Extracorporeal and ex vivo engineering of select cell populations from peripheral blood

Abstract

Disclosed are methods for producing a population of autologous engineered cells to be reinfused into a subject comprising extracorporeally transfecting an internal payload into a population of target cells from a sample obtained from the subject by incubating the population of cells with a lipid nanoparticle (LNP) and / or targeting lipid nanoparticle (tLNP) in an incubation component of an extracorporeal system for no more than 6 hours.

Description

Extracorporeal and Ex Vivo Engineering of Select Cell Populations from Peripheral BloodCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional application number 63 / 510,450, filed June 27, 2023; the disclosures of which is expressly incorporated by reference herein.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] This application contains a Sequence Listing that has been submitted electronically and is hereby incorporated by reference in its entirety. The Sequence Listing was created on June 26, 2024, is named “24-0223-WO_VFinal.xml”, and is 154 kilobytes in size.BACKGROUND

[0003] Peripheral blood cells are the most readily engineered cells of the body due in large part to the ease with which they may be extracted from the body, manipulated, and returned. However, as currently practiced, such procedures involve shipment of cells to and from a central facility, cell separation or selection, extensive cell culture, and viral transduction of genetic material, as typified by generation of CAR-T cells in commercial therapeutics. These procedures impose substantial logistical and financial challenges.

[0004] These challenges are particularly associated with viral vector-based ex vivo transfection. Such procedures can include cellular separation, expansion, and cryopreservation of the donor and / or engineered cells. Ex vivo, as used herein, includes extensive manipulation including extended periods of cell culture and expansion, and / or refrigerated or cryogenic storage or shipment, over several days or longer. Ex vivo manipulation of cells to introduce an engineering agent, for example in the generation of CAR-T cells and the like, at least at the commercial or clinical trial scale, has typically utilized transduction with viral vectors and with integration of the engineering agent into the genome of the engineered cell. Ex vivo methods may also iRECTIFIED SHEET (RULE 91 ) ISA / EPinclude a step of separating a population of leukocytes or HSCs from the blood sample prior to transfection. In certain embodiments, the ex vivo methods can also include additional steps that require additional cell culture and expansion and / or refrigerated or cryogenic storage and / or shipment over several days or longer.

[0005] Therefore, this disclosure provides methods and systems for local expansion-less extracorporeal transfection of select leukocyte populations from peripheral blood to satisfy an urgent need in the field.SUMMARY

[0006] In a first aspect the invention provides methods for producing a population of autologous engineered cells capable of being reinfused into a subject comprising transfecting an internal payload into an extracorporeal or ex vivo population of leukocytes or hematopoietic stem cells (HSC) obtained from the subject by incubating the population of leukocytes or HSCs with a lipid nanoparticle (LNP) or a targeting lipid nanoparticle (tLNP) comprising the internal payload in an incubation component of an extracorporeal system for no more than 6 hours, wherein the LNP comprises the internal payload, and wherein the tLNP comprises the internal payload and a binding moiety capable of binding a molecule on the surface of a leukocyte or HSC.

[0007] In certain embodiments the population of leukocytes is a component of whole blood, is a population of peripheral blood mononuclear cells (PBMCs) or is a population of T cells.

[0008] In additional embodiments the binding moiety comprises an antibody or antigenic binding fragment thereof, in particular a CD2 binding moiety, a CD3 binding moiety, a CD5 binding moiety, a CD7 binding moiety, a CD8 binding moiety, CD28 binding moiety, CD27 binding moiety, CD34 binding moiety, CD38 binding moiety, CD43 binding moiety, CD45 binding moiety (including binding moieties specific for CD45RB, CD45RO and pan CD45 species thereof), CD48 binding moiety, CD1 17 binding moiety, CD127 binding moiety, CD150 binding moiety, 4-1 BB binding moiety, or a Sca-1 binding moiety, and in certain specific embodiments the binding moiety is a CD2 binding moiety, a CD8 binding moiety, CD34 binding moiety, CD45 binding moiety (including binding moieties specific for CD45RB, CD45RO and pan CD45 species thereof), or a CD117 binding moiety.2RECTIFIED SHEET (RULE 91 ) ISA / EP

[0009] The extracorporeal system useful in the practice of the disclosed methods can comprise a closed system, a semi-closed system, or an open system, wherein the incubation component further can comprise an incubation medium in which the population of leukocytes and the LNP or tLNP are incubated.

[0010] The LNP or tLNP disclosed for use in the disclosed methods comprise a payload that delivers to the cell an biological response modifier (BRM) or nucleic acid encoding a BRM that is a cytokine, a chemokine, or immune checkpoint inhibitor, wherein the cytokine is IL-2, IL-7, IL-12, IL15, IL-18, or IL-21 , the chemokine is CCR2, or the immune checkpoint inhibitor is anti-CTLA4. In certain embodiments the payload delivered to the cell is an engineering agent comprising an mRNA that encodes a reprogramming agent comprising a chimeric antigen receptor (CAR), bispecific T cell engager (BiTE), an immune cell engager, a gene or base editing protein, an RNA- guided nuclease, an inactive RNA-guided nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a transposase, a retrotransposase, a gene writer protein, a prime editing protein, or RNA writer, a reverse transcriptase, a DNA nickase, a Cas9 nickase, or a CRISPR nuclease.

[0011] The incubation medium used in the methods disclosed herein can further comprise a biological response modifier (BRM) that is a cytokine, a chemokine, or immune checkpoint inhibitor, wherein the cytokine is IL-2, IL-7, IL-12, IL15, IL-18, or IL-21 , the chemokine is CCR2, or the immune checkpoint inhibitor is anti-CTLA4.

[0012] The extracorporeal system provided herein for use in the disclosed methods can further comprise a collection component comprising a blood collection container or conduit to receive a blood or bone marrow sample obtained from the subject and further comprising an apheresis or separation component to separate the population of leukocytes from a blood or bone marrow sample obtained from the subject wherein the apheresis or separation component comprises photopheresis or leukapheresis, and further wherein the extracorporeal system comprises one or more washing components for washing the population of leukocytes prior to the incubation step and / or after the incubation step.

[0013] In another aspect provided herein are populations of autologous3RECTIFIED SHEET (RULE 91 ) ISA / EPengineered cells designed to express or overexpress a protein, or to prevent or inhibit expression of an endogenous protein wherein the cells are produced by the methods disclosed herein.

[0014] In further aspects provided herein are extracorporeal systems for producing a composition comprising a population of autologous engineered cells comprising: an optional apheresis or separation component for separating a population of leukocytes or HSC from a blood sample obtained from a subject; an incubation component for transfecting the population of leukocytes or HSC with (i) a lipid nanoparticle (LNP) comprising an internal payload or (ii) a targeting lipid nanoparticle (tLNP) comprising an internal payload and a binding moiety to produce a population of autologous engineered cells; and a resuspension component for resuspending the population of autologous engineered cells in a solution which comprises the composition comprising the population of autologous engineered cells for reinfusion into the subject; wherein the extracorporeal system optionally further comprises a collection component comprising a blood collection container or conduit to receive a blood or bone marrow sample obtained from the subject and further comprises an apheresis or separation component to separate the population of leukocytes from a blood or bone marrow sample obtained from the subject wherein the apheresis or separation component comprises photopheresis or leukapheresis, and further wherein the extracorporeal system optionally comprises one or more washing components for washing the population of leukocytes prior to the incubation step and / or after the incubation step, and wherein the extracorporeal system comprises a closed system, a semi-closed system, or an open system.

[0015] In certain embodiments the population of leukocytes is a population of T cells or peripheral blood mononuclear cells (PBMCs). In additional embodiments, the binding moiety is an antibody or antigenic binding fragment thereof.

[0016] In additional embodiments the binding moiety comprises an antibody or antigenic binding fragment thereof, in particular a CD2 binding moiety, a CD3 binding moiety, a CD5 binding moiety, a CD7 binding moiety, a CD8 binding moiety, CD28 binding moiety, CD27 binding moiety, CD34 binding moiety, CD38 binding moiety, CD43 binding moiety, CD45 binding moiety (including binding moieties specific for CD45RB, CD45RO and pan CD45 species thereof), CD48 binding moiety, CD1 174RECTIFIED SHEET (RULE 91 ) ISA / EPbinding moiety, CD127 binding moiety, CD150 binding moiety, 4-1 BB binding moiety, or a Sca-1 binding moiety, and in certain specific embodiments the binding moiety is a CD2 binding moiety, a CD8 binding moiety, CD34 binding moiety, CD45 binding moiety (including binding moieties specific for CD45RB, CD45RO and pan CD45 species thereof), or a CD117 binding moiety, and in certain specific embodiments the binding moiety is a CD2 binding moiety, a CD8 binding moiety, CD34 binding moiety, CD45 binding moiety (including binding moieties specific for CD45RB, CD45RO and pan CD45 species thereof), or a CD117 binding moiety.

[0017] In the extracorporeal systems provided herein LNP or tLNP comprises a payload that delivers to the cell an exogenous protein or nucleic acid encoding a biological response modifier (BRM) that is a cytokine, a chemokine, or immune checkpoint inhibitor, wherein the cytokine is IL-2, IL-7, IL-12, IL15, IL-18, or IL-21 , the chemokine is CCR2, or the immune checkpoint inhibitor is anti-CTLA4. Further, in the extracorporeal systems provided herein the payload delivered to the cell is an engineering agent comprising an mRNA that encodes a reprogramming agent comprising a chimeric antigen receptor (CAR), bispecific T cell engager (BiTE), an immune cell engager, a gene or base editing protein, an RNA-guided nuclease, an inactive RNA-guided nuclease, a zinc finger nuclease (ZFN), a transcription activatorlike effector nuclease (TALEN), a meganuclease, a transposase, a retrotransposase, a gene writer protein, a prime editing protein, or RNA writer, a reverse transcriptase, a DNA nickase, a Cas9 nickase, or a CRISPR nuclease.

[0018] In the extracorporeal systems provided herein the incubation component further comprises an incubation medium in which the population of leukocytes and the LNP or tLNP are incubated, and in specific embodiments the incubation medium further comprises a biological response modifier (BRM) that is a cytokine, a chemokine, or immune checkpoint inhibitor, wherein the cytokine is IL-2, IL-7, IL-12, IL15, IL-18, or IL-21 , the chemokine is CCR2, or the immune checkpoint inhibitor is anti-CTLA4.

[0019] In a further aspect provided herein are treatment regimens comprising: obtaining a blood sample from a subject; optionally separating a population of leukocytes or HSC from the blood sample; incubating the population of leukocytes or HSC with (i) a lipid nanoparticle (LNP) comprising an internal payload or (ii) a targeting5RECTIFIED SHEET (RULE 91 ) ISA / EPlipid nanoparticle (tLNP) comprising an internal payload and a binding moiety to transfect the population of leukocytes or HSC with the LNP or tLNP, to produce a population of autologous engineered cells that express one or more exogenous proteins encoded by the internal payload; resuspending the population of autologous engineered cells in an infusion solution; and providing a dose of the resuspended population of autologous engineered cells to be reinfused into the subject within 6 hours of obtaining the blood sample.

[0020] In certain embodiment the treatment regimen further comprises administering a T cell activating BRM to the subject within 3 days prior obtaining the blood or bone marrow, wherein the T cell activating BRM is a CD3 agonist antibody, or IL-2, IL-7, IL-12, IL-15, IL-18, IL-21 , or other agonists of their receptors.

[0021] In particular embodiments the treatment regimen provided herein further comprises washing the cells after step (b) but before step (c), and / or washing the cells after step (c) but before step (d). In other particular embodiments the treatment regimen further comprises repeating steps (a) to (e) to produce one or more additional doses of the resuspended population of autologous engineered cells for reinfusing into the subject.

[0022] In the treatment regimens disclosed herein the population of leukocytes is a population of T cells or peripheral blood mononuclear cells (PBMCs).

[0023] In further embodiments, the treatment regimens disclosed herein further comprises adding a T cell-activating BRM to the population of leukocytes and the LNP or tLNP during step (c), wherein T cell activating BRM is a cytokine, a chemokine, or immune checkpoint inhibitor, wherein the cytokine is IL-2, IL-7, IL-12, IL15, IL-18, or IL-21 , the chemokine is CCR2, or the immune checkpoint inhibitor is anti-CTLA4.

[0024] In the treatment regimens disclosed herein the binding moiety comprises an antibody or antigenic binding fragment thereof, and in particular embodiments the binding moiety is a CD2 binding moiety, a CD3 binding moiety, a CD5 binding moiety, a CD7 binding moiety, a CD8 binding moiety, CD28 binding moiety, CD27 binding moiety, CD34 binding moiety, CD38 binding moiety, CD43 binding moiety, CD45 binding moiety (including binding moieties specific for CD45RB, CD45RO and pan CD45 species thereof), CD48 binding moiety, CD1 17 binding moiety, CD127 binding6RECTIFIED SHEET (RULE 91 ) ISA / EPmoiety, CD150 binding moiety, 4-1 BB binding moiety, or a Sca-1 binding moiety, and in specific embodiments the binding moiety is a CD2 binding moiety, a CD8 binding moiety, CD34 binding moiety, CD45 binding moiety (including binding moieties specific for CD45RB, CD45RO and pan CD45 species thereof), or a CD1 17 binding moiety.

[0025] In certain embodiments of the treatment regimens disclosed herein the LNP or tLNP comprises a payload that delivers to the cell an exogenous protein or nucleic acid encoding a biological response modifier (BRM) that is a cytokine, a chemokine, or immune checkpoint inhibitor, wherein the cytokine is rlL-2, IL-2, IL-7, IL-12, IL15, IL-18, or IL-21 , the chemokine is CCR2, or the immune checkpoint inhibitor is anti-CTLA4. In additional embodiments, the payload delivered to the cell is an engineering agent comprising an mRNA that encodes a reprogramming agent comprising a chimeric antigen receptor (CAR), bispecific T cell engager (BiTE), an immune cell engager, a gene or base editing protein, an RNA-guided nuclease, an inactive RNA-guided nuclease, a zinc finger nuclease (ZFN), a transcription activatorlike effector nuclease (TALEN), a meganuclease, a transposase, a retrotransposase, a gene writer protein, a prime editing protein, or RNA writer, a reverse transcriptase, a DNA nickase, a Cas9 nickase, or a CRISPR nuclease.

[0026] In another aspect disclosed herein are methods of providing a subject with a population of leukocytes or HSC expressing one or more exogenous proteins comprising: transfecting a nucleic acid into an extracorporeal or ex vivo population of leukocytes or hematopoietic stem cells (HSC) obtained from the subject by contacting the population of leukocytes with (i) a lipid nanoparticle (LNP) comprising an internal payload or (ii) a targeting lipid nanoparticle (tLNP) comprising a nucleic acid, washing the population of leukocytes or HSC, and providing a dose of the resuspended population of leukocytes or HSC to be reinfused into the subject within 6 hours of their extraction. In particular embodiments these methods further comprises performing a leukapheresis technique to obtain the population of leukocytes or HSC substantially free of red blood cells, and in particular embodiments these methods comprise density gradient centrifugation to obtain the population of HSC or leukocytes or a subset of lymphocytes. In certain embodiments the methods comprise immunomagnetic cell separation to obtain a leukocyte subset or lymphocyte subset.

[0027] In certain embodiments the methods for extracorporeally transfecting a7RECTIFIED SHEET (RULE 91 ) ISA / EPpopulation of lymphocytes comprise contacting lymphocytes with the tLNP within a preparation of leukocytes, and in other embodiments the methods comprise contacting lymphocytes with the tLNP in a preparation of lymphocytes. In particular embodiment of these methods the preparation of lymphocytes has been depleted of B lymphocytes and / or CD4+ T lymphocytes, and in certain other embodiments, the methods further comprise contacting the population of leukocytes or HSC with a T cell activating BRM during the extracorporeal transfection step or just prior to the reinfusion step. In these aspects the T cell activating BRM is a cytokine, a chemokine, or immune checkpoint inhibitor, wherein the cytokine is IL-2, IL-7, IL-12, IL15, IL-18, or IL-21 , the chemokine is CCR2, or the immune checkpoint inhibitor is anti-CTLA4. In other embodiments the lymphocytes have been contacted with a T cell activating BRM in vivo within 3 days prior to being removed from the subject’s body, and wherein the BRM is a CD3 agonist antibody, or IL-2, IL-7, IL-12, IL-15, IL-18, IL-21 , or other agonists of their receptors.

[0028] In additional embodiments of the disclosed methods, a component of the tLNP has T cell activating activity or a targeting or binding moiety that binds and activates CD2, CD3, 4-1 BB, or CD28, or an IL-2Ry cytokine, thereby acting as a BRM.

[0029] In certain embodiments of the practice of these methods the payload delivered to the cell is an engineering agent comprising an mRNA that encodes a reprogramming agent comprising a chimeric antigen receptor (CAR), bispecific T cell engager (BiTE), an immune cell engager, a gene or base editing protein, an RNA- guided nuclease, an inactive RNA-guided nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a transposase, a retrotransposase, a gene writer protein, a prime editing protein, or RNA writer, a reverse transcriptase, a DNA nickase, a Cas9 nickase, or a CRISPR nuclease.

[0030] In additional aspects disclosed herein are methods for producing a population of autologous engineered cells capable of being reinfused into a subject, comprising transfecting an internal payload into an enriched extracorporeal or ex vivo population of leukocytes comprising T cells, B cells, NK cells, macrophages, or monocytes, or into hematopoietic stem cells (HSC) by incubating the population of leukocytes or HSCs with a lipid nanoparticle (LNP) in an incubation component of an extracorporeal system for no more than 6 hours, wherein the LNP comprises the8RECTIFIED SHEET (RULE 91 ) ISA / EPinternal payload, extracorporeal system for producing a composition comprising a population of autologous engineered cells comprising: a separation component for separating a subpopulation of leukocytes comprising T cells, B cells, NK cells, macrophages, or monocytes or HSC from a blood sample obtained from a subject; an incubation component for transfecting the subpopulation of leukocytes or HSC with a lipid nanoparticle (LNP) comprising an internal payload to produce a population of autologous engineered cells; a resuspension component for resuspending the population of autologous engineered cells in a solution which comprises the composition comprising the population of autologous engineered cells for reinfusion into the subject, and treatment regimens comprising: obtaining a blood sample from a subject; separating a subpopulation of enriched leukocytes comprising T cells, B cells, NK cells, macrophages, or monocytes or HSC from the blood sample; incubating the subpopulation of enriched leukocytes or HSC with a lipid nanoparticle (LNP) comprising an internal payload to transfect the subpopulation of leukocytes or HSC with the LN P to produce a population of autologous engineered cells that express one or more exogenous proteins encoded by the internal payload; resuspending the population of autologous engineered cells in an infusion solution; and providing a dose of the resuspended population of autologous engineered cells within 6 hours obtaining the blood sample; and methods of providing a subject with a subpopulation of enriched leukocytes comprising T cells, B cells, NK cells, macrophages, or monocytes or HSC expressing one or more exogenous proteins comprising: transfecting a nucleic acid into a subpopulation of enriched of leukocytes, or into hematopoietic stem cells (HSC) obtained from the subject extracorporeally or an ex vivo population thereof by contacting the subpopulation of enriched leukocytes with a lipid nanoparticle (LNP) comprising an internal payload comprising the nucleic acid, washing the subpopulation of enriched leukocytes or HSC, and providing a dose of the resuspended population of enriched leukocytes or HSC to be reinfused into the subject within 6 hours of their extraction.

[0031] In certain embodiments of these methods, systems, or treatment regimens the enriched subpopulation of leukocytes is an enriched subpopulation of T cells

[0032] In certain embodiments of these methods, systems, or treatment9RECTIFIED SHEET (RULE 91 ) ISA / EPregimens the subpopulation is an enriched population of HSCs.

[0033] In certain embodiments of these methods, systems, or treatment regimens the LNP or tLNP comprises a payload that delivers to the cell an exogenous protein or nucleic acid encoding a biological response modifier (BRM) that is a cytokine, a chemokine, or immune checkpoint inhibitor, wherein the cytokine is rlL-2, IL-2, IL-7, IL-12, IL15, IL-18, or IL-21 , the chemokine is CCR2, or the immune checkpoint inhibitor is anti-CTLA4.

[0034] In certain embodiments of these methods, systems, or treatment regimens the payload delivered to the cell is an engineering agent comprising an mRNA that encodes a reprogramming agent comprising a chimeric antigen receptor (CAR), bispecific T cell engager (BiTE), or an immune cell engager, or wherein the engineering agent encodes a gene base editing protein, an RNA-guided nuclease, an inactive RNA-guided nuclease, a zinc finger nuclease (ZFN), a transcription activatorlike effector nuclease (TALEN), a meganuclease, a transposase, a retrotransposase, a gene writer protein, a prime editing protein, or RNA writer, a reverse transcriptase, a DNA nickase, a Cas9 nickase, or a CRISPR nuclease.

[0035] In a further specific aspect disclosed herein are methods for producing a population of autologous engineered cells to be reinfused into a subject, comprising transfecting with an internal payload that is an mRNA or gene editing system a select extracorporeal or ex vivo population of cells that are T cells, CD8+ T cells, NK cells, myeloid cells, B cells, monocytes, macrophages, DCs, or HSC with cell targeting lipid nanoparticles (tLNP) without the need to physically pre-select that subpopulation, in an incubation component of an extracorporeal system for no more than 6 hours, wherein the LNP comprises the internal payload, and wherein the tLNP comprises the internal payload and a binding moiety capable of binding a molecule on the surface of a leukocyte or HSC.

[0036] Particularly provided as disclosed herein the population of cells, leukocytes, or HSC is an extracorporeal population.

[0037] These and other features, objects, and advantages of the present invention will become better understood from the description that follows. In the description, reference is made to the accompanying drawings, which form a part hereof and in ioRECTIFIED SHEET (RULE 91 ) ISA / EPwhich there is shown by way of illustration, not limitation, embodiments of the invention. The description of preferred embodiments is not intended to limit the invention to cover all modifications, equivalents, and alternatives. Reference should therefore be made to the claims recited herein for interpreting the scope of the invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG. 1 shows a schematic of a targeted lipid nanoparticle (tLNP) according to certain embodiments, wherein the tLNP includes a binding moiety specific to CD2 on the surface of a T cell and is loaded with an internal payload that includes a nucleic acid molecule encoding a reprogramming agent; and / or a nucleic acid molecule encoding a gene editing component or is itself a gene editing component; and / or a conditioning agent or a nucleic acid molecule encoding a conditioning agent.

[0039] FIG. 2 shows a schematic of a tLNP according to certain embodiments, wherein the tLNP includes a binding moiety specific to CD3 on the surface of a T cell and is loaded with an internal payload that includes a nucleic acid molecule encoding a reprogramming agent; and / or a nucleic acid molecule encoding a gene editing component or is itself a gene editing component; and / or a conditioning agent or a nucleic acid molecule encoding a conditioning agent.

[0040] FIG. 3 shows a schematic of a tLNP according to certain embodiments, wherein the tLNP includes a binding moiety specific to CD5 on the surface of a T cell and is loaded with an internal payload that includes a nucleic acid molecule encoding a reprogramming agent; and / or a nucleic acid molecule encoding a gene editing component or is itself a gene editing component; and / or a conditioning agent or a nucleic acid molecule encoding a conditioning agent.

[0041] FIG. 4 shows a schematic of a tLNP according to certain embodiments, wherein the tLNP includes a binding moiety specific to CD8 on the surface of a T cell and is loaded with an internal payload that includes a nucleic acid molecule encoding a reprogramming agent; and / or a nucleic acid molecule encoding a gene iiRECTIFIED SHEET (RULE 91 ) ISA / EPediting component or is itself a gene editing component; and / or a conditioning agent or a nucleic acid molecule encoding a conditioning agent.

[0042] FIG. 5 shows a schematic of a tLNP according to certain embodiments, wherein the tLNP includes a binding moiety specific to CD117 on the surface of a hematopoietic stem cell (HSC) and is loaded with an internal payload to encode and / or deliver a gene editing system or component.

[0043] FIG. 6 shows a flowchart of the experiments disclosed herein, in accordance with certain embodiments.

[0044] FIGS. 7A-7D are graphs of anti-CD19 CAR expression in different immune cell subsets using flow cytometry. FIGS. 7A-7D show CAR expression in CD8+(FIG. 7 A) and CD4+ (FIG. 7B) T cell, monocyte (CD14+; FIG. 7C) and B cell (CD20+; FIG. 7D) populations.,

[0045] FIGS. 8A-8F are graphs of mCherry expression detected using flow cytometry after 24h incubation with the tLNPs. FIGS. 8A-8F show percentages of mCherry expression for pan T cell (CD3+; FIG. 8A), CD4+ T cell (FIG. 8B), CD8+ T cell (FIG. 8C), B cell (CD20+; FIG. 8D), NK cell (CD159a+; FIG. 8E), and total monocyte (FIG. 8F) subpopulations in whole blood and PBMC.

[0046] FIGS. 9A-9F show graphs of CAR expression detected using flow cytometry after 24h incubation with the tLNPs. FIGS. 9A-9F show percentages of CAR expression for pan T cell (CD3+; FIG. 8A), CD4+ T cell (FIG. 8B), CD8+ T cell (FIG. 8C), B cell (CD20+; FIG. 8D), NK cell (CD159a+; FIG. 8E), and total monocyte (FIG. 8F) subpopulations in whole blood and PBMC.DETAILED DESCRIPTION

[0047] Provided herein are autologous extracorporeally engineered cells — e.g., engineered immune cells or hematopoietic stem cells (HSC) — produced from a population of cells modified to express one or more exogenous proteins, extracorporeal methods and systems for producing engineered immune cells or HSC, and therapeutic regimens encompassing an extracorporeal transfection method to allow the autologous engineered immune cells or HSC to be delivered to a subject12RECTIFIED SHEET (RULE 91 ) ISA / EPwithin hours of harvesting donor cells — without the need for cellular separation, expansion, or cryopreservation. The engineered immune cells or HSC disclosed herein are autologous, meaning that the cells are derived from donor cells of the same subject that receives the engineered immune cells or HSC as a cellular therapy.

[0048] Also provided herein are autologous ex vivo engineered cells — e.g., engineered immune cells or hematopoietic stem cells (HSC) — produced from a population of cells modified to express one or more exogenous proteins, ex vivo methods for producing engineered immune cells or HSC via tranfection with nucleic acid containing lipid nanoparticles (LNPs) or tLNPs, and therapeutic regimens encompassing an ex vivo transfection method to allow the autologous engineered immune cells or HSC to be delivered to a subject. Such procedures can include cellular separation, expansion, and cryopreservation of the donor and / or engineered cells.

[0049] Prior to setting forth this disclosure in more detail, it may be helpful to provide definitions of certain terms to be used herein. Additional definitions are set forth throughout this disclosure.Definitions

[0050] As used in the specification and claims, the singular form “a,” “an,” and “the” includes plural references unless the context clearly dictates otherwise. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components.

[0051] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[0052] The term “about” as used herein in the context of a number refers to a range centered on that number and spanning 15% less than that number and 15% more than that number. The term “about” used in the context of a range refers to an extended range spanning 15% less than that the lowest number listed in the range and 15% more than the greatest number listed in the range.

[0053] Throughout this disclosure, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth13RECTIFIED SHEET (RULE 91 ) ISA / EPand one hundredth of an integer), unless otherwise indicated. Also, any number range of this disclosure relating to any physical feature, such as polymer subunits, size, or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. Throughout this disclosure, numerical ranges are inclusive of their recited endpoints, unless specifically stated otherwise.

[0054] Unless the context requires otherwise, throughout the present specification and claims, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” As used herein, the terms “include” and “comprise” are used synonymously.

[0055] The phrase “at least one” when followed by a list of items or elements refers to an open-ended set of one or more of the elements in the list, which may, but does not necessarily, include more than one of the elements.

[0056] “Derivative,” as used herein, refers to a chemically or biologically modified version of a compound that is structurally similar to a parent compound and (actually or theoretically) derivable from that parent compound. Generally, a "derivative" differs from an “analogue” in that a parent compound may be the starting material to generate a "derivative," whereas the parent compound may not necessarily be used as the starting material to generate an “analogue.” A derivative may have different chemical or physical properties of the parent compound. For example, a derivative may be more hydrophilic or hydrophobic, or it may have altered reactivity as compared to the parent compound.

[0057] “Artificial sequence,” or “synthetic sequence” as used herein, refers to an amino acid or nucleotide sequence that is devised to serve a specific purpose and that is not derived from a particular sequence existing in nature. The purpose of such sequences can include linkers, spacers, restrictions sites, and untranslated regions, among others.

[0058] As used herein “transfection” or “transfecting” refers to the introduction of nucleic acids into cells by non-viral methods. Transfection can be mediated by calcium phosphate, cationic polymers, magnetic beads, electroporation and lipid-based reagents. In particular embodiments disclosed herein transfection is mediated by solid14RECTIFIED SHEET (RULE 91 ) ISA / EPlipid nanoparticles (LNP) including targeted LNP (tLNP). The term transfection is used in distinction to transduction - transfer of genetic material from cell to cell or virus to cell - and transformation - the uptake of extracellular genetic material by the natural processes of a cell. As used herein, phrases such as “delivering a nucleic acid into a cell” are synonymous with transfection.

[0059] “Reprogramming,” as used herein with respect to immune cells, refers to changing the functionality of an immune cell with respect to antigenic specificity by causing expression of an exogenous T cell receptor (TOR), a chimeric antigen receptor (CAR), or an immune cell engager (collectively termed “reprogramming agents”). As used herein with respect to stem cells, for example hematopoietic stem cells (HSC), hematopoietic stem and progenitor cells (HSPC), or mesenchymal stem cells (MSC), “reprogramming” refers to correction or amelioration of a genetic defect (for example, a hemoglobinopathy) so that the modified or corrected gene and gene product are the reprogramming agents. Reprogramming can be transient or durable depending on the nature of the engineering agent.

[0060] “Engineering agent,” as used herein, refers to agents that confer the expression of a reprogramming agent by an immune cell, particularly a non-B lymphocyte or monocyte. Engineering agents can include nucleic acids, including mRNA, that encode the reprogramming agent. Engineering agents can also include nucleic acids that are or encode components of gene editing systems such as RNA- guided nucleases, guide RNA, and nucleic acid templates for knocking-in or otherwise generating a reprogramming agent or knocking-out an endogenous antigen receptor. Gene editing systems comprise base-editors, prime-editors or gene-writers. RNA- guided nucleases include CRISPR nucleases such as Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-11 , and CasX. For transient expression of a reprogramming agent, such as a CAR, an mRNA encoding the reprogramming agent can be used as the engineering agent. For durable expression of the reprogramming agent, such as an exogenous, modified, or corrected gene (and its gene product), the engineering agent can comprise mRNA-encoded RNA-directed nucleases, guide RNAs, nucleic acid templates and other components of gene / genome editing systems.

[0061] Examples of gene editing components that are encoded by a nucleic acid molecule include an mRNA encoding an RNA-guided nuclease, a gene or base editing15RECTIFIED SHEET (RULE 91 ) ISA / EPprotein, a prime editing protein, a Gene Writer protein (e.g., a modified or modularized non-long terminal repeat (LTR) retrotransposon), a retrotransposase, an RNA writer, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a transposase, a retrotransposon, a reverse transcriptase (e.g., M- MLV reverse transcriptase), a nickase or inactive nuclease (e.g., Cas9, nCas9, dCas9), a DNA recombinase, a CRISPR nuclease (e.g., Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-11 , CasX), a DNA nickase, a Cas9 nickase (e.g., D1 OA or H840A), or any fusion or combination thereof. Other components include a guide RNA (gRNA), a single guide RNA (sgRNA), a prime editing guide RNA (pegRNA), a clustered regularly interspaced short palindromic repeat (CRISPR) RNA (crRNA), a trans-activating clustered regularly interspaced short palindromic repeat (CRISPR) RNA (tracrRNA), or a DNA molecule to be inserted or serve as a template for double-strand break (DSB) repair at a specific genomic locus. Genome-, gene-, and base-editing technology are reviewed in Anzalone et al., Nature Biotechnology 38:824-844, 2020, Sakuma, Gene and Genome Editing 3-4:100017, 2022, and Zhou et al., MedComm 3(3):e155, 2022, each of which is incorporated by reference for all that they teach about the components and uses of this technology to the extent that it does not conflict with the present disclosure.

[0062] “Conditioning agent,” as used herein, refers to a biological response modifier (BRM) that enhances the efficiency of engineering an immune cell, expands the number of immune cells available to be engineered or the number of engineered cells in a target tissue (for example, a tumor, fibrotic tissue, or tissue undergoing autoimmune attack), promotes activity of the engineered cell in a target tissue, or broadens the range of operative mechanisms contributing to a therapeutic immune reaction. A conditioning agent may be provided by delivering an encoding nucleic acid in a tLNP. Exemplary BRMs include cytokines, such as IL-7, IL-15, or IL-18.

[0063] The term “immune cell,” as used herein, can refer to any cell of the immune system. However, particular aspects can exclude polymorphonuclear leukocytes and / or B cells, or be limited to non-B lymphocytes such as T cell and / or NK cells, or to monocytes such as dendritic cells and / or macrophages in their various forms.

[0064] The term “nucleic acid” or “nucleic acid molecule,” as used herein, refers to either an RNA or DNA molecule, especially those encoding an expressible16RECTIFIED SHEET (RULE 91 ) ISA / EPpolypeptide, where context does not dictate otherwise. Description of the disclosed embodiments focuses primarily on improved mRNA molecules having the structure of a canonical mRNA. However, polypeptides can also be encoded in and expressed from circular and self-amplifying (also known as self-replicating) RNA molecules. Accordingly, the sequence of any of the herein disclosed linear mRNA molecules can be incorporated into a circular or self-amplifying / self-replicating RNA molecule. Similarly, each of these RNA molecules can be encoded as a DNA molecule. Each of the disclosed nucleic acid sequences, RNA or DNA, should be understood to disclose the corresponding DNA or RNA sequence, respectively

[0065] As used herein, “antibody” refers to a protein comprising an immunoglobulin domain having hypervariable regions determining the specificity with which the antibody binds antigen, termed complementarity determining regions (CDRs). The term antibody can thus refer to intact, full-length, or whole antibodies as well as antibody fragments and constructs comprising an antigen binding portion of a whole antibody. While the canonical natural antibody has a pair of heavy and light chains, camelids (from camels, alpacas, llamas, and the like) produce antibodies with both the canonical structure and antibodies comprising only heavy chains. The variable region of the camelid heavy chain-only antibody has a distinct structure with a lengthened CDR3 and is referred to as VHH or, when produced as a fragment, a nanobody. Antigen binding fragments and constructs of antibodies include F(ab)2, F(ab), minibodies, Fv, single-chain Fv (scFv), diabodies, and VH. Such elements may be combined to produce bi- and multi-specific reagents, such as BiTEs (bi-specific T-cell engagers). The term “monoclonal antibody” arose out of hybridoma technology but is now used to refer to any singular molecular species of antibody regardless of how it was originated or produced. Antibodies can be obtained through immunization, selection from a naive or immunized library (for example, by phage display), alteration of an isolated antibody-encoding sequence, or any combination thereof. Numerous antibodies that can be used as binding moieties are known in the art. An excellent source of information about antibodies for which an International Non-proprietary Name (INN) has been proposed or recommended, including sequence information, is Wilkinson & Hale, 2022, MAbs 14(1 ):2123299, including its Supplementary Tables, which is incorporated by reference herein for all that it teaches about individual17RECTIFIED SHEET (RULE 91 ) ISA / EPantibodies and the various antibody formats that can be constructed. U.S. Patent No. 1 1 ,326,182 and especially its Table 9 entitled “Cancer, Inflammation and Immune System Antibodies,” is a source of sequence and other information for a wide range of antibodies including many that do not have an INN and is incorporated herein by reference for all that it teaches about individual antibodies.

