhosis is defined as the 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 had at least one (or exactly one) decompensated cirrhosis event.

[0059] In a preferred embodiment, the patient has a MELD score between 10 and 18, or more preferably 10 - 16 or 12 - 18.

[0060] In some embodiments, the liver disease is a disease in which cirrhosis is caused by hepatocyte injury, e.g., is a hepatocyte-derived disease such as those of viral origin (including treated (sustained virologic response) hepatitis C (HCV), hepatitis B), alcohol-induced injury (alcohol-related liver disease (ALD)), or non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis (NASH) (including NASH caused by diabetes or obesity), cryptogenic cirrhosis, hemochromatosis, or alpha-1-antitrypsin deficiency. In some embodiments, the underlying cause has been removed (e.g., a patient who suffered injury due to alcoholism no longer drinks alcohol, or a patient who suffered injury due to HCV no longer has HCV, etc.). In some embodiments, the patient with liver disease is at risk of end-stage renal disease.

[0061] In some embodiments, the liver disease is steatohepatitis (SLD). In some embodiments, the steatohepatitis is metabolic dysfunction-associated steatohepatitis (MASLD), is Met-ALD or cryptogenic SLD. In some embodiments, the metabolic dysfunction-associated steatohepatitis (MASLD) is metabolic-associated steatohepatitis (MASH).

[0062] Cirrhosis can lead to acute-on-chronic liver failure (ACLF). In some embodiments, the liver disease is ACLF. ACLF is a condition distinct from liver decompensation. Liver decompensation is characterized by the presence of ascites, hepatic encephalopathy, gastrointestinal bleeding, or any combination of these conditions in a patient with cirrhosis. In contrast, ACLF is associated with organ failure and has a high short-term mortality of over 15% at 28 days. This syndrome has three main features: ACLF occurs in the context of intense systemic inflammation; ACLF often has a close temporal relationship with pro-inflammatory events 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.

[0063] A diseased patient suitable for treatment or use according to any aspect or embodiment of the present invention can be a patient suffering from a related disease and having a related severity.

[0064] In some embodiments, the subject has experienced their first liver decompensation event. In a preferred embodiment, the subject has been hospitalized after their first liver decompensation event. The subject may exhibit one or more clinical signs of liver decompensation selected from the list consisting of jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, variceal bleeding, and gastrointestinal bleeding. It is expected that the cells, compositions, and methods of the present invention are particularly effective in treating patients who have been hospitalized after their first liver decompensation event. Additionally, the data provided in the examples indicate that the cells, compositions, and methods of the present invention are suitable for treating these specific patients suffering from difficult-to-treat severe diseases.

[0065] Hospitalization after a liver decompensation event is a measure of disease severity and provides a specific clinical situation. Some symptoms of liver decompensation are similar to those of less severe cirrhosis, but when a patient is hospitalized after their first liver decompensation event, this indicates that their disease is severe enough to particularly benefit from the present invention.

[0066] Accordingly, in certain embodiments, the present invention provides cells and compositions for use in a method of treating a patient who exhibits one or more clinical signs of liver decompensation selected from the list consisting of jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, variceal bleeding, and gastrointestinal bleeding, wherein the one or more clinical signs require hospitalization.

[0067] Hospitalisation means admission to hospital for treatment. Thus, hospitalisation generally requires the patient to stay in the hospital for at least 24 hours. Hospitalisation is a measure of disease severity as the symptoms are uncontrollable outside of a hospital environment. Hospitalisation is a recognised measure of disease severity and patient status in the context of cirrhosis. 42(See also, e.g., Balcar et al., United European Gastroenterol J. 2021;9(4):427-437.).

[0068] In a preferred embodiment, once a subject has recovered (recompensated) from their first liver decompensation event, the subject is treated with the cells, compositions, or cell populations of the present invention, optionally, wherein the subject is hospitalized after their first liver decompensation event. In some embodiments, after the subject is discharged from the hospital, the subject is treated with the cells, compositions, or cell populations of the present invention. According to some embodiments, recovery (recompensation) from a liver decompensation event is defined by a physician's clinical assessment and / or by no significant increase in the MELD score between discharge and treatment. In some embodiments, the subject exhibits one or more clinical signs of liver decompensation and / or has recovered from one or more clinical signs of liver decompensation, the one or more clinical signs selected from the list consisting of jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, variceal bleeding, and gastrointestinal bleeding.

[0069] According to a preferred embodiment, the subject is treated after recovering from their first liver decompensation event that required hospitalization and before experiencing another liver decompensation event. As is known in the art, once a subject has more than one decompensation event, the severity and mortality of decompensated cirrhosis increase 42 . Thus, without being bound by theory or mechanism, it is preferred to treat the subject with the cells, compositions, and cell populations of the present invention after recovery from the first decompensation event to increase the subject's chance of not experiencing another liver decompensation event and surviving without the need for a liver transplant. In some embodiments, macrophages engineered to express human IL-10 and human MMP9 can be used to treat cirrhosis.

[0070] In some embodiments, macrophages engineered to express human IL-10 and human MMP9 can be used to treat ACLF.

[0071] In some embodiments, the macrophages have a pro-repair phenotype and are anti-inflammatory and anti-fibrotic. This phenotype is further defined herein.

[0072] 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 technique. Thus, in some embodiments, the macrophages can be engineered with one or more exogenous coding sequences. These exogenous coding sequences can encode IL-10 and / or MMP9 and / or they can encode gene editing proteins such as CRISPR, nickase, etc., which are capable of altering the cell's genome to increase the expression of endogenous IL-10 and / or endogenous MMP9. For example, this can be achieved by editing the promoter or enhancer sequences.

[0073] Known viral vectors for transfecting macrophages include lentivirus, adenovirus, and adeno-associated virus (AAV).

[0074] As used herein, "encoding" refers to the ability of a sequence of nucleotides (such as a gene, cDNA, or mRNA) to serve as a template in a biological process for the synthesis of a macromolecule (such as a defined amino acid sequence). Thus, a coding sequence can be any suitable nucleic acid sequence that provides the instructions for synthesizing the relevant entity (e.g., IL-10 or MMP9). The coding sequences can be included in the same vector / construct or in different vectors / constructs.

[0075] As used herein, "exogenous" refers to any substance, particularly genetic material, that is introduced into or produced outside of a particular cell. As used herein, in some embodiments, the exogenous coding sequence or engineered macrophages encode / express 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 the present invention. Ideally, the genes / coding sequences are human. The genes / coding sequences can be codon-optimized. The genes / coding sequences can be adjusted, and it should be understood that if the reference sequence is RNA, different nucleotides can be present in the DNA vector.

[0076] Preferably, the sequence of the provided IL-10 coding sequence is at least 80% similar or homologous to the NCBI reference sequence: NM_000572.3 (mRNA sequence of human IL10), preferably at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% similar to the native sequence.

[0077] Preferably, the provided MMP9 coding sequence is at least 80% similar or homologous to the NCBI reference sequence: NM_004994.3 (Homo sapiens matrix metallopeptidase 9 (MMP9), mRNA), preferably at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% similar to the native sequence.

[0078] Preferably, the expressed IL-10 protein is preferably at least 80% similar or homologous to the sequence shown in SEQ ID.No.4, preferably at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% similar to SEQ ID No.4.

[0079] Preferably, the expressed MMP9 protein is preferably at least 80% similar or homologous to the sequence shown in SEQ ID.No.6, preferably at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% similar to SEQ ID No.6.

[0080] In some embodiments, the macrophages are non-virally engineered and engineered with a nucleic acid vector. In some embodiments, the macrophages are transfected with a DNA vector, such as a naked DNA vector. Suitably, the DNA vector can 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, the macrophages are transfected with one or more free nucleic acids or vectors. In some embodiments, the macrophages are transfected with an RNA vector. In some embodiments, the macrophages are transfected with mRNA. Suitably, the macrophages are transfected with a single mRNA construct expressing IL-10 and MMP9.

[0081] In some embodiments, IL-10 and MMP9 are provided to the macrophages as mRNA. Thus, the engineered cells are provided with exogenous mRNA molecules. MMP9 and IL-10 can be on the same mRNA molecule or on different mRNA molecules. Preferably, MMP9 and IL-10 are on the same mRNA molecule. Further preferably, the mRNA is a bicistronic vector encoding both MMP9 and IL-10. Suitably, the mRNA molecule comprises the sequence as set forth in any one of SEQ ID No.1 to 3, 10, 13 or 14.

[0082] In some embodiments, the mRNA comprises a sequence encoding IL-10. In some embodiments, the mRNA comprises a sequence that is at least 80% identical to SEQ ID NO:13. In some embodiments, the mRNA comprises a 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 SEQ ID NO:13. In a preferred embodiment, the mRNA comprises the sequence described in SEQ ID NO:13.

[0083] In some embodiments, the mRNA comprises a sequence encoding MMP9. In some embodiments, the mRNA comprises a sequence that is at least 80% identical to SEQ ID NO:14. In some embodiments, the mRNA comprises a sequence that is at least 85%, at least 90%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:14. In a preferred embodiment, the mRNA comprises the sequence described in SEQ ID NO:14.

[0084] In some embodiments, the macrophage is engineered with mRNA encoding both IL-10 and MMP9. In some embodiments, the macrophage is engineered with mRNA comprising both the sequence described in SEQ ID NO:13 and the sequence described in SEQ ID NO:14. In some embodiments, the sequences described in SEQ ID NOs 13 and 14 are on the same mRNA molecule. In some embodiments, the sequences described in SEQ ID NOs 13 and 14 are on different molecules. In a preferred embodiment, the mRNA comprises the sequences described in SEQ ID NOs 13 and 14 on the same mRNA molecule, which are separated by mRNA encoding a self-cleaving linker. In a preferred embodiment, the self-cleaving linker has the amino acid sequence described in SEQ ID NO:9. In a preferred embodiment, the mRNA encoding the self-cleaving linker has the sequence described in SEQ ID NO:15.

[0085] In some embodiments, the mRNA construct expresses IL-10 fused to MMP9 via a cleavable linker. In some embodiments, the linker is p2A. p2A can have the amino acid sequence described in SEQ ID No.9, which can 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. Such peptides are 18-22 amino acids 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, although they are present in the same mRNA molecule.

[0086] In a preferred embodiment, both IL-10 and MMP9 are expressed from the mRNA molecule. In some embodiments, the mRNA molecule comprises a sequence that is at least 80% identical to SEQ ID NO:10. In some embodiments, the mRNA molecule comprises a 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 SEQ ID NO:10. In a preferred embodiment, the mRNA comprises the sequence described in SEQ ID NO:10. In a particularly preferred embodiment, the mRNA comprising the sequence described in SEQ ID NO:10 further comprises a polyA tail, optionally 65 to 250 residues in length, preferably 90 to 120 residues in length, preferably about 90 residues in length, and / or a 5' cap. In a particularly preferred embodiment, the mRNA comprising the sequence described in SEQ ID NO:10 further comprises a polyA tail, optionally 65 to 250 residues in length, preferably 90 to 120 residues in length, such as 90 residues in length, and a 5' cap. In a preferred embodiment, the exogenous mRNA sequence comprises a 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:16. In a particularly preferred embodiment, the exogenous mRNA sequence comprises the sequence listed in SEQ ID NO:16, preferably further comprising a 5' cap.

[0087] In some embodiments, the exogenous mRNA sequence comprises a 5' untranslated region sequence (5'UTR) 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 sequence (5'UTR) 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 the 5'UTR sequence as listed in SEQ ID NO:17.

[0088] In some embodiments, the exogenous mRNA sequence comprises a 3' untranslated region sequence (3'UTR) 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:20. In a preferred embodiment, the exogenous mRNA sequence comprises a 3' untranslated region sequence (3'UTR) 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:18. In a preferred embodiment, the exogenous mRNA sequence comprises the 3'UTR sequence as listed in SEQ ID NO:18. In a preferred embodiment, the exogenous mRNA comprises both the 5'UTR sequence as listed in SEQ ID NO:17 and the 3'UTR sequence as listed in SEQ ID NO:18.

[0089] Alternatively or additionally, an IRES site can be inserted between the coding sequences, which will be understood by those skilled in the art to interrupt expression to form two separate proteins.

[0090] In some embodiments, the mRNA molecule contains a poly A tail. The poly-A tail is a long chain of adenine nucleotides added to the mRNA molecule during RNA processing to increase the stability of the molecule. 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.

[0091] 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 rest of the eukaryotic mRNA by a 5′-5′ triphosphate bridge. This so-called "cap0" particularly contributes to 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. Synthetic anti-reverse cap analogs include additional methylation and trinucleotide AG, methylated at the first adenosine. At least one cap can be a synthetic cap analog, preferably

[0092] In some embodiments, the mRNA is a modified RNA. In some embodiments, mRNA modifications include chemical modifications of uridine and / or chemical modifications of cytidine. In some embodiments, mRNA modifications include pseudouridine, N1-methylpseudouridine, 5-methoxy-uridine, 5-methyl-cytidine, preferably 5-methoxy-uridine.

[0093] In one embodiment, the mRNA is modified with 5-methoxy-uridine and includes at least one

[0094] In some embodiments, 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%, 99% similar or homologous to SEQ ID No.2.

[0095] In some embodiments, the macrophages are engineered with a nucleic acid vector. In some embodiments, the macrophages are transfected by electroporation. Other suitable transfection methods include nucleofection.

[0096] In some embodiments, the mRNA is delivered to the macrophages via nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles, which can be described, for example, in U.S. Patent No. 8,058,069, U.S. Patent No. 8,492,359, U.S. Patent No. 8,822,668, U.S. Patent No. 9,364,435, U.S. Patent No. 9,504,651, and U.S. Patent No. 11,141,378.

[0097] 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.

[0098] In some embodiments, the macrophages are derived from monocytes by culturing the monocytes under suitable conditions. Suitably, these conditions may include:

[0099] An in vitro method for generating macrophages, comprising:

[0100] (b) culturing the monocytes in a medium for 3 - 5 days to generate macrophages, wherein the medium contains one or more growth factors that stimulate macrophage production;

[0101] wherein step (a) is carried out entirely in the same medium.

[0102] Suitably, the medium is suitable for generating macrophages from monocytes. Suitably, the medium is a T cell medium. Suitably, the medium can be selected from: X - Vivo 10, X - Vivo 15, TexMACS, AIMv, RPMI, DMEM, and DMEM / F12. Suitably, the medium is TexMACS (Miltenyi).

[0103] Suitably, the medium is serum - free. Suitably, the medium is xenogeneic - protein - free. Suitably, the medium complies with GMP.

[0104] Suitably, the medium may contain one or more factors. Suitable factors include growth factors, polysaccharides, cytokines, and chemokines. Suitable factors may include: MCSF, GM - CSF. Suitably, thus, the factors are growth factors. Suitably, the one or more factors comply with GMP. In one embodiment, the medium may contain one or more growth factors, which may include MCSF or GM - CSF. Monocytes are most commonly cultured with MCSF or GM - CSF. Culturing monocytes with GM - CSF biases them towards an "inflammatory" phenotype, while culturing monocytes with MCSF biases them towards a "pro - repair" phenotype. In other embodiments, the one or more growth factors do not include a combination of MCSF and GM - CSF. Thus, if M - CSF is used as a growth factor in any method of the present invention to generate macrophages, it may be preferred that GM - CSF is not also used to generate macrophages from monocytes. This applies to the step of culturing monocytes until macrophages are produced.

[0105] According to a second aspect of the present invention, there is provided a population of engineered macrophages as described herein. The population may be suitable for use in the therapies described above.

[0106] According to a third aspect of the present invention, there is provided a composition comprising engineered macrophages. In some embodiments, the engineered macrophage composition is used in the therapies described above, or a population of engineered macrophages is used in the therapies described above. The composition may also comprise other pharmaceutically acceptable components, such as a suitable cell culture medium, excipients, and the like.

[0107] According to any aspect of the present invention, suitably, the use comprises administering to a subject an effective amount of engineered macrophages.

[0108] According to any aspect of the present invention, the use may comprise delivering engineered macrophages to a subject by systemic administration, suitably by systemic injection. In some embodiments, administration is by local injection, for example for the kidney and lung. In some embodiments, administration is to the lung by nebulizer. For example, peripheral intravenous injection is beneficial for liver conditions to avoid invasive procedures in cirrhotic patients. Local injection, such as into the renal artery for kidney conditions, may be better tolerated.

[0109] Preferably, the macrophages used in the therapy are engineered ex vivo and delivered to the patient.

[0110] However, in some embodiments, the macrophages may be engineered in vivo. In some embodiments, the macrophages are engineered in vivo by administering to the subject a formulation of exogenous coding sequences of IL-10 and MMP9 suitable for transfecting macrophages. The formulation may comprise any exogenous coding sequence discussed herein. Suitable delivery vehicles for in vivo engineering may comprise targeting molecules for macrophages due to their cell surface markers. If in vivo transfection of macrophages is contemplated, 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 lung. Such treatment can be formulated as nanoparticles to assist macrophage targeting.

[0111] According to a fourth aspect of the present invention, there is provided a method of improving the migration of monocytes to an inflammatory site, which comprises using the engineered macrophages according to the first aspect of the present invention, a population of engineered macrophages according to the second aspect of the present invention, or a composition according to the third aspect of the present invention.

[0112] 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.

[0113] In some embodiments, one or more of the following markers can be used to describe a pro-repair phenotype: increased CD206 and / or CD163, and decreased inflammatory markers such as CD86 and / or major histocompatibility complex class II (HLA-DR). These increases / decreases are compared to non-polarized (resting) or pro-inflammatory macrophages. In terms of the secretion profile, these macrophages are not expected to express TNFα, IFNγ, 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 non-engineered, non-polarized cells, such as those described in WO2019 / 17559.

[0114] In some embodiments, the engineered macrophages do not secrete TNFα, IFNγ, 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 TNFα, IFNγ, IL1β, IL-12p70, and / or IL-2 at the same or similar levels as non-engineered, non-polarized cells.

[0115] 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 MMP activity 1.5-fold higher than non-engineered, non-polarized cells. In some embodiments, the engineered macrophages secrete IL-6 and / or CXCL8.

[0116] According to another aspect of the present 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 from which they are derived.

