Patent Application
20240002820
The present disclosure relates to polynucleotides, compositions, and methods for genomic editing involving deamination.
Base editing is a genome editing method that directly generates point mutations within a specific region of the genomic DNA without causing double-stranded breaks (DSB). DNA base editors (BEs) comprise fusions between a catalytically impaired Cas nuclease and a base-modification enzyme. Currently, effectors for cytidine-to-thymidine (C-to-T) editing fuse a cytidine deaminase with a nickase and a uracil glycosylase inhibitor (UGI). For example, base editor 3 (BE3) consists of a Cas9 nuclease bearing a mutation that converts it into a nickase (nCas9), fused to an APOBEC1 (apolipoprotein mRNA editing enzyme, catalytic polypeptide 1) deaminase and a UGI (e.g., Wang et al. Cell Research 27:1289-1292 (2017)), and it was reported that an “nCas9-fused UGI domain is still important for achieving high fidelity of base editing, even when high levels of free UGI is present.” As another example, an engineered human APOBEC3A (A3A or apolipoprotein mRNA editing enzyme, catalytic polypeptide 3A) deaminase has been investigated as a replacement for rat APOBEC1 deaminase (RAPO1) in the original BE3 (Gehrke et al., Nature Biotechnology, 36: 977-982 (2018)), but it was noted that the ability of base editors “to edit all Cs within their editing window can potentially have deleterious effects” and that “mutation of the N57 residue in the human A3A deaminase was critical to restoring its native target sequence precision in the context of a base editor and also to lowering its off-target editing activity.” Indeed, APOBEC3A-Class 2 Cas nickase (D10A) base editors have been reported as having a “high degree of mutagenicity” and as showing Cas9-independent off-target base editing (Doman et al., Nature Biotechnology 38:620-628 (2020)). Accordingly, improved compositions and methods for targeted C-to-T base editing using cytidine deaminases (e.g., an APOBEC3A deaminase) and RNA-guided nickase are needed.
Accordingly, the present disclosure provides polynucleotides, compositions, and methods for genomic editing involving a cytidine deaminase (e.g., an APOBEC3A deaminase) and an RNA-guided nickase that induce C-to-T conversions at target nucleotides with greater fidelity and may minimize bystander mutations. The present disclosure is based in part on the findings that by pairing a cytidine deaminase (e.g., an APOBEC deaminase) and an RNA-guided nickase system with UGI in trans (e.g., as a separate mRNA), it is possible to lower the amount of other base editing (C-to-A/G conversions, insertions, or deletions) and increase the purity of C-to-T editing.
Accordingly, the following embodiments are provided.
In some embodiments, a composition is provided, the composition comprising a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase and an RNA-guided nickase, and a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI), wherein the second mRNA is different from the first mRNA. In some embodiments, the first open reading frame does not comprise a sequence encoding a UGI. In some embodiments, the composition comprises a first composition and a second composition, wherein the first composition comprises a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase and an RNA-guided nickase and does not comprise a uracil glycosylase inhibitor (UGI), and the second composition comprises a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI), wherein the second mRNA is different from the first mRNA. In some embodiments, the composition comprises lipid nanoparticles.
In some embodiments, a method of modifying a target gene is provided, the method comprising delivering to a cell a first mRNA comprising a first open reading frame encoding a first polypeptide comprising a cytidine deaminase and an RNA-guided nickase, a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI), wherein the second mRNA is different from the first mRNA, and at least one guide RNA (gRNA).
In some embodiments, a method of modifying at least one cytidine within a target gene in a cell is provided, the method comprising expressing in the cell or contacting the cell with: (i) a first polypeptide comprising a cytidine deaminase and an RNA-guided nickase, wherein the first polypeptide does not comprise a uracil glycosylase inhibitor (UGI); (ii) a UGI polypeptide; and (iii) at least one guide RNA (gRNA) wherein the first polypeptide and gRNA form a complex with the target gene and modify the at least one cytidine in the target gene. In some embodiments, the ratio of the UGI polypeptide to the first polypeptide is from 10:1 to 50:1.
In some embodiments, an mRNA containing an open reading frame (ORF) encoding a polypeptide is provided, the polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase, wherein the polypeptide does not comprise a uracil glycosylase inhibitor (UGI). The polypeptide encoded by the mRNA is also provided. In some embodiments, a method of modifying a target gene is provided, the method comprising delivering an mRNA or a polypeptide described herein to a cell.
In some embodiments, a composition comprises two different mRNAs in which the first mRNA comprises an ORF encoding a cytidine deaminase (e.g., A3A) and an RNA-guided nickase, and the second mRNA comprises an ORF encoding uracil glycosylase inhibitor (UGI). In some embodiments, the first mRNA in the composition does not comprise an ORF encoding UGI. In some embodiments, the molar ratio of the second mRNA to the first mRNA is from 1:1 to 30:1, from 2:1 to 30:1, from 7:1 to 22:1. In some embodiments, the molar ratio of the second mRNA to the first mRNA is 22:1, 7:1, 2:1, or 1:1, 1:4, 1:11, or 1:33.
Further embodiments are provided throughout and described in the claims and Figures.
See the Sequence Table below for the sequences themselves. Transcript sequences may generally include GGG as the first three nucleotides for use with ARCA or AGG as the first three nucleotides for use with CleanCap™. Accordingly, the first three nucleotides can be modified for use with other capping approaches, such as Vaccinia capping enzyme. Promoters and poly-A sequences are not included in the transcript sequences. A promoter such as a U6 promoter (SEQ ID NO: 67) or a CMV Promotor (SEQ ID NO: 68) and a poly-A sequence such as SEQ ID NO: 109 can be appended to the disclosed transcript sequences at the 5′ and 3′ ends, respectively. Most nucleotide sequences are provided as DNA but can be readily converted to RNA by changing Ts to Us.
Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims.
Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a conjugate” includes a plurality of conjugates and reference to “a cell” includes a plurality of cells and the like.
Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings.
The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, or a degree of variation that does not substantially affect the properties of the described subject matter, or within the tolerances accepted in the art, e.g., within 10%, 5%, 2%, or 1%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Unless specifically noted in the above specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of” or “consisting essentially of” the recited components; embodiments in the specification that recite “consisting of” various components are also contemplated as “comprising” or “consisting essentially of” the recited components; and embodiments in the specification that recite “consisting essentially of” various components are also contemplated as “consisting of” or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims).
The section headings used herein are for organizational purposes only and are not to be construed as limiting the desired subject matter in any way. In the event that any literature incorporated by reference contradicts the express content of this specification, including but not limited to a definition, the express content of this specification controls. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.
Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings:
The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed terms preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, CBBA, CABA, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
As used herein, the term “kit” refers to a packaged set of related components, such as one or more polynucleotides or compositions and one or more related materials such as delivery devices (e.g., syringes), solvents, solutions, buffers, instructions, or desiccants.
“Or” is used in the inclusive sense, i.e., equivalent to “and/or,” unless the context requires otherwise.
“Polynucleotide” and “nucleic acid” are used herein to refer to a multimeric compound comprising nucleosides or nucleoside analogs which have nitrogenous heterocyclic bases or base analogs linked together along a backbone, including conventional RNA, DNA, mixed RNA-DNA, and polymers that are analogs thereof. A nucleic acid “backbone” can be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (“peptide nucleic acids” or PNA; PCT No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of a nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, e.g., 2′ methoxy or 2′ halide substitutions. Nitrogenous bases can be conventional bases (A, G, C, T, U), analogs thereof (e.g., modified uridines such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine, or others); inosine; derivatives of purines or pyrimidines (e.g., N4-methyl deoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position (e.g., 5-methylcytosine), purine bases with a substituent at the 2, 6, or 8 positions, 2-amino-6-methylaminopurine, O6-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, and O4-alkyl-pyrimidines; U.S. Pat. No. 5,378,825 and PCT No. WO 93/13121). For general discussion see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992). Nucleic acids can include one or more “abasic” residues where the backbone includes no nitrogenous base for position(s) of the polymer (U.S. Pat. No. 5,585,481). A nucleic acid can comprise only conventional RNA or DNA sugars, bases and linkages, or can include both conventional components and substitutions (e.g., conventional bases with 2′ methoxy linkages, or polymers containing both conventional bases and one or more base analogs). Nucleic acid includes “locked nucleic acid” (LNA), an analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhance hybridization affinity toward complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). RNA and DNA have different sugar moieties and can differ by the presence of uracil or analogs thereof in RNA and thymine or analogs thereof in DNA.
“Polypeptide” as used herein refers to a multimeric compound comprising amino acid residues that can adopt a three-dimensional conformation. Polypeptides include but are not limited to enzymes, enzyme precursor proteins, regulatory proteins, structural proteins, receptors, nucleic acid binding proteins, antibodies, etc. Polypeptides may, but do not necessarily, comprise post-translational modifications, non-natural amino acids, prosthetic groups, and the like.
As used herein, a “cytidine deaminase” means a polypeptide or complex of polypeptides that is capable of cytidine deaminase activity, that is catalyzing the hydrolytic deamination of cytidine or deoxycytidine, typically resulting in uridine or deoxyuridine. Cytidine deaminases encompass enzymes in the cytidine deaminase superfamily, and in particular, enzymes of the APOBEC family (APOBEC1, APOBEC2, APOBEC4, and APOBEC3 subgroups of enzymes), activation-induced cytidine deaminase (AID or AICDA) and CMP deaminases (see, e.g., Conticello et al., Mol. Biol. Evol. 22:367-77, 2005; Conticello, Genome Biol. 9:229, 2008; Muramatsu et al., J. Biol. Chem. 274: 18470-6, 1999); Carrington et al., Cells 9:1690 (2020)). In some embodiments, variants of any known cytidine deaminase or APOBEC protein are encompassed. Variants include proteins having a sequence that differs from wild-type protein by one or several mutations (i.e., substitutions, deletions, insertions), such as one or several single point substitutions. For instance, a shortened sequence could be used, e.g., by deleting N-terminal, C-terminal, or internal amino acids, preferably one to four amino acids at the C-terminus of the sequence. As used herein, the term “variant” refers to allelic variants, splicing variants, and natural or artificial mutants, which are homologous to a reference sequence. The variant is “functional” in that it shows a catalytic activity of DNA editing.
As used herein, the term “APOBEC3A” refers to a cytidine deaminase such as the protein expressed by the human A3A gene. The APOBEC3A may have catalytic DNA editing activity. An amino acid sequence of APOBEC3A has been described (UniPROT accession ID: p31941) and is included herein as SEQ ID NO: 40. In some embodiments, the APOBEC3A protein is a human APOBEC3A protein and/or a wild-type protein. Variants include proteins having a sequence that differs from wild-type APOBEC3A protein by one or several mutations (i.e., substitutions, deletions, insertions), such as one or several single point substitutions. For instance, a shortened APOBEC3A sequence could be used, e.g. by deleting N-terminal, C-terminal, or internal amino acids, preferably one to four amino acids at the C-terminus of the sequence. As used herein, the term “variant” refers to allelic variants, splicing variants, and natural or artificial mutants, which are homologous to an APOBEC3A reference sequence. The variant is “functional” in that it shows a catalytic activity of DNA editing. In some embodiments, an APOBEC3A (such as a human APOBEC3A) has a wild-type amino acid position 57 (as numbered in the wild-type sequence). In some embodiments, an APOBEC3A (such as a human APOBEC3A) has an asparagine at amino acid position 57 (as numbered in the wild-type sequence).
As used herein, a “nickase” is an enzyme that creates a single-strand break (also known as a “nick”) in double strand DNA, i.e., cuts one strand but not the other of the DNA double helix. As used herein, an “RNA-guided nickase” means a polypeptide or complex of polypeptides having DNA nickase activity, wherein the DNA nickase activity is sequence-specific and depends on the sequence of the RNA. Exemplary RNA-guided nickases include Cas nickases. Cas nickases include, but are not limited to, nickase forms of a Csm or Cmr complex of a type III CRISPR system, the Cas10, Csm1, or Cmr2 subunit thereof, a Cascade complex of a type I CRISPR system, the Cas3 subunit thereof, and Class 2 Cas nucleases. Class 2 Cas nickases include Class 2 Cas nuclease variants in which only one of the two catalytic domains is inactivated, which have RNA-guided DNA nickase activity. Class 2 Cas nickases include, for example, Cas9 (e.g., H840A, DOA, or N863A variants of SpyCas9), Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof. Cpf1 protein, Zetsche et al., Cell, 163: 1-13 (2015), is homologous to Cas9, and contains a RuvC-like protein domain. Cpf1 sequences of Zetsche are incorporated by reference in their entirety. See, e.g., Zetsche, Tables S1 and S3. “Cas9” encompasses S. pyogenes (Spy) Cas9, the variants of Cas9 listed herein, and equivalents thereof. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell. 60:385-397 (2015).
Several Cas9 orthologs have been obtained from N. meningitidis (Esvelt et al., NAT. METHODS, vol. 10, 2013, 1116-1121; Hou et al., PNAS, vol. 110, 2013, pages 15644-15649; Edraki et al., Mol. Cell 73:714-726, 2019) (Nme1Cas9, Nme2Cas9, and Nme3Cas9). The Nme2Cas9 ortholog functions efficiently in mammalian cells, recognizes an N4CC PAM, and can be used for in vivo editing (Ran et al., NATURE, vol. 520, 2015, pages 186-191; Kim et al., NAT. COMMUN., vol. 8, 2017, pages 14500). Nme2Cas9 has been shown to be naturally resistant to off-target editing (Lee et al., MOL. THER., vol. 24, 2016, pages 645-654; Kim et al., 2017). See also e.g., WO/2020081568 (e.g., pages 28 and 42), describing an Nme2Cas9 D16A nickase, the contents of which are hereby incorporated by reference in its entirety. Throughout, “NmeCas9” is generic and an encompasses any type of NmeCas9, including, Nme1Cas9, Nme2Cas9, and Nme3Cas9.
As used herein, the term “fusion protein” refers to a hybrid polypeptide which comprises polypeptides from at least two different proteins or sources. One polypeptide may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.
The term “linker,” as used herein, refers to a chemical group or a molecule linking two adjacent molecules or moieties. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein) such as a 16-amino acid residue “XTEN” linker, or a variant thereof (See, e.g., the Examples; and Schellenberger et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat. Biotechnol. 27, 1186-1190 (2009)). In some embodiments, the XTEN linker comprises the sequence SGSETPGTSESATPES (SEQ ID NO: 46), SGSETPGTSESA (SEQ ID NO: 47), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 48). In some embodiments, the linker comprises one or more sequences selected from SEQ ID NOs: 46-59, 61 and 211-272.
As used herein, the term “uracil glycosylase inhibitor”, “uracil-DNA glycosylase inhibitor” or “UGI” refers to a protein that is capable of inhibiting a uracil-DNA glycosylase (UDG) base-excision repair enzyme (e.g., UniPROT ID: P14739; SEQ ID NO: 27; SEQ ID NO:43).
As used herein, “open reading frame” or “ORF” of a gene refers to a sequence consisting of a series of codons that specify the amino acid sequence of the protein that the gene codes for. The ORF generally begins with a start codon (e.g., ATG in DNA or AUG in RNA) and ends with a stop codon, e.g., TAA, TAG or TGA in DNA or UAA, UAG, or UGA in RNA.
“Guide RNA”, “gRNA”, and “guide” are used herein interchangeably to refer to either a crRNA (also known as CRISPR RNA), or the combination of a crRNA and a trRNA (also known as tracrRNA). The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). “Guide RNA” or “gRNA” refers to each type. The trRNA may be a naturally-occurring sequence, or a trRNA sequence with modifications or variations compared to naturally-occurring sequences.
As used herein, a “guide sequence” or “guide region” or “spacer” or “spacer sequence” and the like refers to a sequence within a gRNA that is complementary to a target sequence and functions to direct a gRNA to a target sequence for binding or modification (e.g., cleavage) by an RNA-guided nickase. A guide sequence can be 20 nucleotides in length, e.g., in the case of Streptococcus pyogenes (i.e., Spy Cas9 (also referred to as SpCas9)) and related Cas9 homologs/orthologs. Shorter or longer sequences can also be used as guides, e.g., 15-, 16-, 17-, 18-, 19-, 21-, 22-, 23-, 24-, or 25-nucleotides in length. A guide sequence can be 20-25 nucleotides in length, e.g., in the case of Nme Cas9, e.g., 20-, 21-, 22-, 23-, 24- or 25-nucleotides in length. For example, a guide sequence of 24 nucleotides in length can be used with Nme Cas9, e.g., Nme2 Cas9.
In some embodiments, the target sequence is in a gene or on a chromosome, for example, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the guide sequence and the target region may be 100% complementary or identical. In other embodiments, the guide sequence and the target region may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches, where the total length of the target sequence is at least 17, 18, 19, 20 or more base pairs. In some embodiments, the guide sequence and the target region may contain 1-4 mismatches where the guide sequence comprises at least 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches where the guide sequence comprises 20 nucleotides.
As used herein, a “target sequence” or “genomic target sequence” refers to a sequence of nucleic acid in a target gene that has complementarity to the guide sequence of the gRNA. The interaction of the target sequence and the guide sequence directs an RNA-guided DNA binding agent to bind, and potentially nick or cleave (depending on the activity of the agent), within the target sequence. Target sequences for Cas proteins include both the positive and negative strands of genomic DNA (i.e., the sequence given and the sequence's reverse compliment), as a nucleic acid substrate for a Cas protein is a double stranded nucleic acid. Accordingly, where a guide sequence is said to be “complementary to a target sequence,” it is to be understood that the guide sequence may direct an RNA-guided DNA binding agent (e.g., dCas9 or impaired Cas9) to bind to the reverse complement of a target sequence. Thus, in some embodiments, where the guide sequence binds the reverse complement of a target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., the target sequence not including the PAM) except for the substitution of U for T in the guide sequence.
As used herein, a first sequence is considered to “comprise a sequence with at least X % identity to” a second sequence if an alignment of the first sequence to the second sequence shows that X % or more of the positions of the second sequence in its entirety are matched by the first sequence. For example, the sequence AAGA comprises a sequence with 100% identity to the sequence AAG because an alignment would give 100% identity in that there are matches to all three positions of the second sequence. The differences between RNA and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs such as modified uridines do not contribute to differences in identity or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine as a complement). Thus, for example, the sequence 5′-AXG where X is any modified uridine, such as pseudouridine, N1-methyl pseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5′-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well-known in the art. One skilled in the art will understand what choice of algorithm and parameter settings are appropriate for a given pair of sequences to be aligned; for sequences of generally similar length and expected identity >50% for amino acids or >75% for nucleotides, the Needleman-Wunsch algorithm with default settings of the Needleman-Wunsch algorithm interface provided by the EBI at the www.ebi.ac.uk web server are generally appropriate.
“mRNA” is used herein to refer to a polynucleotide that is not DNA and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs). mRNA can comprise a phosphate-sugar backbone including ribose residues or analogs thereof, e.g., 2′-methoxy ribose residues. In some embodiments, the sugars of an mRNA phosphate-sugar backbone consist essentially of ribose residues, 2′-methoxy ribose residues, or a combination thereof. In general, mRNAs do not contain a substantial quantity of thymidine residues (e.g., 0 residues or fewer than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% thymidine content). An mRNA can contain modified uridines at some or all of its uridine positions.
“Modified uridine” is used herein to refer to a nucleoside other than thymidine with the same hydrogen bond acceptors as uridine and one or more structural differences from uridine. In some embodiments, a modified uridine is a substituted uridine, i.e., a uridine in which one or more non-proton substituents (e.g., alkoxy, such as methoxy) takes the place of a proton. In some embodiments, a modified uridine is pseudouridine. In some embodiments, a modified uridine is a substituted pseudouridine, i.e., a pseudouridine in which one or more non-proton substituents (e.g., alkyl, such as methyl) takes the place of a proton. In some embodiments, a modified uridine is any of a substituted uridine, pseudouridine, or a substituted pseudouridine.
“Uridine position” as used herein refers to a position in a polynucleotide occupied by a uridine or a modified uridine. Thus, for example, a polynucleotide in which “100% of the uridine positions are modified uridines” contains a modified uridine at every position that would be a uridine in a conventional RNA (where all bases are standard A, U, C, or G bases) of the same sequence. Unless otherwise indicated, a U in a polynucleotide sequence of a sequence table or sequence listing in or accompanying this disclosure can be a uridine or a modified uridine.
As used herein, the “minimal uridine codon(s)” for a given amino acid is the codon(s) with the fewest uridines (usually 0 or 1 except for a codon for phenylalanine, where the minimal uridine codon has 2 uridines). Modified uridine residues are considered equivalent to uridines for the purpose of evaluating uridine content.
As used herein, the “uridine dinucleotide (UU) content” of an ORF can be expressed in absolute terms as the enumeration of UU dinucleotides in an ORF or on a rate basis as the percentage of positions occupied by the uridines of uridine dinucleotides (for example, AUUAU would have a uridine dinucleotide content of 40% because 2 of 5 positions are occupied by the uridines of a uridine dinucleotide). Modified uridine residues are considered equivalent to uridines for the purpose of evaluating uridine dinucleotide content.
As used herein, the “minimal adenine codon(s)” for a given amino acid is the codon(s) with the fewest adenines (usually 0 or 1 except for a codon for lysine and asparagine, where the minimal adenine codon has 2 adenines). Modified adenine residues are considered equivalent to adenines for the purpose of evaluating adenine content.
As used herein, the “adenine dinucleotide content” of an ORF can be expressed in absolute terms as the enumeration of AA dinucleotides in an ORF or on a rate basis as the percentage of positions occupied by the adenines of adenine dinucleotides (for example, UAAUA would have an adenine dinucleotide content of 40% because 2 of 5 positions are occupied by the adenines of an adenine dinucleotide). Modified adenine residues are considered equivalent to adenines for the purpose of evaluating adenine dinucleotide content.
As used herein, “indels” refer to insertion/deletion mutations consisting of a number of nucleotides that are either inserted or deleted, e.g., at the site of double-stranded breaks (DSBs) in a target nucleic acid.
As used herein, “knockdown” refers to a decrease in expression of a particular gene product (e.g., protein, mRNA, or both). Knockdown of a protein can be measured either by detecting protein secreted by tissue or population of cells (e.g., in serum or cell media) or by detecting total cellular amount of the protein from a tissue or cell population of interest. Methods for measuring knockdown of mRNA are known and include sequencing of mRNA isolated from a tissue or cell population of interest. In some embodiments, “knockdown” may refer to some loss of expression of a particular gene product, for example a decrease in the amount of mRNA transcribed or a decrease in the amount of protein expressed or secreted by a population of cells (including in vivo populations such as those found in tissues).
As used herein, “knockout” refers to a loss of expression of a particular protein in a cell. Knockout can be measured either by detecting the amount of protein secretion from a tissue or population of cells (e.g., in serum or cell media) or by detecting total cellular amount of a protein a tissue or a population of cells. In some embodiments, the methods of the disclosure “knockout” a target protein one or more cells (e.g., in a population of cells including in vivo populations such as those found in tissues). In some embodiments, a knockout is not the formation of mutant of the target protein, for example, created by indels, but rather the complete loss of expression of the target protein in a cell, i.e., decrease of expression to below the level of detection of the assay used.
As used herein, the terms “nuclear localization signal” (NLS) or “nuclear localization sequence” refers to an amino acid sequence which induces transport of molecules comprising such sequences or linked to such sequences into the nucleus of eukaryotic cells. The nuclear localization signal may form part of the molecule to be transported. In some embodiments, the NLS may be fused to the molecule by a covalent bond, hydrogen bonds or ionic interactions. In some embodiments, the NLS may be fused to the molecule via a linker.
“β2M” or “B2M,” as used herein, refers to nucleic acid sequence or protein sequence of “β-2 microglobulin;” the human gene has accession number NC_000015 (range 44711492 . . . 44718877), reference GRCh38.p13. The B2M protein is associated with MHC class I molecules as a heterodimer on the surface of nucleated cells and is required for MHC class I protein expression.
“CIITA” or “CIITA” or “C2TA,” as used herein, refers to the nucleic acid sequence or protein sequence of “class II major histocompatibility complex transactivator;” the human gene has accession number NC_000016.10 (range 10866208 . . . 10941562), reference GRCh38.p13. The CIITA protein in the nucleus acts as a positive regulator of MHC class II gene transcription and is required for MHC class II protein expression.
As used herein, “MHC” or “MHC molecule(s)” or “MHC protein” or “MHC complex(es),” refers to a major histocompatibility complex molecule (or plural), and includes, e.g., MHC class I and MHC class II molecules. In humans, MHC molecules are referred to as “human leukocyte antigen” complexes or “HLA molecules” or “HLA protein.” The use of terms “MHC” and “HLA” are not meant to be limiting; as used herein, the term “MHC” may be used to refer to human MHC molecules, i.e., HLA molecules. Therefore, the terms “MHC” and “HLA” are used interchangeably herein.
The term “HLA-A,” as used herein in the context of HLA-A protein, refers to the MHC class I protein molecule, which is a heterodimer consisting of a heavy chain (encoded by the HLA-A gene) and a light chain (i.e., beta-2 microglobulin). The term “HLA-A” or “HLA-A gene,” as used herein in the context of nucleic acids refers to the gene encoding the heavy chain of the HLA-A protein molecule. The HLA-A gene is also referred to as “HLA class I histocompatibility, A alpha chain;” the human gene has accession number NC_000006.12 (29942532 . . . 29945870). The HLA-A gene is known to have thousands of different versions (also referred to as “alleles”) across the population (and an individual may receive two different alleles of the HLA-A gene). A public database for HLA-A alleles, including sequence information, may be accessed at IPD-IMGT/HLA: https://www.ebi.ac.uk/ipd/imgt/hla/. All alleles of HLA-A are encompassed by the terms “HLA-A” and “HLA-A gene.”
“HLA-B” as used herein in the context of nucleic acids refers to the gene encoding the heavy chain of the HLA-B protein molecule. The HLA-B is also referred to as “HLA class I histocompatibility, B alpha chain;” the human gene has accession number NC_000006.12 (31353875 . . . 31357179).
“HLA-C” as used herein in the context of nucleic acids refers to the gene encoding the heavy chain of the HLA-C protein molecule. The HLA-C is also referred to as “HLA class I histocompatibility, C alpha chain;” the human gene has accession number NC_000006.12 (31268749 . . . 31272092).
“TRBC1” and “TRBC2” as used herein in the context of nucleic acids refer to two homologous genes encoding the T-cell receptor β-chain. “TRBC” or “TRBC1/2” is used herein to refer to TRBC1 and TRBC2. The human wild-type TRBC1 sequence is available at NCBI Gene ID: 28639; Ensembl: ENSG0000021 1751. T-cell receptor Beta Constant, V_segment Translation Product, BV05SIJ2.2, TCRBC1, and TCRB are gene synonyms for TRBC1. The human wild-type TRBC2 sequence is available at NCBI Gene ID: 28638; Ensembl: ENSG00000211772. T-cell receptor Beta Constant, V_segment Translation Product, and TCRBC2 are gene synonyms for TRBC2.
“TRAC” as used herein in the context of nucleic acids refers to the gene encoding the T-cell receptor α-chain. The human wild-type TRAC sequence is available at NCBI Gene ID: 28755; Ensembl: ENSG00000277734. T-cell receptor Alpha Constant, TCRA, IMD7, TRCA and TRA are gene synonyms for TRAC.
As used herein, the term “homozygous” refers to having two identical alleles of a particular gene.
As used herein, “treatment” refers to any administration or application of a therapeutic for disease or disorder in a subject, and includes inhibiting the disease, arresting its development, relieving one or more symptoms of the disease, curing the disease, or preventing one or more symptoms of the disease, including reoccurrence of the symptom.
As used herein, “delivering” and “administering” are used interchangeably, and include ex vivo and in vivo applications.
Co-administration, as used herein, means that a plurality of substances are administered sufficiently close together in time so that the agents act together. Co-administration encompasses administering substances together in a single formulation and administering substances in separate formulations close enough in time so that the agents act together.
As used herein, the phrase “pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally non-toxic and is not biologically undesirable and that are not otherwise unacceptable for pharmaceutical use. Pharmaceutically acceptable generally refers to substances that are non-pyrogenic. Pharmaceutically acceptable can refer to substances that are sterile, especially for pharmaceutical substances that are for injection or infusion.
As used herein, a “subject” refers to any member of the animal kingdom. In some embodiments, “subject” refers to humans. In some embodiments, “subject” refers to non-human animals. In some embodiments, “subject” refers to primates. In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In certain embodiments, the non-human subject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, a subject may be a transgenic animal, genetically-engineered animal, and/or a clone. In certain embodiments of the present invention the subject is an adult, an adolescent or an infant. In some embodiments, terms “individual” or “patient” are used and are intended to be interchangeable with “subject”.
As used herein, “reduced or eliminated” expression of a protein on a cell refers to a partial or complete loss of expression of the protein relative to an unmodified cell. In some embodiments, the surface expression of a protein on a cell is measured by flow cytometry and has “reduced or eliminated” surface expression relative to an unmodified cell as evidenced by a reduction in fluorescence signal upon staining with the same antibody against the protein. A cell that has “reduced or eliminated” surface expression of a protein by flow cytometry relative to an unmodified cell may be referred to as “negative” for expression of that protein as evidenced by a fluorescence signal similar to a cell stained with an isotype control antibody. The “reduction or elimination” of protein expression can be measured by other known techniques in the field with appropriate controls known to those skilled in the art.
In some embodiments, a nucleic acid is provided, the nucleic acid comprising an open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., A3A) and an RNA-guided nickase, wherein the polypeptide does not comprise a uracil glycosylase inhibitor (UGI). In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, a polypeptide encoded by the mRNA is provided.
In some embodiments, a polypeptide or an mRNA encoding the polypeptide, are provided, the polypeptide comprising a cytidine deaminase and an RNA-guided nickase, wherein the polypeptide does not comprise a UGI. In some embodiments, the cytidine deaminase is A3A. In some embodiments, the RNA-guided nickase does not comprise a uracil glycosylase inhibitor (UGI). In some embodiments, a composition is provided comprising a first polypeptide, or an mRNA encoding a first polypeptide, comprising a cytidine deaminase (e.g., A3A) and an RNA-guided nickase; and a second polypeptide, or an mRNA encoding a second polypeptide, comprising a uracil glycosylase inhibitor (UGI), wherein the second polypeptide is different from the first polypeptide.
In some embodiments, a composition is provided comprising a first nucleic acid comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., A3A) and an RNA-guided nickase, and a second nucleic acid comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI), wherein the second nucleic acid is different from the first nucleic acid. In some embodiments, the first nucleic acid encodes a polypeptide that does not comprise a UGI.
In some embodiments, methods of modifying a target gene are provided comprising administering the compositions described herein. In some embodiments, the method comprises delivering to a cell a first nucleic acid comprising a first open reading frame encoding a first polypeptide comprising a cytidine deaminase (e.g., A3A) and an RNA-guided nickase, and a second nucleic acid comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI), wherein the second nucleic acid is different from the first nucleic acid.
In some embodiments, the methods comprise delivering to a cell a polypeptide comprising a cytidine deaminase (e.g., A3A) and an RNA-guided nickase, or a nucleic acid encoding the polypeptide, and separately (e.g., not via the same nucleic acid construct) delivering to the cell a uracil glycosylase inhibitor (UGI), or a nucleic acid encoding the UGI.
In some embodiments, a molar ratio of the mRNA encoding UGI to the mRNA encoding the cytidine deaminase (e.g., A3A) and the RNA-guided nickase is from about 1:35 to from about 30:1. In some embodiments, the molar ratio is from about 1:25 to about 25:1. In some embodiments, the molar ratio is from about 1:20 to about 25:1. In some embodiments, the molar ratio is from about 1:10 to about 22:1. In some embodiments, the molar ratio is from about 1:5 to about 25:1. In some embodiments, the molar ratio is from about 1:1 to about 30:1. In some embodiments, the molar ratio is from about 2:1 to about 10:1. In some embodiments, the molar ratio is from about 5:1 to about 20:1. In some embodiments, the molar ratio is from about 1:1 to about 25:1. In some embodiments, the molar ratio may be about 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, or 30:1. In some embodiments, the molar ratio is equal to or larger than about 1:1. In some embodiments the molar ratio is about 1:1. In some embodiments the molar ratio is about 2:1. In some embodiments the molar ratio is about 3:1. In some embodiments the molar ratio is about 4:1. In some embodiments the molar ratio is about 5:1. In some embodiments the molar ratio is about 6:1. In some embodiments the molar ratio is about 7:1. In some embodiments the molar ratio is about 8:1. In some embodiments the molar ratio is about 9:1. In some embodiments the molar ratio is about 10:1. In some embodiments the molar ratio is about 11:1. In some embodiments the molar ratio is about 12:1. In some embodiments the molar ratio is about 13:1. In some embodiments the molar ratio is about 14:1. In some embodiments the molar ratio is about 15:1. In some embodiments the molar ratio is about 16:1. In some embodiments the molar ratio is about 17:1. In some embodiments the molar ratio is about 18:1. In some embodiments the molar ratio is about 19:1. In some embodiments the molar ratio is about 20:1. In some embodiments the molar ratio is about 21:1. In some embodiments the molar ratio is about 22:1. In some embodiments the molar ratio is about 23:1. In some embodiments the molar ratio is about 24:1. In some embodiments the molar ratio is about 25:1.
Similarly, in some embodiments, the molar ratio discussed above for the mRNA encoding the UGI protein to the mRNA encoding the cytidine deaminase (e.g., A3A) and the RNA-guided nickase are similar if delivering protein.
For example, in some embodiments, a molar ratio of the UGI protein to be delivered to the cytidine deaminase (e.g., A3A) and the RNA-guided nickase to be delivered is from about 1:35 to from about 30:1. In some embodiments, the molar ratio is from about 1:1 to about 30:1.
In some embodiments, the molar ratio of the UGI peptide and the cytidine deaminase (e.g., A3A) and the RNA-guided nickase is from about 10:1 to about 50:1. In some embodiments, the molar ratio may be about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, or 50:1. In some embodiments, the molar ratio is from about 10:1-about 40:1. In some embodiments the molar ratio is from about 10:1-about 30:1. In some embodiments the molar ratio is about 2:1. In some embodiments the molar ratio is from about 10:1-about 20:1. In some embodiments the molar ratio is from about 10:1-about 15:1. In some embodiments the molar ratio is about 15:1-about 50:1. In some embodiments the molar ratio is about 6:1. In some embodiments the molar ratio is about 20:1-about 50:1. In some embodiments the molar ratio is about 8:1. In some embodiments the molar ratio is about 30:1-about 50:1. In some embodiments the molar ratio is about 30:1-about 40:1. In some embodiments the molar ratio is about 11:1. In some embodiments the molar ratio is about 20:1-about 30:1.
In some embodiments, the composition described herein further comprises at least one gRNA. In some embodiments, a composition is provided that comprises an mRNA described herein and at least one gRNA. In some embodiments, the gRNA is a single guide RNA (sgRNA). In some embodiments, the gRNA is a dual guide RNA (dgRNA).
In some embodiments, the composition is capable of effecting genome editing upon administration to the subject.
A. UGI
Without being bound by any theory, providing a UGI together with a polypeptide comprising a deaminase may be helpful in the methods described herein by inhibiting cellular DNA repair machinery (e.g., UDG and downstream repair effectors) that recognize a uracil in DNA as a form of DNA damage or otherwise would excise or modify the uracil and/or surrounding nucleotides. It should be understood that the use of a UGI may increase the editing efficiency of an enzyme that is capable of deaminating C residues.
Suitable UGI protein and nucleotide sequences are provided herein and additional suitable UGI sequences are known to those in the art, and include, for example, those published in Wang et al., Uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 encodes a binding protein specific for uracil-DNA glycosylase. J. Biol. Chem. 264: 1163-1171(1989); Lundquist et al., Site-directed mutagenesis and characterization of uracil-DNA glycosylase inhibitor protein. Role of specific carboxylic amino acids in complex formation with Escherichia coli uracil-DNA glycosylase. J. Biol. Chem. 272:21408-21419(1997); Ravishankar et al., X-ray analysis of a complex of Escherichia coli uracil DNA glycosylase (EcUDG) with a proteinaceous inhibitor. The structure elucidation of a prokaryotic UDG. Nucleic Acids Res. 26:4880-4887(1998); and Putnam et al., Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with Escherichia coli uracil-DNA glycosylase. J. Mol. Biol. 287:331-346(1999), the entire contents of each are incorporated herein by reference. It should be appreciated that any proteins that are capable of inhibiting a uracil-DNA glycosylase base-excision repair enzyme are within the scope of the present disclosure. Additionally, any proteins that block or inhibit base-excision repair are also within the scope of this disclosure. In some embodiments, a uracil glycosylase inhibitor is a protein that binds uracil. In some embodiments, a uracil glycosylase inhibitor is a protein that binds uracil in DNA. In some embodiments, a uracil glycosylase inhibitor is a single-stranded binding protein. In some embodiments, a uracil glycosylase inhibitor is a catalytically inactive uracil DNA-glycosylase protein. In some embodiments, a uracil glycosylase inhibitor is a catalytically inactive uracil DNA-glycosylase protein that does not excise uracil from the DNA. In some embodiments, a uracil glycosylase inhibitor is a catalytically inactive UDG.
In some embodiments, a uracil glycosylase inhibitor (UGI) disclosed herein comprises an amino acid sequence with at least 80% to SEQ ID NO: 27 or 43. In some embodiments, any of the foregoing levels of identity is at least 90%, at least 95%, at least 98%, at least 99%, or 100%. In some embodiments, the UGI comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 27 or 43. In some embodiments, the UGI comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 27 or 43. In some embodiments, the UGI comprises an amino acid sequence with at least 98% identity to SEQ ID NO: 27 or 43. In some embodiments, the UGI comprises an amino acid sequence with at least 99% identity to SEQ ID NO: 27 or 43. In some embodiments, the UGI comprises the amino acid sequence of SEQ ID NO: 27 or 43.
B. Cytidine Deaminase
Cytidine deaminases encompass enzymes in the cytidine deaminase superfamily, and in particular, enzymes of the APOBEC family (APOBEC1, APOBEC2, APOBEC4, and APOBEC3 subgroups of enzymes), activation-induced cytidine deaminase (AID or AICDA) and CMP deaminases (see, e.g., Conticello et al., Mol. Biol. Evol. 22:367-77, 2005; Conticello, Genome Biol. 9:229, 2008; Muramatsu et al., J. Biol. Chem. 274: 18470-6, 1999); and Carrington et al., Cells 9:1690 (2020)).
In some embodiments, the cytidine deaminase disclosed herein is an enzyme of APOBEC family. In some embodiments, the cytidine deaminase disclosed herein is an enzyme of APOBEC1, APOBEC2, APOBEC4, and APOBEC3 subgroups. In some embodiments, the cytidine deaminase disclosed herein is an enzyme of APOBEC3 subgroup. In some embodiments, the cytidine deaminase disclosed herein is an APOBEC3A deaminase (A3A).
In some embodiments, the cytidine deaminase is:
In some embodiments, the cytidine deaminase is a cytidine deaminase comprising an amino acid sequence having at least 80%, 85% 87%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 40, 41, and 960-1023. In some embodiments, the cytidine deaminase is a cytidine deaminase comprising an amino acid sequence that is at least 80%, 85%, 87%, 90%, 95%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 40, 41, and 960-1013. In some embodiments, the cytidine deaminase is a cytidine deaminase comprising an amino acid sequence that is at least 80%, 85%, 87%, 90%, 95%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In some embodiments, the cytidine deaminase is a cytidine deaminase comprising an amino acid sequence that is at least 80%, 85%, 87%, 90%, 95%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 40, 976, 981, 984, 986, and 1014-1023. In some embodiments, the cytidine deaminase is a cytidine deaminase comprising an amino acid sequence that is at least 80%, 85%, 87%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 976, 977, 993-1006, and 1009.
1. APOBEC3A Deaminase
In some embodiments, an APOBEC3A deaminase (A3A) disclosed herein is a human A3A. In some embodiments, the A3A is a wild-type A3A.
In some embodiment, the A3A is an A3A variant. A3A variants share homology to wild-type A3A, or a fragment thereof. In some embodiments, a A3A variant has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to a wild type A3A. In some embodiments, the A3A variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 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 or more amino acid changes compared to a wild type A3A. In some embodiments, the A3A variant comprises a fragment of an A3A, such that the fragment has at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to the corresponding fragment of a wild-type A3A.
In some embodiments, an A3A variant is a protein having a sequence that differs from a wild-type A3A protein by one or several mutations, such as substitutions, deletions, insertions, one or several single point substitutions. In some embodiments, a shortened A3A sequence could be used, e.g., by deleting N-terminal, C-terminal, or internal amino acids. In some embodiments, a shortened A3A sequence is used where one to four amino acids at the C-terminus of the sequence is deleted. In some embodiments, an APOBEC3A (such as a human APOBEC3A) has a wild-type amino acid position 57 (as numbered in the wild-type sequence). In some embodiments, an APOBEC3A (such as a human APOBEC3A) has an asparagine at amino acid position 57 (as numbered in the wild-type sequence).
In some embodiments, the wild-type A3A is a human A3A (UniPROT accession ID: p319411, SEQ ID NO: 40).
In some embodiments, the A3A disclosed herein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 40. In some embodiments, the level of identity is at least 85%, at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%. In some embodiments, the A3A comprises an amino acid sequence having at least 87% identity to SEQ ID NO: 40. In some embodiments, the A3A comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 40. In some embodiments, the A3A comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 40. In some embodiments, the A3A comprises an amino acid sequence with at least 98% identity to SEQ ID NO: 40. In some embodiments, the A3A comprises an amino acid sequence with at least 99% identity to A3A ID NO: 40. In some embodiments, the A3A comprises the amino acid sequence of SEQ ID NO: 40.
C. Linkers
In some embodiments, the polypeptide comprising the A3A and the RNA-guided nickase described herein further comprises a linker that connects the A3A and the RNA-guided nickase. In some embodiments, the linker is an organic molecule, polymer, or chemical moiety. In some embodiments, the linker is a peptide linker. In some embodiments, the nucleic acid encoding the polypeptide comprising the A3A and the RNA-guided nickase further comprises a sequence encoding the peptide linker. mRNAs encoding the A3A-linker-RNA-guided nickase fusion protein are provided.
In some embodiments, the peptide linker is any stretch of amino acids having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, or more amino acids.
In some embodiments, the peptide linker is the 16 residue “XTEN” linker, or a variant thereof (See, e.g., the Examples; and Schellenberger et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat. Biotechnol. 27, 1186-1190 (2009)). In some embodiments, the XTEN linker comprises a sequence that is any one of SGSETPGTSESATPES (SEQ ID NO: 46), SGSETPGTSESA (SEQ ID NO: 47), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 48). In some embodiments, the XTEN linker consists of the sequence SGSETPGTSESATPES (SEQ ID NO: 46), SGSETPGTSESA (SEQ ID NO: 47), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 48).
In some embodiments, the peptide linker comprises a (GGGGS)n (e.g., SEQ ID NOs: 212, 216, 221, 240), a (G)n, an (EAAAK)n (e.g., SEQ ID NOs: 213, 219, 267), a (GGS)n, an SGSETPGTSESATPES (SEQ ID NO: 46) motif (see. e.g., Guilinger J P, Thompson D B, Liu D R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents are incorporated herein by reference), or an (XP)n motif, or a combination of any of these, wherein n is independently an integer between 1 and 30. See, WO2015089406, e.g., paragraph [0012], the entire content of which is incorporated herein by reference.
In some embodiments, the peptide linker comprises one or more sequences selected from SEQ ID NOs: 46-59, 61 and 211-272. In some embodiments, the peptide linker comprises one or more sequences selected from SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270. SEQ ID NO: 271 and SEQ ID NO: 272. In some embodiments, the peptide linker comprises a sequence of SEQ ID NO: 268.
D. RNA-Guided Nickase
In some embodiments, an RNA-guided nickase disclosed herein is a Cas nickase. In some embodiments, a RNA-guided nickase is from a specific Cas nuclease with its catalytic domain(s) being inactivated. In some embodiments, the RNA-guided nickase is a Class 2 Cas nickase, such as a Cas9 nickase or a Cpf1 nickase. In some embodiments, the RNA-guided nickase is an S. pyogenes Cas9 nickase. In some embodiments, the RNA-guided nickase is Neisseria meningitidis Cas9 nickase.
