Compositions and methods for viral vector integration

Abstract

The present disclosure relates to compositions and methods for treating phenylketonuria (PKU). In particular, the present disclosure relates to AAV donor polynucleotide vectors and LNP compositions comprising a targeted gRNA pair and a nucleic acid encoding a Cas-CLOVER fusion protein, and methods of using the compositions in the treatment of PKU.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 583,701, filed September 19, 2023, which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] The sequence list XML related to this application is submitted electronically in XML file format and is hereby incorporated by reference. The XML file containing the sequence list XML is named "POTH-089_001WO_SeqList.xml". This XML file is 99,347 bytes long, was created on September 9, 2024, and is being submitted electronically through the USPTO Patent Center. Technical Field

[0005] This disclosure generally relates to novel AAV donor polynucleotides designed for site-specific integration into target sequences of the genome into cells, AAV donor polynucleotides comprising expression cassettes or promoterless bidirectional expression cassettes for expressing transgenes (e.g., human phenylalanine hydroxylase (PAH) gene), AAV donor vectors comprising said polynucleotides, compositions comprising AAV donor vectors and lipid nanoparticles (“LNPs”) containing gRNA pairs complementary to the genomic target sequence and mRNA encoding Cas-CLOVER, methods for preparing these polynucleotides and LNPs, and the use of these AAV donor vectors and LNPs for gene therapy applications, particularly for the treatment of phenylketonuria (PKU). Background Technology

[0006] There has long been an unmet need in the art for compositions and methods for delivering nucleic acids to cells in vivo, in vitro, and in vitro, as well as for genetically modifying cells. Widely accepted gene delivery and genetic modification techniques, such as those using viral vectors (including AAV vectors), can cause acute toxicity and harmful side effects in patients. This disclosure provides modified AAV donor polynucleotides designed to integrate site-specifically into target sequences of the cell's genome, AAV donor polynucleotides comprising an expression cassette or promoterless bidirectional expression cassette for expressing transgenes (e.g., the human phenylalanine hydroxylase (PAH) gene), AAV donor vectors comprising said polynucleotides, and compositions comprising AAV donor vectors and lipid nanoparticles (“LNPs”) containing gRNA pairs and mRNA encoding Cas-CLOVER, which enter cells, including hepatocytes, in vivo with high efficiency and low toxicity. Therefore, the compositions and methods of this disclosure are suitable for gene therapy treatment of PKU.

[0007] This disclosure provides AAV donor polynucleotides comprising an expression cassette or promoterless bidirectional expression cassette containing at least one transgenic gene encoding a codon-optimized and modified human phenylalanine hydroxylase (PAH) gene, AAV donor vector compositions comprising polynucleotides, and methods for treating phenylketonuria (PKU) using the AAV donor vector composition in combination with an LNP composition comprising a gRNA pair targeting a specific genomic sequence and at least one mRNA encoding Cas-CLOVER. The compositions and methods are further described in detail herein. Summary of the Invention

[0008] In one aspect, this article provides an adeno-associated virus (AAV) donor polynucleotide comprising, in the 5' to 3' directions: (a) a first AAV ITR sequence; (b) a first targeting sequence homologous to a first region of intron 3 of the albumin gene, the first targeting sequence comprising the sequence of SEQ ID NO: 13; (c) a splice acceptor sequence; (d) a P2A sequence; (e) a sequence encoding a first codon-optimized and modified PAH gene, the sequence comprising the sequence of SEQ ID NO: 25; (f) a DNA spacer sequence; (g) a reverse polyA sequence; (h) a reverse sequence encoding a second codon-optimized and modified PAH gene, the reverse sequence comprising the sequence of SEQ ID NO: 26; (i) a reverse P2A sequence; (j) a reverse splice acceptor sequence; and (k) a second targeting sequence homologous to a second region of intron 3 of the albumin gene, the second targeting sequence comprising SEQ ID NO: 23. The sequence; and (l) the second AAV ITR sequence.

[0009] In some embodiments, the first AAV ITR sequence comprises the sequence of SEQ ID NO: 12; the splice acceptor sequence comprises the sequence of SEQ ID NO: 14; the P2A sequence comprises the sequence of SEQ ID NO: 15; the polyadenylated (poly(A)) sequence comprises the sequence of SEQ ID NO: 17; the DNA spacer sequence comprises the sequence of SEQ ID NO: 18; the reverse poly(A) sequence comprises the sequence of SEQ ID NO: 19; the reverse P2A sequence comprises the sequence of SEQ ID NO: 21; the reverse splice acceptor sequence comprises the sequence of SEQ ID NO: 22; and / or the second AAV ITR sequence comprises the sequence of SEQ ID NO: 24.

[0010] On the other hand, this article provides an adeno-associated virus (AAV) donor polynucleotide comprising, in the 5' to 3' directions: (a) a first AAV ITR sequence; (b) a first targeting sequence homologous to a first region of intron 3 of the albumin gene, the first targeting sequence comprising the sequence of SEQ ID NO: 13; (c) a TTRe promoter sequence; (d) a splice acceptor sequence; (e) a P2A sequence; (f) a sequence encoding a codon-optimized and modified PAH gene, the sequence comprising the sequence of SEQ ID NO: 25; (g) a 3' UTR sequence; (h) a polyadenylated (poly(A)) sequence; (i) a DNA spacer sequence; (j) a second targeting sequence homologous to a second region of intron 3 of the albumin gene, the second targeting sequence comprising the sequence of SEQ ID NO: 23; and (k) a second AAV ITR sequence.

[0011] In some embodiments, the first AAV ITR sequence comprises the sequence of SEQ ID NO: 12; the TTRe promoter sequence comprises the sequence of SEQ ID NO: 27; the splice acceptor sequence comprises the sequence of SEQ ID NO: 14; the P2A sequence comprises the sequence of SEQ ID NO: 15; the 3'UTR sequence comprises the sequence of SEQ ID NO: 10; the polyadenylated (poly(A)) sequence comprises the sequence of SEQ ID NO: 17; the DNA spacer sequence comprises the sequence of SEQ ID NO: 18; the reverse poly(A) sequence comprises the sequence of SEQ ID NO: 19; and / or the second AAV ITR sequence comprises the sequence of SEQ ID NO: 24.

[0012] In some embodiments, the AAV donor polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the AAV donor polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 10.

[0013] On the other hand, this document provides an AAV viral vector comprising the AAV donor polynucleotide described herein. In some embodiments, the AAV viral vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 viral vector. In some embodiments, the AAV viral vector is an AAV8 or AAV9 viral vector.

[0014] On the other hand, this document provides a composition comprising: (a) the AAV viral vector described herein; and (b) at least one LNP composition comprising at least one gRNA pair and at least one mRNA molecule encoding Cas-CLOVER. In some embodiments, the mRNA molecule comprises a 5'-cap. In some embodiments, the at least one gRNA pair comprises a left gRNA containing the sequence of SEQ ID NO: 4 and a right gRNA containing the sequence of SEQ ID NO: 5. In some embodiments, the at least one LNP composition comprises: about 54% ssPalmO-Ph-P4C2 by mole, about 35% cholesterol by mole, about 10% DOPC by mole, and about 1% DMG-PEG2000 by mole.

[0015] On the other hand, this article provides a pharmaceutical composition comprising the AAV viral vector or composition described herein and a pharmaceutically acceptable vector.

[0016] On the other hand, this document provides a method for treating phenylketonuria (PKU) in a subject with this need, the method comprising administering to the subject: (a) at least one therapeutically effective dose of the AAV donor polynucleotide, AAV viral vector, composition, or pharmaceutical composition described herein; and (b) at least one LNP composition comprising at least one gRNA pair and at least one mRNA molecule encoding Cas-CLOVER. In some embodiments, the at least one LNP composition comprises: about 54% ssPalmO-Ph-P4C2 by mole, about 35% cholesterol by mole, about 5% DOPC by mole, about 5% DSPC by mole, and about 1% DMG-PEG2000 by mole. In some embodiments, the mRNA molecule comprises a 5'-cap. In some embodiments, the gRNA pair comprises a left gRNA containing the sequence of SEQ ID NO: 4 and a right gRNA containing the sequence of SEQ ID NO: 5.

[0017] On the other hand, this document provides a method for specifically integrating a transgenic site into the genome of at least one cell of a subject via a viral vector, the method comprising administering to the subject (a) at least one therapeutically effective dose of the AAV donor polynucleotide, AAV viral vector, composition, or pharmaceutical composition described herein; and (b) at least one LNP composition comprising at least one gRNA pair and at least one mRNA molecule encoding Cas-CLOVER, wherein the viral vector DNA is integrated into the genome of at least one cell at a genomic cleavage site generated by Cas-CLOVER. In some embodiments, the at least one LNP composition comprises: about 54% ssPalmO-Ph-P4C2 by mole, about 35% cholesterol by mole, about 5% DOPC by mole, about 5% DSPC by mole, and about 1% DMG-PEG2000 by mole. In some embodiments, the mRNA molecule further comprises a 5'-cap. In some embodiments, the gRNA pair comprises a left gRNA containing the sequence of SEQ ID NO: 4 and a right gRNA containing the sequence of SEQ ID NO: 5. Attached Figure Description

[0018] The above and other features will be more readily understood from the following detailed description taken in conjunction with the accompanying drawings.

[0019] Figure 1A is a schematic diagram of an exemplary AAV donor polynucleotide comprising a promoterless bidirectional luciferase cassette. Figure 1B is a schematic diagram of an exemplary AAV donor polynucleotide comprising a promoterless bidirectional human phenylalanine hydroxylase (PAH) cassette containing the codon-optimized and modified human PAH gene of this disclosure. Figure 1C is a schematic diagram of an exemplary AAV donor polynucleotide comprising a human TTRe-PAH expression cassette containing the TTRe promoter sequence controlling human PAH gene expression.

[0020] Figure 2A shows two plots: one illustrating whole-body luminescence imaging (BLI) and the other illustrating the percentage of albumin gene editing in wild-type treated mice administered low, medium, or high doses of AAV donor DNA containing a promoterless bidirectional luciferase expression cassette and an LNP composition containing albumin targeting gRNA pairs and mRNA encoding Cas-CLOVER. Figure 2B shows two plots: one illustrating the percentage of integration per haploid genome and the other illustrating luciferase mRNA expression in wild-type treated mice administered low, medium, or high doses of AAV donor DNA containing a promoterless bidirectional luciferase expression cassette and an LNP composition containing albumin targeting gRNA pairs and mRNA encoding Cas-CLOVER.

[0021] Figure 3A shows two graphs: one showing the percentage of albumin gene editing and the other showing relative human PAH mRNA expression in wild-type treated mice administered low, medium, or high doses of AAV donor DNA containing a promoterless bidirectional PAH expression cassette or AAV donor DNA containing a TTRe-PAH promoter expression cassette, along with an LNP composition containing an albumin-targeting gRNA pair and mRNA encoding Cas-CLOVER. Figure 3B shows two graphs of the total integration percentage per haploid genome in wild-type treated mice administered low, medium, or high doses of AAV donor DNA containing a promoterless bidirectional PAH expression cassette or AAV donor DNA containing a TTRe-PAH promoter expression cassette, along with an LNP composition containing albumin targeting a gRNA pair and mRNA encoding Cas-CLOVER.

[0022] Figure 4A shows the BTBR PAH enu A diagram showing the relative expression of human PAH mRNA in mice treated with medium or high doses of AAV donor DNA containing a promoterless bidirectional PAH expression cassette and an LNP composition containing albumin targeting gRNA pairs and mRNA encoding Cas-CLOVER. Figure 4B illustrates the expression of PAH mRNA in BTBRPAH mice. enuA graph showing the total percentage of integration of each haploid genome in mice treated with low, medium, or high doses of AAV donor DNA containing a promoterless bidirectional PAH expression cassette or AAV donor DNA containing a TTRe-PAH promoter expression cassette, and an LNP composition containing albumin targeting gRNA pairs and mRNA encoding Cas-CLOVER.

[0023] Figure 5A shows male BTBR PAHs treated with the medium, AAV donor DNA containing a promoterless bidirectional PAH expression cassette, and an LNP composition containing albumin intron 3 targeting gRNA pairs and mRNA encoding Cas-CLOVER at days 0, 7, 14, and 28 post-application. enu A graph showing serum Phe levels (µM) in mice. Figure 5B shows male BTBR PAH mice treated with the medium, AAV donor DNA containing the TTRe-PAH promoter expression cassette, and an LNP composition containing albumin intron 3 targeting gRNA pairs and mRNA encoding Cas-CLOVER at days 0, 7, 14, and 28 post-administration. enu A graph showing serum Phe levels (µM) in mice. Detailed Implementation

[0024] AAV donor polynucleotide

[0025] In certain aspects of this disclosure, an AAV donor polynucleotide is provided comprising a promoterless bidirectional expression cassette for expressing a transgene upon integration into a target sequence in a cellular gene. In some embodiments, the AAV donor polynucleotide comprises first and second AAV ITRs located at the 5' and 3' ends. In some embodiments, the AAV donor polynucleotide comprises a first target sequence and a second target sequence homologous to a gene in the cellular genome and located near the first and second ITRs, respectively. The first and second target sequences are designed to laterally receive portions of target genes upstream and downstream of a site cleaved by a Cas-CLOVER enzyme in the genome to allow site-specific integration of the AAV donor polynucleotide into the cellular genome. Furthermore, the first and second target sequences are complementary to the first and second gRNAs of a gRNA pair, respectively, to allow Cas-CLOVER to cleave the AAV donor polynucleotide, linearizing the polynucleotide and cleaving the first and second AAV ITRs before integration into the cellular genome.

[0026] In some embodiments, the first and second target sequences each comprise nucleic acid sequences homologous to intron sequences of the gene. In some embodiments, the intron is intron 3 of the albumin gene. In some embodiments, the AAV donor polynucleotide targeting the intron comprises a splice acceptor sequence and a P2A ribosomal jumping sequence upstream of the transgene coding sequence, followed by a poly(A) polyadenylation sequence (poly(A)). The two expression cassettes are cloned into the AAV donor polynucleotide in opposite directions (i.e., reverse) to allow transgene expression regardless of whether the AAV donor polynucleotide is integrated at the Cas-CLOVER cleavage site in either a forward or reverse orientation, wherein transgene expression is controlled by regulatory elements and promoters targeting the albumin gene sequence.

[0027] In some aspects, the transgene is a reporter gene. In some embodiments, the transgene encodes a luciferase. In some aspects, the transgene is a human PAH gene. In one embodiment, the nucleotide sequence of the PAH gene is codon-optimized to improve the expression of the encoded human PAH enzyme. In other embodiments, the nucleotide sequence of the human PAH gene is modified to remove any unwanted restriction endonuclease recognition sites; and any putative cryptic splicing sites for cloning the modified sequence into an AAV donor vector. In some embodiments, the nucleotide sequence of the human PAH gene is codon-optimized and further modified as described above. In some embodiments, the human PAH gene further includes a hemagglutinin tag. Two exemplary AAV donor polynucleotides containing codon-optimized and modified PAH genes are schematically shown in Figures 1B and 1C, respectively.

[0028] In certain aspects of this disclosure, the AAV donor polynucleotide comprises a bidirectional luciferase reporter cassette. The AAV donor polynucleotide comprises, in the 5' to 3' direction: a) a first AAV ITR sequence; b) a first target sequence homologous to a first portion of intron 3 of the albumin gene; c) a splice acceptor sequence; d) a P2A sequence; e) a luciferase-coding sequence; f) a poly(A) sequence; g) a DNA spacer sequence; h) a reverse poly(A) sequence; i) a reverse luciferase-coding sequence; j) a reverse P2A sequence; k) a reverse splice acceptor sequence; l) a second target sequence homologous to a second portion of intron 3 of the albumin gene; and m) a second AAV ITR sequence. In one embodiment, the luciferase-coding sequence and the reverse-coding sequence differ in the primary nucleic acid sequence (having synonymous codons) to minimize the potential for internal homologous recombination between the two coding sequences.

[0029] In one embodiment, the first AAV ITR sequence comprises the nucleic acid of SEQ ID NO: 12. In one embodiment, the first target sequence homologous to the upstream portion of intron 3 of the albumin gene comprises the nucleic acid of SEQ ID NO: 13. In one embodiment, the first splice acceptor sequence comprises the nucleic acid of SEQ ID NO: 14. In one embodiment, the P2A sequence comprises the nucleic acid of SEQ ID NO: 15. In some embodiments, the luciferase-coding sequence comprises the nucleic acid of SEQ ID NO: 16. In one embodiment, the poly(A) sequence comprises the nucleic acid of SEQ ID NO: 17. In one embodiment, the DNA spacer sequence comprises the nucleic acid of SEQ ID NO: 18; in one embodiment, the reverse poly(A) sequence comprises the nucleic acid of SEQ ID NO: 19; in one embodiment, the reverse luciferase-coding sequence comprises the nucleic acid of SEQ ID NO: 20. In one embodiment, the reverse P2A sequence comprises the nucleic acid of SEQ ID NO: 21. In one embodiment, the reverse splice acceptor sequence comprises the nucleic acid of SEQ ID NO: 22. In one embodiment, the second target sequence homologous to the second part of intron 3 of the albumin gene contains the nucleic acid of SEQ ID NO: 23; and the second AAV ITR sequence gene contains the nucleic acid of SEQ ID NO: 24.

[0030] In some aspects of this disclosure, the AAV donor polynucleotide comprises a two-way luciferase reporter cassette. In one embodiment, the transgenic PAH nucleotide sequence further comprises a hemagglutinin (HA) tag. In one embodiment, the transgenic PAH nucleotide sequence is codon-optimized. In one embodiment, the transgenic PAH nucleotide sequence is modified. In one embodiment, the transgenic PAH nucleotide sequence comprises a hemagglutinin tag, is codon-optimized, and is further modified as disclosed herein. In one embodiment, the nucleic acid sequence encoding the human PAH gene sequence comprises the nucleic acid of SEQ ID NO: 25. In one embodiment, the PAH gene nucleic acid coding sequence and the reverse PAH nucleic acid coding sequence differ in the primary nucleic acid sequence (having synonymous codons) to minimize the potential for internal homologous recombination between the two coding sequences.

[0031] In certain aspects of this disclosure, the AAV donor polynucleotide comprises a bidirectional PAH expression cassette. The AAV donor polynucleotide comprises, in the 5' to 3' direction: a) a first AAV ITR sequence; b) a first target sequence homologous to a first portion of intron 3 of the albumin gene; c) a splice acceptor sequence; d) a P2A sequence; e) a PAH coding sequence; f) a poly(A) sequence; g) a DNA spacer sequence; h) a reverse poly(A) sequence; i) a reverse PAH coding sequence; j) a reverse P2A sequence; k) a reverse splice acceptor sequence; l) a second target sequence homologous to a second portion of intron 3 of the albumin gene; and m) a second AAV ITR sequence. In one embodiment, the luciferase nucleic acid coding sequence and the reverse nucleic acid coding sequence differ in the primary nucleic acid sequence (having synonymous codons) to minimize the potential for internal homologous recombination between the two coding sequences.

[0032] In one embodiment, the first AAV ITR sequence comprises the nucleic acid of SEQ ID NO: 12. In one embodiment, the first target sequence homologous to the first portion of intron 3 of the albumin gene comprises the nucleic acid of SEQ ID NO: 13. In one embodiment, the first splice acceptor sequence comprises the nucleic acid of SEQ ID NO: 14. In one embodiment, the P2A sequence comprises the nucleic acid of SEQ ID NO: 15. In one embodiment, the PAH coding sequence comprises the nucleic acid of SEQ ID NO: 25. In one embodiment, the poly(A) sequence comprises the nucleic acid of SEQ ID NO: 17. In one embodiment, the DNA spacer sequence comprises the nucleic acid of SEQ ID NO: 18; in one embodiment, the reverse poly(A) sequence comprises the nucleic acid of SEQ ID NO: 19; in one embodiment, the reverse PAH coding sequence comprises the nucleic acid of SEQ ID NO: 26. In one embodiment, the reverse P2A sequence comprises the nucleic acid of SEQ ID NO: 21. In one embodiment, the reverse splice acceptor sequence comprises the nucleic acid of SEQ ID NO: 22. In one embodiment, the second target sequence homologous to the second part of intron 3 of the albumin gene contains the nucleic acid of SEQ ID NO: 23; and the second AAV ITR sequence gene contains the nucleic acid of SEQ ID NO: 24.

[0033] In some aspects of this disclosure, the AAV donor polynucleotide comprises a TTRe-PAH expression cassette. In one embodiment, expression of the PAH gene is controlled by a heterologous TTRe promoter operatively linked to the PAH coding sequence.

[0034] In some embodiments, the AAV donor polynucleotide comprises, in the 5' to 3' direction: a) a first AAV ITR sequence; b) a first target sequence homologous to a first portion of intron 3 of the albumin gene; c) a TTRe promoter sequence; d) a PAH coding sequence; d) a 3'UTR sequence; f) a poly(A) sequence; g) a second target sequence homologous to a second portion of intron 3 of the albumin gene; and h) a second AAV ITR sequence.

[0035] In one embodiment, the first AAV ITR sequence comprises the nucleic acid of SEQ ID NO: 12. In one embodiment, the first target sequence homologous to the upstream portion of intron 3 of the albumin gene comprises the nucleic acid of SEQ ID NO: 13. In one embodiment, the TTRe promoter sequence comprises the nucleic acid of SEQ ID NO: 27. In one embodiment, the PAH coding sequence comprises the nucleic acid of SEQ ID NO: 25. In one embodiment, the 3'-UTR sequence comprises the nucleic acid of SEQ ID NO: 27. In one embodiment, the poly(A) sequence comprises the nucleic acid of SEQ ID NO: 17. In one embodiment, the first target sequence homologous to the second portion of intron 3 of the albumin gene comprises the nucleic acid of SEQ ID NO: 23; and the second AAV ITR sequence gene comprises the nucleic acid of SEQ ID NO: 24.

[0036] lipid nanoparticles

[0037] This disclosure provides a composition comprising at least one lipid nanoparticle containing at least one cationic lipid and at least one nucleic acid molecule. In some aspects, the lipid nanoparticle may further comprise at least one structural lipid. In some aspects, the lipid nanoparticle may further comprise at least one phospholipid. In some aspects, the lipid nanoparticle may further comprise at least one polyethylene glycol-modified lipid.

[0038] Therefore, this disclosure provides a composition comprising at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises at least one cationic lipid, at least one nucleic acid molecule, at least one structural lipid, at least one phospholipid and at least one polyethylene glycol-modified lipid.

[0039] Bioreducible ionizable cationic lipids

[0040] In some respects, cationic lipids can be bioreducible, ionizable cationic lipids.

[0041] Therefore, this disclosure provides compositions comprising at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises at least one bioreducible ionizable cationic lipid.

[0042] As used herein, the term “bioreducible ionizable cationic lipid” is used in its broadest sense to refer to a cationic lipid comprising: at least one tertiary amine, at least one disulfide group, at least one group comprising a bond readily cleaved by thioesterification, and further comprising at least two saturated or unsaturated hydrocarbon chains. Exemplary bioreducible ionizable cationic lipids include, but are not limited to, those described in Akita et al., (2020) Biol. Phar. Bull. 43:1617-1625, the contents of which are incorporated herein by reference in their entirety.

[0043] Other exemplary bioreducible ionizable cationic lipids and methods for preparing such lipids that can be used in the methods of this disclosure include those disclosed in International Patent Application No. PCT / JP2016 / 052690 (published as WO / 2016 / 121942) and International Patent Application No. PCT / JP2019 / 012302 (published as WO / 2019 / 188867), as examples of lipids that can be used in the compositions disclosed herein and methods for preparing the compositions disclosed herein, the contents of each of which are incorporated herein by reference in their entirety.

[0044] Therefore, this disclosure provides compositions comprising at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises any one of the bioreducible ionizable cationic lipids proposed in WO / 2016 / 121942 and WO / 2019 / 188867.

[0045] Therefore, this disclosure provides a composition comprising at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises at least one bioreducible ionizable cationic lipid, at least one nucleic acid molecule, at least one structural lipid, at least one phospholipid and at least one polyethylene glycol-modified lipid.

[0046] In some respects, bioreducible, ionizable cationic lipids can be ssPalmO-Ph-P4C2 with the following structure:

[0047]

[0048] (Formula I)

[0049] See Akita et al., (2020) Biol. Phar. Bull. 43: 1617-1625, as an example of lipids that can be used in the compositions disclosed herein, the contents of which are incorporated herein by reference in their entirety.