[0117] 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 aspect, a method of generating engineered macrophages that express IL-10 such that the engineered macrophages secrete at least 10,000 pg / mL of IL-10 is provided, the method comprising introducing a nucleic acid encoding IL-10 into macrophages or cells from which they are derived. In some embodiments, the method further includes contacting the macrophages with an anti-inflammatory treatment, such as IL-4 and IL-13, after transfection. In some embodiments, the method includes contacting the macrophages with IL-4 and IL-13. In some embodiments, the macrophages are incubated with IL-4, IL-13, and M-CSF.

[0118] In some embodiments, the method includes electroporation. In some embodiments, after electroporation, the macrophages are contacted with an anti-inflammatory treatment.

[0119] In certain embodiments, the present invention provides:

[0120] 1. An engineered macrophage comprising an exogenous coding sequence for IL-10 and an exogenous coding sequence for MMP9.

[0121] 2. The engineered macrophage according to embodiment 1, wherein the expression of the exogenous coding sequence has a synergistic effect in restoring MMP activity and / or in monocyte recruitment of macrophages compared to an engineered macrophage comprising only the exogenous IL-10 sequence.

[0122] 3. The engineered macrophage according to embodiment 1 or embodiment 2, wherein the macrophage and / or the coding sequence is human.

[0123] 4. The 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.

[0124] 5. The engineered macrophage according to embodiment 4, wherein the nucleic acid molecule is a DNA or RNA molecule, preferably an mRNA molecule.

[0125] 6. The engineered macrophage according to embodiment 5, wherein the mRNA molecule contains chemically modified residues, preferably modified uridine residues, and optionally at least one synthetic cap.

[0126] 7. The engineered macrophage according to any of the preceding embodiments, wherein the exogenous coding sequence for IL-10 and the exogenous coding sequence for MMP9 are on the same nucleic acid.

[0127] 8. An engineered macrophage according to any one of the foregoing embodiments, wherein the macrophage is engineered to overexpress IL-10, preferably, wherein at a cell concentration of 4x10 6 / ml, the secreted IL-10 protein level is greater than about 300 pg / ml.

[0128] 9. An engineered macrophage according to any one of the foregoing embodiments, wherein the level of metalloprotease activity is at least 1.5-fold that of the metalloprotease activity of non-engineered macrophages.

[0129] 10. An engineered macrophage according to any one of embodiments 1 to 7, wherein the metalloprotease activity is restored relative to the reduced metalloprotease activity in macrophages engineered with the IL-10 coding sequence alone.

[0130] 11. An engineered macrophage according to any one of the foregoing embodiments, wherein the macrophage is transiently transfected, optionally by electroporation.

[0131] 12. The engineered macrophage according to embodiment 10, wherein the transfection is non-viral.

[0132] 13. An engineered macrophage according to any one of the foregoing embodiments, wherein the macrophage has a pro-repair phenotype.

[0133] 14. A population of engineered macrophages according to any one of the foregoing embodiments.

[0134] 15. A therapeutic composition comprising a population of macrophages according to embodiment 14 and a pharmaceutically acceptable vehicle.

[0135] 16. An engineered macrophage 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 use in therapy.

[0136] 17. The engineered macrophage, population, or composition according to embodiment 16, wherein the therapy is administered to a subject in need thereof.

[0137] 18. An engineered macrophage 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 use in treating an inflammatory condition in a subject.

[0138] 19. The engineered macrophage, population, or composition according to embodiment 17 or embodiment 18, wherein the macrophage is autologous or allogeneic to the subject.

[0139] 20. An engineered macrophage, population, or composition according to embodiment 18 or embodiment 19, wherein the condition is a chronic inflammatory condition having a fibrotic component, optionally, wherein the condition is organ damage associated with chronic inflammation.

[0140] 21. An engineered macrophage, population, or composition according to embodiment 18 or embodiment 19, wherein the condition is acute-on-chronic liver failure (ACLF).

[0141] 22. A method of improving the migration of monocytes to an inflammatory site, the method comprising using an engineered macrophage, population of engineered macrophages, or composition according to any one of the preceding embodiments.

[0142] 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.

[0143] 24. A method of generating an engineered macrophage according to any one of embodiments 1 to 13, the method comprising transiently transfecting macrophages with an mRNA molecule encoding IL-10 and / or MMP9.

[0144] 25. The method according to embodiment 24, the method comprising contacting the macrophages with IL-4 and IL-13 before, during, or after transfection.

[0145] 26. The method according to embodiment 24 or 25, wherein an mRNA molecule encoding IL-10 and MMP9 is co-transfected using a bicistronic vector linked by a p2A linker sequence.

[0146] 27. An engineered macrophage according to any one of embodiments 1 to 13, wherein the macrophage is engineered with an mRNA construct encoding human IL-10 fused to human MMP9 via a cleavable linker.

[0147] 28. A method of treating inflammation and / or fibrosis, the method comprising administering to a subject in need thereof a therapeutically effective amount of an engineered macrophage according to any one of embodiments 1-13.

[0148] The present invention also provides the following embodiments, which can be combined with any other embodiment:

[0149] 1. An engineered macrophage that is engineered to overexpress IL-10.

[0150] 2. The engineered macrophage according to embodiment 1, wherein when at 4x10 6When cultured in vitro at a cell concentration of / ml, the macrophages secrete IL-10 at a concentration of at least 10,000 pg / ml in the culture supernatant.

[0151] 3. The engineered macrophage according to embodiment 1 or embodiment 2, wherein the macrophage is additionally engineered to overexpress MMP9.

[0152] 4. The engineered macrophage according to embodiment 3, wherein the engineered macrophage comprises an exogenous coding sequence of IL-10 and an exogenous coding sequence of MMP9.

[0153] 5. The engineered macrophage according to embodiment 4, wherein the expression of the exogenous coding sequence has a synergistic effect in restoring MMP activity and / or a synergistic effect in monocyte recruitment of macrophages compared to an engineered macrophage comprising only the exogenous IL-10 sequence.

[0154] 6. The engineered macrophage according to any one of embodiments 1-5, wherein the macrophage and / or the coding sequence is human.

[0155] 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 SEQ ID NO:4, optionally, wherein the IL-10 protein comprises the same amino acid sequence as SEQ ID NO:4.

[0156] 8. The engineered macrophage according to any one of embodiments 4-7, wherein the exogenous coding sequence of MMP9 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 SEQ ID NO:6, optionally, wherein the IL-10 protein comprises the same amino acid sequence as SEQ ID NO:6.

[0157] 9. The 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.

[0158] 10. The engineered macrophages according to embodiment 9, wherein the nucleic acid molecule is a DNA or RNA molecule, preferably an mRNA molecule. Optionally, IL-10 and MMP9 are expressed by the same mRNA molecule. Further optionally, the mRNA molecule encodes IL-10 and MMP9 linked by a linker sequence. Further optionally, the linker is a self-cleaving 2A linker. Further optionally, the linker is p2A.

[0159] 11. The engineered macrophages according to embodiment 10, wherein the nucleic acid molecule is an mRNA molecule that comprises a 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 SEQ ID NO:13. Optionally, the nucleic acid comprises SEQ ID NO:13.

[0160] 12. The engineered macrophages according to embodiment 10 or 11, wherein the nucleic acid molecule is an mRNA molecule that comprises a 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 SEQ ID NO:14. Optionally, the nucleic acid comprises SEQ ID NO:14.

[0161] 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 as described in SEQ ID NO:7. Optionally, the protein encoded by the linker sequence comprises the amino acid sequence as described in SEQ ID NO:9.

[0162] 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 linker sequence, and wherein the linker sequence comprises an mRNA having the sequence as described in SEQ ID NO:15.

[0163] The engineered macrophages according to embodiments 10-14, wherein the nucleic acid molecule is an mRNA molecule comprising a sequence that is 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 SEQ ID NO:10. Optionally, the mRNA further comprises a polyA tail that is 65 to 250 residues in length, preferably 90 to 120 residues in length, preferably about, and / or a 5' cap.

[0164] 16. The engineered macrophage according to embodiments 10 - 15, wherein the nucleic acid molecule is an mRNA molecule comprising a sequence that is at least 80% identical to SEQ ID NO: 16, 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 SEQ ID NO: 16, and optionally, wherein the mRNA further comprises a 5' cap.

[0165] 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.

[0166] 18. The engineered macrophage 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.

[0167] 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 modulating the expression of endogenous silencing RNA or introducing an exogenous silencing RNA sequence, and optionally, wherein the silencing RNA is miRNA.

[0168] 20. The engineered macrophage according to any one of the foregoing embodiments, wherein the level of metalloprotease activity is at least 1.5 times that of the metalloprotease activity of non - engineered macrophages.

[0169] 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 to non - engineered, non - polarized cells.

[0170] 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 to non - engineered, non - polarized cells.

[0171] 23. The engineered macrophage according to any one of the foregoing embodiments, wherein the IL - 10 secretion of the engineered macrophage is increased by at least 1000 - fold compared to non - engineered, non - polarized cells.

[0172] 24. The engineered macrophage according to any one of the foregoing embodiments, wherein the MMP3 secretion of the engineered macrophage is increased by at least 10 - fold compared to non - engineered, non - polarized cells.

[0173] 25. The engineered macrophages according to any one of the foregoing embodiments, wherein the secretion of MMP10 by the engineered macrophages is increased by at least 20-fold compared to non-engineered, non-polarized cells.

[0174] 26. The engineered macrophages according to any one of the foregoing embodiments, wherein when cultured in vitro at a cell concentration of 4x10 6 / ml, the macrophages secrete IL-10 at a concentration of at least 10,000 pg / ml in the culture supernatant.

[0175] 27. The engineered macrophages according to any one of the foregoing embodiments, wherein when cultured in vitro at a cell concentration of 4x10 6 / ml, the macrophages secrete MMP9 at a concentration of at least 200 ng / ml in the culture supernatant.

[0176] 28. The engineered macrophages according to any one of the foregoing embodiments, wherein the expression of CD206 by the engineered macrophages is increased by at least 5-fold compared to monocytes.

[0177] 29. The engineered macrophages according to any one of the foregoing embodiments, wherein the expression of 25F9 by the engineered macrophages is increased by at least 5-fold compared to monocytes.

[0178] 30. The engineered macrophages according to any one of the foregoing embodiments, wherein the expression of CD80 by the engineered macrophages is reduced by at least ten percent compared to non-engineered, non-polarized cells.

[0179] 31. The engineered macrophages according to any one of the foregoing embodiments, wherein when cultured in vitro at a cell concentration of 4x10 6 / ml, the macrophages secrete TNF-a at a concentration of up to 40 pg / ml in the culture supernatant.

[0180] 32. The engineered macrophages according to any one of the foregoing embodiments, wherein the engineered macrophages have at least the same phagocytic ability as non-engineered, non-polarized cells.

[0181] 33. The 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 with only the IL-10 coding sequence.

[0182] 34. The engineered macrophages according to any one of the foregoing embodiments, wherein the macrophages are transiently transfected, optionally by electroporation.

[0183] 35. The engineered macrophages according to embodiment 34, wherein the transfection is non-viral.

[0184] 36. The engineered macrophages according to any one of the preceding embodiments, wherein the macrophages have a pro-repair phenotype.

[0185] 37. A population of the engineered macrophages according to any one of the preceding embodiments.

[0186] 38. A therapeutic composition comprising the population of macrophages according to embodiment 37 and a pharmaceutically acceptable medium.

[0187] 39. The engineered macrophages according to any one of embodiments 1 to 36, the population of macrophages according to embodiment 37, or the composition according to embodiment 38, which are used in therapy.

[0188] 40. The engineered macrophages, population, or composition according to embodiment 39, wherein the therapy is administered to a subject in need thereof.

[0189] 41. The engineered macrophages according to any one of embodiments 1 to 36, the population of macrophages according to embodiment 37, or the composition according to embodiment 38, which are used for treating an inflammatory condition of a subject.

[0190] 42. The engineered macrophages, population, or composition according to embodiment 40 or 41, wherein the macrophages are autologous or allogeneic to the subject.

[0191] 43. The engineered macrophages, population, or composition according to embodiment 41, wherein the inflammatory condition is liver injury, optionally chronic liver injury.

[0192] 44. The engineered macrophages, population, or composition according to embodiment 41 or 43, wherein the condition is a chronic inflammatory condition with a fibrotic component, optionally, wherein the condition is organ injury associated with chronic inflammation.

[0193] 45. The engineered macrophages, population, or composition 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.

[0194] 46. The engineered macrophages, population, or composition according to any one of embodiments 43 or 44, wherein the condition is cirrhosis.

[0195] 47. The engineered macrophages, population or composition 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 of the liver, biliary obstruction, autoimmune hepatitis, iron overload, hepatitis B infection (HBV) and hepatitis C infection (HCV).

[0196] 48. The engineered macrophages, population or composition according to embodiment 46, wherein the cirrhosis is caused by steatohepatitis (SLD), optionally, wherein the steatohepatitis is metabolic dysfunction-associated steatohepatitis, metabolic-associated steatohepatitis, Met-ALD or cryptogenic SLD.

[0197] 49. The engineered macrophages, population or composition according to any one of embodiments 46-48, wherein the cirrhosis is selected from compensated cirrhosis and decompensated cirrhosis.

[0198] 50. The engineered macrophages, population or composition according to any one of embodiments 41-49, wherein the condition is acute-on-chronic liver failure (ACLF).

[0199] 51. The engineered macrophages, population or composition according to embodiments 46-49, for treating a subject who has recovered (recompensated) from their first liver decompensation event, optionally, wherein the first liver decompensation event required hospitalization of the subject, preferably, wherein the subject has not experienced another liver decompensation event after recovering from the first liver decompensation event.

[0200] 52. The engineered macrophages, population or composition according to embodiments 46-49 and 51, wherein the subject exhibits one or more clinical signs of liver decompensation, and / or has recovered from one or more clinical signs of liver decompensation, the one or more clinical signs selected from the list consisting of jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, variceal bleeding and gastrointestinal bleeding.

[0201] 53. The engineered macrophages, population or composition according to any one of embodiments 1-52, wherein the macrophages are derived from human monocyte-derived macrophages (hMDM) or stem cells, optionally, wherein the stem cells are induced pluripotent stem cells (iPSC).

[0202] 54. The engineered macrophages, population, or composition according to embodiment 53, wherein the macrophages are derived from iPSCs, and the iPSCs are substantially lacking functional HLA I and II complexes on their surface.

[0203] 55. A method of improving the migration of monocytes to an inflammatory site, comprising using the engineered macrophages, population of engineered macrophages, or composition according to any one of the preceding embodiments.

[0204] 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.

[0205] 57. A method of generating the engineered macrophages according to any one of embodiments 1 to 36, comprising transiently transfecting macrophages with mRNA molecules encoding IL-10 and / or MMP9.

[0206] 58. The method according to embodiment 57, comprising contacting the macrophages with IL-4, IL-13, and M-CSF before, during, or after transfection.

[0207] 59. The method according to embodiment 57 or 58, wherein mRNA molecules encoding IL-10 and MMP9 are co-transfected using a bicistronic vector linked by a p2A linker sequence.

[0208] 60. The engineered macrophages according to any one of embodiments 1 to 36, wherein the macrophages are engineered with an mRNA construct encoding human IL-10 fused to human MMP9 via a cleavable linker.

[0209] 61. A method of treating inflammation and / or fibrosis, comprising administering to a subject in need thereof a therapeutically effective amount of the engineered macrophages according to any one of embodiments 1-36.

[0210] 62. A method of polarizing macrophages to a pro-repair phenotype, wherein the polarized macrophages have increased CD163 and CD206 expression and decreased HLA DR and CD86 expression compared to cells not polarized to the pro-repair phenotype, wherein the method comprises engineering the macrophages to express IL-10 and MMP9 above endogenous levels.

[0211] 63. The method according to embodiment 62, wherein the macrophages are engineered to express IL-10 and MMP9 by introducing exogenous nucleic acid comprising nucleotide sequences encoding IL-10 and MMP9.

[0212] 64. The method according to embodiment 63, wherein the nucleotide sequences encoding IL-10 and MMP9 are present on the same nucleic acid molecule.

[0213] 65. The method according to embodiment 63, wherein the nucleotide sequences encoding IL-10 and MMP9 are present on different nucleic acid molecules.

[0214] 66. The method according to any one of embodiments 63-65, wherein the nucleic acid is mRNA.

[0215] 67. A method of polarizing macrophages to a pro-repair phenotype, wherein the polarized macrophages have increased expression of CD163 and CD206 and decreased expression of HLA-DR and CD86 compared to cells not polarized to the pro-repair phenotype, wherein the method comprises engineering the macrophages to overexpress IL-10, optionally, wherein when cultured in vitro at a cell concentration of 4x10 6 / ml, the macrophages secrete IL-10 at a culture supernatant concentration of at least 10,000 pg / ml.

[0216] 68. The method according to embodiment 67, wherein the macrophages are engineered to express IL-10 by introducing an exogenous nucleic acid comprising a nucleotide sequence encoding IL-10.

[0217] 69. The method according to embodiment 67 or 68, wherein the nucleic acid is mRNA.

[0218] 70. A method of improving the cryo-resistance of macrophages, which comprises incubating macrophages in a medium comprising IL-4, IL-13 and M-CSF.

[0219] 71. A method of cryopreserving macrophages, which comprises incubating macrophages in a medium comprising IL-4, IL-13 and M-CSF before cryopreservation.

[0220] 72. The method according to embodiment 70 or 71, wherein the concentrations of IL-4 and IL-13 in the medium are 20 ng / ml, the concentration of M-CSF is 100 ng / ml, and the concentration of the macrophages is 4x106 cells / ml.

[0221] 73. The method according to embodiments 70-72, wherein the cells are incubated overnight in a medium comprising IL-4, IL-13 and M-CSF.

[0222] 74. Cryopreserved macrophages obtained by the method according to any one of embodiments 70-73.

[0223] According to a preferred embodiment, the present invention provides an engineered macrophage that is engineered to overexpress IL-10 for use in a method of treating cirrhosis in a subject hospitalized after a first liver decompensation event. In a preferred embodiment, the present invention provides an engineered macrophage that is engineered to overexpress IL-10 for use in a method of treating cirrhosis in a subject who has been hospitalized after a first liver decompensation event, wherein the subject is treated with the engineered macrophage once the subject has recovered from their first liver decompensation event and before experiencing another liver decompensation event. In a particularly preferred embodiment, the present invention provides an engineered macrophage that is engineered to overexpress IL-10 and MMP9 for use in a method of treating cirrhosis in a subject who has been hospitalized after a first liver decompensation event, wherein the subject is treated with the engineered macrophage once the subject has recovered from their first liver decompensation event and before experiencing another liver decompensation event.

