Compositions and methods for genome editing

Abstract

Methods and compositions for functional genetic modification at selected sites are disclosed. Cell populations containing targeted integration of one or more exogenous polynucleotides and / or indels at one or more selected genetic loci are also provided.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 486,172, filed February 21, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Electronic Sequence Listing Reference The electronic sequence listing (POTH-079_001WO_SeqListing_ST26.xml, size 179,192 bytes, created on February 12, 2024) is incorporated herein by reference in its entirety.

[0003] Field The present disclosure relates to the field of gene editing and genome engineering. More particularly, the present disclosure relates to compositions and methods for targeted genetic modification. [Background technology]

[0004] Background of the Invention Genome editing refers to methods and technologies for targeted, specific modification of an organism's genetic information (genome). Genome engineering is an active field of research due to its wide range of potential applications, particularly in the field of human health, for example, correcting genes with harmful mutations or investigating gene function. Early technologies developed for inserting transgenes into living cells were often limited by the randomness of the insertion location of new sequences into the genome. Common genome editing methods allow for the modification of specific regions of DNA, thereby increasing the precision of correction or insertion compared to previous techniques. These platforms offer greater reproducibility and reduced levels of unintended effects due to random insertions and deletions in the genome, but limitations remain. There is a need to develop gene editing platforms and fusion proteins with superior efficacy in genome editing. There is also a need to develop safe and effective donor cells using such gene editing platforms to support regenerative medicine and / or cell therapy treatments, including immuno-oncology-related diseases. Summary of the Invention

[0005] Summary of the Invention The present disclosure provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 76. The present disclosure also provides a polynucleotide encoding the polypeptide, a vector comprising the polypeptide, and a pharmaceutical composition comprising the vector and at least one pharmaceutically acceptable excipient or diluent.

[0006] The present disclosure also provides a method for modifying a target sequence in the genome of a plurality of cells, the method comprising introducing into a population of unmodified cells a composition comprising: a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 76 or a polynucleotide encoding the polypeptide of SEQ ID NO: 76; and b) at least one guide RNA (gRNA), thereby causing a modification in the target sequence in the genome, wherein a 1.1-fold to 100-fold greater number of cells comprise a modification in the target sequence in the genome compared to a plurality of modified cells into which a composition comprising the polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or a polynucleotide encoding the polypeptide of SEQ ID NO: 10 has been introduced.

[0007] In some embodiments, 1.6- to 3.2-fold more cells contain an alteration in the target sequence in their genome compared to a plurality of modified cells into which a composition comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or a polynucleotide encoding the polypeptide of SEQ ID NO: 10 has been introduced. In some embodiments, 4- to 75-fold more cells contain an alteration in the target sequence in their genome compared to a plurality of modified cells into which a composition comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or a polynucleotide encoding the polypeptide of SEQ ID NO: 10 has been introduced.

[0008] In some embodiments, the alteration in the target sequence in the genome is a deletion, insertion, substitution, inversion and / or rearrangement.

[0009] In some embodiments, the composition comprises about 40.75 mole % of a compound of Formula (I); TIFF2026507731000001.tif60170About 51.75 mol% cholesterol, about 5 mol% DOPC, The polynucleotide encoding the polypeptide of SEQ ID NO: 76 is an RNA molecule encapsulated in at least one lipid nanoparticle comprising approximately 2.5 mol% DMG-PEG2000, and the ratio of lipid to RNA molecule in the at least one nanoparticle is approximately 120:1 (w / w).

[0010] In some embodiments, the composition is encapsulated in at least one lipid nanoparticle comprising about 54 mol% SS-OP, about 35 mol% cholesterol, about 5 mol% DOPC, about 5 mol% DSPC, and about 1 mol% DMG-PEG2000, wherein the polynucleotide encoding the polypeptide of SEQ ID NO: 76 is an RNA molecule, the ratio of lipid to RNA molecule in the at least one nanoparticle is about 100:1 (w / w), and the total lipid is 25 nM.

[0011] In some embodiments, the plurality of cells comprises (a) hepatocytes, preferably hepatocytes, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal endothelial cells; (b) T cells, preferably activated T cells, resting T cells, or stem memory T cells (T SCM (c) hematopoietic stem cells (HSCs), or T cells.

[0012] The present disclosure also provides a cell modified according to any one of the methods of the present disclosure. The present disclosure also provides a composition comprising a population of cells modified according to any one of the methods of the present disclosure.

[0013] The present disclosure also provides methods for treating at least one disease or disorder in a subject in need thereof, comprising administering to the subject at least one therapeutically effective amount of a composition, pharmaceutical composition, or cell described herein. In some embodiments, at least one disease or disorder is a liver disease or disorder, preferably, the liver disease or disorder is (a) a metabolic liver disorder, (b) a urea cycle disorder (UCD), preferably, the UCD is N-acetylglutamate synthetase (NAGS) deficiency, carbamoyl phosphate synthetase I deficiency (CPSI deficiency), ornithine transcarbamylase (OTC) deficiency, argininosuccinate synthetase deficiency (ASSD) (citrullinemia I), citrin deficiency (citrullinemia II), argininosuccinate lyase deficiency (argininosuccinic aciduria), arginase deficiency (hyperargininemia), ornithine translocase deficiency (HHH syndrome), or any combination thereof. In some embodiments, at least one disease or disorder is cancer. In some embodiments, at least one disease or disorder is hemophilia A.

[0014] All documents cited herein, including cross-references or related patents or applications, are incorporated by reference in their entirety for all purposes unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein, or that it alone, or in combination with any other reference or references, teaches, suggests, or discloses any such invention. Furthermore, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall control. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description of the Invention The present invention provides a method for genetically modifying a genome to include insertions, deletions, and / or substitutions of polynucleotides. In particular, the present disclosure overcomes the problems associated with current technology by providing a method for efficiently genetically modifying a cellular genome to include insertions, deletions, and / or substitutions of polynucleotides. This is advantageous for modifying genes corresponding to disease phenotypes that have important therapeutic implications. This is advantageous for producing a higher yield of modified cells compared to current technology, which is in turn advantageous for producing cells for therapeutic use.

[0016] The present disclosure is based, at least in part, on the discovery that genetic modification of cells with compositions containing mutant forms of Cas-CLOVER results in higher deletion rates compared to wild-type Cas-CLOVER (NLS-dCas9-Clo051-NLS) or conventional CRISPR / Cas9 systems. Structural modeling and sequence analysis of the Clo051 endonuclease domain of Cas-CLOVER relative to other nuclease domains (e.g., the FokI nuclease domain) revealed several conserved amino acid positions that confer hypermobility, thereby resulting in enhanced function. A serine-to-proline modification at a position within a conserved alpha-helical loop has a stabilizing effect because the proline alters the turn of the loop and reduces the degree of freedom at this end of the loop-helix. Furthermore, the proline can still interact with the phosphate backbone of bound DNA within an acceptable hydrogen-bonding distance (e.g., approximately 5 Å or less), maintaining the potential interaction that would have been observed with wild-type Clo051 endonuclease at this position. The use of mutant Cas-CLOVER containing mutant Clo051 endonuclease or a portion thereof provides enhanced functionality such as improved genome editing and knock-in and knock-out efficiency.

[0017] Thus, the present invention provides an efficient, reliable, and targeted approach for transiently or stably incorporating one or more exogenous genes and maintaining high viability and functional response in various cell types, such as expanded iPSCs, as well as differentiated cells derived from modified iPSCs, including, but not limited to, HSCs (hematopoietic stem and progenitor cells), T cell progenitors, NK cell progenitors, T cells, NKT cells, and NK cells, which are useful for the development of safe and effective universal donor cells to support cell therapy treatments (e.g., regenerative medicine or treatment of immune-oncology-related diseases).

[0018] The present disclosure overcomes problems associated with current technology by providing compositions containing engineered fusion molecules (e.g., mutant forms of Cas-CLOVER) for selectively reducing or eliminating gene products in cells for use in in vivo gene therapy. Compositions containing the engineered fusion molecules of the present disclosure are useful for treating genetic diseases. Non-limiting examples include liver diseases, hepatocyte-associated diseases, sickle cell disease, or beta-thalassemia. Accordingly, methods for producing engineered fusion molecules and pharmaceutical formulations thereof (e.g., lipid nanoparticle formulations) for use in in vivo delivery are also provided.

[0019] The present disclosure is based, at least in part, on the discovery that even a small increase in editing activity (e.g., in the range of 1.5- to 3-fold) provided by the disclosed mutant Cas-CLOVER relative to wild-type Cas-CLOVER can have a significant impact on in vivo therapeutic applications. This increase can translate into the difference between a non-useful therapeutic index of efficacy of 25% to 50% and a useful therapeutic index of efficacy of 75%, in which a large proportion of disease (mutant) or disease-associated alleles can be inactivated, including biallelic editing. As a non-limiting example, in the case of HBG locus targeting, the magnitude of improvement provided by the disclosed mutant Cas-CLOVER may exceed a critical therapeutic threshold for fully enabling activation of fetal hemoglobin (HbF) expression, which would result in therapeutic efficacy for functional correction of sickle cell disease or beta-thalassemia. Alternatively, increased activity of the mutant Cas-CLOVER may allow for a reduction in the dosage of the administered therapeutic, with benefits in terms of tolerability and toxicity.

[0020] Methods for targeted genome editing at selected loci

[0021] Gene editing compositions and methods

[0022] The modified cells can be produced by introducing a transgene into the cells. The introducing step can include delivery of the nucleic acid sequence, transgene and / or genome editing construct via a non-transposition delivery system.

[0023] Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ can include one or more of local delivery, adsorption, absorption, electroporation, spinfection, co-culture, transfection, mechanical delivery, sonic delivery, vibration delivery, magnetofection, or nanoparticle-mediated delivery. Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ can include liposome transfection, calcium phosphate transfection, fusion transfection, and dendrimer-mediated transfection. Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ by mechanical transfection can include cell squeezing, cell bombardment, or gene gun techniques. Introducing nucleic acid sequences, transgenes and / or genome editing constructs into cells ex vivo, in vivo, in vitro or in situ by nanoparticle-mediated transfection can include liposome delivery, micelle delivery and polymersome delivery.

[0024] Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ can include non-viral vectors. Non-viral vectors can include nucleic acids. Non-viral vectors can include plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), DoggyBone™ DNA, nanoplasmid, minicircle DNA, single-stranded oligodeoxynucleotides (ssODN), double-stranded oligonucleotides (dsODN), single-stranded mRNA (ssRNA), and double-stranded mRNA (dsRNA). Non-viral vectors can include transposons as described herein.

[0025] Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ can include viral vectors. The viral vector can be a non-integrating, non-chromosomal vector. Non-limiting examples of non-integrating, non-chromosomal vectors include adeno-associated virus (AAV), adenovirus, and herpes virus. The viral vector can be an integrating, chromosomal vector. Non-limiting examples of integrating, chromosomal vectors include adeno-associated vector (AAV), lentivirus, and gammaretrovirus.

[0026] Introducing a nucleic acid sequence, transgene, and / or genome editing construct into a cell ex vivo, in vivo, in vitro, or in situ can involve a combination of vectors. Non-limiting examples of vector combinations include a viral vector and a non-viral vector, multiple non-viral vectors, or multiple viral vectors. Non-limiting examples of vector combinations include a combination of a DNA-based vector and an RNA-based vector, a combination of RNA and reverse transcriptase, a combination of a transposon and a transposase, a combination of a non-viral vector and an endonuclease, and a combination of a viral vector and an endonuclease.

[0027] Genome modification can include introducing a nucleic acid sequence, a transgene, and / or a genome editing construct into a cell ex vivo, in vivo, in vitro, or in situ to stably integrate the nucleic acid sequence, to transiently integrate the nucleic acid sequence, to cause site-specific integration of the nucleic acid sequence, or to cause biased integration of the nucleic acid sequence. The nucleic acid sequence can be a transgene.

[0028] The nucleic acid sequence or transgene can be from about 1 kb to about 15 kb in size. The nucleic acid sequence or transgene can be at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 11 kb, at least 12 kb, at least 13 kb, at least 14 kb, or at least 15 kb in size. The nucleic acid sequence or transgene can be about 1 kb, about 2 kb, about 3 kb, about 4 kb, about 5 kb, about 6 kb, about 7 kb, about 8 kb, about 9 kb, about 10 kb, about 11 kb, about 12 kb, about 13 kb, about 14 kb, or about 15 kb in size.

[0029] Genome modification can include introducing nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ to stably integrate the nucleic acid sequences. Stable chromosomal integration can be random, site-specific, or biased. Site-specific integration can be unassisted or assisted. Assisted site-specific integration is co-delivered with a site-specific nuclease. The site-specific nuclease includes a transgene with 5' and 3' nucleotide sequence extensions containing percentage homology to regions upstream and downstream of the site of genomic integration. A transgene with homologous nucleotide extensions allows for genome integration by homologous recombination, microhomology-mediated end joining, or non-homologous end joining. Site-specific integration can occur at a safe harbor site. A genomic safe harbor site can provide a location for integration of new genetic material in a manner that ensures that the newly inserted genetic element is functional (e.g., expressed at therapeutically effective expression levels) and does not cause deleterious changes to the host genome that pose a risk to the host organism. Non-limiting examples of potential genomic safe harbors include intron sequences of the human albumin gene, the adeno-associated virus site 1 (AAVS1), the naturally occurring integration site of the AAV virus on chromosome 19, the site of the chemokine (CC motif) receptor 5 (CCR5) gene, and the site of the human ortholog of the mouse Rosa26 locus.

[0030] Site-specific transgene integration can occur at a site that disrupts expression of a target gene. Disruption of target gene expression can occur by site-specific integration at an intron, exon, promoter, genetic element, enhancer, suppressor, start codon, stop codon, and response element. Non-limiting examples of target genes targeted by site-specific integration include TRAC, TRAB, PDI, any immunosuppressive gene, and genes involved in allorejection.

[0031] Site-specific transgene integration can occur at sites that result in enhanced expression of the target gene. Enhancement of target gene expression can occur by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.

[0032] Enzymes can be used to generate strand breaks in the host genome to facilitate transgene delivery or integration. The enzymes can generate single-strand or double-strand breaks. Non-limiting examples of cleavage-inducing enzymes include transposases, integrases, endonucleases, CRISPR / Cas9, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), Cas-CLOVER™, and CPF1. Cleavage-inducing enzymes can be delivered to cells encoded by DNA, encoded by mRNA, as proteins, or as nucleoprotein complexes with guide RNAs (gRNAs). Non-limiting examples of cleavage-inducing enzymes are described in International Application Nos. 2016 / 037922, 2018 / 066941, and 2017 / 054799, each of which is incorporated by reference in its entirety. Exemplary mutant Cas-CLOVER cleavage-inducing enzymes of the present disclosure are also described below.

[0033] Site-specific transgene integration can be controlled by vector-mediated integration site bias.

[0034] The site-specific transgene integration site may be a non-stable chromosomal insertion. The integrated transgene may be silenced, removed, excised, or further modified. The genome modification may be a non-stable integration of the transgene. The non-stable integration may be a transient non-chromosomal integration, a semi-stable non-chromosomal integration, a semi-persistent non-chromosomal insertion, or a non-stable chromosomal insertion. The transient non-chromosomal insertion may be epichromosomal or cytoplasmic. In one aspect, the transient non-chromosomal insertion of the transgene is not integrated into a chromosome, and the modified genetic material is not replicated during cell division.

[0035] The genome modification can be semi-stable or persistent non-chromosomal integration of the transgene. The DNA vector encodes a scaffold / matrix attachment region (S-MAR) module that binds to nuclear matrix proteins for episomal retention of the non-viral vector, allowing autonomous replication in the nuclei of dividing cells.

[0036] The genome modification can be non-stable chromosomal integration of the transgene. The integrated transgene can be silenced, removed, excised, or further modified.

[0037] Modification of the genome by transgene insertion can occur via homologous recombination (HR), microhomology-mediated end joining (MMEJ), non-homologous end joining (NHEJ), transposase enzyme-mediated modification, integrase enzyme-mediated modification, endonuclease enzyme-mediated modification, or host cell-directed double-strand break repair (homology-directed repair) by recombinase enzyme-mediated modification. Modification of the genome by transgene insertion can occur via CRISPR / Cas9, TALEN, ZFN, Cas-CLOVER™, and cpfl. Non-limiting examples of cleavage-inducing enzymes are described in International Application No. / US2016 / 037922, International Application No. / US2018 / 066941, and International Application No. / US2017 / 054799, each of which is incorporated by reference in its entirety. Exemplary mutant Cas-CLOVER cleavage-inducing enzymes of the present disclosure are also described herein.

[0038]

[0032] In gene editing systems that involve the insertion of new or existing nucleotides / nucleic acids, an insertion tool (e.g., a DNA template vector, a transposable element (transposon or retrotransposon) must be delivered to the cell in addition to a cleavage enzyme (e.g., a nuclease, recombinase, integrase, or transposase). Examples of such an insertion tool for a recombinase may include a DNA vector. Other gene editing systems require the delivery of an integrase with an insertion vector, the delivery of a transposase with a transposon / retrotransposon, etc. An example of a recombinase that may be used as a cleavage enzyme is CRE recombinase. Non-limiting examples of integrases that may be used in insertion means include viral-based enzymes obtained from any of several viruses, such as AAV, gammaretroviruses, and lentiviruses. Examples of transposons / retrotransposons that may be used in insertion means are described in more detail herein.

[0039] The present disclosure provides a gene editing composition and / or a cell comprising the gene editing composition. The gene editing composition may comprise a sequence encoding a DNA binding domain and a sequence encoding a nuclease protein or its nuclease domain. The sequence encoding the nuclease protein or its nuclease domain may comprise a DNA sequence, an RNA sequence, or a combination thereof. The nuclease or its nuclease domain may comprise one or more of a CRISPR / Cas protein, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), and an endonuclease.

[0040] Exemplary dCas9-Clo051 Fusion Proteins

[0041] The nuclease or nuclease domain thereof may comprise a nuclease-inactivated Cas (dCas) protein and an endonuclease. The endonuclease may comprise a Clo051 nuclease or a nuclease domain thereof. The gene editing composition may comprise a fusion protein. The fusion protein may comprise a nuclease-inactivated Cas9 (dCas9) protein and a Clo051 nuclease or a Clo051 nuclease domain. The gene editing composition may further comprise a guide sequence. The guide sequence comprises an RNA sequence.

[0042] The present disclosure provides compositions comprising a mini-Cas9 (Cas9) operably linked to an effector. The present disclosure provides fusion proteins comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a mini-Cas9 (Cas9). The mini-Cas9 constructs of the present disclosure can include an effector comprising a type IIS endonuclease. A Staphylococcus aureus Cas9 having an active catalytic site comprises the amino acid sequence of SEQ ID NO: 1.

[0043] The present disclosure provides compositions comprising an inactivated mini-Cas9 (dSaCas9) operably linked to an effector. The present disclosure provides fusion proteins comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a mini-inactivated Cas9 (dSaCas9). The mini-inactivated Cas9 (dSaCas9) construct of the present disclosure can include an effector comprising a type IIS endonuclease. The dSaCas9 comprises the amino acid sequence of SEQ ID NO: 2, including D10A and N580A mutations that inactivate the catalytic site.

[0044] The present disclosure provides compositions comprising an inactivated Cas9 (dCas9) operably linked to an effector. The present disclosure provides fusion proteins comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, where the effector comprises an inactivated Cas9 (dCas9). The inactivated Cas9 (dCas9) constructs of the present disclosure can include an effector that comprises a type IIS endonuclease.

[0045] dCas9 can be isolated or derived from Streptococcus pyogenes. dCas9 can include dCas9 with substitutions at amino acid positions 10 and 840 that inactivate the catalytic site. In some embodiments, these substitutions are D10A and H840A. dCas9 can include the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0046] An exemplary Clo051 nuclease domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5. In some embodiments, the Clo051 nuclease domain comprises at least one amino acid substitution. In some embodiments, the amino acid substitution is within the alpha helix loop domain of Clo051 nuclease. In some embodiments, the amino acid substitution is at position 35, 37, 60, 92, 98, 100, or 146 of SEQ ID NO: 5. In some embodiments, the amino acid substitution is at position 37 of SEQ ID NO: 5. In some embodiments, the amino acid substitution is at positions 37 and 92 of SEQ ID NO: 5.

[0047] An exemplary dCas9-Clo051 fusion protein can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 6. An exemplary dCas9-Clo051 fusion protein can be encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 7. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0048] An exemplary dCas9-Clo051 fusion protein can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 8. An exemplary dCas9-Clo051 fusion protein can be encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 9. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0049] Exemplary dCas9-Clo051 fusion proteins of the present disclosure may further comprise at least one nuclear localization sequence (NLS). In some embodiments, dCas9-Clo051 fusion proteins of the present disclosure comprise at least two nuclear localization sequences. In some embodiments, the NLS is at the N-terminus of the dCas9-Clo051 fusion protein (NLS-dCas9-Clo051). In some embodiments, the NLS is at the C-terminus of the dCas9-Clo051 fusion protein (dCas9-Clo051-NLS). In some embodiments, the NLS is at both the N- and C-termini of the dCas9-Clo051 fusion protein ("NLS-dCas9-Clo051-NLS" or "wild-type Cas-CLOVER").

[0050] The NLS-dCas9-Clo051-NLS ("wild-type Cas-CLOVER") fusion protein can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO:10.

[0051] NLS-dCas9-Clo051-NLS amino acid sequence (NLS amino acid sequence is bold and underlined) MA PKKKRKV PKKKRKV SS (SEQ ID NO: 10).

[0052] The nucleic acid encoding the NLS-dCas9-Clo051-NLS ("wild-type Cas-CLOVER") fusion protein can be DNA or RNA. In some embodiments, the NLS-dCas9-Clo051-NLS is encoded by an mRNA sequence comprising, consisting essentially of, or consisting of SEQ ID NO:11.

[0053] NLS-dCas9-Clo051-NLS mRNA sequence (NLS amino acid sequence is bold and underlined) atggca ccaaagaaaaaaagtg cccaagaaaaagcggaaggtg tctagttaatga (SEQ ID NO: 11).

[0054] Exemplary Mutant Cas-CLOVER Fusion Proteins

[0055] In some embodiments, NLS-dCas9-Clo051-NLS ("wild-type Cas-CLOVER") comprises at least one amino acid substitution, which in some embodiments is located in the Clo051 domain of NLS-dCas9-Clo051-NLS.

[0056] In some embodiments, the NLS-dCas9-Clo051-NLS of SEQ ID NO: 10 comprises at least one substitution at amino acid positions 42, 44, 67, 105, 107, or 153. In some embodiments, the amino acid substitution is F42E, F42D, S44E, S44P, R67E, I105Q, Q107A, Q107E, Q107H, Q107D, and / or K153D. In some embodiments, the amino acid substitution is S44P.

[0057] An exemplary S44P mutant NLS-dCas9-Clo051-NLS ("S44P Cas-CLOVER" or "S44P CC" or "S44P") fusion protein can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 35. The S44P Cas-CLOVER fusion protein can be encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 36. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0058] S44P Cas-CLOVER amino acid sequence (SEQ ID NO: 35) MAPKKKRKVE GIKSNISLLK DELRGQISHI SHEYLSLIDL AFDPKQNRLF EMKVLELLVN 60 EYGFKGRHLG GSRKPDGIVY STTLEDNFGI IVDTKAYSEG YSLPISQADE MERYVRENSN 120 RDEEVNPNKW WENFSEEVKK YYFVFISGSF KGKFEEQLRR LSMTTGVNGS AVNVVNLLLG 180 AEKIRSGEMT IEELERAMFN NSEFILKYGG GGSDKKYSIG LAIGTNSVGW AVITDEYKVP 240 SKKFKVLGNT DRHSIKKNLI GALLFDSGET AEATRLKRTA RRRYTRRKNR ICYLQEIFSN 300 EMAKVDDSFF HRLEESFLVE EDKKHERHPI FGNIVDEVAY HEKYPTIYHL RKKLVDSTDK 360 ADLRLIYLAL AHMIKFRGHF LIEGDLNPDN SDVDKLFIQL VQTYNQLFEE NPINASGVDA 420 KAILSARLSK SRRLENLIAQ LPGEKKNGLF GNLIALSLGL TPNFKSNFDL AEDAKLQLSK 480 DTYDDDLDNL LAQIGDQYAD LFLAAKNLSD AILLSDILRV NTEITKAPLS ASMIKRYDEH 540 HQDLTLLKAL VRQQLPEKYK EIFFDQSKNG YAGYIDGGAS QEEFYKFIKP ILEKMDGTEE 600 LLVKLNREDL LRKQRTFDNG SIPHQIHLGE LHAILRRQED FYPFLKDNRE KIEKILTFRI 660 PYYVGPLARG NSRFAWMTRK SEETITPWNF EEVVDKGASA QSFIERMTNF DKNLPNEKVL 720 PKHSLLYEYF TVYNELTKVK YVTEGMRKPA FLSGEQKKAI VDLLFKTNRK VTVKQLKEDY 780 FKKIECFDSV EISGVEDRFN ASLGTYHDLL KIIKDKDFLD NEENEDILED IVLTLTLFED 840 REMIEERLKT YAHLFDDKVM KQLKRRRYTG WGRLSRKLIN GIRDKQSGKT ILDFLKSDGF 900 ANRNFMQLIH DDSLTFKEDI QKAQVSGQGD SLHEHIANLA GSPAIKKGIL QTVKVVDELV 960 KVMGRHKPEN IVIEMARENQ TTQKGQKNSR ERMKRIEEGI KELGSQILKE HPVENTQLQN 1020 EKLYLYYLQN GRDMYVDQEL DINRLSDYDV DAIVPQSFLK DDSIDNKVLT RSDKNRGKSD 1080 NVPSEEVVKK MKNYWRQLLN AKLITQRKFD NLTKAERGGL SELDKAGFIK RQLVETRQIT 1140 KHVAQILDSR MNTKYDENDK LIREVKVITL KSKLVSDFRK DFQFYKVREI NNYHHAHDAY 1200 LNAVVGTALI KKYPKLESEF VYGDYKVYDV RKMIAKSEQE IGKATAKYFF YSNIMNFFKT 1260 EITLANGEIR KRPLIETNGE TGEIVWDKGR DFATVRKVLS MPQVNIVKKT EVQTGGFSKE 1320 SILPKRNSDK LIARKKDWDP KKYGGFDSPT VAYSVLVVAK VEKGKSKKLK SVKELLGITI 1380 MERSSFEKNP IDFLEAKGYK EVKKDLIIKL PKYSLFELEN GRKRMLASAG ELQKGNELAL 1440 PSKYVNFLYL ASHYEKLKGS PEDNEQKQLF VEQHKHYLDE IIEQISEFSK RVILADANLD 1500 KVLSAYNKHR DKPIREQAEN IIHLFTLTNL GAPAAFKYFD TTIDRKRYTS TKEVLDATLI HQSITGLYET RIDLSQLGGD GSPKKKRKVS S

