Targeting Technology to Selectively Express mRNAs in Cardiomyocytes While Avoiding Stimulation of Cardiac Fibroblasts

Abstract

Disclosed is a process of having mRNA selectively adsorbed and expressed in cardiomyocytes, by coupling an aptamer which selectively targets lipid nanoparticles containing the mRNA to cardiomyocytes and does not bind to fibroblasts, to lipid nanoparticles containing the mRNA; and administering the aptamer coupled to the lipid nanoparticles containing the mRNA to a host animal under conditions suitable for expression of the mRNA in cardiomyocytes. One preferred sequence for such an aptamer is: AGCCGTTCTGGGGGGTCGACGTTGCATCGTCA (SEQ ID NO:20), and wherein the mRNA encodes Stemin and/or YAP1(5SA).

Claims

1. A process of having mRNA selectively adsorbed and expressed in cardiomyocytes, comprising: coupling an aptamer which selectively targets lipid nanoparticles containing the mRNA to cardiomyocytes and does not bind to fibroblasts, to lipid nanoparticles containing the mRNA; and administering the aptamer coupled to the lipid nanoparticles containing the mRNA to a host animal under conditions suitable for expression of the mRNA.

2. The process of claim 1 wherein the aptamer has the sequence: AGCCGTTCTGGGGGGTCGACGTTGCATCGTCA (SEQ ID NO:20), or a Variant Sequence thereof.

3. The process of claim 1 wherein the mRNA encodes Stemin and/or YAP1(5SA) or a biologically active variant thereof.

4. The process of claim 1 wherein the mRNA encodes luciferase, a cytokine or a therapeutic molecule.

5. The process of claim 1 wherein the lipid nanoparticles are covalently coupled with the aptamer using a Cu-free click reaction.

6. The process of claim 1 wherein the lipid nanoparticles have surface polyethylene glycol, and some of the surface polyethylene glycol is replaced with a modified polyethylene glycol containing a dibenzylcyclooctyne moiety.

7. The process of claim 6 wherein the dibenzylcyclooctyne moiety has the formula: ##STR00002##

8. The process of claim 6 wherein the dibenzylcyclooctyne moiety reacts with azide in the Cu-free click reaction, and wherein the azide is conjugated to the aptamer.

9. The process of claim 8 wherein either the 3′ end or the 5′ end of the aptamer is conjugated to the azide.

10. The process of claim 1 wherein before uptake by the lipid nanoparticles, the mRNA is complexed with cationic lipids which are neutral under physiological conditions and positively charged at acidic pH.

11. The method of any of claim 1 to 10 wherein the host animal is a human being.

12. A conjugate for carrying mRNA to be selectively adsorbed and expressed in cardiomyocytes, comprising: an aptamer which selectively targets lipid nanoparticles containing the mRNA to cardiomyocytes and does not bind to fibroblasts, coupled to lipid nanoparticles containing the mRNA.

13. The conjugate of claim 12 wherein the aptamer has the sequence: AGCCGTTCTGGGGGGTCGACGTTGCATCGTCA (SEQ ID NO:20), or a Variant Sequence thereof.

14. The conjugate of claim 12 wherein the mRNA encodes Stemin and/or YAP1(5SA) or a biologically active variant thereof.

15. The conjugate of claim 12 wherein the mRNA encodes luciferase, a cytokine or a therapeutic molecule.

16. The conjugate of claim 12 wherein the lipid nanoparticles are covalently coupled with the aptamer using a Cu-free click reaction.

17. The conjugate of claim 12 wherein the lipid nanoparticles have surface polyethylene glycol, and some of the surface polyethylene glycol is replaced with a modified polyethylene glycol containing a dibenzylcyclooctyne moiety.

18. The conjugate of claim 17 wherein the dibenzylcyclooctyne moiety has the formula: ##STR00003##

19. The conjugate of claim 16 wherein the dibenzylcyclooctyne moiety reacts with azide in the Cu-free click reaction, and wherein the azide is conjugated to the aptamer.

20. The conjugate of claim 19 wherein either the 3′ end or the 5′ end of the aptamer is conjugated to the azide.

21. The conjugate of claim 12 wherein before uptake by the lipid nanoparticles, the mRNA is complexed with cationic lipids which are neutral under physiological conditions and positively charged at acidic pH.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0009] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0010] FIG. 1 is an image showing screening results for a selected pool of aptamers displaying binding only to cardiomyocytes; not fibroblasts.

[0011] FIG. 2 depicts results from a microplate fluorescence assay used to distinguish aptamer internalization from binding.

[0012] FIG. 3A depicts a LNP with encapsulated mRNA (orange) and DBCO-PEG lipid incorporated on the surface of the nanoparticle.

[0013] FIG. 3B depicts an azide-functionalized aptamer to allow a Strain-Promoted Azide-Alkyne Click (SPAAC) reaction under mild conditions.

