WO2020168086A1

WO2020168086A1 - Nanocarriers for the delivery of nucleic acids and uses thereof

Info

Publication number: WO2020168086A1
Application number: PCT/US2020/018122
Authority: WO
Inventors: Maurits W. Geerlings; Michiel Lodder; Vidal De La Cruz
Original assignee: Geerlings Maurits W; Michiel Lodder; Vidal De La Cruz
Priority date: 2019-02-13
Filing date: 2020-02-13
Publication date: 2020-08-20

Abstract

Polymers, nanoparticles, and crosslinked polymeric nanoparticles are provided, comprising biologically active components selected from the group of transposons, transposases and/or plasmids and minicircles comprising transposons and/or transposases. Polymers, nanoparticles, and crosslinked polymeric nanoparticles comprising chimeric antigen receptors ("CARs") and/or T-cell receptors ("TCRs") are also provided. Pharmaceutical formulations, transformed cells, and methods of treatment using the polymers, nanoparticles, and crosslinked polymeric nanoparticles are further provided.

Description

TITLE OF THE INVENTION

[0001] NANOCARRIERS FOR THE DELIVERY OF NUCLEIC ACIDS AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No.

62/805,068 filed February 13, 2019 entitled“Nanocarriers for the Delivery of Nucleic Acids and Uses Thereof’, which is incorporated by reference herein in its entirety and for all purposes.

FIELD OF THE INVENTION

[0003] The present invention generally relates to nanoparticles, polymers, and crosslinked polymers prepared therefrom, and, more particularly, to biologically active components such as transposons, transposases, plasmids, and minicircles.

BACKGROUND OF THE INVENTION

[0004] Cationic polymer nanoparticles have been investigated as drug delivery carriers.

However, the cationic polymer systems known from the prior art have several disadvantages. In particular, the need for the presence of excess (cytotoxic) polymer in the therapeutic formulation, the frequently low handling, storage and biological (e.g., in-vivo/in-vitro) stability of the therapeutically loaded nanoparticles, as well as limited efficiencies in endosomal escape and cytosolic unpacking of the therapeutic payload (e.g., transposons, transposases, plasmids, or minicircles) from the complex are still major challenges in this field. As a consequence, various polymeric carriers for efficient delivery of biologically active components, such as DNA, RNA, oligopeptides and proteins have been investigated. In particular, US Patent No. 9,012,424, which is incorporated herein in its entirety, relates to polymeric hybrid core-shell nanocarriers of which the core is designed to bind a therapeutic payload (preferably therapeutic nucleotides, peptides, proteins and small molecule drugs) and the shell is designed to protect the therapeutic payload, stabilize the nanocarrier, provide biocompatibility to the system, enable targeting to specific cells and tissue, and promote efficient intracellular release of the therapeutic payload from the nanocarrier. More in particular, US Patent No. 9,012,424 describes certain PA (PolyAcryl) polymers, nanoparticles and nanogels (described herein as“crosslinked polymeric nanoparticles”), wherein the nanogels may comprise a biologically active component selected from the group of RNA, DNA, oligopeptides, proteins and derivatives or fragments thereof. BRIEF SUMMARY OF THE INVENTION

[0005] One aspect of the present invention relates to novel polymers, nanoparticles and crosslinked polymeric nanoparticles comprising biologically active components selected from the group of transposons, transposases and/or plasmids and minicircles comprising transposons and/or transposases. The polymers, nanoparticles, crosslinked polymeric nanoparticles, plasmids and minicircles can further comprise chimeric antigen receptors and T-cell receptors.

[0006] Another aspect of the present invention relates to processes to prepare the polymers, nanoparticles and crosslinked polymeric nanoparticles comprising biologically active components selected from the group of transposons, transposases and/or plasmids and minicircles comprising transposons and/or transposases. The polymers, nanoparticles, crosslinked polymeric nanoparticles, plasmids and minicircles can further comprise chimeric antigen receptors and T-cell receptors.

[0007] The transposases, chimeric antigen receptors and T-cell receptors can be in DNA, RNA or protein forms.

[0008] Another aspect of the present invention relates to pharmaceutical compositions comprising the polymers, nanoparticles and crosslinked polymeric nanoparticles of the present invention and a pharmaceutically acceptable carrier.

[0009] Another aspect of the present invention relates to the polymers, nanoparticles and crosslinked polymeric nanoparticles of the present invention having a modified surface.

[0010] Another aspect of the present invention relates to the use of the polymers, nanoparticles and crosslinked polymeric nanoparticles of the present invention and the use of the polymers, nanoparticles and polymeric nanoparticles of the present invention for the delivery of a biologically active component to a mammal. Preferably, the mammal is a human.

[0011] Another aspect of the present invention relates to methods of delivering a biologically active component to a mammal, wherein a polymer, nanoparticle or crosslinked polymeric nanoparticle or a pharmaceutical composition comprising such a polymer, nanoparticle or crosslinked polymeric nanoparticle of the present invention is administered to a mammal, preferably a human.

[0012] Another aspect of the present invention relates to methods of treating diseases and disorders with the polymers, nanoparticles and crosslinked polymeric nanoparticles of the present invention. Preferably the disease or disorder is cancer, immune diseases, autoimmune diseases, inflammatory diseases, infectious diseases and genetic disorders.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS [0013] The foregoing summary, as well as the following detailed description of embodiments of the polymer, nanoparticles, crosslinked polymeric nanoparticles, formulations, cells, and methods, will be better understood when read in conjunction with the appended drawings of an exemplary embodiment. It should be understood, however, that the inventions are not limited to the precise arrangements and instrumentalities shown.

[0014] In the drawings:

[0015] Fig. l is a graphical representation of the percentage of GFP-positive cells as a function of time after transfection in accordance with an exemplary embodiment of the present invention;

[0016] Fig. 2 is a graphical representation of the percentage of Mean Fluorescence Intensity (MFI) of the transfected GFP-positive cells as a function of time after transfection in accordance with an exemplary embodiment of the present invention;

[0017] Fig. 3 is a graphical representation of the cell viability of transfected cells in time after transfection in accordance with an exemplary embodiment of the present invention;

[0018] Fig. 4 is a graphical representation of the relative luminescence units (RLU) of cell lysates after transfection with Luciferase mini circle and plasmid DNA in accordance with an exemplary embodiment of the present invention;

[0019] Fig. 5 is a graphical representation of flow cytometry results of human PBMCs transfected with nanoparticles targeted to T cells via coating with an anti-CD3 F(ab’)2-PGA conjugate in accordance with an exemplary embodiment of the present invention; and

[0020] Fig. 6 is a graphical representation of percentages of human PBMCs transfected with nanoparticles targeted to T cells via coating with an anti-CD3 F(ab’)2-PGA using two nanogel preparations (NG-PAA, e.g., NG-1 and NGQ6) in accordance with an exemplary embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention relates to the design and preparation of polymeric hybrid core-shell nanocarriers of which the core is designed to bind transposons, transposases and/or plasmids and minicircles comprising transposon and/or transposases and the shell is designed to protect the payload, stabilize the nanocarrier, provide biocompatibility to the system, enable targeting to specific cells and tissue and promote efficient intracellular release of the payload from the nanocarrier.

[0022] More particularly, the present invention relates to polymers, nanoparticles and crosslinked polymeric nanoparticles, wherein the polymers, nanoparticles and crosslinked polymeric nanoparticles comprise a biologically active component selected from the group of transposons, transposases and/or plasmids and minicircles comprising transposons and/or transposases. The present invention also relates to such polymers, nanoparticles, crosslinked polymeric nanoparticles comprising chimeric antigen receptors (“CARs”) and/or T-cell receptors (“TCRs”).

[0023] The Polymers

[0024] The polymers used for the present invention are represented by the general Formulas (1) and (2):

Formula (2) wherein:

[0025] A is independently selected from a direct carbon-carbon single bond (i.e. a structure wherein A is absent), O, N/R¹) and S;

R¹ is independently selected from H and CFb;

R² is independently selected from the group consisting of:

(a) Ci - C40 alkylene, wherein the alkylene group may be linear or branched and is optionally substituted and/or is optionally (partly) unsaturated and/or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S, and/or wherein the alkylene group is interrupted by one or more -S-S- groups;

(b) C3 - C40 cycloalkylene, wherein the cycloalkylene group is optionally substituted and/or is optionally (partly) unsaturated and/or optionally comprises one or more heteroatoms in the ring, wherein the heteroatoms are independently selected from O, N and S, and/or wherein the cycloalkylene group is interrupted by one or more -S-S- groups outside the ring;

(c) Ce - C4₀ arylene, wherein the arylene group is optionally substituted;

(d) Ce - C4₀ heteroaryl ene, wherein the heteroarylene group comprises one, two or three heteroatoms independently selected from O, N and S and/or wherein the heteroarylene group is optionally substituted;

(e) C7 - C4₀ alkylarylene wherein the alkylarylene group is optionally substituted and/or wherein an alkyl part of the alkylarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S, and/or wherein an alkyl part of the alkylarylene group is interrupted by one or more -S-S- groups;

(f) C7 - C4₀ alkylheteroarylene, wherein the alkylheteroarylene group comprises one, two or three heteroatoms independently selected from O, N and S and/or wherein the alkylheteroarylene group is optionally substituted, and/or wherein an alkyl part of the alkylheteroarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S, and/or wherein an alkyl part of the alkylheteroarylene group is interrupted by one or more -S-S- groups; and

(g) a group wherein two C7 - C4₀ (hetero)arylene groups and/or C7 - C4₀ alkyl(hetero)arylene groups are connected to each other by a -S-S- group, wherein the alkyl part of the

alkyl(hetero)arylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

R³ is independently selected from the group consisting of:

(a) H;

(b) Ci - C10 alkyl, wherein the alkyl group may be linear or branched and is optionally substituted and/or is optionally (partly) unsaturated and/or is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