[0066] A diabody is a dimer of scFv fragment that consists of the VH and VL regions noncovalently connected by a small peptide linker or covalently linked to each other. A BiTE is a fusion protein having two scFvs of different antibodies, usually an antibody for a tumor-associated antigen and antibody for CD3, on a single peptide chain, thus forming a cytolytic synapse between T cells and target antigen-bearing cells. The term "antigen-binding portion" may refer to a portion of an antibody as described that possesses the ability to specifically recognize, associate, unite, or combine with a target molecule. An antigen-binding portion includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a specific antigen. Thus, antibodies and antigen-binding portions thereof constitute means for binding to the surface molecule on a cell. In various embodiments, the cell can be an immune cell, a leukocyte, a lymphocyte, a monocyte, a stem cell, an HSC or an MSG, according to the specificity of the antibody.

[0067] An antibody or other binding moiety (or a fusion protein thereof) “specifically binds” to a target if it binds the target with an affinity or Ka(i.e., an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105M-1, while not significantly binding other components present in a test sample. Binding domains (or fusion proteins thereof) can be classified as “high affinity” binding domains (or fusion proteins thereof) and “low affinity” binding domains (or fusion proteins thereof). “High affinity” binding domains refer to those binding domains with a Kaof at least 108M-1, at least 109M-1, at least 1010M-1, at least 101 1M-1, at least 1012M'1, or at least 1013M-1, preferably at least 108M-1or at least 109M-1. “Low affinity” binding domains refer to those binding domains with a Ka of up to 108M-1, up to 107M-1, up to 106M'1, up to 105M-1. Alternatively, affinity may be defined as an equilibrium dissociation constant (KD) of a particular binding interaction with units of Molarity (M) (e.g., 10'5M to 10-13M). Affinities of binding domain polypeptides and fusion proteins according to this disclosure can be readily determined using conventional techniques18RECTIFIED SHEET (RULE 91 ) ISA / EP(see, e.g., Scatchard et aL, 1949, Ann. N.Y. Acad. Sci. 51 :660; and U.S. Patent Nos. 5,283,173; 5,468,614, or the equivalent).

[0068] In some embodiments, the antibody or antigen-binding portion thereof may be derived from a mammalian species, for example, mice, rats, or human. Antibody variable regions can be those arising from one species, or they can be chimeric, containing segments of multiple species possibly further altered to optimize characteristics such as binding affinity or low immunogenicity. For human applications, it is desirable that the antibody has a human sequence. In the cases where the antibody or antigen-binding portion thereof is derived from a non-human species, the antibody or antigen-binding portion thereof may be humanized to reduce immunogenicity in a human subject. For example, if a human antibody of the desired specificity is not available, but such an antibody from a non-human species is, the non- human antibody can be humanized, e.g., through CDR grafting, in which the CDRs from the non-human antibody are placed into the respective positions in a framework of a compatible human antibody. Less preferred is an antibody in which only the constant region of the non-human antibody is replaced with human sequence. Such antibodies are commonly referred to as chimeric antibodies in distinction to humanized antibodies.

[0069] In some embodiments, the antibody or antigen-binding portion thereof is non-immunogenic. In some embodiments, the antibody may be modified in its Fc region to reduce or eliminate secondary functions, such as FcR engagement, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (GDC).

[0070] As used herein, a “binder”, “binding moiety” or “targeting moiety” refers to a protein, polypeptide, oligopeptide or peptide, carbohydrate, nucleic acid, or combinations thereof capable of specifically binding to a target or multiple targets. A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or another target of interest. Exemplary binding moieties of this disclosure include an antibody or antigen binding domain thereof, a Fab', F(ab')2, Fab, Fv, rlgG, scFv, hcAb (heavy chain antibody), a single domain antibody, VHH, VNAR, sdAb, nanobody, receptor ectodomain or ligand-binding portions thereof, or ligand (e.g., cytokines, chemokines,19RECTIFIED SHEET (RULE 91 ) ISA / EPsaccharides, glycoconjugates). A “Fab” region (fragment antigen-binding region) is the part of an antibody that binds to antigens and includes the variable region and CH1 of the heavy chain linked to the light chain via an inter-chain disulfide bond. In other embodiments, a binding moiety comprises a ligand-binding domain of a receptor or a receptor ligand. In some embodiments, a binding moiety can have more than one specificity including, for example, bispecific or multispecific binders. A variety of assays are known for identifying binding moieties of this disclosure that specifically bind a particular target, including Western blot, ELISA, biolayer interferometry, and surface plasmon resonance. A binding moiety, such as a binding moiety comprising immunoglobulin light and heavy chain variable domains (e.g., scFv), can be incorporated into a variety of protein scaffolds or structures as described herein, such as an antibody or an antigen binding fragment thereof, a scFv-Fc fusion protein, or a fusion protein comprising two or more of such immunoglobulin binding domains.

[0071] As used herein “expansion” refers to proliferation of cells increasing their number. Activating agents can be used to stimulate proliferation (among other metabolic changes) but can also result in activation-induced death upon initial exposure so that there is no immediate expansion. For T cells in vitro, treatment with activating agents such as IL-2 or CD3 / CD28 activators, doubling time can be about 24 hours (which is fairly typical of mammalian cells in vitro generally); in vivo doubling time can be substantially shorter, depending on the presence and type of stimulation. Accordingly, during the limited time of extracorporeal manipulation, even when activating agents are used, such protocols will be effectively expansion-less.

[0072] As used herein an “exogenous protein” refers to a synthetic, recombinant, or other peptide or protein that is not produced by a wild-type cell of that type or is expressed at a lower level in a wild-type cell than in a cell containing the exogenous polypeptide. In some embodiments, an exogenous peptide is a peptide or protein encoded by a nucleic acid that was introduced into the cell, which nucleic acid is optionally not retained by the cell.

[0073] As used herein “extracorporeal” is used to refer to cells, such as peripheral blood or bone marrow cells, harvested or extracted from the body and the manipulation and / or modification of those cells prior to their intended return (reinfusion). Manipulation and modification of cells generally relates to cell separation and / or20RECTIFIED SHEET (RULE 91 ) ISA / EPwashing procedures and / or exposure to activation agents (e.g., biological response modifiers) and transfection agents (e.g., LNPs, tLNPs), over a time interval of several hours, for example, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour; and in space to a single institution. Extracorporeal is used in contradistinction to ex vivo which, as used herein, includes more extensive manipulation including extended periods of cell culture and expansion, and / or refrigerated or cryogenic storage or shipment, over several days or longer. Ex vivo manipulation of cells to introduce an engineering agent, for example in the generation of CAR-T cells and the like, at least at the commercial or clinical trial scale, has typically utilized transduction with viral vectors and with integration of the engineering agent into the genome of the engineered cell.

[0074] The term “recombinant protein” as used herein, refers to a protein, peptide, or portion thereof that is produced by a genetically-engineered organism and / or is encoded by a nucleic acid that is artificially constructed and / or not naturally found in the genome of a host. For example, a recombinant protein may be a fusion protein assembled by splicing or fusing portions of different portions of different genes together to encode the recombinant protein. Another example is a protein that is expressed endogenously or exogenously by a host, but is encoded by a nucleic acid that is intentionally modified or otherwise artificially constructed (e.g., a codon- optimized gene, a gene with insertions, deletions, substitutions or SNPs).RNA

[0075] Certain aspects of this disclosure relate to RNA molecules, such as mRNA molecules that can be produced by in vitro transcription and are suitable for in vivo transfection using an appropriate delivery vehicle, such as a lipid nanoparticle (LNP) or targeted lipid nanoparticle (tLNP) as described in further detail herein. A eukaryotic mRNA typically has the following structural elements in 5’ to 3’ order: a 5’ cap, a 5’ UTR, an open reading frame (ORF) beginning with an initiator codon, one or more termination codons, a 3’ UTR, and a poly(A) tail. Efficiently translatable ORFs typically comprise at least about 80 codons (Aspden et al., 2014, eLife 3: e03528). mRNA molecules used according to various embodiments of this disclosure can comprise particular 5’ untranslated regions (UTRs), particular 3’ UTRs, a particular open reading frame sequence, or any combinations thereof such as those disclosed in21RECTIFIED SHEET (RULE 91 ) ISA / EPPCT / US2024 / 032141 , filed 5 / 31 / 2024, which is incorporated by reference herein for all that it teaches about mRNA structure and delivery that does not conflict with the present disclosure.

[0076] mRNA according to the herein disclosed embodiments can be obtained by in vitro transcription. In particular embodiments, in vitro RNA transcripts are synthesized using a T7 RNA polymerase to generate RNA having one or more or all uridines substituted with a non-natural nucleotide, such as N1-methyl pseudouridine (N1Mi ), a cap (provided by for example, a Cap-AG reagent), a poly(A) tail of approximately 90-110 nucleotides was present in the template plasmid. In some embodiments, RNA molecules generated by in vitro transcription are encapsulated in a LN P or tLNP, which LNPs or tLNPs can be used to transfect cells in vitro, in vivo, extracorporeally, or ex vivo.

[0077] In some embodiments, the RNA comprises at least one modified nucleoside. In some embodiments, the modified nucleoside is pseudouridine, N1- methylpseudouridine, 5-methylcytosine, 5-methyluridine, N6-methyladenosine, 2’-O- methyluridine, or 2-thiouridine. In certain embodiments all of the uridines are substituted with a modified nucleoside. Further disclosure of modified nucleosides and their use can be found in U.S. Patent No. 8,278,036 which is incorporated herein by reference for those teachings.(a) Capped mRNA

[0078] Eukaryotic mRNAs have a 7-methylguanosine (m7G) cap added to their 5’ end through a 5’ to 5’ triphosphate bridge (CapO). Additionally, the 2’ hydroxyl or the ribose of the first one or two templated nucleotides of the mRNA can be methylated (Cap1 and Cap2, respectively). Capping is a post-transcriptional event in vivo but can be either post- or co-transcriptional in vitro. In co-transcriptional capping an oligonucleotide comprising m7G and the first one, two, or more templated nucleotides, which hybridize to the template, is used as a primer. Two or more templated nucleotides that are not the same can be included to assure unidirectional transcription. Cap primers with a variety of templated nucleotides and with additional modifications are commercially available and / or described in the literature. Embodiments of the mRNAs provided herein can incorporate any of these cap22RECTIFIED SHEET (RULE 91 ) ISA / EPstructures or primers. Particular instances incorporate the primer m7G(5')ppp(5')(2'OMeA)pG (Cap AG). Cap AG is well-suited for capping of mRNA encoded by templates using a promoter resembling the T7 c|)2.5 promoter in having the first two templated nucleotides of the mRNA have the sequence AG. Alternatives to Cap AG include m7G(UNA)(5')ppp(5')(2’OMeA)pG (with an “unlocked” structure for the first cap nucleoside, having no C-C bond between the carbons corresponding to the 2’ and 3’ positions) and m7G(5')(ENE)ppp(5')(2’OMeA)pG (with a double-bonded C replacing the 5’ O between the first cap nucleoside and the triphosphate) (Cap4011 and Cap501 1 , respectively, Areterna, Gaithersburg, MD). A further alternative is m7<LNA)G(5')ppp(5')(2'OMeA)pG (Cap AG) (with an “locked” structure for the first cap nucleoside) (ThermoFisher PPT1647 0522).

[0079] In certain embodiments of the above aspects, an mRNA of this disclosure further comprises a co-transcriptional capping structure. Examples of the co- transcriptional capping structures includes without limitation, CapO, ARCA (AntiReverse Cap Analog; a CapO mimic), Cap1 , or Cap2 (Muttach et al., 2017, J Org Chem. 13: 2819-2832).

[0080] A template DNA molecule encoding the mRNA will further comprise a promoter upstream of the coding sequence appropriate to the DNA polymerase to be used for in vitro transcription. In vitro transcription is typically carried out using a phage DNA polymerase, such as T7, T3, or SP6 polymerase.(b) Poly(A) tail of mRNA

[0081] The poly(A) tails of mRNAs produced in living cells are enzymatically added at a poly(A) addition site in the 3’ UTR following transcription and are generally around 200 residues in length. For in vitro transcribed mRNA the necessary enzymes can be added, but alternatively a poly(A) sequence can be included in the DNA template. Such poly(A) tails are often in the range of 90-110 nucleotides, but there are numerous examples in the scientific and patent literature advocating various modifications of length or sequence. Poly(A) tails in a population of mRNA molecules will be heterogenous in length even when templated due to slippage during replication of the template or during transcription.

[0082] With respect to any embodiment comprising or encoding an RNA with a23RECTIFIED SHEET (RULE 91 ) ISA / EPpoly(A) sequence, the poly(A) sequence can have at least about 80 adenosine residues to about 130 or more adenosine residues. In some embodiments the poly(A) sequence has about 80 adenosine residues. In certain embodiments, the poly(A) sequence has about 90 adenosine residues. In certain embodiments, the poly(A) sequence has about 100 adenosine residues. In certain embodiments, the poly(A) sequence has about 110 adenosine residues. In certain embodiments, the poly(A) sequence has about 130 adenosine residues.(c) Untranslated regions (UTRs) of mRNA

[0083] The untranslated regions flanking the coding sequence of an mRNA are known to influence expression. For 5’ UTRs, it is known that excessive or insufficient length, high GC content - contributing to secondary structure formation - and a large number of AUG codons will negatively impact protein expression. Similarly, excessive 3’ UTR length can negatively impact protein expression. But beyond the avoidance of such features, how to choose or alter UTRs to improve protein expression and how 5’ and 3’ UTRs interact are not well understood. It is also known that the inclusion of microRNA (miRNA) binding sites in UTRs will alter expression in tissues that express the cognate miRNA, usually negatively. However, knowledge of which miRNAs are expressed in which tissues and what sequences they can functionally bind to is incomplete. Nonetheless, for known miRNAs with at least partially known expression profiles, it is possible to engineer a binding site into a UTR to suppress expression in a tissue in which expression is not desired (a non-target tissue) thereby conferring a degree of tissue specificity on expression. This miRNA-mediated specificity can augment specificity obtained from targeted delivery of an mRNA, such as with a tLNP or tropic LNP.

[0084] Untranslated regions (UTRs) and codon usages have been shown to modulate mRNA stability and translation activity. Various combinations of UTRs and codon usage in mRNA encoding a CAR were tested to screen mRNAs having an improved level of expression after transfected into a mammalian cell via tLNP delivery.

[0085] The sequences adjoining the UTR core sequences can be altered without disrupting the beneficial effect of the UTR core sequences. In altering these adjoiningRECTIFIED SHEET (RULE 91 ) ISA / EPsequences compatibility with the capping system being used should be maintained as should the functionality of the Kozak sequence. The UTR-adjoining sequences should not form secondary structure within these adjoining sequences or with the core sequence or other adjoining sequence. This can be facilitated by using AU-rich sequences and avoiding complementarity with other portions of the UTR. Additionally, the 5’ UTR adjoining sequences should not contain a start (AUG) codon. Accordingly, some embodiments specify only the core sequences of the UTRs while other embodiments comprise whole UTR sequences, as disclosed herein.

[0086] In addition to linear mRNA, a gene of interest (GOI) can be expressed from other RNA modalities, including but not limited to circular RNAs and self-amplifying RNA (also known as self-replicating RNA or RNA replicon).

[0087] Circular RNA (circRNA) is an RNA molecule without RNA termini, and it does not contain a 5’-cap that is usually present in a linear synthetic mRNA molecule. Instead, circRNA needs at least one sequence moiety that recruits ribosomes where such moiety includes an internal ribosome entry site(s). The sequence of a gene of interest (GOI) is typically inserted downstream of the ribosome recruitment site to ensure efficient gene expression. Depending on the particular internal ribosome entry site used, the 5’ UTR may be wholly retained, just the Kozak sequence retained or be completely replaced by the internal ribosome entry site, Cell-free synthesis of circular RNA is initiated with in vitro transcription to produce a linear RNA followed by a variety of circularization reactions that involve: 1 ) a ribozyme, such as a self-splicing group l / ll intron, 2) a protein enzyme like T4 DNA ligase or RNA ligase 1 / 2, with or without a split oligonucleotide molecule bridging the RNA termini, or 3) Non-enzymatic ligation, such as click chemistry (reviewed in Obi and Chen, The design and synthesis of circular RNAs. Methods. 2021 Dec;196:85-103. doi: 10.1016 / j.ymeth.2021.02.020. Epub 2021 Mar 2. PMID: 33662562; PMCID: PMC8670866, which is incorporated by reference for all that it teaches about the design of circular RNA that is not inconsistent with this disclosure). Due to the circular nature of the final RNA molecule, there is a degree of flexibility on the placement of the 3’ elements and the circularization elements relative to the GOI in the linear precursor RNA.

[0088] Self-amplifying RNA (saRNA), on the other hand, is generated as a linear synthetic RNA molecule with a viral origin chassis that enables its replication within25RECTIFIED SHEET (RULE 91 ) ISA / EPcells. The self-replication machinery encoded in the RNA backbone can be derived from a wide range of single-stranded positive or negative-sense RNA viruses. For instance, a genomic RNA backbone of alphavirus, a positive-stranded RNA virus, is commonly used as an saRNA platform to express an GOI, often for a vaccine application (reviewed in Bloom et al., Self-amplifying RNA vaccines for infectious diseases. Gene Ther. 2021 Apr;28(3-4):1 17-129. doi: 10.1038 / s41434-020-00204-y. Epub 2020 Oct 22. PMID: 33093657; PMCID: PMC7580817, which is incorporated by reference for all that it teaches about the design of saRNA that is not inconsistent with this disclosure). In a typical alphavirus saRNA, the viral structural genes originally present in the alphavirus genome are usually removed and replaced by the GOI(s). Consequently, due to the absence of functional virion formation, the RNA replication remains confined within the cells where it is originally delivered. The non-structural replicase genes, including RNA-dependent RNA polymerase gene, in the alphavirus saRNA are directly expressed from the synthetic genomic transcripts to initiate the replication cycle in cells. In contrast, while encoded in the genomic transcript, a GOI(s) in alphavirus saRNA system is expressed from a subgenomic RNA, which is produced in abundance during RNA replication in cells. In a typical arrangement the viral nonstructural genes and subgenomic promoter (in that order) would be inserted between the 5’ UTR and the GOI of the linear mRNA. Alternatively, the 5’ UTR of the linear mRNA can largely replace the subgenomic promoter. The 3’ UTR of the linear mRNA can be retained but the viral promoter for minus strand synthesis must also be present. The viral non-structural genes contain essential RNA structures that need to be retained by any codon optimization.

[0089] In certain embodiments, the ORF encodes a CAR. In other embodiments, the ORF encodes an immune cell engager such as a BiTE (Bispecific T cell Engager). In some embodiments, the ORF encodes only human or artificial sequences. With respect to these and other nucleic acid aspects, in some embodiments, the mRNA is suitable for expression in a mammalian cell. In some embodiments, the mammalian cell is an immune cell or a hematopoietic stem cell. In some embodiments, any of these cells is a human cell. These mRNAs constitute means for improved expression of a polypeptide, means for improved expression of a polypeptide in an immune cell, means for expression of a polypeptide in a T cell, and the like. Some embodiments26RECTIFIED SHEET (RULE 91 ) ISA / EPare limited to a subset of one or more of the UTR pairings disclosed herein. Other embodiments specifically exclude one or more of these UTR pairings. These mRNAs constitute means for improved expression of a CAR, means for improved expression of a CAR in an immune cell, means for expression of a CAR in a T cell, and the like. Some embodiments are limited to a subset of one or more of these UTR pairings. Other embodiments specifically exclude one or more of these UTR pairings.(d) Opening reading frame (ORF)

[0090] The term “open reading frame” (ORF) refers to a nucleic acid sequence that begins with a start codon and is immediately followed by a stop codon. ORF can encode any protein. For engineered mRNAs, the codons can be those naturally found in mRNA encoding a particular protein or segment of a protein, the codons can be derived from back-translation of the amino acid sequence of a protein without reference to a natural sequence, and in either case these sequences can be subjected to various codon optimizations to favor particular subsets of synonymous codons, adjust GC content, avoid detrimental secondary structures, reduce the occurrence of ribosomal slippery sites (Mulroney et al., 2023, Nature 625: 189-194), and the like, in order to increase the level of translation in a particular species or type of cell. Especially when an mRNA is to be administered to a mammal, it can be advantageous to incorporate one or more modified nucleotides that, while maintaining coding function, increase translation and avoid provoking an innate immune response. In some embodiments, the replaced nucleoside is uridine. In certain embodiments all of the uridines are substituted with a modified nucleoside. In some embodiments, the modified nucleoside is pseudouridine (qj), N1 -methylpseudouridine (N1 Mi ), 5- methoxyuridine (5moU), 5-methylcytosine, 5-methyluridine, N6-methyladenosine, 2’- O-methyluridine, or 2-thiouridine, or combinations thereof. Further disclosure of modified nucleosides and their use can be found in U.S. Patent No. 8,278,036 which is incorporated herein by reference for those teachings. Natural protein-coding open reading frames are immediately followed by a single stop codon however, it is not uncommon for the open reading frame of engineered mRNAs to be followed by multiple in-frame stop codons.27RECTIFIED SHEET (RULE 91 ) ISA / EPCAR constructs

[0091] In some embodiments of the above aspects of the invention disclosed herein, an improved mRNA of this disclosure comprising an ORF encodes a chimeric antigen receptor (CAR). The receptors are chimeric because they combine into a single receptor both an antigen-binding domain of an antibody and one or more T cell activating domains of receptors expressed by T cells. In some embodiments, a CAR of this disclosure is multispecific, for example, bispecific, comprising multiple antigen each specific for a separate antigen. In some embodiments, a CAR can comprise an extracellular binding domain that specifically binds a target antigen, a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, a CAR can further comprise one or more additional elements, including one or more signal peptides, one or more extracellular hinge domains, or one or more intracellular costimulatory domains. Domains can be directly adjacent to one another, or there can be one or more amino acids linking the domains. The signal peptide can be derived from an antibody, a TCR, CD8 or other type 1 membrane protein, preferably a protein expressed in a T cell or other immune cell. The transmembrane domain can be one associated with any of the potential intracellular domains or from another type 1 membrane protein, such as TCR alpha, beta, or zeta chain, CD3 epsilon, CD4, CD8, or CD28, amongst other possibilities known in the art. The transmembrane domain can further comprise a hinge domain located between the extracellular binding domain and the hydrophobic membrane-spanning region of the transmembrane domain. In some but not all embodiments, the hinge domain and transmembrane domain are contiguous sequences in the same source protein. In some instances, the hinge and membrane-spanning domains are derived from CD28. In other instances, the hinge and membrane-spanning domains are derived from CD8a. In still other instances, the hinge domain is derived from an immunoglobulin, for example, the hinge domain of lgG4. The intracellular signaling domain can be derived from the CD3 zeta chain, DAP10, DAP12, FcyRIII, FcsRI, or an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic domain, amongst other possibilities known in the art. The intracellular costimulatory domain can be derived from CD27, CD28, 4-1 BB, 0X40, or ICOS, amongst other possibilities known in the art.28RECTIFIED SHEET (RULE 91 ) ISA / EP

[0092] Further variations on the basic CAR structure and sources for the various domains are described in Zabel et al., Immunol Lett 2019 212:53-69 which is incorporated by reference for all that it teaches about CAR structure and functional domains thereof to the extent it is consistent with this disclosure.

[0093] In some embodiments, a nucleic acid encoding a CAR refers to one or more nucleic acid species encoding one or more CARs; for example, a single or multiple species of nucleic acid encoding a single CAR species, or multiple species of nucleic acid encoding multiple CAR species. In some instances, these multiple CAR species have a same specificity while in other instances they have multiple specificities. In some embodiments, a CAR of this disclosure is multispecific, for example, bispecific, comprising multiple antigen binding moieties each specific for separate antigens.

[0094] Exemplary target antigens against which a CAR, TCR, or ICE can have specificity include, but are not limited to, B cell maturation agent (BCMA)**, CA9**, CD5**, CD19***, CD20 (MS4A1 )***, CD22***, CD23***, CD30 (TNFRSF8)***, CD33***, CD38***, CD44**, CD70***, CD133*, CD174, CD274 (PD-L1)***, CD276 (B7-H3)**, CEACAM5***, CLL1*, CSPG4**, EGFR***, EGFRvlll*, EPCAM***, EPHA2**, ERBB2**, FAP***, FOLH1 , FOLR1 ***, GD2***, GPC3***, GPNMB**, IL1 RAP**,and G-protein coupled receptor family C group 5 member D (GPRC5D)** (associated with leukemias); CD319 (SLAMF7)***, CD38***, CD138**, GPRC5D**, CD267 (TACI)*, and BCMA** (associated with myelomas); and GD2***, HER2***, EGFR***, EGFRvlll*, CD276 (B7H3)**, PSMA***, PSCA*, CAIX (CA9)**, CD171 (L1-CAM)**, CEA**, CSPG4**, EPHA2**, FAP***, LRRC15**, FOLR1 ***, IL-13Ra***, Mesothelin***, MUC1 ***, MUC16***, and ROR1** (associated with solid tumors). (* indicates that exemplary antibodies with the indicated specificity from which a binding moiety could be derived can be found in US Patent 1 1 ,326,182B2 Table 9 or 10. indicates that exemplary antibodies with the indicated specificity from which a binding moiety could be derived can be found in Wilkinson & Hale, 2022. Both references cited and incorporated by reference above. * indicates that exemplary antibodies with the indicated specificity from which a binding moiety could be derived can be found in the29RECTIFIED SHEET (RULE 91 ) ISA / EPTherapeutic Antibody Database (TABS) at tabs.craic.com). Other suitable antibodies can be found in Appendix A. Many of these target antigens are themselves receptors that could bind to their ligand if expressed on an immune cell. Accordingly, in some embodiments, the extracellular binding domain of the CAR comprises a ligand of a receptor expressed on the target cell. In still further embodiments, the extracellular binding domain of the CAR comprises a ligand binding domain of a receptor for a ligand expressed on the target cell. In any of these embodiments, the extracellular binding domain of the CAR can be codon-optimized for expression in a host cell or have variant sequences to increase functions of the extracellular binding domain. The advantages of the aspects and embodiments disclosed herein are independent of the specificity of the binding moiety. As such, the disclosed aspects and embodiments are generally agnostic to binding specificity. In certain embodiments, a particular binding specificity can be required.Exemplary CAR constructs

[0095] In certain embodiments, CARs are used to treat a disease or condition associated with a target cell that expresses the antigen targeted by the CAR as described in the uses and methods of treatment disclosed herein. For example, in some embodiments, an anti-CD19 or anti-CD20 or anti-BCMA CAR can be used to target and treat B cell malignancies or B cell-mediated autoimmune conditions or diseases. In other embodiments, an anti-FAP CAR can be used to target and treat solid tumors or fibrosis (e.g., cardiac fibrosis, cancer-associated fibroblasts). Examples of CARs that can be used in accordance with the embodiments described herein include to those disclosed in US 7,446,190 (anti-CD19), US 10,287,350 (anti- CD19), US2021 / 0363245 (anti-CD19 and anti-CD20), US2023 / 0167184 (anti-BCMA), US2023 / 0277589 (anti-BCMA), US 10,543,263 (anti-CD22), US 10,426,797 (anti- CD33), US 10,844,128 (anti-CD123), US 10,428,141 (anti-ROR1 ), and US2021 / 0087295 (anti-FAP), each of which is incorporated by reference for all that it teaches about CAR structure and function generically and with respect to the CAR’s antigenic specificity and target indications to the extent that it is not inconsistent with this disclosure.

[0096] An mRNA disclosed herein encoding a CAR includes both the mature CAR and a signal peptide. In certain embodiments, a mature CAR minimally comprises an30RECTIFIED SHEET (RULE 91 ) ISA / EPantigen binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, a CAR further comprises one or more co-stimulatory domains in the intracellular portion of the CAR. In some embodiments, a CAR further comprises an extracellular hinge or extension domain between the transmembrane domain and the antigen binding domain; this domain can be derived from the same protein as the transmembrane domain. In some embodiments, a CAR can comprise multiple antigen binding domains. In certain embodiments of the mRNA disclosed herein, the CAR is an anti-CD19 CAR, an anti-CD20 CAR, an anti-BCMA CAR, or an anti-FAP CAR.

[0097] In certain embodiments comprising an anti-CD19 CAR, the anti-CD19 CAR comprises an anti-CD19 binding domain. Some embodiments of an anti-CD19 CAR comprising an anti-CD19 binding domain further comprise a CD28 hinge, transmembrane, and co-stimulatory domains, and a CD3^ signaling domain (e.g., without limitation, CAR2). Some embodiments of an anti-CD19 CAR comprising an anti-CD19 binding domain further comprise a hinge and transmembrane domain from CD8a, a CD28 costimulatory domain, and a CD3^-chain signaling domain (e.g., without limitation, CAR1 ).

[0098] CD20 is an antigen found on the surface of B cells as early as the pro-B phase and progressively at increasing levels until B cell maturity, as well as on the cells of most B-cell neoplasms. CD20 positive cells are also sometimes found in cases of Hodgkin's disease, myeloma, and thymoma. Examples of anti-CD20 CARs include those incorporating a CD20 binding moiety derived from an antibody specific to CD20, including, for example, MB-106 (Fred Hutchinson Cancer Research Center, see Shadman et al., 2019, Blood 134(SuppL1):3235), UCART20 (Cellectis, www.cellbiomedgroup.com), or C-CAR066 (Cellular Biomedicine Group, see Liang et al., 2021 , J. Clin. Oncol. 39(15) suppl:2508). In some embodiments, the extracellular binding domain of the anti-CD20 CAR is derived from an antibody specific to CD20, including, for example, Leu16, 2.1.2, IF5, 1.5.3, rituximab, obinutuzumab, ibritumomab, ofatumumab, tositumumab, odronextamab, veltuzumab, ublituximab, and ocrelizumab. In some embodiments, the extracellular binding domain of the anti- CD20 CAR comprises an scFv derived from the Leu16 monoclonal antibody, which comprises the heavy chain variable region (VH) and the light chain variable region (VL)31RECTIFIED SHEET (RULE 91 ) ISA / EPof Leu16 connected by a linker (See for example, Table A) Wu et al., 2001 , Protein Engineering. 14(12) :1025-1033), such as CAR22 and CAR25 described herein. In some embodiments, the extracellular binding domain of the anti-CD20 CAR comprises an scFv derived from the monoclonal antibody, 2.1.2 (W02006130458A2), which comprises the heavy chain variable region (VH) and the light chain variable region ( VL) of 2.1 .2 connected by a linker, such as CAR7 described herein. The entire contents of each of foregoing references in this paragraph are incorporated by reference for all that they teach about the design, structure, and activity of anti-CD20 CARs.

[0099] In certain embodiments comprising an anti-CD20 CAR, the anti-CD20 CAR comprises a Leu16 scFv. In some embodiments, the anti-CD20 CAR comprising a Leu16 scFv further comprises an lgG4 hinge, CD28 transmembrane domain, 4-1 BB costimulation, and a CD3£ signaling domain. Examples of such an anti-CD20 CAR include, without limitation, CAR25 (SEQ ID NO: 97, or with a signal peptide, SEQ ID NO: 98). In some embodiments, the anti-CD20 CAR comprising a Leu16 scFv (See for example, Table A) further comprises an lgG4 hinge, CD28 transmembrane and costimulation domains, 4-1 BB costimulation, and a CD3^ signaling domain. Examples of such an anti-CD20 CAR include CAR22 (SEQ ID NO: 99, or with a signal peptide, SEQ ID NO: 100).Table A. Exemplary sequences of anti-CD20 scFv and components32RECTIFIED SHEET (RULE 91 ) ISA / EP33RECTIFIED SHEET (RULE 91) ISA / EP34RECTIFIED SHEET (RULE 91) ISA / EP

[0100] In certain embodiments comprising an anti-CD20 CAR, the anti-CD20 CAR comprises a 2.1 .2 scFv. In some embodiments, the anti-CD20 CAR comprising a 2.1 .2 scFv further comprises CD28 hinge, transmembrane, and costimulation domains and a CD3^ signaling domain. Examples of such an anti-CD20 CAR include, without limitation, CAR7 (SEQ ID NO: 101 , or with a signal peptide, SEQ ID NO: 102).

[0101] Each of the CARs with specificity for a particular antigen described herein constitute means for antigen recognition with respect to that antigen and collectively all of the CARs described herein constitute means for antigen recognition. The function may be alternatively stated as antigen recognition by an immune cell or antigen recognition by a T cell and the like.

[0102] In certain embodiments, on ORF can encode a gene-editing protein such as, without limitation Steptococcus pyogenes Cas9 (SpCas9 - SEQ ID NO: 143).LNP and tLNP formulated pharmaceutical compositions

[0103] In certain aspects, this disclosure provides a pharmaceutical composition comprising a nucleic acid payload formulated in an LNP. In some embodiments, the nucleic acid comprises RNA. In some embodiments, the RNA comprises an mRNA. In some embodiments, the RNA comprises a circular RNA or a self-replicating RNA. In some embodiments, the RNA comprises a guide RNA. In some embodiments, the nucleic acid comprises DNA.

[0104] In some embodiments, the LNPs used in the methods described herein can35RECTIFIED SHEET (RULE 91 ) ISA / EPmediate transfection of the target cell for modifying a target immune cell or HSC obtained from a subject, wherein an LNP includes (1) an internal payload and (2) an LNP portion to be delivered to the target immune cell. According to the embodiments disclosed herein, an internal payload includes one or more engineering agents (e.g., one or more nucleic acid molecules that encode a reprogramming agent such as a CAR or BiTE) and / or one or more conditioning agents (e.g., one or more BRM(s), an encoded BRM (or “eBRM”), or one or more nucleic acid molecules encoding an eBRM) as discussed in detail below. In certain embodiments, an LNP includes (1 ) an internal payload; (2) an LNP portion; and (3) a binding moiety conjugated to the surface of the LNP that is capable of binding a surface molecule expressed by the target immune cell. In such embodiments, the LNP that includes a binding moiety is referred to herein as a targeted LNP (tLNP). In some embodiments, the binding moiety may additionally act as a BRM.

[0105] In some embodiments, LNPs and / or tLNPs are used to deliver the payload (e.g., engineering agent(s) and / or conditioning agents(s)) to target immune cells to be modified. The fundamental ability of the tLNP to deliver a payload into the cytoplasm of a cell is agnostic with respect to, and does not depend upon, a particular binding specificity. Of course, a binding moiety is a determinant of which cells a payload is delivered into.

