Bioengineering microbial encapsulin nano-particles as efficacious nanomedicine for nucleic acids

Abstract

The invention relates to a modified encapsulin nano-particle carrier for cell-targeted delivery of one or more therapeutic agent to a mammalian cell, wherein the agent may be a nucleic acid and / or protein capable of modulating the genome and / or modulating the expression of one or more native or non-native genes within said targeted cell. The nano-particles are composed of fusion polypeptides that are capable of binding and encapsulating a cargo of therapeutic agent, as well as cell-specific and intracellular delivery of the cargo to a target mammalian cell.

Description

[0001] TITLE: Bioengineering microbial encapsulin nano-particles as efficacious nanomedicine for nucleic acids

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a nano-particle carrier for cell-targeted delivery of one or more therapeutic agent to a mammalian cell, wherein the agent may be a nucleic acid and / or protein capable of modulating the genome and / or modulating the expression of one or more native or non-native genes within said targeted cell. The nano-particles are composed of fusion polypeptides that are capable of binding and encapsulating a cargo of therapeutic agent, as well as cell-specific and intracellular delivery of the cargo to a target mammalian cell.

[0004] BACKGROUND OF THE INVENTION

[0005] Nano-particles have been explored as a vehicle for drug delivery, since nano-particles have the potential for encapsulating a cargo of drug molecules. Encapsulation can serve to isolate drug molecules that are sensitive to certain environmental factors and degradation (for example, under the action of enzymes or under certain pH conditions), and thereby preserve their biological activity and / or prolong their therapeutic effect. The most extensively explored and tested nano-particles for drug delivery are based on synthetic lipid or polymeric nano-particles.

[0006] Molecular self- and co-assembly of proteins into highly ordered, symmetric supramolecular complexes have more recently been recognized as an elegant and powerful means for the development of advanced biomaterial delivery tools. A supramolecular structure that can both encapsulate a cargo, isolate it from the external environment and / or transport it to the desired location where it will be released to act has potential application for delivery of therapeutic drugs.

[0007] However, nano-particles have struggled to meet clinical expectations due to poor therapeutic benefits. The issues such as cargo purity, low transfection efficiency, lack of specificity, toxicity and high production cost have largely limited their usage.

[0008] Since an effective carrier for delivering therapeutic drugs would open the door to the short and long term treatment of a wide range of diseases, there remains an unmet need to provide improved nano-particle drug carriers, in particular nucleic acids as therapeutic agent.

[0009] The present invention satisfies this unmet need.

[0010] SUMMARY OF THE INVENTION In a first embodiment, the invention provides a modified encapsulin nano-particle comprising fusion polypeptides and a cargo of one or more therapeutic agents, wherein each of said fusion polypeptides comprises:

[0011] (a) an encapsulin polypeptide,

[0012] (b) first peptide inserted within a surface-exposed loop of said encapsulin polypeptide or fused to the C-terminus of said encapsulin polypeptide wherein said first peptide is for binding a targeting ligand on the exterior surface of the nano-particle, and

[0013] (c) a second peptide fused to the N-terminal amino acid of said encapsulin or inserted within the N-terminal region of said encapsulin polypeptide, wherein said second peptide is for capture and / or retention of at least one of said one or more therapeutic agents within the cavity of the nano-particle, wherein said nano-particle is obtained by in vitro self-assembly of said fusion polypeptides.

[0014] In a second embodiment, the invention provides an in vitro method for assembling the modified encapsulin nano-particle of the invention, comprising: i. providing a solution comprising denatured fusion polypeptides derived from at least one disassembled nano-particle comprising the fusion polypeptides of said first embodiment,

[0015] II. mixing the denatured fusion polypeptides in said solution with said one or more therapeutic agents to obtain a mixture, ill. dialyzing the mixture of step (ii) against a neutral pH buffer, such as phosphate buffer, at a temperature of less than 10 °C , such as 4 °C, to allow nano-particle reassembly, iv. recovering reassembled nano-particles obtained in step (iii) by size exclusion filtration, and v. optionally treating the recovered nano-particles with nucleases.

[0016] In a third embodiment, the invention provides a pharmaceutical composition for use in medical treatment, said composition comprising the modified encapsulin nano-particles of the invention, wherein each said nano-particle comprises:

[0017] (a) one for more targeting ligands covalently-bound to said first peptide, and

[0018] (b) at least one nucleic acid therapeutic agent retained within the cavity of the nanoparticle.

[0019] DESCRIPTION OF THE INVENTION Brief description of the figures:

[0020] Figure 1. Cartoon showing a modified encapsulin nano-particle (left panel), composed of self-assembled fusion polypeptides wherein each fusion polypeptide has an encapsulin polypeptide, wherein a second peptide nucleic acid binding domain is fused at the encapsulin N-terminus (upper panel); and a first peptide ligand (Spytag peptide) inserted within a surface-exposed loop of said encapsulin polypeptide (lower panel). The ligand is shown bound to an antibody for cancer cell targeting.

[0021] Figure 2. Cartoon showing (1 and 2) the cloning of a gene encoding an encapsulin fusion protein in a plasmid; (3) in vivo recombinant expression and self-assembly of encapsulin fusion proteins into modified encapsulin nano-particles in a bacterial host cell; (4 - 6) lysis and isolation of said modified nano-particles; (7) in vitro disassembly of said modified nano-particles; (8) nucleic acid (siRNA) cargo loading of said disassembled said modified encapsulin nano-particles; (9) in vitro self-reassembly of cargo-loaded modified encapsulin nano-particles; (10) attaching a targeting ligand to said cargo-loaded modified encapsulin nano-particles.

[0022] Figure 3. Image of a UV-sensitive stain-free SDS-polyacrylamide gel comprising in lane (1) size makers and lanes 2-7 samples derived from purification of isolated denatured nano-particle composed of encapsulin fusion proteins, where the detected single protein migrating with the 35 kD in lanes 2-7 is encapsulin fusion protein.

[0023] Figure 4. Transmission electron microscope (TEM) image of a sample of denatured nanocompartments derived from HPLC purification of isolated denatured nano-particle composed of encapsulin fusion proteins, where intact nano-particles are not observed.

[0024] Figure 5. TEM image of a sample of the isolated cargo-loaded reassembled nanoparticles obtained in 2.3nano-particles (Figure 5).