In some embodiments, the RNA-guided nickase is a modified Class 2 Cas protein or derived from a Class 2 Cas protein. In some embodiments, the RNA-guided nickase is modified or derived from a Cas protein, such as a Class 2 Cas nuclease (which may be, e.g., a Cas nuclease of Type II, V, or VI). Class 2 Cas nuclease include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins and modifications thereof. Examples of Cas9 nucleases include those of the type II CRISPR systems of S. pyogenes. S. aureus, and other prokaryotes (see. e.g., the list in the next paragraph), and modified (e.g., engineered or mutant) versions thereof. See, e.g., US2016/0312198 A1; US 2016/0312199 A1, which is incorporated by reference in its entirety. Other examples of Cas nucleases include a Csm or Cmr complex of a type III CRISPR system or the Cas10, Csm1, or Cmr2 subunit thereof; and a Cascade complex of a type I CRISPR system, or the Cas3 subunit thereof. In some embodiments, the Cas nuclease may be from a Type-IIA, Type-IIB, or Type-IIC system. For discussion of various CRISPR systems and Cas nucleases, see, e.g., Makarova et al., N
A Cas nickase described herein may be a nickase form of a Cas nuclease from the species including, but not limited to, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, Acidaminococcus sp., Lachnospiraceae bacterium ND2006, or Acaryochloris marina.
In some embodiments, the Cas nickase is a nickase form of the Cas9 nuclease from Streptococcus pyogenes. In some embodiments, the Cas nickase is a nickase form of the Cas9 nuclease from Streptococcus thermophilus. In some embodiments, the Cas nickase is a nickase form of the Cas9 nuclease from Neisseria meningitidis. See e.g., WO/2020081568, describing an Nme2Cas9 D16A nickase. In some embodiments, the Cas nickase is a nickase form of the Cas9 nuclease from Staphylococcus aureus. In some embodiments, the Cas nickase is a nickase form of the Cpf1 nuclease from Francisella novicida. In some embodiments, the Cas nickase is a nickase form of the Cpf1 nuclease from Acidaminococcus sp. In some embodiments, the Cas nickase is a nickase form of the Cpf1 nuclease from Lachnospiraceae bacterium ND2006. In further embodiments, the Cas nickase is a nickase form of the Cpf1 nuclease from Francisella tularensis, Lachnospiraceae bacterium, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella disiens, or Porphyromonas macacae. In certain embodiments, the Cas nickase is a nickase form of a Cpf1 nuclease from an Acidaminococcus or Lachnospiraceae. As discussed elsewhere, a nickase may be derived from (i.e. related to) a specific Cas nuclease in that the nickase is a form of the nuclease in which one of its two catalytic domains is inactivated, e.g., by mutating an active site residue essential for nucleolysis, such as D10, H840, or N863 in Spy Cas9. One skilled in the art will be familiar with techniques for easily identifying corresponding residues in other Cas proteins, such as sequence alignment and structural alignment, which is discussed in detail below.
In other embodiments, the Cas nickase may relate to a Type-I CRISPR/Cas system. In some embodiments, the Cas nickase may be a component of the Cascade complex of a Type-I CRISPR/Cas system. In some embodiments, the Cas nickase may be a Cas3 protein. In some embodiments, the Cas nickase may be from a Type-III CRISPR/Cas system.
In some embodiments, a Cas nickase is a nickase form of a Cas nuclease or a modified Cas nuclease in which an endonucleolytic active site is inactivated, e.g., by one or more alterations (e.g., point mutations) in a catalytic domain. See, e.g., U.S. Pat. No. 8,889,356 for discussion of Cas nickases and exemplary catalytic domain alterations.
Wild type S. pyogenes Cas9 has two catalytic domains: RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target strand of DNA. In some embodiments, a Cas nuclease may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015) Cell October 22:163(3): 759-771. In some embodiments, the Cas nuclease may comprise an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015). Further exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the Francisella novicida U112 Cpf1 (FnCpf1) sequence (UniProtKB—AOQ7Q2 (CPF1_FRATN)).
In some embodiments, a Cas nickase such as a Cas9 nickase has an inactivated RuvC or HNH domain. In some embodiments, a nickase is used having a RuvC domain with reduced activity. In some embodiments, a nickase is used having an inactive RuvC domain. In some embodiments, a nickase is used having an HNH domain with reduced activity. In some embodiments, a nickase is used having an inactive HNH domain.
In some embodiments, a Cas9 nickase has an active HNH nuclease domain and is able to cleave the non-targeted strand of DNA, i.e., the strand bound by the gRNA and has an inactive RuvC nuclease domain and is not able to cleave the targeted strand of the DNA, i.e., the strand where base editing by deaminase is desired.
An exemplary Cas9 nickase amino acid sequence is provided as SEQ ID NO: 70. An exemplary Cas9 nickase mRNA ORF sequence, which includes start and stop codons, is provided as SEQ ID NO: 71. An exemplary Cas9 nickase mRNA coding sequence, suitable for inclusion in a fusion protein, is provided as SEQ ID NO: 72.
In some embodiments, the RNA-guided nickase is a Class 2 Cas nickase described herein. In some embodiments, the RNA-guided nickase is a Cas9 nickase described herein.
In some embodiments, the RNA-guided nickase is an S. pyogenes Cas9 nickase described herein.
In some embodiments, the RNA-guided nickase is a D10A SpyCas9 nickase described herein. In some embodiments, the RNA-guided nickase comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% identity to any one of SEQ ID NO: 70, 73, or 76. In some embodiments, the RNA-guided nickase comprises the amino acid sequence of SEQ ID NO: 70.
In some embodiments, the mRNA ORF sequence comprises encoding the RNA-guided nickase, which includes start and stop codons, comprises a nucleotide sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity to the nucleotide sequence of any one of SEQ ID NOs: 71, 74, or 77. In some embodiments, the mRNA sequence encoding the RNA-guided nickase comprises a nucleotide sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity to the nucleotide sequence of any one of SEQ ID NOs: 72, 75, or 78. In some embodiments, the level of identity is at least 90%. In some embodiments, the level of identity is at least 95%. In some embodiments, the level of identity is at least 98%. In some embodiments, the level of identity is at least 99%. In some embodiments, the level of identity is at least 100%. In some embodiments, the sequence encoding the RNA-guided nickase comprises the nucleotide sequence of any one of SEQ ID NOs: 71, 72, 74, 75, 77, or 78.
In some embodiments, the RNA-guided nickase is Neisseria meningitidis (Nme) Cas9 nickase described herein.
In some embodiments, the RNA-guided nickase is a D16A NmeCas9 nickase described herein. In some embodiments, the D16A NmeCas9 nickase is a D16A Nme2Cas9 nickase. In some embodiments, the D16A Nme2Cas9 nickase comprises an amino acid sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 387. In some embodiments, the sequence encoding the D16A Nme2Cas9 comprises a nucleotide sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 388-393.
E. Compositions Comprising a Cytidine Deaminase and an RNA-Guided Nickase
In some embodiments, an mRNA encoding a polypeptide comprising a cytidine deaminase and an RNA-guided nickase is provided, wherein the polypeptide does not comprise a uracil glycosylase inhibitor (UGI).
1. Exemplary Compositions
As described herein, compositions, methods, and uses are provided comprising an mRNA comprising an open reading frame encoding a polypeptide comprising a cytidine deaminase and an RNA-guided nickase, wherein the polypeptide does not comprise a uracil glycosylase inhibitor (UGI). For each exemplary composition described below, the mRNA does not comprise a UGI.
In some embodiments, an mRNA encoding a polypeptide comprising a cytidine deaminase and an RNA-guided nickase is provided. In some embodiments, an enzyme of APOBEC family and an RNA-guided nickase is provided. In some embodiments, the polypeptide comprises an enzyme of APOBEC1 subgroup and an RNA-guided nickase. In some embodiments, the polypeptide comprises an enzyme of APOBEC2 subgroup and an RNA-guided nickase. In some embodiments, the polypeptide comprises an enzyme of APOBEC4 subgroup and an RNA-guided nickase. In some embodiments, the polypeptide comprises an enzyme of APOBEC3 subgroup and an RNA-guided nickase.
In some embodiments, an mRNA encoding a polypeptide comprising a cytidine deaminase and an RNA-guided nickase is provided. In some embodiments, an enzyme of APOBEC family and a D10A SpyCas9 nickase is provided. In some embodiments, the polypeptide comprises an enzyme of APOBEC1 subgroup and a D10A SpyCas9 nickase. In some embodiments, the polypeptide comprises an enzyme of APOBEC2 subgroup and a D10A SpyCas9 nickase. In some embodiments, the polypeptide comprises an enzyme of APOBEC4 subgroup and a D10A SpyCas9 nickase. In some embodiments, the polypeptide comprises an enzyme of APOBEC3 subgroup and a D10A SpyCas9 nickase.
In some embodiments, an mRNA encoding a polypeptide comprising a cytidine deaminase and an RNA-guided nickase is provided. In some embodiments, an enzyme of APOBEC family and a D16A NmeCas9 nickase is provided. In some embodiments, an enzyme of APOBEC family and a D16A Nme2Cas9 nickase is provided. In some embodiments, the polypeptide comprises an enzyme of APOBEC1 subgroup and a D16A Nme2Cas9 nickase. In some embodiments, the polypeptide comprises an enzyme of APOBEC2 subgroup and a D16A Nme2Cas9 nickase. In some embodiments, the polypeptide comprises an enzyme of APOBEC4 subgroup and a D16A Nme2Cas9 nickase. In some embodiments, the polypeptide comprises an enzyme of APOBEC3 subgroup and a D16A Nme2Cas9 nickase.
In some embodiments, the polypeptide lacks a UGI.
In some embodiments, the cytidine deaminase and the RNA-guided nickase are linked via a linker. In some embodiments, the cytidine deaminase and the RNA-guided nickase are linked via a peptide linker. In some embodiments, the peptide linker comprises one or more sequences selected from SEQ ID NOs: 46-59, 61 and 211-272.
In some embodiments, the polypeptide further comprises one or more additional heterologous functional domains. In some embodiments, the polypeptide further comprises one or more nuclear localization sequences (NLSs) (described herein) at the C-terminal of the polypeptide or the N-terminal of the polypeptide.
In some embodiments, an mRNA encoding a polypeptide comprising a cytidine deaminase and an RNA-guided nickase is provided. In some embodiments, an enzyme of APOBEC family and an RNA-guided nickase is provided. In some embodiments, the polypeptide comprises an enzyme of APOBEC1 subgroup and an RNA-guided nickase. In some embodiments, the polypeptide comprises an enzyme of APOBEC2 subgroup and an RNA-guided nickase. In some embodiments, the polypeptide comprises an enzyme of APOBEC4 subgroup and an RNA-guided nickase. In some embodiments, the polypeptide comprises an enzyme of APOBEC3 subgroup and an RNA-guided nickase.
In some embodiments, an mRNA encoding a polypeptide comprising a cytidine deaminase and an RNA-guided nickase is provided. In some embodiments, an enzyme of APOBEC family and a D10A SpyCas9 nickase, wherein the enzyme of APOBEC family and the D10A SpyCas9 nickase are fused via a linker. In some embodiments, the polypeptide comprises an enzyme of APOBEC family and a D10A SpyCas9 nickase, and a nuclear localization sequence (NLS) at the C-terminus of the fused polypeptide. In some embodiments, the polypeptide comprises an enzyme of APOBEC family and a D10A SpyCas9 nickase, and a NLS at the N-terminus of the fused polypeptide. In some embodiments, the polypeptide comprises an enzyme of APOBEC family and a D10A SpyCas9 nickase, wherein the enzyme of APOBEC family and the D10A SpyCas9 nickase are fused via a linker, and a NLS fused to the C-terminus of the D10A SpyCas9 nickase, optionally via a linker. In some embodiments, the polypeptide comprises an enzyme of APOBEC family and a D10A SpyCas9 nickase, wherein the enzyme of APOBEC family and the D10A SpyCas9 nickase are fused via a linker, and a NLS fused to the C-terminus of the D10A SpyCas9 nickase, optionally via a linker.
In some embodiments, the polypeptide comprises an enzyme of APOBEC family and a D16A NmeCas9 nickase, wherein the enzyme of APOBEC family and the D16A NmeCas9 nickase are fused via a linker. In some embodiments, the polypeptide comprises an enzyme of APOBEC family and a D16A Nme2Cas9 nickase, wherein the enzyme of APOBEC family and the D16A Nme2Cas9 nickase are fused via a linker. In some embodiments, the polypeptide comprises an enzyme of APOBEC family and a D16A Nme2Cas9 nickase, and a nuclear localization sequence (NLS) at the C-terminus of the fused polypeptide. In some embodiments, the polypeptide comprises an enzyme of APOBEC family and a D16A Nme2Cas9 nickase, and a NLS at the N-terminus of the fused polypeptide. In some embodiments, the polypeptide comprises an enzyme of APOBEC family and a D16A Nme2Cas9 nickase, wherein the enzyme of APOBEC family and the D16A Nme2Cas9 nickase are fused via a linker, and a NLS fused to the C-terminus of the D16A Nme2Cas9 nickase, optionally via a linker. In some embodiments, the polypeptide comprises an enzyme of APOBEC family and a D16A Nme2Cas9 nickase, wherein the enzyme of APOBEC family and the D16A Nme2Cas9 nickase are fused via a linker, and a NLS fused to the C-terminus of the D16A Nme2Cas9 nickase, optionally via a linker.
In some embodiments, the polypeptide comprises an enzyme of APOBEC1 subgroup and a D10A SpyCas9 nickase, wherein the enzyme of APOBEC1 subgroup and the D10A SpyCas9 nickase are fused via a linker. In some embodiments, the polypeptide comprises an enzyme of APOBEC1 subgroup and a D10A SpyCas9 nickase, and a nuclear localization sequence (NLS) at the C-terminus of the fused polypeptide. In some embodiments, the polypeptide comprises an enzyme of APOBEC1 subgroup and a D10A SpyCas9 nickase, and a NLS at the N-terminus of the fused polypeptide. In some embodiments, the polypeptide comprises an enzyme of APOBEC1 subgroup and a D10A SpyCas9 nickase, wherein the enzyme of APOBEC1 subgroup and the D10A SpyCas9 nickase are fused via a linker, and a NLS fused to the C-terminus of the D10A SpyCas9 nickase, optionally via a linker. In some embodiments, the polypeptide comprises an enzyme of APOBEC1 subgroup and a D10A SpyCas9 nickase, wherein the enzyme of APOBEC1 subgroup and the D10A SpyCas9 nickase are fused via a linker, and a NLS fused to the C-terminus of the D10A SpyCas9 nickase, optionally via a linker.
In some embodiments, the polypeptide comprises an enzyme of APOBEC1 subgroup and a D16A Nme2Cas9 nickase, wherein the enzyme of APOBEC1 subgroup and the D16A Nme2Cas9 nickase are fused via a linker. In some embodiments, the polypeptide comprises an enzyme of APOBEC1 subgroup and a D16A Nme2Cas9 nickase, wherein the enzyme of APOBEC1 subgroup and the D16A Nme2Cas9 nickase are fused via a linker. In some embodiments, the polypeptide comprises an enzyme of APOBEC1 subgroup and a D16A Nme2Cas9 nickase, and a nuclear localization sequence (NLS) at the C-terminus of the fused polypeptide. In some embodiments, the polypeptide comprises an enzyme of APOBEC1 subgroup and a D16A Nme2Cas9 nickase, and a NLS at the N-terminus of the fused polypeptide. In some embodiments, the polypeptide comprises an enzyme of APOBEC1 subgroup and a D16A Nme2Cas9 nickase, wherein the enzyme of APOBEC1 subgroup and the D16A Nme2Cas9 nickase are fused via a linker, and a NLS fused to the C-terminus of the D16A Nme2Cas9 nickase, optionally via a linker. In some embodiments, the polypeptide comprises an enzyme of APOBEC1 subgroup and a D16A Nme2Cas9 nickase, wherein the enzyme of APOBEC1 subgroup and the D16A Nme2Cas9 nickase are fused via a linker, and a NLS fused to the C-terminus of the D16A Nme2Cas9 nickase, optionally via a linker.
In some embodiments, the polypeptide comprises an enzyme of APOBEC3 subgroup and a D10A SpyCas9 nickase, wherein the enzyme of APOBEC3 subgroup and the D10A SpyCas9 nickase are fused via a linker. In some embodiments, the polypeptide comprises an enzyme of APOBEC3 subgroup and a D10A SpyCas9 nickase, and a nuclear localization sequence (NLS) at the C-terminus of the fused polypeptide. In some embodiments, the polypeptide comprises an enzyme of APOBEC3 subgroup and a D10A SpyCas9 nickase, and a NLS at the N-terminus of the fused polypeptide. In some embodiments, the polypeptide comprises an enzyme of APOBEC3 subgroup and a D10A SpyCas9 nickase, wherein the enzyme of APOBEC3 subgroup and the D10A SpyCas9 nickase are fused via a linker, and a NLS fused to the C-terminus of the D10A SpyCas9 nickase, optionally via a linker. In some embodiments, the polypeptide comprises an enzyme of APOBEC3 subgroup and a D10A SpyCas9 nickase, wherein the enzyme of APOBEC3 subgroup and the D10A SpyCas9 nickase are fused via a linker, and a NLS fused to the C-terminus of the D10A SpyCas9 nickase, optionally via a linker.
In some embodiments, the polypeptide comprises an enzyme of APOBEC3 subgroup and a D16A Nme2Cas9 nickase, wherein the enzyme of APOBEC3 subgroup and the D16A Nme2Cas9 nickase are fused via a linker. In some embodiments, the polypeptide comprises an enzyme of APOBEC3 subgroup and a D16A Nme2Cas9 nickase, wherein the enzyme of APOBEC3 subgroup and the D16A Nme2Cas9 nickase are fused via a linker. In some embodiments, the polypeptide comprises an enzyme of APOBEC3 subgroup and a D16A Nme2Cas9 nickase, and a nuclear localization sequence (NLS) at the C-terminus of the fused polypeptide. In some embodiments, the polypeptide comprises an enzyme of APOBEC3 subgroup and a D16A Nme2Cas9 nickase, and a NLS at the N-terminus of the fused polypeptide. In some embodiments, the polypeptide comprises an enzyme of APOBEC3 subgroup and a D16A Nme2Cas9 nickase, wherein the enzyme of APOBEC3 subgroup and the D16A Nme2Cas9 nickase are fused via a linker, and a NLS fused to the C-terminus of the D16A Nme2Cas9 nickase, optionally via a linker. In some embodiments, the polypeptide comprises an enzyme of APOBEC3 subgroup and a D16A Nme2Cas9 nickase, wherein the enzyme of APOBEC3 subgroup and the D16A Nme2Cas9 nickase are fused via a linker, and a NLS fused to the C-terminus of the D16A Nme2Cas9 nickase, optionally via a linker.
In some embodiments, the polypeptide comprises a D10A SpyCas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 268, and a cytidine deaminase comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In some embodiments, the polypeptide comprises a D10A SpyCas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 269, and a cytidine deaminase comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In some embodiments, the polypeptide comprises a D10A SpyCas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 270, and a cytidine deaminase comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In some embodiments, the polypeptide comprises a D10A SpyCas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 271, and a cytidine deaminase comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In some embodiments, the polypeptide comprises a D10A SpyCas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 272, and a cytidine deaminase comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In any of the foregoing embodiments, the D10A SpyCas9 nickase may comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 70, 73, or 76.
In some embodiments, the polypeptide comprises a D16A Nme2Cas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 268, and a cytidine deaminase comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In some embodiments, the polypeptide comprises a D16A Nme2Cas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 269, and a cytidine deaminase comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In some embodiments, the polypeptide comprises a D16A Nme2Cas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 270, and a cytidine deaminase comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In some embodiments, the polypeptide comprises a D16A Nme2Cas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 271, and a cytidine deaminase comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In some embodiments, the polypeptide comprises a D16A Nme2Cas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 272, and a cytidine deaminase comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In any of the foregoing embodiments, the D16A Nme2Cas9 nickase may comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 387.
In some embodiments, the polypeptide comprises a D10A SpyCas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 268, and a cytidine deaminase comprising an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In some embodiments, the polypeptide comprises a D10A SpyCas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 269, and a cytidine deaminase comprising an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In some embodiments, the polypeptide comprises a D10A SpyCas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 270, and a cytidine deaminase comprising an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In some embodiments, the polypeptide comprises a D10A SpyCas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 271, and a cytidine deaminase comprising an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In some embodiments, the polypeptide comprises a D10A SpyCas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 272, and a cytidine deaminase comprising an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In any of the foregoing embodiments, the D10A SpyCas9 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 70, 73, or 76.
In some embodiments, the polypeptide comprises a D16A Nme2Cas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 268, and a cytidine deaminase comprising an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In some embodiments, the polypeptide comprises a D16A Nme2Cas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 269, and a cytidine deaminase comprising an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In some embodiments, the polypeptide comprises a D16A Nme2Cas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 270, and a cytidine deaminase comprising an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In some embodiments, the polypeptide comprises a D16A Nme2Cas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 271, and a cytidine deaminase comprising an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In some embodiments, the polypeptide comprises a D16A Nme2Cas9 nickase, a linker comprising the amino acid sequence of SEQ ID NO: 272, and a cytidine deaminase comprising an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 976, 977, 979, 980, 984-987, 993-1006, and 1009. In any of the foregoing embodiments, the D16A Nme2Cas9 nickase comprises an amino acid sequence that is at least 85%, at least 90%6, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 387.
The polypeptide may be organized in any number of ways to form a single chain. The NLS can be N- or C-terminal, or both N- and C-terminals, and the cytidine deaminase can be N- or C-terminal as compared the RNA-guided nickase. In some embodiments, the polypeptide comprises, from N to C terminus, a cytidine deaminase, an optional linker, an RNA-guided nickase, and an optional NLS. In some embodiments, the polypeptide comprises, from N to C terminus, an RNA-guided nickase, an optional linker, a cytidine deaminase, and an optional NLS. In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, an RNA-guided nickase, an optional linker, and a cytidine deaminase. In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, an RNA-guided nickase, an optional linker, and a cytidine deaminase, and an optional NLS.
In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, an enzyme of APOBEC family, an optional linker, an RNA-guided nickase, and an optional NLS. In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, an RNA-guided nickase, an optional linker, an enzyme of APOBEC family and an optional NLS. In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, an RNA-guided nickase, an optional linker, an enzyme of APOBEC family, and an optional NLS. In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, an RNA-guided nickase, an optional linker, an enzyme of APOBEC family, and an optional NLS.
In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, an enzyme of APOBEC3 subgroup, an optional linker, an RNA-guided nickase, and an optional NLS. In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, an RNA-guided nickase, an optional linker, an enzyme of APOBEC3 subgroup and an optional NLS. In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, an RNA-guided nickase, an optional linker, an enzyme of APOBEC3 subgroup, and an optional NLS. In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, an RNA-guided nickase, an optional linker, an enzyme of APOBEC3 subgroup, and an optional NLS.
In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, an enzyme of APOBEC family, an optional linker, a D10A SpyCas9 nickase or a D16A Nme2Cas9 nickase, and an optional NLS. In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, a D10A SpyCas9 nickase or a D16A Nme2Cas9 nickase, an optional linker, an enzyme of APOBEC family and an optional NLS. In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, a D10A SpyCas9 nickase or a D16A Nme2Cas9 nickase, an optional linker, an enzyme of APOBEC family, and an optional NLS. In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, a D10A SpyCas9 nickase or a D16A Nme2Cas9 nickase, an optional linker, and an enzyme of APOBEC family, and an optional NLS.
In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, an enzyme of APOBEC3 subgroup, an optional linker, a D10A SpyCas9 nickase or a D16A Nme2Cas9 nickase, and an optional NLS. In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, a D10A SpyCas9 nickase or a D16A Nme2Cas9 nickase, an optional linker, an enzyme of APOBEC3 subgroup and an optional NLS. In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, a D10A SpyCas9 nickase or a D16A Nme2Cas9 nickase, an optional linker, an enzyme of APOBEC3 subgroup, and an optional NLS. In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, a D10A SpyCas9 nickase or a D16A Nme2Cas9 nickase, an optional linker, and an enzyme of APOBEC3 subgroup, and an optional NLS.
In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, an enzyme of APOBEC3 subgroup, an optional linker, a D16A Nme2Cas9 nickase.
In some embodiments, the polypeptide comprises, from N to C terminus, (i) an optional NLS; (ii) a cytidine deaminase comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 40, 41, and 960-1023; (iii) a linker comprising one or more sequences selected from SEQ ID NOs: 46-59, 61 and 211-272, (iv) a D10A SpyCas9 nickase or a D16A Nme2Cas9 nickase, and (v) an optional NLS.
In some embodiments, the polypeptide comprises, from N to C terminus, (i) an optional NLS, (ii) a D10A SpyCas9 nickase or a D16A Nme2Cas9 nickase, (iii) a linker comprising one or more sequences selected from SEQ ID NOs: 46-59, 61 and 211-272, (iv) a cytidine deaminase comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 40, 41, and 960-1023, and (v) an optional NLS.
In some embodiments, the polypeptide comprises, from N to C terminus, (i) an optional NLS, (ii) a D10A SpyCas9 nickase or a D16A Nme2Cas9 nickase, (iii) a linker comprising one or more sequences selected from SEQ ID NOs: 46-59, 61 and 211-272, (iv) a cytidine deaminase comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 40, 41, and 960-1023, and (v) an optional NLS.
In some embodiments, the polypeptide comprises, from N to C terminus, (i) an optional NLS, (ii) a D10A SpyCas9 nickase or a D16A Nme2Cas9 nickase, (iii) a linker comprising one or more sequences selected from SEQ ID NOs: 46-59, 61 and 211-272, (iv) cytidine deaminase comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 40, 41, and 960-1023, and (v) an optional NLS.
In some embodiments, the polypeptide comprises, from N to C terminus, (i) an optional NLS, (ii) a cytidine deaminase comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 40, 41, and 960-1023; (iii) a linker comprising one or more sequences selected from SEQ ID NOs: 46-59, 61 and 211-272, (iv) a D10A SpyCas9 nickase or a D16A Nme2Cas9 nickase, and (v) an optional NLS.
In some embodiments, the polypeptide comprises, from N to C terminus, (i) an optional NLS, (ii) a D10A SpyCas9 nickase or a D16A Nme2Cas9 nickase, (iii) a linker comprising one or more sequences selected from SEQ ID NOs: 46-59, 61 and 211-272, (iv) a cytidine deaminase comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 40, 41, and 960-1023, and (v) an optional NLS.
In some embodiments, the polypeptide comprises, from N to C terminus, (i) an optional NLS, (ii) a D10A SpyCas9 nickase or a D16A Nme2Cas9 nickase, (iii) a linker comprising one or more sequences selected from SEQ ID NOs: 46-59, 61 and 211-272, (iv) a cytidine deaminase comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 40, 41, and 960-1023, and (v) an optional NLS.
In some embodiments, the polypeptide comprises, from N to C terminus, (i) an optional NLS, (ii) a D10A SpyCas9 nickase or a D16A Nme2Cas9 nickase, (iii) a linker comprising one or more sequences selected from SEQ ID NOs: 46-59, 61 and 211-272, and (iv) cytidine deaminase comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 40, 41, and 960-1023, and (v) an optional NLS.
2. Compositions Comprising an APOBEC3A Deaminase and an RNA-Guided Nickase
In some embodiments, an mRNA encoding a polypeptide comprising an APOBEC3A deaminase (A3A) and an RNA-guided nickase is provided. In some embodiments, the polypeptide comprises a human A3A and an RNA-guided nickase. In some embodiments, the polypeptide comprises a wild-type A3A and an RNA-guided nickase. In some embodiments, the polypeptide comprises an A3A variant and an RNA-guided nickase. In some embodiments, the polypeptide comprises an A3A and a Cas9 nickase. In some embodiments, the polypeptide comprises an A3A and a D10A SpyCas9 nickase. In some embodiments, the polypeptide comprises a human A3A and a D10A SpyCas9 nickase. In some embodiments, the polypeptide comprises an A3A variant and a D10A SpyCas9 nickase. In some embodiments, the polypeptide lacks a UGI. In some embodiments, the A3A and the RNA-guided nickase are linked via a linker. In some embodiments, the polypeptide further comprises one or more additional heterologous functional domains. In some embodiments, the polypeptide further comprises a nuclear localization sequence (NLS) (described herein) at the C-terminal of the polypeptide or the N-terminal of the polypeptide.
In some embodiments, the polypeptide comprises a human A3A and a D10A SpyCas9 nickase, wherein the human A3A and the D10A SpyCas9 nickase are fused via a linker. In some embodiments, the polypeptide comprises a human A3A and a D10A SpyCas9 nickase, and a nuclear localization sequence (NLS) at the C-terminus of the fused polypeptide. In some embodiments, the polypeptide comprises a human A3A and a D10A SpyCas9 nickase, and a NLS at the N-terminus of the fused polypeptide. In some embodiments, the polypeptide comprises a human A3A and a D10A SpyCas9 nickase, wherein the human A3A and the DOA SpyCas9 nickase are fused via a linker, and a NLS fused to the C-terminus of the DOA SpyCas9 nickase, optionally via a linker. In some embodiments, the polypeptide comprises a human A3A and a D10A SpyCas9 nickase, wherein the human A3A and the DOA SpyCas9 nickase are fused via a linker, and a NLS fused to the C-terminus of the D10A SpyCas9 nickase, optionally via a linker.
The polypeptide may be organized in any number of ways to form a single chain. The NLS can be N- or C-terminal, or both N- and C-terminals. and the A3A can be N- or C-terminal as compared the RNA-guided nickase. In some embodiments, the polypeptide comprises, from N to C terminus, an A3A, an optional linker, an RNA-guided nickase, and an optional NLS. In some embodiments, the polypeptide comprises, from N to C terminus, an RNA-guided nickase, an optional linker, an A3A, and an optional NLS. In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, an RNA-guided nickase, an optional linker, and an A3A. In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, an RNA-guided nickase, an optional linker, and an A3A, and an optional NLS.
In any of the foregoing embodiments, the polypeptide may comprise an amino acid sequence having at least 80% identity to SEQ ID NOs: 3 or 6. In some embodiments, any of the foregoing levels of identity is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%. In some embodiments, the polypeptide disclosed herein may comprise an amino acid sequence with at least 90% identity to SEQ ID NOs: 3 or 6. In some embodiments, the polypeptide disclosed herein may comprise an amino acid sequence with at least 95% identity to SEQ ID NOs: 3 or 6. In some embodiments, the polypeptide disclosed herein may comprise an amino acid sequence with at least 98% identity to SEQ ID NOs: 3 or 6. In some embodiments, the polypeptide disclosed herein may comprise an amino acid sequence with at least 99% identity to SEQ ID NOs: 3 or 6. In some embodiments, the polypeptide disclosed herein may comprise an amino acid sequence of SEQ ID NOs: 3 or 6.
In any of the foregoing embodiments, a nucleic acid sequence comprising an open reading frame encoding the polypeptide disclosed herein may comprise a nucleic acid sequence having at least 80% identity to SEQ ID NOs: 2 or 5. In some embodiments, any of the foregoing levels of identity is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%.
In any of the foregoing embodiments, an mRNA sequence encoding the polypeptide disclosed herein may comprise a nucleic acid sequence having at least 80% identity to SEQ ID NOs: 1 or 4. In some embodiments, any of the foregoing levels of identity is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%.
In any of the foregoing embodiments, the polypeptide may comprise an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 303, 306, 309, or 312. In some embodiments, the polypeptide disclosed herein may comprise an amino acid sequence of SEQ ID NOs: 303, 306, 309, or 312. In any of the foregoing embodiments, a nucleic acid sequence comprising an open reading frame encoding the polypeptide disclosed herein may comprise a nucleic acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: SEQ ID NOs: 302, 305, 308, or 311. In some embodiments, a nucleic acid sequence comprising an open reading frame encoding the polypeptide disclosed herein comprises a nucleic acid sequence of SEQ ID NOs: SEQ ID NOs: 302, 305, 308, or 311. In any of the foregoing embodiments, an mRNA sequence encoding the polypeptide disclosed herein may comprise a nucleic acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 301, 304, 307, or 310. In any of the foregoing embodiments, an mRNA sequence encoding the polypeptide disclosed herein may comprise a nucleic acid sequence of SEQ ID NOs: 301, 304, 307, or 310.
In any of the foregoing embodiments, the A3A may comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 40. In some embodiments, the level of identity is at least 85%, at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%. In some embodiments, the A3A comprises an amino acid sequence of SEQ ID NO: 40.
In any of the foregoing embodiments, the RNA-guided nickase may comprise an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% identity to any one of SEQ ID NOs: 70, 73, or 76. In some embodiments, the level of identity is at least 85%, at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%. In some embodiments, the RNA-guided nickase comprises the amino acid sequence of SEQ ID NO: 70. In some embodiments, the RNA-guided nickase comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the RNA-guided nickase comprises the amino acid sequence of SEQ ID NO: 76.
In any of the foregoing embodiments, the A3A may comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 40 and the RNA-guided nickase may comprise an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% identity to any one of SEQ ID NOs: 70, 73, or 76. In some embodiments, the A3A comprises an amino acid sequence of SEQ ID NO: 40 and the RNA-guided nickase comprises an amino acid sequence of SEQ ID NO: 70.
F. Additional Features
1. Codon-Optimization
In some embodiments, the UGI or polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase) and an RNA-guided nickase is encoded by an open reading frame (ORF) comprising a codon optimized nucleic acid sequence. In some embodiment, the codon optimized nucleic acid sequence comprises minimal adenine codons and/or minimal uridine codons.
A given ORF can be reduced in uridine content or uridine dinucleotide content, for example, by using minimal uridine codons in a sufficient fraction of the ORF. For example, an amino acid sequence for the polypeptide described herein can be back-translated into an ORF sequence by converting amino acids to codons, wherein some or all of the ORF uses the exemplary minimal uridine codons shown below. In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons in the ORF are codons listed in Table 1.
In some embodiments, the ORF may consist of a set of codons of which at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons are codons listed in Table 1.
A given ORF can be reduced in adenine content or adenine dinucleotide content, for example, by using minimal adenine codons in a sufficient fraction of the ORF. For example, an amino acid sequence for the polypeptide described herein can be back-translated into an ORF sequence by converting amino acids to codons, wherein some or all of the ORF uses the exemplary minimal adenine codons shown below. In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons in the ORF are codons listed in Table 2.
In some embodiments, the ORF may consist of a set of codons of which at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons are codons listed in Table 2.
To the extent feasible, any of the features described above with respect to low adenine content can be combined with any of the features described above with respect to low uridine content. So too for uridine and adenine dinucleotides. Similarly, the content of uridine nucleotides and adenine dinucleotides in the ORF may be as set forth above. Similarly, the content of uridine dinucleotides and adenine nucleotides in the ORF may be as set forth above.
A given ORF can be reduced in uridine and adenine nucleotide and/or dinucleotide content, for example, by using minimal uridine and adenine codons in a sufficient fraction of the ORF. For example, an amino acid sequence for the polypeptide described herein can be back-translated into an ORF sequence by converting amino acids to codons, wherein some or all of the ORF uses the exemplary minimal uridine and adenine codons shown below. In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons in the ORF are codons listed in Table 3.
In some embodiments, the ORF may consist of a set of codons of which at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons are codons listed in Table 3. As can be seen in Table 3, each of the three listed serine codons contains either one A or one U. In some embodiments, uridine minimization is prioritized by using AGC codons for serine. In some embodiments, adenine minimization is prioritized by using UCC and/or UCG codons for serine.
In some embodiments, the ORF may have codons that increase translation in a mammal, such as a human. In further embodiments, the mRNA comprises an ORF having codons that increase translation in an organ, such as the liver, of the mammal, e.g., a human. In further embodiments, the ORF may have codons that increase translation in a cell type, such as a hepatocyte, of the mammal, e.g., a human. An increase in translation in a mammal, cell type, organ of a mammal, human, organ of a human, etc., can be determined relative to the extent of translation wild-type sequence of the ORF, or relative to an ORF having a codon distribution matching the codon distribution of the organism from which the ORF was derived or the organism that contains the most similar ORF at the amino acid level. Alternatively, in some embodiments, an increase in translation for a Cas9 sequence in a mammal, cell type, organ of a mammal, human, organ of a human, etc., is determined relative to translation of an ORF with the sequence of SEQ ID NO: 2 or 5 with all else equal, including any applicable point mutations, heterologous domains, and the like. In some embodiments, at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in an ORF are codons corresponding to highly expressed tRNAs (e.g., the highest-expressed tRNA for each amino acid) in a mammal, such as a human. In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in an ORF are codons corresponding to highly expressed tRNAs (e.g., the highest-expressed tRNA for each amino acid) in a mammalian organ, such as a human organ.
Alternatively, codons corresponding to highly expressed tRNAs in an organism (e.g., human) in general may be used.
Any of the foregoing approaches to codon selection can be combined with the minimal uridine and/or adenine codons shown above, e.g., by starting with the codons of Table 1, 2, or 3, and then where more than one option is available, using the codon that corresponds to a more highly-expressed tRNA, either in the organism (e.g., human) in general, or in an organ or cell type of interest (e.g., human liver or human hepatocytes).
In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in an ORF are codons from a codon set shown in Table 4 (e.g., the low U 1, low A, or low A/U codon set). The codons in the low U 1, low G, low A, and low A/U sets use codons that minimize the indicated nucleotides while also using codons corresponding to highly expressed tRNAs where more than one option is available. In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in an ORF are codons from the low U 1 codon set shown in Table 4. In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in an ORF are codons from the low A codon set shown in Table 4. In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in an ORF are codons from the low A/U codon set shown in Table 4.
2. Heterologous Functional Domains; Nuclear Localization Signals (NLS)
In some embodiments, the polypeptide comprising a cytidine deaminase (e.g., A3A) and an RNA-guided nickase further comprises one or more additional heterologous functional domains (e.g., is or comprises a ternary or higher-order fusion polypeptide).
In some embodiments, the heterologous functional domain may facilitate transport of the polypeptide into the nucleus of a cell. For example, the heterologous functional domain may be a nuclear localization signal (NLS). In some embodiments, the polypeptide may be fused with 1-10 NLS(s). In some embodiments, the polypeptide may be fused with 1-5 NLS(s). In some embodiments, the polypeptide may be fused with one NLS. Where one NLS is used, the NLS may be fused at the N-terminus or the C-terminus of the polypeptide sequence. In some embodiments, the polypeptide may be fused C-terminally to at least one NLS. An NLS may also be inserted within the polypeptide sequence. In other embodiments, the polypeptide may be fused with more than one NLS. In some embodiments, the polypeptide may be fused with 2, 3, 4, or 5 NLSs. In some embodiments, the polypeptide may be fused with two NLSs. In certain circumstances, the two NLSs may be the same (e.g., two SV40 NLSs) or different. In some embodiments, the polypeptide is fused to two SV40 NLS sequences at the carboxy terminus. In some embodiments, the polypeptide may be fused with two NLSs, one at the N-terminus and one at the C-terminus. In some embodiments, the polypeptide may be fused with 3 NLSs. In some embodiments, the polypeptide may be fused with no NLS. In some embodiments, the NLS may be a monopartite sequence, such as, e.g., the SV40 NLS, PKKKRKV (SEQ ID NO: 63) or PKKKRRV (SEQ ID NO: 121). In some embodiments, the NLS may be a bipartite sequence, such as the NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 122). In a specific embodiment, a single PKKKRKV (SEQ ID NO: 63) NLS may be fused at the C-terminus of the polypeptide. One or more linkers are optionally included at the fusion site (e.g., between the polypeptide and NLS). In some embodiments, one or more NLS(s) according to any of the foregoing embodiments are present in the polypeptide in combination with one or more additional heterologous functional domains, such as any of the heterologous functional domains described below.
In some embodiments of the mRNA disclosed herein, the cytidine deaminase (e.g., A3A) is located N-terminal to the RNA-guided nickase in the polypeptide. In some embodiments of the mRNA disclosed herein, the encoded RNA-guided nickase comprises a nuclear localization signal (NLS). In some embodiments, the NLS is fused to the C-terminus of the RNA-guided nickase. In some embodiments, the NLS is fused to the C-terminus of the RNA-guided nickase via a linker. In some embodiments, the NLS is fused to the N-terminus of the RNA-guided nickase. In some embodiments, the NLS is fused to the N-terminus of the RNA-guided nickase via a linker (e.g., SEQ ID NO: 61). In some embodiments, the NLS comprises a sequence having at least 80%, 85%, 90%, or 95% identity to any one of SEQ ID NOs: 63 and 110-122. In some embodiments, the NLS comprises the sequence of any one of SEQ ID NOs: 63 and 110-122. In some embodiments, the NLS is encoded by a sequence having at least 80%, 85%, 90%, 95%, 98% or 100% identity to the sequence of any one of SEQ ID NOs: 63 and 110-122.
In some embodiments, the heterologous functional domain may be capable of modifying the intracellular half-life of the A3A and/or the RNA-guided nickase in the polypeptide. In some embodiments, the half-life of the A3A and/or the RNA-guided nickase in the polypeptide may be increased. In some embodiments, the half-life of the A3A and/or the RNA-guided nickase in the polypeptide may be reduced. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the A3A and/or the RNA-guided nickase in the polypeptide. In some embodiments, the heterologous functional domain may be capable of reducing the stability of the A3A and/or the RNA-guided nickase in the polypeptide. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation. In some embodiments, the protein degradation may be mediated by proteolytic enzymes, such as, for example, proteasomes, lysosomal proteases, or calpain proteases. In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the polypeptide may be modified by addition of ubiquitin or a polyubiquitin chain. In some embodiments, the ubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-1 (URM1), neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8, also called Rub1 in S. cerevisiae), human leukocyte antigen F-associated (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitin fold-modifier-1 (UFM1), and ubiquitin-like protein-5 (UBL5).
In some embodiments, the heterologous functional domain may be a marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences. In some embodiments, the marker domain may be a fluorescent protein. Any known fluorescent proteins may be used as the marker domain such as GFP, YFP, EBFP, ECFP, DsRed or any other suitable fluorescent protein. In some embodiments, the marker domain may be a purification tag and/or an epitope tag. Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6×His, 8×His, biotin carboxyl carrier protein (BCCP), poly-His, and calmodulin. In some embodiments, the marker domain may be a reporter gene. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins.
In additional embodiments, the heterologous functional domain may target the polypeptide to a specific organelle, cell type, tissue, or organ. In some embodiments, the heterologous functional domain may target the polypeptide to mitochondria.
3. UTRs; Kozak Sequences
In some embodiments, the nucleic acid (e.g., mRNA) disclosed herein comprises a 5′ UTR, 3′ UTR, or 5′ and 3′ UTRs from Hydroxysteroid 17-Beta Dehydrogenase 4 (HSD17B4 or HSD) or globin such as human alpha globin (HBA), human beta globin (HBB), Xenopus laevis beta globin (XBG), bovine growth hormone, cytomegalovirus (CMV), mouse Hba-al, heat shock protein 90 (Hsp90), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin, alpha-tubulin, tumor protein (p53), or epidermal growth factor receptor (EGFR).
In some embodiments, the nucleic acid disclosed herein comprises a 5′ UTR from HSD and a 3′ UTR from a human albumin gene. In some embodiments, an mRNA disclosed herein comprises a 5′ UTR with at least 90% identity to any one of SEQ ID NO: 93 and a 3′ UTR with at least 90% identity to any one of SEQ ID NO: 69.
In some embodiments, the nucleic acid disclosed herein comprises a 5′ UTR with at least 90% identity to any one of SEQ ID NOs: 91-98. In some embodiments, an mRNA disclosed herein comprises a 3′ UTR with at least 90% identity to any one of SEQ ID NOs: 69, 99-106. In some embodiments, any of the foregoing levels of identity is at least 95%, at least 98%, at least 99%, or 100%. In some embodiments, an mRNA disclosed herein comprises a 5′ UTR having the sequence of any one of SEQ ID NOs: 91-98. In some embodiments, an mRNA disclosed herein comprises a 3′ UTR having the sequence of any one of SEQ ID NOs: 69, 99-106. In some embodiments, the mRNA comprises a 5′ UTR and a 3′ UTR from the same source.
In some embodiments, the nucleic acid described herein does not comprise a 5′ UTR, e.g., there are no additional nucleotides between the 5′ cap and the start codon. In some embodiments, the mRNA comprises a Kozak sequence (described below) between the 5′ cap and the start codon, but does not have any additional 5′ UTR. In some embodiments, the mRNA does not comprise a 3′ UTR, e.g., there are no additional nucleotides between the stop codon and the poly-A tail.