[0106] In some embodiments, an mRNA payload comprises one or more species of mRNA molecule. In some embodiments, the one or more species of mRNA comprises only a single species of mRNA while in other instances the one or more species of mRNA comprises multiple mRNA species, for example, 2, 3, or 4 mRNA species. In some embodiments in which the payload comprises multiple mRNA species, more than one of the species up to and including all of the species encode a polypeptide that is reactive with a same target. For example, in embodiments in which the payload comprises a nucleic acid encoding a CAR or immune cell engager (ICE), the payload can comprise or consist of 1 ) a single nucleic acid species encoding a single species of CAR or ICE, 2) a single nucleic acid species encoding 2 or more species of CAR or ICE (or a mixture of CAR and ICE) such as a bicistronic or multicistronic mRNA in which each CAR and / or ICE has specificity for a same target antigen, 3) a single nucleic acid species encoding 2 or more species of CAR or ICE36RECTIFIED SHEET (RULE 91 ) ISA / EP(or a mixture of CAR and ICE) such as a bicistronic or multicistronic mRNA in which at least one CAR and / or ICE has specificity for a different target antigen than the other(s), 4) two or more nucleic acid species encoding 2 or more species of CAR or ICE (or a mixture of CAR and ICE) in which each CAR and / or ICE has specificity for a same target antigen, 5) two or more nucleic acid species encoding 2 or more species of CAR or ICE (or a mixture of CAR and ICE) in which at least one CAR and / or ICE has specificity for a different target antigen than the other(s). In some instances, a single species of mRNA is any one of the herein disclosed improved mRNA molecules. In other instances, a single species of mRNA has one of the particular combinations of UTRs disclosed herein. When two or more CAR and / or ICE have specificity for a same target antigen, they can have specificity for different epitopes of the same target antigen. Further variations will be apparent to one of skill in the art (e.g., multiple bi- or multicistronic nucleic acids, nucleic acids encoding a TCR, etc., etc., etc.).(a) Binding moiety

[0107] In certain embodiments, the LNP is a tLNP comprising a binding moiety that specifically binds to a cell surface protein. In various embodiments, a binding moiety is an antibody or antigen-binding portion thereof. In certain embodiments, a LNP or tLNP comprises a binding moiety derived from an anti-CD40** antibody, an anti- LRRC15** antibody, an anti-CTSK antibody, an anti- AD AM 12* antibody, an anti- ITGA11 antibody, an anti-FAP*** antibody, an anti-NOX4 antibody, an anti-SGCD antibody, an anti-SYNDIG1 antibody, an anti-CDH1 1* antibody, an anti-PLPP4 antibody, an anti-SLC24A2 antibody, an anti-PDGFRB** antibody, an anti-THY1 * antibody, an anti-ANTXR1 * antibody, an anti-GAS1 antibody, an anti-CALHM5 antibody, an anti-SDC * antibody, an anti-HER2*** antibody, an anti-TROP2*** antibody, an anti-MSLN** antibody, an anti-Nectin4** antibody, or an anti-MUC16*** antibody. In further embodiments, a LNP (or tLNP) comprises a binding moiety specific for an immune cell antigen selected from CD1 , CD2***, CD3***, CD4***, CD5**, CD7**, CD8*, CD1 1 b*, CD14**, CD16, CD25**, CD26**, CD27***, CD28***, CD30***, CD32*, CD38***, CD39*, CD40***, CD40L (CD154)***, CD44**, CD45** (including CD45RA, CD45RB*, CD45RO* and pan CD45* species thereof), CD64**, CD62**, CD68, CD69*, CD73**, CD80**, CD83*, CD86**, CD95*, CD103*, CD119*, CD126*, CD137 (41 BB)**, CD150*, CD153*, CD161*, CD166*, CD183 (CXCR3)*, CD183 (CXCR5)*,37RECTIFIED SHEET (RULE 91 ) ISA / EPCD223 (LAG-3)*™, CD254*, CD275*, CD45RA, CTLA-4****, DEC205, 0X40*, PD- 1 *™, GITR*, TIM-3*™, , FasL**, IL18R1 , IGOS (CD278)*, leu-12, TOR*, TLR1 , TLR2™, TLR3**, TLR4™, TLR6, TREM2*, NKG2D*, CCR, CCR1 (CD191)*, CCR2 (CD192)*™, CCR4(CD194)*™, CCR6(CD196)*, CCR7*, low affinity IL-2 receptor™, IL-7 receptor*, IL-12 receptor*, IL-15 receptor*, IL-18 receptor*, and IL-21 receptor*. In further embodiments, a tLNP comprises a binding moiety specific for an HSC surface molecule selected from CD117*, CD34™, CD44™, CD90 (Thy1) *, CD105*, CD133*, BMPR2*, and Sca-1 ; or specific for an MSC surface molecules selected from CD70**, CD105*, CD73*, Stro-1*, SSEA-3*, SSEA-4*, CD271 *, CD146*, GD2*™, SUSD2, Stro- 4, MSCA-1 , CD56*, CD200*™, PODXL*, CD13*, CD29**, CD44**, CD45™, and CD10*. In particular embodiments, targeting using CD45RO and CD45RB is based on considerations regarding the status of these cell surface molecules as being an earlier marker on CD34+ cells besides T cells. Similarly for targeting T cells, expression of these markers on particular subsets can be considered: CD45RA (naive T cells), RO (memory T cells), and RB (naive, memory T, and effector T cells). (* indicates that exemplary antibodies with the indicated specificity from which a binding moiety could be derived can be found in US Patent 1 1 ,326,182B2 Table 9 or 10. indicates that exemplary antibodies with the indicated specificity from which a binding moiety could be derived can be found in Wilkinson & Hale, 2022. Both references cited and incorporated by reference above. * indicates that exemplary antibodies with the indicated specificity from which a binding moiety could be derived can be found in the Therapeutic Antibody Database (TABS) at tabs.craic.com). Other suitable antibodies can be found in Appendix A.

[0108] In some embodiments, a binding moiety of a tLNP comprises an antigen binding domain of an anti-CD8 antibody. In some embodiments, the anti-CD8 antibody is a humanized mouse antibody. In some instances, the anti-CD8 antibody is a humanized form of a mouse antibody, such as RPA-T8 (also referred to herein as CT8). As expressed on cells in a mammal, CD8 is a dimer, commonly of two a chains or one each of an a and p chain. CT8 recognizes an epitope on the a chain (sometime referred to as CD8a or CD8a). CT8 and its humanized derivatives can bind to both the 02 and op dimers. Further disclosure of CT8 and its humanized derivatives can be found in Patent Application Nos. 63 / 610,917 filed December 15, 2023 and 63 / 654,93038RECTIFIED SHEET (RULE 91 ) ISA / EPfiled on May 31 , 2024, each of which is incorporated by reference in their entirety .(b) LNP compositions

[0109] A variety of LNP and tLNP compositions are known in the art and can be used with the herein disclosed methods and systems for extracorporeal and ex vivo production of autologous engineered cells, and associated methods of delivering of a payload into a cell and methods of treatment. Particular advantageous LNP / tLNP compositions for such purposes are discussed herein below.

[0110] The LNP composition contributes to the formation of stable tLNP, efficient encapsulation of a payload, protection of a payload from degradation until it is delivered into a cell, and promotion of endosomal escape of a payload into the cytoplasm. These functions are primarily independent of the specificity of the binding moiety (or moieties) serving to direct or bias the tLNP to a particular cell type(s).

[0111] As used herein “LNP composition” refers to the lipid components present in an LNP, their molar ratios (e.g., mol%) relative to each other, and the ratio of payload to total lipid. The payload to total lipid ratio can be expressed on a weight to weight (w / w) basis or for nucleic acid molecules as an N / P ratio, wherein an “N / P ratio” means the ratio of positively chargeable lipid amine (N = nitrogen) groups to negatively- charged nucleic acid molecule phosphate (P) groups. The tLNPs of this disclosure are multicomponent compositions comprising an LNP and a binding moiety. As used herein “tLNP composition” refers to the same features as LNP composition with the addition of a binding moiety that serves as a targeting moiety and the binding moiety’s density on the tLNP may be expressed as a ratio to the payload on a w / w basis. As used herein, “LNP formulation” or “tLNP formulation” refers to the complete respective composition plus buffers, carriers, solvents and / or other excipients, which may also be referred to as a pharmaceutical composition.

[0112] In certain embodiments the LNP comprises a lipid composition comprising an ionizable cationic lipid, PEG-lipid comprising functionalized PEG-lipid and nonfunctionalized PEG-lipid, a phospholipid, and a sterol.

[0113] In certain aspects, the instant disclosure provides an LNP or tLNP, wherein the LNP or tLNP comprises about 35 mol% to about 65 mol% of an ionizable cationic lipid, about 0.5 mol% to about 3 mol% of a PEG-lipid (including non-functionalized39RECTIFIED SHEET (RULE 91 ) ISA / EPPEG-lipid and optionally a functionalized PEG-lipid), about 7 mol% to about 13 mol% of a phospholipid, and about 27 mol% to about 50 mol% of a sterol. An mRNA payload is generally encompassed by or in the interior of an LNP or tLNP. As disclosed herein dosages always refer to the amount of mRNA being provided. In some instances, the N / P ratio (the ratio of positively-chargeable lipid amine (N = nitrogen) groups to negatively-charged nucleic acid phosphate (P) groups) is from about 3 to about 9, about 3 to about 7, about 3 to about 6, about 4 to about 6, about 5 to about 6, or about 6. In some instances, the N / P ratio is from 3 to 9, 3 to 7, 3 to 6, 4 to 6, 5 to 6, or 6. In some embodiments, an LNP or tLNP comprises about 40 mol% to about 62 mol% ionizable cationic lipid. In some embodiments, an LNP or tLNP comprises about 1 mol% to about 2 mol% total PEG-lipid. In certain embodiments, an LNP or tLNP comprises about 0.1 mol% to about 0.3 mol%, for example about 0.1 mol%, about 0.2 mol%, or about 0.3 mol% functionalized PEG-lipid. In some embodiments, a binding moiety is conjugated to functionalized PEG-lipid. In certain embodiments, a tLNP is an LNP that further comprises an antibody as the binding moiety that serves as a targeting moiety and the binding moiety’s density on the tLNP may be expressed as a ratio to the payload on a weight by weight (w / w) basis. In certain instances, the binding moiety is a whole antibody (for example, a whole IgG) which is present at an antibody:mRNA ratio (w / w) of about 0.3 to about 1 .0.

[0114] With respect to the LNP or the tLNP, in some embodiments the molar ratio of the lipids is 0.1 to 5 mol% functionalized PEG-lipid: 40 to 65 mol% ionizable cationic lipid: 7 to 30 mol% phospholipid: 20 to 45 mol% sterol: 1 to 30 mol% co-lipid, if present: 0 to 5 mol% further PEG-lipid. In certain instances, the molar ratio of functionalized PEG-lipid is 0.1 to 0.3 mol% and the molar ratio of unfunctionalized PEG-lipid is 1 to 3 mol%, for example, 0.1 and 1.4 mol% respectively. In some embodiments, the functionalized PEG-lipid is conjugated to a binding moiety.

[0115] With respect to the LNP or the tLNP, in some embodiments the ratio of total lipid to nucleic acid is 10:1 to 50:1 on a weight basis. In some embodiments, that ratio of total lipid to nucleic acid is 10:1 , 20:1 , 30:1 , or 40:1 to 50:1 , or 10:1 to 20:1 , 30:1 , 40:1 or 50:1 , or any range bound by a pair of these ratios.

[0116] Particular compositions for precursors to tLNPs and tLNPs are disclosed in US Provisional Patent application Nos. 63 / 505,424 filed May 31 , 2023, 63 / 510,06140RECTIFIED SHEET (RULE 91 ) ISA / EPfiled June 23, 2023, and 63 / 520,303 filed August 17, 2023, as well as PCT Application No. PCT / US23 / 72426 filed August 17, 2023, each of which is incorporated by reference in its entirety. LNP and tLNP compositions can include those of Table B. In various embodiments, N / P can be from 3 to 9 or any integer-bound sub-range in that range or about any integer in that range.(1 ) Phospholipids

[0117] With respect to LNPs or tLNPs of this disclosure, in various embodiments, the phospholipid comprises dimyristoylphosphatidyl glycerol (DMPG), dimyristoylphosphatidyl choline (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), distearoyl-glycero-phosphate (18:0 PA, DSGP), dioleoylphosphatidyl ethanolamine (DOPE), dioleoyl-glycero-phosphate (18:1 PA, DOGP), or diarachidoylphosphotidylcholine (DAPC), or a combination thereof. Phospholipids can contribute to formation of a membrane, whether monolayer, bilayer, or multi-layer, surrounding the core of the LNP or tLNP. Additionally, phospholipids such as DSPC, DMPC, DPPC, DAPC impart stability and rigidity to membrane structure. Phospholipids, such as DOPE, impart fusogenicity. Further phospholipids, such as DMPG, which attains negative charge at physiologic pH, facilitates charge modulation. Thus, phospholipids constitute means for facilitating membrane formation, means for imparting membrane stability and rigidity, means for imparting fusogenicity, and means for charge modulation. Some embodiments specifically include one or more of the above phospholipids while other embodiments specifically exclude one or more of the above phospholipids.

[0118] In some embodiments, an LNP or tLNP has about 7 mol% to about 13 mol% phospholipid, about 7 mol% to about 10 mol% phospholipid, or about 10 mol% to about 13 mol% phospholipid. In certain embodiments, an LNP has about 7 mol%, about 10 mol%, or about 13 mol% phospholipid. In certain instances, the phospholipid is DSPC. In certain instances, the phospholipid is DAPC.(2) Sterols

[0119] The disclosed LNP and tLNP comprise a sterol. Sterol refers to a subgroup of steroids that contain at least one hydroxyl (OH) group. Examples of sterols include,41RECTIFIED SHEET (RULE 91 ) ISA / EPwithout limitation, cholesterol, ergosterol, p-sitosterol, stigmasterol, stigmastanol, 20- hydroxycholesterol, 22-hydroxycholesterol, and the like. With respect to LNPs or tLNPs of this disclosure, in various embodiments, the sterol is cholesterol, 20- hydroxycholesterol, 20(S)-hydroxycholesterol, 22-hydroxycholesterol, or a phytosterol or combinations thereof. In further embodiments, the phytosterol comprises campesterol, sitosterol, or stigmasterol, or combinations thereof. In certain embodiments, the cholesterol is not animal-sourced but is obtained by synthesis using a plant sterol as a starting point. LNPs incorporating C-24 alkyl (such as methyl or ethyl) phytosterols have been reported to provide enhanced gene transfection. The length of the alkyl tail, the flexibility of the sterol ring, and polarity related to a retained C-3 -OH group are important to obtaining high transfection efficiency. While - sitosterol and stigmasterol performed well, vitamin D2, D3 and calcipotriol, (analogs lacking intact body of cholesterol) and betulin, lupeol ursolic acid and olenolic acid (comprising a 5th ring) should be avoided. Sterols serve to fill space between other lipids in the LNP or tLNP and influence LNP or tLNP shape. Sterols also control fluidity of lipid compositions, reducing temperature dependence. Thus, sterols such as cholesterol, ergosterol, 20-hydroxycholesterol, 22-hydroxycholesterol, campesterol, fucosterol, p-sitosterol, and stigmasterol constitute means for controlling LNP shape and fluidity or sterol means for increasing transfection efficiency. Some embodiments specifically include one or more of the above sterols while other embodiments specifically exclude one or more of the above sterols.

[0120] In some embodiments, an LNP or tLNP has about 27 mol% or about 30 mol% to about 50 mol% sterol, or about 30 mol% to about 38 mol% sterol. In certain embodiments, an LNP or tLNP has about 30.5 mol%, about 33.5 mol%, or about 37.5 mol% sterol. In certain instances, the sterol is cholesterol. In certain embodiments the sterol is a mixture of sterols, for example, cholesterol and p-sitosterol or cholesterol and 20-hydroxycholesterol. In some instances, the sterol is about 25 mol% 20- hydroxycholesterol and about 75 mol% cholesterol. In some instances, the sterol is about 25 mol% p-sitosterol and about 75 mol% cholesterol. In some instances, the sterol is about 50 mol% p-sitosterol and about 50 mol% cholesterol. In certain embodiments, an LNP or tLNP has 27 mol% or 30 mol% to 50 mol% sterol or 30 mol% to 38 mol% sterol. In further embodiments, an LNP or tLNP has 30.5 mol%, 33.5 mol%,42RECTIFIED SHEET (RULE 91 ) ISA / EPor 37.5 mol% sterol. In certain instances, a sterol is cholesterol. In certain embodiments, a sterol is a mixture of sterols, for example, cholesterol and [3-sitosterol or cholesterol and 20-hydroxycholesterol. In some instances, a sterol is 25 mol% 20- hydroxycholesterol and 75 mol% cholesterol. In further instances, a sterol is 25 mol% [3-sitosterol and 75 mol% cholesterol. In still further instances, a sterol is 50 mol% [3- sitosterol and 50 mol% cholesterol.(3) Co-Lipids

[0121] With respect to the LNP or the tLNP, in some embodiments, the co-lipid is absent or comprises an ionizable lipid. In some embodiments the ionizable lipid is cholesterol hemisuccinate (CHEMS). In some embodiments, the co-lipid is a charged lipid, such as a quaternary ammonium headgroup-containing lipid. In some instances, the quaternary ammonium headgroup-containing lipid comprises 1 ,2-dioleoyl-3- trimethylammonium propane (DOTAP), N-(1 -(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA), or 3[3-(N-(N',N'- Dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), or combinations thereof. In addition to the chloride salts of the quaternary ammonium headgroup containing lipids, further instances include bromide, mesylate, and tosylate salts.(4) PEG-Lipids

[0122] A PEG-moiety provides a hydrophilic surface on the LNP, inhibiting aggregation or merging of LNP, thus contributing to their stability and reducing polydispersity. Additionally, a PEG moiety impedes binding by the LNP, including binding to plasma proteins. These plasma proteins include apoE which is understood to mediate uptake of LNP by the liver so that inhibition of binding can lead to an increase in the proportion of LNP reaching other tissues. These plasma proteins also include opsonins so that inhibition of binding reduces recognition by the reticuloendothelial system. The PEG-moiety can also be functionalized to serve as an attachment point for a targeting or binding moiety. Conjugating a cell- or tissue-specific binding moiety to the PEG-moiety enables a tLNP to avoid the liver and bind to its target tissue or cell type, greatly increasing the proportion of LNP that reaches the targeted tissue or cell type. PEG-lipid can thus serve as means for inhibiting LNP binding, and PEG-lipid conjugated to a binding moiety can serve as means for LNP-43RECTIFIED SHEET (RULE 91 ) ISA / EPtargeting. However, PEG is not the only moiety that can be attached to a lipid to confer these functions. Other shielding moieties that can be used instead of PEG include polysarcosine, polyglutamic acid, an XTEN polypeptide, a poly(glycerol), a poly(oxaline), poly(vinylpyrrolidone), hyaluronic acid, polysialic acid, a poly(carboxybetaine), apoly(sulfobetaine), and a phosphobetaine-base polymer. Collectively, lipids bearing PEG and these other moieties are referred to herein as shielding lipids and it is to be understood that further embodiments are contemplated throughout this disclosure in which shielding lipid or an alternative shielding moiety lipid is substituted for PEG-lipid.

[0123] In certain embodiments, the LNP is a tLNP comprising one or more functionalized PEG-lipids that has been conjugated to a binding moiety. In certain embodiments, the tLNP also comprises PEG-lipids not functionalized or conjugated with a binding moiety. In some embodiments the functionalization is a maleimide. In some embodiments the functionalization is a bromomaleimide or bromomaleimide amide, alkynylamide, or alkynylimide moiety at the terminal hydroxyl end of the PEG moiety. In some embodiments, the binding moiety comprises an antibody or antigen binding portion thereof. In some embodiments, the binding moiety is a polypeptide comprising a binding domain and an N- or C-terminal extension comprising an accessible thiol group. In some embodiments, the conjugation linkage comprises a reaction product of a thiol in the binding moiety with a functionalized PEG-lipid. In some embodiments the functionalization is a maleimide, azide, alkyne, dibenzocyclooctyne (DBCO), bromomaleimide or bromomaleimide amide, alkynylamide, or alkynylimide. In some embodiments, the binding moiety comprises an antibody or antigen binding portion thereof. In some embodiments, the binding moiety is a polypeptide comprising a binding domain and an N- or C-terminal extension comprising an accessible thiol group.

[0124] The disclosed LNP and tLNP comprise PEG-lipids, that is, a lipid conjugated to a polyethylene glycol. Generally, the lipid is a C14-C20 lipid. In certain embodiments, the PEG-lipid contains a Cis and / or C20 lipid. Common PEG-lipids fall into two classes diacyl glycerols and diacyl phospholipids. Examples of diacyl glycerol PEG-lipids include DMG-PEG (1 ,2-dimyristoyl-glycero-3-methoxypolyethylene glycol), DPG-PEG (1 ,2-dipalmitoyl-glycero-3-methoxypolyethylene glycol), DSG-PEG (1 ,2-distearoyl-44RECTIFIED SHEET (RULE 91 ) ISA / EPglycero-3-methoxypolyethylene glycol), and DOG-PEG (1 ,2-dioleoyl-glycero-3- methoxypolyethylene glycol). Examples of diacyl phospholipids include DMPE-PEG (1 ,2-dimyristoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol), DPPE-PEG (1 ,2-dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol), DSPE-PEG (1 ,2-distearoyl-glycero-3-phosphoethanolamine-3- methoxypolyethylene glycol), and DOPE-PEG (1 ,2-dioleoyl-glycero-3- phosphoethanolamine-3-methoxypolyethylene glycol). The glycerol in these lipids is chiral. Thus, in some embodiments, the PEG-lipid is racemic. Alternatively, optically pure antipodes of the glycerol portion can be employed, that is, the glycerol portion is homochiral. As used herein with respect to glycerol moieties, optically pure means >95% of a single enantiomer (D or L). In some embodiments, the enantiomeric excess is >98%. In some embodiments, the enantiomeric excess is >99%. Further PEG-lipids as well as the use of PEG-lipids and functionalized PEG-lipids and LNP incorporating them are disclosed in International Application Number PCT / US2023 / 017648 (Publication Number WO2023196445A1 ) which is incorporated for all that it teaches about PEG-lipids.

[0125] The above examples are presented as methoxypolyethylene glycols, but the terminus need not necessarily be methoxyl. With respect to any of the PEG-lipids that have not been functionalized, in alternative embodiments, the PEG moiety of the PEG lipids can terminate with a methoxyl, a benzyloxyl, a 4-methoxybenzyloxyl, or a hydroxyl group (that is, an alcohol). The terminal hydroxyl facilitates functionalization. The methoxyl, benzyloxyl, and 4-methoxybenzyloxyl groups are advantageously provided for PEG-lipid that will be used as a component of the LNP without functionalization. However, all four of these alternatives are useful as the (nonfunctionalized) PEG-lipid component of LNPs. The 4-methoxybenzyloxyl group, often used as a protecting group during synthesis of the PEG-lipid, is readily removed to generate the corresponding hydroxyl group. Thus the 4-methoxybenzyloxyl group offers a convenient path to the alcohol when it is not synthesized directly. The alcohol is useful for being functionalized, prior to incorporation of the PEG-lipid into a LNP, so that a binding moiety can be conjugated to it as a targeting moiety for the LNP (making it a tLNP). As used herein, the terminus of the PEG moiety, and similar constructions, refers to the end of the PEG moiety that is not attached to the lipid.45RECTIFIED SHEET (RULE 91 ) ISA / EP

[0126] In some embodiments, an LNP or tLNP comprises about 0.5 mol% to about 3 mol% or 0.5 mol% to 3 mol% PEG-lipid comprising functionalized and nonfunctionalized PEG-lipid. In certain embodiments, an LNP or tLNP comprises DSG- PEG. In other embodiments, an LNP or tLNP comprises DMG-PEG or DPG-PEG. In certain embodiments, an LNP or tLNP comprises DSPE-PEG. In some embodiments, the functionalized and non-functionalized PEG-lipids are not the same PEG-lipid, for example, the non-functionalized PEG-lipid can be a diacylglycerol and the functionalized PEG-lipid a diacyl phospholipid. tLNP with such mixtures have reduced expression in the liver, possibly due to reduced uptake. In certain embodiments the functionalized PEG-lipid is DSPE-PEG and the non-functionalized PEG-lipid is DSG- PEG. In some embodiments, an LNP or tLNP comprises about 0.4 mol% to about 2.9 mol% or about 0.9 mol% to about 1 .4 mol% non-functionalized PEG lipid. In certain embodiments, an LNP or tLNP comprises about 1.4 mol% or 1.4 mol% non- functionalized PEG lipid. In some embodiments, an LNP or tLNP comprises about 0.1 mol% to about 0.3 mol% or 0.1 mol% to 0.3 mol% functionalized lipid. In some instances, the functionalized lipid is DSPE-PEG. In certain instances, an LNP or tLNP comprises about 0.1 mol%, about 0.2 mol%, or about 0.3 mol% DSPE-PEG. In certain instances, an LNP or tLNP comprises 0.1 mol%, 0.2 mol%, or 0.3 mol% DSPE-PEG. In certain instances, the functionalized PEG-lipid is conjugated to a binding moiety. As used herein, the phrase “is conjugated to” and similar constructions are meant to convey a state of being, that is, a structure, and not a process, unless context dictates otherwise.

[0127] In certain aspects, the LNP comprises one or more PEG-lipids and / or functionalized PEG-lipids; when both a functionalized and unfunctionalized PEG-lipid, the PEG-lipid present they can be the same or different; and one or more ionizable cationic lipids; the LNP can further comprise a phospholipid, a sterol, a co-lipid, or any combination thereof. The term “functionalized PEG-lipid” refers to a PEG-lipid in which the PEG moiety has been derivatized with a chemically reactive group that can be used for conjugating a targeting moiety to the PEG-lipid. The functionalized PEG-lipid can be reacted with a binding moiety so that the binding moiety is conjugated to the PEG portion of the lipid. The conjugated binding moiety can thus serve as a targeting moiety for the LNP to constitute a tLNP. In some embodiments, the binding moiety is46RECTIFIED SHEET (RULE 91 ) ISA / EPconjugated to the functionalized PEG-lipid after an LNP comprising the functionalized PEG-lipid is formed. In other embodiments, the binding moiety is conjugated to the PEG-lipid and then the conjugate is inserted into a previously formed LNP.

[0128] PEG can be made in a large range of sizes. In certain embodiments, the PEG of the disclosed LNP and tLNP is PEG-1000 to PEG-5000. It is to be understood that polyethylene preparations of these sizes are polydisperse and that the nominal size indicates an approximate average molecular weight of the distribution. Taking the molecular weight of an individual repeating unit of (OCH2CH2)n to be 44, a PEG molecule with n=22 would have a molecular weight of 986, with n=45 a molecular weight of 1998, and with n=113 a molecular weight of 4990. n~22 to 113 is used to represent PEG-lipids incorporating PEG moieties in the range of PEG-1000 to PEG- 5000 such as PEG-1000, PEG-1500, PEG-2000, PEG-2500, PEG-3000, PEG-3500, PEG-4000, PEG-4500, and PEG-5000, although some molecules from preparations at the average molecular weight boundaries will have n outside that range. For individual preparations n~22 is used to represent PEG-lipids incorporating PEG moieties from PEG-1000, n=45 is used to represent PEG-lipids incorporating PEG moieties from PEG-2000 n»67 is used to represent PEG-lipids incorporating PEG moieties from PEG-3000, n«90 is used to represent PEG-lipids incorporating PEG moieties from PEG-4000, n»113 is used to represent PEG-lipids incorporating PEG moieties from PEG-5000. Some embodiments incorporate PEG moieties in a range bounded by any pair of the foregoing values of n or average molecular weight. Some embodiments incorporate PEG moieties of PEG-1000, PEG-2000, or PEG-5000. Certain embodiments comprise a DSG-PEG, for example, DSG-PEG-2000. Certain embodiments comprise a DSPE-PEG, for example, DSPE-PEG-2000. Certain embodiments comprise both DSG-PEG-2000 and / or DSPE-PEG2000.

[0129] A PEG-moiety provides a hydrophilic surface on the LNP, inhibiting aggregation or merging of LNP, thus contributing to their stability and reducing polydispersity. Additionally, a PEG moiety may impede binding by the LNP, including binding to plasma proteins. These plasma proteins include apoE which is understood to mediate uptake of LNP by the liver so that inhibition of binding can lead to an increase in the proportion of LNP reaching other tissues and cell types. These plasma proteins also include opsonins so that inhibition of binding reduces recognition by the47RECTIFIED SHEET (RULE 91 ) ISA / EPreticuloendothelial system.

[0130] The PEG-moiety can also be functionalized to serve as an attachment point for a binding moiety which serves as a targeting moiety. Conjugating a cell- or tissuespecific binding moiety to the PEG-moiety enables a tLNP to avoid the liver and bind to its target tissue or cell type, greatly increasing the proportion of LNP that reaches the targeted tissue or cell type. PEG-lipid can thus serve as means for inhibiting LNP binding, and PEG-lipid conjugated to a binding moiety can serve as means for LNP- targeting. As used herein, the term “functionalized PEG-lipid” and similar constructions refer generally to both the unreacted and reacted entities. The lipid composition of a LNP may be described referencing the reactive species even after conjugation has taken place (forming a tLNP). For example, a lipid composition may be described as comprising DSPE-PEG-maleimide and may be said to further comprise a binding moiety without explicitly noting that upon reaction to form the conjugate the maleimide will have been converted to a succinimide (or hydrolyzed succinimide). Similarly, if the reactive group is bromomaleimide, after conjugation it will be maleimide. These differences of chemical nomenclature for the unreacted and reacted species are to be implicitly understood even when not explicitly stated. Certain embodiments comprise a DSG-PEG, for example, DSG-PEG-2000. Certain embodiments comprise a functionalized DSPE-PEG, for example, functionalized DSPE-PEG-2000. Certain embodiments comprise both DSG-PEG-2000 and functionalized DSPE-PEG-2000. In some instances, the functionalized PEG-lipid is functionalized with a maleimide moiety, for example, DSPE-PEG-2000-MAL.

[0131] In certain embodiments, the PEG-lipid and / or functionalized PEG-lipid comprises a scaffold selected from Formula S1 , Formula S2, Formula S3, or Formula S4:Formula S1 Formula S2RECTIFIED SHEET (RULE 91 ) ISA / EPFormula S3 Formula S4 whereinrepresents the points of ester connection with a fatty acid, and represents the point of ester (S1 ) or ether (S2, S3, and S4) formation with the PEG moiety. In some embodiments, the fatty acid esters are C14-C20 straight-chain alkyl fatty acids. In some embodiments, the PEG moiety is functionalized and the fatty acid esters are C16-C20 straight-chain alkyl fatty acids. For example, the straightchain alkyl fatty acid is Cu, C15, C16, C17, C18, C19, or C20. In some embodiments, the fatty acid esters are C14-C20 symmetric branched-chain alkyl fatty acids. For example, the branched-chain alkyl fatty acid is C14, C15, C16, C17, C18, C19, or C20. By symmetric it is meant that each alkyl branch has the same number of carbons. In some embodiments, the branch is at the 3, 4, 5, 6, or 7 position of the fatty acid ester. The synthesis and use of PEG-lipids built on scaffolds S1 -S4, and bromomaleimide, alkynylamide, or alkynylimide functionalization and conjugation, is disclosed in WO2023 / 196445A1 which is incorporated by reference for all that it teaches about PEG-lipids and their use.

[0132] In some embodiments, the PEG moiety is PEG-500 to PEG-5000 such as PEG-500, PEG-1000, PEG-1500, PEG-2000, PEG-2500, PEG-3000, PEG-3500,PEG-4000, PEG-4500, and PEG-5000. In some instances, the PEG moiety is PEG- 2000. In some embodiments, the PEG unit has a MW of 2000 Da. In some instances, the MW2000 PEG-lipid comprises DMG-PEG2000 (1 ,2-dimyristoyl-glycero-3- methoxypolyethylene glycol-2000), DPG-PEG2000 (1 ,2-dipalmitoyl-glycero-3- methoxypolyethylene glycol-2000), DSG-PEG2000 (1 ,2-distearoyl-glycero-3- methoxypolyethylene glycol-2000), DGG-PEG2000 (1 ,2-dioleoyl-glycero-3- methoxypolyethylene glycol-2000), DMPE-PEG200 (1 ,2-dimyristoyl-glycero-3- phosphoethanolamine-3-methoxypolyethylene glycol-2000), DPPE-PEG2000 (1 ,2- dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000),RECTIFIED SHEET (RULE 91 ) ISA / EPDSPE-PEG2000 (1 ,2-distearoyl-glycero-3-phosphoethanolamine-3- methoxypolyethylene glycol-2000), DOPE-PEG2000 (1 ,2-dioleoyl-glycero-3- phosphoethanolamine-3-methoxypolyethylene glycol-2000), or combinations thereof. In some embodiments, the glycerol moiety is racemic. Alternatively, optically pure antipodes of the glycerol portion can be employed, that is, the glycerol portion is homochiral. In some embodiments, the PEG moiety is functionalized for conjugation to a binding moiety. The PEG-lipids constitute means for preventing aggregation. The functionalized PEG-lipids constitute means for conjugating a binding moiety. In embodiments comprising both conjugated and unconjugated PEG-lipids, in some the conjugated and unconjugated PEG-lipid are the same and in others the PEG-lipid is different.(5) Conjugation

[0133] Multiple suitable chemistries to conjugate the binding moiety to the PEG of the PEG-lipid, including maleimide (see Parhiz et al., Journal of Controlled Release 291 :106-115, 2018) and click (see Kolb et al., Angewandte Chemie International Edition 40(1 1 ):2004— 2021 , 2001 ; and Evans, Australian Journal of Chemistry 60(6):384-395, 2007) chemistries. Reagents for such reactions include lipid-PEG- maleimide, lipid-PEG-cysteine, lipid-PEG-alkyne, lipid-PEG-dibenzocyclooctyne (DBCO), and lipid-PEG-azide. Further conjugations reactions make use of lipid-PEG- bromomaleimide, lipid- PEG-alkylnoic amide, lipid-PEG-alkynoic imide, and lipid-PEG- alkyne reactions, as disclosed in PCT / US23 / 17648 entitled PEG-Lipids and Lipid Nanoparticles, which is incorporated by reference for all that it teaches about conjugation chemistry and alternative PEG-lipids. On the binding moiety side of the reaction one can use an existing cysteine sulfhydryl, or derivatize the protein by adding a sulfur containing carboxylic acid, for example, to the epsilon amino of a lysine to react with maleimide, bromomaleimide, (collectively, “a maleimide”), alkylnoic amide, or alkynoic imide. Alternatively, one can add an alkyne to a sulfhydryl or an epsilon amino of a lysine to participate in a click chemistry reaction.

[0134] To modify an epsilon amino of a binding moiety lysine to react with a maleimide functionalized PEG-lipid the binding moiety (e.g., an antibody) can be reacted with N-succinimidyl S-acetylthioacetate (SATA). SATA is then deprotected, for example, using 0.5 M hydroxylamine followed by removal of the unreacted50RECTIFIED SHEET (RULE 91 ) ISA / EPcomponents by G-25 Sephadex Quick Spin Protein columns (Roche Applied Science, Indianapolis, IN). The reactive sulfhydryl group on the binding moiety is then conjugated to maleimide moieties on LNPs of the disclosure using thioether conjugation chemistry. Purification can be performed using Sepharose CL-4B gel filtration columns (Sigma-Aldrich). tLNPs (LNPs conjugated with a targeting antibody) can be stored frozen at -80°C until needed. Others have conjugated antibody to free functionalized PEG-lipid and then incorporated the conjugated lipid into pre-formed LNP. However, we have found that this procedure is more controllable and produces more consistent results.

[0135] There are also several approaches to site-specific conjugation. Particularly but not exclusively suitable for truncated forms of antibody, C-terminal extensions of native or artificial sequences containing a particularly accessible cysteine residue are commonly used. Partial reduction of cystine bonds in an antibody, for example, with tris(2-carboxy)phosphine (TCEP), can also generate thiol groups for conjugation which can be site-specific under defined conditions with an amenable antibody fragment. Alternatively, the C-terminal extension can contain a sortase A substrate sequence, LPXTG (SEQ ID NO: 1 12) which can then be functionalized in a reaction catalyzed by sortase A and conjugated to the PEG-lipid, including through click chemistry reactions (see, for example, Moliner-Morro et aL, Biomolecules 10(12):1661 , 2020 which is incorporated by reference herein for all that it teaches about antibody conjugations mediated by the sortase A reaction and / or click chemistry). For whole antibody and other forms comprising an Fc region, site-specific conjugation to either (or both) of two specific lysine residues (Lys248 and Lys288) can be accomplished without any change to or extension of the native antibody sequence by use of one of the AJICAP® reagents (see, for example, Matsuda et aL, Molecular Pharmaceutics 18:4058-4066, 2021 and Fujii et al., Bioconjugate Chemistry https: / / doi.org / 10.1021 / acs.bioconjchem.3c00040, 2023, which are incorporated by reference herein for all that they teach about conjugation of antibodies with AJICAP reagents). The AJICAP reagents are modified affinity peptides that bind to specific loci on the Fc and react with an adjacent lysine residue. The peptide is then cleaved with base to leave behind a thiol-functionalized lysine residue which can then undergo conjugation through maleimide or haloamide reactions, for example).51RECTIFIED SHEET (RULE 91 ) ISA / EPFunctionalization with azide or dibenzocyclooctyne (DBCO) for conjugation by click chemistry is also possible.