[0059] S44P Cas-CLOVER SPECIFICATIONS (36 SPECIFICATIONS) atggctccca agaagaagcg gaaggtcgag ggcatcaaga gcaacatcag cctgctgaag gacgagctga gaggccagat cagccacatc tcccacgagt acctgagcct gatcgacctg gccttcgacc ccaagcagaa ccggctgttc gagatgaagg tgctggaact gctggtcaac gagtacggct tcaagggcag acacctcggc ggcagcagaa agcctgatgg catcgtgtac agcaccacac tcgaggaca cttcggcatc atcgtggaca ccaaggccta cagcgagggc 360. ctatctctca ggccgacgag atggaagat acgtgcgcga gaacagcaac cgcgacgagg aagtgaaccc caacaagtgg tgggagaact tcagcgagg agtcaaaaag 420 tactacttcg tgttcatcag cggcagcttt aagggcaagt tcgaggaaca gctgcggcgg 480 ctgtctatga ccacaggcgt taacggcagc gccgtgaacg tggtcaatct gctgctgggc 540 gccgagaaga ttagaagcgg cgagatgacc atcgaggaac tggaacgggc catgttcaac 600 aacagcgagt tcatcctgaa gtacggcgga ggcggcagcg acaagaagta ctctatcgga 660 ctggccatcg gcaccaactc tgttggatgg gccgtgatca ccgacgagta caaggtgccc 720 agcaagaaat tcaaagtgct gggcaacacc gaccggcaca gcatcaagaa gaatctgatc 780 ggcgccctgc tgttcgactc tggcgaaaca gccgaagcca ccagactgaa gagaaccgcc 840 agacggcggt acaccagaag aaagaaccgg atctgctacc tgcaagagat cttcagcaac 900 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa 960 gaggacaaga agcacgagcg gcaccccatc ttcggaaata tcgtggacga ggtggcctac 1020 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag 1080 gccgacctgc gactgatcta tctggccctg gctcacatga tcaagttccg gggccacttc 1140 ctgatcgagg gcgacctgaa tcctgacaac tccgacgtgg acaagctgtt catccagctg 1200 gtgcagacct acaatcagct gttcgaagag aatcccatca acgcctctgg cgtggacgcc 1260 aaagccatcc tgtctgccag actgagcaag agcagacggc tggaaaaacct gatcgctcag 1320 ctgcccggcg yours tggcctgttc ggcaacctga ttgccctgtc tctgggcctg 1380 acacctaact tcaagtccaa cttcgatctg gccgaggatg ccaaactgca gctgtccaag 1440 gacacctacg acgacgacct ggataacctg ctggcccaga tcggcgatca gtacgccgac 1500 ttgtttctgg ccgccaagaa cctgtctgac gccatcctgc tgagcgacat cctgagagtg 1560 1620 caccaggatc tgaccctgct gaaagctctc gtcaggcagc agctgccaga gaagtaaa 1680 gagattttct tcgaccagag caagaacggc tacgccggct acattgatgg cggagccagc 1740 caagaggaat tctacaagtt catcaagccc atcctcgaga agatggacgg cacagaggaa 1800 1860 tctatccctc accagatcca cctgggagag ctgcacgcca ttctgcggag acaagaggac 1920 ttttacccat tcctgaagga caaccgggaa aagattgaga agatcctgac cttcaggatc 1980 ccctactacg tgggaccact ggccagaggc aatagcagat tcgcctggat gaccagaaag 2040 agcgaggaaa ccatcacacc ctggaacttc gaagaggtgg tggacaaggg cgccagcgct 2100 cagtccttca tcgagcggat gaccaatttc gacaagaatc tgcccaacga gaaagtgctg 2160 cccaagcact ccctgctgta cgagtacttc accgtgtaca acgagctgac caaagtgaaa 2220 tacgtgaccg agggaatgag aaagcccgcc tttctgtccg gcgagcagaa aaaggccatc 2280 gtggatctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac 2340 ttcaagaaaa tcgagtgctt cgactccgtg gaaatcagcg gcgtggaaga tcggttcaat 2400 gccagcctgg gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac 2460 aacgaggaaa acgaggacat ccttgaggac atcgtgctga ccctgacact gttcgaggac 2520 agagagatga tcgaggaaag gctgaaaaca tacgcccacc tgttcgacga caaagtcatg 2580 aagcaactga agcggcggcg ctacacaggc tggggcagac tgtctagaaa gctgatcaac 2640 ggcatccggg acaagcagtc cggcaagacc atcctggact ttctgaagtc cgacggcttc 2700 gccaacagaa acttcatgca gctgattcac gacgacagcc tcaccttcaa agaggacatt 2760 cagaaggccc aggttccgg ccaggggcgat tcttgcacg agcacattgc caatctggcc 2820 ggctctcccg ccattaagaa gggcattctg cagacagtga aagtggtgga tgagctggtc 2880 aaagtgatgg ggagacacaa gcccgagaac atcgtgatcg aaatggccag aggaaccag 2940 accacacaga agggcagaa gaactccccgc gagagaatga agcggatcga gagggaatc 3000 aaagagctgg gggaccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac 3060 gagaagctgt acctgtacta cctccagaac ggccgggata tgtacgtgga ccagagctg 3120 gatacaacc gcctgagcga ctacgatgtg gacgctatcg tgccccagtc ttttctgaaa 3180 gatgactcca tcgacaaca ggtgctgacc agaagcgata agaaccgggg caagcgac 3240 aacgtgccct ctgaagaggt cgtgagaag atgagact actggcgaca gctgctgaac 3300 gccaagctga ttacccagcg gaagttcgat aacctgacca aggccgag aggcggcctg 3360 tctgaactgg ataggccgg cttcatcaag agacagctgg tggaacccg gcagatcacc 3420 aaacacgtgg cacagattct ggactcccgg atgaacacca aatacgatga gaacgacaaa 3480 ctgatccggg aagtgaaagt catcaccctg aagtccaagc tggtgtccga tttccggaag 3540 gatttccagt tctacaaagt gcgggaaatc aacaactacc atcacgccca cgacgcctac 3600 ctgaatgccg ttgttggaac agccctgatc aagaagtatc ccaagctgga aagcgagttc 3660 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaagag 3720 attggaaagg ctaccgccaa atacttcttc tactccaaca tcatgaactt tttcaagaca 3780 gagatcaccc tcgccaacgg cgagatcaga aagcggcctc tgatcgagac aaacggcgaa 3840 accggcgaga ttgtgtggga taagggcaga gactttgcca cagtgcggaa ggtgctcagc 3900 atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag 3960 tccattctgc ctaagcggaa ctccgacaag ctgatcgccc ggaagaagga ctgggacccc 4020 aagaaatacg gcggcttcga tagccctacc gtggcctatt ctgtgctggt ggtggccaaa 4080 gtggaaaagg gaaagtccaa gaagctcaag agcgtcaaag aactcctggg catcaccatc 4140 atggaacggt ccagcttcga gaagaaccct atcgactttc tggaagccaa gggctacaaa 4200 gaagtcaaga aggacctgat catcaagctc cccaagtaca gcctgttcga gctggaaaat 4260 ggccggaagc ggatgctggc ttctgctggc gaactgcaga agggaaacga actggccctg 4320 cctagcaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggcagc 4380 cccgaggaca atgagcagaa gcagctttc gtcgagcagc acaagcacta cctggacgag 4440 atcatcgagc agatctccga gttctccaag agagtgatcc tggccgacgc caacctggac 4500 aaggttctgt ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat 4560 atcatccacc tgtttaccct gaccaacctg ggagcccctg ccgccttcaa gtacttcgac 4620 accaccatcg accggaagcg ctacaccagc accaaagaag tgctggacgc cacactgatc 4680 caccagagca tcaccggcct gtacgagaca cggatcgatc tgtctcagct tggaggcgac 4740 ggcagcccta agaagaagag aaaggtttcc agctaataa 4779

[0060] In some embodiments, the NLS-dCas9-Clo051-NLS of SEQ ID NO: 10 can comprise at least one substitution at amino acid position 99. In some embodiments, the amino acid substitution is E99K or E99R. In some embodiments, the amino acid substitution is E99K. In some embodiments, the amino acid substitution is E99R.

[0061] An exemplary E99K mutant NLS-dCas9-Clo051-NLS ("E99K Cas-CLOVER" or "E99K CC" or "E99K") fusion protein can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 78. The E99K Cas-CLOVER fusion protein can be encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 79. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0062] E99K Cas-CLOVER amino acid sequence (SEQ ID NO: 78) MAPKKKRKVE GIKSNISLLK DELRGQISHI SHEYLSLIDL AFDSKQNRLF EMKVLELLVN 60 EYGFKGRHLG GSRKPDGIVY STTLEDNFGI IVDTKAYSKG YSLPISQADE MERYVRENSN 120 RDEEVNPNKW WENFSEEVKK YYFVFISGSF KGKFEEQLRR LSMTTGVNGS AVNVVNLLLG 180 AEKIRSGEMT IEELERAMFN NSEFILKYGG GGSDKKYSIG LAIGTNSVGW AVITDEYKVP 240 SKKFKVLGNT DRHSIKKNLI GALLFDSGET AEATRLKRTA RRRYTRRKNR ICYLQEIFSN 300 EMAKVDDSFF HRLEESFLVE EDKKHERHPI FGNIVDEVAY HEKYPTIYHL RKKLVDSTDK 360 ADLRLIYLAL AHMIKFRGHF LIEGDLNPDN SDVDKLFIQL VQTYNQLFEE NPINASGVDA 420 KAILSARLSK SRRLENLIAQ LPGEKKNGLF GNLIALSLGL TPNFKSNFDL AEDAKLQLSK 480 DTYDDDLDNL LAQIGDQYAD LFLAAKNLSD AILLSDILRV NTEITKAPLS ASMIKRYDEH 540 HQDLTLLKAL VRQQLPEKYK EIFFDQSKNG YAGYIDGGAS QEEFYKFIKP ILEKMDGTEE 600 LLVKLNREDL LRKQRTFDNG SIPHQIHLGE LHAILRRQED FYPFLKDNRE KIEKILTFRI 660 PYYVGPLARG NSRFAWMTRK SEETITPWNF EEVVDKGASA QSFIERMTNF DKNLPNEKVL 720 PKHSLLYEYF TVYNELTKVK YVTEGMRKPA FLSGEQKKAI VDLLFKTNRK VTVKQLKEDY 780 FKKIECFDSV EISGVEDRFN ASLGTYHDLL KIIKDKDFLD NEENEDILED IVLTLTLFED 840 REMIEERLKT YAHLFDDKVM KQLKRRRYTG WGRLSRKLIN GIRDKQSGKT ILDFLKSDGF 900 ANRNFMQLIH DDSLTFKEDI QKAQVSGQGD SLHEHIANLA GSPAIKKGIL QTVKVVDELV 960 KVMGRHKPEN IVIEMARENQ TTQKGQKNSR ERMKRIEEGI KELGSQILKE HPVENTQLQN 1020 EKLYLYYLQN GRDMYVDQEL DINRLSDYDV DAIVPQSFLK DDSIDNKVLT RSDKNRGKSD 1080 NVPSEEVVKK MKNYWRQLLN AKLITQRKFD NLTKAERGGL SELDKAGFIK RQLVETRQIT 1140 KHVAQILDSR MNTKYDENDK LIREVKVITL KSKLVSDFRK DFQFYKVREI NNYHHAHDAY 1200 LNAVVGTALI KKYPKLESEF VYGDYKVYDV RKMIAKSEQE IGKATAKYFF YSNIMNFFKT 1260 EITLANGEIR KRPLIETNGE TGEIVWDKGR DFATVRKVLS MPQVNIVKKT EVQTGGFSKE 1320 SILPKRNSDK LIARKKDWDP KKYGGFDSPT VAYSVLVVAK VEKGKSKKLK SVKELLGITI 1380 MERSSFEKNP IDFLEAKGYK EVKKDLIIKL PKYSLFELEN GRKRMLASAG ELQKGNELAL 1440 PSKYVNFLYL ASHYEKLKGS PEDNEQKQLF VEQHKHYLDE IIEQISEFSK RVILADANLD 1500 KVLSAYNKHR DKPIREQAEN IIHLFTLTNL GAPAAFKYFD TTIDRKRYTS TKEVLDATLI 1560 HQSITGLYET RIDLSQLGGD GSPKKKRKVS S 1591

[0063] E99K Cas-CLOVER nucleic acid sequences atggctccca agaagaagcg gaaggtcgag ggcatcaaga gcaacatcag cctgctgaag 60 gacgagctga gaggccagat cagccacatc tcccacgagt acctgagcct gatcgacctg 120 gccttcgaca gcaagcagaa ccggctgttc gagatgaagg tgctggaact gctggtcaac gagtacggct tcaagggcag acacctcggc ggcagcagaa agcctgatgg catcgtgtac agcaccacac tcgaggaca cttcggcatc atcgtggaca ccaaggccta cagcaagggc 360. ctatctctca ggccgacgag atggaagat acgtgcgcga gaacagcaac cgcgacgagg aagtgaaccc caacaagtgg tgggagaact tcagcgagg agtcaaaaag 420 tactacttcg tgttcatcag cggcagcttt aagggcaagt tcgaggaaca gctgcggcgg 480 ctgtctatga ccacaggcgt taacggcagc gccgtgaacg tggtcaatct gctgctgggc 540 gccgagaaga ttagaagcgg cgagatgacc atcgaggaac tggaacgggc catgttcaac aacagcgagt tcatcctgaa gtacggcgga ggcggcagcg acaagagta ctctatcgga 660 ctggccatcg gcaccaactc tgttggatgg gccgtgatca ccgacgagta caaggtgccc 720 agcaagaat tcaaagtgct gggcaacacc gaccggcaca gcatcaaga gaatctgatc ggcgccctgc tgttcgactc tggcgaaaca gccgaagcca ccagactgaa gagaaccgcc agacggcggt acaccagaag aaagaaccgg atctgctacc tgcaagagat cttcagcaac 900 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa 960 gaggacaaga agcacgagcg gcaccccatc ttcggaaata tcgtggacga ggtggcctac 1020 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag 1080 gccgacctgc gactgatcta tctggccctg gctcacatga tcaagttccg gggccacttc 1140 ctgatcgagg gcgacctgaa tcctgacaac tccgacgtgg acaagctgtt catccagctg 1200 gtgcagacct acaatcagct gttcgaagag aatcccatca acgcctctgg cgtggacgcc 1260 aaagccatcc tgtctgccag actgagcaag agcagacggc tggaaaacct gatcgctcag 1320 ctgcccggcg agaagaagaa tggcctgttc ggcaacctga ttgccctgtc tctgggcctg 1380 acacctaact tcaagtccaa cttcgatctg gccgaggatg ccaaactgca gctgtccaag 1440 gacacctacg acgacgacct ggataacctg ctggcccaga tcggcgatca gtacgccgac 1500 ttgtttctgg ccgccaagaa cctgtctgac gccatcctgc tgagcgacat cctgagagtg 1560 aacaccgaga tcacaaggc cccctgagc gcctctatga tcaagagata cgacgagcac 1620 caccaggatc tgaccctgct gaagctctc gtcaggcagc agctgccaga gaagtacaa 1680 gagattttct tcgaccagag cagaacggc tacgccggct acatgatgg cggagccagc 1740 cagaggaat tctacaagtt catcaagcccc atcctcgaga agatggacgg cacagaggaa 1800 ctgctcgtga agctgacag agaggacctg ctgcggaagc agcggacctt cgacaatggc 1860 tctatccctc accagatcca cctgggag ctgcacgcca ttctcggag acagaggac 1920 ttttacccat tcctgaagga caccggga aagattgaga agatcctgac cttcaggatc 1980 ccctactacg tgggaccact ggccagaggc atagcagat tcgcctggat gaccagaaag 2040 agcgaggaaa ccatcacacc ctggaacttc gaagaggtgg tggacaaggg cgccagcgct 2100 cagtccttca tcgagcggat gaccaatttc cagagaatc tgcccaacga gaaagtgctg 2160 cccaagcact ccctgctgta cgagtacttc accgtgtaca acgagctgac caagtgaaa 2220 tacgtgaccg agggaatgag aaagcccgcc ttctgtccg gcgagcagaa aaagccac 2280 gtggatctgc tgttcagac siaccggaaa gtgaccgtga agcagctgaa agaggactac 2340 ttcaagaaaa tcgagtgctt cgactccgtg gaatcagcg gcgtggaaga tcggttcaat 2400 gccagcctgg gcacatacca cgatctgctg aaaattatca aggacagga cttcctggac 2460 aacgaggaaa acgaggacat ccttgaggac atcgtgctga ccctgacact gttcgaggac 2520 agagagatga tcgaggaag gctgaaaaca tacgcccacc tgttcgacga caagtcatg 2580 aagcaactga agcggcggcg ctacacaggc tggggcagac tgtctagaaa gctgatcaac 2640 ggcatccggg acaagcagtc cggcaagacc atcctggact ttctgaagtc cgacggctc 2700 gccaacagaa acttcatgca gctgattcac gacgacagcc tcaccttcaa agaggacatt 2760 cagaaggccc aggttccgg ccaggggcgat tcttgcacg agcacattgc caatctggcc 2820 ggctctcccg ccattaagaa gggcattctg cagacagtga aagtggtgga tgagctggtc 2880 aaagtgatgg ggagacacaa gcccgagaac atcgtgatcg aaatggccag aggaaccag 2940 accacacaga agggcagaa gaactccccgc gagagaatga agcggatcga gagggaatc 3000 aaagagctgg ggagccagat cctgaaagaa caccccgtgg aaaacaccca gctccagaac 3060 gagaagctgt acctgtacta cctccagaac ggccgggata tgtacgtgga ccaagagctg 3120 gacatcaacc gcctgagcga ctacgatgtg gacgctatcg tgccccagtc ttttctgaaa 3180 gatgactcca tcgacaacaa ggtgctgacc agaagcgata agaaccgggg caagagcgac 3240 aacgtgccct ctgaagaggt cgtgaagaag atgaagaact actggcgaca gctgctgaac 3300 gccaagctga ttacccagcg gaagttcgat aacctgacca aggccgagag aggcggcctg 3360 tctgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcacc 3420 aaacacgtgg cacagattct ggactcccgg atgaacacca aatacgatga gaacgacaaa 3480 ctgatccggg aagtgaaagt catcaccctg aagtccaagc tggtgtccga tttccggaag 3540 gatttccagt tctacaaagt gcgggaaatc aacaactacc atcacgccca cgacgcctac 3600 ctgaatgccg ttgttggaac agccctgatc aagaagttc ccaagctgga aagcgagttc 3660 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaagag 3720 attggaagg ctaccgccaa atacttcttc tactccaaca tcatgaactt tttcaagaca 3780 gagatcaccc tcgccaacgg cgagatcaga aagcggcctc tgatcgagac aaacggcgaa 3840 accggcgaga ttgtgtggga tagggcaga gactttgcca cagtgcggaa ggtgctcagc 3900 atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag 3960 tccattctgc ctaagcggaa ctccgacaag ctgatcgcccc ggaagaagga ctgggacccc 4020 aagaaatacg gcggcttcga tagccctacc gtggcctatt ctgtgctggt ggtggccaaa 4080 gtggaaaagg gaaagtccaa gaagctcaag agcgtcaaag aactcctggg catcaccatc 4140 atggaacggt ccagcttcga gaagaaccct atcgactttc tggaagccaa gggctacaaa 4200 gaagtcaaga aggacctgat catcaagctc cccaagtaca gcctgttcga gctggaaaat 4260 ggccggaagc ggatgctggc ttctgctggc gaactgcaga agggaacga actggccctg 4320 cctagcaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggcagc 4380 cccgaggaca atgagcagaa gcagcttttc gtcgagcagc acaagcacta cctggacgag 4440 atcatcgagc agatctccga gttctccaag agagtgatcc tggccgacgc caacctggac 4500 aaggttctgt ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat 4560 atcatccacc tgtttaccct gaccaacctg ggagcccctg ccgccttcaa gtacttcgac 4620 accaccatcg accggaagcg ctacaccagc accaaagaag tgctggacgc cacactgatc 4680 caccagagca tcaccggcct gtacgagaca cggatcgatc tgtctcagct tggaggcgac 4740 ggcagcccta agaagaagag aaaggtttcc agctaataa 4779

[0064] An exemplary E99R mutant NLS-dCas9-Clo051-NLS ("E99R Cas-CLOVER" or "E99R CC" or "E99R") fusion protein can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 82. The E99R Cas-CLOVER fusion protein can be encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 83. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0065] E99R Cas-CLOVER amino acid sequence (SEQ ID NO: 82) MAPKKKRKVE GIKSNISLLK DELRGQISHI SHEYLSLIDL AFDSKQNRLF EMKVLELLVN 60 EYGFKGRHLG GSRKPDGIVY STTLEDNFGI IVDTKAYSRG YSLPISQADE MERYVRENSN 120 RDEEVNPNKW WENFSEEVKK YYFVFISGSF KGKFEEQLRR LSMTTGVNGS AVNVVNLLLG 180 AEKIRSGEMT IEELERAMFN NSEFILKYGG GGSDKKYSIG LAIGTNSVGW AVITDEYKVP 240 SKKFKVLGNT DRHSIKKNLI GALLFDSGET AEATRLKRTA RRRYTRRKNR ICYLQEIFSN 300 EMAKVDDSFF HRLEESFLVE EDKKHERHPI FGNIVDEVAY HEKYPTIYHL RKKLVDSTDK 360 ADLRLIYLAL AHMIKFRGHF LIEGDLNPDN SDVDKLFIQL VQTYNQLFEE NPINASGVDA 420 KAILSARLSK SRRLENLIAQ LPGEKKNGLF GNLIALSLGL TPNFKSNFDL AEDAKLQLSK 480 DTYDDDLDNL LAQIGDQYAD LFLAAKNLSD AILLSDILRV NTEITKAPLS ASMIKRYDEH 540 HQDLTLLKAL VRQQLPEKYK EIFFDQSKNG YAGYIDGGAS QEEFYKFIKP ILEKMDGTEE 600 LLVKLNREDL LRKQRTFDNG SIPHQIHLGE LHAILRRQED FYPFLKDNRE KIEKILTFRI 660 PYYVGPLARG NSRFAWMTRK SEETITPWNF EEVVDKGASA QSFIERMTNF DKNLPNEKVL 720 PKHSLLYEYF TVYNELTKVK YVTEGMRKPA FLSGEQKKAI VDLLFKTNRK VTVKQLKEDY 780 FKKIECFDSV EISGVEDRFN ASLGTYHDLL KIIKDKDFLD NEENEDILED IVLTLTLFED 840 REMIEERLKT YAHLFDDKVM KQLKRRRYTG WGRLSRKLIN GIRDKQSGKT ILDFLKSDGF 900 ANRNFMQLIH DDSLTFKEDI QKAQVSGQGD SLHEHIANLA GSPAIKKGIL QTVKVVDELV 960 KVMGRHKPEN IVIEMARENQ TTQKGQKNSR ERMKRIEEGI KELGSQILKE HPVENTQLQN 1020 EKLYLYYLQN GRDMYVDQEL DINRLSDYDV DAIVPQSFLK DDSIDNKVLT RSDKNRGKSD 1080 NVPSEEVVKK MKNYWRQLLN AKLITQRKFD NLTKAERGGL SELDKAGFIK RQLVETRQIT 1140 KHVAQILDSR MNTKYDENDK LIREVQVITL KSKLVSDFRK DFQFYKVREI NNYHHAHDAY 1200 LNAVVGTALI KKYPKLESEF VYGDYKVYDV RKMIAKSEQE IGKATAKYFF YSNIMNFFKT 1260 EITLANGEIR KRPLIETNGE TGEIVWDKGR DFATVRKVLS MPQVNIVKKT EVQTGGFSKE 1320 SILPKRNSDK LIARKKDWDP KKYGGFDSPT VAYSVLVVAK VEKGKSKKKLK SVKELLGITI 1380 MERSSFEKNP IDFLEAKGYK EVKKDLIIKL PKYSLFELEN GRKRMLASAG ELQKGNELAL 1440 PSKYVNFLYL ASHEKLKGS PEDNEQKQLF VEQHKHYLDE IIEQISEFSK RVILADANLD 1500 KVLSAYNKHR DKPIREQAEN IIHLFTLTNL GAPAFKYFD TTIDRKRYTS TKEVLDATLI 1560 HQSITGLYET RIDLSQLGGD GSPKKKRKVS S

[0066] E99R Cas-CLOVER SPECIFICATIONS (83 SPECIFICATIONS) atggctccca agaagaagcg gaaggtcgag ggcatcaaga gcaacatcag cctgctgaag gacgagctga gaggccagat cagccacatc tcccacgagt acctgagcct gatcgacctg gccttcgaca gcaagcagaa ccggctgttc gagatgaagg tgctggaact gctggtcaac gagtacggct tcaagggcag acacctcggc ggcagcagaa agcctgatgg catcgtgtac agcaccacac tcgaggaca cttcggcatc atcgtggaca ccaaggccta cagcaggggc 360. ctatctctca ggccgacgag atggaagat acgtgcgcga gaacagcaac cgcgacgagg aagtgaaccc caacaagtgg tgggagaact tcagcgagg agtcaaaaag 420 tactacttcg tgttcatcag cggcagcttt aagggcaagt tcgaggaaca gctgcggcgg 480 ctgtctatga ccacaggcgt taacggcagc gccgtgaacg tggtcaatct gctgctgggc 540 gccgagaaga ttagaagcgg cgagatgacc atcgaggaac tggaacgggc catgttcaac aacagcgagt tcatcctgaa gtacggcgga ggcggcagcg acaagaagta ctctatcgga 660 ctggccatcg gcaccaactc tgttggatgg gccgtgatca ccgacgagta caaggtgccc 720 agcaagaaat tcaaagtgct gggcaacacc gaccggcaca gcatcaagaa gaatctgatc 780 ggcgccctgc tgttcgactc tggcgaaaca gccgaagcca ccagactgaa gagaaccgcc 840 agacggcggt acaccagaag aaagaaccgg atctgctacc tgcaagagat cttcagcaac 900 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa 960 gaggacaaga agcacgagcg gcaccccatc ttcggaaata tcgtggacga ggtggcctac 1020 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag 1080 gccgacctgc gactgatcta tctggccctg gctcacatga tcaagttccg gggccacttc 1140 ctgatcgagg gcgacctgaa tcctgacaac tccgacgtgg acaagctgtt catccagctg 1200 gtgcagacct acaatcagct gttcgaagag aatcccatca acgcctctgg cgtggacgcc 1260 aaagccatcc tgtctgccag actgagcaag agcagacggc tggaaaacct gatcgctcag 1320 ctgcccggcg yours tggcctgttc ggcaacctga ttgccctgtc tctgggcctg 1380 acacctaact tcaagtccaa cttcgatctg gccgaggatg ccaaactgca gctgtccaag 1440 gacacctacg acgacgacct ggataacctg ctggcccaga tcggcgatca gtacgccgac 1500 ttgtttctgg ccgccaagaa cctgtctgac gccatcctgc tgagcgacat cctgagagtg 1560 1620 caccaggatc tgaccctgct gaaagctctc gtcaggcagc agctgccaga gaagtaaa 1680 gagattttct tcgaccagag caagaacggc tacgccggct acattgatgg cggagccagc 1740 caagaggaat tctacaagtt catcaagccc atcctcgaga agatggacgg cacagaggaa 1800 1860 tctatccctc accagatcca cctgggagag ctgcacgcca ttctgcggag acaagaggac 1920 ttttacccat tcctgaagga caaccgggaa aagattgaga agatcctgac cttcaggatc 1980 ccctactacg tgggaccact ggccagaggc aatagcagat tcgcctggat gaccagaaag 2040 agcgaggaaa ccatcacacc ctggaacttc gaagaggtgg tggacaaggg cgccagcgct 2100 cagtccttca tcgagcggat gaccaatttc gacaagaatc tgcccaacga gaaagtgctg 2160 cccaagcact ccctgctgta cgagtacttc accgtgtaca acgagctgac caaagtgaaa 2220 tacgtgaccg agggaatgag aaagcccgcc tttctgtccg gcgagcagaa aaaggccatc 2280 gtggatctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac 2340 ttcaagaaaa tcgagtgctt cgactccgtg gaaatcagcg gcgtggaaga tcggttcaat 2400 gccagcctgg gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac 2460 aacgaggaaa acgaggacat ccttgaggac atcgtgctga ccctgacact gttcgaggac 2520 agagagatga tcgaggaaag gctgaaaaca tacgcccacc tgttcgacga caaagtcatg 2580 aagcaactga agcggcggcg ctacacaggc tggggcagac tgtctagaaa gctgatcaac 2640 ggcatccggg acaagcagtc cggcaagacc atcctggact ttctgaagtc cgacggcttc 2700 gccaacagaa acttcatgca gctgattcac gacgacagcc tcaccttcaa agaggacatt 2760 cagaaggccc aggttccgg ccaggggcgat tcttgcacg agcacattgc caatctggcc 2820 ggctctcccg ccattaagaa gggcattctg cagacagtga aagtggtgga tgagctggtc 2880 aaagtgatgg ggagacacaa gcccgagaac atcgtgatcg aaatggccag aggaaccag 2940 accacacaga agggcagaa gaactccccgc gagagaatga agcggatcga gagggaatc 3000 aaagagctgg gggaccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac 3060 gagaagctgt acctgtacta cctccagaac ggccgggata tgtacgtgga ccagagctg 3120 gatacaacc gcctgagcga ctacgatgtg gacgctatcg tgccccagtc ttttctgaaa 3180 gatgactcca tcgacaaca ggtgctgacc agaagcgata agaaccgggg caagcgac 3240 aacgtgccct ctgaagaggt cgtgagaag atgagact actggcgaca gctgctgaac 3300 gccaagctga ttacccagcg gaagttcgat aacctgacca aggccgag aggcggcctg 3360 tctgaactgg ataggccgg cttcatcaag agacagctgg tggaacccg gcagatcacc 3420 aaacacgtgg cacagattct ggactcccgg atgacacca atacgatga gaacgacaaa 3480 ctgatccggg aagtgaaagt catcaccctg aagtccaagc tggtgtccga tttccggaag 3540 gatttccagt tctacaaagt gcgggaaatc aacaactacc atcacgccca cgacgcctac 3600 ctgaatgccg ttgttggaac agccctgatc aagaagttc ccaagctgga aagcgagttc 3660 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaagag 3720 attggaagg ctaccgccaa atacttcttc tactccaaca tcatgaactt tttcaagaca 3780 gagatcaccc tcgccaacgg cgagatcaga aagcggcctc tgatcgagac aaacggcgaa 3840 accggcgaga ttgtgtggga tagggcaga gactttgcca cagtgcggaa ggtgctcagc 3900 atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag 3960 tccattctgc ctaagcggaa ctccgacaag ctgatcgcccc ggaagaagga ctgggacccc 4020 aagaaatacg gcggcttcga tagccctacc gtggcctatt ctgtgctggt ggtggccaaa 4080 gtggaaaagg gaaagtccaa gaagctcaag agcgtcaaag aactcctggg catcaccatc 4140 atggaacggt ccagcttcga gaagaaccct atcgactttc tggaagccaa gggctacaaa 4200 gaagtcaaga aggacctgat catcaagctc cccaagtaca gcctgttcga gctggaaaat 4260 ggccggaagc ggatgctggc ttctgctggc gaactgcaga agggaaacga actggccctg 4320 cctagcaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggcagc 4380 cccgaggaca atgagcagaa gcagcttttc gtcgagcagc acaagcacta cctggacgag 4440 atcatcgagc agatctccga gttctccaag agagtgatcc tggccgacgc caacctggac 4500 aaggttctgt ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat 4560 atcatccacc tgtttaccct gaccaacctg ggagcccctg ccgccttcaa gtacttcgac 4620 accaccatcg accggaagcg ctacaccagc accaaagaag tgctggacgc cacactgatc 4680 caccagagca tcaccggcct gtacgagaca cggatcgatc tgtctcagct tggaggcgac 4740 ggcagcccta agaagaagag aaaggtttcc agctaataa 4779

[0067] In some embodiments, the NLS-dCas9-Clo051-NLS of SEQ ID NO: 10 can comprise amino acid substitutions at positions 44 and 99. In some embodiments, the amino acid substitutions are S44P and E99K or S44P and E99R. In some embodiments, the amino acid substitutions are S44P and E99K. In some embodiments, the amino acid substitutions are S44P and E99R.