[0014] It should be understood that the drawings and the associated descriptions below are intended only to illustrate one or more embodiments of the present invention, and not to limit its scope. The drawings are not necessarily to scale.

DETAILED DESCRIPTION

[0015] As used herein, the phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz (1979) Principles of Protein Structure, Springer-Verlag). According to such analyses, groups of amino acids can be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz (1979) supra). Examples of amino acid groups defined in this manner can include: a “charged/polar group” including Glu, Asp, Asn, Gln, Lys, Arg, and His; an “aromatic or cyclic group” including Pro, Phe, Tyr, and Trp; and an “aliphatic group” including Gly, Ala, Val, Leu, Ile, Met, Ser, Thr, and Cys. Within each group, subgroups can also be identified. For example, the group of charged/polar amino acids can be sub-divided into sub-groups including: the “positively-charged sub-group” comprising Lys, Arg and His; the “negatively-charged sub-group” comprising Glu and Asp; and the “polar sub-group” comprising Asn and Gln. In another example, the aromatic or cyclic group can be sub-divided into sub-groups including: the “nitrogen ring sub-group” comprising Pro, His, and Trp; and the “phenyl sub-group” comprising Phe and Tyr. In another further example, the aliphatic group can be sub-divided into sub-groups including: the “large aliphatic non-polar sub-group” comprising Val, Leu, and Ile; the “aliphatic slightly-polar sub-group” comprising Met, Ser, Thr, and Cys; and the “small-residue sub-group” comprising Gly and Ala. Examples of conservative mutations include amino acid substitutions of amino acids within the sub-groups above, such as, but not limited to: Lys for Arg or vice versa, such that a positive charge can be maintained; Glu for Asp or vice versa, such that a negative charge can be maintained; Ser for Thr or vice versa, such that a free —OH can be maintained; and Gln for Asn or vice versa, such that a free —NH2 can be maintained. A “conservative variant” is a polypeptide that includes one or more amino acids that have been substituted to replace one or more amino acids of the reference polypeptide (for example, a polypeptide whose sequence is disclosed in a publication or sequence database, or whose sequence has been determined by nucleic acid sequencing) with an amino acid having common properties, e.g., belonging to the same amino acid group or sub-group as delineated above.

[0016] When referring to a gene, “mutant” means the gene has at least one base (nucleotide) change, deletion, or insertion with respect to a native or wild type gene. The mutation (change, deletion, and/or insertion of one or more nucleotides) can be in the coding region of the gene or can be in an intron, 3′ UTR, 5′ UTR, or promoter region. As nonlimiting examples, a mutant gene can be a gene that has an insertion within the promoter region that can either increase or decrease expression of the gene; can be a gene that has a deletion, resulting in production of a nonfunctional protein, truncated protein, dominant negative protein, or no protein; or, can be a gene that has one or more point mutations leading to a change in the amino acid of the encoded protein or results in aberrant splicing of the gene transcript.

[0017] “Naturally-occurring” or “wild-type” refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or mRNA sequence is a sequence present in an organism, which has not been intentionally modified by human manipulation.

[0018] The terms “percent identity” or “homology” with respect to nucleic acid or polypeptide sequences are defined as the percentage of nucleotide or amino acid residues in the candidate sequence that are identical with the known polypeptides, after aligning the sequences for maximum percent identity and introducing gaps, if necessary, to achieve the maximum percent homology. N-terminal or C-terminal insertion or deletions shall not be construed as affecting homology. Homology or identity at the nucleotide or amino acid sequence level can be determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx (Altschul (1997), Nucleic Acids Res. 25, 3389-3402, and Karlin (1990), Proc. Natl. Acad. Sci. USA 87, 2264-2268), which are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments, with and without gaps, between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified, and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases, see Altschul (1994), Nature Genetics 6, 119-129. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix, and filter (low complexity) can be at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff (1992), Proc. Natl. Acad. Sci. USA 89, 10915-10919), recommended for query sequences over 85 units in length (nucleotide bases or amino acids).

[0019] FIG. 1 shows an aptamer library of FAM labeled DNA aptamers screened by Cell-SELEX positively against human cardiomyocytes and negatively against human ventricular myocytes. After 10 rounds the selected pool of aptamers displayed binding only to cardiomyocytes; not fibroblasts. The selected pool was sequenced and the aptamers were ranked for frequency counts and stability. The 23 highest ranking aptamers were synthesized for subsequent testing.

[0020] In order for a aptamer to be effective in carrying nanoparticle mRNAs into cardiomyocytes, it must not only bind selectively, but also it must be rapidly internalized by cardiomyocytes. To test internalization in the mouse model system a microplate fluorescence assay was used to distinguish internalization from binding (Hernandez L I et al. 2013).