[0136] Accordingly, in some embodiments the binding moiety is conjugated to the PEG moiety of the PEG-lipid through a thiol modified lysine residue. In some embodiments the conjugation is through a cysteine residue in a native or added antibody sequence. In other embodiments, the conjugation is through a sortase A substrate sequence. In still other embodiments, the conjugation is through a specific lysine residue (Lys248 or Lys288) in the Fc region.

[0137] A binder density on the tLNP can be defined according to the ratio of antibody (binder) to mRNA (w / w) either based on the amount of antibody input in the conjugation reaction or as measured in the tLNP. For an intact antibody (e.g., whole IgG), in some embodiments, preferred ratios are about 0.3 to about 1.0, about 0.3 to about 0.7, about 0.3 to about 0.5, about 0.5 to about 1 .0, and about 0.5 to about 0.7 for either the input or final measured binder ratio. In certain embodiments, a tLNP has an antibody ratio of 0.3 to 1 .0, 0.3 to 0.7, 0.3 to 0.5, 0.5 to 1 .0, and 0.5 to 0.7 for either the input or final measured binder ratio. In some embodiments, if the binder is different in size from an intact antibody (for example a scFv, diabody, or minibody, etc.) the w / w ratio is adjusted for the different size of the binder.(6) Ionizable Cationic Lipids

[0138] Ionizable cationic lipids are useful components for complexing with negatively charged payloads and for promoting delivery of the payload into the cytoplasm of a cell following endocytosis. Accordingly, each of the hereinbelow disclosed genera and species of ionizable cationic lipid can be used in defining the scope of embodiments of the herein disclosed LNP and tLNP compositions and pharmaceutical compositions, and methods of using them. In certain embodiments, ionizable cationic lipid(s) of an LNP having a measured pKa of 6 to 7 can remain essentially neutral in the blood stream and interstitial spaces but ionize after uptake into cells as the endosomes acidify. Upon acidification in the endosomal space, the lipid becomes protonated, and associates more strongly with the phosphate backbone of the nucleic acid, which destabilizes the structure of the LNP and promotes nucleic acid release from the LNP into the cell cytoplasm (also referred to as endosomal52RECTIFIED SHEET (RULE 91 ) ISA / EPescape). Thus, the herein disclosed ionizable cationic lipids constitute means for destabilizing LNP structure (when ionized) or means for promoting nucleic acid release or endosomal escape. In some embodiments the ionizable cationic lipid has a c-pKa from 8 to 1 1 and cLogD from 9 to 18 or 11 -14. In some embodiments, the ionizable cationic lipids have branched structure to give the lipid a conical rather than cylindrical shape. Suitable ionizable cationic lipids are known to those of skill in the art. In some embodiments, the ionizable cationic lipid has a structure of Formula 1 , Formula 2, Formula 3, Formula M5, Formula M1 , CICL, CICL-IE, Formula M6 or Formula M2 including species or subgenera thereof, as disclosed in International Application Number PCT / US2023 / 017647 published as WO 2023 / 196444, and US Patent Application Nos. 63 / 632,931 , 63 / 632,937, 63 / 632,940, and 63 / 632,944, also further describing the use of ionizable cationic lipids and LNP incorporating them, which is incorporated by reference in its entirety.

[0139] For simplicity, chemical moieties are defined and referred to throughout primarily as univalent chemical moieties (e.g., alkyl, aryl, and the like). Nevertheless, such terms may also be used to convey corresponding multivalent moieties under the appropriate structural circumstances clear to those skilled in the art. For example, while an “alkyl” moiety generally refers to a monovalent radical (e.g. CH3-CH2-), in certain circumstances a bivalent linking moiety can be “alkyl,” in which case those skilled in the art will understand the alkyl to be a divalent radical (e.g., -CH2-CH2-), which is equivalent to the term “alkylene.” (Similarly, in circumstances in which a divalent moiety is required and is stated as being “aryl,” those skilled in the art will understand that the term “aryl” refers to the corresponding divalent moiety, arylene.) All atoms are understood to have their normal number of valences for bond formation (i.e. , 4 for carbon, 3 for nitrogen, 2 for oxygen, and 2, 4, or 6 for sulfur, depending on the oxidation state of the sulfur atom).

[0140] The term "alkyl" as employed herein refers to saturated straight and branched chain aliphatic groups having from 1 to 12 carbon atoms. As such, “alkyl” encompasses Ci, C2, C3, C4, C5, Ge, Cz, Cs, C9, C10, On and C12 groups.

[0141] The term "alkenyl" as used herein means an unsaturated straight or branched chain aliphatic group with one or more carbon-carbon double bonds, having from 2 to 12 carbon atoms. As such, “alkenyl” encompasses C2, C3, C4, C5, Ce, Cz, Cs,53RECTIFIED SHEET (RULE 91 ) ISA / EPC9, C10, C11 and C12 groups.

[0142] In some embodiments, the hydrocarbon chain is unsubstituted. In other embodiments, one or more hydrogens of the alkyl or alkenyl group may be substituted with the same or different substituents.

[0143] Aryl refers to an aromatic or heteroaromatic ring lacking one hydrogen leaving a bond that connects to another portion of an organic molecule. Examples of aryl include, without limitation, phenyl, naphthalenyl, pyridine, pyrimidine, pyrazine, pyrrole, furan, thiophene, imidazole, thiazole, oxazole, and the like.

[0144] Aryl-alkyl refers to a moiety comprising one or more aryl rings and one or more alkyl moieties. The position of the one or more aryl rings can vary within the alkyl portion of the moiety. For example, the one or more aryl rings may be at an end of the one or more alkyl moieties, be fused into the carbon chain of the one or more alkyl moieties, or substitute one or more hydrogens of one or more alkyl moieties; and the one or more alkyl moieties may substitute one or more hydrogens of the one or more aryl rings. In some embodiments, there is a single ring; while in other embodiments, that are multiple rings.

[0145] Branched alkyl is a saturated alkyl moiety wherein the alkyl group is not a straight chain. Alkyl portions such as methyl, ethyl, propyl, butyl, and the like, can be appended to variable positions of the main alkyl chain. In some embodiments, there is a single branch; while in other embodiments, there are multiple branches.

[0146] Branched alkenyl refers to an alkenyl group comprising at least one branch off the main chain which may be formed by substituting one or more hydrogens of the main chain with the same or different alkyl groups, e.g., without limitation, methyl, ethyl, propyl, butyl, and the like. In some embodiments, a branched alkenyl is a single branch structure, while in other embodiments, a branched alkenyl may have multiple branches.

[0147] Straight chain alkyl is a non-branched, non-cyclic version of the alkyl moiety described above.

[0148] Straight chain alkenyl is a non-branched, non-cyclic version of the alkenyl moiety described above.54RECTIFIED SHEET (RULE 91 ) ISA / EP

[0149] In some embodiments as described herein, the ionizable cationic lipids have a structure of Formula 1 :Formula 1 wherein:Y is O, NH, N-CH3, or CH2, n is an integer from 0 to 4,m is an integer from 1 to 3, o is an integer from 1 to 4, p is an integer from 1 to 4, wherein when p = 1 : each R is independently Ce to Ci6 straight-chain alkyl; Ce to Ci6 branched alkyl; Ce to Ci6 straight-chain alkenyl; Ce to Cie branched alkenyl; C9 to C16 cycloalkyl-alkyl in which the cycloalkyl is C3 to Cs cycloalkyl positioned at either end or within the alkyl chain; or Cs to C18RECTIFIED SHEET (RULE 91 ) ISA / EParyl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain; wherein when p = 2: each R is independently Ce to C14 straight-chain alkyl; Ce to C14 straight-chain alkenyl; Ce to Cu branched alkyl; Ce to C14 branched alkenyl; C9 to Cu cycloalkyl-alkyl in which the cycloalkyl is C3 to Cs cycloalkyl positioned at the either end or within the alkyl chain; or Cs to C16 aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain; wherein when p = 3: each R is independently Ce to Ci 2 straight-chain alkyl; Ce to C12 straight-chain alkenyl; Ce to C12 branched alkyl; Ce to C12 branched alkenyl; C9 to C12 cycloalkyl-alkyl in which the cycloalkyl is C3 to Cs cycloalkyl positioned at either end or within the alkyl chain; or Cs to Cu aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain; and wherein when p = 4: each R is independently Ce to C10 straight-chain alkyl; Ce to C10 straight-chain alkenyl; Ce to C10 branched alkyl; Ce to C10 branched alkenyl; C9 to C10 cycloalkyl- alkyl in which the cycloalkyl is C3 to Cs cycloalkyl positioned at either end or within the alkyl; or Cs to C12 aryl-alky in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain.

[0150] In certain aspects, the ionizable cationic lipids of this disclosure have a structure of the formula M5:RECTIFIED SHEET (RULE 91 ) ISA / EPwherein: each R1is independently selected from a C7-C11 alkyl or a C7-C11 alkenyl,A1is (CH2)I-2,A2is O,A3is (CH2)I-5,X is N, CH, or C-CH3,A4is CH2, C=O, NH, NCH3, or O,A5is absent, O, S, NH, NCH3, or C=O,A6is O, S, NH, NCH3or (CH2)o-2,A7is (CH2)O-6, 3R2is O, R3is C=O and Z is CH or N, or R2is C=O, R3is O and Z is CH;57RECTIFIED SHEET (RULE 91 ) ISA / EPwhereinwherein the number of contiguous atoms present in a span:the range from 7-17.

[0151] As used herein, when a subscript has a value of “0”, the group is absent. For example, when A6is (CH2)o, A6is absent.

[0152] In certain embodiments of formula M5, R2is O, R3is C=O and Z is CH or N. For example, in certain embodiments of formula M5, R2is O, R3is C=O and Z is CH.

[0153] In certain embodiments of formula M5, R2is C=O, R3is O and Z is CH.

[0154] In certain embodiments of formula M5, A1is CH2, A3is (CH2)2-5, X is N, A4is C=O, A5is O, S, NH, NCH3, A6is (CH2)I-2, and A7is (CH2)I-4. For example, in certain embodiments A1is CH2, A3is (CH2)2-5, X is N, A4is C=O, A5is O, A6is (CH2)I-2, and A7is (CH2)I4.

[0155] In certain embodiments of formula M5, A1is CH2, A3is (CH2)I-4, X is CH, A4is CH2, NH, NCH3, O, A5is C=O, A6is O, NH, NCH3, or CH2, and A7is (CH)2.6. In58RECTIFIED SHEET (RULE 91 ) ISA / EPcertain embodiments as described herein, A1is CH2, A3is (CH2)I-4, X is CH, A4is NH, A5is C=O, A6is O, NH, NCH3, or CH2, and A7is (CH) 2-6. For example, in certain embodiments of formula M5, A1is CH2, A3is (CH2)I-4, X is CH, A4is NH, A5is C=O, A6is O, and A7is (CH) 2-6. For example, in certain embodiments of formula M5, A1is CH2, A3is (CH2)I-4, X is CH, A4is NH, A5is C=O, A6is CH2, and A7is (CH) 2-6. In certain embodiments as described herein, A1is CH2, A3is (CH2)I-4, X is CH, A4is CH2, A5is C=O, A6is O, NH, NCH3, or CH2, or A7is (CH) 2-6. For example, in certain embodiments of formula M5, A1is CH2, A3is (CH2)I-4, X is CH, A4is CH2, A5is C=O, A6is O, A7is (CH) 2-6. In certain embodiments as described herein, A1is CH2, A3is (CH2)I-4, X is CH, A4is O, A5is C=O, A6is O, NH, NCH3, or CH2, and A7is (CH) 2-6. For example, in certain embodiments of formula M5, A1is CH2, A3is (CH2)I-4, X is CH, A4is O, A5is C=O, A6is CH2, and A7is (CH) 2-6. In certain embodiments as described herein, A1is CH2, A3is (CH2)I-4, X is CH, A4is NCH3, A5is C=O, A6is O, NH, NCH3, or CH2, and A7is (CH) 2-6. For example, in certain embodiments of formula M5, A1is CH2, A3is (CH2)I-4, X is CH, A4is NCH3, A5is C=O, A6is CH2, and A7is (CH) 2-6.

[0156] In certain embodiments of formula M5, A1is (CH2)2, A3is (CH2)I-4, X is C- CH3, A4is C=O, A5is O, NH, NCH3, A6is (CH2)I-2, or A7is (CH2)I-4. For example, in certain embodiments, A1is (CH2)2, A3is (CH2)I-4, X is C-CH3, A4is C=O, A5is O, A6is (CH2)I-2, or A7is (CH2)I-4.

[0157] In certain embodiments of formula M5, the number of contiguous connective 0 atoms present in a span:is in the range from 7-17. For example, in certain embodiments, the number of contiguous connective atoms present 0 in a span:is in the range of 7-1 1 or 7-10. In certain embodiments, the number of contiguous connective atoms present in a span: 0H A4A5A7A1A3'X' '-A6"A8|Sjn the range of 10-17 (e .g ., in the range of 10-16, or 10-14, or 10-12). For example, in certain embodiments, the number of contiguous connectiveOJ|^ A4A5A7atoms present in a span:a1 a3"a6"a8is 10. For example, in certainRECTIFIED SHEET (RULE 91 ) ISA / EPembodiments, the number of contiguous connective atoms present in a span: -j-hepresent inventors have found that changing thenumber of contiguous connective atoms present in each span can allow for tuning of the pKa of the cationic lipid.

[0158] In some embodiments of formula M5, Y isporexample, in some embodiments of formula M5, Y is

[0159] In some embodiments of formula M5, Ys

[0160] In some embodiments of formula M5, Y i

[0161] In some embodiments of formula M5, Y is

[0162] In some embodiments of formula M5, Y is

[0163] In some embodiments of formula M5, Y is

[0164] In some embodiments of formula M5, Y is

[0165] In some embodiments of formula M5, Y is

[0166] In some embodiments of formula M5, Y is

[0167] In some embodiments of formula M5, Y is

[0168] In some embodiments of formula M5, Y isRECTIFIED SHEET (RULE 91 ) ISA / EP

[0169] In certain aspects, the constrained ionizable cationic lipids of this disclosure have a structure of the formula M6:wherein,andY is O, S, NH, or NCH3;R2is O, R3is C=O and Z is CH or N, or R2is C=O, R3is O and Z is CH; and each R1is independently selected from a C7-C11 alkyl or a C7-C11 alkenyl; each A1, A2, A3, and A4is independently selected from (CH2)o and (CH2)I , A5is selected from (CH2)o-4, CH=CH, and CH2-CH=CH-CH2; and wavy bonds indicate that any relative or absolute stereo-configuration of the corresponding ring atom can be assumed, or a mixture thereof.

[0170] As used herein, when a subscript has a value of “0”, the group is absent. For example, when A1is (CH2)o, A1is absent.RECTIFIED SHEET (RULE 91 ) ISA / EP

[0171] In certain embodiments of formula M6, R2is O, R3is C=O and Z is CH or N. For example, in certain embodiments of formula M6, R2is O, R3is C=O and Z is CH.

[0172] In certain embodiments of formula M6, R2is C=O, R3is O and Z is CH.

[0173] In various embodiments of M6, A1through A4are chosen so that there are only two main chain atoms between the ring nitrogen and each nearest ester oxygen in the nearest tail group.

[0174] In certain embodiments of M6, A1is (CH2)o, A2is (CH2)o, A3is (CH2)I , A4is (CH2)I , and A5is (CH2)I-4 or CH2-CH=CH-CH2.

[0175] In certain embodiments(CH2)O, and A5is (CH2)I .

[0176] In certain embodiments(CH2)O, and A5is (CH2)o.

[0177] In certain embodiments(CH2)O, and A5is (CH2)I .

[0178] In certain embodiments(CH2)O, and A5is (CH2)2or CH=CH.

[0179] In some embodiments of formula M6 as described herein, X is porexample, in some embodiments of formula M6, X is

[0180] In some embodiments of formula M6 as described herein, X is mbodiments of formula M6 as described herein, X is

[0182] In some embodiments of formula M6 as described herein, X isRECTIFIED SHEET (RULE 91 ) ISA / EP

[0183] In some embodiments of formula M6 as described herein, X is embodiments of formula M6 as described herein, X is embodiments of formula M6 as described herein,is embodiments of formula M6 as described herein,is embodiments of formula M6 as described herein, Xembodiments of formula M6 as described herein, X is embodiments of formula M6 as described herein, X is embodiments of formula M6 as described herein, X is

[0191] In some embodiments of formula M6 as described herein, X is63RECTIFIED SHEET (RULE 91 ) ISA / EP

[0192] As described above, in some embodiments of formula M6, Y may be selected from O, S, NH, or NCH3. In some embodiments of formula M6, Y is O. In some other embodiments of formula M6, Y is S.CH3Y (CH2)2-M' v^Li

[0193] In some embodiments of formula M6, X is3and Y is O. InCH3Y (CH2)2-^ some embodiments of formula M6, X is3and Y is S.

[0194] As described above, for both formula M5 and M6, each R1is independently selected from C7-C11 alkyl or C7-C11 alkenyl. In some embodiments of formula M5 and / or M6, each R1is independently selected from C7-C11 alkyl, e.g., C7-C10 alkyl, or C7-C9 alkyl. In certain embodiments of formula M5 and / or M6, each R1is independently selected from a linear C7-C11 alkyl, e.g., a linear C7-C10 alkyl, or a linear C7-C9 alkyl. In some embodiments of formula M5 and / or M6 as described herein, each R1is independently selected from (CH2)6-sCH3. In some of these and other embodiments, R1is (CH2)7CHs. In some embodiments of formula M5 and / or M6, each R1is independently selected from a linear C7-C11 alkenyl, e.g., a linear C7-C10 alkenyl, or a linear C7-C9 alkenyl. For example, in some embodiments of formula M5 and / or M6, each R1is a linear Cs alkenyl. In certain other embodiments of formula M5 and / or M6, each R1is independently selected from a branched C7-C11 alkyl, e.g., C7-Cioalkyl, or C7-C9 alkyl. For example, in some embodiments of formula M5 and / or M6, each R1is a branched Cs alkyl. In certain embodiments of formula M5 and / or M6, each R1is independently selected from a branched C7-C11 alkenyl, e.g., C7-C10 alkenyl, or C7-C9 alkenyl. For example, in some embodiments of formula M5 and / or M6, each R1is a branched Cs alkenyl. In some embodiments of formula M5 and / or M6, wherein R1is a branched alkyl or alkenyl, the branch point is positioned so that ester carbonyls are not in an a position relative to the branch point, for example they are in a p position relative to the branch point.

[0195] In certain embodiments of formula M5 and / or M6 as described herein, each R1is the same. In certain embodiments of formula M5 and / or M6, each R1nearest a common branch point is the same, but those nearest a first common branch point differ from those nearest a second common branch point. In certain embodiments of64RECTIFIED SHEET (RULE 91 ) ISA / EPformula M5 and / or M6, each R1nearest a common branch point is different but the pair of R1s nearest a first common branch point is the same the pair nearest a second common branch point.

[0196] In certain embodiments of formula M6, the ionizable cationic lipid is substantially enantiomerically pure (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%). In certain embodiments of formula M6, the ionizable cationic lipid is a racemic mixture. In certain embodiments of formula M6, the ionizable cationic lipid is a mixture of two or more stereoisomers. In certain embodiments of formula M6, at least two of the two or more stereoisomers are diastereomers. In certain embodiments of formula M6, at least two of the two or more stereoisomers are enantiomers.

[0197] In some embodiments, the ionizable cationic lipid has a structure of Formula 2,wherein Y is O, NH, N-CH3, or CH2, n is an integer from 0 to 4,RECTIFIED SHEET (RULE 91 ) ISA / EPm is an integer from 1 to 3, o is an integer from 1 to 4, p is an integer from 1 to 4, wherein when p is 1 , each R is independently Ce to Ci6 straight-chain alkyl; Ce to CIG straight-chain alkenyl; Ce to C-ie branched alkyl; Ce to C-ie branched alkenyl; C9 to C16 cycloalkyl-alkyl in which the cycloalkyl is C3 to Cs cycloalkyl positioned at either end or within the alkyl chain; or Cs to Cis arylalkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain, wherein when p = 2, each R is independently Ce to C14 straight-chain alkyl; Ce to C14 straight-chain alkenyl; Ce to C14 branched alkyl; Ce to C14 branched alkenyl; C9 to Cu cycloalkyl-alkyl in which the cycloalkyl is C3 to Cs cycloalkyl positioned at the either end or within the alkyl chain; or Cs to C16 arylalkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain, wherein when p = 3, each R is independently Ce to C12 straight-chain alkyl; Ce to C12 straight-chain alkenyl; Ce to C12 branched alkyl; branched Ce to C12 alkenyl; C9 to C12 cycloalkyl-alkyl in which the cycloalkyl is C3 to Cs cycloalkyl positioned at either end or within the alkyl chain; or Cs to C14 arylalkyl in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain, and wherein when p = 4, each R is independently Ce to C10 straight-chain alkyl; straight-chain Ce to C10 alkenyl; Ce to C10 branched alkyl; Ce to C10 branched alkenyl; C9 to C10 cycloalkyl-alkyl in which the cycloalkyl is C3 to Cs cycloalkyl positioned at either end or within the alkyl; or Cs to C12 aryl-alky in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain.

[0198] In some embodiments, the ionizable cationic lipid has a structure of Formula 3,RECTIFIED SHEET (RULE 91 ) ISA / EP(Formula 3) wherein W is C=0 or CH2, n is an integer from 0 to 4,m is an integer from 1 to 3, 0 is an integer from 1 to 4, p is an integer from 1 to 4, wherein when p is 1 , each R is independently Cs to Cis straight-chain alkyl; Cs to Cis straight-chain alkenyl; Cs to Cis branched alkyl; Cs to Gw branched alkenyl; C11 to Cw cycloalkyl-alkyl in which the cycloalkyl is C3 to Cs cycloalkyl positioned at either end or within the alkyl chain; or C10 to C20 aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain, wherein when p = 2, each R is independently Cs to C16 straight-chain alkyl; Cs to C16 straight-chain alkenyl; Cs to C16 branched alkyl; Cs to C16 branched alkenyl; C11 to Cie cycloalkyl-alkyl in which the cycloalkyl is C3 to Cs cycloalkyl positioned at the either end or within the alkyl chain; or C10 to Cis aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain,67RECTIFIED SHEET (RULE 91 ) ISA / EPwherein when p = 3, each R is independently Cs to Ou straight-chain alkyl; Cs to C14 straight-chain alkenyl; Cs to Cu branched alkyl; Cs to CM branched alkenyl; Cn to Cu cycloalkyl-alkyl in which the cycloalkyl is C3 to Cs cycloalkyl positioned at either end or within the alkyl chain; or C10 to C16 aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain, and wherein when p = 4, each R is independently Cs to C12 straight-chain alkyl; Cs to C12 straight-chain alkenyl; Cs to C12 branched alkyl; Cs to C12 branched alkenyl; C11 to Ci2 cycloalkyl-alkyl in which the cycloalkyl is Cs to Cs cycloalkyl positioned at either end or within the alkyl; or C10 to C14 aryl-alky in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain.

[0199] The synthesis of lipids having the structure of Formulas 1 , 2, 3, M1 , M2, M5, CICL, CICL-IE, or M6 is described in US Patent Application Nos. 63 / 632,931 (M2, some M6 embodiments), 63 / 632,937 (M1 , some M5), 63 / 632,940 (CICL-IE, some M5), and 63 / 632,944 (some M6), and US Patent Application Publication No. 2023 / 0320995 (1 , 2, 3, CICL) each of which is incorporated by reference in its entirety for all that it teaches about the synthesis of such lipids, as well as particular subgenera and individual species of lipids.

[0200] In some embodiments, an LNP or tLNP comprises about 35 mol% to about 65 mol%, about 40 mol% to about 62 mol%, or about 54 mol% to about 60 mol% ionizable cationic lipid. In some embodiments, the lipid composition is at least 40 mol% and / or does not exceed 62 mol% ionizable cationic lipid. In certain embodiments, an LNP of tLNP comprises about 54 mol%, about 58 mol%, or about 62 mol% ionizable cationic lipid. In further embodiments an LNP comprises 35 mol% to 65 mol%, 40 mol% to 62 mol%, or 54 mol% to 60 mol% ionizable cationic lipid. In still further embodiments, an LNP has at least 40 mol% or does not exceed 62 mol% ionizable cationic lipid. In certain embodiments, an LNP comprises 54 mol%, 58 mol%, or 62 mol% ionizable cationic lipid.

[0201] With respect to the LNP or the tLNP, in some embodiments the ratio of total lipid to nucleic acid is 10:1 to 50:1 on a weight basis. In some embodiments, that ratio of total lipid to nucleic acid is 10:1 , 20:1 , 30:1 , or 40:1 to 50:1 , or 10:1 to 20:1 , 30:1 , 40:1 or 50:1 , or any range bound by a pair of these ratios.68RECTIFIED SHEET (RULE 91 ) ISA / EP

[0202] Particular compositions for LNP precursors to tLNPs and tLNPs are disclosed in US Provisional Patent application Nos. 63 / 505,424 filed May 31 , 2023, 63 / 510,061 filed June 23, 2023, and 63 / 520,303 filed August 17, 2023, as well as PCT Application No. PCT / US2023 / 72426 (W02024040195A1 ) filed August 17, 2023, each of which is incorporated by reference in its entirety. LNP and tLNP compositions can include those of Table B In various embodiments, N / P can be from 3 to 9 or any integer-bound sub-range in that range or about any integer in that range.Table B. LNP CompositionsRECTIFIED SHEET (RULE 91 ) ISA / EPRECTIFIED SHEET (RULE 91) ISA / EPRECTIFIED SHEET (RULE 91) ISA / EP

[0203] In certain embodiments, the LNP composition is composition F5 CICL1 :DSPC:CHOL:DMG-PEG2000:DSPE-PEG2000-MAL [58:10:30.5:1 .4:0.1 ] or composition F9 CICL1 :DSPC:CHOL:DSG-PEG2000:DSPE-PEG2000-MAL [58:10:30.5:1.4:0.1 ]. The terminal group of the non-functionalized PEG can be methoxy. The N / P ratio (the ratio of positively-chargeable lipid amine (N = nitrogen) groups to negatively-charged nucleic acid phosphate (P) groups) can be 6. After initial LNP formation a SATA-modified anti-CD5 or anti-CD8 antibody can be reacted with72RECTIFIED SHEET (RULE 91 ) ISA / EPthe maleimide moiety to provide the final tLNP. In some embodiments, CD8-targeted tLNP in which the targeting moiety comprises a humanized anti-CD8 antigen binding domain have the lipid composition F9 found in Table B..

[0204] In some embodiments the antibody binding moiety is conjugated to the PEG moiety of the PEG-lipid through a thiol-modified lysine residue. In some embodiments, conjugation is through a cysteine residue in a native or added antibody sequence. In other embodiments, the conjugation is through a sortase A substrate sequence. In still other embodiments, conjugation is through a specific lysine residue (Lys248 or Lys288) in the Fc region, aided by a modified affinity reagent, for example, an AJICAP reagent. In certain embodiments of such embodiments, the humanized anti-CD8 antibody is linked to LNP by N-succinimidyl S-acetylthioacetate (SATA)- maleimide conjugation chemistry to form targeting LNP (tLNP). The antibody is beneficially first modified with SATA to introduce sulfhydryl groups at accessible lysine residues allowing conjugation to maleimide. (Some lysine residues can be buried in the interior of the protein and thus inaccessible to the SATA reagent.) Diabodies and F(ab’)2 can be conjugated by first partially reducing cystine bonds in the antibody with tris(2-carboxy)phosphine (TCEP) to generate thiol groups for conjugation through the maleimide moieties of the LNP.

[0205] In any of the aforementioned tLNP embodiments, certain embodiments include tLNPs encapsulating one or more mRNA(s) encoding an anti-CD19 such as SEQ ID NOS: 103-106, or an anti-CD20 CAR such as SEQ ID NOS: 107-11 1 as its payload. In any of the aforementioned tLNP embodiments, certain embodiments include tLNPs in which the targeting moiety comprises one of the herein disclosed humanized CT8 antigen binding domains, such as one that comprises a VL region having the amino acid sequence of SEQ ID NO: 1 13 and a VH region having the amino acid sequence of one of SEQ ID NOs: 1 14 or 1 15-117. In some such embodiments, the targeting moiety is a whole humanized anti-CD8 antibody comprising heavy chain with a silenced Fc region such as one having the amino acid sequence of SEQ ID NO: 1 18 or 119. In certain instances, the whole humanized anti-CD8 antibody comprises a heavy chain with a silenced Fc region comprises the sequence of CBD1033HC (SEQ ID NO: 120) and / or a light chain comprising the sequence of CBD1033LC (SEQ ID NO: 121). In any of the aforementioned tLNP embodiments encapsulating a herein73RECTIFIED SHEET (RULE 91 ) ISA / EPdisclosed mRNA, in certain embodiments the tLNP comprises as its targeting moiety an antibody or antigen binding portion thereof comprising one of the aforementioned one of the herein disclosed humanized CT8 antigen binding domains.(7) Compositions and Uses of mRNA

[0206] In certain other aspects, this disclosure provides a method of treating a disease or disorder comprising administering an improved mRNA disclosed herein or a pharmaceutical composition of an improved mRNA disclosed herein to a subject in need thereof. In alternative embodiments, a herein disclosed improved mRNA or pharmaceutical composition comprising the improved mRNA is used to transfect a cell ex vivo or extracorporeally, and the transiently transfected cell is administered to the subject in need thereof. In still further alternatives, the improved mRNA or a nucleic acid encoding the improved mRNA is packaged in a viral particle and the viral particle is used to transfect a cell ex vivo or extracorporeally, and the transduced cell is administered to the subject in need thereof. In various embodiments the disease or disorder is cancer or a genetic disease or an autoimmune or fibrotic disease. In some embodiments, the subject is administered a pharmacologically effective dose. In some embodiments, the subject is administered a therapeutically effective dose. In some embodiments, the subject is a human. In some embodiments, administration is by intravenous, subcutaneous, intraperitoneal, or intralesional infusion or injection.

[0207] The term “treating” or “treatment” broadly includes any kind of treatment activity, including mitigation, cure or prevention of disease, or an aspect thereof, in man or other animals, or any activity that otherwise affects the structure or any function of the body of man or other animals. Treatment activity includes administration of the medicaments, dosage forms, and pharmaceutical compositions described herein to a patient, especially according to the various methods of treatment disclosed herein, whether by a healthcare professional, the patient his / herself, or any other person.

[0208] Some embodiments of these methods of treatment comprise administration of an effective amount of an improved mRNA or a composition disclosed herein. Some instances relate to a therapeutically (or prophylactically) effective amount. A therapeutically effective amount is not necessarily a clinically effective amount, that is, while there can be therapeutic benefit as compared to no treatment, a method of74RECTIFIED SHEET (RULE 91 ) ISA / EPtreatment may not be equivalent or superior to a standard of care treatment existing at some point in time. Other instances relate to a pharmacologically effective amount, that is an amount or dose that produces an effect that correlates with or is reasonably predictive of therapeutic (or prophylactic) utility. As used herein, the term “therapeutically effective amount” is synonymous with “therapeutically effective dose” and means at least the minimum dose of an improved mRNA or composition disclosed herein necessary to achieve the desired therapeutic or prophylactic effect. Similarly, a pharmacologically effective dose means at least the minimum dose of an improved mRNA or composition disclosed herein necessary to achieve the desired pharmacologic effect. Some embodiments refer to an amount sufficient to prevent or disrupt a disease process, deplete or ablate a cell population, or to reduce the extent or duration of pathology. Some embodiments refer to a dose sufficient to reduce a symptom associated with the disease or condition being treated. An effective dosage or amount of an improved mRNA or a composition disclosed herein can readily be determined by the person of ordinary skill in the art considering all criteria (for example, the rate of excretion of the compound or composition used, the pharmacodynamics of the improved mRNA or composition used, the nature of the other compounds to be included in the composition, the particular route of administration, the particular characteristics, history and risk factors of the individual, such as, e.g., age, weight, general health and the like, the response of the individual to the treatment, or any combination thereof) and utilizing his / her best judgment on the individual’s behalf. Exemplary dosages are also disclosed in the Examples herein below.

[0209] In further embodiments, a tLNP can be administered in combination with the standard of care for a particular indication, such as corticosteroids (e.g., prednisone) for management of myositis or lupus nephritis. In certain cases, myositis is also treated with methotrexate, which can be combined with immunosuppressive agents (e.g., azathioprine, mycophenolate mofetil, tacrolimus), which are usually required in addition to corticosteroids. For membranous nephropathy, cyclical steroids and cyclophosphamide might be used in combination with tLNPs of this disclosure. In other cases, an anti-IL-6, such as tocilizumab, can also be used as a pretreatment or in combination with tLNPs of this disclosure. These combinations can be administered concurrently or sequentially.75RECTIFIED SHEET (RULE 91 ) ISA / EP

[0210] In some embodiments, the disease or disorder is an autoimmune disease. Examples of autoimmune disease include, without limitation, myocarditis, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, fibrosing alveolitis, multiple sclerosis, rheumatic fever, polyglandular syndromes, agranulocytosis, autoimmune hemolytic anemias, bullous pemphigoid, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis / polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, allergic responses, insulin-resistant diabetes, psoriasis, diabetes mellitus, Addison’s disease, Grave’s disease, diabetes, endometriosis, celiac disease, Chron’s disease, Henoch-Schonlein purpura, ulcerative colitis, Goodpasture's syndrome, thromboangitis obliterans, Sjogren's syndrome, aplastic anemia, rheumatoid arthritis, sarcoidosis, scleritis, a T cell-mediated autoimmunity or a B cell-mediated (antibody- mediated) autoimmunity, a B cell-mediated (antibody-mediated) autoimmune disease, necrotizing myopathy, chronic inflammatory demyelinating polyneuropathy (CIDP), neuromyelitis optica (NMO) myositis, neuromyelitis optica spectrum disorders, pemphigus vulgaris, systemic sclerosis, antisynthetase syndrome (idiopathic inflammatory myopathy), lupus nephritis, membranous nephropathy, severe combined immunodeficiency, Fanconi anemia, and vasculitis.

[0211] In some embodiments, the autoimmune disease is a T cell-mediated autoimmunity or a B cell-mediated autoimmunity. In some instances, the B cell- mediated (antibody-mediated) autoimmune disease is myositis (such as antisynthetase myositis), lupus nephritis, membranous nephropathy, systemic lupus erythematosus, neuromyelitis optica spectrum disorders, myasthenia gravis, pemphigus vulgaris, systemic sclerosis, antisynthetase syndrome (idiopathic inflammatory myopathy), multiple sclerosis, Sjorgen’s syndrome, IgA nephropathy, severe combined immunodeficiency, or Fanconi anemia.

[0212] In some embodiments, the disease or disorder is rejection of an allogeneic organ or tissue graft. Pre-existing antibodies and / or B cells, in their role as antigen presenting cells, can facilitate rapid immune rejection through known mechanisms hence depleting a large number of B cells may help prevent allograft rejection.76RECTIFIED SHEET (RULE 91 ) ISA / EP

[0213] In some embodiments, the disease or disorder is a cancer. Examples of cancers include, without limitation, carcinomas, sarcomas, and hematologic cancers. In some embodiments, the hematologic cancer is a lymphoma, leukemia, or myeloma. In some instances, the hematologic cancer is a B lineage or T lineage cancer. In some instances, the B lineage cancer is multiple myeloma, diffuse large B cell lymphoma, acute myeloid leukemia, Mantle Cell lymphoma, follicular lymphoma, B acute lymphoblastic leukemia, chronic lymphocytic leukemia, or myelodysplastic syndrome. In some embodiments, the cancer is a sarcoma. In some embodiments, the cancer is a carcinoma, such as breast cancer, colon cancer, ovarian cancer, lung cancer, testicular cancer, or pancreatic cancer. In some embodiments, the cancer is melanoma.

[0214] In some embodiments, the disease or disorder is a fibrotic disease or disorder. In some instances, the fibrotic disease is cardiac fibrosis, arthritis, idiopathic pulmonary fibrosis, and nonalcoholic steatohepatitis (also known as metabolic dysfunction-associated steatohepatitis). In other instances, the disorder involves tumor-associated fibroblasts.

