Methods and compositions for reviving immune cells and therapies using revived immune cells

Abstract

Provided herein are methods and compositions for cell viability recovery of immune cells, such as T cells. Cell reviving activity may be achieved by exposing immune cells, such as transient exposure to mRNA encoding a reprogramming factor. Also provided are compositions comprising such revived immune cells, including revived T cells, and the use of the revived immune cells in the treatment of certain diseases and / or disorders, such as cancer and immune disorders.

Description

[0001] Reference to a sequence listing, table or computer program

[0002] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy created on April 11, 2023 is named "111277-0065-8004WO00_SEQ" and is 28,299 bytes in size. Nucleotide sequences related to the present disclosure are provided in Table 1. Technical Field

[0003] The subject matter described herein relates to compositions and methods for cellular rejuvenation of immune cells such as T cells. Cell rejuvenation can be achieved by exposing the immune cells to mRNA encoding reprogramming factors such that expression of the reprogramming factors is transient. Compositions comprising such rejuvenated immune cells, including rejuvenated T cells, and uses of the rejuvenated immune cells in treating certain diseases and / or disorders such as cancer and immune disorders are also provided.

[0004] The subject matter described herein also relates to compositions and methods for suppressing, preventing and / or reversing cell exhaustion of engineered immune cells, such as T cells engineered to express chimeric antigen receptors (CAR T cells). By exposing T cells to mRNA encoding reprogramming factors, the expression of reprogramming factors is transient and retains cell identity, and suppression, prevention and / or reversal of cell exhaustion during CAR T cell manufacturing can be achieved. Compositions comprising such transiently reprogrammed CAR T cells, and their use in treating certain diseases and / or disorders, such as cancer and immune disorders, are also provided. Background Art

[0005] T cells exist in a continuous state of differentiation characterized by the gradual acquisition or loss of functional properties and gene expression patterns, the combined effects of which produce different phenotypic traits. At the opposite end of the differentiation spectrum are naive T cells (Tn or T N ) and terminally differentiated effector T cells (Teff, T E or T EFF In the middle are memory T cells at an intermediate stage of differentiation, and they can further divide into memory stem cells (Tscm or T SCM ), central memory cells (Tcm or T CM ) and effector memory T cells (Tem or T EM ).

[0006] Therapies using functional immune cells, such as T cells, provide potentially effective therapeutic strategies for combating many types of immune cell-related diseases and disorders, including cancer and viral infections. Chimeric antigen receptor (CAR) T cell therapy involves genetically engineering T cells in cancer patients to express chimeric antigen receptors that target one or more antigens found on the surface of tumor cells. These genetically engineered T cells, when infused back into the patient, localize to the tumor site and exert a powerful anti-tumor effect. CAR T cell therapy has achieved remarkable success in treating specific blood cancers, but has not yet fully realized its therapeutic potential in terms of effectiveness or wider application. T cell dysfunction and exhaustion can negatively affect proliferation and T cell-mediated cytotoxicity, and are major challenges that reduce the efficacy and persistence of CAR T cell therapy in patients.

[0007] T cell exhaustion is a state of T cell dysfunction triggered by the continuous stimulation and activation of T cells in the presence of antigens. In the case of cancer, the continuous stimulation of T cells is due to the presence of tumor antigens. T cell exhaustion is characterized by the gradual loss of effector function and reduced expression of cytokines such as interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). It is also characterized by the continuous or increased expression of inhibitory receptors (such as PD1, Tim3 and LAG3). With the progress of T cell exhaustion, the effectiveness of CAR T cell therapy decreases, which leads to ineffective control of cancer.

[0008] The process for making CAR T cells involves the activation of naive T cells that have been isolated from cancer patients. This activation step triggers T cell proliferation and differentiation. However, prolonged CAR T cell activation during the manufacturing process results in a higher percentage of the isolated T cell population having a more differentiated and effector-like phenotype, which is prone to accelerated exhaustion and thus limited in vivo function. Despite high initial response and remission rates, 30% to 60% of patients receiving CAR T-cell therapy experience disease relapse, usually within one year of completing treatment (Xu, X. et al. Mechanisms of Relapse After CD19 CAR T-Cell Therapy for Acute Lymphoblastic Leukemia and Its Prevention and Treatment Strategies. Front. Immunol. 10, 2664 (2019); Vercellino, L. et al. Predictive factors of early progression after CAR T-cell therapy in relapsed / refractory diffuse large B-cell lymphoma. Blood Adv. 4, 5607–5615 (2020); Park, JH et al. Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia. N. Engl. J. Med. 378, 449-459 (2018); Maude, SL et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N. Engl. J. Med. 378, 439–448 (2018)). In most relapsed cases, the target antigen is still expressed on the cancer cells, but the current manufacturing process produces a single therapeutic dose, and the manufacturing cost of a single round is high. In addition, approximately 90% of patients experience CAR T cell-related side effects, which raises concerns among clinicians about patient safety. Overall, these limitations of CAR T cell products and their manufacturing prevent many patients from receiving treatment and durable therapeutic responses.

[0009] It is reported that CAR T cells isolated from patients treated for chronic lymphocytic leukemia (CLL) have a high frequency of memory T cells with a "stem cell-like" phenotype, and these patients have experienced a strong and lasting anti-tumor response. Less differentiated memory T cell phenotypes, such as central memory T cells (Tcm) and stem cell memory T cells (Tscm), have high proliferation capacity, long-term survival, and in vivo persistence. In humans, Tscm rapidly acquire effector functions after TCR stimulation, and they have a long lifespan. Equally important, Tscm is similar to traditional stem cells in terms of its enhanced self-renewal capacity and enhanced proliferation capacity. They can also supplement more differentiated memory T cells and effector T cells. Unfortunately, Tscm only accounts for a very small percentage (2% to 3%) of the total number of circulating T cells.

[0010] Therefore, there is a great need for a means to inhibit, prevent and / or reverse cell exhaustion during CAR T cell manufacturing that mitigates T cell differentiation, increases proliferation capacity, increases tumor cell killing and promotes T central memory (Tcm) and / or T stem cell memory (Tscm) phenotypes.

[0011] In addition, there is a great need for a method for restoring or rejuvenating T cells without dedifferentiation into stem cells. These "rejuvenated" T cells have the activity and properties of younger T cells (in contrast to exhausted T cells), such as antigen-specific killing activity, self-renewal ability, and pluripotency. The rejuvenated T cells also show increased functional persistence and exhibit the same genetic pattern as younger T cells. Therefore, there is a need for a method for rejuvenating immune cells that avoids dedifferentiation and loss of cell identity and provides a convenient and simple treatment paradigm.

[0012] The present disclosure addresses these needs and provides additional benefits as well.

[0013] The foregoing examples of the related art and limitations associated therewith are intended to be illustrative rather than exclusive.Other limitations of the related art will become apparent to those skilled in the art after reading this specification. Summary of the invention

[0014] The following aspects and embodiments thereof described and illustrated below are intended to be exemplary and illustrative, not limiting in scope.

[0015] In one aspect, a method for inhibiting, preventing or reversing the exhaustion of immune cells is provided. In some embodiments, the method comprises exposing the immune cells to messenger RNA (mRNA) encoding one or more reprogramming factors, wherein the exposure achieves expression of the one or more reprogramming factors in the immune cells to inhibit or prevent the exhaustion of the immune cells while retaining their identity.

[0016] In another aspect, a method for enhancing the self-renewal capacity of immune cells is provided. In some embodiments, the method includes exposing immune cells to mRNA encoding one or more reprogramming factors, wherein the exposure results in the expression of one or more reprogramming factors in immune cells to increase their self-renewal capacity. In one embodiment, the increase in self-renewal capacity is determined by measuring the expression level of one or more biomarkers relative to immune cells of the same type or subtype that are not exposed to mRNA encoding one or more reprogramming factors, and the one or more biomarkers are selected from but not limited to: BACH2, CD45RA, CD45RO, CCR7, CD62L, CD28, CD27, IL-7Rα, CXCR3, CD95, CD11a, IL-2Rβ, CD57, CD58, TCF1 and TCF7. In further embodiments, the increase in self-renewal capacity is demonstrated by a two-fold, three-fold, four-fold or greater increase in the expression of one or more biomarkers selected from, but not limited to, BACH2, CD45RA, CD45RO, CCR7, CD62L, CD28, CD27, IL-7Rα, CXCR3, CD95, CD11a, IL-2Rβ, CD58, TCF1 and TCF7. In further embodiments, the increase in self-renewal capacity is demonstrated by an increase in the expression of one or more biomarkers greater than 5%, greater than 10%, greater than 15% or greater than 20%, the one or more biomarkers selected from, but not limited to, BACH2, CD45RA, CD45RO, CCR7, CD62L, CD28, CD27, IL-7Rα, CXCR3, CD95, CD11a, IL-2Rβ and CD58, TCF1 and TCF7. In additional embodiments, the increase in self-renewal capacity is demonstrated by an increase in expression of one or more biomarkers greater than 20%, the one or more biomarkers selected from, but not limited to, BACH2, CD45RA, CD45RO, CCR7, CD62L, CD28, CD27, IL-7Rα, CXCR3, CD95, CD11a, IL-2Rβ, CD58, TCF1, and TCF7. In one embodiment, the increase in self-renewal capacity is demonstrated when the rejuvenated cells show the ability to self-renew for a longer period of time than immune cells of the same type or subtype that were not exposed to mRNA encoding one or more reprogramming factors.

[0017] In another aspect, a method for increasing the pluripotency of immune cells is provided. In some embodiments, the method includes exposing immune cells to mRNA encoding one or more reprogramming factors, wherein the exposure results in the expression of one or more reprogramming factors in immune cells to increase their pluripotency. In one embodiment, the increase in pluripotency is determined by measuring the expression level of one or more biomarkers relative to immune cells of the same type or subtype that are not exposed to mRNA encoding one or more reprogramming factors, the one or more biomarkers are selected from but not limited to: BACH2, CD45RA, CD45RO, CCR7, CD62L, CD28, CD27, IL-7Rα, CXCR3, CD95, CD11a, IL-2Rβ, CD57, CD58, TCF1 and TCF7. In further embodiments, the increase in pluripotency is demonstrated by a two-fold, three-fold, four-fold or greater increase in the expression of one or more biomarkers selected from, but not limited to, BACH2, CD45RA, CD45RO, CCR7, CD62L, CD28, CD27, IL-7Rα, CXCR3, CD95, CD11a, IL-2Rβ, CD58, TCF1 and TCF7. In further embodiments, the increase in pluripotency is demonstrated by an increase in the expression of one or more biomarkers greater than 5%, greater than 10%, greater than 15% or greater than 20%, the one or more biomarkers selected from, but not limited to, BACH2, CD45RA, CD45RO, CCR7, CD62L, CD28, CD27, IL-7Rα, CXCR3, CD95, CD11a, IL-2Rβ and CD58, TCF1 and TCF7. In additional embodiments, the increase in pluripotency is demonstrated by an increase in expression of one or more biomarkers greater than 20%, the one or more biomarkers selected from, but not limited to, BACH2, CD45RA, CD45RO, CCR7, CD62L, CD28, CD27, IL-7Rα, CXCR3, CD95, CD11a, IL-2Rβ, CD58, TCF1, and TCF7. In one embodiment, the increase in pluripotency is demonstrated when the rejuvenated cells show the ability to differentiate into multiple downstream cell types for a longer period of time than the same type or subtype of immune cells not exposed to mRNA encoding one or more reprogramming factors. In one embodiment, the increase in pluripotency is demonstrated when the rejuvenated cells show the ability to differentiate into a greater number of downstream cell types compared to the same type or subtype of immune cells not exposed to mRNA encoding one or more reprogramming factors.In one embodiment, the increase in pluripotency is determined by measuring the expression level of one or more biomarkers selected from, but not limited to, BACH2, CD45RA, CD45RO, CCR7, CD62L, CD28, CD27, IL-7Rα, CXCR3, CD95, CD11a, IL-2Rβ, CD57, CD58, TCF1, and TCF7 relative to the same type or subtype of immune cells that have not been exposed to mRNA encoding one or more reprogramming factors. In a further embodiment, the increase in pluripotency is demonstrated by a two-fold, three-fold, four-fold or greater increase in the expression of one or more biomarkers selected from, but not limited to, BACH2, CD45RA, CD45RO, CCR7, CD62L, CD28, CD27, IL-7Rα, CXCR3, CD95, CD11a, IL-2Rβ, CD58, TCF1, and TCF7. In further embodiments, the increase in pluripotency is demonstrated by an increase in expression of one or more biomarkers greater than 5%, greater than 10%, greater than 15%, or greater than 20%, the one or more biomarkers selected from, but not limited to, BACH2, CD45RA, CD45RO, CCR7, CD62L, CD28, CD27, IL-7Rα, CXCR3, CD95, CD11a, IL-2Rβ, and CD58, TCF1, and TCF7. In additional embodiments, the increase in pluripotency is demonstrated by an increase in expression of one or more biomarkers greater than 20%, the one or more biomarkers selected from, but not limited to, BACH2, CD45RA, CD45RO, CCR7, CD62L, CD28, CD27, IL-7Rα, CXCR3, CD95, CD11a, IL-2Rβ, CD58, TCF1, and TCF7.

[0018] In another aspect, a method for increasing the functional persistence of immune cells is provided. In certain embodiments, the method includes exposing immune cells to mRNA encoding one or more reprogramming factors, wherein the exposure causes the expression of one or more reprogramming factors in immune cells to increase their functional persistence. In one embodiment, when the rejuvenated cells show that they can kill target cells (including but not limited to tumor cells) for a longer period of time than the same type or subtype of immune cells that are not exposed to the mRNA encoding one or more reprogramming factors, the increase of functional persistence is demonstrated. In one embodiment, when the rejuvenated cells produce one or more cytokines (including but not limited to: IL-2, IFN-γ (IFN-gamma), GM-CSF and TNF-α (TNF-alpha)) with increased concentrations, the increase of functional persistence is demonstrated. In further embodiments, the increase in functional persistence is demonstrated when the rejuvenated cells produce increased concentrations of one or more, two or more, or three or more of the following cytokines for a longer period of time than immune cells of the same type or subtype that have not been exposed to mRNA encoding one or more reprogramming factors: IL-2, IFN-γ (IFN-gamma), GM-CSF, and TNF-α (TNF-alpha). In further embodiments, the increase in functional persistence is demonstrated by a two-fold, three-fold, four-fold or greater increase in expression of one or more cytokines selected from, but not limited to, the following for a longer period of time than immune cells of the same type or subtype that have not been exposed to mRNA encoding one or more reprogramming factors: IL-2, IFN-γ (IFN-gamma), GM-CSF, and TNF-α (TNF-alpha). In a further embodiment, the increase in functional persistence is demonstrated by an increase in the expression of one or more, two or more, or three or more cytokines selected from, but not limited to, IL-2, IFN-γ (IFN-gamma), GM-CSF, and TNF-α (TNF-alpha) greater than 5%, greater than 10%, greater than 15%, or greater than 20% for a longer period of time than the same type or subtype of immune cells not exposed to mRNA encoding one or more reprogramming factors. In another embodiment, the increase in functional persistence is demonstrated when the rejuvenated cells show a reduction in the level of one or more exhaustion markers (including but not limited to: TIGIT, LAG-3, TIM-3, PD-1, and 2B4) for a longer period of time than the same type or subtype of immune cells not exposed to mRNA encoding one or more reprogramming factors.In a further embodiment, the increase in functional persistence is demonstrated when the rejuvenated cells show reduced levels of one or more exhaustion markers (selected from the group consisting of: TIGIT, LAG-3, TIM-3, PD-1, and 2B4) for a longer period of time than immune cells of the same type or subtype that were not exposed to mRNA encoding one or more reprogramming factors.

[0019] In another aspect, a method for inducing or enhancing the proliferation of immune cells is provided. In some embodiments, the method comprises exposing immune cells to mRNA encoding one or more reprogramming factors, wherein the exposure achieves expression of one or more reprogramming factors in immune cells to enhance the proliferation of immune cells while retaining their identity. In some embodiments, the method for inducing proliferation does not induce exhaustion. In some embodiments, the proliferation is due to prevention or reversal of exhaustion.

[0020] In another aspect, a method for inducing proliferation is performed before, simultaneously with, or after a method for inhibiting, preventing, or reversing depletion. In some embodiments, a method for inducing proliferation is performed at any time understood by those skilled in the art to provide sufficient proliferation before a method for inhibiting, preventing, or reversing depletion. In some embodiments, a method for inducing proliferation is performed at any time understood by those skilled in the art to provide sufficient proliferation after a method for inhibiting, preventing, or reversing depletion. In some embodiments, a method for inducing proliferation is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days before a method for inhibiting, preventing, or reversing depletion. In some embodiments, a method for inducing proliferation is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after a method for inhibiting, preventing, or reversing depletion.

[0021] In some embodiments, the method of the present technology includes an immune cell, which is a lymphocyte, a granulocyte, a monocyte, a macrophage, a microglia or a dendritic cell. In some embodiments, the lymphocyte is a T cell, a B cell or a natural killer (NK) cell. In some embodiments, the lymphocyte is a tumor infiltrating lymphocyte. In other embodiments, the lymphocyte is a T cell. In some embodiments, the T cell is a cytotoxic T cell (CD8+), a helper T cell (CD4+), an inhibitory or regulatory T cell (Treg), a memory T cell, a natural killer T cell (NKT cell), or a γδT cell. In other embodiments, the helper T cell is a Th1, Th2, Th17, Th9 or Tfh T cell. In other embodiments, the T cell is an initial T cell (Tn or TN) that has not experienced an antigen or a stem cell memory T cell (Tscm or T SCM ), central memory T cells (Tcm or T CM ), effector memory T cells (Tem or T EM ), effector T cells (Teff, T EFF or T E ), exhausted T cell precursors (Tpex or T PEX ) or depleted T cells (Tex or T EX ). In further embodiments, the T cells possess phenotypic markers for their specific subtypes (e.g., Tscm, Tcm, Tem, Teff) as shown in Table 2. In some embodiments, the suppressive or regulatory T cells of the present technology are FOXP3+ (FOXP3 positive) T cells or FOXP3- (FOXP3 negative) T cells. In some embodiments, NKT cells are a subset of CD1d restricted T cells.

[0022] In some embodiments, the granulocytes of the present technology are neutrophils, eosinophils, basophils, or mast cells.

[0023] In other embodiments, the lymphocytes of the present technology are B cells. In some embodiments, the B cells are memory B cells or plasma cells.

[0024] In other embodiments, the immune cell is a monocyte, a macrophage, a microglia, or a dendritic cell.

[0025] In some embodiments, the methods described herein can be used, wherein the immune cells are natural immune cells or engineered immune cells. In some embodiments, the methods described herein are performed in parallel or in series with the methods for engineering immune cells, so that the method is performed before, during, and / or after the engineering of immune cells. In some embodiments, such engineering includes engineering to make immune cells express chimeric antigen receptors. In some embodiments, such chimeric antigen receptors target CD19, CD20, CD22, CD30, CD33, CD123, FLT3, BCMA, GD2, HER2, MUC1, B7-H3, IL13Ra2, TAG72, MUC16, BCMA, or at least one of any other antigens suitable for immunotherapy. In some embodiments, such engineering includes engineering to make immune cells express other proteins or peptides, such as growth factors and cytokines. In some embodiments, the cytokines include IL-7 and / or IL-15. In some embodiments, such engineering of immune cells is performed in vitro, for example, in the manufacture of cell therapy products (such as autologous or allogeneic chimeric antigen receptor (CAR)-T, CAR-NK, CAR-M or CAR-NKT cells). In some embodiments, the CAR-T cells provided herein target at least one of CD19, CD20, CD22, HER2, MUC1, CD30, CD33, CD123, FLT3, B7-H3, IL13Ra2, GD2, TAG72, MUC16 or BCMA via a chimeric antigen receptor. In some embodiments, the CAR-T cells provided herein target at least one of CD19 or BCMA via a chimeric antigen receptor. In some embodiments, the CAR-T cells provided herein target CD19 via a chimeric antigen receptor. In some embodiments, the CAR-T cells provided herein target BCMA via a chimeric antigen receptor. In some embodiments, the CAR-NK cells provided herein target CD19, FLT3, CD20, CD38, CD138, BCMS, CS1, CD3, CD5, CD7, NKG2D, HER2, EGFR, EpCAM, TF, B7-H6, HLA-G, CD24, CD44, CD133, mesothelin, or at least one of αFR through a chimeric antigen receptor. In some embodiments, the CAR-M cells provided herein target at least one of CD19, HER2, CD22, or ALK19 through a chimeric antigen receptor. In some embodiments, the CAR-NK cells provided herein are engineered to express IL-2 and / or IL-15. In some embodiments, the CAR-NKT cells provided herein target GD2 through a chimeric antigen receptor and are engineered to express IL-15. In such embodiments, the immune cell rejuvenation method described herein is performed in vitro during or after the manufacture of a cell therapy product.In other embodiments, such engineering of immune cells is carried out in vitro, for example, in the so-called "in situ" production of CAR engineered cells. In such embodiments, the mRNA encoding CAR or growth factor or cytokine is injected into the subject or patient, for example, for the "in situ" CAR engineering of the patient's immune cells (such as T cells, NK cells, macrophages, tumor infiltrating lymphocytes, dendritic cells and / or NKT cells), i.e., in the patient, without removing cells for ex vivo transfection. In such embodiments, the immune cell rejuvenation method described herein is also carried out in vivo, wherein the mRNA encoding one or more reprogramming factors is injected into the patient before, at the same time or after the mRNA encoding CAR or other cell engineering molecules. In some embodiments, lipids and / or polymer nanoparticles are used to deliver mRNA encoding one or more reprogramming factors in vivo. In some embodiments, lipids and / or polymer nanoparticles are engineered for in vivo targeted delivery to immune cells, such as T cells, NK cells, macrophages, tumor infiltrating cells, dendritic cells and / or NKT cells in vivo. In yet other embodiments, in vivo treatment is performed in the absence of any other in vivo cell engineering to enhance or restore the effectiveness of the immune system and treat diseases associated with immune dysfunction or disorder, such as improving the effectiveness of the immune system to fight cancer or infection, or reducing inflammation.

[0026] In some embodiments, the method described herein includes exposing immune cells to mRNA encoding one or more reprogramming factors, wherein the reprogramming factor is any reprogramming factor capable of rejuvenating cells while retaining identity or not causing identity loss or dedifferentiation. In some embodiments, the method described herein includes exposing immune cells to mRNA encoding one or more reprogramming factors, and the one or more reprogramming factors are selected from the group consisting of Oct, Sox, Klf, Myc, LinNANOG or GLIS1, i.e., any one of these factors alone or in any combination. In other embodiments, the exposure includes exposure to mRNA encoding one or more reprogramming factors selected from the group consisting of Oct4, Sox2, Klf4, cMyc, Lin28, NANOG and GLIS1. In other embodiments, the exposure includes exposure to mRNA encoding Oct4, Sox2, Klf4, cMyc, Lin28 and NANOG. In other embodiments, the exposure includes exposure to mRNA encoding Oct4, Sox2, Klf4, GLIS1, Lin28 and NANOG. In other embodiments, the exposure includes exposure to mRNA encoding Oct4, Sox2, Klf4 and cMyc. In other embodiments, the exposure includes exposure to mRNA encoding Oct4, Sox2, Klf4 and GLIS1. In other embodiments, the exposure includes exposure to mRNA encoding Oct4, Sox2, Lin28 and NANOG. In other embodiments, the exposure includes exposure to mRNA encoding Oct4, Sox2 and Klf4. In some embodiments, one or more reprogramming factors are expressed by a single mRNA molecule. In some embodiments, the single mRNA molecule is a polycistronic. In other embodiments, two or more reprogramming factors are used, and the reprogramming factors are expressed by more than one mRNA molecule, such as wherein each reprogramming factor is expressed by its own mRNA molecule, or wherein two or more reprogramming factors are expressed by the same mRNA molecule. In some embodiments, at least one reprogramming factor is a fusion protein comprising a reprogramming factor or a domain of a reprogramming factor. In some embodiments, at least one reprogramming factor is a reprogramming factor optimized for T cells.

[0027] In some embodiments, the mRNA molecule includes a means for controlling the expression duration of at least one reprogramming factor, such as a switch, a trans or self-amplification system expressed, or a mechanism for controlling the half-life of mRNA or a transcription factor protein transcribed from mRNA. In some embodiments, mRNA contains modifications to increase its half-life. Such means and modifications are known to those skilled in the art. In some embodiments, this control of the expression duration, the increased mRNA half-life, or the increased transcription factor protein half-life allows the frequency of mRNA administration to be lower than the frequency required in the absence of this control of the expression duration. In some embodiments, this control of the expression duration, the mRNA half-life, or the protein transcription factor half-life allows the administration of less mRNA within the dosing interval, such as once, twice, or three times per dosing interval, rather than at least once a day within the dosing interval. In some embodiments, the half-life of linear non-replicative mRNA is controlled by changing the 3' untranslated region, changing the poly (A) tail length, and / or adding a WPRE element to mRNA. By this mechanism, the half-life of mRNA is controlled to 6 minutes to 24 hours. Also by this mechanism, the half-life of mRNA is increased to a maximum of 72 hours. The use of circular RNA provides a half-life of 1-3 days. The use of trans-acting or self-amplifying RNA provides a half-life of 2 days or longer, such as up to 10 days or up to 12 days.

[0028] In some embodiments, the exposure includes providing a composition comprising mRNA, wherein the composition comprises an excipient for transfection. In some embodiments, the composition comprises a lipid and the mRNA is associated with the lipid. In some embodiments, the lipid comprises an ionizable lipid, which can be used in combination with other lipid components such as auxiliary lipids, stabilizing lipids and structural lipids. In some embodiments, the disclosure also provides a lipid-nanoparticle composition comprising such lipids for delivering therapeutic nucleic acids. In other embodiments, the composition comprises a polymer and the mRNA is associated with the polymer. In some embodiments, the polymer is a releasable transporter that changes charge. In some embodiments, the charge-modified releasable transporter contains a mixture of lipophilic blocks in a block (b) or stat (stat) architecture, such as those described in McKinlay et al. 2017 (PNAS 2017 January 24 114 (4) E448-E456), McKinlay et al. 2018 (PNAS 2018 June 26 115 (26) E5859-E5866), or Haabeth et al. 2018 (PNAS 2018 September 25 115 (39) E9153-E9161), which are incorporated herein by reference. In some embodiments, the polymer or lipid forms a nanoparticle, complex, or other nanostructure. In other embodiments, the composition comprises both a polymer and a lipid, and the mRNA is associated with the polymer and / or lipid. In certain embodiments, compared with using different mRNA delivery mechanisms, lipid or polymer delivery mRNA is used, such as in lipid nanoparticles, polymer nanoparticles or hybrid lipid-polymer nanoparticles, resulting in enhanced rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, antipathogenic effect, anticancer effect or anti-inflammatory effect in the immune cells exposed. In certain embodiments, compared with using different mRNA delivery mechanisms, due to the lower toxicity and / or lower physiological impact on cells when compared with different delivery mechanisms, lipid or polymer delivery mRNA is used to cause enhanced rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, antipathogenic effect, anticancer effect or anti-inflammatory effect in the immune cells exposed. In certain embodiments, different delivery mechanisms are electroporation, so that compared with using electroporation, lipid or polymer (including lipid or polymer nanoparticles) delivery mRNA is used to cause enhanced rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, antipathogenic effect, anticancer effect or anti-inflammatory effect in the immune cells exposed. This improvement compared with electroporation can be caused by the toxicity of the reduction of cells compared with electroporation or the physiological impact of the reduction.

[0029] In certain embodiments, the method provided herein realizes transfection of mRNA encoding one or more reprogramming factors into immune cells. In certain embodiments, the exposure realizes transfection of mRNA encoding one or more reprogramming factors into T cells or T cell subsets such as NKT cells or other immune cell types in preference to other immune cell types or other cell types.

[0030] In other embodiments, the methods described herein include exposing immune cells to mRNA encoding one or more reprogramming factor cells in vitro, in vivo or in vitro. In some embodiments, exposure is performed in vitro using a technique selected from microinjection, electroporation, biolistics, optical transfection, microfluid extrusion, chemical transfection or biotransfection. In other embodiments, exposure is performed in vitro, and the method further includes transplanting immune cells into the subject after the exposure. In some embodiments of the present technology, exposure is performed in vivo, and the exposure is performed to transfect mRNA encoding one or more reprogramming factors into immune cells for expressing one or more reprogramming factors in cells. In other embodiments, exposure is performed in vivo, and wherein the exposure or expression is performed before, at the same time or after administering therapeutic antibodies, therapeutic proteins or peptides, vaccine antigens or bispecific antibodies. In some embodiments, therapeutic proteins or peptides are cytokines. In some embodiments, vaccine antigens are cancer vaccine antigens, including but not limited to personalized cancer vaccine antigens. In some embodiments, the methods described herein enhance the efficacy and / or safety of anticancer therapy, anti-inflammatory therapy or cancer vaccine. In some embodiments, such methods are performed ex vivo, i.e., in the manufacture of cell therapy products such as autologous or allogeneic chimeric antigen receptors (CAR)-T, CAR-NK, CAR-M or CAR-NKT cells. In some embodiments, such chimeric antigen receptors target CD19, CD20, CD22, CD30, CD33, CD123, FLT3, BCMA, GD2, HER2, MUC1, B7-H3, IL13Ra2, TAG72, MUC16, BCMA or at least one of any other antigens suitable for immunotherapy. In some embodiments, such engineering includes engineering to make immune cells express other proteins or peptides, such as growth factors and cytokines. In some embodiments, the cytokines include IL-7 and / or IL-15. In some embodiments, such engineering of immune cells is performed ex vivo, for example, in the manufacture of cell therapy products such as autologous or allogeneic chimeric antigen receptors (CAR)-T, CAR-NK, CAR-M or CAR-NKT cells. In some embodiments, the CAR-T cells provided herein target at least one of CD19, CD20, CD22, HER2, MUC1, CD30, CD33, CD123, FLT3, B7-H3, IL13Ra2, GD2, TAG72, MUC16, or BCMA via a chimeric antigen receptor. In some embodiments, the CAR-T cells provided herein target at least one of CD19 or BCMA via a chimeric antigen receptor. In some embodiments, the CAR-T cells provided herein target CD19 via a chimeric antigen receptor.In some embodiments, the CAR-T cells provided herein target BCMA via a chimeric antigen receptor. In some embodiments, the CAR-NK cells provided herein target CD19, FLT3, CD20, CD38, CD138, BCMS, CS1, CD3, CD5, CD7, NKG2D, HER2, EGFR, EpCAM, TF, B7-H6, HLA-G, CD24, CD44, CD133, mesothelin, or at least one of αFR via a chimeric antigen receptor. In some embodiments, the CAR-M cells provided herein target at least one of CD19, HER2, CD22, or ALK19 via a chimeric antigen receptor. In some embodiments, the CAR-NK cells provided herein are engineered to express IL-2 and / or IL-15. In some embodiments, the CAR-NKT cells provided herein target GD2 via a chimeric antigen receptor and are engineered to express IL-15. In such embodiments, the immune cell rejuvenation method described herein is performed in vitro during or after the manufacture of a cell therapy product. In other embodiments, such engineering of immune cells is carried out in vivo, for example, in the so-called "in situ" production of CAR engineered cells. In such embodiments, the mRNA encoding CAR or other cell engineering molecules is injected into the subject or patient, for example, for the "in situ" CAR engineering of the patient's immune cells (such as T cells, NK cells, macrophages, tumor infiltrating lymphocytes, dendritic cells and / or NKT cells), i.e., in the patient, without removing cells for ex vivo transfection. In such embodiments, the immune cell rejuvenation method described herein is also carried out in vivo, wherein the mRNA encoding one or more reprogramming factors is injected into the patient before, at the same time or after the mRNA encoding CAR or other cell engineering molecules. In some embodiments, lipids and / or polymer nanoparticles are used to deliver mRNA encoding one or more reprogramming factors in vivo. In some embodiments, lipids and / or polymer nanoparticles are engineered for in vivo targeted delivery to immune cells, such as T cells, NK cells, macrophages, tumor infiltrating cells, dendritic cells and / or NKT cells in vivo. In yet other embodiments, in vivo treatment is performed in the absence of any other in vivo cell engineering to enhance or restore the effectiveness of the immune system and treat diseases associated with immune dysfunction or disorder, such as improving the effectiveness of the immune system to fight cancer or infection, or reducing inflammation.

[0031] In some embodiments, the method of the present technology includes exposing immune cells to mRNA or to at least one reprogramming factor expressed by transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, for a dosing interval understood by a person of ordinary skill in the art, so that immune cells can be rejuvenated without causing loss of identity or differentiation. In some embodiments, the method of the present technology includes exposing immune cells to mRNA or to at least one reprogramming factor expressed by transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, for a dosing interval of no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 days. In some embodiments, at least one mRNA administration is performed every day during the dosing interval. In some embodiments, mRNA administration is performed once a day during the dosing interval. In some embodiments, mRNA administration is performed twice a day during the dosing interval. In some embodiments, at least two mRNA administrations are performed every day during the dosing interval. In some embodiments, mRNA administration is performed less frequently than once a day during the dosing interval, such as once every two days, once every three days, once every four days, once every x days, where x is a number from 4 to 25. Thus, in such embodiments, for example, administration of mRNA once every 5 days in a 5-day dosing interval means administration of mRNA once in the interval, i.e., once in a total treatment period of 5 days, and administration of mRNA twice a day in a 5-day dosing interval means administration of mRNA 10 times in the interval, i.e., 10 times in 5 days. In some embodiments, the methods of the present technology include exposing immune cells to mRNA or to at least one reprogramming factor expressed by transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, for no more than 21, 18, 14, 10, 7, or 5 consecutive days.

