Compositions and methods of delivery to specific cells

Abstract

The present invention is directed to lipid nanoparticles, compositions, formulations and systems containing lipid-based nanoparticles and methods of treating diseases or conditions with said lipid nanoparticles, compositions, formulations and systems thereof. The present disclosure provides a lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal intein, and (b) a targeting molecule displayed on the outer surface of the lipid nanoparticle. The present disclosure provides a lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule displayed on the outer surface of the lipid nanoparticle.

Description

10063138571Compositions and methods of delivery to specific cells Field of the invention

[0001] The present invention is directed to lipid nanoparticles, compositions, formulations and systems containing lipid-based nanoparticles and methods of treating diseases or conditions with said lipid nanoparticles, compositions, formulations and systems thereof.Cross-reference to earlier application

[0002] This application claims priority to Australian provisional applicationno. 2025900459, the entire content of which is hereby incorporated by reference in its entirety.Background of the invention

[0003] Lipid nanoparticle (LNP) formulations have emerged as a promising delivery system for therapeutics, such as nucleic acid-based therapeutics including small interfering RNA (siRNA), messenger RNA (mRNA), and DNA. LNPs are able to protect the cargo from degradation, facilitate cellular uptake, and promote endosomal escape, thereby enhancing the therapeutic efficacy of the nucleic acid payload.

[0004] However, the clinical development of LNP-based therapies has been hindered by concerns over off-target toxicity. The protein expressed by the mRNA delivered via the LNPs can interact with various cellular pathways and induce unintended effects in non-target organs, tissues and cells. For example, a challenge for LNP-based therapeutics is the accumulation of LNPs in the liver, which is the primary site of clearance for these nanoparticles. The non-specific delivery of LNPs and high expression of the therapeutic protein in the liver can lead to hepatocyte stress, inflammation, and even liver damage.

[0005] Addressing the challenge of toxicity and protecting the cells, tissues and organs from unintended adverse effects are crucial for the successful clinical translation of LNP-based therapies.

[0006] Therefore, there is a need for new and / or improved lipid nanoparticles, and uses thereof, which minimise one or more off-target effects.10063138572

[0007] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.Summary of the invention

[0008] In one aspect, the present disclosure provides a lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle. fR

[0009] In one aspect, the present disclosure provides a lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding both an N and a C-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate N-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle.

[0010] In one aspect, the present disclosure provides a lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate N-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle.

[0011] In any aspect, the nucleotide sequence encoding an N-terminal portion of a polypeptide of interest is linked or fused to the nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation. The nucleotide sequence encoding an N-terminal portion of a polypeptide of interest is 5’ to the nucleotide sequence encoding the N-terminal polypeptide capable of mediating irreversible conjugation.10063138573

[0012] In any aspect, the nucleotide sequence encoding a C-terminal portion of a polypeptide of interest is linked or fused to the nucleotide sequence encoding a C-terminal polypeptide capable of mediating irreversible conjugation. The nucleotide sequence encoding a C-terminal portion of a polypeptide of interest is 3’ to the nucleotide sequence encoding the C-terminal polypeptide capable of mediating irreversible conjugation.

[0013] As used herein, a lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle, may also be referred to as a “first lipid nanoparticle”.

[0014] As used herein, a lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and encoding a C-terminal polypeptide capable of mediating irreversible conjugation, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle, may also be referred to as a “second lipid nanoparticle”.

[0015] In another aspect, the present disclosure provides a composition comprising:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle, and- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate N-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle.10063138574

[0016] In one embodiment, the composition may further comprise:- a third lipid nanoparticle;- a third and fourth lipid nanoparticle;- a third, fourth and fifth lipid nanoparticle;- a third, fourth, fifth and sixth lipid nanoparticle;- a third, fourth, fifth, sixth and seventh lipid nanoparticle;- a third, fourth, fifth, sixth, seventh and eighth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth and ninth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth and tenth lipid nanoparticle; - a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and eleventh lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh and twelfth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and fourteenth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth and fifteenth lipid nanoparticle; or- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth and sixteenth lipid nanoparticle.

[0017] In these embodiments, each lipid nanoparticle comprises a nucleotide sequence encoding a portion of the polypeptide of interest, such that full-length polypeptide of interest is encoded by the totality of each nucleotide sequence encoding each portion of the polypeptide of interest. As used herein, the polypeptide of interest may be encoded by an N-terminal portion and multiple C terminal portions (i.e. portions C-terminal to the N-terminal portion). In other words, where there are 3 lipid nanoparticles - a first, second and third lipid nanoparticles - the polypeptide of interest is encoded by 3 nucleotide sequences, one in each of the 3 lipid nanoparticles. In this instance, the polypeptide of interest may be encoded by a nucleotide sequence encoding an N-terminal portion in the first lipid nanoparticle, the middle portion of the polypeptide in the second lipid nanoparticle and C-terminal portion in the third lipid nanoparticle.10063138575

[0018] In these embodiments, each lipid nanoparticle comprises a nucleotide sequence encoding a polypeptide capable of mediating irreversible conjugation in the presence of a cognate polypeptide. Each lipid nanoparticle comprises at least one, potentially two, polypeptide capable of mediating irreversible conjugation in the presence of a cognate polypeptides wherein when two are present each is a single part of a different orthogonal pair. Where the composition comprises a third lipid nanoparticle, the second nanoparticle further comprises a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide encoded by a nucleotide sequence in the third lipid nanoparticle. Where the composition comprises a fourth lipid nanoparticle, the third nanoparticle further comprises a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide encoded by a nucleotide sequence in the fourth lipid nanoparticle. Where the composition comprises a fifth lipid nanoparticle, the fourth nanoparticle further comprises a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide encoded by a nucleotide sequence in the fifth lipid nanoparticle. Where the composition comprises a sixth lipid nanoparticle, the fifth nanoparticle further comprises a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide encoded by a nucleotide sequence in the sixth lipid nanoparticle. Where the composition comprises a seventh lipid nanoparticle, the sixth nanoparticle further comprises a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide encoded by a nucleotide sequence in the seventh lipid nanoparticle. Where the composition comprises an eighth lipid nanoparticle, the seventh nanoparticle further comprises a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide encoded by a nucleotide sequence in the eighth lipid nanoparticle. Where the composition comprises a ninth lipid nanoparticle, the eighth nanoparticle further comprises a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide encoded by a nucleotide sequence in the ninth lipid nanoparticle. Where the composition comprises a tenth lipid nanoparticle, the ninth nanoparticle further10063138576comprises a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide encoded by a nucleotide sequence in the tenth lipid nanoparticle. Where the composition comprises an eleventh lipid nanoparticle, the tenth nanoparticle further comprises a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide encoded by a nucleotide sequence in the eleventh lipid nanoparticle. Where the composition comprises a twelfth lipid nanoparticle, the eleventh nanoparticle further comprises a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide encoded by a nucleotide sequence in the twelfth lipid nanoparticle. Where the composition comprises a thirteenth lipid nanoparticle, the twelfth nanoparticle further comprises a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide encoded by a nucleotide sequence in the thirteenth lipid nanoparticle. Where the composition comprises a fourteenth lipid nanoparticle, the thirteenth nanoparticle further comprises a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide encoded by a nucleotide sequence in the fourteenth lipid nanoparticle. Where the composition comprises a fifteenth lipid nanoparticle, the fourteenth nanoparticle further comprises a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide encoded by a nucleotide sequence in the fifteenth lipid nanoparticle. Where the composition comprises a sixteenth lipid nanoparticle, the fifteenth nanoparticle further comprises a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide encoded by a nucleotide sequence in the sixteenth lipid nanoparticle.

[0019] Each pair of polypeptides capable of mediating irreversible conjugation may be, and are preferably, orthogonal to another pair.

[0020] In these embodiments, where there are more than 2 nanoparticles, at least 2 nanoparticles display targeting molecules that bind to different portions or epitopes of the same molecule on the surface of a target cell, or that bind to different molecules on the surface of a target cell.10063138577

[0021] In any embodiment, the C-terminal polypeptide capable of mediating irreversible conjugation encoded by a nucleotide sequence of the nth lipid nanoparticle specifically interacts with the N-terminal polypeptide capable of mediating irreversible conjugation encoded by a nucleotide sequence of the (n— 1 )thlipid nanoparticle to covalently link the portion of the polypeptide of interest encoded by the nucleotide sequence of nth lipid nanoparticle with the portion of the polypeptide of interest encoded by the nucleotide sequence of the (n— 1)thlipid nanoparticle. In other words, each nth lipid nanoparticle and (n- 1 )th nanoparticle contain one half of a cognate pair of polypeptides capable of mediating conjugation (e.g. trans-splicing) the result of the conjugation in a cell is the covalent linking of portions of the polypeptide of interest. Therefore, the second lipid nanoparticle and the first lipid nanoparticle each contain one half of a cognate pair of N and C terminal polypeptides capable of mediating irreversible conjugation. Where there is a third lipid nanoparticle, the third lipid nanoparticle and the second lipid nanoparticle each contain one half of a cognate pair of N and C terminal polypeptides capable of mediating irreversible conjugation, and where there is a fourth lipid nanoparticle, the fourth lipid nanoparticle and the third lipid nanoparticle each contain one half of a cognate pair of N and C terminal polypeptides capable of irreversible conjugation, and so on. Preferably each cognate pair is unique such that irreversible conjugation can only occur between the N and C terminal polypeptides of two consecutive lipid nanoparticles.

[0022] In any embodiment or aspect, the polypeptide capable of mediating irreversible conjugation is capable of trans-splicing. Preferably, the polypeptide capable of mediating irreversible conjugation is an intein.

[0023] In any embodiment or aspect, the N-terminal polypeptide capable of mediating irreversible conjugation is an N-terminal intein. Exemplary N-terminal inteins are described in Table 2 (amino acid sequences) and Table 3 (nucleotide sequences).

[0024] In any embodiment or aspect, the C-terminal polypeptide capable of mediating irreversible conjugation is a C-terminal intein. Exemplary C-terminal inteins are described in Table 2 (amino acid sequences) and Table 3 (nucleotide sequences).

[0025] In any embodiment or aspect, the N-terminal and C-terminal inteins are cognate pairs as described in Table 2 (amino acid sequences) and Table 3 (nucleotide sequences).10063138578

[0026] In any embodiment, the C-terminal intein encoded by a nucleotide sequence of the nth lipid nanoparticle specifically interacts with the N-terminal intein encoded by a nucleotide sequence of the (n-1)thlipid nanoparticle to covalently link the portion of the polypeptide of interest encoded by the nucleotide sequence of nth lipid nanoparticle with the portion of the polypeptide of interest encoded by the nucleotide sequence of the (n-1)thlipid nanoparticle.

[0027] In any embodiment or aspect, the polypeptide capable of mediating irreversible conjugation is a SpyTag or SpyCatcher.

[0028] In any embodiment or aspect, the N-terminal polypeptide capable of mediating irreversible conjugation is a SpyTag and the C-terminal polypeptide capable of mediating irreversible conjugation is a SpyCatcher.

[0029] In any embodiment or aspect, the N-terminal polypeptide capable of mediating irreversible conjugation is a SpyCatcher and the C-terminal polypeptide capable of mediating irreversible conjugation is a SpyTag.

[0030] In one embodiment, the present disclosure provides a composition comprising:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle,- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle, and- optionally one or more further lipid nanoparticles.

[0031] In any aspect, the nucleotide sequence encoding the N-terminal intein is arranged relative to the nucleotide sequence encoding the N-terminal portion of the polypeptide of interest such that when the N-terminal intein and N-terminal portion of the polypeptide of interest protein is in the presence of a C-terminal polypeptide of the gene10063138579of interest and a C-terminal intein, the inteins excise themselves resulting in the N and C terminal polypeptides linked covalently via a peptide bond.

[0032] In any aspect, the nucleotide sequence encoding the C-terminal intein is arranged relative to the nucleotide sequence encoding the C-terminal portion of the polypeptide of interest such that when the C-terminal intein and C-terminal portion of the polypeptide of interest protein is in the presence of an N-terminal polypeptide of the gene of interest and an N-terminal intein, the inteins excise themselves resulting in the N and C terminal polypeptides linked covalently via a peptide bond.

[0033] In another aspect, the present disclosure provides a method for expressing a polypeptide of interest in a target cell or tissue in a subject, the method comprising administering to the subject:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle,- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle, and- optionally one or more further lipid nanoparticles,thereby expressing a polypeptide of interest in the target cell or tissue in the subject.

[0034] In any embodiment, the first lipid nanoparticle, second lipid nanoparticle and optionally one or more further lipid nanoparticles are administered separately, for example sequentially or concurrently but by distinct administrations (e.g. either separate routes of administration or the same route but separate administrations).

[0035] In any embodiment, the first lipid nanoparticle and second lipid nanoparticle are administered concurrently (e.g. either same routes of administration and / or in the same administration).100631385710

[0036] In any embodiment, the first lipid nanoparticle, second lipid nanoparticle and optionally one or more further lipid nanoparticles are administered concurrently (e.g. either same routes of administration and / or in the same administration).

[0037] In one embodiment, the first lipid nanoparticle, the second lipid nanoparticle and optionally one or more further lipid nanoparticles may each be administered in a composition of the disclosure.

[0038] In another aspect, the present disclosure provides use of:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle,in the manufacture of a medicament for expressing a polypeptide of interest in a target cell or tissue in a subject, wherein the medicament is to be administered with:- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle, and- optionally one or more further lipid nanoparticles.

[0039] In another aspect, the present disclosure provides use of:- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle,in the manufacture of a medicament for expressing a polypeptide of interest in a target cell or tissue in a subject, wherein the medicament is to be administered with:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal intein, and (b a targeting molecule,100631385711or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle, andoptionally one or more further lipid nanoparticles.

[0040] In another aspect, the present disclosure provides use of:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle,- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle, and- optionally one or more further lipid nanoparticles,in the manufacture of a medicament for expressing a polypeptide of interest in a target cell or tissue in a subject.

[0041] In another aspect, the present disclosure provides a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid first lipid nanoparticle, for use in expressing a polypeptide of interest in a target cell or tissue in a subject, wherein the first lipid nanoparticle is to be administered with:- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle, and- optionally one or more further lipid nanoparticles.100631385712

[0042] In another aspect, the present disclosure provides use of a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle, for use in expressing a polypeptide of interest in a target cell or tissue in a subject, wherein the second nanoparticle is to be administered with:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle, and- optionally one or more further lipid nanoparticles.

[0043] In another aspect, the present disclosure provides:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle,- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle, and- optionally one or more further lipid nanoparticles,for use in expressing a polypeptide of interest in a target cell or tissue in a subject.

[0044] In another aspect, the present invention provides a method for introducing a nucleic acid into a cell, preferably the cell is present in vivo, the method comprising contacting the cell with a first lipid nanoparticle, a second lipid nanoparticle and optionally one or more further lipid nanoparticles of the disclosure, thereby introducing the nucleic acid into the cell.100631385713

[0045] In another aspect, the present invention provides a method for the in vivo delivery of a nucleic acid, the method comprising administering a first lipid nanoparticle, a second lipid nanoparticle and optionally one or more further lipid nanoparticles of the disclosure to a subject in need thereof, thereby delivering a nucleic acid to the subject.

[0046] In another aspect, the present invention provides a method for treating or preventing a disease or condition in a subject in need thereof, the method comprising administering to the subject (a) a first lipid nanoparticle, a second lipid nanoparticle and optionally one or more further lipid nanoparticles of the disclosure, or (a) composition of the disclosure, thereby treating or preventing a disease or condition in a subject in need thereof.

[0047] In another aspect, the present invention provides a (a) a first lipid nanoparticle, a second lipid nanoparticle and optionally one or more further lipid nanoparticles of the disclosure, or (a) composition of the disclosure in the manufacture of a medicament for treating or preventing a disease or condition in a subject in need thereof.

[0048] In another aspect, the present invention provides (a) a first lipid nanoparticle, a second lipid nanoparticle and optionally one or more further lipid nanoparticles of the disclosure, or (a) composition of the disclosure for use in the treatment or prevention of a disease or condition in a subject in need thereof.

[0049] In any embodiment or aspect, the composition, method or use may further comprise:- a third lipid nanoparticle as described herein;- a third and fourth lipid nanoparticle as described herein;- a third, fourth and fifth lipid nanoparticle as described herein;- a third, fourth, fifth and sixth lipid nanoparticle as described herein;- a third, fourth, fifth, sixth and seventh lipid nanoparticle as described herein; - a third, fourth, fifth, sixth, seventh and eighth lipid nanoparticle as described herein;- a third, fourth, fifth, sixth, seventh, eighth and ninth lipid nanoparticle as described herein;- a third, fourth, fifth, sixth, seventh, eighth, ninth and tenth lipid nanoparticle as described herein;100631385714- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and eleventh lipid nanoparticle as described herein;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh and twelfth lipid nanoparticle as described herein;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth lipid nanoparticle as described herein;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and fourteenth lipid nanoparticle as described herein;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth and fifteenth lipid nanoparticle as described herein; or - a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth and sixteenth lipid nanoparticle as described herein.

[0050] In one aspect, the present disclosure provides a lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence encoding a cognate sequence capable of mediating reconstitution, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle.

[0051] In one aspect, the present disclosure provides a lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the first sequence, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle.

[0052] In one aspect, the present disclosure provides a lipid nanoparticle comprising (a) a nucleic acid comprising (i) a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid100631385715comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the first sequence, and (ii) a second nucleotide sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the second sequence, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle.

[0053] In one embodiment, a sequence capable of mediating reconstitution is a DNA or RNA (preferably mRNA) sequence capable of mediating trans-splicing, or other recombination event as described herein, in the presence of a cognate sequence.Typically, the sequence is a hybridization domain (may be referred to as a first hybridization domain), and the hybridization domain is capable of trans-splicing in the presence of a corresponding second hybridization domain.

[0054] In any aspect, the nucleotide sequence comprising, or encoding, an N-terminal portion of a polypeptide of interest is linked or fused to the nucleotide sequence comprising, or encoding, the first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the first sequence.

[0055] In any aspect, the nucleotide sequence comprising, or encoding, a C-terminal portion of a polypeptide of interest is linked or fused to the nucleotide sequence comprising, or encoding, the first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the first sequence.

[0056] In another aspect, the present disclosure provides a composition comprising:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence encoding a cognate sequence capable of mediating100631385716reconstitution, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle, and- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the first sequence, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle.

[0057] In one embodiment, the composition may further comprise:- a third lipid nanoparticle;- a third and fourth lipid nanoparticle;- a third, fourth and fifth lipid nanoparticle;- a third, fourth, fifth and sixth lipid nanoparticle;- a third, fourth, fifth, sixth and seventh lipid nanoparticle;- a third, fourth, fifth, sixth, seventh and eighth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth and ninth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth and tenth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and eleventh lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh and twelfth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and fourteenth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth and fifteenth lipid nanoparticle; or- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth and sixteenth lipid nanoparticle.100631385717

[0058] In these embodiments, each lipid nanoparticle comprises a nucleotide sequence encoding a portion of the polypeptide of interest, such that full-length polypeptide of interest is encoded by the totality of each nucleotide sequence encoding each portion of the polypeptide of interest.

[0059] In these embodiments, each lipid nanoparticle comprises a nucleotide sequence capable of mediating reconstitution in the presence of a cognate sequence. Where the composition comprises a third lipid nanoparticle, the second nanoparticle further comprises a nucleotide sequence capable of mediating reconstitution (e.g. transsplicing) in the presence of a cognate nucleotide sequence in the third lipid nanoparticle. Where the composition comprises a fourth lipid nanoparticle, the third nanoparticle further comprises a nucleotide sequence mediating reconstitution (e.g. trans-splicing) in the presence of a cognate nucleotide sequence in the fourth lipid nanoparticle. Where the composition comprises a fifth lipid nanoparticle, the fourth nanoparticle further comprises a nucleotide sequence capable of mediating reconstitution (e.g. trans-splicing) in the presence of a cognate nucleotide sequence in the fifth lipid nanoparticle. Where the composition comprises a sixth lipid nanoparticle, the fifth nanoparticle further comprises a nucleotide sequence capable of mediating reconstitution (e.g. trans-splicing) in the presence of a cognate nucleotide sequence in the sixth lipid nanoparticle. Where the composition comprises a seventh lipid nanoparticle, the sixth nanoparticle further comprises a nucleotide sequence capable of mediating reconstitution (e.g. trans-splicing) in the presence of a cognate a nucleotide sequence in the seventh lipid nanoparticle. Where the composition comprises an eighth lipid nanoparticle, the seventh nanoparticle further comprises a nucleotide sequence capable of mediating reconstitution (e.g. trans-splicing) in the presence of a cognate nucleotide sequence in the eighth lipid nanoparticle. Where the composition comprises a ninth lipid nanoparticle, the eighth nanoparticle further comprises a nucleotide sequence capable of mediating reconstitution (e.g. trans-splicing) in the presence of a cognate nucleotide sequence in the ninth lipid nanoparticle. Where the composition comprises a tenth lipid nanoparticle, the ninth nanoparticle further comprises a nucleotide sequence capable of mediating reconstitution (e.g. trans-splicing) in the presence of a cognate nucleotide sequence in the tenth lipid nanoparticle. Where the composition comprises an eleventh lipid nanoparticle, the tenth nanoparticle further comprises a nucleotide sequence capable of mediating reconstitution (e.g. trans-splicing) in the presence of a cognate nucleotide sequence in the eleventh lipid100631385718nanoparticle. Where the composition comprises a twelfth lipid nanoparticle, the eleventh nanoparticle further comprises a nucleotide sequence capable of mediating reconstitution (e.g. trans-splicing) in the presence of a cognate nucleotide sequence in the twelfth lipid nanoparticle. Where the composition comprises a thirteenth lipid nanoparticle, the twelfth nanoparticle further comprises a nucleotide sequence capable of mediating reconstitution (e.g. trans-splicing) in the presence of a cognate nucleotide sequence in the thirteenth lipid nanoparticle. Where the composition comprises a fourteenth lipid nanoparticle, the thirteenth nanoparticle further comprises a nucleotide sequence capable of mediating reconstitution (e.g. trans-splicing) in the presence of a cognate a nucleotide sequence in the fourteenth lipid nanoparticle. Where the composition comprises a fifteenth lipid nanoparticle, the fourteenth nanoparticle further comprises a nucleotide sequence capable of mediating reconstitution (e.g. trans-splicing) in the presence of a cognate nucleotide sequence in the fifteenth lipid nanoparticle. Where the composition comprises a sixteenth lipid nanoparticle, the fifteenth nanoparticle further comprises a nucleotide sequence capable of mediating reconstitution (e.g. trans-splicing) in the presence of a cognate nucleotide sequence in the sixteenth lipid nanoparticle.

[0060] In these embodiments, where there are more than 2 nanoparticles, at least 2 nanoparticles display targeting molecules that bind to different portions or epitopes of the same molecule on the surface of a target cell, or that bind to different molecules on the surface of a target cell.

[0061] In any embodiment, the nucleotide sequence capable of mediating reconstitution (e.g. trans-splicing) of the nth lipid nanoparticle specifically interacts with a cognate nucleotide sequence of the (n-1)thlipid nanoparticle to covalently link the portion of the polypeptide of interest encoded by the nucleotide sequence of nth lipid nanoparticle with the portion of the polypeptide of interest encoded by the nucleotide sequence of the (n-1)thlipid nanoparticle. In other words, each nth lipid nanoparticle and (n-1)th nanoparticle contain one half of a cognate pair of nucleotide sequences capable of mediating reconstitution (e.g. trans-splicing) the result of the reconstitution in a cell is the covalent linking of nucleotide sequences that encode the polypeptide of interest. Therefore, the second lipid nanoparticle and the first lipid nanoparticle each contain one half of a cognate pair of nucleotide sequences capable of mediating reconstitution (e.g. trans-splicing). Where there is a third lipid nanoparticle, the third lipid100631385719nanoparticle and the second lipid nanoparticle each contain one half of a cognate pair of nucleotide sequences capable of mediating reconstitution (e.g. trans-splicing), and where there is a fourth lipid nanoparticle, the fourth lipid nanoparticle and the third lipid nanoparticle each contain one half of a cognate pair of nucleotide sequences capable of mediating reconstitution (e.g. trans-splicing), and so on. Preferably each cognate pair is unique such that reconstitution (e.g. trans-splicing) can only occur between the nucleotide sequences of two consecutive lipid nanoparticles.