(c) C3 - C12 cycloalkyl, wherein the cycloalkyl group is optionally substituted and/or is optionally (partly) unsaturated and/or optionally comprises one, two or three heteroatoms in the ring, wherein the heteroatoms are independently selected from O, N and S;

(d) Ce - C12 aryl, wherein the aryl group is optionally substituted; (e) Ce - Ci2 heteroaryl, wherein the heteroaryl group comprises one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S, and wherein the heteroaryl group is optionally substituted;

(f) Ci - Ci4 alkylaryl wherein the alkylaryl group is optionally substituted and/or wherein an alkyl part of the alkylarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S; and

(g) Ci - Ci4 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and/or wherein the alkylheteroarylene group is optionally substituted, and/or wherein an alkyl part of the alkylheteroarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

R⁴ is independently selected from the group consisting of:

(a) H;

(b) Ci - Cio alkyl, wherein the alkyl group may be linear or branched;

(c) C3 - C12 cycloalkyl;

(d) Ce - Ci4 aryl;

(e) C6 - C14 heteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S;

(f) C7 - Ci4 alkylaryl; and

(g) C7 - C14 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S;

R⁵ is independently selected from the group consisting of:

(a) Ci - C12 alkylene, wherein the alkylene group may be linear or branched and is optionally substituted and/or is optionally (partly) unsaturated and/or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S:

(b) C3 - C12 cycloalkylene, wherein the cycloalkylene group is optionally substituted and/or is optionally (partly) unsaturated and/or optionally comprises one or more heteroatoms in the ring, wherein the heteroatoms are independently selected from O, N and S;

(c) Ce - C12 arylene, wherein the arylene group is optionally substituted;

(d) Ce - C12 heteroaryl ene, wherein the heteroarylene group comprises one, two or three heteroatoms independently selected from O, N and S and/or wherein the heteroarylene group is optionally substituted; (e) Ci - Ci2 alkylarylene wherein the alkylarylene group is optionally substituted and/or wherein an alkyl part of the alkylarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S; and

(f) Ci - Ci2 alkylheteroarylene, wherein the alkylheteroarylene group comprises one, two or three heteroatoms independently selected from O, N and S and/or wherein the alkylheteroarylene group is optionally substituted, and/or wherein an alkyl part of the alkylheteroarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

POL represents a polymeric core having a weight average molecular weight M_w of about 300 to about 25000; a = 2 - 64; and b = 1 - 50.

[0026] One aspect of the present invention is a PA (PolyAcryl) polymer according to the general Formulas (1) and (2), wherein the polymer comprises biologically active components selected from the group of transposons, transposases and/or plasmids and minicircles comprising transposons and/or transposases. The polymers, plasmids and minicircles can further comprise chimeric antigen receptors and T-cell receptors.

[0027] The present invention also relates to processes to prepare the polymers comprising the biologically active components of the present invention as well as to such polymers made by the processes.

[0028] The transposases, chimeric antigen receptors and T-cell receptors of the polymer can be in DNA, RNA or protein forms.

The PA Polymer

[0029] According to the present invention, it is preferred that the polymer according to Formulas (1) and (2) has a weight average molecular weight M_w in the range of about 10000 to about

1000000, more preferably in the range of about 20000 to about 500000 g/mol.

[0030] According to a preferred embodiment, A is N(R') or O, most preferably N(R')

According to another preferred embodiment of the present invention, R¹ is H. According to yet another preferred embodiment, R² is selected from:

(al) Ci - C20 alkylene, preferably C2 - C12 alkylene, wherein the alkylene group may be linear or branched and is optionally substituted and/or is optionally (partly) unsaturated and/or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S, and/or wherein the alkylene group is interrupted by one or more -S-S- groups;

(bl) C3 - C20 cycloalkylene, preferably C5 - C12 cycloalkylene, wherein the cycloalkylene group is optionally substituted and/or is optionally (partly) unsaturated and/or optionally comprises 1, 2 or 3 heteroatoms in the ring, wherein the heteroatoms are independently selected from O, N and S, and/or wherein the cycloalkylene group is interrupted by one or more -S-S- groups outside the ring;

(cl) Ce - C20 arylene, preferably Cr> - C 12 arylene, wherein the arylene group is optionally substituted;

(dl) Ce - C20 heteroaryl ene, preferably Ce - C 12 heteroarylene, wherein the heteroarylene group comprises 1, 2 or 3 heteroatoms independently selected from O, N and S and/or wherein the heteroarylene group is optionally substituted;

(el) C7 - C20 alkylarylene, preferably C7 - C 12 alkylheteroarylene wherein the alkylarylene group is optionally substituted and/or wherein an alkyl part of the alkylarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by 1, 2 or 3 heteroatoms, wherein the heteroatoms are independently selected from O, N and S, and/or wherein an alkyl part of the alkylarylene group is interrupted by one or more -S-S- groups;

(fl) C7 - C20 alkylheteroarylene, preferably C7 - C12 alkylheteroarylene, wherein the alkylheteroarylene group comprises 1 - 3 heteroatoms independently selected from O, N and S and/or wherein the alkylheteroarylene group is optionally substituted, and/or wherein an alkyl part of the alkylheteroarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S, and/or wherein an alkyl part of the alkylheteroarylene group is interrupted by one or more -S-S- groups; and

(gl) a group wherein two C7 - C20 (hetero)arylene groups and/or C7 - C20

alkyl(hetero)arylene groups, preferably two C7 - C12 (hetero)arylene groups and/or C7 - C12 alkyl(hetero)arylene groups, are connected to each other by a -S-S- group, wherein the alkyl part of the alkyl(hetero)arylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S.

[0031] According to another preferred embodiment of the present invention, R² is selected from group (al) or group (bl), more preferably group from group (al), and in particular from group (al) wherein the Ci - C40 alkylene group is interrupted by one or more -S-S- groups. [0032] According to a preferred embodiment, when R³ is substituted Ci - Cio alkyl, the alkyl group is substituted by a group selected from -OH, -OR⁷, -ML·; -NH(R⁷), -N(R⁷)2, -C(0)0R⁷, - C(0)R⁷, -C(0)MfR⁷, and -C(0)1S[R⁷2, wherein R⁷ is independently selected from the group consisting of:

(a) H;

(b) Ci - Cio alkyl, wherein the alkyl group may be linear or branched and is optionally substituted and/or is optionally (partly) unsaturated and/or is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

(d) Ce - C12 aryl, wherein the aryl group is optionally substituted;

(e) Ce - C12 heteroaryl, wherein the heteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and/or wherein the heteroaryl group is optionally substituted;

(f) C7 - C14 alkylaryl wherein the alkylaryl group is optionally substituted and/or wherein an alkyl part of the alkylarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S; and

(g) C7 - C14 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and/or wherein the alkylheteroarylene group is optionally substituted, and/or wherein an alkyl part of the alkylheteroarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S.

[0033] According to a preferred embodiment, when R³ is substituted C3 - C12 cycloalkyl, the cycloalkyl group has a pending group selected from -OH, -OR⁷, -ML·; -Mf(R⁷), -N(R⁷)2, - C(0)OR⁷, -C(0)R⁷, -C(0)MfR⁷, and -C(0)NR 2, wherein R⁷ is as defined above.

[0034] According to a preferred embodiment, when R³ is substituted O, - C12 aryl, O, - C12 heteroaryl, C7 - C14 alkylaryl or C7 - C14 alkylheteroaryl, the alkylaryl or alkylheteroaryl group is substituted with, preferably with one, two or three of -OH, -OR⁷, -ML·; -NH(R⁷), -N(R⁷)2, - C(0)OR⁷, -C(0)R⁷, -C(0)MfR⁷, and -C(0)NR 2, wherein R⁷ is as defined above.

[0035] In the crosslinked polymeric nanoparticles according to the present invention which are represented by general Formulas (1) and (2), the polymeric core POL is preferably based on or is selected from a branched, hyperbranched, multi-arm, dentritic or star-type (co)polymer, said (co)polymer preferably having 2 - 64, more preferably 2 - 32, even more preferably 2 - 16 terminal amino groups, preferably primary amino groups. Accordingly, a is 2 - 64, preferably 2 - 32 and in particular 2 - 16. The (co)polymers may comprise different linear or branched spacers which comprise one or more heteroatoms selected from the group consisting of O, N and S, preferably O and N.

[0036] According to the present invention, b is 1 - 50, preferably 1 - 30, and more preferably 1 - 10

[0037] It is well known in the art that dendritic (co)polymers are not always perfectly branched and may therefore have a hyperbranched structure. The degree of branching ( DB ) can be defined by:

wherein D is the number of dendritic units, L the number of linear units, and T the number of terminal units. Perfect dendrimers will have a DB of 1, whereas hyperbranched (co)polymers have typically a DB of 0.4 to 0.5 up to even 0.9. In this patent application, the term“dendrimer” is to be understood as including“perfectly branched dendrimers” as well as“imperfectly branched dendrimers” which are also referred to as“hyperbranched (co)polymers”. Alternatively, the term “hyperbranched (co)polymers” may also comprise“true” hyperbranched (co)polymers. That is, that these macromolecules are purposively prepared as having a hyperbranched structure. The term “dendrimer” is to be understood as comprising both dendrimeric homopolymers and dendrimeric copolymers. The term“copolymer” includes polymers made of at least two different monomers.

[0038] Hyperbranched polymers can be obtained from the random polymerization of monomers in the presence of at least one polyfunctional monomer capable of introducing branching. Such a synthetic scheme is shown by Hawker and Devonport in "Step-Growth Polymers for High- Performance Materials : New Synthetic Methods," Hedrick, J. L. and Labadie, J. W., Eds., Am. Chem. Soc., Washington, D. C., 1996, pp. 191-193. Hult et al. , in "Advances in Polymer Science," Vol. 143 (1999), Roovers, J., Ed., Springer, New York, pp. 1-34, present a review of hyperbranched polymers.