[0032] In some embodiments, the method of the present technology includes exposing immune cells to mRNA or to at least one reprogramming factor expressed by transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, for no more than 18 consecutive days. In some embodiments, the method of the present technology includes exposing immune cells to mRNA or to at least one reprogramming factor expressed by transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, for no more than 14 consecutive days. In some embodiments, the method of the present technology includes exposing immune cells to mRNA or to at least one reprogramming factor expressed by transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, for no more than 10 consecutive days. In an embodiment, the method of the present technology includes exposing immune cells to mRNA or to at least one reprogramming factor expressed by transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, for no more than 7 consecutive days. In an embodiment, the method of the present technology includes exposing immune cells to mRNA or to at least one reprogramming factor expressed by transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, for no more than 5 consecutive days. In some embodiments, the method of the present technology includes exposing immune cells to mRNA or to at least one reprogramming factor expressed from transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, for no more than 5 consecutive days, and in some embodiments, the method further includes interrupting the exposure and repeating the exposure after the interruption. In some embodiments, the repetition is performed any number of times, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, or up to 20 times, or up to 30 times, or more times. For in vivo applications, the repetition can last for any duration.

[0033] In some embodiments, the method of the present technology includes exposing immune cells to mRNA or to at least one reprogramming factor expressed from transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, for no more than 4 consecutive days, and in some embodiments, the method further includes interrupting the exposure and repeating the exposure after the interruption. In some embodiments, the repetition is performed any number of times, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, or up to 20 times, or up to 30 times, or more times. For in vivo applications, the repetition can last for any duration.

[0034] In some embodiments, the method of the present technology includes exposing immune cells to mRNA or to at least one reprogramming factor expressed from transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, for no more than 3 consecutive days, and in some embodiments, the method further includes interrupting the exposure and repeating the exposure after the interruption. In some embodiments, the repetition is performed any number of times, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, or up to 20 times, or up to 30 times, or more times. For in vivo applications, the repetition can last for any duration.

[0035] In some embodiments, the method of the present technology includes exposing immune cells to mRNA or to at least one reprogramming factor expressed from transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, for no more than 2 consecutive days, and in some embodiments, the method further includes interrupting the exposure and repeating the exposure after the interruption. In some embodiments, the repetition is performed any number of times, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, or up to 20 times, or up to 30 times, or more times. For in vivo applications, the repetition can last for any duration.

[0036] In some embodiments, the method of the present technology includes exposing immune cells to mRNA or to at least one reprogramming factor expressed by transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, for only one day, and in some embodiments, further including interrupting the exposure and repeating the exposure after the interruption. In some embodiments, the repetition is performed any number of times, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, or up to 20 times, or up to 30 times, or more times. For in vivo applications, the repetition can last for any duration.

[0037] In other embodiments, the exposure includes interrupting the exposure and repeating the exposure after the interruption. In some embodiments, the exposure includes exposing immune cells to mRNA or to at least one reprogramming factor expressed by transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, for a period of about 2 to 5 days, about 5 to 7 days, about 7 to 10 days, about 10 to 12 days, about 12 to 14 days, about 14 to 17 days, about 17 to 19 days, or about 19 to 21 days, and in some embodiments, further including interrupting the exposure and repeating the exposure after the interruption. In some embodiments, the repetition is performed any number of times, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, or up to 20 times, or up to 30 times, or more times. For in vivo applications, the repetition can be continued for any duration, such as until the disease is successfully treated or cured, or throughout the life of the subject or patient. In some embodiments, the repetition is performed at any time after the interruption, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, up to 20 days, up to 30 days, up to 3 months, up to 6 months or up to 1 year after the interruption. An exposure cycle is considered to be a dosing interval, so that, for example, a sequence of exposure-interruption-repeated exposure includes two dosing intervals. In some embodiments, exposure to mRNA is performed once, twice, three times, four times, five times or six times in a six-day interval. In some embodiments, exposure to mRNA is performed on the first or second day in a six-day interval. In some embodiments, exposure to mRNA is performed on the first or second day in a six-day interval, and then on the fourth or fifth day. In some embodiments, exposure to mRNA is performed on the first or second day in a six-day interval, and then on the third or fourth day, and then on the fifth or sixth day. In some embodiments, exposure to mRNA is performed every day in a six-day interval. In some embodiments, the exposure interval occurs immediately after immune cell activation. In some embodiments, the exposure interval occurs one, two, three, four, five, six, seven, eight, nine or ten days after immune cell activation. In some embodiments, the immune cells are activated with CD3 and / or CD28. In some embodiments, the activation is performed for one, two, three, four, five, six or seven days.

[0038] In some embodiments, the method of the present technology includes exposing immune cells to mRNA or to at least one reprogramming factor expressed by transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, twice a day for no more than 5 consecutive days. In some embodiments, the method of the present technology includes exposing immune cells to mRNA or to at least one reprogramming factor expressed by transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, twice a day for no more than 4 consecutive days. In some embodiments, the method of the present technology includes exposing immune cells to mRNA or to at least one reprogramming factor expressed by transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, twice a day for no more than 3 consecutive days. In some embodiments, the method of the present technology includes exposing immune cells to mRNA or to at least one reprogramming factor expressed by transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, twice a day for no more than 2 consecutive days. In some embodiments, the methods of the present technology include exposing immune cells to mRNA or to at least one reprogramming factor expressed by transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, twice a day for no more than 2 consecutive days. In some embodiments, the methods of the present technology include exposing immune cells to mRNA or to at least one reprogramming factor expressed by transfected mRNA, or expressing at least one reprogramming factor from transfected mRNA, twice a day for no more than 1 day.

[0039] In another aspect, the present technology provides a method for preparing a composition for cell therapy. In some embodiments, the method includes obtaining or providing a sample containing one or more immune cells of the same or different types; treating one or more immune cells with mRNA encoding one or more reprogramming factors, wherein the treatment does not cause loss of identity or loss of differentiation, and wherein the treatment rejuvenates or resuscitates the immune cells, as demonstrated by increased immune cell activity. In some embodiments, the treatment is performed during the manufacturing process of the cell therapy product. In other embodiments, the treatment is performed on the final cell therapy product after the manufacturing process is completed. In other embodiments, the treatment is performed on the final cell therapy product during and after the manufacturing process. In other embodiments, the treatment is performed at the point of care before administering cells to the patient.

[0040] The rejuvenation of immune cells (such as T cells) can allow for the use of fewer cells per administration when administered as a cell therapy product compared to the same immune cells that have not been rejuvenated, thereby reducing the cost per dose and the total cost, allowing the production of more doses, and allowing the storage of autologous cells. Compared to the same immune cells that have not been rejuvenated, the rejuvenation of immune cells (such as T cells) can allow for fewer infusions required. In an embodiment, the immune cells rejuvenated as described herein, such as the rejuvenated T cells described herein, are administered at a dose that is about 2-100 times, 3-100 times, 4-100 times, 5-100 times, 6-100 times, 7-100 times, 8-100 times, 9-100 times, or 10-100 times lower than the dose used clinically when the same immune cells are not rejuvenated. In an embodiment, the immune cells rejuvenated as described herein, such as the rejuvenated T cells, are administered at a dose that is at least 3 times lower than the dose used clinically when the same immune cells are not rejuvenated. In embodiments, immune cells, such as rejuvenated T cells, as described herein are administered at a dose that is about 2-100 fold, 3-100 fold, 4-100 fold, 5-100 fold, 6-100 fold, 7-100 fold, 8-100 fold, 9-100 fold, or 10-100 fold lower than the dose clinically used when the same immune cells are not rejuvenated. In embodiments, immune cells, such as rejuvenated T cells, as described herein are administered at a dose of 0.01-1.5 x 10 8 In an embodiment, the CAR-T cells rejuvenated as described herein are administered at a dose of 0.01-1.5x10 8 In an embodiment, the immune cells rejuvenated as described herein, such as rejuvenated T cells, are administered at a dose of 0.02-1.2 x 10 8 In an embodiment, the CAR-T cells rejuvenated as described herein are administered at a dose of 0.02-1.2 x 10 8 The dose of cells was administered.

[0041] In another aspect, the method for preparing a composition for cell therapy utilizes immune cells, such as lymphocytes, granulocytes, monocytes, macrophages, microglia or dendritic cells. In certain embodiments, lymphocytes are T cells, B cells or natural killer (NK) cells. In certain embodiments, lymphocytes are tumor infiltrating lymphocytes.

[0042] In some embodiments, the immune cell is a T cell, which is a cytotoxic T cell (CD8+), a helper T cell (CD4+), an inhibitory or regulatory T cell (Treg), a memory T cell, a natural killer T cell (NKT cell), or a γδ T cell. In some embodiments, the helper T cell is a Th1, Th2, Th17, Th9, or Tfh T cell. In some embodiments, the T cell is a naive T cell (Tn or T N ), stem cell memory T cells (Tscm or T SCM ), central memory T cells (Tcm or T CM ), effector memory T cells (Tem or T EM ), effector T cells (Teff, T E or T EFF ), exhausted T cell precursors (Tpex or T PEX ) or depleted T cells (Tex or T EX ). In some embodiments, the method uses a T cell having a phenotypic marker for its specific subtype (e.g., Tscm, Tcm, Tem, Teff) as shown in Table 2. In other embodiments, the T cell is a memory T cell, which is a central memory T cell, an effector memory T cell, a tissue-resident memory T cell, or a virtual memory T cell. In other embodiments, the inhibitory or regulatory T cell is a FOXP3+ T cell or a FOXP3- T cell. In some embodiments, NKT cells are part of a subset of CD1d-restricted T cells.

[0043] In other embodiments, the immune cell is a granulocyte, such as a neutrophil, eosinophil, basophil, or mast cell.

[0044] In other embodiments, the immune cell is a monocyte, a macrophage, a microglia, or a dendritic cell.

[0045] In some embodiments, the immune cell is a lymphocyte, such as a B cell, including a memory B cell, or a plasma cell.

[0046] In other embodiments, the method for preparing a composition for cell therapy uses an immune cell that is an engineered immune cell. In some embodiments, the method is carried out in parallel or in series with the engineering of immune cells, that is, the method is carried out before, during and / or after the engineering of immune cells. In some embodiments, immune cells are engineered to express chimeric antigen receptors. In some embodiments, the method for preparing a composition for cell therapy includes processing with mRNA encoding one or more reprogramming factors, and the one or more reprogramming factors are selected from the group consisting of Oct, Sox, Klf, Myc, Lin, NANOG or GLIS1, including but not limited to Oct4, Sox2, Klf4, cMyc, Lin28, NANOG and GLIS1. In other embodiments, the method for preparing a composition for cell therapy includes processing with mRNA encoding Oct4, Sox2, Klf4, cMyc, Lin28 and NANOG. In other embodiments, the method for preparing a composition for cell therapy includes processing with mRNA encoding Oct4, Sox2, Klf4, GLIS1, Lin28 and NANOG. In other embodiments, the method for preparing a composition for cell therapy includes processing with mRNA encoding Oct4, Sox2, Klf4 and cMyc. In other embodiments, the method for preparing a composition for cell therapy includes processing with mRNA encoding Oct4, Sox2, Klf4 and GLIS1. In other embodiments, the method for preparing a composition for cell therapy includes processing with mRNA encoding Oct4, Sox2, Lin28 and NANOG. In other embodiments, the method for preparing a composition for cell therapy includes processing with mRNA encoding Oct4, Sox2 and Klf4. In some embodiments, one or more reprogramming factors are expressed by a single mRNA molecule. In some embodiments, the single mRNA molecule is a polycistronic. In other embodiments, two or more reprogramming factors are used, and the reprogramming factors are expressed by more than one mRNA molecule, such as wherein each reprogramming factor is expressed by its own mRNA molecule, or wherein two or more reprogramming factors are expressed by the same mRNA molecule.

[0047] In certain embodiments, the mRNA molecule used in the method for preparing the composition for cell therapy includes a means for controlling the expression duration of at least one reprogramming factor, such as a switch, a self-amplification system, or a mechanism for controlling the half-life of mRNA or a protein transcribed from mRNA. In certain embodiments, this control of the expression duration allows the frequency of administration of mRNA to be lower than the frequency required in the absence of this control of the expression duration. In certain embodiments, this control of the expression duration allows each dosing interval to be administered once mRNA instead of each dosing interval, for example, once per dosing interval instead of at least once a day within the dosing interval.

[0048] In some embodiments, the method for preparing a composition for cell therapy includes providing a composition comprising mRNA, wherein the composition comprises an excipient for transfection. In some embodiments, the composition comprises a lipid and wherein the mRNA is associated with the lipid. In some embodiments, the lipid is selected from those described in Figures 1 to 18. In some embodiments, the lipid is those in Billingsley et al., Nano Lett.2020 (20, 3, 1578–1589), which are incorporated herein by reference. In other embodiments, the composition comprises a polymer and the mRNA is associated with the polymer. In some embodiments, the polymer is a releasable transporter that changes charge. The charge-modified releasable transporter may contain a mixture of lipophilic blocks in a block (b) or stat (stat) architecture, such as those described in McKinlay et al. 2017 (PNAS 2017 Jan 24 114 (4) E448-E456), McKinlay et al. 2018 (PNAS 2018 Jun 26 115 (26) E5859-E5866), or Haabeth et al. 2018 (PNAS 2018 Sep 25 115 (39) E9153-E9161), which are incorporated herein by reference. In some embodiments, the polymer or lipid forms a nanoparticle, complex, or other nanostructure. In other embodiments, the composition comprises both a polymer and a lipid, and the mRNA is associated with the polymer and / or lipid.

[0049] In some embodiments, the method for preparing a composition for cell therapy includes realizing transfection of mRNA encoding one or more reprogramming factors into immune cells. In some embodiments, the processing realizes transfection of mRNA encoding one or more reprogramming factors into T cells in preference to other immune cells. In some embodiments, the processing is performed in vitro, in vivo or ex vivo. In some embodiments, the processing is performed in vitro using a technology selected from microinjection, electroporation, biolistics, optical transfection, microfluid extrusion, chemical transfection or biotransfection. In other embodiments, the processing is performed ex vivo, and the method further includes transplanting immune cells into a subject after the processing. In some embodiments, the processing is performed in vivo, and the processing realizes transfection of mRNA encoding one or more reprogramming factors into immune cells for expressing one or more reprogramming factors in cells. In other embodiments, the processing is performed in vivo, and wherein the processing is performed before, while or after the administration of a bispecific antibody. In some embodiments, treatment includes treating immune cells with mRNA or with at least one reprogramming factor expressed by transfected mRNA, or with the expression of at least one reprogramming factor from transfected mRNA, for no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 days of dosing interval. In some embodiments, at least one mRNA administration is performed every day during the dosing interval. In some embodiments, mRNA administration is performed during the dosing interval at a frequency lower than once a day, such as once every two days, once every three days, once every four days, once every x days, where x is a number from 4 to 25. Therefore, in such embodiments, for example, in a 5-day dosing interval, mRNA administration once every 5 days means administration of mRNA once in the interval, that is, administration once in the total treatment period of 5 days, and administration of mRNA twice a day in a 5-day dosing interval means administration of mRNA 10 times in the interval, i.e., administration 10 times in 5 days. In some embodiments, the method of the present technology comprises treating immune cells with mRNA or with at least one reprogramming factor expressed by transfected mRNA, or with the expression of at least one reprogramming factor from transfected mRNA, for no more than 21, 18, 14, 10, 7 or 5 consecutive days. In some embodiments, the method of the present technology comprises treating immune cells with mRNA or with at least one reprogramming factor expressed by transfected mRNA, or with the expression of at least one reprogramming factor from transfected mRNA, for no more than 18 consecutive days. In some embodiments, the method of the present technology comprises treating immune cells with mRNA or with at least one reprogramming factor expressed by transfected mRNA, or with the expression of at least one reprogramming factor from transfected mRNA, for no more than 14 consecutive days.In some embodiments, the method of the present technology includes treating immune cells with mRNA or with at least one reprogramming factor expressed by transfected mRNA, or with the expression of at least one reprogramming factor from transfected mRNA, for no more than 10 consecutive days. In an embodiment, the method of the present technology includes treating immune cells with mRNA or with at least one reprogramming factor expressed by transfected mRNA, or with the expression of at least one reprogramming factor from transfected mRNA, for no more than 7 consecutive days. In an embodiment, the method of the present technology includes treating immune cells with mRNA or with at least one reprogramming factor expressed by transfected mRNA, or with the expression of at least one reprogramming factor from transfected mRNA, for no more than 5 consecutive days. In other embodiments, the treatment includes interrupting the treatment and repeating the treatment after the interruption. In some embodiments, the treatment includes treating immune cells with mRNA or with at least one reprogramming factor expressed by transfected mRNA, or with the expression of at least one reprogramming factor from transfected mRNA, for about 2 to 5 consecutive days, about 5 to 7 consecutive days, about 7 to 10 consecutive days, about 10 to 12 consecutive days, about 12 to 14 consecutive days, about 14 to 17 consecutive days, about 17 to 19 consecutive days, or about 19 to 21 consecutive days, and in some embodiments, further comprising interrupting the treatment and repeating the treatment after the interruption. In some embodiments, the repetition is performed any number of times, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, or up to 20 times, or up to 30 times. In some embodiments, the repetition is performed at any time after the interruption, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, up to 20 days, up to 30 days, up to 3 months, up to 6 months or up to 1 year after the interruption. A treatment cycle is considered to be a dosing interval, so that, for example, a sequence of treatment-interruption-repeat treatment includes two dosing intervals. In some embodiments, treatment with mRNA is performed once, twice, three times, four times, five times or six times in a six-day interval. In some embodiments, treatment with mRNA is performed on the first or second day in a six-day interval. In some embodiments, treatment with mRNA is performed on the first or second day in a six-day interval, and then on the fourth or fifth day. In some embodiments, treatment with mRNA is performed on the first or second day in a six-day interval, and then on the third or fourth day, and then on the fifth or sixth day. In some embodiments, treatment with mRNA is performed every day in a six-day interval. In some embodiments, the treatment interval occurs immediately after immune cell activation. In some embodiments, the treatment interval occurs one, two, three, four, five, six, seven, eight, nine or ten days after immune cell activation. In some embodiments, the immune cells are activated with CD3 and / or CD28.In some embodiments, activation is performed for one, two, three, four, five, six, or seven days.

[0050] In another aspect, the present technology relates to immune cell populations prepared according to the methods described herein.

[0051] In another aspect, the present technology relates to the use of immune cell populations prepared according to the methods described herein for treating a disease or disorder. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the disease or disorder is an autoimmune disease, including but not limited to multiple sclerosis, rheumatoid arthritis, lupus, celiac disease, Sjogren's syndrome ( syndrome), ankylosing spondylitis, polymyalgia rheumatica, alopecia areata, vasculitis or temporal arteritis, or T-cell immunodeficiency diseases, including but not limited to severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, ataxia telangiectasia, DiGeorge syndrome (22q11.2 deletion syndrome), immune bone dysplasia, congenital dyskeratosis and chronic mucocutaneous candidiasis. In some embodiments, the disease or illness is fibrosis, including but not limited to fibrosis of the heart, liver, lung, kidney, pancreas, bladder, uterus or gastrointestinal tract. In some embodiments, the disease or illness is associated with inflammation, such as neuroinflammation or inflammation of the heart, liver, lung, kidney, pancreas, bladder, uterus or gastrointestinal tract. In some embodiments, the disease or illness is dementia, Alzheimer's disease, Parkinson's disease or spinal cord injury. In some embodiments, the disease is associated with viral or bacterial infection, such as sepsis, hepatitis or COVID-19. In some embodiments, the disease or condition is associated with aging and / or chronic tissue damage, such as diabetes, fibrosis, cancer, neurodegeneration, arthritis, sarcopenia. In some embodiments, the immune cell population prepared according to the methods described herein is used to destroy or inhibit senescent cells, such as for senotherapy, senolysis, senolytics, or senolytics.

[0052] In another aspect, the present technology relates to a method for rejuvenating immune cells, the method comprising: introducing mRNA encoding one or more reprogramming factors into immune cells for expressing one or more reprogramming factors, thereby producing immune cells expressing one or more reprogramming factors to obtain rejuvenated immune cells. In some embodiments, the immune cells are lymphocytes or granulocytes. In other embodiments, the lymphocytes are T cells, B cells or natural killer (NK) cells. In some embodiments, the lymphocytes are tumor infiltrating lymphocytes. In other embodiments, the lymphocytes are T cells, such as cytotoxic T cells (CD8+), helper T cells (CD4+), inhibitory or regulatory T cells (Treg), memory T cells or natural killer T cells (NKT cells). In some embodiments, helper T cells are Th1, Th2, Th17, Th9 or TfhT cells. In other embodiments, helper T cells are Th1, Th2, Th17, Th9 or Tfh T cells. In other embodiments, T cells are initial T cells (Tn or TN) or stem cell memory T cells (Tscm or T) that have not experienced antigens. SCM ), central memory T cells (Tcm or T CM ), effector memory T cells (Tem or T EM ), effector T cells (Teff, T EFF or T E ), exhausted T cell precursors (Tpex or T PEX ) or depleted T cells (Tex or T EX ). In a further embodiment, the T cell has a phenotypic marker for its specific subtype (e.g., Tscm, Tcm, Tem, Teff) as shown in Table 2. In other embodiments, the T cell is a memory T cell, such as a central memory T cell, an effector memory T cell, a tissue-resident memory T cell, or a virtual memory T cell. In other embodiments, the T cell is an inhibitory or regulatory T cell, which is a FOXP3+T cell or a FOXP3-T cell. In other embodiments, the immune cell is a granulocyte, such as a neutrophil, an eosinophil, a basophil, or a mast cell. In some embodiments, the immune cell is a monocyte, a macrophage, a microglia, or a dendritic cell. In some embodiments, the immune cell is a lymphocyte, such as a B cell, including a memory B cell or a plasma cell. In some embodiments, the immune cell is a natural immune cell. In some embodiments, the immune cell is an engineered immune cell. In some embodiments, the engineered immune cell is engineered in parallel or serially with the rejuvenation method so that rejuvenation occurs before, at the same time, or after engineering. In some embodiments, immune cells are engineered to express a chimeric antigen receptor.

[0053] In some embodiments, the method for rejuvenating immune cells includes introducing mRNA encoding one or more reprogramming factors, which can rejuvenate cells without causing loss of identity or dedifferentiation. In some embodiments, the method for rejuvenating immune cells includes introducing mRNA encoding one or more reprogramming factors, which are selected from the group consisting of Oct, Sox, Klf, Myc, Lin, NANOG or GLIS1, including but not limited to Oct4, Sox2, Klf4, cMyc, Lin28, NANOG and GLIS1. In other embodiments, the method for rejuvenating immune cells includes introducing mRNA encoding Oct4, Sox2, Klf4, cMyc, Lin28 and NANOG. In other embodiments, the method for rejuvenating immune cells includes introducing mRNA encoding Oct4, Sox2, Klf4, GLIS1, Lin28 and NANOG. In other embodiments, the method for rejuvenating immune cells includes introducing mRNA encoding Oct4, Sox2, Klf4, GLIS1, Lin28 and NANOG. In other embodiments, the method for rejuvenating immune cells includes introducing mRNA encoding Oct4, Sox2, Klf4 and cMyc. In other embodiments, the method for rejuvenating immune cells includes introducing mRNA encoding Oct4, Sox2, Klf4 and GLIS1. In other embodiments, the method for rejuvenating immune cells includes introducing mRNA encoding Oct4, Sox2, Lin28 and NANOG. In other embodiments, the method for rejuvenating immune cells includes introducing mRNA encoding Oct4, Sox2 and Klf4. In certain embodiments, one or more reprogramming factors are expressed by a single mRNA molecule. In certain embodiments, the single mRNA molecule is a polycistronic. In other embodiments, two or more reprogramming factors are used, and the reprogramming factors are expressed by more than one mRNA molecule, such as wherein each reprogramming factor is expressed by its own mRNA molecule, or wherein two or more reprogramming factors are expressed by the same mRNA molecule.

[0054] In certain embodiments, the mRNA molecule introduced includes the means for controlling the expression duration of at least one reprogramming factor, such as the switch expressed, self-amplification system or control mRNA or the mechanism of the half-life of the protein transcribed from mRNA. In certain embodiments, this control of expression duration allows the frequency of application of mRNA to be lower than the frequency required in the absence of this control of expression duration. In certain embodiments, this control of expression duration allows each dosing interval to apply mRNA once instead of each dosing interval to apply multiple times, such as each dosing interval once instead of at least once a day in the dosing interval.

[0055] In some embodiments, introducing mRNA includes providing a composition comprising mRNA, wherein the composition comprises an excipient for transfection. In some embodiments, the composition comprises a lipid and the mRNA is associated with the lipid. In some embodiments, the lipid is those described in Figures 1 to 18. In some embodiments, the lipid is those in Billingsley et al., NanoLett.2020 (20, 3, 1578–1589), which are incorporated herein by reference. In other embodiments, the composition comprises a polymer and the mRNA is associated with the polymer. In some embodiments, the polymer is a releasable transporter that changes charge. In some embodiments, the charge-modified releasable transporter contains a mixture of lipophilic blocks in block (b) or stat (stat) structures, i.e., such as those described in McKinlay et al. 2017 (PNAS 2017 Jan 24 114 (4) E448-E456), McKinlay et al. 2018 (PNAS 2018 Jun 26 115 (26) E5859-E5866), or Haabeth et al. 2018 (PNAS 2018 Sep 25 115 (39) E9153-E9161). In some embodiments, the polymer or lipid forms a nanoparticle, complex, or other nanostructure. In other embodiments, the composition comprises both a polymer and a lipid, and the mRNA is associated with the polymer and / or lipid. In some embodiments, compared with using different mRNA delivery mechanisms, lipids or polymers are used to deliver mRNA, such as in lipid nanoparticles, polymer nanoparticles or hybrid lipid-polymer nanoparticles, resulting in enhanced rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, antipathogenic effects, anticancer effects or anti-inflammatory effects in the immune cells exposed. In some embodiments, compared with using different mRNA delivery mechanisms, due to the lower toxicity and / or lower physiological effects on cells when compared with different delivery mechanisms, lipids or polymers are used to deliver mRNA resulting in enhanced rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, antipathogenic effects, anticancer effects or anti-inflammatory effects in the immune cells exposed. In some embodiments, different delivery mechanisms are electroporation, that is, compared with using electroporation, lipids or polymers (such as lipid or polymer nanoparticles) are used to deliver mRNA resulting in enhanced rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, antipathogenic effects, anticancer effects or anti-inflammatory effects in the immune cells exposed. This improvement compared with electroporation can be caused by the toxicity of the reduction of cells compared with electroporation or the physiological effects of the reduction.

[0056] In some embodiments, mRNA is introduced to realize transfection of mRNA encoding one or more reprogramming factors into immune cells. In some embodiments, mRNA is introduced to realize transfection of mRNA encoding one or more reprogramming factors into T cells in preference to other immune cells or other cell types. In some embodiments, mRNA is introduced in vitro, in vivo or ex vivo. In some embodiments, mRNA is introduced using an ex vivo technique selected from microinjection, electroporation, biolistics, optical transfection, microfluid extrusion, chemical transfection or biotransfection. In other embodiments, mRNA is introduced ex vivo, and the method further includes transplanting immune cells into the subject after the introduction. In some embodiments, mRNA is introduced in vivo, and the introduction realizes transfection of mRNA encoding one or more reprogramming factors into immune cells for expressing one or more reprogramming factors in cells. In some embodiments, introduction is performed in vivo and before, simultaneously or after the administration of bispecific antibodies. In some embodiments, such engineering of immune cells is performed ex vivo, i.e., in the manufacture of cell therapy products (such as autologous or allogeneic chimeric antigen receptors (CAR)-T, CAR-NK, CAR-M or CAR-NKT cells). In some embodiments, such chimeric antigen receptors target CD19, CD20, CD22, CD30, CD33, CD123, FLT3, BCMA, GD2, HER2, MUC1, B7-H3, IL13Ra2, TAG72, MUC16, BCMA, or at least one of any other antigens suitable for immunotherapy. In some embodiments, such engineering includes engineering to allow immune cells to express other proteins or peptides, such as growth factors and cytokines. In some embodiments, the cytokines include IL-7 and / or IL-15. In some embodiments, such engineering of immune cells is performed ex vivo, for example, in the manufacture of cell therapy products such as autologous or allogeneic chimeric antigen receptors (CAR)-T, CAR-NK, CAR-M or CAR-NKT cells. In some embodiments, the CAR-T cells provided herein target at least one of CD19, CD20, CD22, HER2, MUC1, CD30, CD33, CD123, FLT3, B7-H3, IL13Ra2, GD2, TAG72, MUC16, or BCMA via a chimeric antigen receptor. In some embodiments, the CAR-T cells provided herein target at least one of CD19 or BCMA via a chimeric antigen receptor. In some embodiments, the CAR-T cells provided herein target CD19 via a chimeric antigen receptor. In some embodiments, the CAR-T cells provided herein target BCMA via a chimeric antigen receptor.In some embodiments, the CAR-NK cells provided herein target CD19, FLT3, CD20, CD38, CD138, BCMS, CS1, CD3, CD5, CD7, NKG2D, HER2, EGFR, EpCAM, TF, B7-H6, HLA-G, CD24, CD44, CD133, mesothelin, or at least one of αFR through a chimeric antigen receptor. In some embodiments, the CAR-M cells provided herein target at least one of CD19, HER2, CD22, or ALK19 through a chimeric antigen receptor. In some embodiments, the CAR-NK cells provided herein are engineered to express IL-2 and / or IL-15. In some embodiments, the CAR-NKT cells provided herein target GD2 through a chimeric antigen receptor and are engineered to express IL-15. In such embodiments, the immune cell rejuvenation method described herein is performed in vitro during or after the manufacture of a cell therapy product. In other embodiments, such engineering of immune cells is performed in vitro, for example, in the so-called "in situ" production of CAR engineered cells. In such embodiments, the mRNA encoding CAR or other cell engineering molecules is injected into the subject or patient, for example, for the patient's immune cells (such as T cells, NK cells, macrophages, tumor infiltrating lymphocytes, dendritic cells and / or NKT cells) "in situ" CAR engineering, that is, in the patient, without removing cells for ex vivo transfection. In such embodiments, the immune cell rejuvenation method described herein is also carried out in vivo, wherein the mRNA encoding one or more reprogramming factors is injected into the patient before, at the same time or after the mRNA encoding CAR or other cell engineering molecules. In some embodiments, lipids and / or polymer nanoparticles are used to deliver mRNA encoding one or more reprogramming factors in vivo. In some embodiments, lipids and / or polymer nanoparticles are engineered for in vivo targeted delivery to immune cells, such as T cells, NK cells, macrophages, tumor infiltrating cells, dendritic cells and / or NKT cells in vivo. In yet other embodiments, in vivo treatment is performed in the absence of any other in vivo cell engineering to enhance or restore the effectiveness of the immune system and treat diseases associated with immune dysfunction or disorder, such as improving the effectiveness of the immune system to fight cancer or infection, or reducing inflammation.