[0062] In another aspect, the present disclosure provides a method for expressing a polypeptide of interest in a target cell or tissue in a subject, the method comprising administering to the subject:- a first lipid nanoparticle (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence encoding a cognate sequence capable of mediating reconstitution, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle, and - a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the first sequence, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle,- optionally one or more further lipid nanoparticles,thereby expressing a polypeptide of interest in the target cell or tissue in the subject.

[0063] In any embodiment, the first lipid nanoparticle, second lipid nanoparticle and optionally further lipid nanoparticle are administered separately, for example sequentially or concurrently but by distinct administrations (e.g. either separate routes of administration or the same route but separate administrations).100631385720

[0064] In any embodiment, the first lipid nanoparticle, second lipid and optionally further nanoparticle are administered concurrently (e.g. either same route but distinct administration and / or in the same administration).

[0065] In any embodiment, the first lipid nanoparticle, second lipid nanoparticle and optionally one or more further lipid nanoparticles are administered concurrently (e.g. either same routes of administration and / or in the same administration).

[0066] In one embodiment, the first lipid nanoparticle, the second lipid nanoparticle and optionally the further lipid nanoparticle may each be administered in a composition of the disclosure.

[0067] In another aspect, the present disclosure provides use of:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence encoding a cognate sequence capable of mediating reconstitution, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle,in the manufacture of a medicament for expressing a polypeptide of interest in a target cell or tissue in a subject, wherein the medicament is to be administered with:- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the first sequence, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle, and- optionally one or more further lipid nanoparticles.

[0068] In another aspect, the present disclosure provides use of:100631385721- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the first sequence, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle,in the manufacture of a medicament for expressing a polypeptide of interest in a target cell or tissue in a subject, wherein the medicament is to be administered with:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence encoding a cognate sequence capable of mediating reconstitution, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle, and- optionally one or more further lipid nanoparticles.

[0069] In another aspect, the present disclosure provides use of:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence encoding a cognate sequence capable of mediating reconstitution, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle, and- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of100631385722mediating reconstitution with the first sequence, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle, and- optionally one or more further lipid nanoparticlesin the manufacture of a medicament for expressing a polypeptide of interest in a target cell or tissue in a subject.

[0070] In another aspect, the present disclosure provides a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence encoding a cognate sequence capable of mediating reconstitution, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle, for use in expressing a polypeptide of interest in a target cell or tissue in a subject, wherein the first lipid nanoparticle is to be administered with:- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the first sequence, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle, and- optionally one or more further lipid nanoparticles.

[0071] In another aspect, the present disclosure provides use of a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the first sequence, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle, for use in expressing100631385723a polypeptide of interest in a target cell or tissue in a subject, wherein the second nanoparticle is to be administered with:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence encoding a cognate sequence capable of mediating reconstitution, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle,- optionally one or more further lipid nanoparticles.

[0072] In another aspect, the present disclosure provides:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence encoding a cognate sequence capable of mediating reconstitution, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle, and- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the first sequence, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle, and- optionally one or more further lipid nanoparticles,for use in expressing a polypeptide of interest in a target cell or tissue in a subject.100631385724

[0073] In one embodiment, the targeting molecule displayed on the outer surface of the first lipid nanoparticle, the targeting molecule displayed on the outer surface of the second lipid nanoparticle, and the targeting molecule displayed on the outer surface of the optionally one or more further lipid nanoparticles bind to the same molecule on the surface of a target cell.

[0074] In one embodiment, the targeting molecule displayed on the outer surface of the first lipid nanoparticle, the targeting molecule displayed on the outer surface of the second lipid nanoparticle, and the targeting molecule displayed on the outer surface of the optionally one or more further lipid nanoparticles bind to different portions or epitopes of the same molecule on the surface of a target cell.

[0075] In one embodiment, the targeting molecule displayed on the outer surface of the first lipid nanoparticle, the targeting molecule displayed on the outer surface of the second lipid nanoparticle and the targeting molecule displayed on the outer surface of the optionally one or more further lipid nanoparticles bind to different molecules on the surface of a target cell.

[0076] In these embodiments, where there are more than 2 lipid nanoparticles, at least 2 lipid nanoparticles display targeting molecules that bind to different portions or epitopes of the same molecule on the surface of a target cell, or that bind to different molecules on the surface of a target cell.

[0077] In one embodiment, the capture binding domain displayed on the outer surface of the first lipid nanoparticle, the capture binding domain displayed on the outer surface of the second lipid nanoparticle, and the capture binding domain displayed on the outer surface of the optionally one or more further lipid nanoparticles bind to targeting molecules that bind to the same molecule on the surface of a target cell.

[0078] In one embodiment, the capture binding domain displayed on the outer surface of the first lipid nanoparticle, the capture binding domain displayed on the outer surface of the second lipid nanoparticle, and the capture binding domain displayed on the outer surface of the optionally one or more further lipid nanoparticles bind to targeting molecules that bind to different portions or epitopes of the same molecule on the surface of a target cell.100631385725

[0079] In one embodiment, the capture binding domain displayed on the outer surface of the first lipid nanoparticle, the capture binding domain displayed on the outer surface of the second lipid nanoparticle, and the capture binding domain displayed on the outer surface of the optionally one or more further lipid nanoparticles bind to targeting molecules that bind to different molecules on the surface of a target cell.

[0080] In these embodiments, where there are more than 2 lipid nanoparticles, the capture binding domain displayed on the outer surface of at least 2 lipid nanoparticles bind to targeting molecules that bind to different portions or epitopes of the same molecule on the surface of a target cell, or that bind to different molecules on the surface of a target cell.

[0081] As used herein, the targeting molecule on the outer surface of the first lipid nanoparticle may be referred to as a first targeting molecule.

[0082] As used herein, the targeting molecule on the outer surface of the second lipid nanoparticle may be referred to as a second targeting molecule.

[0083] As used herein, the targeting molecule on the outer surface of the third lipid nanoparticle may be referred to as a third targeting molecule.

[0084] As used herein, the targeting molecule on the outer surface of the nth lipid nanoparticle may be referred to as a nth targeting molecule.

[0085] In any embodiment, the target cell is a mammalian cell, preferably a human cell. In certain embodiments, the target cell is one or more of T cells (such as pan T cells, CD4+ T cells, CD8+ T cells and CD25+ T cells), NK cells, CD14+ monocytes, B cells (including CD25+ B cells), dendritic cells, NK T cells, tumour cells (such as lymphoma including T cell lymphoma, breast, colon, pancreatic, ovarian and lung cancer cells), bone marrow progenitor cells, kidney stroma, and macrophages.Preferably, the target cell is any one or more of CD4+ T cells, CD8+ T cells, NK cells or B cells.

[0086] In any embodiment, the molecule on the surface of a target cell which the targeting molecule binds is a protein, preferably a cell surface receptor.

[0087] In any embodiment of this aspect, the targeting molecule is any molecule that binds to a molecule expressed on the surface of a target cell. Exemplary targeting100631385726molecules and molecules expressed on the surface of a target cell (e.g. “targets”) to which the targeting molecules bind are described herein.

[0088] In one embodiment, the targeting molecule is an antibody or antibody fragment, such as a nanobody or single chain variable fragment, an affibody, an aptamer or a peptide, preferably a nanobody.

[0089] In one embodiment, the composition is a pharmaceutical composition and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

[0090] In one embodiment, the lipid nanoparticle comprises a plurality of targeting molecules.

[0091] In one embodiment, the lipid nanoparticle comprises a plurality of capture binding domains displayed on the outer surface of the nanoparticle, wherein each capture binding domain is linked to the lipid nanoparticle through a site-specific linkage such that each capture binding domain is displayed in substantially the same orientation and capable of capturing a targeting molecule in an orientation that allows the targeting molecule to interact with its target.

[0092] In one embodiment, each capture binding domain of the plurality of capture binding domains is linked via the same site to the lipid nanoparticle. For example, the location on the capture binding domain that forms the site-specific linkage is the same on each capture binding domain.

[0093] In one embodiment, each capture binding domain is linked to the lipid nanoparticle through a single site-specific linkage.

[0094] In one embodiment, each capture binding domain is linked to the lipid nanoparticle at a position distal to the site of the capture binding domain which interacts with the targeting molecule. For example, where the capture binding domain is an antibody or antigen binding fragment thereof, the site of the capture binding domain which interacts with the targeting molecule is one or more complementary determining regions (CDRs) and the antibody or antigen binding fragment thereof is linked to the lipid nanoparticle particle distal to the one or more CDRs.100631385727

[0095] In one embodiment, each capture binding domain of the plurality of binding domains is linked via a modified amino acid side chain to the lipid nanoparticle.

[0096] In one embodiment, each capture binding domain is not linked via its N or C-terminus to the lipid nanoparticle.

[0097] In one embodiment, each capture binding domain is linked to the lipid nanoparticle at a position that is not part of a p-sheet or a-helix.

[0098] In one embodiment, capture binding domain is linked to the lipid nanoparticle at a position that is on the outer surface of the binding domain in a sterically unhindered position.

[0099] In one embodiment, the site on the capture binding domain for the site-specific linkage has been determined to allow the capture binding domain to be displayed in an optimised orientation when the capture binding domain is linked to the lipid nanoparticle. In one embodiment, determining the location on the capture binding domain for the sitespecific linkage to allow the capture binding domain to be displayed in an optimised orientation may be by any method described herein.

[0100] In one embodiment, the site on the capture binding domain for the site-specific linkage has been determined to allow a targeting molecule to which the capture binding domain binds to be displayed in an optimised orientation when the capture binding domain linked to the lipid nanoparticle is bound to its targeting molecule.

[0101] In one embodiment, the site-specific linkage is formed between a first coupling group on the lipid nanoparticle and a second coupling group on the capture binding domain.

[0102] In one embodiment, the first coupling group is on a hydrophobic molecule, preferably a lipid. Typically, the lipid is a phospholipid, a structural lipid, a PEGylated lipid or a cationic or ionizable lipid. Preferably, the lipid is a PEGylated lipid, for example DSPE-PEG.

[0103] In one embodiment, the first coupling group is an alkene or an alkyne dipolarophile, or a thiol or sulfhydryl or a maleimide or a trans-cyclooctene or a tetrazine or a thiol-ene. Exemplary, dipolarophiles are strained cycloalkenes or cycloalkynes. Preferred strained cycloalkenes or cycloalkynes include a cyclooctyne, a100631385728dibenzocyclooctyne, a monofluorinated cyclcooctyne, a difluorinated cyclooctyne or a biaryl azacyclooctynone.

[0104] In one embodiment, the second coupling group is a 1 ,3-dipole, a thiol or sulfhydryl, a maleimide, a trans-cyclooctene, a tetrazine or a thiol-ene. Typically, the 1 ,3-dipole is an azide, a nitrile oxide, a nitrone, or an isocyanide, preferably an azide.

[0105] In one embodiment, the azide is an azido-phenylalanine (azPhe) where the capture binding domain has been modified to include the azPhe. In one embodiment, the thiol or sulfhydryl is provided by a cysteine residue. Typically, the cysteine residue is introduced into the capture binding domain.

[0106] Where the first and second coupling groups are a thiol or sulfhydryl the sitespecific linkage is a disulphide bond.

[0107] Where the first coupling group is a maleimide and second coupling group is a thiol or sulfhydryl, and the site-specific linkage is a thiosuccinimide.

[0108] In one embodiment, each capture binding domain is an antibody or antibody fragment, such as a nanobody or single chain variable fragment, an affibody, an aptamer, a peptide, protein A, protein G, protein L or spyCatcher. Preferably, the capture binding domain is a nanobody.

[0109] In one embodiment, where the capture binding domain is an antibody, the sitespecific linkage may be at a position from, or equivalent to, amino acid 12 to 17 in FR1 of the antibody; a position from, or equivalent to, amino acid 84 to 91 in FR3 of the antibody; or a position from, or equivalent to, amino acid 117 to 119 in FR4 of the antibody. A non-natural or cysteine amino acid may be introduced (e.g. by mutation of an existing residue or insertion of an additional residue) into the antibody at any position from, or equivalent to, amino acid 12 to 17 in FR1 of the antibody; a position from, or equivalent to, amino acid 84 to 91 in FR3 of the antibody; or a position from, or equivalent to, amino acid 117 to 119 in FR4 of the antibody, except where the amino acid is a proline.

[0110] In any aspect or embodiment, each capture binding domain binds to the Fc region of an antibody, preferably a CH2 or CH3 domain.100631385729

[0111] In any aspect or embodiment, each capture binding domain binds to the framework region of antibody, affibody or fragment thereof, such as an scFv or nanobody.

[0112] In any aspect or embodiment, the capture binding domain is any Fc binding antibody as described herein.

[0113] In any aspect or embodiment, there is at least about 10, 20, 40, 50, 80, 100, 120, 140, 160, 180, 200, 300, 400, or 800 capture binding domains per lipid nanoparticle. Preferably, there is at least about 10, 20, 40, 50, 80, 100, 120, 140, 160, 180 or 200 capture binding domains per lipid nanoparticle.

[0114] In one embodiment, a plurality of targeting molecules are displayed on the outer surface of the nanoparticle, wherein each targeting molecule is linked to the lipid nanoparticle through a site-specific linkage such that each targeting molecule is displayed in substantially the same orientation and capable of binding to a target on a cell surface.

[0115] In any embodiment, each targeting molecule of the plurality of targeting molecules is linked via the same site to the lipid nanoparticle. For example, the location on the targeting molecule that forms the site-specific linkage is same on each targeting molecule.

[0116] In any embodiment, each targeting molecule is linked to the lipid nanoparticle through a single site-specific linkage.

[0117] In any embodiment, each targeting molecule is linked to the lipid particle at a position distal to the target binding site. For example, where the targeting molecule is an antibody or antigen binding fragment thereof, the target binding site is one or more CDRs and the antibody or antigen binding fragment thereof is linked to the lipid-based nanoparticle distal to the one or more CDRs.

[0118] In any embodiment, each targeting molecule of the plurality of targeting molecules is linked via a modified amino acid side chain to the lipid nanoparticle.

[0119] In any embodiment, each targeting molecule is not linked via its N or C-terminus to the lipid nanoparticle.100631385730

[0120] In any embodiment, each targeting molecule is linked to the lipid nanoparticle at a position that is not part of a p-sheet or a-helix.

[0121] In any embodiment, each targeting molecule is linked to the lipid nanoparticle at a position that is on the outer surface of the binding domain in a sterically unhindered position.

[0122] In any embodiment, the site on the targeting molecule for the site-specific linkage has been determined to allow the targeting molecule to be displayed in an optimised orientation when the targeting molecule is linked to the lipid nanoparticle.

[0123] In any embodiment, the site-specific linkage is formed between a first coupling group on the lipid nanoparticle and a second coupling group on the targeting molecule.

[0124] In any embodiment, the first coupling group is on a hydrophobic molecule, preferably a lipid. Typically, the lipid is a phospholipid, a structural lipid, a PEGylated lipid or a cationic or ionizable lipid. Preferably, the lipid is a PEGylated lipid, for example DSPE-PEG.

[0125] In any embodiment, the first coupling group is an alkene or an alkyne dipolarophile, or a thiol or sulfhydryl or a maleimide or a trans-cyclooctene or a tetrazine or a thiol-ene. Exemplary, dipolarophiles are strained cycloalkenes or cycloalkynes. Preferred strained cycloalkenes or cycloalkynes include a cyclooctyne, a dibenzocyclooctyne, a monofluorinated cyclcooctyne, a difluorinated cyclooctyne or a biaryl azacyclooctynone.

[0126] In any embodiment of this aspect, the second coupling group is a 1 ,3-dipole, or a thiol or sulfhydryl or a maleimide or a trans-cyclooctene or a tetrazine or a thiol-ene. Typically, the 1 ,3-dipole is an azide, a nitrile oxide, a nitrone, or an isocyanide, preferably an azide. In one embodiment, the azide is an azido-phenylalanine (azPhe) where the capture binding domain has been modified to include the azPhe. In one embodiment, the thiol or sulfhydryl is provided by a cysteine residue. Typically, the cysteine residue is introduced into the capture binding domain.

[0127] Where the first and second coupling groups are a thiol or sulfhydryl the sitespecific linkage is a disulphide bond.100631385731

[0128] Where the first coupling group is a maleimide and second coupling group is a thiol or sulfhydryl, and the site-specific linkage is a thiosuccinimide.

[0129] In any embodiment, where the targeting molecule is an antibody, the sitespecific linkage may be at a position from, or equivalent to, amino acid 12 to 17 in FR1 of the antibody; a position from, or equivalent to, amino acid 84 to 91 in FR3 of the antibody; or a position from, or equivalent to, amino acid 117 to 119 in FR4 of the antibody. A non-natural or cysteine amino acid may be introduced (e.g. by mutation of an existing residue or insertion of an additional residue) into the antibody at any position from, or equivalent to, amino acid 12 to 17 in FR1 of the antibody; a position from, or equivalent to, amino acid 84 to 91 in FR3 of the antibody; or a position from, or equivalent to, amino acid 117 to 119 in FR4 of the antibody, except where the amino acid is a proline.

[0130] In any embodiment or aspect, the capture binding domain is bound to a targeting molecule, or plurality of capture binding domains are bound to a plurality of targeting domains.

[0131] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0132] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.Brief description of the drawings

[0133] Figure 1 : Schematic illustration of intein-mediated protein splicing and targeted mRNA delivery to minimize off-target effects via two targeted lipid nanoparticles (LNPs). (a) Split intein segments are fused to complementary halves of a functional protein, enabling expression as separate polypeptides. Functional protein reconstitution occurs exclusively upon intein dimerization, which triggers self-excision (splicing) and ligation of the split protein into a continuous, active structure, (b) Targeted delivery of two split mRNA constructs encoding each protein-intein half via antibody-functionalized LNPs. Dual-receptor specificity ensures that only cells co-expressing both100631385732designated surface markers internalize both mRNA components, enabling cytosolic translation and subsequent intein-mediated protein assembly. In non-targeted cells, single mRNA delivery results in expression of an incomplete protein lacking functional activity, thereby reducing off-target effects.

[0134] Figure 2: LoxP-STOP-LoxP-mScarlet-l HEK cells were transfected with Full length Cre or SpLiT Cre mRNA via MessengerMax reagent. Cells were transfected full length Cre or one part of split Cre or both parts of split Cre mRNA via MessengerMax reagent. The percentage of mScarlet-l-positive cells were measured at 24 hours post transfection by flow cytometry.

[0135] Figure 3: Programmed cell death in Jurkat cells is induced by codelivering DTA_N and DTA_C through targeting two distinct surface receptors. CD5+CD7+ Jurkat cells were transfected with untargeted LNPs encapsulating the full diphtheria toxin A (DTA), two separate untargeted LNPs each encapsulating a part of DTA, CD5-targeted LNPs with full DTA, CD7-targeted LNPs with full DTA, and a combination of CD5-targeted LNPs with DTA_N and CD7-targeted LNPs with DTA_C. Cell death was assessed after 24 hours using flow cytometry with a live / dead dye.

[0136] Figure 4: SpLiT diphtheria toxin A (DTA) delivered via various targeted LNPs demonstrates targeted depletion of T cell populations in human PBMC samples ex vivo, without affecting other cell populations, (a) CD4-positive T cells, (b) CD8-positive T cells, and (c) NK cells. Purified PBMCs were incubated with LNPs for 24 hours. A panel of phenotyping antibodies was used to identify CD4 / CD8-positive T cells and NK cells. Data presented as the percentage of single cell population.

[0137] Figure 5: Dual targeting (CD5 and CD7) with DTA_SpLiT mRNA demonstrated precise T cell targeting in PBMC samples, (a) CD4-positive T cells, (b) CD8-positive T cells, and (c) NK cells. PBMCs were purified and incubated with various targeted LNPs for 24 hours. Cells were then washed and stained with a panel of phenotyping antibodies to identify the CD4 / CD8-positive T cells and NK cell populations. Absolute cell counts were calculated.

[0138] Figure 6: B cell depletion was achieved by delivering CD22 receptor on the surface purified PBMCs were incubated with LNPs for 24 hours. A panel of100631385733phenotyping antibodies was used to identify CD4 / CD8-positive T cells and NK cells. Data presented as the percentage of single cells.

[0139] Figure 7: Immunostaining of liver and spleen sections indicating the delivery of Cre_SpLiT mRNA (Cre_N and Cre_C) with CD3-targeted LNPs significantly reduced non-specific signals while maintaining targeting specificity towards T cells, (a) Liver section and (g) spleen section of mice dosed with untargeted LNP-Cre (untargeted LNP containing the full Cre mRNA). (b) Liver and (h) spleen sections of mice dosed with CD3-targeted LNP-Cre. (c, d) Liver and (i, j) spleen sections of mice dosed with two CD3-targeted LNPs formulated with Cre_SpLiT mRNA. All doses were 0.5 mg / kg of mRNA administered intravenously. At the end of the experiments, mice were sacrificed, and liver and spleen samples were prepared for immunostaining. Zoomed-in images were selected to show the expression of tdTomato with different thresholds on the fluorescence signal, indicating the successful translation of functional Cre recombinase by delivering Cre_SpLiT mRNA.

[0140] Figure 8. Image analysis of the percentage of tdTomato-positive and tdTomato-negative hepatocytes. Images from Figure 7 representing (a) untargeted LNP with full Cre, (b) CD3-targeted LNP with full Cre, and (c) a mixture of CD3-targeted LNPs with either Cre_N and Cre_C were analyzed using the cell counting package in CellProfiler. Nuclei staining and tdTomato expression were counted to identify tdTomato-positive and tdTomato-negative cells.

[0141] Figure 9: By delivering Cre_SpLiT mRNA via CD3 targeted LNPs, T cells showed an increase percentage of tdTomato-positive population. Mice were administered with 0.5mg per kg of CD3_targeted LNPs with Cre_SpLiT mRNA intravenously. Immune cells were isolated and enriched from spleen, liver and blood 48 hours later. Major immune cell types were phenotyped with an antibody cocktail. N = 4 mice.

[0142] Figure 10: Targeted depletion of T cells in mice with DTA_ SpLiT via CD3 targeted LNP showed minimal effect on bystander cells and induced no weight loss in mice up to four days. Mice were administered with either 0.05mg per kg of CD3-targeted LNPs with DTA_SpLiT mRNA, CD3-targeted LNPs with DTA mRNA or CD3-targeted LNPs with control mRNA intraperitoneally. Immune cells were isolated and enriched from spleen 24 hours later. Immune cell types were phenotyped as each100631385734sub population (a) CD3-positive T cell, (b) CD19-positive B cells, (c) dendritic cells, (d) neutrophils, (e) monocytes, (f) macrophage cells, and (g) natural killer cells, n = 4 mice. Absolute cell number was quantified by adding CountBright™ Plus Absolute Counting Beads and calculated according to the manufacturer’s instructions, (h) Mice were administered with either 0.05mg per kg of CD3-targeted LNPs with DTA_SpLiT mRNA, or CD3-targeted LNPs with control mRNA intraperitoneally. Mice were monitored and weighted for addition three days. The weight differences were calculated from the predose weight.

[0143] Figure 11 : Survival rate and weight monitoring demonstrated that DTA_SpLiT is a viable option for targeted depletion, (a) Mice were administered 0.05 mg / kg of either full-length DTA or split DTA intraperitoneally. Mice receiving CD3-targeted LNPs with full-length DTA exhibited lethargy and were deemed to reach the ethical endpoint of the experiment, while mice receiving DTA_SpLiT remained healthy, (b) Mice were administered 0.05 mg / kg of either split DTA or split DTA_N intraperitoneally and were monitored for weight changes over four days. Weight changes were calculated based on pre-dose weight.

