Means and methods for delivery of agents to immune effector cells

Abstract

The present invention relates to functional delivery of nucleic acid to (resting) T-cells of mRNA and DNA, i.e. delivery resulted in translation from the mRNA and / or allowed for integration and / or expression from the delivered DNA. This delivery is achieved using nanoparticles, carrying a targeting moiety binding a T-cell, and, another moiety activating a T-cell. Preferably lipid nanoparticles are used, and advantageous compositions provided. These aspects of the invention, provide for highly efficient delivery of nucleic acid to T-cells, including resting T-cells. The nanoparticle technology thus provided herein allows for highly advantageous medical treatments, wherein a (human) patients whole blood can be treated (e.g. ex vivo), or wherein a patient can be administered with nanoparticles such as described directly into the bloodstream, therewith effectively delivering nucleic acid encoding therapeutic genes of interest to resting T-cells.

Description

[0001] Title: Means and methods for delivery of agents to immune effector cells

[0002] Introduction

[0003] In recent years, pharmaceutical products have been developed for the delivery of agents such as nucleic acids to cells of a subject. For example, recently, vaccines for COVID-19 have been developed based on lipid nanoparticles comprising RNA agents, such as tozinameran. Approved products of lipid nanoparticles comprising siRNAs, such as patisiran have furthermore been developed. In addition to nanoparticles, gene therapy vector products have been made and approved for (stable) delivery of transgenes, based e.g. on AAV or lentivirus. Hence, vehicles, such as lipid nanoparticles and viral vectors, have been developed that can carry an agent such as a DNA or an RNA molecule, which can be used to deliver such molecules to cells of a subject. Such delivery subsequently serves for evoking an immune response or treatment of a disorder in the subject, e.g. by being transcribed and / or translated into a therapeutic or antigenic protein, or, when an siRNA is delivered, by reducing expression of a target gene. Moreover, lipid nanoparticles are and may likewise be used to deliver further useful or therapeutic agents other than nucleic acids such as small molecules to cells of interest.

[0004] Nevertheless, delivery of an agent, such as a nucleic acid which includes an mRNA or DNA, remains a challenge. The delivery vehicles containing the agent, serve to protect the agents, e.g. from degradation, and are to facilitate uptake of the agent by cells and delivery thereof in the cell reaching the site within the cell where these are to have its action. For example, when the agent is a DNA encoding a transgene, the DNA is to be delivered across the cellular membrane and subsequently is to be transported to the nucleus where the transcription machinery of the cell resides. Delivery to the nucleus is a hurdle for compounds such as DNA. When the agent is to be released in the cytoplasm, such as with an mRNA, to have its action in the cytoplasm, the mRNA needs to be delivered across the cellular membrane and released, e.g. via endosomal escape, in the cytoplasm. Furthermore, delivery of agents to defined target cells remains to be a challenge as well. In spite of advancements made in the art, there is still a need in the art for improved delivery of agents to cells. There is a need in the art to provide for targeted delivery of agents to cells, in particular of nucleic acids such as DNA.

[0005] Summary

[0006] The present inventors now provide for highly improved nanoparticles, such as LNP formulations, that allows for highly efficient delivery of DNA to T-cells. Such highly improved nanoparticles allow for such efficient delivery to T-cells that CAR-T cells can be generated both ex vivo and in vivo, which are shown to expand and which can effectively control and eradicate cancer cells. The highly improved nanoparticles allow for a very low amount of nucleic acid required to generate functional and therapeutically effective CAR-T-cells.

[0007] First, of all, it was shown that the combining a targeting moiety for binding a T-cell and a T-cell activation moiety at the surface of a nanoparticle, allowed for highly efficient delivery of nucleic acid, including DNA and expression thereof, to both resting and activated T-cells. A highly preferred and advantageous targeting moiety for binding a T-cell includes targeting moieties capable of binding CD7. A highly preferred advantageous T-cell activation moiety includes T-cell activation moieties capable of binding the T-cell receptor complex. For example, LNPs targeting CD7 which is expressed on T cell, via a conjugated VHH as described herein, and targeting the T-cell receptor complex (CD3E, CD3y, CD35, chain, a chain p chain), e.g. with a scFv for CD3E as a T-cell activation moiety, were shown to have highly improved transfection efficiency over targeting CD7 alone or CD3 alone. The combination of a targeting moiety for binding a T-cell and a T-cell activation moiety at the surface of a nanoparticle, were shown to work synergistically. Particular densities both moieties combined and relative ratios thereof were shown to be even more advantageous.

[0008] Moreover, various compositions of lipid nanoparticles were furthermore intensively analyzed. Lipid nanoparticles comprise ionizable lipid, helper lipid, PEG-lipid and a sterol. Highly surprisingly it was observed that in the context of present invention, delivery of nucleic acid to (resting) T-cells, branched ionizable lipids were preferred, and helper lipids with a PC head and a saturated tail were preferred as well. Moreover, the present inventors have observed that it appears to be very beneficial to have at least one branched tail and / or a spacer length (S) of at least 5 in an ionizable lipid. Suitable branched ionizable lipids can be selected from SM102, Lipid A9, ALC-0315, Lipid 5, CL4F8_[35, and CL4F12_E-6. A suitable branched ionizable lipids can be SM102 or CL4F8_[35. In particular suitable helper lipids are selected from DSPC, DMPC, PDDC and DAPC. Such LNP compositions comprising these lipids were find to be highly advantageous for delivery of nucleic acid to (resting) T-cells, i.e. transfection. Delivery of nucleic acid to a cell, may be referred to as transfection.

[0009] The delivery of nucleic acid to (resting) T-cells of mRNA or of DNA was shown to be functional, i.e. resulted in translation from the mRNA and resulted in expression from the transfected DNA. These aspects of the invention, preferably combined, provide for highly efficient delivery of nucleic acid to T-cells, including resting T-cells. The nanoparticle technology thus provided herein allows for highly advantageous medical treatments, wherein a (human) patients whole blood can be treated (e.g. ex vivo), or wherein a patient can be administered with nanoparticles such as described herein directly into the bloodstream, therewith effectively delivering nucleic acid to resting T-cells. When DNA constructs are delivered using such nanoparticles, combined with a means for integration of the DNA constructs in the resting T- cells, highly advantageously, CAR-T cells are generated which have stably integrated into their genome the DNA construct, which CAR-T cells have the capacity to clonally expand and control and / or eradicate cancer in a subject. Likewise, highly advantageous LNPs are provided that allow for highly advantageous and efficient delivery of mRNA and / or DNA to T-cells (e.g. an mRNA encoding a transposase and a DNA with a transposable DNA construct), which can either be performed ex vivo or in vivo, to therewith obtain engineered T cells, that can expand, which, when engineered to express e.g. a CAR or other therapeutic gene, can be useful in medical treatments, e.g. against cancer.

[0010] Figures

[0011] Figure 1. shows a Venn diagram displaying important components for highly efficient nucleic acid delivery to T cells using the tLNP system.

[0012] Figure 2. Illustrates a schematic of an anti-CD7 VHH and anti-CD3 ScFv dual-targeted lipid nanoparticle.

[0013] Figure 3. shows an SDS-PAGE gel loaded with nanoparticles targeted with DSPE-PEG conjugated to anti-CD3 ScFv is particle, DSPE-PEG-CD3 ScFv conjugate and CD3 ScFv protein alone. These materials were purified using either 20 kDa dialysis or 100 kDa Amicon spin filtration.

[0014] Figure 4. Show are the transfection of activated and non-activated T cells with LNPs targeted towards CD3 and / or CD7 at various different binder densities and various different CD7:CD3 ratios as measured by flow cytometry. A) Particle-associated anti-CD3 ScFv can activate T cells and this effect is enhanced by the presence of particle associated anti-CD7 VHH. B) The addition of particle associated anti-CD3 ScFv to particle associated anti-CD7 VHH targeted lipid nanoparticle enhances the transfection of activated T cells. C) The combination of anti- CD7 VHH and anti-CD3 ScFv targeting allows for the transfection of T Cells without the use of additional sources of activation. D) Particle associated anti-CD3 ScFv activation allows anti- CD7 VHH targeted lipid nanoparticles to transfect T cells without using an additional source of activation across a wide range of total binder densities. E)The optimal anti-CD7 VHH to anti- CD3 ScFv ratio is between 1 :2 and 1 :6

[0015] Figure 5. Demonstrates that the tLNP system can be used to generate stable and functional CAR-T cells. A) Flow cytometry analysis of T cells treated with various different tLNP formulations containing different ionizable lipids demonstrating the generation of stable CAR-T cells. B) Killing assay analysis of tLNP generated stable CAR-T cells with CD19- K562 target cells. C) Killing assay analysis of tLNP generated stable CAR-T cells with CD19+ Nalm-6 target cells. D) Killing assay analysis of tLNP generated stable CAR-T cells with CD19+ Raji target cells.

[0016] Figure 6. Depicts the expression of CD19-CAR in cytokine induced killer (CIK) cells (gated on CD3+CD56+) at multiple time points during culture. Flow cytometry was performed on days 5, 12, and 17 post-transfection using dual-targeted LNPs. The data show stable expression of CAR over time, indicating successful delivery of mcDNA by the dual-targeted LNPs and stable integration mediated by the SB100X transposase in the engineered CIK cells.

[0017] Figure 7. Demonstrate that the tLNP system can be used to generate functional CAR-T cells in vivo. A) Flow cytometry analysis of mouse blood at various time points post-tLNP injection demonstrates in vivo T cell transfection with mcDNA CAR and mcDNA eGFP . B) Bioluminescence imaging signal of mice treated with tLNPs containing either control mcDNA eGFP or mcDNA CAR. Tumor grows exponentially in vehicle control mice while increasing and then plateaus and drops in CAR mcDNA treated mice. C) Kaplan-Meier survival curves of mcDNA CAR treated mice and vehicle control mice showing highly significant increase in survival in treated mice.

[0018] Figure 8. Demonstrates transfection of T cells after short incubation with tLNPs in whole blood. A) Schematic representation of whole blood transfection procedure. B) CAR-T expression at different times after 6 hour tLNP transfection in whole blood at different doses. C) CAR-T expression at different times after 2 hour tLNP transfection in whole blood at different doses. Nalm-6 target cells were added to the transfected cells on day 10 in order to demonstrate target specific expansion on day 25.

[0019] Figure 9. Demonstrate the significance of ionizable lipid branching in tLNP-mediated transfection of T cells. A) The molecular structures of branched and non-branched lipids ionizable lipids. B) T cell transfection efficiencies of tLNPs comprised of branched tail or nonbranched tail ionizable lipids. C) T cell transfection efficiencies of tLNPs comprised of ionizable lipids with various alkyl chain lengths before point of branching.

[0020] Figures 10. Demonstrate that the tLNPs made using the ionizable lipid CL4F8-[35 can be used to generate functional CAR-T cells in vivo at doses of 0.04mg / kg and 0.004mg / kg. A) Overview of experimental timeline. B) Flow cytometry analysis of mouse blood at various time points post-tLNP injection demonstrates in vivo T cell transfection with mcDNA CAR and mcDNA eGFP. C) Bioluminescence imaging signal of mice treated with tLNPs containing either control mcDNA eGFP or mcDNA CAR. Tumor grows exponentially in vehicle control mice while increasing and then plateaus and drops in CAR mcDNA treated mice. D) Kaplan-Meier survival curves of mcDNA CAR treated mice and vehicle control mice showing highly significant increase in survival in treated mice.

[0021] Figures 11. tLNPs produced using helper lipids with a PC head group and saturated lipid tails outperform other helper lipid types in terms of DNA and RNA transfection efficiency of activated T cells. A) Flow cytometry analysis of mcDNA eGFP expression in T cells treated with tLNPs comprised of SM 102 and various different helper lipids. B) Flow cytometry analysis of mCherry mRNA expression in T cells treated with tLNPs comprised of SM102 and various different helper lipids.

[0022] Figure 12 Untargeted LNPs produced using helper lipids with a PC head group and saturated lipid tails are outperformed by LNPs produced using lipids of other types in terms of T cell transfection efficiency. Figure shows flow cytometry analysis of mcDNA eGFP expression in T cells treated with untargeted LNPs comprised of SM102 and various different helper lipid types and tLNPs comprised of SM102 and DSPC.

[0023] Figure 13. Untargeted LNPs produced using helper lipids with a PC head group and saturated lipid tails are outperformed by other helper lipid types in terms of DNA and RNA transfection efficiency of HEK293 cells. A) Flow cytometry analysis of eGFP mcDNA expression in HEK293 cells treated with untargeted LNPs comprised of SM102 and various different helper lipids. B) Flow cytometry analysis of mCherry mRNA expression in HEK293 cells treated with untargeted LNPs comprised of SM102 and various different helper lipids

[0024] Figure 14. Untargeted LNPs produced using helper lipids with a PC head group and saturated lipid tails are outperformed by LNPs produced using lipids of other types in transfection of CD7+ HEK293 cells. In contrast, targeted LNPs produced using helper lipids with a PC head group and saturated lipid tails perform comparably LNPs produced using lipids of other types in transfection of CD7+ HEK293 cells. A) Flow cytometry analysis of mCherry mRNA expression in CD7+ HEK293 cells treated with untargeted LNPs comprised of SM102 and various different helper lipids. B) Flow cytometry analysis of mCherry mRNA expression in CD7+ HEK293 cells treated with untargeted tLNPs comprised of SM102 and various different helper lipids.

[0025] Figure 15. tLNPs containing a helper lipid with both a PC head group and a saturated lipid tail outperform LNPs containing helper lipids with only a saturated lipid tail with a non-PC head group. Flow cytometry analysis of mcDNA expression in T cells treated with tLNPs containing the helper lipids DSPC, DSPE and DSPG.

[0026] Figure 16 . Binding of different VHH clones on Jurkat cell line. Jurkat cells were incubated with a titration 10x concentration range from 0.00001 - 1000 nM of the different VHH clones and subsequently with a fluorophore labeled anti-VHH antibody. Data is shown as (A) percentage of cells positive for VHH or (B) as mean fluorescence intensity (MFI) of the total cell population, as a measure for VHH binding. The VHH antibody clone H7 is, was also included in the experiments.

[0027] Figure 17. Functional assessment and comparison of CD7 targeting VHH clones. Isolated T cells were activated with CD3 / CD28 beads and transfected with LNPs loaded with GFP encoding mcDNA and post inserted with different CD7 targeting VHH clones. eGFP expression was measured using flow cytometry (A). Conjugation efficiency of the different VHH clones was measured by SDS-Page and expressed as percentage (B)

[0028] Figure 18. Shown is physical characterization data for two exemplary tLNP formulations containing CD19 CAR mcDNA and SB1 OOx transposase mRNA or an inert stuffer RNA. Particle size (A) and polydispersity indices (B) as measured by dynamic light scattering (DLS). Encapsulation efficiencies as measured by ribogreen (C).

[0029] Figure 19. Liver toxicity assessment of tLNPs in BALB / c mice after tLNP administration. Alanine aminotransferase (ALT) (A) and Aspartate transaminase (AST) (B) levels in the blood of mice was measured using ELISA 1 day post-tLNP administration. ALT levels were again measured using ELISA 7 days post-tLNP administration to produce a time course of ALT levels in the blood.

[0030] Figure 20. illustrates a comparison of point of care manufacturing to extracorporeal administration of tLNP, in accordance with an exemplary embodiment of the present disclosure;

[0031] Figures 21A and 21 B collectively illustrate an example of tLNP transfection of T-cells in whole blood after short incubation times, in accordance with an exemplary embodiment of the present disclosure.

[0032] Figures 22A and 22B collectively illustrate flow plots of GFP expression in T cells (22A) and B cells (22B) following incubation of whole blood with tLNP comprising minicircle DNA encoding a GFP gene, in accordance with an exemplary embodiment of the present disclosure. Figure 23 illustrates T and NK cell specificity targeting with CD7 targeted LNPs, in accordance with an exemplary embodiment of the present disclosure.

[0033] Figure 24 illustrates efficient generation of proliferative CAR-T cells in whole blood at a range of LNP doses within 6 hours, in accordance with an exemplary embodiment of the present disclosure.

[0034] Figures 25A, 25B, 25C, 25D, and 25 E collectively illustrate efficient LNP mediated T cell transfection within 1 hour in whole blood leading to generation of proliferative CAR-T cells, in accordance with an exemplary embodiment of the present disclosure. The legend indicate with Fig. 25A / B / C / D / E as depicted applies to all Figures 25A, 25B, 25C, 25D, and 25 E.

[0035] Figure 26 illustrates efficient LNP mediated T cell transfeciton within 4 hours in isolated PBMCs leading to generation of CAR-T cells at varying cell densities and particle doses, in accordance with an exemplary embodiment of the present disclosure.

[0036] Figure 27 illustrates stable CAR expression induced by LNP mediated T cell transfection in isolated PBMCs using 10 min incubation, in accordance with an exemplary embodiment of the present disclosure.

[0037] Figure 28 shows flow cytometry analysis of non-activated T cells treated with a tLNP formulation generated using separate anti-CD7 VHH and anti-CD3 ScFv targeting ligands and tLNP formulations in which these targeting ligands are combined in a fusion protein

[0038] Figure 29A shows tumor outgrowth measured using BLI for humanised mice treated with tLNP formulations targeted using either separate anti-CD7 VHH and anti-CD3 ScFv ligands or tLNP formulations in which targeting ligands are combined into a single fusion protein

[0039] Figure 29B shows CAR-T expression as measured by flow cytometry for humanised mice treated with tLNP formulations targeted using either separate anti-CD7 VHH and anti-CD3 ScFv ligands or tLNP formulations in which targeting ligands are combined into a single fusion protein.

[0040] Figure 30 shows that tLNPs can efficiently transfect the T cell line Jurkat, with an EC50 of around 50ng, while no transfection of the B- or liver cell line is seen even at the highest doses.

[0041] Figure 31A shows luciferase expression from mRNA in various organs 5 hours after as single iv tLNP injection in PBMC humanized NXG mice .

[0042] Figure 31 B shows luciferase expression from DNA 21 various organs 21 days after as single iv tLNP injection in PBMC humanized NXG mice.

[0043] Figure 32A shows that while DNA vector copies are detected in most tested organs, both on- and off- target, integrated gene copies and mRNA transcripts are only found in on-target organs spleen and lung

[0044] Figures 32B and 32C show that integrated CAR DNA copies and mRNA transcripts can only be found in spleen (B) and lung (C) upon treatment with the tLNP but not with the untargeted LNP.

[0045] Detailed description

[0046] Accordingly, the present invention provides, in one embodiment, for a nanoparticle comprising: at its surface, a targeting moiety for binding a T-cell; at its surface, a T-cell activation moiety; and, a nucleic acid.

[0047] It is understood that in accordance with the invention, a nanoparticle refers to a particle of a size within the range of about 10 - 1000 nm, preferably in the range of 10 nm to 500 nm, which may be useful to contain nucleic acid as described herein, and which can be provided with a targeting moiety for T-cells and a T-cell activation moiety, in accordance with the invention. Examples of nanoparticles include e.g. lipid nanoparticles which are composed of lipids and well known pharmaceutical formulations for drug delivery. A nanoparticle can hence have an envelope structure, e.g. comprising lipids, or a lipid bilayer, in which envelope agents can be contained. A nanoparticle may also have a solid structure. Nanoparticles may include dendrimers, liposomes, polymeric nanoparticles, or inorganic nanoparticles. Polymeric nanoparticles can e.g. comprise polyethyleneimine (PEI) or poly-L-lysine (PLL). Inorganic nanoparticles can comprise calcium phosphates. A nanoparticle may also comprise a capsid structure or the like. Hence, any suitable nanoparticle may be contemplated in which nucleic acid can be contained and which can be provided with a T-cell targeting moiety and a T-cell activation moiety, i.e. functionalized, in accordance with the invention.

[0048] In accordance with the invention, suitable nanoparticles in accordance with the invention may include non-viral vectors and viral vectors. Vectors are compositions of matter which comprise a nucleic acid and which can be used to deliver the nucleic acid to a cell. Vectors can be non-viral vectors and viral vectors. Viral vectors are based on viruses, utilizing the properties of the virus from which they are derived for delivery of a nucleic acid, which can be inserted in the viral vector genome. Viral vectors in accordance with the invention may include retroviral vectors and lentiviral vectors, and adenoviral vectors and adeno-associated viral vectors. Providing such viral vectors with a targeting moiety in accordance with the invention can be via covalent binding, or non-covalent interactions, which are well known in the art. For example, a targeting moiety may be comprised in a viral vector capsid, and / or may be associated with a viral envelop. It may be preferred in accordance with the invention to select non-viral vectors. As shown in the examples, nanoparticles in accordance with the invention which may in particular be suitable and that may be highly preferred, are lipid nanoparticles.

[0049] PBMCs include lymphocytes (T cells, B cells, and NK cells), monocytes, and dendritic cells. Hence, T-cells are found in peripheral blood, and are peripheral blood mononuclear cells (PBMCs). In humans, the frequencies of cell populations in PBMCs can vary across individuals, lymphocytes may be in the range of 70-90 %, monocytes from 10 to 20 %, while dendritic cells are rare, accounting for only 1-2 %. T cells, also referred to as T lymphocytes are CD3+, i.e. positive for Cluster of Differentiation 3. T cells can be divided into helper T cells (CD3+CD4+) and cytotoxic T cells (CD3+CD8+), which function in the adaptive immune response. T cells are of interest as these can be engineered to express a CAR, a chimeric antigen receptor (likewise of course a TCR, a T cell receptor) therewith specifically targeting malignant cells, e.g. in cancer. A targeting moiety for binding a T-cell in accordance with the invention relates to a functional group of a molecule which serves to target or direct the nanoparticle to a particular cell type, in this instance a T-cell. In some embodiments, the targeting moiety is an antibody or a fragment thereof, a cell surface receptor ligand or fragment thereof, a peptide or a nucleic acid, such as e.g. an aptamer. Any molecule that is suitable to function as a targeting moiety may be contemplated. For example, ligands, or parts thereof (such as a CD7 ligand or the like) may be contemplated, which may have further modifications. The targeting moiety can be attached to the nanoparticle using suitable linking chemistry techniques known to those skilled in the art. In some embodiments, the targeting moiety is covalently bound to the nanoparticle. In other embodiments, the targeting moiety is associated with the nanoparticle by non-covalent bonding interactions such as ionic or by van der Waals forces

[0050] A T-cell activation moiety in accordance with the invention relates to a functional group of a molecule which serves to bind with a T-cell and, upon binding, induces activation of the T- cell. In some embodiments, the T-cell activation moiety is an antibody or a fragment thereof, a T-cell receptor complex ligand or fragment thereof, a peptide or a nucleic acid, such as e.g. an aptamer. Any molecule that may be suitable to function as a T-cell activation moiety may be contemplated. For example, CD3 binding arms such as used T-cell engagers may be contemplated, which may have further modifications. The T-cell activation moiety can be attached to the nanoparticle using suitable linking chemistry techniques known to those skilled in the art. In some embodiments, the T-cell activation moiety is covalently bound to the nanoparticle. In other embodiments, the T-cell activation moiety is associated with the nanoparticle by non-covalent bonding interactions such as ionic or by van der Waals forces

[0051] As long as the targeting moiety for binding a T-cell and the T-cell activation moiety in accordance with the invention allow for binding the nanoparticle to the T-cell and activation of the T-cell, and which can be provided to the surface of the nanoparticle, i.e. functionalized, in accordance with the invention, such can be contemplated.

[0052] As outlined herein, in particular the combination of a T-cell activation moiety and a targeting moiety for binding a T-cell were found to be highly advantageous. T-cell activation moiety

[0053] CD3 is present on all T cells and consists of subunits designated y, 6, E, and q. Combined with the TCR receptor, consisting of the TCR-alpha and TCR-beta, it forms the T- cell receptor complex. The cytoplasmic tail of CD3 is sufficient to transduce the signals necessary for T cell activation in the absence of the other components of the TCR receptor complex. Normally, activation of T cells depends first on binding of the TCR with a major histocompatibility complex (MHC) protein, itself presenting a foreign antigen, located on a separate cell. In a normal situation, only when initial TCR-MHC binding has taken place can the CD3 dependent signally cascade responsible for T cell clonal expansion and, ultimately, T cell cytotoxicity ensue.

[0054] T-cell activation refers to one or more cellular response of a T lymphocyte, particularly a CD4+ or CD8+ T cell, selected from: proliferation, differentiation, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. Suitable assays to measure T cell activation are known in the art. In particular aspects, T cell activation is the expression of activation markers, particularly expression of CD25 and / or CD69 (optionally as measured by flow cytometry). In particular aspects, T cell activation is determined by measuring expression of CD25 and / or CD69 on the T cell, e.g. by flow cytometry. As shown in the examples herein, activation can also be measured by an increase in cell side and forward scatter.

[0055] T cell activation moieties in accordance with the invention are capable of interaction with the CD3 receptor to therewith induce T cell activation. T-cell activation moieties in accordance with the invention may thus include e.g. a complex of an antigenic peptide and a major histocompatibility complex (MHC). However, such a T-cell activation moiety, though it can be contemplated, requires that the T-cel with which the nanoparticle with the T-cell activation moiety is to interact, needs to have a TCR that is capable of recognizing the peptide presented by the MHC. Hence, instead, and preferably, the T cell activation in accordance with the invention is a moiety that is capable of binding with the T-cell receptor complex, and through its binding induces T-cell activation. Such binding of T cell activation moieties in accordance with the invention to the T-cell receptor complex, is thus to induce CD3 signalling, inducing at least expansion of cells.

[0056] Accordingly, preferably in one embodiment, the nanoparticle in accordance with the invention, thus comprises a T-cell activation moiety induces T cell receptor signalling. In another and / or a further embodiment, the T-cell activation moiety is a T-cell receptor complex binding moiety. It is understood that a T cell receptor complex binding moiety comprises a CD3E, CD3y, CD35, chain, TCR a chain, and a TCR p chain. A T-cell receptor complex binding moiety that is capable of inducing T-cell activation in accordance with the invention thus comprises a binding moiety capable of binding one or more of the CD3E, CD3y, CD35, chain, TCR a chain, and TCR p chain, as comprised in a T cell receptor complex as presented by a T-cell at its cell surface. It is understood that the T-cell activation moiety most preferably is to engage with a human T cell receptor complex.

[0057] This way, by T-cell activation, advantageously, the cell nucleus because of at least inducing proliferation and hence cell division, becomes accessible for the nucleic acid delivered by the nanoparticle. This is in particular advantageous for DNA, as delivery to the nucleus and / or contact with the genome allows for integration of DNA into the genome of the cell and / or expression from the DNA.

[0058] T cell activation moieties, which may be suitable in accordance with the invention, are known in the art, e.g. as such moieties are comprised in Bispecific T-cell Engagers (BiTE) as presently prescribed in the clinic in the treatment of cancer. Examples of clinically approved T- cell engagers include blinatumomab, epcoritamab, and solitomab, which target with one arm the CD3 receptor, and with the other arm, a target antigen. The arms (e.g. antigen binding domains) of such BiTEs targeting the CD3 receptor, or the T cell receptor complex, can induce T cell activation. Suitable T-cell activation moieties can thus be selected from T-cell receptor complex binding domains as typically used in, and suitable for, BiTEs.

[0059] Hence, T-cell activation binding moieties may be selected from or can be derived from SP34-2, OKT3, and UCHT1 antibodies, or the like. These antibodies originate from mouse and target human CD3, and are well capable of inducing T-cell activation. Hence, in a further embodiment, the T-cell activation moiety in accordance with the invention is an anti-CD3 binding moiety. It is understood that an anti-CD3 binding moiety is capable of binding one or more of the CD3E, CD3y, CD35, and chain, as comprised and as presented by a T-cell as part of the T-cell receptor complex.