[0021] AM-labeled aptamers were incubated with mouse cultured cardiomyocytes for 1 hour in a multi-well plate and then washed with a high salt buffer to remove surface bound aptamer. Fluorescence was recorded for each aptamer and normalized to protein. The results are depicted in FIG. 2, showing internalization of 23 aptamers and a control consisting of an unrelated sequence of the same size.

[0022] As seen in FIG. 2, one aptamer, #20, with a sequence of AGCCGTTCTGGGGGGTCGACGTTGCATCGTCA (SEQ ID NO:20), was taken up significantly more than others. Aptamer Nos. 1 (SEQ ID NO:1) and 19 (SEQ ID NO:19) in Table I also showed high uptake in cardiomyocytes.

[0023] This aptamer and Variant Sequences thereof will be coupled to mRNA encapsulated lipid nanoparticles to facilitate selective mRNA targeting to cardiomyocytes. Sequences of other aptamers tested are shown in Table I, and Variant Sequences of any of the following aptamers could also be tested the same way.

TABLE-US-00001 TABLE I  1 Z83A3G GAGAGAGTCGTGGGGTGGGGCGGGCGGCAGTG (SEQ ID NO: 1)  2 AxAS3K GCCAGATAGTCATGTCAGCGCAAAATCACTTA (SEQ ID NO: 2)  3 AxAS4T GTGTTGCGGCAGAAGTGATGTGAGTTCGTGGG (SEQ ID NO: 3)  4 AxAS32 GGAAGAGGTGGGGAATAGGTTCGGTACATTAA (SEQ ID NO: 4)  5 AxASYU AAATCACGCTGTGTGAAGGTCCTTCTCTCCAA (SEQ ID NO: 5)  6 AxAS1S CCATGATCACATACGCGTACATTACACGAACA (SEQ ID NO: 6)  7 AxASZQ AGGTCAGTGTGTTCGATAGTTCGTGGATGGTA (SEQ ID NO: 7)  8 Z8VCHB ACCTCGCTCTCCCCCCGGCTCCGCGAAATTGA (SEQ ID NO: 8)  9 AxAS25 GACGATCGGATGTTGTTGCAGAAGTTCACTAC (SEQ ID NO: 9) 10 AxAS39 GGGAATGGCGCTCGGTAGTTGTACGTTCTCGG (SEQ ID NO: 10) 11 AxAS5E TATCCCTGGGGTCCGGTCAAACACGTAATAGA (SEQ ID NO: 11) 12 AxAS5U TCTAGCACTCGCGTGTGGACGGTAAACCGTCT (SEQ ID NO: 12) 13 AxAS3A GAGCCAGTGACGTTGAAATACGGTCATGGCGG (SEQ ID NO: 13) 14 AxAS5P TCCGTTCCGATTCTGGGAACGTACAGAATTCA (SEQ ID NO: 14) 15 AxASZU AGTGACGTGCGACGTGCACTGAAGCAAGAGCA (SEQ ID NO: 15) 16 AxAS3T GCGGCGAGGTGATATTACCTCACGGCTGTATT (SEQ ID NO: 16) 17 AxAS5Q TCGAACCAGCAGTAAACGTTACTGTGATGGAC (SEQ ID NO: 17) 18 AxAS1C CAACGTGTGTAATGGATATCCATTACTGGTAT (SEQ ID NO: 18) 19 AxAS5F TATGGCGGACAGGGAGGACCACTCAGTTACAG (SEQ ID NO: 19) 20 AxASZJ AGCCGTTCTGGGGGGTCGACGTTGCATCGTCA (SEQ ID NO: 20) 21 AxAS3V GCGTGATGCGTTTCAACGACTGTAGACGGTGA (SEQ ID