[0039] Highly branched dendritic polymers are for example discussed in "Polymeric Materials Encyclopedia", Vol. 5 (1996), J.C. Salamone, Ed., CRC Press, New York, pp. 3049-3053. Dendritic polymers have generally a non-linear architecture and are intrinsically globular in shape. Discrete, stepwise synthetic methods are used to prepare highly branched pure compounds or dendrimers. As discussed by Hawker and Devonport in "Step-Growth Polymers for High-Performance Materials: New Synthetic Methods", Hedrick, J. L. and Labadie, J. W., Eds., Am. Chem. Soc., Washington, D. C., 1996, pp. 186-196, if the macromolecule has highly regular branching which follows a strict geometric pattern, it is a dendrimer. Dendrimers are typically monodisperse and are prepared in a multi-step approach with purifications at each stage. The architecture of dendrimers is also discussed by Roovers and Comanita in "Advances in Polymer Science", Vol. 142 (1999), Roovers, J., Ed., Springer, New York, pp. 179 - 228. Dendrimers consist of a core molecule which defines the centre of symmetry of the molecule, and branching layers. Tomalia et al ., Angew. Chem. Int. Ed. Eng., 29 (1990), 138-175 disclose "starburst" dendrimers which consist of an initiator core and branching groups.

[0040] Preferably, the polymer core POL is based on PEI (commercially available from e.g. Denditrech, Inc.), Astramol® polymers (DSM), JEFF AMINE® polymers (Huntsman), PAMAM polymers (sometimes also called PAN AM polymers), PPI polymers, PE AN polymers and PEAC polymers. The term“PEI” refers to polyethyleneimines. The term“PAMAM” refer to poly(amido amine) polymers which are commercially available under de trade name Starburst®. The term“PPI” means polypropylene imine polymers. The term“PEAN” refers to poly(ester amine) polymers. The term“PEAC” refers to poly (ether amine) polymers. All these polymers are well known in the art. Accordingly, it is preferred that the polymer core POL is based on or is selected from the group consisting of PEI, PAMAM, PPI, PEAN and PEAC.

[0041] According to the present inventions, it is preferred that the average molecular weight M_w of the polymer core POL is about 300 to about 5000, and more preferably about 600 to about 5000.

[0042] Preferred polymers used for the polymer core POL are the polymers (polyether amines) of the JEFF AMINE® T series which are commercially available with a weight average molecular weight Mw in the range of about 440 to about 5000. Suitable types of JEFF AMINE® T polymers include T-403 (M_w = 440) and T-3000 (M_w = 3000).

[0043] Another group of preferred polymers that can be used for the polymer core POL are PEFs. PEI can have either a linear or branched structure. Linear PEI is commercial available (jetPEI, Polyplus-Transfection Co.; Exgen 500, Fermentas Co.) and is usually prepared by hydrolysis of poly(2-ethyl-2-oxazoline). Branched PEFs are prepared from aziridine and these polymers have a highly branched structure and comprise about 25% primary amine groups, about 50% secondary amine groups, and about 25% tertiary amine groups. Preferably, the PEI has a weight average molecular weight M_n of about 600 to about 3000, more preferably about 600 to about 2000. Linear PEFs may be represented by the general Formula (5a):

(Formula 5a)

[0044] Branched PEFs may be represented by the general Formula (5b):

(Formula 5b) wherein n is such that the PEI has a weight average molecular weight M_n of about 300 to about 5000, more preferably about 300 to about 3000, even more preferably about 600 to about 2500.

Such PEFs are for example available from Sigma- Aldrich.

[0045] Yet another group of preferred polymers used for the polymer core POL are poly(amido amine) hyperbranched polymers and dendrimers, preferably those of the 1^st to the 4^th generation, more preferably of the 1st and/or the 3^rd generation. These polymers are commercially available from Dentritech, Inc., and have a weight average molecular weight M_w in the range of about 1400 (1^st generation) to about 15000 (4^th generation).

[0046] Yet another group of preferred polymers used for the polymer core POL are the polymers represented by the general Formulas (6) - (9): N(R⁸)3-n[(CR⁹2)m-N(R¹⁰R^U)]n

(Formula 6)

[(R¹⁰R¹¹)N-(CR⁹2)m]2N-P-N[(CR⁹2)m-N(R¹⁰R¹¹)]2 (Formula 7)

N(R⁸)3-n[(CR⁹2)m-C(0)N(R⁹)-(CR⁹2)m-N(R¹²R¹³)]n

(Formula 8)

[(R¹²R¹³)N-(CR⁹2)m-N(R⁹)C(0)-(CR⁹2)m]2N-P-N[(CR⁹2)-C(0)NH-(CR⁹2)m-N(R¹²R¹³)]2

(Formula 9) wherein: R⁸ is a hydrogen atom, a linear or branched Ci - C20 alkyl group or a -[(CR¹⁴2)q-X]o-R¹⁵ group, wherein X is O or N(R⁸);

m is 2, 3 or 4;

n is 2 or 3;

o is 1 - 10;

q is 2, 3 or 4;

P is -(CR⁹2)m-, a Ce - C12 arylene group, a Ce - C12 cycloalkylene group or a -[(CR¹⁴2)q-X]p-C(R¹⁴)2]- group, wherein X is O or N(R⁸) and p is 1 - 10;

R⁹ is a hydrogen atom or a linear or branched Ci - Ce alkyl group;

R¹⁰ and R^{1 1} are independently a hydrogen atom, a linear or branched Ci - Ce alkyl group or a group of the formula -(CR¹⁴2)qNR¹⁶R¹⁷, provided that R¹⁰ and R^{1 1} are not both a linear or branched Ci - Ce alkyl group;

R¹⁶ and R¹⁷ are independently a hydrogen atom, a linear or branched Ci - Ce alkyl group or a group of the formula -(CR¹⁴2)qNR¹⁸R¹⁹, provided that R¹⁶ and R¹⁷ are not both a linear or branched Ci - Ce alkyl group;

R¹⁸ and R¹⁹ are independently a hydrogen atom, a linear or branched Ci - Ce alkyl group or a group of the formula -(CR¹⁴2)qNR²⁰R²¹, provided that R¹⁸ and R¹⁹ are not both a linear or branched Ci - Ce alkyl group;

R²⁰ and R²¹ are independently a hydrogen atom or a linear or branched Ci - Ce alkyl group, provided that R²⁰ and R²¹ are not both a linear or branched Ci - Ce alkyl group;

R²² is a hydrogen atom or a methyl group, provided that at least one R²² is a hydrogen atom;

R¹⁵ is a hydrogen or linear or branched Ci - C20 alkyl group or a -[(CR¹⁴2)q-X]o-R¹⁵ group as defined above; R¹² and R¹³ are independently a hydrogen atom, a linear or branched Ci - G alkyl group or a group of the formula -(CR⁹2)m-C(0)NH-(CR⁹2)m-N(R²³R²⁴), provided that R¹² and R¹³ are not both a linear or branched Ci - G alkyl group;

R²³ and R²⁴ are independently a hydrogen atom, a linear or branched Ci - Ce alkyl group or a group of the formula -(CR⁹2)m-C(0)NH-(CR⁹2)m-N(R²⁵R²⁶), provided that R²³ and R²⁴ are not both a linear or branched Ci - G, alkyl group;

R²⁵ and R²⁶ are independently a hydrogen atom, a linear or branched Ci - G alkyl group or a group of the formula -(CR⁹2)m-C(0)NH-(CR⁹2)m-N(R²⁷R²⁸), provided that R²⁵ and R²⁶ are not both a linear or branched Ci - G, alkyl group;

R²⁷ and R²⁸ are independently a hydrogen atom or a linear or branched Ci - G, alkyl group, provided that R²⁷ and R²⁸ are not both a linear or branched Ci - G, alkyl group.

[0047] In Formulas (6) - (9), a preferred group polymers used for the polymer core POL is the group wherein:

R⁸ and R¹⁵ is a hydrogen atom or a -[(CR¹⁴2)q-X]o-R¹⁵ group, wherein X is NH;

R⁹, R²⁰ _, R²¹, and R²¹ are a hydrogen atom;

R¹⁰ and R¹¹ are independently a hydrogen atom or a group of the formula -(CR¹⁴2)qNR¹⁶R¹⁷;

R¹⁶ and R¹⁷ are independently a hydrogen atom or a group of the formula -(CR¹⁴2)qNR¹⁸R¹⁹;

R¹⁸ and R¹⁹ are independently a hydrogen atom or a group of the formula -(CR¹⁴2)qNR²⁰R²¹;

R¹² and R¹³ are independently a hydrogen atom or a group of the formula -(CR⁹2)m-C(0)NH- (CR⁹ ₂)m-N(R²³R²⁴);

R²³ and R²⁴ are independently a hydrogen atom or a group of the formula -(CR⁹2)m-C(0)NH- (CR⁹ ₂)m-N(R²⁵R²⁶);

R²⁵ and R²⁶ are independently a hydrogen atom or a group of the formula -(CR⁹2)m-C(0)NH- (CR⁹ ₂)m-N(R²⁷R²⁸); and

R²⁷ and R²⁸ are independently a hydrogen atom.