[0025] Figure 6. Histograms showing the fluorescence emission of cy3-labelled mRNA cargo- loaded modified encapsulin nano-particles detected by excitation at 550 nm and emission at 570 nm. The nano-particles were in vitro self-assembled from fusion encapsulin polypeptides, where the N-terminally fused nucleic acid binding domain is one of: a synthetic binding domain of either KR [SEQ ID NO. : 1] , RR [SEQ ID NO.: 2] or KK [SEQ ID NO. : 3] : or a native nucleic acid binding domain of either Hu-A [SEQ ID NO. : 4], csrA [SEQ ID NO. : 5] , H-NS [SEQ ID NO. : 6] , HFQ [SEQ ID NO. : 7] , Cro [SEQ ID NO. : 8] , Cl [SEQ ID NO. : 9] or control modified encapsulin nano-particles composed of fusion encapsulin polypeptides lacking an N-terminally fused nucleic acid binding domain. Figure 7. Histogram showing the fluorescence emission of HEK293 cells transfected with modified encapsulin nano-particles loaded with GFP mRNA. The nano-particles were in vitro self-assembled from fusion encapsulin polypeptides, where the N- terminally fused nucleic acid binding domain is one of: a synthetic binding domain of either KR [SEQ ID NO. : 1] , RR [SEQ ID NO. : 2] or KK [SEQ ID NO. : 3]: or a native nucleic acid binding domain of either Hu-A [SEQ ID NO. : 4], csrA [SEQ ID NO. : 5], H- NS [SEQ ID NO. : 6] , HFQ [SEQ ID NO. : 7] , Cro [SEQ ID NO. : 8], or Cl [SEQ ID NO. : 9], or control modified encapsulin nano-particles composed of fusion encapsulin polypeptides lacking an N-terminally fused nucleic acid binding domain..

[0026] Figure 8. Image of GFP expression by HEK293 cells transfected with modified encapsulin nano-particles loaded with GFP mRNA, where the N-terminally fused nucleic acid binding domain is KKRKKRKKRKKRRK.

[0027] Figure 9. [A] Image of GFP-mediated fluorescence by T-cells following incubation with modified encapsulin nano-particles comprising fusion encapsulin polypeptides having a Spy-catcher-GFP peptide fused within their C-terminal domain. The nano-particles were conjugated with anti-CD3 antibodies. [B] Histogram showing GFP-mediated fluorescence by T-cells following incubation with control native encapsulin nanoparticles; or control native encapsulin nano-particles mixed with SpyCatcher-GFP peptide, or modified encapsulin nano-particles comprising SpyCatcher-GFP peptide inserted into the C-terminal domain of the encapsulin fusion polypeptides.

[0028] Figure 10. Cartoon illustrating the use of the modified encapsulin nanaoparticles for the treatment of breast cancer, where the nano-particle are loaded with MDR1 siRNAs and are targeted to breast cancer cells by means of breast cancer cell specific ligands on the nano-particles when administered to the patient.

[0029] Figure 11. Image of a 2% agarose gel showing the composition of UV detectable nucleic acids released from encapsulin nanoparticles by using an SDS-PAGE sample buffer. The encapsulin nanoparticles were either loaded with GFP mRNA molecules in vitro (lane 1) or in vivo (lane 3), as indicated. The central lane 2 was loaded with nucleic acid size markers, where the 750 nt length marker migrates closely with GFP mRNA.

[0030] Figure 12. Image of a 2% agarose gel showing the composition of UV detectable nucleic acids released from encapsulin nanoparticles by using SDS-PAGE sample buffer. The encapsulin nanoparticles were assembled and loaded in vitro with either no nucleic acid as control (lane 1); 2.7 kB plasmid DNA (lane 2); or 2.3 kB plasmid DNA (lane 3). Abbreviations, terms and definitions:

[0031] Nano-particle: as defined herein is a particle of matter that is between 100 and 500 nanometers in diameter.

[0032] Cargo molecule: in the context of the present invention a cargo molecule is one that can be carried as a cargo by a nano-particle of the invention; where the molecule is further one that can be protected from the environment by virtue of encapsulation within the nano-particle. Furthermore, the cargo molecule is one that can both be encapsulated by the nano-particle and subsequently released from the nano-particle, without significant loss of its functional properties. When the cargo molecule is a therapeutic molecule, it may be a nucleic acid molecule; or a protein such as a peptide or polypeptide molecule; or a small molecule.

[0033] Covalently linked: provides a linkage between two molecules or between two group or members of a chain of atoms by means of one or more pi bonds (double bonds).

[0034] Cell-selective targeting ligand: ligand to a cell receptor, aptamer, peptide, antibody (polyclonal or monoclonal antibody, scFv and Fab fragment, and nanobody).

[0035] Immune cell surface receptor ligand: is a ligand to a cell surface receptor of an immune cell, where the immune cell is a monocyte or a lymphocyte selected from among a T cell (e.g. CD4-, CD8-T cell; gamma-delta-T cell [y6-T cell]), B cell and NK cell, or a combination thereof.

[0036] SDS-PAGE: Sodium dodecyl-sulfate polyacrylamide gel electrophoresis.

[0037] Target cell(s): is a cancer cell; or alternatively a monocyte or a lymphocyte selected from among a T cell (e.g. CD4-, CD8-T cell; gamma-delta-T cell [y6-T cell]), B cell and NK cell, or a combination thereof.

[0038] Detailed description of the invention:

[0039] Encapsulins are a class of protein nanocage found inside many archea and bacteria that have the capacity to self-assemble in vivo from identical protein subunits into hollow nanocages within their native host cell. The present invention is based on modifying these native protein subunits to confer them with properties needed for their use in drug delivery

[0040] I: A nano-particle of the invention In a first embodiment, the invention provides an in vitro self-assembled nano-particle comprising fusion polypeptides and a cargo of one or more therapeutic agents, wherein each of said fusion polypeptides comprises:

[0041] (a) an encapsulin polypeptide,

[0042] (b) a first peptide inserted within a surface-exposed loop of said encapsulin polypeptide or fused to the C-terminus of said encapsulin polypeptide wherein said first peptide is for binding a targeting ligand on the exterior surface of the nanoparticle, and

[0043] (c) a second peptide fused to the N-terminal amino acid of said encapsulin polypeptide or inserted within the N-terminal region of said encapsulin polypeptide, wherein said second peptide is for capture and / or retention of at least one of said one or more therapeutic agents within the cavity of the nano-particle.

[0044] The encapsulin polypeptide is a polypeptide capable of self-assembly with identical polypeptides to form a nano-particle, both in vivo in a host cell in which it can be expressed and in vitro. The self-assembled nano-particle comprises at least 60 subunits, for example from 60 to 240 subunits. The form of the nanoparticle may be icosahedral. The amino acid sequence of a suitable encapsulin polypeptide has at least 60, 65, 70, 75, 80, 85, 90, 95, 97, 99, 100% amino acid sequence identity to a polypeptide selected from among SEQ ID NO.: 2 (A0A0F5HPP7) (derivable from Quasibacillus thermotolerans; SEQ ID NO.: 4 (Q9WZP2) (derivable from Thermotoga maritima) and SEQ ID NO. : 6 (Q1D6H4) (derivable from Myxococcus xanthus). Preferably, the amino acid sequence of each encapsulin polypeptide of the nanoparticle has at least 70, 75, 80, 85, 90, 95, 97, 99, 100% % amino acid sequence identity to the Type 1 encapsulin protein derivable from Quasibacillus thermotolerans having SEQ ID NO. : 2 (A0A0F5HPP7).