NO: 21) 22 AxAS5Z TGCGCGATAGTCGAGAATTGGGTTCTCTCGTC (SEQ ID NO: 22) 23 AxAS44 TACGTCGATAAATCGAGCGGAAAGCATACGCT (SEQ ID NO: 23) 24 AxAS4N GTCGCCGTAGGCATTTGGACGTGGGGAACGTT (SEQ ID NO: 24) 25 AxAS6J TTGGGGCACCCAATTTCCTGGTAGGGACAAAT (SEQ ID NO: 25) 26 AxAS56 TGGCCTAGTCAGGGCGTGCGGCTGTCGGGTTC (SEQ ID NO: 26) 27 AxASZT AGTGAAGTCCGTCATACTCGTGACCAGGACGA (SEQ ID NO: 27) 28 AxAS1H CACGGCCAACTTCGTCCAATTGGCAGTACCCA (SEQ ID NO: 28) 29 AxAS24 GACCTCCAGGATGAGTTTCACGAGAGTACTCG (SEQ ID NO: 29) 30 AxAS1T CCCAGCCGGTTTATTAGGTAGAACCGTAAAGC (SEQ ID NO: 30) 31 AxAS5W TGCAGAGTGGATCGGTTGGGGGTAATGAACCA (SEQ ID NO: 31) 32 AxASZ1 ATCGCATACTCGGTCAGACTTTCCTCGCCGAG (SEQ ID NO: 32) 33 AxAS1J CAGATAGTAGCCTCCCGGGGCCCTATCGATCA (SEQ ID NO: 33) 34 AxAS1W CCCGTCTCCGACGAGCTGAACAAGGGAGCTAT (SEQ ID NO: 34) 35 AxAS19 CGCAAGGGCGAAACGGGACAGATCGATGAGTC (SEQ ID NO: 35) 36 AxASZA ACGCAGCGCCCAGGCTCCGGGAGCTATCCCCT (SEQ ID NO: 36) 37 AxASZK AGCGGCTCCTGTAGAATAGGTGGGCATCGCTC (SEQ ID NO: 37) 38 AxAS3W GCTACGGGAATGCCCGCACACATGAATTTCGT (SEQ ID NO: 38) 39 AxASZB ACGCCAGGCAGGATGCGGATCAGCATTCCTTT (SEQ ID NO: 39) 40 AxAS2Y CTGTGTGTGGGGTCCGCATCCGTAACATGCGA (SEQ ID NO: 40) 41 AxAS2U CTCGGAACGTTTTGCTGGGGGGCCCAGGTATA (SEQ ID NO: 41) 42 AxASYX AAGGCGAGAAGTAAGTTGAAGGTTCTGGCCGC (SEQ ID NO: 42) 43 AxAS3I GCATCCCACTGCACGGCTAAACAACTTAGCAT (SEQ ID NO: 43) 44 AxAS2M CGTGTAGAACTCATGCGACCGCTGGGTCCATA (SEQ ID NO: 44) 45 AxASZN AGGCTTCAACCCGGCTTTAGCGTGAAAAGCAA (SEQ ID NO: 45) 46 AxAS2D CGGAACGGGCCAGCTGGAAGGCGGGTGCTTCG (SEQ ID NO: 46) 47 AxAS1N CAGTGTGAATGGCGGTTCGTCCGATGAGACGT (SEQ ID NO: 47) 48 AxASYT AAACTCGAGAGCAATCGGCGAACCGTGTACGG (SEQ ID NO: 48) 49 AxAS2G CGGAGGGATATTTACGTCGTTCCGTGGAGTTA (SEQ ID NO: 49) 50 AxAS3U GCGGCTCCGGACCGAGCGGTTCTATGAGGTTC (SEQ ID NO: 50) 51 AxAS35 GGAGTAAAGGCGGAAACAGTCCGTGCACAACT (SEQ ID NO: 51) 52 AxASZ9 ATTTGCGGGAGGCTCACGTGCTCTGGCTCGGT (SEQ ID NO: 52) 53 AxAS2B CGCGGGGTCGCTGTAGACTTACTGGGATATCC (SEQ ID NO: 53) 54 AxAS12 CCTGCAGAGGACCGGTCCAGCGCCTCCCCCAG (SEQ ID NO: 54) 55 AxAS2Q CTAGGAGGGAGCGATCCGCCCATAGTTGGATT (SEQ ID NO: 55) 56 AxAS21 GAAATGCACCGCCTTCTACGGACGGCACAATT (SEQ ID NO: 56) 57 AxASZE ACTGGCATCAAGGCACCCAACTGCAAGGTTGC (SEQ ID NO: 57) 58 AxAS31 GCTGAACGTCACTATGGTGCTGGACCGGCATC (SEQ ID NO: 58) 59 AxAS37 