[0050] Therefore, this disclosure provides a composition comprising at least one lipid nanoparticle, the lipid nanoparticle comprising at least one bioreducible ionizable cationic lipid, wherein the at least one bioreducible ionizable cationic lipid comprises ssPalmO-Ph-P4C2.

[0051] As will be understood by those skilled in the art, ssPalmO-Ph-P4C2 may also be referred to as Coatsome® SS-OP, ssPalmO-Phe-P4C2, ssPalmO-Phenyl-P4C2, ssPalmO-Phe, and ssPalmO-Ph. Therefore, ssPalmO-Ph-P4C2, Coatsome® SS-OP, ssPalmO-Phe-P4C2, ssPalmO-Phenyl-P4C2, ssPalmO-Phe, and ssPalmO-Ph are used interchangeably herein to refer to bioreducible ionizable cationic lipids having the chemical structure presented in Formula I.

[0052] As described herein, the LNP compositions of this disclosure, comprising at least one bioreducible ionizable cationic lipid, advantageously exhibit significantly reduced toxicity in animals compared to LNP compositions comprising non-bioreducible ionizable cationic lipids. In particular, administration of the LNP compositions of this disclosure surprisingly does not result in any weight loss. In some respects, certain LNP compositions of this disclosure are sufficiently non-toxic that animals administered LNP actually gain weight, even if the amount of LNP administered exceeds the lethal dose of LNP compositions comprising non-bioreducible ionizable cationic lipids.

[0053] LNP components

[0054] In some aspects, the LNP of this disclosure may comprise, on a molar basis, at least one bioreducible ionizable cationic lipid of about 2.5%, or about 5%, or about 7.5%, or about 10%, or about 12.5%, or about 15%, or about 17.5%, or about 20%, or about 22.5%, or about 25%, or about 27.5%, or about 30%, or about 32.5%, or about 35%, or about 37.5%, or about 40%, or about 42.5%, or about 45%, or about 47.5%, or about 50%, or about 52.5%, or about 55%, or about 57.5%, or about 60%, or about 62.5%, or about 65%, or about 67.5%, or about 70%.

[0055] In some aspects, the LNP of this disclosure may comprise at least one bioreducible ionizable cationic lipid in amounts of at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10%, or at least about 12.5%, or at least about 15%, or at least about 17.5%, or at least about 20%, or at least about 22.5%, or at least about 25%, or at least about 27.5%, or at least about 30%, or at least about 32.5%, or at least about 35%, or at least about 37.5%, or at least about 40%, or at least about 42.5%, or at least about 45%, or at least about 47.5%, or at least about 50%, or at least about 52.5%, or at least about 55%, or at least about 57.5%, or at least about 60%, or at least about 62.5%, or at least about 65%, or at least about 67.5%, or at least about 70% on a molar basis.

[0056] In some aspects, the LNP of this disclosure may further comprise, on a molar basis, at least 2.5%, or about 5%, or about 7.5%, or about 10%, or about 12.5%, or about 15%, or about 17.5%, or about 20%, or about 22.5%, or about 25%, or about 27.5%, or about 30%, or about 32.5%, or about 35%, or about 37.5%, or about 40%, or about 42.5%, or about 45%, or about 47.5%, or about 50%, or about 52.5%, or about 55%, or about 57.5%, or about 60%, or about 62.5%, or about 65%, or about 67.5%, or about 70% of at least one structural lipid.

[0057] In some aspects, the LNP of this disclosure may comprise at least one structural lipid in a molar amount of at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10%, or at least about 12.5%, or at least about 15%, or at least about 17.5%, or at least about 20%, or at least about 22.5%, or at least about 25%, or at least about 27.5%, or at least about 30%, or at least about 32.5%, or at least about 35%, or at least about 37.5%, or at least about 40%, or at least about 42.5%, or at least about 45%, or at least about 47.5%, or at least about 50%, or at least about 52.5%, or at least about 55%, or at least about 57.5%, or at least about 60%, or at least about 62.5%, or at least about 65%, or at least about 67.5%, or at least about 70%.

[0058] In some aspects, the LNP disclosed herein may comprise at least one phospholipid in a molar amount of about 2.5%, or about 5%, or about 7.5%, or about 10%, or about 12.5%, or about 15%, or about 17.5%, or about 20%, or about 22.5%, or about 25%, or about 27.5%, or about 30%, or about 32.5%, or about 35%, or about 37.5%, or about 40%, or about 42.5%, or about 45%, or about 47.5%, or about 50%, or about 52.5%, or about 55%, or about 57.5%, or about 60%, or about 62.5%, or about 65%, or about 67.5%, or about 70%.

[0059] In some aspects, the LNP disclosed herein may contain at least one phospholipid in a molar amount of at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10%, or at least about 12.5%, or at least about 15%, or at least about 17.5%, or at least about 20%, or at least about 22.5%, or at least about 25%, or at least about 27.5%, or at least about 30%, or at least about 32.5%, or at least about 35%, or at least about 37.5%, or at least about 40%, or at least about 42.5%, or at least about 45%, or at least about 47.5%, or at least about 50%, or at least about 52.5%, or at least about 55%, or at least about 57.5%, or at least about 60%, or at least about 62.5%, or at least about 65%, or at least about 67.5%, or at least about 70%.

[0060] In some aspects, the LNP disclosed herein may contain at least one polyethylene glycol-modified lipid in an amount of about 0.25%, or about 0.5%, or about 0.75%, or about 1.0%, or about 1.25%, or about 1.5%, or about 1.75%, or about 2.0%, or at least about about 2.5%, or about 5% on a molar basis.

[0061] In some aspects, the LNP of this disclosure may contain at least one polyethylene glycol-modified lipid in an amount of at least about 0.25%, or at least about 0.5%, or at least about 0.75%, or at least about 1.0%, or at least about 1.25%, or at least about 1.5%, or at least about 1.75%, or at least about 2.0%, or at least about 2.5%, or at least about 5% on a molar basis.

[0062] Structural lipids

[0063] In some respects, structured lipids can be steroids. In some respects, structured lipids can be sterols. In some respects, structured lipids can include cholesterol. In some respects, structured lipids can include ergosterol. In some respects, structured lipids can be phytosterols.

[0064] Phospholipids

[0065] As used in this article, the term "phospholipid" is used in its broadest sense to refer to any amphiphilic molecule containing a polar (hydrophilic) head group (containing a phosphate ester and two hydrophobic fatty acid chains).

[0066] In some aspects of the lipid nanoparticles disclosed herein, phospholipids may include dioleoylphosphatidylethanolamine (DOPE).

[0067] In some aspects of the lipid nanoparticles disclosed herein, phospholipids may include DOPC (1,2-dioleoyl-sn-glycerol-3-phosphocholine).

[0068] In some aspects of the lipid nanoparticles disclosed herein, phospholipids may include DSPC (1,2-distearate-sn-glycerol-3-phosphocholine).

[0069] In some respects, phospholipids may include DDPC (1,2-didecanoyl-sn-glycerol-3-phosphate choline), DEPA-NA (1,2-disurenoyl-sn-glycerol-3-phosphate (sodium salt)), DEPC (1,2-disurenoyl-sn-glycerol-3-phosphate choline), DEPE (1,2-disurenoyl-sn-glycerol-3-phosphate ethanolamine), DEPG-NA (1,2-disurenoyl-sn-glycerol-3[phosphate-racemic-(1-glycerol) (sodium salt)), DLOPC (1,2-dilinoleoyl-sn-glycerol-3-phosphate choline), DLPA-NA (1,2-dilauroyl-sn-glycerol-3-phosphate (sodium salt)), DLPC (1 DLPE (1,2-dilauroyl-sn-glycerol-3-phosphate ethanolamine), DLPG-NA (1,2-dilauroyl-sn-glycerol-3-phosphate racemic-(1-glycerol) (sodium salt)), DLPG-NH4 (1,2-dilauroyl-sn-glycerol-3-phosphate racemic-(1-glycerol) (ammonium salt)), DLPS-NA (1,2-dilauroyl-sn-glycerol-3-phosphate serine (sodium salt)), DMPA-NA (1,2-dimyristoyl-sn-glycerol-3-phosphate (sodium salt)), DMPC (1,2-dimyristoyl-sn-glycerol-3-phosphate choline). DMPE (1,2-Dimyristicoyl-sn-glycerol-3-phosphate ethanolamine), DMPG-NA (1,2-Dimyristicoyl-sn-glycerol-3-[phosphate-racemic-(1-glycerol) (sodium salt)), DMPG-NH4 (1,2-Dimyristicoyl-sn-glycerol-3-[phosphate-racemic-(1-glycerol) (ammonium salt)), DMPG-NH4 / NA (1,2-Dimyristicoyl-sn-glycerol-3-[phosphate-racemic-(1-glycerol) (sodium salt / ammonium salt)), DMPS-NA (1,2-Dimyristicoyl-sn-glycerol-3-phosphoserine (sodium salt)), DOPA-NA (1,2-dioleoyl-sn-glycerol-3-phosphate (sodium salt)), DMPG-NA (1,2-dioleoyl-sn-glycerol-3-phosphate (sodium salt)). Sodium salt), DOPC (1,2-dioleoyl-sn-glycerol-3-phosphate choline), DOPE (1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine), DOPG-NA (1,2-dioleoyl-sn-glycerol-3-phosphate racemic-(1-glycerol) (sodium salt)), DOPS-NA (1,2-dioleoyl-sn-glycerol-3-phosphate serine (sodium salt)), DPPA-NA (1,2-dipalmitoyl-sn-glycerol-3-phosphate (sodium salt)), DPPC (1,2-dipalmitoyl-sn-glycerol-3-phosphate choline), DPPE (1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine), DPPG-NA (1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine), DPPG-NA (1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine), DPPG-NA (1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine), DPPC (1,2-dipalmitoyl-sn-glycerol-3-phosphate choline), DPPE (1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine), DPPG-NA ... serine), DPPC (1,2-dipalmitoyl-sn-glycerol-3-phosphate serine), DPPG-NA (1,2-dipalmitoyl-sn-glycerol-3-phosphate serine), DPPG-NA (1,2-dipalmitoyl-sn2-Dipalmitoyl-sn-glycerol-3-[phosphate-racemic-(1-glycerol) (sodium salt)), DPPG-NH4 (1,2-dipalmitoyl-sn-glycerol-3-[phosphate-racemic-(1-glycerol) (ammonium salt)), DPPS-NA (1,2-dipalmitoyl-sn-glycerol-3-phosphoserine (sodium salt)), DSPA-NA (1,2-distearyl-sn-glycerol-3-phosphate (sodium salt)), DSPC (1,2-distearyl-sn-glycerol-3-phosphocholine), DSPE (1,2-distearyl-sn-glycerol-3-phosphocholine) DSPG-NA (1,2-distearyl-sn-glycerol-3-phosphate ethanolamine), DSPG-NH4 (1,2-distearyl-sn-glycerol-3-phosphate ethanolamine), DSPS-NA (1,2-distearyl-sn-glycerol-3-phosphate racemic-(1-glycerol) (sodium salt)), EPC (egg phosphatidylcholine), HEPC (hydrogenated egg phosphatidylcholine), HSPC (hydrogenated soybean phosphatidylcholine), LYSOPC MYRISTIC (1-myristoyl-sn-glycerol-3-phosphate choline), LYSOPC PALMITIC (1-palmitoyl-sn-glycerol-3-phosphate choline), LYSOPC STEARIC (1-stearoyl-sn-glycerol-3-phosphocholine), milk sphingomyelin (MPPC; 1-myristoyl-2-palmitoyl-sn-glycerol-3-phosphocholine), MSPC (1-myristoyl-2-stearoyl-sn-glycerol-3-phosphocholine), PMPC (1-palmitoyl-2-myristoyl-sn-glycerol-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine). POPG-NA (1-palmitoyl-2-oleoyl-sn-glycerol-3-[phosphate-racemic-(1-glycerol)] (sodium salt)), PSPC (1-palmitoyl-2-stearoyl-sn-glycerol-3-phosphocholine), SMPC (1-stearoyl-2-myristoyl-sn-glycerol-3-phosphocholine), SOPC (1-stearoyl-2-oleoyl-sn-glycerol-3-phosphocholine), SPPC (1-stearoyl-2-palmitoyl-sn-glycerol-3-phosphocholine), or any combination thereof.

[0070] PEGylated lipids

[0071] As used herein, the term "polyethylene glycolated lipid" is used to refer to any modified lipid (e.g., covalently linked to at least one polyethylene glycol molecule). In some aspects, polyethylene glycolated lipids may include 1,2-dimyristoyl-racemic-glycerol-3-methoxy polyethylene glycol-2000, hereinafter referred to as DMG-PEG2000.

[0072] Nucleic acid

[0073] In some respects, lipid nanoparticles may contain at least one nucleic acid molecule. In some respects, lipid nanoparticles may contain multiple nucleic acid molecules. In some respects, at least one or more nucleic acid molecules may be formulated in lipid nanoparticles.

[0074] In some aspects, the nucleic acid molecule may be a synthetic nucleic acid molecule. In some aspects, the nucleic acid molecule may be a non-naturally occurring nucleic acid molecule. In some aspects, a non-naturally occurring nucleic acid molecule may contain at least one non-naturally occurring nucleotide. The at least one non-naturally occurring nucleotide may be any non-naturally occurring nucleotide known in the art. In some aspects, the nucleic acid molecule may be a modified nucleic acid molecule. In some aspects, a modified nucleic acid molecule may contain at least one modified nucleotide. The at least one modified nucleotide may be any modified nucleic acid known in the art. In some embodiments, the modified nucleic acid is gRNA or a gRNA pair. In some embodiments, the modified nucleic acid is mRNA encoding Cas-CLOVER.

[0075] In some respects, lipid nanoparticles may contain a specified ratio (by weight) of lipids and nucleic acids.

[0076] In some aspects, lipid nanoparticles comprising at least one nucleic acid molecule can be in ratios of about 5:1 to about 15:1, or about 10:1 to about 20:1, or about 15:1 to about 25:1, or about 20:1 to about 30:1, or about 25:1 to about 35:1, or about 30:1 to about 40:1, or about 35:1 to about 45:1, or about 40:1 to about 50:1, or about 45:1 to about 55:1, or about 50:1 to about 60:1, or about 55:1 to about 65:1, or about 60:1 to about 70:1, or about 65:1 to about 75:1, or about 70:1 to about 80:1, or about 75:1 to about 85:1, or about 80:1 to about 90:1, or about 85:1 to about 95:1, or about 90:1. The ratio of lipids to nucleic acids, weight / weight, to about 100:1, or about 95:1 to about 105:1, or about 100:1 to about 110:1, or about 105:1 to about 115:1, or about 110:1 to about 120:1, or about 115:1 to about 125:1, or about 120:1 to about 130:1, or about 125:1 to about 135:1, or about 130:1 to about 140:1, or about 135:1 to about 145:1, or about 140:1 to about 150:1 (lipids:nucleic acids, weight / weight) includes both lipids and nucleic acids.

[0077] In some aspects, lipid nanoparticles may comprise a lipid:nucleic acid weight / weight ratio of about 5:1, or about 10:1, or about 15:1, or about 20:1, or about 25:1, or about 30:1, or about 35:1, or about 40:1, or about 45:1, or about 50:1, or about 55:1, or about 60:1, or about 65:1, or about 70:1, or about 75:1, or about 80:1, or about 85:1, or about 90:1, or about 95:1, or about 100:1, or about 105:1, or about 110:1, or about 115:1, or about 120:1, or about 125:1, or about 130:1, or about 135:1, or about 140:1, or about 145:1, or about 150:1, or about 200:1. Lipids and nucleic acids.

[0078] In some respects, lipid nanoparticles may comprise lipids and nucleic acids in a lipid:nucleic acid weight / weight ratio of about 10:1, or about 17.5:1, or about 25:1.

[0079] In some respects, nucleic acid molecules can be RNA molecules. Therefore, in some respects, lipid nanoparticles can contain at least one RNA molecule. In some respects, the RNA molecule can be an mRNA molecule. In some respects, the mRNA molecule can contain a 5'-cap.

[0080] In some respects, mRNA molecules can be capped using any method and / or capping motif known in the art. mRNA molecules can be capped using the m7G(5')ppp(5')G motif. The m7G(5')ppp(5')G motif is also referred to herein as “CapO”. mRNA molecules can be capped using the CleanCap® motif. The CleanCap® motif may contain the m7G(5')ppp(5')(2'OMeA) (CleanCap® AG) motif. The CleanCap® motif may contain the m7G(5')ppp(5')(2'OMeG) (CleanCap® GG) motif. mRNA molecules can be capped using the anti-reverse cap analogue (ARCA®) motif. The ARCA® motif may contain the m7(3'-O-methyl)G(5')ppp(5')G motif. mRNA molecules can be capped using the CleanCap® 3'OMe motif (CleanCap®+ARCA®).

[0081] In some respects, mRNA molecules may contain at least one modified nucleic acid.

[0082] Modified nucleic acids may include, but are not limited to, 5-methoxyuridine (5 moU) and N1-methylpseuuridine (me 1 ψ), pseudouridine (Y), and 5-methylcytidine (5-MeC).

[0083] In some respects, nucleic acid molecules can be DNA molecules. Therefore, in some respects, lipid nanoparticles can contain at least one DNA molecule. In some respects, the DNA molecule can be a circular DNA molecule, such as, but not limited to, a DNA plasmid. In some respects, lipid nanoparticles can contain a DNA plasmid. In some respects, the DNA molecule can be a linearized DNA molecule, such as, but not limited to, a linearized DNA plasmid. In some respects, the DNA molecule can be a DoggyBone DNA molecule. In some respects, the DNA molecule can be a DNA nanoparticle.

[0084] The DNA plasmid length can be at least about 0.25 kb, or at least about 0.5 kb, or at least about 0.75 kb, or at least about 1.0 kb, or at least about 1.25 kb, or at least about 1.5 kb, or at least about 1.75 kb, or at least about 2.0 kb, or at least about 2.25 kb, or at least about 2.5 kb, or at least about 2.75 kb, or at least about 3.0 kb, or at least about 3.25 kb, or at least about 3.5 kb, or at least about 3.75 kb, or at least about 4.0 kb, or at least about 4.25 kb, or at least about 4.5 kb, or at least about 4.75 kb, or at least about 5.0 kb, or at least about 5.25 kb, or at least about 5.5 kb, or at least about 5.75 kb, or at least about 6.0 kb, or at least about 6.25 kb. kb, or at least about 6.5 kb, or at least about 6.75 kb, or at least about 7.0 kb, or at least about 7.25 kb, or at least about 7.5 kb, or at least about 7.75 kb, or at least about 8.0 kb, or at least about 8.25 kb, or at least about 8.5 kb, or at least about 8.75 kb, or at least about 9.0 kb, or at least about 9.25 kb, or at least about 9.5 kb, or at least about 9.75 kb, or at least about 10.0 kb, or at least about 10.25 kb, or at least about 10.5 kb, or at least about 10.75 kb, or at least about 11.0 kb, or at least about 11.25 kb, or at least about 11.5 kb, or at least about 11.75 kb, or at least about 12 kb, or at least about 12.25 kb, or at least about 12.5 kb. kb, or at least about 12.75 kb, or at least about 13.0 kb, or at least about 13.25 kb, or at least about 13.5 kb, or at least about 13.75 kb, or at least about 14.0 kb, or at least about 14.25 kb, or at least about 14.5 kb, or at least about 14.75 kb, or at least about 15.0 kb.

[0085] LNP Composition

[0086] In some aspects, lipid nanoparticles may comprise at least one nucleic acid molecule, at least one bioreducible ionizable cationic lipid, and at least one structural lipid. In some aspects, lipid nanoparticles may comprise at least one nucleic acid molecule, at least one bioreducible ionizable cationic lipid, and at least one polyethylene glycol-modified lipid. In some aspects, the at least one bioreducible ionizable cationic lipid may be ssPalmO-Ph-P4C2.

[0087] In some aspects, at least one structural lipid may be a mixture of two structural lipids. In some aspects, at least one polyethylene glycol-modified lipid may be a mixture of two polyethylene glycol-modified lipids.

[0088] In some aspects, lipid nanoparticles may comprise at least one nucleic acid molecule, at least one bioreducible ionizable cationic lipid, at least one structural lipid, at least one polyethylene glycol-modified lipid, or any combination thereof. In some aspects, the at least one bioreducible ionizable cationic lipid may be ssPalmO-Ph-P4C2.

[0089] In some respects, lipid nanoparticles may comprise at least one nucleic acid molecule, at least one bioreducible ionizable cationic lipid, at least one structural lipid, and at least one polyethylene glycol-modified lipid. In some respects, the at least one bioreducible ionizable cationic lipid may be ssPalmO-Ph-P4C2.

[0090] In some aspects, lipid nanoparticles may comprise at least one nucleic acid molecule, at least one bioreducible ionizable cationic lipid, at least one structural lipid, at least one phospholipid, at least one polyethylene glycol-modified lipid, or any combination thereof. In some aspects, the at least one bioreducible ionizable cationic lipid may be ssPalmO-Ph-P4C2.

[0091] In some aspects, lipid nanoparticles may comprise at least one nucleic acid molecule, at least one bioreducible ionizable cationic lipid, at least one structural lipid, at least one phospholipid, and at least one polyethylene glycol-modified lipid. In some aspects, the at least one bioreducible ionizable cationic lipid may be ssPalmO-Ph-P4C2.

[0092] In some aspects, the nucleic acid molecule is an RNA molecule. Therefore, in some aspects, lipid nanoparticles containing at least one nucleic acid molecule may contain approximately 54% ssPalmO-Ph-P4C2 (by mole), approximately 35% cholesterol (by mole), approximately 5% DOPC (by mole), approximately 5% DSPC (by mole), and approximately 1% DMG-PEG2000 (by mole), wherein the lipid nanoparticle further contains at least one mRNA molecule. In some aspects, the mRNA molecule further contains a 5'-cap. In some aspects, the lipid to nucleic acid ratio in at least one nanoparticle may be approximately 100:1 (w / w).

[0093] In some aspects, lipid nanoparticles comprising at least one nucleic acid molecule may comprise ssPalmO-Ph-P4C2 at a molar weight of about 44% to 64%; cholesterol at a molar weight of about 25% to 45%; DOPC at a molar weight of about 0.1% to 20%; DSPC at a molar weight of about 0.1% to 20%; and DMG-PEG2000 at a molar weight of about 0.1% to 11%, wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule. In some aspects, lipid nanoparticles comprising at least one nucleic acid molecule may comprise, on a molar basis, ssPalmO-Ph-P4C2 at a molar basis of about 49% to 59%; cholesterol at a molar basis of about 30% to 40%; DOPC at a molar basis of about 5% to 15%; DSPC at a molar basis of about 5% to 15%; and DMG-PEG2000 at a molar basis of about 0.5% to 6%, wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule. In some aspects, the mRNA molecule further comprises a 5'-cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be from about 75:1 to about 100:1 (w / w).

[0094] In some aspects, the nucleic acid molecule is a DNA molecule. Therefore, the lipid nanoparticles comprising at least one nucleic acid molecule provided in this disclosure may comprise about 54% ssPalmO-Ph-P4C2 (by mole), about 35% cholesterol (by mole), about 5% DOPC (by mole), about 5% DSPC (by mole), and about 1% DMG-PEG2000 (by mole), wherein the lipid nanoparticles further comprise at least one DNA molecule. In some aspects, the at least one DNA molecule may be a DoggyBone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoparticle. In some aspects, the at least one DNA molecule may be terminally covalently closed DNA (see WO / 2020 / 154645). In some aspects, the ratio of lipid to nucleic acid in the nanoparticles may be about 100:1 (w / w).

[0095] In some aspects, lipid nanoparticles comprising at least one nucleic acid molecule may comprise ssPalmO-Ph-P4C2 at a molar weight of about 44% to 64%; cholesterol at a molar weight of about 25% to 45%; DOPC at a molar weight of about 0.1% to 20%; DSPC at a molar weight of about 5% to 15%; and DMG-PEG2000 at a molar weight of about 0.1% to 11%, wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one DNA molecule. In some aspects, lipid nanoparticles comprising at least one nucleic acid molecule may comprise, on a molar basis, ssPalmO-Ph-P4C2 at a concentration of about 49% to 59%; cholesterol at a concentration of about 30% to 40%; DOPC at a concentration of about 5% to 10%; DSPC at a concentration of about 5% to 10%; and DMG-PEG2000 at a concentration of about 0.5% to 6%, wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a DoggyBone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoparticle. In some aspects, the at least one DNA molecule may be terminally covalently closed DNA. In some aspects, the lipid to nucleic acid ratio in the at least one nanoparticle may be about 75:1 to about 100:1 (w / w).