[0045] The first peptide in the fusion polypeptide is fused to the C-terminus or within a surface- exposed loop of said encapsulin polypeptide, wherein said loop and said C-terminus are located on the external surface of the re-assembled nanoparticle. By virtue of the contact between the inserted first peptide and the surrounding environment of the nanoparticle, the first peptide can come in contact with and covalently bind a selected targeting ligand on the exterior surface of the nano-particle. For example, the first peptide is either fused to the C-terminal amino acid of the encapsulin polypeptide or within a surface-exposed loop of said encapsulin polypeptide, wherein the amino acid sequence of the encapsulin polypeptide has at least 70, 75, 80, 85, 90, 95, 97, 99, 100% % amino acid sequence identity to is selected from among SEQ ID NO. : 2 (A0A0F5HPP7), (derived from Quasibacillus thermotolerans; SEQ ID NO. 4 (Q9WZP2) (derived from Thermotoga maritime') and SEQ ID NO.: 6 (Q1D6H4) (derived from Myxococcus xanthus).

[0046] In one embodiment, the first peptide is inserted between amino acid residues 67 and 68 located in an external loop region of the encapsulin polypeptide having SEQ ID NO. : 2 (A0A0F5HPP7); whereby the amino acid sequence of the resulting fusion is found within SEQ ID NO. : 8.

[0047] In alternative embodiment, the first peptide is fused to the C-terminus of the encapsulin polypeptide having SEQ ID NO. : 2 (A0A0F5HPP7); whereby the amino acid sequence of the resulting fusion is found within SEQ ID NO. : 10.

[0048] In a preferred embodiment the first peptide, when fused to the C-terminus of the encapsulin polypeptide or inserted into an external loop region of the encapsulin poypeptide, further comprises a flexible amino acid linker, located between the amino acid residues of the first peptide and the adjacent amino residues of the encapsulin polypeptide. Preferably said linker is composed of 5 - 10 amino acid residues, and is composed of amino acid residues that confer flexibility, such as glycine and serine, (e.g. having amino acid sequence GGGGSG (SEQ ID NO. : 23).

[0049] As stated, the first peptide is capable of covalently binding with a selected targeting ligand. By way of example, the first peptide may be a spy-tag, wherein the amino acid sequence is SEQ ID NO. : 13 (PDB ID NO. 4MLI) or may be the core domain of spy-tag whose amino acid sequence is SEQ ID NO. : 12. Alternatively the first peptide may be a recognition sequence inserted into the encapsulin polypeptide that is capable of binding the targeting ligand such as: a recognition peptide for sortase-based peptide ligation (sequence motif LPXTG (where X is any amino acid) and the SEQ ID NO.: 14, or a first peptide comprising a self-cleaving mini-intein-based affinity tag (e.g. as described by M.-Q. Xu, S. Chong, T.C. Evans Jr. 2001, Protein Splicing in Encyclopedia of Genetics, Pages 1565-1567).

[0050] In one embodiment, a cysteine amino acid that is either inserted or already present in the encapsulin polypeptide can be used for attaching the targeting ligand using chemical linkers.

[0051] The targeting ligand suitable for cell-specific targeting of the nanoparticle may be selected from among an aptamer, peptide, polyclonal or monoclonal antibody, scFv and Fab fragment, and nanobody, wherein the ligand may be selective for a receptor on a cancer cell or an immune cell receptor. To enhance non-specific binding of nanoparticles of the invention to target cells, a cellpenetrating peptide (such as TAT SEQ ID NO. : 16) can be included in the encapsulin polypeptide, either fused to the C-terminus of the expressed encapsulin, or inserted into a loop region of the encapsulin nanoparticle between amino acids 67-68 of SEQ ID NO. 2 (A0A0F5HPP7), (derived from Quasibacillus thermotolerans'), whereby the cellpenetrating peptide is subsequently exposed on the exterior surface of the re-assembled encapsulin nanoparticle.

[0052] The second peptide in the fusion polypeptide is fused to the N-terminus or inserted into the N-terminal region of said encapsulin polypeptide, wherein said N-terminus or said N-terminal region is located on an internal surface of the re-assembled nanoparticle. The second peptide preferably has a net positive charge of at least + 6, thereby creating a positive charge within the nanoparticle cavity thereby enhancing the trapping / binding of nucleic acid molecules within the nanoparticle. Preferably the second peptide is composed of at least 5 amino acid residues, for example from 5 to 25 amino acid residues. By way of example the second peptide comprises an amino acid sequence selected from among:

[0053] (a) KKRKKRKKRKKRRK (SEQ ID NO.: 18 ),

[0054] (b) RRRRRRRRRRRRRR (SEQ ID NO. : 20) and

[0055] (C) KKKKKKKKKKKKKK (SEQ ID NO. : 22).

[0056] Surprisingly, nanoparticles of the invention comprising any of these three synthetic second peptides have an elevated nucleic acid loading capacity when compared to control nanoparticles lacking said second peptide or compared to nanoparticles differing only in having a second peptide comprising a nucleic acid binding domain derived from nature (as detailed in example 3).

[0057] In a preferred embodiment the second peptide, when fused to the N-terminus of the encapsulin polypeptide, further comprises a flexible amino acid linker, located between those amino acid residues of the second peptide capable of capture and / or retention of at least one of said one or more therapeutic agents within the cavity of the nano-particle and the N-terminal residue of the encapsulin polypeptide. Preferably said linker is composed of 5 - 10 amino acid residues, and is composed of amino acid residues that confer flexibility, such as glycine and serine, (e.g. having amino acid sequence GGGGSG (SEQ ID NO.: 23).

[0058] The nano-particle comprises a cargo disposed within the nano-particle, wherein at least one of said agents is a prophylactic or therapeutic agent. The one or more cargo molecules include nucleic acid molecule(s), peptide(s), polypeptide(s), small molecule(s) or any combination thereof. When the cargo molecule is a nucleic acid molecule, it may be a deoxyribonucleic acid molecule including single-stranded DNA, double-stranded DNA, plasmid DNA, mini circle DNA and an oligodeoxynucleotide, or any combination thereof. Alternatively the nucleic acid molecule may be a ribonucleic acid molecule or nucleoside-modified RIMA, including interfering RNA (RNAi), short hairpin RNA (shRNA), double-stranded RNA (dsRNA), RNA polymerase III transcribed RNA, messenger RNA (mRNA), and antisense ribonucleic acid molecule, gRNA, or any combination thereof.

[0059] When the nucleic acid molecule is nucleoside-modified RNA, for example by comprising pseudo-uridine in substitution for uridine, the nucleoside-modified RNA may be synthesized by T7 phage RNA polymerase.