[0060] In another and / or further embodiment, the T-cell activation moiety is an immunoglobulin, such as an antibody, or a fragment thereof. Preferably, the T-cell activation moiety comprises an antigen binding region. The term immunoglobulin as used herein refers to a class of structurally related glycoproteins involved in binding, recognition and adhesion. An immunoglobulin can be a T cell receptor, or an antibody. T cell receptor and antibodies generate diversity via genetic recombination of DNA-encoded segments in individual lymphocytes by somatic V(D)J recombination. The V(D)J segments are comprised in the variable region and interspersed with framework regions. In humans, a T cell receptor is composed of an alpha (VJ) and a beta (VDJ) chain, the combination of the variable regions forming an antigen binding domain. Antibodies in humans are composed of two pairs of heavy and light chains, all four inter-connected by disulfide bonds. Such antibodies thus having two antigen binding domains, and thus are bivalent. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2, and CH3. The hinge region is the region between the CH1 and CH2 domains of the heavy chain and is highly flexible. Disulfide bonds in the hinge region are part of the interactions between two heavy chains in an IgG molecule. Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL or CL). The light chain constant region is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and / or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J Mol Biol 1987; 196:90117). Other species, such as camels, llamas and sharks, have a different antibody structure composed of only heavy chains. Hence, such antibodies are bivalent as well, and an antigen binding region of such an antibody is based on a variable region of the heavy chain only, such antigen binding regions referred to as VHH. It is understood that an immunoglobulin, or a fragment thereof, in accordance with the invention also encompass alternative formats known in the art. Alternative formats may include single chain formats, such as scFv or the like, and may include heavy chain only or light chain only formats. A format with a single VH and a single VL, preferably in single chain format such as scFv can be contemplated. Provided of course whichever format selected allows for binding of the nanoparticle to a T cell receptor complex as presented by a T-cell at its cell surface and allows for T-cell activation, in accordance with the invention. The skilled person is well aware of means and methods for designing and selecting different immunoglobulin formats.

[0061] Preferably, the nanoparticle in accordance with the invention comprises a T-cell activation moiety which comprises an immunoglobulin, or a fragment thereof, in a VHH format or a VH / VL format, most preferably in a VH / VL format such as a scFv.

[0062] It is understood that with regard to the CDR regions of antigen binding regions as part of an immunoglobulin, or fragment thereof, as comprised in a moiety-cell activation moiety, are highly important for the binding of antigen binding region to its target antigen. As is understood, CDR sequences of antibodies comprising a heavy and a light chain can be defined in accordance with IMGT rules (Brochet X., Nucl Acids Res 2008;36: W503-508; Lefranc MP., Nucl Acids Res 1999;27:209-12; www.imgt.org / ). Likewise, CDR sequences of llama antibodies or the like can be determined as well. It is understood that determined CDR regions may vary depending on the algorithm used. Hence, in one embodiment, the nanoparticle according to the invention comprising a T-cell activation moiety, said activation moiety comprising an immunoglobulin or part thereof, comprises CDR regions derived from a VHH domain capable of T-cell activation. It is understood that such an immunoglobulin, or part thereof, preferably comprises a suitable format for the CDRs from which they are derived , e.g. CDRs derived from VHH may preferably be comprised in a VHH format as well. In another embodiment, said immunoglobulin, or a part thereof, comprises CDR regions derived from a VH / VL.

[0063] Highly advantageously, and as shown in the examples, having the antigen binding domain of the immunoglobulin, or fragment thereof, in a VH / VL format, such as an scFV, originating e.g. from a BiTE or the like, was highly effective. In a preferred embodiment, the T- cell activation moiety thus comprises an antigen binding domain. In a further embodiment, the antigen binding domain is in a VH / VL format, more preferably in a scFv format. In particular, and as shown in the examples herein, the T-cell activation moiety comprises an antigen binding domain derived from UCHT1) More preferably, the antigen binding domain comprises complementarity determining regions CDR1 , CDR2, CDR3 from VH, and CDR1 , CDR2, CDR3 from VL from UCHT 1 , these are preferably in an scFV format, such as provided in SEQ ID NO. 37 as listed in table 2, which sequence is humanized. In another embodiment, the CDR1 , CDR2, CDR3 from VH, corresponds with SEQ ID NOs. 38-40, respectively, and the CDR1 , CDR2, CDR3 from VL corresponds with SEQ ID Nos. 41-43, respectively.

[0064] Suitable CDR sequences as provided from antibodies generated in animals may be used in the context of the framework regions in which they were identified. However, as in vivo administration of nanoparticles with targeting moieties may be contemplated, or of cells contacted with such nanoparticles, it may be preferred to have the CDR sequences in the context of human sequences, i.e. human framework regions, and, if applicable, human constant regions, as it is well established that when non-human antibodies are (repeatedly) administered to human subjects, this may evoke an immune response thereto. Hence, in order to avoid immunogenicity, humanization of e.g. antigen binding regions is contemplated. A humanized immunoglobulin in accordance with the invention refers to a genetically engineered nonhuman immunoglobulin, which contains human constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the non-human antibody CDRs, which together form the antigen binding site, onto a homologous human acceptor framework region (FR) (see WO92 / 22653 and EP0629240). In order to fully reconstitute the binding affinity and specificity of the parental immunoglobulin, such as a mouse antibody or llama antibody or the like, the substitution of framework residues from the parental antibody (i.e., the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modelling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized immunoglobulin may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back-mutations, may be applied to obtain a humanized immunoglobulin with preferred characteristics, such as affinity and biochemical properties. In any case, the skilled person is well aware of means and methods for designing different immunoglobulin formats that are suitable for human use. The UCHT1 antibody is a mouse antibody. The UCHT 1 scFv antibody as listed herein as SEQ ID NO. 37 in table 2, is a humanized scFv derived from the UCHT1 mouse antibody based on the CDR regions of the VH and VL chains of the UCHT1 mouse antibody. Y1

[0065] Alternatively, or in addition, it may also be contemplated to de-immunize an amino acid sequence of an antigen binding domain for use as a T-cell activation moiety in accordance with the invention. De-immunization involves the identification of T-cell or B-cell epitopes in amino acid sequence and removal thereof by amending the sequence. Such identification can be performed by in silico analysis of amino acid sequences, and de-immunization services are widely available. It is understood that humanization of immunoglobulin sequences may, and often will, include de-immunization, as the objective and preferred feature of humanization of non-human immunoglobulin sequences is to avoid or at least severely reduce (possible) immune responses against such a T-cell activation moiety, while at the same time retaining advantageous T-cell receptor complex binding and subsequent T-cell activation properties. a targeting moiety for binding a T-cell

[0066] It is understood that the T-cell activation moiety as described above may be regarded to be a targeting moiety for binding a T-cell. The targeting moiety for binding a T-cell that is to be comprised at the surface of the nanoparticle in addition, is understood to not be the same moiety as the T-cell activation moiety. Hence, the nanoparticles in accordance with the invention preferably are to comprise at least two different moieties, one targeting moiety for binding a T cell and another moiety which is a T-cell activation moiety. The targeting moiety for binding a T-cell and the T-cell activation moiety are thus different moieties. The targeting moiety for binding a T-cell can thus be understood to not be a T-cell activation moiety.

[0067] Suitable targeting moieties for binding a T-cell can be selected from moieties capable of binding with known T cell markers present at the cell surface of T-cells: CD2, CD4, CD5, CD7, CD8, CD25, CD127, CD152 and CD28. Moieties capable of binding a T-cell thus preferably are antibodies, or antigen binding domains therefrom, capable of binding CD2, CD4, CD5, CD7, CD8 or CD28. Such moieties preferably comprise antigen binding domains, such as in a VHH or a scFV format, or the like. Hence, in one embodiment, the nanoparticles in accordance with the invention, comprise a targeting moiety for binding a T-cell, selected from moieties capable of binding CD2, CD4, CD5, CD7, CD8, CD25, CD127, CD152 and CD28. In a further embodiment, such targeting moieties are preferably immunoglobulins or fragments thereof, such as comprising antigen binding regions capable of binding CD2, CD4, CD5, CD7, CD8, CD25, CD127, CD152 and CD28. It is understood that such targeting moieties are to be capable of binding such molecules as presented at the surface of T-cells. It is also understood that preferably such T-cell markers are human, i.e. the targeting moiety for binding a T-cell is preferably capable of binding human CD2, CD4, CD5, CD7, CD8, CD25, CD127, CD152 and CD28.

[0068] A targeting moiety for binding a T-cell highly preferably targets CD7. As shown in the examples herein, targeting CD7 was shown to highly efficiently deliver nucleic acid to T cells. Preferably, human CD7 is targeted. Human CD7, i.e. Cluster of Differentiation 7, is a protein that in humans is encoded by the CD7 gene. This gene encodes a transmembrane protein which is a member of the immunoglobulin superfamily. This protein is a cell surface costimulatory molecule expressed on human T and natural killer (NK) cells and on cells in the early stages of T-, B-, and myeloid cell differentiation. A targeting moiety capable of binding human CD7 in accordance with the invention is capable of binding to the extracellular portion of a human CD7 protein, e.g., as set forth in GENBANK No. AAH13297.1.

[0069] In one embodiment, the targeting moiety is an immunoglobulin or a fragment thereof. As shown in the examples herein, a VHH (schematically depicted in Fig. 1c), conjugated to LNPs, was shown to be effective in targeting T cells. Hence, it is understood that the targeting moiety may be an immunoglobulin, such as an antibody, or a fragment thereof, and it is understood that the targeting moiety comprising the immunoglobulin or fragment thereof may be covalently linked or bound via noncovalent interactions with the nanoparticle.

[0070] The term immunoglobulin as used herein has been outlined above. As said, it is understood that an immunoglobulin, or a fragment thereof, in accordance with the invention also encompass alternative formats known in the art. Alternative formats may include single chain formats, such as scFv or the like, and may include heavy chain only or light chain only formats. A format with a single VH and a single VL, preferably in single chain format such as scFv can be contemplated. Provided of course such a format allows for binding to a T-cell, such as to human CD7, and allows for delivery of an nucleic acid with a nanoparticle in accordance with the invention. The skilled person is well aware of means and methods for designing different immunoglobulin formats for targeting moieties that bind with a T-cell.

[0071] Preferably, the targeting moiety for binding a T-cell as comprised in a nanoparticle in accordance with the invention comprises an antigen binding region which is in a VHH format. Highly advantageously, and as shown in the examples, the antigen binding region preferably originates from a Llama antibody, i.e. a VHH such as described in the examples herein. VHHs with highly advantageous properties could be in particular be generated via LNPs, which were formulated to express full length human CD7, which were used to immunize llama’s, and subsequently selected from a phage library using human CD7 expressing cells. Such VHHs selected in this fashion were found to be highly useful for providing suitable antigen binding regions to be comprised in a targeting moiety in a nanoparticle in accordance with the invention.

[0072] In one embodiment, preferably, the nanoparticle according to the invention comprises a targeting moiety for binding T-cells, said targeting moiety comprises an immunoglobulin or part thereof, comprising CDR regions derived from the VHH as defined by SEQ ID NO.1. In one embodiment, said immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.1. In a further embodiment, said CDR regions of SEQ ID NO.1 are CDR1 , CDR2 and CDR3 of SEQ ID NO.1 as defined by SEQ ID NO. 2, 3 and 4, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.1 was identified.

[0073] In another embodiment, the nanoparticle according to the invention comprises a targeting moiety, said targeting moiety comprises an immunoglobulin or part thereof, comprising CDR regions derived from the VHH as defined by SEQ ID NO. 1 ,

[0074] In one embodiment, a targeting moiety for binding T-cells, comprises an immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.5. In a further embodiment, said CDR regions of SEQ ID NO.1 are CDR1 , CDR2 and CDR3 of SEQ ID NO.5 as defined by SEQ ID NO. 6, 7 and 8, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.5 was identified.

[0075] In one embodiment, a targeting moiety for binding T-cells, comprises an immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.9. In a further embodiment, said CDR regions of SEQ ID NO.9 are CDR1 , CDR2 and CDR3 of SEQ ID NO.9 as defined by SEQ ID NO. 10, 11 , 12, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.9 was identified. In one embodiment, a targeting moiety for binding T-cells, comprises an immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.13. In a further embodiment, said CDR regions of SEQ ID NO.13 are CDR1 , CDR2 and CDR3 of SEQ ID NO.13 as defined by SEQ ID NO. 14, 15 and 16, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.13 was identified.

[0076] In one embodiment, a targeting moiety for binding T-cells, comprises an immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.17. In a further embodiment, said CDR regions of SEQ ID NO.17 are CDR1 , CDR2 and CDR3 of SEQ ID NO.17 as defined by SEQ ID NO. 18, 19 and 20, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.17 was identified.

[0077] In one embodiment, a targeting moiety for binding T-cells, comprises an immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.21. In a further embodiment, said CDR regions of SEQ ID NO.21 are CDR1 , CDR2 and CDR3 of SEQ ID NO.21 as defined by SEQ ID NO. 22, 23, and 24, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.21 was identified.

[0078] In one embodiment, a targeting moiety for binding T-cells, comprises an immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.25. In a further embodiment, said CDR regions of SEQ ID NO.25 are CDR1 , CDR2 and CDR3 of SEQ ID NO.25 as defined by SEQ ID NO. 26, l and 28, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.25 was identified.

[0079] In one embodiment, a targeting moiety for binding T-cells, comprises an immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.29. In a further embodiment, said CDR regions of SEQ ID NO.29 are CDR1 , CDR2 and CDR3 of SEQ ID NO.29 as defined by SEQ ID NO. 30, 31 , and 32, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.29 was identified. In one embodiment, a targeting moiety for binding T-cells, comprises an immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.33. In a further embodiment, said CDR regions of SEQ ID NO.33 are CDR1 , CDR2 and CDR3 of SEQ ID NO.33 as defined by SEQ ID NO. 34, 35 and 36, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.33 was identified.

[0080] In addition, and as described in the examples, of the MT-701 antibody, which is a mouse antibody capable of binding with human CD7, CDR regions derived therefrom may be contemplated to be comprised in a targeting moiety for binding T-cells in accordance with the invention as well. In one embodiment, said targeting moiety which comprises an immunoglobulin, or a part thereof, comprises CDR regions of the MT-701 antibody. In a further embodiment, the nanoparticle according to the invention comprises a targeting moiety, comprising a humanized immunoglobulin, or part thereof, comprising the CDR regions of the MT-701 antibody. The CDR1 , CDR2, CDR3 from VH, and the CDR1 , CDR2, CDR3 from VL corresponds of the MT-701 are used in accordance with the invention, to provide for a VH / VL binding domain, preferably in an scFv format or the like. Such an immunoglobulin or part thereof may be conjugated to e.g. a lipid nanoparticle similar to the conjugation of VHH.

[0081] Suitable CDR sequences as provided from antibodies generated in animals may be used in the context of the framework regions in which they were identified. However, as outlined above herein, and as known in the art, such sequences may be advantageously humanized and / or de-immunized. Hence, in one embodiment, a nanoparticle in accordance with the invention is provided, wherein the amino acid sequence of said immunoglobulin or a fragment thereof as comprised in an antigen binding region of a targeting moiety for binding a T-cell, is humanized and / or de-immunized.

[0082] As shown in the examples, the EC50 of an immunoglobulin or part thereof, as selected for a T-cell targeting moiety, for binding with T-cells expressing human CD7, may be in the range of 1 pM - 1 mM, more preferably in the range of 100 pM - 100 nM. Such EC50 may preferably be in the range of 10-6to 10-12M. More preferably, said EC50 of such an immunoglobulin is in the range of 10-6to 10-11, or 10-6to 10-10, or 10-7to 10-9M. In one embodiment, the EC50 may be selected to be in the range of 10-7to 10-9M. Such EC50 preferably is determined on isolated primary T-cells. As is understood, in case of humanization of immunoglobulin sequences, such as of the VHH of SEQ ID NO.1 , the EC50 of such a humanized immunoglobulin preferably may be selected to be in these ranges.

[0083] Nucleic acid

[0084] Nucleic acid refers to any compound and / or substance that comprises a polymer of nucleotides (i.e. of nucleotide monomers). These polymers are referred to as polynucleotides. Thus, the terms "nucleic acid" and "polynucleotide" can be used interchangeably. Nucleic acids may be or may include, for example, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs). RNAs may include mRNAs or the like. DNAs may include double stranded DNA. It is understood that RNAs or mRNAs may include artificial or modified nucleic acids, such as threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a [3-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA), or chimeras or combinations thereof. The skilled artisan will appreciate that polynucleotide sequences may recite "T"s in a representative DNA sequence but where such a sequence represents RNA (e.g., mRNA), the "T"s would be substituted for "U"s. Thus, RNA polynucleotides encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each "T" of the DNA sequence is substituted with "U."

[0085] The nanoparticles in accordance with the invention comprise a nucleic acid, though other agents may be contemplated to be included as well. In accordance with the invention, it is understood that in particular the nucleic acid is a DNA or an RNA. In particular, as shown in the example section, an RNA that may be contemplated may include an mRNA, encoding for a protein of interest, and a DNA that may be contemplated may encode likewise for a gene of interest such as a protein as well.

[0086] As shown in the example section, highly advantageously, mRNA can be highly efficiently transferred to resting T cells, and to activated T-cells as well. The basic components of an mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly-A tail. Further regulatory sequences may be included and contemplated in mRNA molecules. Hence, the nanoparticles in accordance with the invention are in particular useful for delivery of RNA, in particular for delivery thereof to resting T cells and activated T-cells. Any RNA may be contemplated in accordance with the invention, including non-coding RNAs such as antisense RNA, siRNA, miRNA, and coding RNA, such as mRNA, which may be synthetized or isolated, and may comprise naturally occurring sequences or engineered sequences. RNA that may be contemplated may be selected from the group consisting of an mRNA, an siRNA and a microRNA. It may be preferred to select an mRNA. Hence, in some embodiments, a nucleic acid of the present invention may function as messenger RNA (mRNA). A messenger RNA (mRNA) refers to any polynucleotide that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in a cell. Polynucleotides of the present disclosure may function as mRNA but may be distinguished from wild-type mRNA in their functional and / or structural design features, which may serve to allow for effective polypeptide expression using nucleic-acid based therapeutics.

[0087] As likewise also shown in the example section, DNA was shown to be effectively transferred to activated and resting T cells. Such DNA may comprise e.g. an expression cassette for a gene of interest, such as a CAR. Such DNA may also comprise e.g. an expression cassette for a gene of interest, such as a TCR. Preferably, such a DNA is provided as a double stranded DNA, for example as a minicircle DNA or the like. The DNA may be in the form of a double stranded DNA. The DNA furthermore can be in the form of a minicircle DNA. A minicircle DNA in accordance with the invention can be defined as having a circular structure and is double stranded. A minicircle DNA may typically and preferably not comprise any bacterial derived DNA sequences. A minicircle DNA may be derived from DNA, such as a plasmid DNA, comprising a selection marker and an origin of replication for production of the plasmid DNA in a suitable (bacterial) host. To provide for the minicircle DNA, the selection marker and origin of replication may be excised from the plasmid DNA and the circle religated to thereby providing a minicircle DNA without the (bacterial) selection marker and origin of replication sequences. Any length of DNA may be selected that is suitable to be comprised in an LNP. In one embodiment, the length of the DNA contemplated may depending on the size of the gene sequence of interest that is to be comprised in the DNA, and the type of construct, i.e. elements that may need to be contained therein, that is to be delivered. In one embodiment, the length of the minicircle DNA is at most 10 kB. In another embodiment, the length of the minicircle DNA is at most 6 kB. As said, and as shown in the examples, in accordance with the invention the nucleic acid, i.e. a DNA such as a mcDNA, can highly advantageously encode for a CAR or a TCR. As shown in the examples herein, by utilizing nanoparticles in accordance with the invention, highly advantageously, DNA can be efficiently be transferred to both resting and activated T-cells, and allow for expression of transgenes as encoded by the DNA.

[0088] With regard to CAR or a TCR, it is understood that a CAR is a chimeric antigen receptor that is to mimic a TCR or the like. CARs are engineered. The first generation of CAR were provided with an antigen recognition part often an antibody derived region (e.g. a scFv) fused to a transmembrane region and intracellular region chain. Later generations combined intracellular signalling domains from various costimulatory protein receptors (e.g., CD28, 41 BB, ICOS) incorporated in the cytoplasmic tail of the CAR to enhance signalling further. Further generations also incorporated in their design an inducible release of transgenic immune modifiers, such as IL-12, to shape the tumor environment by augmenting e.g. T-cell activation, attracting and activating innate immunity. As CARs often have antibody variable regions (i.e. antigen binding domains) incorporated, these can target e.g. receptors themselves that are presented at the surface of a cell (e.g. Her2, CD19 etc.), or can also target antigens presented by MHC, derived e.g. from proteins intracellular processed by the ubiquitin- proteasome system. Such peptides presented by MHC include proteins that are processed internally and presented by MHC, which can be derived from receptors, secreted proteins, intracellular proteins or internalized proteins. Of course, instead of a CAR, likewise, identified suitable TCR receptors (humanized or human derived) may be selected instead, or any other suitable engineered receptor. Suitable (engineered) TCR receptors may include single chain TCR receptors, e.g. comprising a single chain VaV[3-domain. Such single chain TCR receptors (scTv) may be highly useful as these can avoid potential misparing with endogenous TCR chains. Suitable (engineered) TCR receptors may include TCRa and TCRp chains, with engineered constant regions of both TCRa and TCRp chains, that prevent mispairing and can increase TCR expression and stability.

[0089] Nucleic acids in accordance with the invention, in some embodiments, are codon optimized. Codon optimization methods are known in the art and may be used as provided herein. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove / add post translation modification sites in encoded protein (e.g. glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art - non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and / or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms.

[0090] In highly preferred embodiments, the nanoparticle comprises a DNA which is to be integrated into the genome of activated or resting T cells. Such a DNA may e.g. comprise homology arms to allow for targeted integration. The nanoparticle may also comprise a means for enabling integration of the DNA. For example, an integrase. The nanoparticle may also comprise an mRNA that encodes for a protein facilitating DNA integration, such that upon delivery of the nanoparticle in T cells, such as in particular resting T cells, the DNA can be stably integrated into the genome of the T cells. The DNA may hence include suitable DNA sequences that accommodate integration, for example by interaction with the expressed protein. Integration of DNA may include homologous recombination and / or nicking of an endogenous target DNA sequence. Suitable (recombinant) DNA integration technologies are known in the art which may be employed. Such technologies include gene editing technology such as CRISPR / CAS, and Transposon technology such as sleeping beauty, piggyBac transposon vectors.

[0091] In any case, the nanoparticle in accordance with the invention may include agents or any means that provide for the necessary components to insert a transgene into the genome of the targeted cells, i.e. (resting) T cells. Hence, preferably, in case the nucleic acid comprised in a nanoparticle is a DNA with an expression cassette which is to be integrated, further means can be comprised in the nanoparticle to facilitate integration, and the DNA may further comprise the necessary elements therefor. For example, in case of a transposon system, an mRNA encoding for a transposase may be included in the nanoparticle, and the DNA may have flanking its expression cassette inverted terminal repeats (ITRs) compatible with the selected transposase. Hence, it is understood that an LNP in accordance with the invention may comprise both an RNA, such as an mRNA and a DNA.

[0092] In another embodiment, as outlined in the examples herein, the nanoparticle, such as an LNP, in accordance with the invention, comprises an mRNA encoding a transposase and a DNA encoding a CAR and / or a TCR with flanking ITRs compatible with the transposase, wherein preferably, the transposase is a sleeping beauty transposase. In another embodiment, the transposase is selected from the group consisting of Piggy Bac, SB100x, SB11 , TcBuster, Tol2, Frog prince, SpinON, Himarl, Passport, Minos, HAT, Hsnarl , Harbinger, Harbinger-3-DR, AciDs, and PIF.

[0093] The nanoparticle in accordance with the invention can thus comprise mRNA to DNA in any suitable amounts. Suitable amounts may be selected that allow for the mRNA and DNA to serve their function, i.e. have from the mRNA a protein expressed, e.g. a transposase, to have its action i.e. integrate the DNA, and a suitable amount of DNA such that the transposase can interact with the DNA to integrate it in the genome. The molecular weight ratio of the mRNA encoding the transposase to the DNA comprising the CAR expression cassette as used in the examples was about 1 :5. Suitable weight ratios of mRNA to DNA that can be contemplated in accordance with the invention range from 1 : 100 to 100 : 1 . Suitable weight ratio’s that can be contemplated can further range from 1 :25 to 25: 1. Suitable molar ratios of mRNA to DNA can be in the range of 400: 1 to 1 :25, or, can range from 100:1 to 1 :6.

[0094] Lipid nanoparticles

[0095] In a preferred embodiment, the nanoparticle in accordance with the invention is a lipid nanoparticle. Lipid nanoparticles in accordance with the invention can be defined as comprising a lipid composition with an ionizable cationic lipid and / or a targeting moiety conjugated to a lipid. Lipids are fatty substances which may be naturally occurring or synthetic and include a broad group of molecules including fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), fatty acids, and their derivatives, such as monoglycerides, diglycerides, triglycerides, and phospholipids. Lipids suitable for the preparation of lipid nanoparticles are widely available. Advantageous lipids to be included are ionizable lipids, which can be key components of lipid nanoparticles as they can encapsulate nucleic acid through electrostatic interactions. Preferably the lipid nanoparticles in accordance with the invention comprise the T-cell targeting moiety, and the T-cell activation moiety, conjugated to lipids such that these are presented at the surface of the lipid nanoparticle and capable of binding to their targets as presented on the surface of a T cell such that these can have their action.

[0096] As shown in the examples, a variety of different lipid nanoparticle compositions were shown to be effective in accordance with the invention. Hence, the lipid nanoparticle may not be understood to be restricted by any defined lipid composition or lipid components, as long as the lipid nanoparticle can comprise the T-cell targeting moiety, and the T-cell activation moiety, i.e. present it at its surface, any lipid nanoparticle composition capable of transferring its contents to a cell may be contemplated in accordance with the invention. Of course, certain lipid nanoparticle formulations may be further advantageous and combined with the targeting moiety for binding a T-cell and the T-cell activation moiety may synergistically more efficiently deliver e.g. nucleic acid to T-cells. Although as shown in the examples, the delivery of nucleic acids to, T cells, in particular to resting T cells, was found to be highly advantageous utilizing the lipid nanoparticles in accordance with the invention, other agents may be contemplated to be delivered instead to T-cells with the (lipid) nanoparticles in accordance with the invention.

[0097] The lipid nanoparticle (LNP) preferably comprises: a) an ionizable lipid; b) a helper lipid; c) a PEG lipid; and d) a sterol. The lipid nanoparticle may comprise between 30 and 60 mole% of said ionizable lipid, between 0 and 30 mole% of said helper lipid, between 0 and 10 mole% of said PEG lipid, and between 15 and 50 mole% of said sterol, each of these ranges being based on the combined molar quantities of (a)-(d) being 100 mole%. In another embodiment, the lipid nanoparticle in accordance with the invention comprises 35-45 mole % of ionizable lipid, 15 to 25 mole % of helper lipid, 0.1 to 5 mole % of PEG lipid, and 40 - 60 mole % of a sterol.

[0098] Generally, ionizable lipids comprise an ionizable head group, for example an amine group, that can be protonated or deprotonated based on pH value, allowing these lipids to become positively charged in acidic conditions. In addition, an ionizable lipid generally comprises one or more hydrophobic domains, usually one or more hydrophobic tails (e.g. hydrocarbon tails), which enable them to integrate into lipid bilayers or form lipid nanoparticles. The present ionizable lipids may remain largely non-charged at physiological pH (7.4) and acquire a charge under acidic conditions (e.g., pH 5-6). Such pH-sensitivity can be beneficial for endosomal escape in drug delivery.