[0096] Exemplary LNP compositions that can be used in the methods of this disclosure and methods for preparing such LNP compositions include those disclosed in International Patent Application No. PCT / US2022 / 017570 (published as WO / 2022 / 182792), the contents of which are incorporated herein by reference in their entirety.

[0097] Therefore, this disclosure provides LNP compositions comprising any LNP composition proposed in WO / 2022 / 182792, examples of LNPs that can be used in the compositions provided herein and are incorporated herein by reference in their entirety. In some aspects, lipid nanoparticles comprising at least one nucleic acid molecule may comprise, on a molar basis, between about 54% and 59% ssPalmO-Ph-P4C2, on a molar basis, between about 30% and 40% cholesterol, on a molar basis, between about 5% and 10% DOPC, DSPC, or DOPE, and on a molar basis, about 1% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5'-cap. In some aspects, the ratio of lipid to nucleic acid in at least one nanoparticle may be between about 75:1 and about 100:1 (w / w). In some aspects, an mRNA encodes Cas-CLOVER. In some aspects, the lipid nanoparticle further comprises at least one gRNA. In some respects, the lipid nanoparticles further comprise at least one gRNA pair, comprising a left gRNA and a right gRNA.

[0098] In some aspects, lipid nanoparticles containing at least one nucleic acid molecule may contain about 54% ssPalmO-Ph-P4C2 (by mole), about 35% cholesterol (by mole), about 10% DOPC (by mole), and about 1% DMG-PEG2000 (by mole), wherein the lipid nanoparticles further contain at least one mRNA molecule. In some aspects, the mRNA molecule further contains a 5'-cap. In some aspects, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w). In some aspects, one mRNA encodes Cas-CLOVER. In some aspects, the lipid nanoparticles further contain at least one gRNA. In some aspects, the lipid nanoparticles further contain at least one gRNA pair, comprising a left gRNA and a right gRNA.

[0099] In some aspects, lipid nanoparticles containing at least one nucleic acid molecule may contain about 54% ssPalmO-Ph-P4C2 (by mole), about 35% cholesterol (by mole), about 10% DOPC (by mole), and about 1% DMG-PEG2000 (by mole), wherein the lipid nanoparticles further contain at least one mRNA molecule. In some aspects, the mRNA molecule further contains a 5'-cap. In some aspects, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 75:1 (w / w). In some aspects, one mRNA encodes Cas-CLOVER. In some aspects, the lipid nanoparticles further contain at least one gRNA. In some aspects, the lipid nanoparticles further contain at least one gRNA pair, comprising a left gRNA and a right gRNA.

[0100] In some aspects, lipid nanoparticles containing at least one nucleic acid molecule may contain about 56.5% ssPalmO-Ph-P4C2 (by mole), about 32.5% cholesterol (by mole), about 10% DOPC (by mole), and about 1% DMG-PEG2000 (by mole), wherein the lipid nanoparticles further contain at least one mRNA molecule. In some aspects, the mRNA molecule further contains a 5'-cap. In some aspects, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w). In some aspects, one mRNA encodes Cas-CLOVER. In some aspects, the lipid nanoparticles further contain at least one gRNA. In some aspects, the lipid nanoparticles further contain at least one gRNA pair, comprising a left gRNA and a right gRNA.

[0101] In some aspects, lipid nanoparticles containing at least one nucleic acid molecule may contain about 56.5% ssPalmO-Ph-P4C2 (by mole), about 32.5% cholesterol (by mole), about 10% DOPC (by mole), and about 1% DMG-PEG2000 (by mole), wherein the lipid nanoparticles further contain at least one mRNA molecule. In some aspects, the mRNA molecule further contains a 5'-cap. In some aspects, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 75:1 (w / w). In some aspects, one mRNA encodes Cas-CLOVER. In some aspects, the lipid nanoparticles further contain at least one gRNA. In some aspects, the lipid nanoparticles further contain at least one gRNA pair, comprising a left gRNA and a right gRNA.

[0102] In some aspects, lipid nanoparticles containing at least one nucleic acid molecule may contain about 59% ssPalmO-Ph-P4C2 (by mole), about 30% cholesterol (by mole), about 10% DOPC (by mole), and about 1% DMG-PEG2000 (by mole), wherein the lipid nanoparticles further contain at least one mRNA molecule. In some aspects, the mRNA molecule further contains a 5'-cap. In some aspects, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w). In some aspects, one mRNA encodes Cas-CLOVER. In some aspects, the lipid nanoparticles further contain at least one gRNA. In some aspects, the lipid nanoparticles further contain at least one gRNA pair, comprising a left gRNA and a right gRNA.

[0103] In some aspects, lipid nanoparticles containing at least one nucleic acid molecule may contain about 59% ssPalmO-Ph-P4C2 (by mole), about 30% cholesterol (by mole), about 10% DOPC (by mole), and about 1% DMG-PEG2000 (by mole), wherein the lipid nanoparticles further contain at least one mRNA molecule. In some aspects, the mRNA molecule further contains a 5'-cap. In some aspects, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 75:1 (w / w). In some aspects, one mRNA encodes Cas-CLOVER. In some aspects, the lipid nanoparticles further contain at least one gRNA. In some aspects, the lipid nanoparticles further contain at least one gRNA pair, comprising a left gRNA and a right gRNA.

[0104] In some aspects, lipid nanoparticles containing at least one nucleic acid molecule may contain about 54% ssPalmO-Ph-P4C2 (by mole), about 40% cholesterol (by mole), about 5% DOPC (by mole), and about 1% DMG-PEG2000 (by mole), wherein the lipid nanoparticles further contain at least one mRNA molecule. In some aspects, the mRNA molecule further contains a 5'-cap. In some aspects, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w). In some aspects, one mRNA encodes Cas-CLOVER. In some aspects, the lipid nanoparticles further contain at least one gRNA. In some aspects, the lipid nanoparticles further contain at least one gRNA pair, comprising a left gRNA and a right gRNA.

[0105] In some aspects, lipid nanoparticles containing at least one nucleic acid molecule may contain about 54% ssPalmO-Ph-P4C2 (by mole), about 40% cholesterol (by mole), about 5% DOPC (by mole), and about 1% DMG-PEG2000 (by mole), wherein the lipid nanoparticles further contain at least one mRNA molecule. In some aspects, the mRNA molecule further contains a 5'-cap. In some aspects, the lipid to nucleic acid ratio in at least one nanoparticle may be about 75:1 (w / w). In some aspects, one mRNA encodes Cas-CLOVER. In some aspects, the lipid nanoparticles further contain at least one gRNA. In some aspects, the lipid nanoparticles further contain at least one gRNA pair, comprising a left gRNA and a right gRNA.

[0106] In some aspects, lipid nanoparticles containing at least one nucleic acid molecule may contain about 56.5% ssPalmO-Ph-P4C2 (by mole), about 37.5% cholesterol (by mole), about 5% DOPC (by mole), and about 1% DMG-PEG2000 (by mole), wherein the lipid nanoparticles further contain at least one mRNA molecule. In some aspects, the mRNA molecule further contains a 5'-cap. In some aspects, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w). In some aspects, one mRNA encodes Cas-CLOVER. In some aspects, the lipid nanoparticles further contain at least one gRNA. In some aspects, the lipid nanoparticles further contain at least one gRNA pair, comprising a left gRNA and a right gRNA.

[0107] In some aspects, lipid nanoparticles containing at least one nucleic acid molecule may contain about 56.5% ssPalmO-Ph-P4C2 (by mole), about 37.5% cholesterol (by mole), about 5% DOPC (by mole), and about 1% DMG-PEG2000 (by mole), wherein the lipid nanoparticles further contain at least one mRNA molecule. In some aspects, the mRNA molecule further contains a 5'-cap. In some aspects, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 75:1 (w / w). In some aspects, one mRNA encodes Cas-CLOVER. In some aspects, the lipid nanoparticles further contain at least one gRNA. In some aspects, the lipid nanoparticles further contain at least one gRNA pair, comprising a left gRNA and a right gRNA.

[0108] In some aspects, the lipid nanoparticles of this disclosure can be produced using a microfluidic mixing platform. In some aspects, the microfluidic mixing platform can be a non-turbulent microfluidic mixing platform.

[0109] In some aspects, microfluidic mixing platforms can produce the lipid nanoparticles of this disclosure by using a microfluidic device to combine a miscible solvent phase containing a lipid component of nanoparticles and an aqueous phase containing lipid nanoparticle cargo (e.g., nucleic acids, DNA, mRNA, etc.). In some aspects, the miscible solvent phase and the aqueous phase are mixed in a microfluidic device under laminar flow conditions that do not allow for immediate mixing of the two phases. As the two phases move in laminar flow within the microfluidic channels, the microscopic features within the channels enable controlled and uniform mixing to produce the lipid nanoparticles of this disclosure.

[0110] In some respects, microfluidic mixing platforms may include, but are not limited to, NanoAssemblr® Spark (Precision NanoSystems), NanoAssemblr® Ignite™ (Precision NanoSystems), NanoAssemblr® Benchtop (Precision NanoSystems), NanoAssemblr® Blaze (Precision NanoSystems), or NanoAssemblr® GMP system (Precision NanoSystems).

[0111] In some aspects, the lipid nanoparticles of this disclosure can be produced using a microfluidic mixing platform, wherein the microfluidic mixing platform mixes at a rate of at least about 2.5 ml / min, or at least about 5 ml / min, or at least about 7.5 ml / min, or at least about 10 ml / min, or at least about 12.5 ml / min, or at least about 15 ml / min, or at least about 17.5 ml / min, or at least about 20 ml / min, or at least about 22.5 ml / min, or at least about 25 ml / min, or at least about 27.5 ml / min, or at least about 30 ml / min.

[0112] In some aspects, the lipid nanoparticles of this disclosure can be produced using a T-mixer, wherein the T-mixer mixes at a rate of at least about 2.5 ml / min, or at least about 5 ml / min, or at least about 7.5 ml / min, or at least about 10 ml / min, or at least about 12.5 ml / min, or at least about 15 ml / min, or at least about 17.5 ml / min, or at least about 20 ml / min, or at least about 22.5 ml / min, or at least about 25 ml / min, or at least about 27.5 ml / min, or at least about 30 ml / min.

[0113] In some aspects, the lipid nanoparticles of this disclosure can be produced using a microfluidic mixing platform, wherein the microfluidic mixing platform mixes a miscible solvent phase and an aqueous phase in a solvent:water (v:v) ratio of about 10:1, or about 9:1, or about 8:1, or about 7:1, or about 6:1, or about 5:1, or about 4:1, or about 3:1, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4, or about 1:5, or about 1:6, or about 1:7, or about 1:8, or about 1:9, or about 1:10.

[0114] In some aspects, the lipid nanoparticles of this disclosure can be produced using a T-mixer, wherein the T-mixer mixes a miscible solvent phase and an aqueous phase at a solvent:water (v:v) ratio of about 10:1, or about 9:1, or about 8:1, or about 7:1, or about 6:1, or about 5:1, or about 4:1, or about 3:1, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4, or about 1:5, or about 1:6, or about 1:7, or about 1:8, or about 1:9, or about 1:10.

[0115] Adeno-associated virus (AAV) donor polynucleotide

[0116] This disclosure provides compositions comprising adeno-associated virus (AAV) donor polynucleotides.

[0117] In some aspects, the AAV donor polynucleotide comprising a promoterless bidirectional luciferase expression cassette may contain at least one AAV inverted terminal repeat (ITR) sequence. In some aspects, the AAV donor polynucleotide may contain at least first and second target sequences, which contain nucleic acid sequences homologous to genomic sequences in the cell genome. In some aspects, the AAV donor polynucleotide may contain at least one splicing sequence. In some aspects, the AAV donor polynucleotide may contain at least one P2A sequence. In some aspects, the AAV donor polynucleotide may contain at least one luciferase transgene sequence. In some aspects, the AAV donor polynucleotide may contain at least one poly(A) sequence. In some aspects, the AAV donor polynucleotide may contain at least one DNA spacer sequence.

[0118] AAV donor polynucleotides may include a first AAV ITR sequence, a first target sequence containing a nucleic acid sequence homologous to a first genomic sequence in the cell genome, a first splice acceptor sequence, a luciferase-encoding sequence, a poly(A) sequence, a second target sequence containing a nucleic acid sequence homologous to a second genomic sequence in the cell genome, and a second AAV ITR sequence.

[0119] AAV donor polynucleotides may include a first AAV ITR sequence, a first target sequence containing a nucleic acid sequence homologous to a first genomic sequence in the cell genome, a first splice acceptor sequence, a P2A sequence, a luciferase-encoding sequence, a poly(A) sequence, a second target sequence containing a nucleic acid sequence homologous to a second genomic sequence in the cell genome, and a second AAV ITR sequence.

[0120] AAV donor polynucleotides may include a first AAV ITR sequence, a first target sequence containing a nucleic acid sequence homologous to a first genomic sequence in the cell genome, a first splice acceptor sequence, a P2A sequence, a luciferase-coding sequence, a poly(A) sequence, a DNA spacer sequence; a reverse poly(A) sequence and a reverse luciferase-coding sequence, a reverse P2A sequence, a reverse splice acceptor sequence, a second target sequence containing a nucleic acid sequence homologous to a second genomic sequence in the cell genome; and a second AAV ITR sequence.

[0121] The AAV donor polynucleotide may include a first AAV ITR sequence, a first target sequence containing a nucleic acid sequence homologous to the first sequence of intron 3 of the albumin gene, a first splice acceptor sequence, a P2A sequence, a luciferase-coding sequence, a poly(A) sequence, a DNA spacer sequence; a reverse poly(A) sequence and a reverse luciferase-coding sequence, a reverse P2A sequence, a reverse splice acceptor sequence, a second target sequence containing a nucleic acid sequence homologous to the second sequence of intron 3 of the albumin gene; and a second AAV ITR sequence.

[0122] In some aspects, the AAV donor polynucleotide comprising a promoterless bidirectional PAH expression cassette may contain at least one AAV inverted terminal repeat (ITR) sequence. In some aspects, the AAV donor polynucleotide may contain at least a first target sequence and a second target sequence containing nucleic acid sequences homologous to first and second genomic sequences in the cell genome. In some aspects, the AAV donor polynucleotide may contain at least one splicing sequence. In some aspects, the AAV donor polynucleotide may contain at least one P2A sequence. In some aspects, the AAV donor polynucleotide may contain at least one PAH transgene sequence. In some aspects, the AAV donor polynucleotide may contain at least one poly(A) sequence. In some aspects, the AAV donor polynucleotide may contain at least one DNA spacer sequence.

[0123] AAV donor polynucleotides may include a first AAV ITR sequence, a first target sequence containing a nucleic acid sequence homologous to a first genomic sequence in the cell genome, a first splice acceptor sequence, a PAH coding sequence, a poly(A) sequence, a second target sequence containing a nucleic acid sequence homologous to a second genomic sequence in the cell genome, and a second AAV ITR sequence.

[0124] AAV donor polynucleotides may include a first AAV ITR sequence, a first target sequence containing a nucleic acid sequence homologous to a first genomic sequence in the cell genome, a first splice acceptor sequence, a P2A sequence, a PAH coding sequence, a poly(A) sequence, a second target sequence containing a nucleic acid sequence homologous to a second genomic sequence in the cell genome, and a second AAV ITR sequence.

[0125] AAV donor polynucleotides may include a first AAV ITR sequence, a first target sequence containing a nucleic acid sequence homologous to a first genomic sequence in the cell genome, a first splice acceptor sequence, a P2A sequence, a PAH coding sequence, a poly(A) sequence, a DNA spacer sequence; a reverse poly(A) sequence and a reverse PAH coding sequence, a reverse P2A sequence, a reverse splice acceptor sequence, a second target sequence containing a nucleic acid sequence homologous to a second genomic sequence in the cell genome; and a second AAV ITR sequence.

[0126] The AAV donor polynucleotide may include a first AAV ITR sequence, a first target sequence containing a nucleic acid sequence homologous to the first sequence of intron 3 of the albumin gene, a first splice acceptor sequence, a P2A sequence, a PAH coding sequence, a poly(A) sequence, a DNA spacer sequence; a reverse poly(A) sequence and a reverse PAH coding sequence, a reverse P2A sequence, a reverse splice acceptor sequence, a second target sequence containing a nucleic acid sequence homologous to the second sequence of intron 3 of the albumin gene; and a second AAV ITR sequence.

[0127] In some aspects, the AAV donor polynucleotide containing the PAH expression cassette may comprise an AAV donor polynucleotide comprising a first AAV ITR sequence, a first target sequence containing a nucleic acid sequence homologous to a first genomic sequence in the cell genome, at least one promoter sequence, a PAH coding sequence, a poly(A) sequence, a second target sequence containing a nucleic acid sequence complementary to a second genomic sequence in the cell genome, and a second AAV ITR sequence.

[0128] In some aspects, the AAV donor polynucleotide containing the PAH expression cassette may comprise an AAV donor polynucleotide comprising a first AAV ITR sequence, a first target sequence containing a nucleic acid sequence homologous to a first genomic sequence in the cell genome, a TTRe promoter sequence, a PAH coding sequence, a poly(A) sequence, a second target sequence containing a nucleic acid sequence homologous to a second genomic sequence in the cell genome, and a second AAV ITR sequence.

[0129] In some aspects, the AAV donor polynucleotide containing the PAH expression cassette may comprise an AAV donor polynucleotide comprising a first AAV ITR sequence, a first target sequence containing a nucleic acid sequence homologous to a first sequence of intron 3 of the albumin gene, a TTRe promoter sequence, a PAH coding sequence, a poly(A) sequence, a second target sequence containing a nucleic acid sequence homologous to a second sequence of intron 3 of the albumin gene, and a second AAV ITR sequence.

[0130] In the aforementioned non-limiting examples of AAV donor polynucleotides, at least one transgenic sequence may contain a nucleic acid sequence encoding a human phenylalanine hydroxylase (hPAH) polypeptide. In some aspects, the nucleotide sequence encoding hPAH is codon-optimized. In some aspects, the codon-optimized PAH gene is further modified to remove i) any unwanted restriction endonuclease recognition sites; and ii) putative cryptic splicing sites for cloning the modified sequence into the AAV donor vector. These non-limiting examples of AAV donor polynucleotides are shown in Figures 1B and 1C.

[0131] In some aspects, the AAV donor polynucleotide comprising a promoterless bidirectional luciferase expression cassette may comprise, consist substantially of, or consist of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage therebetween) the sequence shown in SEQ ID NO: 6.

[0132] In some aspects, the AAV donor polynucleotide comprising a promoterless bidirectional PAH expression cassette may comprise, substantially comprise, or comprise of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage therebetween) the sequence shown in SEQ ID NO: 8. In some aspects, the AAV donor polynucleotide comprising a promoterless bidirectional PAH expression cassette may comprise, substantially comprise, or comprise of the sequence shown in SEQ ID NO: 8.

[0133] In some aspects, the AAV donor polynucleotide containing the PAH TTRe expression cassette may comprise, consist substantially of, or consist of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage therebetween) the sequence shown in SEQ ID NO: 10.

[0134] AAV ITR sequence

[0135] The vector described herein may comprise one or more AAV ITR sequences. The AAV ITR sequence may comprise any suitable AAV ITR sequence known in the art or described herein. In some embodiments, the AAV ITR sequence is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 ITR sequence. In some aspects, the AAV ITR sequence may comprise, substantially comprise, or comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage therebetween) the same nucleic acid sequence as, substantially comprise, or comprise of, any of the sequences shown in SEQ ID NO: 12 and 24. In some aspects, the AAV ITR sequence may comprise, substantially comprise, or comprise of any of the sequences shown in SEQ ID NO: 12 and 24.

[0136] In some aspects, the first AAV ITR sequence may comprise, substantially consist of, or consist of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage therein) of the nucleic acid sequence of SEQ ID NO: 12, and the second AAV ITR sequence may comprise, substantially consist of, or consist of, the nucleic acid sequence of SEQ ID NO: 24, and the second AAV ITR sequence may comprise, substantially consist of, or consist of, the nucleic acid sequence of SEQ ID NO: 24.

[0137] Splice acceptor sequence

[0138] In some respects, the AAV donor polynucleotide includes one or more splice acceptor sequences to facilitate the expression of transgenes integrated into gene introns. In some respects, the splice acceptor sequence may include any suitable splice acceptor sequence known in the art or derived from mammalian or viral splice acceptor sequences.

[0139] In some aspects, the splice acceptor sequence may comprise, consist substantially of, or consist of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage therebetween) the nucleic acid sequence shown in SEQ ID NO: 14 or 22.

[0140] promoter sequence

[0141] In some respects, the promoter sequence may comprise any suitable promoter sequence known in the art. In some respects, the promoter sequence may comprise any liver-specific promoter sequence known in the art.

[0142] In some aspects, the promoter sequence may comprise, substantially comprise, or comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage therebetween) the same nucleic acid sequence as, substantially comprise, or comprise of the sequence shown in SEQ ID NO: 27. In some embodiments, the promoter sequence may comprise, substantially comprise, or comprise of the sequence shown in SEQ ID NO: 27.

[0143] In some respects, the promoter sequence may contain the TTRe promoter sequence. The TTRe promoter sequence may contain, consist substantially of, or consist of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage therebetween) the nucleic acid sequence shown in SEQ ID NO: 27.

[0144] Transgenic sequence

[0145] In some aspects, the transgenic sequence may comprise a nucleic acid sequence encoding a human phenylalanine hydroxylase (hPAH) polypeptide. In some aspects, the nucleic acid sequence encoding the hPAH polypeptide may comprise, consist substantially of, or consist of the same nucleic acid sequence as, substantially of, or consist of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage therebetween) of the nucleic acid sequence shown in SEQ ID NO: 25. In some aspects, the nucleic acid sequence encoding the hPAH polypeptide may comprise, consist substantially of, or consist of the nucleic acid sequence shown in SEQ ID NO: 25.

[0146] In some respects, transgenic sequences may contain tags, such as hemagglutinin (HA) tags. Such tags are well known in the art and can be used for protein purification.

[0147] In some respects, the transgenic sequence can be codon optimized using methods known in the art.

[0148] In some respects, the nucleic acid sequence encoding a polypeptide (e.g., hPAH) may be a codon-optimized nucleic acid sequence encoding the polypeptide. The codon-optimized nucleic acid sequence encoding the polypeptide may contain, consist substantially, or consist of no more than 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage between these percentages) the same nucleic acid sequence as, substantially consist of, or consist of the same nucleic acid sequence as the wild-type human nucleic acid sequence encoding the polypeptide.

[0149] SEQ ID NO:25 is a unique codon-optimized PAH nucleic acid sequence that may be included in polynucleotides, vectors, and compositions disclosed herein.

[0150] In some aspects, the codon-optimized nucleic acid sequence encoding the polypeptide, such as the nucleic acid sequence shown in SEQ ID NO: 25, may not contain splicing sites in the donor DNA. In some aspects, the codon-optimized nucleic acid sequence encoding the polypeptide may contain no more than about one, or about two, or about three, or about four, or about five, or about six, or about seven, or about eight, or about nine, or about ten splicing sites. In some aspects, compared to the wild-type human nucleic acid sequence encoding the polypeptide, the codon-optimized nucleic acid sequence encoding the polypeptide contains at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten fewer splicing sites. It has been found that the removal of splicing sites in the donor DNA of the codon-optimized nucleic acid sequence unexpectedly and unpredictably increased polypeptide expression in vivo by preventing recessive splicing. Furthermore, covert splicing can vary among different subjects, meaning that the expression levels of peptides containing donor splicing sites can vary unpredictably among different subjects.

[0151] In some respects, codon-optimized nucleic acid sequences encoding peptides, such as the one shown in SEQ ID NO: 25, may have GC contents different from those of wild-type human nucleic acid sequences encoding peptides. In some respects, the GC contents of codon-optimized nucleic acid sequences encoding peptides are more uniformly distributed throughout the nucleic acid sequence compared to wild-type human nucleic acid sequences encoding peptides. Unwilling to be bound by theory, codon-optimized nucleic acid sequences are expected to exhibit a more uniform melting temperature (“Tm”) along the length of the transcript due to the more uniform distribution of GC contents throughout the nucleic acid sequence. This uniformity of melting temperature unexpectedly leads to increased expression of codon-optimized nucleic acids in human subjects because transcription and / or translation of the nucleic acid sequence occur with less stasis of polymerase and / or ribosomes.