[0057] In some embodiments, mRNA or at least one reprogramming factor expressed by transfected mRNA is introduced into immune cells or expressed in immune cells, and the dosing interval of continuous 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 days is continued. In some embodiments, at least one mRNA administration is performed every day during the dosing interval. In some embodiments, mRNA administration is performed during the dosing interval at a frequency lower than once a day, for example, once every two days, once every three days, once every four days, once every x days, where x is a number of 4 to 25. Therefore, in such embodiments, for example, in a 5-day dosing interval, mRNA is administered once every 5 days, i.e., once in the total treatment period of 5 days, and twice a day in a 5-day dosing interval means that mRNA is administered 10 times in the interval, i.e., 10 times in 5 days. In some embodiments, the method of the present technology includes introducing immune cells into mRNA or at least one reprogramming factor expressed by transfected mRNA, or introducing or expressing at least one reprogramming factor from transfected mRNA, for no more than 21, 18, 14, 10, 7 or 5 consecutive days. In some embodiments, the method of the present technology includes introducing immune cells into mRNA or at least one reprogramming factor expressed by transfected mRNA, or introducing or expressing at least one reprogramming factor from transfected mRNA, for no more than 18 consecutive days. In some embodiments, the method of the present technology includes introducing immune cells into mRNA or at least one reprogramming factor expressed by transfected mRNA, or introducing or expressing at least one reprogramming factor from transfected mRNA, for no more than 14 consecutive days. In some embodiments, the method of the present technology includes introducing immune cells into mRNA or at least one reprogramming factor expressed by transfected mRNA, or introducing or expressing at least one reprogramming factor from transfected mRNA, for no more than 10 consecutive days. In an embodiment, the method of the present technology includes introducing an immune cell into mRNA or at least one reprogramming factor expressed by a transfected mRNA, or introducing or expressing at least one reprogramming factor from a transfected mRNA, for no more than 7 consecutive days. In an embodiment, the method of the present technology includes introducing an immune cell into mRNA or at least one reprogramming factor expressed by a transfected mRNA, or introducing or expressing at least one reprogramming factor from a transfected mRNA, for no more than 5 consecutive days. In other embodiments, the exposure includes interrupting the introduction and repeating the introduction after the interruption.In some embodiments, the introduction includes introducing immune cells into mRNA or at least one reprogramming factor expressed by transfected mRNA, or introducing or expressing at least one reprogramming factor from transfected mRNA, for about 2 to 5 days, about 5 to 7 days, about 7 to 10 days, about 10 to 12 days, about 12 to 14 days, about 14 to 17 days, about 17 to 19 days, or about 19 to 21 days, and in some embodiments, further including interrupting the introduction and repeating the introduction after the interruption. In some embodiments, the repetition is performed any number of times, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, or up to 20 times, or up to 30 times. In some embodiments, the repetition is performed at any time after the interruption, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, up to 20 days, up to 30 days, up to 3 months, up to 6 months or up to 1 year after the interruption. The repetition can last for any duration, for example until the disease is successfully treated or cured, or throughout the life of the subject or patient. An exposure cycle is considered to be a dosing interval, so that, for example, a sequence of introduction-interruption-repeat introduction includes two dosing intervals. In some embodiments, exposure to mRNA is performed once, twice, three times, four times, five times or six times in a six-day interval. In some embodiments, exposure to mRNA is performed on the first or second day in a six-day interval. In some embodiments, exposure to mRNA is performed on the first or second day in a six-day interval, and then on the fourth or fifth day. In some embodiments, exposure to mRNA is performed on the first or second day in a six-day interval, and then on the third or fourth day, and then on the fifth or sixth day. In some embodiments, exposure to mRNA is performed every day in a six-day interval. In some embodiments, the exposure interval occurs immediately after immune cell activation. In some embodiments, the exposure interval occurs one, two, three, four, five, six, seven, eight, nine or ten days after immune cell activation. In some embodiments, immune cells are activated with CD3 and / or CD28. In some embodiments, activation is performed for one, two, three, four, five, six or seven days.

[0058] In an embodiment, at least one reprogramming factor is expressed by the mRNA of the transfection in the cell, and the cell is exposed to at least one reprogramming factor expressed by the mRNA of the transfection, and it continues for no more than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day, or less than 1 day. In an embodiment, at least one reprogramming factor is expressed by the mRNA of the transfection in the cell, or the cell is exposed to at least one reprogramming factor expressed by the mRNA of the transfection, and it continues for no more than about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day, or less than one day. In an embodiment, at least one reprogramming factor is expressed by the mRNA of the transfection in the cell, or the cell is exposed to at least one reprogramming factor expressed by the mRNA of the transfection, and it continues for at least about 2 days and no more than about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days. In an embodiment, at least one reprogramming factor is expressed by the mRNA of the transfection in the cell, or the cell is exposed to at least one reprogramming factor expressed by the mRNA of the transfection, and it continues for no more than about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day, or less than one day.In an embodiment, at least one reprogramming factor is expressed by the mRNA of the transfection in the cell, or the cell is exposed to at least one reprogramming factor expressed by the mRNA of the transfection, and it continues for at least about 2 days and no more than about 10, 9, 8, 7, 6, 5, 4, 3 or 2 days.In an embodiment, at least one reprogramming factor is expressed by the mRNA of the transfection in the cell, or the cell is exposed to at least one reprogramming factor expressed by the mRNA of the transfection, and it continues for no more than about 7, 6, 5, 4, 3, 2 or 1 day, or less than one day.In an embodiment, at least one reprogramming factor is expressed by the mRNA of the transfection in the cell, or the cell is exposed to at least one reprogramming factor expressed by the mRNA of the transfection, and it continues for at least about 2 days and no more than about 7, 6, 5, 4, 3 or 2 days. In an embodiment, at least one reprogramming factor is expressed by transfected mRNA in the cell, or the cell is exposed to at least one reprogramming factor expressed by transfected mRNA, for no more than about 5, 4, 3, 2 or 1 days, or less than one day. In an embodiment, at least one reprogramming factor is expressed by transfected mRNA in the cell, or the cell is exposed to at least one reprogramming factor expressed by transfected mRNA, for at least about 2 days and no more than about 5, 4, 3 or 2 days. In an embodiment, the rejuvenated cells have a phenotype or activity spectrum similar to that of young cells. The phenotype or activity spectrum includes one or more of transcriptome profiles, gene expression of one or more nuclear and / or epigenetic markers, proteolytic activity, mitochondrial health and function, SASP cytokine expression and methylation landscape.

[0059] As described herein, cells can be rejuvenated by transient reprogramming with mRNA encoding one or more cell reprogramming factors transfected into cells. In certain embodiments, transient reprogramming is completed by transfecting cells with non-integrative mRNA for no more than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day, or less than 1 day. In certain embodiments, transient reprogramming is completed by transfecting cells with non-integrative mRNA for no more than about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day, or less than 1 day. In certain embodiments, transient reprogramming is completed by transfecting cells with non-integrative mRNA for no more than about 9, 8, 7, 6, 5, 4, 3, 2 or 1 day, or less than 1 day. In an embodiment, transient reprogramming is accomplished by transfecting cells with non-integrative mRNA for no more than about 6, 5, 4, 3, 2 or 1 days, or less than 1 day. In some embodiments, transient reprogramming is accomplished by expressing at least one reprogramming factor from a non-integrative mRNA transfected in a cell, or exposing the cell to at least one reprogramming factor expressed from a non-integrative mRNA transfected in a cell for no more than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day, or less than 1 day. In some embodiments, transient reprogramming is accomplished by expressing at least one reprogramming factor from a non-integrative mRNA transfected in a cell, or exposing the cell to at least one reprogramming factor expressed from a non-integrative mRNA transfected in a cell for at least 2 days and no more than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days. In some embodiments, transient reprogramming is performed by expressing at least one reprogramming factor from a non-integrating mRNA transfected in a cell, or exposing the cell to at least one reprogramming factor expressed from a non-integrating mRNA transfected in a cell for no more than about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day. In some embodiments, transient reprogramming is performed by expressing at least one reprogramming factor from a non-integrating mRNA transfected in a cell, or exposing the cell to at least one reprogramming factor expressed from a non-integrating mRNA transfected in a cell for at least 2 days and no more than about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. In some embodiments, transient reprogramming is performed by expressing at least one reprogramming factor from a non-integrating mRNA transfected in a cell, or exposing the cell to at least one reprogramming factor expressed from a non-integrating mRNA transfected in a cell for no more than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day.In some embodiments, transient reprogramming is performed by expressing at least one reprogramming factor from a non-integrating mRNA transfected in a cell, or exposing the cell to at least one reprogramming factor expressed from a non-integrating mRNA transfected in a cell for at least 2 days and no more than about 10, 9, 8, 7, 6, 5, 4, 3 or 2 days. In an embodiment, transient reprogramming is performed by expressing at least one reprogramming factor from a non-integrating mRNA transfected in a cell, or exposing the cell to at least one reprogramming factor expressed from a non-integrating mRNA transfected in a cell for no more than about 7, 6, 5, 4, 3, 2 or 1 day, or less than 1 day. In an embodiment, transient reprogramming is performed by expressing at least one reprogramming factor from a non-integrating mRNA transfected in a cell, or exposing the cell to at least one reprogramming factor expressed from a non-integrating mRNA transfected in a cell for at least 2 days and no more than about 7, 6, 5, 4, 3 or 2 days. In an embodiment, transient reprogramming is performed by expressing at least one reprogramming factor from a non-integrating mRNA transfected in a cell, or exposing the cell to at least one reprogramming factor expressed from a non-integrating mRNA transfected in a cell for no more than about 5, 4, 3, 2 or 1 day, or less than 1 day. In an embodiment, transient reprogramming is performed by expressing at least one reprogramming factor from a non-integrating mRNA transfected in a cell, or exposing the cell to at least one reprogramming factor expressed from a non-integrating mRNA transfected in a cell for at least 2 days and no more than about 5, 4, 3 or 2 days. In an embodiment, transient reprogramming of cells eliminates various signs of aging while avoiding complete dedifferentiation of cells into stem cells.

[0060] In some embodiments, exposure includes providing a composition comprising mRNA, and administering mRNA 1, 2, 3, 4, 5 or 6 times to immune cells within a time period of 1, 2, 3, 4, 5 or 6 days. For example, mRNA can be administered once on the first or second day of a five-day or six-day cycle, or it can be administered once on the first day of a five-day or six-day cycle and once on the third day, or it can be administered once within a one-day cycle. In some embodiments, mRNA is administered after the immune cell activation step. In some embodiments, the immune cell activation step includes activating immune cells for 1, 2 or 3 days. In some embodiments, the immune cell activation step includes activating immune cells using at least one of CD3, CD28 and IL-2. In some embodiments, immune cells are activated with CD3 and CD28. In some embodiments, the mRNA administration phase occurs immediately after the immune cell activation step. In some embodiments, the mRNA administration phase occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days after the immune cell activation step. In some embodiments, the administration of the mRNA encoding the reprogramming factor reverses the immune cell exhaustion caused by the immune cell activation step. In some embodiments, the administration of mRNA encoding reprogramming factors reverses immune cell exhaustion in immune cells from elderly patients or donors. In some embodiments, mRNA administration is performed during the manufacturing process of preparing immune cells (e.g., CAR-T, CAR-M or CAR-NK cells) for transplantation.

[0061] In some embodiments, methods and compositions related to rejuvenating immune cells such as T cells include exposing immune cells to messenger RNA (mRNA) encoding one or more reprogramming factors, wherein the reprogramming factors encoding mRNA encode polypeptides encoded by polynucleotides, which polynucleotides have at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID No: 1-14 (Table 1). In some embodiments, methods and compositions related to rejuvenating immune cells such as T cells include exposing immune cells to messenger RNA (mRNA) encoding one or more reprogramming factors, wherein the reprogramming factors encoding mRNA encode polypeptides encoded by polynucleotides, which polynucleotides have about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID No: 1-14 (Table 1).

[0062] In some embodiments, methods and compositions related to rejuvenating immune cells such as T cells include exposing immune cells to messenger RNA (mRNA) encoding one or more reprogramming factors, wherein the reprogramming factors encoding mRNA encode polypeptides encoded by polynucleotides, which polynucleotides have at least about 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID No: 1-14 (Table 1). In some embodiments, methods and compositions related to rejuvenating immune cells such as T cells include exposing immune cells to messenger RNA (mRNA) encoding one or more reprogramming factors, wherein the reprogramming factors encoding mRNA encode polypeptides encoded by polynucleotides, which polynucleotides have about 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID No: 1-14 (Table 1).

[0063] Table 1

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075] Table 2 - Exemplary phenotypic markers of memory T cells

[0076] <![CDATA[T N ]]> <![CDATA[T SCM ]]> <![CDATA[T CM ]]> <![CDATA[T EM ]]> <![CDATA[T EFF ]]> CD45RA + + - - + CD45RO - - + + - CCR7 + + + - - CD62L + + + - - CD27 + + + + / - - CD28 + + + + / - - IL-7Rα + + + + / - - CXCR3 - + + - - CD95 - + + + + CD11a - + + + + IL-2Rβ - + + + + CD57 - - - + / - + CD58 - + + + +

[0077]

[0078] In another aspect, a method for inhibiting, preventing and / or reversing exhaustion of engineered immune cells is provided. In some embodiments, the method comprises exposing the immune cells to mRNA encoding one or more reprogramming factors, wherein the exposure achieves expression of the one or more reprogramming factors in the immune cells to inhibit, prevent and / or reverse exhaustion of the immune cells while retaining their identity.

[0079] In another aspect, a method for reducing T cell exhaustion occurring during the manufacture of CAR T cells is provided. In some embodiments, the method includes activating T cells by exposing T cells to CD3 and / or CD28; transfecting the activated T cells with mRNA encoding one or more reprogramming factors; wherein each transfection uses 1ng to 500ug of mRNA per million T cells, wherein the transfection achieves the expression of one or more reprogramming factors in the T cells to increase their cytotoxicity or proliferation capacity compared to untreated or control T cells. In certain embodiments, the method uses 1ng to 400ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 300ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 250ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 200ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 150ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 100ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 50ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 25ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 20ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 15ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 10ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 9ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 8ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 7ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 6ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 5ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 4ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 3ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 2ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ng to 1ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 10ng to 10ug of mRNA per million T cells per transfection.In certain embodiments, the method uses 50ng to 10ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 100ng to 10ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 150ng to 10ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 200ng to 10ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 300ng to 10ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 350ng to 10ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 400ng to 10ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 500ng to 10ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 600ng to 10ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 750ng to 10ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 900ng to 10ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ug to 10ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 2ug to 10ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 2ug to 6ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ug to 9ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ug to 8ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ug to 7ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ug to 6ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ug to 5ug of mRNA per million T cells per transfection. In certain embodiments, the method uses 1ug to 4ug of mRNA per million T cells for each transfection. In certain embodiments, the method uses 1ug to 3ug of mRNA per million T cells for each transfection. In certain embodiments, the method uses 1ug to 2ug of mRNA per million T cells for each transfection. In certain embodiments, mRNA encoding one or more reprogramming factors is transfected into T cells at least once a day for about 2 to 5 consecutive days. In certain embodiments, mRNA encoding one or more reprogramming factors is transfected into T cells at least once a day for 5 consecutive days. In certain embodiments, mRNA encoding one or more reprogramming factors is transfected into T cells at least once a day for 4 consecutive days.In certain embodiments, mRNA encoding one or more reprogramming factors is transfected into T cells, at least once a day, for 3 consecutive days. In certain embodiments, mRNA encoding one or more reprogramming factors is transfected into T cells, at least once a day, for 2 consecutive days. In certain embodiments, mRNA encoding one or more reprogramming factors is transfected into T cells, at least once a day, for 1 day. In certain embodiments, mRNA encoding one or more reprogramming factors is transfected into T cells, once a day, for 5 consecutive days. In certain embodiments, mRNA encoding one or more reprogramming factors is transfected into T cells, once a day, for 4 consecutive days. In certain embodiments, mRNA encoding one or more reprogramming factors is transfected into T cells, once a day, for 3 consecutive days. In certain embodiments, mRNA encoding one or more reprogramming factors is transfected into T cells, once a day, for 2 consecutive days.

[0080] In another aspect, a method for reducing T cell exhaustion occurring during the manufacture of CAR T cells is provided. In some embodiments, the method includes activating T cells by exposing T cells to CD3 and / or CD28; transfecting the activated T cells with mRNA encoding one or more reprogramming factors; wherein each transfection uses 1ng to 500ug of mRNA per million T cells, wherein the transfection achieves the expression of one or more reprogramming factors in the T cells to increase their cytotoxicity or proliferation capacity compared to untreated or control T cells. In certain embodiments, mRNA encoding one or more reprogramming factors is transfected into T cells once a day for 1 day. In certain further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells is about 1ng to 100ug, and the mRNA encodes reprogramming factors OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG. In certain other further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells is about 50ug, and the mRNA encodes reprogramming factors OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG. In additional further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells is about 25ug, and the mRNA encodes reprogramming factors OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG. In some further embodiments, the mRNA encodes reprogramming factors OCT4, SOX2, KLF4, c-MYC, LIN28, NANOG and Gls1

[0081] In another aspect, a method for reducing T cell exhaustion occurring during the manufacture of CAR T cells is provided. In some embodiments, the method includes activating T cells by exposing T cells to CD3 and / or CD28; transfecting the activated T cells with mRNA encoding one or more reprogramming factors; wherein each transfection uses 1ng to 500ug of mRNA per million T cells, wherein the transfection achieves the expression of one or more reprogramming factors in the T cells to increase their cytotoxicity or proliferation capacity compared to untreated or control T cells. In certain embodiments, the mRNA encoding one or more reprogramming factors is transfected into T cells twice a day for about 2 to 5 consecutive days. In certain further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells: (a) for the first transfection in twice a day, about 1ng to 100ug mRNA per million T cells; and (b) for the second transfection in twice a day, about 1ng to 100ug mRNA per million T cells. In certain other further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells: (a) for the first transfection in two transfections per day, about 100ng to 10ug mRNA per million T cells; and (b) for the second transfection in two transfections per day, about 100ng to 10ug mRNA per million T cells. In certain other further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells: (a) for the first transfection in two transfections per day, about 10ng to 10ug mRNA per million T cells; (b) for the second transfection in two transfections per day, about 10ng to 8ug mRNA per million T cells; and the mRNA encoding OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG for the first transfection in two transfections per day; and the mRNA encoding OCT4, c-MYC and NANOG for the second transfection in two transfections per day.

[0082] In another aspect, a method for reducing T cell exhaustion occurring during the manufacture of CAR T cells is provided. In some embodiments, the method includes activating T cells by exposing T cells to CD3 and / or CD28; transfecting the activated T cells with mRNA encoding one or more reprogramming factors; wherein each transfection uses 1ng to 500ug of mRNA per million T cells, wherein the transfection achieves the expression of one or more reprogramming factors in the T cells to increase their cytotoxicity or proliferation capacity compared to untreated or control T cells. In certain embodiments, mRNA encoding one or more reprogramming factors is transfected into T cells twice a day for 5 consecutive days. In certain further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells: (a) for the first transfection in twice a day, about 1ng to 100ug mRNA per million T cells; and (b) for the second transfection in twice a day, about 1ng to 100ug mRNA per million T cells. In certain other further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells: (a) for the first transfection in two transfections per day, about 100ng to 10ug mRNA per million T cells; and (b) for the second transfection in two transfections per day, about 100ng to 10ug mRNA per million T cells. In certain other further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells: (a) for the first transfection in two transfections per day, about 10ng to 10ug mRNA per million T cells; (b) for the second transfection in two transfections per day, about 10ng to 8ug mRNA per million T cells; and the mRNA encoding OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG for the first transfection in two transfections per day; and the mRNA encoding OCT4, c-MYC and NANOG for the second transfection in two transfections per day.

[0083] In another aspect, a method for reducing T cell exhaustion occurring during the manufacture of CAR T cells is provided. In some embodiments, the method includes activating T cells by exposing T cells to CD3 and / or CD28; transfecting the activated T cells with mRNA encoding one or more reprogramming factors; wherein each transfection uses 1ng to 500ug of mRNA per million T cells, wherein the transfection achieves the expression of one or more reprogramming factors in the T cells to increase their cytotoxicity or proliferation capacity compared to untreated or control T cells. In certain embodiments, mRNA encoding one or more reprogramming factors is transfected into T cells twice a day for 4 consecutive days. In certain further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells: (a) for the first transfection in twice a day, about 1ng to 100ug mRNA per million T cells; and (b) for the second transfection in twice a day, about 1ng to 100ug mRNA per million T cells. In certain other further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells: (a) for the first transfection in two transfections per day, about 100ng to 10ug mRNA per million T cells; and (b) for the second transfection in two transfections per day, about 100ng to 10ug mRNA per million T cells. In certain other further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells: (a) for the first transfection in two transfections per day, about 10ng to 10ug mRNA per million T cells; (b) for the second transfection in two transfections per day, about 10ng to 8ug mRNA per million T cells; and the mRNA encoding OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG for the first transfection in two transfections per day; and the mRNA encoding OCT4, c-MYC and NANOG for the second transfection in two transfections per day.

[0084] In another aspect, a method for reducing T cell exhaustion occurring during the manufacture of CAR T cells is provided. In some embodiments, the method includes activating T cells by exposing T cells to CD3 and / or CD28; transfecting the activated T cells with mRNA encoding one or more reprogramming factors; wherein each transfection uses 1ng to 500ug of mRNA per million T cells, wherein the transfection achieves the expression of one or more reprogramming factors in the T cells to increase their cytotoxicity or proliferation capacity compared to untreated or control T cells. In certain embodiments, mRNA encoding one or more reprogramming factors is transfected into T cells twice a day for 3 consecutive days. In certain further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells: (a) for the first transfection in twice a day, about 1ng to 100ug mRNA per million T cells; and (b) for the second transfection in twice a day, about 1ng to 100ug mRNA per million T cells. In certain other further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells: (a) for the first transfection in two transfections per day, about 100ng to 10ug mRNA per million T cells; and (b) for the second transfection in two transfections per day, about 100ng to 10ug mRNA per million T cells. In certain other further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells: (a) for the first transfection in two transfections per day, about 10ng to 10ug mRNA per million T cells; (b) for the second transfection in two transfections per day, about 10ng to 8ug mRNA per million T cells; and the mRNA encoding OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG for the first transfection in two transfections per day; and the mRNA encoding OCT4, c-MYC and NANOG for the second transfection in two transfections per day. In certain other further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells is: (a) about 10 ng to 10 ug mRNA per million T cells for the first transfection of two transfections per day; and (b) about 10 ng to 8 ug mRNA per million T cells for the second transfection of two transfections per day; and the mRNA used for the first transfection of two transfections per day encodes OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG; and the mRNA used for the second transfection of two transfections per day encodes OCT4, c-MYC and NANOG.

[0085] In another aspect, a method for reducing T cell exhaustion occurring during the manufacture of CAR T cells is provided. In some embodiments, the method includes activating T cells by exposing T cells to CD3 and / or CD28; transfecting the activated T cells with mRNA encoding one or more reprogramming factors; wherein each transfection uses 1ng to 500ug of mRNA per million T cells, wherein the transfection achieves the expression of one or more reprogramming factors in the T cells to increase their cytotoxicity or proliferation capacity compared to untreated or control T cells. In certain embodiments, mRNA encoding one or more reprogramming factors is transfected into T cells twice a day for 2 consecutive days. In certain further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells: (a) for the first transfection in twice a day, about 1ng to 100ug mRNA per million T cells; and (b) for the second transfection in twice a day, about 1ng to 100ug mRNA per million T cells. In certain other further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells: (a) for the first transfection in two transfections per day, about 100ng to 10ug mRNA per million T cells; and (b) for the second transfection in two transfections per day, about 100ng to 10ug mRNA per million T cells. In certain other further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells: (a) for the first transfection in two transfections per day, about 10ng to 10ug mRNA per million T cells; (b) for the second transfection in two transfections per day, about 10ng to 8ug mRNA per million T cells; and the mRNA encoding OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG for the first transfection in two transfections per day; and the mRNA encoding OCT4, c-MYC and NANOG for the second transfection in two transfections per day. In certain other further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells is: (a) about 1 ng to 100 ug mRNA per million T cells for the first transfection of two transfections per day; and (b) about 1 ng to 100 ug mRNA per million T cells for the second transfection of two transfections per day; and the mRNA used for the first transfection of the two transfections per day encodes OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG; and the mRNA used for the second transfection of the two transfections per day encodes OCT4, c-MYC and NANOG.

[0086] In another aspect, a method for reducing T cell exhaustion occurring during the manufacture of CAR T cells is provided. In some embodiments, the method includes activating T cells by exposing T cells to CD3 and / or CD28; transfecting the activated T cells with mRNA encoding one or more reprogramming factors; wherein each transfection uses 1ng to 500ug of mRNA per million T cells, wherein the transfection achieves the expression of one or more reprogramming factors in the T cells to increase their cytotoxicity or proliferation capacity compared to untreated or control T cells. In certain embodiments, mRNA encoding one or more reprogramming factors is transfected into T cells twice a day for 1 day. In certain further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells: (a) for the first transfection in twice a day, about 1ng to 100ug mRNA per million T cells; and (b) for the second transfection in twice a day, about 1ng to 100ug mRNA per million T cells. In certain other further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells: (a) for the first transfection in two transfections per day, about 100ng to 10ug mRNA per million T cells; and (b) for the second transfection in two transfections per day, about 100ng to 10ug mRNA per million T cells. In certain other further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells: (a) for the first transfection in two transfections per day, about 10ng to 10ug mRNA per million T cells; (b) for the second transfection in two transfections per day, about 10ng to 8ug mRNA per million T cells; and the mRNA encoding OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG for the first transfection in two transfections per day; and the mRNA encoding OCT4, c-MYC and NANOG for the second transfection in two transfections per day. In certain other further embodiments, the amount of mRNA encoding one or more reprogramming factors used per million T cells is: (a) about 1 ng to 100 ug mRNA per million T cells for the first transfection of two transfections per day; and (b) about 1 ng to 100 ug mRNA per million T cells for the second transfection of two transfections per day; and the mRNA used for the first transfection of the two transfections per day encodes OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG; and the mRNA used for the second transfection of the two transfections per day encodes OCT4, c-MYC and NANOG.

[0087] In another aspect, a method for reducing T cell exhaustion occurring during the manufacture of CAR T cells is provided. In certain embodiments, the method includes activating T cells by exposing T cells to CD3 and / or CD28; transfecting the activated T cells with mRNA encoding one or more reprogramming factors; wherein each transfection uses 1ng to 500ug of mRNA per million T cells, wherein the transfection achieves the expression of one or more reprogramming factors in the T cells to increase the percentage of Tcm or Tscm cells compared to untreated or control T cells. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is less than 200%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is less than 150%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is less than 125%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is less than 100%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is less than 80%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is less than 60%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is less than 40%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is less than 30%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is less than 20%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is less than 10%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between about 10% and 40%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between about 10% and 35%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between about 5% and 40%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between about 5% and 35%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between about 5% and 30%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between about 5% and 25%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between about 10% and 40%.

[0088] In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between about 10% and 50%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between about 5% and 50%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between about 5% and 65. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between about 5% and 75%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between 1% and 50%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between 1% and 40%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between 1% and 25%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between 20% and 80%. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between about 2-fold and 10-fold. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between about 2-fold and 8-fold. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between about 2-fold and 6-fold. In certain embodiments, the increase in the percentage of Tcm or Tscm cells is between about 1-fold and 4-fold. In certain further embodiments, the mRNA molecule comprises a self-amplifying RNA exhibiting a half-life of 2 days or more. In certain other further embodiments, the mRNA molecule is a self-amplifying RNA exhibiting a half-life of 2 days or more. In certain other further embodiments, the mRNA molecule comprises a trans-amplifying RNA exhibiting a half-life of 2 days or more. In certain other further embodiments, the mRNA molecule is a trans-amplifying RNA exhibiting a half-life of 2 days or more. In certain other further embodiments, the mRNA molecule comprises a trans- or self-amplifying RNA exhibiting a half-life of up to 10 days.

[0089] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by studying the following description.

[0090] Additional embodiments of the methods and compositions of the present invention, etc. will become apparent from the following description, examples, and claims. As can be understood from the foregoing and following descriptions, each and every feature described herein and each and every combination of two or more such features are included within the scope of the present disclosure, provided that the features included in such combinations are not mutually contradictory. In addition, any feature or combination of features may be expressly excluded from any embodiment of the present disclosure. Additional aspects and advantages of the present disclosure are set forth in the following description and claims, particularly when considered in conjunction with the accompanying examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] The patent or application file contains at least one drawing drawn in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0092] FIG. 1 depicts an ionizable lipid of formula (I) Figure 1A ) and its exemplary lipid structure ( Figure 1B and Figure 1C ), which can be used in some embodiments of the present technology, for example, for transfecting immune cells.

[0093] FIG2 depicts an ionizable lipid of formula (IA) Figure 2A ) and its exemplary lipid structure ( Figure 2B ), which can be used in some embodiments of the present technology, for example, for transfecting immune cells.

[0094] FIG3 depicts an ionizable lipid of formula (IB) Figure 3A ) and its exemplary lipid structure ( Figure 3B ), which can be used in some embodiments of the present technology, for example, for transfecting immune cells.

[0095] FIG4 depicts an ionizable lipid of formula (II) ( Figure 4A ) and its exemplary lipid structure ( Figure 4B and Figure 4C ), which can be used in some embodiments of the present technology, for example, for transfecting immune cells.

[0096] FIG5 depicts an ionizable lipid of formula (III) ( Figure 5A ) and its exemplary lipid structure ( Figure 5B and Figure 5C ), which can be used in some embodiments of the present technology, for example, for transfecting immune cells.

[0097] FIG6 depicts an ionizable lipid of formula (VI) Fig. 6A ) and its exemplary lipid structure ( Figure 6B and Figure 6C ), which can be used in some embodiments of the present technology, for example, for transfecting immune cells.

[0098] FIG. 7 depicts an ionizable lipid of formula (V) ( Fig. 7A ) and its exemplary lipid structure ( Figure 7B ), which can be used in some embodiments of the present technology, for example, for transfecting immune cells.

[0099] FIG8 depicts an ionizable lipid of formula (VI) Fig. 8A ) and its exemplary lipid structure ( Figure 8B and Figure 8C ), which can be used in some embodiments of the present technology, for example, for transfecting immune cells.

[0100] FIG. 9 depicts an ionizable lipid of formula (VII) Fig. 9A ) and its exemplary lipid structure ( Fig. 9B and Fig. 9C ), which can be used in some embodiments of the present technology, for example, for transfecting immune cells.

[0101] FIG. 10 depicts an ionizable lipid of formula (VIII) ( Fig. 10A ) and its exemplary lipid structure ( Fig. 10B ), which can be used in some embodiments of the present technology, for example, for transfecting immune cells.

[0102] FIG. 11 depicts an ionizable lipid of formula (IX) ( Fig.11A ) and its exemplary lipid structure ( Fig. 11B and Fig. 11C ), which can be used in some embodiments of the present technology, for example, for transfecting immune cells.

[0103] FIG. 12 depicts an ionizable lipid of formula (X) Fig. 12A ) and its exemplary lipid structure ( Fig. 12B and Fig. 12C ), which can be used in some embodiments of the present technology, for example, for transfecting immune cells.

[0104] FIG. 13 depicts an ionizable lipid of formula (XI) Fig.13A ) and its exemplary lipid structure ( Fig. 13B ), which can be used in some embodiments of the present technology, for example, for transfecting immune cells.

[0105] FIG. 14 depicts an ionizable lipid of formula (XII) ( Fig.14A ) and its exemplary lipid structure ( Fig. 14B ), which can be used in some embodiments of the present technology, for example, for transfecting immune cells.

[0106] FIG. 15 depicts an ionizable lipid of formula (XIII) ( Fig.15A ) and its exemplary lipid structure ( Fig. 15B ), which can be used in some embodiments of the present technology, for example, for transfecting immune cells.

[0107] FIG. 16 depicts an ionizable lipid of formula (XIV) Fig.16A ) and its exemplary lipid structure ( Fig. 16B ), which can be used in some embodiments of the present technology, for example, for transfecting immune cells.

[0108] FIG. 17 depicts an ionizable lipid of formula (XV) ( Fig.17A ) and its exemplary lipid structure ( Fig. 17B and Fig. 17C ), which can be used in some embodiments of the present technology, for example, for transfecting immune cells.

[0109] FIG. 18 depicts an ionizable lipid of formula (XVI) Fig.18A ) and its exemplary lipid structure ( Fig.18B and Fig. 18C ), which can be used in some embodiments of the present technology, for example, for transfecting immune cells.

[0110] Figures 19(A) to 19(F) provide marker expression data at day 15 of manufacturing of rejuvenated T cells expressing OSKMLN reprogramming factors as described herein.

[0111] Figure 20 (A) to 20 (F) provides T cell-mediated cytotoxicity data of rejuvenated T cells expressing OSKLMN reprogramming factors as described herein compared to unrejuvenated control T cells. Starting from the third addition of tumor cells (also referred to as "the third target binding" or "the third binding"), compared with untreated T cell controls, T cells expressing reprogramming factors show an increased efficiency in killing tumor cells. Using the cytotoxicity of ERA-treated T cells as a 100% baseline value, the cytotoxicity of untreated cells is only 20% to 30% of the T cells treated by ERA, as shown in the double arrows in Figure 20 (C) to Figure 20 (F). In addition, this increase in the efficiency of killing tumor cells is a lasting benefit because it maintains a high efficiency from the third to the sixth addition of tumor cells.

[0112] Figures 21(A) to 21(B) provide proliferation data of rejuvenated T cells expressing OSKMLN reprogramming factors as described herein compared to non-rejuvenated control T cells. Fig.21B As highlighted in the shaded portion of , T cells expressing OSKLMN reprogramming factors have a three to five-fold higher proliferation rate than untreated control T cells. For example, at the second target binding, T cells expressing OSKLMN reprogramming factors had a 20-fold increase in proliferation compared to untreated control T cells (Figure 21 (B); p<0.01 for ERA-treated T cells vs. "untreated" control T cells). The T cells used in this proliferation experiment were obtained from a 63-year-old donor. Treatment with OSKLMN reprogramming factors significantly increased T cell proliferation in this elderly patient, who would otherwise not be eligible for CAR-T therapy.

[0113] Fig. 21CT cell-mediated cytotoxicity data of rejuvenated T cells obtained from the same 63-year-old donor as in Figure 21 (A) and (B) are provided. At the third combination / third addition of tumor cells, their cytotoxicity was compared with unrejuvenated control T cells. T cells expressing OSKLMN reprogramming factors were 4-5 times more efficient in killing tumor cells (Figure 21 (C); p < 0.05 for ERA-treated T cells vs. "untreated" control T cells). Treatment with OSKLMN reprogramming factors significantly increased tumor cell killing in this elderly patient, who would otherwise not be eligible for CAR-T therapy.