[0048] In Formulas (6) - (9), it is also preferred that m and q is 2:

[0049] A more preferred class of the PA polymers according to the present invention can be represented by general Formulas (10) and (11):

(Formula 11)

wherein:

R¹ is independently selected from H and CFb;

Y is O or N/R¹);

r is 2, 3 or 4;

Z is -S-S-;

R²⁹ is independently selected from the group consisting of:

(a) H;

(b) Ci - Cio alkyl, wherein the alkyl group may be linear or branched and is optionally substituted and/or is optionally (partly) unsaturated and/or is optionally interrupted by 1, 2 or 3 heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

(c) C3 - C12 cycloalkyl, wherein the cycloalkyl group is optionally substituted and/or is optionally (partly) unsaturated and/or optionally comprises 1, 2 or 3 heteroatoms in the ring, wherein the heteroatoms are independently selected from O, N and S;

(d) Ce - C12 aryl, wherein the aryl group is optionally substituted;

(e) Ce - C12 heteroaryl, wherein the heteroaryl group comprises 1, 2 or 3 heteroatoms independently selected from O, N and S and/or wherein the heteroaryl group is optionally substituted;

(f) C7 - C 14 alkylaryl wherein the alkylaryl group is optionally substituted and/or wherein an alkyl part of the alkylarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by 1, 2 or 3 heteroatoms, wherein the heteroatoms are independently selected from O, N and S; and (g) Ci - Ci4 alkylheteroaryl, wherein the alkylheteroaryl group comprises 12 or 3 heteroatoms independently selected from O, N and S and/or wherein the alkylheteroarylene group is optionally substituted, and/or wherein an alkyl part of the alkylheteroarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by 1, 2 or 3 heteroatoms, wherein the heteroatoms are independently selected from O, N and S; the

alkylheteroaryl group optionally being substituted, preferably with 1, 2 or 3 of -OH, -OR²⁹, -NH2; - NH(R²⁹) or -N(R²⁹)₂;

R³⁰ is independently selected from the group consisting of:

(a) H;

(d) Ce - C12 aryl, wherein the aryl group is optionally substituted;

(g) C7 - C14 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and/or wherein the alkylheteroarylene group is optionally substituted, and/or wherein an alkyl part of the alkylheteroarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

R³¹ is independently selected from the group consisting of:

(a) H;

(b) Ci - C10 alkyl, wherein the alkyl group may be linear or branched;

(c) C3 - C12 cycloalkyl;

(d) Ce - C12 aryl; (e) Ce - Ci2 heteroaryl, wherein the heteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S;

(f) C7 - Ci4 alkyl aryl; and

R³² is independently selected from the group consisting of:

(a) Ci - C12 alkylene, wherein the alkylene group may be linear or branched and is optionally substituted and/or is optionally (partly) unsaturated and/or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

(c) Ce - C12 arylene, wherein the arylene group is optionally substituted;

(d) Ce - C12 heteroaryl ene, wherein the heteroarylene group comprises one, two or three heteroatoms independently selected from O, N and S and/or wherein the heteroarylene group is optionally substituted;

(e) C7 - C14 alkylarylene wherein the alkylarylene group is optionally substituted and/or wherein an alkyl part of the alkylarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S; and

(f) C7 - C14 alkylheteroarylene, wherein the alkylheteroarylene group comprises one, two or three heteroatoms independently selected from O, N and S and/or wherein the alkylheteroarylene group is optionally substituted, and/or wherein an alkyl part of the alkylheteroarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S; and

a = 2 - 64; and

b = 1 to 50.

[0050] According to a preferred embodiment, when R³⁰ is substituted Ci - C10 alkyl, the alkyl group is substituted by a group selected from -OH, -OR⁷, -NH2; -NH(R⁷), -N(R⁷)2, -C(0)OR⁷, - C(0)R⁷, -C(0)NHR⁷, and -C(0)NR⁷2, wherein R⁷ is independently selected from the group consisting of:

(a) H; (b) Ci - Cio alkyl, wherein the alkyl group may be linear or branched and is optionally substituted and/or is optionally (partly) unsaturated and/or is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

(d) Ce - C12 aryl, wherein the aryl group is optionally substituted;

[0051] According to a preferred embodiment, when R³⁰ is substituted C3 - C12 cycloalkyl, the cycloalkyl group has a pending group selected from -OH, -OR⁷, -NH2; -NH(R⁷), -N(R⁷)2, - C(0)OR⁷, -C(0)R⁷, -C(0)NHR⁷, and -C(0)NR⁷2, wherein R⁷ is as defined above.

[0052] According to a preferred embodiment, when R³⁰ is substituted O, - C12 aryl, O, - C12 heteroaryl, C7 - C14 alkylaryl or C7 - C14 alkylheteroaryl, the alkylaryl or alkylheteroaryl group is substituted with, preferably with one, two or three of -OH, -OR⁷, -NH2; -NH(R⁷), -N(R⁷)2, - C(0)OR⁷, -C(0)R⁷, -C(0)NHR⁷, and -C(0)NR⁷2, wherein R⁷ is as defined above.

[0053] In a more preferred class of the PA polymers according to general Formulas (10) and

(11):

R¹ is independently selected from H and CH3;

Y is N(R³);

r is 2;

Z is -S-S-; R²⁹ is independently selected from H and Ci - Cio alkyl, wherein the alkyl group may be linear or branched and is optionally substituted and/or is optionally (partly) unsaturated and/or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

R³⁰ is independently selected from H and Ci - Cio alkyl, wherein the alkyl group may be linear or branched and is optionally substituted and/or is optionally (partly) unsaturated and/or is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

R^{3 1} is independently selected from H and Ci - Cio alkyl, wherein the alkyl group may be linear or branched; and

R³² is Ci - Cio alkylene, wherein the alkylene group may be linear or branched and is optionally substituted and/or is optionally (partly) unsaturated and/or is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S.

[0054] In an even more preferred class of the PA polymers according to general Formulas (10) and (11):

R¹ is H;

Y is N(R³);

r is 2;

Z is -S-S-;

R²⁹ is H;

R³⁰ is independently selected from H and Ci - Cio alkyl, wherein the alkyl group is linear and is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

R³¹ is H; and

R³² is Ci - Cio alkylene, wherein the alkylene group may be linear or branched.

[0055] In yet an even more preferred class of the PA polymers according to general Formulas (10) and (11):

R¹ is H;

Y is N(R³);

r is 2;

Z is -S-S-;

R²⁹ is H; R³⁰ is Ci - Cio alkyl, wherein the alkyl group is linear and is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

R³¹ is H; and

R³² is Ci - Cio alkylene, wherein the alkyl group may be linear or branched.

[0056] In a most preferred class of the PA polymers according to general Formulas (10) and

(11):

Y is NH;

r is 2;

Z is -S-S-;

R²⁹ is H;

R³⁰ is Ci - Ce alkyl;

R³¹ is H; and

R³² is Ci - Cio alkylene, wherein the alkylene group is linear.

[0057] In all these preferred classes of the PA polymers according to Formulas (10) and (11), it is preferred that when R³⁰ is substituted Ci - Cio alkyl, the alkyl group is substituted by a group selected from -OH, -OR⁷, -NH₂; -NH(R⁷), -N(R⁷)₂, -C(0)OR⁷, -C(0)R⁷, -C(0)NHR⁷, and - C(0)NR⁷ ₂, wherein R⁷ is as defined above. Preferably, the alkyl group is substituted by -OH, -NH₂, - NH(R⁷), or -N(R⁷)₂; more preferably by substituted-OH or -NH₂.

Process for Preparing the PA Polymer

[0058] Methods for preparting the PA Polymers are known in the art and, in particular, methods for preparing the PA Polymers are also described in US Patent No. 9,012,424, which is incorporated herein in its entirety.

[0059] According to an embodiment of the present invention, the PA polymers according to the present invention as represented by general Formulas (1) and (2) may be prepared by a process which comprises the steps of:

(1) reacting a monomer (I) according to general Formula (12):

(Formula 12) wherein R¹, A and R² are as defined above, with a monomer (II) according to general Formula (13) or a monomer (III) according to Formula (14):

(H₂N)-R³

(Formula 13)

HR³N-R⁵-NR³H

(Formula 14) wherein R³ and R⁵ are as defined above, in a molar ratio of monomer (I) : monomer (II) of from about 1.5 : 1 to about 10 : 1 to form a macromer according to general Formulas (15) or (16):

(Formula 16)

wherein b is as defined above; and

(2) reacting the macromer according to general Formulas (15) or (16) with a polymer according to Formula (17);

[(R⁴2)N]a-POL

(Formula 17) wherein a, POL and R⁴ are as defined above and wherein at least one R⁴ is H. [0060] Hence, in this reaction, a -C(0)-C(R¹)=C group reacts with a (R⁴ ₂)N group of POL under the formation of a -C(0)-CH(R¹)-CH₂-N(R⁴)- moiety (Michael addition) as will be clear to the person skilled in the art.

[0061] Accordingly, the present invention also relates to PA polymer according to general Formulas (1) and (2) which is obtainable by this process.

[0062] According to a preferred embodiment of the present invention, the PA polymers according to the present invention as represented by general Formulas (10) and (11) may also be prepared by this process which comprises the steps of:

(1) reacting a monomer (III) according to general Formula (18):

(Formula 18) wherein R¹, Y, R²⁹ and r are as defined above, with a monomer (IV) according to general Formula (19) or a monomer (V) according to Formula (20):

(H₂N)-R³⁰

(Formula 19)

HR³⁰N-R³²-NR³⁰H

(Formula 20) wherein R³⁰ and R³² are as defined above, in a molar ratio of monomer (I) : monomer (II) of from about 1.5 : 1 to about 10 : 1 to form a macromer according to general Formulas (21) or (22):

(Formula 21)

(Formula 22)

wherein b is as defined above; and

(2) reacting the macromer according to general Formulas (21) or (22) with a polymer according to Formula (23);

[(R³¹ ₂)N]a-POL

(Formula 23) wherein a, POL and R³¹ are as defined above and wherein at least one R³¹ is H.

[0063] According to the present invention, it is preferred that step (1) of the process is performed at a temperature ranging from ambient temperature to about 100°C, preferably from about 30° to about 80°C.

[0064] According to the present invention, it is preferred that step (2) of the process is performed at a temperature ranging from ambient temperature to about 100°C, preferably from about 30° to about 80°C.