[0224] According to a further preferred embodiment, the present invention provides an engineered macrophage derived from iPSC that is engineered to overexpress IL-10. Preferably, the engineered iPSC-derived macrophages are used to treat cirrhosis, particularly cirrhosis in subjects hospitalized after a first liver decompensation event. In a particularly preferred embodiment, the engineered iPSC-derived macrophages are used to treat cirrhosis, particularly cirrhosis in a subject who has been hospitalized after a first liver decompensation event, wherein the subject is treated with the engineered macrophage once the subject has recovered from their first liver decompensation event and before experiencing another liver decompensation event.

[0225] According to another preferred embodiment, the present invention provides an engineered macrophage engineered to overexpress IL-10 and MMP9, wherein the macrophage comprises exogenous mRNA encoding IL-10 and MMP9, optionally separated by a cleavable linker. Preferably, the exogenous mRNA comprises the sequence shown in SEQ ID NO:10, an optional polyA tail 65 - 250 residues in length, preferably 90 - 120 residues in length, 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 macrophage is used to treat cirrhosis, particularly cirrhosis in subjects hospitalized after a first liver decompensation event. In a particularly preferred embodiment, the engineered macrophage is used to treat cirrhosis, particularly cirrhosis in a subject who has been hospitalized after a first liver decompensation event, wherein the subject is treated with the engineered macrophage once the subject has recovered from their first liver decompensation event and before experiencing another liver decompensation event.

[0226] According to another preferred embodiment, the present invention provides an engineered macrophage, which is derived from iPSC and engineered to overexpress IL-10 and MMP9, wherein the macrophage contains exogenous mRNA encoding IL-10 and MMP9, optionally separated by a cleavable linker. Preferably, the exogenous mRNA contains the sequence shown in SEQ ID NO:10, an optional polyA tail 65-250 residues in length, preferably 90 to 120 residues in length, preferably approximately 90 residues in length, 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 for the treatment of liver cirrhosis, particularly liver cirrhosis in subjects hospitalized after the first liver decompensation event. In a particularly preferred embodiment, the iPSC-derived engineered macrophages are used for the treatment of liver cirrhosis, particularly liver cirrhosis in subjects who have been hospitalized after the first liver decompensation event, wherein the subject is treated with the engineered macrophages once the subject has recovered from their first liver decompensation event and before experiencing another liver decompensation event.

[0227] According to another preferred embodiment, the present invention provides an engineered macrophage that is engineered to overexpress IL-10, wherein when cultured in vitro at a cell concentration of 4x10 6 / ml, the macrophage secretes IL-10 at a concentration of at least 10,000 pg / ml in the culture supernatant, for use in a method of treating liver cirrhosis, particularly in subjects hospitalized after the first liver decompensation event. In a particularly preferred embodiment, the present invention provides an engineered macrophage that is engineered to overexpress IL-10, wherein when cultured in vitro at a cell concentration of 4x10 6 / ml, the macrophage secretes IL-10 at a concentration of at least 10,000 pg / ml in the culture supernatant, for use in a method of treating liver cirrhosis, particularly for treating subjects who have been hospitalized after the first liver decompensation event, wherein the subject is treated with the engineered macrophages once the subject has recovered from their first liver decompensation event and before experiencing another liver decompensation event.

[0228] According to another preferred embodiment, the present invention provides engineered macrophages engineered to overexpress IL-10 and MMP9, wherein the engineered macrophages: have a metalloprotease activity level that is at least 1.5 times that of non-engineered macrophages; have a CD86 expression reduced by at least two-fold compared to non-engineered, non-polarized cells; have an HLA-DR expression reduced by at least two-fold compared to non-engineered, non-polarized cells; have an IL-10 secretion increased by at least 1000-fold compared to non-engineered, non-polarized cells; have an MMP3 secretion increased by at least 10-fold compared to non-engineered, non-polarized cells; have an MMP10 secretion increased by at least 20-fold compared to non-engineered, non-polarized cells; have a CD206 expression increased by at least 5-fold compared to monocytes; have a 25F9 expression increased by at least 5-fold compared to monocytes; have a CD80 expression reduced by at least ten percent compared to non-engineered, non-polarized cells; when cultured in vitro at a cell concentration of 4x10 6 / ml, secrete IL-10 at a culture supernatant concentration of at least 10,000 pg / ml; when cultured in vitro at a cell concentration of 4x10 6 / ml, secrete MMP9 at a culture supernatant concentration of at least 200 ng / ml; and when cultured in vitro at a cell concentration of 4x10 6 / ml, secrete TNF-a at a culture supernatant concentration of up to 40 pg / ml. In a particularly preferred embodiment, the engineered macrophages are derived from iPSCs, and wherein the macrophages comprise exogenous mRNAs encoding IL-10 and MMP9, optionally separated by a cleavable linker. Preferably, the iPSC-derived engineered macrophages are used for the treatment of liver cirrhosis, particularly liver cirrhosis in subjects hospitalized after the first liver decompensation event. The present invention will now be further described with reference to the following titled sections. Any feature under any section can be combined with any aspect or embodiment of the present invention in any feasible order.

[0229] Description

[0230] The following definitions are provided.

[0231] As used herein, "payload" is one or more genes with a therapeutic purpose, which are introduced by transfection to test their effects on macrophages.

[0232] As used herein, "non-polarized macrophages" refers to mature macrophages that have not received any further stimulation to induce specific functional capabilities. Non-polarized macrophages can also refer to naive or unactivated macrophages.

[0233] "Mature macrophages" refer to macrophages that express mature cell surface markers, preferably CCR2-, CD14+ and 25F9+.

[0234] Macrophages can acquire various states, termed "polarization", which are generally but simply divided into two main extremes, "pro-inflammatory" (or classically activated, "M1", "M1-like") and "pro-regenerative" (or "pro-reparative", alternatively activated, "M2", "M2-like", anti-inflammatory or anti-fibrotic). However, macrophages may adopt states that lie between these two extremes, potentially being "unpolarized", resting or naive (M0), or points more towards an anti-inflammatory or pro-inflammatory state.

[0235] It is generally considered that M1 macrophages are pro-inflammatory, while M2 macrophages are responsible for immune regulation and wound healing responses. However, it has become increasingly clear that this binary classification does not account for the greater complexity of heterogeneity in vivo, where macrophages adopt different phenotypes and even switch between phenotypes in response to the myriad stimuli to which they are exposed. These in vivo macrophage phenotypes cannot be precisely recapitulated in tissue culture models, which emphasizes 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 mainly mediate wound healing and tissue regeneration.

[0236] Due to the complexity of macrophage biology, the classification of "M1" and "M2" can be considered too simplistic. For example, "M2" macrophages are actually a spectrum that depends on their environment. Thus, attempts have been made to further classify the pro-regenerative M2-like states, such as the following sub-classification proposed by Gharavi, A.T et al., "The role of macrophage subtypes and exosomes in immunomodulation", Cell Mol Biol Lett 27, 83 (2022). In this sub-classification, for example:

[0237] "M2a" cells are considered anti-inflammatory, pro-fibrotic, and play a role in allergy 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, stabilin 1, factor XIII-A, Ly6C, TREM-2 and DC-SIGN. The M2a state may be acquired under the action of factors such as IL-4, IL-13, IL-10 and PPARg.

[0238] “M2b” cells are thought to be involved in the activation of T helper type 2 (Th2) responses, immune regulation, and promotion of tumor progression. These cells are classified by the expression of IL-6, TNF-a, CD86, and SPHK1. The M2b state may be acquired under the action of IL-1b or upon exposure to LPS.

[0239] “M2c” cells are associated with immunosuppression, phagocytosis, tissue repair, and extracellular matrix remodeling. These cells are classified by the expression of IL-10, CXCL13, CD163, CD206, CXCR4, TGF-b, and MerTK. The M2c state may be acquired under the action of IL-10, glucocorticoids, IL-6, IL-10, TNF-a, and TLR stimulation.

[0240] “M2d” cells are associated with tumor progression, angiogenesis, and clearance of apoptotic tissues. These cells are classified by the expression of IL-10, VEGF, and TGF-b. The M2d state may be acquired upon exposure to LPS.

[0241] Among the markers associated with each of these pro-repair states, the engineered macrophages of the present invention can generally express IL-10, CCL22, CD11b, CD45, CD206, CD86, CD163, and CXCR4. The engineered macrophages of the present invention can generally also express MHC II, which is more commonly associated with pro-inflammatory macrophages. Thus, it can be seen that the engineered macrophages of the present invention are different from the pro-repair, anti-inflammatory macrophages described in the literature because they express markers associated with multiple subtypes, such as M2a and M2c-like cells. However, in all aspects of the present invention, the engineered macrophages of the present invention can express markers consistent with functional human monocyte-derived macrophages, such as CD45, CD14, CD206, CCR2, CD163, CD169, and 25F9.

[0242] The engineered macrophages of the present invention (comprising IL-10 and MMP9) can function as “pro-repair” macrophages. As described, for example, in Ramachandran et al, Proc Natl Acad Sci USA. 2012 Nov 13;109(46):E3186–E3195, pro-repair macrophages have a loss of pro-inflammatory gene expression, an increase in matrix-degrading enzyme expression, and enrichment of phagocytosis-related genes. In addition, the phenotype of pro-repair macrophages does not fall within the M1 / M2 paradigm, highlighting the limitations of this classification in the in vivo context. Pro-repair macrophages play an important role in tissue remodeling, such as fibrosis resolution.

[0243] Macrophages reported by Ramachandran et al. have high MMP activity. In addition, in Figure 5 , the authors showed several genes regulated in pro-repair macrophages. Notably, they saw upregulation of Mrc1 (now CD206), which is also expressed in the macrophages of the examples described herein. In addition, they showed low levels of TNFα and IL1b, which were also confirmed in the examples given herein. Thus, this may be an indication of the pro-repair phenotype. However, these macrophages described herein naturally express low levels of IL-10.

[0244] As used herein, "GMP-compliant" means that the method complies with the principles of good manufacturing practice and can be used interchangeably with "GMP-compatible" and "GMP-grade". For example, a GMP-compliant culture medium must be serum-free, antibiotic-free, animal-free, and free of xenoproteins. The requirements of the WHO for good manufacturing practice provide guidance: "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 updated edition). WHO Press. pp. 17–18. ISBN 9789241547086.

[0245] As used herein, "UT", "NTRx", or "UT N / T" refers to untransfected macrophages that are differentiated from monocytes in the same process used to differentiate transfected macrophages but are not further incubated with additional factors and / or transfection. Such macrophages are similar to the non-polarized, untransfected cells used in the MATCH study described in WO2019175595 (thus UT can also be described as MATCH-like cells in this application), however, the macrophages in the MATCH study were matured for 7 days, while the UT macrophages in this application were matured for 5 days.

[0246] As used herein, "UT+TR", "NTRx+Tr", or "UT T" refers to untransfected cells (as described above) that have been treated similarly to transfected cells (i.e., incubated with IL-4 + IL-13 + M-CSF after mock transfection). The incubation with IL-4 + IL-13 + M-CSF is described in more detail in the examples herein.

[0247] The present text also describes hMDMs transfected with constructs encoding specific products, such as MMP9 and / or IL-10, which are 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 referred to as "IL-10TRx".

[0248] The present text also describes hMDMs transfected with constructs encoding specific products and further treated with IL-4 + IL-13 + M-CSF. Such cells are designated as "TRx+TR", such as "IL-10TRx+TR". The present text also describes hMDMs transfected with an exogenous bicistronic mRNA construct expressing IL-10 and MMP9. Such cells are designated as "RTX001". Without wishing to be bound by theory or mechanism, overexpression of IL-10 and MMP9 means that RTX001 cells can be polarized into an anti-inflammatory and / or pro-repair phenotype.

[0249] It should be noted that the term "a" or "an" entity refers to one or more of that entity.

[0250] "About" means + / - 10%, + / - 9%, + / - 8%, + / - 7%, + / - 6%, + / - 5%, + / - 4%, + / - 3%, + / - 2%, + / - 1% of a given value, unless otherwise stated.

[0251] Engineered Macrophages for Cell Therapy

[0252] 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.

[0253] The present 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 comprising exogenous coding sequences. The exogenous coding sequences can be extrachromosomal or integrated into the cell genome. The engineered macrophage expresses exogenous coding sequences of IL-10 and MMP9.

[0254] In some embodiments, the engineered macrophage carries one or more exogenous sequences that can turn on the 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 native macrophage genome) is an engineered macrophage according to the present invention.

[0255] The engineered macrophages can be used for treatment, for example, for treating inflammatory and / or fibrotic conditions in a subject.

[0256] Preferably, the administration of the engineered macrophages to a subject is not associated with an inflammatory response. In particular, the administration of the engineered macrophages to a subject is preferably not associated with an increase in the concentration of inflammatory cytokines such as IL-1β and / or TNF-α in the plasma.

[0257] As used herein, "macrophage" refers to a phagocytic cell responsible for detecting, phagocytosing, and destroying pathogens and apoptotic cells, and the phagocytic cell is produced by the differentiation of monocytes. The "engineered macrophages" of the present invention are macrophages that have been engineered to express IL-10 and / or MMP9. In particular, the expression is higher than the endogenous level such that the engineered macrophages express IL-10 and / or MMP9 at a higher level than non-engineered cells. The macrophages can be engineered to express these proteins by including an exogenous coding sequence. In some embodiments, when cultured in vitro at a cell concentration of 4×106 / ml, the macrophages secrete IL-10 at a culture supernatant concentration of at least 10,000 pg / ml. In some embodiments, when cultured in vitro at a cell concentration of 4×106 / ml, the macrophages secrete MMP9 at a culture supernatant concentration of at least 200 ng / ml.

[0258] In some embodiments, the amino acid sequence of IL-10 is encoded by an mRNA comprising the sequence of SEQ ID NO.1. The native mRNA sequence of IL-10 mRNA is provided at: NCBI Reference Sequence: NM_000572.3., which also includes variants and homologs of this sequence.

[0259] 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 metalloproteases (MMPs), also known as matrix metalloproteinases or matrixins, are a family of peptidases that can collectively cleave all components of the extracellular matrix (ECM). MMPs can also process bioactive mediators such as growth factors, cytokines, chemokines, and cell surface receptors. Twenty-five mammalian MMPs have been identified, which have different roles in the maintenance of the extracellular matrix and tissue repair processes, and have inhibitory and stimulatory effects in fibrosis.

[0260] In some embodiments, the MMP9 coding sequence is human MMP9 as described in the NCBI reference sequence: NM_004994.3. In some embodiments, the amino acid sequence is encoded by an mRNA comprising the sequence of SEQ ID NO.3 or a variant that is at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% similar to SEQ ID No.3.

[0261] In some embodiments, the mRNA is modified. In some embodiments, the mRNA modification is selected from chemical modifications of uridine and / or chemical modifications of 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 uridines are replaced by 5-methoxy-uridine.

[0262] In some embodiments, the mRNA comprises a poly-A tail at the 3’ end. A suitable poly-A tail is 65-250 residues in length, preferably 90 to 120 residues in length. 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 polyA tail is 120 residues in length. In a preferred embodiment, the polyA tail is longer than the endogenous polyA tail. In a particularly preferred embodiment, the polyA tail is 90 residues in length. In a particularly preferred embodiment, the mRNA comprises the sequence of SEQ ID No.10 or a variant that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to SEQ ID NO:10. In another preferred embodiment, the exogenous mRNA sequence comprises a 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:16. In a particularly preferred embodiment, the exogenous mRNA sequence comprises the sequence listed in SEQ ID NO:16. As described herein, the endogenous uridines in SEQ ID NO 10 and 16 are replaced by 5-methoxy-uridine. In a preferred embodiment, the mRNA comprises the sequence of SEQ ID NO:10, a 90-residue-long polyA tail and a 5’ cap. In a preferred embodiment, the mRNA comprises the sequence of SEQ ID NO:16 and a 5’ cap.

[0263] In some embodiments, the mRNA is degradation-resistant. In some embodiments, the mRNA is non-immunogenic.

[0264] 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 a fusion protein encoded by a single mRNA molecule and linked by a cleavable linker.

[0265] 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%, 99% similar to SEQ ID No.2.

[0266] In some embodiments, either or both of the 5' and 3' ends of the mRNA have been modified by any means that enhance stability and / or the ability to express the encoded protein.

[0267] 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 to 5-methoxy-uridine.

[0268] In some embodiments, one or both caps of the mRNA molecule are synthetic, preferably

[0269] Overexpression is understood as "excessive" or higher-level expression of a gene, such as caused by an increased frequency of gene transcription. Thus, it can also be regarded as expression above the wild-type or normal level. Overexpression can be defined with reference to the amount of protein produced by a cell population, or by reference to the fold increase over the wild-type or normal expression level. The expression level can be described as the amount of protein secreted per unit volume of cell culture. However, an increase in transcription does not necessarily result in an increase in the amount of secreted protein.

[0270] Cells overexpressing IL-10 and / or MMP9 contain coding sequences that express IL-10 and / or MMP9 at higher levels than non-engineered cells. As described above, overexpression can be achieved by introducing exogenous nucleic acids encoding IL-10 and / or MMP9, such as mRNA, or by genetic modification that stimulates the expression of IL-10 and / or MMP9 from endogenous coding sequences. Engineered macrophages overexpressing IL-10 and / or MMP9 do not necessarily secrete a greater amount of IL-10 and / or MMP9 than non-engineered macrophages.

[0271] As used herein, overexpression involves an increase in the artificial expression level of a gene 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, the expression level was quantified between 16 and 24 hours after transfection. The expression level described herein is given for a macrophage population at a concentration of 4x106 / ml (equivalent to 2x106 cells per cm2). In a preferred embodiment, overexpression is achieved by introducing exogenous mRNA into macrophages. In the examples, 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. Equivalent conditions suitable for determining the concentration of secreted proteins will be known to those skilled in the art. The following levels were determined experimentally under these conditions:

[0272] In an embodiment, overexpression of IL-10 means that the level of secreted IL-10 protein is higher than about 300 pg / ml. Suitably, the 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 level of secreted IL-10 protein is greater than 10,000 pg / ml.