In some embodiments, the nucleic acid herein comprises a Kozak sequence. The Kozak sequence can affect translation initiation and the overall yield of a polypeptide translated from an mRNA. A Kozak sequence includes a methionine codon that can function as the start codon. A minimal Kozak sequence is NNNRUGN wherein at least one of the following is true: the first N is A or G and the second N is G. In the context of a nucleotide sequence, R means a purine (A or G). In some embodiments, the Kozak sequence is RNNRUGN, NNNRUGG, RNNRUGG, RNNAUGN, NNNAUGG, RNNAUGG, or GCCACCAUG. In some embodiments, the Kozak sequence is rccRUGg, rccAUGg, gccAccAUG, gccRccAUGG (SEQ ID NO: 107) or gccgccRccAUGG (SEQ ID NO: 108), with zero mismatches or with up to one or two mismatches to positions in lowercase.
4. Poly-A Tail
In some embodiments, the nucleic acid disclosed herein further comprises a poly-adenylated (poly-A) tail. The poly-A tails may comprise at least 8 consecutive adenine nucleotides, but also comprise one or more non-adenine nucleotide. As used herein, “non-adenine nucleotides” refer to any natural or non-natural nucleotides that do not comprise adenine. Guanine, thymine, and cytosine nucleotides are exemplary non-adenine nucleotides. Thus, the poly-A tails on the nucleic acid described herein may comprise consecutive adenine nucleotides located 3′ to nucleotides encoding a polypeptide of interest. In some instances, the poly-A tails on mRNA comprise non-consecutive adenine nucleotides located 3′ to nucleotides encoding a polypeptide comprising a cytidine deaminase (e.g., A3A) and an RNA-guided nickase or a sequence of interest, wherein non-adenine nucleotides interrupt the adenine nucleotides at regular or irregularly spaced intervals.
In some embodiments, the poly-A tail is encoded in the plasmid used for in vitro transcription of mRNA and becomes part of the transcript. The poly-A sequence encoded in the plasmid, i.e., the number of consecutive adenine nucleotides in the poly-A sequence, may not be exact, e.g., a 100 poly-A sequence in the plasmid may not result in a precisely 100 poly-A sequence in the transcribed mRNA. In some embodiments, the poly-A tail is not encoded in the plasmid, and is added by PCR tailing or enzymatic tailing, e.g., using E. coli poly(A) polymerase.
In some embodiments, the one or more non-adenine nucleotides are positioned to interrupt the consecutive adenine nucleotides so that a poly(A) binding protein can bind to a stretch of consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotide(s) is located after at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides. In some embodiments, the one or more non-adenine nucleotide is located after 8-50 consecutive adenine nucleotides. In some embodiments, the one or more non-adenine nucleotide is located after 8-100 consecutive adenine nucleotides.
In some embodiments, the poly-A tail comprises or contains one non-adenine nucleotide or one consecutive stretch of 2-10 non-adenine nucleotides.
In some embodiments, the non-adenine nucleotide is guanine, cytosine, or thymine. In some instances, where more than one non-adenine nucleotide is present, the non-adenine nucleotide may be selected from: a) guanine and thymine nucleotides; b) guanine and cytosine nucleotides; c) thymine and cytosine nucleotides; or d) guanine, thymine and cytosine nucleotides. An exemplary poly-A tail comprising non-adenine nucleotides is provided as SEQ ID NO: 109.
5. Modified Nucleotides
In some embodiments, the nucleic acid disclosed herein comprises a modified uridine at some or all uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5 position, e.g., with a halogen or C1-C3 alkoxy. In some embodiments, the modified uridine is a pseudouridine modified at the 1 position, e.g., with a C1-C3 alkyl. The modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof.
In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the uridine positions in the nucleic acid disclosed herein are modified uridines. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA disclosed herein are modified uridines, e.g., 5-methoxyuridine, 5-iodouridine, N1-methyl pseudouridine, pseudouridine, or a combination thereof.
In some embodiments, at least 10% of the uridine is substituted with a modified uridine. In some embodiments, 15% to 45% of the uridine is substituted with the modified uridine. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the uridine is substituted with the modified uridine.
6. 5′ Cap
In some embodiments, the nucleic acid disclosed herein comprises a 5′ cap, such as a Cap0, Cap1, or Cap2. A 5′ cap is generally a 7-methylguanine ribonucleotide (which may be further modified, as discussed below e.g., with respect to ARCA) linked through a 5′-triphosphate to the 5′ position of the first nucleotide of the 5′-to-3′ chain of the nucleic acid, i.e., the first cap-proximal nucleotide. In Cap0, the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2′-hydroxyl. In Cap1, the riboses of the first and second transcribed nucleotides of the mRNA comprise a 2′-methoxy and a 2′-hydroxyl, respectively. In Cap2, the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2′-methoxy. See, e.g., Katibah et al. (2014) Proc Natl Acad Sci USA 111(33):12025-30; Abbas et al. (2017) Proc Natl Acad Sci USA 114(11):E2106-E2115. Most endogenous higher eukaryotic nucleic acids, including mammalian nucleic acids such as human nucleic acids, comprise Cap1 or Cap2. Cap0 and other cap structures differing from Cap1 and Cap2 may be immunogenic in mammals, such as humans, due to recognition as “non-self” by components of the innate immune system such as IFIT-1 and IFIT-5, which can result in elevated cytokine levels including type I interferon. Components of the innate immune system such as IFIT-1 and IFIT-5 may also compete with eIF4E for binding of a nucleic acids with a cap other than Cap1 or Cap2, potentially inhibiting translation of the nucleic acid.
A cap can be included co-transcriptionally. For example, ARCA (anti-reverse cap analog; Thermo Fisher Scientific Cat. No. AM8045) is a cap analog comprising a 7-methylguanine 3′-methoxy-5′-triphosphate linked to the 5′ position of a guanine ribonucleotide which can be incorporated in vitro into a transcript at initiation. ARCA results in a Cap0 cap or a Cap0-like cap in which the 2′ position of the first cap-proximal nucleotide is hydroxyl. See, e.g., Stepinski et al., (2001) “Synthesis and properties of mRNAs containing the novel ‘anti-reverse’ cap analogs 7-methyl(3′-O-methyl)GpppG and 7-methyl(3′deoxy)GpppG,” RNA 7: 1486-1495. The ARCA structure is shown below.
CleanCap™ AG (m7G(5′)ppp(5′)(2′OMeA)pG; TriLink Biotechnologies Cat. No. N-7113) or CleanCap™ GG (m7G(5′)ppp(5′)(2′OMeG)pG; TriLink Biotechnologies Cat. No. N-7133) can be used to provide a Cap1 structure co-transcriptionally. 3′-O-methylated versions of CleanCap™ AG and CleanCap™ GG are also available from TriLink Biotechnologies as Cat. Nos. N-7413 and N-7433, respectively. The CleanCap™ AG structure is shown below. CleanCap™ structures are sometimes referred to herein using the last three digits of the catalog numbers listed above (e.g., “CleanCap™ 113” for TriLink Biotechnologies Cat. No. N-7113).
Alternatively, a cap can be added to an RNA post-transcriptionally. For example, Vaccinia capping enzyme is commercially available (New England Biolabs Cat. No. M2080S) and has RNA triphosphatase and guanylyltransferase activities, provided by its D1 subunit, and guanine methyltransferase, provided by its D12 subunit. As such, it can add a 7-methylguanine to an RNA, so as to give Cap0, in the presence of S-adenosyl methionine and GTP. See, e.g., Guo, P. and Moss, B. (1990) Proc. Natl. Acad. Sci. USA 87, 4023-4027; Mao, X. and Shuman, S. (1994) J. Biol. Chem. 269, 24472-24479. For additional discussion of caps and capping approaches, see, e.g., WO2017/053297 and Ishikawa et al., Nucl. Acids. Symp. Ser. (2009) No. 53, 129-130.
G. Guide RNA (gRNA)
In some embodiments, the compositions comprise at least one guide RNA (gRNA), and the methods comprise delivering at least one gRNA, wherein the gRNA directs the editor to a desired genomic location. In some embodiments, a composition comprises an mRNA described herein and at least one gRNA. In some embodiments, a composition comprises a polypeptide described herein and at least one gRNA. In some embodiments, the gRNA is a single guide RNA (sgRNA). In some embodiments, the gRNA is a dual guide RNA (dgRNA).
A gRNA disclosed herein may comprise a guide sequence that directs a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase) and an RNA-guided nickase to a cytosine (C) located in any region of a gene (e.g., within the coding region of a gene) for cytosine (C) to thymine (T) conversion (“C-to-T conversion”).
In some embodiments, the C-to-T conversion alters a DNA sequence, such as a human genetic sequence. In some embodiments, the C-to-T conversion alters the coding sequence of a gene. In some embodiments, the C-to-T conversion generates a stop codon, for example, a premature stop codon within the coding region of a gene. In some embodiments, the C-to-T conversion eliminates a stop codon. In some embodiments, the C-to-T conversion alters the regulatory sequence of a gene (e.g., a gene promotor or gene repressor). In some embodiments, the C-to-T conversion alters the splicing of a gene. In some embodiments, the C-to-T conversion corrects a genetic defect associated with a disease or disorder.
In some embodiments, a guide RNA (gRNA) comprises a guide sequence that directs a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase) and an RNA-guided nickase to a splice donor or acceptor site in a gene. In some embodiments, the splice donor or acceptor is a splice donor site. In some embodiments, the splice donor or acceptor site is a splice acceptor site.
In some embodiments, a guide RNA (gRNA) comprises a guide sequence that directs a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase) and an RNA-guided nickase to an acceptor splice site boundary. In some embodiments, a guide RNA (gRNA) comprises a guide sequence that directs a polypeptide comprising a cytidine deaminase (e.g., A3A) and an RNA-guided nickase to a donor splice site boundary.
In some embodiments, a guide RNA (gRNA) comprises a guide sequence that directs a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase) and an RNA-guided nickase to make a single-strand cut in a gene at a cut site 3′ of an acceptor splice site boundary or 5′ of an acceptor splice site boundary. In this and the following discussion, 3′ and 5′ indicate directions in the sense of the strand being cut.
In some embodiments, a guide RNA (gRNA) disclosed herein comprises a guide sequence that directs a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase) and an RNA-guided nickase for making a single-strand cut in a gene at a cut site that is 3′ of a donor splice site boundary or 5′ of a donor splice site boundary.
A “splice site,” as used herein, refers to the three nucleotides that make up an acceptor splice site or a donor splice site (defined below), or any other nucleotides known in the art that are part of a splice site. See e.g., Burset et al., Nucleic Acids Research 28(21):4364-4375 (2000) (describing canonical and non-canonical splice sites in mammalian genomes). The three nucleotides that make up an “acceptor splice site” are two conserved residues (e.g., AG in humans) at the 3′ of an intron and a boundary nucleotide (i.e., the first nucleotide of the exon 3′ of the AG). The three nucleotides that make up a “donor splice site” are two conserved residues (e.g., GT (gene) or GU (in RNA such as pre-mRNA) in human) at the 5′ end of an intron and a boundary nucleotide (i.e., the first nucleotide of the exon 5′ of the GT).
In some embodiments, a composition comprising at least one gRNA is provided in combination with a nucleic acid (e.g., an mRNA) disclosed herein. In some embodiments, one or more gRNA is provided as a separate molecule from the nucleic acid (e.g., an mRNA) disclosed herein. In some embodiments, a gRNA is provided as a part, such as a part of a UTR, of the nucleic acid disclosed herein.
In some embodiments, a composition is provided comprising a polypeptide comprising a cytidine deaminase and an RNA-guided nickase, and a gRNA. In some embodiments, a ribonucleoprotein complex (RNP) is provided, the RNP comprising a polypeptide comprising a cytidine deaminase and an RNA-guided nickase and a gRNA. In some embodiments, the polypeptide does not comprise a UGI.
The gRNA comprises a guide sequence targeting a particular gene or genetic sequence. In some embodiments, the gRNA is a Cas nickase guide. In some embodiments, the gRNA is a Class 2 Cas nickase guide. In further embodiments, the gRNA is a Cpf1 or Cas9 guide. In some embodiments, the gRNA is a Nme nickase guide. In some embodiments, the Nme nickase is a Nme1, Nme2, or Nme3 nickase. In some embodiments, the gRNA comprises a guide sequence 5′ of an RNA that forms two or more hairpin or stem-loop structures. CRISPR/Cas gRNA structures are known in the art and vary with their cognate Cas nuclease. In general, the gRNA used together with any particular Cas9 or Nme nickase described herein must function with that nickase. For example, when the polypeptide disclosed herein comprises a SpyCas9 nickase, the gRNA provided is a SpyCas9 guide RNA (as described herein). When the polypeptide disclosed herein comprises a NmeCas9 nickase, the guide RNA is a NmeCas9 guide RNA (as described herein).
In some embodiments, the gRNA comprises a guide sequence that direct an RNA-guided nickase (e.g., Cas9 nickase), to a target DNA sequence in a target locus, such as a target gene. Targets and exemplary target sequences targeting each gene are exemplified herein and include, but are not limited to, targets and guide sequences disclosed in e.g., WO2017185054 (for trinucleotide repeats in transcription factor four (TCF4)); WO 2018119182 A1 (targeting SERPINA1); WO 2019/067872 (targeting transthyretin (7TR); WO 2020/028327 A1 (targeting hydroxyacid oxidase 1 (HA01), the contents of each of which are hereby incorporated by reference in their entirety. One skilled in the art will be familiar with suitable guide sequences for targeting other genes or loci of interest.
The gRNA may comprise a crRNA comprising 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a guide sequence. The gRNA may further comprise a trRNA. In each composition and method embodiment described herein, the crRNA and trRNA may be associated as a single RNA (sgRNA), or may be on separate RNAs (dgRNA). In the context of sgRNAs, the crRNA and trRNA components may be covalently linked, e.g., via a phosphodiester bond or other covalent bond.
In each of the composition, use, and method embodiments described herein, the gRNA may comprise two RNA molecules as a “dual guide RNA” or “dgRNA”. The dgRNA comprises a first RNA molecule comprising a crRNA comprising a guide sequence, and a second RNA molecule comprising a trRNA. The first and second RNA molecules may not be covalently linked, but may form a RNA duplex via the base pairing between portions of the crRNA and the trRNA.
In each of the composition, use, and method embodiments described herein, the gRNA may comprise a single RNA molecule as a “single guide RNA” or “sgRNA”. The sgRNA may comprise a crRNA (or a portion thereof) comprising a guide sequence covalently linked to, e.g., a trRNA. The sgRNA may comprise 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a guide sequence. In some embodiments, the crRNA and the trRNA are covalently linked via a linker. In some embodiments, the sgRNA forms a stem-loop structure via the base pairing between portions of the crRNA and the trRNA. In some embodiments, the crRNA and the trRNA are covalently linked via one or more bonds that are not a phosphodiester bond.
In some embodiments, the trRNA may comprise all or a portion of a trRNA sequence derived from a naturally-occurring CRISPR/Cas system. In some embodiments, the trRNA comprises a truncated or modified wild type trRNA. The length of the trRNA depends on the CRISPR/Cas system used. In some embodiments, the trRNA comprises or consists of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 nucleotides. In some embodiments, the trRNA may comprise certain secondary structures, such as, for example, one or more hairpin or stem-loop structures, or one or more bulge structures.
The gRNAs provided herein can be useful for recognizing (e.g., hybridizing to) a target sequence in the gene. In some embodiments, the selection of the one or more gRNAs is determined based on target sequences within the gene. In some embodiments, a gRNA complementary or having complementarity to a target sequence within the target locus is used to direct a polypeptide comprising a cytidine deaminase (e.g., A3A) and an RNA-guided nickase to a particular location in the locus. The target locus may be recognized and nicked by a Cas nickase comprising a gRNA.
In some embodiments, the guide sequence is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a target sequence. In some embodiments, the target sequence may be complementary to the guide sequence of the gRNA. In some embodiments, the degree of complementarity or identity between a guide sequence of a gRNA and its corresponding target sequence may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the target sequence and the guide sequence of the gRNA may be 100% complementary or identical. In other embodiments, the target sequence and the guide sequence of the gRNA may contain at least one mismatch. For example, the target sequence and the guide sequence of the gRNA may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches, where the total length of the target sequence is at least about 17, 18, 19, 20 or more base pairs. In some embodiments, the target sequence and the guide sequence of the gRNA may contain 1, 2, 3, 4, 5, or 6 mismatches where the guide sequence is 20 nucleotides.
The gRNA may comprise a guide sequence linked to additional nucleotides to form a crRNA, e.g., with the following exemplary nucleotide sequence following the guide sequence at its 3′ end: GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 139).
In the case of an sgRNA, the guide sequence may be linked to additional nucleotides to form a sgRNA, e.g., with the following exemplary nucleotide sequence following the 3′ end of the guide sequence: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 140) in 5′ to 3′ orientation.
In some embodiments, the sgRNA comprises the modification pattern shown below in SEQ ID NO: 141, where N is any natural or non-natural nucleotide, and where the totality of the N's comprise a guide sequence as described herein and the modified sgRNA comprises the following sequence: mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmGmAmAmAmU mAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAm AmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 141), where “N” may be any natural or non-natural nucleotide. For example, encompassed herein is SEQ ID NO: 141, where the N's are replaced with any of the guide sequences disclosed herein. The modifications remain as shown in SEQ ID NO: 141 despite the substitution of N's for the nucleotides of a guide. That is, although the nucleotides of the guide replace the “N's”, the first three nucleotides are 2′OMe modified and there are phosphorothioate linkages between the first and second nucleotides, the second and third nucleotides and the third and fourth nucleotides.
In some embodiments, a conserved portion of the sgRNA is a conserved region of a spyCas9 or a spyCas9 equivalent. In some embodiments, a conserved portion of the sgRNA is not from S. pyogenes Cas9, such as Staphylococcus aureus Cas9 (“saCas9”). Further description of regions of exemplary sgRNAs are provided in WO2019/237069 published Dec. 12, 2019, the entire contents of which are incorporated herein by reference.
The SpyCas9 gRNA may comprise internal linkers. In some embodiments, the internal linker may have a bridging length of about 3-30, optionally 12-21 atoms, and the linker substitutes for at least 2 nucleotides of the gRNA. In some embodiments, the internal linker has a bridging length of about 6-18 atoms, optionally about 6-12 atoms, and the linker substitutes for at least 2 nucleotides of the gRNA.
In some embodiments the internal linker comprises at least two ethylene glycol subunits covalently linked to each other. In some embodiments, the internal linker comprises a PEG-linker.
In some embodiments, the internal linker comprises a PEG-linker having from 1 to 10 ethylene glycol units. In some embodiments, the internal linker comprises a PEG-linker having from 3 to 6 ethylene glycol units. In some embodiments, the internal linker comprises a PEG-linker having 3 ethylene glycol units. In some embodiments, the internal linker comprises a PEG-linker having 6 ethylene glycol units.
In some embodiments, the conserved portion of a spyCas9 guide RNA comprises a repeat-anti-repeat region, a hairpin 1 region, and a hairpin 2 region, and further comprises at least one of:
Exemplary locations of the linkers in the spyCas9 guide RNA are as shown in the following: NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUA(L1)UAGCAAGUUAAAAUAAG GCUAGUCCGUUAUCAACUU(L1)AAGUGGCACCGAGUCGGUGCUUUUU (SEQ ID NO: 524) where N are nucleotides encoding a guide sequence.
As used herein, “Linker 1” or “L1” refers to an internal linker having a bridging length of about 15-21 atoms. As used herein, “Linker 2” or “L2” refers to an internal linker having a bridging length of about 6-12 atoms.
In some embodiments, the spyCas9 guide RNA comprising internal linkers may be chemically modified. Exemplary modifications include a modification pattern of the following sequence:
In some embodiments, the gRNA comprises a 3′ tail. In some embodiments, the 3′ tail consists of a nucleotide comprising a uracil or modified uracil. In some embodiments, the 3′ terminal nucleotide is a modified nucleotide. In some embodiments, wherein the 3′ tail comprises a modification of any one or more of the nucleotides present in the 3′ tail. In further embodiments, wherein the modification of the 3′ tail is one or more of 2′-O-methyl (2′-OMe) modified nucleotide and a phosphorothioate (PS) linkage between nucleotides. penultimate nucleotide.
1. Short-Single Guide RNA (Short-sgRNA)
In some embodiments, an sgRNA provided herein is a short-single guide RNAs (short-sgRNAs), e.g., comprising a conserved portion of an sgRNA comprising a hairpin region, wherein the hairpin region lacks at least 5-10 nucleotides or 6-10 nucleotides. In some embodiments, the 5-10 nucleotides or 6-10 nucleotides are consecutive.
In some embodiments, a short-sgRNA lacks at least nucleotides 54-58 (AAAAA) of the conserved portion of a spyCas9 sgRNA. In some embodiments, a short-sgRNA is a non-spyCas9 sgRNA that lacks nucleotides corresponding to nucleotides 54-58 (AAAAA) of the conserved portion of a spyCas9 as determined, for example, by pairwise or structural alignment.
Structural alignment is useful where molecules share similar structures despite considerable sequence variation. Structural alignment involves identifying corresponding residues across two (or more) sequences by (i) modeling the structure of a first sequence using the known structure of the second sequence or (ii) comparing the structures of the first and second sequences where both are known, and identifying the residue in the first sequence most similarly positioned to a residue of interest in the second sequence. Corresponding residues are identified in some algorithms based on distance minimization given position (e.g., nucleobase position 1 or the 1′ carbon of the pentose ring for polynucleotides, or alpha carbons for polypeptides) in the overlaid structures (e.g., what set of paired positions provides a minimized root-mean-square deviation for the alignment). When identifying positions in a non-spyCas9 gRNA corresponding to positions described with respect to spyCas9 gRNA, spyCas9 gRNA can be the “second” sequence. Where a non-spyCas9 gRNA of interest does not have an available known structure, but is more closely related to another non-spyCas9 gRNA that does have a known structure, it may be most effective to model the non-spyCas9 gRNA of interest using the known structure of the closely related non-spyCas9 gRNA, and then compare that model to the spyCas9 gRNA structure to identify the desired corresponding residue in the non-spyCas9 gRNA of interest. There is an extensive literature on structural modeling and alignment for proteins; representative disclosures include U.S. Pat. Nos. 6,859,736; 8,738,343; and those cited in Aslam et al., Electronic Journal of Biotechnology 20 (2016) 9-13. For discussion of modeling a structure based on a known related structure or structures, see, e.g., Bordoli et al., Nature Protocols 4 (2009) 1-13, and references cited therein. See also
In some embodiments, the short-sgRNA described herein comprises a conserved portion comprising a hairpin region, wherein the hairpin region lacks 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides. In some embodiments, the lacking nucleotides are 5-10 lacking nucleotides or 6-10 lacking nucleotides. In some embodiments, the lacking nucleotides are consecutive. In some embodiments, the lacking nucleotides span at least a portion of hairpin 1 and a portion of hairpin 2. In some embodiments, the 5-10 lacking nucleotides comprise or consist of nucleotides 54-58, 54-61, or 53-60 of SEQ ID NO: 140.
In some embodiments, the short-sgRNA described herein further comprises a nexus region, wherein the nexus region lacks at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in the nexus region). In some embodiments, the short-sgRNA lacks each nucleotide in the nexus region.
In some embodiments, the SpyCas9 short-sgRNA described herein comprises a sequence of NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAA GGCUAGUCCGUUAUCACGAAAGGGCACCGAGUCGGUGCU (SEQ ID NO: 521). In some embodiments, the short-sgRNA described herein comprises a modification pattern as shown in SEQ ID NO: 520: mN*mN*mN*GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUA AGGCUAGUCCGUUAUCACGAAAGGGCACCGAGUCGGmUmGmC*mU (SEQ ID NO: 520), where A, C, G, U, and N are adenine, cytosine, guanine, uracil, and any ribonucleotide, respectively, unless otherwise indicated. An m is indicative of a 2′O-methyl modification, and an * is indicative of a phosphorothioate linkage between the nucleotides.
In some embodiments, a gRNA described herein is an N. meningitidis Cas9 (NmeCas9) gRNA comprising a conserved portion comprising a repeat/anti-repeat region, a hairpin 1 region, and a hairpin 2 region, wherein one or more of the repeat/anti-repeat region, the hairpin 1 region, and the hairpin 2 region are shortened. Exemplary wild-type NmeCas9 guide RNA comprises a sequence of (N)20-25 GUUGUAGCUCCCUUUCUCAUUUCGGAAACGAAAUGAGAACCGUUGCUACAAU AAGGCCGUCUGAAAAGAUGUGCCGCAACGCUCUGCCCCUUAAAGCUUCUGCUU UAAGGGGCAUCGUUUA (SEQ ID NO: 512). (N)20-25 as used herein represent 20-25, i.e., 20, 21, 22, 23, 24, or 25 consecutive N. A, C, G, and U represent nucleotides having adenine, cytosine, guanine, and uracil bases, respectively. In some embodiments, (N)20-25 has 24 nucleotides in length. N is any natural or non-natural nucleotide, and where the totality of the N's comprises a guide sequence.
In some embodiments, the conserved portion of the NmeCas9 short-gRNA comprises:
In some embodiments, the NmeCas9 short-gRNA comprises one of the following sequences in 5′ to 3′ orientation:
In some embodiments, at least 10 nucleotides of the conserved portion of the NmeCas9 short-sgRNA are modified nucleotides.
In some embodiments, the NmeCas9 short-sgRNA comprises a conserved region comprising one of the following sequences in 5′ to 3′ orientation: GUUGmUmAmGmCUCCCmUmGmAmAmAmCmCGUUmGmCUAmCAAU*AAGmGm CCmGmUmCmGmAmAmAmGmAmUGUGCmCGCmAmAmCmGCUCUmGmCCmUmU mCmUGmGCmAmUC*mG*mU*mU (SEQ ID NO: 516); or GUUGmUmAmGmCUCCCmUmGmAmAmAmCmCGUUmGmCUAmCAAU*AAGmGm CCmGmUmCmGmAmAmAmGmAmUGUGCmCGmCAAmCGCUCUmGmCCmUmUmC mUGGCAUCG*mU*mU (SEQ ID NO: 517).
The shortened NmeCas9 gRNA may comprise internal linkers disclosed herein.
“Internal linker” as used herein describes a non-nucleotide segment joining two nucleotides within a guide RNA. If the gRNA contains a spacer region, the internal linker is located outside of the spacer region (e.g., in the scaffold or conserved region of the gRNA). For Type V guides, it is understood that the last hairpin is the only hairpin in the structure, i.e., the repeat-anti-repeat region. In some embodiments, the internal linker comprises a PEG-linker disclosed herein.
Exemplary locations of the linkers are as shown in the following: (N)20-25 GUUGUAGCUCCCUUC(L1)GACCGUUGCUACAAUAAGGCCGUC(L1)GAUGU GCCGCAACGCUCUGCC(L1)GGCAUCGUU (SEQ ID NO: 518). As used herein, (L1) refers to an internal linker having a bridging length of about 15-21 atoms.
In some embodiments, the shortened NmeCas9 guide RNA comprising internal linkers may be chemically modified. Exemplary modifications include a modification pattern of the following sequence: mN*mN*mN*mNmNNNmNmNNmNNmNNNNNmNNNNmNNNmGUUGmUmAmGmC UCCCmUmUmC(L1)mGmAmCmCGUUmGmCUAmCAAU*AAGmGmCCmGmUmC(L1) mGmAmUGUGCmCGmCAAmCGCUCUmGmCC(L1)GGCAUCG*mU*mU (SEQ ID NO: 519).
2. Modifications
In some embodiments, the gRNA (e.g., sgRNA, short-sgRNA, dgRNA, or crRNA) is modified. The term “modified” or “modification” in the context of a gRNA described herein includes, the modifications described above, including, for example, (a) end modifications, e.g., 5′ end modifications or 3′ end modifications, including 5′ or 3′ protective end modifications, (b) nucleobase (or “base”) modifications, including replacement or removal of bases, (c) sugar modifications, including modifications at the 2′, 3′, and/or 4′ positions, (d) internucleoside linkage modifications, and (e) backbone modifications, which can include modification or replacement of the phosphodiester linkages and/or the ribose sugar. A modification of a nucleotide at a given position includes a modification or replacement of the phosphodiester linkage immediately 3′ of the sugar of the nucleotide. Thus, for example, a nucleic acid comprising a phosphorothioate between the first and second sugars from the 5′ end is considered to comprise a modification at position 1. The term “modified gRNA” generally refers to a gRNA having a modification to the chemical structure of one or more of the base, the sugar, and the phosphodiester linkage or backbone portions, including nucleotide phosphates, all as detailed and exemplified herein (see the modification patterns shown in e.g., SEQ ID NOs: 142-145, 181-185 and 191-203).
Further description and exemplary patterns of modifications are provided in in Table 1 of WO2019/237069 published Dec. 12, 2019, the entire contents of which are incorporated herein by reference.
In some embodiments, a gRNA comprises modifications at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more YA sites. In some embodiments, the pyrimidine of the YA site comprises a modification (which includes a modification altering the internucleoside linkage immediately 3′ of the sugar of the pyrimidine). In some embodiments, the adenine of the YA site comprises a modification (which includes a modification altering the internucleoside linkage immediately 3′ of the sugar of the adenine). In some embodiments, the pyrimidine and the adenine of the YA site comprise modifications, such as sugar, base, or internucleoside linkage modifications. The YA modifications can be any of the types of modifications set forth herein. In some embodiments, the YA modifications comprise one or more of phosphorothioate, 2′-OMe, or 2′-fluoro. In some embodiments, the YA modifications comprise pyrimidine modifications comprising one or more of phosphorothioate, 2′-OMe, 2′-H, inosine, or 2′-fluoro. In some embodiments, the YA modification comprises a bicyclic ribose analog (e.g., an LNA, BNA, or ENA) within an RNA duplex region that contains one or more YA sites. In some embodiments, the YA modification comprises a bicyclic ribose analog (e.g., an LNA, BNA, or ENA) within an RNA duplex region that contains a YA site, wherein the YA modification is distal to the YA site.
In some embodiments, the guide sequence (or guide region) of a gRNA comprises 1, 2, 3, 4, 5, or more YA sites (“guide region YA sites”) that may comprise YA modifications. In some embodiments, one or more YA sites located at 5-end, 6-end, 7-end, 8-end, 9-end, or 10-end from the 5′ end of the 5′ terminus (where “5-end”, etc., refers to position 5 to the 3′ end of the guide region, i.e., the most 3′ nucleotide in the guide region) comprise YA modifications. A modified guide region YA site comprises a YA modification.
In some embodiments, a modified guide region YA site is within 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 9 nucleotides of the 3′ terminal nucleotide of the guide region. For example, if a modified guide region YA site is within 10 nucleotides of the 3′ terminal nucleotide of the guide region and the guide region is 20 nucleotides long, then the modified nucleotide of the modified guide region YA site is located at any of positions 11-20. In some embodiments, a modified guide region YA site is at or after nucleotide 4, 5, 6, 7, 8, 9, 10, or 11 from the 5′ end of the 5′ terminus.
In some embodiments, a modified guide region YA site is other than a 5′ end modification. For example, a sgRNA can comprise a 5′ end modification as described herein and further comprise a modified guide region YA site. Alternatively, a sgRNA can comprise an unmodified 5′ end and a modified guide region YA site. Alternatively, a short-sgRNA can comprise a modified 5′ end and an unmodified guide region YA site.
In some embodiments, a modified guide region YA site comprises a modification that at least one nucleotide located 5′ of the guide region YA site does not comprise. For example, if nucleotides 1-3 comprise phosphorothioates, nucleotide 4 comprises only a 2′-OMe modification, and nucleotide 5 is the pyrimidine of a YA site and comprises a phosphorothioate, then the modified guide region YA site comprises a modification (phosphorothioate) that at least one nucleotide located 5′ of the guide region YA site (nucleotide 4) does not comprise. In another example, if nucleotides 1-3 comprise phosphorothioates, and nucleotide 4 is the pyrimidine of a YA site and comprises a 2′-OMe, then the modified guide region YA site comprises a modification (2′-OMe) that at least one nucleotide located 5′ of the guide region YA site (any of nucleotides 1-3) does not comprise. This condition is also always satisfied if an unmodified nucleotide is located 5′ of the modified guide region YA site.
In some embodiments, the modified guide region YA sites comprise modifications as described for YA sites above. The guide region of a gRNA may be modified according to any embodiment comprising a modified guide region set forth herein.
Conserved region YA sites 1-10 are illustrated in
In some embodiments, the modified conserved region YA sites comprise modifications as described for YA sites above. Any embodiments set forth elsewhere in this disclosure may be combined to the extent feasible with any of the foregoing embodiments.
In some embodiments, the 5′ and/or 3′ terminus regions of a gRNA are modified.
In some embodiments, the terminal (i.e., last) 1, 2, 3, 4, 5, 6, or 7 nucleotides in the 3′ terminus region are modified. Throughout, this modification may be referred to as a “3′ end modification”. In some embodiments, the terminal (i.e., last) 1, 2, 3, 4, 5, 6, or 7 nucleotides in the 3′ terminus region comprise more than one modification. In some embodiments, the 3′ end modification comprises or further comprises any one or more of the following: a modified nucleotide selected from 2′-O-methyl (2′-O-Me) modified nucleotide, 2′-O-(2-methoxyethyl) (2′-O-moe) modified nucleotide, a 2′-fluoro (2′-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, an inverted abasic modified nucleotide, or combinations thereof. In some embodiments, the 3′ end modification comprises or further comprises modifications of 1, 2, 3, 4, 5, 6, or 7 nucleotides at the 3′ end of the gRNA. In some embodiments, the 3′ end modification comprises or further comprises one PS linkage, wherein the linkage is between the last and second to last nucleotide. In some embodiments, the 3′ end modification comprises or further comprises two PS linkages between the last three nucleotides. In some embodiments, the 3′ end modification comprises or further comprises four PS linkages between the last four nucleotides. In some embodiments, the 3′ end modification comprises or further comprises PS linkages between any one or more of the last 2, 3, 4, 5, 6, or 7 nucleotides. In some embodiments, the gRNA comprising a 3′ end modification comprises or further comprises a 3′ tail, wherein the 3′ tail comprises a modification of any one or more of the nucleotides present in the 3′ tail. In some embodiments, the 3′ tail is fully modified. In some embodiments, the 3′ tail comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, or 1-10 nucleotides, optionally where any one or more of these nucleotides are modified. In some embodiments, a gRNA is provided comprising a 3′ end modification, wherein the 3′ end modification comprises the 3′ end modification as shown in any one of SEQ ID Nos: 141-145. In some embodiments, a gRNA is provided comprising a 3′ protective end modification. In some embodiments, the 3′ tail comprises between 1 and about 20 nucleotides, between 1 and about 15 nucleotides, between 1 and about 10 nucleotides, between 1 and about 5 nucleotides, between 1 and about 4 nucleotides, between 1 and about 3 nucleotides, and between 1 and about 2 nucleotides. In some embodiments, the gRNA does not comprise a 3′ tail.
In some embodiments, the 5′ terminus region is modified, for example, the first 1, 2, 3, 4, 5, 6, or 7 nucleotides of the gRNA are modified. Throughout, this modification may be referred to as a “5′ end modification”. In some embodiments, the first 1, 2, 3, 4, 5, 6, or 7 nucleotides of the 5′ terminus region comprise more than one modification. In some embodiments, at least one of the terminal (i.e., first) 1, 2, 3, 4, 5, 6, or 7 nucleotides at the 5′ end are modified. In some embodiments, both the 5′ and 3′ terminus regions (e.g., ends) of the gRNA are modified. In some embodiments, only the 5′ terminus region of the gRNA is modified. In some embodiments, only the 3′ terminus region (plus or minus a 3′ tail) of the conserved portion of a gRNA is modified. In some embodiments, the gRNA comprises modifications at 1, 2, 3, 4, 5, 6, or 7 of the first 7 nucleotides at a 5′ terminus region of the gRNA. In some embodiments, the gRNA comprises modifications at 1, 2, 3, 4, 5, 6, or 7 of the 7 terminal nucleotides at a 3′ terminus region. In some embodiments, 2, 3, or 4 of the first 4 nucleotides at the 5′ terminus region, and/or 2, 3, or 4 of the terminal 4 nucleotides at the 3′ terminus region are modified. In some embodiments, 2, 3, or 4 of the first 4 nucleotides at the 5′ terminus region are linked with phosphorothioate (PS) bonds. In some embodiments, the modification to the 5′ terminus and/or 3′ terminus comprises a 2′-O-methyl (2′-O-Me) or 2′-O-(2-methoxyethyl) (2′-O-moe) modification. In some embodiments, the modification comprises a 2′-fluoro (2′-F) modification to a nucleotide. In some embodiments, the modification comprises a phosphorothioate (PS) linkage between nucleotides. In some embodiments, the modification comprises an inverted abasic nucleotide. In some embodiments, the modification comprises a protective end modification. In some embodiments, the modification comprises a more than one modification selected from protective end modification, 2′-O-Me, 2′-O-moe, 2′-fluoro (2′-F), a phosphorothioate (PS) linkage between nucleotides, and an inverted abasic nucleotide. In some embodiments, an equivalent modification is encompassed. In some embodiments, a gRNA is provided comprising a 5′ end modification, wherein the 5′ end modification comprises a 5′ end modification as shown in any one of SEQ ID Nos: 141-145.
In some embodiments, a gRNA is provided comprising a 5′ end modification and a 3′ end modification. In some embodiments, the gRNA comprises modified nucleotides that are not at the 5′ or 3′ ends.
In some embodiments, a sgRNA is provided comprising an upper stem modification, wherein the upper stem modification comprises a modification to any one or more of US1-US12 in the upper stem region. In some embodiments, a sgRNA is provided comprising an upper stem modification, wherein the upper stem modification comprises a modification of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all 12 nucleotides in the upper stem region. In some embodiments, an sgRNA is provided comprising an upper stem modification, wherein the upper stem modification comprises 1, 2, 3, 4, or 5 YA modifications in a YA site. In some embodiments, the upper stem modification comprises a 2′-OMe modified nucleotide, a 2′-O-moe modified nucleotide, a 2′-F modified nucleotide, and/or combinations thereof. Other modifications described herein, such as a 5′ end modification and/or a 3′ end modification may be combined with an upper stem modification.
In some embodiments, the sgRNA comprises a modification in the hairpin region. In some embodiments, the hairpin region modification comprises at least one modified nucleotide selected from a 2′-O-methyl (2′-OMe) modified nucleotide, a 2′-fluoro (2′-F) modified nucleotide, and/or combinations thereof. In some embodiments, the hairpin region modification is in the hairpin 1 region. In some embodiments, the hairpin region modification is in the hairpin 2 region. In some embodiments, the hairpin modification comprises 1, 2, or 3 YA modifications in a YA site. In some embodiments, the hairpin modification comprises at least 1, 2, 3, 4, 5, or 6 YA modifications. Other modifications described herein, such as an upper stem modification, a 5′ end modification, and/or a 3′ end modification may be combined with a modification in the hairpin region.
In some embodiments, a gRNA comprises a substituted and optionally shortened hairpin 1 region, wherein at least one of the following pairs of nucleotides are substituted in the substituted and optionally shortened hairpin 1 with Watson-Crick pairing nucleotides: H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, and/or H1-4 and H1-9. “Watson-Crick pairing nucleotides” include any pair capable of forming a Watson-Crick base pair, including A-T, A-U, T-A, U-A, C-G, and G-C pairs, and pairs including modified versions of any of the foregoing nucleotides that have the same base pairing preference. In some embodiments, the hairpin 1 region lacks any one or two of H1-5 through H1-8. In some embodiments, the hairpin 1 region lacks one, two, or three of the following pairs of nucleotides: H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10 and/or H1-4 and H1-9. In some embodiments, the hairpin 1 region lacks 1-8 nucleotides of the hairpin 1 region. In any of the foregoing embodiments, the lacking nucleotides may be such that the one or more nucleotide pairs substituted with Watson-Crick pairing nucleotides (H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, and/or H1-4 and H1-9) form a base pair in the gRNA.
In some embodiments, the gRNA further comprises an upper stem region lacking at least 1 nucleotide, e.g., any of the shortened upper stem regions indicated in Table 7 of U.S. Application No. 62/946,905, the contents of which are hereby incorporated by reference in its entirety, or described elsewhere herein, which may be combined with any of the shortened or substituted hairpin 1 regions described herein.
In some embodiments, the gRNA described herein further comprises a nexus region, wherein the nexus region lacks at least one nucleotide.
3. Chemical Modifications of gRNAs
In some embodiments, the gRNA is chemically modified. A gRNA comprising one or more modified nucleosides or nucleotides is called a “modified” gRNA or “chemically modified” gRNA, to describe the presence of one or more non-naturally and/or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues. Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) wholesale replacement of the phosphate moiety with “dephospho” linkers (an exemplary backbone modification); (iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); (v) replacement or modification of the ribose-phosphate backbone (an exemplary backbone modification); (vi) modification of the 3′ end or 5′ end of the oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, cap or linker (such 3′ or 5′ cap modifications may comprise a sugar and/or backbone modification); and (vii) modification or replacement of the sugar (an exemplary sugar modification).
Chemical modifications such as those listed above can be combined to provide modified gRNAs comprising nucleosides and nucleotides (collectively “residues”) that can have two, three, four, or more modifications. For example, a modified residue can have a modified sugar and a modified nucleobase. In some embodiments, every base of a gRNA is modified, e.g., all bases have a modified phosphate group, such as a phosphorothioate group. In certain embodiments, all, or substantially all, of the phosphate groups of an gRNA molecule are replaced with phosphorothioate groups. In some embodiments, modified gRNAs comprise at least one modified residue at or near the 5′ end of the RNA. In some embodiments, modified gRNAs comprise at least one modified residue at or near the 3′ end of the RNA.
In some embodiments, the gRNA comprises one, two, three or more modified residues. In some embodiments, at least 5% (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the positions in a modified gRNA are modified nucleosides or nucleotides.
In some embodiments of a backbone modification, the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified residue, e.g., modified residue present in a modified nucleic acid, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate group as described herein. In some embodiments, the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.
Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. Such modifications may comprise backbone and sugar modifications. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.
The modified nucleosides and modified nucleotides can include one or more modifications to the sugar group, i.e. at sugar modification. For example, the 2′ hydroxyl group (OH) can be modified, e.g. replaced with a number of different “oxy” or “deoxy” substituents. In some embodiments, modifications to the 2′ hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2′-alkoxide ion. Examples of 2′ hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20. In some embodiments, the 2′ hydroxyl group modification can be 2′-O-Me. In some embodiments, the 2′ hydroxyl group modification can be a 2′-fluoro modification, which replaces the 2′ hydroxyl group with a fluoride. In some embodiments, the 2′ hydroxyl group modification can include “locked” nucleic acids (LNA) in which the 2′ hydroxyl can be connected, e.g., by a C1-6 alkylene or C1-6 heteroalkylene bridge, to the 4′ carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges. In some embodiments, the 2′ hydroxyl group modification can included “unlocked” nucleic acids (UNA) in which the ribose ring lacks the C2′-C3′ bond. In some embodiments, the 2′ hydroxyl group modification can include the methoxyethyl group (MOE), (OCH2CH2OCH3, e.g., a PEG derivative).
“Deoxy” 2′ modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially dsRNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH)nCH2CH2— amino (wherein amino can be, e.g., as described herein), —NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino as described herein.
The sugar modification can comprise a sugar group which may also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The modified nucleic acids can also include abasic sugars. These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g. L-nucleosides.
The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified base, also called a nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or wholly replaced to provide modified residues that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine analog, or pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base.
In embodiments employing a dual guide RNA, each of the crRNA and the tracr RNA can contain modifications. Such modifications may be at one or both ends of the crRNA and/or tracr RNA. In embodiments comprising an sgRNA, one or more residues at one or both ends of the sgRNA may be chemically modified, or the entire sgRNA may be chemically modified. Certain embodiments comprise a 5′ end modification. Certain embodiments comprise a 3′ end modification. In certain embodiments, one or more or all of the nucleotides in single stranded overhang of a gRNA molecule are deoxynucleotides.
In some embodiments, the gRNAs disclosed herein comprise one of the modification patterns disclosed in WO2018/107028 A1, published Jun. 14, 2018 the contents of which are hereby incorporated by reference in their entirety.
The terms “mA,” “mC,” “mU,” or “mG” may be used to denote a nucleotide that has been modified with 2′-O-Me. The terms “fA,” “fC,” “fU,” or “fU” may be used to denote a nucleotide that has been substituted with 2′-F. A “*” may be used to depict a PS modification. The terms A*, C*, U*, or G* may be used to denote a nucleotide that is linked to the next (e.g., 3′) nucleotide with a PS bond. The terms “mA*,” “mC*,” “mU*,” or “mG*” may be used to denote a nucleotide that has been substituted with 2′-O-Me and that is linked to the next (e.g., 3′) nucleotide with a PS bond.