[0114] Figures 22 (A) and 22 (B) provide marker expression data for rejuvenated T cells expressing OSKLMN reprogramming factors as described herein at the end of manufacturing and at the time of Daudi target cell binding. Compared to untreated control T cells, the expression of Tscm marker CD28 in reprogrammed T cells was 20% higher, and Figure 22 (C) shows that the expression of Tscm marker CD95 was 20% higher than that of reprogrammed T cells compared to untreated control T cells. These increased Tscm marker levels support increased proliferation and increased killing capacity.

[0115] Figures 22(C) and 22(D) provide exhaustion marker expression data for the same rejuvenated T cells expressing OSKLMN reprogramming factors as described in Figures 22(A) and 22(B). After the second round of tumor cell addition, the levels of exhaustion markers TIGIT and LAG-3 decreased by 25% and 80%, respectively.

[0116] Figures 22(E) and 22(F) also provide exhaustion marker expression data for the same rejuvenated T cells expressing OSKLMN reprogramming factors as described in Figures 22(A) and 22(B). After the third round of tumor cell addition, the expression levels of exhaustion markers TIGIT and LAG-3 decreased by 50% and 70%, respectively.

[0117] Figure 23 (A) provides proliferation data of rejuvenated T cells obtained from a 34-year-old donor. T cells expressing OSKMLN reprogramming factors as described herein were compared to non-rejuvenated control T cells. Fig.23AAs highlighted by the shaded portion of , T cells expressing OSKLMN reprogramming factors have a 4- to 6-fold increase in proliferation compared to untreated control T cells and T cells expressing GFP but not reprogramming factors. For example, at the 4th target binding, "ERA_2" T cells expressing OSKLMN reprogramming factors have an approximately 30-fold increase in proliferation compared to untreated control T cells and compared to GFP control T cells (Figure 21 (B); p < 0.001). Treatment with OSKLMN reprogramming factors significantly increased T cell proliferation.

[0118] Figure 23 (B) provides T cell-mediated cytotoxicity data of rejuvenated T cells obtained from the same 34-year-old donor as Figure 23 (A). Their cytotoxicity during the 5th tumor cell addition / target binding was compared with unrejuvenated control T cells (for "ERA_1") or with T cells expressing GFP rather than reprogramming factors (for "ERA_2"). The efficiency of T cells expressing OSKLMN reprogramming factors in killing tumor cells was 4 to 5 times higher (Figure 23 (B); p < 0.01, for "ERA_2" T cells and GFP, and for "ERA_1" T cells and untreated control T cells). Treatment with OSKLMN reprogramming factors also significantly increased tumor cell killing in this young donor patient, who will be eligible for CAR-T therapy.

[0119] Fig.24 Describe the method of incorporating ERA-treated T cells into the manufacturing process of any cell-based immunotherapy to reduce or eliminate manufacturing-induced differentiation and / or exhaustion. Primary T cells are activated with CD3 and / or CD28, and the addition of mRNA encoding one or more reprogramming factors as described herein results in an increase in the number of T cells, at least one or more of the following characteristics: proliferation, tumor cell killing, self-renewal, pluripotency and / or functional persistence. In contrast, activated T cells that are not treated with mRNA encoding one or more reprogramming factors as described herein are more differentiated, have limited proliferation capacity, and are prone to exhaustion.

[0120] Fig.25 Describes a three-week, 2-group comparative study conducted to demonstrate that treatment with mRNA encoding one or more reprogramming factors enhances T cell proliferation and T cell-mediated cytotoxicity. The study employed a long-term killing assay using a CD19-CD3 bispecific antibody to mimic repeated engagement between T cells and tumor cells in vivo.

[0121] Figures 26 (A) and 26 (B) provide CCR7 and CD27 marker expression data of rejuvenated T cells obtained from a 32-year-old donor expressing OSKLMN reprogramming factors as described herein. Compared to untreated control T cells, the expression of CCR7 in reprogrammed T cells was approximately 1.3 times higher (Figure 26 (A)).

[0122] Figure 27 provides CCR7 and CD27 marker expression data for rejuvenated T cells obtained from a 73-year-old donor expressing OSKLMN reprogramming factors as described herein. CCR7 expression was more than 2-fold higher in reprogrammed T cells compared to untreated control T cells (Figure 27 (A)).

[0123] FIG. 28 provides cytokine concentrations of IL-2, GM-CSF, IFNγ, and TNF-α as measured using the Luminex assay on rejuvenated T cells obtained from a 57 year old donor.

[0124] Figure 29 provides CCR7 and CD45RA expression data after the mRNA mixture encoding OSKLMN reprogramming factors was transfected into CD3 / CD28 activated T cells by electroporation. Increased CCR7 expression indicates that the expression of reprogramming factors enhances central memory (Tcm) and stem memory T cell (Tscm)-like phenotypes. Increased percentages of Tcm and Tscm populations will lead to higher proliferation and longer persistence in patients. 1X ERA and 2X ERA represent different doses of ERA treatment. EoM = End of Manufacturing. T N-SCM (T initial stem cell memory), T CM (T Central Memory), T EM (T effect memory), T E (T effect).

[0125] Sequence Description

[0126] In some embodiments, methods and compositions related to rejuvenating immune cells such as T cells comprise exposing the immune cells to messenger RNA (mRNA) encoding one or more reprogramming factors, wherein the reprogramming factor encoding mRNA encodes a polypeptide encoded by a polynucleotide having at least 95% sequence identity to any one of SEQ Id NO: 1-14 (Table 1).

[0127] In some aspects, reprogramming factors encoding a polynucleotide having at least 95% sequence identity to any one of SEQ Id NOs: 1-14 are contemplated for use in conjunction with the methods and compositions provided herein comprising rejuvenated immune cells.

[0128] In another aspect, a reprogramming factor protein or polypeptide encoded by a polynucleotide having at least 95% sequence identity to any one of SEQ Id NOs: 1-14 is contemplated for use in conjunction with the methods and compositions provided herein comprising rejuvenated immune cells.

[0129] In another aspect, RNA vectors comprising one or more reprogramming factor polynucleotide sequences are contemplated for use in conjunction with the methods and compositions provided herein comprising rejuvenated immune cells, wherein the one or more polynucleotide sequences comprise at least 95% sequence identity with any one of SEQ Id NOs: 1-14.

[0130] In some embodiments, the present technology relates to mRNA encoding one or more reprogramming factors, wherein the reprogramming factor encoding mRNA encodes a polypeptide encoded by a polynucleotide having at least about 70%, about 75%, about 80%, about 85%, 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to any one of SEQ Id NO:1-14 (Table 1).

[0131] In some embodiments, the present technology relates to mRNA encoding one or more reprogramming factors, wherein the reprogramming factor encoding the mRNA encodes a polypeptide encoded by a polynucleotide having about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity to any one of SEQ Id NO:1-14 (Table 1). DETAILED DESCRIPTION

[0132] I. definition

[0133] Various aspects will now be described more fully below. However, such aspects may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; on the contrary, these embodiments are provided to make this disclosure thorough and complete, and these embodiments will fully convey its scope to those skilled in the art.

[0134] Where a range of values ​​is provided, it is intended that each intervening value between the upper and lower limits of the range, as well as any other stated or intervening value in the stated range, is encompassed within the disclosure. For example, if a range of 1 μm to 8 μm is stated, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, and 7 μm are also expressly disclosed, as well as ranges of values ​​greater than or equal to 1 μm and ranges of values ​​less than or equal to 8 μm.

[0135] Unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "a polymer" includes a single polymer as well as two or more of the same or different polymers, reference to "an excipient" includes a single excipient as well as two or more of the same or different excipients, and so forth.

[0136] The term "about", particularly when referring to a given amount, is intended to encompass a deviation of plus or minus 5 percent; when used in reference to a given range or set of values, it encompasses a deviation of plus or minus 5 percent and applies to each stated value within the given range or set of values.

[0137] The compositions of the present disclosure may comprise, consist essentially of, or consist of the disclosed components.

[0138] All percentages, parts and ratios are based upon the total weight of the topical composition and all measurements made are at about 25°C, unless otherwise specified.

[0139] As used herein, the term "cell" refers to a naturally occurring or modified intact living cell. A cell can be isolated from other cells, mixed with other cells in culture or in a tissue (partial or complete) or organism. The methods described herein can be performed on a sample, for example, comprising a single cell, a cell population, or a tissue or organ comprising a cell.

[0140] As used herein, the term "mRNA" refers to a ribonucleic acid (RNA) encoding at least one amino acid (e.g., a peptide or protein). The ribonucleic acid is synthesized according to methods known in the art. The ribonucleic acid can simulate or reproduce the natural messenger RNA encoding the reprogramming factor, or it can have different structures or forms, such as polycistronic RNA, circular RNA, self-amplification RNA, or chemically modified RNA. In this article, all such RNAs are referred to as "mRNA".

[0141] As used herein, the term "immune cell" refers to a cell that is part of the immune system and helps the body resist infection and other diseases. In some cases, immune cells develop from stem cells in the bone marrow and become different types of white blood cells. The immune cells provided herein include, but are not limited to, lymphocytes, granulocytes (neutrophils, eosinophils, basophils, mast cells), monocytes, macrophages, microglia, dendritic cells, T cells (cytotoxic T cells (CD8+), helper T cells (CD4+), inhibitory or regulatory T cells (Tregs), memory T cells, Th1 T cells, Th2 T cells, Th17 T cells, Th9 T cells, Tfh T cells, initial T cells that have not experienced antigens (Tn or TN), stem cell memory T cells (Tscm or T SCM ), central memory T cells (Tcm or T CM ), effector memory T cells (Tem or T EM ), effector T cells (Teff, T EFF or T E ), exhausted T cell precursors (Tpex or T PEX ) or depleted T cells (Tex or T EX ), central memory T cells, effector memory T cells, tissue-resident memory T cells, virtual memory T cells, natural killer T cells (NKT cells), FOXP3+ T cells, FOXP3- T cells), B cells (memory B cells or plasma cells), natural killer (NK) cells, tumor-infiltrating lymphocytes, etc., including natural immune cells or engineered immune cells of all the aforementioned immune cell types.

[0142] In certain embodiments, cell is non-adherent immune cell.In certain embodiments, non-adherent immune cell is processed, instantaneously reprogrammed, rejuvenated or manufactured in a manner where the cell remains non-adherent, and is not attached to a tissue culture substrate or forms or produces cells or cell colonies attached to a tissue culture substrate.In certain embodiments, reprogramming intervals and factors are selected so that the cell is rejuvenated while retaining cell identity, wherein the cell remains non-adherent, and is not attached to a tissue culture substrate or forms or produces cells or cell colonies attached to a tissue culture substrate.Therefore, in certain embodiments, the present technology provides a reprogramming method, wherein the cell, including any non-adherent cell and / or non-adherent immune cell (e.g., non-adherent T cells, NK cells, macrophages, tumor infiltrating cells, dendritic cells and / or NKT cells), is reprogrammed in a manner where the cell is rejuvenated while retaining cell identity, and wherein the cell remains suspended and non-adherent, and they also will not become or produce cells attached to the wall, become attached, or form an adherent colony.

[0143] As used herein, the term "engineered cell" refers to a cell that has been altered to contain exogenous DNA, RNA, protein or polypeptide compared to an unmodified cell of the same type. Merely isolating or purifying a cell from an organism found in the wild does not convert it into an engineered cell.

[0144] As used herein, the term "transfection" refers to the uptake of exogenous DNA or RNA by cells such as immune cells. When exogenous DNA or RNA has been introduced into the cell membrane, the cell has been "transfected". Various transfection techniques are generally well known in the art. See, for example, Graham et al. (1973) Virology, 52: 456, Sambrook et al. (2001) Molecular Cloning, alaboratory manual, 3rd Supplementary Edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd Supplementary Edition, McGraw-Hill, and Chu et al. (1981) Gene 13: 197. Such techniques can be used to introduce one or more exogenous DNA or RNA molecules into cells. The term refers to the stable and transient uptake of DNA or RNA molecules. For example, transfection can be used to transiently uptake mRNA encoding cell reprogramming factors into cells that need to be rejuvenated.

[0145] As used herein, the term "transient reprogramming" refers to exposing cells (such as immune cells including T cells) to cell reprogramming factors for a period of time sufficient to rejuvenate the cells (i.e., eliminate all or some signs of aging) but not sufficient to cause loss of identity or dedifferentiation. This transient reprogramming results in the cells being rejuvenated and retaining their identity (i.e., differentiated cell type).

[0146] As used herein, the term "rejuvenated immune cell" refers to an aged, exhausted, living immune cell that has been treated with one or more cell reprogramming factors or transiently reprogrammed such that the immune cell has the transcriptomic profile, epigenetic profile or "clock" or function of a younger or non-exhausted cell, while still retaining one or more markers of cellular identity.

[0147] As used herein, the term "mammalian cell" refers to any cell derived from a mammalian subject that is suitable for transplantation into the same or a different subject or a cell present in a mammalian subject. Cells suitable for transplantation may be xenogeneic, autologous, or allogeneic. The cell may be a primary cell obtained directly from a mammalian subject. The cell may also be a cell derived from the culture and expansion of a cell obtained from a subject. In some embodiments, the cell has been genetically engineered to express a recombinant protein and / or nucleic acid.

[0148] As used herein, the term "stem cell" refers to a cell that retains the ability to renew itself through mitotic cell division and can be differentiated into a wide range of specialized cell types. Mammalian stem cells can be divided into four major categories: embryonic stem cells, which are derived from blastocysts; induced pluripotent stem cells, which are produced by the dedifferentiation of somatic cells, adult stem cells or umbilical cord blood stem cells; adult stem cells, which are found in adult tissues; and umbilical cord blood stem cells, which are found in the umbilical cord. In the developing embryo, stem cells can differentiate into all specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as the body's repair system by supplementing specialized cells or by secreting trophic factors, cytokines and / or signaling molecules. Totipotent stem cells are produced by the fusion of egg and sperm cells. The cells produced by the first few divisions of the fertilized egg are also omnipotent. These cells can differentiate into embryonic and extraembryonic cell types. Pluripotent stem cells are descendants of omnipotent cells and can differentiate into cells derived from any of the three germ layers. Pluripotent stem cells can only produce cells of closely related cell families (for example, hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). Unipotent cells can only give rise to one cell type, but have the property of self-renewal, which distinguishes them from non-stem cells. Induced pluripotent stem cells are a type of pluripotent stem cell derived from an adult cell that has been reprogrammed to an embryonic-like pluripotent state. Induced pluripotent stem cells can be derived, for example, from adult somatic cells, such as skin or blood cells.

[0149] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, salts, compositions, dosage forms, etc. which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and / or other mammals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. In some aspects, "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals (e.g., animals), and more particularly in humans.

[0150] As used herein, the term "cell reprogramming factor" or "reprogramming factor" refers to any factor that can rejuvenate a cell without causing loss of identity or dedifferentiation. In an embodiment, a cell reprogramming factor or reprogramming factor is a transcription factor and groups and combinations thereof that can convert adult or differentiated cells into pluripotent stem cells. Exemplary reprogramming factors include OCT4, SOX2, KLF4, c-MYC, LIN28, NANOG, and / or GLIS1. Other exemplary reprogramming factors include CMYC, DPPA2, DPPA4, ESRRB, GDF3, GLIS1, KLF2, KLF4, KLF5, LIN28, LMYC, NANOG, NMYC, NR5A1, NR5A2, OCT-4, RCOR2, SALL1, SALL4, SOX1, SOX2, SOX3, TDRD12, TET1, TH2A, TH2B, UTF1, ZFP42, MDM2, CyclinD1, SV40 large T antigen, SIRT6, TCL1A, and RARy.

[0151] In some cases, "cell reprogramming factors" or "reprogramming factors" are selected from the group consisting of Oct, Sox, Klf, Myc, Lin or NANOG, i.e., any of these factors alone or in any combination, including in combination with any additional factors capable of rejuvenating cells without causing loss of identity or dedifferentiation. In some cases, "cell reprogramming factors" or "reprogramming factors" are selected from the group consisting of Oct4, Sox2, Klf4, cMyc, Lin28 and NANOG.

[0152] "Cell reprogramming factors" or "reprogramming factors" described herein may include proteins, such as transcription factors, which play a role in converting adult or differentiated cells (such as immune cells) into pluripotent stem cells. The terms "cell reprogramming factors" and "reprogramming factors" further include any analog molecules that mimic the function of the factor. In an embodiment, the reprogramming factor is a factor from the Oct family, the Sox family, the Klf family, the Myc family, the Nanog family, or the Lin family.

[0153] The compositions provided herein include reprogramming factor polynucleotides, proteins, polypeptides, and RNA and DNA vectors containing the reprogramming factor polynucleotides for producing the reprogramming factor proteins and polypeptides; for example, for expressing such reprogramming factors in cells such as mammalian cells.

[0154] The term "treatment" is used herein, for example, with respect to a method for treating a cell, tissue, or subject, and generally includes administering a compound or composition that reduces the frequency or amplitude of symptoms or markers of aging or medical conditions in a cell, tissue, or subject, or delays the onset of the compound or composition, relative to cells, tissues, or subjects that have not received the compound or composition. This can include reversing, reducing, or preventing symptoms, clinical signs, and potential pathologies of the condition in a manner that improves or stabilizes the condition of the subject. Treatment also includes administering a compound or composition that reduces transcriptome, proteome, and / or epigenetic markers of aging and / or depletion of immune cells, and / or enhances the function of immune cells, including but not limited to proliferation, antipathogen activity, antitumor activity, and anti-inflammatory activity.

[0155] As used herein, the term "rejuvenated cells" refers to cells that have been treated with one or more cell reprogramming factors or transiently reprogrammed such that the cells have a transcriptome profile of younger cells while still retaining one or more cell identity markers. In some embodiments, the treated cells are rejuvenated and reprogrammed to express markers and transcriptome profiles of younger cells while still retaining cell identity markers, such as rejuvenated cells are reprogrammed to express an increase / improvement in expression of at least one rejuvenation marker compared to untreated cells by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or more than 75%.

[0156] The terms "inhibit" or "reduce" are non-limiting expressions used in methods that affect cancer and / or tumor development or progression, slow tumor growth, and / or cause tumor size to stagnate or decrease, such as, but not limited to, inhibiting and / or reducing tumor growth in a population (e.g., reducing the size of a tumor) compared to an untreated control population. These terms are also used to refer to methods that affect other diseases (e.g., fibrosis) of cell proliferation, such as, but not limited to, inhibiting and / or reducing fibrosis in a population (e.g., reducing the amount of fibrotic tissue) compared to an untreated control population. These terms are also used to refer to methods that affect other diseases (e.g., fibrosis) of cell proliferation, such as, but not limited to, inhibiting and / or reducing fibrosis in a population (e.g., reducing the amount of fibrotic tissue) compared to an untreated control population. These terms are also used to refer to methods that affect autoimmune or inflammatory diseases, such as, but not limited to, inhibiting and / or reducing autoimmunity or inflammation in a population (e.g., reducing the amount of autoimmunity or inflammation) compared to an untreated control population. These terms are also used to refer to methods of affecting senescent cells, such as, but not limited to, inhibiting and / or reducing senescence in a population compared to an untreated control population (e.g., by senescence disruption of senescent cells, senescence modulation of a senescent cell phenotype, or senescence blocking inhibition of senescence).

[0157] Less than the entirety of the disclosure may be claimed for any reason by reserving the right to limit or exclude any individual member of any such group that may be claimed by range or in any similar manner, including any subrange or combination of subranges within said group. Thus, less than the entirety of the disclosure may be claimed for any reason by reserving the right to limit or exclude any individual substituent, analog, compound, ligand, structure, or group thereof or any member of a claimed group.

[0158] Throughout this disclosure, various patents, patent applications and publications are cited. The disclosures of these patents, patent applications and publications are incorporated herein by reference in their entirety to more fully describe the prior art known to those skilled in the art as of the date of this disclosure. In the event of any inconsistency between the cited patents, patent applications and publications and this disclosure, the present disclosure shall prevail.

[0159] For convenience, certain terms employed in the specification, examples, and claims are collected here. Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0160] II. Immune cell rejuvenation

[0161] Cell aging process is considered to be caused by the loss of hereditary and epigenetic information. The loss of genetic information causing cell aging usually occurs in the form of genetic mutation, such as replacement and deletion in the genome of an organism. The loss or change of epigenetic information associated with cell aging can take the form of covalent modification to DNA, such as 5-methylcytosine (5mC), hydroxymethylcytosine (5hmeC), 5-formylcytosine (fC) and 5-carboxylcytosine (caC) and adenine methylation, and the form of covalent modification to some proteins, such as lysine acetylation of histones, lysine and arginine methylation, serine and threonine phosphorylation and lysine ubiquitination and threonylation. The loss and change of epigenetic information may lead to the disorder of cellular process, including the process of maintaining cell identity, causing immune cells to show the character associated with aging, such as aging.

[0162] Immune cell exhaustion is a broad term used to describe the response of immune cells (such as T cells) to chronic antigenic stimulation, for example in chronic viral infection and in response to tumors, or in the aging process. Many characteristics and pathways are thought to have a critical impact on immune cell exhaustion, including checkpoint blockade, and have important implications for immune cell therapies (such as adoptive T cell transfer therapy). In some cases, immune cell exhaustion may indicate a complete lack of effector function, while in other cases, immune cells that exhibit or begin to exhibit exhaustion may show altered / reduced immune cell function.

[0163] The disclosed methods, compositions and kits rejuvenate immune cells by preventing and reversing cellular causes of aging, and / or by preventing and / or reversing cell exhaustion. The disclosed methods, compositions and kits rejuvenate immune cells by restoring epigenetic information, which includes epigenetic information lost due to aging process, cell therapy manufacturing process, damage or disease, for example, as described in WO2019178296, the entire contents of which are incorporated herein by reference. The disclosed methods, compositions and kits rejuvenate immune cells by preventing and reversing the exhaustion of immune cells. The disclosed methods, compositions and kits enhance the function of immune cells by increasing the proliferation of immune cells and their anti-pathogen activity and anti-tumor activity, or in the case of suppressive or regulatory T cells, by enhancing their anti-inflammatory activity.

[0164] The method for rejuvenating immune cells provided herein includes transfecting immune cells with one or more mRNAs, such as mRNAs encoding one or more cell reprogramming factors, thereby producing rejuvenated immune cells. In an embodiment, immune cells are contacted with mRNA, exposed to mRNA, or transfected with mRNA, and continue to be no more than about 21 days, 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days or 2 days. In an embodiment, immune cells are contacted with mRNA, exposed to mRNA, or transfected with mRNA, and continue to be no more than about 14 days, 10 days, 7 days, 6 days, 5 days, or 4 days. In an embodiment, immune cells are contacted with mRNA, exposed to mRNA, or transfected with mRNA, and continue to be no more than about 14 days, 10 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day or less than 1 day. In embodiments, the immune cells are contacted with, exposed to, or transfected with mRNA for no more than about 7, 6, 5, or 4 days. In embodiments, the immune cells are contacted with, exposed to, or transfected with mRNA for no more than about 3, 2, 1, or less than 1 day. In embodiments, the immune cells are contacted with, exposed to, or transfected with mRNA for no more than 6 days. In embodiments, the immune cells are contacted with, exposed to, or transfected with mRNA for 1, 2, 3, 4, 5, or 6 days. In embodiments, the immune cells are contacted with, exposed to, or transfected with mRNA for less than one day. In embodiments, the rejuvenated immune cells have a phenotype or activity profile similar to that of young immune cells or non-exhausted immune cells. The phenotypic or activity profile includes one or more of a transcriptomic profile, a proteomic profile, a secretomic profile, gene expression of one or more nuclear and / or epigenetic markers, proteolytic activity, mitochondrial health, cellular function, SASP cytokine expression, and a methylation landscape (e.g., a methylation marker or a methylation clock, such as the Horvath methylation clock).

[0165] In some embodiments, rejuvenated immune cells have transcriptome profiles, proteome and / or secretome profiles that are more similar to such profiles of young immune cells. For example, with respect to T cells, exhausted T cells exhibit continuous phenotypic and functional changes. Exhausted T cells express an array of inhibitory molecules as well as a unique pattern of cytokine receptors, transcription factors, and effector molecules that distinguish these cells from traditional effector, memory, and anergic T cells. For example, changes in the expression pattern of GranB, granzyme B; IFN-γ, interferon-γ; IL-2, interleukin-2; TNF-α, tumor necrosis factor-α may indicate T cell exhaustion and / or rejuvenation. Exhausted T (T EX) cells express high levels of the inhibitory receptors PD-1, TIM-3, LAG-3, and TIGIT.

[0166] On the other hand, changes in CD62L, CCR7 and / or TCF7 expression, particularly increases in expression, may be associated with "stemness" characteristics of T cells, and therefore with rejuvenation and higher and more sustained anti-pathogen, anti-cancer or anti-inflammatory activity. In some embodiments, interleukin-2 (IL-2) production is one of the first effector activities to be eliminated, followed by tumor necrosis factor-α (TNF-α) production. However, these changes in gene expression patterns represent only a small part of the overall alterations that manifest as the exhausted state develops.

[0167] In embodiments, the rejuvenated cells have a transcriptome profile that is more similar to the transcriptome profile of young cells. In embodiments, the transcriptome profile of the rejuvenated cells includes an increase in gene expression of one or more genes selected from RPL37, RHOA, SRSF3, EPHB4, ARHGAP18, RPL31, FKBP2, MAP1LC3B2, Elf1, Phf8, Pol2s2, Taf1, and Sin3a. In embodiments, the transcriptome profile of the rejuvenated cells includes an increase in gene expression of RPL37. In embodiments, the transcriptome profile of the rejuvenated cells includes an increase in gene expression of RHOA. In embodiments, the transcriptome profile of the rejuvenated cells includes an increase in gene expression of SRSF3. In embodiments, the transcriptome profile of the rejuvenated cells includes an increase in gene expression of EPHB4. In embodiments, the transcriptome profile of the rejuvenated cells includes an increase in gene expression of ARHGAP18. In embodiments, the transcriptome profile of the rejuvenated cells includes an increase in gene expression of RPL31. In embodiments, the transcriptome profile of the rejuvenated cells comprises an increase in gene expression of FKBP2. In embodiments, the transcriptome profile of the rejuvenated cells comprises an increase in gene expression of MAP1LC3B2. In embodiments, the transcriptome profile of the rejuvenated cells comprises an increase in gene expression of Elf1. In embodiments, the transcriptome profile of the rejuvenated cells comprises an increase in gene expression of Phf8. In embodiments, the transcriptome profile of the rejuvenated cells comprises an increase in gene expression of Pol2s2. In embodiments, the transcriptome profile of the rejuvenated cells comprises an increase in gene expression of Taf1. In embodiments, the transcriptome profile of the rejuvenated cells comprises an increase in gene expression of Sin3a. In embodiments, the transcriptome profile of the rejuvenated cells comprises an increase in gene expression of RPL37, RHOA, SRSF3, EPHB4, ARHGAP18, RPL31, FKBP2, MAP1LC3B2, Elf1, Phf8, Pol2s2, Taf1, and Sin3a.

[0168] In embodiments, the rejuvenated cells exhibit increased gene expression of one or more nuclear and / or epigenetic markers compared to a reference value. In embodiments, the one or more nuclear and / or epigenetic markers are selected from HP1γ, H3K9me3, laminar support protein LAP2α, and SIRT1 protein. In embodiments, the rejuvenated cells exhibit increased gene expression of HP1γ. In embodiments, the rejuvenated cells exhibit increased gene expression of H3K9me3. In embodiments, the rejuvenated cells exhibit increased gene expression of laminar support protein LAP2α. In embodiments, the rejuvenated cells exhibit increased gene expression of SIRT1 protein. In embodiments, the rejuvenated cells exhibit increased gene expression of HP1γ, H3K9me3, laminar support protein LAP2α, and SIRT1 protein.

[0169] In an embodiment, immune cells are rejuvenated by transient reprogramming with mRNA encoding one or more cell reprogramming factors. In certain embodiments, transient reprogramming is completed by transfecting immune cells with non-integrative mRNA for at least about two days and no more than about 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 days, or no more than about 10, 9, 8, 7, 6, 5 or 4 days, or no more than about 5 or 4 days. In an embodiment, such transfection is carried out at least once a day or less than once a day, for example, every two days, three days, four days, five days, six days, seven days or more days. In an embodiment, the transient reprogramming of immune cells eliminates various marks of aging or exhaustion, or enhances immune cell function, while avoiding cell identity loss or dedifferentiation.

[0170] In an embodiment, transfection of immune cells with messenger RNA can be completed by transfection methods, including but not limited to non-viral techniques, such as Lipofectamine transfection reagent, Polyplus transfection reagent, Fugene transfection reagent, LT-1 mediated transfection, dextran mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, electroporation, microfluid extrusion of cells, mRNA encapsulated in liposomes, lipid nanoparticle compositions and / or direct microinjection. Transfection can be achieved using lipid nanoparticles, polymer nanoparticles, polymer polymers or polymer anion complexes. In some embodiments, lipid nanoparticles include ionizable or cationic lipids, uncharged lipids (including but not limited to phospholipids and cholesterol), lipids and / or biodegradable lipids conjugated or modified with parts such as polyethylene glycol for stability. In some embodiments, lipids are those described in Figures 1 to 18. In some embodiments, lipids are those in Billingsley et al., Nano Lett.2020 (20, 3, 1578–1589), which are incorporated herein by reference. In some embodiments, the polymer includes a cationic polymer; a biodegradable polymer; a natural polymer such as a peptide, a protein or a polysaccharide; and / or a releasable transporter that changes charge. In some embodiments, the polymer is a releasable transporter that changes charge. The releasable transporter that changes charge may contain a mixture of lipophilic blocks in a block (b) or statistical (stat) architecture, that is, it may be at least one "block CART" or "stat CART", such as those described in McKinlay et al. 2017 (PNAS January 24, 2017 114 (4) E448-E456), McKinlay et al. 2018 (PNAS June 26, 2018 115 (26) E5859-E5866) or Haabeth et al. 2018 (PNAS September 25, 2018 115 (39) E9153-E9161), which are incorporated herein by reference. In some embodiments, lipids are combined with polymers to produce hybrid nanoparticles or micelles. In some embodiments, delivery of mRNA using lipids or polymers, such as in lipid nanoparticles, polymer nanoparticles, or hybrid lipid-polymer nanoparticles, results in enhanced rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, anti-pathogenic effects, anti-cancer effects, or anti-inflammatory effects in exposed immune cells compared to using a different mRNA delivery mechanism. In some embodiments, delivery of mRNA using lipids or polymers results in enhanced rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, anti-pathogenic effects, anti-cancer effects, or anti-inflammatory effects in exposed immune cells compared to using a different mRNA delivery mechanism due to lower toxicity and / or lower physiological effects on cells when compared to a different delivery mechanism.In some embodiments, the different delivery mechanism is electroporation, i.e., the use of lipids or polymers to deliver mRNA results in enhanced rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, anti-pathogenic effects, anti-cancer effects, or anti-inflammatory effects in exposed immune cells compared to the use of electroporation. Such improvements compared to electroporation can be caused by reduced toxicity or reduced physiological effects on cells compared to electroporation.

[0171] Compositions, methods and kits as described herein relate to delivery of nucleic acid and other therapeutic molecules to target cells and / or tissues. For example, compositions, methods and kits as described herein may relate to delivery of DNA and / or mRNA to cells by various gene delivery systems. "Gene delivery" refers to any method or system for introducing an exogenous nucleic acid sequence into a target (such as a target cell or tissue, including diseased and / or aging cells or tissues). In certain embodiments, gene delivery systems can be classified as: virus-based, non-viral-based and combined hybrid systems. In certain embodiments, non-viral gene delivery systems provide alternatives to virus-based systems. One of the most important advantages of these systems is to improve safety by enhancing control of transgenic expression and dosage and lacking chromosomal integration. Such systems include systems for delivering mRNA.

[0172] Some non-viral gene delivery systems can be referred to as physical or chemical methods. In certain embodiments, physical methods include but are not limited to microinjection, jet injection, electroporation, ultrasound, gene guns and fluid dynamic systems. In general, physical methods refer to delivering genes via applying physical force to increase the permeability of cell membranes. On the contrary, chemical methods utilize natural or synthetic carriers to deliver genes into cells. In certain embodiments, polymers, cationic polymers, liposomes, lipids, cationic lipids, auxiliary lipids, spacer lipids, ionizable lipids, dendrimers or nanoparticle compositions formed by any of the above or any combination of the above can be combined with compositions, methods and kits as described herein as gene delivery systems. Such gene delivery systems can be used for delivering mRNA as described herein. Such nanoparticle compositions can also be used for delivering non-gene therapeutic molecules, such as proteins, peptides or small molecules.

[0173] In some embodiments, lipid-based delivery systems such as, but not limited to, lipid nanoparticles (LNPs) can provide methods for stabilizing and delivering nucleic acids and other therapeutic molecules such as proteins, peptides, and / or small molecules. Design features such as optimal particle size, high encapsulation efficiency, robust manufacturing processes, and optimal lipophilicity and surface charge are included to provide effective lipid-based delivery systems for nucleic acids and other therapeutic molecules.