[0144] Figure 12: Cytotoxicity is observed when cells were targeted delivered with both parts of split DTA with intein pair gp41-8. Targeted LNPs encapsulating either DTA_N-gp41-8_N or gp41-8_C-DTA_C split constructs were formulated. Jurkat cells were treated for 24 h with CD5 / CD7-targeted gp41-8 split-DTA targeted LNPs (tLNPs) or an isotype-control gp41-8 split-DTA tLNP. Cell viability was assessed by flow cytometry, and the percentage of live cells was quantified.

[0145] Figure 13: Split mRNA delivery system is compatible with different intein pairs. Split mScarlet-l mRNA with M86 intein were formulated into LNPs. Jurkat cells were treated with untargeted, isotype control or CD5 / CD7 targeted LNPs containing either split mScarlet mRNA. After 24 hours, cells were analysed on flow cytometry for mScarlet expression.

[0146] Figure 14: Split mRNA delivery system supports multi-part reassembly by targeting three different receptors. mScarlet-l mRNA was split into three parts and formulated into LNP. Jurkat cells were treated with untargeted, isotype control or CD5 / CD7 / TfR targeted LNP. After 24 hours, cells were analysed on flow cytometry for mScarlet expression.100631385735

[0147] Figure 15: Percentage of tdTomato-positive immune cell populations in the spleen following in vivo delivery of split Cre via targeted LNPs. Bar graph showing tdTomato+frequencies across splenic immune subsets, including CD4+T cells, CD8+T cells, macrophages, NK cells, monocytes, neutrophils, and CD19+B cells, after administration of split-Cre LNPs with different targeting combo. Treatment groups include CD3 / TCRp-targeted, CD3 / CD4-targeted, CD3 / CD8-targeted, isotype-control, and non-targeted Cre LNP formulations with full length Cre mRNA. Each targeted LNP contained only one half of the split Cre mRNA. Data points represent individual mice, with n = 3-4 per group.

[0148] Figure 16: Percentage of mScarlet-positive immune cell populations in the spleen following in vivo delivery of split mScarlet via targeted LNPs.The graph showing the proportion of mScarlet+cells across major splenic immune subsets, including CD4+T cells, CD8+T cells, macrophages, NK cells, monocytes, neutrophils, dendritic cells, and CD19+B cells. Mice were treated with CD3 / TCRP-targeted split mScarlet tLNPs, CD3 / CD5-targeted split mScarlet tLNPs, or I gG 1 isotypecontrol split mScarlet LNPs. Each LNP contained only one half of the split mScarlet mRNA. Data points represent individual mice, with n = 3-4 per group. X axis corresponds to mScarlet-l expression level.

[0149] Figure 17: Representative flow cytometry analysis of split-mScarlet delivery in vivo. CD4 versus CD8a gating strategy is shown in panel a. Representative plots for four immune cell populations — CD4+T cells (b, f, j, n), CD8+T cells (c, g, k, o), macrophages (d, h, I, p), and dendritic cells (e, i, m, q) — are displayed for each treatment group. Panels b-e show mice treated with CD3 / TCRp-targeted tLNPs, panels f-i with CD3 / CD5-targeted tLNPs, panels j-m with the lgG1 isotype control LNPs, and panels n-q with PBS controls. mScarlet-positive events illustrate functional reconstitution of the split mRNA only in receptor-defined targeted cell subsets.

[0150] Figure 18: Absolute numbers of splenic immune cell populations following in vivo delivery of split DTA via CD3 / CD4-targeted LNPs. Wild-type mice were intravenously administered 0.05 mg / kg split DTA formulated in CD3 / CD4-targeted tLNPs, lgG1 isotype-control LNPs, or PBS. After 24 hours, spleens were harvested and absolute cell numbers were quantified using counting beads. Panels show absolute numbers of CD4+T cells (a), CD8+T cells (b), B cells (c), macrophages (d),100631385736monocytes (e), neutrophils (f), and dendritic cells (g). Statistical analysis was performed using one-way ANOVA. n = 5 mice per group.

[0151] Figure 19: Split mRNA delivery system is compatible with different LNP formulations. CD5 / CD7 or lgG1 split mScarlet N / C were formulated with either SM102, ALC0315 or ALC0366 as ionizable lipids. Jurkat cells were treated with either CD5 / CD7 or I gG 1 combination for 24 hours. The expression of mScarlet was measure via flow cytometry.Sequence informationTable 1. Sequences of an Fc binding antibody (TP1107)100631385737Table 2. Intein sequences and proteins of interests that are designed as SpLiT. Underlined text refers to intein sequences (N or C termini)*100631385738100631385739100631385740100631385741100631385742100631385743* The N-terminal methionine (M) in any one or more of SEQ ID NOs: 23, 26, 30, 32, 34, 36, 38, 42, 44, 75-81 , 83-92, 94-105, 191-193, and 195-200 may be absent, e.g. due to post-translational removal. Table 3. Exemplary nucleic acid sequences* - when the sequences represent RNA, the thymidine (“t”) in each sequence refers to uracil (“u”)100631385744100631385745100631385746100631385747100631385748100631385749100631385750100631385751100631385752100631385753100631385754100631385755100631385756* A start codon (e.g. “atg”) may be added to any one or more of the nucleic acid sequences that does not begin with a start codon (e.g. “atg”).Detailed description of the embodiments

[0152] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

[0153] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.100631385757

[0154] All of the patents and publications referred to herein are incorporated by reference in their entirety.

[0155] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0156] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present invention. For purposes of the present invention, the following terms are defined.

[0157] The terms "a," "an," or "the" as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the agent" includes reference to one or more agents known to those skilled in the art, and so forth. For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0158] The inventor(s) have developed a new split system that involves delivery of at least two pieces, potentially 16 pieces, of nucleic acid (e.g. mRNA), containing truncated DNA or mRNA, by two, potentially 16, separate LNPs. These LNPs then combine their nucleic acid (e.g. mRNA) payloads at the target cell, after binding of one, or ideally two or more separate, cell surface molecule(s) (e.g. receptor(s)) on the cell surface. Any stray LNPs reaching off-target cells / tissue would be carrying only one half or part / portion of the nucleic acid (e.g. mRNA) required to express the active product, therefore this system reduces the risk of off-target toxicity in the delivery of mRNA therapeutics (of particular concern in the liver).

[0159] In a particularly prefer form of the invention, the split system involves delivery of two or more truncated pieces of nucleic acid (e.g. mRNA) by two or more separate populations of LNPs, where each population of LNP binds to a different cell surface molecule on the surface of the target cell. Each LNP contains a nucleic acid encoding only one portion of the polypeptide of interest or active product.100631385758

[0160] Advantages of at least an embodiment of the invention described herein include increasing the specificity by which a single population of cell can be targeted, and further a significant increase in switching off off-target effects (e.g. background level of off-targeting is almost turned off completely).

[0161] The split system takes advantage of the ability of cognate or paired sequences to mediate an event such as trans-splicing for example in the case of protein the intrinsic ability of split inteins to mediate protein trans-splicing to reconstitute full-length proteins after their fragmentation or truncation into at least two (potentially 16) split intein-flanked polypeptides. Where there are 3 portions of the polypeptide of interest, the 3 portions are transpliced together by 2 pairs of cognate DNA, RNA or protein sequences. In other words, where there are X portions of the polypeptide of interest, the X portions are transpliced together by X-1 pairs of cognate DNA, RNA or protein sequences.

[0162] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.Definitions

[0163] As used herein, the terms "approximately" and "about," as applied to one or more values of interest, refer to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). For example, when used in the context of an amount of a given nucleic acid in a lipid component of a nanoparticle composition, "about" may mean + / -10% of the recited value. For instance, a nanoparticle composition including a lipid component having about 40% of a given nucleic acid may include 30-50% of the nucleic acid.

[0164] As used herein, the term '"delivering" means providing an entity to a destination. For example, delivering a therapeutic and / or prophylactic to a subject may100631385759involve administering a lipid nanoparticle(s) or composition(s) to the subject (e.g., by an intravenous, intra-muscular, intradermal, or subcutaneous route). Administration of a lipid nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition.

[0165] As used herein, the term "enhanced delivery" means delivery of more (e.g., at least 1.5 fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) of a therapeutic and / or prophylactic by a nanoparticle to a target tissue of interest compared to the level of delivery of a therapeutic and / or prophylactic by a control nanoparticle to a target tissue of interest. The level of delivery of a nanoparticle to a particular tissue may be measured by comparing the amount of protein produced in a tissue to the weight of said tissue, comparing the amount of therapeutic and / or prophylactic in a tissue to the weight of said tissue, comparing the amount of protein produced in a tissue to the amount of total protein in said tissue, or comparing the amount of therapeutic and / or prophylactic in a tissue to the amount of total therapeutic and / or prophylactic in said tissue. It will be understood that the enhanced delivery of a nanoparticle to a target tissue need not be determined in a subject being treated, it may be determined in a surrogate such as an animal model (e.g., a mouse model or a rat model).

[0166] As used herein, the term "specific delivery," "specifically deliver," or "specifically delivering" means delivery of more (e.g., at least 1.5 fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) of a therapeutic and / or prophylactic by a nanoparticle to a target tissue of interest (e.g., mammalian spleen) compared to an off-target tissue (e.g., mammalian liver). The level of delivery of a nanoparticle to a particular tissue may be measured by comparing the amount of protein produced in a tissue to the weight of said tissue, comparing the amount of therapeutic and / or prophylactic in a tissue to the weight of said tissue, comparing the amount of protein produced in a tissue to the amount of total protein in said tissue, or comparing the amount of therapeutic and / or prophylactic in a tissue to the amount of total therapeutic and / or prophylactic in said tissue.

[0167] As used herein, "encapsulation" may refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement.100631385760

[0168] For the purposes for the present disclosure, the term “antibody” includes a protein capable of specifically binding to one or a few closely related antigens by virtue of an antigen binding domain contained within a Fv. This term includes four chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanised antibodies, human antibodies, CDR-grafted antibodies, primatised antibodies, de-immunised antibodies, synhumanised antibodies, halfantibodies).

[0169] An antibody generally comprises constant domains, which can be arranged into a constant region or constant fragment or fragment crystallisable (Fc). Exemplary forms of antibodies comprise a four-chain structure as their basic unit. Full-length antibodies comprise two heavy chains (~50 to 70 kD) covalently linked and two light chains (~23 kDa each). A light chain generally comprises a variable region (if present) and a constant domain and in mammals is either a K light chain or a A light chain. A heavy chain generally comprises a variable region and one or two constant domain(s) linked by a hinge region to additional constant domain(s). Heavy chains of mammals are of one of the following types a, 5, E, y, or p. Each light chain is also covalently linked to one of the heavy chains. For example, the two heavy chains and the heavy and light chains are held together by inter-chain disulfide bonds and by non-covalent interactions. The number of inter-chain disulfide bonds can vary among different types of antibodies. Each chain has an N-terminal variable region (VH or VL wherein each are -110 amino acids in length) and one or more constant domains at the C- terminus. The constant domain of the light chain (CL which is -110 amino acids in length) is aligned with and disulfide bonded to the first constant domain of the heavy chain (CHI which is 330 to 440 amino acids in length). The light chain variable region is aligned with the variable region of the heavy chain. The antibody heavy chain can comprise 2 or more additional CH domains (such as, CH2, CH3 and the like) and can comprise a hinge region between the CHI and CH2 constant domains. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGi, lgG2, IgGs, lgG4, IgAi and lgA2) or subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (such as, human) antibody. In one example the antibody heavy chain is missing a C-terminal lysine residue. In one example, the antibody is humanised, synhumanised, chimeric, CDR-grafted or deimmunised.

[0170] The terms "full-length antibody", "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an100631385761antigen binding fragment of an antibody. Specifically, whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

[0171] As used herein, “variable region” refers to the portions of the light and / or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and, includes amino acid sequences of complementarity determining regions (CDRs); i.e., CDRi, CDR2, and CDR3, and framework regions (FRs). For example, the variable region comprises three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0172] As used herein, the term “complementarity determining regions” (syn. CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable region the presence of which are major contributors to specific antigen binding. Each variable region domain (VH or L) typically has three CDRs identified as CDR1, CDR2and CDR3. The CDRs of VH are also referred to herein as CDR Hi , CDR H2 and CDR H3, respectively, wherein CDR Hi corresponds to CDR 1 of VH, CDR H2 corresponds to CDR 2 of VH and CDR H3 corresponds to CDR 3 of VH. Likewise, the CDRs of VL are referred to herein as CDR Li , CDR L2 and CDR L3, respectively, wherein CDR Li corresponds to CDR 1 of VL, CDR L2 corresponds to CDR 2 of VL and CDR L3 corresponds to CDR 3 of VL. In one example, the amino acid positions assigned to CDRs and FRs are defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as “the Kabat numbering system”). In another example, the amino acid positions assigned to CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html). The present invention is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including the canonical numbering system or of Chothia and Lesk J. Mol. Biol. 196: 901-917, 1987; Chothia et al., Nature 342: 877-883, 1989; and / or Al-Lazikani et al., J. Mol. Biol. 273: 927-948, 1997; the numbering system of Honnegherand Plukthun J. Mol. Biol.309: 657-670, 2001; or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res.25: 206-211 1997.100631385762

[0173] "Framework regions" (FRs) are those variable region residues other than the CDR residues. The FRs of VH are also referred to herein as FR Hi , FR H2, FR H3 and FR H4, respectively, wherein FR Hi corresponds to FR 1 of VH, FR H2 corresponds to FR 2 of VH, FR H3 corresponds to FR 3 of H and FR H4 corresponds to FR 4 of VH. Likewise, the FRs of VL are referred to herein as FR Li, FR L2, FR L3 and FR L4, respectively, wherein FR Li corresponds to FR 1 of VL, FR L2 corresponds to FR 2 of VL, FR L3 corresponds to FR 3 of VL and FR L4 corresponds to FR 4 of VL.

[0174] As used herein, the polypeptide of interest may be encoded by an N-terminal portion and multiple C terminal portions (i.e. portions C-terminal to the N-terminal portion). In other words, where there are 3 lipid nanoparticles - a first, second and third lipid nanoparticles - the polypeptide of interest is encoded by 3 nucleotide sequences, one in each of the 3 lipid nanoparticles. In this instance, the polypeptide of interest may be encoded by a nucleotide sequence encoding an N-terminal portion in the first lipid nanoparticle and C-terminal portions in each of the second and third lipid nanoparticles. An N-terminal portion and one or more C-terminal portions comprise the full-length polypeptide or active polypeptide of interest. Alternatively, the portion encoded by the nucleotide sequence in the second lipid nanoparticle may be referred to as a middle portion. Where there are multiple middle portions, e.g. the second and third lipid nanoparticles when four lipid nanoparticles are present, the portion encoded by the nucleotide sequence in the second lipid nanoparticle may be referred to as a first middle portion and the portion encoded by the nucleotide sequence in the third lipid nanoparticle may be referred to as a second middle portion (and so on).

[0175] Reference herein to a “one or more further lipid nanoparticles” may be reference to any one of:- a third lipid nanoparticle as described herein;- a third and fourth lipid nanoparticle as described herein;- a third, fourth and fifth lipid nanoparticle as described herein;- a third, fourth, fifth and sixth lipid nanoparticle as described herein;- a third, fourth, fifth, sixth and seventh lipid nanoparticle as described herein; - a third, fourth, fifth, sixth, seventh and eighth lipid nanoparticle as described herein;- a third, fourth, fifth, sixth, seventh, eighth and ninth lipid nanoparticle as described herein;100631385763- a third, fourth, fifth, sixth, seventh, eighth, ninth and tenth lipid nanoparticle as described herein;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and eleventh lipid nanoparticle as described herein;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh and twelfth lipid nanoparticle as described herein;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth lipid nanoparticle as described herein;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and fourteenth lipid nanoparticle as described herein;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth and fifteenth lipid nanoparticle as described herein; or - a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth and sixteenth lipid nanoparticle as described herein.Lipid nanoparticle

[0176] A lipid nanoparticle (LNP) is typically a nanoparticle complex comprising of ionizable cationic lipids, zwitterionic structural lipids, PEGylated lipids, cholesterol and nucleic acids. They typically have a diameter between 40-300 nm. S but can be smaller and or larger. A targeted LNP may comprise ionizable cationic lipids, zwitterionic structural lipids, PEGylated lipids, cholesterol and nucleic acids, as well as a targeting molecule, which may be a protein, polypeptide, small molecule, sugar or lipid.

[0177] The use of lipid formulations is contemplated for the introduction of the at least one nucleic acid into a host cell (in vitro, ex vivo or in vivo). In another aspect, the at least one nucleic acid may be associated with a lipid. The at least one nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a lipid nanoparticle or liposome, interspersed within the lipid bilayer of a lipid nanoparticle or liposome, attached to a lipid nanoparticle or liposome via a linking molecule that is associated with both the lipid nanoparticle or liposome and nucleic acid (e.g. the oligonucleotide), entrapped in a lipid nanoparticle or liposome, complexed with a lipid nanoparticle or liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / nucleic acid or lipid / expression100631385764vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape.

[0178] In various embodiments, the lipid nanoparticles may comprise lipids or a derivative thereof. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, aldehydes, and polymers (e.g. PEGylated lipids).

[0179] In any aspect or embodiment, the lipid nanoparticle may have a composition or property as described herein including the Examples. For example, the lipid nanoparticle may be a low-specific LNP as described in the Examples.

[0180] The mean size of a nanoparticle of the invention may be greater than, or equal to about 40nm or 100nm e.g., measured by dynamic light scattering (DLS). For example, the mean size may be greater than, or equal to about, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205nm, 210nm, 215 nm, 220nm, 225 nm, 230 nm, 235 nm, 240 nm, 245 nm, 250 nm, 255 nm, 260 nm, 265 nm, 270 nm, 275 nm, 280 nm, 285 nm, 290 nm, 295 nm, 300 nm, 305 nm, 310 nm, 315 nm, 320 nm, 325 nm, 330 nm, 335 nm, 340 nm, 345 nm, 350 nm, 355 nm, 360 nm, 365 nm, 370 nm, 375 nm, 380 nm, 385 nm, 390 nm, 395 nm, 400 nm, 405 nm, 410 nm, 415nm, 420 nm, 425 nm, 430 nm, 435nm, 440 nm, 445 nm, 450 nm, 455 nm, 460 nm, 465 nm, 470 nm, 475 nm, 480 nm, 485 nm, 490 nm, or 500 nm.

[0181] In some embodiments, the mean size of a nanoparticle of the invention may be from about 40 nm to about 500 nm, from about 45 nm to about 500nm, from about 50 nm to about 500nm, from about 55 nm to about 500 nm, from about 60 nm to about 500 nm, from about 65 nm to about 500 nm, from about 70 nm to about 500 nm, from about 75 nm to about 500 nm, from about 80nm to about 500nm, from about 90nm to about 500nm, from about 100nm to about 500nm, from about 110nm to about 500nm, from about 120nm to about 500nm, from about 130nm to about 500nm, from about100631385765140nm to about 500nm, from about 150nm to about 500nm, from about 160nm to about 500nm, from about 170nm to about 500nm, from about 180nm to about 500nm, from about 190nm to about 500nm, from about 200nm to about 500nm, from about 210nm to about 500nm, from about 220nm to about 500nm, from about 230nm to about 500nm, from about 240nm to about 500nm, from about 250nm to about 500nm, from about 260nm to about 500nm, from about 270nm to about 500nm, from about 280nm to about 500nm, from about 300nm to about 500nm, from about 310nm to about 500nm, from about 320nm to about 500nm, from about 330nm to about 500nm, from about 340nm to about 500nm, from about 350nm to about 500nm, from about 360nm to about 500nm, from about 370nm to about 500nm, from about 380nm to about 500nm, from about 390nm to about 500nm, from about 400nm to about 500nm, from about 410nm to about 500nm, from about 420nm to about 500nm, from about 430nm to about 500nm, from about 440nm to about 500nm, from about 450nm to about 500nm, from about 460nm to about 500nm, from about 470nm to about 500nm, from about 480nm to about 500nm, or from about 490nm to about 500nm.

[0182] In some embodiments, the mean size of a nanoparticle of the invention may be from about 40 nm to about 490 nm, from about 40nm to about 480nm, from about 40nm to about 470nm, from about 40nm to about 460nm, from about 40nm to about 450nm, from about 40nm to about 440nm, from about 40nm to about 430nm, from about 40nm to about 420nm, from about 40nm to about 430nm, from about 40nm to about 420nm, from about 40nm to about 410nm, from about 40nm to about 400nm, from about 40nm to about 390nm, from about 40nm to about 380nm, from about 40nm to about 370nm, from about 40nm to about 360nm, from about 40nm to about 350nm, from about 400nm to about 340nm, from about 40nm to about 330nm, from about 40nm to about 320nm, from about 40nm to about 310nm, from about 40nm to about 300nm, from about 40nm to about 290nm, from about 40nm to about 280nm, from about 40nm to about 270nm, from about 40nm to about 260nm, from about 40nm to about 250nm, from about 40nm to about 240nm, from about 40nm to about 230nm, from about 40nm to about 220nm, from about 40nm to about 210nm, from about 40nm to about 200nm, from about 40nm to about 190nm, from about 40nm to about 180nm, from about 40nm to about 170nm, from about 40nm to about 160nm, from about 40nm to about 150nm, from about 40nm to about 140nm, from about 40nm to about 130nm, from about 40nm to about 120nm, from about 40nm to about 110nm, from about 40nm to about 100nm, from about 40nm to about 95nm, from about 40nm to about 90nm, from about 40nm to100631385766about 85nm, from about 40nm to about 80nm, from about 40nm to about 75nm, from about 40nm to about 70nm, from about 40nm to about 65nm, from about 40nm to about 60nm, from about 40nm to about 55nm, from about 40nm to about 50nm, or from about 40nm to about 45nm.

[0183] A nanoparticle may be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle compositions. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a nanoparticle composition may be from about 0.10 to about 0.20.

[0184] As used herein, the "zeta potential" is the electrokinetic potential of a lipid, e.g., in a nanoparticle.

[0185] The zeta potential of a nanoparticle may be used to indicate the electrokinetic potential of the particle. For example, the zeta potential may describe the surface charge of a nanoparticle. In some embodiments, the lipid nanoparticles may have unusually highly negative zeta potential. In some embodiments, the zeta potential of a nanoparticle may be from about -50 mV to about +10 mV, preferably about -10mV to about +5mV. In some embodiments, the zeta potential of a nanoparticle may be from -50 mV to +10 mV, preferably -10mV to +5mV. Further, in some embodiments, the zeta potential of a nanoparticle may be from about -20mV to about -5mV, from about -15mV to about -5m V, from about -10mV to about -5m V, from about -20m V to about -10mV. Further, in some embodiments, the zeta potential of a nanoparticle may be from -20mV to -5mV, from -15mV to -5mV, from -10mV to -5mV, from -20mV to -10mV. Further, in some embodiments, the zeta potential of a nanoparticle may be about -5mV, about -10MV, about -15mV or about -20mV. Further, in some embodiments, the zeta potential of a nanoparticle may be -5mV, -10mV, -15mV or -20mV.

[0186] The lipid nanoparticle may be any one described herein including those listed in the Examples, such as Examples 1-2.100631385767Cationic and / or ionizable lipid

[0187] Any of a variety of cationic lipids may be used in the lipid nanoparticles described herein.

[0188] Cationic lipids which are useful in the present invention can be any of a number of lipid species which carry a net positive charge at physiological pH. Such lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioeoyloxy-3-(dimethylamino)propane (DODAP), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1 ,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N — (N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3.beta.-oxy)-3'-oxapentoxy)-3-dimethy-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1 ,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1 ,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 4-Hydroxybutyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1 -octylnonyl ester (SM-102) and mixtures thereof. A number of these lipids and related analogs have been described in U.S. Patent Publication Nos. 20060083780 and 20060240554; U.S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO 96 / 10390, the disclosures of which are each herein incorporated by reference in their entirety for all purposes. Additionally, a number of commercial preparations of cationic lipids are available and can be used in the present invention. These include, e.g., LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and DOPE, from GIBCO / BRL, Grand Island, N.Y., USA); LIPOFECTAMINE® (commercially available cationic liposomes comprising DOSPA and DOPE, from GIBCO / BRL); and100631385768TRANSFECTAM® (commercially available cationic liposomes comprising DOGS from Promega Corp., Madison, Wis., USA).