[0099] The nanoparticle may comprises one or more ionizable lipids comprising an ionizable head group (Y) and at least one hydrocarbon tail (X) connected via a spacer (S) to said ionizable head group.

[0100] In particular, the ionizable lipid may have a structure according to the formula: Rz-Y-(S-(L-X)i)kwherein

[0101] R is a substituent z is 0 or an integer from the group of 1-2

[0102] Y is an ionizable head group each S is a spacer individually selected from the group of spacers (S1 , S2, S3, S4 etc.); each L is a bond or a linker individually selected from the group of linkers (L1 , L2, L3, L4 etc.); each X is a hydrocarbon tail individually selected from the group of hydrocarbon tails (X1 , X2, X4, X5 etc.);

[0103] I and k are each integers selected from the group of 1-10, preferably 1-6.

[0104] The R substituent may for example be an octyl-carbonyl group (-C(=O)(CH2)7CH3)).

[0105] The ionizable lipid may have a structure according to the formula Rz-Y-(S-(L-X)i)k wherein z = 0 and I = 1 and k = 2, being (X1-L1-S1)-Y-(S2 -L2-X2) or in a specific embodiment wherein S1 = S2 and L1 = L2 and X1 = X2, the formula is as follows: Y-(S1 -L1-X1)2.

[0106] The ionizable lipid may have a structure according to the formula Rz-Y-(S-(L-X)i)kwherein z = 0 and I = 2 and k = 1 , being Y-((X1-L1)-S1-(L2-X2)) or in a specific embodiment wherein S1 = S2 and L1 = L2 and X1 = X2, the formula is as follows: Y-S1-(L1-X1)2.

[0107] In an embodiment, said ionizable lipid having a structure according to the formula Rz-Y- (S-(L-X)i)kwherein z = 0 and I = 1 and k = 3, being Y-[(S1-L1-X1)]-[(S2-L2-X2)]-[(S3-L3-X3)] or in a specific embodiment wherein S1 = S2 = S3 and L1 = L2 = L3 and X1 = X2 = X3, the formula is as follows: Y-S1-(L1-X1)3.

[0108] In an embodiment, said ionizable lipid having a structure according to the formula Rz-Y- (S-(L-X)i)kwherein z = 0 and I = 1 and k = 6, being Y-[(S1-L1-X1)]-[(S2-L2-X2)]-[(S3-L3-X3)] - [(S4-L4-X4)] -[(S5-L5-X5)] -[(S6-L6-X6)] or in a specific embodiment wherein S1 = S2 = S3 = S4= S5=S6 and L1 = L2 = L3 = L4 = L5 = L6 and X1 = X2 = X3 = X4 = X5 = X6, the formula is as follows: Y-S1-(L1-X1)6.

[0109] The ionizable lipid a) may be selected from the group consisting of an unsaturated tail ionizable lipid, a multi-tail ionizable lipid, a polymeric ionizable lipid, a biodegradable ionizable lipid and a branched-tail ionizable lipid, preferably a branched-tail ionizable lipid.

[0110] An unsaturated tail ionizable lipid according to the present description comprises an ionizable head group (Y) and at least one hydrocarbon tail with at least one carbon-carbon double bond.

[0111] An multi-tail ionizable lipid according to the present description comprises an ionizable head group (Y) and at least two hydrocarbon tails.

[0112] An polymeric ionizable lipid according to the present description comprises a polymer or dendrimer. A biodegradable ionizable lipid according to the present description contains a biodegradable bond, such as an ester bond.

[0113] A branched-tail ionizable lipid according to the present description comprises an ionizable head group (Y) and at least one hydrocarbon tail with at least one branching. A branched tail features one or more alkyl groups or other substituent branches attached to its main carbon chain.

[0114] A ionizable lipid may be a combination of the above, so it may be a unsaturated tail and / or multi-tail and / or polymeric and / or biodegradable and / or branched tail ionizable lipid. The branched-tail ionizable lipid comprising a spacer S comprises preferably at least 5 carbon atoms, such as between 5 and 20 or between 5 and 16, preferably at least 7 carbon atoms, such as between 7 and 20 or between 7 and 12.

[0115] The spacer S (preferably each spacer S, such as S1 , S2, S3, S4 etc.) in the ionizable lipid may be an alkyl substituted or unsubstituted alkyl, substituted or unsubstituted alkylalkenyl, substituted or unsubstituted alky lalkynyl, substituted or unsubstituted alkylaryl, and cycloalkyl, more preferably unsubstituted alkyl.

[0116] The linker L (preferably each spacer L, such as L1 , L2, L3, L4 etc.) may be an ester selected from -C(=O)-O- and -O-C(=O)-.

[0117] The branched-tail ionizable lipid may comprise two branched hydrocarbon tails (X, namely X1 and X2). This may for example be in accordance with formula Rz-Y-(S-(L-X)i)k wherein I = 2 and / or k = 2. The branched-tail ionizable lipid may comprise two spacers (S, namely S1 and S2) and two linkers (L, namely L1 and L2).

[0118] This may for example be in accordance with formula Rz-Y-(S-(L-X)i)kwherein k = 2. The at least one hydrocarbon tail X (preferably each hydrocarbon tail X, such as X1 , X2, X3) may be (individually) selected from: wherein Ra, Rb, Rc, Rd, Reand Rfare each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkylalkenyl, substituted or unsubstituted alkylalkynyl, substituted or unsubstituted alkylaryl, and cycloalkyl, and wherein m and n are each integers selected from the group of 1-20, preferably between 4 and 12.

[0119] More specifically the at least one hydrocarbon tail X (preferably each hydrocarbon tail X, such as X1 , X2, X3) may be (individually) selected from: wherein m and n are each integers selected from the group of 1-20, preferably between 4 and 12.

[0120] The at least one hydrocarbon tail X (preferably each hydrocarbon tail X, such as X1 , X2, X3) may be (individually) selected from

[0121] 9-(C17) 7-(C15)

[0122]

[0123] 2-butyloctyl 7-(C13)

[0124] Y may be selected from the group consisting of: wherein p is an integer selected from 1 to 6, preferably p is 2 or 4. wherein q is an integer selected from 1 to 20, preferably q is 7, wherein r is an integer selected from 1 to 6, preferably r is 3, wherein s is an integer selected from 1 to 8, preferably s is 4. wherein t, u, v are each independently an integer selected from 1 to 6, preferably t, u, and v are 3, wherein R1, R2, R3and R4are each independently selected from Ci-C8substituted or unsubstituted alkyl groups, optionally R1and R2are methyl, optionally R3and R4are n-propyl.

[0125] More specifically, Y may be selected from the group consisting of:

[0126] The branched chain ionizable lipid may be selected from the group consisting of: 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1 -octylnonyl ester (SM102),

[0127] 7-[(2-Hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]heptyl 2-octyldecanoate (Lipid 5), [(4-Hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), octan-4-yl9-[3-[[3,5-bis[3-[bis(9-octan-4-yloxy-9-oxononyl)amino]propylcarbamoyl]benzoyl] amino]propyl-(9-octan-4-yloxy-9-oxononyl)amino]nonanoate (FTT5), bis(2-butyloctyl) 10-(N-(3-(dimethylamino)propyl) nonanamido) nonadecanedioate (Lipid A9),

[0128] 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1 ,13-diyl bis(2-hexyloctanoate) (CL4F 8- 6),

[0129] 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1 ,13-diylbis(3-pentyloctanoate) (CL4F8J35),

[0130] 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1 ,13-diylbis(6-propylundecanoate) (CL4F 11_s-3), and

[0131] 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1 ,13-diyl bis(6-hexyldodecanoate) (CL4F 12_E-6).

[0132] SM-102 is in accordance with formula Rz-Y-(S-(L-X)i)kwherein z = 0, k = 2, I = 1 : being according to formula (X1-L1-S1)-Y-(S2-L2-X2), wherein Y is -N-ethyl-OH, S1 a linear C5- spacer, S2 is a linear C7-spacer, L1 = L2 = -C(=O)O-, X1 is a branched C17 tail, attached to S1 at C9, and X2 is a linear C11 tail.

[0133] Lipid 5 is in accordance with formula Rz-Y-(S-(L-X)i)kwherein z = 0, k = 2, I = 1 : being according to formula (X1-L1-S1)-Y-(S2-L2-X2), wherein Y is -N-ethyl-OH, S1 a linear C8- spacer, S2 is a linear C7-spacer, L1 = -C(=O)O-, L2 = -O-C(=O), X1 is a branched C17 tail, attached to S1 at C9, and X2 is a linear C9 tail. ALC-0315 is in accordance with formula Rz-Y-(S-(L-X)i)kwherein z = 0, k = 2, I = 1 : being according to formula (X1-L1-S1)-Y-(S2-L2-X2), wherein Y is -N-butyl-OH, S1 = S2 = a linear

[0134] C6-spacer, L1 = L2 = -O-C(=O), X1 = X2 is a branched C15 tail, attached to S1 at C7.

[0135] ALC-0315 FTT5 is in accordance with formula Rz-Y-(S-(L-X)i)k wherein z = 0, k = 6, I = 1 , according to formula Y-(S1-L1-X1)6wherein Y is

[0136] S1 a linear C8-spacer, L1 -C(=O)O-, X1 is a branched C8 tail, attached to S1 at C4.

[0137] Lipid A9 is in accordance with formula Rz-Y-(S-(L-X)i)kwherein z = 0, I = 1 and k =, according to formula Y-(S1-L1-X1)2 wherein Y is wherein q = 6, r = 3, R1=R2 = methyl, and wherein S1 a linear C8-spacer, L1 -C(=O)O-, X1 is Lipid A9

[0138] CL4F 8-6 is in accordance with formula Rz-Y-(S-(L-X)i)kwherein z = 0, I = 1 and k = 2, according to formula Y-(S1-L1-X1)2wherein Y is wherein s = 4 and R3 and R4 are propyl, and wherein S1 a linear C6-spacer, L1 -O-C(=O)-, X1 is a branched C13 tail, attached to S1 at C7.

[0139] CL4F 8-6 CL4F8_p5 is in accordance with formula Rz-Y-(S-(L-X)i)kwherein z = 0, I = 1 and k = 2, according to formula Y-(S1-L1-X1)2wherein Y is wherein s = 4 and R3 and R4 are propyl, and wherein S1 a linear C6-spacer, L1 -O-C(=O)-, X1 is a branched C12 tail, being 2-pentylheptyl.

[0140] CL4F 11_E-3 is in accordance with formula Rz-Y-(S-(L-X)i)kwherein z = 0, I = 1 and k = 2, according to formula Y-(S1-L1-X1)2wherein Y is wherein s = 4 and R3 and R4 are propyl, and wherein S1 a linear C6-spacer, L1 -O-C(=O)-, X1 is a branched C13 tail, being 5-propyldecyl.

[0141] CL4F 11 E-3 CL4F 12_E-6 is in accordance with formula Rz-Y-(S-(L-X)i)kwherein z = 0, I = 1 and k = 2, according to formula Y-(S1-L1-X1)2wherein Y is wherein s = 4 and R3 and R4 are propyl, and wherein S1 a linear C6-spacer, L1 -O-C(=O)-, X1 is a branched C17 tail, being 5-hexylundecyl.

[0142] CL4F 12_E-6

[0143] The present inventors have observed that there are several structural parameters that play a role in the selection of suitable ionizable lipids, such as i) the presence of at least one branched tail, ii) the number of branched tails, iii) the length of the branched tail, iv) possibly an additional spacer in between the linker and the point of branching in the branched tail; v) the spacer length between the head group and the branched tail; vi) the structure of the ionizable headgroup; vii) the type of linker in between the spacer and the branching tail. Each of these may affect the effect of an LNP. The present inventors have observed that it appears to be very beneficial to have at least one branched tail and / or a spacer length (S) of at least 5 in an ionizable lipid. Such ionizable lipids when comprised in a nanoparticle, i.e. a lipid nanoparticle in accordance with the invention, were found to be in particular advantageous when targeting T-cells. Of course, other structural parameters may have in addition effect on the capacity of such LNPs targeting T-cells. Such other structural parameters may be selected using the information above and experimentally as shown herein.

[0144] In another embodiment, the lipid nanoparticle in accordance with the invention, preferably has an N / P ratio in the range of 2 - 12. The N / P Ratio of lipid nanoparticles refers to the molar ratio of ionizable amine head groups (N+) to phosphate groups (P) on the encapsulated nucleic acid. The N / P ratio can be in the range of 2 - 12, 3 - 12, 4 - 12, 5 - 12, 2 - 10, 2 - 8, 4 - 8, or, 5 - 7. The N / P ratio can be in the range of 2 - 12, more preferably 3 -10, more preferably 4 - 8, most preferably 5 - 7. It is understood that an N / P ratio of 3, corresponds to 3 mole of ionizable head groups (N+) to 1 mole of phosphate groups (P), i.e. 3 / 1 = 3. As shown in the examples herein, lipid nanoparticles comprising nucleic acids with such N / P ratios were found to highly efficiently delivery both mRNA and DNA to (resting) T-cells in accordance with the invention.

[0145] Helper lipids are important components of lipid nanoparticles (LNPs) and may enhance membrane structure and bilayer stability. The helper lipids are preferably phospholipids. They mimic cell membranes, allowing LNPs to fuse more easily with cell membranes, facilitating cellular uptake. Helper lipids work synergistically with ionizable lipids to enhance the stability, delivery efficiency, and overall performance of LNPs in targeted drug delivery.

[0146] The helper lipid preferably comprises a phosphatidylcholine (PC) head group. The helper lipid may comprises saturated lipid tails, for example saturated hydrocarbon tails, e.g. a substituted or unsubstituted alkyl. The saturated lipid tail preferably has a length of between 14 and 20 carbon atoms.

[0147] Examples of suitable helper lipids include helper lipids with saturated lipids tail, for example DSPC (1 ,2-distearoyl-sn-glycero-3-phosphocholine) having C18:0 tails, DMPC (1 ,2- dimyristoyl-sn-glycero-3-phosphocholine) having C14:0 tails, DPPC (1 ,2-dipalmitoyl-sn- glycero-3-phosphocholine) having C16:0 tails and DAPC (1 ,2-diarachidoyl-sn-glycero-3- phosphocholine) having C20:0 tails.

[0148] Examples of other helper lipids include helper lipids with unsaturated lipids tail, such as DOPC (1 ,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine) having C18:1 tails and egg- PC (L-a-phosphatidylcholine) having C18:1 and C16:0 tails.

[0149] Other helper lipids, with different head groups include, DOPE (1 ,2-di-(9Z-octadecenoyl)- sn-glycero-3-phosphoethanolamine) having C18:1 tails, DOPG (1 ,2-di-(9Z-octadecenoyl)-sn- glycero-3-phospho-(1'-rac-glycerol)) having C18:1 tails. DSPE (1 ,2-dioctadecanoyl-sn-glycero- 3-phosphoethanolamine) having C18:0 tails, and DSPG (1 ,2-dioctadecanoyl-sn-glycero-3- phospho-(l'-rac-glycerol)) having C18:0 tails, and DPyPE (a phosphatidylethanolamine lipid composed of polyisoprene alkyl chains).

[0150] They differ in their fatty acid chain lengths, saturation levels and head groups, which influence their biophysical properties and suitability for specific applications.

[0151] The inventors observed that the helper lipids with unsaturated tails and / or head groups different from PC appear to have less activity in targeting T-cells. Hence, helper lipids with PC head groups and saturated tails are preferred in accordance with the invention.

[0152] Sterol lipids, for example cholesterol and sitosterol, stabilize the lipid nanoparticle by filling spaces between lipid molecules, increasing rigidity, and enhancing durability. It also helps to improve the encapsulation efficiency of the therapeutic payload, such as mRNA. As an example cholesterol (cholest-5-en-3ll-ol) may be mentioned.

[0153] PEG-Lipids (Polyethylene Glycol Lipids): PEGylated lipids, create a hydrophilic outer layer that may aid in increasing LNP stability and circulation time in the bloodstream. PEG- lipids can prevent or reduce opsonization (binding of immune proteins) and may aid in reducing immune clearance. Any suitable PEG lipid may be contemplated in accordance with the invention. A PEG lipid in accordance with the invention preferably may be DMG-PEG-2000 (1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), or PEG-DSPE (1 ,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[carbonyl-amino(polyethylene glycol)-2000). It is understood that the lipid nanoparticles in accordance with the invention may comprise one or more PEG lipids.

[0154] (Lipid) nanoparticle with a targeting moiety for binding a T-cell and a T-cell activation moiety The lipid nanoparticles in accordance with the invention may be prepared with methods and / or devices known in the art. As shown in the example section, LNPs can be prepared using microfluidic devices. Nucleic acid can be provided e.g. in aqueous phase, and mixed with an organic phase comprising the lipid composition, e.g. the ionizable lipid, helper lipid, PEG lipid and sterol, and mixed with a microfluidic device. Lipid nanoparticles in accordance with the invention in one embodiment have a diameter in the range of 50 - 200 nm, allow for efficient encapsulation of >95% and / or can have a neutral charge at pH 7.4. As outlined above, in one embodiment, a lipid nanoparticle in accordance with the invention comprises ionizable lipid, helper lipid, PEG-lipid and a sterol, wherein ionizable lipid can be an SM102, Lipid A9, ALC-0315, Lipid 5, CL4F8_[35, or CL4F12_E-6, and wherein the helper lipid is DSPC. In one embodiment, a lipid nanoparticle in accordance with the invention comprises ionizable lipid, helper lipid, PEG-lipid and a sterol, wherein ionizable lipid can be an SM102, Lipid A9, ALC-0315, Lipid 5, CL4F8J35, or CL4F12_E-6, and wherein the helper lipid is DMPC. In yet another embodiment, a lipid nanoparticle in accordance with the invention comprises ionizable lipid, helper lipid, PEG-lipid and a sterol, wherein ionizable lipid can be an SM102, Lipid A9, ALC-0315, Lipid 5, CL4F8J35, or CL4F12_E-6, and wherein the helper lipid is PDDC. In yet another embodiment, a lipid nanoparticle in accordance with the invention comprises ionizable lipid, helper lipid, PEG-lipid and a sterol, wherein ionizable lipid can be an SM102, Lipid A9, ALC-0315, Lipid 5, CL4F8J35, or CL4F12_E-6, and wherein the helper lipid is DAPC. In another embodiment, a lipid nanoparticle in accordance with the invention comprises ionizable lipid, helper lipid, PEG-lipid and a sterol, wherein ionizable lipid can be an SM102, Lipid A9, ALC-0315, Lipid 5, CL4F8J35, or CL4F12_E-6, and wherein the helper lipid comprises a PC head and a saturated tail. In one embodiment, a lipid nanoparticle in accordance with the invention comprises ionizable lipid, helper lipid, PEG-lipid and a sterol, wherein ionizable lipid is a branched ionizable lipid with at least one branched tail and a spacer length (S) of at least 5, and wherein the helper lipid comprises a PC head and a saturated tail. In another embodiment, a lipid nanoparticle in accordance with the invention comprises ionizable lipid, helper lipid, PEG-lipid and a sterol, wherein ionizable lipid is a branched ionizable lipid with at least one branched tail and a spacer length (S) of at least 5, and wherein the helper lipid is DSPC, DMPC, PDDC or DAPC.

[0155] Such lipid nanoparticles as outlined herein, are understood to be highly suitable for delivery of nucleic acids, in particular to resting T-cells in accordance with the invention, when provided at their surface with a targeting moiety for binding a T-cell and a T-cell activation moiety at the surface of a nanoparticle. In a further embodiment, such lipid nanoparticles comprise a T-cell activation moiety which is capable of binding the T-cell receptor complex, such as targeting CD3, and comprises a targeting moiety for binding a T-cell selected from the group consisting of a moiety targeting CD2, CD4, CD5, CD7, CD8, CD25, CD127, CD152 and CD28. In another further embodiment, such lipid nanoparticles comprise a T-cell activation moiety and comprise a targeting moiety for binding a T-cell which targets CD7. In yet another further and preferred embodiment, such lipid nanoparticles comprise a T-cell activation moiety which is capable of binding the T-cell receptor complex, such as a moiety targeting CD3, and comprises a targeting moiety for binding a T-cell which targets CD7.

[0156] Once the lipid nanoparticles have been prepared comprising the nucleic acid, the moieties i.e. the T-cell activation moiety and T-cell targeting moiety, can be subsequently applied thereto, e.g. via conjugation of the moieties to the surface of the lipid nanoparticle, or via post-insertion of a moiety linked to a lipid anchor via a click chemistry approach. Preferably this is via conjugation to a lipid-PEG. It is understood that the term lipid-PEG or PEG-lipid refers to the same type of molecule, i.e. a polymer of ethylene glycol, i.e. (CH2-CH2-O)n, covalently bound with a lipid. The PEG moiety may further comprise a linker molecule to which via appropriate reactions a T-cell targeting or T-cell activation moiety can be covalently bound thereto. As said, the structure of such a PEG lipid can be described by lipid-PEG-linker, i.e. [ lipid ] - (CH2 - CH2-O)n - [linker ]. When the moiety (either T-cell targeting or activation) is conjugated to a PEG-lipid this can be described by [lipid] - (CH2 - CH2-O)n - [linker] - [moiety] , or, [lipid] - (CH2 - CH2-O)n - [moiety]. For example, a human CD7 targeting moiety such as described in the example section having a VHH format or the like, may have a cysteine, e.g. a C-terminal cysteine, that can be conjugated with a lipid-PEG-maleimide compound via a maleimide-thiol reaction resulting in a stable thioether covalent bond. Likewise, similarly applies to a T-cell activation moiety. Such a specific conjugation may be preferred, however, it is understood that further types of conjugation, i.e. a reaction between a chemical group included in the moiety (either T-cell activation or targeting) and a linker moiety that results in the formation of a covalent bond, may be contemplated instead and such are known in the art.

[0157] As said, the targeting moiety for binding a T-cell and the T-cell activation moiety may be conjugated to a lipid via a linker molecule. It is understood that each moiety preferably is conjugated to a different lipid molecule. It is understood that the different lipid molecules can be the same type of lipid molecule or can be different types of lipid molecules. Hence, as said, PEG-lipids can improve stability of LNPs, e.g. in vivo, and may thus further advantageously be used for conjugation of the targeting moiety via a linker, i.e. a lipid PEG linker may be defined as [lipid] - (CH2 - CH2-O)n - [linker], wherein the - (CH2 - CH2-O)n - group refers to PEG, i.e. poly ethylene glycol.

[0158] It is hence understood that the LNP in addition to comprising PEG-lipid in accordance with the invention, thus further may comprise a lipid-PEG conjugated to a T-cell targeting moiety and a lipid-PEG conjugated to a T-cell activation moiety. In one embodiment, a range from 0.01 mole % - 5 mole %, preferably 0.02 mole % - 1 mole %, for each lipid-PEG-(linker)- moiety may be selected. In another embodiment, a range from 0.01 mole % - 5 mole %, preferably 0.02 mole % - 1 mole %, for each lipid- linker)- moiety may be selected. It is understood that these mole percentages are expressed relative to the lipid constituents of a lipid nanoparticle the sum of which surmounts to 100 mole percent, which lipid nanoparticle may thus comprise ionizable lipid, helper lipid, PEG lipid, (PEG)lipid with T-cell targeting moiety, and (PEG) lipid with a T- cell activation moiety, and a sterol.

[0159] Hence, a lipid nanoparticle comprising the T-cell targeting moiety and a T-cell activation moiety in accordance with the invention, preferably comprises 30 - 60 mole % of ionizable lipid, 10 - 30 mole % of helper lipid, 0.1 - 10 mole % of PEG lipid, and 15 - 50 mole % of a sterol, and 0.01 - 5 mole % of lipids conjugated with said T-cell targeting moiety and 0.01 - 5 mole % of lipids conjugated with a T-cell activation moiety. It is understood that the lipid conjugated with the moiety, preferably consists of a PEG-lipid conjugated with the moiety. Hence, phrased differently, the lipid nanoparticle in accordance with the invention, preferably comprises 30 - 60 mole % of ionizable lipid, 10 - 30 mole % of helper lipid, 0.1 - 10 mole % of PEG lipid, and 15 - 50 mole % of a sterol, 0.01 - 5 mole % of PEG-lipid conjugated with said T-cell targeting moiety and 0.01 - 5 mole % of PEG lipid conjugated with a T-cell activation moiety.

[0160] It is understood that the PEG-lipids and PEG-lipids conjugated with said moiety (i.e. T- cell targeting moiety or a T-cell targeting moiety) from the viewpoint of the PEG and / or lipid portion may be the same or different. For instance, as shown in the examples, a DSPE- PEG2000-Maleimide (i.e. lipid - PEG - linker) may be used for conjugation with the moiety, whereas the lipid-PEG in a lipid nanoparticle may be DMG-PEG2000. Hence, the lipid portion of the PEG-lipid may be different, e.g. DMG, as compared with the lipid of the lipid-PEG-(linker)- moiety, e.g. DSPE. Of course, conversely, the lipid of the PEG-lipid may also be the same as the lipid of the lipid-PEG-(linker)-targeting moiety. With regard to the PEG portion this may be the same (like PEG2000 used for both PEG-lipid and lipid-PEG-(linker)-targeting moiety), or this may be different. It is understood that the mole percentages of LNPs are expressed relative to all the lipid constituents, including ionizable lipid, helper lipid, PEG lipid, PEG-lipid-(linker)- moieties, and sterol, the sum of which surmounts to 100 mole percent. As outlined above, and as shown in the example section, it is preferred that the T-cell targeting and T-cell activation moieties are conjugated to the nanoparticle. Conjugation is understood to encompass covalent binding with the nanoparticle, e.g. via a compound comprised in the nanoparticle such as to a Lipid-PEG. Conjugation of the targeting moiety can be well controlled, e.g. by conjugation of the targeting moiety to a compound to be comprised in the nanoparticle, before preparing the nanoparticle.

[0161] Conjugation can also be performed by conjugation of the targeting moiety to already prepared nanoparticles. The targeting moiety conjugated with a compound, such as a lipid- PEG-targeting moiety as described herein, can also be inserted in the (lipid) nanoparticles after a (lipid) nanoparticle is produced. Methods for insertion of lipid-PEGs conjugated with a compound of interest, such as a T-cell targeting moiety or T-cell activation moiety as described herein, after production of a lipid nanoparticle are well known in the art, and such may be preferred.

[0162] Hence, in any case, (lipid) nanoparticles with conjugated moieties can be prepared with methods known in the art, involving well known established chemical reactions, and may also involve, e.g. a linker, to provide a targeting moiety conjugated with the nanoparticle, having the targeting moiety bind to the nanoparticle via a linker (as described above), including well known insertion methods for lipid nanoparticles. Nevertheless, in other embodiments, the targeting moiety is associated with the nanoparticle by non-covalent bonding interactions such as ionic or by van der Waals forces, instead of covalent binding. Such an association may be via e.g. an adapter molecule covalently bound with the lipid nanoparticle, wherein the adapter molecule has a specific non-covalent binding interaction with the targeting moiety.

[0163] The T-cell activation moiety preferably is conjugated to a PEG lipid. The targeting moiety for binding a T-cell preferably is conjugated to a PEG lipid. The lipid nanoparticle in accordance with the invention preferably comprises 0.01 mole % - 5 mole % of PEG lipid conjugated with the T-cell activation moiety. The lipid nanoparticle in accordance with the invention preferably comprises 0.01 mole % - 5 mole % of PEG lipid conjugated with targeting moiety for binding a T-cell. These mole % are relative to the total amount of lipid. It is understood that the total amount of PEG lipid (which comprises PEG-lipids conjugated to moieties and PEG-lipids not conjugated to moieties) preferably surmounts to at most 10%. Preferably the total amount of PEG-lipids surmounts to at most 5%, or at most 4%, or at most 3%.