[0273] In an embodiment, "relative overexpression" of IL-10 in an IL-10 engineered macrophage culture means an increase in the level of IL-10 protein secreted in the culture of about 100 - 300 pg / ml, or greater than about 300 pg / ml, relative to the average wild-type protein secretion of a wild-type macrophage culture grown 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 level of secreted IL-10 protein is greater than 10,000 pg / ml. Suitably, these IL-10 protein levels can be measured by culturing macrophages as described above, where the concentration of macrophages in the medium is 4x10 6 cells / ml, equivalent to 2x10 6 cells / cm 2 , and measuring the concentration of protein in the medium. Thus, when cultured in vitro at a cell concentration of 4x10 6 / ml, the macrophages secrete IL-10 at a culture supernatant concentration of at least 10,000 pg / ml. In some embodiments, the engineered macrophages secrete IL-10 at a level 1000-fold higher than non-engineered, non-polarized hMDMs. Non-engineered, non-polarized hMDMs are described in the art, for example in WO2019175595.

[0274] In an embodiment, expression of MMP9 means that the level of secreted MMP9 protein is from about 200 ng / ml to 2000 ng / ml. Suitably, the 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 in the range of about 200 ng / ml - 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 (comprising IL-10 and MMP9) is higher than the average level of MMP9 protein secreted by macrophages engineered with IL-10 alone. Suitably, the overall MMP activity of the engineered macrophages of the present invention is also higher than the overall MMP activity of macrophages engineered with IL-10 alone. In a preferred embodiment, the overall MMP activity of the engineered macrophages is at least 1.5-fold higher than that of untransfected macrophages.

[0275] In an embodiment, "relative overexpression of MMP9" in a MMP9-engineered macrophage culture means an increase in the level of secreted MMP9 protein in the culture by about 200 ng / ml to 2000 ng / ml, relative to the average wild-type protein expression in a wild-type macrophage culture cultured under the same conditions. Suitably, the increase in 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 1,000 ng / ml, relative to the average wild-type protein expression in a wild-type macrophage culture cultured under the same conditions. Suitably, relative to the average wild-type protein expression in a wild-type macrophage culture cultured under the same conditions, the level of secreted MMP9 protein in the culture increases by about 200 ng / ml to 2000 ng / ml. Suitably, these MMP9 protein levels can be measured by culturing macrophages as described above, wherein the concentration of macrophages in the medium is 4x10 6 cells / ml, equivalent to 2x10 6 cells / cm 2 , and measuring the concentration of protein in the medium. Thus, when at 4x10 6When cultured in vitro at a cell concentration of / ml, the macrophages secrete MMP9 at a concentration of at least 200 ng / ml in the culture supernatant. In a preferred embodiment, the secreted MMP9 level is greater than 200 ng / ml. Suitably, relative to the average wild-type protein expression of wild-type macrophage cultures cultured under the same conditions, the increase in the level of MMP9 secreted in IL-10-MMP9 engineered macrophage (i.e., containing IL-10 and MMP9) cultures is greater than the average increase in the MMP9 protein cultures secreted in macrophages engineered with MMP9 alone. Suitably, the overall MMP activity of the engineered macrophages of the present invention is also higher than the overall MMP activity of macrophages engineered with IL-10 alone. In some embodiments, these increases are synergistic. In a preferred embodiment, the overall MMP activity of the engineered macrophages is at least 1.5 times higher than that of untransfected macrophages.

[0276] In an embodiment, "relatively low expression of MMP9" in IL-10 engineered macrophage cultures means that the level of MMP9 protein secreted in the culture is reduced by about 50 ng / ml to 100 ng / ml, about 100 ng / ml to 200 ng / ml, about 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, the reduction in the level of secreted MMP9 protein is greater than: about 100 ng / ml or 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 1,000 ng / ml, relative to the average wild-type protein expression of wild-type macrophage cultures cultured under the same conditions. Suitably, the level of MMP9 protein secreted in the culture is reduced by about 200 ng / ml to 500 ng / ml, relative to the average wild-type protein expression of wild-type macrophage cultures cultured under the same conditions.

[0277] The engineered macrophages of the present invention (containing IL-10 and MMP9) secrete MMP9 at a higher level than the MMP9 expressed by macrophages engineered with IL-10 alone (see: Figure 1 : Comparison of IL-10-MMP9 Trx with IL-10Trx). 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 promoter, terminator, poly(A) signal sequence, etc.

[0278] A "promoter" is a nucleotide sequence that initiates and regulates the transcription of a polynucleotide. A promoter may include an inducible promoter (wherein the expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), a repressible promoter (wherein the expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and a constitutive promoter. The term "promoter" or "control element" includes the full-length promoter region and functional (e.g., controlling transcription or translation) fragments of these regions.

[0279] In one embodiment, the DNA vector may include one or more liver-specific promoters or liver cirrhosis-specific promoters. In some embodiments, the DNA vector may comprise a CX3CR1 promoter, insulin-like growth factor 1 (IGF1), or a CD1IB promoter.

[0280] "Operably linked" refers to the arrangement of elements wherein the components so described are configured to perform their usual functions. Thus, a given promoter operably linked to a nucleic acid sequence is capable of affecting the expression of that sequence when the appropriate enzymes are present. The promoter need not be adjacent to the sequence so long as it can direct its expression. Thus, for example, there may be intervening untranslated but transcribed sequences between the promoter sequence and the nucleic acid sequence, and the promoter sequence may still be considered "operably linked" to the coding sequence. Thus, the term "operably linked" is intended to include any spacing or orientation of promoter elements and DNA sequences of interest that allows transcription of the DNA sequence of interest to be initiated when the transcription complex recognizes the promoter elements.

[0281] "Treatment" as used in the present invention refers to an intervention in a physiological condition that prevents, alleviates, or eliminates clinical symptoms associated with a given physiological condition of a subject.

[0282] A "therapeutically effective amount" of the macrophages described in this specification is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes the regression of a disease, as demonstrated by a decrease in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or the prevention of injury or disability caused by the affliction of the disease. The ability of a therapeutic agent to promote the regression of a disease can be evaluated using a variety of methods known to those of skill in the art, such as in human subjects during clinical trials, in animal model systems predictive of human efficacy, or by assaying the activity of the therapeutic agent in in vitro assays. The therapeutically effective amount and dosage regimen can be determined empirically by testing in known in vitro or in vivo (e.g., animal model) systems.

[0283] "Subject", "individual", "animal", or "patient" refers to any subject, particularly a mammalian subject, in need of diagnosis, prognosis, or treatment, unless the subject is defined as a "healthy subject". Mammalian subjects include humans; domestic animals; farm animals; such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, etc.

[0284] Suitably, the subject may be in need of treatment. Thus, suitably, the subject may have a disease, condition, or disorder, or be at risk of developing a disease, condition, or disorder. Suitably, the subject may exhibit one or more symptoms of a disease, condition, or disorder.

[0285] The engineered macrophages of the present invention can be used for treatment. The macrophages of the present invention can be used to treat inflammatory and / or fibrotic conditions in a subject. As described above, treatment here can refer to prevention, reduction, or elimination of inflammation / fibrosis / organ damage. For example, engineered macrophages can be administered to a subject in the acute inflammation phase for the purpose of preventing chronic inflammatory conditions. Engineered macrophages can also be administered to a subject in the chronic inflammation phase for the purpose of preventing / reducing chronic fibrosis. Engineered macrophages can also be administered to a subject experiencing a slow plus acute inflammatory state, such as acute-on-chronic liver failure (ACLF).

[0286] Suitably, an acute disease or injury can be classified as a disease or injury with a disease onset time of less than 24 weeks. Appropriately, a chronic disease can be classified as a disease or injury that persists for more than 6 months. Suitably, a slow plus acute disease can be classified as a disease or injury with a disease onset time of less than 24 weeks from the causative agent in a patient who already has a chronic disease that has persisted for more than 6 months. Suitably, the engineered macrophages of the present invention can be administered to a subject with an acute onset to prevent the transition or increase of chronic inflammation and fibrosis.

[0287] Fibrosis refers to the deposition of extracellular matrix and connective tissue after tissue injury, which may lead to replacement of parenchymal tissue and, if excessive, ultimately result in scarring. In some embodiments, the condition is fibrosis.

[0288] In some embodiments, the condition is present 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, or a combination thereof.

[0289] In some embodiments, the condition is chronic organ damage associated with chronic inflammation. Suitably, the condition involves the kidney, liver, or lung. For example, the condition can be inflammatory liver injury, inflammatory kidney injury, or inflammatory lung injury.

[0290] Suitably, the present invention relates to a cell therapy product for inflammatory organ injury, which is based on monocyte-derived macrophages genetically modified with a payload that induces a pro-repair phenotype.

[0291] Suitably, the engineered macrophages have a pro-repair phenotype and are anti-inflammatory and anti-fibrotic.

[0292] The M2-like phenotype is pro-repair, while the M1-like phenotype is pro-inflammatory.

[0293] The engineered macrophages comprise exogenous coding sequences of IL-10 and MMP9. Such exogenous coding sequences can be provided in any suitable manner. This can be in the form of a nucleic acid vector, regardless of how it is delivered, or gene modification. For example, using viral or non-viral vectors, DNA or RNA constructs, or gene editing using any suitable technique.

[0294] 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 technique.

[0295] The macrophages can be engineered by viruses. In such a genetic engineering method, a virus, such as a lentivirus, an adenovirus, or an AAV, is used to introduce the exogenous coding sequence / payload into the macrophages. The virus can provide the exogenous coding sequence / payload as an "episomal" construct, or the gene can be integrated into the genome of the macrophages. As documented in the art, viral engineering of macrophages requires careful techniques to prevent macrophages from phagocytosing the virus.

[0296] Suitably, the engineered macrophages are non-virally engineered, for example, using a nucleic acid vector comprising an exogenous coding sequence. The nucleic acid can be any suitable nucleic acid, including DNA and RNA. Suitably, the macrophages are transfected with a DNA vector. Suitably, the DNA vector is a naked DNA vector such that it is not bound to proteins and / or lipids. Suitably, 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 macrophages. Alternatively, the nucleic acid vector is a messenger RNA (mRNA) molecule.

[0297] Suitably, the DNA vector contains at least one sequence encoding IL-10 and / or MMP9, which is operably linked to a promoter.

[0298] As used herein, "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 a macromolecule, such as a protein, in a cell. If the transcription and translation of the mRNA corresponding to a gene result in the production of a protein in a cell, then that gene encodes the protein.

[0299] Suitably, the macrophage is a genetically engineered macrophage that contains a nucleic acid construct that overexpresses IL-10 and expresses MMP9.

[0300] Suitably, the mRNA molecule expresses IL-10 and / or MMP9. In other words, the mRNA includes the coding sequences of IL-10 and MMP9, which are exogenous to the macrophage. The mRNA can 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 herein.

[0301] Any suitable transfection method can be used to introduce the nucleic acid vector into the cell, such as, but not limited to: cationic liposome-mediated transfection, lipofection, polymer encapsulation, peptide-mediated transfection, or a bioparticle delivery system, such as a "gene gun". Suitably, the macrophage is transfected with the nucleic acid by electroporation. Other suitable transfection methods include nucleofection.

[0302] The macrophage can be suitably engineered by genetically editing the genome of the macrophage. Genetic editing allows for the permanent insertion of exogenous coding sequences. Many genetic editing techniques are known, including those that require the use of nucleases. From base editing techniques, primer editing techniques to gene editing, various nucleases are known to be useful for modifying the genome. Many nucleases are known - such as Zinc finger, TALEN, 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 macrophage itself can be genetically edited, or the progenitor cell can be genetically edited before being transformed into a macrophage.

[0303] Suitably, the macrophage is transfected with one or more free nucleic acids or vectors.

[0304] Suitably, the macrophage is transfected with one or more exogenous coding sequences of IL-10 and MMP9.

[0305] Suitably, the macrophage has at least one exogenous coding sequence of IL-10 and at least one exogenous coding sequence of MMP9.

[0306] Suitably, the macrophage is engineered to overexpress IL-10 and express MMP9.

[0307] Suitably, the macrophages are autologous or allogeneic to the subject.

[0308] The invention also relates to a population of engineered macrophages as described herein. The population can be used as a cell therapy.

[0309] Suitably, the use includes administering an effective amount of the engineered macrophages to a subject.

[0310] The invention also relates to a composition comprising the engineered macrophages of the invention or a population thereof. Suitably, the composition is a pharmaceutical composition.

[0311] 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 immiscible liquid.

[0312] Suitably, the engineered macrophages are for administration to a subject by any route. Delivery to the subject can be by local or systemic administration. In some embodiments, administration is by, for example, local injection, nebulizer, systemic injection. Suitably, the engineered macrophages are for administration to a subject by infusion. Suitably, the engineered macrophages are for administration to a subject by parenteral, suitably intravenous injection. Suitably, the engineered macrophages are for administration to a subject by injection or infusion. Suitably, the engineered macrophages are for administration to a subject by intravenous infusion.

[0313] The invention also relates to a method of improving the migration of monocytes towards inflammation, which comprises using the engineered macrophages according to the first aspect of the invention, a population of macrophages according to the second aspect of the invention or a composition according to the third aspect of the invention.

[0314] Suitably, the engineered macrophages, the engineered macrophages for treatment or the engineered macrophages in the method of improving the migration of monocytes towards inflammation are chemotactic for monocytes. The engineered macrophages are chemotactic for monocytes in vitro or in vivo. Recruiting host monocytes to the site of inflammation / damaged organ is beneficial for treating inflammatory conditions. In some embodiments, the engineered macrophages are specifically chemotactic for monocytes and not for other immune cell types. In some embodiments, when administered to a subject with fibrosis, such as a subject with a chronic inflammatory condition having a fibrotic component, the engineered macrophages localize to the fibrotic site, and / or recruit monocytes to the fibrotic site. As described in the examples herein, the presence of engineered macrophages or monocytes at the fibrotic site can be determined by flow cytometry. In some embodiments, the fibrotic site can be the lung or the liver.

[0315] Generation of Engineered Macrophages Expressing IL-10 and MMP9

[0316] The present invention also relates to a method for generating engineered macrophages that express IL-10 and MMP9, which comprises transiently transfecting macrophages with a combined mRNA construct comprising at least one sequence encoding IL-10 and at least one sequence encoding MMP9. In some embodiments, after transfection, the macrophages are contacted with an anti-inflammatory therapeutic agent. 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 regain MMP activity due to co-expression with MMP9 (otherwise MMP activity is inhibited by IL-10). Suitably, the engineered macrophages are pro-reparative. Suitably, the generated engineered macrophages can be used in cell therapy.

[0317] Notably, macrophages engineered to overexpress IL-10, or IL-10 and MMP9, exhibit a pro-regenerative phenotype without further anti-inflammatory treatment after transfection. Suitably, the generated engineered macrophages are manufactured according to GMP-compliant standards. Thus, it is suitable that the engineered macrophages and their populations are GMP-compliant.

[0318] In other embodiments, the method for generating engineered macrophages comprises introducing a sequence encoding IL-10 and / or MMP9 into the genome of the macrophages.

[0319] In a preferred embodiment, in a population of engineered macrophages generated by this method, at least 80% of the macrophages secrete IL-10.

[0320] The present invention also provides a method for improving the cryoresilience of macrophages, which comprises incubating macrophages in a medium containing IL-4, IL-13 and M-CSF. According to some embodiments, incubate with IL-4, IL-13 and M-CSF before cryopreservation. According to some embodiments, remove IL-4, IL-13 and M-CSF from the medium before cryopreservation such that the cryopreservation solution is free of IL-4, IL-13 and M-CSF. According to some embodiments, the present invention provides a method for cryopreserving macrophages, which comprises incubating macrophages in a medium containing IL-4, IL-13 and M-CSF before cryopreservation. According to some embodiments, the method for improving cryoresilience or the method for cryopreserving macrophages further comprises removing IL-4, IL-13 and M-CSF from the medium before 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 viable cells after freezing in cryopreserved macrophages. In some embodiments, the concentrations of IL-4 and IL-13 in the medium are 20 ng / ml, the concentration of M-CSF is 100 ng / ml, and the concentration of the macrophages is 4 x 106 cells / ml. In some embodiments, the cells are incubated overnight in a medium containing IL-4, IL-13 and M-CSF.

[0321] Macrophages for engineering

[0322] Suitably, the macrophages for engineering are generated from any suitable progenitor cells. Suitably, the macrophages are generated in vitro.

[0323] Macrophages suitable for engineering are monocyte-derived. Suitably, they are human monocyte-derived macrophages (hMDM). Monocyte-derived means macrophages differentiated from monocytes. Monocytes are the natural precursors of macrophages and dendritic cells; they are present in blood and bone marrow. Suitably, the macrophages are derived from peripheral blood monocytes, suitably, the macrophages are peripheral blood monocyte-derived macrophages. Suitably, the macrophages are human peripheral blood monocyte-derived macrophages. Suitably, the monocytes are isolated from a human subject.

[0324] Suitably, the macrophages are derived from monocytes by suitably culturing monocytes in vitro. Suitably, the macrophages are derived from monocytes by any suitable culturing method.

[0325] Suitably, the macrophages are generated in vitro from monocytes by a culture method lasting for 3 to 8 days, optionally 4 to 8 days. Suitably, the macrophages are generated in vitro from monocytes by a culture method lasting for 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 for 3 - 5 days, 4 or 5 days or 7 days, which are respectively referred to as the day 5 method or the day 7 method. 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 No. PCT / GB2021 / 051294 (the content of which is incorporated herein by reference).

[0326] Suitably, the macrophages are generated by the "day 5" method, which comprises:

[0327] (a) Culturing monocytes in a medium for 3 - 5 or 4 - 5 days to generate macrophages, wherein the medium contains one or more growth factors that stimulate macrophage production;

[0328] wherein step (a) is carried out entirely in the same medium.