[0189] Additionally, cationic lipids of Formula I having the following structures may be used in the present invention.(I)wherein R1and R2are independently selected and are H, OH or C1-C5 alkyls, R3and R4are independently selected and are alkyl groups having from about 4 to about 20 carbon atoms, and at least one of R3and R4comprises at least two sites of unsaturation. In certain instances, R3and R4are both the same, i.e., R3and R4are both linoleyl (Cis), etc. In certain other instances, R3and R4are different, i.e., R3is tetradectrienyl (C14) and R4is linoleyl (Cis). In a preferred embodiment, the cationic lipid of Formula I is symmetrical, i.e., R3and R4are both the same. In another preferred embodiment, both R3and R4comprise at least two sites of unsaturation. In some embodiments, R3and R4are independently selected from the group consisting of dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl. In a preferred embodiment, R3and R4are both linoleyl. In some embodiments, R3and R4comprise at least three sites of unsaturation and are independently selected from, e.g., dodecatrienyl, tetradectrienyl, hexadecatrienyl, linolenyl, and icosatrienyl. In particularly preferred embodiments, the cationic lipid of Formula I is 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) or 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0190] Furthermore, cationic lipids of Formula II having the following structures may be used in the present invention.QI)wherein R1and R2are independently selected and are H or C1-C5 alkyls, R3and R4are independently selected and are alkyl groups having from about 10 to about 20 carbon100631385769atoms, and at least one of R3and R4comprises at least two sites of unsaturation. In certain instances, R3and R4are both the same, i.e., R3and R4are both linoleyl (Cis), etc. In certain other instances, R3and R4are different, i.e., R3is tetradectrienyl (Cu) and R4is linoleyl (Cis). In a preferred embodiment, the cationic lipids of the present invention are symmetrical, i.e., R3and R4are both the same. In another preferred embodiment, both R3and R4comprise at least two sites of unsaturation. In some embodiments, R3and R4are independently selected from the group consisting of dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl. In a preferred embodiment, R3and R4are both linoleyl. In some embodiments, R3and R4comprise at least three sites of unsaturation and are independently selected from, e.g., dodecatrienyl, tetradectrienyl, hexadecatrienyl, linolenyl, and icosatrienyl.

[0191] Moreover, cationic lipids of Formula III having the following structures (or salts thereof) may be used in the present invention.(in)Wherein R1and R2are either the same or different and independently optionally substituted C12-C24 alkyl, optionally substituted C12-C24 alkenyl, optionally substituted C12-C24 alkynyl, or optionally substituted C12-C24 acyl; R3and R4are either the same or different and independently optionally substituted C1-C6 alkyl, optionally substituted C1-Ce alkenyl, or optionally substituted C1-C5 alkynyl or R3and R4may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen; R5is either absent or hydrogen or C1-C6 alkyl to provide a quaternary amine; m, n, and p are either the same or different and independently either 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1 , 2, 3, or 4; and Y and Z are either the same or different and independently O, S, or NH.

[0192] In some embodiments, the cationic lipid of Formula III is 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; “XTC2”), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1 ,3]-dioxolane (DLin-K-C3-DMA), 2 ,2-dil inoleyl-4-(4-dimethylaminobutyl)-[1 ,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-100631385770dimethylaminomethyl-[1 ,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1 ,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1 ,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1 ,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.C1), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.C1), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), or mixtures thereof. In preferred embodiments, the cationic lipid of Formula III is DLin-K-C2-DMA (XTC2).

[0193] Preferably, the cationic lipid is DODAP, DLin-DMA, DLin-K-DMA, DLin-K2-DMA, DLin-MC3-DMA, DLin-MC2-DMA, DLin-MC4-DMA, DLin-KC4-DMA, ATX-0114, ATX-0126, CL-1, Lipid 14, Lipid 5, BP-114, ALC-0315, ALC-0366, 4A3-SC8, C12-200, 98N12-5, or SM102. In one embodiment, the cationic lipid is selected from ALC-0315, ALC-0366 and SM102. In one embodiment, the cationic lipid is any one or more of ALC-0315, ALC-0366 or SM102.

[0194] The cationic lipid typically comprises from about 40 mol % to about 60 mol %, from about 40 mol % to about 55 mol %, from about 40 mol % to about 50 mol %, from about 40 mol % to about 45 mol %, from about 45 mol % to about 60 mol %, from about 50 mol % to about 60 mol %, or from about 55 mol % to about 60 mol % of the total lipid present in the particle.

[0195] The cationic lipid typically comprises about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 mol % of the total lipid present in the particle.Phospholipid

[0196] As used herein, a "phospholipid" is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. A phospholipid may include one or more multiple (e.g., double or triple) bonds (e.g., one or more100631385771unsaturations). Particular phospholipids may facilitate fusion to a membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow one or more elements of a lipid-containing composition to pass through the membrane permitting, e.g., delivery of the one or more elements to a cell.

[0197] The lipid component of a lipid nanoparticle or composition may include one or more phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids may include a phospholipid moiety and one or more fatty acid moieties. For example, a phospholipid may be a lipid according to Formula (IV):in which RPrepresents a phospholipid moiety and Ri and R2 represent fatty acid moieties with or without unsaturation that may be the same or different.

[0198] A phospholipid moiety may be selected from the non-limiting group consisting of: phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.

[0199] A fatty acid moiety may be selected from the non-limiting group consisting of: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0200] Non-natural species including natural species with modifications and substitutions including branch-ing, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid may be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is100631385772replaced with a triple bond). Under appropriate reaction conditions, an alkyne group may undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions may be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).

[0201] Contemplated are phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0202] In some embodiments, a nanoparticle composition includes DSPC. In certain embodiments, a nanoparticle composition includes DOPE. In some embodiments, a nanoparticle composition includes both DSPC and DOPE.

[0203] The phospholipid typically comprises from about 5 mol % to about 20 mol %, from about 5 mol % to about 15 mol %, from about 5 mol % to about 10 mol %, from about 10 mol % to about 20 mol %, or from about 15 mol % to about 20 mol % of the total lipid present in the particle.100631385773

[0204] The phospholipid typically comprises from 5 mol % to 20 mol %, from 5 mol % to 15 mol %, from 5 mol % to 10 mol %, from 10 mol % to 20 mol %, or from 15 mol % to 20 mol % of the total lipid present in the particle.Structural lipid

[0205] The lipid component of a nanoparticle composition may include one or more structural lipids. Structural lipids can be selected from the group consisting of, but are not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof. Further, the structural lipid may be squalene, squalene or combination thereof.

[0206] The structural lipid may include lipids containing geranyl acetate, farnesyl acetate or geranyl-geranyl, or ether, ester, or other derivatives.

[0207] The structural lipid typically comprises from about 30 mol % to about 50 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 30 mol % to about 35 mol %, from about 35 mol % to about 50 mol %, from about 40 mol % to about 50 mol %, or from about 45 mol % to about 50 mol % of the total lipid present in the particle.

[0208] The structural lipid typically comprises from 30 mol % to 50 mol %, from 30 mol % to 45 mol %, from 30 mol % to 40 mol %, from 30 mol % to 35 mol %, from 35 mol % to 50 mol %, from 40 mol % to 50 mol %, or from 45 mol % to 50 mol % of the total lipid present in the particle.PEGylated lipid

[0209] The lipid component of a lipid nanoparticle or composition may include one or more PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. As used herein, a “PEG lipid” or “PEGylated lipid” refers to a lipid comprising a polyethylene glycol component. A PEG lipid may be selected from the non-limiting group consisting of: PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines,100631385774PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0210] In another embodiment, the PEGylated lipid may be 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, also known as DMG-PEG.

[0211] In another embodiment, the PEGylated lipid may be 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0212] The PEGylated lipid may have a PEG component that has a molecular weight of any molecular mass as practically desired, including but not limited to, from about 100 Daltons (Da) to 10,000 Da or more as desired (including but not limited to, sometimes 0.1-10 kDa). The molecular weight of PEG may be of a wide range, including but not limited to, between about 100 Da and about 10,000 Da ormore. PEG may be between about 100 Da and about 100,000 Da, including but not limited to 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, PEG is between about 100 Da and 10,000 Da, between about 1000 Da and 9,000 Da, between about 1000 Da and 8,000 Da, between about 1000 Da and 7,000 Da, between about 1000 Da and 6,000 Da, between about 1000 Da and 5,000 Da, between about 1000 Da and 4,000 Da, between about 1000 Da and 3,000 Da, or between about 1000 Da and 2,000 Da. In some embodiments, PEG is between about 1,000 Da and 5,000 Da. In some embodiments, PEG is between about 2,000 Da and 5,000 Da.

[0213] The PEGylated lipid typically comprises from about 0.05 mol % to about 10 mol %, from about 0.06 mol % to about 10 mol %, from about 0.07 mol % to about 10 mol %, from about 0.08 mol % to about 10 mol %, from about 0.09 mol % to about 10 mol %, from about 0.1 mol % to about 10 mol %, from about 0.15 mol % to about 10 mol %, from about 0.2 mol % to about 10 mol %, from about 0.25 mol % to about 10 mol %, from about 0.3 mol % to about 10 mol %, from about 0.3 mol % to about 10 mol %, from about 0.35 mol % to about 10 mol %, from about 0.4 mol % to about 10 mol %, from about 0.45 mol % to about 10 mol %, from about 0.5 mol % to about 10 mol %, from about 0.55 mol % to about 10 mol %, from about 0.6 mol % to about 10 mol %, from about 0.65 mol % to about 10 mol %, from about 0.7 mol % to about 10 mol %, from100631385775about 0.75 mol % to about 10 mol %, from about 0.8 mol % to about 10 mol %, from about 0.85 mol % to about 10 mol %, from about 0.9 mol % to about 10 mol %, from about 0.95 mol % to about 10 mol %, from about 1.0 mol % to about 10 mol %, from about 1.5 mol % to about 10 mol %, from about 2.0 mol % to about 10 mol %, from about 2.5 mol % to about 10 mol %, from about 3.0 mol % to about 10 mol %, from about 3.5 mol % to about 10 mol %, from about 4.0 mol % to about 10 mol %, from about 4.5 mol % to about 10 mol %, from about 5.0 mol % to about 10 mol %, from about 6.0 mol % to about 10 mol %, from about 7.0 mol % to about 10 mol %, from about 8.0 mol % to about 10 mol %, or from about 9.0 mol % to about 10 mol % of the total lipid present in the particle.

[0214] Alternatively, the PEGylated lipid typically comprises from about from about 0.05 mol % to about 10 mol %, from about 0.05 mol % to about 9 mol %, from about 0.05 mol % to about 8 mol %, from about 0.05 mol % to about 7 mol %, from about 0.05 mol % to about 6 mol %, from about 0.05 mol % to about 5 mol %, from about 0.05 mol % to about 4.5 mol %, from about 0.05 mol % to about 4 mol %, from about 0.05 mol % to about 3.5 mol %, from about 0.05 mol % to about 3 mol %, from about 0.05 mol % to about 2.5 mol %, from about 0.05 mol % to about 2 mol %, from about 0.05 mol % to about 1.5 mol %, from about 0.05 mol % to about 1 mol %, from about 0.05 mol % to about 0.95 mol %, from about 0.05 mol % to about 0.9 mol %, from about 0.05 mol % to about 0.85 mol %, from about 0.05 mol % to about 0.8 mol %, from about 0.05 mol % to about 0.75 mol %, from about 0.05 mol % to about 0.7 mol %, from about 0.05 mol % to about 0.65 mol %, from about 0.05 mol % to about 0.6 mol %, from about 0.05 mol % to about 0.55 mol %, from about 0.05 mol % to about 0.5 mol %, from about 0.05 mol % to about 0.45 mol %, from about 0.05 mol % to about 0.4 mol %, from about 0.05 mol % to about 0.35 mol %, from about 0.05 mol % to about 0.3 mol %, from about 0.05 mol % to about 0.25 mol %, from about 0.05 mol % to about 0.2 mol %, from about 0.05 mol % to about 0.15 mol %, from about 0.05 mol % to about 0.1 mol %, from about 0.05 mol % to about 0.09 mol %, from about 0.05 mol % to about 0.08 mol %, from about 0.05 mol % to about 0.07 mol %, or from about 0.05 mol % to about 0.06 mol % of the total lipid present in the particle.

[0215] The PEGylated lipid typically comprises from 0.05 mol % to 10 mol %, from 0.06 mol % to 10 mol %, from 0.07 mol % to 10 mol %, from 0.08 mol % to 10 mol %, from 0.09 mol % to 10 mol %, from 0.1 mol % to 10 mol %, from 0.15 mol % to 10 mol100631385776%, from 0.2 mol % to 10 mol %, from 0.25 mol % to 10 mol %, from 0.3 mol % to 10 mol %, from 0.3 mol % to 10 mol %, from about 0.35 mol % to 10 mol %, from 0.4 mol % to 10 mol %, from 0.45 mol % to 10 mol %, from 0.5 mol % to 10 mol %, from 0.55 mol % to 10 mol %, from 0.6 mol % to 10 mol %, from 0.65 mol % to 10 mol %, from 0.7 mol % to 10 mol %, from 0.75 mol % to 10 mol %, from 0.8 mol % to 10 mol %, from 0.85 mol % to 10 mol %, from 0.9 mol % to 10 mol %, from 0.95 mol % to 10 mol %, from 1.0 mol % to 10 mol %, from 1.5 mol % to 10 mol %, from 2.0 mol % to 10 mol %, from 2.5 mol % to 10 mol %, from 3.0 mol % to 10 mol %, from 3.5 mol % to 10 mol %, from 4.0 mol % to 10 mol %, from 4.5 mol % to 10 mol %, from 5.0 mol % to 10 mol %, from 6.0 mol % to 10 mol %, from 7.0 mol % to 10 mol %, from 8.0 mol % to 10 mol %, or from 9.0 mol % to 10 mol % of the total lipid present in the particle.

[0216] The PEGylated lipid typically comprises 0.05 mol %, 0.06 mol %, 0.07 mol %, 0.08 mol %, 0.09 mol %, 0.1 mol %, 0.15 mol %, 0.2 mol %, 0.25 mol %, 0.3 mol %, 0.35 mol %, 0.4 mol %, or 0.45 mol % of the total lipid present in the particle.

[0217] As used herein, mole % and mol % are used interchangeably.Capture binding domains

[0218] As used herein, a capture binding domain may be any molecule that is capable of binding to another molecule. For example, the capture binding domain may be an antibody or antigen binding fragment thereof, such as a nanobody or single chain variable fragment, protein A, protein G, protein L or spyCatcher.

[0219] In particular, a capture binding domain as used in the present invention binds or specifically binds to a targeting molecule as described herein.Such targeting molecules are typically moieties that have affinity for cell surface targets (e.g., membrane receptors). The capture binding domain can be any peptide or protein (e.g. antibodies or antibody fragments, including nanobodies) capable of binding to the targeting molecule. Particular embodiments of suitable capture binding domain includes nanobodies, such as single domain antibodies.

[0220] The capture binding domain binds or specifically binds to the targeting molecule such that it does not substantially interfere with the binding activity of the targeting molecule.100631385777

[0221] The capture binding domain may bind or specifically bind to a single site on the targeting molecule, or may bind to multiple sites.

[0222] The binding site of the capture binding domain on the targeting molecule is selected such that it orientates the targeting molecule, exposing the targeting molecule binding domain allowing it to bind to its target.

[0223] When bound to the plurality of capture binding domain, each bound targeting molecule is presented in a non-random orientation on the outer surface of the lipid nanoparticle to substantially expose and / or provide accessibility of each targeting molecule’s binding domain to its target i.e. an “optimal” or “optimised” orientation. The non-random orientation of the targeting molecule is achieved by linking the capture binding domain to the lipid nanoparticle via a site-specific linkage. This site-specific linkage orientates each capture binding protein on the outer surface of the lipid nanoparticle such that when the targeting molecules are bound to the capture binding domain, each targeting molecule has substantially the same non-random orientation projecting from the lipid nanoparticle. The resulting orientation of each capture binding domain and its bound targeting molecule provides enhanced delivery of the lipid nanoparticle to a specific target cell or cell population compared to if the capture binding domain is randomly oriented on the outer surface of the lipid nanoparticle.

[0224] Advantageously, in some methods of the present invention the targeting molecule when bound to the capture binding domain facilitates the delivery of the capture binding domain and any compounds attached thereto, i.e. a lipid nanoparticle, to the site for which the targeting molecule’s ligand or target is present.

[0225] A capture binding domain may bind to the Fc region of an antibody, preferably a CH2 or CH3 domain.

[0226] Alternatively, a capture binding domain binds to the framework region of antibody, affibody or fragment thereof, such as an scFv or nanobody.

[0227] In one embodiment, the LNPs may be modified with a highly specific antibodycapturing nanobody, TP1107. The orientation of TP1107 on the LNP surface may be controlled by site-specifical ly incorporating an azide-bearing synthetic amino acid p-azido-phenylalanine (azPhe) using codon reassignment of the amber stop codon100631385778(UAG). In one embodiment, the TP1107azPhe may be modified with DSPE-PEG2000-DBCO, allowing insertion into the LNP.

[0228] The capture binding domain includes but not limited to antibodies, antibody fragments, e.g. Fab2, Fab, scFV, VHH domains, and other proteins or peptides.

[0229] According to a particular embodiment of the present invention, the capture binding domain comprises complementarity determining regions that bind to the targeting molecule.

[0230] In any embodiment, the capture binding domain comprises a sequence of any of the complementarity determining regions (CDRs) having an amino acid sequence as described in Table 1. Preferably, the framework regions have an amino acid sequence also as described in Table 1.

[0231] In another preferred embodiment, the capture binding domain comprises a CDRH1 , a CDRH2 and / or a CDRH3 of a single domain antibody having a variable heavy chain as defined in SEQ ID NO: 1.

[0232] In another preferred embodiment, the capture binding domain comprises a heavy chain variable region that comprises:a CDRH1 as set forth in SEQ ID NO: 2; a CDRH2 as set forth in SEQ ID NO: 3; and a CDRH3 as set forth in SEQ ID NO: 4;a CDRH1 as set forth in SEQ ID NO: 9 a CDRH2 as set forth in SEQ ID NO: 10; and a CDRH3 as set forth in SEQ ID NO: 11 ; ora CDRH1 as set forth in SEQ ID NO: 15 a CDRH2 as set forth in SEQ ID NO: 16; and a CDRH3 as set forth in SEQ ID NO: 17.

[0233] In another preferred embodiment, the capture binding domain comprises a heavy chain variable region that comprises or consists of the sequence of SEQ ID NO: 1.Targeting molecules

[0234] Targeting molecules may include any suitable binding agent which is capable of specifically interacting with and binding to a target cell ligand on the surface of a100631385779target cell or tissue. The targeting molecules may be naturally occurring or engineered. Targeting molecules may include, but are not limited to, proteins, peptides, antibodies or antibody fragments, immunoglobulins or immunoglobulin fragments, small molecules, aptamers, vitamins, nucleic acid molecules, and the like.

[0235] The target cell ligand may include endogenous ligands occurring on the surface of a cell or in the extracellular space outside of a cell, such as carbohydrates, lipids, polysaccharides, proteins, glycoproteins, glycolipids, peptides, cell membrane components (e.g., cholesterol) or the like.

[0236] In certain embodiments, the endogenous ligands on the target cell are specific for the target cell, i.e. , are expressed and / or are contained only on the target cell, or at least, are minimally present in cells that are not the target cells. In other embodiments, the target ligand on the target cells can be an engineered or otherwise non-naturally occurring ligand, e.g., a genetically modified target cell that expresses a non-naturally occurring surface cell protein. Suitable targeting ligands can be selected so that the unique properties of the target cell are utilized, thus allowing the composition to differentiate between target and non-target cells.

[0237] Alternatively, only one of a first targeting molecule and a second targeting molecule binds to a target molecule that is specific for the target cell i.e., is expressed and / or is contained only on the target cell, or at least, is minimally present in cells that are not the target cells. In other words, in some embodiments, for the first and second lipid nanoparticles to reduce off-target effects only one of the lipid nanoparticles needs to bind, or specifically bind to, a target that is specific for the target cell.

[0238] Further, each of the first and second nanoparticles may bind to a target molecule that is not specific for the target cell, i.e. each target molecule is expressed on cells other than the target cell, however only the target cell expresses, or expresses to a moderate or high level, both target molecules. In other words, the target cell specificity arises from a combination of the first and second targeting molecules.

[0239] In certain embodiments, the target cells are one or more of T cells (such as pan T cells, CD4+ T cells, CD8+ T cells and CD25+ T cells), NK cells, CD14+ monocytes, B cells (including CD25+ B cells), dendritic cells, NK T cells, tumour cells (such as lymphoma including T lymphoma, breast, colon, pancreatic, ovarian and lung100631385780cancer cells), bone marrow progenitor cells, kidney stroma, and macrophages.Preferably, the target cells are one or more of CD4+ T cells, CD8+ T cells, NK cells or B cells.

[0240] Exemplary targeting molecules are listed in Table 4 below.Table 4. Exemplary targeting molecules - the amino acid of the CDRs, and the full VH and / or VL domains of each of the antibodies listed below are hereby incorporated by reference.

[0241] In particular embodiments, the targeting molecules are selected from: an anti-CD3 antibody, anti-CD3e antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD22 antibody, or any combination thereof, preferably the antibody is a monoclonal antibody.100631385781

[0242] In one embodiment, 2 distinct molecules are targeted by the targeting molecules, for example, the cell surface molecule targets may be CD3 and TCRp (beta); CD3 and CD4; CD3 and CD5; CD5 and CD7; or CD3 and CD8 (or CD8a).

[0243] In one embodiment, 3 distinct molecules are targeted by the targeting molecules, for example, the cell surface molecules targets may be CD5, CD7, and TfR.

[0244] In one aspect of the invention, the capture binding domains bind to a targeting molecule (or equivalently “targeting domains”, “targeting moieties” or “targeting ligands”) which function to target the delivery vehicle (e.g., LNP) to a cell or cell population. In this aspect, the targeting molecule binds the cell surface molecule on the target cell and the capture binding domain binds to the targeting molecules. In this aspect, the targeting molecule may be non-covalently bound to the lipid-based nanoparticle via the capture domain.

[0245] Also contemplated is the targeting molecule linked directly, not via a capture domain, to the lipid-nanoparticle. In this aspect, the targeting molecule is covalently linked to the lipid-based nanoparticle.

[0246] When directly linked to the lipid-based nanoparticle the plurality of the targeting molecule is presented in a non-random orientation on the outer surface of the lipid-based nanoparticle to substantially expose and / or provide accessibility of each of the plurality of targeting molecule’s binding domain to its target i.e. an “optimal” or “optimised” orientation. The non-random orientation of the targeting molecule is achieved by linking the targeting molecule to the lipid-based nanoparticle via a sitespecific linkage. This site-specific linkage orientates each targeting molecule on the outer surface of the lipid-based nanoparticle such that each targeting molecule has substantially the same non-random orientation projecting from the lipid-based nanoparticle. The resulting orientation of each targeting molecule provides enhanced delivery of the lipid-based nanoparticle to a specific target cell or cell population compared to if the targeting molecule is randomly oriented on the outer surface of the lipid-based nanoparticle.

[0247] No limit is meant to be placed on the targeting molecules contemplated herein so long as any particular targeting molecule may be (a) bound by a capture binding domain (either covalently or non-covalently) and / or (b) is capable of causing or100631385782facilitating the localization or targeting of the delivery vehicle to a target cell or tissue by the binding or otherwise interaction between the targeting molecule, optionally bound by the capture binding domain, and a target cell ligand on a target cell or tissue.Conjugation of capture binding domains

[0248] In various embodiments of the invention, a capture binding domain is conjugated to the lipid via a site-specific linkage. Exemplary methods of conjugation can include formation of a covalent bond. In one embodiment, the conjugation is a reversible conjugation, such that the delivery vehicle can be disassociated from the targeting domain upon exposure to certain conditions or chemical agents. In another embodiment, the conjugation is an irreversible conjugation, such that under normal conditions the delivery vehicle does not dissociate from the targeting domain.