[0164] As shown in the examples herein, the molar ratio of the targeting moiety for binding a T-cell, to the T-cell activation moiety was shown to be effective from the range of 1 :1 to 1 :35. Preferably, the molar ratio is selected from 1 :1 to 1 :6, more preferably from 1 :2 to 1 :6, as such molar ratio’s provided for further improved delivery to (resting) T-cells. Without being bound by theory, this molar ratio may equally be advantageous for nanoparticles in general, and not necessarily limited to LNPs. Nevertheless, the molar ratios were found to be in particular advantageous when used in LNPs as shown in the examples herein. Preferably, for such molar ratio’s, in an LNP, the molar percentage of a lipid conjugated with a targeting moiety for binding a T-cell, and a lipid conjugated the T-cell activation moiety is preferably in the range of 0.05 - 2 mole %, more preferably in the range of 0.1 -1 mole %.

[0165] It is understood that a nanoparticle, preferably a lipid nanoparticle, in accordance with the invention may be provided with the T-cell activation moiety and the targeting moiety for binding a T-cell, either as separate moieties, or combined e.g. in a single molecule. Such a single molecule may comprise e.g. the T-cell activation moiety and the targeting moiety for binding a T-cell conjugated to each other, such that the T-cell activation moiety or the targeting moiety for binding a T-cell can exert its respective function, or such that both the T-cell activation moiety and the targeting moiety for binding a T-cell exert their function. In one embodiment, such a single molecule may be a fusion protein comprising a T-cell activation moiety such as outlined herein, fused with a targeting moiety for binding a T-cell such as outlined herein.

[0166] Hence, in one embodiment, a nanoparticle in accordance with the invention is provided, wherein the nanoparticle comprises a fusion protein comprising the T-cell activation moiety and the targeting moiety for binding a T-cell. In a preferred embodiment, a lipid nanoparticle in accordance with the invention is provided, wherein the lipid nanoparticle comprises a fusion protein comprising the T-cell activation moiety and the targeting moiety for binding a T-cell. As is understood in accordance with the invention, conjugation to a lipid, such as preferably a PEG lipid, of the T-cell activation moiety and the targeting moiety for binding a T-cell separately, preferably comprises conjugation at the C-terminus, e.g. via a cysteine or the like, as outlined herein, which allows for providing lipid nanoparticles with the T-cell activation moiety and the targeting moiety for binding a T-cell conjugated to lipids, e.g. via post-insertion methods as outlined in the examples herein. Hence, polypeptides can be provided with further peptide sequences at the C-terminus that accommodate such conjugation with a lipid to allow for incorporation in a lipid nanoparticle and presenting at its surface the T-cell activation moiety and the targeting moiety for binding a T-cell. Likewise, preferably, in embodiments wherein a fusion protein comprising the T-cell activation moiety and the targeting moiety for binding a T- cell, are used, conjugation of the fusion protein to lipids may be similarly employed. Hence, in one embodiment, a lipid nanoparticle is provided in accordance with the invention comprising a fusion protein comprising the T-cell activation moiety and the targeting moiety for binding a T-cell, wherein the fusion protein is conjugated to a lipid, comprised in the lipid nanoparticle. In a further embodiment, the fusion protein is conjugated with a PEG lipid, or, is conjugated at the C-terminus of the fusion protein. In yet a further embodiment, the fusion protein is conjugated with a PEG lipid and is conjugated at the C-terminus of the fusion protein. Any suitable conjugation method can be contemplated, as long as the conjugation to the (PEG) lipid allows for the T-cell activation moiety and / or the targeting moiety for binding a T-cell, to exert their respective function, such a conjugation can be contemplated. In one embodiment, a nanoparticle in accordance with the invention is provided, wherein the lipid nanoparticle comprises the fusion protein comprising the T-cell activation moiety and the targeting moiety for binding a T-cell, wherein the fusion protein is conjugated to a lipid, preferably conjugated to a PEG lipid.

[0167] Fusion proteins can be generated with means and methods known in the art. Such may include incorporating suitable linker sequences that can be placed in between the T-cell activation moiety and the targeting moiety for binding a T-cell. Linkers may be selected of any suitable length. Such linker sequences may comprise e.g. glycine and serine residues. Suitable linkers that may be contemplated include repeats of GGGS, indicated as (GGGS)n, wherein n denotes the number of repeated units. Linkers may have a length in the range of at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids. Linkers may have a length of at most 100 amino acids, at most 90 amino acids, or at most 80 amino acids, or at most 70 amino acids, or at most 60 amino acids, or at most 50 amino acids, or at most 40 amino acids, or at most 30 amino acids. Linkers may have a length selected from the range of 3 - 100 amino acids, or from the range of 3 - 90 amino acids, or from the range of 3 - 80 amino acids, or from the range of 3 - 70 amino acids, or from the range of 3 - 60 amino acids, or from the range of 3 - 50 amino acids, or from the range of 3 - 40 amino acids. In one embodiment, a nanoparticle in accordance with the invention is provided, wherein the nanoparticle comprises a fusion protein comprising the T-cell activation moiety and the targeting moiety for binding a T-cell, which are separated by a linker, preferably a linker such as (GGGS)n. In one embodiment, the fusion protein comprises a linker with a sequence as defined by (GGGS)n, wherein n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. Preferably, in one embodiment, the linker is (GGGS)n, wherein n = is selected from the range of 1 - 8. More preferably, in one embodiment, the linker is (GGGS)n, wherein n = is selected from the range of 2 - 6.

[0168] It is understood that whichever T-cell activation moiety and targeting moiety for binding a T-cell, as described herein can be contemplated to be combined in a in a single molecule, such as in a fusion protein as outlined herein. In one embodiment, the T-cell activation moiety interacts with CD3 to therewith induce activation, or, the targeting moiety for binding a T-cell is a CD7 binder, which, is incorporated in a fusion protein as outlined herein for use in (lipid) nanoparticles. Preferably, the T-cell activation moiety interacts with CD3 to therewith induce activation, and, preferably, the targeting moiety for binding a T-cell is a CD7 binder, which are combined a in a single molecule, such as in a fusion protein, for use in a nanoparticle or lipid nanoparticle as outlined herein. As said, T-cell activation binding moieties may be selected from or can be derived from SP34-2, OKT3, and UCHT1 antibodies, or the like. In particular, fusion proteins comprising a T-cell activation moiety such as UCHT1 , or the like, can be contemplated, and a targeting moiety for binding a T-cell, targeting CD7, such as listed in table 1 and 4. It is understood that CDR regions may be selected to be incorporated in a suitable scaffold or full length sequence or a fragment thereof, as listed in tables 1 , 2 and 4. Preferably, a fusion protein can comprise the CDR regions as defined by table 1 (SEQ ID NO. 2-4), and / or the CDR regions as defined by table 2 (SEQ ID NO. 38-43). Preferably, a fusion protein can comprise SEQ ID NO.1 of table 1 and / or SEQ ID NO. 37 of table 2. It is understood that the order of the T-cell activation moiety and the targeting moiety for binding a T-cell, can be either way, one can be at the C-terminus, the other at the N-terminus of the fusion protein and vice versa. In another embodiment, fusion proteins that can be accordingly contemplated include a fusion protein selected from table 5, i.e. SEQ ID NOs. 44 - 49. As said, the single molecule or fusion protein with the T-cell activation moiety and the targeting moiety for binding a T-cell as outlined herein, preferably is conjugated with a PEG lipid. Such a PEG lipid preferably comprises a maleimide, which is compatible for conjugation, e.g. such as with a cysteine. In one embodiment, the PEG lipid used for conjugation of the single molecule or fusion protein with the T-cell activation moiety and the targeting moiety for binding a T-cell is DSPE-PEG-2000 maleimide (1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[carbonyl-amino(polyethyleneglycol)-2000-N’-(3- maleimidopropionyl)]). It is understood that the fusion protein or single molecule may be inserted in a lipid nanoparticle (as comprised in a lipid - fusion protein / single molecule conjugate) and / or conjugated to a nanoparticle. Hence, in one embodiment, a nanoparticle in accordance with the invention is provided wherein the fusion protein (or single molecule) comprising the targeting moiety for binding a T-cell and the T-cell activation moiety conjugated to the lipid, was inserted in and / or conjugated to the lipid nanoparticle.

[0169] Because in these embodiments with a single molecule or fusion protein, the T-cell activation moiety and the targeting moiety for binding a T-cell are present in a 1 :1 ratio, this means that these preferably are present in a 1 :1 ratio in the (lipid) nanoparticle in accordance with the invention as well.

[0170] In another embodiment, a lipid nanoparticle in accordance with the invention is provided, wherein the fusion protein (or single molecule) comprising the T-cell activation moiety and the targeting moiety for binding a T-cell is conjugated a lipid, preferably a PEG lipid, and said lipid nanoparticle comprises 0.01 mole % - 5 mole % of the total lipid content, of lipid conjugated with the fusion protein. Preferably, in an LNP, the molar percentage of a lipid conjugated with a fusion protein (or single molecule) with a targeting moiety for binding a T-cell and a T-cell activation moiety is preferably in the range of 0.05 - 2 mole %, more preferably in the range of 0.1 -1 mole %.

[0171] Uses of (lipid) nanoparticles

[0172] As described in the examples herein, the nanoparticles in accordance with the invention are highly useful for delivery of nucleic to a T-cell. More in particular, the nanoparticles through having at its surface a combination of a targeting moiety for binding a T-cell and a T-cell activation moiety, highly efficiency allows for delivery of nucleic acid to T-cells, much more efficiently as compared with either a targeting moiety for binding a T-cell alone, or, a T-cell activation moiety alone. Moreover, as shown in the examples herein, this was shown to be highly efficient in resting T-cells as well, allowing for efficient delivery of DNA and expression and / or integration of DNA constructs in resting T-cells as well. Such delivery of DNA and expression and / or integration of DNA constructs was much more efficient as compared with either targeting moiety for binding a T-cell alone, or, a T-cell activation moiety alone. Without being bound by theory, the combination of a targeting moiety for binding a T-cell alone, and, a T-cell activation moiety, enhances delivery of the nucleic acid to the T-cell while at the same time induces sufficient activation to allow for at least some cell division / proliferation to occur to therewith allow DNA to be comprised in the nucleus, consequently allowing for expression and / or integration of DNA into the genome of the T-cell. Preferably, the T-cell activation moiety is capable of binding CD3, and / or the T-cell targeting moiety is capable of binding CD7.

[0173] Hence, in one embodiment, the present invention provides for the use of a nanoparticle as defined herein, for delivery of a nucleic acid to a T-cell. In another embodiment, such delivery of a nucleic acid is to a resting T-cell. As said, it is understood that because of the combined action of targeting moiety for binding a T-cell and a T-cell activation moiety, at the surface of the nanoparticle, to the resting T-cells, DNA can be delivered to the nucleus, to therewith allow for expression of a gene of interest from a delivered DNA and / or allows for integration of a delivered DNA into the genome of the T-cell. Hence, accordingly, in another embodiment, the nanoparticle in accordance with the invention is capable of activating a resting T cell. In another embodiment, the nanoparticle in accordance with the invention allows for delivery of DNA to the nucleus of a T-cell, to therewith allow for expression of a gene of interest DNA and / or allows for integration of a DNA into the genome of the T-cell. In yet another embodiment, the nucleic acid comprises a DNA, preferably comprising an expression cassette, wherein the use comprises integration of the DNA construct into the genome of the T-cell and / or expression from the DNA construct, wherein preferably the T-cell is a resting T-cell. Highly advantageously, the DNA construct, when encoding a CAR or a TCR, this allows for generating CAR-T cells, or T-cells with an exogenous TCR, which have stably integrated into their genome an expression cassette encoding a CAR or an (engineered) TCR. As shown herein, upon becoming in contact with their target antigen as expressed on a target cell, such engineered T-cells, e.g. CAR-T cells, thus generated can be further stimulated and expanded. Such nanoparticles as described herein are highly useful for generation CAR-T cells, either from isolated T-cells, from PBMCs, and even in whole blood. Such nanoparticles as described herein are highly useful for generation T-cells expressing an (engineered) TCR, either from isolated T-cells, from PBMCs, and even in whole blood. Such use of nanoparticles in accordance with the invention, comprises contacting the nanoparticles, for up to about 8 hours, preferably up to six hours, e.g. in the range of 0 - 2 hours, 1 - 8 hours or 2 - 6 hours, with whole blood, isolated PBMCs or isolated T-cells. Such whole blood, isolated PBMCs or isolated T-cells highly preferably is human whole blood, isolated human PBMCs or isolated human T-cells. CAR-T cells, or composition comprising such CAR-T cells (or the like, such as T-cells expressing (engineered) TCRs) thus prepared are highly useful, in a medical treatment of human patients, e.g. when suffering from cancer.

[0174] Medical treatments

[0175] As outlined above, the nanoparticles in accordance with the invention allow for highly efficient delivery of nucleic acid to (resting) T-cells. Such delivery can be in vivo or can be ex vivo. Accordingly, in one embodiment, the invention provides for a nanoparticle as described herein, wherein said nanoparticle is for use in the treatment of a disease, wherein said treatment comprises administering the nanoparticle to the patient. It is understood that when reference is made to the administration of the nanoparticle to the patients, this does not comprise the administration of a single nanoparticle, but includes the administration of a plurality of, normally, the same type of nanoparticle. The singular term merely implicating that a single type of nanoparticle is to be administered. A suitable amount is to be administered with which the nucleic acid can be efficiently delivered to resting T-cells of the patient. Hence, it is understood that in one embodiment, the nanoparticles are administered directly into the bloodstream.

[0176] In one embodiment, the nanoparticle for use in the treatment of a disease such as described herein, is administered directly to the blood. In a further embodiment, such a nanoparticle, delivers a DNA encoding a gene of interest and a means for integration of the DNA, wherein said DNA integrates in resting T-cells. In yet a further embodiment, said delivery of the nanoparticle does not result in integration of the DNA in other tissues, such as the heart, liver and kidney. Preferably, such targeted nanoparticles are LNPs such as described herein. More preferably such targeted nanoparticles target both CD3 and CD7 as described herein. Preferably, such DNA encoding a gene of interest is an mcDNA and the means of integration a transposase encoded by an mRNA as described herein and as exemplified in the examples. Such medical treatments of a disease, e.g. aimed to provide T-cells with a suitable CAR or TCR and utilizing a CD3 / CD7 targeted LNP, allow to provide for a substantial integrated vector copy number in genomic DNA, as determined in spleen tissue and / or lung tissue, which tissues contain a high concentration of T-cells. Hence, advantageously, nanoparticles as outlined herein and as shown in the examples, allow for highly specific delivery of DNA to T-cells and subsequent integration of the DNA in the genome of the T-cells, which nanoparticles are administered directly to the patient i.e. via an injection into the bloodstream (e.g. intravenously). In another embodiment, nanoparticles as outlined herein and as shown in the examples, allow for highly specific delivery of DNA to T-cells and subsequent integration of the DNA in the genome of the T-cells, which nanoparticles are administered directly to the patient i.e. via an injection into the bloodstream (e.g. intravenously), wherein said administration does not result in detectable integration of the DNA in other tissues, such as the heart, liver and kidney.

[0177] In another embodiment, the nanoparticle in accordance with the invention is provided, wherein said nanoparticle is for use in the treatment of a disease, wherein said treatment comprises obtaining T cells from the patient, contacting the nanoparticle with the obtained T cells ex vivo and allowing delivery of the nucleic acid to the T cells, and subsequently administering the T cells to the patient. Hence, alternatively, instead of administering directly into patients, one can also treat patients T-cells from a patient ex vivo. Such a treatment may be referred to as an extracorporeal treatment, wherein the obtained T cells from the patient can be comprised in whole blood or can be first isolated from whole blood. It is understood that such an extracorporeal treatment may be performed in-line in a closed fluid circuit. Of course, such an extracorporeal treatment may be performed on collected blood from a patient, and upon the treatment with the nanoparticles, can be reinfused back into the patient, so, the treatment may be optionally in-line in a closed circuit. Hence, in one embodiment, a nanoparticle in accordance with the invention is provided for use in a medical treatment, wherein the medical treatment comprises an extracorporeal treatment which comprises:

[0178] (a) obtaining whole blood from a subject;

[0179] (b) collecting the fraction of blood containing T cells;

[0180] (c) contacting the fraction of blood containing T cells with the nanoparticles; and (d) reinfusing the contacted fraction of blood containing T cells into the patient, wherein steps

[0181] (a)-(d) are performed in-line in a closed fluid circuit.

[0182] In yet another embodiment, a nanoparticle in accordance with the invention is provided for use in a medical treatment, wherein the medical treatment comprises an extracorporeal treatment comprising:

[0183] (a) obtaining whole blood from a subject;

[0184] (b) contacting the whole blood with the nanoparticles; and

[0185] (c) reinfusing the contacted whole blood into the patient, wherein steps (a)-(c) are performed in-line in a closed fluid circuit.

[0186] In one embodiment, a nanoparticle in accordance with the invention is provided for use in a medical treatment, wherein the medical treatment comprises an extracorporeal treatment which comprises:

[0187] (a) obtaining whole blood from a subject;

[0188] (b) collecting the fraction of blood containing T cells;

[0189] (c) contacting the fraction of blood containing T cells with the nanoparticles; and

[0190] (d) reinfusing the contacted fraction of blood containing T cells into the patient. In yet another embodiment, a nanoparticle in accordance with the invention is provided for use in a medical treatment, wherein the medical treatment comprises an extracorporeal treatment comprising:

[0191] (a) obtaining whole blood from a subject;

[0192] (b) contacting the whole blood with the nanoparticles; and

[0193] (c) reinfusing the contacted whole blood into the patient.

[0194] It is understood that the contacting step may be for a suitable time that allows for delivery of the nucleic acid to the T-cells, a suitable may be selected from a 0 up to 8 hours, preferably, the contacting step may be up to 6 hours. For example, as shown in the examples herein, having a contacting step in the range of about 1 - 8 hours, was shown to be at least efficient. Moreover, it was observed by the inventors that already incubation times of 30 minutes, and thus likely shorter incubation times as well, were efficient. Hence, in one embodiment, the nanoparticle in accordance with the invention, for use in a medical treatment as describe above, may comprise a contacting step selected from the range of 0 - 8 hours, preferably selected from the range of 0 - 2 hours, more preferably up to 1 hour, more preferably up to 30 minutes. Incubation times even less than 30 minutes may be contemplated. It is understood that the medical treatments that can be contemplated in accordance with the invention can comprise any type of medical treatment in which engineering of T-cells would be contemplated. In particular, medical treatments, which are presently approved and / or are under development and which comprise CAR-T cell therapy, can be likewise be accommodated by the advantageous nanoparticle delivery technology as provided herein. Hence, any suitable expression cassette as presently designed and / or used in the clinic and as contained e.g. in a retroviral or lentiviral vector, may easily be used in the context of the presently provided advantageous nanoparticle delivery technology. Such clinically approved CAR-T cell therapies comprise CARs targeting CD19, for the treatment of B-cell acute lymphoblastic leukemia (ALL), B-cell non-Hodgkin lymphoma (NHL), Follicular lymphoma, Mantel cell lymphoma (MCL). Such clinically approved CAR-T cell therapies comprise CARs targeting BCMA for the treatment of multiple myeloma. The CAR expression cassette, or the like, can be highly advantageously be stably delivered to T-cells of a subject using the means and methods as outlined herein. Preferably, as shown in the examples herein, subjects suffering from a cancer can be eligible for treatment, provided a suitable CAR or TCR expression cassette, or the like would be available for such a treatment. Such treatment of cancer preferably comprising the treatment of a cancer of blood cells, and / or of a B-cell, such as a lymphoma. In the treatment of a B-cell cancer, the CAR or TCR preferably targets CD19, CD20, or CD22.

[0195] Extracorporeal treatments

[0196] As outlined above, the present invention provides i.a. for highly advantageous means and methods for extracorporeal transfection of immune effector cells. Provided therefor are targeted LNPs, containing DNA encoding CAR, or a TCR and the like, and mRNA encoding transposase as described herein.

[0197] As used herein “extracorporeal” is used in reference to cells, such as peripheral blood or bone marrow cells, harvested or extracted from the body and the modification of those cells prior to their intended return (reinfusion). Modification of cells generally relates to cell separation and washing procedures and exposure to transfection agents (e.g., LNPs, tLNPs), over a time interval of several hours. Such procedures are typically conducted within a single institution. Extracorporeal typically involves minimal manipulation or modification of subject cells, including limited periods of cell culture and expansion. It also includes the avoidance of refrigerated or cryogenic storage or shipment over extended periods spanning multiple days or longer.

[0198] 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 preferred embodiments disclosed herein the transfection is mediated by nanoparticles (NP) including targeted lipid nanoparticles LNPs (tLNPs).

[0199] In some embodiments, the present disclosure provides herein a method for extracorporeal transfection of an immune effector cell collected from a subject in need thereof. The immune effector cell is present in the blood drawn from the subject, and the extracorporeal transfection is achieved by contacting blood, or a cell-containing fraction thereof, from the subject with a nanoparticle ex vivo. In some embodiments, the nanoparticle contains a surface-exposed immune effector binding moiety; and a deoxyribonucleic acid encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR).

[0200] In some embodiments, the contacting of the blood, or a cell-containing fraction thereof, with a nanoparticle comprises drawing blood from the subject and admixing a solution comprising the nanoparticle with the drawn blood that contains the immune effector cells.

[0201] In some embodiments, the blood is drawn into a closed system and the admixing comprises adding the solution into the closed system, for example, a closed vein-to-vein system including but not limited to apheresis system. In some embodiments of the manufacturing processes described herein, immune effector cells are collected using standard apheresis equipment, such as Cobe® Spectra, Spectra Optia®, Fenwal™ Amicus® or equivalent. In some embodiments, cells from the circulating blood of a subject are obtained by leukopheresis, erythrocytapheresis, thrombapheresis, thrombocytapheresis, plasmapheresis, or plateletpheresis. In some embodiments, the leukopheresis process yielded approximately 100-500 mL of apheresis product from a patient (i.e. , a subject). The apheresis product is subjected to the transfection on-site, namely, transfection at the point-of- care, such as bedside.

[0202] As a non-limiting example, an apheresis device can include one or more connections configured to move whole blood and / or blood components to and from blood component separation devices. Blood component separation devices may be housed within the apheresis devices and often includes centrifuges. The apheresis device may be used for whole blood separation processes, where whole blood is withdrawn form a donor or a subject or a patient or source and provided to a blood component separation device where the whole blood is separated into various compounds, components, or elements some of which are collected and a remainder is returned to the donor or the subject or the patient or the source.

[0203] Exemplary description of apheresis and equivalent systems and their use can be found in U.S. Patent Publication No. 20250041501 , U.S. Patent No. 8,123,713; 7,780,618; 10,704,023; 9,435,736, 8,057,376, each of which is hereby incorporated by reference in its entireties.

[0204] In some embodiments, during blood drawing, the blood is collected into an apheresis bag and admixed by adding a solution containing the nanoparticle of the present disclosure to the apheresis bag.

[0205] In some embodiments, the drawn blood is processed and enriched during apheresis.

[0206] In some embodiments, the apheresis product is a “leukopheresis” product. As used herein, the term “leukopheresis” refers to the bulk mononuclear cells present in the blood, namely the separation and collection of leukocytes, white blood cells (WBCs), from plasma and red blood cells.

[0207] In some embodiments, the enriched apheresis product comprises about 5% to about 25% of the total peripheral blood mononuclear cell component. In some embodiments, the enriched apheresis product is a population of lymphoid cells or a lymphoid cell. In this embodiment, the lymphoid cell is selected from the group consisting of a T cell, a B cell, a natural killer (NK) cell, a CD8+ T cell, a CD4+ T cell, a cytotoxic T lymphocyte, a regulatory T cell, and any combination thereof.

[0208] In some embodiments, the enriched apheresis product has a predetermined volume and / or a predetermined hematocrit regardless of the number of PBMC collection cycles executed by the Apheresis system and / or the number of pre-products used to produce the enriched apheresis product.

[0209] In some embodiments, the predetermined volume is about 120 ml to about 400 mL. In some embodiments, the predetermined volume is about 120 ml, about 150 ml, about 175 ml, about 180 ml, about 200 ml, about 225 ml, about 250 ml, about 275 ml, about 300 ml, about 325 ml, about 350 ml, about 375 ml, or about 400 ml or less. In some embodiments, the apheresis is configured with a specific target yield of PBMCs to be collected and treated. The specific target yield of PBMCs to be collected and treated may be assessed by the apheresis system and / or by entering the subject's PBMC pre-count. Based on the target PBMC yield and the number of PBMCs collected during each mononuclear collection cycle, the controller of the apheresis system may determine the number of collection cycles to execute. As an example, in some embodiments, if the target PBMC yield is about 5x109PBMCs, the apheresis system will collect about 1 x109PBMCs per mononuclear collection cycle, then the controller will determine that it is appropriate to execute the mononuclear collection cycle five times.

[0210] In some embodiments, the target PBMC yield is at least about 0.1 xio7, at least about 0.2x107, at least about 0.3x107, at least about 0.4x107, at least about 0.5x107, at least about 0.6X 107, at least about 0.7X 107, at least about 0.8x107, at least about 0.9X 107, at least about 1 x 1 o7, at least about 2x 107, at least about 4x 107, at least about 6x 107, at least about 8x 107, at least about 9x107, at least about 1 X 108, at least about 2X 108, at least about 3x108, at least about 4X 108, or at least about 5x108cells / mL. In some embodiments, the target PBMC yield is from about 0.5x106cells / mL to about 4X 106cells / mL. In some embodiments, target PBMC yield is from about 0.5x106cells / mL to about 1 xio8cells / mL. In some embodiments, target PBMC yield is from about 4.0x106cells / mL to about 1 xio8cells / mL.

[0211] In some embodiments, the predetermined hematocrit is about 0% to about 10%. In another embodiment, the predetermined hematocrit is about 2%. In some embodiments, the predetermined volume is approximately 200 mL and the predetermined hematocrit is approximately 2%. The predetermined volume and / or the predetermined hematocrit may vary without departing from the scope of the present disclosure.

[0212] In some embodiments, an anticoagulant is added to the solution comprising the nanoparticle or the drawn blood prior to, during, or after the admixing. In some embodiments, an anticoagulant is added to the solution comprising the nanoparticle or the drawn blood. In some embodiments, an anticoagulant is added to the drawn blood. In some embodiments, such anticoagulant is added through an anticoagulant pump to draw anticoagulant from an anticoagulant bag and mix the anticoagulant with the content in the apheresis system. In some embodiments, the anticoagulant is sodium citrate. Sodium citrate is widely accepted because healthy donors are able to handle it better. Heparin can also be used, for example, for sicker populations. Using heparin will maintain a static state of calcium ion activity. Citrate based anticoagulants bind with calcium ions. Decrease in calcium ions may cause increase in cardiac adverse events such as arrhythmias. For this reason, heparin has been safely used in cardiac applications for many years. Heparin is standard for the care for patients undergoing vascular procedures. In another embodiment, the anticoagulant can include ethylenediaminetetraacetate (EDTA), and / or fluoride.

[0213] In some embodiments, the apheresis of blood drawn from a subject, and the admixing of the blood with a nanoparticle composition / solution described herein, are performed sequentially, or concurrently.