H. Lipids; Formulation; Delivery
Disclosed herein are various embodiments using lipid nucleic acid assembly compositions comprising nucleic acids(s), or composition(s) described herein. In some embodiments, the lipid nucleic acid assembly composition comprises a nucleic acid (e.g., mRNA) comprising an open reading frame encoding a first polypeptide comprising a cytidine deaminase (e.g., A3A) and an RNA-guided nickase. In some embodiments, the lipid nucleic acid assembly composition comprises a first nucleic acid comprising an open reading frame encoding a first polypeptide comprising a cytidine deaminase (e.g., A3A) and an RNA-guided nickase and a second nucleic acid encoding a UGI.
As used herein, a “lipid nucleic acid assembly composition” refers to lipid-based delivery compositions, including lipid nanoparticles (LNPs) and lipoplexes. LNP refers to lipid nanoparticles <100 nM. LNPs are formed by precise mixing a lipid component (e.g., in ethanol) with an aqueous nucleic acid component and LNPs are uniform in size. Lipoplexes are particles formed by bulk mixing the lipid and nucleic acid components and are between about 100 nm and 1 micron in size. In certain embodiments the lipid nucleic acid assemblies are LNPs. As used herein, a “lipid nucleic acid assembly” comprises a plurality of (i.e. more than one) lipid molecules physically associated with each other by intermolecular forces. A lipid nucleic acid assembly may comprise a bioavailable lipid having a pKa value of <7.5 or <7. The lipid nucleic acid assemblies are formed by mixing an aqueous nucleic acid-containing solution with an organic solvent-based lipid solution, e.g., 100% ethanol. Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, ethanol, chloroform, diethylether, cyclohexane, tetrahydrofuran, methanol, isopropanol. A pharmaceutically acceptable buffer may optionally be comprised in a pharmaceutical formulation comprising the lipid nucleic acid assemblies, e.g., for an ex vivo therapy. In some embodiments, the aqueous solution comprises an RNA, such as an mRNA or a gRNA. In some embodiments, the aqueous solution comprises an mRNA encoding an RNA-guided DNA binding agent, such as Cas9.
As used herein, lipid nanoparticle (LNP) refers to a particle that comprises a plurality of (i.e., more than one) lipid molecules physically associated with each other by intermolecular forces. The LNPs may be, e.g., microspheres (including unilamellar and multilamellar vesicles, e.g., “liposomes”—lamellar phase lipid bilayers that, in some embodiments, are substantially spherical—and, in more particular embodiments, can comprise an aqueous core, e.g., comprising a substantial portion of RNA molecules), a dispersed phase in an emulsion, micelles, or an internal phase in a suspension. Emulsions, micelles, and suspensions may be suitable compositions for local and/or topical delivery. See also, e.g., WO2017173054A1, the contents of which are hereby incorporated by reference in their entirety. Any LNP known to those of skill in the art to be capable of delivering nucleotides to subjects may be utilized with the guide RNAs and the nucleic acid encoding an RNA-guided nickase and the nucleic acid encoding a cytidine deaminase described herein.
In some embodiments, the aqueous solution comprises a nucleic acid encoding a polypeptide comprising an A3A and an RNA-guided nickase. A pharmaceutical formulation comprising the lipid nucleic acid assembly composition may optionally comprise a pharmaceutically acceptable buffer.
In some embodiments, the lipid nucleic acid assembly compositions include an “amine lipid” (sometimes herein or elsewhere described as an “ionizable lipid” or a “biodegradable lipid”), together with an optional “helper lipid”, a “neutral lipid”, and a stealth lipid such as a PEG lipid. In some embodiments, the amine lipids or ionizable lipids are cationic depending on the pH.
1. Amine Lipids
In some embodiments, lipid nucleic acid assembly compositions comprise an “amine lipid”, which is, for example an ionizable lipid such as Lipid A or its equivalents, including acetal analogs of Lipid A.
In some embodiments, the amine lipid is Lipid A, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate. Lipid A can be depicted as:
Lipid A may be synthesized according to WO2015/095340 (e.g., pp. 84-86). In some embodiments, the amine lipid is an equivalent to Lipid A.
In some embodiments, an amine lipid is an analog of Lipid A. In some embodiments, a Lipid A analog is an acetal analog of Lipid A. In particular lipid nucleic acid assembly compositions, the acetal analog is a C4-C12 acetal analog. In some embodiments, the acetal analog is a C5-C12 acetal analog. In additional embodiments, the acetal analog is a C5-C10 acetal analog. In further embodiments, the acetal analog is chosen from a C4, C5, C6, C7, C9, C10, C11, and C12 acetal analog.
Amine lipids and other “biodegradable lipids” suitable for use in the lipid nucleic acid assemblies described herein are biodegradable in vivo or ex vivo. The amine lipids have low toxicity (e.g., are tolerated in animal models without adverse effect in amounts of greater than or equal to 10 mg/kg). In some embodiments, lipid nucleic acid assemblies comprising an amine lipid include those where at least 75% of the amine lipid is cleared from the plasma or the engineered cell within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days. In some embodiments, lipid nucleic acid assemblies comprising an amine lipid include those where at least 50% of the nucleic acid, e.g., mRNA or gRNA, is cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days. In some embodiments, lipid nucleic acid assemblies comprising an amine lipid include those where at least 50% of the lipid nucleic acid assembly is cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days, for example by measuring a lipid (e.g. an amine lipid), nucleic acid, e.g., RNA/mRNA, or other component. In some embodiments, lipid-encapsulated versus free lipid, RNA, or nucleic acid component of the lipid nucleic acid assembly is measured.
Biodegradable lipids include, for example the biodegradable lipids of WO/2020/219876, WO/2020/118041, WO/2020/072605, WO/2019/067992, WO/2017/173054, WO2015/095340, and WO2014/136086, and LNPs include LNP compositions described therein, the lipids and compositions of which are hereby incorporated by reference.
Lipid clearance may be measured as described in literature. See Maier, M. A., et al. Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Ther. 2013, 21(8), 1570-78 (“Maier”). For example, in Maier, LNP-siRNA systems containing luciferases-targeting siRNA were administered to six- to eight-week old male C57Bl/6 mice at 0.3 mg/kg by intravenous bolus injection via the lateral tail vein. Blood, liver, and spleen samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, and 168 hours post-dose. Mice were perfused with saline before tissue collection and blood samples were processed to obtain plasma. All samples were processed and analyzed by LC-MS. Further, Maier describes a procedure for assessing toxicity after administration of LNP-siRNA formulations. For example, a luciferase-targeting siRNA was administered at 0, 1, 3, 5, and 10 mg/kg (5 animals/group) via single intravenous bolus injection at a dose volume of 5 mL/kg to male Sprague-Dawley rats. After 24 hours, about 1 mL of blood was obtained from the jugular vein of conscious animals and the serum was isolated. At 72 hours post-dose, all animals were euthanized for necropsy. Assessments of clinical signs, body weight, serum chemistry, organ weights and histopathology were performed. Although Maier describes methods for assessing siRNA-LNP formulations, these methods may be applied to assess clearance, pharmacokinetics, and toxicity of administration of lipid nucleic acid assembly compositions of the present disclosure.
Ionizable and bioavailable lipids for LNP delivery of nucleic acids known in the art are suitable. Lipids may be ionizable depending upon the pH of the medium they are in. For example, in a slightly acidic medium, the lipid, such as an amine lipid, may be protonated and thus bear a positive charge. Conversely, in a slightly basic medium, such as, for example, blood where pH is approximately 7.35, the lipid, such as an amine lipid, may not be protonated and thus bear no charge.
The ability of a lipid to bear a charge is related to its intrinsic pKa. In some embodiments, the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.1 to about 7.4. In some embodiments, the bioavailable lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.1 to about 7.4, such as from about 5.5 to about 6.6, from about 5.6 to about 6.4, from about 5.8 to about 6.2, or from about 5.8 to about 6.5. For example, the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.8 to about 6.5. Lipids with a pKa ranging from about 5.1 to about 7.4 are effective for delivery of cargo in vivo, e.g. to the liver. Further, it has been found that lipids with a pKa ranging from about 5.3 to about 6.4 are effective for delivery in vivo, e.g. to tumors. See, e.g., WO2014/136086.
2. Additional Lipids
“Neutral lipids” suitable for use in a lipid nucleic acid assembly composition of the disclosure include, for example, a variety of neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), pohsphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In one embodiment, the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoyl phosphatidyl ethanolamine (DMPE). In another embodiment, the neutral phospholipid may be distearoylphosphatidylcholine (DSPC).
“Helper lipids” include steroids, sterols, and alkyl resorcinols. Helper lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate. In one embodiment, the helper lipid may be cholesterol. In one embodiment, the helper lipid may be cholesterol hemisuccinate.
“Stealth lipids” are lipids that alter the length of time the nanoparticles can exist in vivo (e.g., in the blood). Stealth lipids may assist in the formulation process by, for example, reducing particle aggregation and controlling particle size. Stealth lipids used herein may modulate pharmacokinetic properties of the lipid nucleic acid assembly or aid in stability of the nanoparticle ex vivo. Stealth lipids suitable for use in a lipid nucleic acid assembly composition of the disclosure include, but are not limited to, stealth lipids having a hydrophilic head group linked to a lipid moiety. Stealth lipids suitable for use in a lipid nucleic acid assembly composition of the present disclosure and information about the biochemistry of such lipids can be found in Romberg et al., Pharmaceutical Research, Vol. 25, No. 1, 2008, pg. 55-71 and Hoekstra et al., Biochimica et Biophysica Acta 1660 (2004) 41-52. Additional suitable PEG lipids are disclosed, e.g., in WO 2006/007712.
In one embodiment, the hydrophilic head group of stealth lipid comprises a polymer moiety selected from polymers based on PEG. Stealth lipids may comprise a lipid moiety. In some embodiments, the stealth lipid is a PEG lipid.
In one embodiment, a stealth lipid comprises a polymer moiety selected from polymers based on PEG (sometimes referred to as poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyaminoacids and poly[N-(2-hydroxypropyl)methacrylamide].
In one embodiment, the PEG lipid comprises a polymer moiety based on PEG (sometimes referred to as poly(ethylene oxide)).
The PEG lipid further comprises a lipid moiety. In some embodiments, the lipid moiety may be derived from diacylglycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having alkyl chain length independently comprising from about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups such as, for example, an amide or ester. In some embodiments, the alkyl chain length comprises about C10 to C20. The dialkylglycerol or dialkylglycamide group can further comprise one or more substituted alkyl groups. The chain lengths may be symmetrical or asymmetrical.
Unless otherwise indicated, the term “PEG” as used herein means any polyethylene glycol or other polyalkylene ether polymer. In one embodiment, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In one embodiment, PEG is unsubstituted. In one embodiment, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In one embodiment, the term includes PEG copolymers such as PEG-polyurethane or PEG-polypropylene (see, e.g., J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)); in another embodiment, the term does not include PEG copolymers. In one embodiment, the PEG has a molecular weight of from about 130 to about 50,000, in a sub-embodiment, about 150 to about 30,000, in a sub-embodiment, about 150 to about 20,000, in a sub-embodiment about 150 to about 15,000, in a sub-embodiment, about 150 to about 10,000, in a sub-embodiment, about 150 to about 6,000, in a sub-embodiment, about 150 to about 5,000, in a sub-embodiment, about 150 to about 4,000, in a sub-embodiment, about 150 to about 3,000, in a sub-embodiment, about 300 to about 3,000, in a sub-embodiment, about 1,000 to about 3,000, and in a sub-embodiment, about 1,500 to about 2,500.
In some embodiments, the PEG (e.g., conjugated to a lipid moiety or lipid, such as a stealth lipid), is a “PEG-2K,” also termed “PEG 2000,” which has an average molecular weight of about 2,000 daltons. PEG-2K is represented herein by the following formula (I), wherein n is 45, meaning that the number averaged degree of polymerization comprises about 45 subunits
However, other PEG embodiments known in the art may be used, including, e.g., those where the number-averaged degree of polymerization comprises about 23 subunits (n=23), and/or 68 subunits (n=68). In some embodiments, n may range from about 30 to about 60. In some embodiments, n may range from about 35 to about 55. In some embodiments, n may range from about 40 to about 50. In some embodiments, n may range from about 42 to about 48. In some embodiments, n may be 45. In some embodiments, R may be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R may be unsubstituted alkyl. In some embodiments, R may be methyl.
In any of the embodiments described herein, the PEG lipid may be selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG) (catalog #GM-020 from NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE) (catalog #DSPE-020CN, NOF, Tokyo, Japan), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, and PEG-distearoylglycamide, PEG-cholesterol (1-[8′-(Cholest-5-en-3[beta]-oxy)carboxamido-3′,6′-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol)ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG) (cat. #880150P from Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (cat. #880120C from Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2k-DSG; GS-020, NOF Tokyo, Japan), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), and 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In one embodiment, the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid may be PEG2k-DSG. In one embodiment, the PEG lipid may be PEG2k-DSPE. In one embodiment, the PEG lipid may be PEG2k-DMA. In one embodiment, the PEG lipid may be PEG2k-C-DMA. In one embodiment, the PEG lipid may be compound S027, disclosed in WO2016/010840 (paragraphs [00240] to [00244]). In one embodiment, the PEG lipid may be PEG2k-DSA. In one embodiment, the PEG lipid may be PEG2k-C11. In some embodiments, the PEG lipid may be PEG2k-C14. In some embodiments, the PEG lipid may be PEG2k-C16. In some embodiments, the PEG lipid may be PEG2k-C18.
3. Formulations
The lipid nucleic acid assembly may contain (i) a biodegradable lipid, (ii) an optional neutral lipid, (iii) a helper lipid, and (iv) a stealth lipid, such as a PEG lipid. The lipid nucleic acid assembly may contain a biodegradable lipid and one or more of a neutral lipid, a helper lipid, and a stealth lipid, such as a PEG lipid.
The lipid nucleic acid assembly may contain (i) an amine lipid for encapsulation and for endosomal escape, (ii) a neutral lipid for stabilization, (iii) a helper lipid, also for stabilization, and (iv) a stealth lipid, such as a PEG lipid. The lipid nucleic acid assembly may contain an amine lipid and one or more of a neutral lipid, a helper lipid, also for stabilization, and a stealth lipid, such as a PEG lipid.
The mRNAs required to achieve the described functional effects described herein may be delivered to a cell in one or more lipid nucleic acid assembly composition(s). For example, one lipid nucleic acid assembly composition may be formulated for delivery comprising mRNA encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase and additional mRNAs encoding, for example, one or more UGIs and one or more gRNAs. Alternatively, the mRNA encoding the polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase, mRNA encoding one or more UGI, and mRNA encoding one or more gRNAs may be formulated in separate lipid nucleic acid assembly compositions. As such, one or multiple lipid nucleic acid assembly composition(s) may be delivered to a cell in vitro or in vivo.
In some embodiments, a method of modifying a target gene in a cell is provided, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising:
In some embodiments, parts (a) and (b) are in separate lipid nucleic acid assembly compositions. In some embodiments, parts (a) and (b) are in the same lipid nucleic acid assembly composition. In some embodiments, parts (a) and (c) are in separate lipid nucleic acid assembly compositions. In some embodiments, parts (a) and (c) are in the same lipid nucleic acid assembly composition. In some embodiments, parts (b) and (c) are in separate lipid nucleic acid assembly compositions. In some embodiments, parts (a) and (c) are in the same lipid nucleic acid assembly composition, and part (b) is in a separate lipid nucleic acid assembly composition. In some embodiments, parts (a), (b), and (c) are each in separate lipid nucleic acid assembly compositions. In some embodiments, parts (a), (b), and (c) are in the same lipid nucleic acid assembly composition. In some embodiments, the one or more guide RNAs are each in separate lipid nucleic acid assembly compositions.
In some embodiments, the method further comprise delivering one or more guide RNAs in one or more lipid nucleic acid assembly compositions that are separate from the lipid nucleic acid assembly compositions comprising the A3A and UGI.
In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 lipid nucleic acid assembly compositions are delivered to the cell. In some embodiments, the at least one lipid nucleic acid assembly composition comprises lipid nanoparticle (LNPs). In some embodiments, all lipid nucleic acid assembly compositions comprise LNPs. In some embodiments, at least one lipid nucleic acid assembly composition is a lipoplex composition.
In some embodiments, the lipid nucleic acid assembly composition, e.g., LNP composition, comprises an mRNA that encodes a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase, as described herein. In some embodiments, the lipid nucleic acid assembly composition, e.g. LNP composition, comprises an mRNA that encodes a polypeptide comprising an APOBEC3A deaminase (A3A) and an RNA-guided nickase; and a gRNA.
In some embodiments, the lipid nucleic acid assembly composition comprises a first lipid nucleic acid assembly composition comprising an mRNA encoding a first polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase. In some embodiments, the lipid nucleic acid assembly composition further comprises a second lipid nucleic acid assembly composition comprising a gRNA.
In some embodiments, the lipid nucleic acid assembly composition comprises a first composition comprising a mRNA encoding a first polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; and one or more second mRNA encoding a uracil glycosylase inhibitor (UGI). In some embodiments, the lipid nucleic acid assembly composition further comprises one or more gRNA.
In some embodiments, the lipid nucleic acid assembly composition further comprises a second lipid nucleic acid assembly composition comprising a gRNA. In some embodiments, the lipid nucleic acid assembly composition comprises first and second lipid nucleic acid assembly compositions, wherein the first composition comprises an mRNA encoding polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; and the second composition comprises one or more mRNA encoding a uracil glycosylase inhibitor (UGI). In some embodiments, the first lipid nucleic acid assembly composition or the second lipid nucleic acid assembly composition further comprises one or more gRNA. In some embodiments, the lipid nucleic acid assembly composition further comprises a third lipid nucleic acid assembly composition comprising one or more gRNA.
In some embodiments, the lipid nucleic acid assembly composition comprises a first lipid nucleic acid assembly composition comprising an mRNA comprising an open reading frame encoding a first polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase and a second lipid nucleic acid assembly composition comprising one or more guide RNA (gRNA).
In some embodiments, the lipid nucleic acid assembly composition comprises a first composition comprising a first mRNA comprising a first open reading frame encoding a first polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; and a second mRNA comprising one or more second open reading frame encoding a uracil glycosylase inhibitor (UGI). In some embodiments, the lipid nucleic acid assembly composition further comprises one or more gRNA. In some embodiments, the lipid nucleic acid assembly composition further comprises a second lipid nucleic acid assembly composition comprising one or more gRNA.
In some embodiments, the lipid nucleic acid assembly composition comprises first and second lipid nucleic acid assembly compositions, wherein the first composition comprises a first mRNA comprising a first open reading frame encoding a first polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; the second composition comprises a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI). In some embodiments, the first lipid nucleic acid assembly composition or the second lipid nucleic acid assembly composition further comprises a gRNA. In some embodiments, the lipid nucleic acid assembly composition further comprises a third lipid nucleic acid assembly composition comprising a gRNA.
In some embodiments, the lipid nucleic acid assembly composition comprises a first composition comprising a first mRNA comprising a first open reading frame encoding a first polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; and the second composition comprises a uracil glycosylase inhibitor (UGI). In some embodiments, the first lipid nucleic acid assembly composition or the second lipid nucleic acid assembly composition further comprises a gRNA. In some embodiments, the lipid nucleic acid assembly composition further comprises a third lipid nucleic acid assembly composition comprising a gRNA.
In some embodiments, the lipid nucleic acid assembly composition comprises first and second lipid nucleic acid assembly compositions, wherein the first composition comprises a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; the second composition comprises a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI). In some embodiments, the first lipid nucleic acid assembly composition or the second lipid nucleic acid assembly composition further comprises a gRNA. In some embodiments, the lipid nucleic acid assembly composition further comprises a third lipid nucleic acid assembly composition comprising a gRNA.
In certain embodiments, a lipid nucleic acid assembly composition may comprise mRNA, optionally a gRNA, an amine lipid, a helper lipid, a neutral lipid, and a stealth lipid. In certain lipid nucleic acid assembly compositions, the helper lipid is cholesterol. In other lipid nucleic acid assembly compositions, the neutral lipid is DSPC. In additional embodiments, the stealth lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the lipid nucleic acid assembly composition comprises Lipid A or an equivalent of Lipid A; a helper lipid; a neutral lipid; a stealth lipid. In certain compositions, the amine lipid is Lipid A. In certain compositions, the amine lipid is Lipid A or an acetal analog thereof; the helper lipid is cholesterol; the neutral lipid is DSPC; and the stealth lipid is PEG2k-DMG.
Embodiments of the present disclosure also provide lipid nucleic acid assembly compositions described according to the molar ratio between the positively charged amine groups of the amine lipid (N) and the negatively charged phosphate groups (P) of the nucleic acid to be encapsulated. This may be mathematically represented by the equation N/P. In some embodiments, a lipid nucleic acid assembly composition may comprise a lipid component that comprises an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid; and a nucleic acid component, wherein the N/P ratio is about 3 to 10. In some embodiments, a lipid nucleic acid assembly composition may comprise a lipid component that comprises an amine lipid, a helper lipid, and a PEG lipid; and a nucleic acid component, wherein the N/P ratio is about 3 to 10. In some embodiments, a lipid nucleic acid assembly composition may comprise a lipid component that comprises an amine lipid, a helper lipid, a neutral lipid, and a helper lipid; and an RNA component, wherein the N/P ratio is about 3 to 10. In some embodiments, a lipid nucleic acid assembly composition may comprise a lipid component that comprises an amine lipid, a helper lipid, and a PEG lipid; and an RNA component, such as an mRNA or gRNA, wherein the N/P ratio is about 3 to 10. In one embodiment, the N/P ratio may be about 5 to 7. In one embodiment, the N/P ratio may be about 3 to 7. In one embodiment, the N/P ratio may be about 4.5 to 8. In one embodiment, the N/P ratio may be about 6. In one embodiment, the N/P ratio may be 6±1. In one embodiment, the N/P ratio may be 6±0.5. In some embodiments, the N/P ratio will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target N/P ratio. In certain embodiments, LNP inter-lot variability will be less than 15%, less than 10% or less than 5%.
In some embodiments, lipid nucleic acid assembly compositions are formed by mixing an aqueous RNA solution with an organic solvent-based lipid solution, e.g., 100% ethanol. Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, ethanol, chloroform, diethylether, cyclohexane, tetrahydrofuran, methanol, isopropanol. A pharmaceutically acceptable buffer, e.g., for in vivo administration of lipid nucleic acid assembly compositions, may be used. In certain embodiments, a buffer is used to maintain the pH of the composition comprising lipid nucleic acid assembly compositions at or above pH 6.5. In certain embodiments, a buffer is used to maintain the pH of the composition comprising LNPs at or above pH 7.0. In certain embodiments, the composition has a pH ranging from about 7.2 to about 7.7. In additional embodiments, the composition has a pH ranging from about 7.3 to about 7.7 or ranging from about 7.4 to about 7.6. In further embodiments, the composition has a pH of about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7. The pH of a composition may be measured with a micro pH probe. In certain embodiments, a cryoprotectant is included in the composition. Non-limiting examples of cryoprotectants include sucrose, trehalose, glycerol, DMSO, and ethylene glycol. Exemplary compositions may include up to 10% cryoprotectant, such as, for example, sucrose. In certain embodiments, the lipid nucleic acid assembly composition may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% cryoprotectant. In certain embodiments, the lipid nucleic acid assembly composition may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% sucrose. In some embodiments, the lipid nucleic acid assembly composition may include a buffer. In some embodiments, the buffer may comprise a phosphate buffer (PBS), a Tris buffer, a citrate buffer, and mixtures thereof. In certain exemplary embodiments, the buffer comprises NaCl. In certain embodiments, NaCl is omitted. Exemplary amounts of NaCl may range from about 20 mM to about 45 mM. Exemplary amounts of NaCl may range from about 40 mM to about 50 mM. In some embodiments, the amount of NaCl is about 45 mM. In some embodiments, the buffer is a Tris buffer. Exemplary amounts of Tris may range from about 20 mM to about 60 mM. Exemplary amounts of Tris may range from about 40 mM to about 60 mM. In some embodiments, the amount of Tris is about 50 mM. In some embodiments, the buffer comprises NaCl and Tris. Certain exemplary embodiments of the lipid nucleic acid assembly compositions contain 5% sucrose and 45 mM NaCl in Tris buffer. In other exemplary embodiments, compositions contain sucrose in an amount of about 5% w/v, about 45 mM NaCl, and about 50 mM Tris at pH 7.5. The salt, buffer, and cryoprotectant amounts may be varied such that the osmolality of the overall formulation is maintained. For example, the final osmolality may be maintained at less than 450 mOsm/L. In further embodiments, the osmolality is between 350 and 250 mOsm/L. Certain embodiments have a final osmolality of 300+/−20 mOsm/L.
In some embodiments, microfluidic mixing, T-mixing, or cross-mixing is used. In certain aspects, flow rates, junction size, junction geometry, junction shape, tube diameter, solutions, and/or nucleic acids and lipid concentrations may be varied. Lipid nucleic acid assembly compositions may be concentrated or purified, e.g., via dialysis, tangential flow filtration, or chromatography. The lipid nucleic acid assembly compositions may be stored as a suspension, an emulsion, or a lyophilized powder, for example. In some embodiments, an lipid nucleic acid assembly composition is stored at 2-8° C., in certain aspects, the LNP compositions are stored at room temperature. In additional embodiments, a lipid nucleic acid assembly composition is stored frozen, for example at −20° C. or −80° C. In other embodiments, a lipid nucleic acid assembly composition is stored at a temperature ranging from about 0° C. to about −80° C. Frozen lipid nucleic acid assembly compositions may be thawed before use, for example on ice, at room temperature, or at 25° C.
The lipid nucleic acid assembly compositions may be, e.g., microspheres, a dispersed phase in an emulsion, micelles, or an internal phase in a suspension.
Moreover, in some embodiments, the lipid nucleic acid assembly compositions are biodegradable, in that they do not accumulate to cytotoxic levels in vivo at a therapeutically effective dose. In some embodiments, the lipid nucleic acid assembly compositions do not cause an innate immune response that leads to substantial adverse effects at a therapeutic dose level. In some embodiments, the lipid nucleic acid assembly compositions provided herein do not cause toxicity at a therapeutic dose level.
The LNPs disclosed herein may have a size (e.g., Z-average diameter) of about 1 to about 150 nm. In some embodiments, the LNPs have a size of about 10 to about 200 nm. In some embodiments, the LNPs have a size of about 50 to about 100 nm. In some embodiments, the LNPs have a size of about 60 to about 100 nm. In some embodiments, the LNPs have a size of about 75 to about 100 nm. In some embodiments, the LNP composition comprises a population of the LNP with an average diameter of about 20-100 nm. In some embodiments, the LNP composition comprises a population of the LNP with an average diameter of about 50-100 nm. In some embodiments, the LNP composition comprises a population of the LNP with an average diameter of about 60-100 nm. In some embodiments, the LNP composition comprises a population of the LNP with an average diameter of or about 75-100 nm. Unless indicated otherwise, all sizes referred to herein are the average sizes (diameters) of the fully formed nanoparticles, as measured by dynamic light scattering on a Malvern Zetasizer. The nanoparticle sample is diluted in phosphate buffered saline (PBS) so that the count rate is approximately 200-400 kcps. The data is presented as a weighted-average of the intensity measure (Z-average diameter).
In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 50% to about 100%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 50% to about 70%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 70% to about 90%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 90% to about 100%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 75% to about 95%.
In some embodiments, the LNPs are formed with an average molecular weight ranging from about 1.00E+05 g/mol to about 1.00E+10 g/mol. In some embodiments, the LNPs are formed with an average molecular weight ranging from about 5.00E+05 g/mol to about 7.00E+07 g/mol. In some embodiments, the LNPs are formed with an average molecular weight ranging from about 1.00E+06 g/mol to about 1.00E+10 g/mol. In some embodiments, the LNPs are formed with an average molecular weight ranging from about 1.00E+07 g/mol to about 1.00E+09 g/mol. In some embodiments, the LNPs are formed with an average molecular weight ranging from about 5.00E+06 g/mol to about 5.00E+09 g/mol.
In some embodiments, the polydispersity (Mw/Mn; the ratio of the weight averaged molar mass (Mw) to the number averaged molar mass (Mn)) may range from about 1.000 to about 2.000. In some embodiments, the Mw/Mn may range from about 1.00 to about 1.500. In some embodiments, the Mw/Mn may range from about 1.020 to about 1.400. In some embodiments, the Mw/Mn may range from about 1.010 to about 1.100. In some embodiments, the Mw/Mn may range from about 1.100 to about 1.350.
Dynamic Light Scattering (“DLS”) can be used to characterize the polydispersity index (“pdi”) and size of the LNPs of the present disclosure. DLS measures the scattering of light that results from subjecting a sample to a light source. PDI, as determined from DLS measurements, represents the distribution of particle size (around the mean particle size) in a population, with a perfectly uniform population having a PDI of zero. In some embodiments, the pdi may range from 0.005 to 0.75. In some embodiments, the pdi may range from 0.01 to 0.5. In some embodiments, the pdi may range from 0.02 to 0.4. In some embodiments, the pdi may range from 0.03 to 0.35. In some embodiments, the pdi may range from 0.1 to 0.35. In some embodiments, the pdi may range about zero to about 0.4, such as about zero to about 0.35. In some embodiments, the pdi may range from about zero to about 0.35, about zero to about 0.3, about zero to about 0.25, or about zero to about 0.2. In some embodiments, the pdi is less than about 0.08, 0.1, 0.15, 0.2, or 0.4.
In some embodiments, LNPs disclosed herein have a size of 1 to 250 nm. In some embodiments, the LNPs have a size of 10 to 200 nm. In further embodiments, the LNPs have a size of 20 to 150 nm. In some embodiments, the LNPs have a size of 50 to 150 nm. In some embodiments, the LNPs have a size of 50 to 100 nm. In some embodiments, the LNPs have a size of 50 to 120 nm. In some embodiments, the LNPs have a size of 75 to 150 nm. In some embodiments, the LNPs have a size of 30 to 200 nm. Unless indicated otherwise, all sizes referred to herein are the average sizes (diameters) of the fully formed nanoparticles, as measured by dynamic light scattering on a Malvern Zetasizer. The nanoparticle sample is diluted in phosphate buffered saline (PBS) so that the count rate is approximately 200-400 kcts. The data is presented as a weighted-average of the intensity measure. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from 50% to 100%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from 50% to 70%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from 70% to 90%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from 90% to 100%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from 75% to 95%.
Electroporation is also a well-known means for delivery of cargo, and any electroporation methodology may be used for delivery of any one of the RNAs disclosed herein.
In some embodiments, the methods comprises a method for delivering a composition comprising the mRNA disclosed herein to an ex vivo cell, wherein the mRNA is encapsulated in an LNP. In some embodiments, the composition comprises the mRNA and one or more additional RNAs disclosed herein encapsulated in the LNP.
In some embodiments, a lipid nucleic acid assembly composition comprises a lipid component, wherein the lipid component comprises an amine lipid, a neutral lipid, a helper lipid, and a stealth lipid; and wherein the N/P ratio is about 1-10.
In some instances, the lipid component comprises Lipid A or its acetal analog, cholesterol, DSPC, and PEG-DMG; and wherein the N/P ratio is about 1-10. In some embodiments, the lipid component comprises: about 40-60 mol-% amine lipid; about 5-15 mol-% neutral lipid; and about 1.5-10 mol-% PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the lipid nucleic acid assembly composition is about 3-10. In some embodiments, the lipid component comprises about 50-60 mol-% amine lipid; about 8-10 mol-% neutral lipid; and about 2.5-4 mol-% PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the lipid nucleic acid assembly composition is about 3-8. In some instances, the lipid component comprises: about 50-60 mol-% amine lipid; about 5-15 mol-% DSPC; and about 2.5-4 mol-% PEG lipid, wherein the remainder of the lipid component is cholesterol, and wherein the N/P ratio of the lipid nucleic acid assembly composition is about 3-8. In some instances, the lipid component comprises: 48-53 mol-% Lipid A; about 8-10 mol-% DSPC; and 1.5-10 mol-% PEG lipid, wherein the remainder of the lipid component is cholesterol, and wherein the N/P ratio of the lipid nucleic acid assembly composition is 3-8±0.2.
In some embodiments, the lipid component comprises about 50-60 mol-% amine lipid such as Lipid A, about 8-10 mol-% neutral lipid; and about 2.5-4 mol-% stealth lipid (e.g., a PEG lipid), wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the lipid nucleic acid assembly composition is about 6. In some embodiments, the lipid component comprises about 50-60 mol-% amine lipid such as Lipid A; about 27-39.5 mol-% helper lipid; about 8-10 mol-% neutral lipid; and about 2.5-4 mol-% stealth lipid (e.g., a PEG lipid), wherein the N/P ratio of the lipid nucleic acid assembly composition is about 5-7 (e.g., about 6). In some embodiments, the lipid component comprises about 50-60 mol-% amine lipid such as Lipid A; about 5-15 mol-% neutral lipid; and about 2.5-4 mol-% Stealth lipid (e.g., a PEG lipid), wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the lipid nucleic acid assembly composition is about 3-10. In some embodiments, the lipid component comprises about 40-60 mol-% amine lipid such as Lipid A; about 5-15 mol-% neutral lipid; and about 2.5-4 mol-% Stealth lipid (e.g., a PEG lipid), wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the lipid nucleic acid assembly composition is about 6. In some embodiments, the lipid component comprises about 50-60 mol-% amine lipid such as Lipid A; about 5-15 mol-% neutral lipid; and about 1.5-10 mol-% Stealth lipid (e.g., a PEG lipid), wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the lipid nucleic acid assembly composition is about 6. In some embodiments, the lipid component comprises about 40-60 mol-% amine lipid such as Lipid A; about 0-10 mol-% neutral lipid; and about 1.5-10 mol-% Stealth lipid (e.g., a PEG lipid), wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the lipid nucleic acid assembly composition is about 3-10. In some embodiments, the lipid component comprises about 40-60 mol-% amine lipid such as Lipid A; less than about 1 mol-% neutral lipid; and about 1.5-10 mol-% Stealth lipid (e.g., a PEG lipid), wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the lipid nucleic acid assembly composition is about 3-10. In some embodiments, the lipid component comprises about 40-60 mol-% amine lipid such as Lipid A; and about 1.5-10 mol-% Stealth lipid (e.g., a PEG lipid), wherein the remainder of the lipid component is helper lipid, wherein the N/P ratio of the lipid nucleic acid assembly composition is about 3-10, and wherein the lipid nucleic acid assembly composition is essentially free of or free of neutral phospholipid. In some embodiments, the lipid component comprises about 50-60 mol-% amine lipid such as Lipid A; about 8-10 mol-% neutral lipid; and about 2.5-4 mol-% Stealth lipid (e.g., a PEG lipid), wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the Lipid nucleic acid assembly composition is about 3-7.
In some embodiments, the amine lipid is present at about 50 mol-%. In some embodiments, the neutral lipid is present at about 9 mol-%. In some embodiments, the stealth lipid is present at about 3 mol-%. In some embodiments, the helper lipid is present at about 38 mol-%.
In some embodiments, the lipid component comprises, consists essentially of, or consists of: about 50 mol-% amine lipid such as Lipid A; about 9 mol-% neutral lipid such as DSPC; about 3 mol-% of a stealth lipid such as a PEG lipid, such as PEG2k-DMG, and the remainder of the lipid component is helper lipid such as cholesterol, wherein the N/P ratio of the lipid nucleic acid assembly composition is about 6. In some embodiments, the amine lipid is Lipid A. In some embodiments, the neutral lipid is DSPC. In some embodiments, the stealth lipid is a PEG lipid. In some embodiments, the stealth lipid is a PEG2k-DMG. In some embodiments, the helper lipid is cholesterol. In some embodiments, the lipid comprises a lipid component and the lipid component comprises: about 50 mol-% Lipid A; about 9 mol-% DSPC; about 3 mol-% of PEG2k-DMG, and the remainder of the lipid component is cholesterol wherein the N/P ratio of the lipid nucleic acid assembly composition is about 6.
In some embodiments, the lipid component comprises, consists essentially of, or consists of: about 25 to 45 mol-% amine lipid such as Lipid A; about 10 to 30 mol-% neutral lipid such as DSPC; about 1.5 to 3.5 mol-% of a stealth lipid such as a PEG lipid, such as PEG2k-DMG, and about 25 to 65 mol % helper lipid such as cholesterol, wherein the N/P ratio of the lipid nucleic acid assembly composition is about 6. In some embodiments, the lipid component comprises, consists essentially of, or consists of: about 35 mol-% amine lipid such as Lipid A; about 15 mol-% neutral lipid such as DSPC; about 2.5 mol-% of a stealth lipid such as a PEG lipid, such as PEG2k-DMG, and the remainder of the lipid component is helper lipid such as cholesterol, wherein the N/P ratio of the lipid nucleic acid assembly composition is about 6. In some embodiments, the amine lipid is Lipid A. In some embodiments, the neutral lipid is DSPC. In some embodiments, the stealth lipid is a PEG lipid. In some embodiments, the stealth lipid is a PEG2k-DMG. In some embodiments, the helper lipid is cholesterol. In some embodiments, the lipid comprises a lipid component and the lipid component comprises: about 35 mol-% Lipid A; about 15 mol-% DSPC; about 2.5 mol-% of PEG2k-DMG, and the remainder of the lipid component is cholesterol wherein the N/P ratio of the lipid nucleic acid assembly composition is about 6.
In some embodiments, a nucleic acid (e.g., mRNA), polypeptide, composition, or lipid nucleic acid assembly composition disclosed herein is for use in genome editing, e.g., editing a target gene, or modifying a target gene. In some embodiments, a nucleic acid (e.g., mRNA), polypeptide, composition, or lipid nucleic acid assembly composition disclosed herein is for use in modifying a target gene, e.g., altering its sequence or epigenetic status. In some embodiments, the nucleic acid (e.g., mRNA), polypeptide, composition, or lipid nucleic acid assembly composition disclosed herein is for use in the manufacture of a medicament for genome editing or modifying a target gene.
In some embodiments, the use of a nucleic acid (e.g., mRNA), polypeptide, composition, or lipid nucleic acid assembly composition disclosed herein is provided for the preparation of a medicament for genome editing, e.g., editing a target gene. In some embodiments, the use of a nucleic acid (e.g., mRNA), polypeptide, composition, or lipid nucleic acid assembly composition is provided for the preparation of a medicament for modifying a target gene, e.g., altering its sequence or epigenetic status. In some embodiments, the use of a nucleic acid (e.g., mRNA), polypeptide, composition, or lipid nucleic acid assembly composition disclosed herein is provided for the preparation of a medicament for causing C-to-T conversion within a target gene.
In some embodiments, a method of genome editing or modifying a target gene is provided, the method comprising delivering to a cell the mRNA, composition, or lipid nanoparticle(s) described herein.
In some embodiments, the method generates a cytosine (C) to thymine (T) conversion within a target gene.
In some embodiments, the method causes at least 50% C-to-T conversion relative to the total edits in the target sequence. As used herein, the “total edits in the target sequence” is the sum of each read with an indel or at least one conversion, wherein an indel can comprise more than one nucleotide. Indel is calculated as the total number of sequencing reads with one or more base inserted or deleted within the 20 bp scoring region divided by the total number of sequencing reads, including wild type. C-to-T conversions or C-to-A/G conversions were scored in a 40 bp region including 10 bp upstream and 10 bp downstream of the 20 bp sgRNA target sequence. Any sequencing methods (e.g., NGS) that allow reading of sequences diverged from the wild-type alignment may be used. In some embodiments, the method causes at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% C-to-T conversion relative to the total edits in the target sequence.
In some embodiments, the ratio of C-to-T conversion to unintended edits is larger than 1:1. As used herein, an “unintended edit” is any edit in the target region that is not a C-to-T conversion. In some embodiments, the ratio of C-to-T conversion to unintended edits is larger than 2:1, larger than 3:1, larger than 4:1, larger than 5:1, larger than 6:1, larger than 7:1, or larger than 8:1. In some embodiments, the ratio of C-to-T conversion to unintended edits is from 2:1 to 99:1. In some embodiments, the ratio of C-to-T conversion to unintended edits is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1.
In some embodiments, the method causes the A3A to make a base edit corresponding to any one of positions −1 to 10 relative to the 5′ end of the guide sequence.
In some embodiments, the method causes the A3A to make a base edit at a position 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5′ end of the guide sequence.
In some embodiments, the nickase is a SpyCas9 nickase, and the method causes the cytidine deaminase to make a base edit at a cytidine present at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 nucleotides from the 5′ end of the guide sequence.
In some embodiments, the nickase is a NmeCas9 nickase, and the method causes the cytidine deaminase to make a base edit at a cytidine present at position 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5′ end of the guide sequence.
In some embodiments, the composition comprises a first mRNA comprising a first open reading frame encoding a polypeptide comprising an A3A and an RNA-guided nickase, and a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI), and a gRNA; and the first mRNA, the second mRNA, and the gRNA if present, delivered at a ratio of about 6:2:3 (w:w:w). In some embodiments, the target gene is in a subject, such as a mammal, such as a human.
In some embodiments, methods are provided for modifying a target gene comprising delivering to a cell a first mRNA comprising a first open reading frame encoding a first polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase, a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI), wherein the second mRNA is different from the first mRNA, and at least one guide RNA (gRNA).
In some embodiments, methods are provided for modifying a target gene in a cell, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising: (a) a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; (b) a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI); and (c) one or more guide RNAs. In some embodiments, the one or more guide RNAs are each in separate lipid nucleic acid assembly compositions.
In some embodiments, methods are provided for modifying a target gene in a cell, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising: (a) a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; (b) a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI); and (c) one or more guide RNAs, wherein the method comprises one gRNA that targets a gene that reduces or eliminates MHC class I expression on the surface of a cell, and/or one gRNA that targets a gene that reduces or eliminates MHC class II expression on the surface of a cell, and/or one gRNA that targets a gene that reduces or eliminates endogenous TCR expression.
In some embodiments, methods are provided for modifying a target gene in a cell, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising: (a) a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; (b) a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI); and (c) one or more guide RNAs, wherein the method comprises at least two gRNAs selected from: one gRNA that targets a gene that reduces or eliminates MHC class I expression on the surface of a cell, one gRNA that targets a gene that reduces or eliminates MHC class II expression on the surface of a cell, and one gRNA that targets a gene that reduces or eliminates endogenous TCR expression.
In some embodiments, methods are provided for modifying a target gene in a cell, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising: (a) a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; (b) a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI); and (c) one or more guide RNAs, wherein the method comprises one gRNA that targets a gene that reduces or eliminates MHC class I expression on the surface of a cell, one gRNA that targets a gene that reduces or eliminates MHC class II expression on the surface of a cell, and one gRNA that targets a gene that reduces or eliminates endogenous TCR expression.
In some embodiments, methods are provided for modifying a target gene in a cell, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising: (a) a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; (b) a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI); and (c) one or more guide RNAs, wherein the method comprises one gRNA that targets a gene that reduces or eliminates expression of HLA-A on the surface of a cell, and/or one gRNA that targets a gene that reduces or eliminates MHC class II expression on the surface of a cell, and/or one gRNA that targets a gene that reduces or eliminates endogenous TCR expression.
In some embodiments, methods are provided for modifying a target gene in a cell, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising: (a) a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; (b) a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI); and (c) one or more guide RNAs, wherein the method comprises at least two gRNAs selected from: one gRNA that targets a gene that reduces or eliminates expression of HLA-A on the surface of a cell, one gRNA that targets a gene that reduces or eliminates MHC class II expression on the surface of a cell, and one gRNA that targets a gene that reduces or eliminates endogenous TCR expression.
In some embodiments, methods are provided for modifying a target gene in a cell, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising: (a) a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; (b) a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI); and (c) one or more guide RNAs, wherein the method comprises one gRNA that targets a gene that reduces or eliminates expression of HLA-A on the surface of a cell, one gRNA that targets a gene that reduces or eliminates MHC class II expression on the surface of a cell, and one gRNA that targets a gene that reduces or eliminates endogenous TCR expression.
In some embodiments, methods are provided for modifying a target gene in a cell, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising: (a) a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; (b) a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI); and (c) one or more guide RNAs, wherein the method comprises one gRNA selected from a gRNA that targets TRAC, TRBC, B2M, HLA-A, or CIITA. In some embodiments, one gRNA targets TRAC. In some embodiments, one gRNA targets TRBC. In some embodiments, one gRNA targets B2M. In some embodiments, one gRNA targets HLA-A. In some embodiments, one gRNA targets CIITA.