[0249] In some embodiments, the conjugation comprises a covalent bond between a lipid and a capture binding domain. The lipid is activated via functionalization with a first coupling group. In one embodiment, lipid comprises a first coupling group capable of reacting with a second coupling group. In one embodiment, the activated lipid is an activated PEGylated lipid. In one embodiment, the first coupling group is bound to the polyethylene glycol portion of the PEGylated lipid. In one embodiment, the second functional group is covalently attached to or part of the capture binding domain.

[0250] The first coupling group and second coupling group can be any functional groups known to those of skill in the art to together form a covalent bond, for example under mild reaction conditions or physiological conditions. In some embodiments, the first coupling group or second coupling group are selected from the group consisting of maleimides, phosphines, hydroxymethyl phosphines, psoralen, imidoesters, pyridyl disulfide, isocyanates, vinyl sulfones, alpha-haloacetyls, aryl azides, acyl azides, alkyl azides, tetrazine, methyltetrazine, trans-cyclooctene, methylcyclopropene, norborene, diazirines, benzophenone, epoxides, carbonates, anhydrides, sulfonyl chlorides, cyclooctyne, aldehydes, and sulfhydryl groups. In some embodiments, the first coupling group or second coupling group is selected from the group consisting of free sulfhydryl groups (-SH), azide group, strained alkyne, hydrazides, and alkoxyamines. In some embodiments, the first coupling group is a functional group that is reactive toward sulfhydryl groups, such as maleimide, pyridyl disulfide, or a haloacetyl. In one embodiment, the first coupling group is a maleimide. In some embodiments, the first100631385783coupling group is a functional group that is reactive toward azide groups, such as dibenzocyclooctyne, 4-Dibenzocyclooctynol, or an alkyne. In one embodiment, the first coupling group is a dibenzocyclooctyne.

[0251] In one embodiment, the second coupling group is a sulfhydryl group. The sulfhydryl group can be installed on the capture binding domain using any method known to those of skill in the art. In one embodiment, the sulfhydryl group is present on a free cysteine residue.

[0252] In one embodiment, the second coupling group is an azide group. The azide group can be installed on the capture binding domain using any method known to those of skill in the art. In one embodiment, the azide group is present on an azidophenylalanine residue.

[0253] In some embodiments, the lipid and capture binding domain are functionalized with groups used in “click” chemistry. Bioorthogonal “click” chemistry comprises the reaction between a functional group with a 1 ,3-dipole, such as an azide, a nitrile oxide, a nitrone, an isocyanide, and the link, with an alkene or an alkyne dipolarophiles.Exemplary dipolarophiles include any strained cycloalkenes and cycloalkynes known to those of skill in the art, including, but not limited to, cyclooctynes, dibenzocyclooctynes, monofluorinated cyclcooctynes, difluorinated cyclooctynes, and biarylazacyclooctynone.Nucleic acids and polypeptides

[0254] Lipid nanoparticles may include one or more nucleic acid. The disclosure features methods of delivering a nucleic acid to a mammalian cell or organ, producing a polypeptide of interest in a mammalian cell, and / or treating a disease or disorder in a mammal in need thereof comprising administering to a mammal and / or contacting a mammalian cell with a lipid nanoparticle(s) including a nucleic acid.

[0255] In certain embodiments, lipid nanoparticles of the present invention are associated with a nucleic acid, resulting in a nucleic acid-lipid nanoparticle (e.g., NALP). In some embodiments, the nucleic acid is fully encapsulated in the lipid particle.

[0256] As used herein, the term “nucleic acid” includes any oligonucleotide or polynucleotide, with fragments containing up to 60 nucleotides generally termed oligonucleotides, and longer fragments termed polynucleotides.100631385784

[0257] In the context of this invention, the terms “polynucleotide” and “oligonucleotide” refer to a polymer or oligomer of nucleotide or nucleoside monomers consisting of naturally-occurring bases, sugars and intersugar (backbone) linkages. The terms “polynucleotide” and “oligonucleotide” also include polymers or oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.

[0258] Oligonucleotides are generally classified as deoxyribooligonucleotides or ribooligonucleotides. A deoxyribooligonucleotide consists of a 5-carbon sugar called deoxyribose joined covalently to phosphate at the 5' and 3' carbons of this sugar to form an alternating, unbranched polymer. A ribooligonucleotide consists of a similar repeating structure where the 5-carbon sugar is ribose.

[0259] The nucleic acid that is present in a nucleic acid-lipid nanoparticle according to this invention includes any form of nucleic acid that is known. The nucleic acids used herein can be single-stranded DNA or RNA (e.g. pre-mRNA, mature mRNA, mRNA), or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples of double-stranded DNA are described herein and include, e.g., structural genes, genes including control and termination regions, and self-replicating systems such as viral or plasmid DNA.Examples of double-stranded RNA are described herein and include, e.g., siRNA and other RNAi agents such as aiRNA and pre-miRNA. Single-stranded nucleic acids include, e.g., antisense oligonucleotides, ribozymes, mature miRNA, and triplex-forming oligonucleotides. Nucleic acids of the invention may also include nucleotide analogues (e.g., Brdll, dllTP, 7-deaza-dGTP), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In certain embodiments, the nucleic acid may further include non-nucleic acid conjugates.

[0260] The nucleic acid contained in or encapsulated by the lipid nanoparticles of the present invention may, once reconstituted (e.g. via trans-splicing), encode a polypeptide of interest. Preferably, the nucleic acid is capable of being translated in the cell to produce the polypeptide of interest. The polypeptide of interest may be any antigenic or immunogenic polypeptide, such as that that is used to stimulate the humoral (e.g. B cell or T cell) immune system. The polypeptide of interest may be useful for the therapeutic or prophylactic immunisation of a mammal, preferably human. The polypeptide of100631385785interest may be useful for providing a therapeutic or prophylactic effect against a disease or condition, preferably wherein the condition is an infection. The infection may be by any microorganism, such as a bacteria, virus, fungi or protozoa.

[0261] In another preferred embodiment, the polypeptide of interest may be useful for the therapeutic or prophylactic treatment of a disease in a mammal, preferably human. For example, the polypeptide of interest may be a cytotoxic molecule including bacterial toxin molecules such as Pseudomonas exotoxin or Diphtheria toxin, a suicide agent, or an apoptotic gene.

[0262] In some embodiments, the N-terminal fragment of a polypeptide of interest may be linked to an N-terminal intein via a peptide linker, preferably a flexible peptide linker. Alternatively, the N-terminal fragment of a polypeptide of interest may be linked to an N-terminal intein in the absence of any peptide linker.

[0263] In some embodiments, the middle portion of a polypeptide of interest may be linked to an N-terminal intein via a peptide linker, preferably a flexible peptide linker; and / or the middle portion of the polypeptide of interest may be linked to a C-terminal intein via a peptide linker, preferably a peptide flexible peptide linker. Alternatively, the middle portion of a polypeptide of interest may be linked to an N-terminal intein in the absence of any peptide linker; and / or the middle portion of the polypeptide of interest may be linked to a C-terminal intein in the absence of any peptide linker.

[0264] In some embodiments, the C-terminal fragment of a polypeptide of interest may be linked to a C-terminal intein via a peptide linker, preferably a flexible peptide linker. Alternatively, the C-terminal fragment of a polypeptide of interest may be linked to a C-terminal intein in the absence of any peptide linker.

[0265] A peptide linker may be a peptide having a length of up to 20 amino acids. The term “linked to” or “fused to” may refer to a linkage via a covalent bond, e.g., a peptide bond, formed between two moieties in the absence of a peptide linker; or a linkage via a peptide linker. In the context of the present disclosure the peptide linker may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more amino acids.

[0266] The skilled person will be familiar with the design and use of various peptide linkers comprised of various amino acids, and of various lengths, which would be100631385786suitable for use as linkers in accordance with the present disclosure. The peptide linker may comprise various combinations of repeated amino acid sequences. The peptide linker may be a flexible peptide linker (such as those comprising repeats of glycine and serine residues), a rigid peptide linker (such as those comprising glutamic acid and lysine residues, flanking alanine repeats) and / or a cleavable peptide linker (such as sequences that are susceptible by protease cleavage). Any peptide linker known in the art or disclosed herein may be used. Preferably, the linker comprises the amino acid sequence of GSAGSAAGSG (SEQ ID NO: 211), HDG, CSG, VDA or SDL.

[0267] In some embodiments, the cytotoxic molecule is a bacterial toxin molecule, preferably a Diphtheria toxin. In a preferred embodiment, the Diphtheria toxin is provided as an N-terminal fragment (e.g. SEQ ID NO: 76) linked to an N-terminal intein (e.g. SEQ ID NO: 22) and a C-terminal fragment (e.g. SEQ ID NO: 77) linked to a C-terminal intein (e.g. SEQ ID NO: 23 or 24). In a more preferred embodiment, the Diphtheria toxin is provided as SEQ ID NOs: 78 and 79. In some embodiments, the Diphtheria toxin is provided as an N-terminal fragment (e.g. SEQ ID NO: 76) linked to an N-terminal intein (e.g. SEQ ID NO: 33) and a C-terminal fragment (e.g. SEQ ID NO: 77) linked to a C-terminal intein (e.g. SEQ ID NO: 34). In a preferred embodiment, the Diphtheria toxin is provided as SEQ ID NOs: 191 and 192.

[0268] In some embodiments, the cytotoxic molecule is a suicide agent, preferably HSV-tk. In a preferred embodiment, the HSV-tk is provided as an N-terminal fragment (e.g. SEQ ID NO: 102) linked to an N-terminal intein (e.g. SEQ ID NO: 22) and a C-terminal fragment (e.g. SEQ ID NO: 103) linked to a C-terminal intein (e.g. SEQ ID NO: 23 or 24). In a more preferred embodiment, the HSV-tk is provided as SEQ ID NOs: 104 and 105.

[0269] In some embodiments, the cytotoxic molecule is an apoptotic protein, preferably Puma or Bax. In a preferred embodiment, the Puma is provided as an N-terminal fragment (e.g. SEQ ID NO: 92) linked to an N-terminal intein (e.g. SEQ ID NO: 22) and a C-terminal fragment (e.g. SEQ ID NO: 93) linked to a C-terminal intein (e.g. SEQ ID NO: 23 or 24). In a more preferred embodiment, the Puma is provided as SEQ ID NOs: 94 and 95. In another preferred embodiment, the Bax is provided as an N-terminal fragment (e.g. SEQ ID NO: 97) linked to an N-terminal intein (e.g. SEQ ID NO: 22) and a C-terminal fragment (e.g. SEQ ID NO: 98) linked to a C-terminal intein100631385787(e.g. SEQ ID NO: 23 or 24). In a more preferred embodiment, the Bax is provided as SEQ ID NOs: 99 and 100.

[0270] In another preferred embodiment, the polypeptide of interest may be useful for the imaging and tracing of cells in a mammal, preferably human. For example, the polypeptide of interest may be a fluorescent protein such as a red fluorescent protein, a green fluorescent protein or a blue fluorescent protein. Preferably, the fluorescent protein is mScarlet or mScarlet-l. In some embodiments, the mScarlet or mScarlet-l is provided as an N-terminal fragment (e.g. SEQ ID NO: 87) linked to an N-terminal intein (e.g. SEQ ID NO: 22) and a C-terminal fragment (e.g. SEQ ID NO: 88) linked to a C-terminal intein (e.g. SEQ ID NO: 23 or 24). In a preferred embodiment, the mScarlet or mScarlet-l is provided as SEQ ID NOs: 89 and 90.

[0271] In some embodiments, the mScarlet or mScarlet-l is provided as an N-terminal fragment (e.g. SEQ ID NO: 87) linked to an N-terminal intein (e.g. SEQ ID NO: 27) and a C-terminal fragment (e.g. SEQ ID NO: 88) linked to a C-terminal intein (e.g. SEQ ID NO: 28). Optionally, the N-terminal fragment of mScarlet or mScarlet-l is linked to the N-terminal intein via a peptide linker, preferably a flexible peptide linker, more preferably a peptide linker comprising the amino acid sequence of VDA; and / or the C-terminal fragment of mScarlet or mScarlet-l is linked to the C-terminal intein via a peptide linker, preferably a flexible peptide linker, more preferably a peptide linker comprising the amino acid sequence of SDL. In a preferred embodiment, the mScarlet or mScarlet-l is provided as SEQ ID NOs: 199 and 200.

[0272] In some embodiments, the mScarlet or mScarlet-l is provided as: an N-terminal fragment (e.g. SEQ ID NO: 193) linked to a first N-terminal intein (e.g. SEQ ID NO: 55), a middle portion (e.g. SEQ ID NO: 194) linked to a first C-terminal intein (e.g. SEQ ID NO: 56) and a second N-terminal intein (e.g. SEQ ID NO: 22), and a C-terminal fragment (e.g. SEQ ID NO: 195) linked to a second C-terminal intein (e.g. SEQ ID NO: 23 or 24), wherein the first N-terminal intein is cognate to the first C-terminal intein (or vice versa), the second N-terminal intein is cognate to the second C-terminal intein (or vice versa), and the pair of the first N-terminal intein and the first C-terminal intein is different to the pair of the second N-terminal intein and the second C-terminal intein. Optionally, the N-terminal fragment of mScarlet or mScarlet-l is linked to the first N-terminal intein via a peptide linker, preferably a flexible peptide linker, more preferably a peptide linker comprising the amino acid sequence of HDG; the middle portion of100631385788mScarlet or mScarlet-l is linked to the first C-terminal intein via a peptide linker, preferably a flexible peptide linker, more preferably a peptide linker comprising the amino acid sequence of CSG; the middle portion of mScarlet or mScarlet-l is linked to the second N-terminal intein via a peptide linker, preferably a flexible peptide linker; and / or the C-terminal fragment of mScarlet or mScarlet-l is linked to the second C-terminal intein via a peptide linker, preferably a flexible peptide linker. In a preferred embodiment, the mScarlet or mScarlet-l is provided as SEQ ID NOs: 196-198.

[0273] In another preferred embodiment, the polypeptide of interest may be useful for gene editing in a mammal, preferably human. For example, the polypeptide of interest may be a CRISPR associated (Cas) protein such as Cas9, a zinc finger nuclease, a transcription activator-like effector nucleases (TALENs), or a tyrosine recombinase enzyme such as a ere recombinase. Preferably, the polypeptide of interest is a ere recombinase. In some embodiments, the ere recombinase is provided as an N-terminal fragment (e.g. SEQ ID NO: 81) linked to an N-terminal intein (e.g. SEQ ID NO: 22) and a C-terminal fragment (e.g. SEQ ID NO: 82) linked to a C-terminal intein (e.g. SEQ ID NO: 23 or 24). In a preferred embodiment, the ere recombinase is provided as SEQ ID NO: 83 and SEQ ID NO: 84 or 85.

[0274] Nucleic acids of the invention may be of various lengths, generally dependent upon the particular form of nucleic acid. For example, in particular embodiments, plasmids or genes may be from about 100 to about 100,000 nucleotide residues in length. In particular embodiments, oligonucleotides may range from about 10 to about 100 nucleotides in length. In various related embodiments, oligonucleotides, both single-stranded, double-stranded, and triple-stranded, may range in length from about 10 to about 60 nucleotides, from about 15 to about 60 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides, or from about 20 to about 30 nucleotides in length.

[0275] It will be understood that an N-terminal methionine (M) residue of a polypeptide encoded by a start codon may be removed from the polypeptide during post-translational processing. It is therefore contemplated that the N-terminal methionine of the disclosed amino acid sequences may be removed after translation. Therefore, the disclosure encompasses not only the full-length amino acid sequences as disclosed, but also corresponding sequences where the N-terminal methionine is absent. For example, the methionine at position 1 of SEQ ID NO: 23 (encoded by e.g.100631385789SEQ ID NO: 107) may be removed during post-translational processing. Accordingly, in any aspects and embodiments of the present disclosure, the N-terminal methionine in SEQ ID NOs: 23, 26, 30, 32, 34, 36, 38, 42, 44, 75-81, 83-92, 94-105, 191-193 and 195-200 may be absent.

[0276] A start codon (e.g., atg) may be added to the 5’ terminus of a nucleic acid of the disclosure, or a nucleic acid encoding a polypeptide of the disclosure (see e.g.SEQ ID NOs: 107 and 200), if the nucleic acid or the nucleic acid encoding the polypeptide does not start with a start codon at the 5’ terminus. Therefore, the disclosure encompasses not only the nucleic acid sequences as disclosed, but also corresponding sequences further comprising a start codon (e.g., atg) at the 5’ terminus.

[0277] The present invention provides novel lipid nanoparticles comprising one or more nucleic acids, methods of making the lipid particles, and methods of delivering and / or administering the lipid nanoparticles (e.g., for the treatment of a disease or disorder).

[0278] The amount of nucleic acid in a lipid nanoparticle may depend on the size, sequence, and other characteristics of the nucleic acid. The amount of nucleic acid in a lipid nanoparticle may also depend on the size, composition, desired target, and other characteristics of the lipid nanoparticle. The relative amounts of nucleic acid and other elements (e.g., lipids) may also vary. In some embodiments, the wt / wt ratio of the lipid component to a nucleic acid in a nanoparticle composition may be from about 5:1 to about 50:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, and 50:1. For example, the wt / wt ratio of the lipid component to a nucleic acid may be from about 10: 1 to about 40: 1. The amount of nucleic acid in a nanoparticle composition may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0279] In some embodiments, the wt / wt ratio of the lipid component to the nucleic acid in the nanoparticle composition is from about 5:1 to about 50:1. In certain embodiments, the wt / wt ratio is from about 10:1 to about 40:1.

[0280] In some embodiments, the one or more nucleic acids, lipids, and amounts thereof may be selected to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of100631385790phosphate groups in a nucleic acid. In general, a lower N:P ratio is preferred. The one or more nucleic acids, lipids, and amounts thereof may be selected to provide an N:P ratio from about2:1 to about 8:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, and 8:1. In certain embodiments, the N:P ratio may be from about 2:1 to about 5:1. In preferred embodiments, the N:P ratio may be about 4:1. In other embodiments, the N:P ratio is from about 5:1 to about 8:1. For example, the N:P ratio may be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1.

[0281] In some embodiments, the N:P ratio of the nanoparticle composition is from about 2:1 to about 8:1. In particular embodiments, the N:P ratio is from about 2:1 to about 5:1. In preferred embodiments, the N:P ratio is about 4:1. In certain embodiments, the N:P ratio is from about 5:1 to about 8:1. For example, the N:P ratio may be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1.Intein

[0282] In one aspect of the present invention, the lipid nanoparticle may comprise a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal intein or a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and encoding a C-terminal intein.

[0283] As used herein, “intein” refers an auto-catalytic protein segments capable of excising itself from a larger precursor protein, enabling the flanking extein (external protein) sequences to be ligated through the formation of a new peptide bond (e.g., protein splicing). Inteins may include a protein domain sequence that can spontaneously splice (e.g., splice from protein flanking N- and C-terminal domains) and excise itself from a sequence to become a mature protein. Inteins have also been called protein introns, by analogy with (RNA) introns. Inteins may mediate protein splicing or trans-splicing via cognate or paired inteins.

[0284] Inteins can be classified on many criteria, e.g.:- Based on how they splice themselves out, they can be classified into cis-splicing (which means that they splice themselves out) or trans-splicing (which means they need outside help). Split inteins usually involve two halves helping each other out, so they are trans-splicing.100631385791- Based on whether they contain the endonuclease domain. An intein that has an endonuclease domain is called a "maxi-intein", otherwise a "mini-intein".- Based on their splicing mechanism, which can be partially inferred based on the sequence. Class 1 intein are marked by a cysteine or serine as the first residue. Class 2 intein, or "alanine intein" has alanine as the first residue and no WCT motif. Class 3 intein has alanine as the first residue and a non-contiguous "WCT" motif. It has also been proposed that inteins that start with a serine and contain a "WCT" motif should be also classed as class 3.

[0285] The process of an intein excising itself and joining the remaining portions of the protein is termed "protein splicing" or "intein-mediated protein splicing". In some embodiments, an intein of a precursor protein (an intein containing protein prior to intein-mediated protein splicing) comes from two genes. Such intein is referred to herein as a split intein. For example, in cyanobacteria, DnaE, the catalytic subunit a of DNA polymerase III, is encoded by two separate genes, dnaE-n (an N-terminal intein) and dnaE-c (a C-terminal intein). The intein encoded by the dnaE-n gene is herein referred as "intein-N" or “N-terminal intein”. The intein encoded by the dnaE-c gene is herein referred as "intein-C" or “C-terminal intein”.

[0286] Other intein systems may also be used. For example, a synthetic intein based on the dnaE intein, the Cfa-N and Cfa-C intein pair, has been described (e.g., in Stevens et al., J Am Chem Soc. 2016 Feb 24; 138(7):2162-5, incorporated herein by reference). Non-limiting examples of intein pairs that may be used in accordance with the present disclosure include: Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, TerThyX intein, Rma DnaB intein and Cne Prp8 intein (e.g., as described in US Patent 8,394,604, incorporated herein by reference).

[0287] In some embodiments, the nucleic acid of the present invention may comprise a nucleotide sequence encoding inteins of Npu, Cfa, M86, NpuSsp, gp41-1, gp41-8, NrdJ-1, IMPDH-1, SspDnaX, SspGyrB, TvoVMA, PhoRadA, Cth-Ter, MP-M-DnaB, SaP-dpol, NrdA-2, and / or Mja-KIbA as shown in Table 2 or in the Examples, or encode one or more inteins as shown in Table 3. In some embodiments, the N-terminal intein may comprise a polypeptide sequence as defined in SEQ ID NOs: 22, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or 55. The C-terminal intein may comprise a polypeptide sequence as defined in SEQ ID NOs: 23, 24, 26, 28, 30, 32, 34, 36, 38, 40,10063138579242, 44, 46, 48, 50, 52, 54 or 56. Preferably, the C-terminal intein as defined in SEQ ID NO: 23 or 24 is cognate to the N-terminal intein as defined in SEQ ID NO: 22 (or vice versa); the C-terminal intein as defined in SEQ ID NO: 26 is cognate to the N-terminal intein as defined in SEQ ID NO: 25 (or vice versa); the C-terminal intein as defined in SEQ ID NO: 28 is cognate to the N-terminal intein as defined in SEQ ID NO: 27 (or vice versa); the C-terminal intein as defined in SEQ ID NO: 30 is cognate to the N-terminal intein as defined in SEQ ID NO: 29 (or vice versa); the C-terminal intein as defined in SEQ ID NO: 32 is cognate to the N-terminal intein as defined in SEQ ID NO: 31 (or vice versa); the C-terminal intein as defined in SEQ ID NO: 34 is cognate to the N-terminal intein as defined in SEQ ID NO: 33 (or vice versa); the C-terminal intein as defined in SEQ ID NO: 36 is cognate to the N-terminal intein as defined in SEQ ID NO: 35 (or vice versa); the C-terminal intein as defined in SEQ ID NO: 38 is cognate to the N-terminal intein as defined in SEQ ID NO: 37 (or vice versa); the C-terminal intein as defined in SEQ ID NO: 40 is cognate to the N-terminal intein as defined in SEQ ID NO: 39 (or vice versa); the C-terminal intein as defined in SEQ ID NO: 42 is cognate to the N-terminal intein as defined in SEQ ID NO: 41 (or vice versa); the C-terminal intein as defined in SEQ ID NO: 44 is cognate to the N-terminal intein as defined in SEQ ID NO: 43 (or vice versa); the C-terminal intein as defined in SEQ ID NO: 46 is cognate to the N-terminal intein as defined in SEQ ID NO: 45 (or vice versa); the C-terminal intein as defined in SEQ ID NO: 48 is cognate to the N-terminal intein as defined in SEQ ID NO: 47 (or vice versa); the C-terminal intein as defined in SEQ ID NO: 50 is cognate to the N-terminal intein as defined in SEQ ID NO: 49 (or vice versa); the C-terminal intein as defined in SEQ ID NO: 52 is cognate to the N-terminal intein as defined in SEQ ID NO: 51 (or vice versa); the C-terminal intein as defined in SEQ ID NO: 54 is cognate to the N-terminal intein as defined in SEQ ID NO: 53 (or vice versa); and / or the C-terminal intein as defined in SEQ ID NO: 56 is cognate to the N-terminal intein as defined in SEQ ID NO: 55 (or vice versa).