[0214] In some embodiments, apheresis of blood drawn from a subject separates the blood components, thereby generating a first blood fraction containing immune effector cells and a second blood fraction free of immune effector cells. The blood fraction contains immune effector cells, including peripheral blood mononuclear cells (PBMCs), is used for transfection. In some embodiments, the separation is followed by admixing a solution comprising the nanoparticle with the first blood fraction containing the immune effector cells. In some embodiments, the apheresis is leukopheresis. In some embodiments, the first blood fraction is fractionated into an apheresis bag and the admixing comprises adding the solution into the pheresis bag.

[0215] In another embodiments, the contacting and transfection of the immune effector cells is achieved by admixing a solution comprising the nanoparticle with whole blood drawn from the subject, then followed by apheresis of the admixed whole blood, thereby generating a first blood fraction comprising immune effector cells and a second blood fraction free of immune effector cells. The first blood fraction comprises immune effector cells transfected with the nanoparticle described herein. In some embodiments, the apheresis is leukopheresis. In some embodiments, the first blood fraction is fractionated into an apheresis bag and the admixing comprises adding the solution into the pheresis bag.

[0216] In some embodiments, following the contacting and transfection, the immune effector cells in the first blood fraction are separated from free nanoparticles, including isolated / unbound nanoparticles, and / or other unwanted components from the apheresis. In some embodiments, the cells in the first blood fraction collected by apheresis are washed to remove the free / unbound nanoparticles, the plasma fraction, and other undesirable components, and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations, subsequent modifications, or re-infusion back to the subject.

[0217] As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer’s instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+-free, Mg2+ free PBS, PlasmaLyte A, or another saline solution with or without a buffer.

[0218] In some embodiments, the immune effector cells in the first blood fraction are separated by plasmapheresis, filtering with a spinning membrane filter, or centrifugation, or a combination of any of these procedures. In some embodiments, the immune effector cells in the first blood fraction are separated by plasmapheresis. In some embodiments, the immune effector cells in the first blood fraction are separated by a spinning membrane filter. In some embodiments, the immune effector cells in the first blood fraction are separated by centrifugation.

[0219] In some embodiments, the immune effector cells in the first blood fraction are further separated by plasmapheresis, a process that selectively removes plasma while retaining cellular components, thereby allowing for the enrichment of immune effector cells. Plasmapheresis is advantageous in maintaining cell integrity and reducing unwanted plasma proteins that may interfere with subsequent processing. In some embodiments, the immune effector cells in the first blood fraction are separated by a spinning membrane filter, which utilizes a semi-permeable membrane and centrifugal force to selectively filter out smaller plasma components while retaining immune effector cells. This technique can provide a high- purity fraction of immune cells while minimizing shear stress that could impact cell functionality. In some embodiments, the immune effector cells in the first blood fraction are separated by centrifugation, wherein the differential densities of blood components are exploited to isolate the immune effector cells. Centrifugation conditions, such as speed, duration, and gradient media, may be optimized to maximize recovery while minimizing cell damage. Density gradient centrifugation, for example, may be employed to enhance the separation of lymphocytes, monocytes, and other immune effector cells from red blood cells and platelets.

[0220] In some embodiments, a combination of plasmapheresis, spinning membrane filtration, and centrifugation may be used sequentially or in parallel to achieve optimal separation of immune effector cells. This multi-step approach may be beneficial in cases where high-purity cell fractions are required for therapeutic applications, such as cell infusion or genetic modification.

[0221] In some embodiments, the transfection of the immune effector cells collected comprises admixing the cells, the drawn blood comprising the immune effector cells, or a fraction of the blood comprising such cells, with the nanoparticles of the present disclosure. In some embodiments, the admixing may be performed in the apheresis system during the apheresis. In some embodiments, the admixing may be performed prior to the apheresis. In other embodiments, the admixing may be performed subsequent to the apheresis. In yet other embodiments, the admixing may be performed in a closed system, such as a closed vein-to-vein system.

[0222] In some embodiments, the admixing is performed at a cell density from about 250,000 cells per mL to about 1 million cells per mL. In some embodiments, the cell density is from about 250,000 cells per mL to about 1 million cells per mL. In some embodiments, the cell density is from about 250,000 cells per mL to about 500,000 cells per mL. In some embodiments, the cell density is from about 500,000 cells per mL to about 750,000 cells per mL. In some embodiments, the cell density is from about 750,000 cells per mL to about 1 million cells per mL. In some embodiments, the admixing is performed at a cell density of from about 1 million cells per mL to about 20 million cells per mL. In some embodiments, the admixing is performed at a cell density of at least 5 million cells per mL. In some embodiments, the admixing is performed at a cell density of at least 10 million cells per mL. In some embodiments, the admixing is performed at a cell density of at least 15 million cells per mL. In some embodiments, the admixing is performed at a cell density of at least 20 million cells per mL.

[0223] In some embodiments, the cell density for admixing is within a specific range selected from the group consisting of: between 250,000 cells per mL and 500,000 cells per mL; between 250,000 cells per mL and 750,000 cells per mL; between 250,000 cells per mL and 1 million cells per mL; between 250,000 cells per mL and 5 million cells per mL; between 250,000 cells per mL and 10 million cells per mL; between 250,000 cells per mL and 15 million cells per mL; between 250,000 cells per mL and 20 million cells per mL; between 500,000 cells per mL and 750,000 cells per mL; between 500,000 cells per mL and 1 million cells per mL; between 500,000 cells per mL and 5 million cells per mL; between 500,000 cells per mL and 10 million cells per mL; between 500,000 cells per mL and 15 million cells per mL; between 500,000 cells per mL and 20 million cells per mL; between 750,000 cells per mL and 1 million cells per mL; between 750,000 cells per mL and 5 million cells per mL; between 750,000 cells per mL and 10 million cells per mL; between 750,000 cells per mL and 15 million cells per mL; between 750,000 cells per mL and 20 million cells per mL; between 1 million cells per mL and 5 million cells per mL; between 1 million cells per mL and 10 million cells per mL; between 1 million cells per mL and 15 million cells per mL; between 1 million cells per mL and 20 million cells per mL; between 5 million cells per mL and 10 million cells per mL; between 5 million cells per mL and 15 million cells per mL; between 5 million cells per mL and 20 million cells per mL; between 10 million cells per mL and 15 million cells per mL; between 10 million cells per mL and 20 million cells per mL; and between 15 million cells per mL and 20 million.

[0224] In some embodiments, the contacting is performed at a defined ratio of DNA copy number to cells. It is understood that the DNA copy number is to refer to the total number of DNA molecules, such as the total number of double stranded DNA as comprised in a nanoparticle in accordance with the invention, such as in an LNP or tLNP. For example in the context of minicircle DNA comprised in a tLNP, copy number refers to the number of double stranded minicircle DNA molecules. The copy number of a nanoparticle can easily be calculated, either based on the amount of DNA that is incorporated in a preparation, or by determining the DNA concentration (e.g. via qPCR) and thus DNA copy number concentration and / or total DNA copy number comprised in an tLNP dose (i.e. amount of DNA comprised in a defined volume of tLNP prepared). Hence, the ratio of DNA copy number per cell can easily be selected or determined, when admixing a certain amount or defined dose of a tLNP preparation and cells contained in a certain volume e.g. whole blood, having a certain cell concentration (cell / mL). Hence, in some embodiments, the contacting is performed at a ratio of DNA copy number to cells of at least 6*102DNA copies per cell, at least 8*102DNA copies per cell, at least 6x103DNA copies per cell, at least 8X 103DNA copies per cell, at least 6x104DNA copies per cell, at least 8x104DNA copies per cell, at least 1 x-|05DNA copies per cell, at least 2X 105DNA copies per cell, at least 4X 105DNA copies per cell, at least 6x105DNA copies per cell, at least 8x105DNA copies per cell, at least 1 x-|06DNA copies per cell, 3x106DNA copies per cell, at least 6x106DNA copies per cell, at least 8x106DNA copies per cell, or at least 1 X 107DNA copies per cell.

[0225] In some embodiments, the contacting is performed at a ratio of DNA copy number to cells of 8x102DNA copies per cell to 8X 103DNA copies per cell; 8X 102DNA copies per cell to 8x104DNA copies per cell; 8x102DNA copies per cell to 2x105DNA copies per cell; 8x102DNA copies per cell to 4x105DNA copies per cell; 8x102DNA copies per cell to 8x105DNA copies per cell; 8X 102DNA copies per cell to 1 x-|06DNA copies per cell; 8X 102DNA copies per cell to 3x106DNA copies per cell; 8X 102DNA copies per cell to 6x10® DNA copies per cell; 8x103DNA copies per cell to 8x104DNA copies per cell; 8x103DNA copies per cell to 2x10sDNA copies per cell; 8x103DNA copies per cell to 4x105DNA copies per cell; 8x103DNA copies per cell to 8x105DNA copies per cell; 8x103DNA copies per cell to 1 x-|06DNA copies per cell; 8X 103DNA copies per cell to 3x10® DNA copies per cell; 8X 103DNA copies per cell to 6x106DNA copies per cell; 8X 104DNA copies per cell to 2x10sDNA copies per cell; 8x104DNA copies per cell to 4x105DNA copies per cell; 8x104DNA copies per cell to 8x10sDNA copies per cell; 8x104DNA copies per cell to 1 x10® DNA copies per cell; 8x104DNA copies per cell to 3x106DNA copies per cell; 8x104DNA copies per cell to 6x106DNA copies per cell; 2x10sDNA copies per cell to 4x10sDNA copies per cell; 2x10sDNA copies per cell to 8x10sDNA copies per cell; 2x10sDNA copies per cell to 1 xio6DNA copies per cell; 2x10sDNA copies per cell to 3x106DNA copies per cell; 2x10sDNA copies per cell to 6x106DNA copies per cell; 4x10sDNA copies per cell to 8x10sDNA copies per cell; 4x10sDNA copies per cell to 1 X 106DNA copies per cell; 4x10sDNA copies per cell to 3x106DNA copies per cell; 4x10sDNA copies per cell to 6x10® DNA copies per cell; 8x10sDNA copies per cell to 1 xio6DNA copies per cell; 8x10sDNA copies per cell to 3x10® DNA copies per cell; 8x10sDNA copies per cell to 6x10® DNA copies per cell; 1 xio6DNA copies per cell to 3x106DNA copies per cell; 1 x10® DNA copies per cell to 6x106DNA copies per cell; or 3x106 DNA copies per cell to 6*106DNA copies per cell. In some embodiments, the contacting is performed at a defined nanoparticle dose of total nucleic acid (in weight) to cells (number). Nanoparticles, in accordance with the invention are to comprise nucleic acid, e.g. an mRNA and DNA, such as a combination of an mRNA encoding a transposase and an mcDNA with compatible ITRs. The total amount of nucleic acid that is comprised in a nanoparticle can easily be calculated, either based on the amount of nucleic acid (such as DNA and RNA) that is incorporated in a preparation, or by determining the nucleic acid concentration (e.g. via qPCR) and thus nucleic acid copy number concentration, while knowing the length of the nucleic acids (e.g. of mRNA and e.g. minicircle DNA), or any other suitable means (e.g. utilizing spectrophotometry and / or suitable dyes). Hence, the nanoparticle dose (i.e. ng of nucleic acid per cell) can easily be selected or determined, when admixing a certain amount or defined dose of a tLNP preparation and cells contained in a certain volume e.g. whole blood, having a certain cell concentration (cell / mL). Preferably, the total dose of nucleic acid comprises DNA to mRNA in a weight ratio of about 1 :1. Total nucleic acid may thus preferably comprise mcDNA and an mRNA encoding transposase, or the like, and as described herein and as shown in the examples, in a 1 :1 ratio by weight of about 1 :1. More preferably, the ratio is 1 :1.

[0226] In some embodiments, the contacting is performed at a nanoparticle dose of at least 2*1 O'6ng of nucleic acid per cell, at least 5x10-6ng of nucleic acid per cell, at least 2*10'5ng per cell, at least 5xW5ng nucleic acid per cell, at least 2xW4ng nucleic acid per cell, at least 5x10-4ng nucleic acid per cell, at least 0.00125 ng nucleic acid per cell, at least 0.00125 ng nucleic acid per cell, at least 0.0025 ng nucleic acid per cell, at least 0.005 ng nucleic acid per cell, at least 0.01 ng nucleic acid per cell, at least 0.02 ng nucleic acid per cell, or at least 0.04 ng nucleic acid per cell.

[0227] In some embodiments, the contacting is performed at a nanoparticle dose within a range of 2x10“6ng nucleic acid per cell to 5x10“6ng nucleic acid per cell; 2x10“6ng nucleic acid per cell to 2x10“5ng nucleic acid per cell; 2x10“6ng nucleic acid per cell to 5x10“5ng nucleic acid per cell; 2x10“6ng nucleic acid per cell to 2x10“4ng nucleic acid per cell; 2x10“6ng nucleic acid per cell to 5x10“4ng nucleic acid per cell; 2x10“6ng nucleic acid per cell to 0.00125 ng nucleic acid per cell; 2x10“6ng nucleic acid per cell to 0.0025 ng nucleic acid per cell; 2x10“6ng nucleic acid per cell to 0.005 ng nucleic acid per cell; 2x10“6ng nucleic acid per cell to 0.01 ng nucleic acid per cell; 2*10“6ng nucleic acid per cell to 0.02 ng nucleic acid per cell; 2*10“6ng nucleic acid per cell to 0.04 ng nucleic acid per cell; 5*10“6ng nucleic acid per cell to 2*10“5ng nucleic acid per cell; 5x10“6ng nucleic acid per cell to 5*10“5ng nucleic acid per cell; 5*10“6ng nucleic acid per cell to 2*10“4ng nucleic acid per cell; 5*10“6ng nucleic acid per cell to 5*10“4ng nucleic acid per cell; 5*10“6ng nucleic acid per cell to 0.00125 ng nucleic acid per cell; 5*10“6ng nucleic acid per cell to 0.0025 ng nucleic acid per cell; 5*10“6ng nucleic acid per cell to 0.005 ng nucleic acid per cell; 5x10“6ng nucleic acid per cell to 0.01 ng nucleic acid per cell; 5x10“6ng nucleic acid per cell to 0.02 ng nucleic acid per cell; 5x10“6ng nucleic acid per cell to 0.04 ng nucleic acid per cell; 2x10“5ng nucleic acid per cell to 5x10“5ng nucleic acid per cell; 2x10“5ng nucleic acid per cell to 2x10“4ng nucleic acid per cell; 2x10“5ng nucleic acid per cell to 5x10“4ng nucleic acid per cell; 2x10“5ng nucleic acid per cell to 0.00125 ng nucleic acid per cell; 2x10“5ng nucleic acid per cell to 0.0025 ng nucleic acid per cell; 2x10“5ng nucleic acid per cell to 0.005 ng nucleic acid per cell; 2x10“5ng nucleic acid per cell to 0.01 ng nucleic acid per cell; 2x10“5ng nucleic acid per cell to 0.02 ng nucleic acid per cell; or 2x10“5ng nucleic acid per cell to 0.04 ng nucleic acid per cell. In some embodiments, the contacting comprises admixing a solution comprising the nanoparticle with the drawn blood at a weight-to-volume ratio of nanoparticle dose to drawn blood of at least 0.1 ng nucleic acid / mL, at least 1 ng nucleic acid / mL, at least 5 ng nucleic acid / mL, at least 10 ng nucleic acid / mL, at least 25 ng nucleic acid / mL, at least 50 ng nucleic acid / mL, at least 100 ng nucleic acid / mL, at least 200 ng nucleic acid / mL, at least 400 ng nucleic acid / mL, at least 800 ng nucleic acid / mL, at least 1000 ng nucleic acid / mL, at least 1200 ng nucleic acid / mL, at least 1400 ng nucleic acid / mL, at least 1600 ng nucleic acid / mL, at least 1800 ng nucleic acid / mL, or at least 2000 ng nucleic acid / mL.

[0228] In some embodiments, the contacting comprises admixing a solution comprising the nanoparticle with the drawn blood at a weight-to-volume ratio of nanoparticle dose to drawn blood within a range of 0.1 ng nucleic acid / mL to 1 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 5 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 10 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 25 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 50 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 100 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 200 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 400 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 800 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 1000 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 1200 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 1400 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 1600 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 1800 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 2000 ng nucleic acid / mL; 1 ng nucleic acid / mL to 5 ng nucleic acid / mL; 1 ng nucleic acid / mL to 10 ng nucleic acid / mL; 1 ng nucleic acid / mL to 25 ng nucleic acid / mL; 1 ng nucleic acid / mL to 50 ng nucleic acid / mL; 1 ng nucleic acid / mL to 100 ng nucleic acid / mL; 1 ng nucleic acid / mL to 200 ng nucleic acid / mL; 1 ng nucleic acid / mL to 400 ng nucleic acid / mL; 1 ng nucleic acid / mL to 800 ng nucleic acid / mL; 1 ng nucleic acid / mL to 1000 ng nucleic acid / mL; 1 ng nucleic acid / mL to 1200 ng nucleic acid / mL; 1 ng nucleic acid / mL to 1400 ng nucleic acid / mL; 1 ng nucleic acid / mL to 1600 ng nucleic acid / mL; 1 ng nucleic acid / mL to 1800 ng nucleic acid / mL; 1 ng nucleic acid / mL to 2000 ng nucleic acid / mL; 5 ng nucleic acid / mL to 10 ng nucleic acid / mL; 5 ng nucleic acid / mL to 25 ng nucleic acid / mL; 5 ng nucleic acid / mL to 50 ng nucleic acid / mL; 5 ng nucleic acid / mL to 100 ng nucleic acid / mL; 5 ng nucleic acid / mL to 200 ng nucleic acid / mL; 5 ng nucleic acid / mL to 400 ng nucleic acid / mL; 5 ng nucleic acid / mL to 800 ng nucleic acid / mL; 5 ng nucleic acid / mL to 1000 ng nucleic acid / mL; 5 ng nucleic acid / mL to 1200 ng nucleic acid / mL; 5 ng nucleic acid / mL to 1400 ng nucleic acid / mL; 5 ng nucleic acid / mL to 1600 ng nucleic acid / mL; 5 ng nucleic acid / mL to 1800 ng nucleic acid / mL; or 5 ng nucleic acid / mL to 2000 ng nucleic acid / mL.

[0229] Tabulated below is a list of exemplary lipid nanoparticle (LNP) doses calculated in weight-to-cell ratio (ng / cell) or DNA copy number (DNA copy number per cell). Herein the mcDNA has a size of about 3 kB. In these exemplary nanoparticles, RNA and mcDNA is packaged in a 1 :1 ratio by weight. The tLNP dose listed in the table is the total weight of nucleic acid (i.e. RNA and mcDNA) as contained in lipid nanoparticles which is contacted with a defined amount of cells, i.e. 20.000 cells. From the dose contacted with this number of cells, the dose nucleic acid per cell can easily be calculated. For the number of DNA copies per cell, if one assumes that the total dose of nucleic acid corresponds with 100% mcDNA, this means 1 ng nucleic acid corresponds with 3.26 x 108copies of mcDNA. As in this scenario, the ratio of mcDNA to RNA is 1 :1 by weight, this means the actual copy number in the dose needs to be needs to be divided by 2, to get the actual copy number. For the 1 ng, this means a DNA copy number of 1 .63 x 108. When brought into contact with 20.000 cells, this translates to 8.150 DNA copies per cell.

[0230] In some embodiments, the admixing immediately follows drawing the blood from the subject, ensuring there is minimal delay between blood collection and processing. Immediate admixing may help preserve cell viability and functionality by preventing prolonged exposure of immune effector cells to potentially suboptimal conditions.

[0231] In some embodiments, the admixing occurs within twenty-four hours of drawing the blood from the subject, twelve hours of drawing the blood from the subject, within four hours of drawing the blood from the subject, within an hour of drawing the blood from the subject. In some embodiments, the admixing occurs within a specific range of time intervals selected from the group consisting of twenty-four hours and twelve hours, twenty-four hours and four hours, twenty-four hours and one hour, twelve hours and four hours, twelve hours and one hour, and four hours and one hour of drawing the blood from the subject.

[0232] In other embodiments, the admixing is performed within 1 hour, forth-five minutes, thirty minutes, fifteen minutes, ten minutes, five minutes, or one minute of drawing the blood from the subject. In some embodiments, the admixing occurs within a specific range of time intervals selected from the group consisting of one hour and forty-five minutes, one hour and thirty minutes, one hour and fifteen minutes, one hour and ten minutes, one hour and five minutes, one hour and one minute, forty-five minutes and thirty minutes, forty-five minutes and fifteen minutes, forty-five minutes and ten minutes, forty-five minutes and five minutes, forty-five minutes and one minute, thirty minutes and fifteen minutes, thirty minutes and ten minutes, thirty minutes and five minutes, thirty minutes and one minute, fifteen minutes and ten minutes, fifteen minutes and five minutes, fifteen minutes and one minute, ten minutes and five minutes, ten minutes and one minute, or five minutes and one minute after drawing the blood from the subject.

[0233] In yet other embodiments, the admixing is performed concurrently with the drawing of the blood from the subject. In some embodiment, the concurrent admixing is achieved by a real-time or inline mixing approach, for example by using specialized blood collection devices or systems that introduce the reagents immediately as the blood is drawn from a subject. This method could help further minimize processing time and reduce handling procedure steps, thereby ensuring rapid and efficient preparation of the treated blood fraction.

[0234] After the transfection, the immune effector cells, or the drawn blood or a fraction thereof containing the immune effector cells, are returned to the subject by infusion.

[0235] In some embodiments, the immune effector cells, or the drawn blood or a fraction thereof containing the immune effector cells, are infused to the subject within thirty-six hours, twenty-four hours, twelve hours, six hours, four hours, two hours, or one hour of drawing the blood from the subject. In some embodiments, the infusion occurs within a specific range of time interval selected from the group consisting of between thirty-six hours and twenty-four hours, between thirty-six hours and twelve hours, between thirty-six hours and six hours, between thirty-six hours and four hours, between thirty-six hours and two hours, between thirty-six hours and one hour, between twenty-four hours and twelve hours, between twenty- four hours and six hours, between twenty-four hours and four hours, between twenty-four hours and two hours, between twenty-four hours and one hour, between twelve hours and six hours, between twelve hours and four hours, between twelve hours and two hours, between twelve hours and one hour, between six hours and four hours, between six hours and two hours, between six hours and one hour, between four hours and two hours, between four hours and one hour, and between two hours and one hour, of drawing the blood from the subject.

[0236] Examples mcDNA & mRNA

[0237] Minicircle DNA (Mayrhofer, Blaesen, Schleef, & W, 2008) was produced in bacteria (TB1 E.coli strain) and purified using a Machery-Nagel maxiprep kit for plasmid purification. mRNA was produced by Trilink using modified nucleotides or was produced using in vitro transcription with modified nucleotides using a PCR product as a template and purified using the Invitrogen MEGAclear transcription cleanup kit. SB100x mRNA was 1305 bases in length, corresponding to 443 kDa, eGFP mcDNA to 2971 bp, corresponding to 1931 kDA, and the CD19 CAR encoding mcDNA was 3786 bp, corresponding with 2460 kDa. The mcDNA was compatible with the SB100x transposase to allow for integration of expression cassettes. anti-CD7 VHH and anti-CD3 ScFv binders

[0238] The anti-CD7 VHH clone (see table 1 below) was identified through phage display. Briefly, Llamas were immunized with LNPs constructed with SM-102 ionizable lipid, containing mRNA encoding full length human CD7 antigen as set forth in GEN BANK Accession No. AAH13297.1. Binding EC50 of the anti CD7 VHH on primary T lymphocytes was determined to be 10.94 nM based on a flow cytometry titration assay. This VHH clone was fused to a C-terminal tag including a cysteine for conjugation to PEG-lipid and an EPEA sequence for affinity purification and was produced in yeast cells (Saccharomyces cerevisiae) by QVQ (Utrecht, The Netherlands). An anti-human CD3 recombinant antibody scFv fragment (see table 2 below), based on the CD3 antibody clone UCHT1 was obtained from Creative Biolabs. The scFv fragment was made by fusion of the variable heavy and light chain using a flexible glycineserine linker (VH-4(G4S)-VL), the fragment was functionalised with a C-terminal cysteine for conjugation to PEG-lipid, included a C-terminal 6xHIS tag for affinity purification, and was expressed in mammalian cells.

[0239] Table 1. anti-CD7 VHH

[0240] Table 2. anti-human CD3 scFv

[0241] Lipid nanoparticle constituents

[0242] Ionizable lipids used in the experiments were the following:

[0243] SM102 (2089251-47-6 ) (8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1 -octylnonyl ester) was obtained from Cayman chemicals.

[0244] Lipid A9 (2036272-50-9) (b / s(2-butyloctyl) 10-(N-(3-

[0245] (dimethylamino)propyl)nonanamido)nonadecanedioate) was obtained from DC chemicals.

[0246] ALC-0315 (2036272-55-4) (2-hexyl-decanoic acid, 1 ,1'-[[(4-hydroxybutyl)imino]di-6,1- hexanediyl] ester) was obtained from DC chemicals. - Lipid 5 (2089251-33-0) (8-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]-octanoic acid, 1 -octylnonyl ester) was obtained from Cayman Chemicals.

[0247] C12:200 (1220890-25-4) (1 ,1 '-[[2-[4-[2-[[2-[b / s[(2S)-2-hydroxydodecyl]amino]ethyl][(2S)- 2-hydroxydodecyl]amino]ethyl]-1-piperazinyl]ethyl]imino]b / s-2-dodecanol) was obtained from DC Chemicals. DLin-DMA-MC3 (1224606-06-7 )(O-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-

[0248] (N,N-dimethylamino))

[0249] DLin-MC3-DMA and

[0250] 93-017S (2227008-67-3)

[0251] (O=C(CCN(CCCN1C=NC=C1)CCC(OCCSCCCCCCCCCCCCCC)=O)OCCSCCCCCCCCCC CCCC) were obtained from DC Chemicals.

[0252] CKK:E12 (1432494-65-9) (3,6-b / s[4-[b / s(2-hydroxydodecyl)amino]butyl]-2,5- piperazinedione) was obtained from Cayman chemicals. CKK-E12

[0253] CL4F 1 1_E-3 (7-(4-(dipropylamino)butyl)-7-hydroxytridecane1 ,13-diyl bis(6- propylundecanoat), CL4F 12_E-6 (7-(4-(dipropylamino)butyl)-7-hydroxytridecane1 ,13-diyl bis(6- hexyldodecanoate)), CL4F8_[35 (7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1 ,13-diylbis(3- pentyloctanoate),

[0254] Tot-3 (2-(methylpiperidine-4-carboxamido)-2-(oleoxy)methyl)propane-1 ,3-diyldioleate) and

[0255] Tot-19 (2-(1-isopropylpiperidine-4-carboxamido)-2-((oleoyloxy)methyl)propane-1 ,3- diyldioleate were all obtained from the Group of Yusuke Sato at Hokkaido University. - LP-01 (9Z,12Z-octadecadienoic acid, 3-[4,4-bis(octyloxy)-1-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl ester) was obtained from Cayman chemical.

[0256] Helper lipids used in the experiments were the following:

[0257] DSPC (816-94-4 ) (1 ,2-distearoyl-sn-glycero-3-phosphocholine),

[0258] DMPC (18194-24-6 ) (1 ,2-dimyristoyl-sn-glycero-3-phosphocholine),

[0259] DPPC (63-89-8 ) (1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine),

[0260] DAPC (61596-53-0) (1 ,2-diarachidoyl-sn-glycero-3-phosphatidylcholine),

[0261] DOPC (4235-95-4) (1 ,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine),

[0262] Egg-PC (97281-44-2) (L-a-phosphatidylcholine),

[0263] DOPE (4004-05-1) (1 ,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine), DOPG (67254-28-8) (1 ,2-di-(9Z-octadecenoyl)-sn-glycero-3-phospho-(1'-rac- glycerol));

[0264] DSPE (1069-79-0) (1 ,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine) and DSPG (200880-42-8) (1 ,2-dioctadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) ) were all obtained from Avanti Research.