[0065] The Nanoparticles

[0066] The nanoparticles used for the present invention are represented by the general Formulas (3) and (4):

Formula (3)

Formula (4) wherein R¹, R², R³, R⁴, R⁵, A, POL, a and b are as set forth above with respect to the Polymers, and wherein R⁶ is selected from the group consisting of:

(a) Ci - C40 alkylene, wherein the alkylene group may be linear or branched and is optionally substituted and/or is optionally (partly) unsaturated and/or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S, and/or wherein the alkylene group is interrupted by one or more -S-S- groups; or

(b) C3 - C40 cycloalkylene, wherein the cycloalkylene group is optionally substituted and/or is optionally (partly) unsaturated and/or optionally comprises one or more heteroatoms in the ring, wherein the heteroatoms are independently selected from O, N and S, and/or wherein the cycloalkylene group is interrupted by one or more -S-S- groups outside the ring; and

wherein FG is a Functional Group.

[0067] The present invention also relates to nanoparticles according to the general Formulas (3) and (4) wherein the nanoparticles further comprise biologically active components selected from the group of transposons, transposases and/or plasmids and minicircles comprising transposons and/or transposases. The nanoparticles, plasmids and minicircles can further comprise chimeric antigen receptors and T-cell receptors.

[0068] The present invention also relates to processes to prepare the nanoparticles comprising the biologically active components of the present invention as well as to such nanoparticles made by the processes.

[0069] The transposases, chimeric antigen receptors and T-cell receptors of the nanoparticle can be in DNA, RNA or protein forms.

[0070] According to a preferred embodiment, the nanoparticle is represented by general Formulas (24) and (25):

(Formula 25) wherein R¹, R⁶, R²⁹, R³⁰, R³¹, R³², Y, Z, POL, FG, a, b and r as defined above.

[0071] The functional group FG is a substituent that is capable of forming a covalent bond with a complementary functional group (CFG) of a reagent for post-modification. The group FG is preferably selected from functional groups that enable the formation of a covalent bond with a group CFG, preferably under biocompatible reaction conditions, in particular under conditions of physiological pH and ambient temperature and in aqueous systems. Such groups FG are well known to the person skilled in the art. In particular, the group FG is selected from the group consisting of a group selected from -OH, -OR⁷, -ML; -NH(R⁷), -N(R⁷)₂, -C(0)OR⁷, -C(0)R⁷, -C(0)NHR⁷, and - C(0)NR⁷2, wherein R⁷ is independently selected from the group consisting of:

(a") H;

(b") Ci - Cio alkyl, wherein the alkyl group may be linear or branched and is optionally substituted and/or is optionally (partly) unsaturated and/or is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

(c") C3 - C12 cycloalkyl, wherein the cycloalkyl group is optionally substituted and/or is optionally (partly) unsaturated and/or optionally comprises one, two or three heteroatoms in the ring, wherein the heteroatoms are independently selected from O, N and S;

(d") Ce - C12 aryl, wherein the aryl group is optionally substituted; (e") Ce - Ci2 heteroaryl, wherein the heteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and/or wherein the heteroaryl group is optionally substituted;

(f") Ci - Ci4 alkylaryl wherein the alkylaryl group is optionally substituted and/or wherein an alkyl part of the alkylarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S; and

(g") Ci - Ci4 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and/or wherein the alkylheteroarylene group is optionally substituted, and/or wherein an alkyl part of the alkylheteroarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S.

[0072] The CFG is preferably selected from the group consisting of -OH, -OR⁷, -NH2; -NH(R⁷), -N(R⁷)2, -C(0)OR⁷, -C(0)R⁷, -C(0)NHR⁷, and -C(0)NR⁷2, wherein R⁷ is as defined above for FG. For example, the FG may be -OH and the CFG may be -COOH.

[0073] The nanoparticles according to general Formulas (3), (4), (24) and (25) may be prepared by a process wherein a PA polymer according to general Formulas (1), (2), (10) or (11),

respectively, are reacted with a reagent according to Formula (26a) or Formula (26b):

FG-R⁶-NHR³⁰

(Formula 26a)

FG-R⁶-SH

(Formula 26b) wherein FG and R⁶ are as defined above, FG optionally being hydrogen. Other useful FG’s include biologically or pharmacologically active groups, e.g. oligo- and polypeptides. Suitable examples for the reagents according to Formula (26a) and (26b) are ethylene diamine, di ethylene triamine, tri ethylene tetramine, tetraethylene pentamine, 1,6-diamino hexane, amine terminated PEG, amine terminated PPO, thiol terminated PEG and thiol terminated PPO.

[0074] However, as will be apparent to those skilled in the art, the reagent according to Formula (26b) can not only react with the acrylate groups of the PA polymers according to any one of

Formulas (1), (2), (10) and (11), but also with the -S-S- groups (when present) in R² of the PA polymer according to Formula (1) or Formula (2) or with the -S-S- group as represented by Z in the PA polymers according to Formula (10) or Formula (11). Additionally, since R³ of the PA polymer according to Formula (1) or Formula (2) and R³⁰ of the PA polymer according to Formula (10) or Formula (11) may comprise functional groups, it can also be envisaged that the reagent according to Formula (26a) can react with these functional groups. Furthermore, instead of the reagent according to Formula (26a), also reagents according to the Formula FG-R⁶-RG can be used, wherein RG is selected from the group consisting of -OH, -OR⁷, -C(0)OR⁷, -C(0)R⁷, -C(0)NHR⁷, -C(0)NR⁷2 and -SO2CI, wherein R⁷ is as defined above, when R³ and R³⁰ comprise e.g. a primary or secondary amino group.

Methods for preparting the nanoparticles are known in the art and, in particular, methods for preparing the nanoparticles are also described in US Patent No. 9,012,424.

[0075] The Crosslinked Polymeric Nanoparticles

[0076] Another aspect of the present invention is a crosslinked polymeric nanoparticle comprising a PA (PolyAcryl) polymer according to the general Formulas (1), (2), (10) and (12) wherein the crosslinked polymeric nanoparticle is formed by cross-linking the PA polymer and wherein the crosslinked polymeric nanoparticle further comprises biologically active components selected from the group of transposons, transposases and/or plasmids and minicircles comprising transposons and/or transposases. The crosslinked polymeric nanoparticles, plasmids and minicircles can further comprise chimeric antigen receptors and T-cell receptors.

[0077] The present invention also relates to processes to prepare the crosslinked polymeric nanoparticles comprising the biologically active components of the present invention as well as to such crosslinked polymeric nanoparticles made by the processes.

[0078] The transposases, chimeric antigen receptors and T-cell receptors of the crosslinked polymeric nanoparticles can be in DNA, RNA or protein forms.

[0079] The polymers, nanoparticles and crosslinked polymeric nanoparticles comprising the biologically active components according to the present invention can have important advantages as they can be stable in dissolved and dispersed form. The polymers, nanoparticles and crosslinked polymeric nanoparticles according to the present invention can also be storage stable. The solutions of the polymers, nanoparticles and crosslinked polymeric nanoparticles can be frozen without losing their integrity and can be freeze-dried to a powder form which can easily be reconstituted to a solution without loss of activity or integrity. [0080] The polymers, nanoparticles and crosslinked polymeric nanoparticles of the present invention may be functionalized with peptides containing microtubule-associated sequences (MTAS) and nuclear localization signals (NLS) as a means to facilitate nuclear import of their genetic cargo via the microtubule transport machinery by processes known in the art (see, e.g., Narayanan K., et ah, Sci Rep. 2013; 3:2184, which is incorporated in its entirety herein by reference).

[0081] The polymers, nanoparticles and crosslinked polymeric nanoparticles of the present invention may be further engineered to minimize off-target binding by anchoring cell specific targeting ligands to their surfaces and by shielding the payloads they carry with a negatively charged coating, for example a polyglutamic acid (“PGA”) or hyaluronic acid (“HA”) coating.

[0082] The crosslinked polymeric nanoparticles of the present invention are prepared by cross- linking the PA polymer according to general Formulas (1), (2), (10) and (12) wherein the cross- linking is preferably conducted by UV radiation, preferably UV radiation with a wave length of about 365 nm. The cross-linking reaction is preferably performed in the presence of a photo initiator. The cross-linking reaction is also preferably performed in a water-in-oil emulsion. It is further preferred that the cross-linking reaction is performed at a pH of less than 7, preferably less than about 6. Preferably, the pH is higher than about 1, preferably higher than about 2.

[0083] The present invention therefore also relates to a crosslinked polymeric nanoparticle which is obtainable by the above described process which comprises the step of cross-linking a PA polymer according to general Formulas (1), (2), (10 and (12), preferably by subjecting the PA polymer to UV radiation.

[0084] Methods for preparing the nanoparticles are also known in the art and, in particular, methods for preparing the nanoparticles are also described in US Patent No. 9,012,424.

Loaded Polymers, Nanoparticles and Crosslinked Polymeric Nanoparticles

[0085] According to the invention, it is preferred that the polymers, nanoparticles and crosslinked polymeric nanoparticles further comprise a biologically active component selected from the group of transposons, transposases, plasmids and minicircles described herein.

[0086] The loading of the polymers, nanoparticles and crosslinked polymeric nanoparticles with the biologically active component s) is performed by contacting the biologically active

component s) with the polymers, nanoparticles or crosslinked polymeric nanoparticle in an aqueous solvent system, wherein the aqueous solvent system preferably has a physiological pH. Methods for loading polymers, nanoparticles and crosslinked polymeric nanoparticles are known in the art and, in particular, methods for loading the crosslinked polymeric nanoparticles are also described in US Patent No. 9,012,424.