[0288] In some embodiments, the N-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 22, and the C-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 23 or 24. In some embodiments, the N-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 25, and the C-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 26. In some embodiments, the N-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 27, and the C-terminal intein comprises a polypeptide sequence100631385793as defined in SEQ ID NO: 28. In some embodiments, the N-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 29, and the C-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 30. In some embodiments, the N-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 31, and the C-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 32. In some embodiments, the N-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 33, and the C-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 34. In some embodiments, the N-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 35, and the C-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 36. In some embodiments, the N-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 37, and the C-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 38. In some embodiments, the N-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 39, and the C-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 40. In some embodiments, the N-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 41, and the C-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 42. In some embodiments, the N-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 43, and the C-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 44. In some embodiments, the N-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 45, and the C-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 46. In some embodiments, the N-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 47, and the C-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 48. In some embodiments, the N-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 49, and the C-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 50. In some embodiments, the N-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 51, and the C-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 52. In some embodiments, the N-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 53, and the C-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 54. In some embodiments, the N-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 55, and the C-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 56.100631385794Preferably, the N-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 22, and the C-terminal intein comprises a polypeptide sequence as defined in SEQ ID NO: 23 or 24.

[0289] In any aspect, the nucleotide sequence encoding an N-terminal portion of a polypeptide of interest is linked or fused to the nucleotide sequence encoding an N-terminal intein.

[0290] In any aspect, the nucleotide sequence encoding a C-terminal portion of a polypeptide of interest is linked or fused to the nucleotide sequence encoding a C-terminal intein.

[0291] In any aspect, the nucleotide sequence encoding a middle portion of a polypeptide of interest is linked or fused to the nucleotide sequence encoding an N-terminal intein and a nucleotide sequence encoding a C-terminal intein, wherein the N-terminal intein is not cognate to the C-terminal intein (and vice versa). The nucleotide sequence encoding the middle portion of the polypeptide of interest is flanked by the nucleotide sequence encoding the N-terminal intein and the nucleotide encoding the C-terminal intein - i.e. the nucleotide comprises from 5’ terminus to 3’ terminus: (a) the nucleotide sequence encoding the N-terminal intein; (b) the nucleotide sequence encoding the middle portion of the polypeptide of interest; and (c) the nucleotide encoding the C-terminal intein.

[0292] In any aspect, the nucleotide sequence encoding the N-terminal intein (or one part of an intein pair) is arranged relative to the nucleotide sequence encoding the N-terminal portion of the polypeptide of interest such that when the N-terminal intein and N-terminal portion of the polypeptide of interest protein is in the presence of a C-terminal polypeptide of the gene of interest and a C-terminal intein (the cognate or other part of the intein pair), the inteins excise themselves resulting in the N and C terminal polypeptides linked covalently via a peptide bond.

[0293] In any aspect, the nucleotide sequence encoding the C-terminal intein (or one part of an intein pair) is arranged relative to the nucleotide sequence encoding the C-terminal portion of the polypeptide of interest such that when the C-terminal intein and C-terminal portion of the polypeptide of interest protein is in the presence of an N-terminal polypeptide of the gene of interest and an N-terminal intein (the cognate or100631385795other part of the intein pair), the inteins excise themselves resulting in the N and C terminal polypeptides linked covalently via a peptide bond.

[0294] In preferred embodiments, the nucleic acid of the invention comprises a nucleotide sequence encoding the inteins from Nostoc punctiforme including Nostoc punctiforme PCC73102, such as SEQ ID NOs: 22-24.

[0295] In preferred embodiments, the nucleic acid of the invention comprises a nucleotide sequence encoding the inteins from Synechocystis sp. PCC 6803, such as SEQ ID NOs: 27-28.

[0296] In preferred embodiments, the nucleic acid of the invention comprises a nucleotide sequence encoding the inteins from a metagenome including those derived from environmental metagenomic sequence data (e.g., the global ocean sampling metagenomic dataset, also known as the GOS metagenomic dataset), such as SEQ ID NOs: 31-32 (gp41-1) or SEQ ID NOs: 33-34 (gp41-8).

[0297] In preferred embodiments, the nucleic acid of the invention comprises a nucleotide sequence encoding the inteins from Methanococcus jannaschii, such as SEQ ID NOs: 55-56.

[0298] In some embodiments, the nucleic acid of the invention comprises:- a nucleotide sequence encoding an N-terminal fragment of a cytotoxic molecule, preferably a bacterial toxin molecule such as Pseudomonas exotoxin or Diphtheria toxin, a suicide agent such as HSV-tk, or an apoptotic protein such as Puma or Bax, and a nucleotide sequence encoding an N-terminal intein;- a nucleotide sequence encoding a C-terminal fragment of a cytotoxic molecule, preferably a bacterial toxin molecule such as Pseudomonas exotoxin or Diphtheria toxin, a suicide agent such as HSV-tk, or an apoptotic protein such as Puma or Bax, and a nucleotide sequence encoding a C-terminal intein;- a nucleotide sequence encoding an N-terminal intein, a nucleotide sequence encoding a middle portion of a cytotoxic molecule, preferably a bacterial toxin molecule such as Pseudomonas exotoxin or Diphtheria toxin, a suicide agent such as HSV-tk, or an apoptotic protein such as Puma or Bax, and a nucleotide sequence encoding a C-terminal intein, wherein the N-terminal intein is not cognate to the C-terminal intein (and vice versa);100631385796- a nucleotide sequence encoding an N-terminal fragment of a fluorescent protein such as a red fluorescent protein, a green fluorescent protein or a blue fluorescent protein, and a nucleotide sequence encoding an N-terminal intein; - a nucleotide sequence encoding a C-terminal fragment of a fluorescent protein such as a red fluorescent protein, a green fluorescent protein or a blue fluorescent protein, and a nucleotide sequence encoding a C-terminal intein; - a nucleotide sequence encoding an N-terminal intein, a nucleotide sequence encoding a middle portion of a fluorescent protein such as a red fluorescent protein, a green fluorescent protein or a blue fluorescent protein, and a nucleotide sequence encoding a C-terminal intein, wherein the N-terminal intein is not cognate to the C-terminal intein (and vice versa);- a nucleotide sequence encoding an N-terminal fragment of a CRISPR associated (Cas) protein such as Cas9, a zinc finger nuclease, a transcription activator-like effector nucelases (TALENs), or a tyrosine recombinase enzyme such as a ere recombinase, and a nucleotide sequence encoding an N-terminal intein;- a nucleotide sequence encoding a C-terminal fragment of a CRISPR associated (Cas) protein such as Cas9, a zinc finger nuclease, a transcription activator-like effector nucelases (TALENs), or a tyrosine recombinase enzyme such as a ere recombinase, and a nucleotide sequence encoding a C-terminal intein; or - a nucleotide sequence encoding an N-terminal intein, a nucleotide sequence encoding a middle portion of a CRISPR associated (Cas) protein such as Cas9, a zinc finger nuclease, a transcription activator-like effector nucelases (TALENs), or a tyrosine recombinase enzyme such as a ere recombinase, and a nucleotide sequence encoding a C-terminal intein, wherein the N-terminal intein is not cognate to the C-terminal intein (and vice versa).

[0299] In a preferred embodiment, the nucleic acid of the invention comprises:- a nucleotide sequence encoding an N-terminal fragment of Diphtheria toxin (e.g.SEQ ID NO: 76) and a nucleotide sequence encoding an N-terminal intein (e.g. SEQ ID NO: 22 or 33);- a nucleotide sequence encoding a C-terminal fragment of Diphtheria toxin (e.g.SEQ ID NO: 77) and a nucleotide sequence encoding a C-terminal intein (e.g. SEQ ID NO: 23, 24 or 34);100631385797- a nucleotide sequence encoding an N-terminal fragment of HSV-tk (e.g. SEQ ID NO: 102) and a nucleotide sequence encoding an N-terminal intein (e.g. SEQ ID NO: 22);- a nucleotide sequence encoding a C-terminal fragment of HSV-tk (e.g. SEQ ID NO: 103) and a nucleotide sequence encoding a C-terminal intein (e.g. SEQ ID NO: 23 or 24);- a nucleotide sequence encoding an N-terminal fragment of Puma (e.g. SEQ ID NO: 92) and a nucleotide sequence encoding an N-terminal intein (e.g. SEQ ID NO: 22);- a nucleotide sequence encoding a C-terminal fragment of Puma (e.g. SEQ ID NO: 93) and a nucleotide sequence encoding a C-terminal intein (e.g. SEQ ID NO: 23 or 24);- a nucleotide sequence encoding an N-terminal fragment of Bax (e.g. SEQ ID NO: 97) and a nucleotide sequence encoding an N-terminal intein (e.g. SEQ ID NO: 22);- a nucleotide sequence encoding a C-terminal fragment of Bax (e.g. SEQ ID NO: 98) and a nucleotide sequence encoding a C-terminal intein (e.g. SEQ ID NO: 23 or 24);- a nucleotide sequence encoding an N-terminal fragment of mScarlet or mScarlet- I (e.g. SEQ ID NO: 87) and a nucleotide sequence encoding an N-terminal intein (e.g. SEQ ID NO: 22 or 27);a nucleotide sequence encoding a C-terminal fragment of mScarlet or mScarlet-l (e.g. SEQ ID NO: 88) and a nucleotide sequence encoding a C-terminal intein (e.g. SEQ ID NO: 23, 24 or 28);- a nucleotide sequence encoding an N-terminal fragment of mScarlet or mScarlet- I (e.g. SEQ ID NO: 196) and a nucleotide sequence encoding an N-terminal intein (e.g. SEQ ID NO: 55);- a nucleotide sequence encoding a C-terminal intein (e.g. SEQ ID NO: 56), a nucleotide sequence encoding a middle portion of mScarlet or mScarlet-l (e.g. SEQ ID NO: 197) and a nucleotide sequence encoding an N-terminal intein (e.g. SEQ ID NO: 22);- a nucleotide sequence encoding a C-terminal fragment of mScarlet or mScarlet-l (e.g. SEQ ID NO: 198) and a nucleotide sequence encoding a C-terminal intein (e.g. SEQ ID NO: 23 or 24);100631385798- a nucleotide sequence encoding an N-terminal fragment of a ere recombinase (e.g. SEQ ID NO: 81) and a nucleotide sequence encoding an N-terminal intein (e.g. SEQ ID NO: 22); or- a nucleotide sequence encoding a C-terminal fragment of a ere recombinase (e.g. SEQ ID NO: 82) and a nucleotide sequence encoding a C-terminal intein (e.g. SEQ ID NO: 23 or 24).

[0300] In another preferred embodiment, the nucleic acid of the invention comprises:- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 78;- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 79;- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 191;- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 192;- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 104;- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 105;- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 94;- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 95;- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 99;- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 100;- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 89;- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 90;100631385799- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 199;- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 200;- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 196;- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 197;- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 198;- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 83; or- a nucleotide sequence encoding a polypeptide sequence as defined in SEQ ID NO: 84 or 85.

[0301] In any embodiment, the skilled person would be able to determine a nucleic acid sequence that encodes a polypeptide sequence of any aspect of the present invention, such as those in Table 3. In addition to the DNA sequences shown herein, particularly Table 3, the specification also contemplates RNA equivalents of those sequences, particularly those in Table 3.Recombination of nucleic acids

[0302] In one aspect, the present disclosure provides a lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence encoding a cognate sequence capable of mediating reconstitution, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle.

[0303] In one aspect, the present disclosure provides a lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first1006313857100sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the first sequence, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle.

[0304] In one embodiment, a sequence capable of mediating reconstitution is a DNA or RNA (preferably mRNA) sequence capable of mediating trans-splicing, or other recombination event as described herein, in the presence of a a cognate sequence. Typically, the sequence is a hybridization domain (may be referred to as a first hybridization domain), and the hybridization domain is capable of trans-splicing in the presence of a corresponding second hybridization domain.

[0305] In some embodiments, the reconstitution occurs at the DNA level and is mediated by (1) trans-splicing of the DNA, (2) overlapping of the DNA, or (3) a combination of both based on a highly recombinogenic exogenous sequence to the trans-splicing vectors / nucleic acids to increase recombination efficiency. In some embodiments, the trans-splicing may be mediated by the inherent ability of inverted terminal repeats (ITRs) included in the first sequence and the second sequence (or cognate sequence pair) to concatemerise to reconstitute full-length sequences. As another example, the overlapping may be mediated by homologous recombination between an overlapping sequence of the coding sequence (CDS) included in the first sequence and the second sequence (or cognate sequence pair).

[0306] In some embodiments, the reconstitution occurs at the RNA level, and is mediated by a hybridisation domain, complementary binding domains (e.g. as described in Riedmayr et al., Nature Communications 14:6578, 2023, incorporated by reference herein) or ribozyme-activated mRNA trans-ligation (e.g. as described in Lindley et al., Science 386:762-767 , 2024, incorporated by reference herein). For example, the first sequence and the second sequence (or cognate sequence pair) may share an intronic hybridisation domain that facilitate trans-splicing, leading to joining of the two halftranscripts (or more than two transcripts) into an intact full-length mRNA. As another example, the first sequence and the second sequence (or cognate sequence pair) may comprise a promoter, complementary binding domains (e.g. derived from the intronic sequence of the human rhodopsin gene), splice sites and poly adenylation signal that enable transcript expression and subsequent mRNA trans-splicing. As yet another1006313857101example, ribozymes may be expressed on either end of the first mRNA and the second mRNA, which will activate mRNA trans-ligation and translation.Pharmaceutical Compositions

[0307] Lipid nanoparticles may be formulated in whole or in part as pharmaceutical compositions. Pharmaceutical compositions may include one or more nanoparticles. For example, a pharmaceutical composition may include one or more nanoparticles including one or more different nucleic acids. Pharmaceutical compositions may further include one or more pharmaceutically acceptable excipients or accessory ingredients such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington's The Science and Practice of Pharmacy, 21stEdition, A. R. Gennaro;Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and accessory ingredients may be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory ingredient may be incompatible with one or more components of a lipid nanoparticle. An excipient or accessory ingredient may be incompatible with a component of a lipid nanoparticle if its combination with the component may result in any undesirable biological effect or otherwise deleterious effect.

[0308] In some embodiments, one or more excipients or accessory ingredients may make up greater than 50% of the total mass or volume of a pharmaceutical composition including a lipid nanoparticle. For example, the one or more excipients or accessory ingredients may make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical convention. In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0309] Relative amounts of the one or more lipid nanoparticles, the one or more pharmaceutically acceptable excipients, and / or any additional ingredients in a1006313857102pharmaceutical composition in accordance with the present disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, a pharmaceutical composition may comprise between 0.1% and 100% (wt / wt) of one or more lipid nanoparticles.

[0310] In certain embodiments, the lipid nanoparticles and / or pharmaceutical compositions of the invention are refrigerated or frozen for storage and / or shipment (e.g., being stored at a temperature of 4° C. or lower, such as a temperature between about -150° C. and about 0° C. or between about -80° C. and about -20° C. (e.g., about -5° C., -10° C., -15° C., -20° C., -25° C., -30° C., -40° C., -50° C., -60° C., -70° C., -80° C., -90° C., -130° C. or -150° C.)

[0311] In certain embodiments, the pharmaceutical composition of the invention comprises a lipid nanoparticle disclosed herein and a pharmaceutically acceptable carrier selected from one or more of Tris, an acetate (e.g., sodium acetate), a citrate (e.g., sodium citrate), saline, PBS, and sucrose. In certain embodiments, the pharmaceutical composition of the disclosure has a pH value between about 7 and 8 (e.g., 6.86.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0, or between 7.5 and 8 or between 7 and 7.8). For example, a pharmaceutical composition of the disclosure comprises a nanoparticle composition disclosed herein, Tris, saline and sucrose, and has a pH of about 7.5-8, which is suitable for storage and / or shipment at, for example, about -20° C. For example, a pharmaceutical composition of the disclosure comprises a lipid nanoparticle disclosed herein and PBS and has a pH of about 7-7.8, suitable for storage and / or shipment at, for example, about 4° C. or lower. “Stability,” “stabilized,” and “stable” in the context of the present disclosure refers to the resistance of nanoparticle compositions and / or pharmaceutical compositions disclosed herein to chemical or physical changes (e.g., degradation, particle size change, aggregation, change in encapsulation, etc.) under given manufacturing, preparation, transportation, storage and / or in-use conditions, e.g., when stress is applied such as shear force, freeze / thaw stress, etc.

[0312] In a preferred embodiment, the lipid nanoparticles of the invention are formulated with one or more disaccharides, or disaccharide containing molecules, such as sucrose, lactose, maltose, trehalose, maltitol or lactitol in any one or more of the1006313857103buffers described herein, including Tris HCI, Tris Acetate, TT / AA. Lipid nanoparticles formulated with sucrose can be reconstituted after thawing without aggregation.

[0313] Lipid nanoparticles and / or pharmaceutical compositions including one or more lipid nanoparticles may be administered to any patient or subject, including those patients or subjects that may benefit from a therapeutic effect provided by the delivery of nucleic acids to one or more particular cells, tissues, organs, or systems or groups thereof, such as the renal system. Although the descriptions provided herein of lipid nanoparticles and pharmaceutical compositions including lipid nanoparticles are principally directed to compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other mammal. Modification of compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the compositions is contemplated include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, hoses, sheep, cats, dogs, mice, and / or rats.

[0314] A pharmaceutical composition including one or more lipid nanoparticles may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients, and then, if desirable or necessary, dividing, shaping, and / or packaging the product into a desired single- or multi-dose unit.

[0315] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient (e.g., lipid nanoparticle). The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.1006313857104

[0316] Pharmaceutical compositions may be prepared in a variety of forms suitable for a variety of routes and methods of administration. For example, pharmaceutical compositions may be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical and / or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms.

[0317] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include additional therapeutic and / or prophylactics, additional agents such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and / or perfuming agents. In certain embodiments for parenteral administration, compositions are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof.

[0318] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and / or emulsions in nontoxic parenterally acceptable diluents and / or solvents, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.1006313857105

[0319] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0320] Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing compositions with suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

[0321] Solid dosage forms for oral administration include capsules, tablets, pills, films, powders, and granules. In such solid dosage forms, an active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and / or fillers or extenders (e.g. starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g., glycerol), disintegrating agents (e.g., agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarding agents (e.g., paraffin), absorption accelerators (e.g., quaternary ammonium compounds), wetting agents (e.g., cetyl alcohol and glycerol monostearate), absorbents (e.g., kaolin and bentonite clay, silicates), and lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents.

[0322] Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include lipid substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.1006313857106

[0323] Dosage forms for topical and / or transdermal administration of a composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and / or patches. Generally, an active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and / or any needed preservatives and / or buffers as may be required. Additionally, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms may be prepared, for example, by dissolving and / or dispensing the compound in the proper medium. Alternatively or additionally, rate may be controlled by either providing a rate controlling membrane and / or by dispersing the compound in a polymer matrix and / or gel.

[0324] Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositions may be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in PCT publication WO 99 / 34850 and functional equivalents thereof. Jet injection devices which deliver liquid compositions to the dermis via a liquid jet injector and / or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Jet injection devices are described, for example, in U.S. Pat. Nos.5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911;5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627; 5,064,413;5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; and PCT publications WO 97 / 37705 and WO 97 / 13537. Ballistic powder / particle delivery devices which use compressed gas to accelerate vaccine in powder form through the outer layers of the skin to the dermis are suitable. Alternatively or additionally, conventional syringes may be used in the classical Mantoux method of intradermal administration.

[0325] Formulations suitable for topical administration include, but are not limited to, liquid and / or semi liquid preparations such as liniments, lotions, oil in water and / or water in oil emulsions such as creams, ointments and / or pastes, and / or solutions and / or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (wt / wt) active ingredient, although the concentration of active ingredient may be as high as the solubility limit of the active ingredient in the solvent.1006313857107Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

[0326] A pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder and / or using a self-propelling solvent / powder dispensing container such as a device comprising the active ingredient dissolved and / or suspended in a low-boiling propellant in a sealed container. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0327] Low boiling propellants generally include liquid propellants having a boiling point of below 65° F. at atmospheric pressure. Generally the propellant may constitute 50% to 99.9% (wt / wt) of the composition, and active ingredient may constitute 0.1% to 20% (wt / wt) of the composition. A propellant may further comprise additional ingredients such as a liquid non-ionic and / or solid anionic surfactant and / or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).

[0328] Pharmaceutical compositions formulated for pulmonary delivery may provide an active ingredient in the form of droplets of a solution and / or suspension. Such formulations may be prepared, packaged, and / or sold as aqueous and / or dilute alcoholic solutions and / or suspensions, optionally sterile, comprising active ingredient, and may conveniently be administered using any nebulization and / or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and / or a preservative such as methylhydroxybenzoate. Droplets provided by this route of administration may have an average diameter in the range from about 1 nm to about 200 nm.

[0329] Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration is a coarse powder comprising the active1006313857108ingredient and having an average particle from about 0.2 pm to 500 pm. Such a formulation is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.

[0330] Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (wt / wt) and as much as 100% (wt / wt) of active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and / or lozenges made using conventional methods, and may, for example, 0.1% to 20% (wt / wt) active ingredient, the balance comprising an orally dissolvable and / or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and / or an aerosolized and / or atomized solution and / or suspension comprising active ingredient. Such powdered, aerosolized, and / or aerosolized formulations, when dispersed, may have an average particle and / or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.

[0331] A pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1 / 1.0% (wt / wt) solution and / or suspension of the active ingredient in an aqueous or oily liquid excipient. Such drops may further comprise buffering agents, salts, and / or one or more other of any additional ingredients described herein. Other ophthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and / or in a liposomal preparation. Ear drops and / or eye drops are contemplated as being within the scope of this present disclosure.Methods of Treating Diseases and Disorders

[0332] The lipid nanoparticles of the present invention may be useful for treating a disease, disorder, or condition. For example, such compositions may be useful in treating a disease, disorder, or condition characterized by missing or aberrant protein or polypeptide activity. As another example, the compositions may be useful in treating a disease, disorder, or condition responsive to an exogenous protein or polypeptide such1006313857109as a bacterial toxin molecule, a Cas protein, a zinc finger nuclease, TALENs or a tyrosine recombinase enzyme. As yet another example, the compositions may be useful for labelling or tracing the cells associated with a disease, disorder, or condition by delivering a nucleic acid encoding a fluorescent protein to the cell. For example, lipid nanoparticles each comprising an N or C-terminal portion of a nucleic acid, such as mRNA encoding a missing or aberrant polypeptide, may be administered or delivered to a cell. Subsequent translation of the reconstituted mRNA, or trans-splicing of the N and C-terminal portions of the polypeptide of interest may produce the full-length polypeptide or functional fragment thereof, thereby reducing or eliminating an issue caused by the absence of or aberrant activity caused by the polypeptide. Because translation may occur rapidly, the methods and compositions may be useful in the treatment of acute diseases, disorders, or conditions such as sepsis, stroke, and myocardial infarction. A nucleic acid included in a nanoparticle composition may also be capable of altering the rate of transcription of a given species, thereby affecting gene expression.

[0333] Diseases, disorders, and / or conditions characterized by dysfunctional or aberrant protein or polypeptide activity for which a composition may be administered include, but are not limited to, rare diseases, infectious diseases (as both vaccines and therapeutics), cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and renovascular diseases, and metabolic diseases. Multiple diseases, disorders, and / or conditions may be characterized by missing (or substantially diminished such that proper protein function does not occur) protein activity. Such proteins may not be present, or they may be essentially non-functional. A specific example of a dysfunctional protein is the missense mutation variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which produce a dysfunctional protein variant of CFTR protein, which causes cystic fibrosis.