[0265] DPyPE (201036-16-0) (1 ,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine)

[0266] PGPR (29894-35-7) (1 ,2,3-Propanetriol, homopolymer, (9Z,12R)-12-hydroxy-9- octadecenoate)

[0267] E Sphingomyelin (Lipoid E SM) (85187-10-6)

[0268] GM1 (monosialotetrahexosylganglioside) (37758-47-7) ((2S,4S,5R,6R)-5-acetamido-2- [(2S,3R,4R,5S,6R)-5-[(2S,3R,4R,5R,6R)-3-acetamido-5-hydroxy-6-(hydroxymethyl)-4- [(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-2- [(2 / ?,3S,4 / ?,5R,6 / ?)-4,5-dihydroxy-6-[(E,2 / ?,3S)-3-hydroxy-2-(icosanoylamino)icos-4- enoxy]-2-(hydroxymethyl)oxan-3-yl]oxy-3-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-4- hydroxy-6-[(1 R,2R)-1 ,2,3-trihydroxypropyl]oxane-2-carboxylic acid)

[0269] GM3 (124579-05-1 )(monosialodihexosylganglioside) ((2S,4S,5R,6R)-5-acetamido-2- [(2S,3R,4S,5S,6R)-2-[(2R,3S,4R,5R,6R)-4,5-dihydroxy-2-(hydroxymethyl)-6- [(E,2S,3R)-3-hydroxy-2-(octadecanoylamino)octadec-4-enoxy]oxan-3-yl]oxy-3,5- dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-4-hydroxy-6-[(1 R,2R)-1 ,2,3- trihydroxypropyl]oxane-2-carboxylic acid)

[0270] Lysobisphosphatidic acid (LBPA) (288089-44-1) ((S,S) Bisoleoyl-lysobisphosphatidic acid) and

[0271] Cardiolipin (CL) (1 ,3-bis(sn-3’-phosphatidyl)-sn-glycerol) were all obtained from the group of Raymond Schiffelers at UMC Utrecht.

[0272] Cholesterol (57-88-5) (cholest-5-en-3ll-ol) was purchased from Merck.

[0273] DMG-PEG-2000 (160743-62-4) (1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) was purchased from Avanti, and;

[0274] DSPE-PEG(2000) Maleimide (474922-22-0) (1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[carbonyl-amino(polyethylene glycol)-2000-N’-(3- maleimidopropionyl)]) was purchased from Avanti.

[0275] LNP production

[0276] LNP formulations were prepared using a Nanoassemblr microfluidics system. Nucleic acid was diluted in 100mM acetate buffer at pH 4 and mixed with an organic phase containing ionizable lipid, helper lipid, PEG lipid and cholesterol at a flow rate ratio of 2:1 at a total flow rate of 6ml / minute (aqueous: lipid phase). All LNPs were formulated with an N / P ratio between 5.6 to 6.6. The N / P Ratio of lipid nanoparticles refers to the molar ratio of ionizable amine head groups (N+) to phosphate groups (P) on the encapsulated nucleic acid. It is understood that an N / P ratio of 6, corresponds to 6 moles of ionizable head groups (N+) to 1 mole of phosphate groups (P), i.e. 6 / 1 = 6. The molar ratio of lipids was 40:20:39:1 (Ionizable lipid : helper lipid : cholesterol : PEG-Lipid). Various different ionizable lipids and helper lipids were used as indicated. DMG-PEG-2000 was used as a PEG lipid. LNPs were produced using a total lipid concentration of between 2 to 5 mM. LNPs were produced by mixing the lipid containing organic phase diluted in ethanol and the nucleic acid containing aqueous phase prepared in 100mM acetate buffer pH 4 using the Nanoassemblr Benchtop microfluidic mixing system followed by overnight dialysis against phosphate-buffered saline (PBS) at 4°C. Particles were produced in a volume of between 1ml to 10ml. Particles were provided with one or both targeting moieties by first conjugatinga C-terminal cysteine which had been engineered onto the c-terminus of the anti-CD7 VHH and anti-CD3 ScFv binders to a reactive maleimide group on DSPE-PEG-2000- maleimide. Lipid conjugate (targeting CD3 and / or CD7) was post-inserted into the LNP after LNP production at a range of molar ratios ranging from 0.05%: 100% to 1 %: 100% of (Conjugate lipid%:Total LNP lipid%) according to a procedure described (Swart et al. Int J Pharm, 2022, May 25, Vol. 620 121741). Such lipid nanoparticels with T-cell activation moieties and / or T-cell binding moieties, can be referred to as tLNP herein (i.e. targeted particles). LNPs were then purified and concentrated using a 100kDA spin filters (Amicon) with three washing steps to remove unconjugated anti-CD3 ScFv binder and anti-CD7 VHH binder. Final LNP formulations were formulated in PBS. The nucleic concentration of the final LNP product typically ranged from 10ng / pl to 150ng / pl.

[0277] LNP Characterization

[0278] The encapsulated nucleic acid concentration and encapsulation efficiency (EE%) of tLNPs and LNPs was determined using the Quant-iT Ribogreen Assay kit which was used according to manufacturer’s instructions. LNPs and tLNPs were characterised using dynamic light scattering using a Malvern Nano S Zetasizer. All LNPs and tLNPs used here possessed desirable characteristics with encapsulation efficiencies in excess of 90% with sizes between 80 to 150nm with PDIs <0.3 (see exemplary Fig. 18).

[0279] T cell isolation and transfection

[0280] For transfection of T cells PBMCs were isolated from healthy donor blood using Ficoll-PAque (GE healthcare) and SepMate PBMC isolation tubes (stem cell) according to manufacturer’s protocol. T cells (including CD3+CD4+ and CD3+CD8+ cells) were isolated from the PBMCs using EasySep Human T cell isolation kit (stem cell) using manufacturer’s protocol. T cells were plated 60.000 cells per well in a 96 well F-bottom plate in 170 ul T cell medium with 50 iU / ml IL2 (Miltenyi Biotec) and 1 ug / ml Apo lipoprotein-E (Abeam), if indicated, the T cells were activated by CD3 / CD28 dynabeads (Gibco) at a 1 :1 bead to T cell ratio. LNPs were added at the indicated total NA (nucleic acid) dose and incubated for 96 hours for DNA transfection. Total NA doses typically ranged from 50 to 1000 ng. This is equivalent to a nucleic acid dose per cell of 0.0008 ng to 0.01 ng which in case of solely mcDNA corresponds to roughly 1 x 105to 2 x 106copies per cell.

[0281] Flow Cytometry analysis

[0282] T cell activation status was monitored by flow cytometry based on an increase in cell side and forward scatter. Transfection efficiency was also measured by flow cytometry. mcDNA expression was assessed using minicircle DNA encoding either CD19 CAR or eGFP. mRNA expression was assessed using expression of mCherry or BFP reporter mRNAs. CAR was detected using CD19 CAR detection reagent (Miltenyi Biotec) and streptavidin-APC-cy7 (BD). Samples were measured on either a FACs Canto or LSRFortessa flow cytometer (BD). Results were analyzed using FlowJo software.

[0283] T cell killing assay

[0284] A luciferase-based killing assay was performed to evaluate the functionality of tLNP-engineered CD19 CAR T cells against CD19+ target cells. Prior to the assay, CD19 CAR expression was confirmed and only CAR+ T cells were counted as effectors. On day 14 post-transfection, CAR T cells were co-cultured with 5,000 target cells (K562, Raji, or Nalm-6) at varying effector-to- target (E:T) ratios (3:1 , 1.5:1 , 0.75:1 , and 0.35:1) for 24 hours and for 4 days. Before measurement, Luciferin was added to each well and luminescence using a Spectramax iD3 device. Cell numbers were normalized to CD19 CAR expression, and untransfected T cells from the same donor were used as negative controls. Specific lysis was calculated using the standard formula.

[0285] In vivo experiments

[0286] Immunodeficient NXG mice were humanized by tail vein injection of 5 million human PBMCS at day -11 . On day - 8, mice were injected with 500,000 luciferase expressing Nalm-6 cells. At day 0, mice were injected with either PBS or tLNPs containing mcDNA CAR and SB100x transposase mRNA at a dose of 0.04mg / kg or 0.004mg / kg total nucleic acid via the tail vein. Tumor growth was assessed using bioluminescence imaging. CAR expression was assessed using flow cytometry of blood samples. For toxicity assessment, BALB / c mice were injected with tLNPs at dose of 0.25mg / kg, 0.05mg / kg, 0.01mg / kg or 0.005mg / kg. ALT and AST levels were then assessed using ELISA 1 and 7 days post-tLNP administration.

[0287] The combination of anti-CD3 and anti-CD7 synergistically increases nucleic acid delivery to activated and resting T cells, and allows transfer and stable delivery and expression of DNA to resting T cells (in vitro, in whole blood and in vivo) via particle mediated activation

[0288] Example 1

[0289] Removal of free binder and demonstration of association of binder to particle.

[0290] We first set out to study how the combination of T cell targeting ligands and T cell activating binders on our LNPs could interact to influence T cell transfection and activation. Before we could start to study this process it was important to separate free anti-CD3 scFv protein from LNP associated anti-CD3 scFv as both free and particle associated scFv are capable of T cell activation. Therefore, before we could start to investigate the influence of particle associated anti-CD3 scFv, it was important to ensure that residual unconjugated anti-CD3 scFv was removed from the particle preparation, as its presence would confound results. To study this, an LNP formulation was prepared using SM102 according to the methods section and postinserted with CD3-ScFv conjugate at a total binder density of 1%.

[0291] We found that anti-CD3 scFv could be efficiently removed from the particle during preparation by three washes on a 100 kDa Amicon spin filter. As can be seen in the left most lane of Figure 3, when a preparation of unpurified anti-CD3 scFv targeted-LNPs was run on an SDS-PAGE gel, 3 bands corresponding to unconjugated anti-CD3 ScFv dimers, DSPE-PEG conjugated anti-CD3 scFv and unconjugated anti-CD3-scFv monomers were observed. The same bands were observed in the preparation after overnight dialysis into PBS using 20 kDa dialysis cassettes. In contrast, when the particles were repeatedly washed using 100 kDa Amicon spin filters, only the conjugated form was retained, demonstrating the removal of contaminating unconjugated anti-CD3 scFv monomers and dimers. In addition, we also demonstrated that non-particle associated anti-CD3 scFv conjugate is removed from the particle mix. When a preparation of conjugated DSPE-PEG CD3 scFv at an identical concentration to that found in the particle mix was analyzed on an SDS-PAGE gel, the same three bands observed in the particle mix corresponding to unconjugated anti-CD3 scFv dimers, DSPE-PEG conjugated anti-CD3 scFv and unconjugated anti-CD3 scFv monomers were observed. 20 kDa dialysis of the conjugate did not remove these bands. In contrast to the particle mix, when the conjugate mix is washed three times on the amicon spinfilter, all three bands, including the conjugate band, were no longer observed. The same pattern is observed when unconjugated anti-CD3 ScFv protein was treated in the same manner.

[0292] Together, these observations demonstrate that only particle-associated anti-CD3 scFv is retained after 100 kDa spin filter purification. This allowed us to conclude that functional results obtained with particles purified using this method were due to particle-associated binder rather than contaminating residual protein.

[0293] Example 2

[0294] Activation and transfection of activated and resting T cells by anti-CD3 and anti-CD7 targeted LNP formulation - a synergistic combination

[0295] To study if and how anti-CD7 T cell targeting and anti-CD3 scFv activation aid in targeting T cells, LNPs were formulated with mcDNA encoding GFP protein as a nucleic acid payload using SM102 as an ionizable lipid and DSPC as a helper lipid according to the LNP production section as described above. The LNPs were functionalized with anti-CD7 VHH, anti-CD3 scFv or both anti-CD7 VHH and anti-CD3 scFv at various different molar ratios. The total binder density of all these particles was 0.5 mol% of total lipid. As a control for residual unconjugated activating binder, untargeted particles were prepared and unconjugated anti-CD3 scFv protein was spiked in at an identical concentration to which it is found in targeted particles. These particles were then purified using 100kDa spin-filtration to ensure removal of unconjugated activating binder.

[0296] As shown in Figure 4A, activation of the transfected resting T cells was measured using an increase in side scatter as a measure of activation. When particles targeted with anti-CD7 VHH only were added to T cells, no significant increase in activation above baseline was observed. Similarly, untargeted particles with unconjugated anti-CD3 scFv spiked-in prior to particle purification also did not substantially increase activation above baseline. Particles targeted with anti-CD3 scFv alone induced a substantial increase in activation levels with activation peaking at 40%. Interestingly, the addition of anti-CD7 VHH increased the ability of particles to activate T cells even further. Particles targeted with both binders at a range of ratios were able to activate up to over 60% of total T cells, in spite of the particles containing a relatively lower amount of total anti-CD3 scFv activating binder. These results demonstrate CD7 and CD3 targeting act synergistically in terms of T cell activation. This was highly unexpected as particles targeted with anti-CD7 VHH alone did not activate T cells. This synergy is of significance as T cells in vivo are in a resting state and their activation can be highly important and advantageous to allow expression of cargo DNA, i.e. packaged DNA.

[0297] GFP expression, resulting from DNA delivery was also measured after administration of tLNPs to either resting or activated T-cells. Interestingly, Figure 4B and Figure 4C, show that both anti-CD7 VHH and anti-CD3 scFv function synergistically as well with regard to increasing T cell transfection in both activated (Figure 4B) and resting T-cells (Figure 4C).

[0298] Figure 4B clearly shows the synergistic effects of dual CD3 and CD7 targeting on tLNP mediated transfection of activated T cells. As can be seen in figure 4B, untargeted particles are barely capable of T cell transfection in activated cells. Furthermore, particles targeted with only anti-CD7 VHH or anti-CD3 scFv were only capable of modest levels of transfection with peak transfection percentages of 10% and 15% respectively. Interestingly, when the targeting moieties were combined at various ratios, transfection percentages could be substantially increased. This was surprising as the anti-CD3 scFv was initially included for the purpose of T cell activation, rather than for increased transfection. However, the results shown in figure 4B demonstrate that the synergistic function of dual CD3 and CD7 targeting applies to the transfection of activated cells.

[0299] Figure 4C clearly shows that when non-activated T cells are treated with untargeted LNPs or LNPs targeted with anti-CD7 VHH only, no DNA transfection is observed, i.e. no GFP expression was detected. In view of the observations mentioned above in Figure 4A in which targeting CD7 alone did not activate T cells, this implies that DNA in non-activated cells does not reach the nucleus, which is a requirement for GFP expression from the DNA construct transferred. In contrast, when T cells are treated with activating anti-CD3 scFv targeted particles, a small amount of transfection was observed, with around 2% of cells displaying GFP signal. When anti-CD7 VHH targeting is added to these particles, the transfection levels increase substantially. This is true for a wide range of anti-CD7: anti-CD3 binder ratios, with an increase in transfection being observed at all tested ratios. This means that in order for particles to transfer DNA to resting T-cells and provide for expression, activation of T-cells is required. These results further demonstrate the synergistic effects of dual CD3 and CD7 targeting, for both T-cell activation and DNA delivery, translate into highly effective and synergistic transfection of resting cells.

[0300] Given the observation that anti-CD3 scFv, anti-CD7 VHH coated LNPs - at a binder density of 0.5 mol % of total lipid - effectively transfect resting T-cells, a wide range of binder densities on the LNP surface were tested. Results showed that this technology was successful in transfecting T cells from as little as 0.05% total binder density up to 0.8 molar percent of total lipid (Figure 4D). LNPs produced over this range of binder densities were characterized for their physical characteristics, such as their size and PDI, as well as their nucleic acid encapsulation efficiencies (EE%). All produced LNPs within that range displayed desirable physical characteristics and EE% higher than 96%.

[0301] In a separate experiment, a range of anti-CD7 to anti-CD3 binder ratios were tested in a tLNP formulation comprised of SM102 as an ionizable lipid with DSPC as a helper lipid. To this end, LNP formulations with a total binder density of 0.5% of total lipid, made with ratios ranging from 1 :1 to 1 :6 anti-CD7 : anti-CD3 binder were produced, encapsulating minicircle encoding for GFP protein. These particles were applied to resting T cells and T cell DNA transfection was visualized using flow cytometry to detect GFP+ cells. Again, the addition of anti-CD7 VHH targeting increased the transfection of T cells (Figure 4E) as transfection levels were substantially higher when CD3 and CD7 targeting was combined as compared to particles targeted to CD3 only.

[0302] Transfection levels peaked with particles targeted with anti-CD7 and anti-CD3 binder at a ratio of 1 :4. Transfection levels were slightly lower at a ratio of 1 :1 as compared to 1 :2, 1 :4 and 1 :6. In the data shown in figure 4C, transfection levels also drop slightly when the anti-CD7 to anti-CD3 binder ratio is decreased to 1 :10. Taken together, it appears likely that the optimal anti-CD7 to anti-CD3 binder ratio is between 1 :2 to 1 :6. Example 3

[0303] Anti-CD3 scFv, anti-CD7 VHH targeted LNPs effectively deliver mcDNA encoding a chimeric antigen receptor to resting T-cells generating functional T-cells.

[0304] In order to demonstrate that the findings disclosed herein can be used to generate functional and stable CAR-T cells, tLNPs containing different ionizable lipids were prepared according to the methods section as described above using DSPC as a helper lipid encapsulating mcDNA encoding CD19 CAR and SB100x mRNA at a 1 :1 weightweight ratio and were administered to 60,000 isolated primary activated T-cells in a 96 well plate at a total dose of 600ng total nucleic acid which is equivalent to 0.009 ng / cell.CAR expression was measured over time by flow cytometry. 4 different ionizable lipids were evaluated in this setup being SM-102, CL4F8- [35, CL4F12-E6 and CL4F11-E3. Figure 5A clearly shows that tLNPs formulated with all ionizable lipids show mcDNA CAR expression. After prolonged cultivation and expansion of activated T-cells, approximately ~1 / 3 of initial expression remains stable. The potency of T- cells transfected by tLNPs formulated with lipids CL4F8-[35, CL4F12-E6 and CL4F11-E3 was evaluated in a killing assay as shown in Figure 5 B,C,D. The killing assay confirmed reactivity of the CAR-T cells towards CD19 positive target cells at multiple effector to target ratio’s but not to CD19 negative control cells.

[0305] Example 4

[0306] Engineering CAR-CIK cells using dual-Targeted LNPs

[0307] In order to demonstrate another possible utility of the tLNP technology, we investigated the ability of tLNPs to stably transfect NK cells to produce cytokine-induced killer (CIK) cells. Peripheral blood mononuclear cells (PBMCs) were transfected with LNPs targeted using a a 1 :1 ratio of anti-CD3 scFv and CD7-specific VHH at a total binder density of 0.05%. These LNPs were loaded with minicircle DNA (mcDNA) encoding a CD19-CAR and mRNA encoding the SB100X transposase, facilitating the generation of engineered cytokine-induced killer cells expressing CAR.

[0308] Briefly, freshly isolated PBMCs from healthy donor blood were cultured in T cell medium supplemented with IFN-y and OKT-3 and transfection was performed using dual-targeted LNPs carrying mcDNA encoding the CD19-CAR and mRNA for SB100X transposase. After 24 hours, IL-2 was added to the medium. On day 5, flow cytometry analysis confirmed the expression of the CD19 CAR (55%) (Figure 6). Continued culture over 20 days demonstrated sustained CAR expression, indicative of stable integration mediated by the SB100X transposase (Figure 6). Importantly, the transfection with dual-targeted LNPs did not affect the expansion of CIK (cytokine-induced killer) cells during the culture period.

[0309] Example 5

[0310] Anti-CD3 scFv, anti-CD7 VHH targeted LNPs effectively transfect T-cells in huPBMC engrafted NSG mice resulting in tumour control

[0311] In order to demonstrate that tLNP formulations were capable of functional mcDNA delivery in vivo, anti-CD3 scFv, anti-CD7 VHH at a total binder density of 0.05 molar percentage of total lipid targeted tLNPs were prepared using SM102 as an ionizable lipid and DSPC as a helper lipid according to the methods section. tLNPs encapsulating either mcDNA encoding CAR targeting hCD19 and SB100x mRNA (1 :1 w / w%) or mcDNA encoding GFP and Sb100X mRNA (1 :1 w / w%) were produced. These tLNPs were administered at a dose of 0.25 mg total nucleic acid / kg to an NXG mouse model of human leukaemia. Briefly, this model involved tail vein injection of 500,000 luciferase expressing Nalm-6 cells, two days later mice were humanized by the tail vein injection of 5,000,000 human PBMCs. One week after Nalm-6 tumor injection, tLNPs were injected via tail vein injection. After administration, mcDNA expression in peripheral blood T lymhpocytes was measured over time by flow cytometry (Figure 7A) and expressed as CAR positive T cells per 100 pL of blood. Both CAR and eGFP signal could be detected on T cells in the blood 12 days post-tLNP administration. Interestingly, the number of circulating T cells massively increased overtime, while the number of eGFP positive T cells did not. This demonstrates CAR-T cell specific expansion in vivo when the CAR T cells generated in vivo from the transfer and integration of the DNA into (initially) resting T-cells encountered their CD19+ tumor target cells.

[0312] Nalm-6-luc tumor outgrowth was measured by bio-luminescent imaging. Figure 7B shows tumour outgrowth as measured by bioluminescence, expressed as averaged signal area. tLNP-eGFP mcDNA (control), clearly shows tumor outgrowth, whereas tLNP-CAR mcDNA clearly shows initial tumour outgrowth followed by a stable phase and eventually decline of tumor signal. This is also clearly reflected in the survival curve (Figure 7C). At D30, all animals from the control group have died as a consequence of tumour development whereas all animals of the treatment group are still alive. This means that not only did the CAR-T cells that were generated in vivo with the administration of tLNPs expand, these also were apparently able to highly effectively control and eradicate the tumor cells, showing the tLNPs were highly effective in the treatment of cancer, when injected directly into the subjects bloodstream.

[0313] Example 6

[0314] Anti-CD3 scFv, anti-CD7 VHH targeted LNPs effectively transfect T-cells in whole blood Anti-CD3 scFv and anti-CD7 VHH targeted LNPs were used to transfect T cells directly in whole blood, this procedure is schematically represented in Figure 8A. In short, the tLNPs are added to whole blood directly at different nucleic acid doses, and incubated for 2-6 hours while shaking. After the indicated incubation time, PBMCs were isolated and kept in culture, transfection was measured at different timepoints using flow cytometry (Figure 8B). tLNPs were generated with the CL4F8-[35 or SM102 ionizable lipid and DSPC as a helper lipid according to the methods section above, and were loaded with mcDNA encoding hCAR targeting CD19 and SB100x mRNA (1 :1 w / w%). The tLNPs transfected T cells within 6 hours with a dose as low as 50 ng total nucleic acid in 500 pl sodium citrate anti-coagulated blood, initial transfection, measured after 5 days, showed 1-10% CAR expressing cells, which further increased during the culture period to 40-60% of T cells being CAR+. Figure 8C shows that an incubation time as short as 2 hours is also sufficient to generate CAR-T cells, and that these CAR-T cells can further expand upon the addition of antigen specific stimulation, in the form of NALM6 tumor cells, to the culture. This experiment confirms the in vivo experiment described above in example 5, and shows that the tLNPs can also be used to deliver effectively to T-cells, DNA encoding a CAR and allows for stable integration and expression thereof, which CAR-T cells appear functional and expand when brought into contact with tumor cells expressing the target antigen for the CAR. This shows that in addition to injecting tLNPs directly in the bloodstream, tLNPs can also be used in ex vivo treatments.

[0315] Nucleic acid delivery to T cells is strongly enhanced when using ionizable lipids with branched tails with a long alkyl chain between ionizable head group and point of branching Example 7

[0316] Branched ionizable lipids are superior in mcDNA transfection of T-cells

[0317] A typical lipid nanoparticle is comprised of four lipid components. Helper phospholipids and cholesterol contribute to particle structure and PEGylated lipids contribute to particle stability. A further crucial component of LNP formulations is the ionizable lipid. Ionizable lipids possess a charged head group which can interact with negatively charged nucleic acid during particle formation and a lipid tail which facilitates the nucleic acid’s packaging into the LNP particle (Mehta, et al. 2023).

[0318] There are a diverse range of ionizable lipids that have been utilised in LNP formulations and these have been categorised into 5 separate classes based on structure in a recent review. These classes are unsaturated, multi-tail, polymeric, biodegradable and branched-tail (Han, et al. 2021)

[0319] Multiple LNPs were prepared containing different Ionizable Lipids. The structure of these lipids is shown in Figure 9A We have stratified the tested ionizable lipids in this case in two different classes: ionizable lipids that contain a branched lipid tail (branched ionizable lipids) and ionizable lipids that have unbranched lipid tails (non-branched). Different tLNPs were administered to activated T-cells derived from different donors in various different transfections at a dose of 200ng total nucleic acid and transfection efficacy of mcDNA was measured by flow cytometry. The results show that branched tail ionizable lipids generally provide for a higher transfection efficiency than ionizable lipids from other non-branched classes (Figure 9B). It should be noted that as these data are pooled from various different experiments the observed higher transfection efficiencies of branched chain ionizable lipids should be observed as a trend but lipids in this figure should not be compared one to one. Although branched tail ionizable lipids are widely used in LNP formulations designed for the transfection of other cell types, this observed difference in efficiency between branched and non-branched ionizable lipids appears to be somewhat T cell specific. For instance, the multi-tailed non-branched lipids CKK-E12 and C12-200 have been used with great success to deliver siRNA to hepatocytes (Han, et al. 2021). Furthermore, the unsaturated ionizable lipid DLin-DMA-MC3 is used in the clinically approved treatment of transthyretin-mediated amyloidosis Onpattro (Akinc, Maier and Manoharan 2019). However, in our system these lipids are barely functional and are capable of only very low levels of T cell transfection. Hence, branched ionizable lipids may be generally preferred over other types of ionizable lipids when the aim is to target T cells, such as with CD3 x CD7 targeting as outlined herein.

[0320] Example 8

[0321] The branching point of branched ionizable lipids can affect transfection of T-cells by tLNPs

[0322] An interesting trend was observed from various different experiments with multiple donor T cells in that it appeared the point at which the tail branches in a branched ionizable lipid appeared to influence T cell transfection efficiency. Hence different branched ionizable lipids with different spacer lengths were compared in a transfection experiment, wherein an equal dose of 200ng nucleic acid was formulated in tLNPs provided with anti-CD7 VHH only and tested on T cells obtained from a single donor allowing direct comparison between different lipids used in this experiment. As shown in figure 9C (a), the longer the alkyl chain length (length of the spacer S) between the point at which the lipid tails split from the ionizable head group to the point at which the branch is attached, the more efficient the resulting tLNP transfection. The exact component of the ionizable lipid molecule described here is shown in figure 9C (b).

[0323] The results depicted in Figure 9C indicated that the length of the spacer S is an important feature when selecting ionizable lipids for targeted T-cell transfection with LNPs. In subsequent selections of ionizable lipid, including spacer lengths of at least 5 carbon atoms, appeared to confirm this to be a highly suitable selection criterium (data not shown). Hence the inventors have observed that it may be beneficial to have a spacer length (S) of at least 5 carbon atoms, such as at least 6 carbon atoms.