[0174] In some embodiments, the lipids provided herein for transfecting immune cells such as T cells relate to ionizable lipids of formula (I):

[0175]

[0176] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0177] L 1 is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 3 and R 4 Each independently is H

[0178] or C1-C3 alkyl; q1 is absent or is 1; and q2 is absent or is 1 ( Figure 1A ).

[0179] In other embodiments, the ionizable lipid of formula (I) has one of the following structures: Figure 1B and Figure 1C ):

[0180]

[0181] In other embodiments, the lipids provided herein for transfection of immune cells relate to ionizable lipids of formula (IA):

[0182]

[0183] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0184] L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; R 2 C6-C 20 Alkyl; R 3 and R 4 Each independently represents H or C1-C3 alkyl; R 7 C4-C 20 alkyl; and R 8 C4-C 20 alkyl( Figure 2A ).

[0185] In other embodiments, the ionizable lipid of formula (IA) has the following structure ( Figure 2B ):

[0186]

[0187] In one embodiment, the lipid is an ionizable lipid of formula (IB)

[0188]

[0189] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0190] L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; L 3 is C1-C8 alkylene; R 3 and R 4 Each independently

[0191] H or C1-C3 alkyl; R 6 C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R 8 C4-C 20 alkyl; and R 10 C4-C 20 alkyl( Figure 3A ).

[0192] In another embodiment, the ionizable lipid of formula (IB) has the following structure ( Figure 3B ):

[0193]

[0194] In another embodiment, the lipid is an ionizable lipid of formula (II)

[0195]

[0196] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0197] L 1 is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 3 and R 4 are each independently H or C1-C3 alkyl; and R 5 is H or C1-C3 alkyl ( Figure 4A ).

[0198] In other embodiments, the ionizable lipid of formula (II) has one of the following structures: Figure 4B and Figure 4C ):

[0199]

[0200]

[0201] In another embodiment, the lipid is an ionizable lipid of formula (III)

[0202]

[0203] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0204] L 1 is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 3 and R 4 are each independently H or C1-C3 alkyl; and R 5 is H or C1-C3 alkyl ( Figure 5A ).

[0205] In other embodiments, the ionizable lipid of formula (III) has one of the following structures: Figure 5B and Figure 5C ):

[0206] In another embodiment, the lipid is an ionizable lipid of formula (IV)

[0207]

[0208] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0209] L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; R 2 C6-C 20 Alkyl; R 3 and R 4 Each independently represents H or C1-C3 alkyl; R 5 is H or C1-C3 alkyl; R 7 C4-C 20 alkyl; and R 8 C4-C 20 alkyl( Fig. 6A ).

[0210] In other embodiments, the ionizable lipid of formula (IV) has one of the following structures: Figure 6B and Figure 6C ):

[0211]

[0212] In another embodiment, the lipid is an ionizable lipid of formula (V)

[0213]

[0214] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0215] L 1 is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 3 and R 4 are each independently H or C1-C3 alkyl; and

[0216] R 12 C6-C 20 Alkenyl ( Fig. 7A ).

[0217] In other embodiments, the ionizable lipid of formula (V) has the following structure ( Figure 7B ):

[0218]

[0219] In another embodiment, the lipid is an ionizable lipid of formula (VI)

[0220]

[0221] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0222] L 1 is C1-C6 alkylene; L 1 ' is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 1 ' is C6-C 20 Alkenyl; R 9 H, C1-C6 alkyl or –(CH2) n OH; R 12 C6-C 20 Alkenyl; R 12 ' is C6-C 20 alkenyl; and n is 2, 3 or 4 ( Fig. 8A ).

[0223] In other embodiments, the ionizable lipid of formula (VI) has one of the following structures: Figure 8B and Figure 8C ):

[0224]

[0225] In another embodiment, the lipid is an ionizable lipid of formula (VII)

[0226]

[0227] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0228] L 1 is C1-C6 alkylene; L 1 ' is C1-C6 alkylene; R 2 C6-C 20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 9 H, C1-C6 alkyl or –(CH2) n OH; R 11 H or –CH2)OC(=O)R 16 ; R 14 C6-C 20 alkyl;

[0229] R 14 ' is C6-C 20 Alkyl; R 15 C6-C 20 Alkyl; R 16 C6-C 20 Alkyl; and n is 2, 3 or 4 ( Fig. 9A ).

[0230] In another embodiment, the ionizable lipid of formula (VII) has one of the following structures: Fig. 9B and Fig. 9C ):

[0231]

[0232] In another embodiment, the lipid is an ionizable lipid of formula (VIII)

[0233]

[0234] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0235] L 1 is C1-C6 alkylene; L 1 ' is C1-C6 alkylene; R 2 C6-C 20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 9 H, C1-C6 alkyl or –(CH2)n OH; R 14 C6-C 20 Alkyl; R 14 ' is C6-C 20 Alkyl; R 15 For C6-

[0236] C 20 Alkyl; and n is 2, 3 or 4 ( Fig. 10A ).

[0237] In some embodiments, the ionizable lipid of formula (VIII) has the following structure ( Fig. 10B ):

[0238]

[0239] In another embodiment, the lipid is an ionizable lipid of formula (IX)

[0240]

[0241] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0242] R 1 C6-C 20 Alkenyl; R 9 H, C1-C6 alkyl or –(CH2) n OH; R 12 C6-C 20 alkenyl; and n is

[0243] 2, 3, or 4 ( Fig.11A ).

[0244] In some embodiments, the ionizable lipid of formula (IX) has one of the following structures: Fig. 11B and Fig. 11C ):

[0245]

[0246] In another embodiment, the lipid is an ionizable lipid of formula (X)

[0247]

[0248] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0249] L 4 L is absent or is C1-C6 alkylene; 5 does not exist or is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 9 H, C1-C6 alkyl or –(CH2) n OH; R 12 C6-C 20 alkenyl; and n is 2, 3 or 4 ( Fig. 12A ).

[0250] In some embodiments, the ionizable lipid of formula (X) has one of the following structures: Fig. 12B and Fig. 12C ):

[0251]

[0252] In another embodiment, the lipid is an ionizable lipid of formula (XI)

[0253]

[0254] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0255] L 4 L is absent or is C1-C6 alkylene; 5 does not exist or is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 12 C6-C 20 Alkenyl; R 13 H, C1-C6 alkyl, –(CH2) n OH

[0256] or –(CH2) q N(CH3)2; n is 2, 3 or 4; and q is 2, 3 or 4 ( Fig.13A ).

[0257] In some embodiments, the ionizable lipid of formula (XI) has the following structure ( Fig. 13B ):

[0258]

[0259] In another embodiment, the lipid is an ionizable lipid of formula (XII)

[0260]

[0261] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0262] L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 7 C4-C 20 Alkyl; R 7 ' is C4-C 20 Alkyl; R 8 C4-C 20 Alkyl; R 8 ' is C4-C 20 Alkyl; R 13 H, C1-C6 alkyl, –(CH2) n OH or –(CH2) q N(CH3)2;

[0263] n is 2, 3 or 4; and q is 2, 3 or 4 ( Fig.14A ).

[0264] In some embodiments, the ionizable lipid of formula (XII) has the following structure ( Fig. 14B ):

[0265]

[0266] In another embodiment, the lipid is an ionizable lipid of formula (XIII)

[0267]

[0268] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0269] L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 7 C4-C 20 Alkyl; R 7 ' is C4-C 20 Alkyl; R 8 C4-C 20 Alkyl; R 8 ' is C4-C 20 Alkyl; R 13 H, C1-C6 alkyl, –(CH2) n OH or –(CH2) q N(CH3)2;

[0270] n is 2, 3 or 4; and q is 2, 3 or 4 ( Fig.15A ).

[0271] In some embodiments, the ionizable lipid of formula (XIII) has the following structure ( Fig. 15B ):

[0272]

[0273] In another embodiment, the lipid is an ionizable lipid of formula (XIV)

[0274]

[0275] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0276] L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; R 3 and R 4 Each independently is H or C1-C3 alkyl;

[0277] R 6 C4-C 20 alkyl; and R 7 C4-C 20 alkyl( Fig.16A ).

[0278] In some embodiments, the ionizable lipid of formula (XIV) has the following structure ( Fig. 16B ):

[0279]

[0280] In another embodiment, the lipid is an ionizable lipid of formula (XV)

[0281]

[0282] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0283] L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 6 C4-C 20 Alkyl; R 6 ' is C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R 7 ' is C4-C 20 Alkyl; R 8 C4-C 20 Alkyl; R 8 ' is C4-C 20 Alkyl; R 10 For C4-

[0284] C 20 Alkyl; R 10 ' is C4-C 20 Alkyl; R 13 H, C1-C6 alkyl, –(CH2) n OH or –(CH2) q N(CH3)2;

[0285] n is 2, 3 or 4; p1 is absent or is 1; p2 is absent or is 1; and q is 2, 3 or 4 ( Fig.17A ).

[0286] In some embodiments, the ionizable lipid of formula (XV) has one of the following structures ( Fig. 17B and Fig. 17C ):

[0287]

[0288] In another embodiment, the lipid is an ionizable lipid of formula (XVI)

[0289]

[0290] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0291] L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 6 C4-C 20 Alkyl; R 6 ' is C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R 7 ' is C4-C 20 Alkyl; R 13 H, C1-C6 alkyl, –(CH2) n OH or –(CH2) q N(CH3)2;

[0292] n is 2, 3 or 4; p1 is absent or is 1; p2 is absent or is 1; and q is 2, 3 or 4 ( Fig.18A ).

[0293] In some embodiments, the ionizable lipid of formula (XVI) has one of the following structures: Fig.18B and Fig. 18C ):

[0294]

[0295] In one aspect, the lipids described herein, such as transfection lipids, relate to lipid-nanoparticle compositions comprising an ionizable lipid of any one of formula (I) to formula (XVI). The lipid-nanoparticle composition may further comprise a helper lipid, a stabilizing lipid, a structural lipid, and a nucleic acid.

[0296] In some embodiments, the helper lipid in the lipid-nanoparticle composition is selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearo ... Phosphocholine (DSPC), 1,2-heneicosanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), l-oleoyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-docosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dialinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-racemic-(1-glycerol) sodium salt (DOPG) and mixtures thereof.

[0297] In some embodiments, the stabilizing lipid in the lipid-nanoparticle composition is 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG) with an average PEG molecular weight of 2000.

[0298] In some embodiments, the structural lipid in the lipid-nanoparticle composition is selected from the group consisting of cholesterol, cholesterol derivatives, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof.

[0299] In some embodiments, the nucleic acid, such as for transfection into immune cells, is selected from the group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), micro RNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, and mixtures thereof.

[0300] In other embodiments, the virus-mediated gene delivery system is composed of viruses that can be modified to replication-defective but can deliver nucleic acids for exogenous expression. In certain embodiments, adenovirus, retrovirus, RNA virus such as Sendai virus and slow virus can be used as a virus system for delivering exogenous nucleic acid sequences. For example, in some embodiments, Sendai virus and / or related paramyxovirus can be used as a carrier for nucleic acid delivery. Such viruses can be used to deliver mRNA by the RNA in its genome in the case of RNA viruses, or deliver mRNA by the mRNA expressed by the DNA in its genome in the case of DNA viruses.

[0301] In some embodiments, the present disclosure relates to a transfection composition comprising a nanoparticle composition and a nucleic acid for transfection into an immune cell such as a T cell.

[0302] Cell age reversal or rejuvenation is achieved by transient overexpression of one or more mRNAs encoding cell reprogramming factors. Such cell reprogramming factors may include transcription factors, epigenetic remodeling factors, or small molecules that affect mitochondrial function, proteolytic activity, heterochromatin levels, histone methylation, laminar polypeptides, cytokine secretion, or aging. In an embodiment, the cell reprogramming factor includes one or more of OCT4, SOX2, KLF4, c-MYC, LIN28, NANOG, and GLIS1. In an embodiment, the cell reprogramming factors are applied in different mass or molar ratios, such as OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG in a mass or molar ratio of a:b:c:d:e:f, wherein a, b, c, d, e and f can all be the same number (e.g., 1:1:1:1:1:1), some are the same number and some are different numbers (e.g., 3:1:1:1:1:1, 2:1:1:1:1:1, 2:2:1:1:1:1, 2:2:2:1:1:1:1, 7:1-7:1-7:1-7:1-7 (or in the case of a combination of less than 6 factors, 1-7:1-7:1-7:1-7:1-7, 1-7:1-7:1-7:1-7, 1-7:1-7:1-7, 1-7:1-7:1-7 or 1-7:1).

[0303] In some embodiments, mRNA encoding OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG is used in a mass ratio of (1-5:1-5:1-5:1-5:1-5:1-5), and mRNA encoding OCT4, c-MYC and NANOG is used in a mass ratio of (1-5:1-5:1-5); for example, mRNA encoding OSKMLN is used in a mass ratio of 5:1:1:1:1:1, and mRNA encoding OMN is used in a mass ratio of 5:1:1. In some further embodiments, mRNA encoding OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG is used in a mass ratio of (1-4:1-4:1-4:1-4:1-4:1-4), and mRNA encoding OCT4, c-MYC and NANOG is used in a mass ratio of (1-4:1-4:1-4); for example, mRNA encoding OSKMLN is used in a mass ratio of 4:1:1:1:1:1, and mRNA encoding OMN is used in a mass ratio of 4:2:1. In some further embodiments, mRNA encoding OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG is used in a mass ratio of (1-3:1-3:1-3:1-3:1-3:1-3), and mRNA encoding OCT4, c-MYC and NANOG is used in a mass ratio of (1-3:1-3:1-3); for example, mRNA encoding OSKMLN is used in a mass ratio of 3:1:1:1:1:1, and mRNA encoding OMN is used in a mass ratio of 1:1:1.

[0304] In some embodiments, mRNA encoding OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG is used in a molar ratio of (1-5:1-5:1-5:1-5:1-5:1-5), and mRNA encoding OCT4, c-MYC and NANOG is used in a molar ratio of (1-5:1-5:1-5); for example, mRNA encoding OSKMLN is used in a molar ratio of 5:1:1:1:1:1, and mRNA encoding OMN is used in a molar ratio of 5:1:1. In some further embodiments, mRNA encoding OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG is used in a molar ratio of (1-4:1-4:1-4:1-4:1-4:1-4), and mRNA encoding OCT4, c-MYC and NANOG is used in a molar ratio of (1-4:1-4:1-4); for example, mRNA encoding OSKMLN is used in a molar ratio of 4:1:1:1:1:1, and mRNA encoding OMN is used in a molar ratio of 4:2:1. In some further embodiments, mRNA encoding OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG is used in a molar ratio of (1-3:1-3:1-3:1-3:1-3:1-3), and mRNA encoding OCT4, c-MYC and NANOG is used in a molar ratio of (1-3:1-3:1-3); for example, mRNA encoding OSKMLN is used in a molar ratio of 3:3:2:1:1:1, and mRNA encoding OMN is used in a molar ratio of 2:1:1.

[0305] In an embodiment, a cytokine or mRNA expressing a cytokine is administered to an immune cell before, simultaneously with, or after a reprogramming factor. In some embodiments, the cytokine is IL-2. In some embodiments, the cytokine is a combination of IL-7 and IL-15. In some embodiments, the combination of IL-7 and IL-15 enhances T cell response or activity or is used to enhance T cell response or activity in a method.

[0306] In an embodiment, the method provided herein can be applied to any type of immune cell. The method disclosed herein can be used to rejuvenate immune cells (e.g., in vitro or in vitro) in culture to improve the function and efficacy for immune cell therapy. The immune cells used to treat patients can be autologous or allogeneic. Immune cells can be derived from patients or matching donors. For example, in ex vivo therapy, immune cells are obtained directly from patients to be treated, transfected with mRNA encoding cell reprogramming factors, as described herein, and re-implanted into the patient. Such immune cells can be obtained, for example, from a biopsy or surgical operation performed on a patient. Alternatively, mRNA encoding cell reprogramming factors can be used to directly transfect immune cells that need to be rejuvenated in vivo. In some embodiments, such engineering of immune cells is performed in vitro, i.e., in the manufacture of cell therapy products (such as autologous or allogeneic chimeric antigen receptors (CAR)-T, CAR-NK, CAR-M or CAR-NKT cells). In some embodiments, such chimeric antigen receptors target CD19, CD20, CD22, CD30, CD33, CD123, FLT3, BCMA, GD2, HER2, MUC1, B7-H3, IL13Ra2, TAG72, MUC16, BCMA, or at least one of any other antigens suitable for immunotherapy. In some embodiments, such engineering includes engineering to allow immune cells to express other proteins or peptides, such as growth factors and cytokines. In some embodiments, the cytokines include IL-7 and / or IL-15. In some embodiments, such engineering of immune cells is performed ex vivo, for example, in the manufacture of cell therapy products such as autologous or allogeneic chimeric antigen receptors (CAR)-T, CAR-NK, CAR-M or CAR-NKT cells. In some embodiments, the CAR-T cells provided herein target at least one of CD19, CD20, CD22, HER2, MUC1, CD30, CD33, CD123, FLT3, B7-H3, IL13Ra2, GD2, TAG72, MUC16, or BCMA via a chimeric antigen receptor. In some embodiments, the CAR-T cells provided herein target at least one of CD19 or BCMA via a chimeric antigen receptor. In some embodiments, the CAR-T cells provided herein target CD19 via a chimeric antigen receptor. In some embodiments, the CAR-T cells provided herein target BCMA via a chimeric antigen receptor.In some embodiments, the CAR-NK cells provided herein target CD19, FLT3, CD20, CD38, CD138, BCMS, CS1, CD3, CD5, CD7, NKG2D, HER2, EGFR, EpCAM, TF, B7-H6, HLA-G, CD24, CD44, CD133, mesothelin, or at least one of αFR through a chimeric antigen receptor. In some embodiments, the CAR-M cells provided herein target at least one of CD19, HER2, CD22, or ALK19 through a chimeric antigen receptor. In some embodiments, the CAR-NK cells provided herein are engineered to express IL-2 and / or IL-15. In some embodiments, the CAR-NKT cells provided herein target GD2 through a chimeric antigen receptor and are engineered to express IL-15. In such embodiments, the immune cell rejuvenation method described herein is performed in vitro during or after the manufacture of a cell therapy product. In other embodiments, such engineering of immune cells is performed in vitro, for example, in the so-called "in situ" production of CAR engineered cells. In such embodiments, the mRNA encoding CAR or other cell engineering molecules is injected into the subject or patient, for example, for the patient's immune cells (such as T cells, NK cells, macrophages, tumor infiltrating lymphocytes, dendritic cells and / or NKT cells) "in situ" CAR engineering, that is, in the patient, without removing cells for ex vivo transfection. In such embodiments, the immune cell rejuvenation method described herein is also carried out in vivo, wherein the mRNA encoding one or more reprogramming factors is injected into the patient before, at the same time or after the mRNA encoding CAR or other cell engineering molecules. In some embodiments, lipids and / or polymer nanoparticles are used to deliver mRNA encoding one or more reprogramming factors in vivo. In some embodiments, lipids and / or polymer nanoparticles are engineered for in vivo targeted delivery to immune cells, such as T cells, NK cells, macrophages, tumor infiltrating cells, dendritic cells and / or NKT cells in vivo. In yet other embodiments, in vivo treatment is performed in the absence of any other in vivo cell engineering to enhance or restore the effectiveness of the immune system and treat diseases associated with immune dysfunction or disorder, such as improving the effectiveness of the immune system to fight cancer or infection, or reducing inflammation.

[0307] Composition

[0308] In one aspect, provided herein are pharmaceutical compositions comprising rejuvenated immune cells, obtained by transfecting immune cells with one or more messenger RNA encoding one or more cell reprogramming factors, or by expressing at least one reprogramming factor from transfected mRNA in immune cells for no more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 consecutive days, transiently reprogramming immune cells to rejuvenate. In one aspect, provided herein are pharmaceutical compositions comprising rejuvenated immune cells, obtained by transfecting immune cells with one or more messenger RNA encoding one or more cell reprogramming factors, or by expressing at least one reprogramming factor from transfected mRNA in immune cells for no more than 3, 4, 5, or 6 consecutive days, transiently reprogramming immune cells to rejuvenate.

[0309] The compositions provided herein include reprogramming factor polynucleotides, proteins, polypeptides, and RNA and DNA vectors containing the reprogramming factor polynucleotides for producing the reprogramming factor proteins and polypeptides. In certain embodiments, the RNA vector is mRNA and transfected into immune cells using lipid or polymer nanoparticles. In certain embodiments, the RNA vector is an RNA viral vector. In certain embodiments, the DNA vector is a DNA viral vector, such as an adenovirus, a slow virus, or an adeno-associated virus, wherein the DNA viral vector expresses the mRNA encoding the reprogramming factor. In such embodiments, the method may involve processing, introducing immune cells, or exposing immune cells to mRNA, wherein the mRNA is provided by a DNA viral vector. RNA vectors (such as mRNA) are superior to viral vectors because they are non-integrative and provide better control of dosage and expression time. Additionally, they are less toxic to cells, thereby having a greater rejuvenation effect. Therefore, compared with using a viral vector, using mRNA to express reprogramming factors can lead to enhanced rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, anti-pathogenic effects, anti-cancer effects, or anti-inflammatory effects in exposed immune cells. In some embodiments, the reprogramming factors provided herein provide more robust cell rejuvenation because the reprogramming factors have been engineered to reduce any triggered immune response to the protein / polypeptide / RNA / DNA, increase the stability of the protein / polypeptide / RNA / DNA, or alter the activity of the protein / polypeptide or the activity of the protein / polypeptide expressed from the RNA / DNA, such as increased activity when compared to the wild-type reprogramming factor.

[0310] In some embodiments, the composition further comprises B18R polynucleotides, proteins, polypeptides, and RNA and DNA vectors containing the reprogramming factor polynucleotides for producing the B18R proteins and polypeptides. B18R is a B18R protein encoded by vaccinia virus, which acts as a soluble receptor for IFNα and IFNβ. The protein can exist as a soluble extracellular protein and a cell surface binding protein, and has a high affinity for type I IFN. Therefore, the binding of B18R protein can block the autocrine and paracrine functions of type I IFN. In addition, it can also bind to the cell surface of uninfected and infected cells, thereby reducing inflammatory signals. Because the synthetic mRNA induces cellular immune responses, recombinant B18R protein can be applied to avoid cellular immune activation and block the activity of type I IFN. In previous studies, the addition of recombinant B18R protein in long-term cell reprogramming experiments with synthetic mRNA resulted in increased cell viability and successful reprogramming of cells into induced pluripotent stem cells. In particular, codon-optimized B18R polynucleotide sequences that increase RNA expression in T cells can be used.

[0311] In some embodiments, polynucleotides encoding reprogramming factors from the Oct, Sox, Klf, Lin, Nanog, GLIS1 or Myc families may be codon optimized to increase mRNA expression levels in any immune cell of interest without altering the wild-type amino acid sequence of those reprogramming factors. In further embodiments, the polynucleotide sequences listed in Table 1 and / or provided with any sequence listing submitted with the present application are codon optimized.

[0312] In other embodiments, the polynucleotide sequence encoding the reprogramming factor from the Oct, Sox, Klf, Lin, Nanog, GLIS1 or Myc family has been altered to modulate the function of the encoded reprogramming factor. In some embodiments, the polynucleotide sequence has been altered so that when compared to its corresponding wild-type or unchanged reprogramming factor, the reprogramming factor encoded thereby triggers a reduced immune response, is more stable, and / or induces a more desirable activity. For example, Oct4MyoD comprises a transcriptional activation domain from the myogenic determinant gene MyoD, which can enhance its transcriptional activity.

[0313] In other embodiments, the polynucleotide sequence encoding a reprogramming factor from the Oct, Sox, Klf, Lin, Nanog, GLIS1 or Myc family is at least 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence in Table 1. In other embodiments, the polynucleotide sequence encoding a reprogramming factor from the Oct, Sox, Klf, Lin, Nanog, GLIS1 or Myc family is at least 98% or 99% identical to the polynucleotide sequence in Table 1. In yet other embodiments, the polynucleotide sequence encoding a reprogramming factor from the Oct, Sox, Klf, Lin, Nanog, GLIS1 or Myc family is at least 99% identical to the polynucleotide sequence in Table 1.

[0314] In other embodiments, the polynucleotide sequence encoding the reprogramming factor from the Oct, Sox, Klf, Lin, Nanog, GLIS1 or Myc family is about 90%, 85%, 80%, 75% or 70% identical to the polynucleotide sequence in Table 1. In other embodiments, the polynucleotide sequence encoding the reprogramming factor from the Oct, Sox, Klf, Lin, Nanog, GLIS1 or Myc family is about 80% or 85% identical to the polynucleotide sequence in Table 1. In yet other embodiments, the polynucleotide sequence encoding the reprogramming factor from the Oct, Sox, Klf, Lin, Nanog, GLIS1 or Myc family is about 75% identical to the polynucleotide sequence in Table 1. In other embodiments, the polynucleotide sequence encoding the reprogramming factor from the Oct, Sox, Klf, Lin, Nanog, GLIS1 or Myc family is about 70% identical to the polynucleotide sequence in Table 1.

[0315] In an embodiment, the reprogramming factor is a protein, such as a transcription factor, which plays a role in converting adult or differentiated immune cells into pluripotent stem cells. The term "reprogramming factor" further includes any analog molecules that simulate the function of the factor. In an embodiment, the reprogramming factor is a factor from the Oct family, the Sox family, the Klf family, the Myc family, the Nanog family, or the Lin family.

[0316] The "Oct family" refers to a family of octamer ("Oct") transcription factors that play a key role in maintaining pluripotency. POU5F1 (POU domain, class 5, transcription factor 1), also known as Oct3 / 4, is a representative of the Oct family. The absence of Oct3 / 4 in Oct-3 / 4+ cells (such as blastomeres and embryonic stem cells) leads to spontaneous trophoblast differentiation, while the presence of Oct-3 / 4 thus causes the pluripotency and differentiation potential of embryonic stem cells. Exemplary Oct3 / 4 proteins are proteins encoded by the mouse Oct3 / 4 gene (Genbank accession number NM_013633) and the human Oct3 / 4 gene (Genbank accession number NM_002701). Thus, the terms "Oct3 / 4", "Oct4", "OCT4", "Oct4 protein", "OCT4 protein", etc. refer to any naturally occurring form of the Octomer 4 transcription factor, or a variant thereof that maintains Oct4 transcription factor activity (e.g., within at least 50%, 80%, 90% or 100% activity compared to wild-type Oct4 as measured by methods known in the art). In some embodiments, the variant has at least 90% amino acid sequence identity over its entire sequence compared to a naturally occurring Oct4 polypeptide. In other embodiments, the Oct4 protein is a protein as identified by Genbank reference ADW77327.1. In other embodiments, the Oct4 protein is Oct4MyoD, which contains a transactivation domain of MyoD for enhancing transcription factor activity. In other embodiments, the Oct4 polynucleotide sequence is codon optimized for immune cell expression (such as T cell expression).

[0317] Oct reprogramming factor refers to any naturally occurring member of the octamer family of transcription factors, or a variant thereof that maintains similar transcription factor activity (at least 50%, 80% or 90% activity) compared to the closest related naturally occurring Oct family member, or a polypeptide comprising at least the DNA binding domain of a naturally occurring Oct family member, and may further comprise a transcriptional activation domain. Exemplary Oct polypeptides include Oct-1, Oct-2, Oct-3 / 4, Oct-6, Oct-7, Oct-8, Oct-9 and Oct-11. For example, Oct3 / 4 (referred to herein as "Oct4") contains a POU domain, i.e., a 150 amino acid sequence conserved in Pit-1, Oct-1, Oct-2 and uric-86. See, Ryan, AK and Rosenfeld, MG Genes Dev.11, 1207-1225 (1997). In some embodiments, the variant has at least 85%, 90% or 95% amino acid sequence identity over its entire sequence compared to a naturally occurring Oct polypeptide family member, such as those listed above or such as those listed in Genbank Accession Nos. NP002692.2 (human Oct4) or NP038661.1 (mouse Oct4). The Oct polypeptide (e.g., Oct3 / 4) can be from humans, mice, rats, cows, pigs or other animals.

[0318] In some embodiments, the OCT4 reprogramming factor protein / polypeptide provided herein is encoded by a codon-optimized polynucleotide sequence of SEQ ID NO: 1. Therefore, the polynucleotide of SEQ ID NO: 1 is different from the polynucleotide sequence of wild-type OCT4, but the amino acid sequence encoded by it is not different. In other embodiments, codon-optimized nucleotide sequences, such as SEQ ID NO: 4, can also be modified to encode, for example, a more robust OCT4 reprogramming factor, which, when compared to a protein or polypeptide corresponding to a wild-type nucleotide sequence, causes a smaller triggering immune response, is more stable and / or provides a more desirable level of activity. In some embodiments, the OCT4 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO: 1. In some embodiments, the OCT4 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 1. In some embodiments, the OCT4 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence consisting essentially of SEQ ID NO: 1. In some embodiments, the OCT4 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence consisting of SEQ ID NO: 1.

[0319] "Sox family" refers to genes encoding SRY (sex determining region Y)-box 2 (also known as SOX2) associated with maintaining pluripotency. Exemplary Sox2 proteins are proteins encoded by the mouse Sox2 gene (Genbank accession number NM_011443) and the human Sox2 gene (Genbank accession number NM_003106). Therefore, the terms "Sox2", "SOX2", "Sox2 protein", "SOX2 protein", etc. as mentioned herein include any naturally occurring form of Sox2 transcription factor, or a variant thereof that maintains Sox2 transcription factor activity (e.g., as measured by methods known in the art, compared to wild-type Sox2, at least 50%, 80%, 90% or 100% activity). In some embodiments, the variant has at least 90% amino acid sequence identity over its entire sequence compared to a naturally occurring Sox2 polypeptide. In other embodiments, the Sox2 protein is a protein as identified by NCBI reference NP_003097.1. In other embodiments, the Sox2 protein and / or polynucleotide sequence is optimized for immune cell expression and / or activity, such as T cell expression and / or activity.

[0320] Sox reprogramming factor refers to any naturally occurring member of the SRY-related HMG-box (Sox) transcription factor, characterized by the presence of a high mobility group (HMG) domain, or a variant thereof that maintains similar transcription factor activity (within at least 50%, 80% or 90% activity) compared to the closest related naturally occurring family member, or a polypeptide comprising at least the DNA binding domain of a naturally occurring family member, and may further comprise a transcriptional activation domain. See, e.g., Dang, DT et al., Int. J. Biochem. Cell Biol. 32: 1103-1121 (2000). Exemplary Sox polypeptides include, for example, Sox1, Sox-2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox-21, and Sox30. In some embodiments, the variant has at least 85%, 90% or 95% amino acid sequence identity over its entire sequence compared to a naturally occurring Sox polypeptide family member, such as those listed above or such as those listed in Genbank Accession No. CAA83435 (human Sox2). The Sox polypeptide (e.g., Sox1, Sox2, Sox3, Sox15 or Sox18) can be from human, mouse, rat, cow, pig or other animal.

[0321] In some embodiments, the SOX2 reprogramming factor protein / polypeptide provided herein is encoded by a codon-optimized polynucleotide sequence of SEQ ID NO:2. Therefore, the polynucleotide of SEQ ID NO:2 is different from the polynucleotide sequence of wild-type SOX2, but the amino acid sequence encoded by it is not different. In other embodiments, codon-optimized nucleotide sequences, such as SEQ ID NO:2, can also be modified to encode a more robust SOX2 reprogramming factor, which, when compared to a protein or polypeptide corresponding to a wild-type nucleotide sequence, causes a smaller triggering immune response, is more stable and / or provides a more desirable level of activity. In some embodiments, the SOX2 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO:2. In some embodiments, the SOX2 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity to SEQ ID NO: 2. In some embodiments, the SOX2 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 2. In some embodiments, the SOX2 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence consisting essentially of SEQ ID NO: 2. In some embodiments, the SOX2 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence consisting of SEQ ID NO: 2.

[0322] "Klf family" refers to Kruppel-like factor 4 or the "Klf" gene encoding the Klf4 protein is a protein encoded by the mouse klf4 gene (Genbank accession number NM_010637) and the human klf4 gene (Genbank accession number NM_004235). Therefore, the terms "KLF4", "KLF4 protein", etc. as mentioned herein include any naturally occurring form of the KLF4 transcription factor, or a variant thereof that maintains KLF4 transcription factor activity (e.g., as measured by methods known in the art, compared to wild-type KLF4, within at least 50%, 80%, 90% or 100% activity). In some embodiments, the variant has at least 90% amino acid sequence identity over its entire sequence compared to the naturally occurring KLF4 polypeptide. In other embodiments, the KLF4 protein is a protein as identified by NCBI reference NP_004226.3. In other embodiments, the Klf4 protein and / or polynucleotide sequence is optimized for immune cell expression and / or activity (such as T cell expression and / or activity).