In some embodiments, methods are provided for modifying a target gene in a cell, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising: (a) a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; (b) a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI); and (c) one or more guide RNAs, wherein the method comprises at least two gRNAs selected from a gRNA that targets TRAC, TRBC, or B2M, wherein the two guide RNAs do not target the same gene. In some embodiments, the gRNAs are each in separate lipid nucleic acid assembly compositions.
In some embodiments, methods are provided for modifying a target gene in a cell, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising: (a) a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; (b) a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI); and (c) one or more guide RNAs, wherein the method comprises at least two gRNAs selected from a gRNA that targets TRAC, TRBC, or HLA-A wherein the two guide RNAs do not target the same gene. In some embodiments, the gRNAs are each in separate lipid nucleic acid assembly compositions.
In some embodiments, methods are provided for modifying a target gene in a cell, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising: (a) a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; (b) a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI); and (c) one or more guide RNAs, wherein the method comprises at least two gRNAs selected from a gRNA that targets TRAC, TRBC, HLA-A, wherein the two guide RNAs do not target the same gene. In some embodiments, the gRNAs are each in separate lipid nucleic acid assembly compositions.
In some embodiments, methods are provided for modifying a target gene in a cell, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising: (a) a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; (b) a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI); and (c) one or more guide RNAs, wherein the method comprises one guide RNA that targets TRAC, and one gRNA that targets TRBC. In some embodiments, the gRNAs are each in separate lipid nucleic acid assembly compositions.
In some embodiments, methods are provided for modifying a target gene in a cell, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising: (a) a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; (b) a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI); and (c) one or more guide RNAs, wherein the method comprises one guide RNA that targets B2M, and one gRNA that targets CIITA. In some embodiments, the gRNAs are each in separate lipid nucleic acid assembly compositions.
In some embodiments, methods are provided for modifying a target gene in a cell, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising: (a) a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; (b) a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI); and (c) one or more guide RNAs, wherein the method comprises one guide RNA that targets HLA-A, and one gRNA that targets CIITA. In some embodiments, the gRNAs are each in separate lipid nucleic acid assembly compositions. In some embodiments, the cell is homozygous for HLA-B and homozygous for HLA-C.
In some embodiments, methods are provided for modifying a target gene in a cell, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising: (a) a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; (b) a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI); and (c) one or more guide RNAs, wherein the method comprises one guide RNA that targets TRAC, and one gRNA that targets TRBC, and one gRNA that targets B2M. In some embodiments, the gRNAs are each in separate lipid nucleic acid assembly compositions.
In some embodiments, methods are provided for modifying a target gene in a cell, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising: (a) a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; (b) a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI); and (c) one or more guide RNAs, wherein the method comprises one guide RNA that targets TRAC, and one gRNA that targets TRBC, and one gRNA that targets HLA-A. In some embodiments, the gRNAs are each in separate lipid nucleic acid assembly compositions. In some embodiments, the cell is homozygous for HLA-B and homozygous for HLA-C.
In some embodiments, methods are provided for modifying a target gene in a cell, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising: (a) a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; (b) a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI); and (c) one or more guide RNAs, wherein the method comprises one guide RNA that targets TRAC, and one gRNA that targets TRBC, one gRNA that targets B2M, and one gRNA that targets CIITA. In some embodiments, the gRNAs are each in separate lipid nucleic acid assembly compositions.
In some embodiments, methods are provided for modifying a target gene in a cell, comprising delivering to the cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising: (a) a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase; (b) a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI); and (c) one or more guide RNAs, wherein the method comprises one guide RNA that targets TRAC, and one gRNA that targets TRBC, one gRNA that targets HLA-A, and one gRNA that targets CIITA. In some embodiments, the gRNAs are each in separate lipid nucleic acid assembly compositions. In some embodiments, the cell is homozygous for HLA-B and homozygous for HLA-C.
In some embodiments, a cell is provided comprising a composition comprising a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase, and a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI), wherein the second mRNA is different from the first mRNA.
In some embodiments, an engineered cell is provided comprising at least one base edit and/or indel, wherein the base edit and/or indel is made by contacting a cell with a composition comprising a first mRNA comprising a first open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase, and a second mRNA comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI), wherein the second mRNA is different from the first mRNA.
In some embodiments the cell is a human cell. In some embodiments the genetically modified cell is referred to as an engineered cell. An engineered cell refers to a cell (or progeny of a cell) comprising an engineered genetic modification, e.g. that has been contacted with a gene editing system and genetically modified by the gene editing system. The terms “engineered cell” and “genetically modified cell” are used interchangeably throughout. The engineered cell may be any of the exemplary cell types disclosed herein. In some embodiments, the cell is an allogeneic cell.
In some embodiments, the cell is an immune cell. As used herein, “immune cell” refers to a cell of the immune system, including e.g., a lymphocyte (e.g., T cell, B cell, natural killer cell (“NK cell”, and NKT cell, or iNKT cell)), monocyte, macrophage, mast cell, dendritic cell, or granulocyte (e.g., neutrophil, eosinophil, and basophil). In some embodiments, the cell is a primary immune cell. In some embodiments, the immune system cell may be selected from CD3+, CD4+ and CD8+ T cells, regulatory T cells (Tregs), B cells, NK cells, and dendritic cells (DC). In some embodiments, the immune cell is allogeneic. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is an adaptive immune cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is a NK cell. In some embodiments, the lymphocyte is allogeneic.
In some embodiments, the genome editing or modification of the target gene is in vivo. In some embodiments, the genome editing or modification of the target gene is in an isolated or cultured cell.
In some embodiments, the target gene is in an organ, such as a liver, such as a mammalian liver, such as a human liver. In some embodiments, the target gene is in a liver cell, such as a mammalian liver cell, such as a human liver cell. In some embodiments, the target gene is in a hepatocyte, such as a mammalian hepatocyte, such as a human hepatocyte. In some embodiments, the liver cell or hepatocyte is in situ. In some embodiments, the liver cell or hepatocyte is isolated, e.g., in a culture, such as in a primary culture.
In some embodiments, the genome editing or modification of the target gene inactivates a splice donor or splice acceptor site.
Also provided are methods corresponding to the uses disclosed herein, which comprise administering the nucleic acid (e.g., mRNA), polypeptide, composition, or lipid nucleic acid assembly composition disclosed herein to a subject or contacting a cell such as those described above with the nucleic acid (e.g., mRNA), polypeptide, composition, or lipid nucleic acid assembly composition disclosed herein.
In some embodiments the nucleic acid (e.g., mRNA), polypeptide, composition, or lipid nucleic acid assembly composition disclosed herein is administered intravenously for any of the uses discussed above concerning organisms, organs, or cells in situ.
In any of the foregoing embodiments involving a subject, the subject can be mammalian. In any of the foregoing embodiments involving a subject, the subject can be human. In any of the foregoing embodiments involving a subject, the subject can be a cow, pig, monkey, sheep, dog, cat, fish, or poultry.
In some embodiments, the nucleic acid (e.g., mRNA), polypeptide, composition, or lipid nucleic acid assembly composition disclosed herein is administered intravenously or for intravenous administration.
In some embodiments, the genome editing or modification of the target gene knocks down expression of the target gene. In some embodiments, the genome editing or modification of the target gene knocks down expression of the target gene by at least 50%, 55%, 60%, 65%, 70%, 75%, or 80%. In some embodiments, the genome editing or modification of the target gene produces a missense mutation in the gene.
In some embodiments, a single administration of a nucleic acid (e.g., mRNA), polypeptide, composition, or lipid nucleic acid assembly composition disclosed herein is sufficient to knock down expression of the target gene product. In some embodiments, a single administration of a nucleic acid (e.g., mRNA), polypeptide, composition, or lipid nucleic acid assembly composition disclosed herein is sufficient to knock out expression of the target gene product. In other embodiments, more than one administration of a nucleic acid (e.g., mRNA), polypeptide, composition, or lipid nucleic acid assembly composition disclosed herein may be beneficial to maximize editing via cumulative effects.
In some embodiments, the efficacy of treatment with a nucleic acid (e.g., mRNA), polypeptide, composition, or lipid nucleic acid assembly composition disclosed herein is seen at 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years after delivery.
In some embodiments, treatment slows or halts disease progression.
In some embodiments, treatment results in improvement, stabilization, or slowing of change in organ function or symptoms of disease of an organ.
In some embodiments, efficacy of treatment is measured by increased survival time of the subject.
1. Exemplary Guide RNAs, Compositions, Methods, and Engineered Cells for TRAC and TRBC Editing
The disclosure provides a guide RNA that target TRAC. Guide sequences targeting the TRAC gene are shown in Table 5A at SEQ ID NOs: 706-721.
The disclosure provides a guide RNA that target TRBC. Guide sequences targeting the TRBC gene are shown in Table 5B at SEQ ID NOs: 618-669.
In some embodiments, the guide sequences are complementary to the corresponding genomic region shown in the tables below, according to coordinates from human reference genome hg38. Guide sequences of further embodiments may be complementary to sequences in the close vicinity of the genomic coordinate listed in any of Tables 5A and 5B. For example, guide sequences of further embodiments may be complementary to sequences that comprise 15 consecutive nucleotides±10 nucleotides of a genomic coordinate listed in any of Tables 5A an 5B.
As described in the preceding sections, each of the guide sequences shown in Table 5A and Table 5B may further comprise additional nucleotides to form a crRNA, e.g., with the following exemplary nucleotide sequence following the guide sequence at its 3′ end: GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 139) in 5′ to 3′ orientation. In the case of a sgRNA, the guide sequences may further comprise additional nucleotides to form a sgRNA, e.g., with the following exemplary nucleotide sequence following the 3′ end of the guide sequence: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 140) in 5′ to 3′ orientation. The guide sequences may further comprise additional nucleotides to form a sgRNA, e.g.,
In some embodiments, the sgRNA comprises the modification pattern shown below in SEQ ID NO: 141, where N is any natural or non-natural nucleotide, and where the totality of the N's comprise a guide sequence as described herein and the modified sgRNA comprises the following sequence: mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmGmAmAmAmU mAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAm AmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 141), where “N” may be any natural or non-natural nucleotide. For example, encompassed herein is SEQ ID NO: 141, where the N's are replaced with any of the guide sequences disclosed herein. The modifications remain as shown in SEQ ID NO: 141 despite the substitution of N's for the nucleotides of a guide. That is, although the nucleotides of the guide replace the “N's”, the first three nucleotides are 2′OMe modified and there are phosphorothioate linkages between the first and second nucleotides, the second and third nucleotides and the third and fourth nucleotides.
In some embodiments, the gRNA targeting TRAC comprises a guide sequence chosen from: i) SEQ ID NOs: 706-721; ii) at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 706-721; iii) a guide sequence at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 706-721; iv) a sequence that comprises 10 contiguous nucleotides±10 nucleotides of a genomic coordinate listed in Table 5A; v) at least 17, 18, 19, or 20 contiguous nucleotides of a sequence from (iv); or vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (v).
In some embodiments, the guide sequence comprises SEQ ID NO: 706. In some embodiments, the guide sequence comprises SEQ ID NO: 707. In some embodiments, the guide sequence comprises SEQ ID NO: 708. In some embodiments, the guide sequence comprises SEQ ID NO: 709. In some embodiments, the guide sequence comprises SEQ ID NO: 710. In some embodiments, the guide sequence comprises SEQ ID NO: 711. In some embodiments, the guide sequence comprises SEQ ID NO: 712. In some embodiments, the guide sequence comprises SEQ ID NO: 713. In some embodiments, the guide sequence comprises SEQ ID NO: 714. In some embodiments, the guide sequence comprises SEQ ID NO: 715. In some embodiments, the guide sequence comprises SEQ ID NO: 716. In some embodiments, the guide sequence comprises SEQ ID NO: 717. In some embodiments, the guide sequence comprises SEQ ID NO: 718. In some embodiments, the guide sequence comprises SEQ ID NO: 719. In some embodiments, the guide sequence comprises SEQ ID NO: 720. In some embodiments, the guide sequence comprises SEQ ID NO: 721.
In some embodiments, the gRNA targeting TRBC comprises a guide sequence chosen from: i) SEQ ID NOs: 618-669; ii) at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 618-669; iii) a guide sequence at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 618-669; iv) a sequence that comprises 10 contiguous nucleotides±10 nucleotides of a genomic coordinate listed in Table 5B; v) at least 17, 18, 19, or 20 contiguous nucleotides of a sequence from (iv); or vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (v).
In some embodiments, the guide sequence comprises SEQ ID NO: 618. In some embodiments, the guide sequence comprises SEQ ID NO: 619. In some embodiments, the guide sequence comprises SEQ ID NO: 620. In some embodiments, the guide sequence comprises SEQ ID NO: 621. In some embodiments, the guide sequence comprises SEQ ID NO: 622. In some embodiments, the guide sequence comprises SEQ ID NO: 623. In some embodiments, the guide sequence comprises SEQ ID NO: 624. In some embodiments, the guide sequence comprises SEQ ID NO: 625. In some embodiments, the guide sequence comprises SEQ ID NO: 626. In some embodiments, the guide sequence comprises SEQ ID NO: 627. In some embodiments, the guide sequence comprises SEQ ID NO: 628. In some embodiments, the guide sequence comprises SEQ ID NO: 629. In some embodiments, the guide sequence comprises SEQ ID NO: 630. In some embodiments, the guide sequence comprises SEQ ID NO: 631. In some embodiments, the guide sequence comprises SEQ ID NO: 632. In some embodiments, the guide sequence comprises SEQ ID NO: 633. In some embodiments, the guide sequence comprises SEQ ID NO: 634. In some embodiments, the guide sequence comprises SEQ ID NO: 635. In some embodiments, the guide sequence comprises SEQ ID NO: 636. In some embodiments, the guide sequence comprises SEQ ID NO: 637. In some embodiments, the guide sequence comprises SEQ ID NO: 638. In some embodiments, the guide sequence comprises SEQ ID NO: 639. In some embodiments, the guide sequence comprises SEQ ID NO: 640. In some embodiments, the guide sequence comprises SEQ ID NO: 641. In some embodiments, the guide sequence comprises SEQ ID NO: 642. In some embodiments, the guide sequence comprises SEQ ID NO: 643. In some embodiments, the guide sequence comprises SEQ ID NO: 644. In some embodiments, the guide sequence comprises SEQ ID NO: 645. In some embodiments, the guide sequence comprises SEQ ID NO: 646. In some embodiments, the guide sequence comprises SEQ ID NO: 647. In some embodiments, the guide sequence comprises SEQ ID NO: 648. In some embodiments, the guide sequence comprises SEQ ID NO: 649. In some embodiments, the guide sequence comprises SEQ ID NO: 650. In some embodiments, the guide sequence comprises SEQ ID NO: 651. In some embodiments, the guide sequence comprises SEQ ID NO: 652. In some embodiments, the guide sequence comprises SEQ ID NO: 653. In some embodiments, the guide sequence comprises SEQ ID NO: 654. In some embodiments, the guide sequence comprises SEQ ID NO: 655. In some embodiments, the guide sequence comprises SEQ ID NO: 656. In some embodiments, the guide sequence comprises SEQ ID NO: 657. In some embodiments, the guide sequence comprises SEQ ID NO: 658. In some embodiments, the guide sequence comprises SEQ ID NO: 659. In some embodiments, the guide sequence comprises SEQ ID NO: 660. In some embodiments, the guide sequence comprises SEQ ID NO: 661. In some embodiments, the guide sequence comprises SEQ ID NO: 662. In some embodiments, the guide sequence comprises SEQ ID NO: 663. In some embodiments, the guide sequence comprises SEQ ID NO: 664. In some embodiments, the guide sequence comprises SEQ ID NO: 665. In some embodiments, the guide sequence comprises SEQ ID NO: 666. In some embodiments, the guide sequence comprises SEQ ID NO: 667. In some embodiments, the guide sequence comprises SEQ ID NO: 668. In some embodiments, the guide sequence comprises SEQ ID NO: 669.
In some embodiments, the disclosure provides a method of altering a DNA sequence within a TRAC gene, comprising delivering a composition disclosed herein to a cell. The composition may comprise:
In some embodiments, the disclosure provides a method of reducing the expression of a TRAC gene, comprising delivering a composition disclosed herein to a cell. The composition may comprise:
In some embodiments, the disclosure provides a method of immunotherapy comprising administering a composition disclosed herein to a subject, an autologous cell thereof, and/or an allogeneic cell. The composition may comprise:
In some embodiments, a cell altered by the method disclosed herein is provided. The cell may be altered ex vivo. The cell may be a T cell, a CD4+ or CD8+ cell. The cell may be a mammalian, primate, or human cell. The cell may be used for immunotherapy of a subject.
In some embodiments, a compositions is provided, comprising: a gRNA comprising a guide sequence chosen from: i) SEQ ID NOs: 706-721; ii) at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 706-721; iii) a guide sequence at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 706-721; iv) a sequence that comprises 10 contiguous nucleotides±10 nucleotides of a genomic coordinate listed in Table 5A; v) at least 17, 18, 19, or 20 contiguous nucleotides of a sequence from (iv); or vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (v). The composition may optionally further comprise any one of a nucleic acid (e.g., mRNA), polypeptide, composition, or lipid nucleic acid assembly composition disclosed herein.
In certain embodiments, the composition disclosed herein is used for altering a DNA sequence within the TRAC gene in a cell. In certain embodiments, the composition disclosed herein is used for reducing the expression of the TRAC gene in a cell. In some embodiments, the composition disclosed herein is used for immunotherapy of a subject.
In some embodiments, the disclosure provides a method of altering a DNA sequence within a TRBC1 and/or TRBC2 gene, comprising delivering a composition disclosed herein to a cell. The composition may comprise:
In some embodiments, the disclosure provides a method of reducing the expression of a TRBC1 and/or TRBC2 gene, comprising delivering a composition disclosed herein to a cell. The composition may comprise:
In some embodiments, the disclosure provides a method of immunotherapy comprising administering a composition disclosed herein to a subject, an autologous cell thereof, and/or an allogeneic cell. The composition may comprise:
In some embodiments, a cell may be provided, being altered by the method disclosed herein. The cell may be altered ex vivo. The cell is a T cell, a CD4+ or CD8+ cell. The cell may be a mammalian, primate, or human cell. The cell may be used for immunotherapy of a subject.
In some embodiments, a compositions is provided, comprising: a gRNA comprising a guide sequence chosen from: i) SEQ ID NOs: 618-669; ii) at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 618-669; iii) a guide sequence at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 618-669; iv) a sequence that comprises 10 contiguous nucleotides±10 nucleotides of a genomic coordinate listed in Table 5B; v) at least 17, 18, 19, or 20 contiguous nucleotides of a sequence from (iv); or vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (v). The composition may optionally further comprise any one of a nucleic acid (e.g., mRNA), polypeptide, composition, or lipid nucleic acid assembly composition disclosed herein.
In certain embodiments, the composition disclosed herein is used for altering a DNA sequence within the TRBC1 and/or TRBC2 gene in a cell. In certain embodiments, the composition disclosed herein is used for reducing the expression of the TRBC1 and/or TRBC2 gene in a cell. In some embodiments, the composition disclosed herein is used for immunotherapy of a subject.
J. Exemplary DNA Molecules, Vectors, Expression Constructs, Host Cells, and Production Methods
In certain embodiments, the disclosure provides a DNA molecule comprising a sequence encoding a polypeptide described herein. In some embodiments, the DNA molecule further comprises nucleic acids that do not encode the polypeptide. Nucleic acids that do not encode the polypeptide disclosed herein include, but are not limited to, promoters, enhancers, regulatory sequences, and nucleic acids encoding a gRNA.
In some embodiments, the DNA molecule further comprises a nucleotide sequence encoding a crRNA, a trRNA, or a crRNA and trRNA. In some embodiments, the nucleotide sequence encoding the crRNA, trRNA, or crRNA and trRNA comprises or consists of a guide sequence flanked by all or a portion of a repeat sequence from a naturally-occurring CRISPR/Cas system. The nucleic acid comprising or consisting of the crRNA, trRNA, or crRNA and trRNA may further comprise a vector sequence wherein the vector sequence comprises or consists of nucleic acids that are not naturally found together with the crRNA, trRNA, or crRNA and trRNA. In some embodiments, the crRNA and the trRNA are encoded by non-contiguous nucleic acids within one vector. In other embodiments, the crRNA and the trRNA may be encoded by a contiguous nucleic acid. In some embodiments, the crRNA and the trRNA are encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and the trRNA are encoded by the same strand of a single nucleic acid.
In some embodiments, the DNA molecule further comprises a promoter operably linked to the sequence encoding any of the mRNAs encoding the polypeptide described herein. In some embodiments, the DNA molecule is an expression construct suitable for expression in a mammalian cell, e.g., a human cell or a mouse cell, such as a human hepatocyte or a rodent (e.g., mouse) hepatocyte. In some embodiments, the DNA molecule is an expression construct suitable for expression in a cell of a mammalian organ, e.g., a human liver or a rodent (e.g., mouse) liver. In some embodiments, the DNA molecule is a plasmid or an episome. In some embodiments, the DNA molecule is contained in a host cell, such as a bacterium or a cultured eukaryotic cell. Exemplary bacteria include proteobacteria such as E. coli. Exemplary cultured eukaryotic cells include primary hepatocytes, including hepatocytes of rodent (e.g., mouse) or human origin; hepatocyte cell lines, including hepatocytes of rodent (e.g., mouse) or human origin; human cell lines; rodent (e.g., mouse) cell lines; CHO cells; microbial fungi, such as fission or budding yeasts, e.g., Saccharomyces, such as S. cerevisiae; and insect cells.
In some embodiments, a method of producing an mRNA disclosed herein is provided. In some embodiments, such a method comprises contacting a DNA molecule described herein with an RNA polymerase under conditions permissive for transcription. In some embodiments, the contacting is performed in vitro, e.g., in a cell-free system. In some embodiments, the RNA polymerase is an RNA polymerase of bacteriophage origin, such as T7 RNA polymerase. In some embodiments, NTPs are provided that include at least one modified nucleotide as discussed above. In some embodiments, the NTPs include at least one modified nucleotide as discussed above and do not comprise UTP.
In some embodiments, an mRNA disclosed herein alone or together with one or more gRNAs, may be comprised within or delivered by a vector system of one or more vectors. In some embodiments, one or more of the vectors, or all of the vectors, may be DNA vectors. In some embodiments, one or more of the vectors, or all of the vectors, may be RNA vectors. In some embodiments, one or more of the vectors, or all of the vectors, may be circular. In other embodiments, one or more of the vectors, or all of the vectors, may be linear. In some embodiments, one or more of the vectors, or all of the vectors, may be enclosed in a lipid nanoparticle, liposome, non-lipid nanoparticle, or viral capsid. Non-limiting exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, minichromosomes, transposons, viral vectors, and expression vectors.
Non-limiting exemplary viral vectors include adeno-associated virus (AAV) vector, lentivirus vectors, adenovirus vectors, helper dependent adenoviral vectors (HDAd), herpes simplex virus (HSV-1) vectors, bacteriophage T4, baculovirus vectors, and retrovirus vectors. In some embodiments, the viral vector may be an AAV vector. In other embodiments, the viral vector may a lentivirus vector. In some embodiments, the lentivirus may be non-integrating. In some embodiments, the viral vector may be an adenovirus vector. In some embodiments, the adenovirus may be a high-cloning capacity or “gutless” adenovirus, where all coding viral regions apart from the 5′ and 3′ inverted terminal repeats (ITRs) and the packaging signal (T) are deleted from the virus to increase its packaging capacity. In yet other embodiments, the viral vector may be an HSV-1 vector. In some embodiments, the HSV-1-based vector is helper dependent, and in other embodiments it is helper independent. For example, an amplicon vector that retains only the packaging sequence requires a helper virus with structural components for packaging, while a 30 kb-deleted HSV-1 vector that removes non-essential viral functions does not require helper virus. In additional embodiments, the viral vector may be bacteriophage T4. In some embodiments, the bacteriophage T4 may be able to package any linear or circular DNA or RNA molecules when the head of the virus is emptied. In further embodiments, the viral vector may be a baculovirus vector. In yet further embodiments, the viral vector may be a retrovirus vector. In embodiments using AAV or lentiviral vectors, which have smaller cloning capacity, it may be necessary to use more than one vector to deliver all the components of a vector system as disclosed herein. For example, one AAV vector may contain sequences encoding a Cas protein, while a second AAV vector may contain one or more guide sequences.
In some embodiments, the vector may be capable of driving expression of one or more coding sequences, such as the coding sequence of an mRNA disclosed herein, in a cell. In some embodiments, the cell may be a prokaryotic cell, such as, e.g., a bacterial cell. In some embodiments, the cell may be a eukaryotic cell, such as, e.g., a yeast, plant, insect, or mammalian cell. In some embodiments, the eukaryotic cell may be a mammalian cell. In some embodiments, the eukaryotic cell may be a rodent cell. In some embodiments, the eukaryotic cell may be a human cell. Suitable promoters to drive expression in different types of cells are known in the art. In some embodiments, the promoter may be wild type. In other embodiments, the promoter may be modified for more efficient or efficacious expression. In yet other embodiments, the promoter may be truncated yet retain its function. For example, the promoter may have a normal size or a reduced size that is suitable for proper packaging of the vector into a virus.
In some embodiments, the vector system may comprise one copy of a nucleotide sequence encoding a polypeptide disclosed herein. In other embodiments, the vector system may comprise more than one copy of a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the nucleotide sequence encoding a polypeptide disclosed herein may be operably linked to at least one transcriptional or translational control sequence. In some embodiments, the nucleotide sequence encoding the protein may be operably linked to at least one promoter.
In some embodiments, the promoter may be constitutive, inducible, or tissue-specific. In some embodiments, the promoter may be a constitutive promoter. Non-limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-alpha (EF1a) promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, a functional fragment thereof, or a combination of any of the foregoing. In some embodiments, the promoter may be a CMV promoter. In some embodiments, the promoter may be a truncated CMV promoter. In other embodiments, the promoter may be an EF1a promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low basal (non-induced) expression level, such as, e.g., the Tet-On® promoter (Clontech).
In some embodiments, the promoter may be a tissue-specific promoter, e.g., a promoter specific for expression in the liver.
The vector may further comprise a nucleotide sequence encoding at least one gRNA. In some embodiments, the vector comprises one copy of the gRNA. In other embodiments, the vector comprises more than one copy of the gRNA. In embodiments with more than one gRNA, the gRNAs may be non-identical such that they target different target sequences, or may be identical in that they target the same target sequence. In some embodiments where the vectors comprise more than one gRNA, each gRNA may have other different properties, such as activity or stability within a complex with the polypeptide comprising a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) and an RNA-guided nickase disclosed herein. In some embodiments, the nucleotide sequence encoding the gRNA may be operably linked to at least one transcriptional or translational control sequence, such as a promoter, a 3′ UTR, or a 5′ UTR. In one embodiment, the promoter may be a tRNA promoter, e.g., tRNALys3, or a tRNA chimera. See Mefferd et al., RNA. 2015 21:1683-9; Scherer et al., Nucleic Acids Res. 2007 35: 2620-2628. In some embodiments, the promoter may be recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters include U6 and H1 promoters. In some embodiments, the nucleotide sequence encoding the gRNA may be operably linked to a mouse or human U6 promoter. In other embodiments, the nucleotide sequence encoding the gRNA may be operably linked to a mouse or human H1 promoter. In embodiments with more than one gRNA, the promoters used to drive expression may be the same or different. In some embodiments, the nucleotide encoding the crRNA of the gRNA and the nucleotide encoding the trRNA of the gRNA may be provided on the same vector. In some embodiments, the nucleotide encoding the crRNA and the nucleotide encoding the trRNA may be driven by the same promoter. In some embodiments, the crRNA and trRNA may be transcribed into a single transcript. For example, the crRNA and trRNA may be processed from the single transcript to form a double-molecule gRNA. Alternatively, the crRNA and trRNA may be transcribed into a single-molecule gRNA. In other embodiments, the crRNA and the trRNA may be driven by their corresponding promoters on the same vector. In yet other embodiments, the crRNA and the trRNA may be encoded by different vectors.
In some embodiments, the compositions comprise a vector system, wherein the system comprises more than one vector. In some embodiments, the vector system may comprise one single vector. In other embodiments, the vector system may comprise two vectors. In additional embodiments, the vector system may comprise three vectors. When different gRNAs are used for multiplexing, or when multiple copies of the gRNA are used, the vector system may comprise more than three vectors.
In some embodiments, the vector system may comprise inducible promoters to start expression only after it is delivered to a target cell. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low basal (non-induced) expression level, such as, e.g., the Tet-On® promoter (Clontech).
In additional embodiments, the vector system may comprise tissue-specific promoters to start expression only after it is delivered into a specific tissue.
In some embodiments, the vector may be delivered systemically. In some embodiments, the vector may be delivered into the hepatic circulation.
The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way.
As used in the Examples below, the term “editor” refers to an agent comprising a polypeptide that is capable of deaminating a base within a DNA molecule and it is a base editor. The editor may be capable of deaminating a cytidine (C) in DNA. The editor may include an RNA-guided nickase (e.g., Cas9 nickase) fused to a cytidine deaminase (e.g., an APOBEC3A deaminase (A3A)) by an optional linker. In some cases, the editor includes a UGI. In some embodiments, the editor lacks a UGI.
An exemplary editor used in the Examples below is BC22n (SEQ ID NO:3) which consists of a H. sapiens APOBEC3A fused to S. pyogenes-D10A SpyCas9 nickase by an XTEN linker, and mRNA encoding BC22n. An mRNA encoding BC22n (SEQ ID NO: 1) is also used.
In general, the lipid components were dissolved in 100% ethanol at various molar ratios. The RNA cargos (e.g., Cas9 mRNA and sgRNA) were dissolved in 25 mM citrate buffer, 100 mM NaCl, pH 5.0, resulting in a concentration of RNA cargo of approximately 0.45 mg/mL.
Unless otherwise specified, the lipid nucleic acid assemblies contained ionizable Lipid A ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate), cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:3 molar ratio, respectively. The lipid nucleic acid assemblies were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 6, and a ratio of gRNA to mRNA of 1:1 by weight, unless otherwise specified.
LNPs were prepared using a cross-flow technique utilizing impinging jet mixing of the lipid in ethanol with two volumes of RNA solutions and one volume of water. The lipids in ethanol were mixed through a mixing cross with the two volumes of RNA solution. A fourth stream of water was mixed with the outlet stream of the cross through an inline tee (See WO2016010840
1.2. In Vitro Transcription (“IVT”) of mRNA
Capped and polyadenylated mRNA containing N1-methyl pseudo-U was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. Plasmid DNA containing a T7 promoter, a sequence for transcription, and a polyadenylation region was linearized by incubating at 37° C. for 2 hours with XbaI with the following conditions: 200 ng/μL plasmid, 2 U/μL XbaI (NEB), and 1× reaction buffer. The XbaI was inactivated by heating the reaction at 65° C. for 20 min. The linearized plasmid was purified from enzyme and buffer salts. The IVT reaction to generate modified mRNA was performed by incubating at 37° C. for 1.5-4 hours in the following conditions: 50 ng/μL linearized plasmid; 2-5 mM each of GTP, ATP, CTP, and N1-methyl pseudo-UTP (Trilink); 10-25 mM ARCA (Trilink); 5 U/μL T7 RNA polymerase (NEB); 1 U/μL Murine RNase inhibitor (NEB); 0.004 U/μL Inorganic E. coli pyrophosphatase (NEB); and 1× reaction buffer. TURBO DNase (ThermoFisher) was added to a final concentration of 0.01 U/μL, and the reaction was incubated for an additional 30 minutes to remove the DNA template. The mRNA was purified using a MegaClear Transcription Clean-up kit (ThermoFisher) or a RNeasy Maxi kit (Qiagen) per the manufacturers' protocols. Alternatively, the mRNA was purified through a precipitation protocol, which in some cases was followed by HPLC-based purification. Briefly, after the DNase digestion, mRNA is purified using LiCl precipitation, ammonium acetate precipitation and sodium acetate precipitation. For HPLC purified mRNA, after the LiCl precipitation and reconstitution, the mRNA was purified by RP-IP HPLC (see, e.g., Kariko, et al. Nucleic Acids Research, 2011, Vol. 39, No. 21 e142). The fractions chosen for pooling were combined and desalted by sodium acetate/ethanol precipitation as described above. In a further alternative method, mRNA was purified with a LiCl precipitation method followed by further purification by tangential flow filtration. RNA concentrations were determined by measuring the light absorbance at 260 nm (Nanodrop), and transcripts were analyzed by capillary electrophoresis by Bioanlayzer (Agilent).
Nme2Cas9 mRNA was generated from plasmid DNA encoding an open reading frame according to SEQ ID NOs: 360 (see sequences in Table 5C). Streptococcus pyogenes (“Spy”) Cas9 mRNA was generated from plasmid DNA encoding an open reading frame according to SEQ ID NOs: 8, 11, or 23 (see sequences in Table 5C). BC22n mRNA was generated from plasmid DNA encoding an open reading frame according to SEQ ID NOs: 2 or 5. BC22 mRNA was generated from plasmid DNA encoding an open reading frame according to SEQ ID NO: 20. BC22 with 2×UGI mRNA was generated from plasmid DNA encoding an open reading frame according to SEQ ID No: 29. UGI mRNA was generated from plasmid DNA encoding an open reading frame according to SEQ ID NOs: 26 or 35. BE3 mRNA was generated from plasmid DNA encoding an open reading frame according to SEQ ID NO: 14 or 17. BE4Max mRNA was generated from plasmid DNA encoding an open reading frame according to SEQ ID NO: 32. When the sequences cited in this paragraph are referred to below with respect to RNAs, it is understood that Ts should be replaced with Us (which were N1-methyl pseudouridines as described above). Messenger RNAs used in the Examples include a 5′ cap and a 3′ polyadenylation region, e.g., up to 100 nts.
Genomic DNA was extracted using QuickExtract™ DNA Extraction Solution (Lucigen, Cat. QE09050) according to the manufacturer's protocol. To quantitatively determine the efficiency of editing at the target location in the genome, deep sequencing was utilized to identify the presence of insertions and deletions introduced by gene editing. PCR primers were designed around the target site within the gene of interest (e.g., TRAC) and the genomic area of interest was amplified. Primer sequence design was done as is standard in the field.
Additional PCR was performed according to the manufacturer's protocols (Illumina) to add chemistry for sequencing. The amplicons were sequenced on an Illumina MiSeq instrument. The reads were aligned to the human reference genome (e.g., hg38) after eliminating those having low quality scores. Reads that overlapped the target region of interest were re-aligned to the local genome sequence to improve the alignment. Then the number of wild type reads versus the number of reads which contain C-to-T mutations, C-to-A/G mutations or indels was calculated. Insertions and deletions were scored in a 20 bp region centered on the predicted Cas9 cleavage site. Indel percentage is defined as the total number of sequencing reads with one or more base inserted or deleted within the 20 bp scoring region divided by the total number of sequencing reads, including wild type. C-to-T mutations or C-to-A/G mutations were scored in a 40 bp region including 10 bp upstream and 10 bp downstream of the 20 bp sgRNA target sequence. The C-to-T editing percentage is defined as the total number of sequencing reads with either one or more C-to-T mutations within the 40 bp region divided by the total number of sequencing reads, including wild type. The percentage of C-to-A/G mutations are calculated similarly.
APOBEC3A deaminase-Cas9D10A editor was evaluated for efficiency of C-to-T conversion activity and the span of the C-to-T conversion window. C-to-T conversion activity and window results were compared against construct BC27 encoding BE3 (Komor A C, Kim Y B, Packer M S, Zuris J A, Liu D R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016; 533(7603):420-424.) Constructs were each evaluated in triplicate against 5 sgRNA in a single experiment.
Plasmid A, using a pUC19 backbone (GenBank® Accession Number U47119), expresses an S. pyogenes single-guide RNA (sgRNA) from a U6 promoter. Plasmid B, called pCI, expresses base editor constructs from a CMV promoter which are comprised of the candidate deaminase fused to S. pyogenes-D10A-Cas9 by an XTEN linker which is subsequently fused to one copy of UGI and one copy of SV40 NLS. U-2OS cells growing in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) in 96-well plates were transfected using Mirus TransIT-X2® with 100 ng each of plasmid A and plasmid B. Cells were washed and resuspended in fresh media 24 hours after initial transfection. After an additional 48 hours, the media was removed and the cells were lysed with QuickExtract™ DNA Extraction Solution (Lucigen, Cat. QE09050).
Construct BC22 encoding the H. sapiens APOBEC3A deaminase (Uniprot ID P31941) in the fusion had significantly greater C-to-T conversion activity than BC27 encoding the rat APOBEC1 deaminase (Uniprot ID P38483) with more than one guide (sg000296: P value 0.0303; sg001373, P value 0.0263). The average activity of BC22 was comparable to that of BC27 across all guides (Table 6,
Across all 5 sgRNAs tested, BC22 was significantly more likely to convert all target cytosines to thymidines than BC27 (Table 7,
BC22 converted a significantly larger proportion and positional range of target cytosine than BC27 (
The effects of additional UGI gene expression in trans on C-to-T conversion activity relative to unwanted base excision repair activity were investigated in various cell lines.
U-2OS and HuH-7 cells grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS in 96-well plates were co-transfected using Mirus TransIT-X2® with 100 ng of BC22 or BC27, 100 ng of another CMV-driven overexpression plasmid (pcDNA3.1) or a control plasmid (pMAX) and 6.25 pmol of single-guide G000297. The ORFs tested in tandem with BC22 and BC27 were green fluorescent protein (pMAX-GFP, negative control), Uracil DNA glycosylase (pCDNA3.1-UNG), Single-Strand-Selective Monofunctional Uracil-DNA Glycosylase 1 (pcDNA3.1-SMUG1) and Uracil Glycosylase Inhibitor (pCDNA3.1-UGI). UNG and SMUG1 are base excision repair proteins that remove uracil from DNA. UGI is the small protein that binds and inhibits UDG. It has been reported that when UGI is fused to the constructs it increases the rate of C-to-T mutations relative to the other outcomes of C-to-A/G mutations and indels (Liu et al, Nature 2016). To determine whether the addition of UNG transcript knockdown further enriched for C-to-T-only edits, an additional transfection condition included either BC22 or BC27, pcDNA3.1-UGI, G000297 and a pool of siRNA targeting UNG (Dharmacon, #M-011795-00). Three days after transfection, media was removed and the cells were lysed with QuickExtract™ DNA Extraction Solution (Lucigen, Cat. QE09050).
In HuH-7 cells, overexpressing base excision repair proteins UNG or SMUG1 with either BC22 or BC27 led to a 1.3-1.7-fold significant increase in the proportion of C-to-A/G mutations and indels relative to C-to-T only mutations when compared to overexpression of the negative control GFP (Table 11,
The mRNA encodes a fusion protein, BC22n (SEQ ID NO: 3), which is, from N-terminus to C-terminus, a H. sapiens APOBEC3A, an XTEN linker, a D10ACas9 nickase, a linker, and an SV40 NLS. Notably the BC22n polypeptide lacks a UGI. T cells were edited with BC22n using and a variable amount of UGI mRNA to determine the impact of trans UGI levels on editing profile. This experiment was performed using 2 different UGI mRNAs each encoding the same protein using a different open reading frame (SEQ ID Nos: 26 and 35).
Healthy human donor apheresis was obtained commercially (Hemacare), and cells were washed and re-suspended in CliniMACS® PBS/EDTA buffer (Miltenyi Biotec Cat. No. 130-070-525) on the LOVO device. T cells were isolated via positive selection using CD4 and CD8 magnetic beads (Miltenyi Biotec Cat. No. 130-030-401/130-030-801) using the CliniMACS® Plus and CliniMACS® LS disposable kit. T cells were aliquoted into vials and cryopreserved in a 1:1 formulation of Cryostor® CS10 (StemCell Technologies Cat. No. 07930) and Plasmalyte A (Baxter Cat. No. 2B2522X) for future use. Upon thaw, T cells were plated at a density of 1.0×10{circumflex over ( )}6 cells/mL in T cell basal media composed of X-VIVO 15™ serum-free hematopoietic cell medium (Lonza Bioscience) containing 5% (v/v) of fetal bovine serum, 50 μM of 2-Mercaptoethanol, 10 mM of N-Acetyl-L-(+)-cysteine, 10 U/mL of Penicillin-Streptomycin, in addition to 1× cytokines (200 U/mL of recombinant human interleukin-2, 5 μg/mL of recombinant human interleukin-7 and 5 μg/mL of recombinant human interleukin-15). T cells were activated with TransAct™ (1:100 dilution, Miltenyi Biotec). Cells were expanded in T cell basal media for 72 hours prior to electroporation.
3.2 Electroporation of T Cells Using mRNA and sgRNA
A solution containing mRNAs encoding BC22n (SEQ ID NO: 2) and one species of UGI (SEQ ID NO: 26 or 35) was prepared in sterile water. 50 μM TRAC targeting sgRNA G016017 (SEQ ID NO: 184) were removed from their storage plates and denatured for 2 minutes at 95° C. before cooling on ice. Seventy-two hours post activation, T cells were harvested, centrifuged, and resuspended at a concentration of 12.5×10{circumflex over ( )}6 T cells/mL in P3 electroporation buffer (Lonza). For each well to be electroporated, 1×10{circumflex over ( )}5 T cells were mixed with 200 ng of BC22n mRNA, 20 pmols of sgRNA and UGI mRNA ranging from 0.02 ng to 26 ng (dilution factor of 12.24), in a final volume of 20 μL of P3 electroporation buffer. This mRNA+sgRNA+T cell mix was transferred in triplicate to a 96-well Nucleofector™ plate and electroporated using the manufacturer's pulse code. Electroporated T cells were immediately rested in 80 μL of T cell basal media without cytokines for 10 minutes before being transferred to a new flat-bottom 96-well plate containing an additional 100 μL of T cell basal media with 2× concentration of cytokines. Electroporated T cells were subsequently cultured for 3 additional days and were collected for NGS sequencing as described in Example 1.
The constant dose of 200 ng of BC22n mRNA drove high total editing (>92%) across all conditions (Tables 12 and 13, sum of indel, C-to-A/G and C-to-T). The proportion of edits that were C-to-T-only increased in a dose-responsive manner to UGI mRNA concentration in the electroporation. At the highest dose of either UGI mRNAs SEQ ID NOs: 25 or 34, the percentage of sequencing reads containing indels and C-to-A/G mutations dropped to 7.4±0.4, 7.4±0.6 and 2.9±0.2, 3.6±0.3, respectively (Tables 12 and 13).
LNPs formulated with editor mRNA and sgRNA G000282 at a 1:1 RNA weight ratio were tested for editing efficacy and editing outcomes in vivo. Experimental groups included TSS buffer only; mRNA encoding cleavase-spCas9 (SEQ ID NO: 8), mRNA encoding BC22 which includes human APOBEC3A fused to D10A SpyCas9 and one copy of UGI (SEQ ID NO: 20); and mRNAs encoding BE3 which includes rat APOBEC1 fused to D10A SpyCas9 and one copy of UGI (SEQ ID NOs: 14 and 17).
CD-1 female mice ranging from 6-10 weeks of age (n=5/group) were used in this study. LNPs were administered intravenously via tail vein injection at a dose of 1 mg/kg of total RNA to body weight. The animals were periodically observed for adverse effects for at least 24 hours post dose. Six days after treatment, animals were euthanized by cardiac puncture under isoflurane anesthesia; blood and liver tissue were collected for downstream analysis. Blood was collected into serum separator tubes or into tubes containing buffered sodium citrate for plasma. Liver punches weighting between 5 and 15 mg were collected for isolation of genomic DNA and total RNA.
Table 14 and
In vivo editing profiles of deaminase containing constructs were compared to Cas9 when UGI was delivered in trans (as a separate mRNA). The constructs used encoded a fusion protein including D10A SpyCas9 with a deaminase. Further, gene expression differences between these editing conditions were analyzed through transcriptomic analysis.
Forty-five commercially available CD-1 female mice ranging from 6-10 weeks of age (n=5 per group) were used in this study. Animals were weighed pre-dose for dosing calculations. Each RNA species was formulated separately in an LNP. Formulations containing editor mRNA, UGI mRNA and sgRNA were mixed in a w/w ratio of RNA cargos of 6:3:2 (editor mRNA:sgRNA:UGI mRNA). The formulation mixture for Group 3 contained only editor mRNA and sgRNA and these were mixed in a w/w ratio of 2:1 (editor mRNA:sgRNA). Apart from the negative control group, which was dosed with TSS buffer only, all groups received sgRNA G000282. Formulations were administered intravenously via tail vein injection according to the doses listed in Table 15. Animals were periodically observed for adverse effects for at least 24 hours post-dose. Six days after treatment, animals were euthanized by cardiac puncture under isoflurane anesthesia; liver tissue were collected for downstream analysis. Liver punches weighing between 5 and 15 mg were collected for isolation of genomic DNA and total RNA. Genomic DNA samples were analyzed with NGS sequencing as described in Example 1.