[0060] When the RNA is mRNA, this may be obtained by in vitro transcription (IVT) of a DNA template. The RNA preferably has 5' and 3' UTRs whose length can be modified in order to achieve optimal translation efficiency following transfection and delivery of the transcribed RNA encapsulated within the nanoparticle. The 5'UTR may comprise a Kozak sequence to enhance translation efficiency. In a preferred embodiment, the RNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell. In one embodiment, the one or more cargo molecules comprise mRNA molecules encoding one or more therapeutic proteins.

[0061] The one or more cargo molecules may comprise one or more components of a CRISPR- Cas system, where a guide RNA (gRNA) targeted to a gene encoding a target molecule, and a CRISPR-associated (Cas) peptide form a complex to induce mutations within a selected gene within the cell to which the nano-particle is targeted. In one embodiment, the one or more cargo molecules comprise a gRNA or a nucleic acid molecule encoding a gRNA. In one embodiment, the one or more cargo molecules comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.

[0062] When the cargo molecule is a polypeptide, it may be selected from among an antibody (e.g. IgA, IgD, IgE, IgG, IgM, mAB or derivative bispecific antibody / T cell engager) or antibody fragment (e.g. FAB, nanobodies etc), pattern recognition receptors ((PRRs) e.g. Toll-like receptors (TLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), C-type lectin receptors (CLRs), and absent in melanoma-2 (AIM2)-like receptors (ALRs)) and CRISPR- Cas9 endonuclease.

[0063] When the cargo molecule is a small molecule, it may be selected from among a cytokine, small molecule modulator of immune response / pathway such as IL2 cytokine (16 kDa), IL2 / IL15, check point inhibitors, TGF-beta signaling inhibitor. The one or more cargo molecules, encapsulated within the nano-particles of the invention, are obtainable by mixing the one or more cargo molecules with the denatured fusion polypeptide subunits of the nano-particle of the invention (Example 2) and allowing re-assembly of the nano-particle. The encapsulated cargo molecules are thereby protected against chemical and enzymatic degradation, such as following transfection into a mammalian cell.

[0064] II. Method for preparing nano-particles of the invention

[0065] In a second embodiment, the invention provides a method for preparing all embodiments of the nano-particle of the invention described in section I above, wherein the nanoparticle comprises a cargo of one or more therapeutic agents disposed therein.

[0066] The method, as further detailed in example 1 and 2, comprises the following steps; i. providing a solution comprising denatured fusion polypeptides derived from at least one disassembled nano-particle of the invention,

[0067] II. mixing the denatured fusion polypeptides in said solution with said one or more therapeutic agents to obtain a mixture, ill. dialyzing the mixture of step (ii) against a neutral pH buffer, such as phosphate buffer, at a temperature of less than 10 °C , such as 4 °C, to allow nano-particle reassembly, iv. recovering reassembled nano-particles obtained in step (iii) by size exclusion filtration v. optionally treating the recovered nano-particles with nucleases.

[0068] The denatured fusion polypeptides may be derived from fusion polypeptides that are both synthesized and assembled into modified encapsulin nano-particles in vivo in a suitable bacterial host cell. The in vivo assembled modified encapsulin nano-particles are released from the host cell, (e.g. by cell lysis), and the nano-particles are then purified, optionally by affinity-based purification. The purified nanoparticles are then denatured into their component fusion polypeptide subunits, for example by incubation in a solution comprising at least 5M guanidine hydrochloride (GuHCI), where-after they can be used for in vitro binding of one or more therapeutic agent and in vitro re-assembly as describe above.

[0069] The modified encapsulin nano-particles according to the invention, and obtainable by the above method, are able to transfect mammalian cells (as shown in example 4), whereby their cargo molecules are effectively delivered and expressed in the target cells. This is evidenced by the expression of proteins encoded by an mRNA cargo molecule, as demonstrated in example 4.2.

[0070] The modified encapsulin nano-particles, are obtained by in vitro re-assembly, for example by the above described method. Said nanoparticles, according to the invention, can be distinguished from a modified encapsulin nano-particles assembled in vivo by a number of measurable parameters.

[0071] Firstly the cargo of nanoparticles, according to the invention, is precisely defined by the composition of the cargo that is combined in vitro with the denatured fusion polypeptides prior to re-assembly. In this manner, the encapsulation of contaminant cargo is avoided, that cannot be avoided when the nanoparticles are loaded and re-assembled in vivo, particularly where the cargo comprises nucleic acid molecules. The purity of the cargo of self-assembled modified encapsulin nano-particles can be measured by denaturing the self-assembled modified encapsulin nano-particles to release their cargo, and analyzing the composition of their cargo by Liquid chromatography-mass spectrometry (LC-MS) or gel electrophoresis (as shown in example 6 (figures 11 and 12).

[0072] Secondly the structure of in vitro re-assembled modified encapsulin nano-particles can be distinguished from in vivo self-assembled modified encapsulin nano-particles by measurable parameters (structure, elasticity and fragility) detected by atomic force microscopy and or TEM, as described in example 5. in. A pharmaceutical composition comprising modified encapsulin nanoparticles of the invention

[0073] In a third embodiment, the invention provides a pharmaceutical composition for use as a medicament in a method of therapeutic treatment, comprising one or more nanoparticle according to the invention, wherein said one of more cargo molecule is a prophylactic or therapeutic active agent, and wherein the targeting ligand is a cancer cell surface ligand, or immune cell surface receptor ligand.

[0074] The immune cell surface receptor ligand conjugated to the nano-particle is a ligand to a cell surface receptor of a target immune cell, where the target cell may be a monocyte or a lymphocyte selected from among a T cell (e.g. CD4-, CD8-T cell; gamma-delta-T cell [y6-T cell]), B cell and NK cell. Cell surface receptors recognized by said ligands include any of CD-3, CD4, CD5, CD8, CD14, CD19, CTLA-4, TCR, and TCRa where the ligand may be a cell surface receptor-specific antibody (e.g. anti-CD4 antibody, or an anti-CD8a F(ab')2 fragment). At least one of the one or more cargo molecules encapsulated within said nano-particles is an active agent having a prophylactic or therapeutic effect when administered to mammalian subject in need of treatment for a disease.

[0075] The active agent may have a prophylactic or therapeutic effect on a disease selected from among cancer, autoimmune disease, infectious disease, heart or vascular disease and immune-related disease.

[0076] Diseases treatable by administration of the nano-particles of the invention include:

[0077] Cancer: for example a hematological malignancy, where a nano-particle comprising the cargo molecule 'CD19CAR' for targeting lymphoblasts expressing CD19 is used for treatment of a mammalian subject suffering from a hematological malignancy; and where the treatment may be performed by ex vivo treatment of lymphocytes derived from the subject and subsequently infused back onto the subject. Alternatively, the treatment may be performed by direct administration to the subject.

[0078] Solid tumor cancer: by in vivo administration of a nano-particle comprising the cargo molecule 'OT1 TCR' for targeting an OVA-CT26 tumor in a mammalian subject suffering from a solid tumor cancer.