GGCAGATTCCATCTGAATTATTCGACGTAGCG (SEQ ID NO: 59) 60 AxAS4Z TACACATAGCGTTCGTCAAGGTGTGTACGGAC (SEQ ID NO: 60) 61 AxAS2W CTGCAAGCCTCCGCCCTAAAAGTTAAGCGAGG (SEQ ID NO: 61) 62 AxASY8 ACCTTAATGGAAACGTTAGGAGGCAGGCCTAA (SEQ ID NO: 62) 63 AxAS5Y TGCGATACTCCCGATACTCGGTGGCAAAACTT (SEQ ID NO: 63) 64 AxASZI AGCAGGGTTTCCCTTGTTCCGCGCTGAGTGTG (SEQ ID NO: 64) 65 AxAS4K GTCACGTCACAGAGGTGTGGTTTCCAGTATGT (SEQ ID NO: 65) 66 AxAS5S TCGGTTCTGCAACCCGGGCAAGCTGTGGTTCG (SEQ ID NO: 66) 67 AxAS6E TTAGGAGGGACCAGTCTGGTGGCAACTGCTGG (SEQ ID NO: 67) 68 AxAS1Y CCGACCTGTAAATGAATCGGCGCACACCGTAT (SEQ ID NO: 68) 69 AxAS2C CGCGTGGAGAAGGCCCAACCGGCTGGATGGTG (SEQ ID NO: 69) 70 AxAS5T TCTAAGTGTAGCTCCCGGTTTGGGTTTCTTAG (SEQ ID NO: 70) 71 AxAS4L GTCAGTCACGGTTTCAGGGAGCGGACTCGTAT (SEQ ID NO: 71) 72 AxAS5V TCTCGGGTCGCCGCAACGAACCTTACTAACTG (SEQ ID NO: 72) 73 AxAS3Z GCTCTCGGGACCAACTGGTGTGTCGTCTGCCG (SEQ ID NO: 73) 74 AxAS6K TTGGTAAACAAAAGTGTCACCACCTTAGCACT (SEQ ID NO: 74) 75 AxASZD ACTGCGTCAGTGGATTCTTCGGGAATTATTGT (SEQ ID NO: 75) 76 AxAS11 CCTCGGCGTCCTCAGTTAACGTGCGACCCCGG (SEQ ID NO: 76) 77 AxAS3R GCGCAAATTCGATGTTTGTCCAAGAGCGAGGG (SEQ ID NO: 77) 78 AxAS2L CGTGGGGTGCCAGTCTTCTTAGGACCATGAGC (SEQ ID NO: 78) 79 AxAS5A TAGTAAGTGTCCGAGGTGAATCCTCAAGGCGA (SEQ ID NO: 79) 80 AxASZ4 ATGCACAGGCAAAGATGGAGCAGTCTTTTTCA (SEQ ID NO: 80) 81 AxAS3D GAGTTATAAGCGACGGCTTAAGATTTTATGCA (SEQ ID NO: 81) 82 AxASZS AGTCGAGGGACCTCACACTAAAGTTCCGACGG (SEQ ID NO: 82) 83 AxAS1V CCCGGTTCAACGTGCAATGGCGAGTACCAAGC (SEQ ID NO: 83) 84 AxAS3P GCGAACAGCTTTCACTAATGATGTATTGCTCG (SEQ ID NO: 84) 85 AxAS4F GGTACGGGGAATTGTGACCGTCTTTGGGCAAA (SEQ ID NO: 85) 86 AxAS1Q CATTGAGAAAGGGTTTCTTCCCAGGCTATTCC (SEQ ID NO: 86) 87 AxAS3G GCAAGAGTCCCCGGATAAACGGCTCAAGAGAA (SEQ ID NO: 87) 88 AxAS22 GACATGACCCATCCGGAATGTCTAACGTAATT (SEQ ID NO: 88) 89 AxAS3X GCTACGTCAAATCATGGGACTGTCGTACGCAG (SEQ ID NO: 89) 90 AxAS2K CGGTGGGCGTCGATCCCTGGCTTGTGTGAAGT (SEQ ID NO: 90) 91 AxAS4A GGGGACAAACTACCGGCTTATGCTATGTCTCC (SEQ ID NO: 91) 92 AxAS4X TAAGCGCCATTATGGATCTCTTTGGGAGTGGG (SEQ ID NO: 92) 93 AxAS6B TGTATTCTGCTATGTCGGTGCGTCCGTTGGCT (SEQ ID NO: 93) 94 AxASZ5 ATGCCCCACCGAGCCTAATACGGTATATGTTA (SEQ ID NO: 94) 95 AxAS1D CAAGGACGGAAGAACCGGCGACCTCAATTCAT (SEQ ID NO: 95) 96 AxAS4Q GTGCGATCTTCAATCCCAGGCCGGCGTGGGTC (SEQ ID NO: 96)