[0068] An exemplary S44P and E99K mutant NLS-dCas9-Clo051-NLS ("S44P and E99K Cas-CLOVER," "S44P / E99K Cas-CLOVER," "S44P and E99K CC," "S44P / E99K CC," or "S44P / E99K") fusion protein can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 76. The S44P Cas-CLOVER fusion protein can be encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 77. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0069] S44P / E99K Cas-CLOVER amino acid sequence (SEQ ID NO: 76) MAPKKKRKVE GIKSNISLLK DELRGQISHI SHEYLSLIDL AFDPKQNRLF EMKVLELLVN 60 EYGFKGRHLG GSRKPDGIVY STTLEDNFGI IVDTKAYSKG YSLPISQADE MERYVRENSN 120 RDEEVNPNKW WENFSEEVKK YYFVFISGSF KGKFEEQLRR LSMTTGVNGS AVNVVNLLLG 180 AEKIRSGEMT IEELERAMFN NSEFILKYGG GGSDKKYSIG LAIGTNSVGW AVITDEYKVP 240 SKKFKVLGNT DRHSIKKNLI GALLFDSGET AEATRLKRTA RRRYTRRKNR ICYLQEIFSN 300 EMAKVDDSFF HRLEESFLVE EDKKHERHPI FGNIVDEVAY HEKYPTIYHL RKKLVDSTDK 360 ADLRLIYLAL AHMIKFRGHF LIEGDLNPDN SDVDKLFIQL VQTYNQLFEE NPINASGVDA 420 KAILSARLSK SRRLENLIAQ LPGEKKNGLF GNLIALSLGL TPNFKSNFDL AEDAKLQLSK 480 DTYDDDLDNL LAQIGDQYAD LFLAAKNLSD AILLSDILRV NTEITKAPLS ASMIKRYDEH 540 HQDLTLLKAL VRQQLPEKYK EIFFDQSKNG YAGYIDGGAS QEEFYKFIKP ILEKMDGTEE 600 LLVKLNREDL LRKQRTFDNG SIPHQIHLGE LHAILRRQED FYPFLKDNRE KIEKILTFRI 660 PYYVGPLARG NSRFAWMTRK SEETITPWNF EEVVDKGASA QSFIERMTNF DKNLPNEKVL 720 PKHSLLYEYF TVYNELTKVK YVTEGMRKPA FLSGEQKKAI VDLLFKTNRK VTVKQLKEDY 780 FKKIECFDSV EISGVEDRFN ASLGTYHDLL KIIKDKDFLD NEENEDILED IVLTLTLFED 840 REMIEERLKT YAHLFDDKVM KQLKRRRYTG WGRLSRKLIN GIRDKQSGKT ILDFLKSDGF 900 ANRNFMQLIH DDSLTFKEDI QKAQVSGQGD SLHEHIANLA GSPAIKKGIL QTVKVVDELV 960 KVMGRHKPEN IVIEMARENQ TTQKGQKNSR ERMKRIEEGI KELGSQILKE HPVENTQLQN 1020 EKLYLYYLQN GRDMYVDQEL DINRLSDYDV DAIVPQSFLK DDSIDNKVLT RSDKNRGKSD 1080 NVPSEEVVKK MKNYWRQLLN AKLITQRKFD NLTKAERGGL SELDKAGFIK RQLVETRQIT 1140 KHVAQILDSR MNTKYDENDK LIREVKVITL KSKLVSDFRK DFQFYKVREI NNYHHAHDAY 1200 LNAVVGTALI KKYPKLESEF VYGDYKVYDV RKMIAKSEQE IGKATAKYFF YSNIMNFFKT 1260 EITLANGEIR KRPLIETNGE TGEIVWDKGR DFATVRKVLS MPQVNIVKKT EVQTGGFSKE 1320 SILPKRNSDK LIARKKDWDP KKYGGFDSPT VAYSVLVVAK VEKGKSKKLK SVKELLGITI 1380 MERSSFEKNP IDFLEAKGYK EVKKDLIIKL PKYSLFELEN GRKRMLASAG ELQKGNELAL 1440 PSKYVNFLYL ASHYEKLKGS PEDNEQKQLF VEQHKHYLDE IIEQISEFSK RVILADANLD 1500 KVLSAYNKHR DKPIREQAEN IIHLFTLTNL GAPAAFKYFD TTIDRKRYTS TKEVLDATLI 1560 HQSITGLYET RIDLSQLGGD GSPKKKRKVS S 1591

[0070] S44P / E99K Cas-CLOVER nucleic acid sequence(sequential number 77) atggctccca agaagaagcg gaaggtcgag ggcatcaaga gcaacatcag cctgctgaag 60 gacgagctga gaggccagat cagccacatc tcccacgagt acctgagcct gatcgacctg gccttcgacc ccaagcagaa ccggctgttc gagatgaagg tgctggaact gctggtcaac gagtacggct tcaagggcag acacctcggc ggcagcagaa agcctgatgg catcgtgtac agcaccacac tcgaggaca cttcggcatc atcgtggaca ccaaggccta cagcaagggc 360. ctatctctca ggccgacgag atggaagat acgtgcgcga gaacagcaac cgcgacgagg aagtgaaccc caacaagtgg tgggagaact tcagcgagg agtcaaaaag 420 tactacttcg tgttcatcag cggcagcttt aagggcaagt tcgaggaaca gctgcggcgg 480 ctgtctatga ccacaggcgt taacggcagc gccgtgaacg tggtcaatct gctgctgggc 540 gccgagaaga ttagaagcgg cgagatgacc atcgaggaac tggaacgggc catgttcaac aacagcgagt tcatcctgaa gtacggcgga ggcggcagcg acaagagta ctctatcgga 660 ctggccatcg gcaccaactc tgttggatgg gccgtgatca ccgacgagta caaggtgccc 720 agcaagaat tcaaagtgct gggcaacacc gaccggcaca gcatcaaga gaatctgatc ggcgccctgc tgttcgactc tggcgaaaca gccgaagcca ccagactgaa gagaaccgcc 840 agacggcggt acaccagaag aaagaaccgg atctgctacc tgcaagagat cttcagcaac 900 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa 960 gaggacaaga agcacgagcg gcaccccatc ttcggaaata tcgtggacga ggtggcctac 1020 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag 1080 gccgacctgc gactgatcta tctggccctg gctcacatga tcaagttccg gggccacttc 1140 ctgatcgagg gcgacctgaa tcctgacaac tccgacgtgg acaagctgtt catccagctg 1200 gtgcagacct acaatcagct gttcgaagag aatcccatca acgcctctgg cgtggacgcc 1260 aaagccatcc tgtctgccag actgagcaag agcagacggc tggaaaacct gatcgctcag 1320 ctgcccggcg agaagaagaa tggcctgttc ggcaacctga ttgccctgtc tctgggcctg 1380 acacctaact tcaagtccaa cttcgatctg gccgaggatg ccaaactgca gctgtccaag 1440 gacacctacg acgacgacct ggataacctg ctggcccaga tcggcgatca gtacgccgac 1500 ttgttttctgg cctgtctgac gccatccctgc tgagcgacat cctgagagtg 1560 aacaccgaga tcacaaggc cccctgagc gcctctatga tcaagagata cgacgagcac 1620 caccaggatc tgaccctgct gaagctctc gtcaggcagc agctgccaga gaagtacaa 1680 gagattttct tcgaccagag cagaacggc tacgccggct acatgatgg cggagccagc 1740 cagaggaat tctacaagtt catcaagcccc atcctcgaga agatggacgg cacagaggaa 1800 ctgctcgtga agctgacag agaggacctg ctgcggaagc agcggacctt cgacaatggc 1860 tctatccctc accagatcca cctgggag ctgcacgcca ttctcggag acagaggac 1920 ttttacccat tcctgaagga caccggga aagattgaga agatcctgac cttcaggatc 1980 ccctactacg tgggaccact ggccagaggc atagcagat tcgcctggat gaccagaaag 2040 agcgaggaaa ccatcacacc ctggaacttc gaagaggtgg tggacaaggg cgccagcgct 2100 cagtccttca tcgagcggat gaccaatttc cagagaatc tgcccaacga gaaagtgctg 2160 cccaagcact ccctgctgta cgagtacttc accgtgtaca acgagctgac caagtgaaa 2220 tacgtgaccg agggaatgag aaagcccgcc ttctgtccg gcgagcagaa aaagccac 2280 gtggatctgc tgttcagac siaccggaaa gtgaccgtga agcagctgaa agaggactac 2340 ttcaagaaaa tcgagtgctt cgactccgtg gaatcagcg gcgtggaaga tcggttcaat 2400 gccagcctgg gcacatacca cgatctgctg aaaattatca aggacagga cttcctggac 2460 aacgaggaaa acgaggacat ccttgaggac atcgtgctga ccctgacact gttcgaggac 2520 agagagatga tcgaggaag gctgaaaaca tacgcccacc tgttcgacga caagtcatg 2580 aagcaactga agcggcggcg ctacacaggc tggggcagac tgtctagaaa gctgatcaac 2640 ggcatccggg acaagcagtc cggcaagacc atcctggact ttctgaagtc cgacggctc 2700 gccaacagaa acttcatgca gctgattcac gacgacagcc tcaccttcaa agaggacatt 2760 cagaaggccc aggttccgg ccaggggcgat tcttgcacg agcacattgc caatctggcc 2820 ggctctcccg ccattaagaa gggcattctg cagacagtga aagtggtgga tgagctggtc 2880 aaagtgatgg ggagacacaa gcccgagaac atcgtgatcg aaatggccag aggaaccag 2940 accacacaga agggccagaa gaactcccgc gagagaatga agcggatcga agagggaatc 3000 aaagagctgg ggagccagat cctgaaagaa caccccgtgg aaaacaccca gctccagaac 3060 gagaagctgt acctgtacta cctccagaac ggccgggata tgtacgtgga ccaagagctg 3120 gacatcaacc gcctgagcga ctacgatgtg gacgctatcg tgccccagtc ttttctgaaa 3180 gatgactcca tcgacaacaa ggtgctgacc agaagcgata agaaccgggg caagagcgac 3240 aacgtgccct ctgaagaggt cgtgaagaag atgaagaact actggcgaca gctgctgaac 3300 gccaagctga ttacccagcg gaagttcgat aacctgacca aggccgagag aggcggcctg 3360 tctgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcacc 3420 aaacacgtgg cacagattct ggactcccgg atgaacacca aatacgatga gaacgacaaa 3480 ctgatccggg aagtgaaagt catcaccctg aagtccaagc tggtgtccga tttccggaag 3540 gatttccagt tctacaaagt gcgggaaatc aacaactacc atcacgccca cgacgcctac 3600 ctgaatgccg ttgttggaac agccctgatc aagaagttc ccaagctgga aagcgagttc 3660 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaagag 3720 attggaagg ctaccgccaa atacttcttc tactccaaca tcatgaactt tttcaagaca 3780 gagatcaccc tcgccaacgg cgagatcaga aagcggcctc tgatcgagac aaacggcgaa 3840 accggcgaga ttgtgtggga tagggcaga gactttgcca cagtgcggaa ggtgctcagc 3900 atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag 3960 tccattctgc ctaagcggaa ctccgacaag ctgatcgcccc ggaagaagga ctgggacccc 4020 aagaaatacg gcggcttcga tagccctacc gtggcctatt ctgtgctggt ggtggccaaa 4080 gtggaaaagg gaaagtccaa gaagctcaag agcgtcaaag aactcctggg catcaccatc 4140 atggaacggt ccagcttcga gaagaaccct atcgactttc tggaagccaa gggctacaaa 4200 gaagtcaaga aggacctgat catcaagctc cccaagtaca gcctgttcga gctggaaaat 4260 ggccggaagc ggatgctggc ttctgctggc gaactgcaga agggaacga actggccctg 4320 cctagcaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggcagc 4380 cccgaggaca atgagcagaa gcagcttttc gtcgagcagc acaagcacta cctggacgag 4440 atcatcgagc agatctccga gttctccaag agagtgatcc tggccgacgc caacctggac 4500 aaggttctgt ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat 4560 atcatccacc tgtttaccct gaccaacctg ggagcccctg ccgccttcaa gtacttcgac 4620 accaccatcg accggaagcg ctacaccagc accaaagaag tgctggacgc cacactgatc 4680 caccagagca tcaccggcct gtacgagaca cggatcgatc tgtctcagct tggaggcgac 4740 ggcagcccta agaagaagag aaaggtttcc agctaataa 4779

[0071] An exemplary S44P and E99R mutant NLS-dCas9-Clo051-NLS ("S44P and E99R Cas-CLOVER," "S44P / E99R Cas-CLOVER," "S44P and E99R CC," "S44P / E99R CC," or "S44P / E99R") fusion protein can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 80. The S44P and E99R Cas-CLOVER fusion protein can be encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 81. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0072] S44P / E99R Cas-CLOVER amino acid sequence (SEQ ID NO: 80) MAPKKKRKVE GIKSNISLLK DELRGQISHI SHEYLSLIDL AFDPKQNRLF EMKVLELLVN 60 EYGFKGRHLG GSRKPDGIVY STTLEDNFGI IVDTKAYSRG YSLPISQADE MERYVRENSN 120 RDEEVNPNKW WENFSEEVKK YYFVFISGSF KGKFEEQLRR LSMTTGVNGS AVNVVNLLLG 180 AEKIRSGEMT IEELERAMFN NSEFILKYGG GGSDKKYSIG LAIGTNSVGW AVITDEYKVP 240 SKKFKVLGNT DRHSIKKNLI GALLFDSGET AEATRLKRTA RRRYTRRKNR ICYLQEIFSN 300 EMAKVDDSFF HRLEESFLVE EDKKHERHPI FGNIVDEVAY HEKYPTIYHL RKKLVDSTDK 360 ADLRLIYLAL AHMIKFRGHF LIEGDLNPDN SDVDKLFIQL VQTYNQLFEE NPINASGVDA 420 KAILSARLSK SRRLENLIAQ LPGEKKNGLF GNLIALSLGL TPNFKSNFDL AEDAKLQLSK 480 DTYDDDLDNL LAQIGDQYAD LFLAAKNLSD AILLSDILRV NTEITKAPLS ASMIKRYDEH 540 HQDLTLLKAL VRQQLPEKYK EIFFDQSKNG YAGYIDGGAS QEEFYKFIKP ILEKMDGTEE 600 LLVKLNREDL LRKQRTFDNG SIPHQIHLGE LHAILRRQED FYPFLKDNRE KIEKILTFRI 660 PYYVGPLARG NSRFAWMTRK SEETITPWNF EEVVDKGASA QSFIERMTNF DKNLPNEKVL 720 PKHSLLYEYF TVYNELTKVK YVTEGMRKPA FLSGEQKKAI VDLLFKTNRK VTVKQLKEDY 780 FKKIECFDSV EISGVEDRFN ASLGTYHDLL KIIKDKDFLD NEENEDILED IVLTLTLFED 840 REMIEERLKT YAHLFDDKVM KQLKRRRYTG WGRLSRKLIN GIRDKQSGKT ILDFLKSDGF 900 ANRNFMQLIH DDSLTFKEDI QKAQVSGQGD SLHEHIANLA GSPAIKKGIL QTVKVVDELV 960 KVMGRHKPEN IVIEMARENQ TTQKGQKNSR ERMKRIEEGI KELGSQILKE HPVENTQLQN 1020 EKLYLYYLQN GRDMYVDQEL DINRLSDYDV DAIVPQSFLK DDSIDNKVLT RSDKNRGKSD 1080 NVPSEEVVKK MKNYWRQLLN AKLITQRKFD NLTKAERGGL SELDKAGFIK RQLVETRQIT 1140 KHVAQILDSR MNTKYDENDK LIREVKVITL KSKLVSDFRK DFQFYKVREI NNYHHAHDAY 1200 LNAVVGTALI KKYPKLESEF VYGDYKVYDV RKMIAKSEQE IGKATAKYFF YSNIMNFFKT 1260 EITLANGEIR KRPLIETNGE TGEIVWDKGR DFATVRKVLS MPQVNIVKKT EVQTGGFSKE 1320 SILPKRNSDK LIARKKDWDP KKYGGFDSPT VAYSVLVVAK VEKGKSKKLK SVKELLGITI 1380 MERSSFEKNP IDFLEAKGYK EVKKDLIIKL PKYSLFELEN GRKRMLASAG ELQKGNELAL 1440 PSKYVNFLYL ASHYEKLKGS PEDNEQKQLF VEQHSHELF IIEQISEFSK RVILADANLD KVLSAYNKHR DKPIREQAEN IIHLFTLTNL GAPAAFKYFD TTIDRKRYTS TKEVLDATLI HQSITGLYET RIDLSQLGGD GSPKKKRKVS S

[0073] S44P / E99R Cas-CLOVER MECHANICAL SYSTEM(SYSTEM 81) atggctccca agaagaagcg gaaggtcgag ggcatcaaga gcaacatcag cctgctgaag gacgagctga gaggccagat cagccacatc tcccacgagt acctgagcct gatcgacctg gccttcgacc ccaagcagaa ccggctgttc gagatgaagg tgctggaact gctggtcaac gagtacggct tcaagggcag acacctcggc ggcagcagaa agcctgatgg catcgtgtac agcaccacac tcgaggaca cttcggcatc atcgtggaca ccaaggccta cagcaggggc 360. ctatctctca ggccgacgag atggaagat acgtgcgcga gaacagcaac cgcgacgagg aagtgaaccc caacaagtgg tgggagaact tcagcgagg agtcaaaaag 420 tactacttcg tgttcatcag cggcagcttt aagggcaagt tcgaggaaca gctgcggcgg 480 ctgtctatga ccacaggcgt taacggcagc gccgtgaacg tggtcaatct gctgctgggc 540 gccgagaaga ttagaagcgg cgagatgacc atcgaggaac tggaacgggc catgttcaac 600 aacagcgagt tcatcctgaa gtacggcgga ggcggcagcg acaagaagta ctctatcgga 660 ctggccatcg gcaccaactc tgttggatgg gccgtgatca ccgacgagta caaggtgccc 720 agcaagaaat tcaaagtgct gggcaacacc gaccggcaca gcatcaagaa gaatctgatc 780 ggcgccctgc tgttcgactc tggcgaaaca gccgaagcca ccagactgaa gagaaccgcc 840 agacggcggt acaccagaag aaagaaccgg atctgctacc tgcaagagat cttcagcaac 900 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa 960 gaggacaaga agcacgagcg gcaccccatc ttcggaaata tcgtggacga ggtggcctac 1020 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag 1080 gccgacctgc gactgatcta tctggccctg gctcacatga tcaagttccg gggccacttc 1140 ctgatcgagg gcgacctgaa tcctgacaac tccgacgtgg acaagctgtt catccagctg 1200 gtgcagacct acaatcagct gttcgaagag aatcccatca acgcctctgg cgtggacgcc 1260 aaagccatcc tgtctgccag actgagcaag agcagacggc tggaaaacct gatcgctcag 1320 ctgcccggcg agaagaagaa tggcctgttc ggcaacctga ttgccctgtc tctgggcctg 1380 acacctaact tcaagtccaa cttcgatctg gccgaggatg ccaaactgca gctgtccaag 1440 gacacctacg acgacgacct ggataacctg ctggcccaga tcggcgatca gtacgccgac 1500 ttgtttctgg ccgccaagaa cctgtctgac gccatcctgc tgagcgacat cctgagagtg 1560 aacaccgaga tcacaaaggc ccctctgagc gcctctatga tcaagagata cgacgagcac 1620 caccaggatc tgaccctgct gaaagctctc gtcaggcagc agctgccaga gaagtacaaa 1680 gagattttct tcgaccagag caagaacggc tacgccggct acattgatgg cggagccagc 1740 caagaggaat tctacaagtt catcaagccc atcctcgaga agatggacgg cacagaggaa 1800 ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaatggc 1860 tctatccctc accagatcca cctgggagag ctgcacgcca ttctgcggag acaagaggac 1920 ttttacccat tcctgaagga caaccgggaa aagattgaga agatcctgac cttcaggatc 1980 ccctactacg tgggaccact ggccagaggc aatagcagat tcgcctggat gaccagaaag 2040 agcgaggaaa ccatcacacc ctggaacttc gaagaggtgg tggacaaggg cgccagcgct 2100 cagtccttca tcgagcggat gaccaatttc gacaagaatc tgcccaacga gaaagtgctg 2160 cccaagcact ccctgctgta cgagtacttc accgtgtaca acgagctgac caaagtgaaa 2220 tacgtgaccg agggaatgag aaagcccgcc tttctgtccg gcgagcagaa aaaggccatc 2280 gtggatctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac 2340 ttcaagaaaa tcgagtgctt cgactccgtg gaaatcagcg gcgtggaaga tcggttcaat 2400 gccagcctgg gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac 2460 aacgaggaaa acgaggacat ccttgaggac atcgtgctga ccctgacact gttcgaggac 2520 agagagatga tcgaggaaag gctgaaaaca tacgcccacc tgttcgacga caaagtcatg 2580 aagcaactga agcggcggcg ctacacaggc tggggcagac tgtctagaaa gctgatcaac 2640 ggcatccggg acaagcagtc cggcaagacc atcctggact ttctgaagtc cgacggctc 2700 gccaacagaa acttcatgca gctgattcac gacgacagcc tcaccttcaa agaggacatt 2760 cagaaggccc aggttccgg ccaggggcgat tcttgcacg agcacattgc caatctggcc 2820 ggctctcccg ccattaagaa gggcattctg cagacagtga aagtggtgga tgagctggtc 2880 aaagtgatgg ggagacacaa gcccgagaac atcgtgatcg aaatggccag aggaaccag 2940 accacacaga agggcagaa gaactccccgc gagagaatga agcggatcga gagggaatc 3000 aaagagctgg gggaccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac 3060 gagaagctgt acctgtacta cctccagaac ggccgggata tgtacgtgga ccagagctg 3120 gatacaacc gcctgagcga ctacgatgtg gacgctatcg tgccccagtc ttttctgaaa 3180 gatgactcca tcgacaaca ggtgctgacc agaagcgata agaaccgggg caagcgac 3240 aacgtgccct ctgaagaggt cgtgagaag atgagact actggcgaca gctgctgaac 3300 gccaagctga ttacccagcg gaagttcgat aacctgacca aggccgag aggcggcctg 3360 tctgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcacc 3420 aaacacgtgg cacagattct ggactcccgg atgaacacca aatacgatga gaacgacaaa 3480 ctgatccggg aagtgaaagt catcaccctg aagtccaagc tggtgtccga tttccggaag 3540 gatttccagt tctacaaagt gcgggaaatc aacaactacc atcacgccca cgacgcctac 3600 ctgaatgccg ttgttggaac agccctgatc aagaagtatc ccaagctgga aagcgagttc 3660 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaagag 3720 attggaaagg ctaccgccaa atacttcttc tactccaaca tcatgaactt tttcaagaca 3780 gagatcaccc tcgccaacgg cgagatcaga aagcggcctc tgatcgagac aaacggcgaa 3840 accggcgaga ttgtgtggga taagggcaga gactttgcca cagtgcggaa ggtgctcagc 3900 atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag 3960 tccattctgc ctaagcggaa ctccgacaag ctgatcgccc ggaagaagga ctgggacccc 4020 aagaaatacg gcggcttcga tagccctacc gtggcctatt ctgtgctggt ggtggccaaa 4080 gtggaaaagg gaaagtccaa gaagctcaag agcgtcaaag aactcctggg catcaccatc 4140 atggaacggt ccagcttcga gaagaaccct atcgactttc tggaagccaa gggctacaaa 4200 gaagtcaaga aggacctgat catcaagctc cccaagtaca gcctgttcga gctggaaaat 4260 ggccggaagc ggatgctggc ttctgctggc gaactgcaga agggaaacga actggccctg 4320 cctagcaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggcagc 4380 cccgaggaca atgagcagaa gcagctttc gtcgagcagc acaagcacta cctggacgag 4440 atcatcgagc agatctccga gttctccaag agagtgatcc tggccgacgc caacctggac 4500 aaggttctgt ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat 4560 atcatccacc tgtttaccct gaccaacctg ggagcccctg ccgccttcaa gtacttcgac 4620 accaccatcg accggaagcg ctacaccagc accaaagaag tgctggacgc cacactgatc 4680 caccagagca tcaccggcct gtacgagaca cggatcgatc tgtctcagct tggaggcgac 4740 ggcagcccta agaagaagag aaaggtttcc agctaataa 4779

[0074] An amino acid sequence alignment of the wild-type Cas-CLOVER amino acid sequence (SEQ ID NO: 10), the mutant S44P Cas-CLOVER amino acid sequence (SEQ ID NO: 35), the mutant S44P and E99K Cas-CLOVER amino acid sequence (SEQ ID NO: 76), and the mutant S44P and E99R Cas-CLOVER amino acid sequence (SEQ ID NO: 80) is shown below. MAPKKKRKVEGIKSNISLLKDELRGQISHISHEYLSLIDLAFDSKQNRLFEMKVLELLVN 60 MAPKKKRKVEGIKSNISLLKDELRGQISHISHEYLSLIDLAFDPKQNRLFEMKVLELLVN 60 MAPKKKRKVEGIKSNISLLKDELRGQISHISHEYLSLIDLAFDPKQNRLFEMKVLELLVN 60 MAPKKKRKVEGIKSNISLLKDELRGQISHISHEYLSLIDLAFDPKQNRLFEMKVLELLVN 60 ******************************************* **************** EYGFKGRHLGGSRKPDGIVYSTTLEDNFGIIVDTKAYSEGYSLPISQADEMERYVRENSN 120 EYGFKGRHLGGSRKPDGIVYSTTLEDNFGIIVDTKAYSEGYSLPISQADEMERYVRENSN 120 EYGFKGRHLGGSRKPDGIVYSTTLEDNFGIIVDTKAYSKGYSLPISQADEMERYVRENSN 120 EYGFKGRHLGGSRKPDGIVYSTTLEDNFGIIVDTKAYSRGYSLPISQADEMERYVRENSN 120 ************************************** ********************* RDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAVNVVNLLLG 180 RDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAVNVVNLLLG 180 RDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAVNVVNLLLG 180 RDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAVNVVNLLLG 180 ***************************************************************** AEKIRSGEMTIEELERAMFNNSEFILKYGGGGSDKKYSIGLAIGTNSVGWAVITDEYKVP 240 AEKIRSGEMTIEELERAMFNNSEFILKYGGGGSDKKYSIGLAIGTNSVGWAVITDEYKVP 240 AEKIRSGEMTIEELERAMFNNSEFILKYGGGGSDKKYSIGLAIGTNSVGWAVITDEYKVP 240 AEKIRSGEMTIEELERAMFNNSEFILKYGGGGSDKKYSIGLAIGTNSVGWAVITDEYKVP 240 ***************************************************************** SKKFKVLGNTDRHSIKKNLIGALLFDSGTATAEATRLKRTARRRYTRRKNRICYLQEIFSN 300 SKKFKVLGNTDRHSIKKNLIGALLFDSGTATAEATRLKRTARRRYTRRKNRICYLQEIFSN 300 SKKFKVLGNTDRHSIKKNLIGALLFDSGTATAEATRLKRTARRRYTRRKNRICYLQEIFSN 300 SKKFKVLGNTDRHSIKKNLIGALLFDSGTATAEATRLKRTARRRYTRRKNRICYLQEIFSN 300 ***************************************************************** EMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDK 360 EMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDK 360 EMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDK 360 EMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDK 360 ***************************************************************** ADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA 420 ADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA 420 ADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA 420 ADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA 420 ***************************************************************** KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK 480 KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK 480 KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK 480 KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK 480 ********************************************************************** DTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEH 540 DTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEH 540 DTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEH 540 DTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEH 540 ********************************************************************** HQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEE 600 HQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEE 600 HQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEE 600 HQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEE 600 ********************************************************************** LLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI 660 LLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI 660 LLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI 660 ***************************************************************** PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL 720 PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL 720 PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL 720 PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL 720 ***************************************************************** PKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDY 780 PKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDY 780 PKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDY 780 PKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDY 780 ***************************************************************** FKKIECFDSWEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED 840 FKKIECFDSWEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED 840 FKKIECFDSWEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED 840 FKKIECFDSWEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED 840 ***************************************************************** REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGF 900 REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGF 900 REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGF 900 REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGF 900 ***************************************************************** ANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV 960 ANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV 960 ANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV 960 ***************************************************************** KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQN 1020 KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQN 1020 KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQN 1020 KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQN 1020 ***************************************************************** EKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSD 1080 EKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSD 1080 EKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSD 1080 EKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSD 1080 ***************************************************************** NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQIT 1140 NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQIT 1140 NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQIT 1140 NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQIT 1140 ***************************************************************** KHVAQILDSRMNTKYDENDKLIREVKVITLSKLVSDFRKDFQFYKVREINNYHHAHDAY 1200 KHVAQILDSRMNTKYDENDKLIREVKVITLSKLVSDFRKDFQFYKVREINNYHHAHDAY 1200 KHVAQILDSRMNTKYDENDKLIREVKVITLSKLVSDFRKDFQFYKVREINNYHHAHDAY 1200 KHVAQILDSRMNTKYDENDKLIREVKVITLSKLVSDFRKDFQFYKVREINNYHHAHDAY 1200 ***************************************************************** LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT 1260 LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT 1260 LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT 1260 LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT 1260 ***************************************************************** EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE 1320 EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE 1320 EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE 1320 EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE 1320 ***************************************************************** SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI 1380 SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI 1380 SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI 1380 SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI 1380 ***************************************************************** MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELAL 1440 MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELAL 1440 MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELAL 1440 MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELAL 1440 ***************************************************************** PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD 1500 PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD 1500 PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD 1500 PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD 1500 ***************************************************************** KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI 1560 KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI 1560 KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI 1560 KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI 1560 ***************************************************************** HQSITGLYETRIDLSQLGGDGSPKKKRKVSS 1591 (SEQ ID NO: 10) HQSITGLYETRIDLSQLGGDGSPKKKRKVSS 1591 (SEQ ID NO: 35) HQSITGLYETRIDLSQLGGDGSPKKKRKVSS 1591 (SEQ ID NO: 76) HQSITGLYETRIDLSQLGGDGSPKKKRKVSS 1591 (SEQ ID NO: 80) *******************************

[0075] The cells containing the gene-editing composition can stably or transiently express the gene-editing composition.