Editing data are shown in Table 16 and
Lowering the lipid dose from 0.3 to 0.1 mg/kg led to lower editing levels 31.39% C-to-T conversions and 3.67% indel).
Liver punches were mixed with 800 μL of TRIzol reagent (Thermo Fisher Scientific, Cat No. 15596026) in Lysing Matrix D tubes (MPBio, Cat. No. 116913100) which contain ceramic beads. Tissue was homogenized in a bead beater for 45 seconds and transferred to ice. Lysing Matrix D tubes were spun down at maximum speed for 5 min at 4C and ˜600 μL of TRIzol (without tissue debris) was mixed with an equal volume of absolute ethanol. The mixture was loaded in the Directzol RNA miniprep column (Zymo Research, Cat No. R2051) and RNA was extracted following the manufacturer's protocol. Purified RNA samples were quantified in a NanoDrop™ 8000 spectrophotometer (Thermo Fisher Scientific) and diluted to 41.67 ng/μL using nuclease-free water. From each experimental group, 2 samples were randomly chosen for further transcriptomic analysis. 500 ng (12 μL) of purified total RNA were depleted of ribosomal RNA (rRNA) components using the NEBNext® rRNA Depletion Kit (New England Biolabs, Cat. No. E6350L) according to the manufacturer's instructions. rRNA-depleted samples were converted into double-stranded DNA libraries using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, Cat No. E7765S) following the manufacturer's protocol. Amplified libraries were quantified in a Qubit 4 fluorometer and the average fragment size of each library was obtained by capillary electrophoresis. Libraries were pooled at an equimolar concentration of 4 nM and pair-end sequenced using a high-output 300-cycle kit (Illumina, Cat No. 20024908) in a NextSeq550 sequencing platform (Illumina).
Data Processing for Differential Gene Expression Analysis
Sequencing reads in FASTQ format were generated and demultiplexed using the bcl2fastq program (Illumina, v2.20). Reads were assigned to a sample if the Hamming distance (Hamming, R. W. Bell Syst. Tech. J. 29, 147-160) between each index read and the sample indexes was less than or equal to one. The sequencing quality was examined with FastQC program (v0.11.9) (Andrews S. Babraham Inst.). Ribosomal RNA reads were identified by aligning all reads to human rRNA sequences (GenBank U13369.1) with Bowtie2 (v2.3.5.1) (Langmead, B. and Salzberg, S. L. Nat. Methods 9, 357-359). Transcriptome quantification was performed using Salmon (v0.14.1) (Patro R., et al. Nat. Methods 14, 417-419) with non-ribosomal RNA reads. Differential gene expression analysis was carried out using DESeq2 (v1.26.0) (Love, M. I., et al. Genome Biol. 15, 550) on the outputs of Salmon. Genes or transcripts with Benjamini-Hochberg adjusted p-values less than 0.05 were determined to be differentially expressed.
RNA-Seq analysis revealed that treatment with BC22n mRNA and UGI mRNA in trans led to only 53 differentially expressed genes, compared to 223 events in those animals treated with BC22n alone (i.e. no UGI mRNA) and 127 events in animals treated with both Cas9 and UGI mRNA in trans (
6.1 Editing in T Cells
T cells were edited at the CIITA locus with UGI in trans and either BC22n or Cas9 to assess the impact on editing type on MHC II antigens.
Healthy human donor apheresis was obtained commercially (Hemacare), and cells were washed and re-suspended in CliniMACS® PBS/EDTA buffer (Miltenyi Biotec Cat. No. 130-070-525) on the LOVO device. T cells were isolated via positive selection using CD4 and CD8 magnetic beads (Miltenyi Biotec Cat. No. 130-030-401/130-030-801) using the CliniMACS® Plus and CliniMACS® LS disposable kit. T cells were aliquoted into vials and cryopreserved in a 1:1 formulation of Cryostor® CS10 (StemCell Technologies Cat. No. 07930) and Plasmalyte A (Baxter Cat. No. 2B2522X) for future use. Upon thaw, T cells were plated at a density of 1.0×10{circumflex over ( )}6 cells/mL in T cell basal media composed of X-VIVO 15™ serum-free hematopoietic cell medium (Lonza Bioscience) containing 5% (v/v) of fetal bovine serum, 50 μM of 2-Mercaptoethanol, 10 mM of N-Acetyl-L-(+)-cysteine, 10 U/mL of Penicillin-Streptomycin, in addition to 1× cytokines (200 U/mL of recombinant human interleukin-2, 5 μg/mL of recombinant human interleukin-7 and 5 μg/mL of recombinant human interleukin-15). T-cells were activated with TransAct™ (1:100 dilution, Miltenyi Biotec). Cells were expanded in T cell basal media containing TransAct™ for 72 hours prior to electroporation.
A solution containing mRNAs encoding Cas9 (SEQ ID NO. 11), BC22n (SEQ ID NO: 2) or UGI (SEQ ID NO: 26) was prepared in sterile water. 50 μM CIITA sgRNAs were removed from their storage plates and denatured for 2 minutes at 95° C. before cooling on ice. Seventy-two hours post activation, T cells were harvested, centrifuged, and resuspended at a concentration of 12.5×10{circumflex over ( )}6 T cells/mL in P3 electroporation buffer (Lonza). For each well to be electroporated, 1×10{circumflex over ( )}5 T cells were mixed with 200 ng of editor mRNA, 200 ng of UGI mRNA and 20 pmols of sgRNA as described in Table 17 in a final volume of 20 uL of P3 electroporation buffer. This mix was transferred in triplicate to a 96-well Nucleofector™ plate and electroporated using the manufacturer's pulse code. Electroporated T cells were immediately rested in 80 μL of T cell basal media without cytokines for 10 minutes before being transferred to a new flat-bottom 96-well plate containing an additional 100 μL of T cell basal media supplemented with 2× cytokines. The resulting plate was incubated at 37° C. for 4 days. After 96 hours, T cells were diluted 1:3 into fresh T cell basal media with 1× cytokines. Electroporated T cells were subsequently cultured for 3 additional days and were collected for flow cytometry analysis, NGS sequencing, and transcriptomics as described in Example 1.
On day 7 post-editing, T cells were phenotyped by flow cytometry to determine MHC class II protein expression. Briefly, T cells were incubated in a cocktail of antibodies targeting HLA-DR, DQ, DP-PE (BioLegend® Cat. No. 361704) and Isotype Control-PE (BioLegend® Cat. No. 400234). Cells were subsequently washed, processed on a Cytoflex flow cytometer (Beckman Coulter) and analyzed using the FlowJo software package. T cells were gated based on size, shape, viability, and MHC II expression. DNA samples were subjected to PCR and subsequent NGS analysis, as described in Example 1. Table 17 and
On day 7 post-editing, T cells treated with G018117 and G18078 were harvested and preserved at −80 C for future processing. Total RNA was extracted from samples in TRIzol™ reagent using the Direct-zol RNA microprep kit (Zymo Research, Cat No. R2062) following the manufacturer's protocol. Purified RNA samples were quantified in a NanoDrop™ 8000 spectrophotometer (Thermo Fisher Scientific) and diluted to 41.67 ng/μL using nuclease-free water. From each experimental triplicate shown in
Sequencing reads in FASTQ format were generated and demultiplexed using the bcl2fastq program (Illumina, v2.20). Reads were assigned to a sample if the Hamming distance (Hamming, R. W. Bell Syst. Tech. J. 29, 147-160) between each index read and the sample indexes was less than or equal to one. The sequencing quality was examined with FastQC program (v0.11.9) (Andrews S. Babraham Inst.). Ribosomal RNA reads were identified by aligning all reads to human rRNA sequences (GenBank U13369.1) with Bowtie2 (v2.3.5.1) (Langmead, B. and Salzberg, S. L. Nat. Methods 9, 357-359). Transcriptome quantification was performed using Salmon (v0.14.1) (Patro R., et al. Nat. Methods 14, 417-419) with non-ribosomal RNA reads. Differential gene expression analysis was carried out using DESeq2 (v1.26.0) (Love, M. I., et al. Genome Biol. 15, 550) on the outputs of Salmon. Genes or transcripts with Benjamini-Hochberg adjusted p-values less than 0.05 were determined to be differentially expressed. Lists of differentially expressed genes were analyzed in terms of gene ontology using Metascape (Zhou, Y., et al. Nat. Comm. 10, 1523). Protein-protein interactions were determined using the BioGrid, InWeb_IM and OmniPath8 databases (Li, T., et al. Nat. Methods 14, 61-64; Stark, C., et al. Nucleic Acids Res. 34, 535-539; Tilrei, D., et al. Nat. Methods 13, 966-967). Densely connected networks were identified using the molecular complex detection (MCODE) algorithm (Bader, G. D., et al. BMC Bioinformatics 4, 1-27) and the three best-scoring terms by p-value were retained as the functional description of the corresponding network components.
Compared to samples treated with Cas9 mRNA, T cells electroporated with BC22n mRNA displayed a significantly stronger downregulation of MHC class II genes and the HLA-associated CD74 gene (Table 18 and 19). Minimal effects on class I MHC genes were observed (Table 20 and Table 21). In terms of transcriptome-wide differential gene expression events, treatment with BC22n mRNA led to fewer differentially expressed genes (p. adjusted<0.05) when compared to Cas9 mRNA. In T cells electroporated with sgRNA G018076, a total of 553 and 65 differential gene expression events were observed for Cas9 and BC22n mRNA treatments, respectively (
T Cell Preparation
Healthy human donor apheresis was obtained commercially (Hemacare), and cells were washed and re-suspended in CliniMACS® PBS/EDTA buffer (Miltenyi Biotec Cat. No. 130-070-525) on the LOVO device. T cells were isolated via positive selection using CD4 and CD8 magnetic beads (Miltenyi Biotec Cat. No. 130-030-401/130-030-801) using the CliniMACS® Plus and CliniMACS® LS disposable kit. T cells were aliquoted into vials and cryopreserved in a 1:1 formulation of Cryostor® CS10 (StemCell Technologies Cat. No. 07930) and Plasmalyte A (Baxter Cat. No. 2B2522X) for future use. Upon thaw, T cells were plated at a density of 1.0×10{circumflex over ( )}6 cells/mL in T cell basal media composed of X-VIVO 15™ serum-free hematopoietic cell medium (Lonza Bioscience) containing 5% (v/v) of fetal bovine serum, 50 μM of 2-Mercaptoethanol, 10 mM of N-Acetyl-L-(+)-cysteine, 10 U/mL of Penicillin-Streptomycin, in addition to 1× cytokines (200 U/mL of recombinant human interleukin-2, 5 μg/mL of recombinant human interleukin-7 and 5 μg/mL of recombinant human interleukin-15). T-cells were activated with TransAct™ (1:100 dilution, Miltenyi Biotec). Cells were expanded in T cell basal media containing TransAct™ for 72 hours prior to electroporation.
mRNA and sgRNA Electroporation of T Cells
A solution containing mRNA encoding Cas9 (SEQ ID NO: 11), BC22n (SEQ ID NO: 2) or UGI (SEQ ID NO: 26) was prepared in sterile water. A 50 μM TRAC targeting sgRNA (G016017) was removed from its storage plate and denatured for 2 minutes at 95° C. before cooling on ice. Seventy-two hours post activation, T cells were harvested, centrifuged, and resuspended at a concentration of 12.5×10{circumflex over ( )}6 T cells/mL in P3 electroporation buffer (Lonza). For each well to be electroporated, 1×10{circumflex over ( )}5 T cells were mixed with 200 ng of each mRNA and 20 pmols of sgRNA in a final volume of 20 μL of P3 electroporation buffer. The T cell mix was transferred in triplicate to a 96-well Nucleofector™ plate and electroporated using the manufacturer's pulse code. Electroporated T cells were immediately rested in 80 μL of T cell basal media without cytokines for 10 minutes before being transferred to a new flat-bottom 96-well plate containing an additional 100 μL of T cell basal media supplemented with 2× cytokines. The resulting plate was incubated at 37° C. for 4 days. After 96 hours, T cells were diluted 1:3 into fresh T cell basal media with 1× cytokines. Electroporated T cells were subsequently cultured for 3 additional days and were collected for NGS sequencing.
NGS Sequencing
On day 7 post-editing, DNA samples were subjected to PCR and subsequent NGS analysis, as described in Example 1. This study characterized the on-target TRAC locus in addition to 10 genomic loci previously described as mutational hotspots in tumor samples positive for APOBEC enzymes (Buisson et al., 2019). The chromosomal location of these sites is listed on Table 22.
Tables 23, 24, and 25 show C-to-T, C-to-A/G and indel editing levels in the on-target locus and predicted APOBEC hotspot sites for all sample groups. A graphical representation of these results in shown in
Using LNP delivery to activated human T cells, the potency of single-target and multi-target editing was assessed with either Cas9 or BC22n.
Healthy human donor apheresis was obtained commercially (Hemacare), and cells were washed and re-suspended in CliniMACS® PBS/EDTA buffer (Miltenyi Biotec Cat. No. 130-070-525) on the LOVO device. T cells were isolated via positive selection using CD4 and CD8 magnetic beads (Miltenyi Biotec Cat. No. 130-030-401/130-030-801) using the CliniMACS® Plus and CliniMACS® LS disposable kit. T cells were aliquoted into vials and cryopreserved in a 1:1 formulation of Cryostor® CS10 (StemCell Technologies Cat. No. 07930) and Plasmalyte A (Baxter Cat. No. 2B2522X) for future use. Upon thaw, T cells were plated at a density of 1.0×10{circumflex over ( )}6 cells/mL in T cell basal media composed of X-VIVO 15™ serum-free hematopoietic cell medium (Lonza Bioscience) containing 5% (v/v) of fetal bovine serum, 50 μM of 2-Mercaptoethanol, 10 mM of N-Acetyl-L-(+)-cysteine, 10 U/mL of Penicillin-Streptomycin, in addition to 1× cytokines (200 U/mL of recombinant human interleukin-2, 5 μg/mL of recombinant human interleukin-7 and 5 μg/mL of recombinant human interleukin-15). T cells were activated with TransAct™ (1:100 dilution, Miltenyi Biotec). Cells were expanded in T cell basal media for 72 hours prior to LNP transfection.
Each RNA species, i.e. UGI mRNA, sgRNA or editor mRNA, was formulated separately in an LNP as described in Example 1. Editor mRNAs encoded either BC22n (SEQ ID NO: 5) or Cas9 (SEQ ID NO: 23). Guides targeting B2M (G015995), TRAC (G016017), TRBC1/2 (G016206) and CIITA (G018117 and G016086) were used either singly or in combination. Messenger RNA encoding UGI (SEQ ID NO: 26) is delivered in both Cas9 and BC22n arms of the experiment to normalize lipid amounts. Previous experiments have established UGI mRNA does not impact total editing or editing profile when used with Cas9 mRNA. LNPs were mixed to fixed total RNA weight ratios of 6:3:2 for editor mRNA, guide RNA, and UGI mRNA respectively as described in Table 26. In the 4-guide experiment described in Table 27, doses of LNPs with individual guides are decreased 4-fold to maintain the overall 6:3 editor mRNA:guide weight ratio and to allow comparison to individual guide potency based on total lipid delivery. LNP mixtures were incubated for 5 minutes at 37° C. in T cell basal media substituting 6% cynomolgus monkey serum (Bioreclamation IVT, Cat. CYN220760) for fetal bovine serum.
Seventy-two hours post activation, T cells were washed and suspended in basal T cell media. Pre-incubated LNP mix was added to the each well with 1×10e5 T cells/well. T cells were incubated at 37° C. with 5% C02 for the duration of the experiment. T cell media was changed 6 days and 8 days after activation and on tenth day post activation, cells were harvested for analysis by NGS and flow cytometry. NGS analysis was performed as described in Example 1.
Table 26 and
Table 27 and
On day 10 post-activation, T cells were phenotyped by flow cytometry to determine if editing resulted in loss of cell surface proteins. Briefly, T cells were incubated in a mix of the following antibodies: B2M-FITC (BioLegend, Cat. 316304), CD3-AF700 (BioLegend, Cat. 317322), HLA DR DQ DP-PE (BioLegend, Cat 361704) and DAPI (BioLegend, Cat 422801). A subset of unedited cells was incubated with Isotype Control-PE (BioLegend® Cat. No. 400234). Cells were subsequently washed, processed on a Cytoflex instrument (Beckman Coulter), and analyzed using the FlowJo software package. T cells were gated based on size, shape, viability, and antigen expression.
Table 28 and
Editing profiles were assessed to determine the concentration of UGI mRNA necessary to achieve highly pure C-to-T editing in T cells with different editing constructs. C-to-T editing purity (% of edited reads containing only C-to-T conversions) was measured using a saturating dose of editor mRNA and sgRNA and varying amounts of UGI mRNA. Editor mRNAs included an mRNA encoding BC22n (SEQ ID NO: 5), an mRNA encoding BC22 with a total of 2 fused UGI moieties (SEQ ID NO: 29), and an mRNA encoding BE4Max which includes 2 fused UGI moieties (SEQ ID NO: 32) (Koblan L W, Doman J L, Wilson C, et al. Nat Biotechnol. 2018; 36(9):843-846).
Solutions containing editor mRNA as listed in Table 29 or UGI mRNA (SEQ ID NO: 26) were prepared in sterile water. 50 μM B2M targeting sgRNA G015995 were removed from their storage plates and denatured for 2 minutes at 95° C. before cooling on ice. Seventy-two hours post activation, T cells were harvested, centrifuged, and resuspended at a concentration of 12.5×10{circumflex over ( )}6 T cells/mL in P3 electroporation buffer (Lonza). For each well to be electroporated, 1×10{circumflex over ( )}5 T cells were mixed with 200 ng of editor mRNAs, 20 pmols of sgRNA and different concentrations of UGI mRNA ranging from 0.8 ng to 600.0 ng (0.8 nM to 597.0 nM), in a final volume of 20 uL of P3 electroporation buffer. This mRNA+sgRNA+T cell mix was transferred in triplicate to a 96-well Nucleofector™ plate and electroporated using the manufacturer's pulse code. Electroporated T cells were immediately rested in CTS™ OpTmizer™ T Cell Expansion serum-free media (SFM) (ThermoFisher Cat. A1048501) without cytokines for 10 minutes before being transferred to a new flat-bottom 96-well plate containing an additional 100 uL of the same media with 2× concentration of cytokines. The resulting plate was incubated at 37° C. for 9 days, during which time the cells were split and media refreshed two times. T cells were collected and processed for NGS sequencing as described in Example 1.
Table 29 and
CIITA gRNAs were screened for efficacy in T cells by assessing knockdown of MHC class II cell surface expression using both Cas9 and BC22. The percentage of T cells negative for MHC class II protein was assayed following CIITA editing by electroporation with RNP.
Cas9 editing activity was assessed using electroporation of Cas9 ribonucleoprotein (RNP). Upon thaw, Pan CD3+ T cells (StemCell, HLA-A*02.01/A*03.01) were plated at a density of 0.5×10{circumflex over ( )}6 cells/mL in T cell RPMI media composed of RPMI 1640 (Invitrogen, Cat. 22400-089) containing 5% (v/v) of fetal bovine serum, 1× Glutamax (Gibco, Cat. 35050-061), 50 μM of 2-Mercaptoethanol, 100 uM non-essential amino acids (Invitrogen, Cat. 11140-050), 1 mM sodium pyruvate, 10 mM HEPES buffer, 1% of Penicillin-Streptomycin, and 100 U/mL of recombinant human interleukin-2 (Peprotech, Cat. 200-02). T cells were activated with TransAct™ (1:100 dilution, Miltenyi Biotec). Cells were expanded in T cell RPMI media for 72 hours prior to RNP transfection.
CIITA targeting sgRNAs were removed from their storage plates and denatured for 2 minutes at 95° C. before cooling at room temperature for 10 minutes. RNP mixture of 20 uM sgRNA and 10 uM recombinant Cas9-NLS protein (SEQ ID No. 36) was prepared and incubated at 25° C. for 10 minutes. Five μL of RNP mixture was combined with 100,000 cells in 20 μL P3 electroporation Buffer (Lonza). 22 μL of RNP/cell mix was transferred to the corresponding wells of a Lonza shuttle 96-well electroporation plate. Cells were electroporated in duplicate with the manufacturer's pulse code. T cell RPMI media was added to the cells immediately post electroporation. Electroporated T cells were subsequently cultured and collected for NGS sequencing as described in Example 1 at 2 days post edit.
On day 10 post-edit, T cells were phenotyped by flow cytometry to determine HLA class II protein expression. Briefly, T cells were incubated in cocktails of antibodies targeting HLA-DR, DQ, DP-PE (BioLegend® Cat. No. 361704) and Isotype Control-AF647 (BioLegend® Cat. No. 400234). Cells were subsequently washed, processed on a Cytoflex flow cytometer (Beckman Coulter) and analyzed using the FlowJo software package. T cells were gated based on size, shape, viability, and MHC II expression. DNA samples were subjected to PCR and subsequent NGS analysis, as described in Example 1.
10.3. mRNA Electroporation of T Cells
BC22 editing activity was assayed following CIITA editing by electroporation with mRNA and guide. Upon thaw, Pan CD3+ T cells isolated from a commercially obtained leukopak (StemCell) were plated at a density of 0.5×10{circumflex over ( )}6 cells/mL in T cell R10 media composed of RPMI 1640 (Invitrogen, Cat. 22400-089) containing 10% (v/v) of fetal bovine serum, 2 mM Glutamax (Gibco, Cat. 35050-061), 22 μM of 2-Mercaptoethanol, 100 uM non-essential amino acids (Invitrogen, Cat. 11140-050), 1 mM sodium pyruvate, 10 mM HEPES buffer, 1% of Penicillin-Streptomycin, plus 100 U/mL of recombinant human interleukin-2 (Peprotech, Cat. 200-02). T cells were activated with Dynabeads® Human T-Activator CD3/CD28 (ThermoFisher). Cells were expanded in T cell for 72 hours prior to mRNA transfection.
CIITA sgRNAs were removed from their storage plates and denatured for 2 minutes at 95° C. before cooling at room temperature for 10 minutes. Fifty microliter electroporation mix was prepared with 100,00 T cells in P3 buffer (Lonza) and 10 ng/uL mRNA encoding UGI (SEQ ID NO: 26), 10 ng/uL mRNA encoding BC22 (SEQ ID NO: 20) and 2 uM sgRNA. This mix was transferred to the corresponding wells of a Lonza shuttle 96-well electroporation plate. Cells were electroporated in duplicate wells using Lonza shuttle 96 w program with the manufacturer's pulse code. R10 media with IL-2 was added to the cells immediately post electroporation. Electroporated T cells were subsequently cultured and collected for NGS sequencing and flow cytometry 10 days post edit. Flow cytometry was performed as described for Cas9 RNP treated cells above. DNA samples were subjected to PCR and subsequent NGS analysis, as described in Example 1.
Table 31 and
HLA-A guide RNAs were screened for efficacy in T cells by assessing loss of HLA-A cell surface expression. The percentage of T cells negative for HLA-A protein in an HLA-A2 background (“% HLA-A2-”) was assayed by flow cytometry following HLA-A editing by mRNA delivery.
11.1. mRNA Electroporation of T Cells
Cas9 and BC22n editing activity was assessed using electroporation of mRNA encoding Cas9 (SEQ ID NO: 11), mRNA encoding BC22n (SEQ ID NO: 2), or mRNA encoding UGI (SEQ ID NO: 26), as provided below. Upon thaw, Pan CD3+ T cells (StemCell, HLA-A*02.01/A*02.01) were plated at a density of 1×10{circumflex over ( )}6 cells/mL in TCGM composed of CTS OpTmizer T Cell Expansion SFM (Thermofisher, Cat. A3705001) supplemented with 5% human AB serum (Gemini, Cat. 100-512), 1× GlutaMAX (Thermofisher, Cat. 35050061), 10 mM HEPES (Thermofisher, Cat. 15630080), 1× of Penicillin-Streptomycin, further supplemented with 200 U/mL IL-2 (Peprotech, Cat. 200-02), 10 ng/ml IL-7 (Peprotech, Cat. 200-07), 10 ng/ml IL-15 (Peprotech, Cat. 200-15). T cells were activated with TransAct™ (1:100 dilution, Miltenyi Biotec). Cells were expanded in T cell RPMI media for 72 hours at 37° C. prior to mRNA electroporation.
HLA-A sgRNAs were removed from their storage plates and denatured for 2 minutes at 95° C. before incubating at room temperature for 5 minutes. BC22n electroporation mix was prepared with 100,000 T cells in P3 buffer (Lonza), 200 ng of mRNA encoding UGI, 200 ng of mRNA encoding BC22n and 20 pmoles of sgRNA. Cas9 electroporation mix was prepared with 100,000 T cells in P3 buffer (Lonza), 200 ng of mRNA encoding UGI, 200 ng of mRNA encoding Cas9 and 20 pmoles of sgRNA. Each mix was transferred to the corresponding wells of a Lonza shuttle 96-well electroporation plate. Cells were electroporated in duplicate using Lonza shuttle 96 w using manufacturer's pulse code. Immediately post electroporation, cells were recovered in pre-warmed TCGM without cytokines and incubated at 37° C. for 15 minutes. Electroporated T cells were subsequently cultured in TCGM with further supplemented with 200 U/mL IL-2 (Peprotech, Cat. 200-02), 10 ng/ml IL-7 (Peprotech, Cat. 200-07), 10 ng/ml IL-15 (Peprotech, Cat. 200-15) and collected for flow cytometry 8 days post edit.
On day 8 post-edit, T cells were phenotyped by flow cytometry to determine HLA-A protein expression. Briefly, T cells were incubated with antibodies targeting HLA-A2, (eBioscience Cat. No. 17-9876-42). Cells were subsequently washed, processed on a Cytoflex flow cytometer (Beckman Coulter) and analyzed using the FlowJo software package. T cells were gated based on size, shape, viability, and HLA-A2 expression. Table 32 shows the percentage of cells negative for HLA-A surface proteins following genomic editing of HLA-A with BC22n or Cas9.
T cells were edited at the CIITA locus with UGI in trans and either BC22 or Cas9 to assess the impact on editing type on MHC class II antigens.
T cells were prepared from a leukopak using the EasySep Human T cell Isolation Kit (Stem Cell Technology, Cat. 17951) following the manufacturers protocol. T cells were cryopreserved in Cryostor CS10 freezing media (Cat. 07930) for future use. Upon thaw, T cells were plated at a density of 1.0×10{circumflex over ( )}6 cells/mL in T cell R10 media composed of RPMI 1640 (Corning, Cat. 10-040-CV) containing 10% (v/v) of fetal bovine serum, 2 mM Glutamax (Gibco, Cat. 35050-061), 22 μM of 2-Mercaptoethanol, 100 uM non-essential amino acids (Corning, Cat. 25-025-Cl), 1 mM sodium pyruvate, 10 mM HEPES buffer, 1% of Penicillin-Streptomycin, plus 100 U/mL of recombinant human interleukin-2 (Peprotech, Cat. 200-02). T cells were activated with Dynabeads® Human T-Activator CD3/CD28 (Gibco, Cat. 11141D). Cells were expanded in T cell media for 72 hours prior to mRNA transfection.
12.2 T Cell Editing with RNA Electroporation
Solutions containing mRNA encoding Cas9 protein (SEQ ID NO: 8), BC22 (SEQ ID NO: 20) or UGI (SEQ ID NO 26) were prepared in sterile water. 50 μM CIITA targeting sgRNAs were removed from their storage plates and denatured for 2 minutes at 95° C. before cooling on ice. Seventy-two hours post activation, T cells were harvested, centrifuged, and resuspended at a concentration of 12.5×10{circumflex over ( )}6 T cells/mL in P3 electroporation buffer (Lonza). For each well to be electroporated, 1×10{circumflex over ( )}5 T cells were mixed with 200 ng of editor mRNA, 200 ng of UGI mRNA and 20 pmols of sgRNA as described in Table 33 in a final volume of 20 uL of P3 electroporation buffer. This mix was transferred in duplicate to a 96-well Nucleofector™ plate and electroporated using the manufacturer's pulse code. Electroporated T cells were rested in 180 ul of R10 media plus 100 U/mL of recombinant human interleukin-2 before being transferred to a new flat-bottom 96-well plate. The resulting plate was incubated at 37° C. for 4 days. On day 10 post-editing cells were collected for flow cytometry analysis and NGS sequencing.
On day 10 post-editing, T cells were phenotyped by flow cytometry to determine MHC class II protein expression as described in Example 6 using antibodies targeting HLA-DR, DQ, DP-PE (BioLegend® Cat. No. 361704) and Isotype Control-PE (BioLegend® Cat. No. 400234). DNA samples were subjected to PCR and subsequent NGS analysis, as described in Example 1. Table 33 shows CIITA gene editing and MHC class II negative results for cells edited with BC22. Table 34 shows CIITA gene editing and MHC class II negative results for cells edited with Cas9.
Three guides from Table 33, G016086, G016092, and G016067, were further characterized for editing efficacy with increasing amounts of guide and in combination with guides targeting TRAC (G013009, G016016, or G016017) and B32M (G015991, G015995, or G015996).
Cell preparation, activation, and electroporation were performed as described in Example 6 with the following deviations. Editing was performed using two miRNA species encoding BC22 (SEQ ID NO: 20) and UGI (SEQ ID NO:26) respectively. Editing was assessed at multiple concentrations of sgRNA, as indicated in Table 35 and Table 36. When multiple guides were used in a single reaction, each guide represented one quarter of the total guide concentration.
On day 10 post-editing, T cells were phenotyped by flow cytometry to determine MHC class II protein expression as described in Example 6. In addition, B32M detection was performed with B2M-FITC antibody (BioLegend, Cat. 316304) and CD3 expression was assayed using CD3-BV605 antibody (BioLegend, Cat. 317322).
DNA samples were subjected to PCR and subsequent NGS analysis, as described in Example 1. Table 35 provides MHC Class II negative flow cytometry results and NGS editing for cells edited with BC22 and individual guides targeting CIITA, with
TRAC gRNAs were screened for efficacy in T cells by assessing knockdown of CD3 surface expression using both Cas9 with UGI in trans and BC22 with UGI in trans. The percentage of T cells negative for CD3 protein and the percentage of editing at the TRAC locus was assayed following TRAC editing by electroporation with mRNA and gRNA.
T cells were prepared from a leukopak using the EasySep Human T cell Isolation Kit (Stem Cell Technology, Cat. 17951) following the manufacturers protocol. T cells were cryopreserved in Cryostor CS10 freezing media (Cat. 07930) for future use. Upon thaw, T cells were plated at a density of 1.0×10{circumflex over ( )}6 cells/mL in T cell R10 media composed of RPMI 1640 (Corning, Cat. 10-040-CV) containing 10% (v/v) of fetal bovine serum, 2 mM Glutamax (Gibco, Cat. 35050-061), 22 μM of 2-Mercaptoethanol, 100 uM non-essential amino acids (Corning, Cat. 25-025-Cl), 1 mM sodium pyruvate, 10 mM HEPES buffer, 1% of Penicillin-Streptomycin, plus 100 U/mL of recombinant human interleukin-2 (Peprotech, Cat. 200-02). T cells were activated with Dynabeads® Human T-Activator CD3/CD28 (Gibco, Cat. 11141D). Cells were expanded in T cell media for 72 hours prior to mRNA transfection.
Solutions containing mRNA encoding Cas9 protein (SEQ ID NO: 8), BC22 (SEQ ID NO: 20) or UGI (SEQ ID NO 26) were prepared in sterile water. 50 μM TRAC targeting sgRNAs were removed from their storage plates and denatured for 2 minutes at 95° C. before cooling on ice. Seventy-two hours post activation, T cells were harvested, centrifuged, and resuspended at a concentration of 12.5×10{circumflex over ( )}6 T cells/mL in P3 electroporation buffer (Lonza). For each well to be electroporated, 1×10{circumflex over ( )}5 T cells were mixed with 200 ng of editor mRNA, 200 ng of UGI mRNA and 20 pmols of sgRNA as described in Table 37 in a final volume of 20 uL of P3 electroporation buffer. This mix was transferred in triplicate to a 96-well Nucleofector™ plate and electroporated using the manufacturer's pulse code. Electroporated T cells were rested in 180 ul of R10 media plus 100 U/mL of recombinant human interleukin-2 before being transferred to a new flat-bottom 96-well plate. The resulting plate was incubated at 37° C. for 6 days. On day 9 post-editing cells were collected for flow cytometry analysis and NGS sequencing.
On day 9 post-editing, T cells were phenotyped by flow cytometry to determine CD3 protein expression as described in Example 6 using antibodies targeting CD3 (BioLegend® Cat. No. 317322) and Isotype Control-PE (BioLegend® Cat. No. 400269). DNA samples were subjected to PCR and subsequent NGS analysis, as described in Example 1. The mean percentage of each editing type at the TRAC locus and the mean number of CD3 negative cells after editing with BC22 and UGI are showing in Table 37; results after editing with Cas9 and UGI are shown in Table 38. C-to-T editing purity is calculated as the percentage of edited reads containing only C-to-T conversions.
TRBC gRNAs were screened for efficacy in T cells by assessing knockdown of CD3 surface expression using both Cas9 with UGI in trans and BC22 with UGI in trans. The percentage of T cells negative for CD3 protein and the percentage of editing of each type at the TRBC1 locus was assayed following TRBC editing by electroporation with mRNA and gRNA.
T cells were prepared from a leukopak using the EasySep Human T cell Isolation Kit (Stem Cell Technology, Cat. 17951) following the manufacturers protocol. T cells were cryopreserved in Cryostor CS10 freezing media (Cat. 07930) for future use. Upon thaw, T cells were plated at a density of 1.0×10{circumflex over ( )}6 cells/mL in T cell R10 media composed of RPMI 1640 (Corning, Cat. 10-040-CV) containing 10% (v/v) of fetal bovine serum, 2 mM Glutamax (Gibco, Cat. 35050-061), 22 μM of 2-Mercaptoethanol, 100 uM non-essential amino acids (Corning, Cat. 25-025-Cl), 1 mM sodium pyruvate, 10 mM HEPES buffer, 1% of Penicillin-Streptomycin, plus 100 U/mL of recombinant human interleukin-2 (Peprotech, Cat. 200-02). T cells were activated with Dynabeads® Human T-Activator CD3/CD28 (Gibco, Cat. 11141D). Cells were expanded in T cell media for 72 hours prior to mRNA transfection.
Solutions containing mRNA encoding Cas9 protein (SEQ ID NO: 8), BC22 (SEQ ID NO: 20) or UGI (SEQ ID NO 26) were prepared in sterile water. 50 μM TRBC targeting sgRNAs were removed from their storage plates and denatured for 2 minutes at 95° C. before cooling on ice. Seventy-two hours post activation, T cells were harvested, centrifuged, and resuspended at a concentration of 12.5×10{circumflex over ( )}6 T cells/mL in P3 electroporation buffer (Lonza). For each well to be electroporated, 1×10{circumflex over ( )}5 T cells were mixed with 200 ng of editor mRNA, 200 ng of UGI mRNA and 20 pmols of sgRNA as described in Table 39 in a final volume of 20 uL of P3 electroporation buffer. This mix was transferred in triplicate to a 96-well Nucleofector™ plate and electroporated using the manufacturer's pulse code. Electroporated T cells were rested in 180 ul of R10 media plus 100 U/mL of recombinant human interleukin-2 before being transferred to a new flat-bottom 96-well plate. The resulting plate was incubated at 37° C. for 6 days. On day 9 post-editing cells were collected for flow cytometry analysis and NGS sequencing.
On day 9 post-editing, T cells were phenotyped by flow cytometry to determine CD3 protein expression as described in Example 6 using antibodies targeting CD3 (BioLegend® Cat. No. 317322) and Isotype Control-PE (BioLegend® Cat. No. 400269). DNA samples were subjected to PCR and subsequent NGS analysis, as described in Example 1. The mean percentage of each editing type at the TRAC locus and the mean number of CD3 negative cells after editing with BC22 and UGI are showing in Table 39; results after editing with Cas9 and UGI are shown in Table 40. C-to-T editing purity is calculated as the percentage of edited reads containing only C-to-T conversions.
Healthy human donor apheresis was obtained commercially (Hemacare), and cells were washed and re-suspended in CliniMACS® PBS/EDTA buffer (Miltenyi Biotec Cat. No. 130-070-525) on the LOVO device. T cells were isolated via positive selection using CD4 and CD8 magnetic beads (Miltenyi Biotec Cat. No. 130-030-401/130-030-801) using the CliniMACS®) Plus and CliniMACS®) LS disposable kit. T cells were aliquoted into vials and cryopreserved in Cryostor® CS10 (StemCell Technologies Cat. 07930) for future use. Upon thawing, T cells were plated at a density of 1.0×10{circumflex over ( )}6 cells/nmL in T cell growth (TCG) media consisting of CTS OpTmiizer T cell expansion serum-free media (Thermofisher, Cat. A3705001) supplemented with 5% human AB serum (Gemini, Cat. 100-512), 1× GlutaMAX (Thermofisher, Cat. 35050061), 10 mM HEPES (Thermofisher, Cat. 15630080) and 1× of Penicillin-Streptomycin, further supplemented with 200 U/mL IL-2 (Peprotech, Cat. 200-02), 10 ng/ml IL-7 (Peprotech, Cat. 200-07), 10 ng/ml IL-15 (Peprotech, Cat. 200-15). T cells were activated with TransAct™ (1:100 dilution, Miltenyi Biotec, Cat. 130-111-160). Cells were expanded for 72 hours at 37° C. prior to mRNA electroporation.
A solution containing mRNA encoding Cas9 (SEQ ID NO: 11), BC22n (SEQ ID NO: 2), UGI (SEQ ID NO: 26) or BE4MAX (SEQ ID NO: 32) was prepared in sterile water. A 50 μM B2M targeting sgRNA (G015995) was removed from its storage plate and denatured for 2 minutes at 95° C. before cooling at room temperature. Seventy-two hours post activation, T cells were harvested, centrifuged, and resuspended at a concentration of 12.5×10{circumflex over ( )}6 T cells/mL in P3 electroporation buffer (Lonza). For each well to be electroporated, 1×10{circumflex over ( )}5 T cells were mixed with 200 ng of mRNAs and 20 pmols of sgRNA in a final volume of 20 uL of P3 electroporation buffer. The T cell mix was transferred in 5 replicates to a 96-well Nucleofector™ plate and electroporated using the manufacturer's pulse code. Electroporated T cells were immediately rested in 80 μL of TCG media without cytokines for 15 minutes before being transferred to a new flat-bottom 96-well plate containing an additional 100 μL of TCG media supplemented with 2× cytokines cited in Section 16.1.
To evaluate RNA off-targets when expression level of BC22n peaks, a fraction of edited T cells was collected 24 h post-electroporation. This fraction was further divided into 2 plates, one of which was subjected to cell lysis, PCR amplification and NGS analysis, while the other fraction was used for RNA extraction and transcriptome sequencing. On day 3 post-electroporation, the remaining T cells were collected for cell lysis and NGS sequencing, which enabled confirmation of maximum B2M editing in these samples at a timepoint when editing is normally complete.
At 24 and 72 h post-electroporation, T cells were subjected to lysis, PCR amplification of the B2M locus and subsequent NGS analysis, as described in Example 1. Table 41 and
At 24 h post-electroporation, T cells were centrifuged, and the cell pellet was resuspended in 200 uL of TRIzol™ reagent (Thermo Fisher Scientific, Cat No. 15596026) which was frozen at −80 C for future processing. Total RNA was extracted from samples in TRIzol™ reagent using the Direct-zol RNA microprep kit (Zymo Research, Cat No. R2062) following the manufacturer's protocol. Purified RNA samples were quantified in a NanoDrop™ 8000 spectrophotometer (Thermo Fisher Scientific) and diluted to 18.18 ng/uL using nuclease-free water. From each experimental group shown in
Paired-end reads were aligned to human genome GRCh38 with STAR v2.7.1a (Dobin et al., 2013). PCR duplicates were removed with Picard MarkDuplicates v2.19.0 (BroadInstitute, 2019). GATK tools SplitNCigarReads, BaseRecalibrator, ApplyBQSR v4.1.8.1 were deployed consecutively to preprocess alignments. Variations were called with GATK HaplotypeCaller (Auwera et al., 2013; DePristo et al., 2011; McKenna et al., 2010). Variants discovered from replicates of the same sample were merged using bcftools v1.8 (Li, 2011). Sample specific variants were retrieved by excluding variants discovered in controls using vcflib v1.0.0 (Garrison, 2016). Relative C to U frequencies were calculated by dividing the number of C to U variants by the total number of SNVs for each sample. Treatment groups were compared using unpaired t-tests and the statistical significance was determined using the Holm-Sidak method with an alpha of 0.05, without assuming a consistent standard deviation.
Compared to samples treated with Cas9 mRNA, T cells electroporated with both Cas9 and UGI mRNAs, BC22n and UGI mRNAs or BE4MAX and UGI mRNAs showed no statistically significant (p<0.05) increase in the frequency of C to U transitions, demonstrating the absence of detectable homology-independent cytosine deamination events in the transcriptome of these samples (Table 42 and
Healthy human donor apheresis was obtained commercially (Hemacare), and cells were washed and re-suspended in CliniMACS® PBS/EDTA buffer (Miltenyi Biotec Cat. No. 130-070-525) on the LOVO device. T cells were isolated via positive selection using CD4 and CD8 magnetic beads (Miltenyi Biotec Cat. No. 130-030-401/130-030-801) using the CliniMACS® Plus and CliniMACS® LS disposable kit. T cells were aliquoted into vials and cryopreserved in Cryostor® CS10 (StemCell Technologies Cat. 07930) for future use. Upon thawing, T cells were plated at a density of 1.0×10{circumflex over ( )}6 cells/mL in T cell growth (TCG) media consisting of CTS OpTmizer T cell expansion serum-free media (Thermofisher, Cat. A3705001) supplemented with 5% human AB serum (Gemini, Cat. 100-512), 1× GlutaMAX (Thermofisher, Cat. 35050061), 10 mM HEPES (Thermofisher, Cat. 15630080) and 1× of Penicillin-Streptomycin, further supplemented with 200 U/mL IL-2 (Peprotech, Cat. 200-02), 10 ng/ml IL-7 (Peprotech, Cat. 200-07), 10 ng/ml IL-15 (Peprotech, Cat. 200-15). T cells were activated with TransAct™ (1:100 dilution, Miltenyi Biotec, Cat. 130-111-160). Cells were expanded for 72 hours at 37° C. prior to mRNA electroporation.
A solution containing mRNA encoding Cas9 (SEQ ID NO: 11), BC22n (SEQ ID NO: 2) or UGI (SEQ ID NO: 26) was prepared in sterile water. A 50 μM B2M targeting sgRNA (G015995) was removed from its storage plate and denatured for 2 minutes at 95° C. before cooling at room temperature. Seventy-two hours post activation, T cells were harvested, centrifuged, and resuspended at a concentration of 12.5×10{circumflex over ( )}6 T cells/mL in P3 electroporation buffer (Lonza). For each well to be electroporated, 1×10{circumflex over ( )}5 T cells were mixed with 200 ng of mRNAs and 20 pmols of sgRNA in a final volume of 20 uL of P3 electroporation buffer. The T cell mix was transferred in 8 replicates to a 96-well Nucleofector™ plate and electroporated using the manufacturer's pulse code. Electroporated T cells were immediately rested in 80 μL of TCG media without cytokines for 15 minutes before being transferred to a new flat-bottom 96-well plate containing an additional 100 IL of TCG media supplemented with 2× cytokines.
To promote expansion, T cells were split at the ratios of 1:4 and 1:3 on days 3 and 6 post-electroporation, respectively, using fresh TCG media with 1× cytokines. On day 7 post-electroporation, a fraction of cells was collected for flow cytometry and NGS sequencing, while the remaining cells were frozen for subsequent single-cell whole genome amplification and sequencing.