[0024] One approach to determine if mRNAs expressing Stemin and YAP1(5SA) can be taken up and expressed in cardiomyocytes, is to model mRNA uptake as follows. Exemplary mRNAs, preferably encoding luciferase, will be encapsulated into lipid nanoparticles (LNPs) using the NanoAssemblr Platform (Precision NanoSystems, Vancouver, BC) and covalently coupled with the cardiomyocyte targeted aptamer number 20 using a Cu-free click reaction. The aptamer-coupled LNPs containing luciferase mRNA will be tested to determine cellular uptake, through luciferase expression, in mouse hearts. The luciferase expression system can also be used to initially determine the optimal aptamers, followed by luciferase expression testing in animals for further in vivo optimization. The mRNAs complexed with cationic lipids are neutral under physiological conditions and positively charged at acidic pH to enable efficient entrapment of mRNAs. When internalized, the low endosomal pH allows for protonation of the ionizable lipid, destabilization of the endosome and escape of the mRNA into the cytoplasm (Kulkarni J A et al. 2019). FIG. 3A.

[0025] Approximately 2% of the surface polyethylene glycol (PEG) of the LNP will be replaced with a modified PEG containing a dibenzylcyclooctyne (DBCO) moiety, preferably having the formula:

##STR00001##

[0026] The aptamer will be functionalized with an azide at the 3′ or 5′ ends to test which orientation of the aptamer produces the most luciferase expression. The DBCO-azide reaction combines high reactivity and adequate hydrophilicity to allow a rapid and complete Strain-Promoted Azide-Alkyne Click (SPAAC) reaction under mild conditions, at neutral pH in aqueous solution (Debets M F et al. 2010). FIG. 3B.

Formulations

[0027] After preparation of a suitable functionalized LNP, it can be prepared in a formulation for administration to a subject. A lyophilized formulation is preferred, which as a first step, requires preparing a pre-lyophilized formulation. The amount of mRNA in the pre-lyophilized formulation is determined taking into account the desired dose volumes, mode(s) of administration, etc. The preferred buffer is histidine as it can have lyoprotective properties. Succinate is also a useful buffer.

[0028] A lyoprotectant is added to the pre-lyophilized formulation. In preferred embodiments, the lyoprotectant is a non-reducing sugar such as sucrose or trehalose. The amount of lyoprotectant in the pre-lyophilized formulation is generally such that, upon reconstitution, the resulting formulation will be isotonic, as preferred, though hypertonic reconstituted formulations may also be suitable. In addition, the amount of lyoprotectant must not be too low such that an unacceptable amount of degradation/aggregation of the protein occurs upon lyophilization.

[0029] It may be desirable to add a surfactant to the pre-lyophilized formulation. Alternatively, or in addition, the surfactant may be added to the lyophilized formulation and/or the reconstituted formulation. Exemplary surfactants include nonionic surfactants such as polysorbates (e.g. polysorbates 20 or 80); poloxamers (e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palnidopropyl-, or isostearamidopropyl-betaine (e.g lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 etc). The amount of surfactant added is such that it reduces aggregation of the LNP and minimizes the formation of particulates after reconstitution.

[0030] A mixture of the lyoprotectant (such as sucrose or trehalose) and a bulking agent (e.g. mannitol or glycine) may be used in the preparation of the pre-lyophilization formulation. The bulking agent may allow for the production of a uniform lyophilized cake without excessive pockets therein.

[0031] Other pharmaceutically acceptable carriers, excipients or stabilizers such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) may be included in the pre-lyophilized formulation (and/or the lyophilized formulation and/or the reconstituted formulation) provided that they do not adversely affect the desired characteristics of the formulation. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include; additional buffering agents; preservatives; co-solvents; antioxidants including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g. Zn-protein complexes); biodegradable polymers such as polyesters; and/or salt-forming counterions such as sodium.

[0032] The pharmaceutical compositions and formulations described herein are preferably stable, so as to retain its physical and chemical stability and integrity upon storage. Stability can be measured at a selected temperature for a selected time period.

[0033] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to, or following, lyophilization and reconstitution. Alternatively, sterility of the entire mixture may be accomplished by autoclaving the ingredients, at about 120° C. for about 30 minutes.

[0034] After the LLNP, lyoprotectant and other optional components are mixed together, the formulation is lyophilized. Many different freeze-dryers are available for this purpose such as Hu1150® (Hull, USA) or GT20® (Leybold-Heraeus, Germany) freeze-dryers. Freeze-drying is accomplished by freezing the formulation and subsequently subliming ice from the frozen content at a temperature suitable for primary drying. Under this condition, the product temperature is below the eutectic point or the collapse temperature of the formulation.

[0035] Typically, the shelf temperature for the primary drying will range from about −30 to 25° C. (provided the product remains frozen during primary drying) at a suitable pressure, ranging typically from about 50 to 250 mTorr. The formulation, size and type of the container holding the sample (e.g., glass vial) and the volume of liquid will mainly dictate the time required for drying, which can range from a few hours to several days (e.g. 40-60 hours). A secondary drying stage may be carried out at about 0-40° C., depending primarily on the type and size of container and the type of LNP employed. For example, the shelf temperature throughout the entire water removal phase of lyophilization may be from about 15-30° C. (e.g., about 20° C.). The time and pressure required for secondary drying will be that which produces a suitable lyophilized cake, dependent, e.g., on the temperature and other parameters. The secondary drying time is dictated by the desired residual moisture level in the product and typically takes at least about 5 hours (e.g. 10-15 hours). The pressure may be the same as that employed during the primary drying step. Freeze-drying conditions can be varied depending on the formulation and vial size.