[0079] Autoimmune Disease: by administration of a nano-particle comprising the cargo molecule CRISPR / CAS9 CD3 KO to silencing OT-1 T cells in a mammalian subject suffering from Ova induce Inflammatory bowel disease (IBD); and where the treatment may be performed by ex vivo treatment of OT-1 T cells derived from the subject and subsequently infused back onto the subject. Alternatively the treatment may be performed by direct administration to the subject.

[0080] Administration of the nano-particles of the invention to a mammalian subject can be by any route, including but not limited to intravenous, intranodal, intradermal, transdermal, subcutaneous, intramuscular, inhalation (e.g., via an aerosol), buccal (e.g., sub-lingual), topical (i.e., both skin and mucosal surfaces, including airway surfaces), intrathecal, intraarticular, intraplural, intracerebral, intra-arterial, intraperitoneal, oral, intralymphatic, intranasal, rectal or vaginal administration, by perfusion through a regional catheter, or by direct intralesional injection.

[0081] In a preferred embodiment the mammalian subject in need of prophylactic or therapeutic treatment for a disease is a human. EXAMPLES

[0082] Example 1: Synthesis of encapsulin fusion polypeptides

[0083] Biosynthesis of encapsulin fusion polypeptides are set out below with reference to Figure 1 and 2.

[0084] 1.1 Domain structure of an encapsulin fusion polypeptide.

[0085] In one example the fusion polypeptide has a core comprising the amino acid sequence of a Quasibacillus thermotolerans encapsulin polypeptide having the amino acid sequence of SEQ ID NO. 2 (A0A0F5HPP7); and having a second peptide capable of nucleic acid binding (e.g. KR nucleotide binding domain of SEQ ID NO. : 18) fused at the N-terminal amino acid residue of the encapsulin polypeptide via an amino acid linker; and a first peptide (e.g. Spy-tag of SEQ ID NO. : 12) for binding a targeting ligand fused to the C-terminus of the encapsulin polypeptide via amino acid linkers, followed by a C-terminal (6xH) histidine tag. The complete amino acid sequence of this encapsulin fusion polypeptide has SEQ ID NO. : 10.

[0086] In one example, the first peptide (e.g. core Spy-tag of SEQ ID NO. : 12) for binding a targeting ligand and having N- and C- terminal amino acid linker sequences is fused within an external loop region of the Quasibacillus thermotolerans encapsulin polypeptide by insertion between amino acid residues 67 [Q] and 68 [G] of SEQ ID NO. 2 (A0A0F5HPP7), rather than at the C-terminus. The complete amino acid sequence of this encapsulin fusion polypeptide is SEQ ID NO. : 8.

[0087] A DNA molecule encoding the fusion polypeptide of SEQ ID NO. : 10 having nucleic acid sequence SEQ ID NO. : 9; was cloned into the multiple cloning site of plasmid pETDuet behind the isopropyl-p-d-thiogalactopyranoside- [IPTG] inducible promoter, T7 promoter having SEQ ID NO. : 24 (https: / / www.ncbi.nlm.nih.gov / nuccore / MK659889 REGION: 158...201 (GeneBank)).

[0088] 1.2 Gene expression and purification

[0089] Plasmid constructs, each comprising a gene whose nucleic acid sequence was optimized for expression in E. coll and that encodes an encapsulin fusion polypeptide, or encodes alternative or control encapsulin fusion proteins, were individually transformed into cells of E. coll BL21, where the cells were induced to express encapsulin with 1 mM IPTG overnight at 30 °C. The cells were then lysed with sonication. The nano-compartments in the cell lysate were then purified with Ni-NTA kit in denaturing conditions to remove any potential nucleic acids associated with the host cells-protomers in denatured form, so that contamination with such residual nucleic acids derived from the lysed cells was avoided in the reassembled nano-particles. To this end, 6M GuHcl were added to all the buffers in purification.

[0090] 1.3 Purity of isolated encapsulin fusion proteins

[0091] The purity of the isolated denatured nano-particle composed of encapsulin fusion proteins was assessed by SDS-polyacrylamide gel electrophoresis, where a single Coomassie stained protein migrating with the 35 kD of the encapsulin fusion protein, is shown in Figure 3.

[0092] The complete dissociation of the nano-particles into their component encapsulin fusion proteins mediate by GuHcl-denaturation is confirmed by the absence of detectable nano-particles as assessed by TEM (figure 4).

[0093] Example 2: In vitro loading and self-assembly of encapsulin fusion polypeptides into modified encapsulin nano-particles.

[0094] Loading and self-assembly of cargo-loaded encapsulin fusion polypeptides is set out below with reference to Figure 2.

[0095] In one example, the denatured encapsulin fusion polypeptides expressed, isolated and denatured as described in Example 1, were loaded with three examples of nucleic acid cargo, as follows:

[0096] 2.1 siRNA cargo loading:

[0097] A sample of the denatured fusion encapsulin polypeptides (1 mg / ml) were transferred to a dialysis membrane (with 3 KD cut off), and 200 ng of AllStars HS siRNA pool was added to the denatured fusion encapsulin polypeptides. To reassemble the encapsulin nanocompartment and load the nucleic acid, the mixture was dialyzed against PBS at 4 °C for 1 hour. Then, the outside buffer was changed to a fresh PBS, and the dialysis was continued for 4 hours. Next, the re-assembled nanocompartments were treated with RNase for 1 hour at 25 °C to digest any residual non-capsulated siRNA molecules, and finally the RNase-treated nano-particles were purified with amicon filter columns (100 KD) or Superose 6 (10 / 300) GL column.

[0098] 2.2 mRNA cargo loading:

[0099] A sample of the denatured fusion encapsulin polypeptides (0.5-1 mg / ml) were transferred to the dialysis membrane (with 3 KD cut off), and 1 ug of mRNA having ORF SEQ ID NO. : 25 (with a full length of 720-mer) was added to the denatured fusion encapsulin polypeptides. The mixture was dialyzed against PBS at 4 °C for 1 hour. Then, the outside buffer was changed to a fresh PBS, and the dialysis was continued for 4 hours. Then, the reassembled nanocompartments were treated with RNase for 1 hour at 25 °C to digest the non-capsulated mRNAs and finally the RNase-treated nanoparticles were re-purified with Superose 6 (10 / 300) GL column.

[0100] 2.3 Plasmid DNA cargo loading:

[0101] A sample of the denatured fusion encapsulin polypeptides (0.5-1 mg / ml) were transferred to the dialysis membrane (with 3 KD cut off), and 1-4 ug of targeted plasmid (with a length of 4000 bp) was added to the denatured encapsulin. The mixture was dialyzed against PBS at 4 °C for 1 hour. Next, the outside buffer was changed to a fresh PBS, and the dialysis was continued for 4 hours. Then, the reassembled nanocompartments were treated with DNase for 1 hour at 25 °C to digest the non-capsulated mRNAs. The particles were re-purified with Superose 6 (10 / 300) GL column.