[0334] The present disclosure provides a method for treating such diseases, disorders, and / or conditions in a subject by administering a lipid nanoparticle of the invention including a nucleic acid, including RNA, wherein the RNA may be an mRNA encoding a polypeptide that antagonizes or otherwise overcomes an aberrant protein activity present in the cell of the subject.1006313857110

[0335] The disclosure provides methods involving administering lipid nanoparticles including one or more nucleic acids and pharmaceutical compositions including the same. The terms therapeutic and prophylactic can be used interchangeably herein with respect to features and embodiments of the present disclosure. Therapeutic compositions, or imaging, diagnostic, or prophylactic compositions thereof, may be administered to a subject using any reasonable amount and any route of administration effective for preventing, treating, diagnosing, or imaging a disease, disorder, and / or condition and / or any other purpose. The specific amount administered to a given subject may vary depending on the species, age, and general condition of the subject; the purpose of the administration; the particular composition; the mode of administration; and the like. Compositions in accordance with the present disclosure may be formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of a lipid nanoparticle or pharmaceutical composition of the present disclosure will be decided by an attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or otherwise appropriate dose level (e.g., for imaging) for any particular patient will depend upon a variety of factors including the severity and identify of a disorder being treated, if any; the one or more nucleic acids employed; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific pharmaceutical composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific pharmaceutical composition employed; and like factors well known in the medical arts.

[0336] A lipid nanoparticle including one or more nucleic acids may be administered by any route. In some embodiments, compositions, including prophylactic, diagnostic, or imaging compositions including one or more lipid nanoparticles described herein, are administered by one or more of a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, trans- or intra-dermal, interdermal, rectal, intravaginal, intraperitoneal, intraocular, subretinal, intravitreal, topical (e.g. by powders, ointments, creams, gels, lotions, and / or drops), mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual, intranasal; by intratracheal instillation, bronchial instillation, and / or inhalation; as an oral spray and / or powder, nasal spray, and / or aerosol, and / or through a portal vein catheter. In some embodiments, a composition may be administered intravenously, intramuscularly,1006313857111intradermally, intra-arterially, intratumorally, subcutaneously, intraocularly, subretinally, intravitreally, or by inhalation. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the nanoparticle composition including one or more nucleic acids (e.g., its stability in various bodily environments such as the bloodstream and gastrointestinal tract), the condition of the patient (e.g., whether the patient is able to tolerate particular routes of administration), etc.

[0337] In certain embodiments, compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg / kg to about 10 mg / kg, from about 0.001 mg / kg to about 10 mg / kg, from about 0.005 mg / kg to about 10 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.05 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 10 mg / kg, from about 2 mg / kg to about 10 mg / kg, from about 5 mg / kg to about 10 mg / kg, from about 0.0001 mg / kg to about 5 mg / kg, from about 0.001 mg / kg to about 5 mg / kg, from about 0.005 mg / kg to about 5 mg / kg, from about 0.01 mg / kg to about 5 mg / kg, from about 0.05 mg / kg to about 5 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 1 mg / kg to about 5 mg / kg, from about 2 mg / kg to about 5 mg / kg, from about 0.0001 mg / kg to about 2.5 mg / kg, from about 0.001 mg / kg to about 2.5 mg / kg, from about 0.005 mg / kg to about 2.5 mg / kg, from about 0.01 mg / kg to about 2.5 mg / kg, from about 0.05 mg / kg to about 2.5 mg / kg, from about 0.1 mg / kg to about 2.5 mg / kg, from about 1 mg / kg to about 2.5 mg / kg, from about 2 mg / kg to about 2.5 mg / kg, from about 0.0001 mg / kg to about 1 mg / kg, from about 0.001 mg / kg to about 1 mg / kg, from about 0.005 mg / kg to about 1 mg / kg, from about 0.01 mg / kg to about 1 mg / kg, from about 0.05 mg / kg to about 1 mg / kg, from about 0.1 mg / kg to about 1 mg / kg, from about 0.0001 mg / kg to about 0.25 mg / kg, from about 0.001 mg / kg to about 0.25 mg / kg, from about 0.005 mg / kg to about 0.25 mg / kg, from about 0.01 mg / kg to about 0.25 mg / kg, from about 0.05 mg / kg to about 0.25 mg / kg, or from about 0.1 mg / kg to about 0.25 mg / kg of a nucleic acid in a given dose, where a dose of 1 mg / kg (mpk) provides 1 mg of a nucleic acid per 1 kg of subject body weight. In some embodiments, a dose of about 0.001 mg / kg to about 10 mg / kg of a nucleic acid of a nanoparticle composition may be administered. In other embodiments, a dose of about 0.005 mg / kg to about 2.5 mg / kg of a nucleic acid may be administered. In certain embodiments, a dose of about 0.1 mg / kg to about 1 mg / kg may be administered. In other embodiments, a dose of about 0.05 mg / kg to about 0.25 mg / kg may be administered. A dose may be1006313857112administered one or more times per day, in the same or a different amount, to obtain a desired level of mRNA expression and / or therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be delivered, for example, three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). In some embodiments, a single dose may be administered, for example, prior to or after a surgical procedure or in the instance of an acute disease, disorder, or condition.Examples

[0338] The following examples provide extensive experimental data directly supporting non-limiting embodiments of the disclosure including, but not limited to:• different intein pairs;• different proteins of interest;• proteins of interest fragmented into 1 or 2 pairs (i.e. a protein of interest in 2 or 3 fragments that can be reconstructed using inteins and lipid nanoparticles of the disclosure);• different molecules targeted on the specific target cell type;• different targeting molecules;• lipid nanoparticles with different lipid compositions; and• both in in vitro and in vivo efficacy.Example 1: Materials and methodsmRNA synthesis

[0339] All IVT mRNA were synthesized from a PCR template containing a T7 promoter upstream followed by the codon optimized ORF. All constructs contain a 5’IITR, 3’IITR and 125 polyA tail. All mRNA was transcribed using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs). Capping was performed cotranscriptionally using CleanCap Reagent AG (TriLink Biotechnologies). All uridine was replaced with N1-Methylpseudouridine (TriLink Biotechnologies). IVT reaction was treated with DNase to eliminate the template and dsRNA was removed by cellulose1006313857113clean-up methods (Baiersddrfer M et al., Molecular Therapy-Nucleic Acids, 2019, 15:26-35). The final product was purified by sodium acetate precipitation.LNP formulation

[0340] A lipid mixture consisting of ionizable lipids (e.g., SM102, ALC-0366 or ALC-0315), DSPC (Avanti Polar Lipids), cholesterol (Sigma) and 18:0 PEG2000 PE (DSPE-PEG2000) (Avanti Polar Lipids) was prepared in ethanol as a 20 mM stock. The molar composition used was 50:10:38.5:1.5 molar ratio. The lipid solution was mixed by flowing through a micro fluidic mixing device Nanoassemblr® (Precision Nanosystems, Vancouver BC) with an aqueous mRNA solution in 10 mM citrate buffer (pH 4) at a 1 :3 organic to aqueous volume ratio, 4 ml per min total flow rate. The resulting LNPs were then diluted twice with PBS (pH 7.4) immediately and further dialysed overnight. Then next day, LNPs were filtered through a 0.22 micron filter.Targeting antibody

[0341] Targeting antibodies used in in vitro and ex vivo studies include mouse anti-hCD5 (UCHT2, Thermo Fisher), mouse anti-hCD7 (124-1 D1, ThermoFisher), mouse anti-human TfR (Clone OKT9), and mouse anti-hCD22 (eBio4KB128 (4KB128), Thermo Fisher). Targeting antibodies used in in vivo studies include mouse anti-mouse CD3E (QA17A05, Biolegend), mouse anti-mouse CD4 (YTS.191.1, absolute antibody), rabbit anti-mouse CD5 (YTS.121.5.2, absolute antibody), mouse anti-mouse CD8a (VHHCD8, absolute antibody), and rat anti-mouse TCRbeta (QA18A18, Biolegend).Cell culture maintenance

[0342] LoxP-STOP-LoxP-mScarlet-l HEK cells were maintained in DMEM media, and Jurkat cells were maintained with RPMI media (Gibco). Both supplied with 10% fetal bovine serum and penicillin-streptomycin (100 U / mL). Cells were cultured at 37 °C in a humidified incubator with 5% atmospheric CO2 along with routine testing or mycoplasma contamination.Mouse models

[0343] Male and female B6.Cg-Gf(ROSA)26SomM^G fd7’omafo^ze / J mice (Ai14 mice, Jackson Laboratory, Bar Harbor, ME) were procured and maintained under specific-pathogen-free (SPF) conditions at the Monash Animal Research Platform (MARP).1006313857114C57BL / 6J mice (hereafter C57BL / 6) were sourced directly from MARP and acquired on-demand for experiments. Mice aged 6-14 weeks were housed in ventilated cages under standardized conditions (22 ± 1°C, 12-hour light / dark cycle). All experimental protocols were approved by the Monash University Institutional Animal Care and Use Committee. Human PBMC collection and purification

[0344] Healthy donors aged between 18-50 years old of both sexes were recruited voluntarily after an invitation to participate. The ethics is approved by Monash University Human Research Ethics Committee. 10-30 mL of heparinised human blood was collected and diluted with PBS before carefully layered on Ficoll-Paque PLUS density gradient media with 1:1 v / v. PBMC layer was collected after 400g, 40 mins spin and washed with prewarmed RPMI media twice. PBMCs were either used for experiments or frozen in cell frozen media at -80oC.Cells association and transfection assay with functionalized LNPs

[0345] To assess the delivery and depletion efficiency of functionalized lipid nanoparticles (LNPs) in Jurkat cells, approximately 50,000 cells were added to individual wells in a 96-well plate. A final LNP concentration equivalent to 0.5 ng / pL of mRNA (total amount) was added to the cells and incubated at 37°C overnight.Subsequently, cells were washed three times with 2% FBS-PBS following centrifugation at 400x g for 5 minutes. Cells were then resuspended in 80 pL of 2% FBS-PBS and stained with Live / Dead dye, and live cell population was quantified using a Stratedigm S1000EXi flow cytometer.Human PBMC association and transfection assay with functionalized LNPs

[0346] To assess the delivery and depletion efficiency of functionalized lipid nanoparticles (LNPs), approximately 500,000 PBMC were added to individual wells in a 96-well plate with functionalized LNPs equivalent to a final mRNA concentration of 2ng / pL. Then cells were incubated at 37°C for 24 hours. PBMC then were washed thrice with 2% FBS-PBS after centrifugation at 400x g for 5 minutes. To phenotype the sub-populations, cells were stained against CD3-PE mAb (clone OKT3, Biolegend), CD4-BV510 mAb (clone OKT4, Biolegend), CD8-BV786 mAb (clone SK1, Biolegend), CD19-BV421 mAb (clone HIB19, Biolegend), CD56-BV605 mAb (clone 5.1H11, Biolegend), and viability dye (eBioscience™ Fixable Viability Dye eFluor™ 780,1006313857115Thermofisher) on ice for 30 min. Antibodies were all used at 1:200 dilutions with Human TruStain FcX™ (Biolegend) according to the manufacturer’s protocol. After washing away the excessive antibody, cells were resuspended with 100 pL 2% FBS-PBS for the flow analysis (Cytek Aurora 5 lasers full spectrum cytometer). Cells were identified by a combination of surface markers: CD4+ T cells (CD3+ and CD4+), CD8+ T cells (CD3+ and CD8+), NK cells (CD3-, CD19-, CD14- and CD56+), and B cells (CD3- and CD19+). The absolute cell count were calculated with CountBrite beads and following its manufacturing protocols.In vivo assessment of split targeting of LN Ps to T cells cross multiple organs

[0347] Ai 14 or C57BL6 mice were injected intravenously with unmodified lipid nanoparticles (LNPs), targeted LNPs, or isotype control LNPs loaded with Cre mRNA. Isotype control antibodies used in the Examples were purchased from ThermoFisher Scientific (eg, clone P3.6.2.8.1). After 48 hours, blood was collected via cardiac puncture, and the mice underwent transcardiac perfusion with PBS to remove circulating blood. Red blood cells were lysed using ammonium-chloride-potassium (ACK) buffer (Thermo Fisher, USA) at a 1:10 (v / v) ratio twice, followed by washing with 2% FBS-PBS. The liver, spleen, and lymph nodes (inguinal, iliac, and cervical) were collected and processed as follows:

[0348] The liver was minced and digested using a gentleMACS™ dissociator with 2.8 mg / mL Collagenase H and 0.28 mg / mL DNase. The digested mixture was filtered to remove undigested material and subjected to a slow spin at 60g. The supernatant was collected and spun down to collect the pellet. The pellet was resuspended in 30% Percoll media and spun to remove hepatocytes, followed by resuspension with ACK lysis buffer and washing with 2% FBS HBSS before antibody staining.

[0349] The spleen was minced with 1 mg / mL Collagenase III and 0.28 mg / mL DNase and digested by constant gentle mixing until fully digested. Cells were filtered and red blood cells lysed using ACK buffer.

[0350] Lymph nodes were collected and homogenized by passing through a 0.45 pm filter. Dissociated cells were collected and washed with media.

[0351] All immune cell pellets were stained with a flow cytometry panel containing the following antibodies: CD3e-BV650 mAb (clone 145-2C11, BD Biosciences), CD90.2-1006313857116BV650 mAb (clone 53-2.1, BD Biosciences), CD4-APC-Cy7 mAb (clone GK1.5, BioLegend), CD8-BV711 mAb (clone 53-6.7, BD Biosciences), CD19-BV786 mAb (clone 1D3, BD Biosciences), CD49b-FITC mAb (clone HMa2, BioLegend), CD11b-BV421 mAb (clone M1 / 70, BioLegend), Ly-6C-BUV661 mAb (clone HK1.4.rMAb, BD Biosciences), Ly-6G-BV605 mAb (clone 1A8, BD Biosciences), CD45-Pacific Blue mAb (clone S18009F, BioLegend), I-A / I-E-BV510 mAb (clone M5 / 114.15.2, BioLegend), F4 / 80-PE / Dazzle mAb (clone BM8, BioLegend), and CD11c-Alexa Fluor 700 mAb (clone N418, BioLegend). Additionally, Mouse BD Fc Block™ and viability dye (LIVE / DEAD™ Fixable Blue Dead Cell Stain Kit, Thermo Fisher) were included.Samples were incubated on ice for 30 minutes, followed by washing to remove excess antibody.

[0352] Flow cytometry was performed using a Cytek Aurora 5 laser cytometer, and data were analyzed using FlowJo (BD Biosciences). Leukocyte phenotyping was conducted using the following markers: CD4+ T cells (CD45+, CD11b-, CD3e+, CD4+); CD8+ T cells (CD45+, CD11b-, CD3e+, CD8+); dendritic cells (CD45+, CD3e-, CD19-, CD11c+, MHCII+); monocytes (CD45+, CD11b+, Ly6C+, Ly6G-); neutrophils (CD45+ CD11b+, Ly6C+, Ly6G+); NK cells (CD45+ CD11b+, Ly6C low, Ly6G-, F4 / 80+, CD49b+); macrophages (CD45+ CD11b+, Ly6C low, Ly6G-, F4 / 80+, SSA low); and CD19+ B cells (CD45+, CD11b-, CD3-, CD19+ or MHCII+).Tissue sample preparation, sectioning and imaging

[0353] Liver and spleen tissues were harvested at the experimental endpoint and immediately immersed in freshly prepared 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS; pH 7.4) for 24 hours at 4°C to ensure complete fixation. Fixed tissues were subsequently rinsed three times with PBS to remove residual PFA.Tissues were sequentially incubated in 15% sucrose (w / v in PBS) for 24 hours, followed by 30% sucrose (w / v in PBS) for an additional 24 hours at 4°C. After cryoprotection, tissues were trimmed to optimal dimensions, rinsed in PBS to remove excess sucrose, and embedded in cryoembedding molds (1.5 x 1.5 cm) using optimal cutting temperature (OCT) compound (Tissue-Tek). Embedded blocks were rapidly frozen in a dry ice-isopentane slurry and stored at -80°C until sectioning. Frozen tissues were sectioned at a thickness of 20 pm using a cryostat (Leica CM 1950) maintained at -20°C. Sections were mounted onto Superfrost Plus adhesion slides (Thermo Fisher Scientific) and stored at -80°C until staining. For CD3+ T-cell visualization, tissue1006313857117sections were permeabilized with 0.3% Triton X-100 in PBS for 30 minutes at room temperature (RT) and blocked with 5% normal donkey serum (NDS) in PBS for 1 hour. Sections were incubated with a primary antibody targeting CD3 (rat anti-mouse CD3E, clone 17A2; 1:200 dilution in PBS containing 1% NDS and 0.1% Triton X-100) for 16-24 hours at 4°C. After three PBS washes, sections were incubated with a fluorophore-conjugated secondary antibody (donkey anti-rat IgG Alexa Fluor 647; 1:200 dilution in PBS with 1% NDS) for 2 hours at RT. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; 1 pg / mL) for 5 minutes. Finally, sections were coverslipped using Dako Folurescence Mounting Medium (Dako). Fluorescent images were acquired using a Leica DMi8 inverted widefield microscope equipped with a *20 / 0.75 NA objective and a scientific CMOS camera (Leica DFC9000 GT), controlled by LAS AF software (version 3.5.5). Excitation / emission wavelengths were configured as follows: 405 nm / 490 nm (DAPI), 550 nm / 600 nm (tdTomato), and 600 nm / 700 nm (Alexa 647). Image dimensions were standardized to 665.60 * 665.60 pm per field.Image Analysis

[0354] Raw images were processed using Imaged (Fiji distribution, NIH) for contrast adjustment, and channel alignment. Quantification of tdTomato-positive hepatocytes cells was performed using CellProfiler 4.2.1 with a customized pipeline: nuclei were identified via DAPI signal (threshold: 0.1-1.0 intensity), and tdTomato-positive cells were defined as cells with tdTomato signal objects (threshold: 0.2-1.0 intensity) colocalized with nuclear staining. Data were presented in GraphPad Prism 9.0.Statistical analysis

[0355] Data are presented as mean ± standard deviation based on the data obtained from at least n = 3 independent experiments or well or mice. Statistical significance was determined using GraphPad Prism 9.0.Example 2: SpLiT mRNA-LNP technology enhances the specific mRNA delivery to cells and eliminates the non-specific expression of the cargo

[0356] The inventors have developed a technology as an innovative way to deliver messenger RNA to targeted cells with limited off-targeting effect. For example, the inventors developed an approach that involves delivering a single mRNA in two or more segments, incorporating additional intein sequences at the N or C termini of the mRNA1006313857118sequence, referred to as mRNA_N and mRNA_C. As used in this example, “SpLiT” means that the mRNA was delivered with split mRNAs (ie mRNA_N and mRNA_C). Upon co-delivery of mRNA_N and mRNA_C to the same cells via different targeted LNPs, translation results in protein_N and protein_C, which subsequently undergo intein-mediated splicing to form the full-length, active protein. Conversely, since nonspecific cells receive likely only one mRNA segment (either N or C), the resulting protein_N or protein_C remains inactive. Coupled with a precise and efficient targeted LNP delivery system, this method allows for control over cargo delivery to target cells expressing the appropriate surface receptors, significantly minimising non-specific mRNA delivery. A schematic diagram showing one exemplary embodiment of the present invention is provided in Figure 1.

[0357] As an example, protein and nucleic acid sequences used in this study are outlined in Tables 2 and 3.

[0358] LoxP-STOP-LoxP-mScarlet-l HEK cells were transfected with either Cre_C, Cre_N, Cre or Cre_SpLiT mRNA for 24 hours. About 70% of the cell population were expressing mScarlet-l upon Cre_SpLiT transfection (Figure 2), indicating that Cre_SpLiT was successfully translated and underwent intein-mediated splicing to form an active Cre recombinant protein.

[0359] As another example, the inventors have split the mRNA of diphtheria toxin unit A (Tables 2 and 3). Diphtheria toxin is a bacterial protein composed of units A and B. Unit B facilitates receptor binding and internalization into target cells, while unit A inhibits protein synthesis, leading to the death of susceptible cells. Here, the inventors replaced the entry route of this mechanism with targeted lipid nanoparticle (LNP) technology to deliver either the full-length diphtheria toxin unit A (DTA) or its split version.

[0360] Jurkat cells, a human T lymphocyte cell line expressing CD5 and CD7 receptors on the surface, were treated with either untargeted DTA LNP or untargeted split DTA. Toxicity was observed only at relatively high concentrations (1 ng-0.1 ng / pL). By targeting either CD5 or CD7 with full-length DTA, toxicity was observed at concentrations as low as 0.0001 ng / pL, which is 10,000 times lower as compared to the controls (Figure 3). When cells were treated with split DTA in CD5 and CD7 targeted LNPs (each particle containing one part of the sequence), cell viability was reduced at10063138571190.003 ng / pL. This demonstrates that the split targeting system can deliver functional protein to the cells and exhibits higher efficiency as compared to the untargeted system.

[0361] Next, targeted depletion in human peripheral blood mononuclear cell (PBMC) models was examined. PBMCs contain multiple immune cell types and represent a more physiologically relevant environment. Delivery of full DTA with either CD5 or CD7 targeting LNPs reduced the percentage of T cells, as T cells naturally express both receptors (Figure 4a and b). However, a reduction in NK cells was observed with CD7-targeted LNPs due to unintended depletion, as CD7 is also expressed by NK cells (Figure 4c). This unwanted effect was circumvented by delivering split DTA with a dual targeting profile. No reduction in NK cells was observed when PBMC samples were treated with T cell-targeted split DTA (Figure 4c), while T cell populations were depleted (Figure 4a and b). This advantage is considered to arise from the high selectivity of the targeted delivery system, where the cargos only function when both parts have recombined within a cell.

[0362] The same experiments were conducted with CD5 / CD7 targeted LNPs containing one part of the DTAJSpLiT mRNA, further demonstrating that the depletion of targeted T cells population was achieved by delivering both parts of DTA_SpLiT mRNA via two different receptors on T cells (Figure 5). These results indicates that SpLiT targeting system enhanced the precision of the targeting delivery by splitting the targeting mRNA into two targeted LNP unit with unique targeting property thereby avoided unwanted, non-specific protein expression.

[0363] To expand the range of targetable cell populations, the inventors have demonstrated the effective depletion of B cells using CD22-targeted LNPs with either full-length DTA or DTA_SpLiT (Figure 6).

[0364] In vivo, hepatocytes are considered one of the primary destinations for LNPs due to the high level of LDL receptor expression on their surface. CD3-targeted LNPs exhibit superior ability to deliver mRNA to T cells in vivo via a receptor-mediated mechanism; however, substantial non-specific mRNA delivery was detected in hepatocytes (Figure 7a and b). Delivering Cre_SpLiT mRNA via two CD3-targeted LNPs resulted in negligible off-target effects in hepatocytes while retaining targeting properties towards T cells (Figure 7c and d). Further quantification analysis showed that more than 80% and 60% hepatocytes showed expression of tdTomato in1006313857120untargeted LNP and CD3 targeted LNP treated mice, respectively (Figure 8a and b). In contrast, below 1% hepatocytes was tdTomato-positive in mice dosed with Cre_SpLiT (Figure 8c).

[0365] In the spleen, tdTomato expression after dosing with untargeted LNPs showed a distinct distribution pattern on CD3 staining in other words a non-specific distribution pattern as compared to the corresponding CD3 staining (Figure 7g), while CD3-targeted LNPs with full Cre showed clear expression in CD3-positive T cells in other words colocalisation of tdTomato expression and CD3 staining (Figure 7h). However, some CD3-negative cells also exhibited strong tdTomato expression (Figure 7h, square). This off-target expression was largely reduced when samples were dosed with Cre_SpLiT with CD3 targeting (Figure 7i and j).

[0366] To further analyse the efficiency and specificity of Cre_SpLiT mRNA delivery, multicolor flow cytometry on the isolated immune cells was performed (Figure 9). The antibody cocktail used for phenotyping included markers for T cells, B cells, NK cells, and other immune cell subsets. The results demonstrated a significant increase in the percentage of tdTomato-positive T cells in the spleen, liver, and blood, indicating successful transfection and expression of Cre recombinase in T cells. Importantly, despite this significant reduction in hepatocyte delivery, T cells still expressed the tdTomato signal.