[0152] In some respects, codon-optimized nucleic acid sequences encoding peptides, such as those shown in SEQ ID NO: 25, exhibit at least a 5%, 10%, 20%, 30%, 50%, 75%, 100%, 200%, 300%, 500%, or 1000% increase in expression in human subjects, relative to wild-type or non-codon-optimized nucleic acid sequences encoding peptides.

[0153] In some respects, at least one transgenic sequence can be operatively linked to at least one promoter sequence present in the same polynucleotide.

[0154] Poly(A) sequence

[0155] In some respects, the AAV donor polynucleotide contains a poly(A) sequence. In some respects, the AAV donor polynucleotide contains an inverse poly(A) sequence. In some respects, the AAV donor polynucleotide contains both a poly(A) sequence and an inverse poly(A) sequence.

[0156] In some aspects, the poly(A) sequence may comprise any poly(A) sequence known in the art or described herein. Non-limiting examples of poly(A) sequences include, but are not limited to, the SV40 poly(A) sequence and the bGH poly(A) sequence. In some aspects, the poly(A) sequence may comprise, substantially comprise, or comprise, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage therebetween) the nucleic acid sequence shown in SEQ ID NO: 17. In some aspects, the poly(A) sequence may comprise, substantially comprise, or comprise, the nucleic acid sequence shown in SEQ ID NO: 17. In some aspects, the reverse poly(A) sequence may comprise, substantially consist of, or consist of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage therebetween) the nucleic acid sequence shown in SEQ ID NO: 19.

[0157] DNA spacer sequence

[0158] In some respects, the AAV donor polynucleotide contains one or more spacer sequences. In some respects, the DNA spacer sequence may contain, consist substantially of, or consist of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% (or any percentage therebetween) the same nucleic acid sequence as, substantially consist of, or consist of the sequence shown in SEQ ID NO: 18.

[0159] The DNA spacer sequence can be located anywhere within the AAV donor polynucleotide. In some respects, the DNA spacer sequence is located between the poly(A) sequence and the inverse poly(A) sequence.

[0160] Enhanced subsequence

[0161] In some respects, the AAV donor polynucleotide contains one or more enhancer sequences. In some respects, the enhancer sequence may contain any suitable enhancer sequence known in the art. In some respects, the enhancer sequence may contain any suitable liver-specific enhancer sequence known in the art.

[0162] In some aspects, the enhancer sequence may comprise, consist substantially of, or consist of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage therebetween) the nucleic acid sequence shown in either SEQ ID NO: 5 or 6.

[0163] In some respects, the enhancer sequence may include a TTR enhancer sequence. The TTR enhancer sequence may contain, consist substantially of, or consist of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage therebetween) the nucleic acid sequence shown in SEQ ID NO: 6.

[0164] 3' UTR sequence

[0165] In some aspects, the AAV donor polynucleotide contains a 3' untranslated region (UTR) sequence. In some aspects, the 3' UTR sequence may contain, substantially consist of, or consist of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage therebetween) the nucleic acid sequence shown in SEQ ID NO: 10. In some aspects, the 3' UTR sequence may contain, substantially consist of, or consist of the nucleic acid sequence shown in SEQ ID NO: 10.

[0166] In some aspects of the aforementioned transposon, the first 3' UTR sequence may be an AES-mtRNR 3' UTR sequence. The AES-mtRNR 3' UTR sequence may comprise, consist substantially of, or be composed of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage between) the same nucleic acid sequence as, substantially of, or composed of the same nucleic acid sequence as SEQ ID NO: 10, or may comprise any suitable UTR sequence known in the art, particularly those known to enhance translation and / or stabilize RNA by slowing its degradation. In some embodiments, the first 3' UTR sequence comprises the nucleic acid sequence shown in SEQ ID NO: 10.

[0167] Exemplary dCas9-Clo051 (Cas-CLOVER) fusion protein

[0168] On the other hand, this document provides a fusion protein comprising a nuclease or nuclease domain and a restriction endonuclease. The nuclease or its nuclease domain may comprise a nuclease-inactivated Cas (dCas) protein and a restriction endonuclease. The restriction endonuclease may comprise a Clo051 nuclease or its nuclease domain. Gene editing compositions may comprise a fusion protein. The fusion protein may comprise a nuclease-inactivated Cas9 (dCas9) protein and a Clo051 nuclease or a Clo051 nuclease domain. Gene editing compositions may further comprise a guide sequence. The guide sequence comprises an RNA sequence.

[0169] This disclosure provides compositions comprising a small Cas9 (Cas9) operatively linked to an effector. This disclosure provides a fusion protein comprising, substantially comprising, or comprising of an effector molecule, wherein the effector comprises a small Cas9 (Cas9). The small Cas9 construct of this disclosure may comprise an effector containing an IIS-type endonuclease. Staphylococcus aureus Cas9 with an active catalytic site may comprise the amino acid sequence of SEQ ID NO: 30.

[0170] This disclosure provides compositions comprising an inactivated small Cas9 (dSaCas9) operatively linked to an effector. This disclosure provides a fusion protein comprising a DNA positioning component and an effector molecule, wherein the effector comprises an inactivated small Cas9 (dSaCas9). The small inactivated Cas9 (dSaCas9) construct of this disclosure may comprise an effector containing an IIS-type endonuclease. In some embodiments, dSaCas9 comprises the amino acid sequence of SEQ ID NO: 31, including D10A and N580A mutations that inactivate the catalytic site. In some embodiments, dSaCas9 comprises at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 31.

[0171] This disclosure provides compositions comprising inactivated Cas9 (dCas9) operatively linked to an effector. This disclosure also provides a fusion protein comprising a DNA positioning component and an effector molecule, wherein the effector comprises inactivated Cas9 (dCas9). The inactivated Cas9 (dCas9) construct of this disclosure may comprise an effector containing an IIS-type endonuclease.

[0172] dCas9 can be isolated from or derived from *Streptococcus pyogenes*. dCas9 may contain dCas9 with substitutions at amino acid positions 10 and 840, which inactivates the catalytic site. In some aspects, these substitutions are D10A and H840A. dCas9 may contain the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33. In some embodiments, dCas9 contains at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO 32 or 33.

[0173] The exemplary Clo051 nuclease domain comprises, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 34. In some embodiments, the Clo051 nuclease domain comprises at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 34. In some aspects, the Clo051 nuclease domain comprises at least one amino acid substitution. In some aspects, the amino acid substitution is in the α-helical loop domain of the Clo051 nuclease. In some aspects, the amino acid substitution is at positions 35, 37, 60, 92, 98, 100, or 146 of SEQ ID NO: 34. In some aspects, the amino acid substitution is at position 37 of SEQ ID NO: 34.

[0174] An exemplary dCas9-Clo051 (Cas-CLOVER) fusion protein may comprise, consist substantially of, or consist of the amino acid sequence of SEQ ID NO: 37. In some embodiments, the Cas-CLOVER fusion protein comprises at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence shown in SEQ ID NO: 37. An exemplary dCas9-Clo051 fusion protein may be encoded by a polynucleotide comprising, consist substantially of, or consist of the nucleic acid sequence of SEQ ID NO: 38. In some embodiments, the Cas-CLOVER fusion protein is encoded by a polynucleotide comprising at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence shown in SEQ ID NO: 38. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0175] An exemplary dCas9-Clo051 (Cas-CLOVER) fusion protein may comprise, consist substantially of, or consist of the amino acid sequence of SEQ ID NO: 39. In some embodiments, the Cas-CLOVER fusion protein comprises at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence shown in SEQ ID NO: 39. An exemplary dCas9-Clo051 fusion protein may be encoded by a polynucleotide comprising, consist substantially of, or consist of the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the Cas-CLOVER fusion protein is encoded by a polynucleotide comprising at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence shown in SEQ ID NO: 40. The nucleic acid encoding the dCas9-Clo051 fusion protein may be DNA or RNA.

[0176] The exemplary dCas9-Clo051 fusion protein (Cas-CLOVER) of this disclosure may further comprise at least one nuclear localization sequence (NLS). In some embodiments, the dCas9-Clo051 fusion protein of this disclosure comprises at least two nuclear localization sequences. In some embodiments, the NLS is located at the N' terminus of the dCas9-Clo051 fusion protein (NLS-dCas9-Clo051). In some embodiments, the NLS is located at the C' terminus of the dCas9-Clo051 fusion protein (dCas9-Clo051-NLS). In some embodiments, the NLS is located at both the N' and C' terms of the dCas9-Clo051 fusion protein (“NLS-dCas9-Clo051-NLS” or “wild-type Cas-CLOVER”).

[0177] The NLS-dCas9-Clo051-NLS (“wild-type Cas-CLOVER”) fusion protein may comprise, consist substantially of, or consist of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the NLS-dCas9-Clo051-NLS fusion protein comprises at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence shown in SEQ ID NO: 28.

[0178] NLS-dCas9-Clo051-NLS amino acid sequence (NLS amino acid sequence in bold and underlined):

[0179] The NLS-dCas9-Clo051-NLS fusion protein may comprise, consist substantially of, or consist of the amino acid sequence of SEQ ID NO: 3. In some embodiments, the NLS-dCas9-Clo051-NLS fusion protein comprises at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence shown in SEQ ID NO: 3.

[0180]

[0181] The nucleic acid encoding the NLS-dCas9-Clo051-NLS (“wild-type Cas-CLOVER”) fusion protein can be DNA or RNA. In some embodiments, the dCas9-Clo051 fusion protein comprising two NLS regions is encoded by an mRNA sequence comprising, substantially comprising, or comprising of SEQ ID NO: 29. In some embodiments, the dCas9-Clo051 fusion protein comprising two NLS regions is encoded by a polynucleotide comprising at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence shown in SEQ ID NO: 29.

[0182] NLS-dCas9-Clo051-NLS mRNA sequence (NLS amino acid sequence in bold and underlined):

[0183]

[0184] Exemplary mutant Cas-CLOVER fusion protein

[0185] In some respects, NLS-dCas9-Clo051-NLS (“wild-type Cas-CLOVER”) contains at least one amino acid substitution. In some respects, the amino acid substitution is located in the Clo051 domain of NLS-dCas9-Clo051-NLS.

[0186] In some aspects, NLS-dCas9-Clo051-NLS of SEQ ID NO: 28 may contain at least one substitution at amino acid positions 42, 44, 67, 105, 107, and / or 153. In some aspects, the amino acid substitution is F42E, F42D, S44E, S44P, R67E, I105Q, Q107A, Q107E, Q107H, Q107D, and / or K153D. In some aspects, the amino acid substitution is S44P.

[0187] An exemplary S44P mutant NLS-dCas9-Clo051-NLS (“S44P Cas-CLOVER”, “S44P CC”, or “S44P”) fusion protein may comprise, consist substantially of, or consist of the amino acid sequence of SEQ ID NO: 35. In some embodiments, the S44P mutant NLS-dCas9-Clo051-NLS fusion protein comprises at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence shown in SEQ ID NO 35.

[0188] The S44P Cas-CLOVER fusion protein may be encoded by a polynucleotide comprising, substantially comprising, or comprising the nucleic acid sequence of SEQ ID NO:36. In some embodiments, the S44P mutant NLS-dCas9-Clo051-NLS fusion protein is encoded by a polynucleotide comprising at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO:36. The nucleic acid encoding the dCas9-Clo051 fusion protein may be DNA or RNA.

[0189] S44P Cas-CLOVER amino acid sequence (SEQ ID NO: 35):

[0190]

[0191] S44P Cas-CLOVER nucleic acid sequence (SEQ ID NO: 36)

[0192]

[0193] genetically modified

[0194] In some respects, the compositions described herein can be used to deliver genetically modified organisms into cells. Cells containing the gene-editing composition can stably or transiently express the gene-editing composition.

[0195] Transgenic structures may contain sequences encoding therapeutic agents. Therapeutic agents may be proteins or RNA that provide therapeutic benefit when administered to cells or subjects. Therapeutic agents may be therapeutic proteins or therapeutic RNA. Examples of therapeutic agents include human β-globin (HBB), T87Q human β-globin (HBB T87Q), BAF chromatin remodeling complex subunit (BCL11A) shRNA, insulin-like growth factor 2 binding protein 1 (IGF2BP1), interleukin 2 receptor γ (IL2RG), α-galactosidase A (GLA), α-L-idurosidase (IDUA), iduronate 2-sulfatase (IDS), and cystinosin lysosomal cysteine ​​transporter (CTNS). Transgenic structures may contain sequences of factor VIII or factor IX. Transgenic structures may contain sequences encoding chimeric antigen receptors (CARs). The transgene may contain a sequence encoding a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an extracellular domain containing an activating component isolated from or derived from a first protein; (b) a transmembrane domain; and (c) an intracellular domain containing at least one signal transduction domain isolated from or derived from a second protein; wherein the first protein and the second protein are not identical. In one aspect, the transgene may contain both a CAR sequence and a CSR sequence. In one aspect, a transgene containing a CAR or CSR specifically binds to BCMA, PSMA, MUC1-C, CD133, c-KIT, CD19, or CD20. The transgene may contain a sequence encoding an inducible pro-apoptotic polypeptide comprising (a) a ligand-binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not contain a non-human sequence. The transgene may be integrated into the genome of an HSC. Integration can be stable or temporary.

[0196] Factor VIII (FVIII) deficiency leads to the development of hemophilia A. Factor IX (FIX) deficiency leads to the development of hemophilia B. Prior to the compositions and methods of this disclosure, standard treatment for hemophilia B involved infusions of recombinant FIX every 2 to 3 days, costing approximately $250,000 per year. In stark contrast to this standard treatment regimen, the iPSCs of this disclosure can differentiate into any cell type, including HSCs, and remain in the human body for decades.

[0197] gRNA

[0198] In some respects, guide RNA is used to deliver the transgenes provided in this article to target cells.

[0199] The guide RNA may contain a sequence complementary to a target sequence within the genomic DNA sequence. The target sequence within the genomic DNA sequence may be a target sequence located within a safe harbor site of the genomic DNA sequence. Exemplary target sequences include, but are not limited to, ALB, HBB, TRAC, B2M, TCRb, GAPDH, or SOX17.

[0200] The guide RNA may contain a sequence complementary to at least one target sequence on a transposon, plasmid, or vector. In some respects, the complementary sequence of the guide RNA on the transposon, plasmid, or vector is located within the transgene for targeted nucleic acid insertion. In some respects, the complementary sequence on the transposon, plasmid, or vector facilitates binding to gRNA that binds to the effector molecule, thereby tethering all components. In some respects, the effector molecule is a Cas-CLOVER. In some respects, the Cas-CLOVER further contains at least one NLS sequence. In some respects, the NLS sequence of the Cas-CLOVER facilitates the tethering of components to the nucleus. This facilitates the localization of all components required for gene editing (Cas-CLOVER, gRNA, and transposon, plasmid, or vector) to the nucleus, thereby increasing the efficiency of gene editing.

[0201] As used herein, the term “guide sequence” in the context of the Cas-CLOVER or CRISPR-Cas9 system includes any polynucleotide sequence that is sufficiently complementary to the target nucleic acid sequence to hybridize with it and guides the nucleic acid targeting complex to bind sequence-specifically to the target nucleic acid sequence. The guide sequence may form a double helix with the target sequence. The double helix may be a DNA double helix, an RNA double helix, or an RNA / DNA double helix. The terms “guide molecule,” “guide RNA,” and “single guide RNA” are used interchangeably herein to refer to an RNA-based molecule capable of forming a complex with a Cas-CLOVER or CRISPR-Cas protein and containing a guide sequence that is sufficiently complementary to the target nucleic acid sequence to hybridize with it and guides the complex to bind sequence-specifically to the target nucleic acid sequence. A guide molecule or guide RNA may encompass an RNA-based molecule with one or more chemical modifications (e.g., by chemically linking two ribonucleotides or by replacing one or more ribonucleotides with one or more deoxyribonucleotides), as described herein.

[0202] The terms “target region,” “target sequence,” or “prespacer sequence,” used interchangeably herein, refer to the region of the target gene targeted by the Cas-Clover system or a CRISPR / Cas9-based system. A Cas-CLOVER or CRISPR / Cas9-based system may include at least one gRNA that targets a different DNA sequence. Target DNA sequences may overlap. A Cas-CLOVER system may include at least two gRNAs that target different DNA sequences. Following the target sequence or prespacer sequence is a PAM sequence at the 3' end of the prespacer sequence. Different type II systems have different PAM requirements. For example, the Streptococcus pyogenes type II system uses the “NGG” sequence, where “N” can be any nucleotide.

[0203] The guide RNA, or the guide RNA of the Cas-Clover or CRISPR-Cas protein, may comprise a tracr chaperone sequence (in the context of an endogenous CRISPR system, this refers to a "directed repeat sequence") and a guide sequence (also referred to as a "spacer region" in the context of an endogenous CRISPR system). In some embodiments, the Cas-CLOVER or CRISPR-Cas system or complex described herein does not contain and / or is independent of the presence of the tracr sequence. In some embodiments, the guide molecule may comprise, consist substantially of, or consist of a directed repeat sequence fused to or linked to the guide sequence or spacer sequence.

[0204] In some embodiments, the guide sequence or spacer of the guide molecule is 15 to 50 nucleotides long. In some embodiments, the spacer of the guide RNA is at least 15 nucleotides long. In some embodiments, the spacer length is 15 to 17 nucleotides, 17 to 20 nucleotides, 20 to 24 nucleotides, 23 to 25 nucleotides, 24 to 27 nucleotides, 27 to 30 nucleotides, 30 to 35 nucleotides, or greater than 35 nucleotides.

[0205] In some embodiments, the guidance sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54. 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides in length.

[0206] In some embodiments, the sequence of the guide molecule (homogeneous repeats and / or spacers) is selected to reduce the degree of secondary structure within the guide molecule. In some embodiments, about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1% or less of the nucleotides targeting the guide RNA participate in self-complementary base pairing at optimal folding. Optimal folding can be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimum Gibbs free energy. An example of such an algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example of a folding algorithm is RNAfold, an online web server developed by the Institute of Theoretical Chemistry at the University of Vienna, which uses a centroid structure prediction algorithm (see, for example, AR Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).

[0207] As described above, the Cas-Clover system and the CRISPR / Cas9 system utilize targeting gRNAs and shuttle gRNAs that provide targeting for both the Cas-Clover system and the CRISPR / Cas9-based system. The gRNA can be a fusion of two non-coding RNAs: crRNA and tracrRNA. The sgRNA can target any desired DNA sequence by exchanging sequences encoding a 20 bp prespacer sequence, which imparts targeting specificity through complementary base pairing with the desired DNA target. The gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in type II effector systems. This duplex can include, for example, a 42-nucleotide crRNA and a 75-nucleotide tracrRNA, serving as a guide for Cas9 cleavage of the target nucleic acid.

[0208] In some embodiments, the gRNA is a left gRNA and targets a region upstream of the target gene cleavage site (e.g., intron 3 of the albumin locus), for example, a region upstream of the target gene between 0 and 1000 bp. In some embodiments, the gRNA targets a region upstream of the transcription start site of the target gene, ranging from 0 to 50 bp, 0 to 100 bp, 0 to 150 bp, 0 to 200 bp, 0 to 250 bp, 0 to 300 bp, 0 to 350 bp, 0 to 400 bp, 0 to 450 bp, 0 to 500 bp, 0 to 550 bp, 0 to 600 bp, 0 to 650 bp, 0 to 700 bp, 0 to 750 bp, 0 to 800 bp, 0 to 850 bp, 0 to 900 bp, 0 to 950 bp, or 0 to 1000 bp. In some embodiments, the gRNA targets a region approximately 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 1100 bp, 1200 bp, 1300 bp, 1400 bp, or 1500 bp upstream of the target gene.

[0209] In some embodiments, the gRNA is a right gRNA and targets a region downstream of the target gene cleavage site, such as a region downstream of the target gene between 0 and 1000 bp. In some embodiments, the gRNA targets a region downstream of the target gene between 0 and 50 bp, 0 and 100 bp, 0 and 150 bp, 0 and 200 bp, 0 and 250 bp, 0 and 300 bp, 0 and 350 bp, 0 and 400 bp, 0 and 450 bp, 0 and 500 bp, 0 and 550 bp, 0 and 600 bp, 0 and 650 bp, 0 and 700 bp, 0 and 750 bp, 0 and 800 bp, 0 and 850 bp, 0 and 900 bp, 0 and 950 bp, or 0 and 1000 bp. In some embodiments, the gRNA targets a region approximately 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 1100 bp, 1200 bp, 1300 bp, 1400 bp, or 1500 bp downstream of the target gene.

[0210] In some embodiments, the left and right gRNAs of the gRNA pair are designed with appropriate spacing to allow dimerization of the Clo051 nuclease domain when the left and right gRNAs bind to the target sequence of the target region. In some embodiments, the first and second gRNA sequences are complementary to the first and second target sequences of the AAV donor polynucleotide, respectively, and are designed with appropriate spacing to allow Cas-CLOVER to target the AAV donor polynucleotide to cleave the AAV donor polynucleotide, thereby linearizing the polynucleotide and removing the terminal AAV ITR sequence.

[0211] gRNAs can be divided into target-binding regions and Cas9-binding regions. The target-binding region hybridizes with the target region in the target gene. Methods for designing such target-binding regions are known in the art, see, for example, Doench et al., Nat Biotechnol. (2014) 32: 1262-7; and Doench et al., Nat Biotechnol. (2016) 34: 184-91, which are incorporated herein by reference in their entirety. Design tools are available, for example, from the following: targetFinder from Feng Zhang's laboratory, Target Finder (E-CRISP) from Michael Boutros' laboratory, RGEN Tools (Cas-OFFinder), CasFinder, and CRISPR Optimal Target Finder. In some embodiments, the length of the target binding region may be between about 15 and about 50 nucleotides (lengths of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 nucleotides). In some embodiments, the length of the target binding region may be between about 19 and about 21 nucleotides. In one embodiment, the length of the target binding region is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

[0212] In one embodiment, the target-binding region is complementary to the target region in the target gene, for example, completely complementary. In one embodiment, the target-binding region is substantially complementary to the target region in the target gene. In one embodiment, the target-binding region contains no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides that are not complementary to the target region in the target gene.

[0213] The exemplary sgRNAs of this disclosure include, but are not limited to, sequences for targeting intron 3 of the albumin gene. The exemplary sgRNAs of this disclosure comprise, are substantially composed of, or are composed of the sequences shown in Table 1.

[0214] Table 1: Exemplary sgRNAs of this disclosure

[0215]

[0216] The carrier of this disclosure

[0217] This disclosure provides compositions comprising a vector, wherein the vector comprises at least one adeno-associated virus (AAV) donor polynucleotide. A vector comprising at least one adeno-associated virus (AAV) donor polynucleotide is referred to herein as an "AAV donor vector".

[0218] The vectors disclosed herein may be viral vectors or recombinant vectors. Viral vectors may contain sequences isolated from or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. Viral vectors may contain sequences isolated from or derived from adeno-associated virus (AAV). Viral vectors may contain recombinant AAV (rAAV).

[0219] Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids (e.g., AAV2 / 5, AAV-DJ, and AAV-DJ8) containing the genome of one serotype and the capsid of another serotype. Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, AAV8, AAV9, rAAV-LK03, AAV-KP-1 (also known as AAV-KP1; described in detail in Kerun et al. JCI Insight, 2019; 4(22): el31610), and AAV-NP59 (described in detail in Paulk et al. Molecular Therapy, 2018; 26(1): 289-303).

[0220] This disclosure provides a composition comprising a plurality of AAV-KP-1 particles, each particle containing at least one adeno-associated virus (AAV) donor polynucleotide. This disclosure also provides a composition comprising a plurality of AAV-NP59 particles, each particle containing at least one adeno-associated virus (AAV) donor polynucleotide.

[0221] This disclosure provides a composition comprising a plurality of AAV-NP59 (described in detail in Paulk et al., Molecular Therapy, 2018; 26 (1): 289-303).