[0076] The transgene can include a sequence encoding a therapeutic agent. The therapeutic agent can be a protein or RNA that provides a therapeutic benefit when administered to a cell or a subject. The therapeutic agent can be a therapeutic protein or a therapeutic RNA. The therapeutic agent can be human beta globin (HBB), T87Q human beta globin (HBB T87Q), BAF chromatin remodeling complex subunit (BCL11A) shRNA, insulin-like growth factor 2 binding protein 1 (IGF2BP1), interleukin 2 receptor gamma (IL2RG), alpha galactosidase A (GLA), alpha-L-iduronase (IDUA), iduronate 2-sulfatase (IDS), or cystinosine lysosomal cysteine ​​transporter (CTNS). The transgene can include a sequence encoding factor VIII or factor IX. The transgene can include a sequence encoding a chimeric antigen receptor (CAR). The transgene may comprise a sequence encoding a non-naturally occurring chimeric stimulating receptor (CSR) comprising: (a) an ectodomain comprising an activating component, wherein the activating component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein, wherein the first protein and the second protein are not identical. In one embodiment, the transgene may comprise a CAR sequence and a CSR sequence. In one embodiment, the CAR or CSR-containing transgene specifically binds to BCMA, PSMA, MUC1-C, CD133, c-KIT, CD19, or CD20. The transgene may comprise a sequence encoding an inducible pro-apoptotic polypeptide comprising (a) a ligand-binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise non-human sequences. The transgene may be integrated into the genome of the HSC. Integration may be stable or transient.

[0077] Factor VIII (FVIII) deficiency leads to the development of hemophilia A. Factor IX (FIX) deficiency leads to the development of hemophilia B. Prior to the compositions and methods of the present disclosure, standard treatment for hemophilia B required infusions of recombinant FIX every 2-3 days at a cost of approximately $250,000 per year. In sharp contrast to this standard treatment option, the iPSCs of the present disclosure can be differentiated into any cell type, including HSCs, and can be maintained in humans for decades.

[0078] The guide RNA may comprise a sequence complementary to a target sequence in a genomic DNA sequence. The target sequence in a genomic DNA sequence may be a target sequence in a safe harbor site of the genomic DNA sequence. Exemplary target sequences include, but are not limited to, HBB, TRAC, B2M, TCRb, GAPDH, or SOX17.

[0079] The guide RNA can comprise a sequence complementary to at least one target sequence on a transposon, plasmid, or vector. In some embodiments, the complementary sequence to the guide RNA on the transposon, plasmid, or vector is located within a transgene for targeted nucleic acid insertion. In some embodiments, the complementary sequence to the guide RNA on the transposon, plasmid, or vector is located within a transgene for targeted nucleic acid insertion. In some embodiments, the complementary sequence on the transposon, plasmid, or vector promotes binding of a gRNA bound to an effector molecule, thereby tethering all components. In some embodiments, the effector molecule is Cas-CLOVER. In some embodiments, Cas-CLOVER further comprises at least one NLS sequence. In some embodiments, the NLS sequence of Cas-CLOVER promotes localization of the tethered components to the nucleus. This promotes localization of all components required for gene editing (Cas-CLOVER, gRNA, and transposon, plasmid, or vector) to the nucleus, thereby improving the efficiency of gene editing.

[0080] gRNA

[0081] As used herein, the term "guide sequence" in the context of the Cas-CLOVER or CRISPR-Cas9 system includes any polynucleotide sequence that hybridizes with a target nucleic acid sequence and has sufficient complementarity with the target nucleic acid sequence to direct sequence-specific binding of a nucleic acid-targeting complex to the target nucleic acid sequence. The guide sequence can form a duplex with the target sequence. This duplex can be a DNA duplex, an RNA duplex, or an RNA / DNA duplex. The terms "guide molecule," "guide RNA," and "single guide RNA" are used interchangeably herein to refer to an RNA-based molecule that can form a complex with a Cas-CLOVER or CRISPR-Cas protein and that includes a guide sequence that hybridizes with a target nucleic acid sequence and has sufficient complementarity with the target nucleic acid sequence to direct sequence-specific binding of the complex to the target nucleic acid sequence. A guide molecule or guide RNA can include RNA-based molecules with one or more chemical modifications (e.g., by chemically linking two ribonucleotides or by replacing one or more ribonucleotides with one or more deoxyribonucleotides), as described herein.

[0082] The terms "target region," "target sequence," or "protospacer," used interchangeably herein, refer to the region of a target gene targeted by a Cas-CLOVER system or a CRISPR / Cas9-based system. A Cas-CLOVER or CRISPR / Cas9-based system can include at least one gRNA, where the gRNAs target different DNA sequences. The target DNA sequences can overlap. A Cas-CLOVER system can include at least two gRNAs, where the gRNAs target different DNA sequences. At the 3' end of the protospacer, the target sequence or protospacer is followed by a PAM sequence. Different type II systems have different PAM requirements. For example, the Streptococcus pyogenes type II system uses the "NGG" sequence, where "N" can be any nucleotide.

[0083] The guide RNA or guide RNAs of a Cas-CLOVER protein or CRISPR-Cas protein can comprise a tracr-mate sequence (which encompasses a "direct repeat" in the context of an endogenous CRISPR system) and a guide sequence (also referred to as a "spacer" in the context of an endogenous CRISPR system). In some embodiments, the Cas-CLOVER or CRISPR-Cas systems or complexes described herein do not comprise and / or are not dependent on the presence of a tracr sequence. In certain embodiments, a guide molecule can comprise, consist essentially of, or consist of a direct repeat sequence fused or linked to a guide sequence or spacer sequence.

[0084] In some embodiments, the guide sequence or spacer length of the guide molecule is 15 to 50 nucleotides. In some embodiments, the spacer length of the guide RNA is at least 15 nucleotides. In some embodiments, the spacer length is 15 to 17 nucleotides, 17 to 20 nucleotides, 20 to 24 nucleotides, 23 to 25 nucleotides, 24 to 27 nucleotides, 27 to 30 nucleotides, 30 to 35 nucleotides, or more than 35 nucleotides.

[0085] In some embodiments, the guide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 , 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides in length.

[0086] In some embodiments, the sequence of the guide molecule (direct repeats and / or spacers) is selected to reduce the degree of secondary structure within the guide molecule. In some embodiments, when optimally folded, approximately 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1% or less of the nucleotides of the nucleic acid-targeting guide RNA participate in self-complementary base pairing. Optimal folding can be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculations of minimum Gibbs free energy. One example of such an algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another exemplary folding algorithm is the online web server RNAfold, developed at the Institute of Theoretical Chemistry, University of Vienna, using a centroid structure prediction algorithm (see, e.g., A.R. Gruber et al., 2008, Cell 106(1):23-24, and P.A. Carr and G.M. Church, 2009, Nature Biotechnology 27(12):1151-62).

[0087] As described above, the Cas-CLOVER and CRISPR / Cas9 systems utilize a targeting gRNA and a shuttling gRNA to provide targeting for the Cas-CLOVER and CRISPR / Cas9-based systems. The gRNA can be a fusion of two non-coding RNAs, a crRNA and a tracrRNA. The sgRNA can target any desired DNA sequence by exchanging sequences encoding a 20-bp protospacer, which confers targeting specificity through complementary base pairing with the desired DNA target. The gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in Type II effector systems. This duplex, which can include, for example, a 42-nucleotide crRNA and a 75-nucleotide tracrRNA, acts as a guide for Cas9 to cleave the target nucleic acid.

[0088] In some embodiments, the gRNA targets a region upstream of a target gene (e.g., the HBB, B2M, TRAC, or GAPDH locus), e.g., a region 0 to 1000 bp upstream of the target gene. In some embodiments, the gRNA targets a region 0 to 50 bp, 0 to 100 bp, 0 to 150 bp, 0 to 200 bp, 0 to 250 bp, 0 to 300 bp, 0 to 350 bp, 0 to 400 bp, 0 to 450 bp, 0 to 500 bp, 0 to 550 bp, 0 to 600 bp, 0 to 650 bp, 0 to 700 bp, 0 to 750 bp, 0 to 800 bp, 0 to 850 bp, 0 to 900 bp, 0 to 950 bp, or 0 to 1000 bp upstream of the transcription start site of the target gene. In some embodiments, the gRNA targets a region within about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, or about 1500 bp upstream of the target gene.

[0089] In some embodiments, the gRNA targets a region downstream of a target gene (e.g., the HBB, B2M, TRAC, or GAPDH locus), e.g., 0 to 1000 bp downstream of the target gene. In some embodiments, the gRNA targets a region 0 to 50 bp, 0 to 100 bp, 0 to 150 bp, 0 to 200 bp, 0 to 250 bp, 0 to 300 bp, 0 to 350 bp, 0 to 400 bp, 0 to 450 bp, 0 to 500 bp, 0 to 550 bp, 0 to 600 bp, 0 to 650 bp, 0 to 700 bp, 0 to 750 bp, 0 to 800 bp, 0 to 850 bp, 0 to 900 bp, 0 to 950 bp, or 0 to 1000 bp downstream of the target gene. In some embodiments, the gRNA targets a region within about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, or about 1500 bp downstream of the target gene.

[0090] gRNA can be divided into target binding region and Cas9 binding region.Target binding region hybridizes with target region in target gene.Methods for designing such target binding region are known in the art, for example, see Doench et al., Nat Biotechnol.(2014)32:1262-7 and Doench et al., Nat Biotechnol.(2016)34:184-91, the entire contents of which are incorporated herein by reference.Design tools are available, for example, in Feng Zhang lab's Target Finder, Michael Boutros lab's Target Finder (E-CRISP), RGEN tool (Cas-OF Finder), CasFinder, and CRISPR Optimal Target Finder. In certain embodiments, the target binding region can be about 15 to about 50 nucleotides in length (about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 nucleotides in length). In certain embodiments, the target binding region can be about 19 to about 21 nucleotides in length. In one embodiment, the target binding region is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

[0091] In one embodiment, the target binding region is complementary, e.g., perfectly complementary, to a target region in the target gene. In one embodiment, the target binding region is substantially complementary to a target region in the target gene. In one embodiment, the target binding region comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides that are not complementary to the target region in the target gene.

[0092] Exemplary sgRNAs of the present disclosure include, but are not limited to, sequences for targeting the HBB, B2M, TRAC, or GAPDH loci. Exemplary sgRNAs of the present disclosure also include, but are not limited to, sequences for targeting hemoglobin, albumin, TTR, APOC3, PCSK9, and KLKB1. Exemplary sgRNAs of the present disclosure comprise, consist essentially of, or consist of the sequences set forth in Table 1.

[0093] [Table 1]

[0094] Gene editing compositions comprising Cas-CLOVER and methods of using these compositions for gene editing are described in detail in International Application No. / US2016 / 037922, International Application No. / US2018 / 066941, International Application No. / US2017 / 054799, U.S. Patent Application Publication Nos. 2017 / 0107541, 2017 / 0114149, 2018 / 0187185, and U.S. Patent No. 10,415,024, each of which is incorporated herein by reference in its entirety. Exemplary gene editing compositions comprising mutant Cas-CLOVER and methods of using these compositions for gene editing are described herein.

[0095] Gene editing tools can also be delivered to cells using one or more poly(histidine)-based micelles. Poly(histidine) (e.g., poly(L-histidine)) is a pH-sensitive polymer due to the imidazole ring that provides a lone pair of electrons on the unsaturated nitrogen. That is, poly(histidine) has amphoteric properties due to protonation-deprotonation. In particular, at a certain pH, poly(histidine)-containing triblock copolymers can assemble into micelles with positively charged poly(histidine) units on the surface, thereby enabling complexation with negatively charged gene editing molecules. Using these nanoparticles to bind and release proteins and / or nucleic acids in a pH-dependent manner can provide an efficient and selective mechanism for achieving desired genetic modifications. In particular, this micelle-based delivery system offers substantial flexibility in terms of charged materials, as well as large payload capacity and selective release of nanoparticle payloads. In one example, site-specific cleavage of double-stranded DNA is enabled by the delivery of nucleases using poly(histidine)-based micelles. Without wishing to be bound by any particular theory, it is believed that in micelles formed by various triblock copolymers, the hydrophobic blocks aggregate to form a core, leaving the hydrophilic and poly(histidine) blocks at the ends to form one or more surrounding layers.

[0096] In one aspect, the present disclosure provides a triblock copolymer made of a hydrophilic block, a hydrophobic block, and a charged block. In some aspects, the hydrophilic block can be poly(ethylene oxide) (PEO) and the charged block can be poly(L-histidine). An exemplary triblock copolymer that can be used is PEO-b-PLA-b-PHIS, where the number of repeating units in each block varies by design.

[0097] Diblock copolymers that can be used as intermediates for preparing triblock copolymers can comprise hydrophilic, biocompatible poly(ethylene oxide) (PEO), which is chemically equivalent to PEG, and are conjugated to various hydrophobic aliphatic poly(anhydrides), poly(nucleic acids), poly(esters), poly(orthoesters), poly(peptides), poly(phosphazenes), and poly(saccharides), including, but not limited to, poly(lactide) (PLA), poly(glycolide) (PLGA), poly(lactic-co-glycolic acid) (PLGA), poly(ε-caprolactone) (PCL), and poly(trimethylene carbonate) (PTMC). Polymeric micelles composed of 100% PEGylated surfaces have improved in vitro chemical stability, increased in vivo bioavailability, and prolonged blood circulation half-lives.

[0098] Polymeric vesicles, polymersomes, and poly(histidine)-based micelles, such as those comprising triblock copolymers, and methods for making them, are described in further detail in U.S. Pat. Nos. 7,217,427, 7,868,512, 6,835,394, 8,808,748, 10,456,452, U.S. Patent Application Publication Nos. 2014 / 0363496, 2017 / 0000743, and 2019 / 0255191, and WO 2019 / 126589, each of which is incorporated by reference in its entirety.

[0099] Gene editing compositions (e.g., mutant Cas-CLOVER) can also be delivered to cells using one or more lipid nanoparticle compositions and methods of making the same, as described in International Application No. / US2023 / 061005 and International Publication No. 2022 / 182792, which are incorporated by reference in their entireties.

[0100] In some embodiments, the composition is encapsulated in at least one lipid nanoparticle comprising about 40.75 mol% terpene lipidoid compound, about 51.75 mol% cholesterol, about 5 mol% DOPC, and about 2.5 mol% DMG-PEG2000, wherein the polynucleotide encoding the mutant Cas-CLOVER is an RNA molecule, and the ratio of lipid to RNA molecule in the at least one nanoparticle is about 120:1 (w / w).

[0101] In some embodiments, the terpene lipidoid compound is HMA-404. TIFF2026507731000003.tif46170Thus, in some embodiments, the gene editing composition is encapsulated in at least one lipid nanoparticle comprising about 40.75 mol% HMA-404, about 51.75 mol% cholesterol, about 5 mol% DOPC, and about 2.5 mol% DMG-PEG2000, wherein the polynucleotide encoding the mutant Cas-CLOVER is an RNA molecule, and the ratio of lipid to RNA molecule in the at least one nanoparticle is about 120:1 (w / w).

[0102] In some embodiments, the composition is encapsulated in at least one lipid nanoparticle comprising about 54 mol% SS-OP, about 35 mol% cholesterol, about 5 mol% DOPC, about 5 mol% DSPC, and about 1 mol% DMG-PEG 2000. The lipid to nucleic acid ratio in the nanoparticle was about 100:1 (wt / wt), and the total lipid was 25 mM.

[0103] Metastasis

[0104] The present disclosure also provides a composition comprising a transposon. In a preferred embodiment, the composition comprising a transposon further comprises a plasmid comprising a nucleotide sequence encoding a transposase. The nucleotide sequence encoding the transposase can be a DNA sequence or an RNA sequence. Preferably, the sequence encoding the transposase is an mRNA sequence.

[0105] The transposon of the present disclosure can be a piggyBac™ (PB) transposon. In some embodiments where the transposon is a PB transposon, the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, or a Super piggyBac™ (SPB) transposase. The sequence encoding the SPB transposase is an mRNA sequence.

[0106] The transposon of the present disclosure can be a Footprint-Free™ transposon. In some embodiments, the transposase is a PBx transposase. The sequence encoding the PBx transposase is an mRNA sequence. In some embodiments, the PBx transposase facilitates Footprint-Free™ removal of a nucleic acid cassette in a transposon, plasmid, or vector.

[0107] Non-limiting examples of PB transposons and PB, PBL, and SPB transposases are described in detail in U.S. Pat. No. 6,218,182, U.S. Pat. No. 6,962,810, U.S. Pat. No. 8,399,643, and WO 2010 / 099296, WO 2010 / 099301, WO 2013 / 012824, each of which is incorporated herein in its entirety.

[0108] PB, PBL, and SPB transposases recognize transposon-specific inverted terminal repeats (ITRs) at the ends of transposons and insert their contents between the ITRs at the sequence 5'-TTAT-3' (TTAT target sequence) or the sequence 5'-TTAA-3' (TTAA target sequence) within the chromosomal site. The target sequences of PB or PBL transposon are 5'-CTAA-3', 5'-TTAG-3', 5'-ATAA-3', 5'-TCAA-3', 5'AGTT-3', 5'-ATTA-3', 5'-GTTA-3', 5'-TTGA-3', 5'-TTTA-3', 5' -TTAC-3', 5'-ACTA-3', 5'-AGGG-3', 5'-CTAG-3', 5'-TGAA-3', 5'-AGGT-3', 5'-ATCA-3', 5'-CTCC-3', 5'-TAAA-3', 5'-TCTC-3', 5'TGAA-3', 5'- The PB or PBL transposon system may comprise or consist of the following sequences: AAAT-3', 5'-AATC-3', 5'-ACAA-3', 5'-ACAT-3', 5'-ACTC-3', 5'-AGTG-3', 5'-ATAG-3', 5'-CAAA-3', 5'-CACA-3', 5'-CATA-3', 5'-CCAG-3', 5'-CCCA-3', 5'-CGTA-3', 5'-GTCC-3', 5'-TAAG-3', 5'-TCTA-3', 5'-TGAG-3', 5'-TGTT-3', 5'-TTCA-3', 5'-TTCT-3', and 5'-TTTT-3'. The PB or PBL transposon system does not impose any restrictions on the payload for the gene of interest that can be included between the ITRs.

[0109] Exemplary amino acid sequences of one or more PB, PBL, and SPB transposases are disclosed in US Pat. No. 6,218,185, US Pat. No. 6,962,810, and US Pat. No. 8,399,643.

[0110] As described herein, in certain embodiments, the invention features integration-defective piggyBac transposons. Integration-defective is intended to refer to transposons that integrate into a host genome at a lower frequency than the corresponding wild-type transposon. In certain exemplary embodiments, the transposons of the invention integrate via conventional integration mechanisms.

[0111] In certain exemplary embodiments, the integration-defective piggyBac transposon is derived from the wild-type piggyBac sequence, SEQ ID NO: 16. In exemplary embodiments, the integration-defective piggyBac transposon comprises an alteration in SEQ ID NO: 16 selected from R372A or K375A. In certain preferred embodiments, the integration-defective piggyBac transposon comprises an amino acid sequence selected from SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19. In one embodiment, the amino acid change in SEQ ID NO: 16 comprises R372A and corresponds to SEQ ID NO: 17. The integration-defective mutant encoded by SEQ ID NO: 17 corresponds to a nucleotide change from CGA to GCA in SEQ ID NO: 20, corresponding to SEQ ID NO: 21. In another embodiment, the amino acid change in SEQ ID NO: 16 comprises K375A and corresponds to SEQ ID NO: 18. The integration-defective mutant encoded by SEQ ID NO: 18 corresponds to a nucleotide change from AAA to GCA in SEQ ID NO: 20, corresponding to SEQ ID NO: 50. In another embodiment, the amino acid changes in SEQ ID NO: 2 comprise R372A, K375A and correspond to SEQ ID NO: 19. The integration-defective mutant encoded by SEQ ID NO:19 corresponds to the CGA to GCA / AAA to GCA nucleotide change in SEQ ID NO:20, which corresponds to SEQ ID NO:22.

[0112] In exemplary embodiments, the integration-defective piggyBac transposase comprises an alteration selected from at least R372A or K375A and D450N in SEQ ID NO: 16. In some aspects, the PBx transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 23.

[0113] In exemplary embodiments, the integration-defective piggyBac transposase comprises an alteration selected from at least R372A or K375A and D450N in SEQ ID NO: 16. In some aspects, the PBx transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 24.

[0114] In some embodiments, the PB transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:25.

[0115] The PB or PBL transposase may comprise or consist of an amino acid sequence having an amino acid substitution at two or more positions, at three or more positions, or at each of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 25. The transposase may be an SPB transposase comprising or consisting of the amino acid sequence of SEQ ID NO: 25, wherein the amino acid substitution at position 30 may be a substitution of valine (V) for isoleucine (I), the amino acid substitution at position 165 may be a substitution of serine (S) for glycine (G), the amino acid substitution at position 282 may be a substitution of valine (V) for methionine (M), and the amino acid substitution at position 538 may be a substitution of lysine (K) for asparagine (N). In preferred embodiments, the SPB transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:26.

[0116] In certain embodiments, where the transposase comprises the above-described mutations at positions 30, 165, 282, and / or 538, the PB, PBL, and SPB transposases may comprise any of the mutations at positions 3, 46, 82, 103, 119, 125, 173, 182, 193, 203, 219, 225, 230, 240, 253, 265, 270, 282, 293, 304, 310, 320, 332, 340, 353, 360, 370, 380, 390, 410, 420, 430, 440, 453, 460, 470, 482, 503, 510, 520, 532, 540, 553, 560, 570, 580, 590, 603, 610, 620, 630, 640, 653, 660, 670, 680, 690, 703, 710, 720, 730, 740, 753, 760, 770, 780, 790, 803, 810, 820, 830, 840, 853, 860, 870, 880, 890, 903, 910, 920, 930, 940, 953, 960, 970, 980, 990, 1003, 1050, 1060 and further comprising an amino acid substitution at one or more of positions 7, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570, and 591.

[0117] The PB, PBL, or SPB transposase can be isolated or derived from an insect, vertebrate, crustacean, or urochordate, as described in further detail in WO 2019 / 173636 and WO 2019 / 049816. In a preferred embodiment, the PB, PBL, or SPB transposase is isolated or derived from the insect Trichoplusia ni (GenBank Accession No. AAA87375) or Bombyx mori (GenBank Accession No. BAD11135).

[0118] A hyperactive PB or PBL transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In a preferred embodiment, the hyperactive PB or PBL transposase is isolated or derived from Bombyx mori or Xenopus tropicalis. Examples of hyperactive PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185, U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643, and WO 2019 / 173636. A list of hyperactive amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0119] In some embodiments, the PB or PBL is integration-defective. An integration-defective PB or PBL transposase is a transposase that can excise a corresponding transposon but integrates the excised transposon less frequently than a corresponding wild-type transposase. Examples of integration-defective PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185, U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643, and WO 2019 / 173636. A list of integration-defective amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0120] In some embodiments, the PB or PBL transposase is fused to a nuclear localization signal. Examples of PB or PBL transposases fused to nuclear localization signals are disclosed in U.S. Patent No. 6,218,185, U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643, and WO 2019 / 173636.

[0121] The transposon of the present disclosure can be a Sleeping Beauty transposon. In some embodiments, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (e.g., as disclosed in U.S. Pat. No. 9,228,180) or a hyperactive Sleeping Beauty (SB100X) transposase. In preferred embodiments, the Sleeping Beauty transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:27. In preferred embodiments, the hyperactive Sleeping Beauty (SB100X) transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:28.

[0122] The transposon of the present disclosure may be a Helraiser transposon. Exemplary Helraiser transposons include Helibat1, which comprises or consists of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:29. In some embodiments, when the transposon is a Helraiser transposon, the transposase is a Helitron transposase (e.g., as disclosed in WO 2019 / 173636). In preferred embodiments, the Helitron transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:30.

[0123] The transposon of the present disclosure may be a Tol2 transposon. Exemplary Tol2 transposons, including inverted repeat sequences, minor terminal sequences, and a Tol2 transposase, comprise or consist of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 31. In some embodiments, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (e.g., as disclosed in WO 2019 / 173636). In preferred embodiments, the Tol2 transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 32.

[0124] The transposon of the present disclosure can be a TcBuster transposon. In some embodiments, when the transposon is a TcBuster transposon, the transposase is a TcBuster transposase or a hyperactive TcBuster transposase (e.g., as disclosed in WO 2019 / 173636). The TcBuster transposase can comprise or consist of a naturally occurring or non-naturally occurring amino acid sequence. In preferred embodiments, the TcBuster transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 33. The polynucleotide encoding the TcBuster transposase can comprise or consist of a naturally occurring or non-naturally occurring nucleic acid sequence. In preferred embodiments, the TcBuster transposase is encoded by a polynucleotide comprising or consisting of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:34.

[0125] In some embodiments, the mutant TcBuster transposase comprises one or more sequence mutations when compared to the wild-type TcBuster transposase, as described in more detail in WO 2019 / 173636 and WO 2019 / 049816.

[0126] The transposon can be a nanotransposon. The nanotransposon can comprise, essentially comprise, or consist of: (a) a transposon insert encoding sequence including a first inverted terminal repeat (ITR) encoding sequence, a second inverted terminal repeat (ITR) encoding sequence, and an intra-ITR sequence; (b) a backbone encoding sequence including a sequence encoding an origin of replication having 1 to 450 nucleotides, including the endpoints, and a sequence encoding a selectable marker having 1 to 200 nucleotides, including the endpoints; and (c) an inter-ITR sequence. In some embodiments, the inter-ITR sequence of (c) comprises the sequence of (b). In some embodiments, the intra-ITR sequence of (a) comprises the sequence of (b).

[0127] A selectable marker having 1 to 200 nucleotides inclusive of the endpoint can comprise a sequence encoding a sucrose selectable marker. The sequence encoding the sucrose selectable marker can comprise a sequence encoding an RNA-OUT sequence. The sequence encoding the RNA-OUT sequence can comprise or consist of 137 base pairs (bp). A selectable marker having 1 to 200 nucleotides inclusive of the endpoint can comprise a sequence encoding a fluorescent marker. A selectable marker having 1 to 200 nucleotides inclusive of the endpoint can comprise a sequence encoding a cell surface marker.

[0128] The sequence encoding an origin of replication having 1 to 450 nucleotides inclusive of the endpoint can comprise a sequence encoding a mini origin of replication. In some embodiments, the sequence encoding an origin of replication having 1 to 450 nucleotides inclusive of the endpoint comprises a sequence encoding an R6K origin of replication. The R6K origin of replication can comprise an R6K gamma origin of replication. The R6K origin of replication can comprise an R6K miniorigin of replication. The R6K origin of replication can comprise an R6K gamma miniorigin of replication. The R6K gamma miniorigin of replication can comprise or consist of 281 base pairs (bp).

[0129] In some aspects of the nanotransposon, the backbone-encoding sequence does not include recombination sites, excision sites, ligation sites, or combinations thereof. In some aspects, neither the nanotransposon nor the backbone-encoding sequence includes products of recombination sites, excision sites, ligation sites, or combinations thereof. In some aspects, neither the nanotransposon nor the backbone-encoding sequence is derived from recombination sites, excision sites, ligation sites, or combinations thereof.

[0130] In some embodiments of the nanotransposon, the recombination sites comprise sequences that result from a recombination event. In some embodiments, the recombination sites comprise sequences that are the product of a recombination event. In some embodiments, the recombination event comprises the activity of a recombinase (e.g., a recombinase site).

[0131] In some embodiments of the nanotransposon, the backbone-encoding sequence does not further comprise a sequence encoding foreign DNA.

[0132] In some embodiments of the nanotransposon, the inter-ITR sequence does not comprise a recombination site, an excision site, a ligation site, or a combination thereof. In some embodiments, the inter-ITR sequence does not comprise a product of a recombination event, an excision event, a ligation event, or a combination thereof. In some embodiments, the inter-ITR sequence is not derived from a recombination event, an excision event, a ligation event, or a combination thereof. In some embodiments, the inter-ITR sequence comprises a sequence encoding foreign DNA. In some embodiments, the intra-ITR sequence comprises at least one sequence encoding an insulator and a sequence encoding a promoter capable of expressing the exogenous sequence in a mammalian cell. The mammalian cell can be a human cell. In some embodiments, the intra-ITR sequence comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing the exogenous sequence in a mammalian cell, and a second sequence encoding an insulator. In some embodiments, the sequence within the ITRs comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing the exogenous sequence in mammalian cells, a polyadenosine (polyA) sequence, and a second sequence encoding the insulator. In some embodiments, the sequence within the ITRs comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing the exogenous sequence in mammalian cells, at least one exogenous sequence, a polyadenosine (polyA) sequence, and a second sequence encoding the insulator.

[0133] Nanotransposons are described in further detail in International Application No. WO / US2019 / 067758, which is incorporated herein by reference in its entirety.

[0134] Vector

[0135] The vectors of the present disclosure may be viral vectors or recombinant vectors. Viral vectors may comprise sequences isolated or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. Viral vectors may comprise sequences isolated or derived from adeno-associated viruses (AAV). Viral vectors may comprise recombinant AAV (rAAV). Exemplary adeno-associated viruses and recombinant adeno-associated viruses comprise two or more inverted terminal repeat (ITR) sequences located adjacent to the sequence encoding the scFv or CAR of the present disclosure. Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids, such as the genome of one serotype and the capsid of another serotype (e.g., AAV2 / 5, AAV-DJ, and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, rAAV-LK03.