[0329] Suitably, the medium contains one or more growth factors selected from the CSF family, preferably M - CSF. Suitably, the medium contains M - CSF at a concentration between 25 - 150 ng / mL. Suitably, the medium contains 100 ng / mL of GMP - grade recombinant human macrophage colony - stimulating factor 1 (rhM - CSF - 1; also referred to as "rh(recombinant human)CSF - 1").

[0330] In other embodiments, the macrophages are generated from stem cells, such as pluripotent stem cells or multipotent stem cells. In a particular embodiment, the macrophages are generated from induced pluripotent stem cells (iPSCs).

[0331] Anti - inflammatory treatment

[0332] In one embodiment, a method for generating engineered macrophages engineered with a combination of IL - 10 and MMP9 comprises transiently transfecting macrophages with exogenous coding sequences of IL - 10 and MMP9. These coding sequences can be provided by transfecting with nucleic acids, such as one or more mRNA molecules.

[0333] The macrophages have at least one exogenous coding sequence of IL - 10 and at least one exogenous coding sequence of MMP9.

[0334] In some embodiments, after transfection with an exogenous coding sequence, the macrophages are contacted with an anti-inflammatory therapeutic agent. Suitably, the anti-inflammatory therapeutic agent comprises an anti-inflammatory cytokine. In some embodiments, the macrophages are contacted with an anti-inflammatory therapeutic agent comprising IL-4 and IL-13.

[0335] In some embodiments, the anti-inflammatory therapeutic agent can be added in the method of transfecting macrophages.

[0336] The transfected macrophages can be contacted with these anti-inflammatory cytokines (IL4 and IL13) for a period of about 2 hours to about 48 hours, suitably 4 hours to 40 hours, suitably 12 hours to 24 hours, optionally about 16 hours.

[0337] The transfected macrophages can be contacted with these anti-inflammatory cytokines (IL4 and IL13) at a cytokine concentration of 2 ng / mL to 200 ng / mL, suitably 5 ng / mL to 150 ng / mL, suitably 10 ng / mL to 100 ng / mL, suitably 15 ng / mL to 75 ng / mL, suitably 20 ng / mL to 50 ng / mL.

[0338] Suitably, the anti-inflammatory therapeutic agent is used as a solution.

[0339] For the step of contacting macrophages with IL4 + IL13, the cells are suitably plated as follows: 2x10 6 hMDM / cm 2 in 4x10 6 / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0340] The present invention will now be described with reference to the following drawings.

[0341] Figure 1 - Graph depicting the experimental results of transfecting macrophages with IL-10 and IL-10 + MMP9 - Macrophages secrete high levels of IL-10. A: IL-10 transfected hMDM show reduced MMP9 expression levels. B: IL-10 + MMP9 transfected hMDM show elevated MMP9 levels. The dashed line represents the minimum level required for the product.

[0342] Figure 2 - Graph depicting the experimental results of transfecting macrophages with multiple constructs. Depicted is the percentage (%) of IL-10 secreting cells within a 2-hour time frame using a flow cytometry capture assay. Both constructs produce a high percentage of secreting cells. Each symbol represents an independent donor.

[0343] Figure 3- Graph depicting the experimental results of transfecting macrophages with multiple constructs in terms of cell surface markers. By flow cytometry, flow cytometry analysis of identity cell surface markers of hMDM. The black solid line is the normalized level of expression on non-engineered cells. Each graph represents a different macrophage surface marker: A: CD45, B: CD14, C: CD206; D: CCR2, E: CD163, F: CD169, and G: 25F9.

[0344] Figure 4 - Graph depicting the experimental results of transfecting macrophages with multiple constructs in terms of pro-inflammatory markers. Measured by flow cytometry, the pro-inflammatory markers CD86 (A) and HLA-DR (B) were significantly downregulated in engineered cells. Other inflammatory markers were essentially unchanged compared to un-Trx hMDM. The expression of CD80 was ideally no more than 20% or 10 - 15% higher than the non-genetically engineered (NTx) level. The black solid line represents the level of un-Trx cells. The red dashed line represents the maximum desired level. Each symbol represents an independent donor.

[0345] Figure 5 - Graph depicting the experimental results of transfecting macrophages with multiple constructs in terms of phagocytic ability. Phagocytosis was measured using pH-sensitive (pHrodo) beads coated with Escherichia coli. The percentage of phagocytic macrophages was measured by flow cytometry. Each symbol represents an independent donor. The dashed line represents the minimum desired percentage of phagocytic macrophages.

[0346] Figure 6 - Graph depicting the experimental results of transfecting macrophages with multiple constructs in terms of M1 (A and B) and M2 markers (C and D). M1 and M2 markers were used as indicators of the pro-inflammatory and anti-inflammatory phenotypes of macrophages generated in vitro. Flow cytometry analysis of M1- and M2-type cell surface markers in macrophages treated overnight with supernatants from un-Trx, un+Trx+ treated, IL-10Trx, and IL-10+MMP9 Trx hMDM. M2 = positive control: macrophages from the same donor were polarized using a high level of IL-10 (dashed line). Panel A shows the results for CD86, panel B shows the results for HLA DR. Panel C shows the results for CD206, and panel D shows the results for CD163.

[0347] Figure 7 - Graph depicting the experimental results of transfecting macrophages with multiple constructs in terms of the effect on the migration of other cells. Results of PBMC migration assay measured by flow cytometry. Only monocytes showed significant migration. The data were normalized to Ntrx cells (black line), and the minimum desired increase is represented by the dashed line.

[0348] Figure 8 - A figure depicting the experimental results of transfecting macrophages with multiple constructs in terms of MMP expression. It shows the MMP activity assays in the supernatants of NTrx, NTrx + treatment, IL-10Trx, IL-10 + MMP9 Trx hMDM tested. The dashed line represents 1.5 times the activity level measured in the NTrx hMDM supernatant. Each symbol represents an independent donor.

[0349] Figure 9 - A figure depicting the experimental results of transfecting macrophages with multiple constructs in terms of the localization at the site of in vivo injury. It shows the percentage (%) of viable (7AAD-) human macrophages measured in the digests of fibrotic livers using flow cytometry. Each point represents a different mouse. Data are reported as mean ± standard deviation (SD). Panel A is localized in the lung and Panel B is localized in the liver.

[0350] Figure 10 - A figure depicting the experimental results of transfecting macrophages with multiple constructs in terms of the in vivo expression of IL-10 and MMP9. It shows the measurement of human IL-10 and human MMP9 in the circulation of mice with chronic liver fibrosis by ELISA at different time points after cell injection. Each point represents a different mouse. Data are reported as mean ± SD. Panel A is IL-10 in plasma and Panel B is MMP9 in plasma.

[0351] Figure 11 - A figure depicting the experimental results after injecting macrophages transfected with multiple constructs into a mouse model. The inflammatory cytokines IL1b and TNFa in mouse plasma were measured by ELISA to verify the inflammation (if any) induced by injecting engineered cells and non-engineered 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. Panel A describes the results of mIL-1β and Panel B describes the results of mTNF-α.

[0352] Figure 12 - As shown, human monocytes were recruited by conditioned media from non-engineered hMDM or hMDM transfected with various genes. The assay was performed as described in Materials and Methods, and the 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.

[0353] Figure 13 - Conditioned media from hMDM transfected with IL-10 specifically recruited monocytes. The data shown were obtained using the same as Figure 12Generated by the same protocol, but shows the migration of individual cell types. Each symbol represents conditioned media from an independent donor. Data are reported as single-donor scatter, mean, and standard deviation.

[0354] Figure 14 - Conditioned media from IL-10-transfected macrophages polarize unpolarized macrophages to a pro-repair phenotype. Assays were performed as described in part (A) - macrophages on day 5 of culture were treated with conditioned media (culture method as described in the examples), the conditioned media from non-engineered cells treated with IL-4, IL-13, and M-CSF, cells engineered with the payload of IL-10 or MERTK, or a polarization medium (containing 50 ng / ml of MexMACS and IL-10). As described in the examples, flow cytometry was used to evaluate the expression of HLA-DR (B), CD86 (C), 25F9 (D), CD206 (E), and CD163 (F).

[0355] Figure 15 - Conditioned media from IL-10-transfected macrophages rescue pro-inflammatory macrophages and promote a pro-repair phenotype. Assays were performed as described in part (A) - macrophages on day 5 of culture were treated with conditioned media (culture method as described in the examples), the conditioned media from non-engineered cells treated with IL-4, IL-13, and M-CSF, cells engineered with the payload of IL-10 or MERTK, or a polarization medium (containing 50 ng / ml of MexMACS and IL-10). As described in the examples, flow cytometry was used to evaluate the expression of HLA-DR (B), CD86 (C), 25F9 (D), CD206 (E), and CD163 (F).

[0356] Figure 16 - Macrophages transfected with IL-10 alone have a secretome comparable to that of non-engineered, non-polarized macrophages. Cells were treated as shown: untransfected cells, which were treated (UT TR) or not treated (UT) after transfection with IL-4, IL-13, and M-CSF, or cells transfected with a specific payload (IL-10, MERTK, MMP9, or MMP12, as shown on the x-axis). 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) were measured in the culture supernatants, where the cells were cultured at a concentration of 4x10 6 cells / ml, as described herein.

[0357] 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, cells transfected with IL-10 or PBS were intravenously administered to mice with a CCl4-induced fibrotic liver injury model.

[0358] Figure 18 - Macrophages transfected with MMP9 showed increased MMP activity. As described in the examples, total MMP activity was measured in untransfected macrophages with or without administration of stimulator of interferon genes inhibitor (iSTING) or macrophages transfected with a specific payload. Data are represented as mean ± SD. ***p ≤ 0.005.

[0359] Figure 19 - MMP9-transfected macrophages increased the expression of other matrix metalloproteinases. The expression of MMP1, 3, 7, 8, and 10 was measured in untransfected (NT), macrophages with or without administration of STING inhibitor (STINGi), or macrophages transfected with MMP9 alone. Deeper color intensity represents stronger expression.

[0360] Figure 20 - RTX 001 macrophages recruited monocytes in vitro. RTX001 macrophages were 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. The results were normalized to untransfected (NTrx) cells.

[0361] Figure 21 - RTX 001 macrophages recruited monocytes in vivo. The recruitment of total myeloid cells was evaluated by detecting the proportion of CD11b+Tim4- cells among total CD45+ cells using flow cytometry (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 vehicle were injected into mice, and recruitment was evaluated 24 hours after administration.

[0362] Figure 22 - RTX001 macrophages promoted an anti-inflammatory environment in vivo. Mouse IL-10 in liver homogenates was measured 24 hours after administration of RTX001 macrophages, untransfected macrophages, or PBS control to a CCL4-induced liver fibrosis mouse model.

[0363] Figure 23- Macrophages engineered to express either single IL-10, or both IL-10 and MMP9 exhibit similar repair-promoting secretomes. The amounts of secreted IL-2 (A), IL-12p70 (B), IFNγ (C), TNF-α (D) and IL-1β (E) were measured in the supernatant of the culture medium, where the engineered cells were cultured at a concentration of 4x10 6 cells / ml.

[0364] Figure 24 - Administration of RTX001 macrophages reduced scar-forming cells in vivo. RTX001 macrophages, untransfected macrophages or PBS vehicle were administered to a CCl4-induced liver fibrosis mouse model. Tissue sections were taken from the mice 1 week after administration and stained for α-SMA (left panel). The percentage area of the α-SMA positive region was calculated and the results are shown in the right panel.

[0365] Figure 25 . RTX001 macrophage CM significantly reduced the expression of α-SMA in LX-2. (A) Fold change in the mean fluorescence intensity (MFI) of α-SMA relative to the TGF-β-stimulated control group (without CM). (B) Chart comparing the percentage of cells expressing α-SMA. The mean of three experimental replicates was taken, and the data are represented as mean ± SD (n = 1). **p ≤ 0.01.

[0366] Figure 26 - Optimized bicistronic mRNA results in improved repair-promoting properties. Cells were transfected with unoptimized mRNA (SEQ ID NO:2, also containing a 120-residue long 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 detected, and MMP activity was measured as described herein. MMP activity was normalized to untransfected cells.

[0367] Figure 27-RTX001 is stable in an inflammatory environment. In engineered macrophages exposed to a specific concentration of IFN-γ, changes in CD80 and CD86 (inflammatory markers) and CD206 (pro-repair marker and general macrophage identity marker) were measured. No significant changes were observed in the pro-inflammatory markers (CD80 and CD86) or the macrophage identity 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 hours. The mean fluorescence intensity (MFI) of the pro-inflammatory markers HLA-DR and CD80 was evaluated using flow cytometry. The data shown are fold changes relative to Ntrx cells cultured without IFN-γ (B and C).

[0368] Figure 28 . Table showing the expression of macrophage markers or cytokine secretion in specific 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-10MMP9 TRx) or treated with IL-4, IL-13, and M-CSF after transfection (IL-10MMP9 TRx+TR). The mean fluorescence intensity was measured by flow cytometry as described herein.

[0369] Figure 29 -Post-transfection treatment did not result in further polarization of engineered macrophages. The expression of cD86 (left) and MHC II (right) in macrophage products was detected by flow cytometry. 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).

[0370] Figure 30 -Treatment with IL-4, IL-13, and M-CSF improved cryo-resistance. The viability of macrophage product populations was determined after cryopreservation. Macrophage products consisted of: untransfected macrophages, untreated (NTx) or further treated with IL-4, IL-13, and M-CSF (NTrx+Tr); macrophages transfected with both MMP9 and IL-10, untreated (Trx) or further treated with IL-4, IL-13, and M-CSF after transfection (Trx+Tr). Viability was evaluated by measuring the proportion of cells that remained viable after cryopreservation relative to the total number of viable cells before cryopreservation.

[0371] Figure 31 - Summary of the protocol used to measure efficacy in the mouse-on-mouse model in Example 25.

[0372] Figure 32 -Schematic diagram outlining the mechanisms thought to underlie the therapeutic effects of engineered macrophages.

[0373] The data shown in these figures indicate that the inventors have shown that the surprising and effective combination of expression of two specific genes (IL-10 and MMP9) in series has an unforeseen and surprising benefit to the phenotype of engineered macrophages. Surprisingly, the expression of MMP9 alone can rescue the suppressed MMP expression / activity observed in IL-10 transfected macrophages. As shown above, this effect appears to be synergistic. In addition, the excellent recruitment of monocytes by combined overexpression was not predicted because entities such as cytokines are conventionally shown to be involved in the recruitment process.

[0374] Genetic engineering of macrophages allows for overexpression of IL-10 in combination with MMP9, providing many of the features and functions desired for cell therapy, which can be used for inflammatory conditions, such as organ injury regeneration. The inventors have shown that the combination of IL-10 and MMP9 produces:

[0375] - Reliable macrophage characterization without interference from engineering processes.

[0376] -Strong anti-inflammatory phenotype.

[0377] - Ability to pattern primitive macrophages into a pro-repair phenotype.

[0378] -Excellent swallowing ability.

[0379] - Reassuring safety and biodistribution profile, including penetration into compromised organs and rapid clearance / loss in other organs.

[0380] The above features are shared with macrophages engineered with IL-10 alone that the inventors previously studied. However, the combination of IL-10 and MMP9 provides some specific and surprising features that are key to providing the desired therapeutic effects, such as:

[0381] -Strong ability to attract monocytes, which are then patterned into a pro-repair phenotype.

[0382] - Ability to restore MMP activity (surrogate for fibrosis / extracellular matrix (ECM) remodeling) that was engineered to be abolished by IL-10 alone.

[0383] Accordingly, the present inventors believe that engineering macrophages with a combination of IL-10 and MMP9 will provide an effective product capable of having anti-inflammatory and anti-fibrotic functions in therapies, such as in several acute and chronic organ injury situations. In the case of acute injury, the remodeling of ECM components is crucial to ensure tissue restitution and proper regeneration.

[0384] Equivalents

[0385] Those skilled in the art will recognize or be able to determine equivalents of the specific embodiments described herein using only routine experimentation. These equivalents are intended to be encompassed by the appended claims. Any combination of the embodiments disclosed in any multiple dependent claims or multiple examples is considered to be within the scope of this disclosure.

[0386] Incorporation by reference

[0387] The disclosures of each and every patent, patent application publication, and scientific publication cited herein are hereby incorporated by reference in their entirety, as are the contents of their drawings.

[0388] Sequence Listing

[0389] Italic – Encoded protein

[0390] Bold – Linker

[0391] In all mRNA sequences, uridine is transcribed as thymine.

[0392] SEQ ID No.1

[0393] 1. Single hIL10 mRNA

[0394]

[0395] SEQ ID No.2

[0396] 2. Bicistronic hIL10-P2A-hMMP9

[0397]

[0398]

[0399] SEQ ID No.3

[0400] 3. MMP9

[0401]

[0402]

[0403] SEQ ID No.4

[0404] Protein sequence of human IL10 (bold = open reading frame)

[0405]

[0406] SEQ ID No.5

[0407] Protein sequence of IL10-p2a-MMP9 (bold = open reading frame, underlined = linker)

[0408]

[0409]

[0410] SEQ ID No.6

[0411] Protein sequence of human MMP9 (bold = open reading frame)

[0412]

[0413] SEQ ID No.7

[0414] p2A motif

[0415] DXEXNPGP

[0416] SEQ ID No.8

[0417] Non-optimized p2A mRNA sequence

[0418] GGAAGCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCT

[0419] SEQ ID No.9

[0420] p2A amino acid sequence

[0421] GSGATNFSLLKQAGDVEENPGP

[0422] SEQ ID No.10

[0423] Optimized bicistronic mRNA coding sequence (linker sequence underlined). All uridines (transcribed as thymines here) are 5-methoxy-uridine.