[0323] In other embodiments, the Klf reprogramming factor refers to any naturally occurring member of the Kruppel-like factor (Klf) family, a zinc finger protein containing an amino acid sequence similar to the amino acid sequence of the Drosophila embryonic pattern regulator Kruppel, or a variant of a naturally occurring member that maintains similar transcription factor activity (at least 50%, 80% or 90% activity) compared to the closest related naturally occurring family member, or a polypeptide comprising at least the DNA binding domain of a naturally occurring family member, and may further comprise a transcriptional activation domain. See, Dang, DT, Pevsner, J. and Yang, VW, Cell Biol. 32, 1103-1121 (2000). Exemplary Klf family members include Klf1, Klf2, Klf3, Klf-4, Klf5, Klf6, Klf7, Klf8, Klf9, Klf10, Klf11, Klf12, Klf13, Klf14, Klf15, Klf16 and Klf17. In some embodiments, the variant has at least 85%, 90%, or 95% amino acid sequence identity over its entire sequence compared to a naturally occurring Klf polypeptide family member, such as those listed above or such as those listed in Genbank Accession Nos. CAX16088 (mouse Klf4) or CAX14962 (human Klf4). The Klf polypeptides (e.g., Klf1, Klf4, and Klf5) can be from humans, mice, rats, cows, pigs, or other animals.

[0324] In some embodiments, the KLF4 reprogramming factor protein / polypeptide provided herein is encoded by a codon-optimized polynucleotide sequence of SEQ ID NO:4. Therefore, the polynucleotide of SEQ ID NO:4 is different from the polynucleotide sequence of wild-type KLF4, but the amino acid sequence encoded by it is not different. In other embodiments, codon-optimized nucleotide sequences, such as SEQ ID NO:4, can also be modified to encode, for example, a more robust KLF4 reprogramming factor, which, when compared to a protein or polypeptide corresponding to a wild-type nucleotide sequence, causes a smaller triggering immune response, is more stable and / or provides a more desirable level of activity. In some embodiments, the KLF4 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO:4. In some embodiments, the KLF4 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity to SEQ ID NO: 4. In some embodiments, the KLF4 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 4. In some embodiments, the KLF4 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence consisting essentially of SEQ ID NO: 4. In some embodiments, the KLF4 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence consisting of SEQ ID NO: 4.

[0325] The factor of the Myc family refers to a factor encoded by the myc proto-oncogene related to cancer.Exemplary c-Myc protein is a protein encoded by the mouse c-myc gene (Genbank accession number NM_010849) and the human c-myc gene (Genbank accession number NM_002467).N-Myc or L-myc are also used as possible reprogramming factors to replace c-Myc.Therefore, the terms "c-Myc", "C-MYC", "c-Myc protein", "C-MYC protein" etc. as mentioned herein include any naturally occurring form of cMyc transcription factor, or a variant thereof maintaining cMyc transcription factor activity (e.g., as measured by methods known in the art, compared to wild-type cMyc, at least 50%, 80%, 90% or 100% activity).In some embodiments, compared to naturally occurring c-Myc polypeptides, variants have at least 90% amino acid sequence identity over their entire sequence. In other embodiments, the c-Myc protein is the protein identified as NCBI reference NP_002458.2.

[0326] The Myc family of cellular genes consists of c-myc, N-myc and L-myc, and reference to Myc refers to any naturally occurring member of the Myc family (see, e.g., Adhikary, S. and Eilers, M. Nat. Rev. Mol. Cell Biol. 6:635-645 (2005)), or a variant thereof that maintains similar transcription factor activity (within at least 50%, 80% or 90% activity) compared to the closest related naturally occurring family member, or a polypeptide comprising at least the DNA binding domain of a naturally occurring family member, and may further comprise a transcriptional activation domain. Exemplary Myc polypeptides include, e.g., c-Myc, N-Myc and L-Myc. In some embodiments, the variant has at least 85%, 90% or 95% amino acid sequence identity over its entire sequence compared to naturally occurring Myc polypeptide family members, such as those listed above or such as those listed in Genbank Accession No. CAA25015 (human Myc). The Myc polypeptide (eg, c-Myc) can be from human, mouse, rat, cow, pig, or other animals.

[0327] In some embodiments, the c-Myc reprogramming factor protein / polypeptide provided herein is encoded by a codon-optimized polynucleotide sequence of SEQ ID NO:3. Therefore, the polynucleotide of SEQ ID NO:3 is different from the polynucleotide sequence of wild-type c-Myc, but the amino acid sequence encoded by it is not different. In other embodiments, codon-optimized nucleotide sequences, such as SEQ ID NO:3, can also be modified to encode, for example, a more robust c-Myc reprogramming factor, which, when compared to a protein or polypeptide corresponding to a wild-type nucleotide sequence, causes a smaller triggering immune response, is more stable and / or provides a more desirable level of activity. In some embodiments, c-Myc reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO:3. In some embodiments, c-Myc reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO:3. In some embodiments, c-Myc reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO:3. In some embodiments, c-Myc reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence consisting essentially of SEQ ID NO:3. In some embodiments, c-Myc reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence consisting of SEQ ID NO:3. In other embodiments, c-Myc protein and / or polynucleotide sequence is optimized for immune cell expression and / or activity (such as T cell expression and / or activity).

[0328] The term "Nanog" or "nanog" refers to a transcription factor that is closely related to the self-renewal of undifferentiated embryonic stem cells. In humans, the protein is encoded by the NANOG gene. Exemplary nanog is a protein encoded by the mouse gene (Genbank Accession No. XM.sub.13 132755) and the human Nanog gene (Genbank Accession No. NM_024865). The term "Nanog" or "nanog" and the like include any naturally occurring form of the Nanog transcription factor, or a variant thereof that maintains the activity of the Nanog transcription factor (e.g., as measured by methods known in the art, within at least 50%, 80%, 90% or 100% activity compared to the wild-type Nanog). In some embodiments, the variant has at least 90% amino acid sequence identity throughout its entire sequence compared to the naturally occurring Nanog polypeptide. In other embodiments, the Nanog protein is a protein as identified by NCBI reference NP_079141. In other embodiments, the Nanog protein and / or polynucleotide sequence is optimized for immune cell expression and / or activity, such as T cell expression and / or activity.

[0329] In some embodiments, the Nanog reprogramming factor protein / polypeptide provided herein is encoded by a codon-optimized polynucleotide sequence of SEQ ID NO: 6. Therefore, the polynucleotide of SEQ ID NO: 6 is different from the polynucleotide sequence of wild-type Nanog, but the amino acid sequence encoded by it is not different. In other embodiments, codon-optimized nucleotide sequences, such as SEQ ID NO: 6, can also be modified to encode, for example, a more robust Nanog reprogramming factor that triggers a smaller triggering immune response, is more stable and / or provides a more desirable level of activity when compared to a protein or polypeptide corresponding to a wild-type nucleotide sequence. In some embodiments, the Nanog reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO: 6. In some embodiments, the Nanog reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity to SEQ ID NO: 6. In some embodiments, the Nanog reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 6. In some embodiments, the Nanog reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence consisting essentially of SEQ ID NO: 6. In some embodiments, the Nanog reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence consisting of SEQ ID NO: 6.

[0330] The term "Lin28" or "Lin-28 homolog A" is a protein encoded by the LIN28 gene in humans. Exemplary Lin28 is a protein encoded by a mouse gene (Genbank accession number NM_145833) and a human Lin28 gene (Genbank accession number NM_024674). Therefore, the term "Lin28" or "Lin28 homolog A" and the like as referred to herein include any naturally occurring form of Lin28 transcription factor, or a variant thereof that maintains Lin28 transcription factor activity (e.g., within at least 50%, 80%, 90% or 100% activity compared to wild-type Lin28 as measured by methods known in the art). In some embodiments, the variant has at least 90% amino acid sequence identity over its entire sequence compared to a naturally occurring Lin28 polypeptide. In other embodiments, the Lin28 protein is a protein as identified by NCBI reference NP_078950. In other embodiments, the Lin28 protein and / or polynucleotide sequence is optimized for immune cell expression and / or activity, such as T cell expression and / or activity.

[0331] In some embodiments, the Lin28 reprogramming factor protein / polypeptide provided herein is encoded by a codon-optimized polynucleotide sequence of SEQ ID NO:5. Therefore, the polynucleotide sequence of SEQ ID NO:5 is different from the polynucleotide sequence of wild-type Lin28, but the amino acid sequence encoded by it is not different. In other embodiments, codon-optimized nucleotide sequences, such as SEQ ID NO:5, can also be modified to encode, for example, a more robust Lin28 reprogramming factor, which, when compared to a protein or polypeptide corresponding to a wild-type nucleotide sequence, causes a smaller triggering immune response, is more stable and / or provides a more desirable level of activity. In some embodiments, the Lin28 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO:5. In some embodiments, the Lin28 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity to SEQ ID NO: 5. In some embodiments, the Lin28 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 5. In some embodiments, the Lin28 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence consisting essentially of SEQ ID NO: 5. In some embodiments, the Lin28 reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence consisting of SEQ ID NO: 5.

[0332] In some embodiments, the reprogramming factor protein / polypeptide provided herein in SEQ ID NO.7-14 is encoded by a nucleotide sequence that has been codon-optimized for expression in T cells. Therefore, in some embodiments, the polynucleotides of SEQ ID NO:7-14 are different from the polynucleotide sequences of the corresponding wild-type reprogramming factors, although the amino acid sequences they encode are not different. In other embodiments, the codon-optimized nucleotide sequences of the reprogramming factors described herein, such as SEQ ID NO:7-14, can also be modified to encode, for example, more robust T cell reprogramming factors, which, when compared to proteins or polypeptides corresponding to wild-type nucleotide sequences, trigger a smaller triggering immune response, are more stable and / or provide a more desirable level of activity. In some embodiments, the reprogramming factor protein / polypeptide optimized by T cells is encoded by a polynucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with any one of the sequences of SEQ ID NO:7-14. In some embodiments, the T cell optimized reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with any one of the sequences of SEQ ID NO:7-14. In some embodiments, the T cell optimized reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence comprising any one of the sequences of SEQ ID NO:7-14. In some embodiments, the T cell optimized reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence consisting essentially of any one of the sequences of SEQ ID NO:7-14. In some embodiments, the T cell optimized reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence consisting of any one of the sequences of SEQ ID NO:7-14.

[0333] In some embodiments, the T cell optimized reprogramming factor comprises OCT4MyoD for T cells (T-OCT4MyoD, SEQ ID NO: 7), or the reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO: 7. In some embodiments, the T cell optimized reprogramming factor is encoded by a polynucleotide sequence having about 96% sequence identity with SEQ ID NO: 7. In some embodiments, the T cell optimized reprogramming factor is encoded by a polynucleotide sequence having about 97% sequence identity with SEQ ID NO: 7. In some embodiments, the T cell optimized reprogramming factor is encoded by a polynucleotide sequence having about 98% sequence identity with SEQ ID NO: 7. In some embodiments, the T cell optimized reprogramming factor is encoded by a polynucleotide sequence having about 99% sequence identity with SEQ ID NO: 7. In some embodiments, the T cell optimized reprogramming factor is encoded by a polynucleotide sequence comprising SEQ ID NO: 7. In some embodiments, the T cell optimized reprogramming factor is encoded by a polynucleotide sequence consisting of SEQ ID NO:7.

[0334] In some embodiments, the T cell optimized reprogramming factor comprises OCT4MyoD for T cells (T-OCT4MyoD, SEQ ID NO: 7) or the reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO: 7. In some embodiments, the T cell optimized reprogramming factor comprises B18R for T cells (T-B18R, SEQ ID NO: 8), or the reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO: 8. In some embodiments, the T cell optimized reprogramming factor comprises B18R for T cells (T-B18R, SEQ ID NO: 8), or the reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO: 8. In some embodiments, the T cell optimized reprogramming factor comprises KLF4 for T cells (T-KLF4, SEQ ID NO: 9), or the reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO: 9. In some embodiments, the T cell optimized reprogramming factor comprises KLF4 for T cells (T-KLF4, SEQ ID NO: 9), or the reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO: 9. In some embodiments, the T cell optimized reprogramming factor comprises LIN28 for T cells (T-LIN28, SEQ ID NO: 10), or the reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO: 10.In some embodiments, the T cell optimized reprogramming factor comprises LIN28 for T cells (T-LIN28, SEQ ID NO: 10), or the reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO: 10. In some embodiments, the T cell optimized reprogramming factor comprises NANOG for T cells (T-NANOG, SEQ ID NO: 11), or the reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO: 11. In some embodiments, the T cell optimized reprogramming factor comprises NANOG for T cells (T-NANOG, SEQ ID NO: 11), or the reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO: 11. In some embodiments, the T cell optimized reprogramming factor comprises OCT4 for T cells (T-OCT4, SEQ ID NO: 12), or the reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO: 12. In some embodiments, the T cell optimized reprogramming factor comprises OCT4 for T cells (T-OCT4, SEQ ID NO: 12), or the reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO: 12. In some embodiments, the T cell optimized reprogramming factor comprises SOX2 for T cells (T-SOX2, SEQ ID NO: 13), or the reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO: 13.In some embodiments, the T cell optimized reprogramming factor comprises SOX2 for T cells (T-SOX2, SEQ ID NO: 13), or the reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO: 13. In some embodiments, the T cell optimized reprogramming factor comprises cMYC for T cells (T-cMyc, SEQ ID NO: 14), or the reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with SEQ ID NO: 14. In some embodiments, the T cell optimized reprogramming factor comprises cMYC for T cells (T-cMyc, SEQ ID NO: 14), or the reprogramming factor protein / polypeptide is encoded by a polynucleotide sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity to SEQ ID NO: 14.

[0335] In one embodiment, the RNA vector provided herein encodes the expression of a combination of one, two, three, four, five, six or more reprogramming factors. In one embodiment, the reprogramming factor is selected from Oct4, Klf4, Sox2, c-Myc (or L-myc), Lin28 and Nanog. In one embodiment, the reprogramming factor is Oct4, Klf4, Sox2, c-Myc (or L-myc), Lin28 and Nanog. In one embodiment, the reprogramming factor is Oct4, Klf4, Sox2, c-Myc (or L-myc). In one embodiment, the reprogramming factor is Oct4, Klf4, Sox2. In one embodiment, the reprogramming factor is Oct4, Sox2, Lin28 and Nanog.

[0336] In certain embodiments, the composition comprising rejuvenated immune cells for cell therapy may further comprise one or more additional factors, such as nutrients, cytokines, growth factors, vaccine antigens (including cancer vaccine antigens), extracellular matrix (ECM) components, antibiotics, antioxidants or immunosuppressants to improve immune cell function or viability. The composition may further comprise a pharmaceutically acceptable carrier.

[0337] Examples of growth factors include, but are not limited to, fibroblast growth factor (FGF), insulin-like growth factor (IGF), transforming growth factor β (TGF-B), epiregulin, epidermal growth factor ("EGF"), endothelial cell growth factor ("ECGF"), nerve growth factor ("NGF"), leukemia inhibitory factor ("LIF"), bone morphogenetic protein-4 ("BMP-4"), hepatocyte growth factor ("HGF"), vascular endothelial growth factor-A ("VEGF-A"), and cholecystokinin octapeptide.

[0338] Examples of ECM components include, but are not limited to, proteoglycans (e.g., chondroitin sulfate, heparan sulfate, and keratan sulfate), non-proteoglycan polysaccharides (e.g., hyaluronic acid), fibers (e.g., collagen and elastin), and other ECM components (e.g., fibronectin and laminin).

[0339] Examples of immunosuppressants include, but are not limited to, steroidal (e.g., prednisone) or nonsteroidal (e.g., sirolimus (Rapamune, Wyeth-Ayerst Canada), tacrolimus (Prograf, Fujisawa Canada), and anti-IL2R daclizumab (Zenapax, Roche Canada). Other immunosuppressants include 15-deoxyspergualin, cyclosporine, methotrexate, rapamycin, Rapamune (sirolimus / rapamycin), FK506, or lisofylline (LSF).

[0340] One or more pharmaceutically acceptable excipients may also be included. Examples include, but are not limited to, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.

[0341] For example, an antimicrobial agent for preventing or stopping microbial growth may be included. Non-limiting examples of antimicrobials suitable for the present disclosure include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimerosal (thimersol) and combinations thereof. Antimicrobials also include antibiotics that can also be used to prevent bacterial infections. Example antibiotics include amoxicillin, penicillin, sulfonamides, cephalosporins, erythromycin, streptomycin, gentamicin, tetracycline, methylerythromycin, ciprofloxacin, azithromycin, etc. Antifungal agents are also included, such as miconazole (myconazole) and terconazole (terconazole).

[0342] Various antioxidants may also be included, such as molecules with thiol groups, such as reduced glutathione (GSH) or its precursors, glutathione or glutathione analogs, glutathione monoesters and N-acetylcysteine. Other suitable antioxidants include superoxide dismutase, catalase, vitamin E, Trolox, lipoic acid, lazaroids, butylated hydroxyanisole (BHA), vitamin K, etc.

[0343] Excipients suitable for injectable compositions include water, alcohol, polyol, glycerol, vegetable oil, phospholipid and surfactant. Carbohydrates such as sugars, derivative sugars such as sugar alcohols, aldonic acid, esterified sugars and / or sugar polymers can be present as excipients. Specific carbohydrate excipients include, for example: monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, inositol, etc. Excipients can also include inorganic salts or buffers such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium dihydrogen phosphate, disodium hydrogen phosphate and combinations thereof.

[0344] Acid or alkali can also exist as excipient.The non-limiting examples of operable acid include the acid selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid and combinations thereof.The example of suitable alkali includes but is not limited to the alkali selected from the group consisting of the following: sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate and combinations thereof.

[0345] Generally, the optimum amount of any independent excipient is determined by routine experiments, i.e., by preparing the composition containing different amounts of excipients (ranging from low to high), checking stability and other parameters, then determining to obtain the best performance without the scope of significant side effects. However, usually the amount of excipient in the composition is about 1 % by weight to about 99 % by weight of the excipient, preferably about 5 % by weight to about 98 % by weight, more preferably about 15 % by weight to about 95 % by weight, including a concentration less than 30 % by weight in some embodiments. These aforementioned pharmaceutical excipients and other excipients are described in " Remington: The Science & Practice of Pharmacy ", 19th edition, Williams & Williams, (1995), " Physician ' s Desk Reference ", 52nd edition, Medical Economics, Montvale, NJ (1998) and Kibbe, AH, Handbook of Pharmaceutical Excipients, 3rd edition, American Pharmaceutical Association, Washington, DC, 2000.

[0346] How to use

[0347] The methods and compositions provided herein can be used to treat or alleviate any condition, disease or disorder associated with immune cell function, such as, but not limited to, neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, dementia, spinal cord injury and stroke), cardiovascular and peripheral vascular diseases (e.g., atherosclerosis, peripheral arterial disease (PAD), hematoma, calcification, thrombosis, embolism and aneurysm), ocular diseases (e.g., age-related macular degeneration, glaucoma, cataracts, dry eyes, diabetic retinopathy, vision loss), dermatological diseases (dermal atrophy and thinning, elastic fiber lysis and skin wrinkling, sebaceous gland hyperplasia or hypoplasia, senile lentigo and other pigmentation abnormalities, graying, hair loss or thinning and chronic skin ulcers), autoimmune diseases (e.g., polymyalgia rheumatica (PMR), giant cell arteritis (GCA), rheumatoid arthritis (RA), crystalline arthropathy and spinal cord injury), and other conditions, such as leukoderma, ... The invention relates to diseases and conditions of the present invention, including, but not limited to, osteoarthritis, spondylolisthesis, osteoporosis, diabetes mellitus, adrenal insufficiency, various forms of hypogonadism, and endocrine malignancies, musculoskeletal disorders (e.g., arthritis, osteoporosis, myeloma, gout, Paget's disease, fractures, bone marrow failure syndrome, ankylosing skeletal hypertrophy, hematogenous osteomyelitis, muscular atrophy, peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, primary lateral sclerosis, and myasthenia gravis), digestive disorders (e.g., cirrhosis, liver fibrosis, Barrett's esophagus), respiratory disorders (e.g., pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, chronic bronchitis, pulmonary embolism (PE), lung cancer, and infections), conditions associated with cell proliferation, and any other diseases and conditions associated with aging. The methods and compositions provided herein can be used to treat fibrosis, including but not limited to fibrosis of the heart, liver, lung, kidney, pancreas, bladder, uterus, or gastrointestinal tract. The methods and compositions provided herein can be used to treat any disease or illness associated with inflammation, such as neuroinflammation or inflammation of the heart, liver, lung, kidney, pancreas, bladder, uterus, or gastrointestinal tract. The methods and compositions provided herein can be used to treat diseases or illnesses associated with aging and / or chronic tissue damage, such as diabetes, fibrosis, cancer (including solid tumors), neurodegeneration, arthritis, sarcopenia. The methods and compositions provided herein can be used to destroy, inhibit, or rejuvenate senescent cells, for example, for aging therapy, aging effects, aging effects, or aging blocking.

[0348] The terms "neoplasm" and "tumor" are used interchangeably herein and refer to an abnormal mass of tissue, wherein the growth of the mass exceeds the growth of normal tissue and is not coordinated with the growth of normal tissue. A neoplasm or tumor may be "benign" or "malignant", depending on the following characteristics: degree of cell differentiation (including morphology and function), growth rate, local invasion and metastasis. "Benign tumors" are usually well differentiated, characteristically grow slower than malignant tumors and remain confined to the site of origin. In addition, benign tumors do not have the ability to infiltrate, invade or metastasize to distant sites. Exemplary benign tumors include, but are not limited to, lipomas, chondromas, adenomas, soft droops, senile hemangiomas, seborrheic keratosis, lentigo and sebaceous hyperplasia. In some cases, certain "benign" tumors may subsequently cause malignant tumors, which may be caused by additional genetic changes in a subpopulation of tumor cells of the tumor, and these tumors are referred to as "premalignant tumors". An exemplary premalignant tumor is a teratoma. In contrast, "malignant tumors" are usually poorly differentiated (degenerative) and have a characteristic rapid growth accompanied by progressive infiltration, invasion and destruction of surrounding tissues. In addition, malignant tumors usually have the ability to transfer to distant sites.Term " transfer (metastasis) ", " metastatic " or " transfer (metastasize) " refers to that cancer cells spread or migrate to another organ or tissue from primary or original tumor, and can usually be identified by " secondary tumor " or " secondary cell mass " of primary or original tumor tissue type rather than " secondary tumor " or " secondary cell mass " of the organ or tissue where secondary (metastatic) tumor is located.For example, prostate cancer that has migrated to bone is considered as metastatic prostate cancer, and includes cancerous prostate cancer cells grown in bone tissue.

[0349] "Cancer" refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. See, e.g., Stedman's Medical Dictionary, 25th ed.; Hensyl, ed.; Williams & Wilkins: Philadelphia, 1990. Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendothelioma, hemangioendothelioma); appendix cancer; benign monoclonal gammopathy; bile duct cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., breast adenocarcinoma, breast papillary carcinoma, breast cancer, medullary breast cancer); brain cancer (e.g., meningioma, glioblastoma, glioma (e.g., astrocytoma, oligodendroglioma); glioma), medulloblastoma); bronchogenic carcinoma; carcinoid tumors; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial cancer; ependymoma; endothelial sarcomas (e.g., Kaposi sarcoma, multiple idiopathic hemorrhagic sarcomas); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., esophageal adenocarcinoma, Barrett's gland cancer); Ewing's sarcoma; eye cancer (e.g., intraocular melanoma, retinoblastoma); familial hypereosinophilia; gallbladder cancer; stomach cancer (e.g., gastric adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), laryngeal cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal carcinoma, oropharyngeal cancer)); hematopoietic cancer (e.g., leukemia, such as acute lymphoblastic leukemia (ALL) (e.g., B cell ALL, T cell ALL), acute myeloid leukemia (AML) (e.g., B cell AML, T cell AML), chronic myeloid leukemia (CML) (e.g., B cell CML, T cell CML) and chronic lymphocytic leukemia (CLL) (e.g., B cell CLL, T cell CLL);Lymphoma, such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL, such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenstrom's macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, and primary central nervous system (CNS) lymphoma; and T-cell NHL, such as precursor T-lymphocytic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphomas as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharyngeal cancer; inflammatory myofibroblastic blastoma; immune cell amyloidosis; kidney cancer (e.g., Wilms' tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular carcinoma (HCC), malignant liver cancer); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorders (MPD) (e.g., polycythemia vera (PV), essential thrombocythemia (ET), idiopathic myeloid metaplasia (AMM) also known as myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis type 1 or type 2 (NF), schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), islet cell tumor); penile cancer (e.g., Paget's disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT); plasmacytoma; paraneoplastic syndrome; intraepithelial neoplasia;Prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small intestine cancer (e.g., appendix cancer); soft tissue sarcomas (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland cancer; small intestine cancer; sweat gland cancer; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary thyroid carcinoma, papillary thyroid carcinoma (PTC), medullary thyroid carcinoma); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva). ;

[0350] "Therapeutically effective dose or amount" means the amount of rejuvenated immune cells that cause a positive therapeutic response in a subject in need of rejuvenating immune cells, such as the amount of normal anatomy and / or physiology restored at the treatment site. Rejuvenated cells can be produced by transfection in vitro, ex vivo or in vivo with RNA or RNA vectors, which are used to express one or more reprogramming nucleotide sequences encoding one or more cell reprogramming factors, as described herein (including SEQ ID NO: 1-14). Therefore, for example, a "positive therapeutic response" will be an improvement in immune cell-related diseases or conditions associated with rejuvenated immune cell therapy, and / or an improvement in one or more symptoms of immune cell-related diseases or conditions associated with the therapy, such as functional recovery, pain relief, improved endurance, increased strength, increased activity and / or improved cognitive function. The exact amount required (immune cells or mRNA) will vary from subject to subject, depending on the species, age and overall condition of the subject, the severity of the condition being treated, the mode of administration, etc. The appropriate "effective" amount in any individual case can be determined by a person of ordinary skill in the art using routine experiments based on the information provided herein.

[0351] As used herein, the terms "subject," "individual," and "patient" are used interchangeably herein and refer to any vertebrate subject, including, but not limited to, humans and other primates, including non-human primates such as chimpanzees and other apes and monkeys; livestock such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; rodents such as mice, rats, rabbits, hamsters, and guinea pigs; birds, including domestic birds, wild birds, and game birds such as chickens, turkeys and other galliformes, ducks, geese, and the like. In some cases, the methods of the present disclosure can be used for experimental animals, veterinary applications, and the development of animal models of disease. The term does not indicate a particular age. Thus, both adult and neonatal individuals are intended to be covered.

[0352] Reagent test kit

[0353] The present disclosure also provides a test kit including one or more containers equipped with a composition, the composition comprising one or more mRNA encoding one or more cell reprogramming factors for transient reprogramming of immune cells. The test kit may further include a transfection agent, a culture medium for culturing cells, and optionally one or more other factors, such as growth factors, ECM components, antibiotics, etc. The mRNA and / or other compositions encoding the cell reprogramming factors may be in liquid form or lyophilized. Such test kits may also include components for preserving or maintaining mRNA to prevent its degradation. Such components may be RNAse-free or may be protected from the effects of RNAse. Suitable containers for compositions include, for example, bottles, vials, syringes, and test tubes. The container may be formed by various materials (including glass or plastic). The container may have a sterile inlet port (for example, the container may be an intravenous solution bag or a vial with a stopper that may be pierced by a hypodermic needle).

[0354] The kit may further include a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution or dextrose solution. It may also contain other materials useful to the end user, including other pharmaceutically acceptable formulation solutions, such as buffers, diluents, filters, needles and syringes or other delivery devices. The delivery device may be pre-filled with the composition.

[0355] The kit may also include a package insert containing written instructions for treating a method of an immune cell-related disease or condition. The package insert may be an unapproved draft package insert, or may be a package insert approved by the Food and Drug Administration (FDA) or other regulatory agency.

[0356] In certain embodiments, the kit includes T cells and encoding one or more cell reprogramming factors selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, NANOG and GLIS1. In certain embodiments, the kit includes T cells and encoding one or more cell reprogramming factors selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, NANOG and GLIS1. In certain embodiments, the kit includes T cells and encoding two or more cell reprogramming factors selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, NANOG and GLIS1. mRNA. In certain embodiments, the kit includes T cells and encoding three or more cell reprogramming factors selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, NANOG and GLIS1. mRNA. In certain embodiments, the kit includes T cells and coding selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, NANOG and GLIS1 four or more cell reprogramming factors mRNA. In certain embodiments, the kit includes T cells and coding selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, NANOG and GLIS1 five or more cell reprogramming factors mRNA. In certain embodiments, the kit includes T cells and coding selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, NANOG and GLIS1 six kinds of cell reprogramming factors mRNA. In certain embodiments, the kit includes T cells and coding cell reprogramming factors OCT4, SOX2, KLF4, c-MYC, LIN28, NANOG and GLIS1 mRNA. In certain embodiments, the kit includes T cells and mRNA encoding the cell reprogramming factors OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0357] III. Examples

[0358] The following examples are illustrative in nature and are not intended to be limiting in any way.

[0359] Example 1

[0360] Effects of dosing duration and factor combination on T cell rejuvenation, exhaustion, proliferation, and efficacy

[0361] mRNA molecules encoding OCT4 (O), SOX2 (S), KLF4 (K), c-MYC (M), LIN28 (L), and NANOG (N) (i.e., one molecule per factor) were synthesized in vitro from plasmid DNA and purified. Buffy Coat Pan T Cell Isolation Kit, Human (Cat. No. 130-120-001) Pan T cells were isolated from buffy coats obtained from blood samples of young (age 20-39 years), middle-aged (age 40-59 years), and elderly (age > 60 years) individuals, or from commercially available human buffy coats, human buffy coat leukocytes, or human blood samples. Pan T cells were transduced with lentivirus containing anti-CD19-ScFv-CD28 (without CD3ζ), anti-CD19-ScFv-CD28-CD3ζ, and anti-CD19-ScFv-4-1BB-CD3ζ obtained from GenTarget Inc. and cultured on Dynabeads with the T Cell Expansion and Activation Kit (ThermoFisher Cat. No. 11161D). TM Human T activator CD3 / CD28 was activated in X-VIVO-15 (Lonza catalog number: BEBP02-054Q) medium or used in a naive, non-transduced, non-activated state. Under additional conditions, multiple activations were performed, such as 2, 3 or 4 consecutive activations.

[0362] The mRNA molecules in the combination of OSKMLN, OSKM and OSK were prepared as naked mRNA in nuclease-free H2O and applied to T cells using electroporation. The parameters and conditions were as follows: 3 days after activation with Dynabeads, Pan T cells were electroporated with the Neon transfection system at 1350V, 10ms, 3 pulses or 1600V, 10ms, 3 pulses. In each repetition under each condition, 50 nanograms to 10 micrograms of mRNA were used per 1 million cells. Electroporation-only and untreated controls were also included. After electroporation, the cells were returned to the culture conditions. The cells were cultured for a total of 3, 5, 7, 9, 12, 14, 17, 15 and 21 days, and electroporation delivery of mRNA was performed every day, every other day or every three days. 3-18 days after the last electroporation, cells were collected for analysis. In additional conditions, mRNA molecules O, S, K, M, L and N are applied in all single, double, triple, quadruple and penta-factor combinations, such as OSKMLN, OSKML, SKMLN, OSKM, SKML, KMLN, OSLN, KOMN, OMN, MLN, OS, etc. In additional conditions, transfection is performed at the same time intervals, but lipid nanoparticles and polymer nanoparticles are used for transfection instead of electroporation; such nanoparticles are as in Example 3. In some experiments, delivery of mRNA molecules using lipid nanoparticles and / or polymer nanoparticles results in enhanced T cell rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, anti-pathogenic effects, anti-cancer effects or anti-inflammatory effects in exposed immune cells compared to delivery of mRNA molecules using electroporation.

[0363] For analysis of T cell identity, flow cytometry was used to assess the T cell markers CD8 and CD3. Other markers of T cell identity or lineage known in the art may also be used.

[0364] For analysis of rejuvenation, flow cytometry was used to assess stemness markers CCR7, CD62L, TCF7. The Horvath epigenetic clock was also used. Other markers of stemness or rejuvenation known in the art may also be used.

[0365] For the analysis of the prevention and reversal of exhaustion and aging, flow cytometry was used to assess the surface expression of CD39, CD57, PD-1, 2B4, LAG-3 (CD223), KLRG-1 (MAFA), TIGIT, CTLA-4, CD56, CD27, CD28 and TIM-3 (CD366) proteins. Other markers of exhaustion or aging known in the art may also be used.

[0366] For analysis of proliferation, cell numbers were counted by Invitrogen Countess automated cell counter.

[0367] For analysis of tumor cell killing efficacy, cytokine release (IFNγ, TNF-α, GM-CSF, IL-2, IL-6, IL-10, IL-13, GzmB, perforin, IL12p70, and IL-8) was measured using ELISA or Luminex. Additionally, short-term and long-term killing assays are performed according to methods familiar to those skilled in the art, such as described in Sommer et al. (Molecular Therapy, 2019 Jun 5; 27(6): 1126-38), Davenport et al. (Cancer Immunology Research, 2015 May 1; 3(5): 483-94), Zolov et al. (Cytotherapy, 2018 Oct 1; 20(10): 1259-66), Lee et al. (Molecular Therapy, 2022 Feb 2; 30(2): 579-92), Wei et al. (Journal for Immunotherapy of Cancer. 2019 Dec; 7(1): 1-5.), or Konduri et al. (Science Translational Medicine. 2021 May 5; 13(592): eabc3196).