Biologically Active Components

Transposons. Transposases. Plasmids and Minicircles

[0087] The biologically active components of the present invention comprise transposon and transposase gene delivery systems for introducing nucleic acids into the DNA of a cell. Such gene delivery systems may comprise vectors, cDNA, mRNA, plasmids and minicircles known in the art. In particular embodiments, the nucleic acid includes a sequence (e.g., a gene) for expressing a gene editing agent or phenotype-altering protein. Suitable vectors for introducing the biologically active components into cells are preferably plasmids and more preferably standard plasmid and minicircle plasmids that can be used to transfer a gene to a cell. The nucleic acids (e.g., minicircle plasmids) can further include any additional sequence information to facilitate expression in a selectively modified cell. The plasmids and minicircle plasmids are generally well known in the art and can be prepared using conventional techniques. For example, see Kobelt et ah, Mol Biotechnol (2013) 53:80-89; Mayrhofer, et ah, The J Gene Med 2008; 10: 1253-1269; US Patent Nos. 6,143,530, 6,492,164, 8,647,863, 8,911,974, 9,644,211, US Pub. No. US 2006/0211117 and International Published Application WO 2017/158019 all of which are incorporated herein in their entirety by reference. Preferably, the polymer, nanoparticle and crosslinked polymeric nanoparticle genetic material is in the form of minicircles. Preferably, the minicircles are those described in US Patent Nos. 8,647,863, 8,911,974, 9,644,211. Preferably, the minicircles comprise a chimeric antigen receptor as described herein. Preferably, the mini circles further comprise an EFla promoter, WPRE, a PolyA signal, a SB11 transposase and a pT2 transposon. Preferably the chimeric antigen receptor comprises a muring single-chain antibody fragment (scFv) specific for CD 19, a CD8 hinge, a transmembrane region fused to the intracellular signaling domains for 4-1BB (CD37) and CD3 zeta. Preferably the polymers, nanoparticles and crosslinked polymeric nanoparticles comprise a minicircle-DNA chimeric antigen receptor transposon cassette with SB 11 transposase in mRNA format.

[0088] The polymers, nanoparticles and crosslinked polymeric nanoparticles, plasmids and minicircles of the present invention comprise a transposon/transposase system for stably introducing nucleic acid(s) into the genome of a cell. In particular, the transposon/transposase system may comprise the transposase of the transposon system Sleeping Beauty (SB) and gene transfer systems containing the SB system for stably introducing nucleic acid(s) into the DNA of a cell by using the transposase of the transposon system. Various Sleeping Beauty transposons/transposases are known in the art (for example see, Singh, et al., Immunol Rev. 2014 January; 257(1): 181-190; US Patent Nos. 6,489,458, 7,160,682, 8,227,432 and 9,228, 180 and International Published Application WO 2017/158029 all of which are incorporated herein in their entirety by reference). Preferably the transposase is the SB11 transposase in mRNA format. Preferably the transposon is the pT2 transposon. Other transposon/transposase systems for gene delivery, such as CRISPR-cas9, piggyBac and Tol2 are also known in the art (for example, see J. Tipanee, et al., Bioscience Reports, Dec 05, 2017, 37(6); European Patent Publication WO 2020/099301; and US Patent No. 9,790,490 incorporated herein in their entirety by reference). In addition, other genome-editing systems such as designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases are known for producing targeted genomic perturbations.

CARs and TCRs

[0089] The biologically active components of the present invention may also comprise chimeric antigen receptors (CARs) or T-cell receptors (TCRs). CARs and TCRs refer to synthetically designed receptors including at least a binding domain and an effector domain and optionally a spacer domain and/or a transmembrane domain. In particular embodiments, the binding domain is a single chain variable fragment (scFv) of a monoclonal antibody. Preferably, the scFv targets a CD 19 antigen. Most preferably, the biologically active component comprises a CD 19 CAR, an EFla promoter, WPRE, a Poly A signal and the pT2 transposon. Most preferably the CAR comprises a scFv specific for CD 19, a CD8 hinge region and a transmembrane region that is fused to the intracellular signaling domains for 4-1BB (CD37) and CD3 zeta. Such CARs and TCRs are well known in the art. The plasmids and minicircles may also comprise genes encoding CARs or TCRs. Chimeric antigen receptors and T-cell receptors and methods of transfecting cells with CARs and TCRs are generally known in the art (for example, see US Patent No. 7,741,465 (Eshhar);

US2013/0287748 (UPenn/June); US2014/0271635 (UPenn/June); US2015/0306141 (Kite); Eshhar, Human Gene Therapy 25:773-778 (September 2014); Maus, et al., Blood, 24 April 2014, Volume 123, Number 17; Sadelain, et al., Cancer Discovery 2013; 3 :388-398; Eshhar, et al., Proceedings of the National Academy of Sciences of the United States of America, vol. 90, no. 2, pp. 720-724,

1993; Han, et al., J Hematol Oncol. 2013; 6:47; Geiger, et al., Blood, vol. 98, no. 8, pp. 2364-2371, 200; Finney, et al., J. Immunol. 2004; 172: 104-113; Imai, et al., Leukemia 2004; 18: 676-684;

Brentjens, et al., Clinical Cancer Research, vol. 13, no. 18, pp. 5426-5435, 2007; Pule, et al.,

Molecular Therapy, Vol. 12, No. 5, pp. 933-941, November 2005; Tammana, et al., Hum Gene Ther 2010; 21 : 75-86; Hombach, et al., Int. J. Cancer, 8: 115-120 (2000); Nolan et al.; Clinical Cancer Research, 5:3928-3941 (1999); and Narayanavari, et al., Cell & Gene Therapy Insights 2017 all of which are incorporated herein in their entirety by reference).

[0090] Plasmids containing CARs with the Sleeping Beauty transposon/transposase system and their use in clinical trials to introduce CAR transgenes into cells are also known in the art (see, e.g., Kebriaei P., et al., J. Clin Invest. 2016; 126:3363-3376, which is incorporated in its entirety herein by reference).

Surface Modification

[0091] The polymers, nanoparticles and crosslinked polymeric nanoparticles according to the present invention, either in loaded form or in unloaded form, may be further functionalized by surface modification. Methods for surface modification of polymers, nanoparticles and crosslinked polymeric nanoparticles are known in the art and, in particular, methods for surface modification of the crosslinked polymeric nanoparticles are also described in US Patent No. 9,012,424.

[0092] According to a preferred embodiment of the surface modification, the crosslinked polymeric nanoparticle that is obtainable by cross-linking PA polymers according to Formulas (1) or (2), wherein R3 is independently selected from the group consisting of:

(al) substituted Ci - Cio alkyl, wherein the alkyl group may be linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

(bl) substituted C3 - C12 cycloalkyl, wherein the cycloalkyl group is optionally (partly) unsaturated and/or optionally comprises one, two or three heteroatoms in the ring, wherein the heteroatoms are independently selected from O, N and S;

(cl) substituted Ce - C12 aryl;

(dl) substituted O, - C12 heteroaryl, wherein the heteroaryl group comprises one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

(el) substituted C7 - C14 alkylaryl wherein an alkyl part of the alkylarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

(fl) substituted C7 - C 14 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and/or wherein an alkyl part of the alkylheteroarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

[0093] wherein the above groups (al) - (fl) comprise a functional group (FG) as a substituent that is capable of forming a covalent bond with a complementary functional group (CFG) of a reagent for surface modification R and wherein the crosslinked polymeric nanoparticles comprise a biologically active component, is reacted with said reagent R according to the process:

crosslinked polymeric nanoparti cle-FG + R-CFG

[0094] The group CFG is capable of forming a covalent bond with the group FG. Suitable examples for groups FG and CFG are well known to the person skilled in the art. For example, the FG can be a -COOH group whereas the CFG group is a -ML· group.

[0095] According to a preferred embodiment, the functional group FG is selected from the group consisting of a group selected from -OH, -OR⁷, -ML·; -NH(R⁷), -N(R⁷)2, -C(0)OR⁷, -C(0)R⁷, - C(0)M1R⁷, and -C(0)1S[R⁷2, wherein R⁷ is independently selected from the group consisting of:

(a") H;

(d") Ce - C12 aryl, wherein the aryl group is optionally substituted;

(e") Ce - C12 heteroaryl, wherein the heteroaryl group comprises one, two or three

heteroatoms independently selected from O, N and S and/or wherein the heteroaryl group is optionally substituted;

(f") C7 - C 14 alkylaryl wherein the alkylaryl group is optionally substituted and/or wherein an alkyl part of the alkylarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S; and

(g") C7 - C14 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and/or wherein the alkylheteroarylene group is optionally substituted, and/or wherein an alkyl part of the alkylheteroarylene group is linear or branched and is optionally (partly) unsaturated and/or is optionally interrupted by one, two or three heteroatoms, wherein the heteroatoms are independently selected from O, N and S. [0096] According to another preferred embodiment of the surface modification, the crosslinked polymeric nanoparticle that is obtainable by cross-linking PA polymers according to Formulas (10) or (11), is reacted with a reagent for surface modification R’ according to the process:

crosslinked polymeric nanoparticle + R’-SH

[0097] The groups R and R’ are preferably 2-thioethyl, 2-hydroxyethyl, and PEG (polyethylene oxide) residues or PPO (polypropylene oxide) residues having a number average molecular weight Mn of about 500 to about 10000. As will apparent to those skilled in the art, the reagent R’-SH can also react with the -S-S- groups (when present) in the crosslinked polymeric nanoparticle as is described above for PA polymers according to Formulas (1), (2) (10) and Formula (11).

[0098] The present invention therefore further relates to surface modified crosslinked polymeric nanoparticles which are obtainable by a first process comprising the steps of:

(1) cross-linking a PA polymer according to general Formulas (1), (2), (10) or (11), preferably by subjecting the PA polymer to UV radiation to form a crosslinked polymeric nanoparticle;

(2) loading the crosslinked polymeric nanoparticle with a biologically active component selected from the group of transposons, transposases, plasmids and minicircles as described herein; and

(3) reacting the crosslinked polymeric nanoparticle with a reagent R-CFG or R’-SH.

[0099] The surface modified crosslinked polymeric nanoparticles are also obtainable by a second process comprising the steps of:

(G) cross-linking a PA polymer according to general Formulas (1), (2), (10) or (11), preferably by subjecting the PA polymer to UV radiation to form a crosslinked polymeric nanoparticle;

(2’)reacting the crosslinked polymeric nanoparticle with a reagent R-CFG or R’-SH; and

(3’) loading the crosslinked polymeric nanoparticle with a biologically active component selected from the group of transposons, transposases, plasmids and minicircles as described herein.