[0424]

[0425] SEQ ID No.11

[0426] Amino acid sequence encoded by the optimized dicistronic mRNA sequence (IL-10-p2A-MMP9 fusion protein) (the linker sequence is underlined)

[0427] MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN GSGATNFSLLKQAGDVEENPGPMSLWQPLVLVLLVLGCCFAAPRQRQSTLVLFPGDLRTNLTDRQLAEEYLYRYGYTRVAEMRGESKSLGPALLLLQKQLSLPETGELDSATLKAMRTPRCGVPDLGRFQTFEGDLKWHHHNITYWIQNYSEDLPRAVIDDAFARAFALWSAVTPLTFTRVYSRDADIVIQFGVAEHGDGYPFDGKDGLLAHAFPPGPGIQGDAHFDDDELWSLGKGVVVPTRFGNADGAACHFPFIFEGRSYSACTTDGRSDGLPWCSTTANYDTDDRFGFCPSERLYTQDGNADGKPCQFPFIFQGQSYSACTTDGRSDGYRWCATTANYDRDKLFGFCPTRADSTVMGGNSAGELCVFPFTFLGKEYSTCTSEGRGDGRLWCATTSNFDSDKKWGFCPDQGYSLFLVAAHEFGHALGLDHSSVPEALMYPMYRFTEGPPLHKDDVNGIRHLYGPRPEPEPRPPTTTTPQPTAPPTVCPTGPPTVHPSERPTAGPTGPPSAGPTGPPTAGPSTATTVPLSPVDDACNVNIFDAIAEIGNQLYLFKDGKYWRFSEGRGSRPQGPFLIADKWPALPRKLDSVFEERLSKKLFFFSGRQVWVYTGASVLGPRRLDKLGLGADVAQVTGALRSGRGKMLLFSGRRLWRFDVKAQMVDPRSASEVDRMFPGVPLDTHDVFQYREKAYFCQDRFYWRVSSRSELNQVDQVGYVTYDILQCPED-

[0428] SEQ ID No.12

[0429] Mouse mRNA-based transcript sequence introduced into mRTX001.

[0430]

[0431] SEQ ID NO:13

[0432] Optimized sequence of IL-10 mRNA. All uridines (transcribed as thymines here) are 5-methoxy-uridine.

[0433] ATGCACAGCTCCGCCCTGCTGTGCTGCCTGGTGCTGCTGACCGGCGTGCGGGCCAGCCCCGGCCAGGGCACACAGTCCGAGAACAGCTGCACCCACTTCCCAGGCAATCTCCCCAACATGCTGAGAGACCTGAGGGACGCCTTCTCCCGCGTGAAGACATTCTTCCAGATGAAGGACCAGCTGGACAATCTCCTGCTGAAGGAGAGCCTGCTGGAGGACTTCAAGGGCTACCTGGGCTGCCAGGCCCTGTCCGAGATGATCCAGTTCTACCTGGAGGAGGTGATGCCACAGGCCGAGAACCAGGACCCCGACATCAAGGCCCACGTGAACAGCCTGGGCGAGAACCTGAAGACCCTGCGGCTGAGACTGAGGCGCTGCCACCGGTTCCTGCCATGCGAGAACAAGTCCAAGGCCGTGGAACAAGTGAAGAACGCCTTCAACAAGCTGCAGGAGAAGGGCATCTACAAGGCCATGAGCGAGTTCGACATCTTCATCAACTACATCGAGGCCTACATGACAATGAAGATCAGAAAC

[0434] SEQ ID NO:14

[0435] Optimized sequence of MMP9 mRNA. All uridines (transcribed as thymines here) are 5-methoxy-uridine.

[0436]

[0437] SEQ ID NO:15

[0438] mRNA sequence of the optimized linker. All uridines (transcribed as thymines here) are 5-methoxy-uridine.

[0439] GGCTCCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCA

[0440] SEQ ID NO:16

[0441] Optimized bicistronic mRNA (including 5’UTR and 3’UTR). All uridines (transcribed as thymines here) are 5-methoxy-uridine.

[0442]

[0443] SEQ ID NO: 17 - 5' UTR of the optimized dicistronic mRNA construct (all uridines, transcribed as thymidines here, are 5 - methoxy - uridines)

[0444] AGGCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATTCACAACCAGGCCTCCACAACC

[0445] SEQ ID NO: 18 - 3' UTR of the optimized dicistronic mRNA construct (all uridines, transcribed as thymidines here, are 5 - methoxy - uridines)

[0446] ACTCGAGTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTATCCCAGTGTATATTGTCGAC

[0447] SEQ ID NO: 19 - 5' UTR of the un - optimized dicistronic mRNA construct AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC

[0448] SEQ ID NO: 20 - 3' UTR of the un - optimized dicistronic mRNA construct GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG

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[0497] 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.Obeticholicacid for the treatment of non-alcoholic steatohepatitis:interimanalysis froma multicentre,randomised,placebo-controlled phase 3 trial.Lancet(London,England),394(10215),2184–2196.(2019)

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[0499] 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.ARandomized,Controlled Trial of the Pan-PPAR Agonist Lanifibranor in NASH.TheNew England journal of medicine,385(17),1547–1558(2021) Detailed implementation mode

[0500] Example

[0501] Materials and Methods

[0502] The following protocols were used to generate the data described in the following examples.

[0503] Macrophage culture

[0504] We used a Ficoll gradient (GE Healthcare) and then performed magnetic column selection using CliniMACS CD14 reagent (Miltenyi Biotec) to isolate monocytes from buffy coat products of healthy volunteers from the Scottish National Blood Transfusion Service (SNBTS). Then, 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), we matured the monocytes in culture for 1 to 7 days. hMDM were cultured at a density of 2x10 6 / cm 2 in 6-well multiwell plates (Corning Costar) for 5 days. hMDM were counted using an automated cell counter (TC20, BioRad).

[0505] Macrophage engineering

[0506] Small and Medium Scale Transfection - mRNA

[0507] Pellet mature macrophages at 300 x g for 5 minutes, then remove the supernatant and resuspend the cells in supplemented buffer P3 (Lonza) at a density of 50 x 10^6 or 100 x 10^6 cells / ml. Add mRNA of IL10 (798NT) at a concentration of 2 μg / 10^6 cells or IL10-MMP9 (2985NT) at 8 μg / 10^6 cells and mix well by pipetting. Transfer the cell suspension to an electroporation cuvette / cassette (100 μl / 1 ml). Transfect the cells on a Lonza Nucleofector using pulse code CM-137. Collect the cells in a sterile Falcon tube. 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 it to the cell suspension. Place the cells in a TC incubator for 20 minutes. Perform cell counting and adjust the cell density to 4 x 10^6 / ml. Seed the cells at a density of 2 x 10^6 / cm 2 Seed the cells and place them in a cell culture incubator.

[0508] Large Scale Fluidics Transfection - mRNA

[0509] Pellet mature macrophages at 300 x g for 5 minutes. Remove the supernatant and resuspend the cells in supplemented buffer P3 (Lonza) at a density of 200 x 10^6 cells / ml. Transfer mRNA of IL10 (798NT) at a concentration of 2 μg / 10^6 cells or IL10-MMP9 (2985NT) at 8 μg / 10^6 cells to a sterile Falcon tube and fill it up with supplemented 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 appropriate. Connect a cell culture bag 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 cassette to elute the cells. Transfect the cells on a Lonza Nucleofector using pulse code CM-137. Transfer the bag containing the cells to a TC incubator for 20 minutes. Perform cell counting and adjust the cell density to 4 x 10^6 / ml. Seed the cells at a density of 2 x 10^6 / cm 2 Seed the cells and place them in a cell culture incubator.

[0510] Small Scale Transfection – pDNA

[0511] As used herein, "payload" is one or more genes with a therapeutic purpose, which are introduced by transfection to test their effects on macrophages.

[0512] On day 5, human monocyte-derived macrophages (hMDM) were resuspended at a density of 75×10^6 or 150×10^6 cells / ml in electroporation buffer (Miltenyi Biotec, #170-076-625), and 100 - 300 μl of the suspension was transferred to an electroporation cuvette with a gap size of 0.2 cm. 5 μg of plasmid DNA per 5×10^6 cells was added directly to the cuvette, and the plasmid DNA was mixed with the cells by gently shaking the cuvette. The cells were transfected using an electroporator controlled by CliniMACS. The electroporation parameters are listed in Table 1 below (as further described in patent application PCT / GB2021 / 051300 (published as WO2021240167)):

[0513] Table 1 – Electroporation transfection parameters for IL10 expression plasmid in hMDM.

[0514]

[0515] After transfection, the cells were recovered from the cuvette into sterile TexMACS TN GMP medium (Miltenyi Biotec, #170-076-306) using an 18G sterile needle connected to a 1 mL syringe or similar tool. Cell counting was performed using a TC-20 automated cell counter (Bio-Rad). The cells were centrifuged 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 TM 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), and plated at a density of 2×10^6 cells / cm 2 2.

[0516] Macrophages used to generate Figures 1 - 11 and the data shown in 20 - 30 were transfected with RNA constructs. Macrophages used to generate Figures 12 - 16 the data shown in 18 and 19 were transfected with plasmid DNA constructs.

[0517] Low-temperature stress resistance treatment

[0518] To improve the low-temperature stress resistance of engineered macrophages after transfection, the 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), at a cell concentration of 4x10 6 cells / mL and 2x10 6 / cm 2 .

[0519] Phagocytosis assay

[0520] Briefly, macrophages for imaging analysis were prepared by plating macrophages at a density of 150,000 cells / well in a 96-well clear-bottom imaging plate (Grenier). The supernatant was removed, and the cells were stained with 100 μl of PBS+NucBlue (ThermoFisher)+5 μg / ml Cellmask Deep Red plasma membrane stain (Invitrogen) for 30 minutes at 37 °C and 5% CO2. The cells were washed three times with 100 μl of PBS. 50 μl of PBS was added to the cells for T0 analysis on an Opera Phenix high-content screening system. After T0, 50 μl of 0.2 mg / ml pHrodo Red zymosan beads (Life Technologies) was added to the cells. A series of images were obtained over a 96-minute time period to monitor phagocytosis. The images were analyzed using Columbus data imaging software and Tibco Spotfire data analysis system. Graphs were plotted using GraphPad Prism 9.2.0.

[0521] By plating the cells at 2x10 6Cells for flow cytometry analysis were prepared by resuspending them at a concentration of / ml in PBS + 0.5 mM EDTA (Life Technologies). 50 μL of the cell suspension was dispensed into a low-adhesion round-bottom 96-well plate. 50 μL of resuspended pHrodo beads (prepared according to the manufacturer's instructions) was added to the test wells. The cells were incubated at 37 °C in 5% CO2 for 2 hours. At the end of the 1-hour incubation, the plate was spun at 300 x g at 4 °C for 5 minutes, the supernatant was removed, and the pellet was resuspended in 100 μL of 1:100 FcR-blocking PEA solution / well. Subsequently, it was incubated in the dark at 4 °C for 15 minutes, then the antibody (see Table 3) was added to the appropriate test wells and incubated at 4 °C for 20 minutes. The cells were washed with PBS + 0.5 mM EDTA and spun at 300 g for 5 minutes. The supernatant was flicked off and the cells were resuspended in PBS + 0.5 mM EDTA + 1:1000 DRAQ7. They were incubated at 4 °C for 5 minutes. They were washed as described above and then resuspended in 100 μL of PBS + 0.5 mM EDTA + 0.1% human serum. 50 μL of cells was acquired on a Novocyte 3000 or Novocyte Quanteon (Agilent). Flow cytometry analysis was performed on NovoExpress software, and the following gating strategy was used to identify actively phagocytic macrophages: "Cell gate" excluded debris, "singlet gate" excluded doublet cells, "live gate" excluded dead cells, "CD14+ gate" identified iMACS, and "phRodo+ve gate" measured the percentage of phagocytic macrophages.

[0522] IL-10 Capture Assay

[0523] For each condition, 1 x 10 6The cells were resuspended in 80 uL of cold TexMACS, and 20 uL of IL-10 capture reagent was added. The mixture was 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 tube of payload-transfected cells as a negative control. The test group was incubated with continuous rotation at 37 °C for 1.5 hours. The negative control was kept on ice. After incubation, the cells were topped up to 15 mL with cold buffer and spun down at 4 °C. Then the cells were washed again with 10 mL of cold buffer and spun down at 4 °C. The cells were resuspended in 80 uL of cold buffer, 20 uL of detection antibody and 5 uL of CD14 VioBlue were added, and the mixture was incubated on ice for 10 minutes. The cells were washed with 5 mL of cold buffer and spun down at 4 °C. Then the cells were resuspended in 1 mL of 1:1000 Draq7 in cold buffer, and 100 uL of the sample was transferred to a 96-well plate. The plate was spun down, resuspended with 100 uL of cold buffer, and acquired on a flow cytometer.

[0524] Flow cytometry labeling

[0525] The macrophages were resuspended in PBS + 0.5 mM EDTA (Life Technologies) + FcR Block 1:100 (Miltenyi) at a concentration of 1x10 6 / ml. 100 ul of the cells were aliquoted into low-adhesion round-bottom 96-well plates. The cells were incubated for 5 minutes, then the appropriate antibodies (see Table 2) were added to the appropriate test wells and placed at 4 °C for 20 minutes. The cells were washed with PBS + 0.5 mM EDTA and spun at 300 g for 5 minutes. The supernatant was flicked off 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 and then resuspend in 100 ul of PBS + 0.5 mM EDTA + 0.1% human serum. 50 ul of the cells were acquired on a Novocyte 3000 or Novocyte Quanteon (Agilent).

[0526] Table 2 - Antibodies for flow cytometry labeling of hMDM.

[0527]

[0528] MSD V-plex cytokine dosage

[0529] Cytokines in cell culture supernatant were analyzed using the V-PLEX Human Biomarker 10-Plex Kit on a MESO Quickplex SQ 120 according to the manufacturer's instructions (Meso Scale Discovery). 10 μL of supernatant was tested. Results are in pg / mL. Values were adjusted for dilution during testing. All data shown represent secretion over a 24 h period. Data reported are net concentrations calculated by subtracting the amount of a given cytokine in the separate medium (TexMACS) from the amount of cytokine detected in the cell culture supernatant.

[0530] PBMC Recruitment / Migration Assay

[0531] Buffy coat donations were purchased from SNBTS at Sample Governance 20 - 17. Peripheral blood mononuclear cells were isolated from buffy coats using standard methods and cryopreserved at a density of 50 x 10 6 / mL in CryoStor CS10 (STEMCELL) and stored until needed. To set up the migration assay, PBMCs were thawed and resuspended at 4.6 x 10 6 in TexMACS (Miltenyi). 75 μL of cells in TexMACS were added to the top chamber of a 5 μm 96-well transwell (Corning). 150 μL of thawed and frozen conditioned medium was added to the bottom chamber. The transwell plate was placed in an incubator (37 °C, 5% CO2). After 3 h, the top chamber was removed and the migrating cells in the bottom chamber were stained using standard flow cytometry staining procedures with CD45-PerCP, CD14-VioBlue, CD15-Pevio770, CD16-BV605, CD56-PE, CD3-FITC, and CD19-APC and acquired using a Novocyte 3000 or Novocyte Quanteon (Agilent).

[0532] Polarization Assay - Non-Polarized Macrophages

[0533] Day 5 mature macrophages were seeded at 2 x 10 6 / cm 2Cells were seeded at a density of [density value not provided] in TexMACS + 100 ng / ml of MCSF (R&D) in 96-well plates (Corning). 50 ng / ml of IL-10 (R&D) was added to the control wells (M2 polarization medium). After cell attachment (5 hours), the medium was removed and replaced with conditioned medium. The cells were incubated with the conditioned medium in an incubator (37 °C, 5% CO2) for 18 hours, and then stained for CD14-VioBlue, CD45-PerCP, CD206-BV711, 25F9-eF660, HLA-DR-PeCy7, CD86-PE, CD163-FITC using a standard flow cytometry staining protocol. DRAQ7 was used to stain dead cells. Cells were then acquired using a Novocyte3000 or Novocyte Quanteon (Agilent).

[0534] MMP Activity Assay

[0535] MMP activity was confirmed by successful cleavage of a standard MMP peptide. The standard MMP peptide was flanked by a quencher and a fluorescent signal, and did not emit fluorescence when intact. The cleaved peptide no longer quenched the fluorescent signal, thus resulting in fluorescence emission, and this fluorescence 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-ab112146MMP ActivityAssay Kit Fluorometric-Green v4b), testing 25 ul of cell culture supernatant. All data shown represent the activity of MMP secreted over a 24-hour period. Results were plotted as RFU minus the background fluorescence of the medium alone (TexMACS).

[0536] Statistics

[0537] Each reported point was a different donor. Unless otherwise stated, at least 3 donors were analyzed per condition. Where appropriate, data are shown as mean ± SD. Where appropriate, a two-tailed t-test was performed on paired data.

[0538] Results

[0539] Example 1: Efficient Transfection of IL-10 and IL-10 + MMP9 in hMDM

[0540] To generate transfected (Trx) human monocyte-derived macrophages (hMDM) for cell therapy, a significant increase in the expression and secretion levels of the desired payloads needs to be achieved. Based on our internal and published results, we set a minimum threshold for the payloads IL-10 and MMP9 that we selected. The levels of secreted proteins in the cell culture supernatant 24 hours post-transfection were measured by ELISA. Interestingly, both IL-10 and IL1-0+MMP9 (bicistronic vector) transfection led to a significant increase in IL-10 secretion ( Figure 1 ). Flow cytometry based on the IL-10 capture assay also confirmed the increase in IL-10 secretion in engineered macrophages, which monitored the secretion of IL-10 in macrophages over a 2-hour period ( Figure 2 ). However, MMP9 secretion did not increase strikingly: only IL-10 transfection led to a slight decrease in MMP9 secretion, which was at least partially rescued by co-transfection of IL-10 and MMP9 ( Figure 1 ), indicating sufficient transfection efficiency of MMP9. In all experiments presented herein, we always analyzed untransfected (NTrx), NTx+restoring treatment (IL4+IL13), Trx of IL-10 only, and IL-10+MMP9 Trx. All Trx cells received the restoring treatment. IL-10 and MMP9 were co-transfected using a bicistronic vector linked by the p2A linker sequence.

[0541] Example 2: IL-10 and IL-10 + MMP9 Transfected hMDM Show Retention of Macrophage Recognition Markers

[0542] The key to obtaining a safe and effective macrophage therapy is to maintain macrophage surface recognition markers. This indicates that the genetic engineering procedure does not fundamentally alter the cell characteristics. The pan-leukocyte marker CD45 is present in all cell types. In IL-10+MMP9, the expression intensity of the myeloid marker CD14 decreased slightly (MFI fold change), but the percentage of positive cells did not change, so we were satisfied that the cells maintained their myeloid characteristics. The mature macrophage marker 25F9 increased slightly in the engineered cells, which is a positive sign of strong macrophage characteristics. All other markers analyzed (CD206, CD163, CCR2, CD169) did not change or increased slightly in the engineered cells compared to non-engineered cells. These data support the view that our engineering approach is safe and does not interfere with cell characteristics. Data are as Figure 3 shown.