[0036] In some instances, it may be desirable to lyophilize the formulation in the container in which reconstitution is to be carried out in order to avoid a transfer step. The container in this instance may, for example, be a 3, 5, 10, 20, 50 or 100 cc vial. As a general proposition, lyophilization will result in a lyophilized formulation in which the moisture content thereof is less than about 5%, and preferably less than about 3%.

[0037] At the desired stage, typically when it is time to administer it to the patient, the lyophilized formulation may be reconstituted with a diluent such that the LNP concentration in the reconstituted formulation is preferably similar to that of the pre-lyophilized formulation.

[0038] Reconstitution generally takes place at a temperature of about 25° C. to ensure complete hydration, although other temperatures may be employed as desired. The time required for reconstitution will depend, e.g., on the type of diluent, amount of excipient(s) and LNP. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution. The diluent optionally contains a preservative. Exemplary preservatives have been described above, with aromatic alcohols such as benzyl or phenol alcohol being the preferred preservatives. The amount of preservative employed is determined by assessing different preservative concentrations for compatibility with the protein and preservative efficacy testing. For example, if the preservative is an aromatic alcohol (such as benzyl alcohol), it can be present in an amount from about 0.1-2.0% and preferably from about 0.5-1.5%, but most preferably about 1.0-1.2%.

[0039] Alternatively, a non-lyophilized formulation may be used, including any of the well-known carriers, excipients, buffers, stabilizers, preservatives, adjuvants and other additives described herein and well known in the art.

Dosages and Administration

[0040] The formulation described above can be administered to a subject (e.g., a human) in need of the treatment via a suitable route, such as administration by intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intracutaneous, intraarticular, intrasynovial, intrathecal, intradermal, intratumoral, intranodal, intramedulla, oral, inhalation or topical routes; or it may be administered orally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir; and in any case, as a bolus or by continuous infusion over a period of time; or via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.

[0041] Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration. Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution. Alternatively, LNP can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.

[0042] The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.

[0043] An “effective amount” refers to the amount of active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. Effective amounts vary, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors, all of which are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. A lower dose or tolerable dose for medical reasons, psychological reasons or other reasons, is also appropriate.

[0044] Empirical considerations, such as the mRNA half-life, generally will contribute to the determination of the dosage. Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment of the condition. Alternatively, sustained continuous release formulations of antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0045] In one example, dosages may be determined empirically in individuals who have been given one or more administration(s) of the LNP. Individuals are given incremental dosages of the LNP. To assess efficacy, an indicator of the disease can be followed according to routine practice.

[0046] Generally, for administration, an initial candidate dosage can be extrapolated from the experiments. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved. An exemplary dosing regimen comprises administering an initial higher dose, followed by a lower maintenance dose. However, other dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve. For example, dosing from one-four times a week is contemplated. In some embodiments, dosing frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 months, or every 3 months, or longer. The progress of this therapy is easily monitored by conventional techniques and assays. The dosing regimen can vary over time.

[0047] Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the pharmaceutical composition to the subject, depending upon the treatment goal.

[0048] Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like).

[0049] For intravenous injection, a pharmaceutical formulation containing the antibody and a physiologically acceptable excipients can be infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients.

[0050] Intramuscular preparations, e.g., a sterile formulation can be dissolved and administered in a pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or 5% glucose solution.

[0051] In one embodiment, the LNP is administered via site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of the mRNA or local delivery catheters, such as infusion catheters, an indwelling catheter, or a needle catheter, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site specific carriers, direct injection, or direct application. See, e.g., WO 00/53211 and U.S. Pat. No. 5,981,568.

[0052] In another embodiment of the present disclosure, an article of manufacture is provided which contains any of the pharmaceutical compositions and formulations described herein and provides instructions for its use and/or reconstitution. The article of manufacture comprises a container. Suitable containers include, for example, bottles, vials (e.g. dual chamber vials), syringes (such as dual chamber syringes) and test tubes. The container may be formed from a variety of materials such as glass or plastic. The container holds the formulation and the label on, or associated with, the container may indicate directions for reconstitution and/or use. For example, the label may indicate that the formulation is reconstituted to particular concentrations. The container holding the formulation may be a multi-use vial, which allows for repeat administrations (e.g., from 2-6 administrations) of the reconstituted formulation. The article of manufacture may further comprise a second container comprising a suitable diluent (e.g. BWFI). Upon mixing of the diluent and the lyophilized formulation, the final concentration in the reconstituted formulation will generally be set at a threshold. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0053] Example: Testing of mRNA expression from targeted nanoparticles in mouse hearts. In order to verify whether the aptamer-conjugated nanoparticles can bind, enter and express the mRNA contents in a live animal the construct containing luciferase mRNA will be injected into mouse hearts.