[0102] 2.4 Structure of in vitro cargo-loaded self-reassembled modified encapsulin nanoparticles

[0103] Icosahedral structure of the cargo-loaded reassembled nano-particles obtained in 2.3 is shown by TEM imagery of a sample of the isolated nano-particles (Figure 5).

[0104] Example 3: In vitro cargo loading efficiency of self-assembled modified encapsulin nano-particles.

[0105] The loading capacity of the nano-particles produced in example 2 as compared to various comparator (control) nano-particles, was determined using nucleic acid cargos labeled with Cyanine3 (Cy3) dye. Cy3 is a bright, orange-fluorescent dye that can be excited using 550 nm light using a plate reader and detected at an emission wavelength of 570 nm.

[0106] The mRNA binding capacity of nine different modified encapsulin nano-particles loaded with mRNA as described in example 2.3 was compared. The modified encapsulin nanoparticles differed only in the amino acid sequence of the nucleic acid binding domain fused to the N-terminal amino acid of the fusion encapsulin polypeptide.

[0107] The amino acid sequence of the respective nucleic acid binding domains was:

[0108] KR synthetic binding domain: KKRKKRKKRKKRRK [SEQ ID NO 18]

[0109] RR synthetic binding domain: RRRRRRRRRRRRRR [SEQ ID NO 20] KK synthetic binding domain : KKKKKKKKKKKKKK [SEQ ID NO 22]

[0110] Hu-A nucleic acid domain derived from HU DNA binding proteins [SEQ ID NO. 27;

[0111] POACFO] :

[0112] M N KTQLIDVIAEKAELSKTQAKAALESTLAAITESLKEG DAVQLVG FGTFKVN H RAERTG RN PQTG KEI KIAAAN VPAFVSG KALKDAVK

[0113] H-NS nucleic acid domain derived from Histone-like nucleoid-structuring proteins [SEQ

[0114] ID NO. : 29; P0A1S2] :

[0115] MSEALKILNNIRTLRAQARECTLETLEEMLEKLEVWN ERREEESAAAAEVEERTRKLQQYREMLIA DGIDPNELLNSLAAVKSGTKAKRAQRPAKYSYVDENGETKTWTGQGRTPAVIKKAMDEQGKSLD DFLIKQ csrA domain derived from carbon storage regulator A proteins [SEQ ID NO. 31;

[0116] P69913] :

[0117] MLILTRRVGETLMIGDEVTVTVLGVKGNQVRIGVNAPKEVSVHREEIYQRIQAEKSQQSSY

[0118] HFQ domain derived from an RNA binding protein [SEQ ID NO. : 33; P0A6X3] :

[0119] MAKGQSLQDPFLNALRRERVPVSIYLVNGIKLQGQIESFDQFVILLKNTVSQMVYKHAISTWPSR PVSHHSNNAGGGTSSNYHHGSSAQNTSAQQDSEETE

[0120] Cro domain derived from bacteriophage repressor protein [SEQ ID NO. : 35; P03040] : MQTLSERLKKRRIALKMTQTELATKAGVKQQSIQLIEAGVTKRPRFLFEIAMALNCDPVWLQYGTK RGKAA

[0121] CI domain derived from bacteriophage repressor and activator protein [SEQ ID NO. :

[0122] 37; P03034] :

[0123] MSISSRVKSKRIQLGLNQAELAQKVGTTQQSIEQLENGKTKRPRFLPELASALGVSVDWLLNGTS DSNVR

[0124] The results show in Figure 6 demonstrate that the synthetic binding domains (KR, RR and KR) have a higher loading capacity for mRNA molecules than native nucleic acid binding domains.

[0125] Example 4: In vitro cell transfection capacity of mRNA cargo-loaded modified encapsulin nano-particles

[0126] Mammalian cells were transfected with the nine different modified encapsulin nanoparticles as described in example 3, where the nano-particles were loaded with a cargo of mRNA encoding green fluorescent protein (GFP) 4.1 Samples and cell lines

[0127] HEK-293T cells (CRL-11268™) is a derivative of an original parental human cell line that expresses a mutant version of the SV40 large T antigen, which was purchased from American Type Culture Collection, USA.

[0128] 4.2 Transfection of HEK-293T cells mRNA delivery by the engineered nano-particles was assessed by detection of GFP mRNA expression delivery to HEK 293 cell line. To this end, the PBS buffer used for dialysis was substituted with Hanks' Balanced Salt Solution (HBSS) buffer supplemented with 250 mM CaCI2, then 10 ul of the nano-particles (0.5 mg / ml) harboring GFP mRNA were added to the HEK cells (80% confluency) with 90 ul DMEM medium (Pen-strep+10% FBS). The fluorescence emission was measured 16 hours after transfection with plate reader.

[0129] The fluoresce emission of GFP expressed in transfected cells shown in figure 7 reveals that the synthetic nucleic acid binding domains have a higher capacity for mRNA delivery to the HEK cells. Also, among the native binding domains, Hu-A domain showed better mRNA delivery ability to the cells.

[0130] The fluoresce emission of GFP expressed in individual HEK cells transfected with the mRNA loaded modified encapsulin nano-particle after 48 h is seen in figure 8.

[0131] Example 5: Properties of cargo-loaded modified-encapsulin nano-particles prepared by in vitro re-assembly of denatured fusion polypeptides as compared in vivo self-assembled fusion polypeptides.

[0132] The structure of in vitro re-assembled modified encapsulin nano-particles can be distinguished from in vivo self-assembled modified encapsulin nano-particles in terms of their elasticity and fragility that are both measurable parameters detected by atomic force microscopy (AFM), as described by Boyton et al ACS Omega 2022, 7, 823-836 on pages 833 (right column under Atomic Force Microscopy) - 834 (left column).

[0133] Alternatively the structure of nanoparticles are analyzed by TEM, to visualize the morphology, size, and state of in vitro encapsulin re-assembly and to distinguish the re-assembled nanoparticles from in vivo self-assembled modified encapsulin nanoparticles, as follows: TEM was performed using a Tecnai G2 T20 (200 kV accelerating voltage). Briefly, encapsulin samples (0.2 mg / mL) were adsorbed onto formvar / carbon 200 mesh copper grids for 10 min. Prior to imaging, samples were negatively stained for 1 h using UA-Zero non-radioactive EM Stain replacement (Agar scientific Co.), washed with ultrapure water, and allowed to dry for at least 15 min. Example 6: Nucleic acid cargo-loading properties of modified-encapsulin nano-particles prepared by in vitro re-assembly of denatured fusion polypeptides as compared in vivo self-assembled fusion polypeptides

[0134] Modified-encapsulin nanoparticles composed of fusion polypeptides of SEQ ID NO. : 8 comprising the encapsulin polypeptide of SEQ ID NO.: 2; the first peptide of SEQ ID NO. : 12 (Spytag) fused via a flexible amino acid linker to the C-terminus of said encapsulin polypeptide, and a second peptide fused to the N-terminal amino acid of said encapsulin polypeptide of SEQ ID No. : 18 (KKRKKRKKRKKRRK) were loaded with GFP mRNA molecules by either in vivo encapsulation or in vitro encapsulation.