[0222] This disclosure provides a composition comprising a plurality of AAV8 particles containing at least one adeno-associated virus (AAV) donor polynucleotide, and an LNP composition containing a targeting gRNA pair and an mRNA encoding a Cas-CLOVER. In some aspects, the mRNA molecule further comprises a 5'-cap. In some aspects, the Cas-CLOVER is a wild-type Cas-CLOVER sequence, an S44P mutant Cas-CLOVER sequence, or other Cas-CLOVER sequences described herein. In some aspects, at least one LNP composition comprises: about 54% ssPalmO-Ph-P4C2 by mole, about 35% cholesterol by mole, about 5% DOPC by mole, about 5% DSPC by mole, and about 1% DMG-PEG2000 by mole.

[0223] This disclosure provides a composition comprising a plurality of AAV9 particles containing at least one adeno-associated virus (AAV) donor polynucleotide, and an LNP composition containing a targeting gRNA pair and an mRNA encoding a Cas-CLOVER. In some aspects, the mRNA molecule further comprises a 5'-cap. In some aspects, the Cas-CLOVER is a wild-type Cas-CLOVER sequence, an S44P mutant Cas-CLOVER sequence, or other Cas-CLOVER sequences described herein. In some aspects, at least one LNP composition comprises: about 54% ssPalmO-Ph-P4C2 by mole, about 35% cholesterol by mole, about 5% DOPC by mole, about 5% DSPC by mole, and about 1% DMG-PEG2000 by mole.

[0224] The viral vectors and viral particles disclosed herein can be prepared using standard methods known in the art.

[0225] The cell delivery compositions (e.g., polynucleotides, carriers) disclosed herein may comprise nucleic acids encoding therapeutic proteins or therapeutic agents. Examples of therapeutic proteins include those disclosed in PCT Publications WO 2019 / 173636 and WO / 2020 / 051374, each of which is incorporated herein by reference in its entirety as an example of therapeutic proteins that can be delivered using the compositions described herein. Therapeutic proteins may also include, but are not limited to, any of the polypeptides described herein that are part of a transgenic sequence (e.g., hPAH).

[0226] Formulation, dosage and administration

[0227] This disclosure provides formulations, dosages, and methods of administration of the compositions described herein.

[0228] The disclosed compositions and pharmaceutical compositions may further comprise at least one of any suitable adjuvants, such as, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, etc. Pharmaceutically acceptable adjuvants are preferred. Non-limiting examples and methods of preparing such sterile solutions are well known in the art, such as, but not limited to, Gennaro, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co. (Easton, Pa.) 1990 and "Physician's Desk Reference", 52nd edition, Medical Economics (Montvale, NJ) 1998.

[0229] Pharmaceutically acceptable carriers suitable for the administration method, solubility, and / or stability of compositions well known in the art or as described herein can be conventionally selected.

[0230] For example, the LNP compositions disclosed herein may further include a diluent. In some compositions, the diluent may be phosphate-buffered saline (“PBS”). In some compositions, the diluent may be sodium acetate.

[0231] Non-limiting examples of suitable pharmaceutical excipients and additives include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derived sugars, such as sugar alcohols, aldonic acids, esterified sugars, etc.; and polysaccharides or sugar polymers), which may be present alone or in combination, and may constitute 1% to 99.99% by weight or volume, respectively. Non-limiting examples of protein excipients include serum albumin, such as human serum albumin (EISA), recombinant human albumin (rHA), gelatin, casein, etc. Representative amino acid / protein components that may also be used for buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, etc. A preferred amino acid is glycine.

[0232] Non-limiting examples of suitable carbohydrate excipients include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbitol, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melitriose, maltodextrin, dextran, starch, etc.; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucol), inositol, etc. Preferably, the carbohydrate excipient is mannitol, trehalose, and / or raffinose.

[0233] The composition may also include a buffer or pH adjuster; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; tris(hydroxymethyl)aminomethane, tromethamine hydrochloride, or phosphate buffers. Preferred buffers are organic acid salts, such as citrates.

[0234] In addition, the disclosed compositions may include polymeric excipients / additives such as polyvinylpyrrolidone, polysucrose (polymeric sugar), dextrin (e.g., cyclodextrin, such as 2-hydroxypropyl-P-cyclodextrin), polyethylene glycol, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates, such as “TWEEN 20” and “TWEEN 80”), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0235] Therapeutic amounts of the compositions or pharmaceutical compositions disclosed herein can be administered using many known and developed methods. Non-limiting examples of administration methods include bolus, buccal, infusion, intra-articular, intrabronchial, intra-abdominal, intra-sacral, intra-cartilaginous, intracavitary, intra-body cavity, intracerebellum, intravenous, intracolonic, intracervical, intra-nasal, intragastric, intrahepatic, intralesional, intramuscular, intracardiac, intranasal, intraocular, intraosseous, intrapelvic, intrapelvic cavity, intraperitoneal, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intratumoral, intravenous, intravesical, intravesical, intravesical, intravenous, intravesical, oral, extra-gastric, rectal, sublingual, subcutaneous, transdermal, or vaginal administration.

[0236] The compositions disclosed herein can be prepared for parenteral (subcutaneous, intramuscular, or intravenous) or any other administration, particularly in the form of a liquid solution or suspension; for use in vaginal or rectal administration, particularly in semi-solid forms such as, but not limited to, creams and suppositories; for buccal or sublingual administration, such as, but not limited to, tablets or capsules; or for intranasal administration, such as, but not limited to, powders, nasal drops, or aerosols or certain pharmaceutical preparations; or for transdermal administration, such as, but not limited to, gels, ointments, lotions, suspensions, or patch delivery systems, wherein chemical enhancers such as dimethyl sulfoxide are used to modify the skin structure or increase the drug concentration in the transdermal patch (Junginger et al., in “Drug Permeation Enhancement”, Hsieh, DS, ed., pp. 59-90 (Marcel Dekker, Inc. New York 1994)), or using oxidizing agents that enable the application of formulations containing proteins and peptides to the skin (WO 98 / 53847), or applying an electric field to create a transient transport pathway, such as electroporation, or enhancing the flow of charged drugs through the skin, such as iontophoresis, or applying ultrasound, such as ultrasound-enhanced permeation (US Patent Nos. 4,309,989 and 4,767,402) (the above publications and patents are incorporated herein by reference in their entirety).

[0237] For parenteral administration, any composition disclosed herein may be formulated as a solution, suspension, emulsion, granules, powder, or lyophilized powder, in combination with or alone with a pharmaceutically acceptable parenteral carrier. Formulations for parenteral administration may contain sterile water or saline, polyalkylene glycols (such as polyethylene glycol), plant-derived oils, hydrogenated naphthalene, etc., as common excipients. Aqueous or oily suspensions for injection may be prepared using appropriate emulsifiers or humectants and suspending agents according to known methods. Injectables may be non-toxic, non-oral diluents, such as aqueous solutions, sterile injectable solutions, or suspensions in solvents. Water, Ringer's solution, isotonic saline, etc., may be used as suitable carriers or solvents; sterile non-volatile oils may be used as common solvents or suspension solvents. For these purposes, any kind of non-volatile oil and fatty acid may be used, including natural or synthetic or semi-synthetic fatty oils or fatty acids; natural or synthetic or semi-synthetic monoglycerides, diglycerides, or triglycerides. Parenteral administration is known in the art and includes, but is not limited to, conventional injection methods, pneumatic needleless injection devices as described in U.S. Patent No. 5,851,198, and laser perforation devices as described in U.S. Patent No. 5,839,446.

[0238] Formulations intended for oral administration rely on the co-application of adjuvants (e.g., resorcinol and nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether) to artificially increase intestinal permeability, and the co-application of enzyme inhibitors (e.g., pancreatic trypsin inhibitors, diisopropyl fluorophosphate (DFF), and trasylol) to inhibit enzymatic degradation. U.S. Patent No. 6,309,663 describes formulations for delivering hydrophilic pharmaceutical agents (including proteins and protein scaffolds, and combinations of at least two surfactants) intended for oral, buccal, mucosal, nasal, pulmonary, vaginal, transmembrane, or rectal administration. The active ingredient compound in solid dosage forms intended for oral administration may be mixed with at least one additive, including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, arginine, chitin, deacetylated chitosan, pectin, gum arabic, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, and glycerides. These dosage forms may also contain other types of additives, such as inactive diluents, lubricants (e.g., magnesium stearate, parabens), preservatives (e.g., sorbic acid, ascorbic acid, α-tocopherol), antioxidants (e.g., cysteine), disintegrants, binders, thickeners, buffers, sweeteners, flavorings, and aromas.

[0239] Tablets and pills can be further processed into enteric-coated formulations. Liquid formulations for oral administration include emulsions, syrups, elixirs, suspensions, and solutions that are permissible for medical use. These formulations may contain inactive diluents, such as water, commonly used in the aforementioned fields. Liposomes have also been described as drug delivery systems for insulin and heparin (US Patent No. 4,239,754). Recently, synthetic polymer microspheres of mixed amino acids (protein-like substances) have been used for drug delivery (US Patent No. 4,925,673). Furthermore, carrier compounds described in US Patent Nos. 5,879,681 and 5,871,753 for the oral delivery of bioactive agents are known in the art.

[0240] For pulmonary administration, preferably, the compositions or pharmaceutical compositions described herein are delivered at a particle size that effectively reaches the lower respiratory tract, specifically the lungs or sinuses. The compositions or pharmaceutical compositions can be delivered by any of a variety of inhalation or nasal devices known in the art for administering therapeutic agents by inhalation. These devices, capable of depositing nebulized formulations into a patient's sinuses or alveoli, include metered-dose inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers), dry powder generators, nebulizers, etc. All such devices can use formulations suitable for administration to dispense the compositions or pharmaceutical compositions described herein into an aerosol. Such aerosols may contain solutions (both aqueous and non-aqueous) or solid particles. Furthermore, a spray comprising the compositions or pharmaceutical compositions described herein can be generated by forcing a suspension or solution of at least one protein scaffold through a nozzle under pressure. In metered-dose inhalers (MDIs), the propellant, the compositions or pharmaceutical compositions described herein, and any excipients or other additives are contained as a mixture comprising a liquefied compressed gas in a canister. The actuated release of the metering valve is a mixture of aerosols, which preferably contains particles in the size range of less than about 10 pm, preferably from about 1 pm to about 5 pm, and most preferably from about 2 pm to about 3 pm. A more detailed description of the pulmonary administration, formulation and related device is disclosed in PCT Publication No. WO 2019 / 049816.

[0241] For absorption via mucosal surfaces, the composition comprises an emulsion containing multiple submicron particles, mucosal adhesion macromolecules, bioactive peptides, and an aqueous continuous phase, which promotes absorption via mucosal surfaces by achieving mucosal adhesion of the emulsion particles (US Patent No. 5,514,670). Suitable mucosal surfaces for applying the emulsions of this disclosure may include the cornea, conjunctiva, buccal, sublingual, nasal, vaginal, lung, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration (e.g., suppositories) may contain, for example, polyalkylene glycols, petrolatum, cocoa butter, etc., as excipients. Formulations for intranasal administration may be solid and contain, for example, lactose as an excipient, or may be an aqueous or oily solution of nasal drops. For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, etc. (US Patent No. 5,849,695). A more detailed description of mucosal administration and formulations is disclosed in PCT Publication No. WO 2019 / 049816.

[0242] For transdermal administration, the compositions or pharmaceutical compositions disclosed herein are encapsulated in a delivery device, such as liposomes or polymeric nanoparticles, microparticles, microcapsules, or microspheres (collectively referred to as microparticles unless otherwise stated). Many suitable devices are known, including microparticles made of synthetic polymers such as polyhydroxy acids, such as polylactic acid, polyglycolic acid and copolymers thereof, polyorthoesters, polyanhydrides, and polyphosphazenes, as well as natural polymers such as collagen, polyamino acids, albumin and other proteins, alginates and other polysaccharides, and combinations thereof (US Patent No. 5,814,599). A more detailed description of transdermal administration, formulations, and suitable devices is disclosed in PCT Publication No. WO 2019 / 049816.

[0243] It is desirable to deliver the disclosed compound to the subject over a longer period of time (e.g., from one week to one year via a single administration). Various sustained-release, reservoir, or implantable dosage forms may be used. For example, the dosage form may contain a pharmaceutically acceptable, non-toxic salt of a compound having low solubility in bodily fluids, such as (a) an acid addition salt of a polybasic acid, such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene monosulfonic acid or disulfonic acid, polygalacturonic acid, etc.; (b) a salt having a polyvalent metal cation (such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc.) or an organic cation formed from, for example, N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c) a combination of (a) and (b), such as zinc tannate. Additionally, the disclosed compounds, or preferably relatively insoluble salts, such as those just described, can be formulated in gels suitable for injection, for example, aluminum monostearate gel containing, for example, sesame oil. Particularly preferred salts are zinc salts, zinc tannate salts, dihydroxynaphthyl salts, etc. Another type of slow-release reservoir formulation for injection will contain compounds or salts dispersed in a slowly degrading, non-toxic, non-antigenic polymer, such as the polylactic acid / polyglycolic acid polymer described in U.S. Patent No. 3,773,919. Compounds, or preferably relatively insoluble salts, such as those described above, can also be formulated in cholesterol-based silicone rubber pellets, particularly for use in animals. Other slow-release, reservoir, or implantable formulations, such as gaseous or liquid liposomes, are known in the literature (U.S. Patent No. 5,770,222 and “Sustained and Controlled Release Drug Delivery Systems”, edited by JR Robinson, Marcel Dekker, Inc., NY, 1978).

[0244] This type of dosage form is well known in the art. See, for example: Wells et al., eds., Pharmacotherapy Handbook, 2nd edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000); Nursing 2001 Handbook of Drugs, 21st edition, Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, Shannon, Wilson, Stang, eds., Prentice-Hall, Inc., Upper Saddle River, NJ. Preferred dosages may optionally include about 0.1 to 99 mg / kg / dose and / or 100 to 500 mg / kg / dose, or any range, value or fraction thereof, or a single or multiple dose to achieve about 0.1 to 5000 pg / ml. Serum concentration, or any range, value, or fraction thereof. Preferred dosage ranges for the compositions or pharmaceutical compositions disclosed herein are about 1 mg / kg of subject body weight, up to about 3 mg / kg of subject body weight, about 6 mg / kg of subject body weight, or about 12 mg / kg of subject body weight.

[0245] Alternatively, the dosage may vary depending on known factors such as the pharmacodynamic characteristics of the specific drug and its method and route of administration; the recipient's age, health status, and weight; the nature and severity of symptoms; the type, frequency, and desired effect of concurrent treatments. Typically, the dosage of the active ingredient can be from about 0.1 to 100 mg per kilogram of body weight. Generally, 0.1 to 50 mg / kg, and preferably 0.1 to 10 mg / kg, in either direct or sustained-release form, is effective in achieving the desired results.

[0246] As a non-limiting example, treatment in humans or animals may be provided as a single, infusion, or repeated dose, as a single or periodic dose (about 0.1 to 100 mg / kg or any range, value, or fraction thereof) of the compositions or pharmaceutical compositions disclosed herein, for at least one day from day 1 to 40, or alternatively or additionally for at least one day from week 1 to 52, or alternatively or additionally for at least one year from year 1 to 20, or any combination thereof.

[0247] Dosage forms suitable for internal administration typically contain about 0.001 mg to about 500 mg of the active ingredient per unit or container. In these pharmaceutical compositions, the active ingredient is typically present in an amount from about 0.5% by weight to 99.999% by weight based on the total weight of the composition.

[0248] An effective dose may comprise an amount from about 0.001 mg / kg to about 500 mg / kg per single (e.g., high dose), multiple or consecutive administrations, or to achieve a serum concentration of 0.01 pg / ml to 5000 pg / ml per single, multiple or consecutive administrations, or any effective range or value thereof, as performed and determined by known methods, as described herein or known in the relevant art.

[0249] In the aspect of the composition to be administered to subjects in need of this, the cells are modified as disclosed herein, and the cells can be approximately 1 x 102 3 With 1x10 15 Between cells; 1x10 3 With 1x10 15 Between cells; approximately 1x10 4 With 1x10 12 Between cells; approximately 1x10 5 With 1x10 10 Between cells; approximately 1x10 6 With 1x10 9 Between cells; approximately 1x10 6 With 1x10 8 Between cells; approximately 1x10 6 With 1x10 7 Between cells; or approximately 1 x 10 6 With 25xl0 6 Application between individual cells. On one hand, the applied cells are approximately 5 x 10-1 cells. 6 1 cell and 25 x 10 6 Between individual cells.

[0250] A more detailed description of the pharmaceutically acceptable excipients, formulations, dosages and methods of administration of the disclosed compositions and pharmaceutical compositions is disclosed in PCT Publication No. WO 2019 / 049816, which is incorporated herein by reference in its entirety.

[0251] Any use or method of this disclosure may include administering an effective amount of any of the compositions or pharmaceutical compositions disclosed herein to cells, tissues, organs, animals, or subjects in need of such modulation, treatment, or therapy. Such methods may optionally include co-administration or combination therapy for treating such diseases or conditions, wherein administration of any of the compositions or pharmaceutical compositions disclosed herein further includes administration before, concurrently with, and / or after at least one chemotherapeutic agent (e.g., alkylating agents, mitotic inhibitors, radiopharmaceuticals).

[0252] In some respects, no graft-versus-host (GvH) and / or host-versus-graft (HvG) develops in the subject after administration. In one respect, administration is systemic. Systemic administration can be any means known in the art and described in detail herein. Preferably, systemic administration is performed by intravenous injection or infusion. In another respect, administration is local. Local administration can be any means known in the art and described in detail herein. Preferably, local administration is performed by intratumoral injection or infusion, intraspinal injection or infusion, intraventricular injection or infusion, intraocular injection or infusion, or intraosseous injection or infusion.

[0253] In some aspects, the therapeutically effective dose is a single dose. In some aspects, a single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 doses, or any number thereof, manufactured simultaneously. In some aspects, the composition comprises autologous or allogeneic cells, and the dose is sufficient to allow cell implantation and / or sustain for a sufficient time to treat the disease or ailment.

[0254] How to use

[0255] This disclosure provides for the use of the disclosed compositions or pharmaceutical compositions in treating diseases or ailments in cells, tissues, organs, animals, or subjects, such as by administering or exposing a therapeutically effective amount of the disclosed compositions or pharmaceutical compositions to cells, tissues, organs, animals, or subjects, as known in the art or as described herein. In one aspect, the subject is a mammal. Preferably, the subject is a human. The terms “subject” and “patient” are used interchangeably herein.

[0256] This disclosure provides a method for treating phenylketonuria (PKU) in a subject with this need, the method comprising administering to the subject a) at least one therapeutically effective dose of a composition comprising the polynucleotide, carrier, or pharmaceutical composition of this disclosure; and b) at least one LNP composition comprising a gRNA pair targeting and an mRNA encoding a Cas-CLOVER. In some aspects, the mRNA molecule further comprises a 5'-cap. In some aspects, the Cas-CLOVER is a wild-type Cas-CLOVER sequence, an S44P mutant Cas-CLOVER sequence, or other Cas-CLOVER sequences described herein. In some aspects, the at least one LNP composition comprises: about 54% ssPalmO-Ph-P4C2 by mole, about 35% cholesterol by mole, about 5% DOPC by mole, about 5% DOPE by mole, and about 1% DMG-PEG2000 by mole.

[0257] This disclosure provides at least one composition of the present disclosure for treating a subject with PKU, wherein the at least one composition is administered to the subject in at least one therapeutically effective amount.

[0258] This disclosure provides the use of at least one composition of the present disclosure in the preparation of a medicament for treating a subject with PKU, wherein the at least one composition is administered to the subject in at least one therapeutically effective amount.

[0259] This disclosure provides a method for treating at least one disease in a subject, the method comprising administering to the subject at least one therapeutically effective amount of at least one composition of this disclosure, said at least one composition comprising at least one nucleic acid encoding a therapeutic protein.

[0260] In some aspects, the nucleic acid molecule formulated in the compositions disclosed herein may contain at least one transgenic sequence. In some aspects, the transgenic sequence may contain a nucleotide sequence encoding at least one therapeutic protein.

[0261] In some aspects, the nucleic acid molecule formulated in the composition containing AAV donor polynucleotides of this disclosure may contain at least one transgenic sequence. In some aspects, the transgenic sequence may contain a nucleotide sequence encoding at least one therapeutic protein.

[0262] In some aspects, at least one sequence encoding at least one therapeutic protein may be a sequence encoding a human phenylalanine hydroxylase (hPAH) polypeptide, wherein the hPAH polypeptide comprises the nucleic acid sequence of SEQ ID NO: 9. In some aspects, the nucleotide sequence encoding hPAH is codon-optimized.

[0263] In some aspects, the hPAH polypeptide comprises, is substantially composed of, or is composed of the same nucleic acid sequence as, that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage therebetween) of SEQ ID NO: 9. In some aspects, the hPAH polypeptide comprises, is substantially composed of, or is composed of the nucleic acid sequence shown in SEQ ID NO: 9.

[0264] In some aspects of the foregoing methods, a composition comprising a nucleic acid molecule containing a nucleotide sequence encoding at least one gRNA pair and at least one mRNA encoding Cas-CLOVER may be a composition comprising at least one LNP of the present disclosure, wherein the LNP comprises at least one nucleic acid molecule containing a nucleotide sequence encoding at least one Cas-CLOVER. Therefore, the present disclosure provides a method of treating a subject for at least one disease, the method comprising administering to the subject: a) at least one therapeutically effective amount of a composition comprising an AAV donor polynucleotide, wherein the polynucleotide contains a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of the LNP of the present disclosure, wherein the LNP comprises at least one targeting gRNA pair and at least one nucleic acid containing a nucleotide sequence encoding at least one Cas-CLOVER.

[0265] In some aspects of the foregoing methods, a composition comprising a nucleic acid molecule containing a nucleotide sequence encoding at least one AAV donor polynucleotide may be a composition comprising an adeno-associated virus (AAV) viral vector particle containing at least one nucleic acid molecule containing a polynucleotide, wherein the polynucleotide contains a nucleotide sequence encoding at least one therapeutic protein. Therefore, this disclosure provides a method of treating a subject for at least one disease, the method comprising administering to the subject: a) at least one therapeutically effective amount of an AAV viral vector particle containing at least one nucleic acid molecule containing an AAV donor polynucleotide, wherein the polynucleotide contains a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of a composition comprising a nucleic acid molecule containing a targeting gRNA pair and a nucleotide sequence encoding at least one Cas-CLOVER. In some methods, the nucleic acid molecule containing the nucleotide sequence encoding at least one Cas-CLOVER is mRNA.

[0266] In one non-limiting example, an AAV viral vector particle comprising at least one nucleic acid molecule containing a transposon, wherein the transposon contains a nucleotide sequence encoding at least one therapeutic protein, wherein the therapeutic protein is hPAH, may contain, substantially consist of, or consist of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage therebetween) the same nucleic acid sequence as either SEQ ID NO: 18 or 21. In some embodiments, an AAV viral vector particle comprising at least one nucleic acid molecule containing a transposon (where the transposon contains a nucleotide sequence encoding at least one therapeutic protein, wherein the therapeutic protein is hPAH) contains, substantially consists of, or consists of the nucleic acid sequence shown in SEQ ID NO: 18 or 21.

[0267] In some aspects of the foregoing methods, a composition comprising a nucleic acid molecule containing an AAV donor polynucleotide (wherein the polynucleotide contains a nucleotide sequence encoding at least one therapeutic protein) and a composition comprising a nucleic acid molecule containing a targeting gRNA pair and a nucleotide sequence encoding at least one Cas-CLOVER may be applied simultaneously. In some aspects, a composition comprising a nucleic acid molecule containing an AAV donor polynucleotide (wherein the polynucleotide contains a nucleotide sequence encoding at least one therapeutic protein) and a composition comprising a nucleic acid molecule containing a targeting gRNA pair and a nucleotide sequence encoding at least one Cas-CLOVER may be applied sequentially. In some aspects, a composition comprising a nucleic acid molecule containing an AAV donor polynucleotide (wherein the polynucleotide contains a nucleotide sequence encoding at least one therapeutic protein) and a composition comprising a nucleic acid molecule containing a targeting gRNA pair and a nucleotide sequence encoding at least one Cas-CLOVER may be applied approximately in time.