[0367] Additionally, the data showed that the use of CD3-targeted LNPs with Cre_SpLiT mRNA minimised off-target effects in non-T cell populations, such as hepatocytes and CD3-negative immune cells. This high specificity and efficiency of the Cre_SpLiT system highlight its potential for targeted gene delivery and therapeutic applications.

[0368] Next, the ability of the DTA_SpLiT system to specifically deplete T cells in vivo without inducing systemic cell apoptosis was investigated. Mice were administered intraperitoneally with 0.05 mg / kg of either CD3-targeted lipid nanoparticles (LNPs) containing control mRNA, CD3-targeted LNPs with full-length DTA, or CD3-targeted LNPs with DTA_SpLiT. After 24 hours, spleens were harvested, and absolute cell counts were assessed using multi-color flow cytometry (Figure 10).1006313857121

[0369] The results demonstrated that mice treated with DTA_SpLiT-targeted T cells exhibited a significant decrease in T cells and B cells, while dendritic cells and other cell types remained unaffected. This indicates that the DTA_SpLiT system can selectively target and deplete T cells without impacting other immune cells.

[0370] In contrast, the group receiving CD3-targeted LNPs with full-length DTA showed a significant reduction in all major immune cell types, highlighting the nonspecific nature of full-length DTA delivery. Additionally, systemic damage was observed in the liver and gut of these mice, leading to shortened survival (Figure 11a), suggesting that even targeted delivery of full-length DTA is not a viable strategy due to its non-specific effects.

[0371] To further assess the safety of the DTA_SpLiT system, mice that received DTA_SpLiT were monitored for an additional four days to ensure no late-onset damage to the liver or other major organs occurred. These mice showed no obvious weight loss nor decreased activity (Figure 11b), indicating that the SpLiT-DTA system did not induce delayed toxicity.

[0372] By splitting the mRNA and delivering them separately, the inventors achieved targeted depletion of T cells without inducing non-specific toxicity. This result indicates that the targeted LNP with the SpLiT system significantly reduces non-specific delivery and side effects of mRNA-LNPs without compromising therapeutic efficacy. This approach showcases a promising strategy for achieving precise immune cell modulation with minimal off-target effects, paving the way for safer and more effective therapeutic interventions.Example 3: SpLiT mRNA-LNP technology with the gp41-8 intein pair

[0373] To further demonstrate that the cargo can be divided using any intein pair (e.g., those listed in Table 2), DTA was split and fused to the gp41-8 intein pair (SEQ ID NOs: 33-34), producing DTA_N_gp41-8_N and gp41-8_C_DTA_C constructs (SEQ ID NOs: 191-192, encoded by SEQ ID NOs: 201-202). Targeted LNPs were formulated containing either half of the split DTA cargo. Jurkat cells were then treated for 24 hours with CD5 / CD7-targeted gp41-8 split-DTA LNPs or an isotype-control gp41-8 split-DTA LNP. Cell viability was quantified by flow cytometry.1006313857122

[0374] Consistent with prior observations using the Npu intein, the gp41-8 split-DTA system exhibited functional activity only when both DTA halves were delivered via the targeted LNPs, whereas cells receiving both halves through isotype-control LNPs showed minimal cytotoxicity (Figure 12). This supports the broader applicability of multiple intein pairs for split-cargo reconstitution in targeted delivery systems.Example 4: SpLiT mRNA-LNP technology with the M86 intein pair

[0375] As another example, mScarlet-l was split and fused to the M86 intein pair, producing mScarlet_N_M86_N and M86_C_mScarlet_C constructs (SEQ ID NOs: 199-200, encoded by SEQ ID NOs: 209-210). Untargeted or targeted LNPs were formulated containing either half of the split mScarlet cargo. Jurkat cells were then treated for 24 hours with CD5 / CD7-targeted M86 split-mScarlet LNPs, isotype-control M86 split-mScarlet LNPs or untargeted M86 split mScarlet LNPs. The expression level of mScarlet was measured via flow cytometry. Consistent with split DTA, split mScarlet with M86 intein recombined and exhibited fluorescence only when both pairs were delivered to cells with targeted LNPs (Figure 13).

[0376] Again, the data support the broader applicability of multiple intein pairs for split-cargo reconstitution in targeted delivery systems.Example 5: Number of nucleic acid fragments and inteins

[0377] To further demonstrate the potential to split the cargo, in order to further specify the target cell population, an additional example employing model protein mScarlet was generated. mScarlet was split into three fragments and fused to intein pairs Mja-KIbA and Npu, as mScarlet_1_Mja-KlbA_N, Mja-Klba_C_mScarlet_2_NpuN and NpuC_mScarlet_3 (SEQ ID NOs: 196-198, encoded by SEQ ID NOs: 206-208). Untargeted or targeted LNPs were formulated containing one of the split fragments. Jurkat cells were treated with either CD5 / CD7 / TfR split mScarlet or isotype control split mScarlet for 24 hours. The percentage of mScarlet positive cells were measured by flow cytometry.

[0378] As shown in Figure 14, cells produced mScarlet only when all three mRNA fragments were delivered together via targeted LNPs (tLNPs), confirming successful multi-part reassembly. By using antibody against three different surface receptors, the inventors further demonstrated the potential to direct expression into distinct sub-1006313857123populations of cells. The inventors anticipate that further fragments can be incorporated via similar designs.Example 6: In vivo efficacy

[0379] To demonstrate that the split-mRNA delivery system enables cargo expression specifically within cell populations defined by their surface-receptor combinations, a series of in vivo studies were performed using multiple cargos, targeting pairs, and immune cell subsets.Split Cre recombinase

[0380] Cre mRNA was divided into two fragments (Cre_N and Cre_C), and each half was separately encapsulated into targeted LNPs formulated as described above.Distinct receptor combinations were selected to define specific T-cell subsets, including CD3 / TCRbeta for pan T cells, CD3 / CD4 for CD4+T cells, CD3 / CD8 for CD8+T cells, along with corresponding isotype-control formulations. Each targeted LNP contained only one half of the split Cre cargo. tdTomato reporter mice were dosed at 0.5 mg / kg for 48 hours, after which immune cells from spleen were isolated. tdTomato-positive cells were quantified by multicolour flow cytometry as a readout of Cre-mediated recombination (Figure 15).

[0381] Across all targeting pairs, tdTomato activation was confined to the receptor-defined immune subsets. In the spleen, CD3 / TCRp-targeted LNPs induced tdTomato expression in 44% of CD4+T cells and 38% of CD8+T cells, consistent with broad pan-T-cell targeting. CD3 / CD4-targeted LNPs yielded 41% tdTomato-positive CD4+T cells with only minimal tdTomato+CD8+cells (<5%). Conversely, CD3 / CD8-targeted LNPs produced -28% tdTomato-positive CD8+T cells, again with minimal expression in the CD4+compartment (<5%). Non-targeted and isotype-control LNPs generated only background activation in T-cell subsets (<5%).

[0382] In contrast, non-targeted LNPs drove substantial tdTomato activation in irrelevant immune populations. Non-targeted formulations also produced 27% tdTomato-positive monocytes, whereas targeted formulations showed only minimal monocyte activation (5-10%).1006313857124

[0383] Together, these results show that targeted LNPs delivering split Cre mediate functional recombination almost exclusively within the intended cell population, underscoring that cell-type-specific delivery is determined by the selected surfacereceptor combination.Split mScarlet

[0384] mScarlet mRNA was divided into two fragments (mScarlet_N and mScarlet_C), and each half was separately formulated into targeted LNPs. Targeting pairs included CD3 / TCRP for pan-T-cell delivery and CD3 / CD5 for CD5+T cells, alongside lgG1 isotype controls. Each LNP formulation carried only one half of the split mScarlet mRNA. Wild-type mice were administered 1 mg / kg total mRNA intravenously. Immune cells from spleen were collected, and mScarlet-positive populations were quantified by multicolour flow cytometry.

[0385] For the split mScarlet studies, functional activation aligned closely with the receptor-defined targeting scheme (Figure 16). CD3 / TCRp-targeted LNPs produced strong mScarlet expression across the T-cell compartment, with 18% mScarlet-positive CD4+T cells and 17% mScarlet-positive CD8+T cells, consistent with broad pan-T-cell targeting. CD3 / CD5- targeted LNPs yielded 12 - 15 % mScarlet-positive cells within the T cell population, with only minimal expression in CD5“ cells (<5%). In contrast, lgG1 isotype-control LNPs induced only background activation across all T-cell subsets (<5%), consistent with the split cargo not being co-delivered to the correct receptor-defined population.

[0386] Examples of representative flow cytometry plots are shown in Figure 17 to illustrate mScarlet-positive cells within each treatment group. The CD4 versus CD8a gating strategy is presented in Figure 17a. Four immune populations are displayed as examples: CD4+T cells (panels b, f, j, n), CD8+T cells (c, g, k, o), macrophages (d, h, I, p), and dendritic cells (e, i, m, q). Treatment groups are organised as follows:CD3 / TCRp-targeted tLNPs (b-e), CD3 / CD5-targeted tLNPs (f-i), lgG1 isotype controls (j-m), and PBS controls (n-q).

[0387] To assess targeted delivery using the split-mRNA LNP platform, split DTA was formulated into CD3 / CD4- targeted tLNPs and administered intravenously to wild-type mice at 0.05 mg / kg. After 24 hours, spleens were harvested and immune cell subsets1006313857125were quantified using absolute counting beads. Delivery of split DTA via the CD3 / CD4-targeted tLNPs resulted in a selective reduction of CD4+T-cell numbers compared with lgG1 isotype and PBS controls, whereas no depletion was observed in other immune populations, including CD8+T cells, B cells, macrophages, monocytes, neutrophils, and dendritic cells (Figure 17).Discussion

[0388] Across the three cargos, Cre, mScarlet, and DTA, the targeted LNP platform consistently directed functional activity to the intended immune cell population only when both halves of the split mRNA were co-delivered to the same receptor-defined subset. By selecting different surface-receptor pairs, including CD3 / TCRP, CD3 / CD4, CD3 / CD5, and CD3 / CD8, the system enabled precise and programmable targeting of distinct T-cell populations in vivo. Split Cre and split mScarlet showed receptordependent activation with minimal off-target expression, while split DTA induced selective depletion specifically within the CD4+T-cell compartment when delivered via the CD3 / CD4 combination.

[0389] Together, these results demonstrate that the split-mRNA tLNP platform provides a flexible and modular approach to cell-specific delivery, where functional reconstitution occurs only when both mRNA halves are delivered to the same receptor-defined immune cell subset, and where the target population can be deliberately chosen by selecting the corresponding surface-receptor combination.Example 7: LNP type

[0390] To further demonstrate that the split-mRNA delivery system is compatible with multiple lipid nanoparticle formulations, split mScarlet constructs were encapsulated into LNPs formulated with either ALC-0366 or ALC-0315 ionizable lipids. The resulting particles were functionalised with either CD5 / CD7-targeting moieties or isotype-control ligands. Jurkat cells were treated for 24 hours with targeted or isotype-control split-mScarlet LNPs, and the percentage of mScarlet-positive cells and corresponding mean fluorescence intensity were quantified by flow cytometry.

[0391] Consistent with earlier examples formulated using SM-102, targeted LNPs delivering both halves of the split mScarlet transcript achieved successful transfection and functional mScarlet expression in Jurkat cells (Figure 18). The magnitude of1006313857126expression was primarily determined by the intrinsic delivery efficiency of the ionizable lipid employed, which may be adjusted in order to achieve a desirable expression magnitude.

Claims

1006313857127CLAIMS1. A lipid nanoparticle comprising:(a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide, and (b) a targeting molecule, ora capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle; or(a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate N-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle.

2. A lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding both an N and a C-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate N-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle.

3. A composition comprising:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle, - a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle, and1006313857128- optionally one or more further lipid nanoparticles.

4. The composition of claim 3, further comprising one or more further lipid nanoparticles, wherein the one or more further lipid nanoparticles comprise (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding both an N and a C-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate N-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle.

5. The composition of claim 3 or 4, wherein the one or more further lipid nanoparticles comprise:- a third lipid nanoparticle;- a third and fourth lipid nanoparticle;- a third, fourth and fifth lipid nanoparticle;- a third, fourth, fifth and sixth lipid nanoparticle;- a third, fourth, fifth, sixth and seventh lipid nanoparticle;- a third, fourth, fifth, sixth, seventh and eighth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth and ninth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth and tenth lipid nanoparticle; - a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and eleventh lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh and twelfth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and fourteenth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth and fifteenth lipid nanoparticle; or- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth and sixteenth lipid nanoparticle;wherein each lipid nanoparticle comprises a nucleotide sequence encoding a portion of the polypeptide of interest, such that full-length polypeptide of interest is1006313857129encoded by the totality of each nucleotide sequence encoding each portion of the polypeptide of interest.

6. The lipid nanoparticle of claim 1 or 2, or the composition of any one of claims 3 to 5, wherein the polypeptide capable of mediating irreversible conjugation is a SpyTag or SpyCatcher.

7. The lipid nanoparticle of claim 1 or 2, or the composition of any one of claims 3 to 5, wherein the polypeptide capable of mediating irreversible conjugation is capable of trans-splicing.

8. The lipid nanoparticle or composition of claim 7, wherein the polypeptide capable of mediating irreversible conjugation is an intein.

9. The lipid nanoparticle or composition of claim 8, wherein the nucleotide sequence encoding an N-terminal portion of a polypeptide of interest is linked or fused to the nucleotide sequence encoding an N-terminal intein.

10. The lipid nanoparticle or composition of claim 8 or 9, wherein the nucleotide sequence encoding a C-terminal portion of a polypeptide of interest is linked or fused to the nucleotide sequence encoding a C-terminal intein.

11. A lipid nanoparticle or composition of any one of claims 8 to 10, wherein the nucleotide sequence encoding the N-terminal intein is arranged relative to the nucleotide sequence encoding the N-terminal portion of the polypeptide of interest such that when the N-terminal intein and N-terminal portion of the polypeptide of interest protein is in the presence of a C-terminal polypeptide of the gene of interest and a C-terminal intein, the inteins excise themselves resulting in the N and C terminal polypeptides linked covalently via a peptide bond.

12. A lipid nanoparticle or composition of any one of claims 8 to 11 , wherein the nucleotide sequence encoding the C-terminal intein is arranged relative to the nucleotide sequence encoding the C-terminal portion of the polypeptide of interest such that when the C-terminal intein and C-terminal portion of the polypeptide of interest protein is in the presence of an N-terminal polypeptide of the gene of interest and an N-terminal intein, the inteins excise themselves resulting in the N and C terminal polypeptides linked covalently via a peptide bond.100631385713013. A method for expressing a polypeptide of interest in a target cell or tissue in a subject, the method comprising administering to the subject:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle, - a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle, and- optionally one or more further lipid nanoparticles,thereby expressing a polypeptide of interest in the target cell or tissue in the subject.

14. The method of claim 13, further comprising administering to the subject one or more further lipid nanoparticles, wherein the one or more further lipid nanoparticles comprise (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding both an N and a C-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate N-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle,15. The method of claim 13 or 14, wherein the first lipid nanoparticle, second lipid nanoparticle and optionally one or more further lipid nanoparticles are administered separately, for example sequentially or concurrently but by distinct administrations (e.g. either separate routes of administration or the same route but separate administrations); or wherein the first lipid nanoparticle, second lipid nanoparticle and optionally one or more further lipid nanoparticles are administered concurrently (e.g. either same routes of administration and / or in the same administration).100631385713116. The method of any one of claims 13 to 15, wherein the first lipid nanoparticle, the second lipid nanoparticle and optionally one or more further lipid nanoparticles are each administered in a composition.

17. Use of:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle, in the manufacture of a medicament for expressing a polypeptide of interest in a target cell or tissue in a subject, wherein the medicament is to be administered with:- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle, and- optionally one or more further lipid nanoparticles.

18. Use of:- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle,in the manufacture of a medicament for expressing a polypeptide of interest in a target cell or tissue in a subject, wherein the medicament is to be administered with:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing1006313857132a targeting molecule, displayed on the outer surface of the first lipid nanoparticle, and- optionally one or more further lipid nanoparticles.

19. The use of claim 17 or 18, wherein the medicament is to be administered with one or more further lipid nanoparticles, and wherein the one or more further lipid nanoparticles comprise (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding both an N and a C-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate N-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle.

20. Use of:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle, and- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle, and- optionally one or more further lipid nanoparticles,in the manufacture of a medicament for expressing a polypeptide of interest in a target cell or tissue in a subject.

21. Use of:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal polypeptide capable of mediating1006313857133irreversible conjugation in the presence of a cognate C-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle, and- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle, and- one or more further lipid nanoparticles,in the manufacture of a medicament for expressing a polypeptide of interest in a target cell or tissue in a subject.

22. A first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle, for use in expressing a polypeptide of interest in a target cell or tissue in a subject, wherein the first lipid nanoparticle is to be administered with:- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle, and- optionally one or more further lipid nanoparticles.

23. Use of a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle, for use in expressing a polypeptide of interest1006313857134in a target cell or tissue in a subject, wherein the second nanoparticle is to be administered with:- a first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle, and- optionally one or more further lipid nanoparticles.

24. The use of claim 22 or 23, wherein the first or second nanoparticle is to be administered with one or more further lipid nanoparticles, and wherein the one or more further lipid nanoparticles comprise (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding both an N and a C-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate N-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle.

25. A first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle,a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle, andoptionally one or more further lipid nanoparticles,for use in expressing a polypeptide of interest in a target cell or tissue in a subject.100631385713526. A first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal polypeptide capable of mediating irreversible conjugation in the presence of a cognate C-terminal polypeptide, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the first lipid nanoparticle,a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the second lipid nanoparticle, andone or more further lipid nanoparticles,for use in expressing a polypeptide of interest in a target cell or tissue in a subject.

27. The method, use or lipid nanoparticle of any one of claims 13-26, wherein the one or more further lipid nanoparticles comprise:- a third lipid nanoparticle;- a third and fourth lipid nanoparticle;- a third, fourth and fifth lipid nanoparticle;- a third, fourth, fifth and sixth lipid nanoparticle;- a third, fourth, fifth, sixth and seventh lipid nanoparticle;- a third, fourth, fifth, sixth, seventh and eighth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth and ninth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth and tenth lipid nanoparticle; - a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and eleventh lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh and twelfth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and fourteenth lipid nanoparticle;1006313857136- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth and fifteenth lipid nanoparticle; or- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth and sixteenth lipid nanoparticle;wherein each lipid nanoparticle comprises a nucleotide sequence encoding a portion of the polypeptide of interest, such that full-length polypeptide of interest is encoded by the totality of each nucleotide sequence encoding each portion of the polypeptide of interest.

28. A lipid nanoparticle comprising:(a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence encoding a cognate sequence capable of mediating reconstitution, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle; or(a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the first sequence, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle.

29. A lipid nanoparticle comprising (a) a nucleic acid comprising (i) a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the first sequence, and (ii) a second nucleotide sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the second sequence, and (b) a targeting molecule, or a capture binding domain that is1006313857137capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle.

30. A composition comprising:- first lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence encoding a cognate sequence capable of mediating reconstitution, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle., and- a second lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the first sequence, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle; and- optionally one or more further lipid nanoparticles.

31. The composition of claim 30, further comprising one or more further lipid nanoparticles, wherein the one or more further lipid nanoparticles comprise (a) a nucleic acid comprising (i) a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and a nucleotide sequence comprising, or encoding, a first sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the first sequence, and (ii) a second nucleotide sequence capable of mediating reconstitution when in the presence of a nucleic acid comprising a nucleotide sequence comprising, or encoding, a cognate sequence capable of mediating reconstitution with the second sequence, and (b) a targeting molecule, or a capture binding domain that is capable of capturing a targeting molecule, displayed on the outer surface of the lipid nanoparticle.100631385713832. The composition of claim 30 or 31 , wherein the one or more further lipid nanoparticles comprise:- a third lipid nanoparticle;- a third and fourth lipid nanoparticle;- a third, fourth and fifth lipid nanoparticle;- a third, fourth, fifth and sixth lipid nanoparticle;- a third, fourth, fifth, sixth and seventh lipid nanoparticle;- a third, fourth, fifth, sixth, seventh and eighth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth and ninth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth and tenth lipid nanoparticle; - a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and eleventh lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh and twelfth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and fourteenth lipid nanoparticle;- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth and fifteenth lipid nanoparticle; or- a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth and sixteenth lipid nanoparticle;wherein each lipid nanoparticle comprises a nucleotide sequence encoding a portion of the polypeptide of interest, such that full-length polypeptide of interest is encoded by the totality of each nucleotide sequence encoding each portion of the polypeptide of interest.

33. A lipid nanoparticle of claim 28 or 29 or a composition of any one of claims 30 to 32, wherein the first and / or second sequences capable of mediating reconstitution are a hybridization domain.

34. A lipid nanoparticle or composition of any one of claims 28 to 33, wherein the first and / or second sequences capable of mediating reconstitution are a DNA or1006313857139RNA sequence capable of mediating trans-splicing, or other recombination event, in the presence of a corresponding cognate sequence.

35. A lipid nanoparticle or composition of any one of claims 28 to 34, wherein the reconstitution occurs at the DNA level and is mediated by (1) trans-splicing of the DNA, (2) overlapping of the DNA, or (3) a combination of both based on a highly recombinogenic exogenous sequence.

36. A lipid nanoparticle or composition of any one of claims 28 to 34, wherein the reconstitution occurs at the RNA level, and is mediated by a hybridisation domain, complementary binding domains or ribozyme-activated mRNA trans-ligation.

37. A lipid nanoparticle, composition, method or use of any one of claims 1 to 36, wherein the targeting molecule displayed on the outer surface of the first lipid nanoparticle, the targeting molecule displayed on the outer surface of the second lipid nanoparticle, and / or the targeting molecule displayed on the outer surface of the optionally one or more further lipid nanoparticles bind to the same molecule on the surface of a target cell.

38. A lipid nanoparticle, composition, method or use of any one of claims 1 to 36, wherein the targeting molecule displayed on the outer surface of the first lipid nanoparticle, the targeting molecule displayed on the outer surface of the second lipid nanoparticle and / or the targeting molecule displayed on the outer surface of the optionally one or more further lipid nanoparticles bind to different portions or epitopes of the same molecule on the surface of a target cell.

39. A lipid nanoparticle, composition, method or use of any one of claims 1 to 36, wherein the targeting molecule displayed on the outer surface of the first lipid nanoparticle, the targeting molecule displayed on the outer surface of the second lipid nanoparticle and / or the targeting molecule displayed on the outer surface of the optionally one or more further lipid nanoparticles bind to different molecules on the surface of a target cell.

40. A method or use of any one of claims 13 to 27 or the lipid nanoparticle, composition, method or use of any one of claims 37 to 39, wherein the target cell is a mammalian cell, preferably a human cell.100631385714041. A method or use of any one of claims 13 to 27 or the lipid nanoparticle, composition, method or use of any one of claims 37 to 39, wherein the target cell is one or more of T cells (such as pan T cells, CD4+ T cells, CD8+ T cells and CD25+ T cells), NK cells, CD14+ monocytes, B cells (including CD25+ B cells), dendritic cells, NK T cells, tumour cells (such as lymphoma including T lymphoma, breast, colon, pancreatic, ovarian and lung cancer cells), bone marrow progenitor cells, kidney stroma, and macrophages.

42. A lipid nanoparticle, composition, method or use of any one of claims 1 to 41, wherein the molecule on the surface of a target cell which the targeting molecule binds is a protein, preferably a cell surface receptor.

43. A lipid nanoparticle, composition, method or use of any one of claims 1 to 42, wherein the targeting molecule is an antibody or antibody fragment, such as a nanobody or single chain variable fragment, an affibody, an aptamer or a peptide, preferably a nanobody.

44. A lipid nanoparticle, composition, method or use of any one of claims 1 to 43, wherein the lipid nanoparticle comprises a plurality of targeting molecules.

45. A lipid nanoparticle, composition, method or use of any one of claims 1 to 44, wherein the capture binding domain is bound to a targeting molecule.

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