On day 7 post-electroporation, T cells were phenotyped by flow cytometry to evaluate loss of B2M expression levels following editing with sgRNA G015995. Briefly, T cells were incubated for 30 min at 4° C. with a mixture of antibodies against CD3 (BioLegend® Cat. No. 317340), CD4 (BioLegend® Cat. No. 300537), CD8 (BioLegend® Cat. No. 344706) and B2M (BioLegend® Cat. No. 316314), diluted at 1:200 in cell staining buffer (BioLegend® Cat. No. 420201). Cells were subsequently washed, processed on a Cytoflex flow cytometer (Beckman Coulter) and analyzed using the FlowJo software package. T cells were gated based on size, shape and B2M expression. Table 43 and
On day 7 post-electroporation, T cells were also subjected to lysis, PCR amplification of the B2M locus and subsequent NGS analysis, as described in Example 1. Table 44 and
One sample from each group of 8 replicates was randomly chosen for single cell isolation, whole genome amplification and sequencing. Frozen cells from samples 12, 21 and 30 (Table 44) were transferred to a contract research organization (Singulomics Corporation, Inc.) where 10 single T cells were isolated from each sample. These single T cells were lysed, and their genomes were amplified using multiple displacement amplification (MDA), according to previously published methods (Dong et al., Nat Methods, 2017). Amplified genomes were subjected to PCR amplification of the B2M locus and subsequent NGS analysis, as described in Example 1, aiming to confirm the edited genotype in single T cells. From each group of 10 single cells, 6 DNA samples were converted into whole genome sequencing libraries using the KAPA HyperPlus kit (Roche, Cat. 07962410001), following the manufacturer's protocol. The resulting 18 libraries were sequenced in an Illumina NovaSeq 6000 platform using an S4 reagent kit v1.5 (Illumina, Cat. 20028312).
Paired-end reads were aligned to human genome GRCh38 with BWA-MEM v0.7.17 (Li, 2013). PCR duplicates were removed with Picard MarkDuplicates v2.19.0 (Broad Institute, 2019). Subsequently, base scores were corrected using GATK BaseRecalibrator and ApplyBQSR v4.1.8.1 (Auwera et al., 2013; DePristo et al., 2011; McKenna et al., 2010). Variants were called using DeepVariant v1.0.0 (Poplin et al., 2018). Relative C to T frequencies were calculated by dividing the total number of C to T variants by the total number of SNVs for each sample. Treatment groups were compared using unpaired t-tests and the statistical significance was determined using the Holm-Sidak method with an alpha of 0.05, without assuming a consistent standard deviation.
Compared to samples treated with Cas9 mRNA, T cells electroporated with both Cas9 and UGI mRNAs or those treated with BC22n and UGI mRNAs, showed no statistically significant (p<0.05) increase in the frequency of C to T transitions in amplified genomic DNA, demonstrating the absence of detectable homology-independent cytosine deamination events in these samples (Table 45 and
Fully haploid, engineered Hap1 (eHap1) cells were obtained commercially (Horizon Discovery Cat. C669), and cells were cultured in IMDM growth media composed of Iscove's Modified Dulbecco's medium (Thermofisher Cat. 12440053) supplemented with 10% (v/v) of fetal bovine serum (Thermofisher Cat. A3840001) and 1× of Penicillin-Streptomycin (Thermofisher Cat. 15140122). Upon thawing, eHap1 cells were cultured for 48 hours at 37° C. and seeded 24 hours prior to LNP treatments at the density of 6×10{circumflex over ( )}5 cells/well in 6-well plates, which were incubated at 37° C. until treatment.
The B2M-targeting sgRNA G015991 and mRNAs encoding Cas9 (SEQ ID NO: 11), BC22n (SEQ ID NO: 2), UGI (SEQ ID NO: 26) or BE4MAX (SEQ ID NO: 32) were formulated as individual RNA species in LNPs as described in Example 1. LNPs were applied to T cells in different combinations with at the following concentrations of total RNA: 0.104 μg/mL editor mRNA; 0.55 μg/mL UGI mRNA; 0.4175 μg/mL sgRNA. Different LNP combinations (Table 46) were pre-mixed in IMDM growth media supplemented with 10 μg/mL of recombinant human ApoE3 (Peprotech Cat. 350-02) and incubated at 37° C. for 15 minutes. The culture media of eHap1 cells was removed and each well received 3 mL of LNP mixture. Untreated controls received 3 mL of IMDM growth media supplemented with 10 μg/mL of ApoE3 without LNPs. Cells were incubated for 24 hours at 37° C. and the media was removed and replaced by IMDM growth media.
Three and five days after treatment, eHap1 cells were detached, re-seeded at a lower density, and returned to the 37° C. incubator. At the 5-day timepoint, a fraction of cells was stained with anti-B2M antibodies (BioLegend Cat. No. 316304) to evaluate the loss of B2M expression by flow cytometry. These cells were incubated for 30 min at 4° C. with anti-B2M antibodies diluted 1:200 in cell staining buffer (BioLegend® Cat. No. 420201). Cells were subsequently washed, processed on a Cytoflex flow cytometer (Beckman Coulter) and analyzed using the FlowJo software package. eHap1 cells were gated based on size, shape and B2M expression. Table 46 and
Seven days after treatment, eHap1 cells were detached, the replicates from each treatment group were pooled and the resulting cell suspension was centrifuged. A small fraction of cells from each pool was collected for bulk NGS sequencing as described in Example 1. Table 47 and
After 10 days in culture, clones were visually inspected under an inverted fluorescence microscope. Twelve clones from each treatment group were transferred to 6-well plates to enable further expansion. A small fraction of cells from each clone was collected for NGS sequencing as described in Example 1, while the remaining cells were seeded in 6-plates and cultured at 37° C. until confluence was reached. Based on their on-target editing outcomes, 5 clones per group were selected for whole genome sequencing. Cells from all clonal populations, in addition to one non-clonal sample of eHap1 cells, were lysed and their genomic DNA was extracted using the DNeasy Blood & Tissue kit (Qiagen Cat. 69504) following the manufacturer's protocol. DNA samples were converted into whole genome sequencing libraries using the KAPA HyperPlus kit (Roche, Cat. 07962410001), following the manufacturer's instructions. The resulting 36 libraries were sequenced in an Illumina NovaSeq 6000 platform using an S4 reagent kit v1.5 (Illumina, Cat. 20028312).
Reads from each sample were first aligned to the human genome build hg38 with bwa (v0.7.17) (Li, 2013; Li and Durbin, 2010). Alignments were processed with samtools (v1.11) modules fixmate, sort and markdup, consecutively (Kumaran et al., 2019; Li et al., 2009). Variations were called from processed alignments using DeepVariant (v1.0.0) (Poplin et al., 2018). Variants from each sample were then merged using GLnexus (v1.3.1) (Lin et al., 2018; Yun et al., 2021). Variants that occurred in the non-clonal sample of eHap1 cells were excluded from all clonal samples. Variants with read depth below 10 or genotype quality score below 15 were ignored as well. Relative C to T frequencies were calculated by dividing the total number of C to T variants by the total number of SNVs for each sample. Treatment groups were compared using unpaired t-tests and the statistical significance was determined using the Holm-Sidak method with an alpha of 0.05, assuming a consistent standard deviation.
Compared to untreated controls, eHap1 cells treated with Cas9, BC22n or BE4MAX mRNA, in the absence or presence of UGI mRNA, did not show a statistically significant (p<0.05) increase in the frequency of C to T transitions in the genome, demonstrating the absence of detectable homology-independent cytosine deamination events in these samples (Table 48 and
To assess the amount of structural genomic changes associated with delivery conditions and editing by Cas9 or base editor, T cells treated with electroporation to deliver RNP or lipid nanoparticles (LNP) to deliver four guides and either Cas9 or BC22n were analyzed for cell viability, DNA double-stranded breaks, editing, surface protein expression, and chromosomal structural.
Healthy human donor apheresis was obtained commercially (Hemacare), and cells were washed and re-suspended in CliniMACS® PBS/EDTA buffer (Miltenyi Biotec Cat. No. 130-070-525) on the LOVO device. T cells were isolated via negative selection using EasySep™ Human T Cell Isolation Kit (Stemcell Cat. No. 17951). T cells were aliquoted into vials and cryopreserved in a 1:1 formulation of Cryostor® CS10 (StemCell Technologies Cat. No. 07930) and Plasmalyte A (Baxter Cat. No. 2B2522X) for future use.
Upon thaw, T cells were plated at a density of 1.0×10{circumflex over ( )}6 cells/mL in OpTmizer-based media containing CTS OpTmizer T Cell Expansion SFM and T Cell Expansion Supplement (ThermoFisher Cat. A1048501), 5% human AB serum (GeminiBio, Cat. 100-512) 1× Penicillin-Streptomycin, 1× Glutamax, 10 mM HEPES, 200 U/mL recombinant human interleukin-2 (Peprotech, Cat. 200-02), 5 ng/ml recombinant human interleukin 7 (Peprotech, Cat. 200-07), and 5 ng/ml recombinant human interleukin 15 (Peprotech, Cat. 200-15). T cells were activated with TransAct™ (1:100 dilution, Miltenyi Biotec) in this media for 72 hours, at which time they were washed and plated in quadruplicate for editing either by electroporation or lipid nanoparticle.
LNPs were formulated generally as in Example 1 with a single RNA species cargo. Cargo was selected from an mRNA encoding BC22n, an mRNA encoding Cas9, an mRNA encoding UGI, sgRNA G015995 targeting B2M, sgRNA G016017 targeting TRAC, sgRNA G016200 targeting TRBC or sgRNA G016086 targeting CIITA. Each LNP was incubated in OpTmizer-based media with cytokines as described above supplemented with 20 ug/ml recombinant human ApoE3 (Peprotech, Cat. 350-02) for 15 minutes at 37° C. Seventy-two hours post activation, T cells were washed and suspended in OpTmizer media with cytokines without human serum. For single sgRNA editing conditions, pre-incubated LNP mix was added to the each well of 100,000 cells to yield a final concentration of 2.3 ug/ml editor mRNA (BC22n or Cas9), 1.1 ug/mL UGI and 4.6 ug/uL G016017. For four-plex sgRNA editing LNP mix was added to the each well of 100,000 cells to yield a final concentration of 2.3 ug/ml editor mRNA (BC22n or Cas9), 1.1 ug/mL UGI, 1.15 ug/uL G015995, 1.15 ug/uL G016017, 1.15 ug/uL G016200 and 1.15 ug/uL G016086. A control group including unedited T cells (no LNP) was also included. At 16 hours post-delivery, a subset of cells was used to measure cell viability and another subset of cells was processed for imaging of γH2AX foci. The remaining T cells continued to expand in culture. Media was changed 5 days and 8 days after activation and on the eleventh day post activation, cells were harvested for analysis by NGS, flow cytometry and UnIT. NGS was performed as in Example 1.
Electroporation was performed 72 hours post activation. sgRNA G015995 (SEQ ID NO: 182) targeting B2M, sgRNA G016017 (SEQ ID NO: 184) targeting TRAC, sgRNA G016200 (SEQ ID NO:801) targeting TRBC and sgRNA G016086 (SEQ ID NO: 586) were denatured for 2 minutes at 95° C. before cooling at room temperature for 10 minutes. T cells were harvested, centrifuged, and resuspended at a concentration of 12.5×10{circumflex over ( )}6 T cells/mL in P3 electroporation buffer (Lonza). For single sgRNA editing conditions, 1×10≡T cells were mixed with 40 ng/uL of editor mRNA (BC22n or Cas9), 10 ng/uL of UGI mRNA and 80 pmols of sgRNA in a final volume of 20 uL of P3 electroporation buffer. For four-plex sgRNA editing conditions, 1×10{circumflex over ( )}5 T cells were mixed with 40 ng/uL of editor mRNA (BC22n or Cas9), 10 ng/uL of UGI mRNA and 20 pmols of the four individual sgRNA in a final volume of 20 uL of P3 electroporation buffer. This mix was transferred in quadruplicate to a 96-well Nucleofector™ plate and electroporated using a manufacturer's pulse code. Electroporated T cells were rested in 80 ul of OpTmizer-based media with cytokines before being transferred to a new flat-bottom 96-well plate. A control group including unedited T cells (no EP) was also included. At 16 hours post-delivery, a subset of cells was used to measure cell viability and another subset of cells was processed for imaging of γH2AX foci.
Sixteen hours post electroporation or lipid nanoparticle delivery 20 uL of control or edited cells were removed from original plate and added to a new flat-bottom 96-well plate with black walls (Corning Cat. 3904). CellTiter-Glo® 2.0 (Promega Cat. G9241) was added and samples were processed according to manufacturer's protocol. Relative luminescence units (RLU) were readout by the CLARIstar plus (BMG Labtech) plate reader with gain set at 3600. Relative viability as shown in Table 49 and
16 hours post electroporation or lipid nanoparticle delivery T cells were cytospun to a slide using Cytospin 4 (Thermo Fisher). After 5 min pre-extraction in PBS/0.5% Trion X-100 on ice, cells were fixed in 4% paraformaldehyde for 10 min. Then, cells were washed in PBS several times and blocked in PBS/0.1% TX-100/1% BSA for 30 min. Primary antibody (Mouse anti-phospho-Histone H2A.X (Ser139) (Millipore Cat. 05-636) was incubated in the blocking buffer at 4° C. overnight. After washed in PBS/0.05% Tween-20 three times, secondary antibody (Goat anti-Mouse IgG Alexa 568 (Thermo Fisher Cat. A31556) was incubated in the blocking buffer at room temperature for 30 min. Cells were washed in PBS/0.05% Tween-20 and nuclei were counter stained with Hoechst 33342. Images were generated by confocal imaging with the Leica SP8. Image analysis was performed via a custom protocol on Thermo Scientific HCS Studio Cell Analysis Software Spot Detector module. Table 50 and
On day 8 post-editing, T cells were phenotyped by flow cytometry to determine B2M, CD3 and HLA II-DR, DP, DQ protein expression as described in Example 6 using antibodies targeting B2M-APC/Fire™ 750 (BioLegend® Cat. No. 316314), CD3-BV605 (BioLegend® Cat. No. 316314) and HLA II-DR, DP, DQ-PE (BioLegend® Cat. No. 361716). DNA samples were subjected to PCR and subsequent NGS analysis, as described in Example 1. Table 51 and
On day 8 post-editing a subset of T cells from the untreated, LNP-Cas9-4 guides and LNP-BC22n-4 guides samples were collected, spun down and resuspended in 100 uL of PBS. gDNA was isolated from the cells using DNeasy Blood & Tissue Kit (Qiagen Cat. 69504). The UnIT structural variant characterization assay was applied to these gDNA samples. High molecular weight genomic DNA is simultaneously fragmented and sequence-tagged (‘tagmented’) with the Tn5 transposase and an adapter with a partial Illumina P5 sequence and a 12 bp unique molecular identifier (UMI). Two sequential PCRs using a primer to P5 and hemi-nested gene specific primers (GSP) imparting the Illumina the P7 sequence to create two Illumina compatible NGS libraries per sample. Sequencing across both directions of the CRISPR/Cas9 targeted cut site with the two libraries allows the inference and quantification of structural variants in DNA repair outcomes after genome editing. If the two fragments were aligned to different chromosomes, the SV was classified as an “inter-chromosomal translocation.” Structural variation results show that interchromosomal translocations are reduced to background levels when multiplex editing is being conducted by BC22n whereas Cas9 multiplex editing leads to significant increases in structural variation, as shown in Table 53 and
T cells were edited at the CD38 locus with either Cas9 or with BC22n and UGI mRNAs to assess the editing outcomes and the corresponding loss of CD38 expression.
Healthy human donor apheresis was obtained commercially (Hemacare), and cells were washed and resuspended in CliniMACS PBS/EDTA buffer (Miltenyi Biotec, Cat. No. 130-070-525) on the LOVO device. T cells were isolated via positive selection using CD4 and CD8 magnetic beads (Miltenyi Biotec, Cat. No. 130-030-401/130-030-801) using the CliniMACS Plus and CliniMACS LS disposable kit. T cells were aliquoted into vials and cryopreserved in Cryostor CS10 (StemCell Technologies, Cat. No. 07930) for future use. Upon thaw, T cells were plated at a density of 1.0×10{circumflex over ( )}6 cells/mL in T cell X-VIVO 15 expansion media composed of X-VIVO 15 (Lonza, Cat. No. BE02-06Q) containing 5% (v/v) of fetal bovine serum (ThermoFisher, Cat. No. A3160902), 50 μM (1×) 2-Mercaptothanol (ThermoFisher, Cat. No. 31350010), 1% of Penicillin-Streptomycin (ThermoFisher, Cat. No. 15140122), 1 M N-acetyl L-cystine (Fisher, Cat. No. ICN19460325) diluted in phosphate buffered saline (PBS) and normalized to pH 7, supplemented with 100 U/mL of recombinant human interleukin-2 (Peprotech, Cat. No. 200-02), 5 ng/mL recombinant human interleukin-7 (Peprotech, Cat. No. 200-07) and 5 ng/mL recombinant human interleukin-15 (Peprotech, Cat. No. 200-15). T cells were activated with TransAct™ (1:100 dilution, Miltenyi Biotec, Cat. No. 130-111-160). Cells were expanded for 72 hours at 37° C. prior to mRNA electroporation.
Solutions containing mRNA encoding Cas9 protein, BC22n, or UGI were prepared in sterile water. 50 μM CD38 targeting sgRNAs were removed from their storage plates and denatured for 2 minutes at 95° C. and incubated at room temperature for 5 minutes. Seventy-two hours post activation, T cells were harvested, centrifuged, and resuspended at a concentration of 12.5×10{circumflex over ( )}6 T cells/mL in P3 electroporation buffer (Lonza). Messenger RNAs were prepared as described in Example 1. For each well to be electroporated, 1×10{circumflex over ( )}5 T cells were mixed with 200 ng of Cas9 or BC22n mRNAs, 200 ng of UGI mRNA and 20 pmols of sgRNA as described in Table 54 in a final volume of 20 μL of P3 electroporation buffer. This mix was transferred in duplicate to a 96-well Nucleofector™ plate and electroporated using the manufacturer's pulse code. Electroporated T cells were immediately rested in 80 μL of X-VIVO 15 media without cytokines for 15 minutes before being transferred to new flat-bottom 96-well plates containing an additional 90 μL of X-VIVO 15 media supplemented with 2× cytokines. The resulting plates were incubated at 37° C. for 10 days. To promote expansion, T cells were split at the ratios of 1:4 and 1:3 on days 3 and 6 post-electroporation, respectively, using fresh X-VIVO 15 media with 1× cytokines. On day 9 post-electroporation, cells were split 1:2 in 2 U-bottom plates and one plate was collected for NGS sequencing, while the other plate was used for flow cytometry on Day 10.
On day 10 post-editing, T cells were phenotyped by flow cytometry to determine CD38 receptor expression. Briefly, T cells were incubated for 30 min at 4° C. with a mixture of antibodies against CD3 (BioLegend, Cat. No. 317340), CD4 (BioLegend, Cat. No. 300537), CD8 (BioLegend, Cat. No. 344706) diluted at 1:200 and CD38 (BioLegend, Cat. No. 303546), diluted at 1:100 in cell staining buffer (BioLegend, Cat. No. 420201). Cells were subsequently washed and stained with viability antibody DAPI (BioLegend, Cat. No. 422801) diluted at 1:10,000 in cell staining buffer. Cells were then processed on a Cytoflex flow cytometer (Beckman Coulter) and analyzed using the FlowJo software package. T cells were gated based on size, shape, viability, and CD38 expression.
On day 9 DNA samples were subjected to PCR and subsequent NGS analysis, as described in Example 1. Table 54 shows CD38 gene editing and CD38 positive results for cells edited with BC22n or Cas9.
Base editor constructs comprising an APOBEC3A deaminase domain fused to Nme2Cas9 D16A nickase were tested for base conversion efficiency with various guide designs in HepG2 cells.
HepG2 cells constitutively overexpress solute carrier family 10 member 1 (SLC10A1) (HepG2-NTCP, Seeger et al. Mol Ther Nucleic Acids. 2014 Dec.; 3(12): e216) were thawed and resuspended in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) (Media Y) followed by centrifugation. The supernatant was discarded and the cells were resuspended in Media Y and plated at a density of 25,000 cells per well in a 96-well collagen coated plate (Corning, Cat. 354407) in 100 uL of Media Y.
Nme2Cas9 base editor mRNAs were prepared by in vitro transcription essentially as described in Example 1 from plasmids encoding SEQ ID No: 304 (2XNLS N-terminal, lxC-terminal NLS Nme2Cas9 base editor), SEQ ID No: 310 (2XNLS N-terminal, NLS Nme2Cas9 base editor), and SEQ ID No: 301 (1×C-term NLS Nme2Cas9 base editor). SpyCas9 mRNA and uracil glycosylase inhibitor (UGI) mRNA (SEQ ID No: 34) were transcribed from plasmids using the same method.
Chemically modified NmeCas9 sgRNAs targeted to NTCP, with different PAM sequences, (G020927, G020928) or VEGF (G020073) and SpyCas9 sgRNA targeted to NTCP (G020929) were synthesized using routine methods.
The tested NmeCas9 sgRNAs targeting NTCP include a 24 nucleotide guide sequence (as represented by N) and a guide scaffold as follows: mN*mNNNNNNNNmNNNmNNNNNNNNNNNNmGUUGmUmAmGmCUCCCmUmGm AmAmAmCmCGUUmGmCUAmCAAU*AAGmGmCCmGmUmCmGmAmAmAmGmAm UGUGCmCGCmAmAmCmGCUCUmGmCCmUmUmCmUGmGCmAmUC*mG*mU*mU (SEQ ID NO: 522), where A, C, G, U, and N are adenine, cytosine, guanine, uracil, and any ribonucleotide, respectively, unless otherwise indicated. An m is indicative of a 2′O-methyl modification, and an * is indicative of a phosphorothioate linkage between the nucleotides. Unmodified and modified versions of the guides are provided in Table 5C. Guide RNA, editor mRNA, and UGI mRNA were mixed at a 1:1:1 weight ratio with premixed transfection reagent containing Lipid A, cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:3 molar ratio, respectively. Reagents were combined at a lipid amine to RNA phosphate (N:P) molar ratio of about 6.0. RNA-lipid mixture was mixed approximately 1:1 with 10% FBS media and incubated for 10 minutes. Post-incubation, the cells were treated with the RNA-lipid mixture in an 8-point, 2-fold serial dilution starting at 400 ng total editor RNA per well.
At 72 hours post-treatment, cells were lysed for NGS analysis as provided in Example 1.
Dose response of editing efficiency to guide concentration was performed in triplicate. Table 55 shows mean editing percentages calculated [at each guide concentration and a calculated EC50 value. The target site in VEGFA is prone to indel formation due to high GC content. All editor miRNAs achieved the same maximum C to T editing. There were slight differences in EC50 where SEQ ID No: 310 mRNA outperformed SEQ ID No: 304 and SEQ ID No: 301.
Base editor constructs comprising an APOBEC3A deaminase domain fused to Nme2Cas9 nickase were tested for base conversion efficiency with various guide designs in primary mouse hepatocytes (PMH).
PMH (In Vitro ADMET Laboratories, cat #MC148) were thawed and resuspended in 50 mL Cryopreserved Hepatocyte Recovery Media (CHRM) (Invitrogen, CM7000) followed by centrifugation. Cells were resuspended in hepatocyte medium with plating supplements: Williams' E Medium Plating Supplements with FBS content (Gibco, Cat. A13450). Cells were pelleted by centrifugation, resuspended in media and plated at a density of 20,000 cells/well on Bio-coat collagen I coated 96-well plates (Corning #354407). Plated cells were allowed to settle and adhere for 4-6 hours in a tissue culture incubator at 37° C. and 5% CO2 atmosphere. After incubation cells were checked for monolayer formation and were washed once and plated with 100 uL hepatocyte maintenance medium: Williams' E Medium (Gibco, Cat. A12176-01) plus supplement pack (Gibco, Cat. CM3000).
Nme2Cas9 base editor mRNAs, SEQ ID No: 304, SEQ ID No: 310, and SEQ ID No: 301; and uracil glycosylase inhibitor (UGI) mRNA (SEQ ID No: 34) were prepared as described in Example 1 and paired with a series of chemically modified sgRNA targeted to mouse TTR and screened at a single dose of 128 ng of base editor mRNA. At 72 hours post-treatment, cells were lysed for NGS analysis as provided in Example 1. The mean editing of representative guides (ratio of edit types) are shown in Table 56.
The editing efficiency of the modified gRNAs with different mRNAs were tested with a Nme2Cas9 base editor construct in the mouse model.
LNPs were generally prepared as described in Example 1 with a single RNA species as cargo. The LNPs used were prepared with Lipid A, cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:3 molar ratio, respectively. The LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 6. The LNPs were formulated as described in Example 1, except that each component, guide RNA, or mRNA was formulated individually into an LNP, and the LNP were mixed prior to administration as described in Table 57. For Nme2Cas9 and Nme2Cas9 base editor samples, LNPs were mixed at a ratio of 2:1 by weight of gRNA to editor mRNA cargo. For SpyCas9 base editor samples, LNPs were mixed at a ratio of 1:2 by weight of gRNA to editor mRNA cargo. Dose, as indicated in Table 57 and
The tested NmeCas9 gRNA (G021844) including linkers has the following modification pattern of (N)24
where A, C, G, U, and N are adenine, cytosine, guanine, uracil, and any ribonucleotide, respectively, unless otherwise indicated. An m is indicative of a 2′O-methyl modification, and an * is indicative of a phosphorothioate linkage between the nucleotides. Unmodified and modified versions of the guides are provided in Table 5C (Sequence Table).
CD-1 female mice, ranging 6-10 weeks of age were used in each study involving mice (n=5 per group, except TSS control n=4). Formulations were administered intravenously via tail vein injection according to the doses listed in Table 57. Animals were periodically observed for adverse effects for at least 24 hours post-dose. Six days after treatment, animals were euthanized by cardiac puncture under isoflurane anesthesia; liver tissue were collected for downstream analysis. Liver punches weighing between 5 and 15 mg were collected for isolation of genomic DNA and total RNA. Genomic DNA was extracted using a DNA isolation kit (ZymoResearch, D3010) and samples were analyzed with NGS sequencing as described in Example 1. The editing efficiency for LNPs containing the indicated gRNAs are shown in Table 57 and illustrated in
Candidate deamiinase-Cas9 fusion constructs were evaluated on efficiency of C to T conversion activity. All experimental deaminase C to T conversion activity were compared against construct BC27 encoding BE3 (Komor A C, Kim Y B, Packer M S, Zuris J A, Liu D R Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016; 533(7603):420-424.) In total, 56 deaminase-Cas9 fusion constructs as mentioned in Table 58 were each evaluated in triplicate against sg005883 in a single experiment. The constructs were designed and methods performed as described in Example 1A.
Table 59 and
M. musculus
M. musculus
M. smegmatis
B. pseudomallei
M. marinum
B. anthracis
M. tuberculosis
M. leprae
B. subtilis
S. cerevisiae
B. anthracis
M. musculus
H. sapiens
S. cerevisiae
C. elegans
C. elegans
H. sapiens
H. sapiens
N. europaea
H. sapiens
M. musculus
H. sapiens
H. sapiens
H. sapiens
M. musculus
M. musculus
R. norvegicus
M. domestica
H. sapiens
O. cuniculus
M. kandleri
E. coli
V. cholerae
M. musculus
A. thalia
M. mulatta
H. sapiens
C. aethiops
H. sapiens
M. mulatta
H. sapiens
P. troglodytes
H. sapiens
M. musculus
M. musculus
M. musculus
M. musculus
M. musculus
M. musculus
M. musculus
E. coli
M. mulatta
C. acetobutylicum
K. pneumoniae
H. sapiens
H. sapiens
A set of sixty-eight amino acid linkers listed in Table 60 encompassing various lengths and flexibilities were encoded into the region between the N-terminal cytosine deaminase and C-terminal Cas9 nickase of expression plasmid BC27 as mentioned in Example 1A. Selected base editor constructs described in Table 58 were redesigned with the substitution of the XTEN linker between the cytosine deaminase domain and the Spy Cas9 nickase domain with a linker from Table 60. The BC27 linker screen was conducted using sg001373 with target sequence UCCCUGGCUGAGGAUCCCCA (SEQ ID NO: 157). The other 10 deaminase domain linker screens were conducted using sg005883 with target sequence CCCCCCGCCGUGUUUGUGGG (SEQ ID NO: 159)
Plasmid A, uses a pUC19 backbone (GenBank® Accession Number U47119), expresses an S. pyogenes single-guide RNA (sgRNA) from a U6 promoter. Plasmid B, called pCI, expresses base editor constructs from a CMV promoter which are comprised of the candidate deamiinase fused to S. pyogenes-D10A-Cas9 by an experimental linker which is subsequently fused to one copy of UGI and one copy of SV40 NLS. U-2OS cells growing in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) in 96-well plates were transfected using Mirus TransIT-X2® with 100 ng each of plasmid A and plasmid B. 100 uL of fresh media was added 24 hours after initial transfection. After an additional 48 hours, the media was removed and the cells were lysed with QuickExtract™ DNA Extraction Solution (Lucigen, Cat. QE09050). Table 61A and 61B lists the percent of total reads containing at least 1 cytosine to thymidine conversion across all deaminase domains tested and representative data is shown in
Select base editor constructs derived from BC22 but with linkers from Table 60 substituted between the cytosine deaminase and Cas9 nickase were assayed for C to T base editing activity using Lipofectamine transfection of mRNA and gRNA. Tested base editor mRNAs include SEQ ID NOs: 19 and 347-357. Constructs were screened in a dilution series ranging from 150 ng to 1.17 ng of base editor mRNA in Huh-7 cells, co-delivered with a dilution series of SERPINA1 sgRNA (G000255) ranging from 20 to 0.15 nM and UGI mRNA (SEQ ID No: 25) at a dose of 25 to 0.20 ng in 100 uL of media. C to T base editing of each construct was compared to BC22 (SEQ ID No: 19).
Huh-7 cells growing in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) in 96-well plates were transfected using Lipofectamine® RNAiMAX with an 8-point, two-fold dilution series starting with a maximum dose of 150 ng base editor mRNA, 25 ng UGI mRNA and 20 nM sgRNA in 100 uL media.
Seventy-two hours post transfection, Huh-7 cells were subjected to lysis, PCR amplification of the SERPINA1 locus and subsequent NGS analysis, as described in Example 1. Table 62 and
Select base editor constructs derived from BC22n but with linkers from Table 65 substituted between the cytosine deaminase and Cas9 nickase were assayed for C to T base editing activity in PHH. Constructs (SEQ ID NOs: 341-346) were screened in a 12-point dilution series of base editor mRNA in PHH cells, co-delivered with a fixed mass of ANAPC5 sgRNA (G019427) and UGI mRNA (SEQ ID NO: 34). The C to T editing efficiency of each tested based editor construct was compared to BC22n (SEQ ID NO: 1).
PHH cells are thawed and recovered in CHRMs media (Gibco, cat #CM7000). They are then resuspended in Primary Hepatocyte Plating Media (Consisting of William's E media (Gibco, Cat #A1217601) and primary hepatocyte plating supplements (Gibco, Cat #CM3000))) to be plated in collagen-coated 96-well plates at a density of 33,000 cells/well for twenty-four hours. Post twenty-four hours, cells are washed, and fresh primary hepatocyte maintenance media is added on cells. Cells are then transfected simultaneously with separate lipoplexes formed individually with either UGI mRNA (SEQ ID No: 34), ANAPC5 sgRNA G019427 or base editor mRNA.
Lipofection reagent was prepared as mixture of lipids at a ratio of 50/9/38/3 Lipid A, DSPC, cholesterol and PEG2k-DMG as described in Example 1. Lipofection reagent was combined by bulk mixing with separately with each RNA species: base editor mRNA, UGI mRNA or gRNA G019427. The materials were combined at a lipid amine to RNA phosphate (N:P) molar ratio of about 6. The resulting bulk-mixed lipoplex material (lipid kits) was pre-incubated with 10% FBS (Gibco; A3160501) in Primary Hepatocyte Maintenance Media (Consisting of William's E media (Gibco, Cat #A1217601) and primary hepatocyte maintenance supplements (Gibco, Cat #CM4000)), for 15 min before addition to hepatocytes.
Each well received three components in a final volume of 100 uL: base editor mRNAs ranging from 400 ng to 0 ng of mRNAs, 30 ng of UGI mRNA and 5 pmols of G019427 (ANAPC5).
Seventy-two hours post transfection, PHH cells were subjected to lysis, PCR amplification of the ANAPC5 locus and subsequent NGS analysis, as described in Example 1. Table 66 and
C to T base editing for a range of base editor mRNAs doses is shown in Table 67. The EC95 (mass of BC22n mRNA required to edit 95% of maximum C to T edits) was calculated as shown in Table 68 and
Select base editor constructs derived from BC22n but with linkers from Table 65 substituted between the cytosine deaminase and Cas9 nickase were assayed for C to T base editing activity. Constructs (SEQ ID Nos: 341-346) were screened in a 12-point dilution series of base editor mRNA in healthy human T cells, co-delivered with a fixed amount of TRAC sgRNA (G016017) and of UGI mRNA (SEQ ID NO: 34). The C to T editing efficiency of each base editor mRNA construct was compared to BC22n (SEQ ID NO: 1).
Healthy human donor apheresis was obtained commercially (Hemacare), and cells were washed, re-suspended in CliniMACS® PBS/EDTA buffer (Miltenyi Biotec Cat. 130-070-525) and processed in a MultiMACS™ Cell 24 Separator Plus device (Miltenyi Biotec). T cells were isolated via positive selection using a Straight from Leukopak® CD4/CD8 MicroBead kit, human (Miltenyi Biotec Cat. 130-122-352). T cells were aliquoted and cryopreserved for future use in Cryostor® CS10 (StemCell Technologies Cat. 07930).
Upon thaw, T cells were plated at a density of 1.0×10{circumflex over ( )}6 cells/mL in T cell growth media (TCGM) composed of CTS OpTmizer T Cell Expansion SFM and T Cell Expansion Supplement (ThermoFisher Cat. A1048501), 5% human AB serum (GeminiBio, Cat. 100-512) 1× Penicillin-Streptomycin, 1× Glutamax, 10 mM HEPES, 200 U/mL recombinant human interleukin-2 (Peprotech, Cat. 200-02), 5 ng/ml recombinant human interleukin 7 (Peprotech, Cat. 200-07), and 5 ng/ml recombinant human interleukin 15 (Peprotech, Cat. 200-15). T cells were rested in this media for 24 hours, at which time they were activated with T Cell TransAct™, human reagent (Miltenyi, Cat. 130-111-160) added at a 1:100 ratio by volume. T cells were activated for 48 hours prior to use in screening experiments.
A sgRNA targeting TRAC (G016017) was denatured for 2 minutes at 95° C., incubated at room temperature for 5 minutes and stored on ice. 48 hours post-activation, T cells were harvested, centrifuged at 500 g for 5 min, and resuspended at a concentration of 12.5×10{circumflex over ( )}6 T cells/mL in P3 electroporation buffer (Lonza). For each well to be electroporated, 1×10{circumflex over ( )}5 T cells were mixed with a decreasing amount of base editor mRNAs ranging from 400 ng to 0 ng, 200 ng of UGI mRNA and 40 pmols of G016017 in a final volume of 20 uL of P3 electroporation buffer.
The resulting mix was transferred in duplicate to 96-well Nucleofector™ plates (Lonza) and electroporated using the manufacturer's pulse code. Immediately after electroporation, T cells received 80 μL of TCGM and plates were incubated at 37° C. for 15 minutes. After incubation, 80 μL were transferred to new flat-bottom 96-well plates containing 80 μL of TCGM. T cells were incubated at 37° C. for 4 days, at which time they were mixed, split 1:4 with fresh TCGM, and incubated at for 3 additional days prior to phenotypic assessments by flow cytometry.
Four days post electroporation, T cells were subjected to lysis, PCR amplification of the TRAC locus and subsequent NGS analysis, as described in Example 1. Table 69 and
None of the linkers tested between the N-terminal cytosine deaminase and C-terminal Cas9 nickase limit the efficiency of cytosine base editing or impacts the levels of C to T editing purity.
Seven days post electroporation, T cells were assayed by flow cytometry to evaluate loss of CD3 expression. T cells were incubated with a fixable viability dye (Beckman Coulter, Cat. C36628) and an antibody cocktail targeting the following molecules: CD3 (Biolegend, Cat. 317336), CD4 (Biolegend, Cat. 317434) and CD8 (Biolegend, Cat. 301046). Cells were subsequently washed, analyzed on a Cytoflex LX instrument (Beckman Coulter) using the FlowJo software package. T cells were gated on size, viability and CD8 positivity before expression of any markers was determined. The resulting data was plotted on GraphPad Prism v. 9.0.2 and analyzed using a variable slope (four parameter) non-linear regression.
As shown in Table 70, treatment with >100 ng of any of base editor mRNA tested resulted in >95% of CD8+ T cells lacking expression of CD3. The EC90s of the base editor mRNAs tested (i.e., mass of mRNA that leads to 90% of CD8+ T cells lacking CD3) with 95% confidence interval of each non-linear regression are shown in Table 71 and
Select base editor constructs derived from BC22n but with linkers from Table 65 substituted between the cytosine deaminase and Cas9 nickase were assayed for multiple simultaneous base editing efficacy via protein knockdown. Constructs were screened in a 12-point dilution series of base editor mRNA in healthy human T cells, co-delivered with a fixed mass of UGI mRNA (SEQ ID NO: 34) and 4 distinct 91-mer sgRNAs. The potency of each mRNA construct was compared to BC22n mRNA (SEQ ID NO: 1) to evaluate the effects of each linker in terms of phenotypic receptor knockout.
Healthy human donor apheresis was obtained commercially (Hemacare), and cells were washed, re-suspended in CliniMACS® PBS/EDTA buffer (Miltenyi Biotec Cat. 130-070-525) and processed in a MultiMACS™ Cell 24 Separator Plus device (Miltenyi Biotec). T cells were isolated via positive selection using a Straight from Leukopak® CD4/CD8 MicroBead kit, human (Miltenyi Biotec Cat. 130-122-352). T cells were aliquoted and cryopreserved for future use in Cryostor® CS10 (StemCell Technologies Cat. 07930).
Upon thaw, T cells were plated at a density of 1.0×10{circumflex over ( )}6 cells/mL in T cell growth media (TCGM) composed of CTS OpTmizer T Cell Expansion SFM and T Cell Expansion Supplement (ThermoFisher Cat. A1048501), 5% human AB serum (GeminiBio, Cat. 100-512) 1× Penicillin-Streptomycin, 1× Glutamax, 10 mM HEPES, 200 U/mL recombinant human interleukin-2 (Peprotech, Cat. 200-02), 5 ng/ml recombinant human interleukin 7 (Peprotech, Cat. 200-07), and 5 ng/ml recombinant human interleukin 15 (Peprotech, Cat. 200-15). T cells were rested in this media for 24 hours, at which time they were activated with T Cell TransAct™, human reagent (Miltenyi, Cat. 130-111-160) added at a 1:100 ratio by volume. T cells were activated for 48 hours prior to use in screening experiments.
Four 91-mer sgRNAs targeting TRAC (G023520), TRBC1/2 (G023524), CIITA (G023521) and HLA-A (G023523) were denatured for 2 minutes at 95° C., incubated at room temperature for 5 minutes and stored on ice. 48 hours post-activation, T cells were harvested, centrifuged at 500 g for 5 min, and resuspended at a concentration of 12.5×10{circumflex over ( )}6 T cells/mL in P3 electroporation buffer (Lonza). For each well to be electroporated, 1×10{circumflex over ( )}5 T cells were mixed with a dilution series of base editor mRNAs from 400 ng to 0 ng (SEQ ID NOs: 1 and 321-326), 200 ng of UGI mRNA (SEQ ID NO: 34), 3.6 pmols of G023520 (TRAC), 6.96 pmols of G023524 (TRBC1/2), 17.12 pmols of G023521 (CIITA) and 52.24 pmols of G023523 (HLA-A) in a final volume of 20 uL of P3 electroporation buffer.
The resulting mix was transferred in duplicate to 96-well Nucleofector™ plates (Lonza) and electroporated using the manufacturer's pulse code. Immediately after electroporation, T cells received 80 μL of TCGM and plates were incubated at 37° C. for 15 minutes. After incubation, 80 μL were transferred to new flat-bottom 96-well plates containing 80 μL of TCGM. T cells were incubated at 37° C. for 4 days, at which time they split 1:4 with fresh TCGM, and incubated at for 3 additional days prior to phenotypic assessments by flow cytometry.
Seven days post electroporation, T cells were assayed by flow cytometry to evaluate loss of CD3, HLA-A3 and/or HLA-DR, DP, DQ. T cells were incubated with a fixable viability dye (Beckman Coulter, Cat. C36628) and an antibody cocktail targeting the following molecules: CD3 (Biolegend, Cat. 317336), CD4 (Biolegend, Cat. 317434), CD8 (Biolegend, Cat. 301046), HLA-A3 (ThermoFisher, Cat. 12-5754-42) and HLA-DP, DQ, DR (Biolegend, Cat. 361714). Cells were subsequently washed, analyzed on a Cytoflex LX instrument (Beckman Coulter) using the FlowJo software package. T cells were gated on size, viability and CD8 positivity before expression of any markers was determined. The resulting data was plotted on GraphPad Prism v. 9.0.2 and analyzed using a variable slope (four parameter) non-linear regression.
As shown in Table 72 treatment with >100 ng of any of BC22n mRNA tested resulted in >97% of CD8+ T cells lacking expression of CD3, HLA-A3 and HLA-DR, DP, DQ. The EC90s of the base editor mRNAs tested (i.e., mass of mRNA that leads to 90% of CD8+ T cells lacking CD3) with 95% confidence interval of each non-linear regression are shown in Table 73 and
The base editing efficacy of 91-mer sgRNAs as assessed by NGS and/or receptor knockout was compared to that of 100-mer sgRNA controls with the same guides sequences.
The tested 91-mer sgRNAs include a 20 nucleotide guide sequence (as represented by N) and a guide scaffold as follows: mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmGmAmAmAmU mAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCACGAAAGGGCACCGAGUCG GmUmGmC*mU (SEQ ID NO: 520), where A, C, G, U, and N are adenine, cytosine, guanine, uracil, and any ribonucleotide, respectively, unless otherwise indicated. An m is indicative of a 2′O-methyl modification, and an * is indicative of a phosphorothioate linkage between the nucleotides. Unmodified and modified versions of the guides are provided in Table 5C (Sequence Table).
Healthy human donor apheresis was obtained commercially (Hemacare), and cells were washed, re-suspended in CliniMACS® PBS/EDTA buffer (Miltenyi Biotec Cat. 130-070-525) and processed in a MultiMACS™ Cell 24 Separator Plus device (Miltenyi Biotec). T cells were isolated via positive selection using a Straight from Leukopak® CD4/CD8 MicroBead kit, human (Miltenyi Biotec Cat. 130-122-352). T cells were aliquoted and cryopreserved for future use in Cryostor® CS10 (StemCell Technologies Cat. 07930).
Upon thaw, T cells were plated at a density of 1.0×10{circumflex over ( )}6 cells/mL in T cell growth media (TCGM) composed of CTS OpTmizer T Cell Expansion SFM and T Cell Expansion Supplement (ThermoFisher Cat. A1048501), 5% human AB serum (GeminiBio, Cat. 100-512) 1× Penicillin-Streptomycin, 1× Glutamax, 10 mM HEPES, 200 U/mL recombinant human interleukin-2 (Peprotech, Cat. 200-02), 5 ng/ml recombinant human interleukin 7 (Peprotech, Cat. 200-07), and 5 ng/ml recombinant human interleukin 15 (Peprotech, Cat. 200-15). T cells were rested in this media for 24 hours, at which time they were activated with T Cell TransAct™, human reagent (Miltenyi, Cat. 130-111-160) added at a 1:100 ratio by volume. T cells were activated for 48 hours prior to LNP treatments.
Forty-eight hours post-activation, T cells were harvested, centrifuged at 500 g for 5 min, and resuspended at a concentration of 1×10{circumflex over ( )}6 T cells/mL in T cell plating media (TCPM): a serum-free version of TCGM containing 400 U/mL recombinant human interleukin-2 (Peprotech, Cat. 200-02), 10 ng/ml recombinant human interleukin 7 (Peprotech, Cat. 200-07), and 10 ng/ml recombinant human interleukin 15 (Peprotech, Cat. 200-15). 50 μL of T cells in TCPM (5×10{circumflex over ( )}4 T cells) were added per well to be treated in flat-bottom 96-well plates.