[0268] As used herein, the term "time proximity" refers to a time period preceding or following the administration of one therapeutic composition (e.g., a composition containing a transposon) and the administration of another therapeutic composition (e.g., a composition containing a transposase), such that the therapeutic effect of one therapeutic agent overlaps with that of the other. In some embodiments, the therapeutic effects of one therapeutic agent completely overlap with those of the other. In some embodiments, "time proximity" means that the administration of one therapeutic agent occurs within a time period preceding or following the administration of another therapeutic agent, such that there is a synergistic effect between the two therapeutic agents. "Time proximity" can vary depending on a variety of factors, including, but not limited to, the age, sex, weight, genetic background, medical condition, medical history, and treatment history of the subject to be administered the therapeutic agent; the disease or condition to be treated or improved; the therapeutic outcome to be achieved; the dose, frequency, and duration of administration of the therapeutic agent; the pharmacokinetics and pharmacodynamics of the therapeutic agent; and the route of administration of the therapeutic agent. In some embodiments, "time proximity" means within 15 minutes, within 30 minutes, within 1 hour, within 2 hours, within 4 hours, within 6 hours, within 8 hours, within 12 hours, within 18 hours, within 24 hours, within 36 hours, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within 1 week, within 2 weeks, within 3 weeks, within 4 weeks, within 6 weeks, or within 8 weeks. In some embodiments, multiple administrations of one therapeutic agent may occur within time proximity to a single administration of another therapeutic agent. In some embodiments, time proximity may vary during a treatment cycle or within a dosing regimen.

[0269] In one non-limiting example, this disclosure provides a method of treating a subject with PKU, the method comprising administering to the subject: a) at least one therapeutically effective amount of an AAV viral vector particle comprising at least one nucleic acid molecule containing an AAV donor polynucleotide, wherein the polynucleotide contains a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of the LNP of this disclosure, wherein the LNP comprises at least one targeting gRNA pair and at least one RNA molecule containing a nucleotide sequence encoding at least one Cas-CLOVER. In some aspects, the at least one therapeutic protein may comprise a human phenylalanine hydroxylase (hPAH) polypeptide.

[0270] In some aspects of the treatment methods disclosed herein, administration of at least one composition and / or nanoparticles of the present disclosure to a subject may induce the expression of exogenous proteins (e.g., therapeutic proteins, transposases, etc.) in at least one organ and / or tissue of the subject.

[0271] In some aspects, application of at least one composition and / or nanoparticles of this disclosure causes the expression of exogenous proteins in cells of organs and / or tissues of at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99%.

[0272] In some aspects, application of at least one composition and / or nanoparticles of this disclosure causes expression of exogenous proteins in at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of one or more specific subsets of cells in organs and / or tissues.

[0273] In some aspects, the application of at least one composition and / or nanoparticles of this disclosure causes the expression of exogenous proteins in tissues and / or organs for at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days.

[0274] In some aspects, application of at least one composition and / or nanoparticles of this disclosure causes expression of exogenous proteins in one or more specific subsets of cells in tissues and / or organs for at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days.

[0275] In some aspects, the application of at least one composition and / or nanoparticles of this disclosure causes the expression of exogenous proteins in tissues and / or organs for no more than about 1 day, or no more than about 2 days, or no more than about 3 days, or no more than about 4 days, or no more than about 5 days, or no more than about 6 days, or no more than about 7 days, or no more than about 8 days, or no more than about 9 days, or no more than about 10 days.

[0276] In some aspects, the application of at least one composition and / or nanoparticles of this disclosure causes expression of exogenous proteins in one or more specific subsets of cells in tissues and / or organs for no more than about 1 day, or no more than about 2 days, or no more than about 3 days, or no more than about 4 days, or no more than about 5 days, or no more than about 6 days, or no more than about 7 days, or no more than about 8 days, or no more than about 9 days, or no more than about 10 days.

[0277] This disclosure also provides a method for treating at least one disease in a subject who has a missense mutation (e.g., F263S) that inactivates the PAH gene and who exhibits typical PKU, with elevated serum phenylalanine (Phe) levels, cognitive impairment, and maternal PKU syndrome. See, for example, Charron C. et al., Molecular Therapy, Vol. 11, Supplement 1, May 2005, pp. 163-164.

[0278] In some aspects, this disclosure provides a method of treating a subject with PKU in need, the method comprising administering to the subject: a) at least one therapeutically effective amount of an AAV viral vector particle comprising at least one nucleic acid molecule containing an AAV donor polynucleotide, wherein the polynucleotide contains a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of the LNP of this disclosure, wherein the LNP comprises at least one targeting gRNA pair and at least one RNA molecule containing a nucleotide sequence encoding at least one Cas-CLOVER. In some aspects, the at least one therapeutic protein may comprise a human phenylalanine hydroxylase (hPAH) polypeptide.

[0279] The cells and modified cells disclosed herein

[0280] In another aspect, this document provides cells comprising the vectors and compositions described herein, and modified cells. The cells and modified cells disclosed herein may be mammalian cells. Preferably, the cells and modified cells are human cells. In one aspect, the cells targeted for modification using the LNP compositions disclosed herein are hepatocytes, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal endothelial cells. In one embodiment, the LNP composition comprises at least one targeting gRNA pair and at least one RNA molecule containing a nucleotide sequence encoding at least one Cas-CLOVER, and generates modified cells in vivo. In one embodiment, the polynucleotide comprises a nucleotide sequence encoding a therapeutic gene operatively linked to a liver-specific promoter.

[0281] The cells and modified cells disclosed herein can be somatic cells. The cells and modified cells disclosed herein can be differentiated cells. The cells and modified cells disclosed herein can be autologous cells or allogeneic cells. Allogeneic cells are engineered to prevent adverse reactions to transplantation after administration to a subject. Allogeneic cells can be any type of cell. Allogeneic cells can be stem cells or cells derived from stem cells. Allogeneic cells can be differentiated somatic cells.

[0282] Nucleic acid molecules

[0283] The nucleic acid molecules of this disclosure encoding therapeutic proteins may be in the form of RNA, such as mRNA, hnRNA, tRNA, or any other form; or in the form of DNA, including but not limited to cDNA and genomic DNA obtained by cloning or synthesized; or any combination thereof. DNA may be triple-stranded, double-stranded, or single-stranded, or any combination thereof. Any portion of at least one strand of DNA or RNA may be a coding strand (also known as a sense strand), or it may be a non-coding strand (also known as an antisense strand).

[0284] The isolated nucleic acid molecules of this disclosure may include nucleic acid molecules containing an open reading frame (ORF) that optionally contains one or more introns, such as, but not limited to, at least one designated enzymatically active moiety of a therapeutic protein; nucleic acid molecules containing a coding sequence of a therapeutic protein; and nucleic acid molecules containing a nucleotide sequence substantially different from the above but still encoding therapeutic proteins described herein and / or known in the art due to the degeneracy of the genetic code. Of course, the genetic code is well known in the art. Therefore, it will be conventional for those skilled in the art to generate such degenerate nucleic acid variants encoding specific protein scaffolds of this disclosure. See, for example, Ausubel et al., ibid., and such nucleic acid variants are included in this disclosure.

[0285] As illustrated herein, the nucleic acid molecules comprising nucleic acid molecules encoding therapeutic proteins may include, but are not limited to, those encoding amino acid sequences of an enzymatically active fragment of the therapeutic protein itself; the coding sequence of the entire therapeutic protein or a portion thereof; the coding sequence of the therapeutic protein, such as the coding sequence of at least one signal leader or fusion peptide, having or not having the aforementioned additional coding sequences, such as at least one intron, and additional non-coding sequences, including but not limited to non-coding 5' and 3' sequences, such as transcribed non-translated sequences that function in transcription, mRNA processing (including splicing and polyadenylation signals) (e.g., ribosome binding and mRNA stability); and additional coding sequences encoding additional amino acids, such as those providing additional functions. Thus, the sequence encoding the therapeutic protein may be fused with a marker sequence, such as a sequence encoding a peptide that promotes the purification of the therapeutic protein through fusion.

[0286] Nucleic acid construction

[0287] The isolated nucleic acids of this disclosure can be prepared using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques and / or (d) combinations thereof, which are well known in the art.

[0288] Nucleic acids may conveniently contain sequences other than the polynucleotides disclosed herein. For example, a multiple cloning site containing one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in the isolation of polynucleotides. Additionally, a translatable sequence may be inserted to aid in the isolation of the post-translational polynucleotides of this disclosure. For example, a hexahistine marker sequence provides a convenient means of purifying the proteins of this disclosure. The nucleic acids of this disclosure (excluding coding sequences) may optionally be vectors, adaptors, or linkers for cloning and / or expressing the polynucleotides of this disclosure.

[0289] Additional sequences may be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in the isolation of polynucleotides, or to improve the introduction of polynucleotides into cells. The use of cloning vectors, expression vectors, adaptors, and linkers is well known in the art. (See, for example, Ausubel, ibid.; or Sambrook, ibid.).

[0290] Recombinant methods for constructing nucleic acids

[0291] The isolated nucleic acid compositions (such as RNA, cDNA, genomic DNA, or any combination thereof) disclosed herein can be obtained from biological sources using any suitable cloning method known to those skilled in the art. In some aspects, oligonucleotide probes that selectively hybridize with the polynucleotides of this disclosure under stringent conditions will be used to identify desired sequences in cDNA or genomic DNA libraries. The isolation of RNA and the construction of cDNA and genomic libraries are well known to those skilled in the art (see, for example, Ausubel, ibid., or Sambrook, ibid.).

[0292] Nucleic acid screening and isolation methods

[0293] Probes based on the polynucleotide sequences of this disclosure can be used to screen cDNA or genomic libraries. The probes can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes from the same or different organisms. Those skilled in the art will understand that various degrees of hybridization stringency can be employed in the assay; and the hybridization or washing media can be stringent. As hybridization conditions become increasingly stringent, a greater degree of complementarity must be present between the probe and the target to form a double strand. The stringency can be controlled by one or more of temperature, ionic strength, pH, and the presence of a partially denaturing solvent (such as formamide). For example, the stringency of hybridization can be conveniently altered by changing the polarity of the reactant solution, for example, by manipulating the formamide concentration in the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary depending on the stringency of the hybridization and / or washing media. The degree of complementarity will preferably be 100%, or 70% to 100%, or any range or value thereof. However, it should be understood that minor sequence variations in the probe and primers can be compensated for by reducing the stringency of the hybridization and / or washing media.

[0294] Methods for amplifying RNA or DNA are well known in the art and can be used in accordance with this disclosure based on the teachings and instructions provided herein, without requiring extensive experimentation.

[0295] Known DNA or RNA amplification methods include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (see, for example, U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, and 4,965,188 to Mullis et al.; U.S. Patent Nos. 4,795,699 and 4,921,794 to Tabor et al.; U.S. Patent No. 5,142,033 to Innis; U.S. Patent No. 5,122,464 to Wilson et al.; U.S. Patent No. 5,091,310 to Innis; U.S. Patent No. 5,066,584 to Gyllensten et al.; U.S. Patent No. 4,889,818 to Gelfand et al.; U.S. Patent No. 4,994,370 to Silver et al.; U.S. Patent No. 4,766,067 to Biswas; and U.S. Patent No. 4,766,067 to Ringold et al.). U.S. Patent No. 4,656,134 to Malek et al., and RNA-mediated amplification, which uses antisense RNA targeting a target sequence as a template for double-stranded DNA synthesis (U.S. Patent No. 5,130,238 to Malek et al., trade name NASBA), the entire contents of which are incorporated herein by reference. (See, for example, Ausubel, ibid.; or Sambrook, ibid.)

[0296] For example, polymerase chain reaction (PCR) technology can be used to directly amplify the disclosed polynucleotide sequences and related genes from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be used, for example, to clone nucleic acid sequences encoding proteins to be expressed, to prepare nucleic acids for use as probes to detect the presence of desired mRNA in a sample, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to guide a technician through in vitro amplification methods can be found in Berger et al., Sambrook et al., Ausubel et al., U.S. Patent No. 4,683,202 (1987); and Innis et al., eds., PCR Protocols: A Guide to Methods and Applications, Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic PCR amplification are known in the art. See, for example, the Advantage-GC Genomic PCR Kit (Clontech). Additionally, for example, the T4 gene 32 protein (Boehringer Mannheim) can be used to improve the yield of long PCR products.

[0297] Synthetic methods for constructing nucleic acids

[0298] The isolated nucleic acids disclosed herein can also be prepared by direct chemical synthesis using known methods (see, for example, Ausubel et al., ibid.). Chemical synthesis generally produces single-stranded oligonucleotides, which can be converted into double-stranded DNA by hybridization with complementary sequences or by polymerization with DNA polymerase using single strands as templates. Those skilled in the art will recognize that although the chemical synthesis of DNA may be limited to sequences of about 100 or more bases, longer sequences can be obtained by joining shorter sequences.

[0299] Recombinant expression cassette

[0300] This disclosure further provides recombinant expression cassettes containing the nucleic acids of this disclosure. The nucleic acid sequences of this disclosure, such as cDNA or genomic sequences encoding the protein scaffold of this disclosure, can be used to construct recombinant expression cassettes that can be introduced into at least one desired host cell. The recombinant expression cassette typically contains a polynucleotide of this disclosure operatively linked to a transcription initiation regulatory sequence that directs the transcription of the polynucleotide in the target host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct the expression of the nucleic acids of this disclosure.

[0301] In some respects, isolated nucleic acids, serving as promoters, enhancers, or other elements, can be introduced into appropriate locations (upstream, downstream, or within introns) of the non-heterologous polynucleotides of this disclosure in order to upregulate or downregulate the expression of the polynucleotides of this disclosure. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion, and / or substitution.

[0302] Expression vectors and host cells

[0303] This disclosure provides vectors comprising isolated nucleic acid molecules of this disclosure, host cells genetically engineered with recombinant vectors, and the production of at least one therapeutic protein via recombinant technology, as is well known in the art. See, for example, Sambrook et al., ibid.; Ausubel et al., ibid., each incorporated herein by reference in its entirety.

[0304] Polynucleotides can optionally be ligated into vectors containing selective markers for replication in the host. Generally, plasmid vectors are introduced into precipitates (such as calcium phosphate precipitates) or into complexes containing charged lipids. If the vector is a virus, it can be packaged in vitro using appropriate packaging cell lines and then transduced into host cells.

[0305] The DNA insert should be operatively ligated to a suitable promoter. The expression construct will further contain sites for transcription initiation and termination, as well as ribosome-binding sites in the transcribed region for translation. The coding portion of the mature transcript expressed by the construct will preferably include translation initiated at start and stop codons (e.g., UAA, UGA, or UAG) appropriately located at the ends of the mRNA to be translated, wherein UAA and UAG are preferred for mammalian or eukaryotic cell expression.

[0306] The expression vector will preferably, but optionally, include at least one selection marker. Such markers include, for example, but not limited to, ampicillin, zeocin (bleomycin gene from *Streptomyces indicum*), puromycin (pac gene), hygromycin B (hygB gene), G418 / genemycin (neo gene), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), mycophenolic acid or glutamine synthase (GS, US Patent Nos. 5,122,464; 5,770,359; 5,827,739), blastomycin (bsd gene), eukaryotic cell culture resistance genes, and ampicillin, zeocin (Shbla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / genemycin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, and polymyxin B. Or tetracycline resistance genes for culture in *E. coli* and other bacteria (the aforementioned patent is incorporated herein by reference in its entirety). Appropriate culture media and conditions for the aforementioned host cells are known in the art. Suitable vectors will be apparent to those skilled in the art. Vector constructs can be introduced into host cells by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other known methods. Such methods are described in the art, such as those described in Sambrook, ibid., Chapters 1–4 and 16–18; Ausubel, ibid., Chapters 1, 9, 13, 15, and 16.

[0307] The expression vector will preferably, but optionally, include at least one selective cell surface marker for isolating cells modified by the compositions and methods of this disclosure. Selective cell surface markers of this disclosure include surface proteins, glycoproteins, or a proteome that distinguishes one cell or cell subpopulation from another defined cell subpopulation. Preferably, the selective cell surface marker distinguishes those cells modified by the compositions or methods of this disclosure from those not modified by the compositions or methods of this disclosure. Such cell surface markers include, for example, but not limited to, “designated cluster” or “taxonomic cluster” proteins (generally abbreviated as “CD”), such as truncated or full-length forms of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. Cell surface markers further include the suicide gene marker RQR8 (Philip B et al. Blood. 21 August 2014; 124(8):1277-87).

[0308] The expression vector will preferably, but optionally, include at least one selective drug resistance marker for isolating cells modified by the compositions and methods of this disclosure. The selective drug resistance markers of this disclosure may include wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0309] At least one protein scaffold of this disclosure can be expressed in a modified form, such as a fusion protein, and may include not only secretory signals but also additional heterologous functional regions. For example, additional amino acid regions, particularly charged amino acids, may be added to the N-terminus of the protein scaffold to improve stability and persistence in host cells during purification or during subsequent processing and storage. Furthermore, peptide moieties may be added to the protein scaffold of this disclosure to facilitate purification. Such regions may be removed prior to the final preparation of the protein scaffold or at least one fragment thereof. Such methods are described in numerous standard laboratory manuals, such as Sambrook, ibid., Chapters 17.29–17.42 and 18.1–18.74; Ausubel, ibid., Chapters 16, 17, and 18.

[0310] Those skilled in the art are familiar with numerous expression systems that can be used to express nucleic acid molecules encoding proteins of this disclosure. Alternatively, the nucleic acids of this disclosure can be expressed in host cells by opening (through manipulation) in host cells containing endogenous DNA encoding a scaffold of proteins of this disclosure. Such methods are well known in the art, for example, as described in U.S. Patent Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, which are fully incorporated herein by reference.

[0311] Examples of cell cultures that can be used to produce protein scaffolds, specific portions thereof, or variants are bacterial, yeast, and mammalian cells known in the art. Mammalian cell systems are typically in the form of cell monolayers, although mammalian cell suspensions or bioreactors can also be used. Many suitable host cell lines capable of expressing fully glycosylated proteins have been developed in the art, including COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610), and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2 / 0-Agl4, 293 cells, HeLa cells, etc., which are readily available from, for example, the American Center for Type Culture Collection (www.atcc.org), Manassas, Virginia. Preferred host cells include lymphoid cells, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC accession number CRL-1580) and SP2 / 0-Agl4 cells (ATCC accession number CRL-1851). In one preferred aspect, the recombinant cells are P3X63Ab8.653 or SP2 / 0-Agl4 cells.

[0312] The expression vectors for these cells may include one or more of the following expression control sequences, such as, but not limited to, origin of replication; promoters (e.g., late or early SV40 promoters, CMV promoters (US Patent Nos. 5,168,062; 5,385,839), HSV tk promoters, pgk (glycerol phosphokinase) promoters, EF-1α promoters (US Patent No. 5,266,491), at least one human promoter); enhancers and / or processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites (e.g., SV40 large T Ag poly(A) addition sites), and transcription terminator sequences. See, for example, Ausubel et al., ibid., Sambrook et al., ibid. Other cells that can be used to produce the nucleic acids or proteins of this disclosure are known and / or available, for example, from the U.S. Type Culture Collection's Cell Lines and Hybridoma Catalogue (www.atcc.org) or other known or commercial sources.

[0313] When using eukaryotic host cells, polyadenylated or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenylated poly(A) sequence from the bovine growth hormone gene. Sequences for accurate transcript splicing may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague et al., J.Virol. 45:773-781 (1983)). Furthermore, as is known in the art, gene sequences controlling replication in host cells may be incorporated into the vector.

[0314] Amino acid code

[0315] The amino acids constituting the proteins of this disclosure are generally abbreviated. Amino acid names may be indicated by specifying the amino acid by its single-letter code, its three-letter code, its name, or its three-nucleotide codon, as is well known in the art (see Alberts, B. et al., Molecular Biology of The Cell, 3rd ed., Garland Publishing, Inc., New York, 1994). Therapeutic proteins of this disclosure may include one or more amino acid substitutions, deletions, or additions from spontaneous or mutated and / or human manipulation, as specified herein. Functionally essential amino acids in the therapeutic proteins of this disclosure may be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (e.g., Ausubel, ibid., Chapters 8 and 15; Cunningham and Wells, Science 244: 1081-1085 (1989)). The latter procedure introduces a single alanine mutation at each residue in the molecule. The resulting mutant molecule is then tested for biological activity, such as, but not limited to, at least one neutralizing activity. Sites crucial for maintaining the activity of therapeutic proteins can also be identified through structural analysis, such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al., Science 255:306-312 (1992)).

[0316] As those skilled in the art will understand, this disclosure includes at least one bioactive therapeutic protein of this disclosure. The specific activity of the bioactive therapeutic protein is at least 20%, 30%, or 40% of the specific activity of natural (non-synthetic), endogenous, or related and known protein scaffolds, and preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95%-99% or more. Methods for determining and quantifying enzyme activity and substrate specificity are well known to those skilled in the art.

[0317] The fatty acids and fatty acid esters suitable for modifying the therapeutic proteins of this disclosure may be saturated or may contain one or more unsaturated units. Fatty acids suitable for modifying the protein scaffold of this disclosure include, for example, n-dodecanoic acid (C12, lauric acid), n-tetradecanoic acid (C14, myristic acid), n-octadecanoic acid (C18, stearic acid), n-eicosanoic acid (C20, arachidic acid), n-docosahexaenoic acid (C22, behenic acid), n-triacontica (C30), n-triacontica (C40), cis-A9-octadecanoic acid (C18, oleic acid), all cis-Δ5.8.1 1.14-eicosatetraenoic acids (C20, arachidonic acid), octanoic acid, tetradecanoic acid, octadecanoic acid, docosanoic acid, etc. Suitable fatty acid esters include monoesters of dicarboxylic acids containing straight-chain or branched lower alkyl groups. The lower alkyl groups may contain one to about twelve, preferably one to about six carbon atoms.

[0318] Modified therapeutic proteins and fragments can be prepared using suitable methods, such as by reacting with one or more modifiers. As used herein, the term "modifier" refers to a suitable organic group (e.g., a hydrophilic polymer, fatty acid, fatty acid ester) containing an activating group. An "activating group" is a chemical moiety or functional group that can react with a second chemical group under appropriate conditions to form a covalent bond between the modifier and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as toluenesulfonates, methanesulfonates, halogens (chlorine, bromine, fluorine, iodine), N-hydroxysuccinimide esters (NHS), etc. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acryloyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), etc. Aldehyde functional groups can be coupled to molecules containing amines or hydrazides, and azide groups can react with trivalent phosphorus groups to form phosphoramide or phosphorimide bonds. Suitable methods for introducing an activating group into a molecule are known in the art (see, for example, Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). The activating group may bind directly to an organic group (e.g., a hydrophilic polymer, fatty acid, fatty acid ester) or through a linker motif, such as a divalent C1-C12 group, where one or more carbon atoms may be replaced by heteroatoms (e.g., oxygen, nitrogen, or sulfur). Suitable linker motifs include, for example, tetraethylene glycol, —(CH2)3—, —NH—(CH2)6—NH—, —(CH2)2—NH—, and —CH2—O—CH2—CH2—O—CH2—CH2—O—CH—NH—. Modifiers containing a linker moiety can be generated, for example, by reacting a mono-Boc-alkyl diamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylic ester. The primary amine can be exposed by treating the product with trifluoroacetic acid (TFA) to remove the Boc protecting group, which can then be coupled to the other carboxylic ester, or by reacting it with maleic anhydride, and cyclizing the resulting product to produce an activated maleimide derivative of the fatty acid. (See, for example, Thompson et al., WO 92 / 16221, the entire teachings of which are incorporated herein by reference.)

[0319] The modified therapeutic proteins of this disclosure can be produced by reacting a protein scaffold or fragment with a modifying agent. For example, the organic moiety can be bound to the protein scaffold in a non-site-specific manner by using an amine-reactive modifying agent, such as an NHS ester of PEG. Modified therapeutic proteins and fragments comprising organic portions that bind to specific sites of the protein scaffold of this disclosure can be prepared using suitable methods, such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3: 147-153 (1992); Werlen et al., Bioconjugate Chem., 5: 411-417 (1994); Kumaran et al., Protein Sci. 6 (10): 2233-2241 (1997); Itoh et al., Bioorg. Chem., 24 (1): 59-68 (1996); Capellas et al., Biotechnok Bioeng., 56(4): 456-463 (1997)), and the methods described in Hermanson, GT, Bioconjugate Techniques, AcademicPress: San Diego, Calif. (1996).

[0320] definition

[0321] As used throughout this disclosure, unless the context clearly indicates otherwise, the singular forms “a,” “and,” and “the / said” include a plural of the referred objects. Thus, for example, reference to “a method” includes multiple such methods, and reference to “dosage” includes reference to one or more dosages and their equivalents known to those skilled in the art, and so on.