[0136] The vector of the present disclosure can be a nanoparticle. Non-limiting examples of nanoparticle vectors include nucleic acids (e.g., RNA, DNA, synthetic nucleotides, modified nucleotides, or any combination thereof), amino acids (L-amino acids, D-amino acids, synthetic amino acids, modified amino acids, or any combination thereof), polymers (e.g., polymersomes), micelles, lipids (e.g., liposomes), organic molecules (e.g., carbon atoms, sheets, fibers, tubes), inorganic molecules (e.g., calcium phosphate or gold), or any combination thereof. Nanoparticle vectors can be passively or actively transported across cell membranes.

[0137] The cell delivery compositions (e.g., transposons, vectors) disclosed herein can include nucleic acids encoding therapeutic proteins or therapeutic agents. Examples of therapeutic proteins include those disclosed in WO 2019 / 173636 and WO 2019 / 049816.

[0138] nucleic acid molecule

[0139] The nucleic acid molecules of the present disclosure may be in the form of RNA, such as mRNA, hnRNA, tRNA, or any other form, or DNA, such as, but not limited to, cDNA and genomic DNA obtained by cloning or produced synthetically, or any combination thereof. The DNA may be triple-stranded, double-stranded, or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA may be the coding strand, also known as the sense strand, or the non-coding strand, also called the antisense strand.

[0140] Isolated nucleic acid molecules of the present disclosure can include nucleic acid molecules comprising an open reading frame (ORF), optionally with one or more introns, for example, but not limited to, nucleic acid molecules comprising a coding sequence for at least one specific portion of at least one scFv, a protein backbone or loop region that binds to a target protein, and nucleic acid molecules comprising a substantially different nucleotide sequence than those described above, but which, due to the degeneracy of the genetic code, still encode a protein backbone described herein and / or known in the art. Of course, the genetic code is well known in the art. Thus, it would be routine for one of skill in the art to generate such degenerate nucleic acid variants encoding specific scFvs of the present disclosure. See, e.g., Ausubel et al., supra, and such nucleic acid variants are included in the present disclosure.

[0141] As provided herein, nucleic acid molecules of the present disclosure can include, but are not limited to, those that themselves encode the amino acid sequence of an scFv fragment, coding sequences for the entire protein scaffold or a portion thereof, coding sequences for scFvs, fragments, or portions, as well as additional sequences, such as the aforementioned additional coding sequences, e.g., coding sequences for at least one signal leader or fusion peptide, with or without at least one intron, as well as additional non-coding sequences, such as, but not limited to, non-coding 5' and 3' sequences, e.g., transcribed, non-translated sequences that play a role in mRNA processing (e.g., ribosome binding and mRNA stability), such as transcription, splicing, and polyadenylation signals, additional coding sequences encoding additional amino acids, e.g., that provide additional functionality. Thus, the protein scaffold-encoding sequence can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fusion protein scaffold comprising the protein scaffold fragment or portion.

[0142] Polynucleotides that selectively hybridize to the polynucleotides described herein

[0143] The present disclosure provides isolated nucleic acids that hybridize to the polynucleotides disclosed herein under selective hybridization conditions. Thus, the polynucleotides can be used to isolate, detect, and / or quantify nucleic acids containing such polynucleotides. For example, the polynucleotides of the present disclosure can be used to identify, isolate, or amplify partial or full-length clones in a deposited library. The polynucleotides can be genomic or cDNA sequences isolated or complementary to cDNAs from a human or mammalian nucleic acid library.

[0144] Preferably, the cDNA library contains at least 80% of the full-length sequences, preferably at least 85% or 90% of the full-length sequences, and more preferably at least 95% of the full-length sequences. The cDNA library can be normalized to increase the representation of rare sequences. Low or medium stringency hybridization conditions are typically, but not exclusively, used for sequences with reduced sequence identity compared to the complementary sequence. Medium and high stringency conditions can optionally be used for sequences with higher identity. Low stringency conditions allow selective hybridization of sequences with approximately 70% sequence identity and can be used to identify orthologous or paralogous sequences.

[0145] Optionally, the polynucleotide encodes at least a portion of the protein scaffold encoded by the polynucleotide described herein. The polynucleotide comprises a nucleic acid sequence that can be used for selective hybridization to the polynucleotide encoding the protein scaffold of the present disclosure. See, e.g., Ausubel, supra; Colligan, supra, each of which is incorporated herein by reference in its entirety.

[0146] Nucleic acid construction

[0147] Isolated nucleic acids of the disclosure can be produced using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and / or (d) combinations thereof, as are known in the art.

[0148] A nucleic acid can conveniently contain nucleotide sequences in addition to the polynucleotides of the present disclosure. For example, a multiple cloning site containing one or more endonuclease restriction sites can be inserted into the nucleic acid to facilitate isolation of the polynucleotide. Also, a translatable sequence can be inserted to facilitate isolation of the translated polynucleotide of the present disclosure. For example, a hexa-histidine marker sequence provides a convenient means for purifying the proteins of the present disclosure. A nucleic acid of the present disclosure, excluding the coding sequence, is optionally a vector, adapter, or linker for cloning and / or expression of the polynucleotides of the present disclosure.

[0149] Additional sequences can be added to such cloning and / or expression sequences to optimize function in cloning and / or expression, to aid in isolation of the polynucleotide, or to improve introduction of the polynucleotide into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art (see, e.g., Ausubel, supra, or Sambrook, supra).

[0150] Recombinant methods for constructing nucleic acids

[0151] The isolated nucleic acid compositions of the present disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art. In some embodiments, oligonucleotide probes that selectively hybridize under stringent conditions to the polynucleotides of the present disclosure are used to identify desired sequences in cDNA or genomic DNA libraries. The isolation of RNA and the construction of cDNA and genomic libraries are well known to those of skill in the art (see, e.g., Ausubel, supra, or Sambrook, supra).

[0152] Nucleic Acid Screening and Isolation Methods

[0153] Probes based on the polynucleotide sequences of the present disclosure can be used to screen cDNA or genomic libraries. Probes can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes from the same or different organisms. Those skilled in the art will understand that varying degrees of hybridization stringency can be employed in the assay, and that either the hybridization or wash medium can be stringent. The more stringent the hybridization conditions, the greater the degree of complementarity between the probe and target required for duplex formation to occur. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH, and the presence of a partially denaturing solvent such as formamide. For example, hybridization stringency can be conveniently varied by changing the polarity of the reaction solution, e.g., by manipulating the formamide concentration within a range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding varies depending on the stringency of the hybridization medium and / or wash medium. The degree of complementarity is optimally 100%, or 70-100%, or any range or value therebetween, although it should be understood that minor sequence differences between the probe and primer can be compensated for by reducing the stringency of the hybridization and / or wash solutions.

[0154] Methods for amplifying RNA or DNA are well known in the art and can be used in accordance with the present disclosure without undue experimentation, based on the teachings and guidance provided herein.

[0155] Known methods for amplifying DNA or RNA include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 (Mullis et al.), 4,795,699 and 4,921,794 (Tabor et al.), 5,142,033 (Innis), 5,122,464 (Wilson et al.), 5,091,310 (Innis), 5,066,584 (Gyllens, Nos. 4,889,818 (Gelfand et al.), 4,994,370 (Silver et al.), 4,766,067 (Biswas), and 4,656,134 (Ringold)), as well as RNA-mediated amplification (U.S. Pat. No. 5,130,238 (Malek et al.), under the trade name NASBA), which uses antisense RNA against a target sequence as a template for double-stranded DNA synthesis, the entire contents of which are incorporated herein by reference (see, e.g., Ausubel, supra, or Sambrook, supra).

[0156] For example, polymerase chain reaction (PCR) techniques can be used to directly amplify sequences of the disclosed polynucleotides and related genes from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods are also useful for, for example, cloning nucleic acid sequences encoding proteins to be expressed, generating nucleic acids to be used as probes to detect the presence of desired mRNA in a sample, nucleic acid sequencing, and other purposes. Examples of techniques sufficient to guide one of skill in the art through in vitro amplification methods can be found in Berger, Sambrook, Ausubel, and Mullis et al., U.S. Pat. No. 4,683,202 (1987); and Innis et al., PCR Protocols: A Guide to Methods and Applications, Eds., Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic PCR amplification are known in the art. See, for example, the Advantage-GC Genomic PCR Kit (Clontech). Additionally, to improve the yield of long PCR products, the T4 gene 32 protein (Boehringer Mannheim), for example, can be used.

[0157] Synthetic methods for constructing nucleic acids

[0158] The isolated nucleic acids of the present disclosure can also be prepared by direct chemical synthesis using known methods (see, for example, Ausubel et al., supra). Chemical synthesis generally produces a single-stranded oligonucleotide, which can be converted into double-stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. Those skilled in the art will understand that chemical synthesis of DNA is limited to base sequences of about 100 bases, but that longer base sequences can be obtained by ligating shorter base sequences.

[0159] Expression vectors and host cells

[0160] The present disclosure also relates to vectors comprising the isolated nucleic acid molecules of the present disclosure, host cells genetically engineered with the recombinant vectors, and the production of at least one protein scaffold by recombinant techniques, as known in the art (see, e.g., Sambrook et al., supra; Ausubel et al., supra, each of which is incorporated herein by reference in its entirety).

[0161] The polynucleotide can be optionally linked to a vector containing a selectable marker for propagation in a host. Generally, the plasmid vector is introduced in a precipitate such as a calcium phosphate precipitate or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and transduced into host cells.

[0162] The DNA insert must be operably linked to a suitable promoter. The expression construct further contains a transcription initiation site, a termination site, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct preferably includes translation initiation at a start and stop codon (e.g., UAA, UGA, or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG being preferred for mammalian or eukaryotic expression.

[0163] Expression vectors preferably, but optionally, include at least one selectable marker, such as, but not limited to, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), mycophenolic acid, or glutamine synthetase (GS, U.S. Patent Nos. 5,122,464, 5,770,359, and 5,827,739), blasticidin (bsd gene), resistance genes for eukaryotic cell culture, and ampicillin, zeocin (Sh bla gene), for culturing in E. coli and other bacteria or prokaryotes. Examples of suitable host cell resistance genes include genes for resistance to HIV-1 (bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline (the above patents are incorporated herein by reference in their entireties). Appropriate culture media and conditions for the above host cells are known in the art. Suitable vectors will be readily apparent to those skilled in the art. Introduction of vector constructs into host cells can be achieved by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other known methods. Such methods are described in the art, e.g., Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, and 16.

[0164] The expression vector preferably, but optionally, includes at least one selectable cell surface marker for isolating cells modified by the disclosed compositions and methods. The selectable cell surface markers of the present disclosure consist of a surface protein, glycoprotein, or group of proteins that distinguish a cell or a subset of cells from another defined subset of cells. Preferably, the selectable cell surface marker distinguishes cells modified by the disclosed compositions or methods from cells not modified by the disclosed compositions or methods. Examples of such cell surface markers include, but are not limited to, "cluster designator" or "classification determinant" proteins (often abbreviated as "CD"), such as truncated or full-length forms of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or combinations thereof. Cell surface markers include the suicide gene marker RQR8 (Philip B et al. Blood. 2014 Aug 21;124(8):1277-87).

[0165] Preferably, but optionally, the expression vector includes at least one selectable drug resistance marker for isolating cells modified by the compositions and methods of the present disclosure. Selectable drug resistance markers of the present disclosure may include wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0166] At least one protein scaffold of the present disclosure can be expressed in modified forms, such as fusion proteins, and can include not only secretion signals but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the protein scaffold to improve stability and persistence in host cells during purification or subsequent handling and storage. Peptide moieties can also be added to the protein scaffold of the present disclosure to facilitate purification. Such regions can be removed prior to final preparation of the protein scaffold or at least one fragment thereof. Such methods are described in many standard laboratory manuals, e.g., Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17, and 18.

[0167] Those skilled in the art are familiar with the many expression systems available for expressing nucleic acids encoding the proteins of the present disclosure. Alternatively, the nucleic acids of the present disclosure can be expressed in host cells by turning on (by manipulation) endogenous DNA encoding the protein scaffold of the present disclosure in the host cell. Such methods are well known in the art, for example, as described in U.S. Patent Nos. 5,580,734, 5,641,670, 5,733,746 and 5,733,761, which are incorporated herein by reference in their entirety.

[0168] Illustrative examples of cell cultures useful for the production of protein scaffolds, specific portions or variants thereof are bacterial, yeast and mammalian cells known in the art. Mammalian cell systems are often in the form of monolayers of cells, although mammalian cell suspensions or bioreactors can also be used. Several suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, including COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610), and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hepG2 cells, P3X63Ag8.653, SP2 / 0-Ag14, 293 cells, HeLa cells, and the like, which are readily available, for example, from the American Type Culture Collection, Manassas, Va. (www.atcc.org). Preferred host cells include cells of lymphoid origin, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession No. CRL-1580) and SP2 / 0-Ag14 cells (ATCC Accession No. CRL-1851). In a preferred embodiment, the recombinant cell is a P3X63Ab8.653 or SP2 / 0-Ag14 cell.

[0169] Expression vectors for these cells can include one or more of the following expression control sequences, including, but not limited to, an origin of replication, a promoter (e.g., the late or early SV40 promoter, a CMV promoter (U.S. Pat. Nos. 5,168,062, 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alpha promoter (U.S. Pat. No. 5,266,491), at least one human promoter, enhancer, and / or processing information site, such as a ribosome binding site, an RNA splice site, a polyadenylation site (e.g., the SV40 large T Ag polyA addition site), and a transcription terminator sequence. See, e.g., Ausubel et al., supra; Sambrook et al., supra. Other cells useful for producing the nucleic acids or proteins of the disclosure are known and / or available from, for example, the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or other known or commercial sources.

[0170] When eukaryotic host cells are used, a polyadenylation or transcription terminator sequence is typically incorporated into the vector. An example of a terminator sequence is the polyadenylation sequence derived from the bovine growth hormone gene. A sequence for accurate splicing of the transcript can also be included. An example of a splicing sequence is the VP1 intron derived from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). Furthermore, as is known in the art, gene sequences for regulating replication in host cells can be incorporated into the vector.

[0171] scFv purification

[0172] scFvs can be recovered and purified from recombinant cell cultures by well-known methods, including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), e.g., chapters 1, 4, 6, 8, 9, and 10, each of which is incorporated herein by reference in its entirety.

[0173] The scFvs of the present disclosure include purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from prokaryotic or eukaryotic hosts, such as E. coli, yeast, higher plants, insect cells, and mammalian cells. Depending on the host used in a recombinant production procedure, the protein scaffolds of the present disclosure can be glycosylated or non-glycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, sections 17.37-17.42; Ausubel, supra, chapters 10, 12, 13, 16, 18, and 20; and Colligan, Protein Science, supra, chapters 12-14, all of which are incorporated herein by reference in their entireties.

[0174] Amino acid code

[0175] The amino acids comprising the protein scaffolds of the present disclosure are often abbreviated. The designation of an amino acid can be indicated by its single-letter code, its three-letter code, its name, or its three-nucleotide codon, as is well understood in the art (see Alberts, B. et al., Molecular Biology of the Cell, 3rd ed., Garland Publishing, New York, 1994). The protein scaffolds of the present disclosure can contain one or more amino acid substitutions, deletions, or additions due to natural mutations and / or human manipulation, as described herein. Amino acids in the protein scaffolds of the present disclosure that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra, Chapters 8 and 15; Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces a single alanine mutation at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, including, but not limited to, at least one neutralizing activity. Sites important for protein backbone binding can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de Vos, et al., Science 255:306-312 (1992)).

[0176] As will be appreciated by those skilled in the art, the present disclosure includes at least one biologically active protein scaffold of the present disclosure. A biologically active protein scaffold has a specific activity of at least 20%, 30%, or 40%, preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95%-99% or more of the specific activity of a natural (non-synthetic), endogenous, or related known protein scaffold. Methods for assaying and quantifying the degree of enzymatic activity and substrate specificity are well known to those skilled in the art.

[0177] In another aspect, the present disclosure relates to protein scaffolds and fragments described herein that have been modified by the covalent attachment of organic moieties. Such modifications can produce protein scaffold fragments with improved pharmacokinetic properties (e.g., increased in vivo serum half-life). The organic moieties can be linear or branched hydrophilic polymer groups, fatty acid groups, or fatty acid ester groups. In certain aspects, the hydrophilic polymer groups can have a molecular weight of about 800 to about 120,000 daltons and can be polyalkane glycols (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymers, amino acid polymers, or polyvinylpyrrolidone, and the fatty acid or fatty acid ester groups can contain about 8 to about 40 carbon atoms.

[0178] The modified protein scaffolds and fragments of the present disclosure can include one or more organic moieties covalently attached directly or indirectly to an antibody. Each organic moiety attached to a protein scaffold or fragment of the present disclosure can independently be a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" encompasses monocarboxylic and dicarboxylic acids. A "hydrophilic polymer group," as the term is used herein, refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than in octane. Thus, protein scaffolds modified by the covalent attachment of polylysine are included in the present disclosure. Hydrophilic polymers suitable for modifying the protein backbone of the present disclosure can be linear or branched, and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartic acid, etc.), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide, etc.), and polyvinylpyrrolidone. Preferably, the hydrophilic polymer modifying the protein backbone of the present disclosure has a molecular weight of about 800 to about 150,000 daltons as a single molecular entity. For example, PEG5000 and PEG20,000 (the subscripts indicate the average molecular weight of the polymer in daltons) can be used. The hydrophilic polymer group can be substituted with one to about six alkyl, fatty acid, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared by suitable methods. For example, a polymer containing an amine group can be attached to the carboxylate of a fatty acid or fatty acid ester, and an activated carboxylate (e.g., activated with N,N-carbonyldiimidazole) on the fatty acid or fatty acid ester can be attached to a hydroxyl group on the polymer.

[0179] Fatty acids and fatty acid esters suitable for modifying the protein backbone of the present disclosure may be saturated or contain one or more unsaturated units. Fatty acids suitable for modifying the protein backbone of the present disclosure include, for example, n-dodecanoate (C12, laurate), n-tetradecanoate (C14, myristic acid), n-octadecanoate (C18, stearate), n-eicosanoate (C20, arachidate), n-docosanoate (C22, behenate), n-triacontanoate (C30), n-tetracontanoate (C40), cis-Δ9-octadecanoate (C18, oleic acid), all-cis-Δ5,8,11,14-eicosatetraenoate (C20, arachidonic acid), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid esters include monoesters of dicarboxylic acids containing a straight or branched chain lower alkyl group, which can contain from 1 to about 12, preferably from 1 to about 6, carbon atoms.

[0180] Modified protein scaffolds and fragments can be prepared using suitable methods, for example, by reaction with one or more modifying agents. "Modifying agent," as the term is used herein, refers to a suitable organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester) containing an activating group. An "activating group" is a chemical moiety or functional group that can react with a second chemical group under appropriate conditions, thereby forming a covalent bond between the modifying agent and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl ester (NHS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. Aldehyde functional groups can be attached to amine- or hydrazide-containing molecules, and azide groups can react with trivalent phosphorus groups to form phosphoramidate or phosphorimide linkages. Suitable methods for introducing activating groups into molecules are known in the art (see, for example, Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). Activating groups can be attached to organic groups (e.g., hydrophilic polymers, fatty acids, fatty acid esters) directly or via a linker moiety, such as a divalent C1-C12 group in which one or more carbon atoms can be replaced with a heteroatom such as oxygen, nitrogen, or sulfur. Suitable linker moieties include, for example, tetraethylene glycol, -(CH2)3-, -NH-(CH2)6-NH-, -(CH2)2-NH-, and -CH2-O-CH2-CH2-O-CH2-CH2-O-CH-NH-. A modifying agent containing a linker moiety can be generated, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate.The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine, which can be coupled to another carboxylate as described, or reacted with maleic anhydride and the resulting product cyclized to generate an activated maleimide derivative of a fatty acid (see, e.g., WO 92 / 16221 to Thompson et al., the teachings of which are incorporated herein by reference in their entirety).

[0181] The modified protein scaffolds of the present disclosure can be produced by reacting a protein scaffold or fragment with a modifying agent. For example, an amine-reactive modifying agent, such as an NHS ester of PEG, can be used to attach organic moieties to the protein scaffold in a non-site-specific manner. Modified protein scaffolds and fragments containing organic moieties attached to specific sites of the protein scaffold of the present disclosure can be prepared using suitable methods, such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1):59-68 (1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)) and the methods described in Hermanson, G.T., Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996).

[0182] Cells and modified cells of the present disclosure

[0183] The cells and modified cells of the present disclosure can be mammalian cells. The cells and modified cells are human cells. The cells and modified cells of the present disclosure can be immune cells. The immune cells of the present disclosure can be iPSCs, lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T cells), stem memory T cells (TSCM cells), central memory T cells (T CM ), stem cell-like T cells, B lymphocytes (B cells), antigen presenting cells (APCs), cytokine-induced killer (CIK) cells, myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, macrophages, platelets, erythrocytes, red blood cells (RBCs), megakaryocytes, or osteoclasts.

[0184] Immune progenitor cells can include any cell that can differentiate into one or more types of immune cells. Immune progenitor cells can include multipotent stem cells that can self-renew and develop into immune cells. Immune progenitor cells can include hematopoietic stem cells (HSCs) or their progeny. Immune progenitor cells can include progenitor cells that can develop into immune cells. Immune progenitor cells can include hematopoietic progenitor cells (HPCs).

[0185] Hematopoietic stem cells (HSCs) are multipotent, self-renewing cells. All differentiated blood cells from the lymphoid and myeloid lineages arise from HSCs. HSCs can be found in adult bone marrow, peripheral blood, mobilized peripheral blood, peritoneal dialysis effluent, and umbilical cord blood.

[0186] HSCs can be isolated or derived from primary or cultured stem cells. HSCs can be isolated or derived from embryonic stem cells, multipotent stem cells, pluripotent stem cells, adult stem cells, or induced pluripotent stem cells (iPSCs).

[0187] Immune progenitor cells can include HSCs or HSC progeny, including, but not limited to, multipotent stem cells, lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T cells), B lymphocytes (B cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, and macrophages.

[0188] HSCs generated by the disclosed methods are isolated or derived from adult stem cells and are committed to a single lineage, but can retain the characteristics of "primitive" stem cells that share the characteristics of embryonic stem cells. For example, "primitive" HSCs generated by the disclosed methods retain their "stemness" after division and do not differentiate. As a result, as adoptive cell therapy, "primitive" HSCs generated by the disclosed methods not only replenish their numbers but also expand in vivo. "Primitive" HSCs generated by the disclosed methods can be therapeutically effective when administered as a single dose.

[0189] Primitive HSCs can be CD34+. Primitive HSCs can be CD34+ and CD38-. Primitive HSCs can be CD34+, CD38- and CD90+. Primitive HSCs can be CD34+, CD38-, CD90+ and CD45RA-. Primitive HSCs can be CD34+, CD38-, CD90+, CD45RA- and CD49f+. Primitive HSCs can be CD34+, CD38-, CD90+, CD45RA- and CD49f+.

[0190] Primitive HSCs, HSCs, and / or HSC progeny can be modified to express exogenous sequences (e.g., chimeric antigen receptors or therapeutic proteins) according to the disclosed methods. The modified primitive HSCs, modified HSCs, and / or modified HSC progeny can be differentiated to generate modified immune cells, such as, but not limited to, modified T cells, modified natural killer cells, and / or modified B cells.

[0191] The modified immune cells or immune progenitor cells can be NK cells. NK cells can be cytotoxic lymphocytes that differentiate from lymphoid progenitor cells. The modified NK cells can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. In some embodiments, the non-activated NK cells are derived from CD3-depleted leukapheresis (containing CD14 / CD19 / CD56+ cells).

[0192] The modified immune cells or immune precursor cells can be B cells. B cells are a type of lymphocyte that express a B cell receptor on the cell surface. The B cell receptor binds to a specific antigen. The modified B cells can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0193] The modified T cells of the present disclosure can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. Unlike conventional biologics and chemotherapeutic agents, the disclosed modified T cells have the ability to rapidly replicate upon antigen recognition, thereby potentially eliminating the need for repeat treatments. To accomplish this, in some embodiments, the modified T cells not only promote an initial response but also survive in the patient as a stable population of viable memory T cells to prevent potential relapse. Alternatively, in some aspects, the modified T cells do not survive in the patient when undesired.

[0194] Intensive efforts are being made to develop antigen receptor molecules that do not cause T cell exhaustion through antigen-independent (tonic) signaling, as well as to develop molecules that stimulate early memory T cells, especially stem cell memory (T SCM The present disclosure focuses on the development of engineered T cell products containing central memory (T) or stem cell-like T cells. CM ) T cells or T CM -like cells, effector memory (T EM ) and effector T cells (T E ), thereby leading to better tumor eradication and long-term engraftment of engineered T cells. A linear pathway of differentiation leads to the differentiation of these cells, i.e., naive T cells (T N )>T SCM >T CM >T EM >T E >T TE where T N is T SCM directly, and then T CMThe parent progenitor cells that directly give rise to T cells, etc. The T cell compositions of the present disclosure can include one or more of each parent T cell subset, SCM cells are the most abundant (e.g., T SCM >T CM >T EM >T E >T TE ).

[0195] Immune cell precursors include early memory T cells, stem cell-like T cells, and naive T cells (T N ), T SCM , T CM , T EM , T E , or T TE The immune cell precursors can be primitive HSCs, HSCs, or HSC progeny cells of the present disclosure. The immune cells can be early memory T cells, stem cell-like T cells, naive T cells (T N ), T SCM , T CM , T EM , T E , or T TE It could be.

[0196] Modified Cells of the Present Disclosure

[0197] The methods of the disclosure (e.g., using mutant Cas-CLOVER compositions) can modify and / or generate populations of modified immune cells, where at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least In some embodiments, 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% comprise a transgene or a sequence encoding a transgene, and the modified cells have not been subjected to an enrichment protocol. In some embodiments, the modified cells are further enriched using an enrichment protocol.

[0198] The disclosed methods (e.g., using mutant Cas-CLOVER compositions) can modify and / or generate a population of modified cells that have modifications in target sequences at selected sites in the genome. In some embodiments, the disclosed methods can generate about a 1.1-fold to about a 100-fold greater population of modified cells that have modifications in target sequences at selected sites in the genome compared to the number of modified cells not generated using the disclosed methods (e.g., using wild-type Cas-CLOVER or using a CRISPR / Cas9 system).

[0199] In some embodiments, the methods of the disclosure provide an increase in the number of engineered cells by about 1-fold to about 2-fold, about 2-fold to about 3-fold, about 3-fold to about 4-fold, about 4-fold to about 5-fold, about 5-fold to about 6-fold, about 6-fold to about 7-fold, about 7-fold to about 8-fold, about 8-fold to about 9-fold, about 9-fold to about 10-fold, about 10-fold to about 15-fold, about 15-fold to about 20-fold compared to the number of engineered cells not generated using the methods of the disclosure (e.g., using wild-type Cas-CLOVER or using a CRISPR / Cas9 system). It is possible to generate a population of modified cells having modifications in target sequences at selected sites of the genome that are about 20 to about 25 times larger, about 25 to about 30 times larger, about 30 to about 35 times larger, about 35 to about 40 times larger, about 40 to about 45 times larger, about 45 to about 50 times larger, about 50 to about 65 times larger, about 65 to about 70 times larger, about 70 to about 75 times larger, about 75 to about 80 times larger, about 80 to about 85 times larger, about 85 to about 90 times larger, about 90 to about 95 times larger, or about 95 to about 100 times larger. In some embodiments, the methods of the disclosure (e.g., using the Cas-CLOVER system) can generate about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold greater population of modified cells having modifications in target sequences at selected sites in the genome compared to a population of modified cells (e.g., immune cells or hepatocytes) not generated using the methods of the disclosure (e.g., using the CRISPR / Cas9 system). In some embodiments, the methods of the disclosure (e.g., using the Cas-CLOVER system) result in an increase in the number of engineered cells (e.g., immune cells or hepatocytes) of about 1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3.1-fold, about 3.1-fold, about 3.2-fold, about 3.3-fold, about 3.4-fold, about 3.5-fold, about 3.6-fold, about 3.7-fold, about 3.8-fold, about 3.9-fold, about 4.0-fold, about 4.1-fold, about 4.2-fold, about 4.3-fold, about 4.4-fold, about 4.5-fold, about 4.6-fold, about 4.7-fold, about 4.8-fold, about 4.9-fold, about 5.0-fold, about 5.1-fold, about 5.2-fold, about 5.3-fold, about 5.4-fold, about 5.5-fold, about 5.6-fold, about 5.7-fold, about 5.8-fold, about 5.9-fold, about 6.0-fold, about 6.1-fold, about 6.2-fold, about 6.3-fold, about 6.4-fold, about 6.5-fold, about 6.6-fold, about 6.7-fold, about 6.8-fold, about A population of modified cells can be generated that has modifications in target sequences at selected sites in the genome that are 3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3-fold, about 3.1-fold, about 3.2-fold, about 3.3-fold, about 3.4-fold, about 3.5-fold, about 3.6-fold, about 3.7-fold, about 3.8-fold, about 3.9-fold, about 4-fold, about 4.1-fold, about 4.2-fold, about 4.3-fold, about 4.4-fold, about 4.5-fold, about 4.6-fold, about 4.7-fold, about 4.8-fold, about 4.9-fold, or about 5-fold larger.

[0200] The disclosed methods (e.g., using mutant Cas-CLOVER compositions) can modify and / or generate a population of modified cells that have a transgene or a sequence encoding the transgene at a selected site in their genome. In some embodiments, the disclosed methods can generate about 1-fold to about 2-fold, about 2-fold to about 3-fold, about 3-fold to about 4-fold, about 4-fold to about 5-fold, about 5-fold to about 6-fold, about 6-fold to about 7-fold, about 7-fold to about 8-fold, about 8-fold to about 9-fold, or about 9-fold to about 10-fold greater population of modified cells that have a transgene at a selected site in their genome compared to the number of modified cells that have not been subjected to the disclosed methods (e.g., using wild-type Cas-CLOVER or using a CRISPR / Cas9 system). In some embodiments, the methods of the disclosure (e.g., using mutant Cas-CLOVER compositions) can produce about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold greater population of modified cells having a transgene at a selected site in the genome compared to a population of modified cells that have not been subjected to the methods of the disclosure (e.g., using wild-type Cas-CLOVER or using a CRISPR / Cas9 system).