[0054] Study design: C57BL/6J mice will be injected using microCT imaging to locate the left ventricle with luciferase mRNA encapsulated in lipid nanoparticles conjugated with aptamer #20. Two nanoparticle preparations will be tested, one with the aptamer conjugated at the 5′ end and one conjugated at the 3′ end. A total of 100 μg of luciferase mRNA will be administered in 3 separate injections of 20 μl. After 24 hours, 150 mg/kg of D-luciferin will be injected subcutaneously 10 minutes prior to imaging the luciferase signal using an IVIS Bioimager. Detection of a luciferase signal will confirm that the lipid nanoparticle mRNA was able to enter ventricular cells, but it does not prove that the aptamer was able to produce luciferase expression selectively in cardiomyocytes. In order to test specificity, the animals will be sacrificed and the left ventricles removed and fixed with paraformaldehyde. The fixed tissue will be sectioned and stained with luciferase and anti-troponin T antibodies. If luciferase and troponin T staining co-localizes, it will indicate that the aptamer conjugated lipid nanoparticles are selective for cardiomyocytes.

[0055] A similar protocol using the aptamer-coupled lipid nanoparticles carrying mRNA for Stemin and YAP1(5SA) could be used therapeutically for treating heart disease or heart failure. The Stemin sequence is SEQ No. 97 (same as SEQ ID NO: 2 in U.S. Pat. No. 11,179,479 (incorporated by reference; US Publ'n No. 20210069294A1 is also incorporated by reference)). The mRNA sequence for YAP1(5SA) is shown below as SEQ ID NO: 98.

TABLE-US-00002 SEQ ID NO: 98 auggaucccg ggcagcagcc gccgccucaa ccggcccccc agggccaagg gcagccgccu   60 ucgcagcccc cgcaggggca gggcccgccg uccggacccg ggcaaccggc acccgcggcg  120 acccaggcgg cgccgcaggc accccccgcc gggcaucaga ucgugcacgu ccgcggggac  180 ucggagaccg accuggaggc gcucuucaac gccgucauga accccaagac ggccaacgug  240 ccccagaccg ugcccaugag gcuccggaag cugcccguau cccuucuuca agccgccgga  300 gcccaaaucc cacucccgac aggccaguac ugaugcaggc acugcaggag cccugacucc  360 cagcauguuc gagcucauuc cucuccagcu ucucugcagu ugggagcugu uucuccuggg  420 auggaucccg ggcagcagcc gccgccucaa ccggcccccc agggccaagg gcagccgccu  480 ucgcagcccc cgcaggggca gggcccgccg uccggacccg ggcaaccggc acccgcggcg  540 acacugaccc ccacuggagu agucucuggc ccagcagcua cacccacagc ucagcaucuu  600 cgacagucuu cuuuugagaa uaccugauga uguaccucug ccagcgguug ggagauggca  660 aagacaucuu cuggucagag auacuucuua aaucacaucg aucagacaac aacauggcag  720 gaccccagga aggccaugcu gucccagaug aacgucacag cccccaccag uccaccagug  780 cagcagaaua ugaugaacuc ggcuucaggu ccucuuccug auggauggga acaagccaug  840 acucaggaug gagaaauuua cuauauaaac cauaagaaca agaccaccuc uuggcuagac  900 ccaaggcuug acccucguuu ugccaugaac cagagaauca gucagagugc uccagugaaa  960 cagccaccac cccuggcucc ccagagccca cagggaggcg ucaugggugg cagcaacucc 1020 aaccagcagc aacagaugcg acugcagcaa cugcagaugg agaaggagag gcugcggcug 1080 aaacagcaag aacugcuucg gcaggcaaug cggaauauca aucccagcac agcaaauucu 1140 ccaaaauguc aggaguuagc ccugcguagc caguuaccaa cacuggagca ggaugguggg 1200 acucaaaauc cagugucuuc ucccgggaug ucucaggaau ugagaacaau gacgaccaau 1260 agcucagauc cuuuccuuaa caguggcacc uaucacucuc gagaugagag uacagacagu 1320 gggacuaagc augagcagcu acaguguccc ucgaacccca gaugacuucc ugaacagugu 1380 ggaugagaug gauacaggug auacuaucaa ccaaagcacc cugcccucac agcagaaccg 1440 uuucccagac uaccuugaag ccauuccugg gacaaaugug gaccuuggaa cacuggaagg 1500 agauggaaug aacauagaag gagaggagcu gaugccaagu cugcaggaag cuuugaguuc 1560 ugacauccuu aaugacaugg agucuguuuu ggcugccacc aagcuagaua aagaaagcuu 1620 ucuuacaugg uuauag 1636

[0056] The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. Thus, for example, in each instance herein, in embodiments or examples of the present invention, any of the terms “comprising”, “including”, “containing”, “having” and “have” are to be read as synonyms and expansively and without limitation. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims.

[0057] Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.

[0058] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

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