[0215] In some embodiments, the disease or disorder is a genetic disease or disorder such as a monogenic genetic disease. In some instances, the genetic disease or disorder is a hemoglobinopathy, for example, sickle cell disease or p-thalassemia.

[0216] For the purposes of promoting an understanding of the principles of the disclosure, reference will now be made to embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure was thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

[0217] Various exemplary embodiments of compositions and methods according to this invention are now described in the following non-limiting Examples. The Examples are offered for illustrative purposes only and are not intended to limit the scope of this invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the following examples and fall within the77RECTIFIED SHEET (RULE 91 ) ISA / EPscope of the appended claims.Methods of producing autologous engineered cells

[0218] In accordance with the embodiments disclosed herein, methods for extracorporeally modifying immune cells or HSC from a subject are provided to produce a population of autologous engineered immune cells or HSC expressing an exogenous protein rendering the cells capable of eliciting a therapeutic response when reintroduced to the subject. In alternative embodiments, methods for modifying immune cells or HSC, ex vivo, from a subject are provided. Also provided are populations of autologous engineered immune cells or HSC produced using the methods and compositions disclosed herein.

[0219] In some embodiments, the methods for modifying cells described herein include supporting compositions such as lipid nanoparticles (LNPs) or targeted lipid nanoparticles (tLNPs) loaded with an internal payload that achieves selective reprogramming of blood cell populations outside of the body followed by their reinfusion into the subject, allowing for a rapid cell expansion-less process at bedside.

[0220] The term “modifying” or “modification” of cells refers, to reducing or inhibiting the expression or activity of a target gene(s) or gene product(s), increasing the expression or activity of a target gene(s) or gene product(s), or otherwise transfecting a cell with an exogenous nucleic acid molecule to effect a change in the activity or expression of a target gene(s) or gene product(s). For example, “modifying” or “to modify” can mean either reducing or inhibiting the expression or activity of, or alternatively increasing a (relevant or intended) expression or activity of, a target as measured using a suitable in vitro, cellular or in vivo assay (which will usually depend on the target involved), by at least 5%, at least 10%, at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more, compared to expression or activity of the target in the same assay under the same conditions prior to the modification. An “increase” or “decrease” refers to a statistically significant increase or decrease respectively. For the avoidance of doubt, an increase or decrease will be at least 5% relative to a reference, such as at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or more, up to and including at least78RECTIFIED SHEET (RULE 91 ) ISA / EP100% or more, in the case of an increase, for example, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9- fold, at least 10-fold, at least 50-fold, at least 100-fold, or more. “Modifying” can also include causing a cell to express a target gene or gene product at any level when that cell does not normally express that target gene or gene product or causing a change in the sequence, of a target gene or gene product that results in a change in activity, affinity, avidity, specificity and / or selectivity of the target gene or gene product. Again, as will be clear to the skilled person, such an action as an agonist or an antagonist can be determined in any suitable manner and / or using any suitable assay known or described herein (e.g., in vitro or cellular assay), depending on the target involved.

[0221] In some embodiments, the methods provided herein include one or more of the following steps: extracorporeally transfecting a population of leukocytes or HSCs within a blood sample obtained or collected from a subject with one or more nucleic acid engineering agents and / or conditioning agents by incubating or exposing the blood sample to tLNPs carrying the nucleic acid engineering agents and / or conditioning agents as part of an internal payload, and resuspending the transfected population of leukocytes or HSCs for infusion into the subject. The method may also include a step of washing the cells obtained from the blood sample prior to the extracorporeal transfection step. In such embodiments, the starting cell population (i.e., the cells that the engineering agent is added to) for producing the engineered cells includes all cell types found in the subject’s blood sample without performing any separation or apheresis step (e.g., cells in a whole blood sample). While it is also possible to include a step of separating or enriching a mixed population of immune cells (e.g., leukocytes or PBMCs), a subpopulation of immune cells (e.g., T cells, B cells, macrophages, monocytes, or NK cells) or HSCs from the blood sample, the use of tLNPs obviate the need to do so. In addition, in certain embodiments, the sample collected from the subject as described herein can be a bone marrow sample as an alternative to obtaining a blood sample if an HSC is the cell to be modified. However, administering G-CSF or G-CSF and plerixafor prior to blood collection (e.g., for approximately 11 hours prior to collection and / or separation) can facilitate mobilization of HSC into the blood from the bone marrow. As such, the blood sample and starting cell population can include mobilized HSC to be transfected by a tLNP or LNP79RECTIFIED SHEET (RULE 91 ) ISA / EPaccording to their degree of enrichment and in accordance with the methods described herein.

[0222] In certain embodiments, the methods provided herein include one or more of the following steps: separating a population of leukocytes or HSCs from a blood sample obtained or collected from a subject, washing the population of leukocytes or HSCs, extracorporeally transfecting the population of leukocytes or HSCs with one or more nucleic acid engineering agents and / or conditioning agents by incubating or exposing the population of leukocytes or HSCs to LNPs or tLNPs carrying the nucleic acid engineering agents and / or conditioning agents as part of an internal payload, and resuspending the transfected population of leukocytes or HSCs for infusion into the subject. In embodiments where HSCs are modified after separation, either an LNP or tLNP may be used. In embodiments where a population of leukocytes are separated from the blood sample, a tLNP may be used to target a specific type of cell for modification. In certain embodiments, the starting cell population (i.e., the cells that the engineering agent will be added to) for producing the engineered cells in accordance with the method above can be, for example, (i) a mixed population of apheresed cells that have a broad collection of white blood cells or (ii) a mixed population of peripheral blood mononuclear cells (PBMC) (e.g., apheresed cells run on gradient to remove granulocytes and other cells, thereby enriching for lymphocytes and monocytes). In certain embodiments, the starting cell population (i.e., the cells that the engineering agent will be added to) for producing the engineered cells in accordance with the method above can be, for example, a subpopulation of T cells (or “a T cell fraction”) or other immune cells (e.g., B cells, NK cells, macrophages, monocytes) obtained by a suitable separation technique, e.g., apheresis followed by beads using a positive or negative selection process. Although certain embodiments contemplate steps of separating or enriching a subpopulation of immune cells (e.g., a fraction of T cells, B cells, NK cells, macrophages, monocytes, or HSCs) from the mixed population of leukocytes in order to use an LNP, the use of tLNPs obviate the need to do so and are thus optional. In addition, in certain embodiments, the sample collected from the subject as described herein can be a bone marrow sample as an alternative to obtaining a blood sample if an HSC is the cell to be modified. However, obtaining HSC from blood can be facilitated by mobilizing the HSC, for example, by80RECTIFIED SHEET (RULE 91 ) ISA / EPadministering G-CSF or G-CSF and plerixafor prior to blood collection (e.g., for approximately 11 hours prior to collection and / or separation). As such, mobilized HSC can be transfected by a tLNP or LNP according to their degree of enrichment and in accordance with the methods described herein.

[0223] In some embodiments, the methods provided herein include the use of a tLNP in one or more of the following steps: extracorporeally transfecting a target cell within a blood sample obtained or collected from a subject with one or more nucleic acid engineering agents and / or conditioning agents by incubating or exposing the blood sample to tLNPs carrying the nucleic acid engineering agents and / or conditioning agents as part of an internal payload, and resuspending the transfected population of leukocytes or HSCs for infusion into the subject. The method may also include a step of washing the cells obtained from the blood sample prior to the extracorporeal transfection step. In such embodiments, the starting cell population (i.e., the cells that the engineering agent is added to) for producing the engineered cells is a mixed cell population and the targeted cell type is a particular cell type within the mixed cell population. Non-limiting examples of the mixed cell population include, but are not limited to, cells found in a whole blood sample; a population of leukocytes that has been apheresed or otherwise separated from a whole blood sample, a population of PBMCs that has been apheresed or otherwise separated from a whole blood sample or from a population of leukocytes, or any other separated population of mixed cells. Non-limiting examples of the target cells within the mixed population can be a T cells, B cells, NK cells, macrophages, monocytes, or HSC cells or any other cell found within the mixed population. In accordance with these embodiments, the target cell is targeted with a tLNP that binds the target cell with a targeting or binding moiety, as described in detail below. In some embodiments, administering G-CSF or G-CSF and plerixafor prior to blood collection (e.g., for approximately 1 1 hours prior to collection and / or separation) can facilitate mobilization of HSC into the blood from the bone marrow. As such, the blood sample and starting cell population can include mobilized HSC to be transfected by a tLNP or LNP according to their degree of enrichment and in accordance with the methods described herein. In other embodiments, the methods provided herein include the use of a non-targeted LNP in one or more of the aforementioned steps. In such embodiments, the method includes81RECTIFIED SHEET (RULE 91 ) ISA / EPa separation step to separate the target cells (e.g., T cells, B cells, NK cells, macrophages, monocytes, or HSC cells) from the blood sample obtained from the patient.

[0224] In an alternative embodiment, the methods provided herein include an ex vivo procedure that includes one or more of the following steps: transfecting, ex vivo, a population of leukocytes or HSCs within a fractionated or whole blood sample obtained or collected from a subject with one or more nucleic acid engineering agents and / or conditioning agents by incubating or exposing the blood sample to tLNPs carrying the nucleic acid engineering agents and / or conditioning agents as part of an internal payload, and resuspending the transfected population of leukocytes or HSCs for infusion into the subject. Ex vivo methods may also include a step of separating a population of leukocytes or HSCs from the blood sample prior to transfection. In certain embodiments, the ex vivo methods may also include additional steps that allow for additional cell culture and expansion and / or refrigerated or cryogenic storage and / or shipment over several days or longer.

[0225] In certain embodiments in which a population of leukocytes, PBMCs, or other subpopulation (e.g., T cells, B cells, NK cells, macrophages, monocytes, HSCs) is to be harvested or separated from the blood sample prior to incubating with an LNP or tLNP, any suitable separation technique may be used. For example, in some embodiments the separation technique is an apheresis technique and / or a gradient separation technique with a continuous or batched process. In some embodiments, the separation or apheresis technique is used to enrich or separate a particular population of cells from the blood sample. For example, in certain embodiments, the gradient separation or apheresis technique produces a population of PBMCs, or a population of HSCs.

[0226] In some embodiments, cells in a whole blood sample, a population of mixed leukocytes, or a population of mixed PBMCs is exposed and / or incubated with tLNPs for a defined interval of time in a vessel without additional cell selection / separation. In other embodiments, a specific subpopulation of cells (e.g., T cells, NK cells, B cells, macrophages, monocytes, or HSCs) within the whole blood sample or mixed cell population is exposed and / or incubated with tLNPs for a defined interval of time in a vessel without additional cell selection / separation. In other embodiments, a population82RECTIFIED SHEET (RULE 91 ) ISA / EPof separated HSCs is exposed and / or incubated with LNPs or tLNPs for a defined interval of time in a vessel. In other embodiments, a population of separated or enriched immune cells (e.g., T cells, NK cells, B cells, macrophages, monocytes) is exposed and / or incubated with LNPs or tLNPs for a defined interval of time in a vessel.

[0227] According to some embodiments, the interval of time for incubating or exposing cells in the whole blood sample, mixed population of leukocytes, mixed population of PBMCs, or enriched / separated subpopulation of cells (e.g., T cells, NK cells, B cells, macrophages, monocytes, or HSCs) is six hours or less to allow for a bedside extracorporeal procedure. For example, in some embodiments, the interval of time in an extracorporeal procedure includes, but is not limited to, 15 minutes, 30 minutes, 45 minutes, 1 hour, 1 .5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, or 6 hours. The vessel used can be any suitable culture vessel including, but not limited to a flask or a bag. In other embodiments, the interval of time can be longer than 6 hours if an ex vivo procedure is used as opposed to an extracorporeal procedure.

[0228] In some embodiments, the transfected cells are washed as needed before and / or after incubation, and are reinfused to the subject without cryopreservation or the need for off-site processing.

[0229] In some embodiments, the methods can be applied to selectively engineer T cells or T cell subsets to express an exogenous protein such as a reprogramming agent (e.g., a CAR, BiTE) encoded by an mRNA engineering agent; to express an encoded BRM (eBRM) encoded by an mRNA conditioning agent, or to express or carry gene editing components — without the need to physically separate or select those subsets — to deliver and redirect T cells against pathogenic cells or to deliver an edited cell to replace a pathogenic cell. In other embodiments, the methods can be applied to selectively engineer other immune cell subsets, such as B cells, natural killer (NK) cells or macrophages — without the need to physically separate or select those subsets — to express CARs to redirect the NK cells and macrophages against pathogenic cells. In other embodiments, the methods can be applied to selectively engineer hematopoietic stem cells to transiently express or carry gene editing components — without the need to physically separate or select those subsets.83RECTIFIED SHEET (RULE 91 ) ISA / EP

[0230] In some embodiments, an incubation step for extracorporeally transfecting cells in the whole blood sample, mixed population of leukocytes, mixed population of PBMCs, or enriched / separated subpopulation of cells (e.g., T cells, NK cells, B cells, macrophages, monocytes, or HSCs) with a composition comprising the internal payload is provided. In certain aspects, the incubation step is performed by or in an incubation component (for example, a container or conduit in which inculation takes place) of an extracorporeal system (as discussed below). An incubation step and component can also be employed in an ex vivo procedure.

[0231] In accordance with the embodiments described herein, a conditioning agent (i.e., a BRM) may be used before, during, and / or after the incubation of the immune cells with the LNPs or tLNPs for modulating the response of an immune cell. To that end, a BRM may in some embodiments be added directly to the cell culture medium or mixture in which the immune cell is being cultured or suspended. For example, in some embodiments, the BRM is added directly to the incubation mixture or medium during incubation of the immune cell. In other embodiments, the BRM may in some embodiments be added directly to the infusion solution during resuspension of the engineered just prior to reinfusion of the engineered cells.

[0232] In other embodiments, the payload carried within the LNPs or tLNPs includes a BRM and / or a nucleic acid encoding a BRMs resulting in expression of an eBRM by the transfected immune cells. In other embodiments, a tLNP includes a BRM that acts as a targeting or binding moiety. In certain of these embodiments, one or more BRMs and / or one or more nucleic acid molecules that encode an eBRMs, and / or one or more engineering agent(s) may all be delivered by an LNP or tLNP. For example, in some embodiments, the LNP or tLNP may include an internal payload that includes an engineering agent (e.g., a nucleic acid that encodes a CAR or a CRISPR nuclease) and one or more of the following: (i) a BRM; (ii) a nucleic acid molecule that encodes an eBRM (e.g., a nucleic acid molecule that encodes a cytokine), and / or (iii) a targeting or binding moiety attached to a tLNP. In some embodiments, the targeting or binding moiety is a BRM that serves to activate or otherwise modify T cell transcription, metabolism, or other functions that may in turn improve a T cell’s ability to process the tLNP.

[0233] The methods disclosed herein have several advantages. As compared to84RECTIFIED SHEET (RULE 91 ) ISA / EPelectroporation, these methods are gentler on cells and can reprogram selectively only desired cell populations owing to the binders on tLNPs. Moreover, the methods allow for cells in a whole blood sample or mixed cell subpopulation of the blood sample (e.g., leukocytes, PBMCs) to be transfected without the need to physically separate or enrich a particular targeted cell type or subpopulation (e.g., T cells, NK cells, B cells, macrophages, monocytes, or HSCs). In other words, the subpopulation being targeted may be an unselected subpopulation, thereby making the bedside procedure rapid and more efficient. As compared to viral transduction, with tLNP this method can be cell specific and only transfect a specific cell subset. In addition, to achieve selective reprogramming of cell populations, this method obviates the utilization of magnetic beads, similar devices or complex steps. Other methods to accomplish targeted transfection include the use of enriched populations of certain types of cells with an LNP, obviating the need for a tLNP. Further, as compared to methods widely used in the field, this method does not rely on central manufacturing, cell expansion, and product cell cryopreservation - with both logistical and clinical performance implications.

[0234] Other advantages include the use of cell activating / modulating agents (e.g., biological response modifiers) in vitro thereby limiting systemic exposure to those agents. For example, through using binders on tLNP that serve as biological response modifiers (or “BRMs”) or through using other BRMs added during the incubation process that activate or otherwise modify the engineered cells (by way of adding BRMs to the incubation mixture or resuspended cells prior to reinfusion, or by way of LNP or tLNP payload), the performance and safety of the process and clinical results may be enhanced.Methods of Delivering a Payload into a Cell

[0235] In some aspects, disclosed herein are methods of delivering a nucleic acid (or other negatively charged payload) into a cell comprising contacting the cell with tLNP of any of the foregoing aspects. Accordingly, each of the herein disclosed genera, subgenera, and or species of LNP or tLNP disclosed herein including those based on the inclusion or exclusion of particular lipids, particular lipid compositions, particular payloads, and / or particular targeting moieties can be used in defining the scope of the methods of delivering a payload to a cell. In some embodiments the85RECTIFIED SHEET (RULE 91 ) ISA / EPcontacting takes place ex vivo. In some embodiments, the contacting takes place extracorporeally.

[0236] By contacting the LNP or tLNP with the cells to be engineered outside of the body, exposure to the components of the LNP or tLNP can be greatly reduced or eliminated and consequently any toxicity associated with those components will be mitigated. However, the possibility of a toxicity, whether based on residual components of the LNP or tLNP or on the reprogramming agent and its effects, still exists. In various embodiments the toxicity can be described as an observable toxicity, a substantial toxicity, a severe toxicity, or an acceptable toxicity, or a dose-limiting toxicity (such as but not limited to a maximum tolerated dose (MTD)). By an observable toxicity it is meant that while a change is observed the effect is negligible or mild. By substantial toxicity it is meant that there is a negative impact on the patient’s overall health or quality of life. In some instances, a substantial toxicity may be mitigated or resolved with other ongoing medical intervention. By a severe toxicity it is meant that the effect requires acute medical intervention and / or dose reduction or suspension of treatment. The acceptability of a toxicity will be influenced by the particular disease being treated and its severity and the availability of mitigating medical intervention. In some embodiments, toxicity is confined (or largely confined) to an observable toxicity. In some embodiments, toxicity is confined (or largely confined) to grades of 0 or 1 or two.(1 ) Internal Payload

[0237] In some embodiments, the payload is a nucleic acid and the method of delivering is a method of transfecting. In some embodiments, the nucleic acid payload comprises an mRNA, circular RNA, self-amplifying RNA, or guide RNA. Nucleic acid structures and especially mRNA structures, as well as individual RNA molecules encoding particular polypeptides, that are well-adapted to delivery by LNP or tLNP are disclosed in U.S. Provisional Patent Application Number. 63 / 595,753 filed November 2, 2023, U.S. Provisional Patent Application Number. 63 / 61 1 ,092 filed December 15, 2023, and U.S. Provisional Patent Application Number 63 / 654,928, filed May 31 , 2024, each of which is incorporated by reference for all that it teaches about nucleic acid payloads for in vivo transfection and their design.86RECTIFIED SHEET (RULE 91 ) ISA / EP

[0238] In some embodiments, the payload comprises a nucleic acid encoding an immune receptor or immune cell engager and the method of delivering is also a method of reprogramming an immune cell. In some embodiments, the payload comprises a nucleic acid that encodes, or is, a BRM and the method of delivering is also a method of providing a conditioning agent. In various embodiments, the BRM or conditioning agent is a gamma chain receptor cytokine such as IL-2, IL-7, IL-15, IL- 15 / 15Ralpha, IL-21 ; an immune modulating cytokine such as IL-12, IL-18; a chemokine such as RANTES, IP10, MIG; or another BRM such as Flt3, GM-CSF, and G-CSF.

[0239] In some embodiments comprising multiple agents, the nucleic acid can be multicistronic. In other embodiments comprising multiple agents or components, each agent or component is encoded or contained is a separate nucleic acid species. In some embodiments involving multiple payload nucleic acid species, two or more nucleic acid species are packaged together in a single LNP species. In other embodiments, a subset of the payload nucleic acid species to be delivered, (e.g., a single nucleic acid species) is packaged in one LNP or tLNP species while another subset of the nucleic acid species is packaged in another LNP or tLNP species. The different (t)LNP species can differ by only the payload they contain. The different (t)LNP species may be combined in a single formulation or pharmaceutical composition for administration.

[0240] In some embodiments, the payload comprises a nucleic acid encoding a gene / genome editing enzyme and / or a guide RNA or other component of a gene / genome editing system and the method of delivering is also a method of reprogramming a cell. In some instances, the cell is an immune cell. In some instances, the cell is an HSC. In some instances, the cell is an MSG.Engineering agents and Reprogramming agents

[0241] In some embodiments, the LNPs and / or tLNPs for modulating an immune cell carry an internal payload that includes one or more engineering agents for engineering the immune cell or HSC. The term “engineering agent” refers to an agent used to modify or engineer a target immune cell or HSC harvested from or otherwise derived from the recipient. In some embodiments, an engineering agent comprises a87RECTIFIED SHEET (RULE 91 ) ISA / EPnucleic acid molecule. In some embodiments, the nucleic acid is messenger RNA (mRNA), self-replicating RNA, small interfering RNA (siRNA), micro RNA (miRNA), antisense oligonucleotide (ASO), DNA, DNA-RNA hybrid, or a gene editing component (e.g., a gRNA, a sgRNA, or a tracrRNA). In other embodiments, the engineering agent is a gene editing protein component that facilitates a gene editing process. In some embodiments, the engineering agent or agents used in the embodiments described herein include one or more nucleic acids that act to suppress or inhibit expression of one or more target genes. In such embodiments, the nucleic acid may be an siRNA), miRNA, or ASO designed to bind and suppress expression of a target gene.

[0242] In other embodiments, the nucleotide sequence encoding a CAR, TCR, immune cell engager, or other payload discussed in detail below may further comprise miRNA target sequences for inhibition of expression in non-targeted cells to further increase the specificity of targeted expression. For example, inclusion of the target sequence for miRNA122 will suppress translation of the mRNA in hepatocytes as will the target sights for miRNAs 96, 185, and 223. Similarly, inclusion of the target site for miRNA 142 will lead to suppression of translation in Kupffer cells. Expression can be similarly suppressed in myeloid and reticuloendothelial system cells by including a target domain for miRNAs 100, 125a, 125b, 146a, 146b, and 155. The distribution of miRNAs in human tissues is presented in Ludwig et al. Nucleic Acids Research, 44(8): 3865-3877, 2016, and downloadable from the Human miRNA tissue atlas at ccb- web.cs.uni-saarland.de / tissueatlas / , each of which is incorporated by reference in its entirety.

[0243] In some embodiments, the engineering agent or agents used in the embodiments described herein include one or more a nucleic acid molecule(s) that encode one or more reprogramming agents. As used herein, a “reprogramming agent” refers to an agent that, when expressed by or is present in a host or target cell, alters the antigenic specificity of the host or target cell or corrects / ameliorates a genetic defect. In accordance with the embodiments described herein, a reprogramming agent may include, but is not limited to, an immune receptor such as a chimeric antigen receptor (CAR) or a T cell receptor, or an immune cell engager such as a bispecific T cell engager (BITE), a bispecific killer cell engager (BIKE), a trispecific kill cell engager (TriKE), a dual affinity retargeting antibody (DART), a TRIDENT (linking two DART88RECTIFIED SHEET (RULE 91 ) ISA / EPunits or a DART unit and a Fab domain), a macrophage engager (e.g., BiME), an innate cell engager (ICE), and the like. Generally, T lymphocytes and natural killer (NK) cells can be reprogrammed with a TCR, a CAR, or an immune cell engager while only a CAR or an immune cell engager is used in reprogramming monocytes. In accordance with other embodiments described herein, a reprogramming agent may include, but is not limited to, a corrected gene, or non-mutant / healthy protein expressed by a corrected gene. In some embodiments, the reprogramming agent encodes or is a gene / genome editing component. In some embodiments, the gene / genome editing component is a guide RNA for an RNA-directed nuclease or other nucleic acid editing enzyme, clustered regularly interspaced short palindromic repeat RNA (crisprRNA), a trans-activating clustered regularly interspaced short palindromic repeat RNA (tracrRNA). In some embodiments, the gene / genome editing component is a nucleic acid-encoded enzyme, such as RNA-guided nuclease, a gene or base editing protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a transposase, or a CRISPR nuclease (e.g., Cas9 or Cas 12, etc.). In some embodiments, the gene / genome editing component is DNA to be inserted or that serves as a template in gene or genome editing for example a template for repair of a double-strand break.

[0244] In certain embodiments, a reprogramming agent may be transiently expressed by an engineered cell (i.e., a “transient reprogramming agent”). In such embodiments, the engineering agent or agents include one or more nucleic acid molecules (e.g., mRNA) that encode the reprogramming agent(s). In other embodiments, a reprogramming agent may be durably expressed by an engineered cell (i.e., a “durable reprogramming agent”). In such embodiments, the engineering agent or agents can include one or more nucleic acid molecules (e.g., DNA, RNA) that encode the reprogramming agent(s) or a portion thereof, one or more nucleic acid molecules (e.g., mRNA) that encode or are themselves a gene editing component to knock-in or otherwise generate the reprogramming agent.

[0245] In some embodiments, the nucleic acid comprises at least one modified nucleoside. In some embodiments, the modified nucleoside is pseudouridine, N1 - methylpseudouridine, 5-methylcytosine, 5-methyluridine, N6-methyladenosine, 2’-O- methyluridine, or 2-thiouridine.89RECTIFIED SHEET (RULE 91 ) ISA / EP

[0246] The payload may include one more nucleic acid species. Thus, in some embodiments, the payload may include a single engineering agent species that encodes a single type of reprogramming agent (e.g., an mRNA that encodes a CAR, a TCR, or an immune cell engager). In other embodiments, the payload includes multiple types of engineering agents that encode and / or give rise to multiple types of reprogramming agents. In some embodiments involving multiple payload nucleic acids, two or more nucleic acid species are packaged together in a single LNP species. In other embodiments, a subset of the payload nucleic acid species to be delivered, (e.g, a single nucleic acid species) are packaged in one LNP species while another subset of the nucleic acid species are packaged in another LNP species. The different LNP species can differ by only the payload they contain. The different LNP species may be combined in a single composition for administration.

[0247] In some embodiments, the ratio of total lipid to nucleic acid of the LNPs and / or tLNP is between about 10:1 and about 50:1 by weight. In some embodiments, the ratio of total lipid to nucleic acid is about 10:1 , about 20:1 , about 30:1 , about 40:1 , about 50:1 , or in any range bound by any pair of these ratios.Chimeric antigen receptors (CARs)

[0248] In some embodiments, the reprogramming agent is a chimeric antigen receptor (CAR). CARs (also known as chimeric immunoreceptors, chimeric T cell receptors, or artificial T cell receptors) are receptor proteins that have been engineered to give host cells or target cells (e.g., T cells, B cells, NK cells, macrophages, monocytes) the new ability to target a specific protein. The receptors are chimeric because they combine both antigen-binding and T cell activating functions into a single receptor. The LNPs and / or tLNPs of the present technology may be used to deliver their payload comprising the mRNA to immune cells (e.g., T cells, B cells, NK cells, macrophages, monocytes) extracorporeally or ex vivo so that the immune cells would express the CAR encoded by the mRNA after reinfusion, thereby engineering of the immune cells for use in cell-based therapies against various target antigens (e.g., cancer antigens, viral antigens, antigens of autoimmunity-mediating T or B cells, antigens of fibrogenic cells, etc.).

[0249] In some embodiments, the CAR may comprise an extracellular binding90RECTIFIED SHEET (RULE 91 ) ISA / EPdomain that specifically binds a target antigen, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CAR may further comprise one or more additional elements, including one or more signal peptides, one or more extracellular hinge domains, and / or one or more intracellular costimulatory domains. Domains may be directly adjacent to one another, or there may be one or more amino acids linking the domains. The signal peptide can be derived from an antibody, a TCR, CD8 or other type 1 membrane proteins, preferably a protein expressed in a T or other immune cell. The transmembrane domain can be one associated with any of the potential intracellular domains or from another type 1 membrane protein, such as TCR alpha, beta, or zeta chain, CD3 epsilon, CD4, CD8, or CD28, amongst other possibilities known in the art. The transmembrane domain can further comprise a hinge domain located between the extracellular binding domain and the hydrophobic membrane-spanning region of the transmembrane domain. In some but not all embodiments, the hinge domain and transmembrane domain are contiguous sequences in the same source protein. The intracellular signaling domain can be derived from the CD3 zeta chain, FcyRIII, FcsRI, or an immunoreceptor tyrosine- based activation motif (ITAM) bearing cytoplasmic domain, amongst other possibilities known in the art. The intracellular costimulatory domain can be derived from CD27, CD28, 4-1 BB, or DAP12, amongst other possibilities known in the art.

[0250] In certain embodiments, CARs are used to treat a disease or condition associated with a target cell that expresses the antigen targeted by the CAR. For example, in some embodiments, an anti-CD19 or anti-CD20 CAR can be used to target and treat B cell malignancies or B cell-mediated autoimmune conditions or diseases. In other embodiments, an anti-FAP CAR can be used to target and treat solid tumors or fibrosis (e.g., cardiac fibrosis). Examples of CARs that may be used in accordance with the embodiments described herein include, but are not limited to those disclosed in US 7,446,190 (anti-CD19), US 10,287,35 (anti-CD19), US2021 / 0363245 (anti-CD19 and anti-CD20), US 10,543,263 (anti-CD22), US 10,426,797 (anti-CD33), US 10,844,128 (anti-CD123), US 10,428,141 (anti-ROR1), and US2021 / 0087295 (anti-FAP), each of which is incorporated by reference for all that it teaches about CAR structure and function generically and with respect to the CAR’s antigenic specificity and target indications to the extent that it is not inconsistent91RECTIFIED SHEET (RULE 91 ) ISA / EPwith the present disclosure.

[0251] The nucleotide sequence encoding a CAR may be derived from a mammalian sequence, for example, a mouse sequence, a primate sequence, a human sequence, or combinations thereof. In the cases where the nucleotide sequence encoding a CAR is non-human, the sequence of the CAR may be humanized. The nucleotide sequence encoding a CAR may also be codon-optimized for expression in a mammalian cell, for example, a human cell. In any of these embodiments, the nucleotide sequence encoding a CAR may be at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the nucleotide sequences disclosed herein. The sequence variations may be due to codon-optimalization, humanization, restriction enzyme-based cloning scars, and / or additional amino acid residues linking the functional domains.

[0252] In certain embodiments, the CAR may comprise a signal peptide at the N- terminus. Non-limiting examples of signal peptides include CD8a signal peptide, IgK signal peptide, and granulocyte-macrophage colony-stimulating factor receptor subunit alpha (GMCSFR-o, also known as colony stimulating factor 2 receptor subunit alpha (CSF2RA)) signal peptide, and variants thereof, the amino acid sequences of which are provided in Table 1 below.Table 1 . Exemplary sequences of signal peptides

[0253] In certain embodiments, the CAR may comprise an extracellular binding domain, also referred to as a binder. In certain embodiments, the extracellular binding domain may comprise one or more antibodies specific to one target antigen or multiple target antigens. The antibody may be an antibody fragment, for example, an scFv, or a single-domain antibody fragment, for example, a VHH. In certain embodiments, the scFv may comprise a heavy chain variable region (VH) and a light chain variable region92RECTIFIED SHEET (RULE 91 ) ISA / EP(VL) of an antibody connected by a linker. The VH and the VL may be connected in either order, i.e., VH-linker-Vi_ or Vi_-linker-VH. Non-limiting examples of linkers include Whitlow linker, (G4S)n (n can be a positive integer, e.g., 1 , 2, 3, 4, 5, 6, etc.) linker, and variants thereof. In certain embodiments, the antigen may be an antigen that is exclusively or preferentially expressed on tumor cells, or an antigen that is characteristic of an autoimmune or inflammatory disease. Exemplary target antigens include, but are not limited to, B cell maturation agent (BCMA), CA9, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD70, CD133, CD174, CD274, CD276, CEACAM5, CLL1 , CSPG4, EGFR, EGFRvlll, EPCAM, EPHA2, ERBB2, FAP, FOLH1 , FOLR1 , GD2, GPC3, GPNMB, IL1 RAP, IL3BA, IL13RA2, Kappa, KDR, L1 CAM, Lambda, MET, MS4A1 , MSLN, MUC1 , NCAM1 , PDCD1 , PSCA, ROR1 , SDC1 , SLAMF7, TEM1 , TNFRSF8, TNFRSF17, ULBP1 ULBP2, G-protein coupled receptor family C group 5 member D (GPRC5D) (associated with leukemias); CS1 / SLAMF7, CD38, CD138, GPRC5D, TACI, and BCMA (associated with myelomas); GD2, HER2, EGFR, EGFRvlll, B7H3, PSMA, PSCA, CAIX, CD171 , CEA, CSPG4, EPHA2, FAP, FRa, IL-13Ra, Mesothelin, MUC1 , MUC16, and R0R1 (associated with solid tumors). In any of these embodiments, the extracellular binding domain of the CAR can be codon-optimized for expression in a host cell or have variant sequences to increase functions of the extracellular binding domain. In accordance with the advantages of the aspects and embodiments disclosed herein are independent of the specificity of the binder. As such, the disclosed aspects and embodiments are generally agnostic to binding specificity. In certain embodiments, certain species may require a particular binding specificity.

[0254] In certain embodiments, the CAR may comprise a hinge domain, also referred to as a spacer. The terms "hinge" and "spacer" may be used interchangeably in the present disclosure. Non-limiting examples of hinge domains include CD8a hinge domain, CD28 hinge domain, lgG4 hinge domain, lgG4 hinge-CH2-CH3 domain, and variants thereof, the amino acid sequences of which are provided in Table 2 below.Table 2. Exemplary sequences of hinge domains93RECTIFIED SHEET (RULE 91 ) ISA / EP

[0255] In certain embodiments, the CAR may comprise a transmembrane domain. In other embodiments, the transmembrane domain may comprise a transmembrane region of CD3C, CD3s, CD3y, CD35, CD4, CD5, CD8a, CD8f3, CD9, CD16, CD22, CD28, CD32, CD33, CD34, CD37, CD40, CD45, CD64, CD80, CD86, OX40 / CD134, 4-1 BB / CD137, CD40L / CD154, FAS, FcsRIy, FGFR2B, TCRa, TCR|3, or VEGFR2, or a functional variant thereof, including the human versions of each of these sequences. Table 3 provides the amino acid sequences of a few exemplary transmembrane domains.Table 3. Exemplary sequences of transmembrane domains

[0256] In certain embodiments, the CAR may comprise an intracellular signaling domain. In certain embodiments, the intracellular signaling domain may comprise one or more signaling domains selected from B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1 , B7- H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5,94RECTIFIED SHEET (RULE 91 ) ISA / EPICOS / CD278, PD-1 , PD-L2 / B7-DC, PDCD6, 4-1 BB / TNFSF9 / CD137, 4-1 BB Ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF R / TNFRSF13C, CD27 / TNFRSF7, CD27 Ligand / TNFSF7, CD30 / TNFRSF8, CD30 Ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 Ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR Ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, Lymphotoxin-alpha / TNF[3, OX40 / TNFRSF4, 0X40 Ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNFa, TNF RII / TNFRSF1 B, 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, SLAM / CD150, CD2, CD7, CD53, CD82 / Kai-1 , CD90 / Thy1 , CD96, CD160, CD200, CD300a / LMIR1 , HLA Class I, HLA-DR, Ikaros, Integrin alpha 4 / CD49d, Integrin alpha 4 beta 1 , Integrin alpha 4 beta 7 / LPAM-1 , LAG-3, TCL1 A, TCL1 B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function associated antigen-1 (LFA-1 ), NKG2C, CD3^, an immunoreceptor tyrosine- based activation motif (ITAM), CD27, CD28, 4-1 BB, CD134 / 0X40, CD30, CD40, PD- 1 , ICOS, lymphocyte function-associated antigen-1 (LFA-1 ), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and a functional variant thereof including the human versions of each of these sequences. In some embodiments, the intracellular signaling domain comprises one or more signaling domains selected from a CD3^ domain, an ITAM, a CD28 domain, 4-1 BB domain, or a functional variant thereof. Table 4 provides the amino acid sequences of a few exemplary intracellular signaling domains. 4-1 BB, also known as CD137, transmits a potent costimulatory signal to T cells, promoting differentiation and enhancing longterm survival of T lymphocytes. CD28 is another co-stimulatory molecule on T cells. CD3 zeta ( £ ) associates with T cell receptors (TCRs) to produce a signal and contains immunoreceptor tyrosine-based activation motifs (ITAMs). The CD3 £ signaling domain refers to amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit an initial signal necessary for T cell activation. In certain embodiments, as in the case of tisagenlecleucel as described below, the CD3£ signaling domain of SEQ ID NO:13 may have a mutation, e.g., a glutamine (Q) to lysine (K) mutation, at amino acid position 14 (see SEQ ID NO:14).95RECTIFIED SHEET (RULE 91 ) ISA / EPTable 4. Exemplary sequences of intracellular signaling domains

[0257] In certain embodiments where the in vivo engineering agent (e.g., mRNA) encodes two or more CARs, the two or more CARs may comprise the same functional domains, or one or more different functional domains, as described. For example, the two or more CARs may comprise different signal peptides, extracellular binding domains, hinge domains, transmembrane domains, and / or intracellular signaling domains, in order to minimize the risk of recombination due to sequence similarities. Or, alternatively, the two or more CARs may comprise the same functional domains. In the cases where the same domain(s) and / or backbone are used, it is optional to introduce codon divergence at the nucleotide sequence level to minimize the risk of recombination. Overall, the risk of recombination is much reduced clinically when the in vivo engineering agent is mRNA, however, recombination can still be a concern in propagation of the plasmid used in production of the mRNA by in vitro transcription.