[0324] Example 9

[0325] Anti-CD3 scFv, anti-CD7 VHH targeted LNPs containing the ionizable lipid CL4F8- / 35 effectively transfect T-cells in huPBMC engrafted NSG mice resulting in tumour control

[0326] Anti-CD3 scFv, anti-CD7 VHH targeted LNPs were administered at a dose of 0.04 or 0.004 mg mg total nucleic acid I kg to huPBMC engrafted NXG mice (Figure 10A). tLNPs encapsulating either mcDNA encoding CAR targeting hCD19 and SB100x mRNA (1 :1 w / w%) or as a vehicle control, mcDNA encoding GFP and Sb100X mRNA (1 :1 w / w%) were produced. After administration, mcDNA expression on peripheral blood T lymphocytes was measured over time by flow cytometry (Figure 10B) and expressed as CAR-T cells per 100 pL of blood. eGFP expression in the T cells of vehicle control treated mice was also measured and observed (Data not shown). Nalm-6-luc tumor outgrowth was measured by bio-luminescent imaging. Figure 10C shows tumor outgrowth as measured by bioluminescence, expressed as averaged signal area. tLNP-eGFP mcDNA (control), clearly shows tumour outgrowth, whereas tLNP-CAR mcDNA clearly shows initial tumour outgrowth followed by a stable phase and eventually decline of tumor signal. This is also clearly reflected in the survival curve (Figure 10D). At D30, all animals from the control group have died as a consequence of tumor development whereas most animals of the treatment group are still alive.

[0327] It should be noted that in a separate in vivo experiment in which tLNPs were administered to immunocompetent BALB / C mice at similar doses within the effective dose range used in this example, a modest elevation in liver damage markers ALT and AST was observed one day post- tLNP administration. This elevation was transient as ALT and AST levels returned to baseline 7 days post-tLNP administration (figure 19). These ALT and AST measurements from mice injected with tLNP (CD3 x CD7) indicate signs of liver toxicity to be around or close to background levels measured, when amounts of nucleic acids administered are in the effective dose range, indicating the tLNPs in accordance with the invention allow for both safe and effective generation of CAR-T in vivo.

[0328] Nucleic acid delivery to T cells is strongly enhanced when using PC helper lipids with saturated lipid tails

[0329] Example 10

[0330] The influence of helper lipid selection on tLNP transfection efficacy

[0331] The choice of helper lipid is also an important aspect for T cell transfection efficiency. We have screened numerous helper lipids for their ability to function in our tLNP system and have identified features of helper lipids that can be important for T cell transfection efficiency. The first feature is a phosphatidylcholine (PC) head group. The second feature is a saturated lipid tail. Interestingly, these helper lipid features appear to mainly increase transfection efficiency in T cells or cells which express the tLNP target for instance HEK293T cells which have been engineered to express the CD7 target on their surface.

[0332] To assess the influence of different helper lipids on the delivery efficacy to activated T- cells, tLNP particles were formulated with mcDNA encoding an eGFP reporter as well as mRNA encoding BFP mRNA. This allowed the helper lipids influence on both DNA and RNA expression to be assessed.

[0333] Particles were formulated using SM102 ionizable lipid and were targeted with both anti- CD3 scFv and anti-CD7 VHH. As can be seen in figure 11 , particles formulated using helper lipids with a PC head group and saturated lipid tails generally performed better than helper lipids of other types in terms of mcDNA transfection efficiency. (Figure 11 A) Interestingly, unsaturated lipids perform worse as compared to their saturated counterparts (e.g. DSPC vs DOPC). This also applies to RNA expression, as expression of the BFP mRNA reporter is considerably higher in LNPs produced using lipids with a PC head group and saturated lipid tails (Figure 11 B). It also appears that the length of the helper lipid tail was of importance, as the transfection efficacy showed a trend from DSPC>DPPC>DMPC.

[0334] Example 11

[0335] The influence of helper lipid selection on LNP transfection efficacy

[0336] Next we investigated whether saturated PC lipids are the most efficient helper lipids for functional delivery of nucleic acid to T cells in general or whether they are particularly amenable to targeting using our dual anti-CD7 / CD3 targeting system. We again formulated tLNPs and identical LNPs lacking only the dual targeting moieties. Both LNP formulations contained an mcDNA encoding a GFP reporter. To investigate untargeted T cell transfection we applied significantly higher doses of untargeted LNPs to activated T cells. At these very high doses we were able to observe mcDNA transfection in T cells as signal was observed above a dose of 1200ng. Our targeted LNPs displayed signal at all tested doses from 150ng to 600ng (Figure 12).

[0337] Interestingly, the pattern of T cell transfection obtained at high doses using untargeted particles was the opposite as what is observed with targeted particles. In contrast to targeted particles, LNPs formulated using the unsaturated helper lipids DOPE and DOPC resulted in considerably higher levels of transfection as compared to their saturated counterparts (Figure 12).

[0338] This result suggests that the increased T cell transfection efficiency observed when tLNPs are formulated using saturated helper lipids with PC head groups is likely not a result of an increased ability of the LNP to functionally deliver nucleic acid to T cells per se. Rather, it appears that our highly efficient targeting using CD3 and / or CD7 system is particularly compatible with helper lipids of this class.

[0339] Example 12

[0340] Saturated PC helper lipids are not the most efficient helper lipids for nucleic acid delivery to HEK293 cells using untargeted LNPs

[0341] To investigate the extent to which the observation that helper lipids with a PC head group and saturated lipid tails result in improved T cell transfection is specific for tLNPs targeted towards T cells, we formulated untargeted LNPs with exactly the same lipid and nucleic acid composition as used in tLNPs. In this iteration, the LNPs contained an eGFP mcDNA reporter and mCherry reporter mRNA. These LNPs were then applied to HEK293 cells. This cell line is commonly used in the development of nanoparticle formulations and was used to provide a readout of untargeted transfection efficiency in a different cell type. In contrast to targeted formulations on T cells, the untargeted particles formulated using the unsaturated DOPC, DOPE and Egg-PC greatly outperformed the saturated PC lipids both in terms of DNA delivery (Figure 13A) and mRNA delivery (Figure 13B) in this setting. This finding demonstrates that saturated PC lipids are particularly suited to the transfection of T cells using targeted LNP formulations and that these helper lipids are not more efficient in LNP formulations in general or on other cell types.

[0342] Example 13

[0343] Saturated PC helper lipids are the most efficient type of helper lipids when particles are targeted and HEK293 cells are engineered to express CD7 target

[0344] To further strengthen the claim that unsaturated PC helper lipids are the most compatible lipids for our targeting system rather than simply being suited to T cell transfection in general, we again tested our particles in HEK293 cells. However, in this iteration we used HEK293 cells which had been engineered to express CD7 on their surface. We again formulated particles containing eGFP reporter mcDNA and mCherry reporter mRNA. However, we only display mRNA expression data here as the CD7 construct was a CD7-GFP fusion, meaning mcDNA GFP expression was difficult to detect reliably. We produced targeted and untargeted LNP formulations and applied these particles to the CD7+ HEK293 cells to investigate how the choice of helper lipid and targeting would interact in a different target positive cell type.

[0345] As can be seen in figure 14A, when untargeted particles were applied to CD7+ HEK293 cells, LNPs produced using unsaturated and non-PC helper lipids strongly outperformed the saturated PC helper lipids. This is a very similar pattern to what is observed in figures 13A and 13B which display transfection efficiencies in wild type HEK293 cells treated with untargeted particles.

[0346] In contrast, when targeted tLNP particles are applied to the CD7+ HEK293 cells, a pattern resembling what is observed when tLNPs are applied to T cells is observed. In this targeted transfection setting, the transfection efficiencies of tLNPs with saturated PC lipids are greatly increased while the transfection efficiencies of the targeted non-saturated and non-PC lipids are only slightly increased / modestly affected as compared to the non-targeted setting (Figure 14B).

[0347] These findings imply that our targeting system is most compatible with PC helper lipids with saturated lipid tails.

[0348] Example 14

[0349] Saturated helper lipids DSPE and DSPG are less efficient than DSPC in terms of mcDNA delivery efficiency to T cells

[0350] To demonstrate the importance of the PC head group in helper lipids used in our system, we again formulated tLNPs using an SM102 ionizable lipid and tLNPs were provided with both anti-CD3 ScFv and anti-CD7 VHH. These tLNPs contained an eGFP mcDNA reporter. We compared the efficiency of DSPC, which combines a PC head group with a saturated lipid tail to DSPE and DSPG, which have identical C18 saturated lipid tails but are lacking the PC head group.

[0351] As can be seen in figure 15, DSPC greatly outperforms both DSPE and DSPG in terms of mcDNA delivery to T cells. This demonstrates the importance of the PC head group for helper lipids in our system. Taken together with the results shown above, this implies that helper lipids with a saturated lipid tail and a PC head group are particularly suitable for targeted LNP delivery of nucleic acid to T cells which was unexpected.

[0352] Further CD7 binders

[0353] Example 15

[0354] The MT-701 antibody (BD, 555359), which is a mouse antibody capable of binding with human CD7, was also shown to be useful for transfer of nucleic acid to T-cells when bound to an LNP (data not shown). In addition to the anti-CD7 VHH clone H7 used in the examples above, eight further CD7 binding VHH clones were identified from the same Phage library from which the original CD7 clone H7 originated (See Table 3 below). The VHHs were in a test assessed for binding on the T cell line Jurkat by flow cytometry. Briefly, the VHH clones were incubated with Jurkat cells, which express CD7, after which VHH bound to the cells was detected using an anti-VHH secondary antibody which was fluorophore labeled. Figure 16 shows a titration of the different VHH clones in a flow cytometry binding assay, showing percentage VHH labeled positive cells (upper graph of Figure 16) or detected mean fluorescence (lower graph of Figure 16). These results show that while the different VHH clones do not show large differences marking cell populations positive at different VHH concentrations, the different VHH clones in this preliminary test appeared to plateau at different MFIs, indicating that these further candidates differ in binding characteristics (figure 16, and table 3 below).

[0355] Table 3. MFI of 10 nM datapoint shown in Figure 10, lower graph, with values ranked from high to low

[0356] Binding of the VHH clones to primary T cells and CD7 knock-in Flp-ln-Trex 293 cell line, was also confirmed. None of the clones appeared to bind to PBMCs isolated from cynomolgus monkey blood.

[0357] Of the eight additional CD7 VHH clones, in addition to the H7 clone, five were selected for further functional testing. These VHHs were produced at lager scale and used to target tLNPs loaded with mcDNA encoding for eGFP as described above. Briefly, the ionizable lipid used was SM102, and CD7 VHH was conjugated to DSPE-PEG-2000-maleimide at a molar percentage of 0.05% of total lipid. The LNPs were loaded with a minicircle DNA comprising a transposable transgene encoding the reporter gene eGFP. A dose range of LNPs (expressed as total nucleic acid dose) was used to transfect isolated and activated T cells as described. Transfection efficiency was measured by eGFP expression. Figure 17A shows that all of the tested VHH clones boosted transfection of T cells with mcDNA when used to target the LNPs. There was variation observed in in the transfection efficiency. There was also variation observed in conjugation efficiency (Fig 17B). Both are regarded to be highly important. The H7 VHH clone provided the best % of transfection, having a highly similar profile as the 2A- C6 and 5A-C6 clones, while having the highest percentage at the low dose of 75 ng. The conjugation efficiency of the H7 clone was the highest of all VHHs tested. Combined, this indicates that the H7 VHH clone can be preferred.

[0358] Table 4. Further CD7 VHHs

[0359] Example 16

[0360] Methods for extracorporeal transfection of an immune effector cell

[0361] To show the potential of extracorporeal transfection for CAR-T manufacturing using tLNPs, an in vitro test was performed to assess the capability of tLNPs to transfect and activate T cells in whole blood in a short time period. For this purpose, LNPs encapsulating minicircle DNA encoding GFP (used as reporter), with a lipid ratio of 40 / 20 / 39 / 1 (SM102 / DSPC / Cholesterol / DMG-PEG) were generated as described above. The LNPs were post-inserted with PEG lipids conjugated to anti-CD7 VHH and an anti CD3scFv 1 :1 , at a total binder density of 0.05 molar percentage of total lipid. This post-insertion ensures the tLNPs are specifically targeted to T- and NK cells and can induce T cell activation by engaging CD3 on the surface of T cells. These targeted and activating tLNPs were incubated with healthy donor blood for 10 minutes, 2 hours and 6 hours. After the indicated incubation times, PBMC were isolated from the blood using Ficoll Paque density centrifugation. The PBMCs were subjected to rigorous washing after the initial isolation, to ensure removal of the excess tLNPs, that were not bound to- or taken up by cells. Subsequently, PBMCs were cultured for 4 days, to allow GFP expression driven by DNA (Figure 21). Transfection efficiency of T cells was determined by measuring GFP signal. T-cell transfection was achieved in all tested conditions, a tLNP incubation period as short as 10 minutes lead to a transfection efficiency of 5%, which increased up to almost 20% when the incubation was prolonged to 6 hours (Figure 21 , T cells chart). B cell transfection was not seen at any of the time points (Figure 21 , B cells chart).

[0362] Material and Methods tLNP production

[0363] Lipid nanoparticles (LNPs) were generated as described above employing a microfluidic device to combine an aqueous phase containing nucleic acids with an ethanol phase containing lipid and cholesterol components. The aqueous phase comprised nucleic acids such as, minicircle DNA encoding for GFP or CD19 CAR, and SB100X mRNA in a citrate buffer, while the ethanol phase contained specific types of phospholipids, ionizable lipids, cholesterol, and PEGylated lipids, with a lipid composition of SM102 / DSPC / Cholesterol / DMG-PEG and respective ratio of 40 / 20 / 39 / 1. The LNPs were post-inserted with PEG lipids conjugated to anti-CD7 VHH and an anti CD3scFv, at a total binder density of 0.05 molar percentage of total lipid. The aqueous and ethanol phases were subsequently mixed in the microfluidic device at a specific ratio. The resulting LNPs were then targeted with DSPE-PEG-VHH-CD7 and CD3 scFv using a postinsertion method. The concentration of encapsulated nucleic acid and the encapsulation efficiency (EE%) of the targeted LNPs were determined using the Quant-iT Ribogreen Assay kit.

[0364] Whole blood tLNP transfection

[0365] Healthy donor blood was collected using VACUETTE TUBE 2 ml 9NC Coagulation sodium citrate 3.2% (Greiner). The blood was plated 500 pl per well in a 24 well plate and tLNP encapsulating a 3000 base pair minicircle DNA (mcDNA) encoding a green fluorescent protein gene flanked by Sleeping Beauty ITRs and an mRNA encoding the SB100x transposase at a 1 :1 mass ratio was added at an amount of 6.5 pg total nucleic acids, corresponding to a dose of approximately 5.3x10A7 mcDNA per cell. After the indicated transfection time, PBMCs were isolated by ficoll-paque density gradient centrifugation (Gibco) using 15 ml Sepmate Tubes (Stemcell Technologies) according to manufacturer’s protocol. The isolated PBMCs were maintained for 4 days at 37 degrees 5% CO2 in RPMI 1640 (Thermo Scientific) medium containing 2.5% heath inactivated pooled human Serum (Sanquin), 1 % Pen / Strep (Gibco), 2mM 2-mercaptoethanol (Gibco)and 50 ill / ml IL-2 (Mitenyi Biotec). After four days, GFP expression on T- and B cells was measured on FACS Canto, B cells were stained using CD19- PE (Antibodies- online), and T cells using CD3-APC ( BD biosciences), as illustrated in Figure 22.

[0366] See also Nawaz, W. et al. AAV-mediated in vivo CAR gene therapy for targeting human T-cell leukemia. Blood Cancer J 11 , 119 (2021); Rogers, G.L. et al. Optimization of AAV6 transduction enhances site-specific genome editing of primary human lymphocytes. Mol Ther Methods Clin Dev 23, 198-209 (2021); Lam, N. et al. Anti-BCMA chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains. Nat Commun 11 , 283 (2020); Ghassemi, S. et al. Rapid manufacturing of non-activated potent CAR T cells. Nat Biomed Eng 6, 118-128 (2022), each of which is hereby incorporated by reference in its entirety for all purposes.

[0367] Example 17

[0368] Specific targeting to T cells and NK cells with CD7-targeted lipid nanoparticles

[0369] To measure the targeting specificity of LNPs to T cells and NK cells in vitro, mRNA conjugated with fluorescent dye Cy5 was encapsulated in lipid nanoparticles (LNPs). The loaded LNPs were then either functionalized by integrating PEG lipids conjugated to anti-CD7 VHH (tLNPs) or left unfunctionalized (untargeted). with a lipid composition of SM102 / DSPC / Cholesterol / DMG-PEG and respective ratio of 40 / 20 / 39 / 1 , and LNPs were postinserted with PEG lipids conjugated to anti-CD7 VHH at a total binder density of 0.05 molar percentage of total lipid The anti-CD7 VHH is surface exposed in the tLNPs. The functionalized (tLNPs) and unfunctionalized (untargeted) were added to aliquots of peripheral blood mononuclear cells (PBMCs) for 2 hours. Cell transfection following the incubation was determined by measuring the Cy5 signal of each cell type using flow cytometry. Figure 23 illustrates the specific targeting of CD7-targeted LNPs to T cells and / or NK cells. Specifically, in the PMBC population incubated with the untargeted LNPs, less than half of the cells showing Cy5 transfection were either T-cells or NK-cells, consistent with indiscriminate transfection of cell type. However, in the PMBC population incubated with the targeted LNPs (tLNP), nearly all of the cells showing Cy5 transfection were either T-cells or NK-cells, demonstrating selective targeting and transfection of these cell types.

[0370] Efficient generation of proliferative CAR-T cells in whole blood within 6 hours of incubation time; proliferation over time can be induced by the addition of target cells (antigen-induced proliferation)

[0371] To attain stable DNA expression of a chimeric antigen receptor (CAR) in T cells, an LNP formulation co-delivering DNA and mRNA was prepared and targeted to T cells. Briefly, LNPs encapsulating a 3800 base pair minicircle DNA (mcDNA) encoding an anti-CD19 CAR flanked by Sleeping Beauty ITRs and an mRNA encoding the SB100x transposase were formulated, with a lipid composition of CL4F8-[35 / DSPC / Cholesterol / DMG-PEG at a respective ratio of 40 / 20 / 39 / 1. These LNPs were then functionalized with an anti-CD7 variable heavy domain of heavy chain (VHH) and an anti-CD3 single-chain variable fragment (scFv) by inserting lipids conjugated to the anti-CD7 VHH and lipids conjugated to the anti-CD3 scFv into the LNPs, at a total binder density of 0.05 molar percentage of total lipid using a previously described postinsertion method (Swart, L. E. et al. A robust post-insertion method for the preparation of targeted siRNA LNPs. Int J Pharm 620, 121741 (2022)). The mcDNA and mRNAs were encapsulated at a 1 :1 mass ratio. The loaded LNPs were then added to 500 pl of whole human blood at amounts of 50 ng, 100 ng, 400 ng, and 800 ng, measured as total nucleic acid mass. These doses correspond to approximately 3.2x10A5, 6.4x10A5, 2.6x10A6, and 5.2x10A6 mcDNA per cell, respectively. The blood was then incubated while shaking for 6 hours at 37 degrees °C. After the 6-hour incubation period, PBMCs were isolated from the whole blood, washed to further remove any remaining free tLNPs, and resuspended in culture medium.

[0372] To evaluate transfection and integration of the CAR DNA into the genome of T-cells, the percentage of CD3+ cells that expressed the anti-CD19 CAR were determined over time, as CD3+ is a defining marker for T cells. Figure 24 shows these percentages as a function of time post-transfection. As indicated by these results, all four doses of tLNP treatment were effective in transfection and stable genomic integration.

[0373] Efficient LNP-mediated T-cell transfection in whole blood leading to generation of proliferative CAR-T cells within 1 hour of incubation time

[0374] To study conditions for efficient LNP-mediated T-cell transfection to generate proliferative CAR- T cells in whole blood, minicircle CAR CD19 and SB100x mRNA were loaded into CD7 / CD3- targeted LNPs, as described in this example . Different amounts of the loaded LNPs (50 ng, 100 ng, 400 ng, and 800 ng) were added to 500 pl of whole blood and incubated (while shaking) for different durations (30 min, 1 hour, 2 hours, 4 hours, and 6 hours) at 37 degrees °C. After the respective incubation period, PBMCs were isolated from the whole blood, washed, and resuspended in culture medium. To evaluate T-cell proliferation upon presentation of CD19 (antigen-induced proliferation), each culture was split 10-days post incubation and NALM6 tumor cells were added to half of the cultures at a 1 : 1 ratio of T cells in the cultures. NALM6 is a B cell precursor leukemia cell line expressing CD19. The NALM6 cell line is available, for example, under accession number ACC 128 from the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH.

[0375] The percentage of CD3+ cells expressing the anti-CD19 CAR in each culture was determined over time, as described in Example 16. Figure 25 shows these percentages as a function of time post-transfection. Significantly, proliferation of cells treated with all four doses for at least one hour were stimulated by exposure to the NALM6 cells, as indicated by the increase in the percentage of CD3+ cells that were also CAR CD19+.

[0376] Efficient LNP-mediated T-cell transfection in isolated PBMCs leading to generation of CAR-T cells within 4 hours of incubation time

[0377] To study conditions for efficient LNP-mediated T-cell transfection of peripheral blood mononuclear cells (PMBC) cells separated from whole blood to generate proliferative CAR-T cells, minicircle CAR CD19 and SB100x mRNA were loaded into CD7 / CD3-targeted LNPs, as described in this example. PBMCs isolated from whole blood were then incubated with the targeted LNPs (tLNPs) for 4 hours at a range of cell densities and tLNP concentrations. As indicated in Figure 26, incubation was performed using cell densities ranging from 300,000 cells I mL (mio / mL) to 20 mio / mL, at concentrations of tLNP from 0.01 ng I cell to 0.01 ng I cell, measured as total nucleic acids. These correspond to doses of approximately 1.3x10A5 to 1.3x10A6 mcDNA per cell. Incubation (transfection) was done in 100 pl total volume in LNP freezing medium (TBS, 8% sucrose) directly or diluted in PBS. Incubation was done for 4 hours in an incubator (not shaking) at 37 °C. After the incubation, cells were washed and resuspended in culture medium.

[0378] Five days after incubation, the percentage of CD3+ cells expressing the anti-CD19 CAR in each culture was determined, as described in Example 16. Figure 26 shows these percentages as functions of cell density and tLNP dose. The data shown in Figure 26, suggests dosedependent and cell culture density-dependent effects of the tLNP treatment.

[0379] Stable CAR expression induced by LNP-mediated T-cell transfection in isolated PBMCs within 10 minutes of incubation time

[0380] To study and compare conditions for stable CAR expression induced by LNP-mediated T-cell transfection in isolated PBMCs or whole blood, minicircle CAR CD19 and SB100x mRNA were loaded into CD7 / CD3-targeted LNPs, as described in this example. Whole blood transfection was performed by adding 16 pg of loaded LNPs, measured as total nucleic acid mass, into 25 ml of blood. The incubation was performed at room temperature in a 50 ml tube that was rolling. After 4 hours of incubation, PBMCs were isolated from the blood, washed, and kept in the culture. tLNP transfection of PBMC was performed using cells from the same healthy donor as the whole blood transfection. PBMCs were isolated and were transfected at a cell density of 30 million / ml and at a LNP dose of 0.003 ng LNP / cell, measured as total nucleic acid mass, in a total volume of 60 pl in LNP freezing medium (TBS, 8% sucrose). Incubation was performed for 10 minutes at room temperature (no shaking). After the incubation, the cells were washed in PBS and resuspended in the medium. Cells were then cultured for 4 weeks and the percentage of CD3+ cells expressing the anti-CD19 CAR in each culture was determined over time, as described in Example 16. Figure 28 shows these percentages as a function of time. Example 18

[0381] Fusion proteins of CD3 ligand and CD7 ligand for conjugation to LNPs

[0382] Anti CD3 scFV, anti-CD7 VHH binders were shown to be effective when combined in a single fusion protein which performs comparably in vitro and in vivo to particles targeted with separate binders

[0383] To simplify the particle production process, the anti-CD7 VHH and anti-CD3 ScFv binders were combined into a single fusion protein. It was hypothesised that the T cell targeting behaviour and transfection advantages derived from the CD7-VHH and the T cell activating properties of the CD3-ScFv would be maintained when these polypeptides were fused into one molecule. This is advantageous for LNP particle production, i.a. because such an approach only requires a single conjugation and post-insertion step, rather than two.

[0384] Single polypeptides encoding a VHH targeting CD7 (corresponding with H7, SEQ ID NO.1) and scFv targeting CD3 (corresponding with UCHT1 , SEQ ID NO. 37) were fused in either order, i.e. CD7 x CD3 or CD7 x CD3, respectively, with different linker lengths, respectively, i.e. (GGGGS x 2, x 4 or x 6). Note that UCHT 1 also has a linker between VH and VL of GGGGS x 3. Single polypeptide sequences tested are listed in Table 5.

[0385] LNPs were produced as described above under LNP production. The lipid composition in this experiment was 40 / 20 / 39 / 1 molar % of CL4F8_[35 / DSPC / Cholesterol / DMG-PEG, respectively, with lipid conjugates post-inserted. Briefly, single fusion polypeptides targeting both CD7 and CD3 as listed in table 5 were conjugated to a C-terminal cysteine engineered at the C-terminus of the single fusion polypeptides as described and post-inserted into LNPs after LNP production, with molar ranges of 0.25% and 0.05% (conjugate lipid I total lipid x 100%). LNP with fusion polypeptides were compared with LNPs containing both a CD7 and CD3 binder separately.

[0386] As shown in Figure 28, non-activated T cells treated with particles targeted by fusion protein F1 (corresponding with SEQ ID NO. 44 as listed in Table 5 below) at binder densities of 0.05% and 0.25% display similar transfection percentages to cells treated with the binders applied separately at a 1 :1 ratio. All conditions display a peak of expression of around 60% at day 7 post-transfection. This indicates that transfection efficiencies obtained from fusion protein targeted particles are comparable to particles targeted using separate binders. Shown in Figure 28 are the results obtained with the F1 fusion protein. Similar results were obtained with all fusion proteins as listed in Table 1 based on an initial experiment (not shown), which indicated all fusion proteins were similarly active and results were comparable with LNPs having CD3 scFv and CD7 VHH conjugated separately.

[0387] Performance of separately targeted particles and fusion protein targeted particles was also shown to be comparable in vivo. As can be seen in figure Figure 29A tumor outgrowth as measured by bioluminesence imaging of Nalm-6 tumor outgrowth in huPBMC engrafted NXG mice (Janvier biolabs) was completely controlled by particles targeted using separate ligands (0.025% scFV-CD3 and 0.025% VHH-CD7) as well as particles targeted using fusion protein at densities of both 0.05% and 0.25%. This observation is supported by flow cytometry analysis of circulating CAR-T cells in blood which showed that CAR expression levels were roughly equal between all three tested LNP particles (Fig 29B).

[0388] Together, these data indicate that the synergistic effects of anti-CD3 and anti-CD7 targeting as observed when conjugated separately, are maintained when these two targeting ligands are combined into a single fusion protein. This highly advantageously allows to i.a. increase the manufacturing of tLNPs as the CMC complexity of targeting these particles is reduced, which e.g. may allow for providing a more consistent and / or more reproducible LNP product for use in clinical applications.