[0543] Example 3: IL-10 and IL-10 + MMP9 Transfected hMDM Have Significant Anti-Inflammatory Characteristics

[0544] To treat acute and chronic inflammatory conditions associated with organ injury, for example, it is important to obtain highly anti-inflammatory macrophages. Surprisingly, not only did the transfected macrophages retain all the recognition markers (Figure 3 ) and they also showed a decrease in several pro-inflammatory markers such as CD86 and HLA-DR( Figure 4 ). This emphasizes 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 in IL-10+MMP9 is unlikely to be significant in terms of biological impact as it is within 10% of the required level (red dashed line).

[0545] Example 4: IL-10 and IL-10 + MMP9 Transfected hMDM Have Excellent Phagocytic Capacity

[0546] In the context of acute and chronic inflammatory conditions such as those associated with organ injury, another key aspect of effective macrophage therapy is its ability to phagocytose effectively. Here, we report that both engineered and non-engineered macrophages phagocytose effectively and above the minimum required level (dashed line)( Figure 5 ).

[0547] Example 5: IL-10 and IL-10 + MMP9 Transfected hMDM Polarize Naïve Macrophages into a Pro-Repair Phenotype

[0548] During inflammatory organ injury, local macrophages need to acquire 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+ treatment, IL-10Trx, 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 were used as a positive control (red dashed line). The desired outcome was a decrease in the M1 markers CD86 and HLA-DR and a similar or increased level of the M2 markers CD206 and CD163 in macrophages treated with supernatants from engineered hMDM compared to non-engineered hMDM. Supernatants from IL-10Trx and IL-10+MMP9 Trx hMDM were both effective and promoted the conversion of unrelated donor macrophages into a pro-repair phenotype (decrease in CD86 and HLA-DR, increase in CD206, and similar levels of CD163)( Figure 6 ).

[0549] Example 6: IL-10 + MMP9 Transfected hMDM Have Better Monocyte Recruitment Capacity Compared to IL-10 Transfected hMDM

[0550] An important function of macrophages is to recruit new monocytes in situ so that they can be patterned into a pro-repair phenotype mode. Figure 6 Evidence is provided for the ability of IL-10Trx and IL-10+MMP9 Trx hMDM to induce this phenotype in non-polarized macrophages. Figure 7The data presented surprisingly support the view that when tested in a PBMC migration assay, only the supernatant from IL-10+MMP9 Trx hMDM induced significant monocyte migration.

[0551] Example 7: Macrophages Transfected with IL-10 + MMP9 Are Superior to IL-10 Transfected Macrophages in Terms of Metalloprotease Activity

[0552] The last key feature of macrophage therapies aimed at inducing tissue remodeling is their ability to digest extracellular matrix (ECM) components. This assay measures the ability of the total pool of MMPs in cell culture supernatants to digest ECM components using a fluorescent probe. Surprisingly, compared to NTrx hMDM, IL-10Trx greatly reduced (-50%) the total MMP activity measured in the supernatant. Notably, co-transfection of only MMP9 was sufficient to restore this ability in the supernatant and even make it 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 increased the MMP activity of hMDM. This may also emphasize the ability of MMP9 transfection to increase the activation of other MMPs. The data are as Figure 8 shown.

[0553] Example 8: In a Chronic Liver Disease Model, IL-10 and IL-10 + MMP9 Transfected hMDM Localize in the Liver at 24 Hours and 72 Hours after Injection and Are Rapidly Cleared from the Lungs

[0554] For macrophage therapies to be effective, they need to localize to the site of injury after injection. In this experiment, chronic liver disease was induced in immunodeficient mice (NSG strain) by injecting a toxin (CCl4). Four weeks after fibrosis induction, hMDM were injected via the tail vein, and the livers were retrieved at different time points, enzymatically digested to retrieve the non-parenchymal fraction. The results showed that both IL-10Trx and IL-10+MMP9 Trx hMDM initially localized to the lungs and liver but persisted only in the liver for 72 h. As expected, one week after injection, the genetically engineered cells were cleared ( Figure 9 ). Thus, they exhibited pharmacokinetics and distribution compatible with efficacy and safety.

[0555] Example 9: In a Chronic Liver Disease Model, IL-10 and IL-10 + MMP9 Transfected hMDM Maintain Expression of the Payload at 24 Hours after Injection

[0556] In the same experiment described above, circulating human IL-10 and MMP9 were measured in mouse plasma at different time points after injection of the cell therapy. Interestingly, although macrophages were concentrated in the liver and lungs, IL-10 and MMP9 were detected systemically (in the blood circulation) in the expected mouse groups ( Figure 10 ). Appropriate controls were performed to ensure reliable detection of the human proteins without cross-reactivity with their mouse counterparts.

[0557] Example 10: IL-10 and IL-10 + MMP9 Have Good Safety in a Chronic Liver Disease Model

[0558] Finally, for success in the clinical setting, cell therapies need to be safe, both at the time of injection and at various time points thereafter. In particular, it is crucial that cell therapies have no off-target effects in uncompromised organs and are cleared within a safe time frame. Data support the safety of both IL-10Trx and IL-10+MMP9 Trx hMDM. In fact, no embolisms were detected at the time of injection, and at various time points after injection, no systemic inflammation was detected throughout the body of mice with chronic liver disease (Table 3 and Figure 11 ). These data, combined with Figure 10 the rapid clearance observed in

[0559] Table 3 - Summary of safety features and outcomes required to meet safety requirements.

[0560]

[0561] Example 11: Engineered Macrophages Overexpressing IL-10 Specifically Recruit Monocytes. As described herein, PBMC migration from conditioned media of hMDM in response to treatment as Figure 12 described was measured. As described herein, "D6 UT" cells were removed after 6 days of culture and were not transfected, nor transfected and treated with IL-4 or IL-13 (recovery treatment as described herein). As described herein, "D6 UT+TR" cells were removed after 6 days of culture, were not transfected, but were treated with IL-4 / IL-13 (recovery treatment as described herein). The remaining treatment groups were transfected with the indicated genes. Transfection with IL-10 alone induced the greatest monocyte recruitment, and transfection with CCR2 was the only other treatment that produced macrophages capable of significantly recruiting monocytes. Figure 12 Only monocyte recruitment identified by flow cytometry is shown. Figure 13 Macrophage recruitment in other cell types (UT and UT TR correspond to Figure 12 the D6 UT and D6 UT TR cells described in Figure 12 ) treated with a subset of the conditions shown, as identified using flow cytometry (B cells, T cells, NK cells, neutrophils, and monocytes) is shown. Figure 13 IL-10 transfected cells were shown to specifically recruit monocytes and not other cell types. Conditioned media from untransfected macrophages and macrophages overexpressing MMP9 had no effect on monocyte recruitment.

[0562] Example 12: Engineered Macrophages Overexpressing IL-10 Convert Both Non-Polarized and Pro-Inflammatory Macrophages into a Pro-Repair Phenotype.To test the ability of macrophages overexpressing IL-10 to convert monocyte-derived macrophages into pro-repair macrophages, the inventors tested the effect of the CM from macrophages overexpressing IL-10 on the M0 and M1 macrophage phenotypes. 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, substantially as described in WO 2021 / 240162) were incubated with the CM from IL-10 overexpressing cells, they showed a marker profile associated with pro-repair M2 macrophages (i.e., downregulation of HLA-DR & CD86 and upregulation of 25F9, CD206, and CD163). The effect was equivalent to incubating M0 macrophages with a medium containing an M2 polarization medium (TexsMACS + 50 ng / ml IL-10) ( Figure 14 ). Flow cytometry and measurement of mean fluorescence intensity (MFI) were used to evaluate surface markers.

[0563] Similarly, incubation with the CM of macrophages overexpressing IL-10 was able to "rescue" M1 macrophages and convert their phenotype into a pro-repair M2 phenotype. For the test, the CM was incubated with macrophages pre-polarized into the M1 phenotype using 100 ng / ml LPS + 50 ng / ml IFN-γ. This rescue ability was similar to using an M2 polarization medium ( Figure 15 ).

[0564] Example 13: Macrophages Engineered to Overexpress IL-10 Have an Anti-Inflammatory Secretome Profile. The secretion levels of pro-inflammatory cytokines were measured in macrophages overexpressing IL-10 alone, macrophages overexpressing MMP9 alone, macrophages overexpressing IL-10 and MMP9, and untransfected non-polarized macrophages. Macrophages overexpressing IL-10 showed an anti-inflammatory secretome profile. In particular, 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 overexpressing MMP9 alone showed increased secretion of pro-inflammatory cytokines such as IL2, IL12p70, IFNg, TNFa, and IL1β ( Figure 16 ), macrophages overexpressing MMP9 and IL-10 showed secretion levels similar to those of macrophages overexpressing IL-10 alone ( Figure 23)。Similarly, the role of macrophages in systemic inflammation was evaluated in vivo. Briefly, NSG mice were subjected to CCl4 intoxication for 4 - 5 weeks. At SD 23, the mice were randomly administered macrophages overexpressing IL-10 alone (IL-10Trx hMDM) or macrophages overexpressing both IL-10 and MMP9 (IL-10-MMP Trx hMDM). Mice injected with phosphate-buffered saline (PBS) were used as vehicle controls. The levels of IL1β and TNFa in mouse liver homogenates were measured by the MSD assay. Surprisingly, although the pro-inflammatory secretome profile of macrophages expressing MMP9 alone was measured in vitro, it was found that neither macrophages overexpressing IL-10 alone nor macrophages overexpressing both IL-10 and MMP9 induced an increase in inflammatory cytokines compared to the vehicle control ( Figure 11 ).

[0565] Example 14: Engineered Macrophages Can Be Delivered to the Liver and Persist There. Furthermore, human macrophages overexpressing IL-10 were intravenously injected into mice with modeled local liver fibrosis and persisted for at least 72 hours after administration, indicating that they can be delivered to the treatment site of patients with liver fibrosis ( Figure 17 ).

[0566] Example 15: Engineered Macrophages Have MMP and Scar Remodeling Activity. To rescue the ability of macrophages overexpressing IL-10 to induce scar remodeling, the present inventors considered introducing matrix metalloproteinases (MMPs), as MMPs are known to play an important role in degrading scar tissue in the inflamed liver (e.g., Campana et al, Nature Reviews Molecular Cell Biology Vol. 22, pp. 608 - 624 (2021)). Since macrophages overexpressing MMP9 or MMP12 showed the ability to maintain some phagocytic capacity, the present inventors tested the scar remodeling ability of macrophages expressing either MMP. By measuring total MMP activity using a FRET-based fluorophore method, an increase in total MMP activity was observed for both MMP9 and MMP12, with MMP9 inducing a higher increase in total activity ( Figure 1 ).

[0567] Example 16 - Macrophages Engineered to Overexpress Both MMP9 and IL-10 Recruit Monocytes In Vitro and In Vivo Figure 20 RTX001 macrophages were generated by transfecting hMDM with a single bicistronic mRNA containing sequences encoding both MMP9 and IL-10. Figure 21Shows the results of the PBMC migration assay performed as described in Materials and Methods, 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 intoxication) 24 hours after administration. The results of this assay are as Figure 21 shown. NSG mice were subjected to 4 - 5 weeks of CCl4 intoxication. RTX001 and NTrx cells were administered intravenously on SD 23, and readings were collected 24 hours after cell administration. Mice injected with phosphate - buffered saline (PBS) were used as vehicle controls. Flow cytometry counts of the percentage of bone marrow cells (defined as CD45+, SiglecF -, Ly6G -, Tim4 -, CD11b+) and classical monocytes (classical monocytes defined as CD45+, SiglecF -, Ly6G -, Tim4 -, CD11b+, CD64 -, Ly6Chi) recruited out of mouse CD45 white blood cells were performed. In vivo migration was defined as the comparison of cell recruitment to the liver with the vehicle control. As ​ can be seen, compared to mice treated with non - transfected 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 (left panel) recruited from total white blood cells.

[0568] Example 17 - Engineered macrophages convert cells in the liver to a pro - reparative phenotype in vivo. NSG mice were subjected to 4 - 5 weeks of CCl4 intoxication. RTX001 and NTrx cells were administered intravenously on SD 23, and readings were collected 24 hours after cell administration. Mice injected with phosphate - buffered saline (PBS) were used as vehicle controls. Mouse cytokines, including the level of IL - 10 in liver homogenates, were evaluated by MSD assay. As Figure 22 can be seen, administration of human cells overexpressing IL - 10 and MMP9 (RTX001) was able to induce an increase in mouse IL - 10 (as measured in liver homogenates). This indicates that human IL - 10 induced an anti - inflammatory phenotype, and given the well - known polarization properties of IL - 10 (notably, untransfected non - polarized cells did not induce any increase in mouse IL - 10 levels), this suggests that mouse cells were transformed into a pro - repair phenotype.

[0569] Example 18 - RTX 001 macrophages reduce the expression of a - SMA in vitro and in vivo. The beneficial effects of macrophages overexpressing IL-10 and MMP9 have been demonstrated in vivo in a murine model of liver fibrosis (mice poisoned with CCl4 for 4 - 5 weeks) 1 week after administration. When macrophages were injected intravenously into mice, a decrease in the activation of scar-forming cells (i.e., activated hepatic stellate cells, HSCs) was observed within the scar tissue compared to untransfected (NTrx) macrophages and PBS controls (visible by staining for a-SMA, an activation marker of HSCs, which was quantified in the results shown in the right panel)( Figure 24 ).

[0570] To further demonstrate the effect of macrophages expressing IL-10 and MMP9 (RTX001) on 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 the key features of hepatic stellate cells and is thus a suitable model for human liver fibrosis. In this system, LX-2 cells were treated with 50 ng / ml TGF-β in DMEM medium for 24 hours to activate the cells, which occurs during liver fibrosis. The cells were then incubated for an additional 24 hours with DMEM conditioned medium (CM) in which the following macrophages had grown for approximately 18 hours:

[0571] 1. Untreated / Un-transfected / UT human macrophages (Non-Trx CM).

[0572] 2. Human macrophages expressing constructs encoding IL-10 and MMP9 (RTX001 CM).

[0573] 3. Human macrophages expressing constructs encoding IL-10 alone (Control CM).

[0574] For each of these groups, conditioned medium from macrophages from 6 different donors was used as biological replicates. After incubation with the conditioned medium, we measured the number of LX-2 cells expressing a-SMA (Figure B) and the level of a-SMA expression in the cells( Figure 25 A). Since a-SMA is an activation marker of human hepatic stellate cells, a decrease in its level indicates a decrease in activation. Since hepatic stellate cells are associated with the production of liver scars, a decrease in their activation portends a reduction in liver fibrosis. As Figure 25 shown in B, LX-2 cells treated with RTX001 showed a decrease in the number of a-SMA-expressing cells and lower a-SMA expression, indicating the repressive effect of RTX001 on hepatic stellate cell activation.

[0575] Example 19 - Optimized bicistronic mRNA results in improved IL - 10 secretion. As Figure 26 As shown in the right figure, transfection with optimized bicistronic mRNA led to much higher IL-10 secretion. Surprisingly, despite the inhibitory effect of IL-10 on MMP9 secretion, as Figure 1 shown in B, compared with untransfected macrophages (NTRx), the optimized sequence led to much higher MMP9 secretion and much higher MMP activity ( Figure 26 left and lower figures). In these experiments, the conditions and mRNA concentrations used were the same for optimized / unoptimized mRNA.

[0576] Example 20 - Engineered macrophages are stable in an inflammatory environment. 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, for example, Campana et al, 2021).

[0577] To confirm that once macrophages expressing IL-10 + MMP9 are administered to patients and encounter the inflammatory environment of the liver, they do not revert to a pro-inflammatory phenotype, we conducted a stability study. In this study, we confirmed the stability of the pro-repair phenotype of macrophages expressing IL-10 + MMP9 in an inflammatory environment (by modeling the level of hIFN-γ in patients to simulate the liver environment of patients with end-stage chronic liver disease).

[0578] To test the stability of the phenotype, we tested the phenotype of cells after incubation with hIFN-γ using the following experimental protocol:

[0579] To obtain macrophages, PBMC were isolated from steady-state leukapheresis or active blood samples, CD14+ cells were isolated from PBMC, and plated in TexMacs + M-CSF for 5 days to differentiate into macrophages. On the 5th day 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, 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 seeded at 2 × 10 5The density of cells / well was plated in U-bottom ultra-low attachment sterile culture plates (Corning Cat# 7007) in TexMacs with or without stimulation (hIFN-γ). The concentration of hIFN-γ was calculated with the following information: the total IFN-γ in human liver was calculated using the total IFN-γ concentration in damaged mouse liver, which was determined by previous in vivo pharmacology experiments and scaled up to the weight of human liver. Then the total level of IFN-γ was divided by the planned therapeutic cell dose to obtain the IFN-γ level per cell. This was used to establish a concentration gradient to account for the possibility that patients may have IFN-γ levels higher or lower than the calculated concentration. All groups were plated to provide 3 technical replicates. Then the macrophages were cultured for 24 hours and subsequently centrifuged at 300xG for 5 minutes. The culture supernatant was collected and frozen at -20°C, and the cells were used for flow cytometry to evaluate the levels of CD80, CD86, MHCII, 25F9, CD14, CD206, and CD163 present on the cell surface. After staining, the cells were analyzed on a Novocyte Quanteon, and data analysis was performed using NovoExpress and GraphPad Prism software. There were no significant changes in the pro-inflammatory markers (HLA-DR, CD80, and CD86) or the macrophage recognition marker CD206.

[0580] It can be seen from Figure 27 that the phenotypes of cells expressing MMP9 and IL10 are stable in an inflammatory environment.

[0581] Example 21 - 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-10MMP9 TRx) or treated with IL-4, IL-13, and M-CSF after transfection (IL-10MMP9 TRx+TR). As described herein, the mean fluorescence intensity was measured by flow cytometry, and the results are as Figure 28 shown.