[0258] In some embodiments, the CAR may be a commercially or otherwise available CAR, for example, a CD19 CAR, CD20 CAR, CD22 CAR, or BCMA CAR, and in these embodiments, the CAR contains an extracellular binding domain that96RECTIFIED SHEET (RULE 91 ) ISA / EPspecifically binds the specific antigen after which they are named.

[0259] CD19 CAR. In some embodiments, the CAR is a CD19 CAR, and in certain of these embodiments, the CD19 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD19, a hinge domain, a transmembrane domain, and / or an intracellular signaling domain as described herein in tandem.

[0260] In some embodiments, the extracellular binding domain of the CD19 CAR is specific to CD19, for example, human CD19. The extracellular binding domain of the CD19 CAR can be codon-optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an antigen binding portion of an immunoglobulin molecule, for example, an scFv.

[0261] In some embodiments, the extracellular binding domain of the CD19 CAR comprises an scFv derived from the FMC63 monoclonal antibody (FMC63), which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of FMC63 connected by a linker. FMC63 and the derived scFv have been described in Nicholson et al., Mol. Immun. 34(16-17):1157-1165 (1997) and PCT Application Publication Nos. WO2018213337 and WO2015187528, the entire contents of each of which are incorporated by reference herein. In some embodiments, the amino acid sequences of the entire FMC63-derived scFv (also referred to as FMC63 scFv) and its different portions are provided in Table 5 below. In some embodiments, the CD19- specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the CD19-specific scFv may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 17-19 and 22-24. In some embodiments, the CD19-specific scFv may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 17-19. In some embodiments, the CD19-specific scFv may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 22-24. In any of these embodiments, the CD19-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions or comprising a sequence that is at least 80%97RECTIFIED SHEET (RULE 91 ) ISA / EPidentical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the CD19 CAR comprises the one or more CDRs as described herein.

[0262] In some embodiments, the linker linking the VH and the VL portions of the scFv is a Whitlow linker having an amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the Whitlow linker may be replaced by a different linker, for example, a (G4S)n (n can be a positive integer, e.g., 1 , 2, 3, 4, 5, or 6) linker. In some embodiments, the linker is a 3xG4S linker having an amino acid sequence set forth in SEQ ID NO: 26, which gives rise to a different FMC63-derived scFv having an amino acid sequence set forth in SEQ ID NO: 25. In certain of these embodiments, the CD19- specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 25.Table 5. Exemplary sequences of anti-CD19 scFv and componentsRECTIFIED SHEET (RULE 91 ) ISA / EP99RECTIFIED SHEET (RULE 91) ISA / EP

[0263] In some embodiments, the extracellular binding domain of the CD19 CAR is derived from an antibody specific to CD19, including, for example, SJ25C1 (Bejcek et al., Cancer Res. 55:2346-2351 (1995)), HD37 (Pezutto et al., J. Immunol. 138(9):2793-2799 (1987)), 4G7 (Meeker et al., Hybridoma 3:305-320 (1984)), B43 (Bejcek (1995)), BLY3 (Bejcek (1995)), B4 (Freedman et al., 70:418-427 (1987)), B4 HB12b (Kansas & Tedder, J. Immunol. 147:4094-4102 (1991 ); Yazawa et al., Proc. Natl. Acad. Sci. USA 102:15178-15183 (2005); Herbst et al., J. Pharmacol. Exp. Then 335:213-222 (2010)), BU12 (Callard et al., J. Immunology, 148(10): 2983-2987 (1992)), and CLB-CD19 (De Rie Cell. Immunol. 118:368-381 (1989)). In any of these embodiments, the extracellular binding domain of the CD19 CAR can comprise or consist of the VH, the Vi_, and / or one or more CDRs of any of the antibodies.

[0264] In some embodiments, the CD19 CAR is a commercially available embodiment of CD19 CAR. Non-limiting examples of commercially available embodiments of T cells expressing CD19 CARs include tisagenlecleucel, lisocabtagene maraleucel, axicabtagene ciloleucel, and brexucabtagene autoleucel.

[0265] In some embodiments, the CD19 CAR is the CD19 CAR expressed by tisagenlecleucel or portions thereof. Tisagenlecleucel comprises a CD19 CAR with the following components: CD8a signal peptide, FMC63 scFv (VL-3XG4S linker-Vn), CD8a hinge domain, CD8a transmembrane domain, 4-1 BB signaling domain, and CD3^ signaling domain. The nucleotide and amino acid sequence of the CD19 CAR in tisagenlecleucel are provided in Table 6, with annotations of the sequences provided in Table 7.

[0266] In some embodiments, the CD19 CAR is the CD19 CAR expressed by lisocabtagene maraleucel or portions thereof. Lisocabtagene maraleucel comprises a CD19 CAR with the following components: GMCSFR-a or CSF2RA signal peptide, FMC63 scFv (Vi_-Whitlow linker-Vn), lgG4 hinge domain, CD28 transmembrane domain, 4-1 BB signaling domain, and CD3^ signaling domain. The nucleotide and amino acid sequence of the CD19 CAR in lisocabtagene maraleucel are provided in Table 6, with annotations of the sequences provided in Table 8.

[0267] In some embodiments, the CD19 CAR is the CD19 CAR expressed by axicabtagene ciloleucel or portions thereof. Axicabtagene ciloleucel comprises a100RECTIFIED SHEET (RULE 91 ) ISA / EPCD19 CAR with the following components: GMCSFR-a or CSF2RA signal peptide, FMC63 scFv (Vi_-Whitlow linker-Vn), CD28 hinge domain, CD28 transmembrane domain, CD28 signaling domain, and CD3 signaling domain. The nucleotide and amino acid sequence of the CD19 CAR in axicabtagene ciloleucel are provided in Table 6, with annotations of the sequences provided in Table 9.

[0268] In some embodiments, the CD19 CAR is the CD19 CAR expressed by brexucabtagene autoleucel or portions thereof. Brexucabtagene autoleucel comprises a CD19 CAR with the following components: GMCSFR- a signal peptide, FMC63 scFv, CD28 hinge domain, CD28 transmembrane domain, CD28 signaling domain, and CD3^ signaling domain.Table 6. Exemplary sequences of CD19 CARs*101RECTIFIED SHEET (RULE 91 ) ISA / EP102RECTIFIED SHEET (RULE 91) ISA / EP103RECTIFIED SHEET (RULE 91) ISA / EP104RECTIFIED SHEET (RULE 91) ISA / EP105RECTIFIED SHEET (RULE 91) ISA / EP* Nucleotide sequences are presented here (and throughout) as DNA sequences; t should be understood as u for corresponding RNA sequences.Table 7. Annotation of tisagenlecleucel CD19 CAR sequences106RECTIFIED SHEET (RULE 91 ) ISA / EPTable 8. Annotation of lisocabtagene maraleucel CD19 CAR sequencesTable 9. Annotation of axicabtagene ciloleucel CD19 CAR sequences

[0269] CD20 CAR. In some embodiments, the CAR is a CD20 CAR, and in certain of these embodiments, the CD20 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD20, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and / or an intracellular signaling domain as described herein in tandem. CD20 is an antigen found on the surface of B cells as early as the pro-B phase and progressively at increasing levels until B cell maturity, as well as on the cells of most B-cell neoplasms. CD20 positive cells are also sometimes found in cases of Hodgkin's disease, myeloma, and thymoma.

[0270] In some embodiments, the extracellular binding domain of the CD20 CAR is specific to CD20, for example, human CD20. The extracellular binding domain of107RECTIFIED SHEET (RULE 91 ) ISA / EPthe CD20 CAR can be codon-optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv.

[0271] In some embodiments, the extracellular binding domain of the CD20 CAR is derived from an antibody specific to CD20, including, for example, Leu16, IF5, 1 .5.3, rituximab, obinutuzumab, ibritumomab, ofatumumab, tositumumab, odronextamab, veltuzumab, ublituximab, and ocrelizumab. In some embodiments, the CD20 CAR is derived from a CAR specific to CD20, including, for example, MB-106 (Fred Hutchinson Cancer Research Center, see Shadman et al., Blood 134(Suppl.1 ):3235 (2019)) UCART20 (Cellectis, www.cellbiomedgroup.com), or C-CAR066 (Cellular Biomedicine Group, see Liang et al., J. Clin. Oncol. 39(15) suppl:2508 (2021 )). In any of these embodiments, the extracellular binding domain of the CD20 CAR can comprise or consist of the VH, the Vi_, and / or one or more CDRs of any of the antibodies.

[0272] In some embodiments, the extracellular binding domain of the CD20 CAR comprises an scFv derived from the Leu16 monoclonal antibody, which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of Leu16 connected by a linker. See Wu et aL, Protein Engineering. 14(12):1025-1033 (2001). In some embodiments, the linker is a (G4S)n (n can be a positive integer, e.g., 1 , 2, 3, 4, 5, or 6) linker, for example, a 3xG4S linker. In other embodiments, the linker is a Whitlow linker as described herein. In some embodiments, the amino acid sequences of different portions of the entire Leu16-derived scFv (also referred to as Leu 16 scFv) and its different portions are provided in Table 10 below. In some embodiments, the CD20-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO: 33, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 33. In some embodiments, the CD20-specific scFv may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 35-37 and 39-40. In some embodiments, the CD20-specific scFv may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 35-37. In some108RECTIFIED SHEET (RULE 91 ) ISA / EPembodiments, the CD20-specific scFv may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 39-40. In any of these embodiments, the CD20-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the CD20 CAR comprises the one or more CDRs as described herein.Table 10. Exemplary sequences of anti-CD20 scFv and components109RECTIFIED SHEET (RULE 91 ) ISA / EP

[0273] CD22 CAR. In some embodiments, the CAR is a CD22 CAR, and in certain of these embodiments, the CD22 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD22, a hinge domain, a transmembrane domain, and / or an intracellular signaling domain as described herein in tandem. CD22 is a transmembrane protein found mostly on the surface of mature B cells that functions as an inhibitory receptor for B cell receptor (BCR) signaling. CD22 is expressed in 60-70% of B cell lymphomas and leukemias (e.g., B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma) and is not present on the cell surface in early stages of B cell development or on stem cells.

[0274] In some embodiments, the extracellular binding domain of the CD22 CAR is specific to CD22, for example, human CD22. The extracellular binding domain of the CD22 CAR can be codon-optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv.

[0275] In some embodiments, the extracellular binding domain of the CD22 CAR is derived from an antibody specific to CD22, including, for example, SM03, inotuzumab, epratuzumab, moxetumomab, and pinatuzumab. In any of these embodiments, the extracellular binding domain of the CD22 CAR can comprise or noRECTIFIED SHEET (RULE 91 ) ISA / EPconsist of the VH, the Vi_, and / or one or more CDRs of any of the antibodies.

[0276] In some embodiments, the extracellular binding domain of the CD22 CAR comprises an scFv derived from the m971 monoclonal antibody (m971 ), which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of m971 connected by a linker. In some embodiments, the linker is a (G4S)n (n can be a positive integer, e.g., 1 , 2, 3, 4, 5, or 6) linker, for example, a 3xG4S linker. In other embodiments, the Whitlow linker may be used instead. In some embodiments, the amino acid sequences of the entire m971 -derived scFv (also referred to as m971 scFv) and its different portions are provided in Table 1 1 below. In some embodiments, the CD22-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO: 41 , or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 41. In some embodiments, the CD22-specific scFv may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 43-45 and 47-49. In some embodiments, the CD22-specific scFv may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 43-45. In some embodiments, the CD22-specific scFv may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 47-49. In any of these embodiments, the CD22-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the CD22 CAR comprises the one or more CDRs as described herein.

[0277] In some embodiments, the extracellular binding domain of the CD22 CAR comprises an scFv derived from m971 -L7, which is an affinity matured variant of m971 with significantly improved CD22 binding affinity compared to the parental antibody m971 (improved from about 2 nM to less than 50 pM). In some embodiments, the scFv derived from m971 -L7 comprises the VH and the VL of m971 -L7 connected by a (G4S)n (n can be a positive integer, e.g., 1 , 2, 3, 4, 5, or 6) linker, for example, a 3xG4S linker. In other embodiments, the Whitlow linker may be used instead. In some embodiments, inRECTIFIED SHEET (RULE 91 ) ISA / EPthe amino acid sequences of the entire m971 -L7-derived scFv (also referred to as m971 -L7 scFv) and its different portions are provided in Table 11 below. In some embodiments, the CD22-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO: 50, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 50. In some embodiments, the CD22-specific scFv may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 52-54 and 56-58. In some embodiments, the CD22-specific scFv may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 52-54. In some embodiments, the CD22-specific scFv may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 56-58. In any of these embodiments, the CD22-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the CD22 CAR comprises the one or more CDRs as described herein.Table 11 . Exemplary sequences of anti-CD22 CAR, scFv, and components112RECTIFIED SHEET (RULE 91 ) ISA / EP113RECTIFIED SHEET (RULE 91) ISA / EP

[0278] BCMA CAR. In some embodiments, the CAR is a BCMA CAR, and in certain of these embodiments, the BCMA CAR may comprise a signal peptide, an114RECTIFIED SHEET (RULE 91 ) ISA / EPextracellular binding domain that specifically binds BCMA, a hinge domain, a transmembrane domain, and / or an intracellular signaling domain as described herein in tandem. BCMA is a tumor necrosis family receptor (TNFR) member expressed on cells of the B cell lineage, with the highest expression on terminally differentiated B cells or mature B lymphocytes. BCMA is involved in mediating the survival of plasma cells for maintaining long-term humoral immunity. The expression of BCMA has been recently linked to a number of cancers, such as multiple myeloma, Hodgkin's and nonHodgkin’s lymphoma, various leukemias, and glioblastoma.

[0279] In some embodiments, the extracellular binding domain of the BCMA CAR is specific to BCMA, for example, human BCMA. The extracellular binding domain of the BCMA CAR can be codon-optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain.

[0280] In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv. In some embodiments, the extracellular binding domain of the BCMA CAR is derived from an antibody specific to BCMA, including, for example, belantamab, erlanatamab, teclistamab, LCAR-B38M, and ciltacabtagene. In any of these embodiments, the extracellular binding domain of the BCMA CAR can comprise or consist of the VH, the VL, and / or one or more CDRs of any of the antibodies.

[0281] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from C11 D5.3, a Mouse monoclonal antibody as described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013). See also PCT Application Publication No. WO2010104949. The C11 D5.3-derived scFv may comprise the heavy chain variable region (VH) and the light chain variable region ( L) of C1 1 D5.3 connected by the Whitlow linker, the amino acid sequences of which is provided in Table 12 below. In some embodiments, the BCMA-specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 59, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 59. In some embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs:115RECTIFIED SHEET (RULE 91 ) ISA / EP61 -63 and 65-67. In some embodiments, the BCMA-specific extracellular binding domain may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 61-63. In some embodiments, the BCMA-specific extracellular binding domain may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 65-67. In any of these embodiments, the BCMA-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the BCMA CAR comprises the one or more CDRs as described herein.

[0282] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from another Mouse monoclonal antibody, C12A3.2, as described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013) and PCT Application Publication No. WO2010104949, the amino acid sequence of which is also provided in Table 12 below. In some embodiments, the BCMA-specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 68, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 68. In some embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 70-72 and 74-76. In some embodiments, the BCMA-specific extracellular binding domain may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 70-72. In some embodiments, the BCMA-specific extracellular binding domain may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 74-76. In any of these embodiments, the BCMA-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the BCMA CAR116RECTIFIED SHEET (RULE 91 ) ISA / EPcomprises the one or more CDRs as described herein.

[0283] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from a Mouse monoclonal antibody with high specificity to human BCMA, referred to as BB2121 in Friedman et al., Hum. Gene Ther. 29(5):585- 601 (2018). See also, PCT Application Publication No. WO2012163805.

[0284] In some embodiments, the extracellular binding domain of the BCMA CAR comprises single variable fragments of two heavy chains (VHH) that can bind to two epitopes of BCMA as described in Zhao et al., J. Hematol. Oncol. 11 (1):141 (2018), also referred to as LCAR-B38M. See also, PCT Application Publication No. WO201 8028647.

[0285] In some embodiments, the extracellular binding domain of the BCMA CAR comprises a fully human heavy-chain variable domain (FHVH) as described in Lam et al., Nat. Commun. 11 (1 ):283 (2020), also referred to as FHVH33. See also, PCT Application Publication No. WO2019006072. The amino acid sequences of FHVH33 and its CDRs are provided in Table 12 below. In some embodiments, the BCMA- specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 77 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 77. In some embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 78-80. In any of these embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the BCMA CAR comprises the one or more CDRs as described herein.

[0286] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from CT103A (or CAR0085) as described in U.S. Patent No. 11 ,026,975 B2, the amino acid sequence of which is provided in Table 12 below.117RECTIFIED SHEET (RULE 91 ) ISA / EPIn some embodiments, the BCMA-specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 81 , or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 81 . In some embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 83-85 and 87-89. In some embodiments, the BCMA-specific extracellular binding domain may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 83-85. In some embodiments, the BCMA-specific extracellular binding domain may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 87-89. In any of these embodiments, the BCMA-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the BCMA CAR comprises the one or more CDRs as described herein.

[0287] Additionally, CARs and binders directed to BCMA have been described in U.S. Application Publication Nos. 2020 / 0246381 A1 and 2020 / 0339699 A1 , the entire contents of each of which are incorporated by reference herein.Table 12. Exemplary sequences of anti-BCMA binder and components118RECTIFIED SHEET (RULE 91 ) ISA / EP119RECTIFIED SHEET (RULE 91) ISA / EP120RECTIFIED SHEET (RULE 91) ISA / EP121RECTIFIED SHEET (RULE 91) ISA / EP

[0288] In some embodiments, the BCMA CAR is a commercially available embodiment of BCMA CAR, including, for example, idecabtagene vicleucel (ide-cel, also called bb2121) or portions thereof. Idecabtagene vicleucel comprises a BCMA CAR with the following components: the BB2121 binder, CD8a hinge domain, CD8a transmembrane domain, 4-1 BB signaling domain, and CD3^ signaling domain.T cell receptors (TCRs)

[0289] In some embodiments, the engineering agent is a nucleic acid that encodes a T cell receptor (TCR), and the reprogramming agent is the TCR. TCRs specific for a target antigen (e.g., a cancer antigen, a viral antigen, an antigen of autoimmunitymediating T or B cells, an antigen of fibrogenic cells, etc.) may be identified, sequenced, modified, and / or introduced into an immune cell (e.g., a CD8+ T cell) by LNPs and / or tLNPs of the present technology. In some embodiments, the TCR may comprise an alpha chain and a beta chain (most common), or a delta chain and a gamma chain (less common). Each chain may have a variable (V) region and a constant (C) region. The variable region recognizes and binds the target antigen usually presented by an MHC complex of an antigen-presenting cell (APC). The122RECTIFIED SHEET (RULE 91 ) ISA / EPvariable region of each of the chains has three CDRs: CDR1 , CDR2, and CDR3, while CDR3 is thought to be the main CDR responsible for recognizing processed antigens. The nucleotide sequence encoding a TCR may be derived from a mammalian sequence, for example, a mouse sequence, a primate sequence, a human sequence, or combinations thereof. In the cases where the nucleotide sequence encoding a TCR is non-human, the sequence of the TCR may be humanized. The nucleotide sequence encoding a TCR may also be codon-optimized for expression in a mammalian cell, for example, a human cell.Immune cell engagers

[0290] In some embodiments, the engineering agent is a nucleic acid that encodes an immune cell engager, and the reprogramming agent is the immune cell engager, including, for example, a T cell engager (e.g., BiTE, DART), an NK cell engager (e.g., BIKE, TriKE), a macrophage engager (e.g., BIME), and an innate cell engager (e.g., ICE). BiTEs are a class of artificial bispecific monoclonal antibodies that direct a host’s immune system, more specifically the T cells’ cytotoxic activity, against cells bearing target antigens (e.g., cancer cells). BiTEs usually are fusion proteins having two single-chain variable fragments (scFvs) of different antibodies in a single peptide chain. One of the scFvs binds a T cell via the CD3 receptor, and the other to a target cell (e.g., a cancer cell) via a target cell-specific antigen. Unlike CARs and TCRs, BiTEs are secreted molecules. BiTEs can effectively redirect T cells whether secreted from T cells or other cells, whether due to co-targeting, designed targeting to non-T cells, or off-target delivery (such as to hepatocytes). Further immune cell engagers can be constructed by replacing the anti-CD3 moiety with a binding moiety specific for another immune cell surface molecule as disclosed herein to engage a different segment of the immune system. For example, using an anti-CD8 binding moiety instead of anti-CD3 in a BiTE-like protein would generate an immune cell engager limited to engaging just the CD8+ subset of T cells while using an anti-CD2 binding moiety would lead to engagement of both T cells and NK cells. A DART is a heterodimer of two scFv-like polypeptides, one containing the Vi_of a first antibody and the VH of a second antibody, and the other containing the VH of the first antibody and the VL of the second antibody, with the VH and VL regions within each chain connected by a short diabody-like linker to promote interchain pairing. A typical DART also has a123RECTIFIED SHEET (RULE 91 ) ISA / EPC-terminal interchain disulfide bond. The two parental antibodies of a DART have different specificities, typically for a target antigen and CD3, so that the DART is bispecific and serves the same function as a BiTE. BiKEs and TriKEs are analogous to BiTEs but replace the anti-CD3 binding domain with an anti-CD16 binding domain so that instead of engaging T cells they engage NK cells. They may also contain in IL- 15 linker between the scFv units instead of or in addition to the anti-CD16 binding domain to provide further NK activation. An ICE is a tetravalent, bispecific engager comprising an anti-CD16A diabody (e.g., a dimer of anti-CD16A scFv) and an antitumor antigen diabody (e.g., a dimer of anti-tumor antigen scFv) tandemly connected by peptide linkers (e.g., (Gly-Gly-Ser)s linker). The anti-CD16A diabody portion has high binding affinity to CD16A expressed on NK cells and macrophages, while the anti-tumor diabody portion specifically recognizes a surface antigen expressed by a tumor of interest. Thus, the ICE functions to connect innate immune cells (e.g., NK cells, macrophages) and their target tumor cells, thereby activating the killing of the tumor cells through processes such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). See, e.g., Ellwanger et aL, MAbs (2019) 1 1 (5):899-918; Reusch et aL, MAbs (2014) 6(3):727-738, the entire contents of each of which is incorporated by reference herein. In some embodiments, the ICE is one currently in clinical trial for treatment of various cancers, including, for example, AFM13 (targeting CD30-positive lymphomas) and AFM24 (targeting EGFR- expressing tumors). In certain of these embodiments, the anti-CD16A diabody comprises an scFv that binds efficiently and stably to a unique epitope of the CD16A receptor on NK cells and macrophages without competition from the body’s own circulating serum IgG, which has the following amino acids sequences of the VH and VL regions, respectively:QVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPS GGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADY WGQGTLVTVSS (VH region, SEQ ID NO:90);SYVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPS GIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVL (VL region, SEQ ID NO:91 ).

[0291] We refer to an immune cell engager encoded in nucleic acid packaged in a124RECTIFIED SHEET (RULE 91 ) ISA / EPtLNP as a targeted immune cell engager of TICE.Gene editing components and systems

[0292] In some embodiments, the engineering agent is a gene editing component or a nucleic acid molecule that encodes or is itself a gene editing component. Examples of gene editing components that are themselves nucleic acid molecules include, but are not limited to, a guide RNA (gRNA), a prime editing guide RNA (pegRNA), a single guide RNA (sgRNA), a clustered regularly interspaced short palindromic repeat (CRISPR) RNA (crRNA), a trans-activating CRISPR RNA (tracrRNA), and / or a DNA or RNA molecule to be inserted or serve as a template for double-strand break (DSB) repair at a specific genomic locus (i.e., a repair template). In some embodiments, the gene editing component may be an RNA containing the targeting domain(s) of a gene expression cassette (e.g., a promoter, transgene, and polyA signal) in RNA format. In some embodiments, the gene editing component that is itself a nucleic acid molecule can be a DNA containing a DNA transposon to be transposed and inserted into the genome, or an RNA molecule to be used as a reverse transcriptase template to generate DNA and insert the DNA into the genome at a specific genomic locus. The DNA can also be provided by an AAV viral vector in accordance with some embodiments.

[0293] Examples of gene editing components that are encoded by the nucleic acid include, but are not limited to, an mRNA encoding an RNA-guided nuclease, a gene or base editing protein, a prime editing protein, a Gene Writer protein (e.g., a modified or modularized non-long terminal repeat (LTR) retrotransoposon), a retrotransposase, an RNA writer, a zinc finger nuclease (ZFN), a transcription activator- 1 ike effector nuclease (TALEN), a meganuclease, a transposase, a retrotransposon, a reverse transcriptase (e.g., M-MLV reverse transcriptase), a nickase or inactive nuclease (e.g., Cas9, nCas9, dCas9), a DNA recombinase, a CRISPR nuclease (e.g., Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-1 1 ), a DNA nickase, a Cas9 nickase (e.g., D10A or H840A), or any fusion or combination thereof.

[0294] In some embodiments, one or more gene editing components comprise a gene editing system that is used in a gene editing technique. For example, in one embodiment, the gene editing technique is a conventional CRISPR / Cas technique that125RECTIFIED SHEET (RULE 91 ) ISA / EPuses a gene editing system that includes, but is not limited to, one or more of the following gene editing components: a CRISPR nuclease (e.g., Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-1 1), a crRNA, a tracrRNA, an sgRNA, and / or a repair template. In another embodiment, the gene editing technique is a gene writer technique that uses a gene editing system that includes, but is not limited to, one or more of the following gene editing components: a Gene Writer protein and / or DNA or RNA template. In another embodiment, the gene editing technique is a prime editor technique that uses a gene editing system that includes, but is not limited to, one or more of the following gene editing components: a pegRNA, a fusion protein that includes a cas9 H840A nickase fused to a Maloney Murine Leukemia Virus (M-MLV) reverse transcriptase), and an sgRNA. Other gene editing techniques that can be used in accordance with the embodiments described herein include techniques that use a gene editing system that includes, but is not limited to, one or more of the following gene editing components: a TALEN, a Zinc finger nuclease, and / or a meganuclease.

[0295] In certain embodiments, one or more gene editing components or a gene editing system may be used to insert (or “knock-in”) a DNA molecule that encodes a reprogramming agent. For example, in some embodiments, one or more gene editing components may include components used in a conventional CRISPR / Cas technique (e.g., CRISPR / Cas9), a gene writer technique, or a modified prime editor technique wherein the one or more gene editing components or gene editing system is codelivered with an engineering agent (e.g., DNA or mRNA) that encodes a reprogramming agent (e.g., a CAR, TCR, or immune cell engager) for permanent integration of the reprogramming agent.

[0296] In certain embodiments, one or more gene editing components can be used to insert or repair a mutated or deleted gene that is associated with a genetic disorder or disease. In certain embodiments, the one or more gene editing components may include components used in a conventional CRISPR / Cas technique (e.g., CRISPR / Cas9), a gene writer technique, or a modified prime editor technique. For example, the gene editing components or system can be used to replace or repair a mutated HBB gene in a patient suffering from sickle cell disease or Beta-thalassemia (e.g., replace the entire HBB gene or a point mutation responsible for causing sickle cell disease). Other mutated genes that are responsible for genetic diseases that can126RECTIFIED SHEET (RULE 91 ) ISA / EPbe repaired by gene editing components used in methods described herein include, but are not limited to: mutated SERPINA1 gene, which causes Alpha-1 antitrypsin deficiency (AATD); mutated TTR gene, which causes amyloid transthyretin (ATTR) amyloidosis; mutated CFTR gene, which causes cystic fibrosis (CF); mutated G6PC gene, which causes glycogen storage disease la (GSD la); mutated F8 (type A) or F9 (type B) genes, which cause hemophilia types A and B; mutated HTT gene, which causes Huntington's disease; mutated IDUA (type 1) or IDS (type 2) genes, which cause Mucopolysaccharidosis (MPS) types I and II; mutated AGXT gene, which causes primary hyperoxaluria (PH) type 1 ; mutated IL2RG, JAK3, or ZAP70 genes, which cause severe combined immunodeficiency (SCID); mutated USH2A genes, which causes usher syndrome type 2a.

[0297] In certain embodiments, a DNA molecule to be inserted or serve as a template for double-strand break (DSB) repair at a specific genomic locus plate may be co-delivered with the LNP and / or tLNP payload by co-delivering the one or more gene editing components with the DNA molecule. In other embodiments, a DNA molecule to be inserted or serve as a template for double-strand break (DSB) repair at a specific genomic locus plate may be delivered by a delivery vehicle that is separate from the LNPs and / or tLNP payload. For example, in some embodiments, LNPs and / or tLNPs and a population or immune or HSC cells may be co-incubated with a viral vector, where the viral vector is designed to deliver a template for doublestrand break (DSB) repair. In certain embodiments, the viral vector may be an AAV vector, an adenoviral vector, a lentiviral vector, a herpes simplex virus vector, or a vaccinia viral vector.Conditioning agents

[0298] In some embodiments, the LNPs and / or tLNPs for modulating an immune cell carry an internal payload that includes one or more conditioning agents for engineering the immune cell or HSC. The term “conditioning agent” refers to a BRM that alters the environment so that engineering efficiency or the therapeutic and / or physiologic effect of the reprogramming agent, is improved. For example, a BRM can refer to a substance that activates or otherwise modifies the response of an immune cell, or that modifies the immune response upon reinfusion of engineered cells expressing the BRM. In another example, a BRM may promote the immune response127RECTIFIED SHEET (RULE 91 ) ISA / EPto a diseased tissue (e.g., tumor tissue) or suppress or inhibit regulatory responses that would otherwise diminish or block the immune response to diseased tissue. Such a response may occur upon reinfusion of cells engineered to express a BRM.

[0299] The BRM can be provided as part of the internal payload as a protein or peptide, or as an mRNA molecule that encodes the BRM. A BRM that has been expressed by an mRNA provided as part of the internal payload may be referred to herein as an “encoded BRM” or “eBRM.” A BRM may also be delivered as a protein separately from the payload according to some embodiments, as discussed in detail below.

[0300] Accordingly, in some embodiments, the LNPs and / or tLNPs used in accordance with the embodiments described herein carry an internal payload that includes a BRM. In some embodiments, the LNPs and / or tLNP payload comprises a nucleic acid (e.g., an mRNA) that encodes a BRM. When administered systemically to a patient, BRMs can be prohibitively toxic. As such, the use of BRMs in an in vitro setting such as in the extracorporeal methods described herein is advantageous because it allows for activation of cells needed to engineer the applicable cells, but avoids the toxic effects of administering such BRMs systemically. Thus, as discussed below, the cells targeted for modification can be co-treated with potent BRMs without the concern of causing deleterious effects in a patient. As discussed herein, certain embodiments include administering, in vitro, potent BRMs including, but not limited to, gamma chain receptor cytokines, T 1 -biasing cytokines (e.g. IL-12 and family).

[0301] In some embodiments a BRM provided as part of the internal payload and / or an eBRM expressed by an engineered immune cell transfected with nucleic acid(s) provided as part of the internal payload include cytokines, chemokines, and immune checkpoint inhibitors. Cytokines and chemokines are generally receptor agonists, as are some immune checkpoint inhibitors, but some BRMs and / or eBRMs are antagonists or otherwise block receptor activity, most notably many immune checkpoint inhibitors. Consequently, the various BRMs and / or eBRMs referred to herein may be substituted with alternative compounds which are also ligands (agonists or antagonists, as appropriate) of the BRM’s and / or eBRMs receptor.

[0302] In some embodiments, the BRM and / or eBRMs comprises a cytokine. In128RECTIFIED SHEET (RULE 91 ) ISA / EPsome embodiments, the cytokine may be a natural cytokine including, but not limited to, recombinant IL-2 (rlL-2), IL-2, IL-7, IL-12, IL-15, IL-18, or IL-21. In other embodiments, the BRM and / or eBRMs is a cytokine receptor ligand that is not a natural cytokine. In some embodiments, the cytokine receptor ligand may be a cytokine receptor agonist, a cytokine fragment capable of binding its corresponding receptor, an aptamer, or a peptide-mimetic agent. In some embodiments, the BRM and / or eBRMs is an IL-2 receptor or a CD25 (a component of the IL-2 receptor) ligand, an IL- 7 receptor ligand, an IL-12 receptor ligand, an IL-15 receptor ligand, an IL-18 receptor ligand, or an IL-21 receptor ligand.

[0303] In other embodiments, the BRM and / or eBRMs is a chemokine. In some embodiments, the chemokine is selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CCL11 , CXCL1 , CXCL2, CXCL8, CXCL9, CXCL10, and CXCL1 1. In other embodiments, the BRM and / or eBRMs is a chemokine receptor ligand that is not a natural chemokine. In some embodiments, the chemokine receptor ligand may be a chemokine receptor agonist, a chemokine fragment capable of binding its corresponding receptor, an aptamer, or a peptide-mimetic agent. In some embodiments, the BRM and / or eBRMs is a CCL2 receptor ligand, a CCL3 receptor ligand, a CCL4 receptor ligand, a CCL5 receptor ligand, a CCL11 receptor ligand, a CXCL1 receptor ligand, a CXCL2 receptor ligand, a CXCL8 receptor ligand, a CXCL9 receptor ligand, a CXCL10 receptor ligand, or a CXCL11 receptor ligand.

[0304] In other embodiments, the BRM and / or eBRMs is an immune checkpoint inhibitor. Upon reinfusion of an engineered cell that expresses an immune checkpoint inhibitor, inhibition of the immune checkpoint molecules can bring about multiple effects including reducing the number or functionality of Treg cells mediating immune suppressive effects, activation or functional enablement of immune cells (such as T effector cells), promotion of broader immune responses including epitope spreading, and facilitation of redistribution of immune cells to organs or tissues of interest such as the tumor microenvironment. Thus, immune checkpoint inhibitors constitute means for broadening an immune response, means for mobilizing immune effector cells in one or more of the disclosed mobilization facets, as appropriate, and means for reducing immune suppression. Accordingly, an autologous engineered immune cell that expresses an immune checkpoint inhibitor will provide the benefits of activation or129RECTIFIED SHEET (RULE 91 ) ISA / EPfunctional enablement of the reprogrammed cells, a reduction in the number of Treg cells opposing the activity of the reprogrammed cells, recruitment of broader immunity including epitope spreading and mobilization of immune cells into tissues or organs of interest (where the cells targeted by the reprogramming agent reside). In some embodiments, the immune checkpoint inhibitor can block inhibitory signaling (as with anti-CTLA-4) or give a co-stimulatory signal (as with anti-4-1 BB (CD137)). In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a CD166 inhibitor, a cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibitor, a glucocorticoid-induced TNFR-related protein (GITR) inhibitor, a lymphocyteactivation gene 3 (LAG-3) inhibitor, an 0X40 inhibitor, a programed death-1 (PD-1 ) inhibitor, a TIGIT (T cell immunoreceptor with Ig and ITIM domains) inhibitor, or a T cell immunoglobulin and mucin-domain containing-3 (TIM-3) inhibitor.