[0201] The disclosed methods (e.g., using mutant Cas-CLOVER compositions) can modify and / or generate populations of engineered cells (e.g., immune cells or hepatocytes) that have a transgene or a sequence encoding the transgene at a selected site in their genome. In some embodiments, the disclosed methods (e.g., using mutant Cas-CLOVER compositions) can generate a population of viable engineered cells that is about 1-fold to about 2-fold, about 2-fold to about 3-fold, about 3-fold to about 4-fold, about 4-fold to about 5-fold, about 5-fold to about 6-fold, about 6-fold to about 7-fold, about 7-fold to about 8-fold, about 8-fold to about 9-fold, or about 9-fold to about 10-fold greater than the number of engineered cells that have not been subjected to the disclosed methods (e.g., using wild-type Cas-CLOVER or using a CRISPR / Cas9 system). In some embodiments, the methods of the disclosure (e.g., using mutant Cas-CLOVER compositions) can generate a population of viable modified cells that is about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold greater than a population of modified cells that have not been subjected to the methods of the disclosure (e.g., using wild-type Cas-CLOVER or using a CRISPR / Cas9 system).

[0202] The methods of the disclosure can modify and / or generate a population of modified cells (e.g., immune cells or hepatocytes), where at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least In some embodiments, 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% comprise a transgene or a sequence encoding a transgene, and the modified cells have not been subjected to an enrichment protocol. In some embodiments, the modified cells are further enriched using an enrichment protocol.

[0203] a plurality of modified cells (e.g., immune cells or hepatocytes) of the population comprise a transgene or a sequence encoding the transgene, and at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least In some embodiments, 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% comprise a transgene or a sequence encoding a transgene, and the modified cells have not been subjected to an enrichment protocol. In some embodiments, the modified cells are further enriched using an enrichment protocol.

[0204] Compositions and methods for generating and / or expanding immune cells or immune progenitor cells (e.g., the disclosed modified immune cells), as well as buffers for maintaining or enhancing the level of cell viability and / or stem-like phenotype of immune cells or immune progenitor cells (e.g., the disclosed modified immune cells), are disclosed elsewhere herein.

[0205] The cells and engineered immune cells of the present disclosure can be autologous or allogeneic cells. Allogeneic cells are engineered to prevent adverse reactions to engraftment after administration to a subject. Allogeneic cells can be any type of cell. Allogeneic cells can be stem cells or can be derived from stem cells. Allogeneic cells can be differentiated somatic cells.

[0206] Methods for expressing chimeric antigen receptors

[0207] The present disclosure provides methods for expressing a CAR on the surface of a cell, the methods including: (a) obtaining a cell population; (b) contacting the cell population with a composition comprising a CAR or a sequence encoding a CAR under conditions sufficient to translocate the CAR across the cell membrane of at least one cell in the cell population, thereby producing a modified cell population; (c) culturing the modified cell population under conditions suitable for incorporation of the sequence encoding the CAR; and (d) expanding and / or selecting at least one cell from the modified cell population that expresses the CAR on its cell surface.

[0208] In some embodiments, the cell population can comprise leukocytes and / or CD4+ and CD8+ leukocytes. The cell population can comprise CD4+ and CD8+ leukocytes at an optimized ratio. The optimized ratio of CD4+ and CD8+ leukocytes does not naturally occur in vivo. The cell population can comprise tumor cells.

[0209] In some embodiments, the conditions sufficient to translocate the CAR or sequence encoding the CAR, transposon, or vector across the cell membrane of at least one cell in the cell population comprise at least one of the following: application of one or more electrical pulses at a specified voltage, a buffer, and one or more additional factors. In some embodiments, the conditions suitable for integration of the sequence encoding the CAR comprise at least one of a buffer and one or more additional factors.

[0210] The buffer can include PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, human T cell nucleofection buffer, or any combination thereof. The one or more additional factors can include (a) recombinant human cytokines, chemokines, interleukins, or any combination thereof, (b) salts, minerals, metabolites, or any combination thereof, (c) cell culture media, (d) inhibitors of cellular DNA sensing, metabolic, differentiation, signal transduction, one or more apoptotic pathways, or combinations thereof, (e) one or more nucleic acid modifying or stabilizing reagents. The recombinant human cytokine, chemokine, interleukin, or any combination thereof may be IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN-gamma , IL-1alpha / IL-1F1, IL-1beta / IL-1F2, IL-12p70, IL-12 / IL-35p35, IL-13, IL-17 / IL-17A, IL-17A / F heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32beta, IL-32gamma, IL-33, LAP (TGF-beta 1), lymphotoxin-alpha / TNF-beta, TGF-beta, TNF-alpha, TRANCE / TNFSF11 / RANK L, or any combination thereof.The salts, minerals, metabolites, or any combination thereof, can include HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS / FCS, human serum, serum replacers, antibiotics, pH adjusters, Earle's salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, Nucleofector PLUS Supplement, KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca(NO3)2, Tris / HCl, K2HPO4, KH2PO4, polyethyleneimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, crown-5, or any combination thereof. The cell culture medium can include PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC Medium, CTS OpTimizer T Cell Expansion SFM, TexMACS Medium, PRIME-XV T Cell Expansion Medium, ImmunoCult-XF T Cell Expansion Medium, or any combination thereof. Inhibitors of cellular DNA sensing, metabolism, differentiation, signal transduction, and / or one or more apoptosis pathways, or combinations thereof, include inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase-1, Pro-IL1B, PI3K, Akt, Wnt3A, glycogen synthase kinase-3β (GSK-3β) (e.g., TWS119), or any combination thereof. Examples of such inhibitors include bafilomycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK, or any combination thereof.Reagents that modify or stabilize one or more nucleic acids include pH adjusters, DNA binding proteins, lipids, phospholipids, CaPO4, net neutral charge DNA binding peptides with or without NLS sequences, TREX1 enzyme, or any combination thereof.

[0211] The expansion and selection steps can be performed simultaneously or sequentially. Expansion can occur prior to selection. Expansion can occur after selection, optionally followed by a further (i.e., second) round of selection. Simultaneous expansion and selection can occur simultaneously. The expansion and / or selection steps can be performed over a period of 10-14 days, including the endpoint.

[0212] The expansion may include contacting at least one cell of the modified cell population with an antigen to stimulate the at least one cell via the CAR, thereby generating an expanded cell population. The antigen may be presented on the surface of a substrate. The substrate may have any form, including, but not limited to, a surface, a well, beads, or a plurality thereof, and a matrix. The substrate may further include a paramagnetic or magnetic component. The antigen may be presented on the surface of a substrate, where the substrate is a magnetic bead, and a magnet can be used to remove or separate the magnetic bead from the modified and expanded cell population. The antigen may be presented on the surface of a cell or an artificial antigen-presenting cell. Artificial antigen-presenting cells may include, but are not limited to, tumor cells and stem cells.

[0213] In some embodiments where the transposon or vector comprises a selection gene, the selection step comprises contacting at least one cell of the modified cell population with a compound to which the selection gene confers resistance, thereby identifying cells that express the selection gene as surviving the selection and identifying cells that do not express the selection gene as not surviving the selection step.

[0214] The present disclosure provides compositions comprising the modified, expanded, and selected cell populations of the methods described herein.

[0215] A more detailed description of methods for expressing a CAR on the surface of a cell is disclosed in WO 2019 / 049816 and WO 2019 / 049816.

[0216] The present disclosure provides a cell or cell population comprising a composition comprising: (a) an inducible transgene construct comprising a sequence encoding an inducible promoter and a sequence encoding a transgene; and (b) a receptor construct comprising a sequence encoding a constitutive promoter and a sequence encoding an exogenous receptor, such as a CAR; wherein, upon integration of the construct of (a) and the construct of (b) into the genomic sequence of the cell, the exogenous receptor is expressed, and upon binding to a ligand or antigen, the exogenous receptor transduces an intracellular signal that directly or indirectly targets the inducible promoter that regulates expression of the inducible transgene (a), thereby modifying gene expression.

[0217] The compositions can alter gene expression by reducing gene expression. The compositions can alter gene expression by temporarily altering gene expression (e.g., during the period when a ligand binds to an exogenous receptor). The compositions can alter gene expression acutely (e.g., when a ligand reversibly binds to an exogenous receptor). The compositions can alter gene expression chronically (e.g., when a ligand irreversibly binds to an exogenous receptor).

[0218] Exogenous receptors can include endogenous receptors with respect to the genomic sequence of a cell. Exemplary receptors include, but are not limited to, intracellular receptors, cell surface receptors, transmembrane receptors, ligand-gated ion channels, and G-protein coupled receptors.

[0219] The exogenous receptor can include a non-naturally occurring receptor. The non-naturally occurring receptor can be a synthetic, modified, recombinant, mutant, or chimeric receptor. The non-naturally occurring receptor can include one or more sequences isolated or derived from a T cell receptor (TCR). The non-naturally occurring receptor can include one or more sequences isolated or derived from a scaffold protein. In some embodiments, such as embodiments in which the non-naturally occurring receptor does not include a transmembrane domain, the non-naturally occurring receptor interacts with a second transmembrane, membrane-bound, and / or intracellular receptor, which transduce an intracellular signal after contact with the non-naturally occurring receptor. The non-naturally occurring receptor can include a transmembrane domain. The non-naturally occurring receptor can interact with an intracellular receptor that transduce an intracellular signal. The non-naturally occurring receptor can include an intracellular signaling domain. The non-naturally occurring receptor can be a chimeric ligand receptor (CLR). The CLR can be a chimeric antigen receptor (CAR).

[0220] The sequence encoding the inducible promoter includes a sequence encoding the NF-B promoter, a sequence encoding an interferon (IFN) promoter, or a sequence encoding an interleukin-2 promoter. In some embodiments, the IFN promoter is an IFNγ promoter. The inducible promoter can be isolated or derived from a cytokine or chemokine promoter. The cytokine or chemokine can include IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, IL17A / F, IL21, IL22, IL23, transforming growth factor beta (TGFβ), colony-stimulating factor 2 (GM-CSF), interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), LTα, perforin, granzyme C (Gzmc), granzyme B (Gzmb), CC-C motif chemokine ligand 5 (CCL5), CC-C motif chemokine ligand 4 (Ccl4), CC-C motif chemokine ligand 3 (Ccl3), XC motif chemokine ligand 1 (Xcl1), or LIF interleukin 6 family cytokine (Lif).

[0221] Inducible promoters can be isolated from or derived from promoters of genes including surface proteins involved in cell differentiation, activation, exhaustion, and function, hi some embodiments, the genes include CD69, CD71, CTLA4, PD-1, TIGIT, LAG3, TIM-3, GITR, MHCII, COX-2, FASL, or 4-1BB.

[0222] The inducible promoter can be isolated from or derived from the promoters of genes involved in CD4+ metabolism and differentiation, such as Nr4a1, Nr4a3, Tnfrsf9(4-1BB), Sema7a, Zfp3612, Gadd45b, Dusp5, Dusp6, and Neto2.

[0223] In some embodiments, the inducible transgene construct comprises or drives expression of downstream signaling components of inhibitory checkpoint signals, transcription factors, cytokines or cytokine receptors, chemokines or chemokine receptors, cell death or apoptosis receptors / ligands, metabolic sensing molecules, proteins that confer sensitivity to cancer therapy, and oncogenes or tumor suppressor genes, non-limiting examples of which are disclosed in WO 2019 / 173636 and WO 2019 / 049816.

[0224] The present disclosure provides methods for generating a population of engineered T cells, the methods comprising, consisting essentially of, or consisting of introducing a composition comprising a CAR of the present disclosure or a sequence encoding same into a plurality of primary human T cells to generate a plurality of engineered T cells. The present disclosure provides compositions comprising a population of engineered T cells generated by the methods. In some embodiments, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population express a CAR of the present disclosure.

[0225] Transposon and Vector Compositions

[0226] The present disclosure provides compositions and methods for delivering a therapeutic protein (an antibody (e.g., an scFv) or a CAR (e.g., comprising an scFv)) to a cell or cell population. Non-limiting examples of compositions for delivering the compositions of the present disclosure to a cell or cell population include a transposon or a vector. Thus, the present disclosure provides a transposon comprising a therapeutic protein (an antibody (e.g., an scFv) or a CAR (e.g., comprising an scFv)) or a vector comprising a therapeutic protein (an antibody (e.g., an scFv) or a CAR (e.g., comprising an scFv)).

[0227] A transposon comprising a Therapeutic protein of the present disclosure or a vector comprising a Therapeutic protein of the present disclosure can further comprise a sequence encoding an inducible pro-apoptotic polypeptide. Alternatively or additionally, one transposon or one vector can comprise a Therapeutic protein of the present disclosure, and another transposon or another vector can comprise a sequence encoding an inducible pro-apoptotic polypeptide of the present disclosure. Inducible pro-apoptotic polypeptides are described in more detail herein.

[0228] A transposon comprising a Therapeutic protein of the present disclosure or a vector comprising a Therapeutic protein of the present disclosure can further comprise a sequence encoding a chimeric stimulating receptor (CSR). Alternatively or additionally, one transposon or one vector can comprise a CAR of the present disclosure, and another transposon or another vector can comprise a sequence encoding a CSR of the present disclosure. Chimeric stimulating receptors are described in more detail herein.

[0229] A transposon comprising a Therapeutic protein of the present disclosure or a vector comprising a Therapeutic protein of the present disclosure can further comprise a sequence encoding a recombinant HLA-E polypeptide. Alternatively or additionally, one transposon or one vector can comprise a Therapeutic protein of the present disclosure, and another transposon or another vector can comprise a sequence encoding a recombinant HLA-E polypeptide. Recombinant HLA-E polypeptides are described in more detail herein.

[0230] A transposon comprising a Therapeutic protein of the present disclosure or a vector comprising a Therapeutic protein of the present disclosure can further comprise a selection gene. The selection gene can encode a gene product essential for cell viability and survival. The selection gene can encode a gene product essential for cell viability and survival when challenged by selective cell culture conditions. The selective cell culture conditions can include a compound deleterious to cell viability or survival, and the gene product confers resistance to the compound. Non-limiting examples of selection genes include neo (confers resistance to neomycin), DHFR (encodes dihydrofolate reductase, conferring resistance to methotrexate), TYMS (encodes thymidylate synthetase), MGMT (encodes O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encodes aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (encodes RAD51 Paralog C), GCS (encodes glucosylceramide synthase), NKX2.2 (encodes NK2 homeobox 2), or any combination thereof.

[0231] In a preferred embodiment, the selection gene encodes a DHFR mutein enzyme. The DHFR mutein enzyme comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 37. The DHFR mutein enzyme is encoded by a polynucleotide comprising, consists essentially of, or consists of the nucleic acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. The amino acid sequence of the DHFR mutein enzyme can further comprise a mutation at one or more of positions 80, 113, or 153. The amino acid sequence of the DHFR mutein enzyme can comprise one or more of a phenylalanine (F) or leucine (L) substitution at position 80, a leucine (L) or valine (V) substitution at position 113, and a valine (V) or aspartic acid (D) substitution at position 153.

[0232] A transposon comprising a CAR of the present disclosure or a vector comprising a CAR of the present disclosure can further comprise at least one self-cleaving peptide. For example, the self-cleaving peptide can be located between the CAR (e.g., comprising an scFv) and the inducible pro-apoptotic polypeptide, or the self-cleaving peptide can be located between the CAR (e.g., comprising an scFv) and the protein encoded by the selection gene.

[0233] A transposon comprising a CAR of the present disclosure or a vector comprising a CAR of the present disclosure can further comprise at least two self-cleaving peptides. For example, the first self-cleaving peptide is located upstream or immediately upstream of the CAR, and the second self-cleaving peptide is located downstream or immediately downstream of the CAR. Alternatively, the first self-cleaving peptide and the second self-cleaving peptide are adjacent to the CAR. For example, the first self-cleaving peptide is located upstream or immediately upstream of the inducible pro-apoptotic polypeptide, and the second self-cleaving peptide is located downstream or immediately downstream of the inducible pro-apoptotic polypeptide. Alternatively, the first self-cleaving peptide and the second self-cleaving peptide are adjacent to the inducible pro-apoptotic polypeptide. For example, the first self-cleaving peptide is located upstream or immediately upstream of the protein encoded by the selected gene, and the second self-cleaving peptide is located downstream or immediately downstream of the protein encoded by the selected gene. Alternatively, the first self-cleaving peptide and the second self-cleaving peptide are adjacent to the protein encoded by the selected gene.

[0234] Inducible Pro-Apoptotic Polypeptides

[0235] The inducible pro-apoptotic polypeptides disclosed herein are superior to existing inducible polypeptides because the inducible pro-apoptotic polypeptides of the present disclosure are much less immunogenic. The inducible pro-apoptotic polypeptides are recombinant polypeptides and therefore do not occur in nature. Furthermore, they are engineered to produce inducible pro-apoptotic polypeptides that do not contain non-human sequences that can be recognized as "non-self" by the host human immune system, thereby inducing an immune response in a subject receiving the inducible pro-apoptotic polypeptide, cells containing the inducible pro-apoptotic polypeptide, or compositions comprising the inducible pro-apoptotic polypeptide or cells containing the inducible pro-apoptotic polypeptide.

[0236] The present disclosure provides an inducible pro-apoptotic polypeptide comprising a ligand-binding region, a linker, and a pro-apoptotic peptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction site. In certain embodiments, the ligand-binding region can be a multimeric ligand-binding region. In certain embodiments, the pro-apoptotic peptide is a caspase polypeptide. Non-limiting examples of caspase polypeptides include caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, and caspase 14. Preferably, the caspase polypeptide is a caspase 9 polypeptide. The caspase 9 polypeptide can be a cleaved caspase 9 polypeptide. The inducible pro-apoptotic polypeptide can be non-naturally occurring. When the caspase is caspase 9 or cleaved caspase 9, the inducible pro-apoptotic polypeptide can also be referred to as an "iC9 safety switch."

[0237] The inducible caspase polypeptide can comprise (a) a ligand-binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise non-human sequences. In certain embodiments, the inducible caspase polypeptide comprises (a) a ligand-binding region, (b) a linker, and (c) a cleaved caspase 9 polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise non-human sequences.

[0238] The ligand-binding region can comprise an FK506 binding protein 12 (FKBP12) polypeptide. The amino acid sequence of the ligand-binding region comprising the FK506 binding protein 12 (FKBP12) polypeptide can comprise a modification at position 36 of the sequence. The modification can be a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). The FKBP12 polypeptide can comprise, consist essentially of, or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:40. The FKBP12 polypeptide can be encoded by a polynucleotide comprising or consisting of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:41.

[0239] The linker region can comprise, consist essentially of, or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 42, or the linker region can be encoded by a polynucleotide that comprises or consists of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 43. In some embodiments, the nucleic acid sequence encoding the linker does not include any restriction sites.

[0240] The truncated caspase-9 polypeptide can comprise an amino acid sequence that does not include an arginine (R) at position 87 of the sequence. Alternatively, or additionally, the truncated caspase-9 polypeptide can comprise an amino acid sequence that does not include an alanine (A) at position 282 of the sequence. The truncated caspase-9 polypeptide can comprise, consist essentially of, or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:44, or the truncated caspase-9 polypeptide can be encoded by a polynucleotide that comprises or consists of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:45.

[0241] In particular embodiments, when the polypeptide comprises a cleaved caspase-9 polypeptide, the inducible pro-apoptotic polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:46, or the inducible pro-apoptotic polypeptide is encoded by a polynucleotide that comprises or consists of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:47.

[0242] In particular embodiments, when the polypeptide comprises a cleaved caspase-9 polypeptide, the inducible pro-apoptotic polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:48, or the inducible pro-apoptotic polypeptide is encoded by a polynucleotide that comprises or consists of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:49.

[0243] The inducible pro-apoptotic polypeptide may be expressed in a cell under the transcriptional control of any promoter known in the art that is capable of initiating and / or regulating expression of the inducible pro-apoptotic polypeptide in the cell.

[0244] Activation of inducible pro-apoptotic polypeptides can be achieved, for example, by chemically induced dimerization (CID) mediated by an inducer to generate a conditionally regulated protein or polypeptide. Not only are the pro-apoptotic polypeptides inducible, but the induction of these polypeptides is also reversible by degradation of the labile dimerizer or by administration of a monomeric competitive inhibitor.

[0245] Inducible pro-apoptotic peptides and methods for inducing these peptides are described in detail in U.S. Patent Publication No. 2019 / 0225667 and WO 2018 / 068022.

[0246] Formulation, dosage and administration method

[0247] The present disclosure provides formulations, dosages and methods of administration of the compositions described herein.

[0248] The disclosed compositions and pharmaceutical compositions can further comprise at least one of any suitable auxiliary agent, such as, but not limited to, a diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, etc. Pharmaceutically acceptable auxiliary agents are preferred. Non-limiting examples of such sterile solutions and methods for their preparation are well known in the art, for example, but not limited to, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990 and Physician's Desk Reference, 52nd ed., Medical Economics (Montvale, NJ) 1998. Pharmaceutically acceptable carriers suitable for the mode of administration, solubility, and / or stability of the protein scaffold, fragment, or variant composition can be routinely selected, as known in the art or described herein.

[0249] Non-limiting examples of pharmaceutical excipients and additives suitable for use include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars, such as alditols, aldonic acids, and esterified sugars; and polysaccharides or sugar polymers), which may be present alone or in combination and may comprise 1 to 99.99% by weight or volume, alone or in combination. Non-limiting examples of protein excipients include serum albumins, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acids / protein components that may also function as buffers include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

[0250] Non-limiting examples of carbohydrate excipients suitable for use include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc., disaccharides such as lactose, sucrose, trehalose, cellobiose, etc., polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc., and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myo-inositol, etc. Preferably, the carbohydrate excipient is mannitol, trehalose, and / or raffinose.

[0251] The composition can also contain a buffer or pH adjuster, and typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, and phthalic acid, as well as Tris, tromethamine hydrochloride, and phosphate buffers. Preferred buffers are organic acid salts such as citrate.

[0252] Additionally, the disclosed compositions can include polymeric excipients / additives such as polyvinylpyrrolidone, Ficoll (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates, such as "TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0253] Many known and developed modes can be used to administer a therapeutically effective amount of the compositions or pharmaceutical compositions disclosed herein. Non-limiting examples of administration modes include bolus, buccal, injection, intra-articular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intracavitary, intracerebellar, intraventricular, intracavity, intracervical, intragastric, intrahepatic, intralesional, intramuscular, intramyocardial, intranasal, intraocular, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intravesical, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intratumoral, intravenous, intravesical, oral, parenteral, rectal, sublingual, subcutaneous, transdermal or intravaginal means.

[0254] The compositions of the present disclosure may be used for parenteral administration (subcutaneous, intramuscular, or intravenous) or any other administration, particularly in the form of a liquid solution or suspension, for vaginal or rectal administration, particularly in semi-solid form, for example, but not limited to, creams and suppositories, for buccal or sublingual administration, for example, but not limited to, in the form of tablets or capsules, or intranasally, for example, but not limited to, in the form of powders, nasal sprays, or aerosols or specific medications, or transdermally, for example, but not limited to, with chemical enhancers such as dimethyl sulfoxide to modify skin structure or increase drug concentration in transdermal patches (Junginger, et al. In "Drug Permeation Enhancement;" Hsieh, D.S., Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994) or using oxidizing agents that allow application of protein and peptide-containing formulations to the skin (WO 98 / 53847), or the application of an electric field to generate a temporary transport pathway, such as electroporation, or to increase the mobility of charged drugs through the skin, such as iontophoresis, or the application of ultrasound, such as sonophoresis (U.S. Pat. Nos. 4,309,989, 4,767,402) (the above publications and patents are incorporated herein by reference in their entirety).

[0255] For parenteral administration, any composition disclosed herein may be formulated in the form of a solution, suspension, emulsion, particle, powder, or lyophilized powder in combination with a pharmaceutically acceptable parenteral vehicle, or may be provided separately from the parenteral vehicle. Parenteral formulations may contain common excipients such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, and hydrogenated naphthalenes. Aqueous or oily suspensions for injection can be prepared according to known methods using appropriate emulsifiers or wetting agents and suspending agents. Injectable preparations may be non-toxic, non-oral diluents such as aqueous solutions, sterile injectable solutions, or suspensions in solvents. Usable vehicles or solvents include water, Ringer's solution, and isotonic saline. Sterile fixed oils can be used as common solvents or suspension media. For these purposes, any type of fixed oil and fatty acid, including natural, synthetic, or semisynthetic fatty oils or fatty acids, and natural, synthetic, or semisynthetic mono-, di-, or triglycerides, can be used. Parenteral administration is known in the art and includes, but is not limited to, conventional injection means, gas-pressurized needleless injection devices such as those described in U.S. Pat. No. 5,851,198, and laser perforation devices such as those described in U.S. Pat. No. 5,839,446.

[0256] Formulations for oral administration rely on the co-administration of adjuvants (e.g., resorcinol and non-ionic surfactants, such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether) to artificially increase the permeability of the intestinal wall, and enzyme inhibitors (e.g., pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DFF) and trasylol) to inhibit enzymatic degradation. Formulations for delivering hydrophilic agents, including proteins and protein backbones, and combinations of at least two surfactants intended for oral, buccal, mucosal, nasal, pulmonary, transvaginal or rectal administration are described in U.S. Pat. No. 6,309,663. The active ingredient compound in a solid dosage form for oral administration can be mixed with at least one additive such as sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, and glycerides. These dosage forms can also contain other types of additives, such as inert diluents, lubricants such as magnesium stearate and parabens, preservatives such as sorbic acid, ascorbic acid, and alpha-tocopherol, antioxidants such as cysteine, disintegrants, binders, thickeners, buffers, sweeteners, flavorings, and fragrances.

[0257] Tablets and pills can be further processed into enteric-coated preparations. Liquid preparations for oral administration include emulsions, syrups, elixirs, suspensions, and pharmaceutically acceptable solutions. These preparations can contain inert diluents commonly used in the art, such as water. Liposomes have also been described as drug delivery systems for insulin and heparin (U.S. Pat. No. 4,239,754). More recently, microspheres of artificial polymers of mixed amino acids (proteinoids) have been used to deliver pharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, carrier compounds used for oral delivery of biologically active agents, as described in U.S. Pat. Nos. 5,879,681 and 5,871,753, are known in the art.

[0258] For pulmonary administration, the compositions or pharmaceutical compositions described herein are preferably delivered in particle sizes effective to reach the lower respiratory tract of the lungs or paranasal sinuses. The compositions or pharmaceutical compositions can be delivered by any of a variety of inhalation or nasal devices known in the art for administering therapeutic agents by inhalation. These devices, which can deposit aerosolized formulations in a patient's paranasal sinuses or alveoli, include metered-dose inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers, etc.), dry powder generators, sprayers, and the like. All such devices can employ formulations suitable for aerosolizing the compositions or pharmaceutical compositions described herein. Such aerosols can be composed of solutions (both aqueous and non-aqueous) or solid particles. Furthermore, sprays containing the compositions or pharmaceutical compositions described herein can be generated by forcing a suspension or solution of at least one protein scaffold through a nozzle under pressure. In metered-dose inhalers (MDIs), a propellant, a composition or pharmaceutical composition described herein, and excipients or other additives are contained in a canister as a mixture with a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol containing particles preferably in the size range of less than about 10 μm, preferably about 1 μm to about 5 μm, and most preferably about 2 μm to about 3 μm. A more detailed description of pulmonary administration, formulations, and associated devices is disclosed in WO 2019 / 049816.

[0259] For absorption through mucosal surfaces, the composition comprises an emulsion comprising a plurality of submicron particles, a mucoadhesive polymer, a bioactive peptide, and an aqueous continuous phase, which promotes absorption through mucosal surfaces by achieving mucoadhesion of the emulsion particles (U.S. Pat. No. 5,514,670). Mucosal surfaces suitable for application of the emulsions of the present disclosure include the corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, such as suppositories, can contain excipients such as polyalkylene glycols, petrolatum, cocoa butter, etc. Formulations for nasal administration are solid and may contain excipients such as lactose, or may be aqueous or oily solutions for nasal drops. For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, etc. (U.S. Pat. No. 5,849,695). A more detailed description of mucosal administration and formulations is disclosed in WO 2019 / 049816.

[0260] For transdermal administration, the compositions or pharmaceutical compositions disclosed herein are encapsulated in delivery devices such as liposomes or polymeric nanoparticles, microparticles, microcapsules, or microspheres (collectively referred to as microparticles unless otherwise specified). Some suitable devices are known, such as microparticles made of polyhydroxy acids such as polylactic acid, polyglycolic acid and its copolymers, polyorthoesters, polyanhydrides, and polyphosphazenes, as well as synthetic polymers such as collagen, polyamino acids, albumin, and other proteins, alginates, and other polysaccharides, and combinations thereof (U.S. Patent No. 5,814,599). A more detailed description of transdermal administration, formulations, and suitable devices is disclosed in International Publication No. WO 2019 / 049816.