[0368] The data from Example 1 demonstrate reprogramming factors and / or reprogramming durations that provide favorable T cell characteristics and properties. Reprogramming factors and combinations and conditions thereof (such as reprogramming duration) that reduce or reverse T cell exhaustion, increase T cell rejuvenation, increase or prolong self-renewal, increase or prolong pluripotency, increase or prolong functional persistence, provide retention of T cell identity, enhance T cell proliferation, and provide increased efficacy (such as increased tumor killing) may provide attractive candidates for immune cell rejuvenation compositions, methods, kits, and related therapies.

[0369] Example 2

[0370] Effects of dosing interval and factor combination on T cell rejuvenation, exhaustion, proliferation, and efficacy

[0371] mRNA molecules encoding OCT4 (O), SOX2 (S), KLF4 (K), c-MYC (M), LIN28 (L), and NANOG (N) (i.e., one molecule per factor) were synthesized in vitro from plasmid DNA and purified. Buffy Coat Pan T Cell Isolation Kit, Human (Cat. No. 130-120-001) Pan T cells were isolated from buffy coats of blood samples from young (age 20-25 years), middle-aged (age 40-50 years), and elderly (age >65 years) individuals, or from commercially available human buffy coats, human buffy coat leukocytes, or human blood samples. Pan T cells were transduced with lentivirus containing anti-CD19-ScFv-CD28 (no CD3ζ), anti-CD19-ScFv-CD28-CD3ζ, and anti-CD19-ScFv-4-1BB-CD3ζ obtained from GenTarget Inc. and cultured on Dynabeads using the T Cell Expansion and Activation Kit (ThermoFisher Cat. No. 11161D). TM Human T activator CD3 / CD28 was activated in X-VIVO-15 (Lonza catalog number: BEBP02-054Q) medium or used in a naive, non-transduced, non-activated state. Under additional conditions, multiple activations were performed, such as 2, 3 or 4 consecutive activations.

[0372] mRNA molecules in the combination of OSKMLN, OSKM, and OSK were prepared as naked mRNA in nuclease-free H2O and applied to T cells using electroporation with the following parameters and conditions: 3 days after activation with Dynabeads, Pan T cells were electroporated with the Neon transfection system at 1350V, 10ms, 3 pulses or 1600V, 10ms, 3 pulses. In each replicate under each condition, 50 ng to 10 μg of mRNA was used per 1 million cells. Three dosing intervals for electroporation were performed: (1) prevention of depletion on days 4-9, (2) reversal of depletion on days 10-15, and (3) reversal of depletion of cell therapy products on days 16-21. Electroporation-only and untreated controls were also included. After electroporation, the cells were returned to culture conditions. The cells were cultured for a total of 15 or 21 days, and for each interval, mRNA electroporation delivery was performed once a day, every other day, or every three days (i.e., day 4-9, every day, every other day, or every three days; day 10-15, every day, every other day, or every three days; day 16-21, every day, every other day, or every three days). At the end of the culture period (15 or 21 days), cells were collected for analysis. Under additional conditions, transfection was performed at the same time intervals, but lipid nanoparticles and polymer nanoparticles were used for transfection instead of electroporation; such nanoparticles are as in Example 3. In some experiments, delivery of mRNA molecules with lipid nanoparticles and / or polymer nanoparticles resulted in enhanced T cell rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, anti-pathogenic effects, anti-cancer effects, or anti-inflammatory effects in exposed immune cells compared to delivery of mRNA molecules using electroporation.

[0373] For analysis of T cell identity, flow cytometry was used to assess the T cell markers CD8 and CD3. Other markers of T cell identity or lineage known in the art may also be used.

[0374] For analysis of rejuvenation, flow cytometry was used to assess stemness markers CCR7, CD62L, TCF7. The Horvath epigenetic clock was also used. Other markers of stemness or rejuvenation known in the art may also be used.

[0375] For the analysis of the prevention and reversal of exhaustion and aging, flow cytometry was used to assess the surface expression of CD39, CD57, PD-1, LAG-3 (CD223), KLRG-1 (MAFA), TIGIT, CTLA-4, CD56, CD27, CD28, 2B4 and TIM-3 (CD366) proteins. Other markers of exhaustion or aging known in the art may also be used.

[0376] For analysis of proliferation, cell numbers were counted by Invitrogen Countess automated cell counter.

[0377] For analysis of tumor cell killing efficacy, cytokine release (IFNγ, TNF-α, GM-CSF, IL-2, IL-6, IL-10, IL-13, IL12p70, GzmB, perforin, and IL-8) was measured using ELISA or Luminex. TM Luciferase assay system kit (Promega, catalog number: E6110) was used for short-term (20 hours to 3 days) cytotoxicity assay according to the manufacturer's protocol. In short, CD19+Daudi target cells expressing eGFP-FLuc were incubated with anti-CD19 CAR-T cells at different effector: target ratios for 20 hours to 72 hours and placed in 96-well plates. The amount of oxyfluorofluorescein converted to 5'-fluorofluorescein by live Daudi target cells expressing FLuc was measured on a photometer. The measured values ​​in the long-term killing assay were the same as those in the short-term killing assay. However, the starting effector: target ratio was 1: 2, 1: 1, 2: 1 or 4: 1. Every 3 days, half of the culture was transferred to a new plate and the killing percentage was analyzed, and the remaining culture was re-attacked with fresh Daudi target cells.

[0378] Additionally, short-term and long-term killing assays are performed according to methods familiar to those skilled in the art, such as described in Sommer et al. (Molecular Therapy, 2019 Jun 5; 27(6): 1126-38), Davenport et al. (Cancer Immunology Research, 2015 May 1; 3(5): 483-94), Zolov et al. (Cytotherapy, 2018 Oct 1; 20(10): 1259-66), Lee et al. (Molecular Therapy, 2022 Feb 2; 30(2): 579-92), Wei et al. (Journal for Immunotherapy of Cancer. 2019 Dec; 7(1): 1-5.), or Konduri et al. (Science Translational Medicine. 2021 May 5; 13(592): eabc3196).

[0379] The data from the experiments of Example 2 demonstrate reprogramming factors and / or dosing intervals that provide favorable T cell characteristics and properties. Reprogramming factors and combinations thereof and conditions (such as dosing intervals) inhibit, reduce or reverse T cell exhaustion, increase T cell rejuvenation, provide retention of T cell identity, enhance T cell proliferation and provide increased efficacy (such as increased tumor killing), and may provide attractive candidates for immune cell rejuvenation compositions, methods, kits and related therapies.

[0380] Example 3

[0381] Comparison of nanoparticle mRNA transfection and electroporation: effects on T cell rejuvenation, exhaustion, proliferation, and efficacy

[0382] ring

[0383] mRNA molecules encoding OCT4 (O), SOX2 (S), KLF4 (K), c-MYC (M), LIN28 (L), and NANOG (N) (i.e., one molecule per factor) were synthesized in vitro from plasmid DNA and purified. Buffy Coat Pan T Cell Isolation Kit, Human (Cat. No. 130-120-001) Pan T cells were isolated from buffy coats of blood samples from young (age 20-39 years), middle-aged (age 40-59 years), and elderly (age >60 years) individuals, or from commercially available human buffy coats, human buffy coat leukocytes, or human blood samples. Pan T cells were transduced with lentivirus containing anti-CD19-ScFv-CD28 (no CD3ζ), anti-CD19-ScFv-CD28-CD3ζ, and anti-CD19-ScFv-4-1BB-CD3ζ obtained from GenTarget Inc. and cultured on Dynabeads with the T Cell Expansion and Activation Kit (ThermoFisher Cat. No. 11161D). TM Human T activator CD3 / CD28 was activated in X-VIVO-15 (Lonza catalog number: BEBP02-054Q) medium or used in a naive, non-transduced, non-activated state. Under additional conditions, multiple activations were performed, such as 2, 3 or 4 consecutive activations.

[0384] The mRNA molecules in the OSKMLN combination were prepared as naked mRNA in nuclease-free H2O or incorporated into nanoparticles, such as the manufacturer's protocols of Lipofectamine, Fugene or Polyplus or those described in Billingsley et al., NanoLett. 2020 (20, 3, 1578–1589), McKinlay et al. 2017 (PNAS 2017 Jan 24 114 (4) E448-E456), McKinlay et al. 2018 (PNAS 2018 Jun 26 115 (26) E5859-E5866), or Haabeth et al. 2018 (PNAS 2018 Sep 25 115 (39) E9153-E9161), which are incorporated herein by reference. Nanoparticles incorporating lipids selected from those described in Figures 1 to 18 were also used. Naked mRNA was transfected into T cells using electroporation with the following parameters and conditions: 3 days after activation with Dynabeads, Pan T cells were electroporated using the Neon transfection system at 1350 V, 10 ms, 3 pulses or 1600 V, 10 ms, 3 pulses. Nanoparticle-incorporated mRNA was transfected into T cells under conditions as described in the manufacturer's protocols for Lipofectamine, Fugene, or Polyplus, or as described in Billingsley et al., Nano Lett. 2020 (20, 3, 1578–1589), McKinlay et al. 2017 (PNAS 2017 Jan 24 114 (4) E448-E456), McKinlay et al. 2018 (PNAS 2018 Jun 26 115 (26) E5859-E5866), or Haabeth et al. 2018 (PNAS 2018 Sep 25 115 (39) E9153-E9161), which are incorporated herein by reference. In each replicate under each condition, 50 nanograms to 10 micrograms of mRNA were used per 1 million cells. After electroporation or nanoparticle transfection, the cells were returned to culture conditions. Via transfection, cells were exposed to mRNA for 0-10 days and then cultured for 0-18 days. Cells were then harvested for analysis.

[0385] For analysis of T cell identity, flow cytometry was used to assess the T cell markers CD8 and CD3. Other markers of T cell identity or lineage known in the art may also be used.

[0386] For analysis of rejuvenation, flow cytometry was used to assess stemness markers CCR7, CD62L, TCF7. The Horvath epigenetic clock was also used. Other markers of stemness or rejuvenation known in the art may also be used.

[0387] For the analysis of the prevention and reversal of exhaustion and aging, flow cytometry was used to assess the surface expression of CD39, CD57, PD-1, LAG-3 (CD223), KLRG-1 (MAFA), TIGIT, CTLA-4, CD56, CD27, CD28, 2B4 and TIM-3 (CD366) proteins. Other markers of exhaustion or aging known in the art may also be used.

[0388] For analysis of proliferation, cell numbers were counted by Invitrogen Countess automated cell counter.

[0389] For analysis of tumor cell killing efficacy, cytokine release (IFNγ, TNF-α, GM-CSF, IL-2, IL-6, IL-10, IL-13, IL12p70, GzmB, perforin, and IL-8) was measured using ELISA or Luminex. TMLuciferase assay system kit (Promega, catalog number: E6110) was used for short-term (20h to 3 days) cytotoxicity assay according to the manufacturer's protocol. In short, CD19+Daudi target cells expressing eGFP-FLuc were incubated with anti-CD19 CAR-T cells at different effector: target ratios for 20h to 72h and placed in 96-well plates. The amount of oxyfluorofluorescein converted to 5'-fluorofluorescein by live Daudi target cells expressing FLuc was measured on a photometer. The measured values ​​in the long-term killing assay were the same as those in the short-term killing assay. However, the starting effector: target ratio was 1:2, 1:1, 2:1 or 4:1. Every 3 days, half of the culture was transferred to a new plate and the killing percentage was analyzed, and the remaining culture was re-attacked with fresh Daudi target cells. Additionally, short-term and long-term killing assays are performed according to methods familiar to those skilled in the art, such as described in Sommer et al. (Molecular Therapy, 2019 Jun 5; 27(6): 1126-38), Davenport et al. (Cancer Immunology Research, 2015 May 1; 3(5): 483-94), Zolov et al. (Cytotherapy, 2018 Oct 1; 20(10): 1259-66), Lee et al. (Molecular Therapy, 2022 Feb 2; 30(2): 579-92), Wei et al. (Journal for Immunotherapy of Cancer. 2019 Dec; 7(1): 1-5.), or Konduri et al. (Science Translational Medicine. 2021 May 5; 13(592): eabc3196).

[0390] The data from Example 3 were analyzed to demonstrate that delivery of mRNA molecules using lipid nanoparticles and / or polymer nanoparticles resulted in enhanced T cell rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, anti-pathogenic effects, anti-cancer effects, or anti-inflammatory effects in exposed immune cells compared to delivery of mRNA molecules using electroporation. In some experiments, delivery of mRNA molecules using lipid nanoparticles and / or polymer nanoparticles resulted in enhanced T cell rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, anti-pathogenic effects, anti-cancer effects, or anti-inflammatory effects in exposed immune cells compared to delivery of mRNA molecules using electroporation.

[0391] The data from the experiments of Example 3 also demonstrate reprogramming factors that provide favorable T cell characteristics and properties. Reprogramming factors and combinations thereof prevent, reduce or reverse T cell exhaustion, increase T cell rejuvenation, provide retention of T cell identity, enhance T cell proliferation, and provide increased efficacy (such as increased tumor killing), and may provide attractive candidates for immune cell rejuvenation compositions, methods, kits, and related therapies.

[0392] Example 4

[0393] Effects of reprogramming factor ratios on T cell rejuvenation, exhaustion, proliferation, and efficacy

[0394] mRNA molecules encoding OCT4 (O), SOX2 (S), KLF4 (K), c-MYC (M), LIN28 (L), and NANOG (N) (i.e., one molecule per factor) were synthesized in vitro from plasmid DNA and purified. Buffy Coat Pan T Cell Isolation Kit, Human (Cat. No. 130-120-001) Pan T cells were isolated from buffy coats of blood samples from young (age 20-39 years), middle-aged (age 40-60 years), and elderly (age >60 years) individuals, or from commercially available human buffy coats, human buffy coat leukocytes, or human blood samples. Pan T cells were transduced with lentivirus containing anti-CD19-ScFv-CD28 (no CD3ζ), anti-CD19-ScFv-CD28-CD3ζ, and anti-CD19-ScFv-4-1BB-CD3ζ obtained from GenTarget Inc. and cultured on Dynabeads using the T Cell Expansion and Activation Kit (ThermoFisher Cat. No. 11161D). TM Human T activator CD3 / CD28 was activated in X-VIVO-15 (Lonza catalog number: BEBP02-054Q) medium or used in a naive, non-transduced, non-activated state. Under additional conditions, multiple activations were performed, such as 2, 3 or 4 consecutive activations.

[0395] The mRNA molecules were prepared as naked mRNA in nuclease-free H2O and mixed into a mixture of OSKLMN mass ratio of 1-5:1-5:1-5:1-5:1-5:1-5:1-5. Naked mRNA was transfected into T cells using electroporation, and the parameters and conditions were as follows: 3 days after activation using Dynabeads, PanT cells were electroporated with Neon transfection system at 1350V, 10ms, 3 pulses or 1600V, 10ms, 3 pulses. In each repetition under each condition, 50 nanograms to 10 micrograms of mRNA were used for every 1 million cells. Only electroporation and untreated controls were also included. After electroporation, cells were returned to culture conditions. Via electroporation, cells were exposed to mRNA for 0-10 days, and then cultured for 0-18 days. Under additional conditions, transfection was performed at the same time interval, but lipid nanoparticles and polymer nanoparticles were used for transfection instead of electroporation; such nanoparticles are as in Example 3. In some experiments, delivery of mRNA molecules using lipid nanoparticles and / or polymer nanoparticles resulted in enhanced T cell rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, anti-pathogenic effects, anti-cancer effects, or anti-inflammatory effects in exposed immune cells compared to delivery of mRNA molecules using electroporation.

[0396] For analysis of T cell identity, flow cytometry was used to assess the T cell markers CD8 and CD3. Other markers of T cell identity or lineage known in the art may also be used.

[0397] For analysis of rejuvenation, stemness markers CCR7, CD62L, TCF7 were assessed using flow cytometry.

[0398] Other markers of stemness or rejuvenation known in the art may also be used.

[0399] For the analysis of the prevention and reversal of exhaustion and aging, flow cytometry was used to assess the surface expression of CD39, CD57, PD-1, LAG-3 (CD223), KLRG-1 (MAFA), TIGIT, CTLA-4, CD56, CD27, CD28, 2B4 and TIM-3 (CD366) proteins. Other markers of exhaustion or aging known in the art may also be used.

[0400] For analysis of proliferation, cell numbers were counted by Invitrogen Countess automated cell counter.

[0401] For analysis of tumor cell killing efficacy, cytokine release (IFNγ, TNF-α, GM-CSF, IL-2, IL-6, IL-10, IL-13, IL12p70, GzmB, perforin, and IL-8) was measured by ELISA or Luminex, as well as short-term and long-term killing assays. TM Luciferase assay system kit (Promega, catalog number: E6110) was used for short-term (20h to 3 days) cytotoxicity assay according to the manufacturer's protocol. In short, CD19+Daudi target cells expressing eGFP-FLuc were incubated with anti-CD19 CAR-T cells at different effector: target ratios for 20h to 72h and placed in 96-well plates. The amount of oxyfluorofluorescein converted to 5'-fluorofluorescein by live Daudi target cells expressing FLuc was measured on a photometer. The measured values ​​in the long-term killing assay were the same as those in the short-term killing assay. However, the starting effector: target ratio was 1:2, 1:1, 2:1 or 4:1. Every 3 days, half of the culture was transferred to a new plate and the killing percentage was analyzed, and the remaining culture was re-attacked with fresh Daudi target cells. Additionally, short-term and long-term killing assays are performed according to methods familiar to those skilled in the art, such as described in Sommer et al. (Molecular Therapy, 2019 Jun 5; 27(6): 1126-38), Davenport et al. (Cancer Immunology Research, 2015 May 1; 3(5): 483-94), Zolov et al. (Cytotherapy, 2018 Oct 1; 20(10): 1259-66), Lee et al. (Molecular Therapy, 2022 Feb 2; 30(2): 579-92), Wei et al. (Journal for Immunotherapy of Cancer. 2019 Dec; 7(1): 1-5.), or Konduri et al. (Science Translational Medicine. 2021 May 5; 13(592): eabc3196).

[0402] The data from the experiments of Example 4 demonstrate reprogramming factors and / or reprogramming factor ratios that provide favorable T cell characteristics and properties. Reprogramming factors and combinations thereof (including ratios) prevent, reduce or reverse T cell exhaustion, increase T cell rejuvenation, provide retention of T cell identity, enhance T cell proliferation, and provide increased efficacy (such as increased tumor killing), and may provide attractive candidates for immune cell rejuvenation compositions, methods, kits, and related therapies.

[0403] Example 5

[0404] Effects of sustained reprogramming factor exposure over dosing intervals on T cell rejuvenation, exhaustion, proliferation, and efficacy

[0405] mRNA molecules encoding OCT4 (O), SOX2 (S), KLF4 (K), c-MYC (M), LIN28 (L), and NANOG (N) (i.e., one factor per molecule) were synthesized in vitro from plasmid DNA and purified. To control the persistence of exposure to reprogramming factors, linear non-replicative mRNA molecules were used in which the 3' untranslated region was altered, the poly(A) tail length was optimized, and the WPRE element was included, resulting in an mRNA half-life of 6 minutes to 24 hours. Circular RNA with a half-life of 1-3 days was used. Trans- or self-amplifying RNA with a half-life of up to 10-14 days was used. By using Buffy Coat Pan T Cell Isolation Kit, Human (Cat. No. 130-120-001) Pan T cells were isolated from buffy coats of blood samples from young (age 20-39 years), middle-aged (age 40-59 years), and elderly (age >60 years) individuals, or from commercially available human buffy coats, human buffy coat leukocytes, or human blood samples. Pan T cells were transduced with lentivirus containing anti-CD19-ScFv-CD28 (no CD3ζ), anti-CD19-ScFv-CD28-CD3ζ, and anti-CD19-ScFv-4-1BB-CD3ζ obtained from GenTarget Inc. and cultured on Dynabeads using the T Cell Expansion and Activation Kit (ThermoFisher Cat. No. 11161D). TM Human T activator CD3 / CD28 was activated in X-VIVO-15 (Lonza catalog number: BEBP02-054Q) medium or used in a naive, non-transduced, non-activated state. Under additional conditions, multiple activations were performed, such as 2, 3 or 4 consecutive activations.

[0406] The mRNA molecules in the OSKMLN combination were prepared as naked mRNA in nuclease-free H2O and transfected into T cells using electroporation with the following parameters and conditions: 3 days after activation with Dynabeads, Pan T cells were electroporated with the Neon transfection system at 1350V, 10ms, 3 pulses or 1600V, 10ms, 3 pulses. In each replicate under each condition, 50 ng to 10 μg of mRNA was used per 1 million cells. Electroporation-only and untreated controls were also included. The cells are cultured for 3-10 days, and electroporation delivery of mRNA is performed on the 1st day, the 2nd day, the 3rd day, the 4th day, the 5th day, the 6th day, the 7th day, the 8th day, the 9th day and / or the 10th day, depending on the mRNA construct used (i.e., for mRNA with the longest expression, the longest half-life or the expression of the transcription factor with the longest half-life, transfection is performed only on the 1st day, and for mRNA with the shortest expression, transfection is performed every three days, every two days or every day). At each time point, cells are collected for analysis. Under additional conditions, transfection is performed at the same time intervals, but lipid nanoparticles and polymer nanoparticles are used for transfection instead of electroporation; such nanoparticles are as in Example 3. In some experiments, delivery of mRNA molecules with lipid nanoparticles and / or polymer nanoparticles results in enhanced T cell rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, anti-pathogenic effects, anti-cancer effects or anti-inflammatory effects in exposed immune cells compared to delivery of mRNA molecules using electroporation.

[0407] For analysis of T cell identity, flow cytometry was used to assess the T cell markers CD8 and CD3. Other markers of T cell identity or lineage known in the art may also be used.

[0408] For analysis of rejuvenation, flow cytometry was used to assess stemness markers CCR7, CD62L, TCF7. Other markers of stemness or rejuvenation known in the art may also be used.

[0409] For the analysis of the prevention and reversal of exhaustion and aging, flow cytometry was used to assess the surface expression of CD39, CD57, PD-1, LAG-3 (CD223), KLRG-1 (MAFA), TIGIT, CTLA-4, CD56, CD27, CD28, 2B4 and TIM-3 (CD366) proteins. Other markers of exhaustion or aging known in the art may also be used.

[0410] For analysis of proliferation, cell numbers were counted by Invitrogen Countess automated cell counter.

[0411] For analysis of tumor cell killing efficacy, cytokine release (IFNγ, TNF-α, GM-CSF, IL-2, IL-6, IL-10, IL-13, IL12p70, GzmB, perforin, and IL-8) was measured using ELISA or Luminex. TM Luciferase assay system kit (Promega, catalog number: E6110) was used for short-term (20h to 3 days) cytotoxicity assay according to the manufacturer's protocol. In short, CD19+Daudi target cells expressing eGFP-FLuc were incubated with anti-CD19 CAR-T cells at different effector: target ratios for 20h to 72h and placed in 96-well plates. The amount of oxyfluorofluorescein converted to 5'-fluorofluorescein by live Daudi target cells expressing FLuc was measured on a photometer. The measured values ​​in the long-term killing assay were the same as those in the short-term killing assay. However, the starting effector: target ratio was 1:2, 1:1, 2:1 or 4:1. Every 3 days, half of the culture was transferred to a new plate and the killing percentage was analyzed, and the remaining culture was re-attacked with fresh Daudi target cells. Additionally, short-term and long-term killing assays are performed according to methods well known to those skilled in the art, such as described in Sommer et al. (Molecular Therapy, 2019 Jun 5; 27(6): 1126-38), Davenport et al. (Cancer Immunology Research, 2015 May 1; 3(5): 483-94), Zolov et al. (Cytotherapy, 2018 Oct 1; 20(10): 1259-66), Lee et al. (Molecular Therapy, 2022 Feb 2; 30(2): 579-92), Wei et al. (Journal for Immunotherapy of Cancer. 2019 Dec; 7(1): 1-5.), or Konduri et al. (Science Translational Medicine. 2021 May 5; 13(592): eabc3196). Each of these references is incorporated herein.

[0412] The data from the experiments of Example 5 demonstrate reprogramming factors and / or mechanisms of persistent reprogramming factor exposure that provide favorable T cell characteristics and properties. Reprogramming factors and combinations thereof and expression regulation mechanisms (such as sustained expression) prevent, reduce or reverse T cell exhaustion, increase T cell rejuvenation, provide retention of T cell identity, enhance T cell proliferation and provide increased efficacy (such as increased tumor killing), and may provide attractive candidates for immune cell rejuvenation compositions, methods, kits and related therapies.

[0413] Example 6

[0414] Comparison of mRNA transfection and viral transduction: effects on T cell rejuvenation, exhaustion, proliferation, and efficacy

[0415] mRNA molecules encoding OCT4 (O), SOX2 (S), KLF4 (K), c-MYC (M), LIN28 (L), and NANOG (N) (i.e., one factor per molecule) were synthesized in vitro from plasmid DNA and purified. Lentivirus, retrovirus, or Sendai virus vectors encoding O, S, K, M, L, and N were generated according to methods known in the art, such as described in Egusa et al. (PLoS One. 2010 Sep 14; 5(9): e12743). By using Buffy Coat Pan T Cell Isolation Kit, Human (Cat. No. 130-120-001) Pan T cells were isolated from buffy coats of blood samples from young (age 20-39 years), middle-aged (age 40-59 years), and elderly (age >60 years) individuals, or from commercially available human buffy coats, human buffy coat leukocytes, or human blood samples. Pan T cells were transduced with lentivirus containing anti-CD19-ScFv-CD28 (no CD3ζ), anti-CD19-ScFv-CD28-CD3ζ, and anti-CD19-ScFv-4-1BB-CD3ζ obtained from GenTarget Inc. and cultured on Dynabeads using the T Cell Expansion and Activation Kit (ThermoFisher Cat. No. 11161D). TM Human T activator CD3 / CD28 was activated in X-VIVO-15 (Lonza catalog number: BEBP02-054Q) medium or used in a naive, non-transduced, non-activated state. Under additional conditions, multiple activations were performed, such as 2, 3 or 4 consecutive activations.

[0416] The mRNA molecules in the OSKMLN combination are incorporated into nanoparticles, such as the manufacturer's protocols of Lipofectamine, Fugene or Polyplus or those described in Billingsley et al., Nano Lett. 2020 (20, 3, 1578–1589), McKinlay et al. 2017 (PNAS 2017 Jan 24 114 (4) E448-E456), McKinlay et al. 2018 (PNAS 2018 Jun 26 115 (26) E5859-E5866) or Haabeth et al. 2018 (PNAS 2018 Sep 25 115 (39) E9153-E9161), which are incorporated herein by reference. Nanoparticles incorporating lipids selected from those described in Figures 1 to 18 are also used. Nanoparticle-incorporated mRNA was transfected into T cells under conditions as described in the manufacturer's protocols for Lipofectamine, Fugene, or Polyplus, or as described in Billingsley et al., Nano Lett. 2020 (20, 3, 1578–1589), McKinlay et al. 2017 (PNAS 2017 Jan 24 114 (4) E448-E456), McKinlay et al. 2018 (PNAS 2018 Jun 26 115 (26) E5859-E5866), or Haabeth et al. 2018 (PNAS 2018 Sep 25 115 (39) E9153-E9161), which are incorporated herein by reference. In each replicate under each condition, 50 nanograms to 10 micrograms of mRNA were used per 1 million cells. Viral transduction is performed according to methods known in the art, such as described in Egusa et al. (PLoS One. 2010 Sep 14; 5(9): e12743). After nanoparticle transfection or viral transduction, the cells are returned to culture conditions. Via transfection or transduction, the cells are exposed to mRNA for 0-10 days and then cultured for 0-18 days. The cells are then collected for analysis.

[0417] For analysis of T cell identity, flow cytometry was used to assess the T cell markers CD8 and CD3. Other markers of T cell identity or lineage known in the art may also be used.

[0418] For analysis of rejuvenation, flow cytometry was used to assess T cell stemness markers CCR7, CD62L, TCF7. Other markers of T cell stemness or T cell rejuvenation known in the art may also be used.

[0419] For the analysis of the prevention and reversal of exhaustion and aging, flow cytometry was used to assess the surface expression of CD39, CD57, PD-1, LAG-3 (CD223), KLRG-1 (MAFA), TIGIT, CTLA-4, CD56, CD27, CD28, 2B4 and TIM-3 (CD366) proteins. Other markers of T cell exhaustion or T cell aging known in the art may also be used.

[0420] For analysis of proliferation, cell number was counted by Invitrogen Countess automated cell counter. For analysis of tumor cell killing efficacy, cytokine release (IFNγ, TNF-α, GM-CSF, IL-2, IL-6, IL-10, IL-13, IL12p70, GzmB, perforin and IL-8) was measured using ELISA or Luminex. ONE-Glo TMLuciferase assay system kit (Promega, catalog number: E6110) was used for short-term (20h to 3 days) cytotoxicity assay according to the manufacturer's protocol. In short, CD19+Daudi target cells expressing eGFP-FLuc were incubated with anti-CD19 CAR-T cells at different effector: target ratios for 20h to 72h and placed in 96-well plates. The amount of oxyfluorofluorescein converted to 5'-fluorofluorescein by live Daudi target cells expressing FLuc was measured on a photometer. The measured values ​​in the long-term killing assay were the same as those in the short-term killing assay. However, the starting effector: target ratio was 1:2, 1:1, 2:1 or 4:1. Every 3 days, half of the culture was transferred to a new plate and the killing percentage was analyzed, and the remaining culture was re-attacked with fresh Daudi target cells. Additionally, short-term and long-term killing assays are performed according to methods familiar to those skilled in the art, such as described in Sommer et al. (Molecular Therapy, 2019 Jun 5; 27(6): 1126-38), Davenport et al. (Cancer Immunology Research, 2015 May 1; 3(5): 483-94), Zolov et al. (Cytotherapy, 2018 Oct 1; 20(10): 1259-66), Lee et al. (Molecular Therapy, 2022 Feb 2; 30(2): 579-92), Wei et al. (Journal for Immunotherapy of Cancer. 2019 Dec; 7(1): 1-5.), or Konduri et al. (Science Translational Medicine. 2021 May 5; 13(592): eabc3196).

[0421] The data from Example 6 are analyzed to demonstrate that delivery of mRNA molecules with lipid nanoparticles and / or polymer nanoparticles results in enhanced T cell rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, anti-pathogenic effects, anti-cancer effects, or anti-inflammatory effects in exposed immune cells compared to viral gene delivery of reprogramming factors. In some experiments, delivery of mRNA molecules with lipid nanoparticles and / or polymer nanoparticles results in enhanced T cell rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, anti-pathogenic effects, anti-cancer effects, or anti-inflammatory effects in exposed immune cells compared to viral gene delivery of reprogramming factors.

[0422] The data from the experiments of Example 6 demonstrate reprogramming factors that provide favorable T cell characteristics and properties. Reprogramming factors and combinations thereof reduce or reverse T cell exhaustion, increase T cell rejuvenation, provide retention of T cell identity, enhance T cell proliferation, and provide increased efficacy (such as increased tumor killing), and may provide attractive candidates for immune cell rejuvenation compositions, methods, kits, and related therapies.

[0423] Example 7

[0424] Rejuvenation, depletion, proliferation, and efficacy of tumor-infiltrating lymphocytes

[0425] In addition to the specific regimen, conditions and parameters for tumor-infiltrating lymphocytes described below, similar regimens, conditions and parameters as those used for T cells in Examples 1-6 above were used to observe the effects of dosing duration, dosing interval, reprogramming factor combination, reprogramming factor mass or molar ratio, continuous reprogramming factor exposure, nanoparticle-mediated delivery versus electroporation-mediated delivery, and mRNA transfection versus viral transduction on tumor-infiltrating lymphocytes. Tumor infiltrating lymphocytes are obtained and expanded from commercial sources or according to methods known to those skilled in the art, such as described by Wu et al. (Nanomedicine. 2019 Apr; 14(8):955-67), Friedman et al. (Journal of immunotherapy (Hagerstown, Md.: 1997). 2012 Jun; 35(5):400), Belldegrun et al. (Cancer research. 1988 Jan 1; 48(1):206-14), or Rosenberg et al. (New England Journal of Medicine. 1990 Aug 30; 323(9):570-8), which are incorporated herein by reference. Tumor infiltrating lymphocytes are stimulated with IL-2, for example, as described by Rosenberg et al. (New England Journal of Medicine. August 30, 1990; 323(9): 570-8) or Wu et al. (Nanomedicine. April 2019; 14(8): 955-67), or remain unstimulated. Under additional conditions, stimulation is performed at different intervals, for example, for 8 hours to 21 days. In some experiments, delivery of mRNA molecules with lipid nanoparticles and / or polymer nanoparticles results in enhanced tumor infiltrating lymphocyte rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, anti-pathogenic effects, anti-cancer effects, or anti-inflammatory effects in exposed immune cells compared to delivery of mRNA molecules using electroporation.