[0305] In some embodiments, the immune checkpoint inhibitor is a programed death-1 (PD-1 ) inhibitor. Programed death-1 (PD-1 ) is a checkpoint protein on T cells. Antibodies against both PD-1 and its binding partner programmed death-ligand 1 (PD- L1 ) have been used clinically as immune checkpoint inhibitors (PD-1 blockade). Nonlimiting examples of monoclonal antibodies (mAbs) that target PD-1 / PD-L1 include: the anti-PD-1 mAbs nivolumab (OPDIVO®, Bristol-Myers Squibb), pembrolizumab (KEYTRUDA®, Merck & Co.), cemiplimab-rwlc (LIBTAYO®, Regeneron Pharmaceuticals), and the anti-PD-L1 mAbs durvalumab (MEDI4736, IMFINZI™, Medimmune), atezolizumab (MPDL3280A; TECENTRIQ®, Hoffmann-La Roche), avelumab (BAVENCIO®, EMD Serono), and BMS- 936559 (Bristol-Myers Squibb). These may be referred to as means for PD-1 blockade, means for inhibiting PD-1 / PD- L1 binding, or means for immune checkpoint inhibition.

[0306] In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor, and the CTLA-4 inhibitor is an antibody or an antigen-binding portion thereof specific to CTLA-4. CTLA-4 is an immune checkpoint molecule expressed on the surface of CD4+ and CD8+ T cells and on CD25+, FOXP3+ T regulatory (Treg) cells. Non-limiting examples of anti-CTLA-4 antibodies include vudalimab (a PD-1 / CTLA-4 bispecific antibody), nurulimab, cadonilimab (a PD-1 / CTLA-4 bispecific antibody), ipilimumab (YERVOY®), tremelimumab (Medimmune), and any combinations, antigen-binding portions, or fragments thereof.130RECTIFIED SHEET (RULE 91 ) ISA / EP

[0307] In some embodiments, the immune checkpoint inhibitor is a TIM-3 (T-cell immunoglobulin and mucin-domain containing-3) inhibitor. TIM-3 is a molecule selectively expressed on IFN-y-producing CD4+T helper 1 (Th1 ) and CD8+T cytotoxic 1 (Tc1 ) T cells. Non-limiting, exemplary antibodies to TIM-3 are disclosed in U.S. Patent Application Publication 20160075783 which is incorporated by reference herein for all it contains regarding anti-TIM-3 antibodies that is not inconsistent with the present disclosure. Other anti-TIM-3 antibodies include TSR-022 (Tesaro). These may be referred to as means for inhibiting TIM-3, or means for immune checkpoint inhibition.

[0308] In some embodiments, the immune checkpoint inhibitor is a LAG-3 (lymphocyte-activation gene 3; CD223) inhibitor. LAG-3 negatively regulates cellular proliferation, activation, and homeostasis of T cells, in a similar fashion to CTLA-4 and PD- 1 and plays a role in T reg suppressive function. Non-limiting exemplary antibodies to LAG- 3 include GSK2831781 (GlaxoSmithKline), relatlimab (BMS-986016, Bristol- Myers Squibb), and the antibodies disclosed in U.S. Patent Application Publication 2011 / 0150892 which is incorporated by reference herein for all it contains regarding anti-LAG-3 antibodies that is not inconsistent with the present disclosure. These may be referred to as means for inhibiting LAG-3, or means for immune checkpoint inhibition.

[0309] In some embodiments, the immune checkpoint inhibitor is a TIGIT (T cell immunoreceptor with Ig and ITIM domains) inhibitor. TIGIT is an immunoreceptor inhibitory checkpoint that has been implicated in tumor immunosurveillance. It competes with immune activating receptor CD226 (DNAM-1 ) for the same set of ligands: CD155 (PVR or poliovirus receptor) and CD112 (Nectin-2 or PVRL2). Anti- TIGIT antibodies have demonstrated synergy with anti-PD-1 / PD-L1 antibodies in pre- clinical models. Tiragolumab (Roche), etigilimab (OncoMed), vibostolimab (MK-7684; Merck), and EOS- 448 (iTeos Therapeutics) are non-limiting examples of an anti-TIGIT antibodies. They may be referred to as means for inhibiting TIGIT, or means for immune checkpoint inhibition.

[0310] In some embodiments, the immune checkpoint inhibitor is a GITR (glucocorticoid-induced TNFR-related protein) promotes effector T cell functions and inhibits suppression of immune responses by regulatory T cells. As with OX- 40,131RECTIFIED SHEET (RULE 91 ) ISA / EPmentioned above, the checkpoint inhibitor is an agonist of the target, in this case GITR. An agonistic antibody, TRX518 is currently undergoing human clinical trials in cancer. While by itself it may not be sufficient to mediate substantial clinical improvement in advanced cancer, combination with other checkpoint inhibition, such as PD-1 blockade was promising.

[0311] Other immune checkpoint inhibitor targets include, but are not limited to, B- and T-cell attenuator (BTLA), CD40, CD122, inducible T-cell costimulator (ICOS), 0X40 (tumor necrosis factor receptor superfamily, member 4), Siglec-15, B7H3, CD137 (4-1 BB; as with CD40 and 0X40, checkpoint inhibition is accomplished with an agonist) and others are potentially useful in the disclosed methods. Several anti- 0X40 agonistic monoclonal antibodies are in early phase cancer clinical trials including, but not limited to, MEDI0562 and MED16469 (Medimmune), MOXR0916 (Genetech), and PF-04518600 (Pfizer); as is an anti-ICOS agonistic antibody, JTX- 2011 (Jounce Therapeutics). Anti-CD40 agonistic antibodies under clinical investigation include dacetuzumab, CP-870,893 (selicrelumab), and Chi Lob 7 / 4. Anti- siglec-15 antibodies are also known (see, for example, US 8,575,531 ). Anti-CD137 agonistic antibodies include, but are not limited to, urelumab and utomilumab. Additionally, CD122 has been targeted in cancer clinical trials with bempegaldesleukin (NKTR-214, a pegyltated-IL-2 used as a CD122-biased agonist). B7H3 has been targeted both for immune checkpoint inhibition and as a tumor antigen with reagents such as enoblituzumab,131l-omburtamab,177Lu-DTPA-omburtamab,1311-8H9,124l- 8H9, MCG018, and DS-7300a. These may be referred to as means for immune checkpoint inhibition or means for inhibiting (or activating (agonizing), as appropriate) their respective targets. Some embodiments can specifically include or exclude one or more immune checkpoint inhibitor.(2) LNP portion

[0312] Nucleic acid-based therapeutics (e.g., DNA, siRNA, mRNA, miRNA, antisense oligonucleotides (ASO), and self-replicating RNA), for example, the engineering agents described herein, have significant systemic and cellular barriers for efficient delivery into cells. They are highly susceptible to degradation by nucleases in the body and are at risk of rapid clearance by kidneys. Additionally, their negative charge and hydrophilic nature inhibits efficient delivery across the cell membrane.132RECTIFIED SHEET (RULE 91 ) ISA / EPNanoparticle based delivery systems especially those comprising lipids, polymers and / or lipopolymers help overcome these delivery challenges. Amongst these, lipid nanoparticle (LNP)-based systems are most advanced for RNA delivery with 3 currently approved drugs (Onpattro, and two mRNA based COVID-19 vaccines, Comirnaty and Spikevax). Targeted delivery may be accomplished through a specific binding interaction between the binding moiety on the surface of the nanoparticles ( / .e., in the form of a tLNP) and a ligand on the surface of a targeted cell.

[0313] In some embodiments, the LNP portion of the LNP and / or tLNPs may comprise a permanently charged or ionizable cationic lipid, a phospholipid, cholesterol, and / or a PEG-lipid. See, e.g., Hou et al., Nature Reviews Materials (2021 ) 6:1078-1094, which is incorporated by reference for all that it teaches about delivery of nucleic acids by LNPs. Other examples of lipid-based nanoparticles include cationic liposomes or cationic lipoplexes typically comprising a cationic lipid (permanently charged amino lipid) and a co-lipid such as a phospholipid or cholesterol and in some cases a PEG-lipid or LNPs comprising of lipidoids (lipid-like material). Examples of polymer-based nanoparticles for nucleic acid delivery include linear cationic polymers such as polyamino acid-based polymers (e.g., poly-L-Lysine (PLL), polyarginine, polyhistidine), polyethyleneimine (PEI), natural polymers such as chitosan and hyaluronic acid, branched polymer bases systems such as polyamidoamine (PAMAM) dendrimers, and poly-beta amino esters (PBAE). Examples of lipopolymeric or lipidpolymer hybrid nanoparticles include nanoparticles with a polymeric core (e.g., polylactic-co-glycolic acid (PLGA)) and a lipid shell (see, e.g., Byun et al., BioChip J. 2022:1 -18, which is incorporated by reference for all that it teaches about delivery of nucleic acids by nanoparticles including polymer and hybrid nanoparticles that is not inconsistent with the present disclosure). Other such hybrid systems include dendrimeric systems like Janus dendrimers that consist of a lipophilic region of linear or branched alkyl chains and polar ionizable amino heads (Zhang et aL, J. Am. Chem. Soc. (2021 ) 143:12315-12327, which is hereby incorporated by reference for all that it teaches about delivery of nucleic acids by dendrimer nanoparticles).

[0314] In some embodiments, the LNP portion of the LNPs and / or tLNPs may comprise one or more of an ionizable cationic lipid, a phospholipid, a sterol, a co-lipid, and a polyethylene glycol (PEG)-lipid, or combinations thereof, and a functionalized133RECTIFIED SHEET (RULE 91 ) ISA / EPPEG-lipid conjugated to a binding moiety. As used herein, functionalized PEG-lipid refers to a PEG-lipid in which the PEG moiety has been derivatized with a chemically reactive group (such as, maleimide, NHO ester, Cys, azide, alkyne, and the like) that can be used for conjugating a targeting or binding moiety to the PEG-lipid, and thus, to the LNP comprising the PEG-lipid. The functionalized PEG-lipid can be reacted with a binding moiety after the LNP is formed, so that the binding moiety is conjugated to the PEG portion of the lipid.

[0315] In some embodiments, with respect to the LNP or tLNP, the molar ratio of the lipids is 40 to 60 mol% ionizable cationic lipid: 7 to 30 mol% phospholipid: 20 to 45 mol% sterol: 1 to 30 mol% co-lipid, if present: 0 to 5 mol% PEG-lipid: 0.1 to 5 mol% functionalized PEG-lipid, when present.

[0316] In some embodiments, with respect to the LNP or tLNP, the ionizable cationic lipid comprises a lipid with a measured pKa in the LNP of 6 to 7, facilitating ionization in the endosome. In some embodiments, the ionizable cationic lipid has a c-pKa from 8 to 1 1 and cLogD from 9 to 18 or 11 -14. In some embodiments, the ionizable cationic lipid has branched structure to give the lipid a conical rather than cylindrical shape. Suitable ionizable cationic lipids are known to those of skill in the art, including those disclosed in US20130022665, US20180170866, US20160095924, US20120264810, US9,061 ,063, US9,433,681 US9,593,077, US9,642,804US10,196,637, US10,207,010 US10383952, US10,426,737 US1 1 ,066,355 US1 1 ,246,993, WO2012170952, WO2021026647, WO2017004143, and WO201 7075531 , each of which is incorporated by reference for all that it teaches about ionizable cationic lipids that is not inconsistent with the present disclosure.

[0317] In some aspects, provided is a method of making a LNPs and / or tLNP comprising rapid mixing of an aqueous solution of a payload (e.g., a nucleic acid) and an alcoholic solution of the lipids. In some embodiments, the lipid mixture includes functionalized PEG-lipid, for later conjugation to a binding moiety. In other embodiments, the functionalized PEG-lipid is inserted into and LNP subsequent to initial formation of an LNP from other components. In either embodiment, the binding moiety is conjugated to functionalized PEG-lipid after the functionalized PEG-lipid containing LNP is formed. Protocols for conjugation can be found, for example, in Parhiz et al., J. Controlled Release (2018) 291 :106-115, and Tombacz et al., Molecular134RECTIFIED SHEET (RULE 91 ) ISA / EPTherapy (2021 ) 29(1 1 ):3293-3304, each of which is incorporated by reference for all that it teaches about conjugation of PEG-lipids to binding moieties that is not inconsistent with the present disclosure.

[0318] After the LNP are formed they are diluted with buffer, for example phosphate, HEPES, or Tris, in a pH range of 6 to 8.5 to reduce the alcohol (ethanol) concentration. The diluted LNP are purified either by dialysis or ultrafiltration or diafiltration using tangential flow filtration (TFF) against a buffer in a pH range of 6 to 8.5 (for example, phosphate, HEPES, or Tris) to remove the alcohol. Alternatively, one can use size exclusion chromatography. Once the alcohol is completely removed the buffer is exchanged with like buffer containing a cryoprotectant (for example, glycerol or a sugar such as sucrose, trehalose, or mannose). The LNP is concentrated to a desired concentrated, followed by 0.2 pm filtration through, for example, a polyethersulfone (PES) filter and filled into glass vials, stoppered, capped, and stored frozen. In alternative embodiments, a lyoprotectant is used and the LNP lyophilized for storage instead of as a frozen liquid. Further methodologies for making LNPs can be found, for example, in US20200297634, US20130115274, and WO2017 / 048770, each of which is incorporated by reference for all that they teach about the production of LNP that is not inconsistent with the present disclosure.(3) Binding Moiety

[0319] In accordance with the embodiments described herein, a binding moiety may target an immune cell or a particular type or subtype of immune cell due to its binding specificity to the surface molecule, thereby directing tLNPs to the target immune cell to exert the desired effects. In some embodiments, the binding moiety may itself be biologically active upon binding the target cell by acting as a BRM to trigger activation of the target cell. In such embodiments, the tLNPs can achieve the dual functionality of activating and engineering the immune cell in one formulation.

[0320] In some embodiments, the tLNPs for modulating an immune cell comprise a binding moiety conjugated to the surface of the tLNP that binds a surface molecule expressed by the immune cell. The design of the tLNPs, and the selection of the binding moiety, can be based on the immune cell to be targeted and / or modulated and the surface molecule(s) the desired immune cell expresses. The tLNPs can target an135RECTIFIED SHEET (RULE 91 ) ISA / EPimmune cell through the binding specificity of the binding moiety to the surface molecule. In some embodiments, the binding moiety comprises an accessible thiol group for use in conjugation with the tLNP. Alternatively, one can add an alkyne or an azide to a sulfhydryl or an epsilon amino of a lysine to participate in a click chemistry reaction to form a functionalized tNLP. In another alternative, an epsilon amino of a lysine can be reacted with N-succinimidyl S-acetylthioacetate (SATA) to introduce a reactive sulfhydryl group which can then be reacted with a maleimide-modified nanoparticle, for example, a nanoparticle comprising a maleimide-modified PEG-lipid, to form the conjugate. In another alternative, a SATA functionalized lysine in the antibody is reacted with a maleimide reagent, and the maleimide in turn reacts with a functionalized PEG on the LNP.

[0321] Upon binding the surface molecule expressed on the target immune cell, the binding moiety, the tLNP is internalized by the target immune cell, and the following sequence of events begins: endo-lysosomal uptake of the tLNP, cytoplasmic release of the internal payload, nuclear translocation of the internal payload, and initiation of transcriptive and / or translative activities of the immune cell so that the biomolecules encoded by the payload are expressed. These events take place even with binding moieties that do not trigger any additional cellular signaling.

[0322] In some embodiments, in addition to binding the surface molecule expressed on the target immune cell, the binding moiety is an agonist binding moiety that also functions as a BRM through its binding activity with the surface molecule. In some embodiments, the binding moiety may achieve a BRM function of the target immune cell through its specific binding to the surface molecule, which promotes signal transduction by the surface molecule. Such function can enhance or facilitate the events that lead to the expression of nucleic acids that are part of the payload delivered to a cell by an tLNP.

[0323] In some embodiments, binding of an agonist binding moiety to the surface molecule enhances signal transduction by the surface molecule, which leads to increased tLNP internalization, endo-lysosomal uptake of the tLNP, cytoplasmic release of the internal payload, and / or nuclear translocation of the internal payload as compared to other binding moieties.136RECTIFIED SHEET (RULE 91 ) ISA / EP

[0324] In some embodiments, binding of an agonist binding moiety to the surface molecule increases transcription and / or translation of the internal payload. In some embodiments, binding of an agonist binding moiety to the surface molecule promotes signal transduction of the surface molecule, which leads to enhanced transcriptive and / or translative activities of the immune cell so that the biomolecules encoded by the payload are expressed at a higher rate and / or efficiency when compared to other binding moieties.

[0325] In other embodiments, binding of an agonist binding moiety to the surface molecule alters signal transduction by the surface molecule, which leads to at least partial activation of the immune cell, increased susceptibility of the immune cell to the engineering agent, increased metabolic activity of the immune cell, and / or increased polyfunctionality of the immune cell. Because the methods described herein are designed to generate engineered cells in short order (i.e., within hours at a patient’s bedside or in the same building) without the need for cryopreservation, activation of the immune cells will not result in the expansion of the immune cell population (i.e., the process is expansion-less; or the population of immune cells is not expanded). However, the metabolic activation that occurs when inducing polyfunctionality can also elevate the nanoparticle handling capacity of the cell making them more susceptible to engineering, that is, the intrinsic per cell efficiency of engineering is increased.

[0326] In certain embodiments comprising delivering the payload into an immune cell, the binding moiety of a tLNP targeting moiety binds to a lymphocyte surface molecule or a monocyte surface molecule. Lymphocyte surface molecules include CD2, CD3, CD4, CD5, CD7, CD8, CD28, 4-1 BB (CD137), CD166, CTLA-4, 0X40, PD-1 , GITR, LAG-3, TIM-3, CD25, low affinity IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, IL-18 receptor, and IL-21 receptor. Monocyte surface molecules include CD5, CD14, CD16a, CD32, CD40, CD1 1 b (Mac-1 ), CD64, DEC205, CD68, and TREM2. Exemplary antibodies that can provide antigen binding domains to bind these surface molecules are disclosed herein above. Such antibodies, individually and collectively, constitute means for binding to an immune cell (or leukocyte) - or to a lymphocyte or monocyte, as indicated.

[0327] In certain embodiments comprising delivering the payload into a stem cell, the binding moiety of a tLNP targeting moiety binds to a HSC surface molecule or a137RECTIFIED SHEET (RULE 91 ) ISA / EPMSC surface molecule. HSC surface molecules include CD1 17, CD34, CD44, CD90 (Thy1 ), CD105, CD133, BMPR2, and Sca-1. MSC surface molecules include CD70, CD105, CD73, Stro-1 , SSEA-4, CD271 , CD146, GD2, SSEA-3, SUSD2, Stro-4, MSCA-1 , CD56, CD200, PODXL, CD13, CD29, CD44, and CD10. Exemplary antibodies that can provide antigen binding domains to bind these surface molecules are disclosed herein above. Such antibodies, individually and collectively, constitute means for binding to a stem cell - or to an HSC or MSC, as indicated.

[0328] In certain embodiments, an scFv antibody can be derived from a natural antibody by linking the variable regions of the heavy (VH) and light (VL) chains of the immunoglobulin with a short linker peptide. Commonly, a linker peptide is used to connect the C-terminus of one chain to the mature N-terminus of the other to form a scFv. (By mature N-terminus we refer to the N-terminus after removal of the signal sequence.) If the linker has sufficient length and flexibility to allow pairing of the two V domains, the result is a monovalent scFv. Linkers serving this function include GSTSGGGSGGGSGGGGSS (SEQ ID NO:93) and SGGGGSGGGGSGGGG (SEQ ID NO:94). Generally, a linker of 12 to 18 amino acid residues, preferably 14-15 residues, is suitable. In a cys-scFv, the scFv has a short cysteine-containing extension at its C-terminus, for example GGC (SEQ ID NO:95). which can be used to conjugate the scFv to a label, drug, toxin, or lipid, for example by reacting the cysteines with maleimide. In uses not involving conjugation through the cysteine residue it can support dimerization.

[0329] Similarly, a disulfide linked Fv antibody can be generated by linking the VH and VL using an interdomain disulfide bond. If the linker does not allow the two V domains of the single chain to pair, the VL domain of one scFv molecule will pair with the VH domain of a second scFv molecule and vice versa to form a homodimer that is divalent: a diabody. Linkers serving this function include SGGGG (SEQ ID NO:96). In some embodiments, a cys-diabody uses a somewhat longer cysteine-containing extension at its C-terminus, for example GGCPPCPPCPPC (SEQ ID NO:97). The cysteines in the two chains of the diabody can be allowed to form a cystine bond, increasing the stability of the molecule. The cystines bonds can also be partially reduced and used to conjugate the diabody to a label, drug, toxin, or lipid, for example by reacting the reduced cysteines with maleimide. A bi-specific molecule can be138RECTIFIED SHEET (RULE 91 ) ISA / EPobtained by encoding two scFv with different specificities as a single polypeptide. When one of the specificities is for a T cell surface antigen the molecule is a BiTE. A bispecific diabody-like molecule can be made obtained by encoding two scFv with different specificities as a pair of polypeptides, one polypeptide comprising the VL for the first specificity and the VH for the second specificity and the other polypeptide comprising the VL for the second specificity and the VH for the first specificity. These two polypeptides can then pair to form a bispecific heterodimer, termed a DART (for Dual-Affinity Re-Targeting), is formed. Typically, the two polypeptides of a DART are linked by a cystine bond (as described above for diabodies). A minibody is a form of antibody in which the F(ab) portion of the antibody has been replaced with a scFv and the CH2 domain has been deleted, but a hinge region between the scFv and the CH3 domain is present. A similar antibody in which the CH2 domain is not deleted is termed a scFv-Fc. sdAbs consist of only the variable region from either the heavy or light chain and usually are the smallest antigen-binding fragments of antibodies. A VHH antibody is the antigen-binding fragment of heavy chain only.

[0330] In accordance with the embodiments described herein, the binding moiety is capable of binding a molecule on the surface of a leukocyte or HSC. In some embodiments, the binding moiety is a binding moiety that does not have an agonistic or antagonistic activity upon binding a target, (i.e., . does not trigger signal transduction upon binding its target, thus does not serve as a BRM), for example, a CD5 binding moiety or a CD7 binding moiety.

[0331] CD5 binding moiety. In some embodiments, the binding moiety is a CD5 binding moiety that is capable of binding CD5. CD5 is an adhesion molecule that is constitutively expressed on T cells and a subset of B cells. In some embodiments, the binding moiety is an antibody or an antigen-binding portion thereof specific to CD5. Non-limiting examples of anti-CD5 antibodies include 5D7, UCHT2, L17F12, H65, and any combinations, antigen-binding portions, or fragments thereof. Each of these constitutes a means for binding CD5

[0332] CD7 binding moiety. In some embodiments, the binding moiety is a CD7 binding moiety that is capable of binding CD7. CD7 is a transmembrane protein in the immunoglobulin superfamily. CD7 is found on thymocytes and mature T cells and plays an essential role in T cell interactions and in T cell / B cell interaction during early139RECTIFIED SHEET (RULE 91 ) ISA / EPlymphoid development. In some embodiments, the binding moiety is an antibody or an antigen-binding portion thereof specific to CD7. Non-limiting examples of anti-CD7 antibodies include TH-69, 3A1 E, 3A1 F, Huly-m2, WT1 , YTH3.2.6, T3-3A1 , and any combinations, antigen-binding portions, or fragments thereof. Each of these constitutes a means for binding CD7.

[0333] In some embodiments, the binding moiety is an agonist binding moiety (i.e. , a binding moiety that triggers signal transduction upon binding its target, thus serving as a BRM), for example, a CD2 binding moiety, a CD8 binding moiety, CD28 binding moiety, CD27 binding moiety, CD38 binding moiety, CD43 binding moiety, CD48 binding moiety, CD1 17 binding moiety, CD127 binding moiety, CD150 binding moiety, 4-1 BB binding moiety, or a Sca-1 binding moiety. These and other binding moieties are described further below.

[0334] CD2 binding moiety. In some embodiments, the binding moiety is a CD2 binding moiety that is capable of binding cluster of differentiation 2 (CD2). CD2 is a cell adhesion molecule found on the surface of T cells and natural killer (NK) cells and acts as a costimulatory molecule for these cells. In some embodiments, the binding moiety is an antibody or an antigen-binding portion thereof specific to CD2. Nonlimiting examples of anti-CD2 antibodies include OKT11 , RPA-2.10, T11 1 (3T4-8B5), T112 (1OLD2-4C1 ), T113 (1 Mono2A6), siplizumab, HuMCD2, TS2 / 18, TS1 / 8, AB75, LT-2, T6.3MEM-65, BTI-322, OTI4E4, and any combinations, antigen-binding portions, or fragments thereof. Each of these constitutes a means for binding CD2.

[0335] CD8 binding moiety. In some embodiments, the binding moiety is a CD8 binding moiety that is capable of binding CD8 or a subunit thereof. CD8 is expressed on a T cell subset primarily having cytolytic activity and natirual killer cells. In some embodiments, the binding moiety is an antibody or an antigen-binding portion thereof specific to CD8. Non-limiting examples of anti-CD8 antibodies include SP-16, 3B5, SP-16, LT8, 17D8, MEM-31 , MEM-87, RIV11 , UCHT4, or YTC182.20, the anti-CD8a antibodies OKT8, SK1 , RPA-T8, IAB22M (a set of humanized antibody fragments derived from OKT8), (MT-807R1 ), TRX2, HIT8a, C8 / 144B, or RAVB3, or the anti- CD8p antibodies SIDI8BEE, BU88, EPR26538-16, or 2ST8.5H7. USPN 10,414,820, which discloses minibodies and diabodies based on humanized OKT8 including minibodies IAb_M1 b_CD8 and IAb_M2b_CD8, and the diabody IAb_CysDb3b_CD8,140RECTIFIED SHEET (RULE 91 ) ISA / EPis incorporated by reference for all that it teaches about humanized anti-CD8 antibodies that is not inconsistent with the present disclosure. Each of these constitutes a means for binding CD8.

[0336] CD3 binding moiety. In some embodiments, the binding moiety is a CD3 binding moiety that is capable of binding CD3 or a subunit thereof. CD3 is a T cell coreceptor involved in activating both the cytotoxic T cell (CD8+ T cells) and T helper cells (CD4+ T cells). In mammals, CD3 may comprise a CD3 gamma chain, a CD3 delta chain, two CD3 epsilon chains, and a homodimer of CD3 zeta chains. A TCR complex is formed by the association of CD3 with a TCR. Thus, a TCR complex may be composed of a CD3 gamma chain, a CD3 delta chain, two CD3 epsilon chains, a homodimer of CD3 zeta chains, a TCR alpha chain, and a TCR beta chain. Alternatively, a TCR complex may be composed of a CD3 gamma chain, a CD3 delta chain, two CD3 epsilon chains, a homodimer of CD3 zeta chains, a TCR gamma chain, and a TCR delta chain. In some embodiments, the CD3 subunit is CD3 epsilon chain.

[0337] In some embodiments, the binding moiety is an antibody or an antigenbinding portion thereof specific to CD3 or a subunit thereof. Non-limiting examples of anti-CD3 antibodies include teplizumab (TZIELD™, OKT3, PRV-031 , or MGA031 ; targets CD3 epsilon chain), otenlixizumab (TRX4; targets epsilon chain), visilizumab (Nuvion®; targets CD3 epsilon chain), cevostamab (BFCR4350A; a CD3 / FcRH5 bispecific antibody), teclistamab (TECVAYLI™; a CD3 / B-cell maturation antigen (BCMA) bispecific antibody), linvoseltamab (CD3 / BCMA bispecific antibody), elranatamab (PF-06863135; a CD3 / BCMA bispecific antibody), pavurutamab (AMG 701 ; a CD3 / BCMA bispecific antibody), vibecotamab (XmAb®14045; a CD3 / CD123 bispecific antibody), blinatumomab (CD3 / CD19 bispecific antibody), talquetamab (CD3 / GPRC5D bispecific antibody), odronextamab (REGN1979; a CD3 / CD20 bispecific antibody), epcoritamab (CD3 / CD20 bispecific antibody), glofitamab (CD3 / CD20 bispecific antibody), flotetuzumab (CD3 / IL3Ra bispecific antibody), cibisatamab (CD3 / CEA bispecific antibody), cevostamab (CD3 / IRTA2 bispecific antibody), foralumab (TZLS-401 ; targets CD3 epsilon chain), TR66 (targets CD3 epsilon chain), mosunetuzumab (targets CD3 epsilon chain), UCHT1v9 (UCHT1 v9), SP34 (UCHT1v9), L2K, and any combinations, antigen-binding portions, or fragments thereof. Each of these constitutes a means for binding CD3.141RECTIFIED SHEET (RULE 91 ) ISA / EP

[0338] CD27 binding moiety. In some embodiments, the binding moiety is a CD27 binding moiety that is capable of binding CD27. CD27 is a member of the tumor necrosis factor receptor superfamily that is expressed both naive and activated effector T cells, NK cells, and activated B cells. CD27 is a co-stimulatory immune checkpoint molecule. In some embodiments, the binding moiety is an antibody or an antigen-binding portion thereof specific to CD27. Non-limiting examples of anti-CD27 antibodies include varlilumab, hCD27.15, vorsetuzumab, vunakizumab, and any combinations, antigen-binding portions, or fragments thereof. Each of these constitutes a means for binding CD27.

[0339] CD38 binding moiety. In some embodiments, the binding moiety is a CD38 binding moiety that is capable of binding CD38. CD38 is a glycoprotein found on the surface of many leukocytes, including CD4+ and CD8+ T cells, B lymphocytes and natural killer cells. In some embodiments, the binding moiety is an antibody or an antigen-binding portion thereof specific to CD38. Non-limiting examples of anti-CD38 antibodies include daratumumab, isatuximab, felzartamab, mezagitamab, GEN-3014, and any combinations, antigen-binding portions, or fragments thereof. Each of these constitutes a means for binding CD38.

[0340] CD43 binding moiety. In some embodiments, the binding moiety is a CD43 binding moiety that is capable of binding CD43. CD43 is a major sialoglycoprotein on the surface of T lymphocytes, monocytes, granulocytes, and some B lymphocytes, which is important for immune function and may be part of a physiologic ligandreceptor complex involved in T-cell activation. In some embodiments, the binding moiety is an antibody or an antigen-binding portion thereof specific to CD43. Nonlimiting examples of anti-CD43 antibodies include 2A1 1 D6, 84-3C1 , DF-T 1 , MEM-59, SP55, W3 / 13HLK, CO.44B8, DFT-1 , OTI6A4, OTI4G8, and any combinations, antigen-binding portions, or fragments thereof. Each of these constitutes a means for binding CD43.

[0341] CD45 binding moiety. In some embodiments, the binding moiety is a CD43 binding moiety that is capable of binding CD45 generally or to one of its isoforms such as CD45RA, CD45RB, and CD45RO. CD45 is found on lymphocytes and HSCs. Among the isoforms with respect to lymphocytes CD45RA is expressed well on naive T cells, CD45RB on naive, memory and effector T cells, and CD45RO on memory T142RECTIFIED SHEET (RULE 91 ) ISA / EPcells. CD45 RB and CD45RO are also early markers on CD34+ HSCs. Non-limiting examples of anti-CD43 antibodies include apamistamab, BC8-B10, EP322Y, BL-178- 12C7, RM2051 , F10-89-4, HI30, MEM-28, 158-4D3, 4KB5 (CD45RA), SPM504 (CD45RA), MEM-55 (CD45RB), PD7 / 26 (CD45RB), C363016A (CD45RB), UCH-L1 (CD45RO), IL-A1 16 (CD45RO), 190-2F2.5 (CD45RO), T200 / 797 (CD45RO), and any combinations, antigen-binding portions, or fragments thereof. Each of these constitutes a means for binding CD45 as well as the indicated isoform thereof.

[0342] CD48 binding moiety. In some embodiments, the binding moiety is a CD48 binding moiety that is capable of binding CD48. CD48 is a member of the CD2 subfamily of the immunoglobulin superfamily (IgSF) which includes signaling lymphocyte activation molecules (SLAM) proteins, such as CD84, CD150, CD229 and CD244. CD48 is found on the surface of lymphocytes and other immune cells, dendritic cells and endothelial cells, and participates in activation and differentiation pathways in those cells. In some embodiments, the binding moiety is an antibody or an antigen-binding portion thereof specific to CD48. Non-limiting examples of anti- CD48 antibodies include HM48-1 , 156-4H9, OX-45, 341 , ARC2103, 7E1 , MEM-102, 3B8-2C2, and any combinations, antigen-binding portions, or fragments thereof. Each of these constitutes a means for binding CD48.

[0343] CD117 binding moiety. In some embodiments, the binding moiety is a CD1 17 binding moiety that is capable of binding CD1 17. CD1 17 is a cytokine receptor expressed on, among other cells, the surface of hematopoietic stem cells. CD117 is a receptor tyrosine kinase type III, which binds to stem cell factor, also known as "steel factor" or "c-kit ligand". When CD1 17 binds to stem cell factor (SCF) it forms a dimer that activates its intrinsic tyrosine kinase activity that in turn phosphorylates and activates signal transduction molecules that propagate the signal in the cell. After activation, the receptor is ubiquitinated to mark it for transport to a lysosome and eventual destruction. Signaling through CD1 17 plays a role in cell survival, proliferation, and differentiation. In some embodiments, the binding moiety is an antibody or an antigen-binding portion thereof specific to CD117. Non-limiting examples of anti-CD117 antibodies include briquilimab, barzolvolimab, 104D2, 2B8, ACK2, YB5.B8, K45, 104D2, 1 C5, HC34LC14 ST04-99, 22HCLC, G.813.2, 8D7, kit2c75, ACK4, OTI2B12, OTI1 B6, OTI2E3, OTI1 E2, OTI2C1 D5, OTI9A11 , OTI14B1 ,143RECTIFIED SHEET (RULE 91 ) ISA / EP6F2, OTI3F9, UMAB216, 104D2, OTI2B5, 0TI1 F6, OTI2C1 H4, OTI6F8, 53H8L29, 288 / BM, CL1659, T595, and any combinations, antigen-binding portions, or fragments thereof. Each of these constitutes a means for binding CD1 17.

[0344] CD150 binding moiety. In some embodiments, the binding moiety is a CD150 binding moiety that is capable of binding CD150. CD150 belongs to the signaling lymphocytic activation molecule (SLAM) family. The interaction of CD150 promoter and enhancers with the Early B-cell factor 1 (EBF1 ) is required for the expression of CD150 gene in B cells. STAT6, IRF4, and NF-kB factors involved in the transfer of the signals from the B-cell receptor, its co-receptors and IL-4R, also play important role in the regulation of CD150 expression. In some embodiments, the binding moiety is an antibody or an antigen-binding portion thereof specific to CD150. Non-limiting examples of anti-CD150 antibodies include mShad150, A12 (7D4), 9D1 , SLAM.4, 5B4G1 , IPO-3, 1 1 B1 H1 B4, and any combinations, antigen-binding portions, or fragments thereof. Each of these constitutes a means for binding CD150.