[0322] The terms “about” or “approximately” refer to an acceptable range of error for a particular value as determined by a person skilled in the art, the acceptable range of error depending in part on how the value is measured or determined, such as the limitations of the measurement system.

[0323] For example, "about" may mean within one or more standard deviations. Alternatively, "about" may mean a range of up to 20%, 10%, 5%, or 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within a certain order of magnitude of a value, preferably within 5 times, and more preferably within 2 times. Where a specific value is described in this application and claims, unless otherwise stated, the term "about" should be considered to mean within an acceptable margin of error for that specific value.

[0324] It should be understood that the compounds disclosed herein may not be presented in a specified configuration (e.g., not in a specified stereochemical configuration). Such representation is intended to cover all available isomers, tautomers, positional isomers, and stereoisomers of the compound. In some embodiments, the presentation of a compound whose configuration is not specified herein is intended to refer to each of the available isomers, tautomers, positional isomers, and stereoisomers of that compound, or any mixture thereof.

[0325] This disclosure provides isolated or substantially purified polynucleotide or protein compositions. The “isolated” or “purified” polynucleotide or protein, or its biologically active portion, is substantially or substantially free of components typically associated with or interacting with polynucleotides or proteins as seen in their natural environment. Therefore, when produced by recombinant technology, the isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium, or when chemically synthesized, is substantially free of chemical precursors or other chemicals. Preferably, the “isolated” polynucleotide does not contain sequences (preferably protein-coding sequences) naturally flanking the polynucleotide in the genomic DNA of the organism from which the polynucleotide is derived (i.e., sequences located at the 5' and 3' ends of the polynucleotide). For example, in various aspects, the isolated polynucleotide may contain nucleotide sequences of less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb, which are naturally flanked by polynucleotides in the genomic DNA of the cell from which the polynucleotide is derived. Proteins that are substantially free of cellular material include protein formulations containing less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating proteins. When the proteins of this disclosure or their bioactive portions are produced via recombinant synthesis, the culture medium preferably represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-target proteins.

[0326] This disclosure provides fragments and variants of the disclosed DNA sequences and proteins encoded by these DNA sequences. As used throughout this disclosure, the term "fragment" refers to a portion of a DNA sequence or a portion of an amino acid sequence, and therefore to a portion of a protein encoded therein. DNA sequence fragments containing coding sequences may encode protein fragments that retain the biological activity of the native protein, and thus retain DNA recognition or binding activity to target DNA sequences as described herein. Alternatively, DNA sequence fragments that can be used as hybridization probes generally do not encode biologically active proteins or do not retain promoter activity. Thus, DNA sequence fragments may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and at most the full-length polynucleotides of this disclosure.

[0327] "Binding" refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between proteins and nucleic acids). Not all components of a binding interaction need to be sequence-specific (e.g., contact with phosphate residues in the DNA backbone), as long as the entire interaction is sequence-specific.

[0328] As used in this paper in the context of polynucleotides (e.g., gRNA) that are homologous to the target sequence, “homologous to” means that the polynucleotide has sufficient homology to bind to the target sequence.

[0329] The term "comprising" is intended to mean that a composition and method include the listed elements, but does not exclude other elements. When used to define compositions and methods, "consistently composed of" should mean excluding other elements that are of any significance to the composition when used for the intended purpose. Thus, a composition consisting essentially of elements as defined herein does not exclude trace contaminants or inert carriers. "Constitutes of" should mean portions other than trace elements of other components and substantial method steps. Each of these transitional terms is defined within the scope of this disclosure.

[0330] As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotides are derived from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells.

[0331] "Gene expression" refers to the conversion of information contained in a gene into a gene product. Gene products can be direct transcription products of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozymes, shRNA, microRNA, structural RNA, or any other type of RNA) or proteins produced by the translation of mRNA. Gene products also include RNA modified through processes such as capping, polyadenylation, methylation, and editing, as well as proteins modified through processes such as methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0332] The "regulation" or "control" of gene expression refers to changes in gene activity. Regulation of expression can include, but is not limited to, gene activation and gene repression.

[0333] "Operationally linked" or its equivalents (e.g., "operationally linked") mean that two or more molecules are positioned relative to each other so that they can interact to affect the function of one or two molecules or a combination thereof.

[0334] This invention discloses non-covalently linked components and methods for preparing and using them. Various components can take the form of various different entities as described herein. For example, non-covalently linked (i.e., operably linked) proteins can be used to achieve temporary interactions to avoid one or more problems in the art. The ability of non-covalently linked components (such as proteins) to associate and dissociate allows functional association to occur only or primarily when such association is required to achieve the desired activity. This bond can persist long enough to achieve the desired effect.

[0335] This invention discloses a method for directing a protein to a specific locus in the genome of an organism. The method may include the steps of providing a DNA positioning component and providing an effector molecule, wherein the DNA positioning component and the effector molecule are operably linked via non-covalent bonding.

[0336] "Target site" or "target sequence" is a nucleic acid sequence that the binding molecule will bind to, provided that sufficient binding conditions exist.

[0337] The terms "nucleic acid," "oligonucleotide," or "polynucleotide" refer to at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Therefore, nucleic acids may also encompass the complementary strand of the depicted single strand. The nucleic acids of this disclosure also encompass substantially identical nucleic acids and their complements that retain the same structure or encode the same protein.

[0338] The probes disclosed herein may comprise single-stranded nucleic acids capable of hybridizing with a target sequence under stringent hybridization conditions. Therefore, the nucleic acids disclosed herein can refer to probes that hybridize under stringent hybridization conditions.

[0339] The nucleic acids disclosed herein can be single-stranded or double-stranded. Even if the majority of the molecule is single-stranded, the nucleic acids disclosed herein may contain double-stranded sequences. Even if the majority of the molecule is double-stranded, the nucleic acids disclosed herein may contain single-stranded sequences. The nucleic acids disclosed herein may include genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids disclosed herein may contain combinations of deoxyribonucleotides and ribonucleotides. The nucleic acids disclosed herein may contain combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. The nucleic acids disclosed herein can be synthesized to include non-natural amino acid modifications. The nucleic acids disclosed herein can be obtained by chemical synthesis or by recombinant methods.

[0340] The nucleic acids disclosed herein (whether their complete sequence or any part thereof) may be non-naturally occurring. The nucleic acids disclosed herein may contain one or more non-naturally occurring mutations, substitutions, deletions, or insertions, making the entire nucleic acid sequence non-naturally occurring. The nucleic acids disclosed herein may contain one or more repeating, inverted, or repetitive sequences, the resulting sequences of which are not naturally occurring, making the entire nucleic acid sequence non-naturally occurring. The nucleic acids disclosed herein may contain non-naturally occurring modified, artificial, or synthetic nucleotides, making the entire nucleic acid sequence non-naturally occurring.

[0341] Due to the redundancy of the genetic code, multiple nucleotide sequences can encode any particular protein. This article envisions all such nucleotide sequences.

[0342] As used throughout this disclosure, the term "operably linked" means that gene expression is under the control of a promoter to which it is spatially linked. The promoter can be located at the 5' (upstream) or 3' (downstream) of the gene it controls. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene it controls (the gene from which the promoter originates). The distance between the promoter and the gene can be adapted to changes without loss of promoter function.

[0343] As used throughout this disclosure, the term "promoter" refers to a synthetic or naturally derived molecule capable of conferring, activating, or enhancing nucleic acid expression in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter may also contain distal enhancer or repressor elements, which may be located thousands of base pairs from the transcription start site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters may constitutively or differentially regulate the expression of genomic molecules at the cell, tissue, or organ where expression occurs, at the developmental stage where expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the phage T7 promoter, phage T3 promoter, SP6 promoter, lac operon promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, EF-1 α promoter, CAG promoter, SV40 early promoter or SV40 late promoter and CMV IE promoter.

[0344] As used throughout this disclosure, the term “variant” when used to describe a nucleic acid means (i) a portion or fragment of a reference nucleotide sequence; (ii) the complement of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid substantially identical to a reference nucleic acid or its complement; or (iv) a nucleic acid that hybridizes to a reference nucleic acid, its complement, or a sequence substantially identical to it under stringent conditions.

[0345] As used throughout this disclosure, the term "vector" refers to a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, and is preferably a DNA plasmid. A vector can contain amino acids and a DNA sequence, an RNA sequence, or a combination of both DNA and RNA sequences.

[0346] As used throughout this disclosure, the term "variant," when used to describe a peptide or polypeptide, refers to a peptide or polypeptide that differs in its amino acid sequence due to the insertion, deletion, or conserved substitution of amino acids, but retains at least one biological activity. A variant may also mean a protein having a substantially identical amino acid sequence to a reference protein, wherein the amino acid sequence of the reference protein retains at least one biological activity.

[0347] Conservative substitution of amino acids, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), is generally recognized in the art as typically involving minute variations. As understood in the art, these minute variations can be identified in part by taking into account the hydrophilicity index of the amino acid. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydrophilicity index of an amino acid is based on considerations of its hydrophobicity and charge. Amino acids with similar hydrophilicity indices can be substituted and still retain protein function. In one respect, amino acids with a hydrophilicity index of ±2 are substituted. The hydrophilicity of an amino acid can also be used to reveal substitutions that will result in the retention of biological function of the protein. Taking the hydrophilicity of amino acids into account in the context of a peptide allows for the calculation of the peptide's maximum local average hydrophilicity, a available metric that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, which is incorporated herein by reference in its entirety.

[0348] Substitution of amino acids with similar hydrophilicity values ​​can result in the peptide retaining its biological activity, such as immunogenicity. Substitution can be performed using amino acids with hydrophilicity values ​​within ±2. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this observation, biocompatible amino acid substitutions are understood to depend on the relative similarity of the amino acids, and specifically on the side chains of those amino acids, as revealed by properties such as hydrophobicity, hydrophilicity, charge, size, and other characteristics.

[0349] As used herein, “conservative” amino acid substitutions can be defined as shown in Tables A, B, or C below. In some respects, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides include conserved substitutions introduced by modifying polynucleotides encoding polypeptides disclosed herein. Amino acids can be classified according to their physical properties and their contribution to secondary and tertiary protein structures. A conserved substitution is the replacement of one amino acid with another amino acid having similar properties. Exemplary conserved substitutions are listed in Table 2.

[0350] Table 2 Conservative Substitution I

[0351]

[0352] Alternatively, conserved amino acids can be grouped as shown in Table 3, according to Lehninger's description (Biochemistry, 2nd edition; Worth Publishers, Inc. NY, NY (1975), pp. 71-77).

[0353] Table 3: Conservative Substitution II

[0354]

[0355] Alternatively, exemplary conservative alternatives are listed in Table 4.

[0356] Table 4 Conservative Substitution III

[0357]

[0358] It should be understood that the polypeptides disclosed herein are intended to include polypeptides carrying one or more amino acid residues inserted, deleted, or substituted, or any combination thereof, as well as modifications other than the insertion, deletion, or substitution of amino acid residues. The polypeptides or nucleic acids disclosed herein may contain one or more conserved substitutions.

[0359] As used throughout this disclosure, the term "more than one" refers to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more of the aforementioned amino acid substitutions. The term "more than one" can also refer to 2, 3, 4, or 5 of the aforementioned amino acid substitutions.

[0360] The polypeptides and proteins disclosed herein (whether their complete sequence or any part thereof) may be non-natural. The polypeptides and proteins disclosed herein may contain one or more non-natural mutations, substitutions, deletions, or insertions, such that the entire amino acid sequence is non-natural.

[0361] The polypeptides and proteins disclosed herein may contain one or more repeating, inverted, or repetitive sequences, the resulting sequences of which are not naturally occurring, making the entire amino acid sequence non-natural. The polypeptides and proteins disclosed herein may contain modified, artificial, or synthetic amino acids that are not naturally occurring, making the entire amino acid sequence non-natural.

[0362] As used throughout this disclosure, “sequence identity” can be determined using a standalone executable BLAST engine program (bl2seq) for blasting two sequences, with default parameters, which is available from the National Center for Biotechnology Information (NCBI) FTP site (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which is incorporated herein by reference in its entirety). When the terms “identical” or “identical” are used in the context of two or more nucleic acid or polypeptide sequences, they refer to the specified percentage of residues that are the same within a specific region of each sequence. Percentage comparability is calculated by optimally aligning two sequences, comparing specified regions of the two sequences, determining the number of positions in the two sequences where the same residues are present to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. When two sequences have different lengths or the alignment produces one or more staggered ends and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator of the calculation but not in the molecule. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Characterization can be performed manually or using computer sequencing algorithms such as BLAST or BLAST 2.0.

[0363] As used throughout this disclosure, the term "endogenous" refers to a nucleic acid or protein sequence naturally associated with a target gene or a host cell into which the target gene is introduced.

[0364] As used throughout this disclosure, the term “exogenous” means a nucleic acid or protein sequence that is not naturally associated with a target gene or a host cell into which the target gene is introduced, including multiple non-natural copies of naturally occurring nucleic acids, such as DNA sequences, or naturally occurring nucleic acid sequences located at non-natural genomic locations.

[0365] This disclosure provides a method for introducing a polynucleotide construct containing a DNA sequence into a host cell. The term "introduction" refers to presenting the polynucleotide construct to the cell in a manner that allows the construct to enter the host cell. The method of this disclosure is not dependent on a specific method for introducing the polynucleotide construct into the host cell, as long as the polynucleotide construct can enter the interior of a host cell. Methods for introducing polynucleotide constructs into bacteria, plants, fungi, and animals are known in the art, including but not limited to stable transformation, transient transformation, and virus-mediated transformation.

[0366] Example

[0367] Example 1: Construction of an exemplary AAV donor polynucleotide for site-specific genome integration

[0368] A. Promoter-free bidirectional luciferase expression of AAV donor polynucleotides

[0369] The following are non-limiting examples demonstrating the construction of an exemplary AAV donor polynucleotide (which contains a promoterless bidirectional luciferase expression cassette for measuring site-specific integration into intron 3 of the albumin gene) and the luciferase expression level controlled by the endogenous albumin promoter.

[0370] A general schematic diagram of an AAV donor polynucleotide containing a two-way luciferase reporter cassette is shown in Figure 1A. As shown in Figure 1A, the AAV donor polynucleotide contains, in the 5' to 3' direction: a) a first AAV ITR sequence (SEQ ID NO: 12); b) a first target sequence homologous to the upstream portion of intron 3 of the albumin gene (SEQ ID NO: 13); c) a splice acceptor sequence (SEQ ID NO: 14); d) a P2A sequence (SEQ ID NO: 15); e) a sequence encoding luciferase (SEQ ID NO: 16); f) a polyadenylated (poly(A)) sequence (SEQ ID NO: 17); g) a DNA spacer sequence (SEQ ID NO: 18); h) a reverse poly(A) sequence (SEQ ID NO: 19); i) a reverse sequence encoding luciferase (SEQ ID NO: 20); j) a reverse P2A sequence (SEQ ID NO: 21); k) a reverse splice acceptor sequence (SEQ ID NO: 20). 22); l) a second target sequence homologous to the downstream portion of intron 3 of the albumin gene (SEQ ID NO: 23); and m) a second AAV ITR sequence (SEQ ID NO: 24).

[0371] The AAV donor DNA was constructed using Gibson assembly from i) a single DNA fragment containing a bidirectional SA-P2A-luciferase gene poly(A); and ii) a vector backbone containing Cas-CLOVER linearization sites (first and second target sequences) side-attached to the AAV ITR. The complete nucleotide sequence of the AAV donor DNA containing a promoterless bidirectional luciferase expression cassette is provided in SEQ ID NO: 6.

[0372] B. Promoterless bidirectional phenylalanine hydroxylase (PAH) expression of AAV donor vector polynucleotides

[0373] The following are non-limiting examples demonstrating the construction of an exemplary AAV donor polynucleotide (which contains a promoterless bidirectional PAH expression cassette for site-specific integration into intron 3 of the albumin gene) and the PAH expression level controlled by the endogenous albumin promoter.

[0374] A general schematic diagram of an AAV donor polynucleotide containing a bidirectional PAH expression cassette is shown in Figure 1B. Figure 1BAs shown, the AAV donor polynucleotide comprises, in the 5' to 3' direction: a) a first AAV ITR sequence (SEQ ID NO: 12); b) a first target sequence homologous to the upstream portion of intron 3 of the albumin gene (SEQ ID NO: 13); c) a splice acceptor sequence (SEQ ID NO: 14); d) a P2A sequence (SEQ ID NO: 15); e) a sequence encoding a codon-optimized and modified PAH gene (SEQ ID NO: 25); f) a polyadenylated (poly(A)) sequence (SEQ ID NO: 17); g) a DNA spacer sequence (SEQ ID NO: 18); h) a reverse poly(A) sequence (SEQ ID NO: 19); i) a reverse sequence encoding a codon-optimized and modified PAH gene (SEQ ID NO: 26); j) a reverse P2A sequence (SEQ ID NO: 21); k) The reverse splice acceptor sequence (SEQ ID NO: 22); l) a second target sequence homologous to the downstream portion of intron 3 of the albumin gene (SEQ ID NO: 23); and m) a second AAVITR sequence (SEQ ID NO: 24).

[0375] Following instructions from various manufacturers (e.g., ThermoFisher, Blue Heron Biotech, and Integrated DNA Technologies), computer algorithms were used to codon-optimize the nucleotide sequence of the mRNA encoding the human PAH sequence (GenBank U49897.1) to improve PAH expression. After codon optimization, the optimized sequence was further modified to remove: i) any unwanted restriction endonuclease recognition sites; and ii) putative cryptic splicing sites for cloning the modified sequence into the AAV donor vector. The sequence also contains an N-terminal hemagglutinin tag.

[0376] The AAV donor polynucleotide DNA was constructed using Gibson assembly from i) a single DNA fragment containing a bidirectional SA-P2A-HA-PAH gene poly(A); and ii) a vector backbone containing Cas-CLOVER linearization sites (first and second target sequences) flanked by an AAV ITR. The complete nucleotide sequence of the AAV donor polynucleotide containing a promoterless bidirectional PAH expression cassette is provided in SEQ ID NO: 8.

[0377] C. TTRe-PAH reporter gene AAV donor polynucleotide

[0378] The following are non-limiting examples demonstrating the construction of an exemplary AAV donor polynucleotide (which contains a TTRe-PAH promoter expression cassette for enabling site-specific DNA integration into intron 3 of the albumin gene) and the PAH expression level using a heterologous promoter.

[0379] A general schematic diagram of an AAV donor polynucleotide containing a bidirectional PAH expression cassette is shown in Figure 1C. Figure 1C As shown, the AAV donor polynucleotide comprises, in the 5' to 3' direction: a) a first AAV ITR sequence (SEQ ID NO: 12); b) a first target sequence homologous to the upstream portion of intron 3 of the albumin gene (SEQ ID NO: 13); c) a TTRe promoter sequence; d) a splice acceptor sequence (SEQ ID NO: 14); e) a P2A sequence (SEQ ID NO: 15); f) a sequence encoding a codon-optimized and modified PAH gene (SEQ ID NO: 25); g) a 3' UTR sequence; h) a polyadenylated (poly(A)) sequence (SEQ ID NO: 17); i) a DNA spacer sequence (SEQ ID NO: 18); j) a second target sequence homologous to the downstream portion of intron 3 of the albumin gene (SEQ ID NO: 23); and k) a second AAV ITR sequence (SEQ ID NO: 24).

[0380] The first AAV donor PAH reporter gene polynucleotide was constructed using Gibson assembly from i) a single DNA fragment containing the TTRe promoter-HA-PAH gene-poly(A); and ii) a vector backbone containing Cas-CLOVER linearization sites (first and second target sequences) and a UTR, side-attached to an AAV ITR. The complete nucleotide sequence of the first AAV PAH reporter gene donor polynucleotide is provided in SEQ ID NO: 10.

[0381] Example 2 - Preparation of 5'-capped mRNA encoding Cas-CLOVER encapsulated in LNP compositions

[0382] The following are non-limiting examples of the preparation of exemplary mRNA encoding Cas-CLOVER, which can be incorporated into LNP compositions for use in methods in combination with the AAV donor DNA polynucleotides and vectors disclosed herein.

[0383] The DNA plasmid pRTb_Cas-CLOVERv3 encodes Cas-CLOVER (SEQ ID NO: 1), which contains an N-terminal SV40 nuclear localization signal (NLS) and contains the 5'UTR of the human β-globin gene (HBB) and the 3'UTR of the human cytochrome b-245 α chain gene (CYBA).

[0384] This plasmid is used as a template for an in vitro transcription reaction to produce an mRNA encoding Cas-CLOVERv3 and further containing a 5'-cap.

[0385] In short, approximately 300 µg of supercoiled pRTb_Cas-CLOVERv3 was added to a 15 mL Erlenmeyer flask containing 300 µL of CutSmart buffer and 60 µL of restriction enzyme Bbsl-HF (New England Biolabs, catalog number R3539M), for a total volume of 3000 µL. The plasmid DNA was linearized by incubating it overnight at 37°C to ensure complete digestion.

[0386] Following the manufacturer's instructions, the linearized plasmid was purified using the DNA QIAquick PCR Purification Kit (Qiagen, catalog number 28106), and the purified DNA was eluted in 900 µL of nuclease-free water (ThermoFisher, catalog number AM9937). The DNA concentration and purity of the eluent were determined using a NanoDrop microvolume spectrophotometer (ThermoFisher) according to the manufacturer's instructions.

[0387] According to the internal quality control production batch records, purified plasmids were used as DNA templates to generate mRNA using a custom-designed in vitro transcription mMESSAGE mMACHINE T7 transcription kit (ThermoFisher, catalog number AM1345B001). In short, 100 mM stock solutions of nucleotides GTP (ThermoFisher, catalog number R0481), ATP (ThermoFisher, catalog number R0481), CTP (ThermoFisher, catalog number R0481), and N1MeΨTP (N1-methylpseudouridine-5'-triphosphate) (TriLink, catalog number N-1081) and CleanCap reagent AG (m7G(5')ppp(5')(2'OMeA)pG); TriLink, catalog number N-7113) were prepared. Each of 1,485 µL of ATP, UTP, and 5MeC was blended with each of 1,188 µL of GTP and CleanCap reagent AG.

[0388] 153 µg of linearized pRTb_Cas-CLOVERv3 DNA, 1,800 µL of 10X T7 RXN buffer (ThermoFisher, catalog number AM1345B001), 1,800 µL of T7 enzyme mixture (ThermoFisher, catalog number AM1345B001), and 6,831 µL of NTP and capping blend were added to a 50 mL Erlenmeyer flask (final volume 18,000 µL) and incubated at 37 °C for 3 hours. 900 µL of DNase I enzyme (ThermoFisher, catalog number AM1345B001) was added to an aliquot of the sample, and the tubes were further incubated at 37 °C for 15 minutes to degrade the DNA template.

[0389] The poly(A) tail was enzymatically added to the 3' end of 5'-CleanCap®-Cas-CLOVER-N1MeΨ mRNA. 18,000 µL of 5X EPAP buffer (ThermoFisher, catalog number AM1345B001), 9,000 µL of 25 mMnCl2 (ThermoFisher, catalog number AM1345B001), 9,000 µL of ATP solution (ThermoFisher, catalog number AM1345B001), and 3,000 µL of E-PAP (ThermoFisher, catalog number AM1345B001) were added to the IVT reaction (total volume 90,000 µL) and incubated at 37°C for 1 hour. The bulk E-PAP reaction was then aliquoted into three 125 mL PETG vials, each containing 30 mL aliquots.

[0390] Purify 5'-CleanCap®-Cas-CLOVER-poly(A)-N1MeΨ mRNA using the RNeasy Maxi Purification Kit (Qiagen, catalog number 75162) according to the manufacturer's instructions. Briefly, prepare a working stock solution for Buffer RLT using 178.2 mL of Buffer RLT (Qiagen, catalog number 75162) and 1,800 µL of 2-mercaptoethanol (Sigma, catalog number M3148). Add 52.2 mL of BME + RLT solution and 37.8 mL of 100% EtOH (ThermoFisher, catalog number BP2818) to each 30 mL aliquot of mRNA. Elute the purified mRNA product in 52.5 mL of nuclease-free water and store the bulk product at -80°C. Repeat the DNA linearization, IVT, and mRNA purification process until the target yield is achieved.