[0426] For analysis of tumor infiltrating lymphocyte identity, flow cytometry was used to assess tumor infiltrating lymphocyte markers CD8, CD3, CD45, CD4, and FOXP3. Other markers of tumor infiltrating lymphocyte identity or lineage known in the art may also be used.

[0427] For analysis of rejuvenation, flow cytometry was used to assess stemness markers CCR7, CD62L, TCF7. Other markers of stemness or rejuvenation known in the art may also be used.

[0428] For the analysis of the prevention and reversal of exhaustion and aging, flow cytometry was used to assess the surface expression of CD39, CD57, PD-1, LAG-3 (CD223), KLRG-1 (MAFA), TIGIT, CTLA-4, CD56, CD27, CD28 and TIM-3 (CD366) proteins. Other markers of exhaustion or aging known in the art may also be used.

[0429] For analysis of proliferation, cell numbers were counted by Invitrogen Countess automated cell counter.

[0430] For analysis of tumor cell killing efficacy, cytokine release (IFNγ, TNF-α, GM-CSF, IL-2, IL-6, IL-10, IL-13, IL12p70 and IL-8) was measured using ELISA or Luminex. In addition, tumor killing was measured according to methods well known to those skilled in the art, such as Ritthipichai et al. (Clinical Cancer Research. October 15, 2017; 23 (20): 6151-64.), Inozume et al. (Journal of Investigative Dermatology 136.1 (2016): 255-263), Ortegel et al. (Lung cancer. April 1, 2002; 36 (1): 17-25) or Nielsen et al. (Cancer Immunology, Immunotherapy 69, Issue 11 (2020): 2179-219), which are incorporated herein by reference.

[0431] The data from the experiments of Example 7 demonstrate reprogramming factors that provide favorable tumor-infiltrating lymphocyte characteristics and properties. Reprogramming factors and their combinations, mass or molar ratios, reprogramming durations and intervals reduce or reverse tumor-infiltrating lymphocyte depletion, increase tumor-infiltrating lymphocyte rejuvenation, provide retention of tumor-infiltrating lymphocyte identity, enhance tumor-infiltrating lymphocyte proliferation, and provide increased efficacy (such as increased tumor killing), and can provide attractive candidates for immune cell rejuvenation compositions, methods, kits, and related therapies.

[0432] Example 8

[0433] Rejuvenation, exhaustion, proliferation, and efficacy of regulatory T cells (TREGS)

[0434] In addition to the specific scheme, conditions and parameters for tumor infiltrating lymphocytes described below, similar schemes, conditions and parameters for T cells in the above-mentioned examples 1-6 are used to observe the effects of administration duration, administration interval, reprogramming factor combination, reprogramming factor mass or molar ratio, continuous reprogramming factor exposure, nanoparticle-mediated delivery and electroporation-mediated delivery, and mRNA transfection and viral transduction on Treg. Treg is obtained and amplified from commercial sources or according to methods known to those skilled in the art, such as Collison et al. (Regulatory T cells 2011 (pages 21-37). Humana Press, Totowa, NJ), which is incorporated herein by reference. Anti-CD3+ anti-CD28 coated beads as described in Example 1 above, or irradiated splenocytes or purified dendritic cells combined with soluble anti-CD3 or peptides are used to activate Treg, such as Collison et al. (Regulatory T cells 2011 (pages 21-37). Humana Press, Totowa, NJ), or remain unactivated. In some experiments, delivery of mRNA molecules using lipid nanoparticles and / or polymer nanoparticles resulted in enhanced Treg rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, anti-pathogenic effects, anti-cancer effects, or anti-inflammatory effects in exposed immune cells compared to delivery of mRNA molecules using electroporation.

[0435] For analysis of Treg identity, flow cytometry was used to assess Treg markers CD3, CD4, CD25, CD127, IL-2Rα, and FoxP3. Other markers of Treg identity or lineage known in the art may also be used.

[0436] For analysis of rejuvenation, flow cytometry was used to assess the markers CCR7, CD62L, TCF7, and GARP. Other markers of stemness or rejuvenation known in the art may also be used.

[0437] For analysis of the prevention and reversal of exhaustion and senescence, flow cytometry was used to assess the surface expression of CD39, CD57, PD-1, LAG-3 (CD223), KLRG-1 (MAFA), TIGIT, CTLA-4, CD56, CD27, CD28 and TIM-3 (CD366) proteins or CD4, CD25 and CD279. Other markers of exhaustion or senescence known in the art may also be used.

[0438] For analysis of proliferation, cell numbers were counted by Invitrogen Countess automated cell counter.

[0439] For analysis of anti-inflammatory or immunomodulatory efficacy, assays known to those skilled in the art are used, such as inhibition of effector T cell proliferation, as described by Pedersen et al. (Immunopharmacology and Immunotoxicology, Vol. 37, No. 1, 2015) or Court et al. (EMBO Reports (2020) 21: e48052).

[0440] The data from the experiments of Example 8 demonstrate reprogramming factors that provide favorable Treg characteristics and properties. Reprogramming factors and their combinations, ratios, reprogramming durations and intervals reduce or reverse Treg exhaustion, increase Treg rejuvenation, provide retention of Treg identity, enhance Treg proliferation, and provide increased efficacy (such as immune tolerance or reduced inflammation), and may provide attractive candidates for immune cell rejuvenation compositions, methods, kits, and related therapies.

[0441] Example 9

[0442] Rejuvenation, exhaustion, proliferation, and efficacy of γδ T cells

[0443] In addition to the specific protocols, conditions and parameters for γδT cells described below, similar protocols, conditions and parameters for T cells in Examples 1-6 above were used to observe the effects of duration of administration, dosing interval, reprogramming factor combination, reprogramming factor mass or molar ratio, continuous reprogramming factor exposure, nanoparticle-mediated delivery and electroporation-mediated delivery, and mRNA transfection and viral transduction on γδT cells. γδT cells were obtained and expanded from commercial sources or according to methods known to those skilled in the art, such as Pauza et al. (Frontiers in immunology. June 8, 2018; 9: 1305) or Deniger et al. (Clin Cancer Res (2014) 20 (22): 5708–5719), which are incorporated herein by reference. The γδ T cells are stimulated with IL-2 and / or IL-21, for example as described by Pauza et al. (Frontiers inimmunology. 2018 Jun 8; 9: 1305) or Deniger et al. (Clin Cancer Res (2014) 20(22): 5708–5719), or remain unstimulated. In additional conditions, stimulation is performed at different intervals, for example, from 8 hours to 21 days. In some experiments, delivery of mRNA molecules using lipid nanoparticles and / or polymer nanoparticles results in enhanced γδ T cell rejuvenation, proliferation, recovery from exhaustion or prevention of exhaustion, anti-pathogenic effects, anti-cancer effects, or anti-inflammatory effects in exposed immune cells compared to delivery of mRNA molecules using electroporation.

[0444] For analysis of γδ T cell identity, flow cytometry was used to assess CD8 and CD3. Other markers of γδ T cell identity or lineage known in the art may also be used.

[0445] For analysis of rejuvenation, flow cytometry was used to assess stemness markers CCR7, CD62L, TCF7. Other markers of stemness or rejuvenation known in the art may also be used.

[0446] For the analysis of the prevention and reversal of exhaustion and aging, flow cytometry was used to assess the surface expression of CD39, CD57, PD-1, LAG-3 (CD223), KLRG-1 (MAFA), TIGIT, CTLA-4, CD56, CD27, CD28 and TIM-3 (CD366) proteins. Other markers of exhaustion or aging known in the art may also be used.

[0447] For analysis of proliferation, cell numbers were counted by Invitrogen Countess automated cell counter.

[0448] For analysis of tumor cell killing efficacy, cytokine release (IFNγ, TNF-α, GM-CSF, IL-2, IL-6, IL-10, IL-13, IL12p70, IL-8 and IL-17) was determined u...

Claims

1. A method for inhibiting, preventing and / or reversing the exhaustion of immune cells, the method comprising: exposing the immune cells to messenger RNA (mRNA) encoding one or more reprogramming factors, wherein said exposure effects expression of said one or more reprogramming factors in said immune cells to inhibit, prevent and / or reverse exhaustion of said immune cells while preserving their identity.

2. A method for inducing proliferation of immune cells, the method comprising: exposing the immune cells to messenger RNA (mRNA) encoding one or more reprogramming factors, wherein the exposing achieves expression of the one or more reprogramming factors in the immune cells to induce proliferation of the immune cells while retaining their identity.

3. The method according to claim 1 or claim 2, wherein the immune cell is a lymphocyte or a granulocyte.

4. The method of claim 3, wherein the lymphocytes are T cells, B cells or natural killer (NK) cells.

5. The method of claim 2, wherein the lymphocytes are tumor infiltrating lymphocytes.

6. The method of claim 4 or claim 5, wherein the lymphocytes are T cells.

7. The method of claim 6, wherein the T cell is a cytotoxic T cell (CD8+), a helper T cell (CD4+), a suppressor T cell or a memory T cell.

8. The method of claim 7, wherein the helper T cells are Th1, Th2, Th17, Th9 or Tfh T cells.

9. The method of claim 7, wherein the memory T cells are central memory T cells, effector memory T cells, tissue-resident memory T cells, or virtual memory T cells.

10. The method of claim 7, wherein the suppressor T cells are FOXP3 + T cells or FOXP3 - T cells.

11. The method of claim 2, wherein the granulocyte is a neutrophil, eosinophil, basophil, monocyte, macrophage, mast cell or dendritic cell.

12. The method of claim 3 or claim 4, wherein the lymphocytes are B cells.

13. The method of claim 12, wherein the B cell is a memory B cell or a plasma cell.

14. The method according to any one of claims 1 to 12, wherein the immune cell is a natural immune cell or an engineered immune cell.

15. The method according to any one of claims 1 to 14, wherein the exposing comprises exposure to mRNA encoding one or more reprogramming factors selected from the group consisting of Oct, Sox, Klf, Myc, Lin or NANOG.

16. The method according to any one of claims 1 to 14, wherein the exposing comprises exposure to mRNA encoding one or more reprogramming factors selected from the group consisting of Oct4, Sox2, Klf4, cMyc, Lin28 and NANOG.

17. The method of any one of claims 1 to 16, wherein the exposing comprises providing a composition comprising the mRNA, wherein the composition comprises an excipient for transfection.

18. The method of claim 17, wherein the composition comprises a lipid and wherein the mRNA is associated with the lipid.

19. The method according to any one of claims 1 to 18, wherein the exposing achieves transfection of the mRNA encoding one or more reprogramming factors into the immune cells.

20. The method of any one of claims 1 to 18, wherein the exposing achieves transfection of the mRNA encoding one or more reprogramming factors into T cells preferentially over other immune cells.

21. The method of any one of claims 1 to 19, wherein the exposing is performed in vitro, in vivo or ex vivo.

22. The method of claim 21, wherein the exposing is performed ex vivo using a technique selected from microinjection, electroporation, biolistics, optical transfection, microfluidic extrusion, chemical transfection, or biotransfection.

23. The method of claim 21 or claim 22, wherein the exposing is performed ex vivo and the method further comprises transplanting the immune cells into a subject after the exposing.

24. The method according to claim 21, wherein the exposing is performed in vivo, and the exposing achieves transfection of the mRNA encoding one or more reprogramming factors into the immune cells for expressing the one or more reprogramming factors in the cells.

25. The method of any one of claims 1 to 24, wherein the exposing comprises exposing the immune cells to the mRNA at least once a day for no more than 5 consecutive days.

26. The method of claim 25, further comprising interrupting the exposure and repeating the exposure after the interruption.

27. The method of any one of claims 1 to 24, wherein the exposing comprises exposing the immune cells to the mRNA at least once a day for between about 2 and 5 consecutive days.

28. The method of claim 27, further comprising interrupting the exposure and repeating the exposure after the interruption.

29. A method for preparing a composition for cell therapy, the method comprising: obtaining or providing a sample comprising one or more of the same or different types of immune cells; One or more immune cells are treated with messenger RNA (mRNA) encoding one or more reprogramming factors, wherein the treatment does not cause loss of differentiation, and wherein the treatment rejuvenates or resuscitates the immune cells as measured by an immune cell killing assay.

30. The method of claim 29, wherein the immune cell is a lymphocyte or a granulocyte.

31. The method of claim 30, wherein the lymphocyte is a T cell, a B cell, or a natural killer (NK) cell.

32. The method of claim 30, wherein the lymphocytes are tumor infiltrating lymphocytes.

33. A method according to claim 31 or claim 32, wherein the lymphocytes are T cells.

34. The method of claim 33, wherein the T cell is a cytotoxic T cell (CD8+), a helper T cell (CD4+), a suppressor T cell, or a memory T cell.

35. The method of claim 34, wherein the helper T cells are Th1, Th2, Th17, Th9 or Tfh T cells.

36. The method of claim 34, wherein the memory T cell is a central memory T cell, an effector memory T cell, a tissue-resident memory T cell, or a virtual memory T cell.

37. The method of claim 34, wherein the suppressor T cells are FOXP3 + T cells or FOXP3 - T cells.

38. The method of claim 30, wherein the granulocyte is a neutrophil, eosinophil, basophil, monocyte, macrophage, mast cell, or dendritic cell.

39. The method of claim 30 or claim 31 , wherein the lymphocytes are B cells.

40. The method of claim 39, wherein the B cell is a memory B cell or a plasma cell.

41. The method of any one of claims 29 to 40, wherein the immune cell is a natural immune cell or an engineered immune cell.

42. The method of any one of claims 29 to 41, wherein the treatment comprises treatment with mRNA encoding one or more reprogramming factors selected from the group consisting of Oct, Sox, Klf, Myc, Lin or NANOG.

43. The method of any one of claims 29 to 42, wherein the treatment comprises treatment with mRNA encoding one or more reprogramming factors selected from the group consisting of Oct4, Sox2, Klf4, cMyc, Lin28 and NANOG.

44. The method of any one of claims 29 to 43, wherein the processing comprises providing a composition comprising the mRNA, wherein the composition comprises an excipient for transfection.

45. The method of claim 44, wherein the composition comprises a lipid and wherein the mRNA is associated with the lipid.

46. ​​The method of any one of claims 29 to 45, wherein the treatment effects transfection of the mRNA encoding one or more reprogramming factors into the immune cells.

47. The method of any one of claims 29 to 45, wherein the treatment effects transfection of the mRNA encoding one or more reprogramming factors into T cells preferentially over other immune cells.

48. The method according to any one of claims 29 to 46, wherein the treatment is performed in vitro, in vivo or ex vivo.

49. The method of claim 48, wherein the treating is performed ex vivo using a technique selected from microinjection, electroporation, biolistics, optical transfection, microfluidic extrusion, chemical transfection, or biotransfection.

50. The method of claim 48 or claim 49, wherein the treatment is performed ex vivo and the method further comprises transplanting the immune cells into a subject after the treatment.

51. The method according to claim 48, wherein the treatment is performed in vivo, and the treatment achieves transfection of the mRNA encoding one or more reprogramming factors into the immune cells for expressing the one or more reprogramming factors in the cells.

52. The method of claim 48, wherein the treating is performed in vivo, and wherein the treating is performed before, simultaneously with, or after administration of the bispecific antibody.

53. The method of any one of claims 29 to 52, wherein the treatment comprises treating the immune cells with the mRNA at least once a day for no more than 5 consecutive days.

54. The method of claim 53, further comprising interrupting the processing and repeating the processing after the interruption.

55. The method of any one of claims 29 to 52, wherein the treatment comprises treating the immune cells with the mRNA at least once a day for between about 2 and 5 consecutive days.

56. The method of claim 55, further comprising interrupting the processing and repeating the processing after the interruption.

57. An immune cell population prepared according to the method of any one of claims 29 to 56.

58. Use of the immune cell population according to claim 57 for treating a disease or disorder.

59. The use according to claim 58, wherein the disease or disorder is cancer or an autoimmune disease.

60. A method for rejuvenating immune cells, the method comprising: mRNA encoding one or more reprogramming factors is introduced into the immune cells for expressing the one or more reprogramming factors, thereby generating immune cells expressing the one or more reprogramming factors to obtain rejuvenated immune cells.

61. The method of claim 60, wherein the immune cell is a lymphocyte or a granulocyte.

62. The method of claim 30, wherein the lymphocyte is a T cell, a B cell, or a natural killer (NK) cell.

63. The method of claim 30, wherein the lymphocytes are tumor infiltrating lymphocytes.

64. The method of claim 60 or claim 61, wherein the lymphocytes are T cells.

65. The method of claim 64, wherein the T cell is a cytotoxic T cell (CD8+), a helper T cell (CD4+), a suppressor T cell, or a memory T cell.

66. The method of claim 65, wherein the helper T cells are Th1, Th2, Th17, Th9 or Tfh T cells.

67. The method of claim 65, wherein the memory T cell is a central memory T cell, an effector memory T cell, a tissue-resident memory T cell, or a virtual memory T cell.

68. The method of claim 65, wherein the suppressor T cells are FOXP3 + T cells or FOXP3 - T cells.

69. The method of claim 61, wherein the granulocyte is a neutrophil, eosinophil, basophil, monocyte, macrophage, mast cell, or dendritic cell.

70. The method of claim 61 or claim 62, wherein the lymphocytes are B cells.

71. The method of claim 70, wherein the B cell is a memory B cell or a plasma cell.

72. The method of any one of claims 60 to 71, wherein the immune cell is a natural immune cell or an engineered immune cell.

73. The method of any one of claims 60 to 72, wherein the introducing comprises introducing mRNA encoding one or more reprogramming factors selected from the group consisting of Oct, Sox, Klf, Myc, Lin or NANOG.

74. The method of any one of claims 60 to 73, wherein the introducing comprises introducing mRNA encoding one or more reprogramming factors selected from the group consisting of Oct4, Sox2, Klf4, cMyc, Lin28 and NANOG.

75. The method of any one of claims 60 to 74, wherein the introducing comprises providing a composition comprising the mRNA, wherein the composition comprises an excipient for transfection.

76. The method of claim 75, wherein the composition comprises a lipid and wherein the mRNA is associated with the lipid.

77. The method of any one of claims 60 to 76, wherein the introducing is achieved by transfecting the mRNA encoding one or more reprogramming factors into the immune cell.

78. The method of any one of claims 60 to 76, wherein the introducing achieves transfection of the mRNA encoding one or more reprogramming factors into T cells preferentially over other immune cells.

79. The method of any one of claims 60 to 77, wherein the introducing is performed in vitro, in vivo or ex vivo.

80. The method of claim 79, wherein the introducing is performed ex vivo using a technique selected from microinjection, electroporation, biolistics, optical transfection, microfluidic extrusion, chemical transfection, or biotransfection.

81. The method of claim 79 or claim 80, wherein the introducing is performed ex vivo and the method further comprises transplanting the immune cells into a subject after the introducing.

82. The method according to claim 79, wherein the introducing is performed in vivo, and the introducing is achieved by transfecting the mRNA encoding one or more reprogramming factors into the immune cells for expressing the one or more reprogramming factors in the cells.

83. The method of claim 79, wherein the introducing is performed in vivo, and wherein the introducing is performed before, simultaneously with, or after administration of the bispecific antibody.

84. The method of any one of claims 60 to 83, wherein the introducing comprises introducing the mRNA into the immune cells at least once a day for no more than 5 consecutive days.

85. The method of claim 84, further comprising interrupting the introducing and repeating the introducing after the interruption.

86. The method of any one of claims 60 to 83, wherein the introducing comprises introducing the mRNA into the immune cells at least once a day for between about 2 to 5 consecutive days.

87. The method of claim 86, further comprising interrupting the introducing and repeating the introducing after the interruption 88. A method for inhibiting, preventing and / or reversing exhaustion of engineered immune cells, the method comprising: exposing the immune cells to messenger RNA (mRNA) encoding one or more reprogramming factors, wherein said exposure effects expression of said one or more reprogramming factors in said immune cells to inhibit, prevent and / or reverse exhaustion of said immune cells while preserving their identity.

89. A method for reducing T cell exhaustion that occurs during the manufacture of CAR T cells, wherein the method comprises: activating the T cells by exposing the T cells to CD3 and / or CD28; transfecting the activated T cells with mRNA encoding one or more reprogramming factors; For each transfection, 1ng to 500ug of mRNA was used per million T cells. wherein the transfection achieves expression of the one or more reprogramming factors in the T cells to increase their cytotoxic or proliferative capacity compared to untreated or control T cells.

90. The method of claim 89, wherein 1 ng to 400 ug of mRNA is used per million T cells per transfection.

91. The method of claim 89, wherein 1 ng to 300 ug of mRNA is used per million T cells per transfection.

92. The method of claim 89, wherein 1 ng to 250 ug of mRNA is used per million T cells per transfection.

93. The method of claim 89, wherein 1 ng to 200 ug of mRNA is used per million T cells per transfection.

94. The method of claim 89, wherein 1 ng to 150 ug of mRNA is used per million T cells per transfection.

95. The method of claim 89, wherein 1 ng to 100 ug of mRNA is used per million T cells per transfection.

96. The method of claim 89, wherein 1 ng to 50 ug of mRNA is used per million T cells per transfection.

97. The method of claim 89, wherein 1 ng to 25 ug of mRNA is used per million T cells per transfection.

98. The method of claim 89, wherein 1 ng to 20 ug of mRNA is used per million T cells per transfection.

99. The method of claim 89, wherein 1 ng to 15 ug of mRNA is used per million T cells per transfection.

100. The method of claim 89, wherein 1 ng to 10 ug of mRNA is used per million T cells per transfection.

101. The method of claim 89, wherein 1 ng to 9 ug of mRNA is used per million T cells per transfection.

102. The method of claim 89, wherein 1 ng to 8 ug of mRNA is used per million T cells per transfection.

103. The method of claim 89, wherein 1 ng to 7 ug of mRNA is used per million T cells per transfection.

104. The method of claim 89, wherein 1 ng to 6 ug of mRNA is used per million T cells per transfection.

105. The method of claim 89, wherein 1 ng to 5 ug of mRNA is used per million T cells per transfection.

106. The method of claim 89, wherein 1 ng to 4 ug of mRNA is used per million T cells per transfection.

107. The method of claim 89, wherein 1 ng to 3 ug of mRNA is used per million T cells per transfection.

108. The method of claim 89, wherein 1 ng to 2 ug of mRNA is used per million T cells per transfection.

109. The method of claim 89, wherein 1 ng to 1 ug of mRNA is used per million T cells per transfection.

110. The method of claim 89, wherein 10 ng to 10 ug of mRNA is used per million T cells per transfection.

111. The method of claim 89, wherein 50 ng to 10 ug of mRNA is used per million T cells per transfection.

112. The method of claim 89, wherein 100 ng to 10 ug of mRNA is used per million T cells per transfection.

113. The method of claim 89, wherein 150 ng to 10 ug of mRNA is used per million T cells per transfection.

114. The method of claim 89, wherein 200 ng to 10 ug of mRNA is used per million T cells per transfection.

115. The method of claim 89, wherein 300 ng to 10 ug of mRNA is used per million T cells per transfection.

116. The method of claim 89, wherein 350 ng to 10 ug of mRNA is used per million T cells per transfection.

117. The method of claim 89, wherein 400 ng to 10 ug of mRNA is used per million T cells per transfection.

118. The method of claim 89, wherein 500 ng to 10 ug of mRNA is used per million T cells per transfection.

119. The method of claim 89, wherein 600 ng to 10 ug of mRNA is used per million T cells per transfection.

120. The method of claim 89, wherein 750 ng to 10 ug of mRNA is used per million T cells per transfection.

121. The method of claim 89, wherein 900 ng to 10 ug of mRNA is used per million T cells per transfection.

122. The method of claim 89, wherein 1 ug to 10 ug of mRNA is used per million T cells per transfection.

123. The method of claim 89, wherein 2 ug to 10 ug of mRNA is used per million T cells per transfection.

124. The method of claim 89, wherein 2 ug to 6 ug of mRNA is used per million T cells per transfection.

125. The method of claim 89, wherein 1 ug to 9 ug of mRNA is used per million T cells per transfection.

126. The method of claim 89, wherein 1 ug to 8 ug of mRNA is used per million T cells per transfection.

127. The method of claim 89, wherein 1 ug to 7 ug of mRNA is used per million T cells per transfection.

128. The method of claim 89, wherein 1 ug to 6 ug of mRNA is used per million T cells per transfection.

129. The method of claim 89, wherein 1 ug to 5 ug of mRNA is used per million T cells per transfection.

130. The method of claim 89, wherein 1 ug to 4 ug of mRNA is used per million T cells per transfection.

131. The method of claim 89, wherein 1 ug to 3 ug of mRNA is used per million T cells per transfection.

132. The method of claim 89, wherein 1 ug to 2 ug of mRNA is used per million T cells per transfection.

133. The method of claim 89, wherein the mRNA encoding the one or more reprogramming factors is transfected into the T cells at least once a day for between about 2 and 5 consecutive days.

134. The method of claim 89, wherein the mRNA encoding the one or more reprogramming factors is transfected into the T cells at least once a day for 5 consecutive days.

135. The method of claim 89, wherein the mRNA encoding the one or more reprogramming factors is transfected into the T cells at least once a day for 4 consecutive days.

136. The method of claim 89, wherein the mRNA encoding the one or more reprogramming factors is transfected into the T cells at least once a day for 3 consecutive days.

137. The method of claim 89, wherein the mRNA encoding the one or more reprogramming factors is transfected into the T cells at least once a day for 2 consecutive days.

138. The method of claim 89, wherein the mRNA encoding the one or more reprogramming factors is transfected into the T cells at least once a day for 1 day.

139. The method of claim 89, wherein the mRNA encoding the one or more reprogramming factors is transfected into the T cells once a day for 5 consecutive days.

140. The method of claim 89, wherein the mRNA encoding the one or more reprogramming factors is transfected into the T cells once a day for 4 consecutive days.

141. The method of claim 89, wherein the mRNA encoding the one or more reprogramming factors is transfected into the T cells once a day for 3 consecutive days.

142. The method of claim 89, wherein the mRNA encoding the one or more reprogramming factors is transfected into the T cells once a day for 2 consecutive days.

143. The method of claim 89, wherein the mRNA encoding the one or more reprogramming factors is transfected into the T cells once a day for 1 day.

144. The method of claim 89, wherein the mRNA encoding the one or more reprogramming factors is transfected into the T cells twice a day for between about 2 to 5 consecutive days.

145. The method of claim 89, wherein the mRNA encoding the one or more reprogramming factors is transfected into the T cells twice a day for 5 consecutive days.

146. The method of claim 89, wherein the mRNA encoding the one or more reprogramming factors is transfected into the T cells twice a day for 4 consecutive days.

147. The method of claim 89, wherein the mRNA encoding the one or more reprogramming factors is transfected into the T cells twice a day for 3 consecutive days.

148. The method of claim 89, wherein the mRNA encoding the one or more reprogramming factors is transfected into the T cells twice a day for 2 consecutive days.

149. The method of claim 89, wherein the mRNA encoding the one or more reprogramming factors is transfected into the T cells twice a day for 1 day.

150. The method of any one of claims 144 to 149, wherein the amount of mRNA encoding the one or more reprogramming factors used per million T cells is: (a) about 1 ng to 100 ug mRNA per million T cells for the first of two transfections per day; (b) For the second of two transfections per day, approximately 1 ng to 100 ug mRNA per million T cells.

151. The method of any one of claims 144 to 149, wherein the amount of mRNA encoding the one or more reprogramming factors used per million T cells is: (a) about 100 ng to 10 ug mRNA per million T cells for the first of two transfections per day; (b) For the second of two transfections per day, approximately 100 ng to 10 ug mRNA per million T cells.

152. The method of any one of claims 144 to 149, wherein the amount of mRNA encoding the one or more reprogramming factors used per million T cells is: (a) about 10 ng to 10 ug mRNA per million T cells for the first of two transfections per day; (b) about 10 ng to 8 ug mRNA per million T cells for the second of two transfections per day; And among them (c) the mRNA used for the first of two transfections per day encodes OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG; and (d) The mRNAs used for the second of two daily transfections encode OCT4, c-MYC and NANOG.

153. The method of claim 147, wherein the amount of mRNA encoding the one or more reprogramming factors used per million T cells is: (a) about 10 ng to 10 ug mRNA per million T cells for the first of two transfections per day; (b) about 10 ng to 8 ug mRNA per million T cells for the second of two transfections per day; And among them (c) the mRNA used for the first of two transfections per day encodes OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG; and (d) The mRNAs used for the second of two daily transfections encode OCT4, c-MYC and NANOG.

154. The method of claim 148, wherein the amount of mRNA encoding the one or more reprogramming factors used per million T cells is: (a) about 1 ng to 100 ug mRNA per million T cells for the first of two transfections per day; (b) about 1 ng to 100 ug mRNA per million T cells for the second of two transfections per day; and wherein (c) the mRNA used for the first of two transfections per day encodes OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG; and (d) The mRNAs used for the second of two daily transfections encode OCT4, c-MYC and NANOG.

155. The method of claim 149, wherein the amount of mRNA encoding the one or more reprogramming factors used per million T cells is: (a) about 1 ng to 100 ug mRNA per million T cells for the first of two transfections per day; (b) about 1 ng to 100 ug mRNA per million T cells for the second of two transfections per day; and wherein (c) the mRNA used for the first of two transfections per day encodes OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG; and (d) The mRNAs used for the second of two daily transfections encode OCT4, c-MYC and NANOG.

156. The method of claim 143, wherein the amount of mRNA encoding the one or more reprogramming factors used per million T cells is about 1 ng to 100 ug; And wherein the mRNA encodes the reprogramming factors OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

157. The method of claim 143, wherein the amount of mRNA encoding the one or more reprogramming factors used per million T cells is about 50 ug; And wherein the mRNA encodes the reprogramming factors OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

158. The method of claim 143, wherein the amount of mRNA encoding the one or more reprogramming factors used per million T cells is about 25 ug; And wherein the mRNA encodes the reprogramming factors OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

159. A method for reducing T cell exhaustion that occurs during the manufacture of CAR T cells, the method comprising: activating the T cells by exposing the T cells to CD3 and / or CD28; transfecting the activated T cells with mRNA encoding one or more reprogramming factors; For each transfection, 1ng to 500ug of mRNA was used per million T cells. wherein the transfection achieves expression of the one or more reprogramming factors in the T cells to increase the percentage of Tcm or Tscm cells compared to untreated or control T cells.

160. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is less than 200%.

161. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is less than 150%.

162. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is less than 125%.

163. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is less than 100%.

164. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is less than 80%.

165. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is less than 60%.

166. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is less than 40%.

167. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is less than 30%.

168. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is less than 20%.

169. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is less than 10%.

170. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is between about 10% and 40%.

171. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is between about 10% and 35%.

172. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is between about 5% and 40%.

173. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is between about 5% and 35%.

174. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is between about 5% and 30%.

175. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is between about 5% and 25%.

176. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is between about 10% and 40%.

177. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is between about 10% and 50%.

178. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is between about 5% and 50%.

179. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is between about 5% and 65%.

180. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is between about 5% and 75%.

181. The method of claim 155, wherein the increase in the percentage of Tcm or Tscm cells is between 1% and 50%.

182. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is between 1% and 40%.

183. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is between 1% and 25%.

184. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is between 20% and 80%.

185. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is between about 2-fold and 10-fold.

186. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is between about 2-fold and 8-fold.

187. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is between about 2-fold and 6-fold.

188. The method of claim 159, wherein the increase in the percentage of Tcm or Tscm cells is between about 1-fold and 4-fold.

189. The method of any one of claims 1 to 188, wherein the mRNA molecule comprises self-amplifying RNA and provides a half-life of 2 days or longer.

190. The method of any one of claims 1 to 188, wherein the mRNA molecule is a self-amplifying RNA and provides a half-life of 2 days or longer.

191. A method according to any one of claims 1 to 188, wherein the mRNA molecule comprises trans-amplified RNA and provides a half-life of 2 days or longer.

192. A method according to any one of claims 1 to 188, wherein the mRNA molecule is a trans-amplified RNA and provides a half-life of 2 days or longer.

193. The method of any one of claims 1 to 188, wherein the mRNA molecule comprises trans-amplifying RNA or self-amplifying RNA and provides a half-life of up to 10 days.

194. The method of any one of claims 1 to 188, wherein the mRNA molecule comprises trans-amplifying RNA or self-amplifying RNA and provides a half-life of up to 10 days.

195. The method of any one of claims 1 to 195, wherein the half-life of a linear non-replicating mRNA is controlled by altering the 3' untranslated region, changing the poly(A) tail length and / or adding a WPRE element to the mRNA.

196. The method of claim 195, wherein the half-life of the mRNA is controlled to be between 6 minutes and 24 hours.

197. The method of claim 196, wherein the half-life of the mRNA is increased to a maximum of 72 hours.

198. The method of any one of claims 1 to 197, wherein at least one of the one or more reprogramming factors is a T cell-optimized reprogramming factor encoded by a polynucleotide sequence having approximately 95% sequence identity to any one of the sequences of SEQ ID NOs: 7-14.

199. The method of claim 198, wherein the T cell optimized reprogramming factor is encoded by a polynucleotide sequence having about 96% sequence identity to SEQ ID NO:

7.

200. The method of claim 198, wherein the T cell optimized reprogramming factor is encoded by a polynucleotide sequence having about 97% sequence identity to SEQ ID NO:7.

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