[0345] Sca-1 binding moiety. In some embodiments, the binding moiety is a Sca-1 binding moiety that is capable of binding Sca-1. Sca-1 is a glycosyl phosphatidylinositol-anchored cell surface protein (GPI-AP) of the LY6 gene family. It is the common biological marker used to identify hematopoitic stem cell (HSC) along with other markers. In some embodiments, the binding moiety is an antibody or an antigen-binding portion thereof specific to Sca-1. Non-limiting examples of anti-Sca-1 antibodies include 9H19L2, 31 A1067, 74T2, 1 H4L3, SU38-04, CPP32 4-1 -18), 3CSP03 (4.1.18), 31 A893, 3CSP01 (7.1.44), 22HCLC, D7, SR01 -02, 7 HCLC, SG4B2, 31 A1067, SZ02-08, ARC0143, ARC0133, JM53-11 , AC2361 , 5H9T5, CT- 6A / 6E, 5A3, 7E1 1 , OTI3A1 1 , OTI7B8, 4D3, OTI3A11 , OTI3A12, OTI1 D2, AM1 4, and any combinations, antigen-binding portions, or fragments thereof. Each of these constitutes a means for binding Seal .

[0346] CD34 binding moiety. In some embodiments, the binding moiety is a CD34+ binding moiety that is capable of binding CD34. CD34 is express by HSCs. In some embodiments, the binding moiety is an antibody or an antigen-binding portion thereof specific to CD34. Non-limiting examples of anti-CD34 antibodies include h4C8, 9C5, 2E10, 5B12, REA1164, C5B12, C2e10, My10, QBend / 10, as well as those disclosed in W02009079922, WO2023141297, WO2015121383, US8927696, or US8399249,144RECTIFIED SHEET (RULE 91 ) ISA / EPeach of which is incorporated by reference for all that they teach about anti-CD34 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD34.

[0347] TCR binding moiety. In some embodiments, the binding moiety is a TCR binding moiety that is capable of binding a TCR or a fragment thereof. In some embodiments, the binding moiety is an antibody or an antigen-binding portion thereof specific to the TCR or fragment thereof. In some embodiments, the binding moiety is a ligand of the TCR or fragment thereof.

[0348] Costimulatory molecule binding moieties. In some embodiments, the binding moiety is a binding moiety that is capable of binding a costimulatory molecule. In some embodiments, the costimulatory molecule is selected from the group consisting of CD8, CD28, and 4-1 BB.

[0349] In some embodiments, the costimulatory molecule binding moiety is a CD8 binding moiety. CD8 is a transmembrane glycoprotein and serves as a co-receptor for TCR. Along with the TCR, the CD8 co-receptor plays a role in T cell signaling and aiding with cytotoxic T cell-antigen interactions. In some embodiments, the binding moiety is an antibody or an antigen-binding portion thereof specific to CD8. Nonlimiting examples of anti-CD8 antibodies include crefmirlimab, 3B5, SP-16, LT8, 17D8, MEM-31 , MEM-87, RIV1 1 , UCHT4, YTC182.20, RPA-T8, OKT8, SK1 , YTC182.20, 51.1 , TRX2, MT807, IAB22M, HIT8a, C8 / 144B, RAVB3, SIDI8BEE, BU88, EPR26538-16, 2ST8.5H7, and any combinations, antigen-binding portions, or fragments thereof.

[0350] In some embodiments, the costimulatory molecule binding moiety is a CD28 binding moiety. CD28 is expressed on T cells and provides costimulatory signals required for T cell activation and survival. T cell stimulation through CD28 in addition to TCRs promotes production of various interleukins (e.g., IL-6). CD28 is the receptor for CD80 (B7.1 ) and CD86 (B7.2) proteins on APCs. CD80 expression is upregulated in activated APCs, while CD86 expression is constitutive and independent of environmental factors. In some embodiments, the binding moiety is an antibody or an antigen-binding portion thereof specific to CD28. Non-limiting examples of anti-CD28 antibodies include TGN1412, lulizumab, prezalumab, and any combinations, antigen-145RECTIFIED SHEET (RULE 91 ) ISA / EPbinding portions, or fragments thereof. In some embodiments, the binding moiety is a ligand of CD28, including, for example, CD80 and CD86.

[0351] In some embodiments, the costimulatory molecule binding moiety is a 4- 1 BB binding moiety. 4-1 BB (also known as CD137) is a member of the tumor necrosis factor (TNF) receptor superfamily and is expressed by activated CD8+ and CD4+ T cells. 4-1 BB functions as a receptor for its ligand 4-1 BBL (also known as CD137L) expressed on APCs and provides costimulatory signals for T cells, enhancing T cell proliferation, cytokine secretion, survival, and cytolytic activity. In some embodiments, the binding moiety is an antibody or an antigen-binding portion thereof specific to 4- 1 BB. Non-limiting examples of anti-4-1 BB antibodies include YH004, urelumab (BMS- 663513), utomilumab (PF-05082566), ADG106, LVGN6051 , PRS-343, and any combinations, antigen-binding portions, or fragments thereof. In some embodiments, the binding moiety is a ligand of 4-1 BB, including, for example, 4-1 BBL.

[0352] Immune checkpoint protein binding moieties. In some embodiments, the binding moiety is a binding moiety that is capable of binding and / or inhibiting an immune checkpoint protein. Immune checkpoint inhibition can bring about multiple effects including reducing the number or functionality of Treg cells mediating immune suppressive effects, activation or functional enablement of immune cells (such as T effector cells), promotion of broader immune responses including epitope spreading, and facilitation of redistribution of immune cells to organs or tissues of interest such as the tumor microenvironment. Thus, immune checkpoint inhibitors constitute means for broadening an immune response, means for mobilizing immune effector cells in one or more of the disclosed mobilization facets, as appropriate, and means for reducing immune suppression. Accordingly, the use of immune checkpoint inhibitors will provide the benefits of activation or functional enablement of the reprogrammed cells, a reduction in the number of T reg cells opposing the activity of the reprogrammed cells, recruitment of broader immunity including epitope spreading and mobilization of immune cells into tissues or organs of interest (where the cells targeted by the engineering agent reside). In some embodiments, the immune checkpoint protein binding moiety is selected from the group consisting of a CD166 binding moiety, a cytotoxic T lymphocyte-associated protein 4 (CTLA-4) binding moiety, a glucocorticoid-induced TNFR-related protein (GITR) binding moiety, a lymphocyte-146RECTIFIED SHEET (RULE 91 ) ISA / EPactivation gene 3 (LAG-3) binding moiety, a 0X40, programed death-1 (PD-1 ) binding moiety, and a T cell immunoglobulin and mucin-domain containing-3 (TIM-3) binding moiety.

[0353] The agents bringing about immune checkpoint inhibition, whether a binding moiety or a BRM / eBRM, are commonly called immune checkpoint inhibitors and it should be understood that it is the check on T cell development that is being inhibited. Thus, while many immune checkpoint inhibitors also inhibit the interaction of receptorligand pairs (e.g., programmed cell death 1 (PD-1 ) interaction with programmed death ligand 1 (PD-L1 )), other checkpoint inhibitors (such as anti-OX40, anti GITR, anti- CD137, anti-CD122, anti-CD40, and anti-ICOS) act as agonists of their targets which release or otherwise inhibit the check on T cell development, ultimately promoting effector function and / or inhibiting regulatory function.

[0354] In some embodiments, the immune checkpoint protein binding moiety is a CD166 binding moiety, and the binding moiety is an antibody or an antigen-binding portion thereof specific to CD166. CD166 (also known as MEMC and CD6L) is a transmembrane glycoprotein in the immunoglobulin superfamily and is encoded by the ALCAM gene. CD166 binds CD6 and is expressed by activated T cells, activated monocytes, epithelial cells, fibroblasts, neurons, and melanoma cells, and in sweat and sebaceous glands. Non-limiting examples of anti-CD166 antibodies include praluzatamab, 3D9F1 , EPR2759, EJ212 / 007-CI2-5, 3A6, 8E12C7, ARC1720, 1 1 -6, and any combinations, antigen-binding portions, or fragments thereof.

[0355] In some embodiments, the immune checkpoint protein binding moiety is a CTLA-4 binding moiety, and the binding moiety is an antibody or an antigen-binding portion thereof specific to CTLA-4. CTLA-4 is an immune checkpoint molecule expressed on the surface of CD4+ and CD8+ T cells and on CD25+, FOXP3+ T regulatory (Treg) cells. Non-limiting examples of anti-CTLA-4 antibodies include vudalimab (a PD-1 / CTLA-4 bispecific antibody), nurulimab, cadonilimab (a PD- 1 / CTLA-4 bispecific antibody), ipilimumab (YERVOY®), tremelimumab (Medimmune), botensilimab, quavonlimab, tuvonralimab, zalifrelimab and any combinations, antigenbinding portions, or fragments thereof.

[0356] In some embodiments, the immune checkpoint protein binding moiety is a147RECTIFIED SHEET (RULE 91 ) ISA / EPGITR binding moiety, and the binding moiety is an antibody or an antigen-binding portion thereof specific to GITR. GITR promotes effector T cell functions and inhibits suppression of immune responses by regulatory T cells. Non-limiting examples of anti- GITR antibodies include TRX518, DTA-1 , INCAGN01876, ragifilimab, and any combinations, antigen-binding portions, or fragments thereof. In some embodiments, the binding moiety is a ligand of GITR, including, for example, GITRL (TNFSF18), efaprinermin (GITRL-Fc fusion), and efgivanermin (GITRL-Fc fusion).

[0357] In some embodiments, the immune checkpoint protein binding moiety is a LAG-3 binding moiety, and the binding moiety is an antibody or an antigen-binding portion thereof specific to LAG-3. LAG-3 negatively regulates cellular proliferation, activation, and homeostasis of T cells, in a similar fashion to CTLA-4 and PD-1 and plays a role in Treg suppressive function. Non-limiting examples of anti-LAG-3 antibodies include encelimab, ieramilimab, favezelimab, fianlimab, miptenalimab, relatlimab (BMS-986916), LAG525 (IMP701), MK-4280, TSR-033, REGN3767, Sym022, INCAGN02385, FS118, BI75411 1 , MGD013, GSK2831781 , and any combinations, antigen-binding portions, or fragments thereof.

[0358] In some embodiments, the immune checkpoint protein binding moiety is an 0X40 binding moiety, and the binding moiety is an antibody or an antigen-binding portion thereof specific to 0X40. Non-limiting examples of anti-OX40 antibodies include ivuxolimab, cudarolimab, revdofilimab, rocatinlimab, tavolimab, telazorlimab, vonlerolizumab, MEDI6469, MEDI6383, MOXR0916, INCAGN01949, and any combinations, antigen-binding portions, or fragments thereof. In some embodiments, the binding moiety is a ligand of 0X40, including, for example, OX40L.

[0359] In some embodiments, the immune checkpoint protein binding moiety is a PD-1 binding moiety, and the binding moiety is an antibody or an antigen-binding portion thereof specific to PD-1. PD-1 is a checkpoint protein on T cells. Antibodies against both PD-1 and its binding partner, programmed death-ligand 1 (PD-L1 ), have been used clinically as immune checkpoint inhibitors. Non-limiting examples of anti- PD-1 / PD-L1 antibodies include vudalimab (a PD-1 / CTLA-4 bispecific antibody), sasanlimab, pidilizumab, cadonilimab (a PD-1 / CTLA-4 bispecific antibody), lodapolimab, envafolimab, cosibelimab, sugemalimab, adebrelimab, pacmilimab, nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), cemiplimab-rwlc (LIBTAYO®),148RECTIFIED SHEET (RULE 91 ) ISA / EPdurvalumab (MEDI4736, IMFINZI™), atezolizumab (TECENTRIQ®, MPDL3280A), avelumab (BAVENCIO®), BMS-936559, and any combinations, antigen-binding portions, or fragments thereof.

[0360] In some embodiments, the immune checkpoint protein binding moiety is a TIM-3 binding moiety, and the binding moiety is an antibody or an antigen-binding portion thereof specific to TIM-3. TIM-3 is a molecule selectively expressed on IFN-y- producing, CD4+ T helper 1 (Th1 ) and CD8+ T cytotoxic 1 (Tc1 ) T cells. Non-limiting examples of anti-TIM-3 antibodies include sabatolimab, cobolimab, spartalizumab (PDR001 ), surzebiclimab, INCAGN02390, TSR-022, and any combinations or, antigen-binding portions, fragments thereof. In some embodiments, the binding moiety is a ligand of TIM-3, including, for example, galectin-9.

[0361] Cytokine receptor binding moieties. In some embodiments, the binding moiety is a cytokine receptor binding moiety. In some embodiments, the cytokine receptor binding moiety may be a cytokine receptor agonist (e.g., IL-2, IL-7, IL-15), a native cytokine, a cytokine fragment capable of binding its corresponding receptor, or a peptide-mimetic agent. In some embodiments, the cytokine receptor binding moiety is an IL-2 receptor binding moiety. In some embodiments, the binding moiety is an antibody or an antigen-binding portion thereof specific to IL-2 receptor. In some embodiments, the binding moiety is a ligand of IL-2 receptor, including, for example, IL-2. Other cytokine receptor binding moieties include a ligand of IL-7 receptor, including, for example, IL-7; a ligand of IL-15 receptor, including, for example, IL-15; and a ligand of IL-18 receptor, for example, IL-18.

[0362] In some embodiments, the cytokine receptor binding moiety is a CD127 binding moiety that is capable of binding CD127. CD127 is a type I cytokine receptor and is a subunit of the functional lnterleukin-7 receptor and Thymic Stromal Lymphopoietin (TSLP) receptors. In some embodiments, the binding moiety is an antibody or an antigen-binding portion thereof specific to CD127. Non-limiting examples of anti-CD127 antibodies include A7R34, ARC0672, and any combinations, antigen-binding portions, or fragments thereof.Target Cells

[0363] In some embodiments, the immune cells to be modified by the engineering149RECTIFIED SHEET (RULE 91 ) ISA / EPagent(s) and / or conditioning agents according to various embodiments of the present technology are T cells, including, but not limited to, naive T cells, helper T cells (CD4+), cytotoxic T cells (CD8+), regulatory T cells (Treg), central memory T cells (TCM), effector memory T cells (TEM), stem cell memory T cells (TSCM), or any combination thereof. T cells are a type of lymphocyte, which develop in the thymus gland and plays a central role in the immune response. In certain embodiments, the T cells can be naive T cells (not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased expression of CD45RO as compared to TCM), memory T cells (antigen-experienced and long-lived), or effector cells (antigen- experienced, cytotoxic). In particular embodiments, targeting using CD45RO and CD45RB is based on considerations regarding the status of these cell surface molecules as being an earlier marker on CD34+ cells besides T cells. Similarly for targeting T cells, expression of these markers on particular subsets can be considered: CD45RA (naive T cells), RO (memory T cells), and RB (naive, memory T, and effector T cells).

[0364] In some embodiments, the immune cells to be modified by the engineering agent(s) and / or conditioning agents according to various embodiments of the present technology are natural killer (NK) cells. NK cells (also defined as large granular lymphocytes) represent a cell lineage differentiated from the common lymphoid progenitor (which also gives rise to B lymphocytes and T lymphocytes). Unlike T cells, NK cells do not naturally express CD3 at the plasma membrane. Importantly, NK cells do not express TCRs and typically also lack other antigen-specific cell surface receptors. NK cells’ cytotoxic activity does not require sensitization but is enhanced by activation with a variety of cytokines including IL-2. NK cells are cytotoxic, and they balance activating and inhibitory receptor signaling to modulate their cytotoxic activity. For instance, NK cells expressing CD16 may bind to the Fc domain of antibodies bound to an infected cell, resulting in NK cell activation. By contrast, activity is reduced against cells expressing high levels of MHC class I proteins. Similar to T cells, NK cells release proteins such as perforin, and enzymes such as proteases (granzymes) upon contact with a target cell. Perforin can form pores in the cell membrane of a target cell, inducing apoptosis or cell lysis.

[0365] In some embodiments, the immune cells to be modified by the engineering150RECTIFIED SHEET (RULE 91 ) ISA / EPagent(s) and / or conditioning agents according to various embodiments of the present technology are natural killer T (NKT) cells. NKT cells are a heterogeneous group of T cells that share properties of both T cells and NK cells. Many of these cells recognize the non-polymorphic CD1 d molecule, an antigen-presenting molecule that binds self and foreign lipids and glycolipids. They constitute ...

Claims

What is claimed is:1 . A method for producing a population of autologous engineered cells capable of being reinfused into a subject comprising transfecting an internal payload into an extracorporeal or ex vivo population of leukocytes or hematopoietic stem cells (HSC) obtained from the subject by incubating the population of leukocytes or HSCs with a lipid nanoparticle (LNP) or a targeting lipid nanoparticle (tLNP) comprising the internal payload in an incubation component of an extracorporeal or ex vivo system for no more than 6 hours, wherein the LNP comprises the internal payload, and wherein the tLNP comprises the internal payload and a binding moiety capable of binding a molecule on the surface of a leukocyte or HSC.

2. The method of Claim 1 , wherein the population of leukocytes is a component of whole blood, is a population of peripheral blood mononuclear cells (PBMCs) or is a population of T cells.

3. The method of any one of Claims 1 or 2, wherein the binding moiety comprises an antibody or antigenic binding fragment thereof.

4. The method of any one of Claims 1 -3, wherein the binding moiety is a CD2 binding moiety, a CD3 binding moiety, a CD5 binding moiety, a CD7 binding moiety, a CD8 binding moiety, CD28 binding moiety, CD27 binding moiety, CD34 binding moiety, CD38 binding moiety, CD43 binding moiety, CD45 binding moiety (including binding moieties specific for CD45RB, CD45RO and pan CD45 species thereof), CD48 binding moiety, CD117 binding moiety, CD127 binding moiety, CD150 binding moiety, 4-1 BB binding moiety, or a Sca-1 binding moiety.

5. The method of Claim 4, wherein the binding moiety is a CD2 binding moiety, a CD8 binding moiety, CD34 binding moiety, CD45 binding moiety (including binding moieties specific for CD45RB, CD45RO and pan CD45 species thereof), or a CD1 17 binding moiety.

6. The method of any one of Claims 1 to 5 wherein the extracorporeal system comprises a closed system, a semi-closed system, or an open system and249RECTIFIED SHEET (RULE 91 ) ISA / EPwherein the incubation component further comprises an incubation medium in which the population of leukocytes and the LNP or tLNP are incubated.

7. The method of any one of Claims 1 to 6 wherein the LNP or tLNP comprises a payload that delivers to the cell an biological response modifier (BRM) or nucleic acid encoding a BRM that is a cytokine, a chemokine, or immune checkpoint inhibitor, wherein the cytokine is IL-2, IL-7, IL-12, IL15, IL-18, or IL-21 , the chemokine is CCR2, or the immune checkpoint inhibitor is anti-CTLA4.

8. The method of any one of Claims 1 -7, wherein the payload delivered to the cell is an engineering agent comprising an mRNA that encodes a reprogramming agent comprising a chimeric antigen receptor (CAR), bispecific T cell engager (BiTE), an immune cell engager, a gene or base editing protein, an RNA- guided nuclease, an inactive RNA-guided nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a transposase, a retrotransposase, a gene writer protein, a prime editing protein, or RNA writer, a reverse transcriptase, a DNA nickase, a Cas9 nickase, or a CRISPR nuclease.

9. The method of any one of Claims 6 to 8, wherein the incubation medium further comprises a biological response modifier (BRM) that is a cytokine, a chemokine, or immune checkpoint inhibitor, wherein the cytokine is IL-2, IL-7, IL-12, IL15, IL-18, or IL-21 , the chemokine is CCR2, or the immune checkpoint inhibitor is anti-CTLA4.

10. The method of any one of Claims 1 to 9, wherein the extracorporeal system further comprises a collection component comprising a blood collection container or conduit to receive a blood or bone marrow sample obtained from the subject and further comprising an apheresis or separation component to separate the population of leukocytes from a blood or bone marrow sample obtained from the subject wherein the apheresis or separation component comprises photopheresis or leukapheresis, and further wherein the extracorporeal system comprises one or more washing components for washing the population of leukocytes prior to the incubation step and / or after the incubation step.1 1. A population of autologous engineered cells designed to express or250RECTIFIED SHEET (RULE 91 ) ISA / EPoverexpress a protein, or to prevent or inhibit expression of an endogenous protein wherein the cells are produced by the process of any one of Claims 1 to 1012. An extracorporeal system for producing a composition comprising a population of autologous engineered cells comprising:(a) an optional apheresis or separation component for separating a population of leukocytes or HSC from a blood sample obtained from a subject;(b) an incubation component for transfecting the population of leukocytes or HSC with (i) a lipid nanoparticle (LNP) comprising an internal payload or (ii) a targeting lipid nanoparticle (tLNP) comprising an internal payload and a binding moiety to produce a population of autologous engineered cells;(c) a resuspension component for resuspending the population of autologous engineered cells in a solution which comprises the composition comprising the population of autologous engineered cells for reinfusion into the subject; wherein the extracorporeal system optionally further comprises a collection component comprising a blood collection container or conduit to receive a blood or bone marrow sample obtained from the subject and further comprises an apheresis or separation component to separate the population of leukocytes from a blood or bone marrow sample obtained from the subject wherein the apheresis or separation component comprises photopheresis or leukapheresis, and further wherein the extracorporeal system optionally comprises one or more washing components for washing the population of leukocytes prior to the incubation step and / or after the incubation step, and wherein the extracorporeal system comprises a closed system, a semi-closed system, or an open system.

13. The extracorporeal system of Claim 12, wherein the population of leukocytes is a population of T cells or peripheral blood mononuclear cells (PBMCs).

14. The extracorporeal system of Claims 12 or 13, wherein the binding moiety is a CD2 binding moiety, a CD3 binding moiety, a CD5 binding moiety, a CD7 binding moiety, a CD8 binding moiety, CD28 binding moiety, CD27 binding moiety,251RECTIFIED SHEET (RULE 91 ) ISA / EPCD34 binding moiety, CD38 binding moiety, CD43 binding moiety, CD45 binding moiety (including binding moieties specific for CD45RB, CD45RO and pan CD45 species thereof), CD48 binding moiety, CD1 17 binding moiety, CD127 binding moiety, CD150 binding moiety, 4-1 BB binding moiety, or a Sca-1 binding moiety.

15. The extracorporeal system of Claims 12 or 13, wherein the binding moiety is a CD2 binding moiety, a CD8 binding moiety, CD34 binding moiety, CD45 binding moiety (including binding moieties specific for CD45RB, CD45RO and pan CD45 species thereof), or a CD117 binding moiety.

16. The extracorporeal system of any one of Claims 12 to 15, wherein the binding moiety comprises an antibody or antigenic binding fragment thereof.

17. The extracorporeal system of any one of Claims 12 to 16, wherein the LNP or tLNP comprises a payload that delivers to the cell an exogenous protein or nucleic acid encoding a biological response modifier (BRM) that is a cytokine, a chemokine, or immune checkpoint inhibitor, wherein the cytokine is IL-2, IL-7, IL-12, IL15, IL-18, or IL-21 , the chemokine is CCR2, or the immune checkpoint inhibitor is anti-CTLA4.

18. The extracorporeal system of any one of Claims 12 to 17, wherein the payload delivered to the cell is an engineering agent comprising an mRNA that encodes a reprogramming agent comprising a chimeric antigen receptor (CAR), bispecific T cell engager (BiTE), an immune cell engager, a gene or base editing protein, an RNA-guided nuclease, an inactive RNA-guided nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a transposase, a retrotransposase, a gene writer protein, a prime editing protein, or RNA writer, a reverse transcriptase, a DNA nickase, a Cas9 nickase, or a CRISPR nuclease.

19. The extracorporeal system of any one of Claims 12 to 18, wherein the incubation component further comprises an incubation medium in which the population of leukocytes and the LNP or tLNP are incubated.Claim 20. The extracorporeal system of any one of Claims 12 to 19, wherein the incubation medium further comprises a biological response modifier (BRM) that is a cytokine, a chemokine, or immune checkpoint inhibitor, wherein the cytokine is IL-2,252RECTIFIED SHEET (RULE 91 ) ISA / EPIL-7, IL-12, IL15, IL-18, or IL-21 , the chemokine is CCR2, or the immune checkpoint inhibitor is anti-CTLA4.21 . A treatment regimen comprising:(a) obtaining a blood sample from a subject;(b) optionally separating a population of leukocytes or HSC from the blood sample;(c) incubating the population of leukocytes or HSC with (i) a lipid nanoparticle (LNP) comprising an internal payload or (ii) a targeting lipid nanoparticle (tLNP) comprising an internal payload and a binding moiety to transfect the population of leukocytes or HSC with the LNP or tLNP, to produce a population of autologous engineered cells that express one or more exogenous proteins encoded by the internal payload;(d) resuspending the population of autologous engineered cells in an infusion solution; and(e) providing a dose of the resuspended population of autologous engineered cells to be reinfused into the subject within 6 hours of obtaining the blood sample.

22. The treatment regimen of claim 21 , further comprising administering a T cell activating BRM to the subject within 3 days prior obtaining the blood or bone marrow, wherein the T cell activating BRM is a CD3 agonist antibody, or IL-2, IL-7, IL-12, IL- 15, IL-18, IL-21 , or other agonists of their receptors.

23. The treatment regimen of claims 21 or 22, further comprising washing the cells after step (b) but before step (c).

24. The treatment regimen of any one of claims 21 to 23, further comprising washing the cells after step (c) but before step (d).

25. The treatment regimen of any one of claims 21 to 24, further comprising repeating steps (a) to (e) to produce one or more additional doses of the resuspended population of autologous engineered cells for reinfusing into the subject.

26. The treatment regimen of any one of claims 21 to 25, wherein the253RECTIFIED SHEET (RULE 91 ) ISA / EPpopulation of leukocytes is a population of T cells or peripheral blood mononuclear cells (PBMCs).

27. The treatment regimen of of any one of claims 21 to 26, further comprising adding a T cell-activating BRM to the population of leukocytes and the LNP or tLNP during step (c), wherein T cell activating BRM is a cytokine, a chemokine, or immune checkpoint inhibitor, wherein the cytokine is IL-2, IL-7, IL-12, IL15, IL-18, or IL-21 , the chemokine is CCR2, or the immune checkpoint inhibitor is anti-CTLA4.

28. The treatment regimen of of any one of claims 21 to 27, wherein the binding moiety is a CD2 binding moiety, a CD3 binding moiety, a CD5 binding moiety, a CD7 binding moiety, a CD8 binding moiety, CD28 binding moiety, CD27 binding moiety, CD34 binding moiety, CD38 binding moiety, CD43 binding moiety, CD45 binding moiety (including binding moieties specific for CD45RB, CD45RO and pan CD45 species thereof), CD48 binding moiety, CD1 17 binding moiety, CD127 binding moiety, CD150 binding moiety, 4-1 BB binding moiety, or a Sca-1 binding moiety.

29. The treatment regimen of of any one of claims 21 to 27, wherein the binding moiety is a CD2 binding moiety, a CD8 binding moiety, CD34 binding moiety, CD45 binding moiety (including binding moieties specific for CD45RB, CD45RO and pan CD45 species thereof), or a CD117 binding moiety.

30. The treatment regimen of any one of claims 21 to 29 wherein the binding moiety comprises an antibody or antigenic binding fragment thereof.

31. The treatment regimen of any one of claims 21 to 30, wherein the LNP or tLNP comprises a payload that delivers to the cell an exogenous protein or nucleic acid encoding a biological response modifier (BRM) that is a cytokine, a chemokine, or immune checkpoint inhibitor, wherein the cytokine is rlL-2, IL-2, IL-7, IL-12, IL15, IL-18, or IL-21 , the chemokine is CCR2, or the immune checkpoint inhibitor is anti-CTLA4.

32. The treatment regimen of any one of claims 21 to 31 , wherein the payload delivered to the cell is an engineering agent comprising an mRNA that encodes a reprogramming agent comprising a chimeric antigen receptor (CAR),254RECTIFIED SHEET (RULE 91 ) ISA / EPbispecific T cell engager (BiTE), an immune cell engager, a gene or base editing protein, an RNA-guided nuclease, an inactive RNA-guided nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a transposase, a retrotransposase, a gene writer protein, a prime editing protein, or RNA writer, a reverse transcriptase, a DNA nickase, a Cas9 nickase, or a CRISPR nuclease.

33. A method of providing a subject with a population of leukocytes or HSC expressing one or more exogenous proteins comprising:(a) transfecting a nucleic acid into an extracorporeal or ex vivo population of leukocytes or hematopoietic stem cells (HSC) obtained from the subject by contacting the population of leukocytes with (i) a lipid nanoparticle (LNP) comprising an internal payload or (ii) a targeting lipid nanoparticle (tLNP) comprising a nucleic acid,(b) washing the population of leukocytes or HSC, and(c) providing a dose of the resuspended population of leukocytes or HSC to be reinfused into the subject within 6 hours of their extraction.

34. The method of claim 33 further comprising performing a leukapheresis technique to obtain the population of leukocytes or HSC substantially free of red blood cells.

35. The method of claim 33 further comprising density gradient centrifugation to obtain the population of HSC or leukocytes or a subset of lymphocytes.

36. The method of any one of claims 33 or 35, comprising immunomagnetic cell separation to obtain a leukocyte subset or lymphocyte subset.

37. The method of any one of claims 33-36, for extracorporeally transfecting a population of lymphocytes, wherein the lymphocytes are contacted with the tLNP within a preparation of leukocytes.

38. The method of any one of claims 33-37, wherein the lymphocytes are contacted with the tLNP in a preparation of lymphocytes.

39. The method of claim 37, wherein the preparation of lymphocytes has been depleted of B lymphocytes and / or CD4+T lymphocytes.255RECTIFIED SHEET (RULE 91 ) ISA / EP40. The method of any one of claims 33 to 39, further comprising contacting the population of leukocytes or HSC with a T cell activating BRM during the extracorporeal transfection step or just prior to the reinfusion step.41 . The method of claim 40, wherein T cell activating BRM is a cytokine, a chemokine, or immune checkpoint inhibitor, wherein the cytokine is IL-2, IL-7, IL-12, IL15, IL-18, or IL-21 , the chemokine is CCR2, or the immune checkpoint inhibitor is anti-CTLA4.

42. The method of any one of claims 33 to 41 , wherein lymphocytes have been contacted with a T cell activating BRM in vivo within 3 days prior to being removed from the subject’s body, and wherein the BRM is a CD3 agonist antibody, or IL-2, IL- 7, IL-12, IL-15, IL-18, IL-21 , or other agonists of their receptors.

43. The method of any one of claims 33 to 42, wherein a component of the tLNP has T cell activating activity or a targeting or binding moiety that binds and activates CD2, CD3, 4-1 BB, or CD28, or an IL-2Ry cytokine, thereby acting as a BRM.

44. The method of any one of claims 33 to 43, wherein the payload delivered to the cell is an engineering agent comprising an mRNA that encodes a reprogramming agent comprising a chimeric antigen receptor (CAR), bispecific T cell engager (BiTE), an immune cell engager, a gene or base editing protein, an RNA-guided nuclease, an inactive RNA-guided nuclease, a zinc finger nuclease (ZFN), a transcription activatorlike effector nuclease (TALEN), a meganuclease, a transposase, a retrotransposase, a gene writer protein, a prime editing protein, or RNA writer, a reverse transcriptase, a DNA nickase, a Cas9 nickase, or a CRISPR nuclease.

45. A method for producing a population of autologous engineered cells capable of being reinfused into a subject, comprising transfecting an internal payload into an enriched extracorporeal or ex vivo population of leukocytes comprising T cells, B cells, NK cells, macrophages, or monocytes, or into hematopoietic stem cells (HSC) by incubating the population of leukocytes or HSCs with a lipid nanoparticle (LNP) in an incubation component of an extracorporeal or ex vivo system for no more than 6 hours, wherein the LNP comprises the internal payload.

46. An extracorporeal system for producing a composition comprising a population of autologous engineered cells comprising:256RECTIFIED SHEET (RULE 91 ) ISA / EP(a) a separation component for separating a subpopulation of leukocytes comprising T cells, B cells, NK cells, macrophages, or monocytes or HSC from a blood sample obtained from a subject;(b) an incubation component for transfecting the subpopulation of leukocytes or HSC with a lipid nanoparticle (LNP) comprising an internal payload to produce a population of autologous engineered cells;(c) a resuspension component for resuspending the population of autologous engineered cells in a solution which comprises the composition comprising the population of autologous engineered cells for reinfusion into the subject.

47. A treatment regimen comprising:(a) obtaining a blood sample from a subject;(b) separating a subpopulation of enriched leukocytes comprising T cells, B cells, NK cells, macrophages, or monocytes or HSC from the blood sample;(c) incubating the subpopulation of enriched leukocytes or HSC with a lipid nanoparticle (LNP) comprising an internal payload to transfect the subpopulation of leukocytes or HSC with the LNP to produce a population of autologous engineered cells that express one or more exogenous proteins encoded by the internal payload;(d) resuspending the population of autologous engineered cells in an infusion solution; and(e) providing a dose of the resuspended population of autologous engineered cells within 6 hours obtaining the blood sample.

48. A method of providing a subject with a subpopulation of enriched leukocytes comprising T cells, B cells, NK cells, macrophages, or monocytes or HSC expressing one or more exogenous proteins comprising:(a) transfecting a nucleic acid into a subpopulation of enriched of leukocytes, or into hematopoietic stem cells (HSC) obtained from the subject extracorporeally or an ex vivo population thereof by contacting the subpopulation of enriched leukocytes with a lipid nanoparticle (LNP) comprising257RECTIFIED SHEET (RULE 91 ) ISA / EPan internal payload comprising the nucleic acid,(b) washing the subpopulation of enriched leukocytes or HSC, and(c) providing a dose of the resuspended population of enriched leukocytes or HSC to be reinfused into the subject within 6 hours of their extraction.

49. The method, system, or treatment regimen of any one of claims 46 to 48, wherein the enriched subpopulation of leukocytes is an enriched subpopulation of T cells.

50. The method, system, or treatment regimen of any one of claims 46 to 48, wherein the subpopulation is an enriched population of HSCs.

51. The method, system, or treatment regimen of any one of claims 46 to 50 wherein the LNP or tLNP comprises a payload that delivers to the cell an exogenous protein or nucleic acid encoding a biological response modifier (BRM) that is a cytokine, a chemokine, or immune checkpoint inhibitor, wherein the cytokine is rlL-2, IL-2, IL-7, IL-12, IL15, IL-18, or IL-21 , the chemokine is CCR2, or the immune checkpoint inhibitor is anti-CTLA4.

52. The method, system, or treatment regimen of any one of claims 46 to 50, wherein the payload delivered to the cell is an engineering agent comprising an mRNA that encodes a reprogramming agent comprising a chimeric antigen receptor (CAR), bispecific T cell engager (BiTE), or an immune cell engager, or wherein the engineering agent encodes a gene base editing protein, an RNA-guided nuclease, an inactive RNA-guided nuclease, a zinc finger nuclease (ZFN), a transcription activatorlike effector nuclease (TALEN), a meganuclease, a transposase, a retrotransposase, a gene writer protein, a prime editing protein, or RNA writer, a reverse transcriptase, a DNA nickase, a Cas9 nickase, or a CRISPR nuclease.

53. A method for producing a population of autologous engineered cells to be reinfused into a subject, comprising transfecting with an internal payload that is an mRNA or gene editing system a select extracorporeal or ex vivo population of cells that are T cells, CD8+ T cells, NK cells, myeloid cells, B cells, monocytes, macrophages, DCs, or HSC with cell targeting lipid nanoparticles (tLNP) without the need to physically pre-select that subpopulation, in an incubation component of an258RECTIFIED SHEET (RULE 91 ) ISA / EPextracorporeal system for no more than 6 hours, wherein the LNP comprises the internal payload, and wherein the tLNP comprises the internal payload and a binding moiety capable of binding a molecule on the surface of a leukocyte or HSC.

54. The method or system of any one of claims 1 -10 and 33-53, wherein the population of cells, leukocytes, or HSC is an extracorporeal population.259RECTIFIED SHEET (RULE 91 ) ISA / EP

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