[0389] Table 5. Single polypeptide fusion proteins targeting CD7 and CD3

[0390] Example 19

[0391] Limited off-target delivery of LNPs To study if and how anti-CD7 T cell targeting and anti-CD3 scFv activation aid in specific transfection of DNA into T cells, LNPs were formulated with mcDNA encoding GFP protein as a nucleic acid payload using CL4F8-[35 as an ionizable lipid and DSPC as a helper lipid according to the LNP production section as described in example 18. The LNPs were functionalized with anti-CD7 VHH and anti-CD3 scFv at a 1 :1 molar ratio at a binder density of 0.05 mol% of total lipid (i.e. 0.025 mol % and 0.025 mol % respectively, of anti-CD7 VHH and anti-CD3 scFv). The tLNPs (targeted LNPs) were used to transfect Jurkat T cell leukemia line, NALM6 B cell leukemia cell line and HEPG2 hepatocellular carcinoma cell line at different doses, and DNA transfection was quantified after two days by measuring GFP expression. Figure 30 shows tLNPs can efficiently transfect the T cell line Jurkat, with an EC5o of around 50ng nucleic acid, while no transfection of the B- or liver cell line is seen even at the highest doses. Example 20

[0392] Limited off-target expression of payload

[0393] To study in vivo biodistribution of the CD3 / CD7 tLNPs, tLNPs were formulated with mRNA encoding for luciferase as nucleic acid payload (Figure 31A) or with mcDNA carrying a CAR and a luciferase gene, separated by a T2A plus SB100x mRNA (Figure 31 B). tLNPS were made using CL4F8-[35 as an ionizable lipid and DSPC as a helper lipid according to the LNP production section as described in example 18. The LNPs were functionalized with anti-CD7 VHH and anti-CD3 scFv at a 1 :1 molar ratio at a binder density of 0.05 mol% of total lipid (i.e. 0.025 mol % and 0.025 mol % respectively, of anti-CD7 VHH and anti-CD3 scFv). The tLNPs were injected into PBMCs engrafted NXG mice and biodistribution of nucleic acid delivery and expression was assessed by bioluminescence imaging. Luciferase expression from mRNA was visualized 5 hours after LNP injection (Figure 31 A) and from DNA 21 days after injection (Figure 31 B). Showing that addition of CD3 / CD7 targeting to the LNP greatly enhanced on-target functional delivery of nucleic acid to T cell rich organs such as spleen and lung. While low level of off-target mRNA expression can be detected in liver for both targeted and untargeted particles, DNA expression is almost exclusively detected in the on-target organs spleen and lung only for the targeted LNP. This shows that an untargeted LNP does not functionally deliver mcDNA to lung, kidney and heart tissue, and that a corresponding CD3 / CD7 targeted LNP highly efficiently functionally delivers mcDNA to organs (i.e. lung and spleen) which are rich in T-cells, showing both efficient functional mRNA and mcDNA luciferase expression. Off-target expression is not observed from mRNA in kidney and the heart, and is not observed from mcDNA in liver, kidney and the heart.

[0394] Example 21

[0395] Limited off-target integration of payload

[0396] In vivo biodistribution of nucleic acid delivery was further assessed after in vivo injection of tLNPs formulated with mcDNA encoding CAR protein flanked by SB100x compatible ITRs (i.e. transposable DNA, referred to as vector DNA herein) and mRNA for SB100x as a nucleic acid payload using CL4F8-[35 as an ionizable lipid and DSPC as a helper lipid according to the LNP production section as described in example 18 The LNPs were functionalized with anti-CD7 VHH and anti-CD3 scFv or at a 1 :1 molar ratio different molar ratio at a binder density of 0.05 mol% of total lipid (i.e. 0.025 mol % and 0.025 mol % respectively, of anti-CD7 VHH and anti- CD3 scFv). Organs were collected from PBMC engrafted NXG mice 20-30 days after they received a single iv injection of 0.05 mg / kg tLNP. qPCR was used to determine DNA vector copies per 100 ng of genomic DNA of whole tissue extract of several organs, additionally integrated gene copies (i.e. vector copies) were determined by using DPNI enzyme to digest all non-integrated DNA copies making use of difference in methylation prolife. Lastly, RT-PCR was performed to determine CAR mRNA transcript copies. Figure 32A shows that DNA vector copies are detected in most tested organs, both on- and off- target. However, integrated gene copies and mRNA transcripts are only found in on-target organs spleen and lung that are rich in T-cells. The integrated vector copy number per 100 ng of genomic DNA as determined by qPCR in spleen tissue and lung tissue, was respectively about 800 and 600, whereas integration was not detectable in other tissues. This shows that the tLNP has very limited off- target functional DNA delivery to organs such as liver, heart and kidney. To further confirm the contribution of CD3 / CD7 targeting to the specificity of the tLNP the experiment was also conducted using untargeted control particles. Figure 32B and 32C show that integrated CAR DNA copies and mRNA transcripts can only be found in spleen (B) and lung (C) upon treatment with the tLNP but not with the untargeted LNP. Additionally, a targeted LNP without SB100x was used as control particle for which again no DNA integration or mRNA transcription could be detected. Results show that a tLNP targeting carrying a transposable DNA, and an mRNA encoding a transposase, highly efficiently resulted in integrated DNA with no detectable off- target integration in the liver and kidney. Highly advantageously, medical treatments with tLNP allow to provide for an integrated vector copy number per 100 ng of genomic DNA of about 800 and about 600, as determined in spleen tissue and / or lung tissue. Highly advantageously, the integrated vector copy number (i.e. the number of DNA integrations of the transposable DNA) in tissues such as kidney, liver is below the level of detection.

[0397] To conclude, tLNP targeting CD3 / CD7 and carrying a transposable DNA, and an mRNA encoding a transposase, highly efficiently allows for integration of transgene DNA (e.g. a transposable expression cassette comprised in a mcDNA) with no detectable off-target integration in the heart, liver and kidney. These tLNP highly specifically target T-cells.

Claims

1. CLAIMS1.A nanoparticle comprising: o at its surface, a targeting moiety for binding a T-cell; o at its surface, a T-cell activation moiety; and, o a nucleic acid.

2. The nanoparticle in accordance with claim 1 , wherein the T-cell activation moiety is a T cell receptor complex binding moiety.

3. The nanoparticle in accordance with claim 2, wherein the T-cell activation moiety inducesT cell receptor signaling.

4. The nanoparticle in accordance with any of claims 1-3, wherein the T-cell activation moiety is an anti-CD3 binding moiety.

5. The nanoparticle in accordance with any of claims 1-4, wherein the T-cell activation moiety is an immunoglobulin, such as an antibody, or a fraction thereof.

6. The nanoparticle in accordance with any of claims 1-5, T-cell activation moiety comprises an antigen binding region.

7. The nanoparticle in accordance with any of claims 1 -6, wherein the antigen binding region comprises a VH and / or a VL domain.

8. The nanoparticle in accordance with any of claims 1 -7, wherein the antigen binding region comprises a VH with the CDR1 , CDR2, and CDR3 of VH as comprised in SEQ ID NO. 37 and a VL with the CDR1 , CDR2, and CDR3 of VL as comprised in SEQ ID NO. 37, wherein, preferably, the antigen binding region is in an scFv format.

9. The nanoparticle in accordance with any of claims 1-8, wherein the amino acid sequence of said immunoglobulin or a fragment thereof, is humanized and / or de-immunized.

10. The nanoparticle in accordance with any of claims 1 -9, wherein the targeting moiety for binding a T-cell, is selected from targeting moieties selected from binding moieties capable of binding CD2, CD4, CD5, CD7, CD8, CD25, CD127, CD152 and CD28.

11. The nanoparticle in accordance with any of claims 1-10, wherein the targeting moiety for binding a T-cell, is an anti-CD7 binding moiety.

12. The nanoparticle in accordance with any of claims 1-11 , wherein the targeting moiety for binding a T-cell is an immunoglobulin, such as an antibody, or a fragment thereof.

13. The nanoparticle in accordance with any of claims 1-12, wherein the targeting moiety for binding a T-cell comprises an antigen binding region.

14. The nanoparticle in accordance with any of claims 1-13, wherein the targeting moiety for binding a T-cell comprises an antigen binding region, which comprises a VH and / or a VL domain.

15. The nanoparticle in accordance with any of claims 1-13, wherein the targeting moiety for binding a T-cel comprises an antigen binding region, which comprises a VHH.

16. The nanoparticle in accordance with 15, wherein the targeting moiety for binding a T- cell, comprises a VHH, with the CDR1 , CDR2, and CDR3 selected from SEQ ID NO. 1 , 5, 9, 13, 17, 21 , 25, 29 or 33.

17. The nanoparticle in accordance with any of claims 1-16, wherein said nucleic acid comprises a DNA.

18. The nanoparticle in accordance with claim 17, wherein said DNA is a minicircle DNA.

19. The nanoparticle in accordance with claim 17 or claim 18, wherein said DNA is a minicircle DNA of at most 10 kB.

20. The nanoparticle in accordance with any one of claims of 17-19, wherein said DNA encodes a gene of interest.10821. The nanoparticle in accordance with any of claims 17-20, wherein the nanoparticle comprises a means for integrating the DNA, or part thereof, into a genome.

22. The nanoparticle in accordance with any of claims 17-21 , wherein the nanoparticle furthermore comprises an mRNA.

23. The nanoparticle in accordance with claim 21 or claim 22, wherein the means comprises an mRNA encoding a transposase, and wherein the DNA encodes a gene of interest comprises flanking ITRs compatible with the transposase.

24. The nanoparticle in accordance with claim 23, wherein the transposase is selected from the group consisting of Piggy Bac, SB100x, SB11 , TcBuster, Tol2, Frog prince, SpinON, Himarl, Passport, Minos, HAT, Hsnarl , Harbinger, Harbinger-3-DR, AciDs, and PIF.

25. The nanoparticle in accordance with any of claims 20-24, wherein the gene of interest comprises an expression cassette encoding a CAR or a TCR.

26. The nanoparticle in accordance with any of claims 20-24, wherein the gene of interest comprises an expression cassette encoding a CAR or a TCR targeting an antigen of a cancer.

27. The nanoparticle in accordance with any of claims 22-26, wherein the weight ratio of mRNA to DNA is in the range of 1 : 100 to 100 : 1 .

28. The nanoparticle in accordance with any one of claims 1-27, wherein said nanoparticle is a lipid nanoparticle29. The nanoparticle according to claim 28, wherein said lipid nanoparticle comprises:(a) an ionizable lipid ;(b) a helper lipid ;(c) a PEG lipid ; and109(d) a sterol.

30. The nanoparticle in accordance with claim 28 or claim 29, wherein said lipid nanoparticle comprises 30-60 mole % ionizable lipid, 0-30 mole % of helper lipid, 0-10 mole % of PEG lipid, and 15-50 mole % of sterol.

31. The nanoparticle in accordance with any of claims 28-30, wherein said lipid nanoparticle comprises an N / P ratio in the range of 2 - 12.

32. The nanoparticle in accordance with any of claims 29-31 , said ionizable lipid comprising an ionizable head group (Y) and at least one hydrocarbon tail (X) connected via a spacer (S) to said ionizable head group.

33. The nanoparticle in accordance with any of claims 29-31 , said ionizable lipid having a structure according to the formula Rz-Y-(S-(L-X)i)kwherein:R is a substituent z is 0 or 1 or 2Y is an ionizable head groupS is a spacer each individually selected from the group of spacersL is a bond or a linker each individually selected from the group of linkersX is a hydrocarbon tail each individually selected from the group of hydrocarbon tails I and k are each integers selected from the group of 1-10, preferably 1-6.

34. The nanoparticle in accordance with any of claims 29-33, wherein said ionizable lipid is selected from the group consisting of an unsaturated tail ionizable lipid, a multi-tail ionizable lipid, a polymeric ionizable lipid, a biodegradable ionizable lipid and a branched-tail ionizable lipid, preferably a branched-tail ionizable lipid.

35. The nanoparticle in accordance with claim 34, said branched-tail ionizable lipid comprises a spacer S, said spacer S comprising at least 5 carbon atoms, such as between 5 and 20 or between 5 and 16, preferably at least 6 carbon atoms, such as110 between 6 and 20 and 6 and 12, more preferably at least 7 carbon atoms, such as between 7 and 20 or between 7 and 12.

36. The nanoparticle in accordance with any of claims 33-35, wherein said spacer S in said ionizable lipid is an alkyl substituted or unsubstituted alkyl, substituted or unsubstituted alkylalkenyl, substituted or unsubstituted alkylalkynyl, substituted or unsubstituted alkylaryl, and cycloalkyl, more preferably unsubstituted alkyl.

37. The nanoparticle in accordance with any of claims 33-36, wherein said linker L is an ester selected from -C(=O)-O- and -O-C(=O)-.

38. The nanoparticle in accordance with any of claims 33-37, wherein said branched chain ionizable lipid is selected from the group consisting of: 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1 -octylnonyl ester (SM102), 7-[(2-Hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]heptyl 2-octyldecanoate (Lipid 5), [(4-Hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), octan-4-yl9-[3-[[3,5-bis[3-[bis(9-octan-4-yloxy-9-oxononyl)amino]propylcarbamoyl] benzoyl]amino]propyl-(9-octan-4-yloxy-9-oxononyl)amino]nonanoate (FTT5), bis(2-butyloctyl) 10-(N-(3-(dimethylamino)propyl) nonanamido) nonadecanedioate (Lipid A9),7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1 ,13-diyl bis(2-hexyloctanoate) (CL4F8-6), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1 ,13-diyl-bis(3- pentyloctanoate)(CL4F8_p5), 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1 ,13-diyl-bis(6-propylundecanoate) (CL4F 11_E-3), and 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1 ,13-diyl-bis(6-hexyldodecanoate) (CL4F 12_E-6).

39. The nanoparticle in accordance with any of claims 34-38, wherein said branched-tail ionizable lipid comprises two branched hydrocarbon tails (X1 , X2).Ill40. The nanoparticle in accordance with any of claims 39, wherein the branched-tail ionizable lipid comprises two spacers (S1 , S2) and two linkers (L1 , L2).

41. The nanoparticle in accordance with any of claims 33-40, wherein the at least one hydrocarbon tail X is selected from:wherein Ra, Rb, Rc, Rd, Reand Rfare each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkylalkenyl, substituted or unsubstituted alky lalkynyl, substituted or unsubstituted alkylaryl, and cycloalkyl, and wherein m and n are each integers selected from the group of 1-20, preferably between 4 and 12.

42. The nanoparticle in accordance with claim 41 , wherein the at least one hydrocarbon tail X is selected from:wherein m and n are each integers selected from the group of 1-20, preferably between4 and 12.

43. The nanoparticle in accordance with claim 41 or 42, wherein the at least one hydrocarbon tail X is selected from11244. The nanoparticle in accordance with any one of claims 33-43, wherein Y is selected from the group consisting of:wherein p is an integer selected from 1 to 6, preferably p is 2 or 4. wherein q is an integer selected from 1 to 20, preferably q is 7, wherein r is an integer selected from 1 to 6, preferably r is 3, wherein s is an integer selected from 1 to 8, preferably s is 4.113 wherein t, u, v are each independently an integer selected from 1 to 6, preferably t, u, and v are 3, and wherein R1, R2, R3and R4are each independently selected from Ci-C8substituted or unsubstituted alkyl groups, optionally R1and R2are methyl, optionally R3and R4are n-propyl.

45. The nanoparticle in accordance with any one of claims 33-44, wherein Y is selected from the group consisting of:

46. The nanoparticle in accordance with any of claims 28-45, wherein said helper lipid comprises a phosphatidylcholine (PC) head group.

47. The nanoparticle in accordance with any of claims 28-46, wherein said helper lipid comprises a saturated lipid tail.

48. The nanoparticle in accordance with claim 47, wherein said saturated lipid tail has a length of 14 to 20 carbon atoms.11449. The nanoparticle in accordance with any of claims 28-48, wherein said helper lipid is selected from the group consisting of DSPC, DMPC, DPPC or DAPC.

50. The nanoparticle in accordance with any of claims 28-49, wherein PEG lipid comprises one or more PEG lipids, selected from the group consisting of:DMG-PEG-2000 (1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), PEG-DSPE (1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carbonyl- amino(polyethylene glycol)-2000).

51. The nanoparticle in accordance with any of claims 28-50, wherein the lipid nanoparticle comprises a T-cell activation moiety conjugated to a lipid, preferably conjugated to a PEG lipid.

52. The nanoparticle in accordance with any of claims 28-51 , wherein the lipid nanoparticle comprises a targeting moiety for binding a T-cell conjugated to a lipid, preferably to a PEG lipid.

53. The nanoparticle in accordance with claim 51 or claim 52, wherein the PEG lipid used for conjugation is DSPE-PEG-2000 maleimide (1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[carbonyl-amino(polyethyleneglycol)-2000-N’-(3- maleimidopropionyl)]).

54. The nanoparticle in accordance with any of claims 51-53, wherein the targeting moiety for binding a T-cell and / or the T-cell activation moiety conjugated to the lipid, was inserted in and / or conjugated to the lipid nanoparticle.

55. The nanoparticle in accordance with any of claims 51-54, wherein the T-cell activation moiety is conjugated to the PEG lipid, and said lipid nanoparticle comprises 0.01 mole % - 5 mole % of the total lipid content, of PEG lipid conjugated with the T-cell activation moiety.11556. The nanoparticle in accordance with any of claims 51-55, wherein the targeting moiety for binding a T-cell is conjugated to the PEG lipid, and said lipid nanoparticle comprises 0.01 mole % - 5 mole % of the total lipid content, of PEG lipid conjugated with the T-cell activating binder.

57. The nanoparticle in accordance with any of claims 1-56, wherein the molar ratio of the targeting moiety for binding a T-cell, to the T-cell activation moiety is from 1 :1 to 1 :35, preferably from 1 :1 to 1 :6, more preferably from 1 :2 to 1 :6.

58. The nanoparticle in accordance with any of claims 28-50, wherein the lipid nanoparticle comprises a fusion protein comprising the T-cell activation moiety and the targeting moiety for binding a T-cell.

59. The nanoparticle in accordance with claim 58, wherein the lipid nanoparticle comprises the fusion protein comprising the T-cell activation moiety and the targeting moiety for binding a T-cell conjugated to a lipid, preferably conjugated to a PEG lipid.

60. The nanoparticle in accordance with claim 58 or claim 59, wherein the fusion protein comprising the T-cell activation moiety and the targeting moiety for binding a T-cell are separated by a linker, preferably a linker such as (GGGS)n.

61. The nanoparticle in accordance with any of claims 58-59, wherein the fusion protein is selected from SEQ ID NO. 44 - 49.

62. The nanoparticle in accordance with any of claims 59-61 , wherein the PEG lipid used for conjugation is DSPE-PEG-2000 maleimide (1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[carbonyl-amino(polyethyleneglycol)-2000-N’-(3- maleimidopropionyl)]).

63. The nanoparticle in accordance with any of claims 59-62, wherein the fusion protein comprising the targeting moiety for binding a T-cell and the T-cell activation moiety conjugated to the lipid, was inserted in and / or conjugated to the lipid nanoparticle.

64. The nanoparticle in accordance with any of claims 59-63, wherein the fusion protein comprising the T-cell activation moiety and the targeting moiety for binding a T-cell is conjugated a lipid, preferably a PEG lipid, and said lipid nanoparticle comprises 0.01 mole % - 5 mole % of the total lipid content, of lipid conjugated with the fusion protein.

65. Use of the nanoparticle as defined in any of claims 1-64, for delivery of nucleic acid to a T cell.

66. The use according to claim 65, for delivery of a nucleic acid to a resting T cell.

67. The use according to claim 65 or claim 66, for delivery of a nucleic acid to a resting T cell in PBMCs or in whole blood.

68. The use according to claim 67, wherein the nanoparticle is contacted with whole blood for 0 - 8 hours.

69. A nanoparticle in accordance with any of claims 1-64, wherein said nanoparticle is for use in the treatment of a disease, wherein said treatment comprises administering the nanoparticle to the patient.

70. The nanoparticle in accordance with any of claims 1-64, wherein said nanoparticle is for use in the treatment of a disease, wherein said treatment comprises obtaining T cells from the patient, contacting the nanoparticle with the obtained T cells ex vivo and allowing delivery of the nucleic acid to the T cells, and subsequently administering the T cells to the patient.

71. The nanoparticle for use in accordance with claim 70, wherein the treatment comprises an extracorporeal treatment, wherein the obtained T cells from the patient are comprised in whole blood or are isolated from whole blood.

72. The nanoparticle for use in accordance with claim 71 , wherein the extracorporeal treatment comprises:(a) obtaining whole blood from a subject;(b) collecting the fraction of blood containing T cells;(c) contacting the fraction of blood containing T cells with the nanoparticles; and(d) reinfusing the contacted fraction of blood containing T cells into the patient, wherein steps (a)-(d) are performed in-line in a closed fluid circuit.

73. The nanoparticle for use in accordance with claim 71 , wherein the extracorporeal treatment comprises:(a) obtaining whole blood from a subject;(b) contacting the whole blood with the nanoparticles; and(c) reinfusing the contacted whole blood into the patient, wherein steps (a)-(c) are performed in-line in a closed fluid circuit.

74. The nanoparticle for use in accordance with any of claims 70-73, wherein the contacting step is selected from the range of 0 - 8 hours.

75. The nanoparticle for use in accordance with any of claims 70-74, wherein the contacting comprises drawing blood from the subject and admixing a solution comprising the nanoparticle with the drawn blood.

76. The nanoparticle for use in accordance with any of claims 75, wherein the blood is drawn into an apheresis bag and the admixing comprises adding the solution into the apheresis bag.

77. The nanoparticle for use in accordance with any of claims 75-76, wherein an anticoagulant is added to the solution comprising the nanoparticle prior to, during, or after the admixing.

78. The nanoparticle for use in accordance with claim 77, wherein an anticoagulant is sodium citrate.11879. The nanoparticle for use in accordance with any of claims 75-78, further comprising, after the admixing, returning the blood to the subject.

80. The nanoparticle for use in accordance with any of claims 70-74, wherein the contacting step comprises apheresis of blood drawn from the subject, thereby generating a first blood fraction comprising immune effector cells and a second blood fraction free of immune effector cells, followed by admixing a solution comprising the nanoparticle with the first blood fraction.

81. The nanoparticle for use in accordance with any of claims 70-74, wherein the contacting comprises admixing a solution comprising the nanoparticle with blood drawn from the subject, followed by apheresis of the blood, thereby generating a first blood fraction comprising immune effector cells and a second blood fraction free of immune effector cells.

82. The nanoparticle for use in accordance with claim 80 or 81 , wherein the immune effector cells comprise peripheral blood mononuclear cells (PBMCs).

83. The nanoparticle for use in accordance with any of claims 80-82, wherein the apheresis is leukopheresis.

84. The nanoparticle for use in accordance with any of claims 80-83, wherein the first blood fraction is fractionated into an apheresis bag and the admixing comprises adding the solution into the apheresis bag.

85. The nanoparticle for use in accordance with any of claims 80-84, further comprising, after the contacting, separating cells in the first blood fraction from free nanoparticles.

86. The nanoparticle for use in accordance with claim 85, wherein the separating comprises washing the cells.11987. The nanoparticle for use in accordance with any of claims 70 - 85, wherein after admixing, incubating, or contacting the cells and the nanoparticles, and before reinfusion the cells are subjected to a washing step.

88. The nanoparticle for use in accordance with any of claims 85-87, wherein the separating comprises plasmapheresis, filtering with a spinning membrane filter, or centrifugation.

89. The nanoparticle for use in accordance with claim 80-88, further comprising, returning cells from the first blood fraction to the subject.

90. The nanoparticle for use in accordance with any of claims 70-89, wherein the contacting occurs in a closed vein-to-vein system.

91. The nanoparticle for use in accordance with any of claims 70-90, wherein the contacting is performed at a cell density of at least 250,000 cells per mL, at least 1 million cells per mL, at least 5 million cells per mL, or at least 10 million cells per mL.

92. The nanoparticle for use in accordance with any of claims 70-90, wherein the contacting is performed at a cell density of at least 1 million cells per mL.

93. The nanoparticle for use in accordance with any of claims 70-90, wherein the contacting is performed at a cell density of at least 5 million cells per mL.

94. The nanoparticle for use in accordance with any of claims 70-90, wherein the contacting is performed at a cell density of from 1 million cells per mL to 20 million cells per mL.

95. The nanoparticle for use in accordance with any of claims 70-94, wherein the contacting is performed at a ratio of DNA copy number to cells of at least 6x102DNA copies per cell, at least 8x102DNA copies per cell, at least 6X 103DNA copies per cell, at least 8X 103DNA copies per cell, at least 6X 104DNA copies per cell, at least 8X 104DNA copies per cell, at least 1 xio5DNA copies per cell, at least 2X 105DNA copies per cell, at least 4X 105DNA copies per cell, at least 6x105DNA copies per cell, at least 8x105120DNA copies per cell, at least 1 x-|06DNA copies per cell, 3x106DNA copies per cell, or at least 6x106DNA copies per cell.

96. The nanoparticle for use in accordance with any of claims 70-94, wherein the contacting is performed at a ratio of DNA copy number to cells of from 6x102to 6X 107DNA copies per cell.

97. The nanoparticle for use in accordance with any of claims 70-94, wherein the contacting is performed at a ratio of DNA copy number to cells of from 6x103to 6X 107DNA copies per cell.

98. The nanoparticle for use in accordance with any of claims 75-97, wherein the admixing occurs within twenty-four hours of drawing the blood from the subject, within four hours of drawing the blood from the subject, or within an hour of drawing the blood from the subject.

99. The nanoparticle for use in accordance with any of claims 75-97, wherein the admixing occurs within fifteen minutes of drawing the blood from the subject.

100. The nanoparticle for use in accordance with any of claims 75-97, wherein the admixing occurs within five minutes of drawing the blood from the subject.

101. The nanoparticle for use in accordance with any of claims 75-97, wherein the blood, or cell-containing fraction thereof, is contacted with the nanoparticle for no more than six hours.

102. The nanoparticle for use in accordance with any of claims 75-97, wherein the blood, or cell-containing fraction thereof, is contacted with the nanoparticle for no more than two hours.

103. The nanoparticle for use in accordance with any of claims 75-97, wherein the blood, or cell-containing fraction thereof, is contacted with the nanoparticle for no more than one hour.121104. The nanoparticle for use in accordance with any of claims 75-97, wherein the blood, or cell-containing fraction thereof, is contacted with the nanoparticle for no more than 30 minutes.

105. The nanoparticle for use in accordance with any of claims 75-97, wherein the blood, or cell-containing fraction thereof, is contacted with the nanoparticle for no more than 15 minutes.

106. The nanoparticle for use in accordance with any of claims 75-105, wherein the blood or cells from the first blood fraction is returned to the subject within twenty-four hours of drawing the blood from the subject, within twelve hours of drawing the blood from the subject, or within four hours of drawing the blood from the subject.

107. The nanoparticle for use in accordance with any of claims 75-105, wherein the blood or cells from the first blood fraction is returned to the subject within two hours of drawing the blood from the subject.

108. The nanoparticle for use in accordance with any of claims 75-105, wherein the blood or cells from the first blood fraction is returned to the subject within one hour of drawing the blood from the subject.

109. The nanoparticle for use in the treatment of a disease in accordance with any of claims 68-108, wherein the disease is a cancer.

110. The nanoparticle for use in accordance with claim 109, wherein the cancer is a B-cell cancer.

111. The nanoparticle for use in accordance with any of claims 6-110, wherein the nanoparticle comprises a DNA encoding a CAR.

112. The nanoparticle for use in accordance with claim 111 , wherein the CAR targets a B-cell cancer cell.122113. The nanoparticle for use in accordance with claim 111 or 112, wherein the CAR targets CD19, CD20, or CD22.

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