[0582] Example 22 - Post - transfection treatment does not lead to further polarization of engineered macrophages but does improve cold resistance. In cells transfected with a bicistronic construct encoding MMP9 and IL-10, the expression of CD86 (A) and MHC II (B) was measured, without treatment (IL-10-MMP9) or further treatment with IL-4, IL-13, and M-CSF (IL-10-MMP9+TR) after transfection. No differences in the expression of CD86 and MHC II were detected between the IL-10-MMP9 and IL-10-MMP9+TR groups ( Figure 29 )。Therefore, due to the secretion of IL-10, the transfected cells are fully self-polarized (e.g., the decrease in HLA-DR and CD86 is solely due to the secretion of IL-10 by the transfected cells). However, as Figure 30 shown, untransfected (NTRx) or transfected (TRx) macrophages incubated with IL-4 + IL-13 have higher cryo-resistance, which is measured by the percentage of cells that remain viable after cryopreservation.

[0583] Example 23 - Measure the antifibrotic ability of engineered macrophages in an immunosuppressed mouse model of liver fibrosis ability

[0584] The macrophages described herein are of human origin. To understand their behavior in a mammalian system, immunodeficient mice that allow for the transient implantation of human materials in vivo are required. Otherwise, immunocompetent wild-type recipient strains will cause the rapid elimination of the administered human cells through xenogeneic acute rejection mechanisms. The highly immunodeficient "NSG" strain has been genetically altered to eliminate the activity of host T cells, B cells, and NK cells. Without wishing to be bound by theory or mechanism, 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). Although NSG mice can accept the transfer of human cells, they lack several host functional immune cells that provide a secondary immune response after macrophage treatment. Therefore, as expected in humans, the full pharmacological effect cannot be modeled in immunodeficient strains. In addition, due to species differences in receptor binding and downstream signaling pathways, a portion of the human proteins in the macrophage secretome may not function in mice. Thus, the limitations of immunodeficient strains will, at best, underestimate or completely exclude the absolute efficacy demonstration of the macrophages described herein (evidence is a statistically significant reduction in liver fibrosis). However, an in vivo experimental model for testing efficacy is described below.

[0585] The macrophages used in this example are primary human monocyte-derived macrophages (hMDM) that have been phenotypically modified by transient transfection to provide a bicistronic mRNA transcript encoding IL-10 and MMP-9 with a P2A self-cleaving peptide. To demonstrate the anti-fibrotic properties of the test cell therapy, experimental liver fibrosis was modeled in an immunodeficient mouse strain. NSG mice (fully named: NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, from Charles River Laboratories) lack T cells, B cells, and NK cells, which renders the mice immunodeficient and allows for transient engraftment of human cells in vivo. 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-recognized and tractable rodent fibrosis model, and the clinical predictability of obeticholic acid (Fan et al., 2019 47 ; Younossi et al. 2019 48 ) and ranolazine (Wettstein et al., 2017 49 ; Francque et al. 2021 50 ) has been demonstrated. When liver fibrosis was established, 8 weeks after CCl4 administration, the anti-fibrotic properties of the test macrophages were tested by intravenous delivery of the cells (1×10 6 ). Impaired mice receiving PBS alone served as a vehicle control. In addition, mice received untransfected human monocyte-derived macrophages (NTrx hMDM, 1x10 6, Mice damaged by CCl4 (intravenous injection) were used as a comparison group for the engineering steps for control. Twenty-four hours after the 17th dose of CCl4, cells or vehicle alone (100 μL, intravenous injection, in PBS) were administered. All mice continued to receive CCl4 for an additional 4 weeks. All mice were humanely euthanized by bleeding under terminal anesthesia 24 hours after the 24th and last dose of CCl4, prior to cervical dislocation. Whole blood was collected by cardiac puncture and processed to release plasma or serum for liver chemical biomarker assessment. The liver, spleen, lung, heart, and kidney were harvested and fixed for histological analysis. To determine the anti-fibrotic properties of macrophages, picrosirius red (PSR) staining was used in histological liver sections to visualize collagen fibers and quantify liver fibrosis. The PSR-stained sections were digitized by a microscope slide scanner (Zeiss Axioscan, Zeiss AG) and liver fibrosis was quantified by image analysis software (Zen, Zeiss AG). The anti-fibrotic properties of macrophages were evaluated by comparing the percentage of PSR in the group of mice treated with macrophages with the group of mice receiving vehicle alone or NTrx-hMDM treatment.

[0586] Example 24 - Measure the anti - fibrotic ability of engineered macrophages in an immunosuppressed mouse model of fibrotic fatty liver disease

[0587] To demonstrate the anti-fibrotic properties of the engineered macrophages of Example 23, experimental liver fibrosis was modeled in an immunocompromised mouse strain in the context of fatty liver disease. NSG mice (fully named: NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ, from Charles River Laboratories) lack T cells, B cells, and NK cells, which renders the mice immunodeficient, thus allowing for transient engraftment of human cells in vivo. Liver fibrosis was induced by feeding the mice a choline-deficient, amino acid-defined high-fat diet (CDAA HFD) with restricted methionine content. Ad libitum feeding of CDAA HFD induces hepatic steatosis and inflammation, leading to a distinct histological pattern of fibrosis, but no complete weight loss is exhibited in the animals. The CDAA HFD model is valuable because, unlike other fibrosis models, fibrosis induced by CDAA HFD regresses slowly, showing stable fibrosis for at least two weeks after cessation of the dietary insult. Thus, the characteristics of this model allow for the evaluation of test articles in the absence of ongoing injury. Here, NSG mice were provided with CDAA-HFD (ad libitum) for 12 weeks to induce established liver fibrosis in the context of fatty liver disease. After 12 weeks, the mice were switched back to a standard chow diet. After restoration of normal diet, the anti-fibrotic properties of the engineered macrophages were tested after intravenous administration. Impaired mice receiving PBS alone served as vehicle controls. In addition, impaired mice receiving untransfected human monocyte-derived macrophages (NTrx hMDM, 1x10 6 , intravenous injection) were used as a comparison group for the engineering step for control. Twenty-four hours after restoration of the standard (RM3) diet, cells or vehicle alone (100 μL, intravenous injection, in PBS) were administered. All mice continued to receive the standard diet for an additional 4 weeks. At the end of the study, all mice were humanely euthanized by exsanguination prior to cervical dislocation under terminal anesthesia. Whole blood was collected by cardiac puncture and processed to release plasma or serum to evaluate liver chemical biomarkers. The liver, spleen, lung, heart, and kidneys were harvested and fixed for histological analysis. To measure liver fibrosis, histological liver sections were stained with Sirius red (PSR) to visualize collagen fibers. The PSR-stained sections were digitized by a microscope slide scanner (Zeiss Axioscan, Zeiss AG), and liver fibrosis was quantified by image analysis software (QuPath image analysis open-source software). The anti-fibrotic properties of the engineered macrophages were evaluated by comparing the percentage of PSR-positive staining in macrophage-treated mouse samples with samples derived from mice receiving vehicle alone or NTrx-hMDM treatment.

[0588] Example 25 - Use engineered murine macrophages as a substitute for human - engineered macrophages in an immunocompetent liver fibrosis modelTable 4. Exemplary Structural and Functional Characteristics of Engineered Macrophages

[0589]

[0590] Table 5. Further Exemplary Structural and Functional Characteristics of Engineered Macrophages

[0591]

[0592] Conclusion

[0593] In summary, the genetic engineering of IL-10 in combination with MMP9 provides many of the characteristics and functions required for cell therapy, such as for the prevention / treatment of inflammatory conditions and / or the regeneration of organ injury. The combination of IL-10 and MMP9 provides:

[0594] - Reliable macrophage characteristics that are not disrupted by the engineering process.

[0595] - A strong anti-inflammatory phenotype.

[0596] - The ability to pattern naive macrophages into a pro-repair phenotype.

[0597] - Excellent phagocytic ability.

[0598] - Reassuring safety and biodistribution characteristics, including penetration into damaged organs and rapid clearance / absence in other organs.

[0599] The above characteristics are shared with macrophages engineered with IL-10 alone. However, the combination of IL-10+MMP9 provides some specific and surprising characteristics that are key to providing the therapeutic effects we need, such as:

[0600] - A strong ability to attract monocytes, which are then patterned into a pro-repair phenotype.

[0601] - The ability to restore MMP activity (a surrogate for fibrosis / ECM remodeling) that was eliminated by engineering with IL-10 alone.

[0602] Therefore, we believe that engineering macrophages with the combination of IL-10 and MMP9 will provide an effective product that can have anti-inflammatory and anti-fibrotic functions in several acute and chronic organ injury settings (remodeling of ECM components is also crucial in acute injury settings to ensure tissue restitution and proper regeneration).

Claims

1. An engineered macrophage, wherein the engineered macrophage is engineered to overexpress IL-10.

2. The engineered macrophage according to claim 1, wherein when cultured in vitro at a cell concentration of 4x10 6 / ml, the macrophage secretes IL-10 at a culture supernatant concentration of at least 10,000 pg / ml.

3. The engineered macrophage according to claim 1 or 2, wherein the macrophage is additionally engineered to overexpress MMP9.

4. The engineered macrophage according to claim 3, wherein the engineered macrophage comprises an exogenous coding sequence of IL-10 and an exogenous coding sequence of MMP9.

5. The engineered macrophage according to claim 4, wherein the expression of the exogenous coding sequence has a synergistic effect in restoring MMP activity and / or in monocyte recruitment of macrophages as compared to an engineered macrophage comprising only the exogenous IL-10 sequence.

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

7. The engineered macrophage according to any one of claims 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 SEQ ID NO:4, optionally, wherein the IL-10 protein comprises the same amino acid sequence as SEQ ID NO:

4.

8. The engineered macrophage according to any one of claims 4-7, wherein the exogenous coding sequence of MMP9 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 SEQ ID NO:6, optionally, wherein the IL-10 protein comprises the same amino acid sequence as SEQ ID NO:

6.

9. The engineered macrophage according to any one of claims 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.

10. The engineered macrophage according to claim 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 linker sequence, further optionally, wherein the linker is a self-cleaving 2A linker, further optionally, wherein the linker is p2A.

11. The engineered macrophage according to claim 10, wherein the nucleic acid molecule is an mRNA molecule that comprises a 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 SEQ ID NO:13, optionally, wherein the nucleic acid comprises SEQ ID NO:

13.

12. The engineered macrophage according to claim 10 or 11, wherein the nucleic acid molecule is an mRNA molecule comprising a 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 SEQ ID NO: 14, optionally, wherein the nucleic acid comprises SEQ ID NO:

14.

13. The engineered macrophage according to claims 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.

14. The engineered macrophage according to claims 10-13, wherein the nucleic acid molecule is an mRNA molecule encoding IL-10 and MMP9 linked by a linker sequence, and wherein the linker sequence comprises an mRNA having the sequence described in SEQ ID NO:

15.

15. The engineered macrophage according to claims 10-14, wherein the nucleic acid molecule is an mRNA molecule comprising a sequence that is 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 SEQ ID NO: 10, optionally, wherein the mRNA further comprises a polyA tail that is 65 to 250 residues in length, preferably 90 to 120 residues in length, preferably about, and / or a 5' cap.

16. The engineered macrophage according to claims 10-15, wherein the nucleic acid molecule is an mRNA molecule comprising a sequence that is 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 SEQ ID NO: 16, optionally, wherein the mRNA further comprises a 5' cap.

17. The engineered macrophage according to claim 16, wherein the mRNA molecule contains chemically modified residues, preferably modified uridine residues, and optionally at least one synthetic cap.

18. The engineered macrophage according to any one of claims 4 to 17, wherein the exogenous coding sequence of IL-10 and the exogenous coding sequence of MMP9 are on the same nucleic acid.

19. The 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 endogenous silencing RNA or introducing an exogenous silencing RNA sequence, optionally, wherein the silencing RNA is miRNA.

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

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

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

23. The engineered macrophage according to any one of the preceding claims, wherein the IL-10 secretion of the engineered macrophage is increased by at least 1000-fold compared to non-engineered, non-polarized cells.

24. The engineered macrophage according to any one of the preceding claims, wherein the MMP3 secretion of the engineered macrophage is increased by at least 10-fold compared to non-engineered, non-polarized cells.

25. The engineered macrophage according to any one of the preceding claims, wherein the MMP10 secretion of the engineered macrophage is increased by at least 20-fold compared to non-engineered, non-polarized cells.

26. The engineered macrophage according to any one of the preceding claims, wherein when cultured in vitro at a cell concentration of 4×10 6 / ml, the macrophage secretes IL-10 at a culture supernatant concentration of at least 10,000 pg / ml.

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

28. The 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 to monocytes.

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

30. The engineered macrophage according to any one of the preceding claims, wherein the CD80 expression of the engineered macrophage is reduced by at least ten percent compared to non-engineered, non-polarized cells.

31. The engineered macrophage according to any one of the preceding claims, wherein when cultured in vitro at a cell concentration of 4x10 6 / ml, the macrophage secretes TNF-α at a culture supernatant concentration of up to 40 pg / ml.

32. The engineered macrophage according to any one of the preceding claims, wherein the engineered macrophage has at least an equivalent phagocytic ability to non-engineered, non-polarized cells.

33. The engineered macrophage according to any one of claims 1-32, wherein the metalloproteinase activity is restored relative to the reduced metalloproteinase activity in macrophages engineered with the IL-10 coding sequence alone.

34. The engineered macrophage according to any one of the preceding claims, wherein the macrophage is transiently transfected, optionally by electroporation.

35. The engineered macrophage according to claim 34, wherein the transfection is non-viral.

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

37. A population of engineered macrophages according to any one of the preceding claims.

38. A therapeutic composition comprising the population of macrophages according to claim 37 and a pharmaceutically acceptable medium.

39. The engineered macrophage according to any one of claims 1 to 36, the population of macrophages according to claim 37 or the composition according to claim 38, for use in therapy.

40. The engineered macrophages, population or composition according to claim 39, wherein the therapy is administered to a subject in need thereof.

41. The engineered macrophages according to any one of claims 1 to 36, the population of macrophages according to claim 37 or the composition according to claim 38, for use in treating an inflammatory condition in a subject.

42. The engineered macrophages, population or composition according to claim 40 or 41, wherein the macrophages are autologous or allogeneic to the subject.

43. The engineered macrophages, population or composition according to claim 41, wherein the inflammatory condition is liver injury, optionally chronic liver injury.

44. The engineered macrophages, population or composition according to claim 41 or 43, wherein the condition is a chronic inflammatory condition with a fibrotic component, optionally, wherein the condition is organ damage associated with chronic inflammation.

45. The engineered macrophages, population or composition according to any one of claims 41, 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.

46. The engineered macrophages, population or composition according to any one of claims 43 - 45, wherein the condition is cirrhosis.

47. The engineered macrophages, population or composition according to claim 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).

48. The engineered macrophages, population or composition according to claim 46, wherein the cirrhosis is caused by steatohepatitis liver disease (SLD), optionally, wherein the steatohepatitis liver disease is metabolic dysfunction - associated steatohepatitis liver disease, metabolic - associated steatohepatitis, Met - ALD or cryptogenic SLD.

49. The engineered macrophages, population or composition according to claims 46 - 48, wherein the cirrhosis is selected from compensated cirrhosis and decompensated cirrhosis.

50. The engineered macrophages, population or composition according to any one of claims 41 - 49, wherein the condition is acute - on - chronic liver failure (ACLF).

51. The engineered macrophages, populations or compositions according to claims 46 - 49, for treating a subject who has recovered (recompensated) from their first liver decompensation event, optionally, wherein the first liver decompensation event required hospitalization of the subject, preferably, wherein the subject has not experienced an additional liver decompensation event after recovering from the first liver decompensation event.

52. The engineered macrophages, populations or compositions according to claims 46 - 49 and 51, wherein the subject exhibits one or more clinical signs of liver decompensation, and / or has recovered from one or more clinical signs of liver decompensation, the one or more clinical signs selected from the list consisting of jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, variceal bleeding and gastrointestinal bleeding.

53. The engineered macrophages, populations or compositions according to any one of claims 1 - 52, wherein the macrophages are derived from human monocyte - derived macrophages (hMDM) or stem cells, optionally, wherein the stem cells are induced pluripotent stem cells (iPSC).

54. The engineered macrophages, populations or compositions according to claim 53, wherein the macrophages are derived from iPSC, and the iPSC are substantially lacking in functional HLA I and II complexes on their surface.

55. A method of improving the migration of monocytes to an inflammatory site, comprising using the engineered macrophages, populations of engineered macrophages or compositions according to any one of the preceding claims.

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

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

58. The method according to claim 57, comprising contacting the macrophages with IL - 4, IL - 13 and M - CSF before, during or after transfection.

59. The method according to claim 57 or 58, wherein mRNA molecules encoding IL - 10 and MMP9 are co - transfected using a bicistronic vector linked by a p2A linker sequence.

60. The engineered macrophages according to any one of claims 1 to 36, wherein the macrophages are engineered with an mRNA construct encoding human IL - 10 fused to human MMP9 via a cleavable linker.

61. A method of treating inflammation and / or fibrosis, comprising administering to a subject in need thereof a therapeutically effective amount of the engineered macrophages according to any one of claims 1 - 36.

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

63. The method according to claim 62, wherein the macrophages are engineered to express IL-10 and MMP9 by introduction of an exogenous nucleic acid comprising nucleotide sequences encoding IL-10 and MMP9.

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

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

66. The method according to any one of claims 63-65, wherein the nucleic acid is mRNA.

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

68. The method according to claim 67, wherein the macrophages are engineered to express IL-10 by introduction of an exogenous nucleic acid comprising a nucleotide sequence encoding IL-10.

69. The method according to claim 67 or 68, wherein the nucleic acid is mRNA.

70. A method of improving the low-temperature stress resistance of macrophages, which comprises incubating macrophages in a culture medium comprising IL-4, IL-13, and M-CSF.

71. A method of cryopreserving macrophages, which comprises incubating macrophages in a culture medium comprising IL-4, IL-13, and M-CSF before cryopreservation.

72. The method according to claim 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 the macrophages is 4x10 6 cells / ml.

73. The method according to claims 70-72, wherein the cells are incubated overnight in a culture medium comprising IL-4, IL-13, and M-CSF.

74. Cryopreserved macrophages obtained by the method according to any one of claims 70-73.

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