[0135] In vivo loaded modified-encapsulin nanoparticles were prepared using E. coli BL21 transformed with (1) a plasmid encoding the fusion polypeptide of SEQ ID NO.: 8 (as described in Example 1.2. and (2) a plasmid comprising a GFP gene under the control of the T7 promoter; but omitting a ribosomal binding site to prevent translation. Coexpression of fusion polypeptides and GFP mRNA was induced by IPTG as described in Example 1.2. The in vivo loaded modified-encapsulin nanoparticles were then released from the E. coli BL21 host cells by cell lysis, as described in Example 1.2, and then captured intact by His-tag purification and avoiding exposure to a denaturing agent.

[0136] In vitro loaded modified-encapsulin nanoparticles were prepared from encapsulin fusion polypeptides derived from E. coli BL21, as described in example 1.2, followed by re-assembly in the presence of either GFP mRNA, as described in example 2.2 or in the presence of plasmid DNA, as described in example 2.3.

[0137] The nucleic acid content of the in vitro and in vivo loaded modified-encapsulin nanoparticles was analyzed by suspending the nanoparticles in SDS-PAGE sample buffer to release their nucleic acid cargo, and loading the respective samples onto a 2% agarose gel to separate and UV detect the component cargo nucleic acids stained with ethidium bromide.

[0138] As seen in figures 11 and 12, modified-encapsulin nanoparticles according to the invention as capable of in vitro encapsulation of both RNA (mRNA) and DNA (plasmid DNA). Furthermore, unlike the in vivo loaded nanoparticles, the nucleic acids encapsulated in vitro are shown to be free of contaminating (non-target) nucleic acids.

[0139] Embodiments of the inventionA first embodiment provides a modified encapsulin nano-particle comprising fusion polypeptides and a cargo of one or more therapeutic agents, wherein each of said fusion polypeptides comprises: (a) an encapsulin polypeptide;

[0140] (b) a first peptide inserted within a surface-exposed loop of said encapsulin polypeptide or fused to the C-terminus of said encapsulin polypeptide for binding a targeting ligand on the exterior surface of the nano-particle, and

[0141] (c) a second peptide fused to the N-terminal amino acid of said encapsulin polypeptide or inserted within the N-terminal region of said encapsulin polypeptide, wherein said second peptide is for capture and / or retention of at least one of said one or more therapeutic agents within the cavity of said nano-particle, wherein said nano-particle is obtained by in vitro self-assembly of said fusion polypeptides.

[0142] A second embodiment provides the modified encapsulin nano-particle according to the first embodiment, wherein at least one of said therapeutic agents is a nucleic acid molecule.

[0143] A third embodiment provides the modified encapsulin nano-particle according to the first or second embodiment, wherein said one or more therapeutic agents is selected from:

[0144] (a) a deoxyribonucleic acid molecule selected from among a single-stranded DNA, a double-stranded DNA, a mini circle DNA, a plasmid, and an oligodeoxynucleotide, or any combination thereof,

[0145] (b) a ribonucleic acid molecule selected from among an interfering RNA (RNAi), a dsRNA, an RNA polymerase III transcribed RNA, a messenger RNA (mRNA), gRNA, and an antisense ribonucleic acid molecule, or any combination thereof,

[0146] (c) a combination of any deoxyribonucleic acid molecule selected from (a) and any ribonucleic acid molecule selected from (b).

[0147] A fourth embodiment provides the modified encapsulin nano-particle according to any one of the first to third embodiments, wherein said one or more therapeutic agents further includes at least one molecule selected from a therapeutic peptide, a therapeutic polypeptide, a therapeutic small molecule, or any combination thereof.

[0148] A fifth embodiment provides the modified encapsulin nano-particle according to any one of the first to fourth embodiments, wherein each said encapsulin polypeptide comprises an amino acid sequence having at least 70% sequence identity to Type 1 encapsulin protein from Quasibacillus thermotolerans of SEQ ID NO. : 2.

[0149] A sixth embodiment provides the modified encapsulin nano-particle according to any one of the first to fifth embodiments, wherein said first peptide is covalently linked to a cell-specific targeting ligand, such as a SpyTag of SEQ ID NO. : 12.

[0150] A seventh embodiment provides the modified encapsulin nano-particle according to the fifth embodiment, wherein said first peptide is inserted between amino acid residues 67 and 68 of SEQ ID NO. : 2 of said encapsulin polypeptide.

[0151] An eighth embodiment provides the modified encapsulin nano-particle according to any one of the first to seventh embodiments, wherein said second peptide has a net positive charge of at least + 6, preferably comprising an amino acid sequence selected from among:

[0152] (a) KKRKKRKKRKKRRK

[0153] (b) RRRRRRRRRRRRRR and

[0154] (C) KKKKKKKKKKKKKK.

[0155] A ninth embodiment provides the modified encapsulin nano-particle according to any one of the first to eighth embodiments, wherein said first peptide and or second peptide further comprises a flexible amino acid linker, preferably comprising an amino acid sequence of 5 - 10 amino acid residues.

[0156] A tenth embodiment provides the modified encapsulin nano-particle according to any one of the first to ninth embodiments, wherein said nano-particle further comprises a targeting ligand selected from among an aptamer, a peptide, a polyclonal or monoclonal antibody, an scFv and Fab fragment, and a nanobody, wherein the ligand is selective for an immune cell receptor.

[0157] An eleventh embodiment provides an in vitro method for assembling the modified encapsulin nano-particle according to any one of embodiments one to ten, comprising: i. providing a solution comprising denatured fusion polypeptides derived from at least one disassembled modified encapsulin nano-particle comprising the fusion polypeptides of any one of embodiments one to ten,

[0158] II. mixing the denatured fusion polypeptides in said solution with said one or more therapeutic agents to obtain a mixture, ill. dialyzing the mixture of step (ii) against a neutral pH buffer, such as phosphate buffer, at a temperature of less than 10 °C, such as 4 °C, to allow nano-particle reassembly, iv. recovering reassembled nano-particles obtained in step (iii) by size exclusion filtration, and v. optionally treating the recovered nano-particles with nucleases.

[0159] A twelfth embodiment according to the in vitro method of embodiment eleven, further comprising the preceding steps of: i. in vivo synthesis of modified encapsulin nano-particles comprising the fusion polypeptides of any one of embodiments one to ten,

[0160] II. in vitro incubation of said nano-particles in a solution comprising at least 5M guanidine hydrochloride (GuHCI) and ill. optionally affinity-based purification of said fusion polypeptides.