LNPs were prepared as described in Example 1 at a ratio of 35/47.5/15/2.5 (Lipid A/cholesterol/DSPC/PEG2k-DMG). The LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 6. LNPs encapsulated a single RNA species, either a sgRNA as described in Table 74, BC22n mRNA (SEQ ID No: 1), or UGI mRNA (SEQ ID No. 34).
Prior to T cell treatment, LNPs encapsulating a sgRNA were diluted to 6.64 μg/mL in T cell treatment media (TCTM): a version of TCGM containing 20 ug/mL rhApoE3 in the absence of interleukins 2, 5 or 7. These LNPs were incubated at 37° C. for 15 minutes and serially diluted 1:4 using TCTM, which resulted in an 8-point dilution series ranging from 6.64 μg/mL to zero. Similarly, single-cargo LNPs with BC22n mRNA (SEQ ID NO: 1) or UGI mRNA (SEQ ID NO: 34) were diluted in TCTM to 3.32 and 1.67 μg/mL, respectively, incubated at 37° C. for 15 minutes, and mixed 1:1 by volume with sgRNA LNPs serially diluted in the previous step. Last, 50 μL from the resulting mix was added to T cells in 96-well plates at a 1:1 ratio by volume. T cells were incubated at 37° C. for 24 hours, at which time they were harvested, centrifuged at 500 g for 5 min, resuspended in 200 μL of TCGM and returned to the incubator.
Four days post-LNP treatment, T cells were subjected to lysis, PCR amplification of each targeted locus and subsequent NGS analysis, as described in Example 1. Tables 75-80 and
When compared to their 100-mer versions, 91-mer sgRNAs resulted in higher editing frequencies when delivered at the same concentration. This result was observed for all 5 different sets of sgRNAs assayed by NGS, which target 6 different genomic loci. No differences in C to T editing purity were observed between 100-mer and 91-mer sgRNAs. The set of sgRNAs targeting the HLA-A gene were evaluated by flow cytometry instead of NGS due to the hyperpolymorphic nature of the HLA-A locus.
Seven days post LNP treatment, T cells were assayed by flow cytometry to evaluate receptor knockout. T cells were incubated with a fixable viability dye (Beckman Coulter, Cat. C36628) and an antibody cocktail targeting the following molecules: CD3 (Biolegend, Cat. 317336), CD4 (Biolegend, Cat. 317434) and CD8 (Biolegend, Cat. 301046), B2M (Biolegend, Cat. 316306), CD38 (Biolegend, Cat. 303516), HLA-A2 (Biolegend, Cat. 343304) and HLA-DR, DP, DQ (Biolegend, Cat. 361714). Cells were subsequently washed, analyzed on a Cytoflex LX instrument (Beckman Coulter) using the FlowJo software package. T cells were gated on size, viability and CD8 positivity before expression of any markers was determined. The resulting data was plotted on GraphPad Prism v. 9.0.2 and analyzed using a variable slope (four parameter) non-linear regression.
As shown in Tables 81-83 and
In vivo liver editing profiles were evaluated using fixed doses of BC22n (SEQ ID No: 1) and guide RNA targeting ANAPC5 (G019427) along with a serial dilution of UGI mRNA (SEQ ID No: 34).
Fifteen commercially available CD-1 female mice ranging from 6-10 weeks of age (n=3 per group) were used in this study. Animals were weighed pre-dose for dosing calculations. Each RNA species was formulated separately in an LNP. LNPs were formulated generally as described in Example 1. LNPs contained ionizable Lipid A, cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:3 molar ratio, respectively. The lipid nucleic acid assemblies were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 6. LNPs encapsulated a single RNA species, either G019427, BC22n mRNA (SEQ ID No: 1), or UGI mRNA (SEQ ID No. 34).
LNPs encapsulating base editor mRNA and LNPs encapsulating sgRNA were mixed and administered simultaneously at fixed doses of 0.2 mpk and 0.1 mpk RNA by weight, respectively (editor mRNA and sgRNA) along with UGI mRNA doses of either 0.0, 0.03, 0.1, and 0.3 mpk RNA by weight. The negative control group was dosed with TSS buffer only. Formulations were administered intravenously via tail vein injection according to the doses listed in Table 85.
Animals were periodically observed for adverse effects for at least 24 hours post-dose. Six days after treatment, animals were euthanized by cardiac puncture under isoflurane anesthesia; liver tissue were collected for downstream analysis. Liver punches weighing between 5 and 15 mg were collected for isolation of genomic DNA. Genomic DNA samples were analyzed with NGS sequencing as described in Example 1. Editing data and percent C to T purity are shown in Table 86 and
Protein expression levels are determined using HiBiT tagged versions of a protein. To determine the relative amounts of base editor protein and UGI protein expressed for efficient base editing with high C to T purity, parallel editing experiments are performed one of which uses an mRNA encoding a HiBiT-tagged base editor and the other of which uses an mRNA encoding a HiBiT-tagged UGI.
Cell preparation, engineering and editing assays are performed as in Example 9. One arm of the experiment (editor tagged) transfects with an mRNA encoding a HiBiT-tagged BC22n (SEQ ID No. 4) and an mRNA UGI (SEQ ID No: 34). Another arm of the experiment (UGI tagged) transfects with an mRNA encoding base editor (SEQ ID No: 1) and an mRNA encoding a Hibit-tagged UGI.
Twenty-four hours after transfection, the number of live T cells per sample is determined using a cell titer-glo assay (Promega Cat No. G7571) and the number of HiBiT tagged proteins is measured using a Nano-Glo® HiBiT lytic detection assay (Promega Cat No. N3040). NGS sequencing is performed 96 hours post-transfection to assess total editing and C to T purity levels. BC22n and UGI protein levels is plotted against total editing levels to determine the minimum number of each proteins cell that is required to achieve saturating editing levels and C to T purity.
The optimal ratio of BC22n to UGI proteins is calculated by dividing the minimum number of BC22 proteins per cell that is required to achieve saturating editing levels by the minimum number of UGI proteins per cell that is required to achieve maximum C to T purity.
Similar methods could be used to calculate the ratio of base editor to UGI protein that use different cell types, different guides, or different transfection methods.
Base editing is performed in mice to assess overall editing efficiency and C to T purity with lower levels of UGI mRNA. Experimental details are as described in Example 30 with the following exceptions. LNPs encapsulating BC22n mRNA and LNPs encapsulating sgRNA are mixed at fixed doses of 0.2 mg/kg and 0.1 mg/kg of RNA cargos respectively, and combined with UGI mRNA doses of either 0.0, 0.0001, 0.001, 0.003, 0.01 0.03, 0.1, and 0.3 mg/kg. The negative control group is a TSS treated animal. Formulations are administered intravenously via tail vein injection. Six days after treatment, animals are euthanized by cardiac puncture under isoflurane anesthesia; liver tissue are collected for downstream analysis. Liver punches weighing between 5 and 15 mg are collected for isolation of genomic DNA and total RNA. Genomic DNA samples are analyzed with NGS sequencing as described in Example 1 to determine the minimum UGI mRNA dose required for maximum C-to-T editing purity in mouse liver.
Engineered T cells are assayed for cytotoxic susceptibility when targeted by natural killer (NK) cells.
NK cells (Stemcell Technologies) are thawed and resuspended at a cell concentration of 1×10{circumflex over ( )}6 cells/ml into T cell growth media (TCGM) composed of OpTmizer TCGM and further supplemented with 100 U/mL of recombinant human interleukin-2 (Peprotech, Cat. 200-02), 5 ng/ml IL-7 (Peprotech, Cat. 200-07), 5 ng/ml IL-15 (Peprotech, Cat. 200-15). Cells are incubated at 37° C. for 24 hours.
Twenty-four hours post thaw, the NK cells are labelled with 0.5 μM Cell Trace Violet as follows: a vial of Cell Trace Violet (CellTrace™ Violet Cell Proliferation Kit, for flow cytometry, Cat. C34571) is reconstituted in DMSO from the kit to give a 5 mM stock concentration. Two μL of CTV stock is diluted with 18 μL Phosphate-Buffered Saline (Corning, Cat. 21-040-CV) to obtain a concentration of 0.5 mM. NK cells are centrifuged at 500×g for 5 minutes, the media is aspirated, and cells are resuspended in Phosphate-Buffered Saline (PBS) at a concentration of 1×10{circumflex over ( )}6 cells/mL such that the final concentration of CTV dye is 0.5 μM. The cells are mixed with CTV (Cell Trace Violet) dye solution incubated at 37° C. for 20 minutes. Unbound dye is quenched by the addition of TCGM and incubated for 5 minutes. The cells are centrifuged at 500×g for 5 minutes. Cells are resuspended in TCGM supplemented with 100 U/mL of recombinant human interleukin-2 (Peprotech, Cat. 200-02), 5 ng/ml IL-7 (Peprotech, Cat. 200-07), 5 ng/ml IL-15 (Peprotech, Cat. 200-15) at a concentration of 2×10{circumflex over ( )}6 cells/mL. To test a range of effector:target (E:T) ratios, CTV-labelled NK cells aliquoted in 100 ul of media in a 6 point, 2 fold serial dilution with the highest number of cells being 2×10{circumflex over ( )}5 cells Media only samples are included as negative controls.
T cells are engineered using BC22n and UGI mRNA as described in Example 14 using the G023523 targeting HLA-A or G015991 targeting B2M. Unedited (WT) T cells, Unstained, LD heat killed and 7-AAD FMO (2×10{circumflex over ( )}4 unlabelled NK cells and 2×10{circumflex over ( )}4 WT T cells) and CTV+ (2×10{circumflex over ( )}5 CTV labelled NK cells) are also included as controls. T cells are resuspended at a density of 2×10{circumflex over ( )}5 cells of TCGM composed of OpTmizer TCGM and further supplemented with 100 U/mL of recombinant human interleukin-2 (Peprotech, Cat. 200-02), 5 ng/ml IL-7 (Peprotech, Cat. 200-07), 5 ng/ml IL-15 (Peprotech, Cat. 200-15). Twenty thousand T cells are added each well of NK cells and media controls. Cells are incubated at 37° C. for 24 hours.
At twenty-four hours, half of the volume of the cells from the LD Heat Killed well are heat killed and transferred back to the same well in the assay plate. Cells are centrifuged and resuspended in in 80 μL of a 1:200 v/v solution of 7-AAD (BD Biosciences, Cat. 559925) in FACS buffer (PBS+2% FBS (Gibco, Cat. A31605-02)+2 mM EDTA (Invitrogen, Cat. 15-575-020). Data for specific lysis of T cells is acquired by flow cytometry using a Cytoflex LX instrument (Beckman Coulter) and analyzed using the FlowJo software package. Gates are first drawn on the CTV negative population to gate out the NK cells, followed by gating on singlets after which a gate is drawn on the 7-AAD negative population to gate for the live T cells. The percent lysis of T cells is calculated by subtracting the live cell percentage from 100.
Base editing was performed with mRNAs encoding proteins with fused HiBiT tags in primary human hepatocytes to determine the relative amounts of base editor and UGI protein enzymatic units appropriate for high efficiency editing with high C to T purity.
Messenger RNAs encoding BC22n with a C-terminal HiBiT tag (BC22n-HiBIT, SEQ ID NO: 4), BC22-2XUGI (2 tandem copies of UGI in cis) with a C-terminal HiBiT tag (BC22-2XUGI-HibIT, SEQ ID NO: 314), and UGI with a C-terminal HiBiT tag (UGI-HiBiT, SEQ ID NO: 316), were transfected into primary human hepatocytes (PHH) in dose response with a fixed concentration of a guide RNA targeting B2M (G015991; SEQ ID NO: 179) to determine the minimum number of intracellular base editor and UGI protein copies to perform base editing with high activity (total editing %=2×EC90) and high C-to-T purity (C to T purity %=2×EC90). BC22n-HiBiT was titrated across fixed concentrations of B2M guide and UGI (SEQ ID NO: 34), UGI-HiBiT was titrated across fixed concentrations of B2M guide and BC22n (SEQ ID NO: 1), and BC22-2XUGI-HiBiT was titrated across a fixed concentration of B2M guide without any additional UGI mRNA in trans. A HiBiT lytic assay (Promega cat #N3040) was performed twenty hours post transfection to determine intracellular protein levels at an early timepoint. Those protein levels were then related to endpoint editing at the B2M locus, derived from NGS data, ninety-six hours post transfection.
PHH cells (ThermoFisher, Lot HU8284) were thawed and recovered in CHRMs media (Gibco, cat #CM7000). Cells were resuspended in Primary Hepatocyte Plating Media (Consisting of William's E media (Gibco, Cat #A1217601) and primary hepatocyte plating supplements (Gibco, Cat #CM3000)) before being plated in collagen-coated 96-well plates at a density of 30,000 cells/well for twenty-four hours. Cells were washed, and fresh primary hepatocyte maintenance media was added.
Lipofection reagent was prepared as described in Example 1, with a molar ratio of 50/9/38/3 of Lipid A, DSPC, cholesterol, and PEG2k-DMG respectively. Each RNA species (base editor mRNA, UGI mRNA, or gRNA G015991 (SEQ ID NO: 179)) was individually bulk-mixed with lipofection reagent at a lipid amine to RNA phosphate (N:P) molar ratio of about 6. The resulting bulk-mixed lipoplex material was pre-incubated with 10% FBS (Gibco, Cat #A3160501) in Primary Hepatocyte Maintenance Media (Consisting of William's E media (Gibco, Cat #A1217601) and primary hepatocyte maintenance supplements (Gibco, Cat #CM4000)), for 15 minutes before addition to hepatocytes.
For the BC22n-HiBiT titration, each well received three components in a final volume of 100 uL: BC22n-HiBiT mRNA ranging from 100 ng to 0 ng, 11 ng of UGI mRNA and 2 pmols of G015991 (B2M) as described in Table 87. For the UGI-HiBiT titration each well received three components in a final volume of 100 uL: UGI-HiBiT mRNA ranging from 100 ng to 0 ng, 33 ng of BC22n mRNA and 2 pmols of G015991 (B2M) as described in Table 88. For the BC22-2XUGI-HiBiT titration each well received three components in a final volume of 100 uL: BC22-2XUGI-HiBiT mRNA ranging from 100 ng to 0 ng, 11 ng of UGI mRNA and 2 pmols of G015991 (B2M) as described in Table 89.
Ninety-six hours post transfection, separate PHH replicate plates that had been transfected at the same time as the HiBiT plates were subjected to lysis, PCR amplification of the B2M locus, and subsequent NGS analysis, as described in Example 1. All experiments were performed in biological triplicate. Background C-to-T, C-to-A/G and indel edits (all <2%) were subtracted from all wells by calculating the average background rates from a set of untreated wells. Editing results for different BC22n-HiBiT mRNA concentrations are shown in Table 87, and total editing at different BC22n-HiBiT mRNA concentrations are shown in
To facilitate comparisons between mRNAs of different sizes, all figures display mRNA doses in terms of molarity. For these purposes, the molecular weight of BC22n mRNA was considered as 1,724.25 kDa, BC22-2X-UGI mRNA as 1915.12 kDa, UGI mRNA as 287.58 kDa and sgRNAs as 29.66 kDa. The addition of a HiBiT tag to any miRNA increased its molecular weight by 18.24 kDa.
Twenty hours post transfection, protein levels were detected using the Nano-Glo® HiBiT Lytic Detection System (Promega cat #N3040) according to the manufacture's protocol. Promega #N3010, a commercially available control protein with a HiBiT tag (of known concentration and known molecular weight) was serially diluted (1:5) in PBS and spiked into wells containing PHH that had not been transfected as a reference control. One hundred μL of reconstituted HiBiT lytic reagent was added to the standard wells and all other experiment wells. Lysates were moved to white-walled plates, and relative luminescence units (RLU) were read out by the CLARIstar plus (BMG Labtech) plate reader with gain set at 3,600. Background signal was subtracted from all wells, and the number of protein copies per well for each sample was calculated using the linear regression equation derived by the HiBiT standard. HiBiT quantitation is presented in Table 90 and normalized to the expression level of the 3.7 ng BC22-2XUGI-HiBiT mRNA sample.
The data in Table 90 were used to generate hyperbolic curves and interpolate the protein units expressed at the minimum dose required to achieve saturating editing or C-to-T purity levels. The minimum dose as saturating levels is defined here as twice the EC90 (2×EC90), the concentration required to achieve 90% total editing or C-to-T purity, respectively, for each mRNA tested. Table 91 shows the 2×EC90 doses (in ng and nM) of the mRNAs tested in this experiment and the BC22 and UGI peptide units and their ratios at these doses. For base editors and UGI-HiBiT, protein units are equal to peptide units. UGI peptide units for BC22-2XUGI were calculated by multiplying the BC22-2XUGI protein units by a factor of 2. For the samples edited with BC22n and UGI expressed as separate, unfused proteins, the ratio of BC22n peptide units at total editing 2×EC90 to the UGI peptide units at C to T purity 2×EC90 was approximately 1:10. For the samples edited with BC22-2XUGI, the ratio of base editor peptide units at total editing 2×EC90 to the UGI peptide units at C to T purity 2×EC90 was approximately 1:5.
Base editing was performed with mRNAs encoding proteins with fused HiBiT tags in isolated human T cells to determine the relative amounts of base editor and UGI protein enzymatic units appropriate for high efficiency editing with high C to T purity.
Healthy human donor apheresis was obtained commercially (Hemacare), and cells were washed, re-suspended in CliniMACS® PBS/EDTA buffer (Miltenyi Biotec Cat. 130-070-525) and processed in a MultiMACS™ Cell 24 Separator Plus device (Miltenyi Biotec). T cells were isolated via positive selection using a Straight from Leukopak® CD4/CD8 MicroBead kit, human (Miltenyi Biotec Cat. 130-122-352). T cells were aliquoted and cryopreserved for future use in Cryostor® CS10 (StemCell Technologies Cat. 07930).
Upon thaw, T cells were plated at a density of 1.0×10{circumflex over ( )}6 cells/mL in T cell growth media (TCGM) composed of CTS OpTmizer T Cell Expansion SFM and T Cell Expansion Supplement (ThermoFisher Cat. A1048501), 5% human AB serum (GeminiBio, Cat. 100-512) 1× Penicillin-Streptomycin, 1× Glutamax, 10 mM HEPES, 200 U/mL recombinant human interleukin-2 (Peprotech, Cat. 200-02), 5 ng/ml recombinant human interleukin 7 (Peprotech, Cat. 200-07), and 5 ng/ml recombinant human interleukin 15 (Peprotech, Cat. 200-15). After 24 hours in this media. T cells were activated with T Cell TransAct™, human reagent (Miltenyi, Cat. 130-111-160) added at a 1:100 ratio by volume.
Forty-eight hours post-activation, T cells were harvested, centrifuged at 500 g for 5 min, and resuspended at a concentration of 1×10{circumflex over ( )}6 T cells/mL in T cell plating media (TCPM): a serum-free version of TCGM containing 400 U/mL recombinant human interleukin-2 (Peprotech, Cat. 200-02), 10 ng/ml recombinant human interleukin 7 (Peprotech, Cat. 200-07), and 10 ng/ml recombinant human interleukin 15 (Peprotech, Cat. 200-15). Fifty thousand T cells in 50 ul TCPM were added per well to be treated in flat-bottom 96-well plates.
LNPs were generated as described in Example 1 at a molar ratio of 35/47.5/15/2.5 (Lipid A/cholesterol/DSPC/PEG2k-DMG). Prior to T cell treatment, two separate LNP mixes (referred below as mixes “A” and “B”) were prepared in T cell treatment media (TCTM): a version of TCGM containing 20 ug/mL rhApoE3 (Peprotech, Cat. 350-02) in the absence of interleukins 2, 5 or 7.
To determine the minimum levels of BC22n-HiBiT mRNA necessary for saturating total editing, mix “A” consisted of an LNP with BC22n-HiBiT mRNA (SEQ ID NO: 4) diluted to 6.68 μg/mL (3.83 nM), while mix “B” consisted of an LNP with UGI mRNA (SEQ ID NO: 34) diluted to 1.67 μg/mL (5.8 nM) and a multi-cargo LNP containing sgRNAs G023520 (TRAC), G023524 (TRBC1/2), G023521 (CIITA) and G023523 (HLA-A) at the ratio of 4.5/8.7/21.4/65.4 diluted to 6.68 μg/mL (225.19 nM). LNP mixes “A” and “B” were individually incubated at 37° C. for 15 minutes. Mix “A” was serially diluted 1:4 in TCTM, and mixed 1:1 by volume with mix “B”. The resulting solution was added to T cells in 96-well plates at a 1:1 ratio by volume (50 μL per well).
To determine the minimum levels of UGI-HiBiT mRNA necessary for saturating C to T purity, mix “A” consisted of an LNP with UGI-HiBiT mRNA (SEQ ID NO: 316) diluted to 6.68 μg/mL (21.84 nM), while mix “B” consisted of an LNP with BC22n mRNA (SEQ ID NO: 1) diluted to 3.34 μg/mL (1.94 nM) and a multi-cargo LNP containing sgRNAs G023520 (TRAC), G023524 (TRBC1/2), G023521 (CIITA) and G023523 (HLA-A) at the ratio of 4.5/8.7/21.4/65.4 diluted to 6.68 μg/mL (225.19 nM). LNP mixes “A” and “B” were individually incubated at 37° C. for 15 minutes. Mix “A” was serially diluted 1:4 in TCTM, and mixed 1:1 by volume with mix “B”. The resulting solution was added to T cells in 96-well plates at a 1:1 ratio by volume (50 μL per well).
To determine the minimum levels of BC22-2XUGI-HiBiT mRNA necessary for saturating total editing and C to T purity, mix “A” consisted of an LNP with BC22-2XUGI-HiBiT mRNA (SEQ ID NO: 314) diluted to 6.68 μg/mL (3.46 nM), while mix “B” consisted of a multi-cargo LNP containing sgRNAs G023520 (TRAC), G023524 (TRBC1/2), G023521 (CIITA) and G023523 (HLA-A) at the ratio of 4.5/8.7/21.4/65.4 diluted to 6.68 μg/mL (225.19 nM). LNP mixes “A” and “B” were incubated individually at 37° C. for 15 minutes. Mix “A” was serially diluted 1:4 in TCTM, and mixed 1:1 by volume with mix “B”. The resulting solution was added to T cells in 96-well plates at a 1:1 ratio by volume (50 μL per well).
Following the addition of LNPs, T cells were incubated at 37° C. for 24 hours, at which time half of the cells were harvested for cell viability and protein expression assays, and the remaining cells were centrifuged at 500 g for 5 min, resuspended in 200 μL of TCGM and returned to the incubator.
On day 4 post-LNP treatment, T cells were centrifuged at 500 g for 5 min, subjected to lysis, PCR amplification of each targeted locus and subsequent NGS analysis, as described in Example 1. Editing results at the TRAC, TRBC1, TRBC2, and CIITA loci with different concentrations of HiBiT mRNAs are shown in Tables 92-95, respectively.
Tables 92-95 show editing data for 4 different genomic loci (TRAC, TRBC1, TRBC2 and CIITA).
Twenty-four hours post-LNP treatments, all samples were mixed thoroughly and two aliquots of 25 JAL were transferred to white-walled 96-well plates containing 75 μL of TCGM. One plate was subjected to a CellTiter-Glo® 2.0 Cell Viability Assay (Promega Cat No. G9242) and the other plate was subjected to a Nano-Glo® HiBiT Lytic Detection Assay (Promega Cat No. N3040). Both assays were performed following the manufacturer's protocol. Standards curves were prepared using known numbers of donor-matched T cells or a commercially available protein with a HiBiT tag (Promega Cat No. N3010). HiBiT quantitation is presented in Table % normalized to the expression level of the 0.0037 nM BC22n-HiBiT mRNA sample.
Data from Table 96 was used to generate hyperbolic curves and interpolate the the protein units expressed at the minimum doses required to achieve saturating editing or C-to-T purity levels. The minimum dose as saturating levels is defined here as twice the EC90 (2×EC90), the concentration required to achieve 90% total editing or C-to-T purity, respectively, for each mRNA tested.
Table 97 shows the 2×EC90 doses of the mRNAs tested in this experiment and the BC22 and UGI peptide units and their ratios at these doses. For base editors and UGI-HiBiT, protein units are equal to peptide units. UGI peptide units for BC22-2XUGI were calculated by multiplying the BC22-2XUGI protein units by a factor of 2. Table 98 shows the ratio of base editor peptide units at total editing 2×EC90 to the UGI peptide units at C to T purity 2×EC90.
In vivo editing profiles of deaminase containing constructs were compared to Cas9 when UGI was delivered in trans (as a separate mRNA). The constructs used encoded a fusion protein including D10A Cas9 with a deaminase.
Twenty-four commercially available CD-1 female mice ranging from 6-10 weeks of age (n=3 per group) were used in this study. Animals were weighed pre-dose for dosing calculations. Each RNA species was formulated separately in an LNP. Formulations containing editor mRNA, UGI mRNA and G019427 sgRNA were mixed in a w/w ratio of RNA cargos. The formulation mixture for Group 2 contained only editor mRNA and sgRNA and these were mixed in a w/w ratio of 2:1 (editor mRNA:sgRNA). Groups 3-8 contained mRNA:sgRNA at a w/w ratio of 2:1, and UGI mRNA mixed in at w/w ratios of 1:3, 1:10, 1:30, 1:100, 1:300, and 1:3000 (editor mRNA+sgRNA:UGI mRNA). Apart from the negative control group, which was dosed with TSS buffer only, all groups resulted in editing at the ANACP5 locus targeted with G019427. Formulations were administered intravenously via tail vein injection according to the doses listed in Table 99. Animals were periodically observed for adverse effects for at least 24 hours post-dose. Six days after treatment, animals were euthanized by cardiac puncture under isoflurane anesthesia; liver tissue were collected for downstream analysis. Liver punches weighing between 5 and 15 mg were collected for isolation of genomic DNA. Genomic DNA samples were analyzed with NGS sequencing as described in Example 1. Editing data are shown in Table 100 and
Engineered T cells were assayed for cytotoxic susceptibility when targeted by natural killer (NK) cells.
NK cells (Stemcell Technologies) were thawed and resuspended at a cell concentration of 1×10{circumflex over ( )}6 cells/ml into T cell growth media (TCGM) composed of OpTmizer TCGM and further supplemented with 100 U/mL of recombinant human interleukin-2 (Peprotech, Cat. 200-02), 5 ng/mL IL-7 (Peprotech, Cat. 200-07), 5 ng/mL IL-15 (Peprotech, Cat. 200-15). Cells were incubated at 37° C. for 24 hours.
Twenty-four hours post thaw, the NK cells were labelled with 0.5 μM Cell Trace Violet (CTV) as follows: a vial of CTV (CellTrace™ Violet Cell Proliferation Kit, for flow cytometry, Cat. C34571) was reconstituted in DMSO from the kit to give a 5 mM stock concentration. Two μL of CTV stock was diluted with 18 μL Phosphate-Buffered Saline (PBS) (Corning, Cat. 21-040-CV) to obtain a concentration of 0.5 mM. NK cells were centrifuged at 500×g for 5 minutes, the media was aspirated, and cells were resuspended in PBS at a concentration of 1×10{circumflex over ( )}6 cells/mL such that the final concentration of CTV dye was 0.5 μM. The cells were mixed with CTV dye solution incubated at 37° C. for 20 minutes. Unbound dye was quenched by the addition of TCGM and incubated for 5 minutes. The cells were centrifuged at 500×g for 5 minutes. Cells are resuspended in TCGM supplemented with 100 U/mL of recombinant human interleukin-2 (Peprotech, Cat. 200-02), 5 ng/mL IL-7 (Peprotech, Cat. 200-07), 5 ng/mL IL-15 (Peprotech, Cat. 200-15) at a concentration of 2×10{circumflex over ( )}6 cells/mL. To test a range of effector:target (E:T) ratios, CTV-labelled NK cells were aliquoted in 100 μL of media in a 6-point, 2-fold serial dilution with the highest number of cells being 2×10{circumflex over ( )}5 cells. Media-only samples were included as negative controls.
T cells were engineered using BC22n and UGI mRNA as described in Example 14 using G023523 (SEQ ID NO: 501) targeting HLA-A as a test sample and with G023519 (SEQ ID NO: 498) targeting B2M was as a positive control for NK killing. Unedited T cells were assayed as a negative control for NK killing. Other controls for flow cytometry included CTV-labelled NK cells without T cells; a “unstained” sample combining unlabelled NK cells and T cells; and a 1:1 mix of unlabeled heat killed and non-heat killed NK cells and T cells stained with 7AAD. T cells were resuspended at a density of 2×10{circumflex over ( )}5 cells in TCGM composed of OpTmizer TCGM and further supplemented with 100 U/mL of recombinant human interleukin-2 (Peprotech, Cat. 200-02), 5 ng/mL IL-7 (Peprotech, Cat. 200-07), and 5 ng/mL IL-15 (Peprotech, Cat. 200-15). Twenty thousand T cells were added to each well of NK cells and media controls. Cells were incubated at 37° C. for 24 hours.
At 24 hours, half of the volume of the cells from the LD heat killed well were heat killed and transferred back to the same well in the assay plate. Cells were centrifuged and resuspended in 80 μL of a 1:200 v/v solution of 7-AAD (BD Biosciences, Cat. 559925) in FACS buffer (PBS+2% FBS (Gibco, Cat. A31605-02)+2 mM EDTA (Invitrogen, Cat. 15-575-020)). Data for specific lysis of T cells were acquired by flow cytometry using a Cytoflex LX instrument (Beckman Coulter) and analyzed using the FlowJo software package. Gates were first drawn on the CTV negative population to gate out the NK cells, followed by gating on singlets after which a gate was drawn on the 7-AAD negative population to gate for the live T cells. The percent lysis of T cells was calculated by subtracting the live cell percentage from 100. T cells edited using BC22n and HLA-A guide G023523 (SEQ ID NO: 501) were protected from NK cell mediated cytotoxicity as shown in Table 101 and
The range of guide positions available for deamination by base editors designed with Nme2Cas9 nickase or SpyCas9 nickase and APOBEC3a was assayed by evaluating C to T conversion efficacy on a position by position basis across a panel of target sites with cytosine residues in the guide region.
Healthy human donor apheresis was obtained commercially (Hemacare), and cells were washed and resuspended in in CliniMACS® PBS/EDTA buffer (Miltenyi Biotec Cat. 130-070-525) and processed in a MultiMACS™ Cell 24 Separator Plus device (Miltenyi Biotec). T cells were isolated via positive selection using a Straight from Leukopak® CD4/CD8 MicroBead kit, human (Miltenyi Biotec Cat. 130-122-352). T cells were aliquoted and cryopreserved for future use in Cryostor® CS10 (StemCell Technologies Cat. 07930).
Upon thaw, T cells were plated at a density of 1.0×10{circumflex over ( )}6 cells/mL in T cell growth media (TCGM) composed of CTS OpTmizer T Cell Expansion SFM and T Cell Expansion Supplement (ThermoFisher Cat. A1048501), 5% human AB serum (GeminiBio, Cat. 100-512) 1× Penicillin-Streptomycin, 1× Glutamax, 10 mM HEPES, 200 U/mL recombinant human interleukin-2 (Peprotech, Cat. 200-02), 5 ng/mL recombinant human interleukin 7 (Peprotech, Cat. 200-07), and 5 ng/ml recombinant human interleukin 15 (Peprotech, Cat. 200-15). T cells were rested in this media for 24 hours, at which time they were activated with T Cell TransAct™, human reagent (Miltenyi, Cat. 130-111-160) added at a ratio of 1:100 by volume. T cells were activated for 48 hours prior to electroporation.
Solutions containing mRNA encoding Spy BC22n (SEQ ID NO: 1) or Nme2 BC22n (SEQ ID NO: 315) and UGI (SEQ ID NO: 34) were prepared in P3 buffer. Guide RNAs targeting either the SCAP, LINC01588, LSP1, SEC61B, VEGFA, FancF, AAVS1, or ARHGEF9 locus was removed from the storage and denatured for 2 minutes at 95° C. and incubated at room temperature for 5 minutes.
Forty-eight hours post activation, T cells were harvested, centrifuged, and resuspended at a concentration of 12.5×10{circumflex over ( )}6 T cells/mL in P3 electroporation buffer (Lonza). For each well to be electroporated, 1×10{circumflex over ( )}5 T cells were mixed with 200 ng of BC22n or Nme2 base editor mRNA, 200 ng of UGI mRNA and 4 μM of sgRNAs in a final volume of 20 μL of P3 electroporation buffer. This mix was transferred in duplicate to 96-well Nucleofector™ plates and electroporated using the manufacturer's pulse code. Electroporated T cells were immediately rested in 80 μL of CTS Optimizer T cell growth media without cytokines for 15 minutes before being transferred to new flat-bottom 96-well plates containing an additional 80 μL of CTS OpTmizer T cell growth media supplemented with 2× cytokines. The resulting plates were incubated at 37° C. for 4 days. On day 4 post-electroporation, 100 μL of cells was harvested for DNA extraction. DNA samples were subjected to PCR and subsequent NGS analysis with two separate primer sets (technical replicates), as described in Example 1.
Position values were assigned to each DNA base within the protospacer region and its 5′ and 3′ adjacent nucleotides. or SpyCas9, Position 1-20 represent the 20 bases bound by a SpyCas9 guide wherein position 1 is the PAM distal base and position 20 is the PAM proximal base. For Nme2Cas9, positions 1-24 represent the 24 bases bound by a Nme2Cas9 guide wherein position 1 is the PAM distal base and position 24 is the PAM proximal base. The 5′ and 3′ ends outside of protospacer region were assigned to be negative and positive positions, respectively; relative to Position 1.
Define conversion frequency: A guide can be designed to target the reference strand (strand +) or reverse complementary strand (strand −) of a gene. For strand + guides, each wild-type cytosine position in the target region was recorded and conversion frequency (cytosine to thymine) at the position was calculated as ratio of sequencing reads with thymine at the position versus reads with either cytosine or thymine at the position; For strand-guide, each wild-type guanine position in target region was recorded and a conversion frequency (guanine to adenine) at the position is calculated as ratio of sequencing reads with adenine at the position versus reads with adenine and guanine at the position.
Classify guide activity: Four replicates were measured for each guide: two technical replicates for each of two biological replicates. Only positions in a replicate with >500 sequencing reads would be further analysed. For each guide, the mean conversion frequency of a position is the average of the replicates, and the highest mean conversion frequency of all positions for a guide is used to classify this guide's conversion activity: Low, medium, and high activity guides have a highest mean conversion frequency of <50%, 50% & <70%, >70%, respectively. High activity guides for BC22n (n=38) and Nme2 base editor (n=14) were chosen for further window analysis as shown in Table 102.
Define conversion rate for each position across all guides: If a guide has the position recorded, each of its replicates with >500 sequencing reads at the position is considered as a “case”. Total cases were summed for each position of all guides and served as a denominator for conversion rate calculation. In each case, if the conversion frequency is greater than 50%, it is considered as a high conversion “event”, and total events is the sum of high conversion events for each position across all guides and replicates. The “events” serve as numerator for conversion rate calculation. Conversion Rate of a position equals the percentage of high conversion events in all cases for each position.
Table 103 displays the position, cases, events, and conversion rate for the spyBC22n C-to-T editing window.
Different sgRNAs were screened for their potency in knocking out the CIITA gene in human T cells using C to T base editing. The percentage of T cells negative for MHC class II and/or CD74 protein expression was assayed following CIITA editing following electroporation with mRNA and different sgRNAs.
Healthy human donor apheresis was obtained commercially (Hemacare), and cells were washed and resuspended in CliniMACS® PBS/EDTA buffer (Miltenyi Biotec Cat. 130-070-525) and processed in a MultiMACS™ Cell 24 Separator Plus device (Miltenyi Biotec). T cells were isolated via positive selection using a Straight from Leukopak® CD4/CD8 MicroBead kit, human (Miltenyi Biotec Cat. 130-122-352). T cells were aliquoted and cryopreserved for future use in Cryostor® CS10 (StemCell Technologies Cat. 07930).
Upon thaw, T cells were plated at a density of 1.0×10{circumflex over ( )}6 cells/mL in T cell growth media (TCGM) composed of CTS OpTimizer T Cell Expansion SFM and T Cell Expansion Supplement (ThermoFisher Cat. A1048501), 5% human AB serum (GeminiBio, Cat. 100-512) 1× Penicillin-Streptomycin, 1× Glutamax, 10 mM HEPES, 200 U/mL recombinant human interleukin-2 (Peprotech, Cat. 200-02), 5 ng/mL recombinant human interleukin 7 (Peprotech, Cat. 200-07), and 5 ng/mL recombinant human interleukin 15 (Peprotech, Cat. 200-15). T cells were rested in this media for 24 hours, at which time they were activated with T Cell TransAct™, human reagent (Miltenyi, Cat. 130-111-160) added at a 1:100 ratio by volume. T cells were activated for 48 hours prior to electroporation.
Solutions containing mRNA encoding BC22n (SEQ ID NO: 1) and UGI (SEQ ID NO: 34) were prepared in P3 buffer. One hundred μM of CIITA-targeting sgRNAs were removed from their storage plates and denatured for 2 minutes at 95° C. and incubated at room temperature for 5 minutes. Forty-eight hours post activation, T cells were harvested, centrifuged, and resuspended at a concentration of 12.5×10{circumflex over ( )}6 T cells/mL in P3 electroporation buffer (Lonza). For electroporation, 1×10{circumflex over ( )}5 T cells were mixed with 20 ng/μL of BC22n mRNAs, 20 ng/μL of UGI mRNA, and 20 pmols of sgRNA as described in Table 1 in a final volume of 20 μL of P3 electroporation buffer. This mix was transferred in duplicate to a 96-well Nucleofector™ plate and electroporated using the manufacturer's pulse code. Electroporated T cells were immediately rested in 80 μL of CTS Optimizer T cell growth media without cytokines for 15 minutes before being transferred to new flat-bottom 96-well plates containing an additional 80 μL of CTS Optimizer T cell growth media supplemented with 2× cytokines. The resulting plates were incubated at 37° C. for 10 days. On day 4 post-electroporation, cells were split 1:2 in 2 U-bottom plates. One plate was collected for NGS sequencing, while the other plate was replenished with CTS Optimizer fresh media with 1× cytokines. This plate was used for flow cytometry on Day 7.
On day 7 post-editing, T cells were assayed by flow cytometry to determine the surface expression of CD74 and HLA-DR, DP, DQ. Briefly, T cells were incubated for 30 minutes at 4° C. with a mixture of antibodies diluted in cell staining buffer (BioLegend, Cat. No. 420201). Antibodies against CD3 (BioLegend, Cat. No. 317336), CD4 (BioLegend, Cat. No. 317434), CD8 (BioLegend, Cat. No. 301046), and Viakrome (Beckman Coulter, Cat. No. C36628) were diluted at 1:100, and antibodies against HLA II-DR (BioLegend, Cat. No. 327018), HLA II-DP (BD Biosciences Cat No. 750872), HLA II-DQ (BioLegend, Cat. No. 561504), and CD74 (BioLegend, Cat. No. 326808) were diluted at 1:50. Cells were subsequently washed, resuspended in 100 μL of cell staining buffer and processed on a Cytoflex flow cytometer (Beckman Coulter). Flow cytometry data was analyzed using the FlowJo software package. T cells were gated based on size, shape, viability, CD8, HLA II-DP, HLA II-DQ, HLA II-DR, and CD74 expression.
On day 4 post-editing, DNA samples were subjected to PCR and subsequent NGS analysis, as described in Example 1. Table 105 shows CIITA editing outcomes in T cells edited with BC22n.
Highly efficient CIITA sgRNAs identified in Example 41 were further assayed for base editing efficacy at multiple guide concentrations in T cells. The potency of each was assayed for genome editing efficacy by NGS or by disruption of surface protein expression of HLA-DR, DP, DQ by flow cytometry.
Healthy human donor apheresis was obtained commercially (Hemacare), and cells were washed and resuspended in in CliniMACS® PBS/EDTA buffer (Miltenyi Biotec Cat. 130-070-525) and processed in a MultiMACS™ Cell 24 Separator Plus device (Miltenyi Biotec). T cells were isolated via positive selection using a Straight from Leukopak® CD4/CD8 MicroBead kit, human (Miltenyi Biotec Cat. 130-122-352). T cells were aliquoted and cryopreserved for future use in Cryostor® CS10 (StemCell Technologies Cat. 07930).
Upon thaw, T cells were plated at a density of 1.0×10{circumflex over ( )}6 cells/mL in T cell growth media (TCGM) composed of CTS OpTmizer T Cell Expansion SFM and T Cell Expansion Supplement (ThermoFisher Cat. A1048501), 5% human AB serum (GeminiBio, Cat. 100-512), 1× Penicillin-Streptomycin, 1× Glutamax, 10 mM HEPES, 200 U/mL recombinant human interleukin-2 (Peprotech, Cat. 200-02), 5 ng/mL recombinant human interleukin 7 (Peprotech, Cat. 200-07), and 5 ng/mL recombinant human interleukin 15 (Peprotech, Cat. 200-15). T cells were rested in this media for 24 hours, at which time they were activated with T Cell TransAct™ human reagent (Miltenyi, Cat. 130-111-160) added at a 1:100 ratio by volume. T cells were activated for 48 hours prior to electroporation.
Solutions containing mRNAs encoding BC22n (SEQ ID NO: 1) and UGI (SEQ ID NO: 34) were prepared in P3 buffer. 100 μM CIITA targeting sgRNAs were removed from their storage plates and denatured for 2 minutes at 95° C. and incubated at room temperature for 5 minutes. Forty-eight hours post activation, T cells were harvested, centrifuged, and resuspended at a concentration of 12.5×10{circumflex over ( )}6 T cells/mL in P3 electroporation buffer (Lonza). Each sgRNA was serially diluted in ratio of 1:2 in P3 electroporation buffer starting from 60 pmols in a 96-well PCR plate in duplicate. Following dilution, 1×10{circumflex over ( )}5 T cells, 20 ng/μL of BC22n mRNAs, and 20 ng/μL of UGI mRNA were mixed with sgRNA plate to make the final volume of 20 μL of P3 electroporation buffer. This mix was transferred to 4 corresponding 96-well Nucleofector™ plates and electroporated using the manufacturer's pulse code. Electroporated T cells were immediately rested in 80 μL of CTS Optimizer T cell growth media without cytokines for 15 minutes before being transferred to new flat-bottom 96-well plates containing an additional 80 μL of CTS OpTmizer T cell growth media supplemented with 2× cytokines. The resulting plates were incubated at 37° C. for 7 days. On day 4 post-electroporation, cells were
split 1:2 in two U-bottom plates, and one plate was collected for NGS sequencing, while the other plate was replenished with CTS Optimizer fresh media with 1× cytokines. This plate was used for flow cytometry on Day 7.
On day 7 post-editing, T cells were assayed by flow cytometry to determine surface expression of HLA-DR, DP, DQ. Briefly, T cells were incubated for 30 minutes at 4° C. with a mixture of antibodies diluted in cell staining buffer (BioLegend, Cat. No. 420201). Antibodies against CD3 (BioLegend, Cat. No. 317336), CD4 (BioLegend, Cat. No. 317434), CD8 (BioLegend, Cat. No. 301046), and Viakrome (Beckman Coulter, Cat. No. C36628) were diluted at 1:100, and antibodies against HLA II-DR, DP, DQ (BioLegend, Cat. No. 361714) were diluted at 1:50. Cells were subsequently washed, resuspended in 100 μL of cell staining buffer and processed on a Cytoflex flow cytometer (Beckman Coulter). Flow cytometry data was analyzed using the FlowJo software package. T cells were gated based on size, shape, viability, CD8, and HLA-DR, DP, DQ.
Table 106 shows CIITA editing outcomes and the percentage of T cells negative for HLA-DR, DP, DQ in T cells following base editing with BC22n.
The following numbered embodiments provide additional support for and descriptions of the embodiments herein.
The following numbered embodiments provide additional support for and descriptions of the embodiments herein.
This application is a Continuation of International Application No. PCT/US2021/062922, filed Dec. 10, 2021, which claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application No. 63/124,060, filed Dec. 11, 2020; U.S. Provisional Application No. 63/130,104, filed Dec. 23, 2020; U.S. Provisional Application No. 63/165,636, filed Mar. 24, 2021; and U.S. Provisional Application No. 63/275,424, filed Nov. 3, 2021, each of which is herein incorporated by reference in its entirety. This application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled “01155-0016-00US_ST26.XML” created on Jun. 9, 2023, which is 2,996,760 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63275424 | Nov 2021 | US | |
| 63165636 | Mar 2021 | US | |
| 63130104 | Dec 2020 | US | |
| 63124060 | Dec 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2021/062922 | Dec 2021 | US |
| Child | 18332335 | US |