[0261] It may be desirable to deliver the disclosed compounds to a subject over an extended period of time, for example, from one week to one year, with a single administration. A variety of sustained-release, depot, and implant formulations are available. For example, dosage forms may include pharmaceutically acceptable non-toxic salts of compounds with low solubility in body fluids, such as (a) acid addition salts with polybasic acids, such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or disulfonic acid, polygalacturonic acid, etc.; (b) salts with polyvalent metal cations, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc., or salts with organic cations, such as those formed from N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c) a combination of (a) and (b), such as zinc tannate. Furthermore, the disclosed compounds or preferably relatively insoluble salts, such as those just described, can be formulated into gels suitable for injection, such as aluminum monostearate gels containing sesame oil. Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts, and the like. Another type of sustained-release depot formulation for injection contains the compound or salt dispersed for encapsulation in a slowly degrading, non-toxic, non-antigenic polymer, such as a polylactic acid / polyglycolic acid polymer, as described, for example, in U.S. Pat. No. 3,773,919. The compounds or preferably relatively insoluble salts, such as those described above, can also be formulated into cholesterol-matrix silastic pellets, particularly for use in animals. Additional sustained-release, depot, or implant formulations, such as gas or liquid liposomes, are known in the literature (U.S. Pat. No. 5,770,222 and "Sustained and Controlled Release Drug Delivery Systems," J.R. Robinson ed., Marcel Dekker, Inc., NY, 1978).

[0262] Suitable dosages are well known in the art.See, for example, Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, California (2000); Nursing 2001 Handbook of Drugs, 21st Edition, Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc., Upper Saddle River, NJ. Preferred doses can optionally include about 0.1-99 and / or 100-500 mg / kg / administration, or any range, value, or fraction thereof, or can achieve a serum concentration of about 0.1-5000 μg / ml per single or multiple administrations, or per any range, value, or fraction thereof. Preferred dosage ranges for the compositions or pharmaceutical compositions disclosed herein are from about 1 mg / kg to about 3, about 6, or about 12 mg / kg of subject body weight.

[0263] Alternatively, dosages can vary depending on known factors such as the pharmacodynamic properties of the particular drug and its mode and route of administration, the recipient's age, health, and weight, the nature and severity of symptoms, the type of concurrent treatment, the frequency of treatment, and the desired effect. Typically, the dosage of active ingredient can be about 0.1 to 100 milligrams per kilogram of body weight. A dosage of 0.1 to 50, preferably 0.1 to 10 milligrams per kilogram per administration or sustained-release form is usually effective to achieve the desired results.

[0264] By way of non-limiting example, human or animal treatment can be provided as a single or periodic dose of about 0.1 to 100 mg / kg per day, or any range, value, or fraction thereof, of a composition or pharmaceutical composition disclosed herein, at least once on days 1 to 40, or alternatively or additionally, at least once on weeks 1 to 52, or alternatively or additionally, at least once on years 1 to 20, or any combination thereof, using a single dose, an infusion, or multiple doses.

[0265] Dosage forms suitable for internal administration generally contain from about 0.001 milligrams to about 500 milligrams of active ingredient per unit or container. In these pharmaceutical compositions, the active ingredient is usually present in an amount of from about 0.5 to 99.999% by weight based on the total weight of the composition.

[0266] An effective amount can include an amount of about 0.001 to about 500 mg / kg per single (e.g., bolus), multiple, or continuous administration, or an amount to achieve a serum concentration of 0.01 to 5000 μg / ml per single, multiple, or continuous administration, or any effective range or value therein, as performed and determined using known methods described herein or known in the relevant art.

[0267] In embodiments where the composition administered to a subject in need thereof is a modified cell disclosed herein, the cells are about 1 x 10 3 ~1×10 15 cells, approximately 1 x 10 4 ~1×10 12 cells, approximately 1 x 10 5 ~1×10 10 cells, approximately 1 x 10 6 ~1×10 9 cells, approximately 1 x 10 6 ~1×10 8 cells, approximately 1 x 10 6 ~1×10 7 cells, or approximately 1 x 10 6 ~25×10 6 In one embodiment, the cells are administered in amounts of about 5 x 10 6 ~25×10 6It is administered in cells.

[0268] A more detailed description of the pharmaceutically acceptable excipients, formulations, dosages and methods of administration of the disclosed compositions and pharmaceutical compositions is disclosed in WO 2019 / 049816.

[0269] Methods of Using the Compositions of the Present Disclosure

[0270] The present disclosure provides for the use of the disclosed compositions and pharmaceutical compositions for the treatment of a disease or disorder in a cell, tissue, organ, animal, or subject, as known in the art or as described herein, e.g., by administering or contacting the cell, tissue, organ, animal, or subject with a therapeutically effective amount of the composition or pharmaceutical composition. In one aspect, the subject is a mammal. Preferably, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0271] The present disclosure provides methods for modulating or treating at least one malignant disease or disorder in a cell, tissue, organ, animal, or subject. Preferably, the malignant disease is cancer. Non-limiting examples of malignant diseases or disorders include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B-cell, T-cell, or FAB. These include ALL, acute myeloid leukemia (AML), acute myeloid leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, paraneoplastic syndrome / hypercalcemia of malignancy, solid tumors, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary non-polyposis carcinoma, Hodgkin's lymphoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, metastatic disease, cancer-related bone resorption, and cancer-related bone pain.

[0272] The compositions of the present disclosure may be used to treat a variety of conditions, including, but not limited to, osteopetrosis, Parkinson's disease, Hunter syndrome, sickle cell disease, severe combined immunodeficiency, alpha-mannosidosis, sideroblastic anemia, autosomal recessive hyper-IgE syndrome, primary myelofibrosis, cutaneous vasculitis, X-linked protoporphyria, fucosidosis, Maroteaux-Lamy syndrome, WAS-related disease, chronic granulomatous disease, thalassemia major, hereditary angioedema, hereditary lymphedema, hyper-IgM syndrome, Friedrich ataxia, Charcot-Marie-Tooth disease, phenylketonuria, methylmalonic acidemia, adrenoleukodystrophy, Kugelberg-Welder syndrome, retinitis pigmentosa, hydrocephalus, hereditary sensory and autonomic neuropathy type IV, mucoamyloidosis type III, Corneal dystrophy, erythropoietic protoporphyria, Fabry disease, Werdnig-Hoffmann disease, hypophosphatemia, Coats disease, Fanconi anemia, Niemann-Pick disease, Crigler-Najjar syndrome, hemophilia A, hemophilia B, leukodystrophy, Sandhoff disease, Usher syndrome, Wolman disease, Dupuytren's contracture, Wolfram syndrome, X-linked myotubular myopathy, Canavan disease, Ehler syndrome, epidermolysis bullosa, osteogenesis imperfecta, short bowel syndrome, giant axonal neuropathy, paroxysmal nocturnal hemoglobinuria, Ferrand-McDermid syndrome, retinosis, beta-thalassemia, hypophosphatemia, propionic acidemia, cholesterol ester storage disease, cystinosis, glycogen storage disease type II, Pompe disease, mucopolysaccharidosis (MPS) It can be used to treat diseases or disorders such as Mucopolysaccharidosis Type I (Hurler-Shy Syndrome), Mucopolysaccharidosis Type II (Hunter Syndrome), and Mucopolysaccharidosis Type IV (Morquio).

[0273] The compositions of the present disclosure may be used to treat a disease or disorder through the use of a therapeutic transgene encoding an exogenous nucleic acid sequence or an exogenous amino acid sequence. For specific diseases or disorders, the therapeutic transgene "Disease (Therapeutic Transgene)" may be any of the following: beta-thalassemia (HBB T87Q, BCL11A shRNA, IGF2BP1), sickle cell disease (HBB T87Q, BCL11A shRNA, IGF2BP1), hemophilia A (factor VIII), hemophilia B (factor IX), X-linked severe combined immunodeficiency (interleukin-2 receptor gamma (IL2RG)), hypolymphatic leukemia (tissue-nonspecific alkaline phosphatase (TNAP)), osteopetrosis (TCIRG1), glycogen storage disease type II (Pompe disease) (alpha-glucosidase (GAA)), alpha-galactosidase A deficiency (Fabry disease) (alpha-galactosidase A (GLA)), mucopolysaccharidosis type I (MPS) Mucopolysaccharidosis type I (MPS II) (alpha-L-iduronidase (IDUA)), mucopolysaccharidosis type II (MPS II) (iduronate 2-sulfatase (IDS)), mucopolysaccharidosis type IIIA (MPS IIIA) (sulfoglycosamine sulfohydrolase (SGSH)), mucopolysaccharidosis type IIIB (MPS IIIB) (N-alpha-acetylglucosaminidase (NAGLU)), mucopolysaccharidosis type IV A (MPS IVA) (Morquio) (N-acetylgalactosamine-6-sulfate sulfatase (GALNS)), mucopolysaccharidosis type IV B (MPS IVB) beta-galactosidase (GLB1) (beta-galactosidase (GLB1)), cholesteryl ester storage disease (CESD) (lysosomal acid lipase (LIPA)), cystinosis (cystinosinin lysosomal cystine transporter (CTNS)), X-linked chronic granulomatous disease (X-CGD) (CYBB), Wiskott-Aldrich syndrome (WAS) (WAS), X-linked adrenoleukodystrophy (X-ALD) (ABCD1), metachromatic leukodystrophy (MLD) (ARSA), phenylketonuria (PAH), methylmalonic acidemia (MMUT), propionic acidemia (PCCA, PCCB), retinitis pigmentosa (RPE65), Usher syndrome (MYO7A), and Gaucher disease (GBA).

[0274] In a preferred embodiment, the treatment of a malignant disease or disorder involves adoptive cell therapy. For example, in one aspect, the present disclosure provides engineered cells expressing at least one disclosed antibody (e.g., scFv) and / or a CAR comprising the antibody (e.g., scFv), selected and / or expanded for administration to a subject in need thereof. The engineered cells can be formulated for storage at any temperature, such as room temperature or body temperature. The engineered cells can be formulated for cryopreservation and subsequent thawing. The engineered cells can be formulated in a pharmaceutically acceptable carrier for direct administration to a subject from sterile packaging. The engineered cells can be formulated in a pharmaceutically acceptable carrier with indicators of cell viability and / or CAR expression level to ensure a minimum level of cell function and CAR expression. The engineered cells can be formulated in a pharmaceutically acceptable carrier at a prescribed density with one or more reagents to inhibit further proliferation and / or prevent cell death.

[0275] All may comprise administering an effective amount of any of the compositions or pharmaceutical compositions disclosed herein to a cell, tissue, organ, animal, or subject in need of such modulation, treatment, or therapy. Such methods may optionally further comprise co-administration or combination therapy for treating such disease or disorder, and the administration of any of the compositions or pharmaceutical compositions disclosed herein may further comprise the administration of at least one chemotherapeutic agent (e.g., alkylating agent, mitotic inhibitor, radiopharmaceutical) prior to and / or after the simultaneous administration.

[0276] In some embodiments, after administration, the subject does not develop graft versus host (GvH) and / or host versus graft (HvG). In one embodiment, administration is systemic. Systemic administration can be by any means known in the art and described in detail herein. Preferably, systemic administration is intravenous injection or infusion. In one embodiment, administration is local. Local administration can be by any means known in the art and described in detail herein. Preferably, local administration is by intratumoral, intraspinal, intraventricular, intraocular, or intraosseous injection or infusion.

[0277] In some embodiments, the therapeutically effective dose is a single dose. In some embodiments, the single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 doses, or any number of doses therebetween, produced simultaneously. In some embodiments, when the composition is autologous or allogeneic cells, the dose is sufficient to allow the cells to engraft and / or persist for a sufficient time to treat a disease or disorder.

[0278] In one example, the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a composition comprising an antibody (e.g., an scFv) or a CAR comprising the antibody (e.g., an scFv), wherein the antibody or CAR specifically binds to an antigen on a tumor cell. In embodiments where the composition comprises an engineered cell or cell population, the cell or cell population can be autologous or allogeneic.

[0279] In some aspects of the therapeutic methods described herein, treatment can be modified or terminated. Specifically, in aspects in which the composition used in treatment comprises an inducible pro-apoptotic polypeptide, apoptosis can be selectively induced in cells by contacting the cells with an inducer. Treatment can be modified or terminated, for example, in response to signs of recovery, signs of reduced disease severity / progression, signs of disease remission / halt, and / or the occurrence of adverse events. In some aspects, the method includes administering an inhibitor of the inducer to inhibit the modification of the cell therapy, thereby restoring the function and / or effectiveness of the cell therapy (e.g., if signs or symptoms of disease return or increase in severity and / or if adverse events resolve).

[0280] definition

[0281] As used throughout this disclosure, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a plurality of such methods, reference to "a dose" includes a reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0282] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When particular values ​​are described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range of the particular value.

[0283] The present disclosure provides isolated or substantially purified polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its natural environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material and culture medium when produced by recombinant techniques, and substantially free of chemical precursors and other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide is free of sequences that naturally flank the polynucleotide in the genomic DNA of the organism from which the polynucleotide is derived (i.e., sequences located at the 5' and 3' ends of the polynucleotide (optimally, protein-encoding sequences)). For example, in various embodiments, an isolated polynucleotide contains less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein containing less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When a protein of the disclosure or a biologically active portion thereof is recombinantly produced, optimally, the culture medium contains less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

[0284] The present disclosure provides fragments and variants of the disclosed DNA sequences, as well as proteins encoded by these DNA sequences. As used throughout this disclosure, the term "fragment" refers to a portion of a DNA sequence or a portion of an amino acid sequence, and thus to the protein encoded thereby. Fragments of DNA sequences consisting of a coding sequence may encode protein fragments that retain the biological activity of the native protein, as described herein, and thus retain DNA recognition or binding activity to a target DNA sequence. Alternatively, fragments of DNA sequences useful as hybridization probes generally do not encode proteins that retain biological activity or promoter activity. Thus, fragments of DNA sequences can range from at least about 20, 50, or 100 nucleotides, up to the full-length polynucleotides of the present disclosure.

[0285] Nucleic acids or proteins of the present disclosure can be constructed by a modular approach involving preassembly of monomeric and / or repeating units in a target vector, which can then be assembled into a final destination vector. Polypeptides of the present disclosure can be constructed by a modular approach by preassembling repeating units in a target vector, which can be composed of repeating monomers of the present disclosure, which can then be assembled into a final destination vector. The present disclosure provides polypeptides produced by this method, and nucleic acid sequences encoding these polypeptides. The present disclosure provides host organisms and cells comprising nucleic acid sequences encoding the polypeptides produced by this modular approach.

[0286] The term "binding" refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction (e.g., contacts with phosphate residues in a DNA backbone) need be sequence-specific, as long as the interaction as a whole is sequence-specific.

[0287] The term "comprising" is intended to mean that the compounds, compositions, and methods include the recited elements, but do not exclude others. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements that are essential to the combination for the purpose described. Thus, a composition consisting essentially of components defined herein does not exclude trace amounts of contaminants or inert carriers. "Consisting of" is intended to mean excluding more than trace amounts of other components and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0288] The term "epitope" refers to an antigenic determinant of a polypeptide. An epitope can include three amino acids in a spatial conformation unique to that epitope. Generally, an epitope consists of at least four, five, six, or seven such amino acids, and more usually, at least eight, nine, or ten such amino acids. Methods for determining the spatial conformation of amino acids are known in the art and include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.

[0289] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression includes splicing of the mRNA in a eukaryotic cell.

[0290] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, microRNA, structural RNA, or other types of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs that have been modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins that have been modified, for example, by methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0291] "Modulation" or "regulation" of gene expression refers to a change in the activity of a gene. Modulation of expression includes, but is not limited to, gene activation and gene repression.

[0292] The term "operatively linked" or its synonyms (e.g., "linked operatively") means that two or more molecules are positioned relative to each other so that they can interact in a manner that affects the function attributable to one or both molecules, or a combination thereof.

[0293] Non-covalently linked components, as well as methods for making and using non-covalently linked components, are disclosed. The various components can take a variety of different forms, as described herein. For example, non-covalently linked (i.e., operably linked) proteins can be used to allow for transient interactions, avoiding one or more problems in the art. The ability of non-covalently linked components, such as proteins, to associate and dissociate allows for functional association only, or primarily, under circumstances where such association is required for the desired activity. Linkage need only be for a period of time sufficient to achieve the desired effect.

[0294] A method for targeting a protein to a specific locus in the genome of an organism is disclosed. The method may include providing a DNA localization component and providing an effector molecule, wherein the DNA localization component and the effector molecule can be operably linked via a non-covalent linkage.

[0295] A "target site" or "target sequence" is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind when conditions sufficient for binding are present.

[0296] The term "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked. A description of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid can encompass the complementary strand of a described single strand. Nucleic acids of the present disclosure also encompass substantially identical nucleic acids and their complements that retain the same structure or encode the same protein.

[0297] A probe of the present disclosure may consist of a single-stranded nucleic acid capable of hybridizing to a target sequence under stringent hybridization conditions. Thus, a nucleic acid of the present disclosure may refer to a probe that hybridizes under stringent hybridization conditions.

[0298] The nucleic acids of the present disclosure may be single-stranded or double-stranded. The nucleic acids of the present disclosure may be predominantly single-stranded or may contain double-stranded sequences. The nucleic acids of the present disclosure may be predominantly double-stranded or may contain single-stranded sequences. The nucleic acids of the present disclosure may include genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids of the present disclosure may contain a combination of deoxyribonucleotides and ribonucleotides. The nucleic acids of the present disclosure may contain a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. The nucleic acids of the present disclosure may be synthesized to contain unnatural amino acid modifications. The nucleic acids of the present disclosure may be obtained by chemical synthesis or recombinant methods.

[0299] The nucleic acids of the present disclosure may be non-naturally occurring, either in their entirety or in part. The nucleic acids of the present disclosure may contain one or more mutations, substitutions, deletions, or insertions that are not naturally occurring, and the entire nucleic acid sequence may not be naturally occurring. The nucleic acids of the present disclosure may contain one or more duplicated, inverted, or repeated sequences, such that the sequence is not naturally occurring, and the entire nucleic acid sequence may not be naturally occurring. The nucleic acids of the present disclosure may contain modified, artificial, or synthetic nucleotides that are not naturally occurring, and the entire nucleic acid sequence may not be naturally occurring.

[0300] Given the redundancy in the genetic code, more than one nucleotide sequence may encode a particular protein, and all such nucleotide sequences are contemplated herein.

[0301] As used throughout this disclosure, the term "operably linked" refers to the expression of a gene under the control of a spatially linked promoter. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. Changes in the distance between the promoter and the gene can be accommodated without impairing the function of the promoter.

[0302] As used throughout this disclosure, the term "promoter" refers to a synthetic or naturally occurring molecule capable of conferring, activating, or enhancing expression of a nucleic acid in a cell. A promoter can contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter can also contain distal enhancer or repressor elements, located as far away as thousands of base pairs from the transcription start site. Promoters are derived from viruses, bacteria, fungi, plants, insects, animals, etc. Promoters can constitutively or differentially regulate the expression of genetic components with respect to the cell, tissue or organ in which expression occurs, or the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, EF-1 alpha promoter, CAG promoter, SV40 early promoter or SV40 late promoter, and CMV IE promoter.

[0303] As used throughout this disclosure, the term "substantially complementary" refers to a first sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0304] As used throughout this disclosure, the term "substantially identical" refers to first and second sequences or nucleic acids that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, where the first sequence is substantially complementary to the complement of the second sequence.

[0305] As used throughout this disclosure, the term "variant," when used to describe a nucleic acid, refers to: (i) a portion or fragment of a referenced nucleotide sequence; (ii) a complement of the referenced nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to the referenced nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0306] As used throughout this disclosure, the term "vector" refers to a nucleic acid sequence that includes an origin of replication. Vectors include viral vectors, bacteriophages, bacterial artificial chromosomes, and yeast artificial chromosomes. Vectors can be DNA or RNA vectors. Vectors can be self-replicating extrachromosomal vectors, and are preferably DNA plasmids. Vectors are composed of amino acids and DNA sequences, RNA sequences, or a combination of both DNA and RNA sequences.

[0307] As used throughout this disclosure, the term "variant," when used to describe a peptide or polypeptide, refers to a peptide or polypeptide that differs in amino acid sequence by amino acid insertions, deletions, or conservative substitutions, but retains at least one biological activity. A variant can also refer to a protein having an amino acid sequence substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity.

[0308] Conservative amino acid substitutions, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as typically resulting in minor changes. These minor changes can be identified, in part, by considering the hydropathic index of the amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid takes into account its hydrophobicity and charge. Protein function can be maintained when substituted with an amino acid having a similar hydropathic index. In some embodiments, amino acids with a hydropathic index ±2 are substituted. Amino acid hydrophilicity can also be used to identify substitutions that maintain the biological function of a protein. Considering the hydrophilicity of amino acids in the context of a peptide allows for calculation of the peptide's greatest local average hydrophilicity, a useful index that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101 is incorporated herein by reference in its entirety.

[0309] Substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, such as immunogenicity. Substitutions can be made with amino acids whose hydrophilicity values ​​are within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are affected by the specific side chain of that amino acid. Consistent with this observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, particularly their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0310] As used herein, "conservative" amino acid substitutions may be defined as shown in Tables A, B, or C below. In some embodiments, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides contain conservative substitutions introduced by modifying a polynucleotide encoding a polypeptide of the present disclosure. Amino acids can be classified according to their physical properties and contribution to the secondary and tertiary structure of proteins. A conservative substitution is the replacement of one amino acid with another amino acid with similar properties. Exemplary conservative substitutions are shown in Table A.

[0311] [Table A]

[0312] Alternatively, conservative amino acids are described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, NY (1975), pp. 71-77).

[0313] [Table B]

[0314] Alternatively, exemplary conservative substitutions are shown in Table C.

[0315] [Table C]

[0316] It should be understood that the polypeptides of the present disclosure are intended to include polypeptides having one or more insertions, deletions, substitutions, or any combination thereof, of amino acid residues, as well as polypeptides having modifications other than insertions, deletions, or substitutions of amino acid residues. A polypeptide or nucleic acid of the present disclosure may contain one or more conservative substitutions.

[0317] As used throughout this disclosure, the term "two or more" of the foregoing amino acid substitutions refers to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more of the described amino acid substitutions. The term "two or more" can refer to 2, 3, 4, or 5 of the described amino acid substitutions.

[0318] The polypeptides and proteins of the present disclosure may be non-naturally occurring, either in their entirety or in part. The polypeptides and proteins of the present disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not occur in nature, and the entire amino acid sequence may not occur in nature. The polypeptides and proteins of the present disclosure may contain one or more duplicated, inverted, or repeated sequences, such that the sequence does not occur in nature and the entire amino acid sequence does not occur in nature. The polypeptides and proteins of the present disclosure may contain modified, artificial, or synthetic amino acids that do not occur in nature, and the entire amino acid sequence may not occur in nature.

[0319] As used throughout this disclosure, "sequence identity" can be determined using a standalone executable BLAST engine program (bl2seq) for blasting two sequences, which can be obtained from the National Center for Biotechnology Information (NCBI) ftp site using default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250, which is incorporated herein by reference in its entirety). The term "identical" or "identity," when used in the context of two or more nucleic acid or polypeptide sequences, refers to a specific percentage of residues that are the same over a specific region of each sequence. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a designated region, determining the number of positions where identical residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the designated region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths, or if the alignment produces sequences with one or more offset ends, and the specified comparison region contains only a single sequence, the residues of the single sequence are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identification can be done manually or using computer alignment algorithms such as BLAST and BLAST 2.0.

[0320] As used throughout this disclosure, the term "endogenous" refers to a nucleic acid or protein sequence that is naturally associated with the target gene or host cell into which it is introduced.

[0321] As used throughout this disclosure, the term "exogenous" refers to a nucleic acid or protein sequence that is not naturally associated with the target gene or host cell into which it is introduced, and includes non-naturally occurring multiple copies of a naturally occurring nucleic acid, e.g., a DNA sequence, or a naturally occurring nucleic acid sequence located in a non-naturally occurring genomic location.

[0322] The present disclosure provides methods for introducing a polynucleotide construct containing a DNA sequence into a host cell. By "introducing," it is intended to mean presenting the polynucleotide construct to the cell in such a way that the polynucleotide construct has access to the interior of the host cell. The methods of the present disclosure do not depend on a particular method for introducing the polynucleotide construct into the host cell, but only on the polynucleotide construct having access to the interior of one cell of the host. Methods for introducing polynucleotide constructs into bacteria, plants, fungi, and animals are known in the art, including, but not limited to, stable transformation, transient transformation, and virus-mediated methods.

[0323] As used herein, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, total amount, weight, or length that differs by 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a referenced amount, level, value, number, frequency, percentage, dimension, size, total amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, total amount, weight, or length that is within a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the referenced amount, level, value, number, frequency, percentage, dimension, size, total amount, weight, or length.

[0324] As used herein, the term "substantially" or "essentially" refers to an amount, level, value, number, frequency, percentage, dimension, size, total amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more compared to a referenced amount, level, value, number, frequency, percentage, dimension, size, total amount, weight, or length. In one embodiment, the term "essentially the same" or "substantially the same" refers to a range of about the same amount, level, value, number, frequency, percentage, dimension, size, total amount, weight, or length as the referenced amount, level, value, number, frequency, percentage, dimension, size, total amount, weight, or length.

[0325] As used herein, the terms "substantially free" and "essentially free" are used interchangeably and, when used to describe a composition, such as a cell population or culture medium, refer to a composition that is free of a particular substance, e.g., 95% free, 96% free, 97% free, 98% free, 99% free, or where it is undetectable as measured by conventional means. The term "free" or "essentially free" of a particular component or substance in a composition also means that such component or substance (1) is not included in the composition at any concentration, or (2) is functionally inactive but is included in the composition at low concentrations. A similar meaning can be applied to the term "absence," which refers to the absence of a particular substance or source of a particular substance in a composition.

[0326] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" are understood to mean the inclusion of the stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. In certain embodiments, the terms "include," "has," "contains," and "comprise" are used interchangeably.

[0327] As used herein, "consisting of" means including and limited to what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0328] "Consisting essentially of" means including any elements listed after the phrase, limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are necessary or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or function of the listed elements.

[0329] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0330] The term "ex vivo" generally refers to activities performed outside of an organism, such as experiments or measurements performed in or using living tissue in an artificial environment outside the organism, preferably with minimal alteration of natural conditions. In certain embodiments, "ex vivo" procedures involve live cells or tissues removed from an organism and cultured in a laboratory setting, usually under sterile conditions, typically for a few hours or up to about 24 hours, but depending on the circumstances, for example, up to 48 or 72 hours or longer. In certain embodiments, such tissues or cells can be harvested, frozen, and then thawed for ex vivo therapy. Tissue culture experiments or procedures lasting longer than a few days using live cells or tissues are typically considered "in vitro," although in certain embodiments, the term can be used interchangeably with ex vivo.

[0331] The term "in vivo" generally refers to activities that occur inside a living organism.

[0332] As used herein, the terms "reprogramming" or "dedifferentiation" or "increasing cell potential" or "increasing developmental potential" refer to a method of increasing the potential of a cell or dedifferentiating a cell into a less differentiated state. For example, a cell with increased cell potential has greater developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in an unreprogrammed state. In other words, a reprogrammed cell is a cell that is less differentiated than the same cell in an unreprogrammed state.

[0333] As used herein, the term "induced pluripotent stem cells" or iPSCs means that the stem cells are generated from differentiated adult, neonatal, or fetal cells that have been induced or modified, i.e., reprogrammed, into cells that can differentiate into tissues of all three germ layers: mesodermal, endodermal, and ectodermal. The generated iPSCs do not represent the cells as they are found in nature.

[0334] As used herein, the term "subject" refers to any animal, preferably a human patient, livestock, or other domesticated animal.

[0335] "Pluripotency factor" or "reprogramming factor" refers to an agent that can increase the developmental potential of a cell, alone or in combination with other agents. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of a cell. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0336] "Culture" or "cell culture" refers to the maintenance, growth, and / or differentiation of cells in an in vitro environment. "Cell culture media," "culture media" (in each case singular "medium"), "supplement," and "media supplement" refer to nutritional compositions in which cell cultures are cultivated.

[0337] "Culturing" or "maintaining" refers to the maintenance, propagation (growth) and / or differentiation of tissues or cells outside the body, for example, in sterile plastic (or coated plastic) cell culture dishes or flasks. "Culturing" or "maintenance" may utilize the medium as a source of nutrients, hormones and / or other factors useful for propagating and / or maintaining the cells.

[0338] The terms "hematopoietic stem and progenitor cells," "hematopoietic stem cells," "hematopoietic progenitor cells," or "hematopoietic precursor cells" refer to cells committed to the hematopoietic lineage but capable of further hematopoietic differentiation, including multipotent hematopoietic stem cells (hematopoietic blasts), myeloid progenitor cells, megakaryocytic progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all blood lineages, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid (T cells, B cells, NK cells) lines. As used herein, the term "adult hematopoietic stem cells" refers to CD34+ hematopoietic cells that can give rise to both mature myeloid and lymphoid cell types, such as T cells, NK cells, and B cells. Hematopoietic cells also include various subsets of primitive hematopoietic cells that give rise to primitive erythrocytes, megakaryocytes, and macrophages.

[0339] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to the major type of white blood cell that completes maturation in the thymus and has various roles in the immune system, such as identifying specific foreign antigens in the body and activating and inactivating other immune cells. T cells can be any T cell, such as cultured T cells, e.g., primary T cells, or T cells from cultured T cell lines, e.g., Jurkat, SupT1, etc., or T cells obtained from a mammal. T cells can be CD3+ cells. T cells can be any type of T cell, at any stage of development, including, but not limited to, CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (γδ T cells), etc. Further types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Further types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically engineered T cells, for example, T cells modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells can also be differentiated from stem or progenitor cells.

[0340] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by expression of CD56 or CD16 and the absence of T cell receptor (CD3). As used herein, the terms "adaptive NK cells" and "memory NK cells" are used interchangeably and refer to a subset of NK cells that are phenotypically CD3- and CD56+, express NKG2C and CD57, and optionally express CD16, but lack expression of one or more of the following: PLZF, SYK, FceRy, and EAT-2. In some embodiments, an isolated subpopulation of CD56+ NK cells contains expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A, and DNAM-1. CD56+ can be dark or light expressing.