[0161] A thirteenth embodiment provides the modified encapsulin nano-particle according to any one of the fourth to tenth embodiments, wherein said therapeutic polypeptide is selected from among an antibody (e.g. IgA, IgD, IgE, IgG, IgM, mAB or derivative bispecific antibody / T cell engager) or antibody fragment (e.g. FAB, nanobodies), pattern recognition receptors ((PRRs) e.g. Toll-like receptors (TLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), C-type lectin receptor (CLR), and absent in melanoma-2 (AIM2)-like receptors (ALRs)) and CRISPR-Cas9 endonuclease.

[0162] A fourteenth embodiment provides the modified encapsulin nano-particle according to any one of the fourth to tenth embodiments, wherein said therapeutic small molecule is a cytokine, a small molecule modulator of immune response / pathway, a check point inhibitor, and a TGF-beta signaling inhibitor.

[0163] A fifteenth embodiment provides a pharmaceutical composition for use in medical treatment, said composition comprising the modified encapsulin nano-particle of any one of embodiments one to ten, thirteen and fourteen, wherein said nano-particle comprises:

[0164] (a) one for more targeting ligands covalently-bound to said first peptide, and

[0165] (b) at least one nucleic acid therapeutic agent retained within the cavity of the nanoparticle.

Claims

Claims:

1. A modified encapsulin nano-particle comprising fusion polypeptides and a cargo of one or more therapeutic agents, wherein each of said fusion polypeptides comprises:(a) an encapsulin polypeptide wherein amino acid sequence of said polypeptide has at least 90% sequence identity to SEQ ID NO. : 2(b) a first peptide of SEQ ID NO. : 12 or 13 or 14 inserted via a flexible amino acid linker within a surface-exposed loop of said encapsulin polypeptide between amino acid residues 67 and 68 of SEQ ID NO.: 2 or fused via a flexible amino acid linker to the C-terminus of said encapsulin polypeptide for binding a targeting ligand on the exterior surface of the nano-particle, and(c) a second peptide fused to the N-terminal amino acid of said encapsulin polypeptide, wherein said second peptide is 5 to 25 amino acid residues in length and has a net charge of at least +6 and is for capture and / or retention of at least one of said one or more therapeutic agents within the cavity of said nano-particle, wherein at least one of said therapeutic agents is a nucleic acid molecule, and wherein said nano-particle is obtained by in vitro self-assembly of said fusion polypeptides whereby encapsulation of contaminant cargo is avoided when compared with said nanoparticles loaded and reassembled in vivo.

2. The modified encapsulin nano-particle of claim 1, wherein said second peptide has an amino acid sequence selected from among:(a) KKRKKRKKRKKRRK;(b) RRRRRRRRRRRRRR; and(C) KKKKKKKKKKKKKK.

3. The modified encapsulin nano-particle of claim 1, wherein the amino acid sequence of each of said fusion polypeptides is SEQ ID NO. : 8 or 10.

4. The modified encapsulin nano-particle of any one of claims 1 - 3, wherein said at least one nucleic acid molecule is selected from:(a) a deoxyribonucleic acid molecule selected from among a singlestranded DNA, a double-stranded DNA, a mini circle DNA, a plasmid, and an oligodeoxynucleotide, or any combination thereof,(b) a ribonucleic acid molecule selected from among an interfering RIMA (RNAi), a dsRNA, an RNA polymerase III transcribed RNA, a messenger RNA (mRNA), gRNA, and an antisense ribonucleic acid molecule, or any combination thereof,(c) a combination of any deoxyribonucleic acid molecule selected from (a) and any ribonucleic acid molecule selected from (b).

5. The modified encapsulin nano-particle of any one of claims 1 - 4, wherein said one or more therapeutic agents is at least one molecule selected from among a therapeutic peptide, a therapeutic polypeptide, and a therapeutic small molecule, or any combination thereof.

6. The modified encapsulin nano-particle of any one of claims 1 - 5, wherein said first peptide is covalently linked to a cell-specific targeting ligand.

7. The modified encapsulin nano-particle of any one of claims 1-6, wherein said flexible amino acid linker is an amino acid sequence of 5 - 10 amino acid residues.

8. The modified encapsulin nano-particle claim 6, wherein said cell-specific targeting ligand comprises a targeting ligand selected from among an aptamer, a peptide, a polyclonal or monoclonal antibody, an scFv and Fab fragment, and a nanobody, wherein the ligand is selective for an immune cell receptor.

9. The modified encapsulin nano-particle of any one of claims 5 - 8, wherein said therapeutic polypeptide is selected from among an antibody (e.g. IgA, IgD, IgE, IgG, IgM, mAB or derivative bispecific antibody / T cell engager); an antibody fragment (e.g. FAB, nanobodies); a pattern recognition receptor (e.g. Toll-like receptors (TLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), C-type lectin receptor (CLR), and absent in melanoma-2 (AIM2)-like receptors (ALRs)); and a CRISPR-Cas9 endonuclease.

10. The modified encapsulin nano-particle any one of claims 5 -8, wherein said therapeutic small molecule is selected from among a cytokine, a small molecule modulator of immune response / pathway, a check point inhibitor, and a TGF-beta signaling inhibitor.

11. A pharmaceutical composition for use in medical treatment, said composition comprising a plurality of the modified encapsulin nano-particle of any one of claims 1- 10, wherein said nano-particle comprises one for more targeting ligands covalently-bound to said first peptide.

12. An in vitro method for assembling the modified encapsulin nano-particle of any one of claims 1 to 10, comprising: i. providing a solution comprising denatured fusion polypeptides derived from at least one disassembled modified encapsulin nano-particle comprising the fusion polypeptides of any one of claims 1-10,II. mixing the denatured fusion polypeptides in said solution with said one or more therapeutic agents to obtain a mixture, ill. dialyzing the mixture of step (ii) against a neutral pH buffer, such as phosphate buffer, at a temperature of less than 10 °C , such as 4 °C, to allow nano-particle reassembly, iv. recovering reassembled nano-particles obtained in step (iii) by size exclusion filtration, and v. optionally treating the recovered nano-particles with nucleases.

13. The in vitro method of claim 11, further comprising the preceding steps of: i. in vivo synthesis of modified encapsulin nano-particles comprising the fusion polypeptides of any one of claims 1-10,II. in vitro incubation of said nano-particles in a solution comprising at least 5M guanidine hydrochloride (GuHCI) and iii. optionally affinity-based purification of said fusion polypeptides.

14. A method of providing a medical treatment to a subject in need thereof, comprising administering a composition comprising a plurality of the modified encapsulin nano-particle of any one of claims 1- 10, wherein said nano-particle comprises one for more targeting ligands covalently-bound to said first peptide.

15. The method of claim 14, wherein said targeting ligand is to a cell surface receptor of an immune cell, wherein the immune cell is a monocyte or a lymphocyte selected from among a T cell (e.g. CD4-, CD8-T cell; gamma- delta-T cell [y6-T cell]), a B cell and a NK cell, or any combination thereof.

16. The method of claim 15, wherein said cell surface receptor(s) recognized by said ligands are one or more selected from among CD-3, CD4, CD5, CD8, CD14, CD19, CTLA-4, TCR, and TCRa.

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