[00100] The surface modified crosslinked polymeric nanoparticles are also obtainable by a third process comprising the steps of:

(1") reacting the crosslinked polymeric nanoparticles with a reagent R-CFG or R’-SH thereby forming a functionalized crosslinked polymeric nanoparticle;

(2") cross-linking the functionalized crosslinked polymeric nanoparticle, preferably by subjecting the functionalized crosslinked polymeric nanoparticle to UV radiation to form a crosslinked polymeric nanoparticle; and

(3") loading the crosslinked polymeric nanoparticle with a biologically active component selected from the group of transposons, transposases, plasmids and minicircles as described herein. [00101] In the step (1) of the first process and step ( ) of the second process, not all acrylate groups need to be cross-linked so that non-cross-linked acrylate groups can be functionalized in step (3) of the first process and step (2’) of the second process, respectively. Likewise, in step (1”) of the third process, not all acrylate groups need to be functionalized to that non-cross-linked acrylate groups can still be cross-linked. Accordingly, the product obtained in either of these three processes may be very complex. According to the present invention, it is even preferred that when first a functionalization step is carried out not all of the acrylate groups are functionalized thereby enabling further cross-linking of the remaining acrylate groups. Likewise, it is also preferred when first a cross-linking step is carried out not all acrylate groups are cross-linked thereby enabling further functionalization of the remaining acrylate groups. A person skilled in the art will be well capable to select appropriate reaction conditions, in particular by selecting appropriate molar ratios of reactants and starting materials, to control the degree of cross-linking and functionalization. In addition, and as described above, there are other reactive groups within the PA polymers, e.g. -S-S- groups, that may be functionalized in conjunction with the acrylate groups.

[00102] In particular embodiments, the surface modification ot the polymers, nanoparticles and crosslinked polymeric nanoparticles according to the present invention, either in loaded form or in unloaded form, may include a coating the shields the encapsulated biologically active components and reduces or prevents off-target binding. Off-target binding is reduced or prevented by reducing the surface charge of the polymers, nanoparticles and crosslinked polymeric nanoparticles to neutral or negative. As such, surface modification can include neutral or negative polymer- and/or liposomal-based coatings. Particular embodiments utilize polyglutamic acid (PGA) as a surface modification coating. Examples of neutrally charged surface modification coatings include polyethylene glycol (PEG), polypropylene glycol), and polyalkylene oxide copolymers known in the art. When used, the coating need not necessarily coat the entire surface of the polymers, nanoparticles and crosslinked polymeric nanoparticles, but should be sufficient to reduce off-target binding by the polymers, nanoparticles and crosslinked polymeric nanoparticles.

[00103] Particular embodiments include polymers, nanoparticles and crosslinked polymeric nanoparticles according to the present invention that can be targeted to specific selected cells. As such, the biologically active component are delivered to and expressed by one or more selected cell populations. Selected cell targeting ligands can include surface-anchored targeting ligands that selectively bind the polymers, nanoparticles and crosslinked polymeric nanoparticles according to the present invention to selected cells. Selected cell targeting ligands can include antibodies, scFv antibodies and proteins, peptides and/or aptamers. Preferably, the antibodies include whole antibodies, or binding fragments of an antibody, e.g., Fv, Fab, Fab’, F(ab’)2, Fc, and single chain Fv fragments (scFvs) or any biologically effective fragments of an immunoglobulin that bind specifically to a motif expressed by the target cells. Antibodies an antigen binding fragments include all or a portion of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, bispecific antibodies, mini bodies, and linear antibodies. Preferably, the cell targeting ligands are human or humanized anti-CD3 Fab2 fragments anchored to a PGA surface modification coating.

[00104] Applications

[00105] The polymers, nanoparticles and crosslinked polymeric nanoparticles according to the present invention are very suitable as delivery carriers for biologically active components because of their high loading capacity, high stability and responsiveness to environmental factors, such as ionic strength, reduction potential, pH and temperature. In particular, the crosslinked polymeric nanoparticles according to the present invention can have additional benefits for delivery of biologically active components over self-assembled polymeric nanoparticulate systems as delivery vehicles, since in the case of the crosslinked polymeric nanoparticles of the present invention all polymeric constituents are incorporated into the delivery crosslinked polymeric nanoparticle whereas in the self-assembled polymeric nanoparticulate systems usually excess of free polymer in solution is needed due to the equilibrium conditions. This frequently causes undesired cytotoxicity effects and upon dilution of the formulation, destabilization of the nanoparticles.

[00106] The present invention also relates to cells transfected with the polymers, nanoparticles and crosslinked polymeric nanoparticles of the present invention and methods of producing such transfected cells. The method comprises introducing the polymers, nanoparticles and/or crosslinked polymeric nanoparticles of the present invention into said cells. Such methods may be carried out in vitro or in vivo.

[00107] Another aspect of the present invention are kits comprising the polymers, nanoparticles or crosslinked polymeric nanoparticles of the present invention.

[00108] Referring to Figs. 1 and 2, there is shown the percentage of GFP-positive cells and their MFI as a function of time after transfection, respectively. In the first days after transfection, the mock-supplemented pT-GFP-transfected cells contained more GFP-positive cells and had a higher MFI. However, the transfection of these cells was transient and there were no GFP-positive cells remaining by day 14. In contrast, the cells that were transfected with the combination of pT-GFP and the SB 11 transposase plasmid showed a lower initial transfection efficiency, but a GFP-positive population persisted until day 21. At day 21, 12.7% of the COS-7 cells were GFP-positive. These data show that the combined delivery of pT-GFP transposon and SB11 transposase pDNA by the crosslinked polymeric nanoparticles resulted in stable gene expression, indicating that stable gene integration into the genome had occurred.

[00109] Figure 3 shows the cell viability of transfected cells in time after transfection. One day after transfection the cell viability was reduced to 70-80% of the non-transfected control. In time, the relative cell viability remained stable, suggesting that the growth rate of transfected cells was similar to non-transfected cells.

[00110] Figure 4 shows the relative luminescence units (RLU) of cell lysates after transfection with Luciferase minicircle and plasmid DNA. Transfection with equimolar amounts of minicircle and plasmid DNA resulted in similar RLU values, thereby showing that minicircle DNA was functional and delivered successfully by the crosslinked polymeric nanoparticles.

[00111] Another aspect of the present invention relates to methods of treating diseases and disorders with cells genetically engineered with the polymers, nanoparticles and crosslinked polymeric nanoparticles comprising biologically active components of the present invention.

Preferably the genetically engineered cells are T-cells, NK-cells, CIK cells and/or macrophages. Preferably the disease or disorder is cancer, immune diseases, autoimmune diseases, inflammatory diseases, infectious diseases and genetic disorders. Preferably, the cancer is a B cell malignancy (e.g., acute lymphoblastic leukemia (ALL) and diffuse B cell Lymphoma (DCBL)). The genetically engineered cells can also be used to target infected cells displaying viral antigens, including hepatitis B, hepatitis C, cytomegalovirus, and human immunodeficiency virus and to treat associated diseases and disorders. The genetically engineered cells can also be used to target anti-Dsg3 antibody- producing B cells, which are pathogenic for pemphigus vulgaris and to treat associated diseases and disorders. The genetically engineered cells can also be used to target cells with auto-reactivity against auto-antigens and to treat associated diseases and disorders. The genetically engineered cells can also be used for controlling auto-immune diseases and graft-versus-host disease. In addition, such diseases or disorders may be treated by infusing the polymers, nanoparticles and crosslinked polymeric nanoparticles comprising biologically active components of the present invention to a subject in need thereof (in-situ or in-vivo - within the patient) or cells may be genetically engineered with the polymers, nanoparticles and crosslinked polymeric nanoparticles comprising biologically active components of the present invention ex-vivo and subsequently infused into a subject in need thereof. The ex-vivo treatment can be autologous (using the subjects cells) or allogeneic (using cells from a donor). [00112] Examples

Materials and Methods

[00113] Delivery of pT-GFP transposon and SB11 transposase pDNA to COS-7 cells by crosslinked polymeric nanoparticles

[00114] One day prior to transfection, COS-7 cells were plated in 48-wells plates at a density of 8xl0⁴ cells/ml in 200 mΐ. Cells were grown in DMEM + 10% FBS and incubated at 37°C with 5% CO2. pT-GFP transposon, SB11 transposase and mock (pCMV-Luc) pDNA were diluted to 120 pg/ml in water. pT-GFP was mixed with either SB 11 or mock pDNA at a 3 : 1 w/w ratio. pDNA mixtures were added to an equal volume of crosslinked polymeric nanoparticles (e.g, p(CBA- ABOL)/01igoamine Nanogel) at a crosslinked polymer nanoparti cle:pDNA w/w ratio of 25 : 1.

Loaded crosslinked polymeric nanoparticles were equilibrated at room temperature for 15 minutes. Medium on COS-7 cells was changed to serum-free DMEM and 6 pg loaded crosslinked polymeric nanoparticles were added per well in triplicate. Serum-containing DMEM (200 mΐ) was added after four hours, without removal of the crosslinked polymeric nanoparticle-containing medium.

[00115] Transfection efficiency and cell viability were analyzed on day 1, 2, 3, 4, 7, 14 and 21 after transfection. Replicates of transfected cells were analyzed on day 1, 2, 3 and 4. At day 4, 7, 10, 14 and 17 cells were passaged and subsequent analyses were performed on the passaged cells.

[00116] Transfection efficiency was analyzed by flow cytometry. Cells were trypsinized and washed in DMEM + 10% FBS. The percentage of GFP-positive cells and their mean fluorescence intensity (MFI) were measured on a FACS Calibur and analyzed using Flowing Software v2.5.1.