[0391] Before assembly in 500 mL PETG bottles, the bulk mRNA batches were analyzed by gel electrophoresis, and concentration readings were obtained using NanoDrop sampling. Lithium chloride 5X (ThermoFisher, catalog number AM1345B001) was added to the pooled mRNA at 1 / 3 of the total mRNA volume, and then divided into 40 mL aliquots in 50 mL conical tubes and incubated at -20 °C for 45 min. After incubation, the conical tubes were centrifuged at 14,000 g for 30 min at 4 °C. The mRNA particles were washed three times with 70% EtOH (ThermoFisher, catalog number BP8201).

[0392] The washed mRNA particles were dried and then resuspended in nuclease-free water. The mRNA concentration was determined using NanoDrop, and additional nuclease-free water was added as needed to further dilute the product to the target concentration. The mRNA was sterilely filtered using a 0.22 µm PES SteriCup filter (Sigma, catalog number 52GPU05RE) before measuring the final mRNA concentration and purity on NanoDrop.

[0393] Example 3 - Preparation of LNP compositions containing 5'-capped mRNA and gRNA pairs encoding Cas-CLOVER

[0394] The following are non-limiting examples of exemplary methods for formulating LNP compositions comprising 5'-capped mRNA and gRNA pairs encoding Cas-CLOVER for use in combination with AAV donor DNA polynucleotides disclosed herein.

[0395] Individual 25 mg / ml stock solutions were prepared by dissolving lipids in 200-proof HPLC-grade ethanol and stored at -80°C until reconstitution. During reconstitution, the lipid stock solutions were briefly equilibrated to room temperature and then placed on a hot plate maintained at a temperature range of 50°C to 55°C. The heated lipid stock solutions were then combined to obtain the desired final molar percentage.

[0396] Add 1 mg / ml of the 5'CleanCap-N1-CC mRNA solution prepared in Example 2, to be incorporated into the LNP, to 150 mM sodium acetate buffer (pH 5.2) to form a stock solution and store it on ice. Add 1 mg / ml of the gRNA pair to be incorporated into the LNP, dissolved in RNase-free water, to 150 mM sodium acetate buffer (pH 5.2) to form a gRNA stock solution and store it on ice. Mix the mRNA and gRNA stock solutions at a 3:1 ratio to form a nucleic acid stock solution. Following the manufacturer's instructions, use a NanoAssemblr® instrument (Precision Nanosystems, Vancouver, British Columbia, Canada) to mix the lipid phase with the aqueous phase mRNA / gRNA within the microfluidic chip to form an LNP composition containing encapsulated Cas-CLOVER mRNA and a targeting gRNA pair. The Nanoassemblr process parameters for mRNA encapsulation are a flow rate of 20 ml / min and a lipid:RNA ratio (v / v) of 1:3.

[0397] The resulting Cas-CLOVER mRNA-gRNA to LNP composition was then transferred to a Repligen Float-A-Lyzer dialysis unit (Spectrum Chemical Mfg. Corp., CA, USA) with a molecular weight cutoff (MWCO) of 8 to 10 kDa and treated by dialyzing overnight at 4°C with 25 mM sodium acetate (dialysate: dialysis buffer volume at least 1:200 v / v) at pH 5.5 (or alternatively dialyzing at room temperature for at least 4 hours) to remove 25% ethanol and achieve complete buffer exchange. Where appropriate, the LNP composition was further concentrated in an Amicon® Ultra-4 centrifuge filter (MWCO-30 kDa, Millipore Sigma, USA) at approximately 4100 xg. Sucrose was added to the mRNA LNP to a final concentration of 5% (w / v) and then stored at 4°C or frozen at -80°C until further use.

[0398] The average particle size of LNP ranges from approximately 85 nm to 105 nm.

[0399] Example 4 - In vivo site-specific integration of AAV donor polynucleotides from Cas-CLOVER into hepatocytes

[0400] The following are non-limiting examples demonstrating that AAV donor DNA polynucleotides can be specifically integrated into intron 3 of the albumin gene in vivo.

[0401] Adult (10 to 12 weeks old) C57BL / 6 mice (n = 3 mice / group) were treated with the following:

[0402] Treatment #1: Medium;

[0403] Treatment #2: Promoter-free bidirectional luciferase expression of AAV viral vector particles (Example 1A);

[0404] Treatment #3: Promoter-free bidirectional luciferase expression AAV viral vector particles (1E12 vg / kg) combined with 1.5 mg / kg of LNPs encapsulating the mRNA encoding Cas-CLOVER and the gRNA pair targeting intron 3 of the mouse albumin gene;

[0405] Treatment #4: A promoter-free bidirectional luciferase expression AAV viral vector particle (5E12 vg / kg) combined with 1.5 mg / kg of LNPs encapsulating the mRNA encoding Cas-CLOVER and the gRNA pair targeting intron 3 of the mouse albumin gene;

[0406] Treatment #5: Promoter-free bidirectional luciferase expression of AAV viral vector particles (1E13 vg / kg) combined with 1.5 mg / kg of LNPs encapsulating the mRNA encoding Cas-CLOVER and the gRNA pair targeting intron 3 of the mouse albumin gene; and

[0407] Treatment #6: Promoter-free bidirectional luciferase expression AAV viral vector particles (3E13 vg / kg) combined with 1.5 mg / kg of LNPs encapsulating mRNA encoding Cas-CLOVER and gRNA targeting intron 3 of the mouse albumin gene.

[0408] The promoterless bidirectional luciferase expression AAV viral vector particle is an AAV viral vector particle containing the nucleic acid of SEQ ID NO: 6 and is administered post-orbital. The bidirectional cassette allows albumin-regulated luciferase mRNA expression regardless of whether the AAV donor DNA polynucleotide is integrated in the forward or reverse conformation.

[0409] The LNP composition containing gRNA pairs and mRNA encoding Cas-CLOVER prepared in Example 2 comprises the following components: ssPalmO-Ph-P4C2, DOPC, DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 54:5:5:35:1 and has a lipid:RNA ratio of 100:1 (w / w). The LNP composition was administered via tail vein injection.

[0410] Fourteen days after administration, whole-body luminescence imaging (BLI) of control and treated animals was performed, and liver biopsies and blood samples were collected from treated mice to determine the percentage of albumin gene editing, the percentage of AAV donor DNA integration, and luciferase expression levels relative to albumin mRNA expression levels. For genomic DNA isolation, liver samples were mixed with lysis buffer (200 µL lysis buffer + 10 µL proteinase K for 15 mg tissue) and homogenized in a TissueLyser II (Qiagen) using Triple-Pure zirconium beads (Fisher Scientific). The homogenized tissue was then incubated at 56 °C for 30 min and column purified using the Monarch Genomic DNA Purification Kit (New England Biolabs) according to the manufacturer's instructions. Final DNA elution was performed in 50 µL elution buffer (10 mM Tris-Cl, pH 8.5). The concentration and purity of the DNA samples were assessed by measuring absorbance at 260 nm and 280 nm using a Nanodrop device. DNA samples were used for albumin editing and transgene integration quantification. The degree of albumin gene editing observed in Cas-CLOVER mRNA delivered to mice was measured using a drop-off assay with a fluorescent probe hybridizing to the Cas-CLOVER target site via Droplet Digital PCR (ddPCR). Transgene integration at the albumin site was measured using a probe-based detection protocol via ddPCR. A primer binding to the luciferase transgene was paired with a primer binding to albumin genomic DNA near the Cas-CLOVER cleavage site.

[0411] To isolate mRNA, liver samples were mixed with lysis buffer (300 µL lysis buffer + 25 mg tissue in 15 µL proteinase K) and homogenized in a TissueLyser II (Qiagen) using Triple-Pure zirconium beads (Fisher Scientific). The homogenized tissue was then incubated at room temperature for 30 min and column purified using the Quick RNA Miniprep Plus kit (Zymo Research) according to the manufacturer's instructions. Final RNA elution was performed in 50 µL of DNase-free / RNase-free water. The concentration and purity of the DNA sample were assessed using a Nanodrop device by measuring absorbance at 260 nm and 280 nm. Luciferase mRNA was quantified using the RNA sample via quantitative real-time polymerase chain reaction (RT-qPCR).

[0412] The results are depicted in Figures 2A and 2B. Luciferase expression is shown via BLI (Figure 2A), and it was found that the integration percentage and luciferase mRNA expression (Figure 2B) were dose-dependent; and compared to levels observed in mice treated with either the mediator or AAV donor DNA alone, the addition of an LNP containing albumin and Cas-CLOVER mRNA targeting the gRNA pair significantly increased luciferase mRNA expression, integration percentage (Figure 2B), and the percentage of albumin-edited haploid genomes (Figure 2A). These observations suggest that integration is site-specific and that expression is driven by endogenous albumin genes.

[0413] Example 5 - In vivo site-specific integration of AAV donor polynucleotides expressed using Cas-CLOVER and PAH in hepatocytes

[0414] The following is a non-restrictive example demonstrating that AAV donor DNA polynucleotides can be specifically integrated into intron 3 of the albumin gene in adult mouse hepatocytes in vivo, and that PAH is expressed in the integrated hepatocytes.

[0415] Adult (10 to 12 weeks old) C57BL / 6 mice (n = 3 mice / group) were treated with the following:

[0416] Treatment #1: Medium;

[0417] Treatment #2: Promoterless bidirectional PAH reporter gene AAV viral vector particle (Example 1B);

[0418] Treatment #3: Promoterless bidirectional PAH expression AAV viral vector particles (1E12 vg / kg) combined with 1.5 mg / kg of LNPs encapsulating Cas-CLOVER mRNA and gRNA targeting intron 3 of the mouse albumin gene;

[0419] Treatment #4: Promoterless bidirectional PAH expression AAV viral vector particles (3E12 vg / kg) combined with 1.5 mg / kg of LNPs encapsulating Cas-CLOVER mRNA and gRNA targeting intron 3 of the mouse albumin gene;

[0420] Treatment #5: Promoterless bidirectional PAH expression AAV viral vector particles (1E13 vg / kg) combined with 1.5 mg / kg of LNPs encapsulating the mRNA encoding Cas-CLOVER and the gRNA pair targeting intron 3 of the mouse albumin gene; and

[0421] Treatment #6: TTRe-PAH expression of AAV viral vector particles (Example 1C);

[0422] Treatment #7: TTRe-PAH expression of AAV viral vector particles (1E12 vg / kg) combined with 1.5 mg / kg of LNPs encapsulating the mRNA encoding Cas-CLOVER and the gRNA pair targeting intron 3 of the mouse albumin gene;

[0423] Treatment #8: TTRe-PAH expression of AAV viral vector particles (3E12 vg / kg) combined with 1.5 mg / kg of LNPs encapsulating the mRNA encoding Cas-CLOVER and the gRNA pair targeting intron 3 of the mouse albumin gene; and

[0424] Treatment #9: TTRe-PAH expression of AAV viral vector particles (1E13 vg / kg) combined with 1.5 mg / kg of LNPs encapsulated with mRNA encoding Cas-CLOVER and gRNA targeting intron 3 of the mouse albumin gene.

[0425] The promoterless bidirectional PAH reporter gene AAV viral vector particle is an AAV viral vector particle containing the nucleic acid of SEQ ID NO: 8 and is administered post-orbital. The bidirectional cassette allows albumin-regulated PAH mRNA expression regardless of whether the AAV donor polynucleotide is integrated in the forward or reverse conformation.

[0426] The TTRe-PAH-expressing AAV viral vector particle is an AAV viral vector particle containing the nucleic acid of SEQ ID NO: 10 and is administered post-orbital. PAH expression is controlled by a heterologous TTRe promoter.

[0427] The LNP composition containing gRNA pairs and mRNA encoding Cas-CLOVER prepared in Example 2 comprises the following components: ssPalmO-Ph-P4C2, DOPC, DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 54:5:5:35:1 and has a lipid:RNA ratio of 100:1 (w / w). The LNP composition was administered via tail vein injection.

[0428] Fourteen days after administration, liver biopsies and blood samples were collected from treated mice to determine the percentage of albumin gene editing, the percentage of AAV donor DNA integration, and the human PAH expression level relative to mouse PAH expression levels. To isolate genomic DNA, liver samples were mixed with lysis buffer (200 µL lysis buffer + 10 µL proteinase K for 15 mg tissue) and homogenized in a TissueLyser II (Qiagen) using Triple-Pure zirconium beads (Fisher Scientific). The homogenized tissue was then incubated at 56°C. Incubate for 30 minutes and perform column purification using the Monarch Genomic DNA Purification Kit (New England Biolabs) according to the manufacturer's instructions. Final DNA elution is performed in 50 µL of elution buffer (10 mM Tris-Cl, pH 8.5). The concentration and purity of the DNA sample are assessed using a Nanodrop device by measuring absorbance at 260 nm and 280 nm. The DNA sample is used for albumin editing and transgene integration quantification. The degree of albumin gene editing observed against Cas-CLOVER mRNA delivered to mice is measured using a drop-off assay with a fluorescent probe containing a hybridization site to the Cas-CLOVER target site via Droplet Digital PCR (ddPCR). The degree of transgene integration at the albumin site is measured by ddPCR using a probe-based detection protocol. A primer binding within the PAH transgene is paired with a primer binding ALB genomic DNA near the Cas-CLOVER cleavage site.

[0429] To isolate mRNA, liver samples were mixed with lysis buffer (300 µL lysis buffer + 15 µL proteinase K for 25 mg tissue) and homogenized using Triple-Pure zirconium beads (Fisher Scientific) in a TissueLyser II (Qiagen) syringe. The homogenized tissue was then incubated at room temperature for 30 min and column purified using the Quick RNA Miniprep Plus kit (Zymo Research) according to the manufacturer's instructions. Final RNA elution was performed in 50 µL of DNase-free / RNase-free water. The concentration and purity of the DNA sample were assessed using a Nanodrop device by measuring absorbance at 260 nm and 280 nm. Human PAH mRNA was quantified using the RNA sample via quantitative real-time polymerase chain reaction (RT-qPCR).

[0430] The results are shown in Figure 3A As defined in 3B. Robust albumin gene editing was observed in the context of no promoter and a TTRe-PAH AAV donor (1E12; Figure 3A). For both reporter gene AAV donor polynucleotides ( Figure 4A Human PAH mRNA expression is dose-dependent, and the total number of integrations per haploid genome is also dose-dependent (Figure 3B).

[0431] Example 6 - In BTBR PAH enu In a mouse model, co-administration of an AAV donor vector containing a codon-optimized and modified PAH gene and an LNP combination containing a gRNA pair and mRNA encoding Cas-CLOVER restored normal serum phenylalanine levels.

[0432] The following is displayed in BTBR PAH enu Non-limiting examples of restoring normal serum phenylalanine levels and normal hypopigmentation in mouse models by co-administration of an AAV donor polynucleotide comprising a codon-optimized and modified hPAH gene and an LNP composition comprising a gRNA pair and an mRNA encoding Cas-CLOVER.

[0433] Adult males (n = 1 to 3 / group) were given BTBR PAH. enu Mice were treated with the following:

[0434] Treatment #1: Medium;

[0435] Treatment #2: Bidirectional PAH expression AAV viral vector particles without promoter (1E13 vg / kg; Example 1B);

[0436] Treatment #3: Promoterless bidirectional PAH expression AAV viral vector particles (3E12 vg / kg) combined with 1.5 mg / kg of LNPs encapsulating the mRNA encoding Cas-CLOVER and the gRNA pair targeting intron 3 of the mouse albumin gene;

[0437] Treatment #4: Promoterless bidirectional PAH expression AAV viral vector particles (1E13 vg / kg) combined with 1.5 mg / kg of LNPs encapsulating Cas-CLOVER mRNA and gRNA targeting intron 3 of the mouse albumin gene;

[0438] Treatment #5: TTRe-PAH expression of AAV viral vector particles (3E12 vg / kg; Example 1C);

[0439] Treatment #6: PAH TTRe expression of AAV viral vector particles (3E12 vg / kg) combined with 1.5 mg / kg of LNPs encapsulating the mRNA encoding Cas-CLOVER and the gRNA pair targeting intron 3 of the mouse albumin gene; and

[0440] Treatment #7: PAH TTRe expressed AAV viral vector particles (1E13 vg / kg) with 1.5 mg / kg of LNPs encapsulated with mRNA encoding Cas-CLOVER and gRNA targeting intron 3 of the mouse albumin gene. Untreated adult C57BL / 6 wild-type mice (n = 3 / group) were presented in parallel as a positive control for measuring normal serum Phe levels.

[0441] Blood samples were collected from each group of treated and control mice on days 0, 7, 14, and 28, and serum Phe levels were quantified using a colorimetric assay (Abcam). The results are shown in Figures 5A and 5B. Figure 5A As shown, diseased mice treated with a promoterless AAV PAH reporter gene vector and an LNP formulation containing albumin gRNA pairs and mRNA encoding Cas-CLOVER showed a rapid and dramatic decrease in serum Phe levels on day 7, approaching wild-type levels. These serum Phe levels continued to decrease further on days 14 and 28, falling below baseline (Figure 5A).

[0442] A similar decrease in serum Phe levels was observed using the AAV-TTRe-PAH reporter gene vector in the presence of an LNP formulation containing albumin gRNA pairs and mRNA encoding Cas-CLOVER (Figure 5B).

[0443] Therefore, co-administration of the disclosed AAV donor vector containing a codon-optimized PAH gene and an LNP composition containing an albumin intron 3-targeting gRNA pair and mRNA encoding Cas-CLOVER in an in vivo PKU disease model reduced serum Phe levels to levels equal to or lower than those in wild-type mice. These results indicate that Cas-CLOVER integration of an AAV donor polynucleotide containing a codon-optimized and modified PAH gene, followed by stable expression of PAH in integrated hepatocytes, leads to a reduction in serum Phe levels to normal wild-type levels.

Claims

1. An adeno-associated virus (AAV) donor polynucleotide comprising, along the 5' to 3' directions: a) First AAV ITR sequence; b) A first targeting sequence homologous to the first region of intron 3 of the albumin gene, the first targeting sequence comprising the sequence of SEQ ID NO: 13; c) Splice acceptor sequence; d) P2A sequence; e) A sequence encoding a first codon-optimized and modified PAH gene, said sequence comprising the sequence of SEQ ID NO:25; f) DNA spacer sequence; g) Reverse poly-A sequence; h) The reverse sequence encoding a second codon-optimized and modified PAH gene, said reverse sequence comprising the sequence of SEQ ID NO: 26; i) Reverse P2A sequence; j) Backsplicing acceptor sequence; k) A second targeting sequence homologous to the second region of intron 3 of the albumin gene, the second targeting sequence comprising the sequence of SEQ ID NO: 23; and l) Second AAV ITR sequence.

2. The AAV donor polynucleotide according to claim 1, wherein... The first AAV ITR sequence contains the sequence of SEQ ID NO: 12; The splice acceptor sequence includes the sequence of SEQ ID NO: 14; The P2A sequence includes the sequence of SEQ ID NO: 15; The polyadenylation (poly(A)) sequence includes the sequence of SEQ ID NO: 17; The DNA spacer sequence comprises the sequence of SEQ ID NO: 18; The reverse polyA sequence comprises the sequence of SEQ ID NO: 19; The reverse P2A sequence comprises the sequence of SEQ ID NO: 21; The reverse shear acceptor sequence comprises the sequence of SEQ ID NO: 22; and / or The second AAV ITR sequence contains the sequence of SEQ ID NO:

24.

3. An adeno-associated virus (AAV) donor polynucleotide comprising, along the 5' to 3' direction: a) First AAV ITR sequence; b) A first targeting sequence homologous to the first region of intron 3 of the albumin gene, the first targeting sequence comprising the sequence of SEQ ID NO: 13; c) TTRe promoter sequence; d) Splice acceptor sequence; e) P2A sequence; f) A sequence encoding a codon-optimized and modified PAH gene, said sequence comprising the sequence of SEQ ID NO: 25; g) 3'UTR sequence; h) Polyadenylation (poly(A)) sequence; i) DNA spacer sequence; j) A second targeting sequence homologous to the second region of intron 3 of the albumin gene, the second targeting sequence comprising the sequence of SEQ ID NO: 23; and k) Second AAV ITR sequence.

4. The AAV donor polynucleotide according to claim 3, wherein... The first AAV ITR sequence contains the sequence of SEQ ID NO: 12; The TTRe promoter sequence includes the sequence of SEQ ID NO: 27; The splice acceptor sequence includes the sequence of SEQ ID NO: 14; The P2A sequence includes the sequence of SEQ ID NO: 15; The 3' UTR sequence contains the sequence of SEQ ID NO: 10; The polyadenylation (poly(A)) sequence includes the sequence of SEQ ID NO: 17; The DNA spacer sequence comprises the sequence of SEQ ID NO: 18; The reverse polyA sequence contains the sequence of SEQ ID NO: 19; and / or The second AAV ITR sequence contains the sequence of SEQ ID NO:

24.

5. The AAV donor polynucleotide according to claim 1, wherein the AAV donor polynucleotide comprises the nucleic acid sequence of SEQ ID NO:

8.

6. The AAV donor polynucleotide according to claim 3, wherein the AAV donor polynucleotide comprises the nucleic acid sequence of SEQ ID NO:

10.

7. An AAV viral vector comprising an AAV donor polynucleotide according to any one of the preceding claims.

8. The AAV virus vector according to claim 7, wherein the AAV virus vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11 virus vector.

9. The vector according to claim 7, wherein the AAV virus vector is an AAV8 or AAV9 virus vector.

10. A composition comprising: a) an AAV viral vector according to any one of claims 7 to 9; and b) at least one LNP composition comprising at least one gRNA pair and at least one mRNA molecule encoding Cas-CLOVER.

11. The composition of claim 10, wherein the mRNA molecule comprises a 5'-cap.

12. The composition according to claim 10 or 11, wherein the at least one gRNA pair comprises a left gRNA containing the sequence of SEQ ID NO: 4 and a right gRNA containing the sequence of SEQ ID NO:

5.

13. The composition according to any one of claims 10 to 12, wherein the at least one LNP composition comprises: The molar content is approximately 54% ssPalmO-Ph-P4C2. It contains approximately 35% of cholesterol. Approximately 10% of DOPC, and Approximately 1% DMG-PEG2000 per molar.

14. A pharmaceutical composition comprising: an AAV viral vector according to any one of claims 1 to 7 or a composition according to any one of claims 10 to 13, and a pharmaceutically acceptable vector.

15. A method of treating phenylketonuria (PKU) in a subject with this need, the method comprising administering to the subject: a) at least one therapeutically effective dose of an AAV donor polynucleotide according to any one of claims 1 to 6, an AAV viral vector according to any one of claims 7 to 9, a composition according to any one of claims 10 to 13, or a pharmaceutical composition according to claim 14; and b) at least one LNP composition comprising at least one gRNA pair and at least one mRNA molecule encoding Cas-CLOVER.

16. The method of claim 15, wherein the at least one LNP composition comprises: The molar content is approximately 54% ssPalmO-Ph-P4C2. It contains approximately 35% of cholesterol. The molar concentration is approximately 5% DOPC. Molecularly calculated at approximately 5% DSPC, and Approximately 1% DMG-PEG2000 per molar.

17. The method of claim 15 or 16, wherein the mRNA molecule comprises a 5'-cap.

18. The method according to claims 15 to 17, wherein the gRNA pair comprises a left gRNA containing the sequence of SEQ ID NO: 4 and a right gRNA containing the sequence of SEQ ID NO:

5.

19. A method for specifically integrating a transgene site into the genome of at least one cell of a subject via a viral vector, the method comprising administering to the subject a) at least one therapeutically effective dose of an AAV donor polynucleotide according to any one of claims 1 to 6, an AAV viral vector according to any one of claims 7 to 9, a composition according to any one of claims 10 to 13, or a pharmaceutical composition according to claim 14; and b) at least one LNP composition comprising at least one gRNA pair and at least one mRNA molecule encoding Cas-CLOVER, wherein the viral vector DNA is integrated into the genome of at least one cell at a genomic cleavage site generated by Cas-CLOVER.

20. The method of claim 19, wherein the at least one LNP composition comprises: The molar content is approximately 54% ssPalmO-Ph-P4C2. It contains approximately 35% of cholesterol. The molar concentration is approximately 5% DOPC. Molecularly calculated at approximately 5% DSPC, and Approximately 1% DMG-PEG2000 per molar.

21. The method of claim 19 or 20, wherein the mRNA molecule further comprises a 5'-cap.

22. The method according to claims 19 to 21, wherein the gRNA pair comprises a left gRNA containing the sequence of SEQ ID NO: 4 and a right gRNA containing the sequence of SEQ ID NO: 5.

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