[00117] Cell viability was analyzed by MTT assay. Cells were incubated with 0.5 mg/ml MTT in colorless DMEM for 30 to 60 minutes (depending on cell density on day of analysis). The produced formazan was dissolved in DMSO and quantified by its absorbance at 540 nm using a Tecan Infinite Pro plate reader.

[00118] Delivery of Luciferase minicircle and plasmid DNA to COS-7 cells by crosslinked polymeric nanoparticles

[00119] One day prior to transfection, COS-7 cells were plated in a 48-wells plate at a density of 8xl0⁴ cells/ml in 200 mΐ. Cells were grown in DMEM + 10% FBS and incubated at 37°C with 5% CO2. Crosslinked polymeric nanoparticles (e.g, p(CBA-ABOL)/01igoamine Nanogel) were loaded with Luciferase minicircle and plasmid DNA at equimolar ratios, that effectively corresponded to a crosslinked polymeric nanoparticles:DNA w/w ratio of 83 : 1 for the minicircle and 50: 1 for the plasmid. The crosslinked polymeric nanoparticles were loaded by addition of DNA to an equal volume of crosslinked polymeric nanoparticles and loaded crosslinked polymeric nanoparticles were equilibrated at room temperature for 15 minutes. Medium on COS-7 cells was changed to serum- free DMEM and 6 pg loaded crosslinked polymeric nanoparticles were added per well in triplicate. Serum-containing DMEM (200 mΐ) was added after four hours, without removal of the crosslinked polymeric nanoparticle-containing medium.

[00120] Luciferase expression was analyzed three days after transfection using the Pierce™ Firefly Luciferase Glow Assay Kit. Cells were washed in PBS and lysed in 100 mΐ lysis buffer for 30 minutes on a plate shaker. Remaining cell clumps were triturated and lysates were kept on ice from here on. Lysates (10 mΐ) were transferred to a white 96-well plate and 50 mΐ D-Luciferin working solution was added. After 10 minutes incubation at room temperature, luminescence was measured using a Tecan Infinite Pro plate reader. Luminescence was corrected for total protein content of the lysates. To measure protein concentration, 200 mΐ BCA working solution (Pierce™ Rapid Gold BCA Protein Assay Kit) was added to 20 mΐ cell lysate. After 5 minutes incubation at room temperature, 480 nm absorbance was measured on a Tecan Infinite Pro plate reader.

[00121] Human PBMCs transfected with nanoparticles targeted to T cells via coating with an anti-CD3 F(ab’)2-PGA conjugate

[00122] Figure 5 shows flow cytometry results of human PBMCs transfected with nanoparticles targeted to T cells via coating with an anti-CD3 F(ab’)2-PGA conjugate. Crosslinked polymeric nanogel (NG-PAA, e.g., NGQ06) was loaded with eGFP mRNA. The loaded nanogel was then coated with PGA containing PGA-conjugated to Anti-CD3 F(ab’)2 antibody. Both anti-human CD3 and anti-murine CD3 antibodies were used. Nanoparticles were composed of the crosslinked polymeric nanogel (NG-PAA, e.g., NGQ06) with an F(ab’)2 antibody fragment (OKT3 for human CD3, and 145-2C11 for murine CD3) that was conjugated to PGA using click chemistry. F(ab’)2 antibodies were linked to NHS-PEG4-DCBO and then cross-linked to PGA-azide). The coating ratios onto the nanogel were 1 :0.1, 1 :0.3, and 1.1.0 (NG-PAA, e.g., NGQ06 Nanogel :F(ab’)2). Human PBMCs were then transfected with the preparations, then stained with anti-CD3-PE where GFP expression and PE fluorescence were analyzed by flow cytometry 24 hrs later. Results for the 1 :0.1 ratio are shown.

[00123] Results show that GFP expression (shown in“R-4” box along X-axis) was observed only in human PBMCs transfected with the nanoparticles targeted with the anti-human CD3 antibody and not with the anti-murine CD3 antibody. Furthermore, the anti-CD3 PE signal (purple, Y-axis) was decreased only when the human PBMCs were incubated with the anti-human CD3 nanoparticles and not with the anti-murine CD3 or non-targeted nanoparticles. The lower signal in this case is attributed to specific finding of the human CD3 -targeted nanoparticles, which decreased binding of the anti-CD3-PE staining reagent.

[00124] Figure 6 shows similar results using two nanogel preparations (NG-PAA, e.g., NG-1 and NGQ6). As is evident from the GFP-positive cell counts, the NGQ6 nanogel used for mRNA encapsulation was more efficient than the NG01 nanogel in transfecting the human PBMCs. The optimal ratio of nanogel to F(ab’)2 was 1 :01. Furthermore, species specificity of the targeting F(ab’)2 was evident as the human PBMCs were transfected only with the anti-human antibody and not with the anti-mouse antibody or uncoated nanogels.

[00125] It will be appreciated by those skilled in the art that changes could be made to the exemplary embodiments shown and described above without departing from the broad inventive concepts thereof. It is understood, therefore, that this invention is not limited to the exemplary embodiments shown and described, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the claims. For example, specific features of the exemplary embodiments may or may not be part of the claimed invention and various features of the disclosed embodiments may be combined. Unless specifically set forth herein, the terms“a”,“an” and“the” are not limited to one element but instead should be read as meaning“at least one”.

[00126] It is to be understood that at least some of the figures and descriptions of the invention have been simplified to focus on elements that are relevant for a clear understanding of the invention, while eliminating, for purposes of clarity, other elements that those of ordinary skill in the art will appreciate may also comprise a portion of the invention. However, because such elements are well known in the art, and because they do not necessarily facilitate a better understanding of the invention, a description of such elements is not provided herein.

[00127] Further, to the extent that the methods of the present invention do not rely on the particular order of steps set forth herein, the particular order of the steps should not be construed as limitation on the claims. Any claims directed to the methods of the present invention should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the steps may be varied and still remain within the spirit and scope of the present invention.

Claims

CLAIMS I/we claim:

1. A polymer having the general Formula (1) or (2):

Formula (1) Formula (2)

wherein R¹, R², R³, R⁴, R⁵, A, POL, a and b are as defined herein; and wherein the polymer comprises one or more transposons and/or transposases.

2. The polymer of claim 1 comprising chimeric antigen receptors and/or T-cell receptors.

3. The polymer of claim 1, wherein the transposons and/or transposases are in the form of plasmids and/or minicircles.

4. The polymer of claim 2, wherein the chimeric antigen receptors and/or T-cell receptors are in the form of plasmids and/or minicircles.

5. The polymer of claim 1 or claim 2 wherein the transposases, chimeric antigen receptors and T-cell receptors are in the form of DNA, RNA or protein.

6. The polymer of one of claims 1-4, wherein the transposase comprises the transposase of the transposon system Sleeping Beauty (SB).

7. The polymer of claim 5, wherein the transposase comprises the transposase of the transposon system Sleeping Beauty (SB).

8. A nanoparticle having the general Formula (3) or (4):

Formula (4)

wherein R¹, R², R³, R⁴, R⁵, R⁶, A, POL, FG, a and b are as defined herein; and wherein the nanoparticle comprises one or more transposons and/or transposases.

9. The nanoparticle of claim 8 comprising chimeric antigen receptors and/or T-cell receptors.

10. The nanoparticle of claim 8, wherein the transposons and/or transposases are in the form of plasmids and/or minicircles.

11. The nanoparticle of claim 9, wherein the chimeric antigen receptors and/or T-cell receptors are in the form of plasmids and/or minicircles.

12. The nanoparticle of claim 8 or claim 9 wherein the transposases, chimeric antigen receptors and T-cell receptors are in the form of DNA, RNA or protein.

13. The nanoparticles of one of claims 8-11, wherein the transposase comprises the transposase of the transposon system Sleeping Beauty (SB).

14. The nanoparticles of claim 6, wherein the transposase comprises the transposase of the transposon system Sleeping Beauty (SB).

15. The nanoparticles of claim 12, wherein the transposase comprises the transposase of the transposon system Sleeping Beauty (SB).

16. A crosslinked polymeric nanoparticle comprising the PA polymer of general Formula (1) or

(2):

Formula (2)

wherein R¹, R², R³, R⁴, R⁵, A, POL, a and b are as defined herein; and wherein the crosslinked polymeric nanoparticle further comprises one or more transposons and/or transposases.

17. The crosslinked polymeric nanoparticle of claim 16 comprising chimeric antigen receptors and/or T-cell receptors.

18. The crosslinked polymeric nanoparticle of claim 16, wherein the transposons and/or transposases are in the form of plasmids and/or minicircles.

19. The crosslinked polymeric nanoparticle of claim 16, wherein the chimeric antigen receptors and/or T-cell receptors are in the form of plasmids and/or minicircles.

20. The nanoparticle of claim 16 or claim 17 wherein the transposases, chimeric antigen receptors and T-cell receptors are in the form of DNA, RNA or protein.

21. The crosslinked polymeric nanoparticle of one of claims 16-19, wherein the transposase comprises the transposase of the transposon system Sleeping Beauty (SB).

22. The crosslinked polymeric nanoparticle of claim 20, wherein the transposase comprises the transposase of the transposon system Sleeping Beauty (SB).

23. Pharmaceutical formulations comprising the polymers, nanoparticles and crosslinked polymeric nanoparticles of one of claims 1, 8, and 16.

24. Cells modified with the polymers, nanoparticles and crosslinked polymeric nanoparticles of one of claims 1, 8, and 16.

25. Methods of treating a subject in need thereof, comprising administering the polymers, nanoparticles and crosslinked polymeric nanoparticles of one of claims 1, 8, and 16 to the subject.

26. The method of claim 25, wherein the subject is a human.

27. The method of claim 25 wherein the treatment is treatment of cancer, immune disorder, autoimmune disorder, inflammatory diseases, viral infectious diseases and genetic disorders.

28. The method of claim 26 wherein the treatment is treatment of cancer, immune disorder, autoimmune disorder, inflammatory diseases, viral infectious diseases and genetic disorders.