[0341] As used herein, the term "NKT cells" or "natural killer T cells" refers to CD1d-restricted T cells that express the T cell receptor (TCR). Unlike conventional T cells, which detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by the non-classical MHC molecule, CD1d. Two types of NKT cells are currently recognized. Invariant NKT cells, or type I NKT cells, express a very limited TCR repertoire: a standard α chain (Va24-Ja18 in humans) associated with a limited range of β chains (Vβ11 in humans). A second population of NKT cells, termed non-classical or non-invariant type II NKT cells, exhibits more heterogeneous TCRαβ usage. Type I NKT cells are currently considered suitable for immunotherapy. Adaptive or invariant (type I) NKT cells can be identified by expression of at least one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161 and CD56.

[0342] As used herein, terms such as "isolated" refer to a cell or population of cells that has been separated from its original environment; i.e., the environment of an isolated cell is substantially free of at least one component found in the environment in which the "non-isolated" referenced cell resides. This term includes cells that have been removed from some or all of the components of the cell's natural environment, e.g., tissue, biopsy. This term also includes cells that have been removed from at least one, some, or all of the components of the cell's non-naturally occurring environment, e.g., culture, cell suspension. Thus, an isolated cell is partially or completely separated from at least one component, e.g., other substances, cells, or cell populations, as it is found in nature or as it is grown, stored, or persists in the non-naturally occurring environment. Specific examples of isolated cells include partially pure cells, substantially pure cells, and cells cultured in a non-naturally occurring medium. Isolated cells can be obtained by separating a desired cell or population thereof from other substances or cells in the environment, or by removing one or more other cell populations or subpopulations from the environment. As used herein, terms such as "purify" refer to increasing purity. For example, the purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0343] As used herein, the term "encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules, and the biological properties resulting therefrom, in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids. Thus, a gene encodes a protein if the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0344] A "construct" refers to a polymeric or molecular complex containing a polynucleotide that is delivered to a host cell either in vitro or in vivo. As used herein, a "vector" refers to any nucleic acid construct capable of directing the delivery or transfer of foreign genetic material to a target cell and capable of replication and / or expression in the target cell. As used herein, the term "vector" includes the delivered construct. A vector can be a linear or circular molecule. A vector can be integrating or non-integrating. Major types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, etc.

[0345] "Integration" means that one or more nucleotides of a construct are stably inserted into a cell's genome, i.e., covalently linked to a nucleic acid sequence within the cell's chromosomal DNA. "Targeted integration" means that nucleotides of a construct are inserted into the chromosome or mitochondrial DNA of a cell at a preselected site or "integration site." As used herein, the term "integration" also refers to a process that includes the insertion of one or more exogenous sequences or nucleotides of a construct, with or without deletion of the endogenous sequence or nucleotides at the integration site. If there is a deletion at the insertion site, "integration" can further include replacing the endogenous sequence or deleted nucleotides with one or more inserted nucleotides.

[0346] As used herein, the term "exogenous" is intended to mean that the referenced molecule or referenced activity is introduced into a host cell. The molecule can be introduced, for example, by introducing an encoding nucleic acid into the host genetic material, e.g., by integration into a host chromosome, or as non-chromosomal genetic material such as a plasmid. Thus, when used in reference to expression of an encoding nucleic acid, the term refers to introducing the encoding nucleic acid into a cell in an expressible form. The term "endogenous" refers to a referenced molecule or activity that is present in a host cell. Similarly, when used in reference to expression of an encoding nucleic acid, the term refers to expression of an encoding nucleic acid that is contained within the cell and not exogenously introduced.

[0347] As used herein, a "gene of interest" or a "polynucleotide sequence of interest" is a DNA sequence that is transcribed into RNA and, optionally, translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. Genes or polynucleotides of interest can include, but are not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, a gene of interest can encode an miRNA, shRNA, a naturally occurring polypeptide (i.e., a polypeptide found in nature) or a fragment thereof, a variant polypeptide (i.e., a variant of a naturally occurring polypeptide having less than 100% sequence identity with the naturally occurring polypeptide) or a fragment thereof, an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, etc.

[0348] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The sequence of a polynucleotide is composed of the four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T); if the polynucleotide is RNA, thymine is uracil (U). Polynucleotides can include genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotide also refers to both double-stranded and single-stranded molecules.

[0349] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to molecules having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, the term refers to both short chains, e.g., also commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the art as polypeptides or proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins, among others. Polypeptides include naturally occurring polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0350] "Operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence or functional RNA if it is capable of affecting the expression of that coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in either a sense or antisense orientation.

[0351] As used herein, the term "enhanced therapeutic properties" refers to enhanced therapeutic properties of a cell compared to typical immune cells of the same general cell type. For example, NK cells with "enhanced therapeutic properties" have enhanced, improved, and / or enhanced therapeutic properties compared to typical unmodified and / or naturally occurring NK cells. Therapeutic properties of immune cells include, but are not limited to, cell engraftment, trafficking, homing, survival, self-renewal, persistence, immune response regulation and modulation, survival, and cytotoxicity. Therapeutic properties of immune cells are also demonstrated by expression of antigen-targeting receptors, HLA presentation or lack thereof, resistance to the tumor microenvironment, induction and immunomodulation of bystander immune cells, improved on-target specificity with reduced non-tumor effects, and resistance to treatments such as chemotherapy.

[0352] As used herein, the term "engager" refers to a molecule, e.g., a fusion polypeptide, that can form a link between an immune cell, e.g., a T cell, a NK cell, a NKT cell, a B cell, a macrophage, or a neutrophil, and a tumor cell, and activate the immune cell. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTEs), bispecific killer cell engagers (BiKEs), trispecific killer cell engagers, or multispecific killer cell engagers, or universal engagers that are compatible with multiple immune cell types.

[0353] As used herein, the term "safety switch protein" refers to an engineered protein designed to prevent potential toxicity or other adverse effects of cell therapy. In some examples, expression of the safety switch protein is conditionally controlled to address safety concerns of transplanted engineered cells that have permanently integrated a gene encoding the safety switch protein into their genome. This conditional regulation can be variable and can include control through small molecule-mediated post-translational activation and tissue-specific and / or temporal transcriptional regulation. The safety switch can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional gene regulation, and / or antibody-mediated depletion. In some examples, the safety switch protein is activated by an exogenous molecule, e.g., a prodrug, and upon activation, causes apoptosis and / or cell death of the treated cell. Examples of safety switch proteins include suicide genes, such as, but not limited to, caspase 9 (or caspase 3 or 7), thymidine kinase, cytosine deaminase, B cell CD20, modified EGFR, and any combination thereof. In this strategy, a prodrug administered in the event of an adverse event is activated by the suicide gene product, causing the death of the transduced cells.

[0354] As used herein, the term "pharmaceutically active protein or peptide" refers to a protein or peptide capable of achieving a biological and / or pharmaceutical effect on an organism. Pharmaceutically active proteins have curative or palliative properties for diseases and can be administered to ameliorate, alleviate, alleviate, reverse, or reduce the severity of diseases. Pharmaceutically active proteins also have prophylactic properties and are used to prevent the onset of diseases or to reduce the severity of such diseases or pathological conditions when they occur. Pharmaceutically active proteins include whole proteins or peptides or pharmaceutically active fragments thereof. Pharmaceutically active proteins also include pharmaceutically active analogs of proteins or peptides or analogs of fragments of proteins or peptides. The term pharmaceutically active protein also refers to multiple proteins or peptides that act cooperatively or synergistically to provide a therapeutic benefit. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth suppressor proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0355] As used herein, the term "signaling molecule" refers to any molecule that regulates, participates in, inhibits, activates, decreases, or increases cell signaling. Signal transduction refers to the transmission of a molecular signal in the form of a chemical modification through the recruitment of protein complexes along a pathway that ultimately causes a biochemical event within the cell. Signaling pathways are well known in the art and include, but are not limited to, G protein-coupled receptor signaling, tyrosine kinase receptor signaling, integrin signaling, toll gate signaling, ligand-gated ion channel signaling, ERK / MAPK signaling pathway, Wnt signaling pathway, cAMP-dependent pathway, and IP3 / DAG signaling pathway.

[0356] As used herein, the term "targeting modality" refers to molecules, e.g., polypeptides, that are genetically incorporated into cells to facilitate antigen and / or epitope specificity, including, but not limited to, i) antigen specificity associated with a unique chimeric antigen receptor (CAR) or T cell receptor (TCR), ii) engager specificity associated with a monoclonal antibody or bispecific engager, iii) targeting of transformed cells, iv) targeting of cancer stem cells, and v) other targeting strategies in the absence of a particular antigen or surface molecule.

[0357] As used herein, the terms "specific" or "specificity" can be used to refer to the ability of a molecule, e.g., a receptor or engager, to selectively bind to a target molecule, as opposed to non-specific or non-selective binding.

[0358] As used herein, the term "adoptive cell therapy," as used herein, refers to cell-based immunotherapy involving the transfusion of autologous or allogeneic lymphocytes, identified as T cells or B cells, whether genetically modified or not, that are expanded ex vivo prior to transfusion.

[0359] As used herein, a "therapeutically sufficient amount" includes within its meaning a non-toxic yet sufficient and / or effective amount of a particular therapeutic and / or pharmaceutical composition, such amount providing the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient's overall health, the patient's age, and the stage and severity of the condition. In certain embodiments, a therapeutically sufficient amount is sufficient and / or effective to ameliorate, alleviate, and / or improve at least one symptom associated with the disease or condition being treated in the subject.

[0360] When used in the context of genome editing or modification of iPSCs and derived non-pluripotent cells differentiated therefrom, or genome editing or modification of non-pluripotent cells and derived iPSCs reprogrammed therefrom, "functional" refers to (1) at the genetic level, successful gene expression, such as knock-in, knock-out, knock-down gene expression, transgenic, or controlled gene expression, e.g., inducible or transient expression at a desired stage of cellular development, achieved through direct genome editing or modification, or through "passaging" by differentiation or reprogramming of an originally genomically engineered starting cell, or (2) at the cellular level, (i) through direct genome editing. "removal, addition, or alteration of cellular function / characteristics" refers to (i) gene expression modifications obtained in the cell through "passage" by differentiation or reprogramming of the starting cell from which the original genome was engineered; (ii) gene expression modifications maintained in the cell through "passage" by differentiation or reprogramming of the starting cell from which the original genome was engineered; (iii) downstream gene regulation in the cell as a result of gene expression modifications that are only manifest in an earlier developmental stage of the cell or only in the starting cell that gave rise to the cell by differentiation or reprogramming; or (iv) enhanced or newly achieved cellular function or cellular attribute exhibited in the mature cell product initially derived by genome editing or modifications performed in iPSCs, progenitor cell sources, or de-differentiated cell sources.

[0361] "HLA deficiency," including HLA class I deficiency, HLA class II deficiency, or both, refers to cells that exhibit little or no longer maintain surface expression of complete MHC complexes containing HLA class I protein heterodimers and / or HLA class II heterodimers, or that exhibit reduced levels of expression such that the reduced or decreased levels are lower than those naturally detectable by other cells or synthetic methods. HLA class I deficiency can be achieved by functional deletion of any region of the HLA class I locus (chromosome 6p21) or by deletion or reduced expression levels of HLA class I-associated genes, such as, but not limited to, the beta-2 microglobulin (B2M) gene, TAP1 gene, TAP2 gene, and tapasin. HLA class II deficiency can be achieved by functional deletion or reduction of HLA-II-associated genes, such as, but not limited to, RFXANK, CIITA, RFX5, and RFXAP. Prior to the present invention, it was unclear whether HLA complex-deficient iPSCs or modified iPSCs have the ability to develop, mature, and generate functional differentiated cells while retaining regulated activity. Additionally, prior to the present invention, it was unclear whether HLA complex-deficient differentiated cells could be reprogrammed into iPSCs and maintained as pluripotent stem cells while retaining HLA complex deficiency. Unexpected failures in cell reprogramming, maintenance of pluripotency, and differentiation may be related to circumstances such as, but not limited to, developmental stage-specific gene expression or lack thereof, requirements for HLA complex presentation, protein shedding of introduced surface expression patterns, the need for proper and efficient clonal reprogramming, and the need to reconfigure differentiation protocols. [Example]

[0362] Example 1 - S44P and E99K mutations in Cas-CLOVER result in increased in vitro gene editing activity compared to wild-type Cas-CLOVER This example shows that a Cas-CLOVER amino acid sequence with S44P and E99K mutations in the Clo051 nuclease domain exhibits increased in vitro gene editing activity in several cell lines compared to wild-type Cas-CLOVER.

[0363] Gene editing in Huh7 and HepaRG cell lines

[0364] Huh7 or HepaRG cells were seeded at a density of 5E5 cells / ml in 12-well plates and transfected with a mixture containing a non-saturating amount of 0.2 μg / ml of gRNA pairs targeting introns of the albumin, APOC3, or TTR genes, 0.5 μg / ml of wild-type Cas-CLOVER mRNA (encoded by the nucleic acid sequence of SEQ ID NO: 11), Cas-CLOVER mRNA containing the S44P mutation (encoded by the nucleic acid sequence of SEQ ID NO: 36), or Cas-CLOVER mRNA containing the S44P and E99K mutations (encoded by the nucleic acid sequence of SEQ ID NO: 77), and 15 μl / ml of Lipofectamine Messenger MAX Reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 48 hours, the medium was removed, the cells were lysed, and genomic DNA was isolated from the cell extract using a QuickExtract Kit (Lucigen Corp.) according to the manufacturer's instructions.

[0365] The isolated genomic DNA was then analyzed using PCR by designing the following primers flanking specific target sequences: Albumin-Fwd: AAGACGTTGTGGGGATCAG (SEQ ID NO: 51) and Albumin-Rev: GAGCAAAGGCAATCAACACCC (SEQ ID NO: 52), APOC3-Fwd: ctcagccctgctctttcctc (SEQ ID NO: 53) and APOC3-Rev: ctcgcaggatggataggcag (SEQ ID NO: 54), TTR-Fwd: attgaaccccaagaaccacat (SEQ ID NO: 55) and TTR-Rev: ctgcctcctagattcaaggg (SEQ ID NO: 56). PCR products were isolated, and the DNA sequences were determined by direct Sanger sequencing and the indel rates of the wild-type, S44P, and S44P and E99K Cas-CLOVER mutants were quantified using ICE analysis (Synthego Corp.). The percentages of indels for wild type, S44P, and S44P and E99K in Huh7 and HepaRG cell lines are shown in Table 2 .

[0366] [Table 2]

[0367] As shown in Table 2, the S44P / E99K double mutant results in approximately a 1.1- to 1.7-fold improvement in indel rates at the APOC3 and TTR loci compared to the wild-type Cas-CLOVER sequence.

[0368] Gene editing in the HepG2 cell line

[0369] A mixture containing a non-saturating dose of 0.2 μg / ml of a gRNA pair targeting an intron of the albumin gene, APOC3 gene, or TTR gene and 0.5 μg / ml of either wild-type Cas-CLOVER mRNA (encoded by the nucleic acid sequence of SEQ ID NO: 11), Cas-CLOVER mRNA containing the S44P mutation (encoded by the nucleic acid sequence of SEQ ID NO: 36), or Cas-CLOVER mRNA containing the S44P and E99K mutations (encoded by the nucleic acid sequence of SEQ ID NO: 77) was electroporated into 5E5 HepG2 cells using a Lonza Nucleofector according to the manufacturer's instructions. Electroporated cells were seeded at a density of 5E6 cells / ml and incubated in culture medium for 48 hours. After 48 hours, the medium was removed, the cells were lysed, and genomic DNA was isolated from the cell extract using a QuickExtract Kit (Lucigen Corp.) according to the manufacturer's instructions.

[0370] The isolated genomic DNA was then analyzed using PCR by designing the following primers flanking specific gene target sequences: Albumin-Fwd: AAGACGTTGTGGGGATCAG (SEQ ID NO: 51) and Albumin-Rev: GAGCAAAGGCAATCAACACCC (SEQ ID NO: 52), APOC3-Fwd: ctcagccctgctctttcctc (SEQ ID NO: 53) and APOC3-Rev: ctcgcaggatggataggcag (SEQ ID NO: 54), TTR-Fwd: attgaaccccaagaaccacat (SEQ ID NO: 55) and TTR-Rev: ctgcctcctagattcaaggg (SEQ ID NO: 56). The PCR products were isolated, and the DNA sequences were determined by direct Sanger sequencing. The indel rates of the wild-type, S44P, and S44P and E99K Cas-CLOVER mutants were quantified using ICE analysis (Synthego Corp.). The percentages of indels for wild-type, S44P and S44P and E99K Cas-CLOVER mutants in the HepG2 cell line are shown in Table 3.

[0371] [Table 3]

[0372] As shown in Table 3, the S44P / E99K variant results in approximately a 1.5-fold improvement in indel rates at the albumin and APOC3 loci and an approximately 4.4-fold improvement in indel rates at the TTR locus compared to the wild-type Cas-CLOVER sequence.

[0373] Gene editing in hPSCs

[0374] A mixture containing a non-saturating amount of 0.2 μg / ml of a gRNA pair (SEQ ID NO: 67 and SEQ ID NO: 68) targeting the HBG1 gene, 1.5 or 4.0 μg / ml of wild-type Cas-CLOVER mRNA (encoded by the nucleic acid sequence of SEQ ID NO: 11), Cas-CLOVER mRNA containing the S44P mutation (encoded by the nucleic acid sequence of SEQ ID NO: 36), or Cas-CLOVER mRNA containing the S44P and E99K mutations (encoded by the nucleic acid sequence of SEQ ID NO: 77) was electroporated into 5E5 HSPC cells using a Lonza Nucleofector according to the manufacturer's instructions. Electroporated cells were seeded at a density of 5E6 cells / ml and incubated in culture medium for 48 hours. After 48 hours, the medium was removed, the cells were lysed, and genomic DNA was isolated from the cell extract using a QuickExtract Kit (Lucigen Corp.) according to the manufacturer's instructions.

[0375] Gene editing at the HBG locus was measured in HSPC cells using next-generation sequencing (NGS). Briefly, genomic DNA samples were subjected to PCR amplification using the following DNA primers flanking exon 1 of the HBG1 gene (shown in underlined font) that further contained an Illumina partial adapter: FwdACACTCTTTCCCTACACGACGCTCTTCCGATCT GCAGTATCCTCTTGGGGG (SEQ ID NO: 57), and RevGACTGGAGTTCAGACGTGTGCTCTTCCGATCT ACCTCAGACGTTCCAGAAGC(SEQ ID NO: 58). The resulting PCR amplicons were subjected to a second PCR reaction using primers containing Illumina P5 and P7 sequences (Illumina Corp) and a unique index sequence (New England Biolabs). The final amplicons were pooled at equimolar concentrations and analyzed using a Miseq benchtop sequencer according to the standard Amplicon-seq procedure from the manufacturer (Illumina Corp). Sequence data were analyzed using the CRISPResso2 program to determine the frequency of insertions / deletions in each sample. The results are shown in Table 4.

[0376] [Table 4]

[0377] As shown in Table 4, the double S44P and E99K Cas-CLOVER mutant showed approximately a two-fold increase in indel activity at the HBG1 locus compared to wild-type Cas-CLOVER, and showed further improvement compared to the S44P mutant alone.

[0378] Example 2 - S44P and E99K mutations in Cas-CLOVER result in increased in vivo gene editing activity compared to wild-type Cas-CLOVER This example demonstrates that a Cas-CLOVER amino acid sequence with the S44P mutation in the Clo051 nuclease domain exhibits increased in vivo gene editing activity at multiple genomic loci compared to wild-type Cas-CLOVER.

[0379] Editing the Psck9 gene

[0380] A.LNP preparation

[0381] To formulate lipid nanoparticles (LNPs), various ratios of the terpene lipidoid HMA-404, the phospholipid DOPC, the structural lipid cholesterol (Chol), and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (DMG-PEG2000, Avanti Polar Lipids, Alabaster, AL, USA) were combined to prepare the LNP composition. RNA molecules were encapsulated in lipid nanoparticles containing approximately 40.75 mol% HMA-404, approximately 51.75 mol% cholesterol, approximately 5 mol% DOPC, and approximately 2.5 mol% DMG-PEG2000. The lipid to nucleic acid ratio in the nanoparticles was approximately 120:1 (wt / wt), and the total lipid was 25 mM.

[0382] TIFF2026507731000010.tif50170

[0383] Compound HMA-404 was prepared as described in International Application No. / US2023 / 061005. The crude product was purified by silica gel flash chromatography (eluent: 5% MeOH / DCM). 1 H NMR(400MHz,Chloroform-d)δ4.38-4.12(m,16H),2.90-2.78(m,4H),2.71(t,J=6.8Hz,8H),2.61(s,3H) ),2.57-2.36(m,12H),2.35-2.04(m,4H),2.01-1.62(m,17H),1.60-0.96(m,45H),0.94-0.67(m,30H).

[0384] The mRNA molecules were further capped using CleanCap® (TriLink Corp), replacing all cytidine residues in the mRNA with 5-methylcytidine (5-MeC).

[0385] Individual 25 mg / ml stock solutions were prepared by solubilizing lipids in 200-proof HPLC-grade ethanol, and the stock solutions were stored at -80°C until formulation. During formulation, the lipid stock solutions were briefly equilibrated to room temperature and then placed on a hot plate maintained at a temperature range of 50–55°C. The hot lipid stock solutions were then combined to obtain the desired final molar percentages.

[0386] A 1 mg / ml solution of a gRNA pair targeting the first exon of the mouse pcsk9 gene (SEQ ID NO: 59 and SEQ ID NO: 60) and either mRNA encoding 5'-CleanCap-5MeC-Cas-CLOVER (SEQ ID NO: 69), mRNA encoding 5'-CleanCap-5MeC-Cas-CLOVER S44P (SEQ ID NO: 70), or 5'-CleanCap-5MeC-Cas-CLOVER S44P / E99K (SEQ ID NO: 71) was incorporated into LNPs and added separately to 150 mM sodium acetate buffer (pH 5.2) to form stock solutions, which were kept on ice. The lipid phase was mixed with the aqueous mRNA phase in a microfluidic chip using a NanoAssemblr® instrument (Precision Nanosystems, Vancouver, British Columbia, Canada) according to the manufacturer's instructions to form an LNP composition containing the encapsulated mRNA. The NanoAssemblr process parameters for mRNA encapsulation are shown in Table 5.

[0387] [Table 5]

[0388] The resulting mRNA LNP composition was then transferred to a Repligen Float-A-Lyzer dialysis device (Spectrum Chemical Mfg. Corp., CA, USA) with a molecular weight cutoff (MWCO) of 8-10 kDa and processed by dialysis overnight at 4 °C (or alternatively, for at least 4 h at room temperature) against phosphate-buffered saline (PBS) (dialysate:dialysis buffer volume at least 1:200 v / v), pH 7.4, to remove 25% ethanol and achieve complete buffer exchange. In some experiments, the LNPs were further concentrated using Amicon® Ultra-4 centrifugal filter units, MWCO-30 kDa (Millipore Sigma, USA), and spun at approximately 4100 × g in an ultracentrifuge. The mRNA LNPs were then stored at 4 °C until further use. The average particle size of the LNPs was approximately 70 nm.

[0389] B. Pilot Testing

[0390] Adult female BALB / C mice (n=2 / group) were intravenously co-administered with a pair of psck9-targeting gRNAs (SEQ ID NO: 59 and SEQ ID NO: 60) and 1.0 mg / kg of either mRNA encoding 5'-CleanCap-5MeC-Cas-CLOVER (SEQ ID NO: 69) or mRNA encoding 5'-CleanCap-5MeC-Cas-CLOVER S44P (SEQ ID NO: 70) or 5'-CleanCap-5MeC-Cas-CLOVER S44P / E99K (SEQ ID NO: 71), formulated into the LNP composition of Example 2, Section A. One group of mice was treated with vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0391] Mice were euthanized 7 days after administration, and DNA was isolated from the livers of treated and untreated mice. Briefly, after euthanasia, livers were excised, flash-frozen in liquid nitrogen, mixed with lysis buffer (15 mg of tissue in 200 μL of lysis buffer + 10 μL of proteinase K), and ground in a TissueLyser II (Qiagen) using triple-pure zirconium beads (Fisher Scientific). The homogenized tissue was incubated at 56°C for 30 minutes and column-purified using the New England Biolabs Monarch Genomic DNA Purification Kit according to the manufacturer's instructions. The final DNA elution was performed in 50 μL of elution buffer (10 mM Tris-Cl, pH 8.5). The concentration and purity of the DNA samples were assessed by measuring absorbance at 260 and 280 nm.

[0392] The extent of gene editing observed for Cas-CLOVER and Cas-CLOVER S44P / E99K mRNA delivered to mice was measured by droplet digital PCR (ddPCR) using a drop-off assay containing a fluorescent probe that hybridizes to the Cas-CLOVER target site. The percentage of indels for wild-type Cas-CLOVER and Cas-CLOVER S44P / E99K at the psck9 locus in the livers of treated mice is shown in Table 6.

[0393] [Table 6]

[0394] As shown in Table 6, Cas-CLOVER with the S44P mutation or the S44P and E99K mutations each resulted in an approximately 1.6- to 1.8-fold increase in indel percentage compared to wild-type Cas-CLOVER, indicating improved in vivo activity of the S44P and E99K double mutant.

[0395] C. Follow-up

[0396] Adult female BALB / C mice (n=2 / group) were intravenously co-administered with a pair of psck9-targeting gRNAs (SEQ ID NO:59 and SEQ ID NO:60) and either 0.75 mg / kg or 1.5 mg / kg of mRNA encoding 5'-CleanCap-5MeC-Cas-CLOVER (SEQ ID NO:69) or 5'-CleanCap-5MeC-Cas-CLOVER S44P (SEQ ID NO:70) or 5'-CleanCap-5MeC-Cas-CLOVER S44P / E99K (SEQ ID NO:71), formulated into the LNP composition of Example 2A. One group of mice was treated with vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0397] Mice were euthanized 7 days after administration, and DNA was isolated from five tissue types from each group: liver, heart, spleen, lung, and kidney. Briefly, liver biopsies were excised after euthanasia, flash-frozen in liquid nitrogen, mixed with lysis buffer (15 mg of tissue in 200 μL of lysis buffer + 10 μL of proteinase K), and ground in a TissueLyser II (Qiagen) using triple-pure zirconium beads (Fisher Scientific). The homogenized tissue was incubated at 56°C for 30 minutes and column-purified using the New England Biolabs Monarch Genomic DNA Purification Kit according to the manufacturer's instructions. The final DNA elution was performed in 50 μL of elution buffer (10 mM Tris-Cl, pH 8.5). The concentration and purity of the DNA samples were assessed by measuring absorbance at 260 and 280 nm.

[0398] The extent of gene editing observed for wild-type Cas-CLOVER, S44P Cas-CLOVER, and S44P / E99K Cas-CLOVER mRNA delivered to mice was measured by droplet digital PCR (ddPCR) using a drop-off assay containing a fluorescent probe that hybridizes to the Cas-CLOVER target site. The percentage of indels at the psck9 locus for wild-type Cas-CLOVER, S44P Cas-CLOVER, and S44P / E99K Cas-CLOVER in the livers of treated mice is shown in Table 7.

[0399] [Table 7]

[0400] As shown in Table 7, Cas-CLOVER with the S44P mutation resulted in an approximately 3-40-fold increase in indel percentage compared to wild-type Cas-CLOVER, while the double S44P and E99K mutant further increased the indel rate by approximately 4-75-fold, indicating improved in vivo activity of the double S44P and E99K mutant compared to the wild-type.

[0401] Example 3 - S44P and E99K mutations in Cas-CLOVER result in increased in vitro gene editing activity compared to wild-type Cas-CLOVER This example shows that a Cas-CLOVER amino acid sequence with S44P and E99K mutations in the Clo051 nuclease domain exhibits increased in vitro gene editing activity in human hepatocytes compared to wild-type Cas-CLOVER.

[0402] A.LNP preparation

[0403] To formulate lipid nanoparticles (LNPs), various proportions of the ionizable lipid SS-OP, the phospholipids DOPC and DSPC, the structural lipid cholesterol (Chol), and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (DMG-PEG2000, Avanti Polar Lipids, Alabaster, AL, USA) were combined to prepare the LNP composition. LNPs were prepared as described in WO 2022 / 182792. RNA molecules were encapsulated in lipid nanoparticles containing approximately 54 mol% SS-OP, approximately 35 mol% cholesterol, approximately 5 mol% DOPC, approximately 5 mol% DSPC, and approximately 1 mol% DMG-PEG2000. The lipid to nucleic acid ratio in the nanoparticles was approximately 100:1 (wt / wt), and the total lipid was 25 mM. The mRNA molecules were further capped using CleanCap® (TriLink Corp), replacing all cytidine residues in the mRNA with 5-methylcytidine (5-MeC).

[0404] Individual 25 mg / ml stock solutions were prepared by solubilizing lipids in 200-proof HPLC-grade ethanol, and the stock solutions were stored at -80°C until formulation. During formulation, the lipid stock solutions were briefly equilibrated to room temperature and then placed on a hot plate maintained at a temperature range of 50–55°C. The hot lipid stock solutions were then combined to obtain the desired final molar percentages.

[0405] A 1 mg / ml solution of a gRNA pair to be incorporated into LNPs targeting either the human KLKB1 gene (SEQ ID NO:72 and SEQ ID NO:73) or the mouse klkb1 gene (SEQ ID NO:74 and SEQ ID NO:75) and either the mRNA encoding 5'-CleanCap-5MeC-Cas-CLOVER (SEQ ID NO:69) or the mRNA encoding 5'-CleanCap-5MeC-Cas-CLOVER S44P+E99K (SEQ ID NO:71) was added individually to 150 mM sodium acetate buffer (pH 5.2) to generate a stock solution, which was kept on ice. The lipid phase was mixed with the aqueous mRNA phase in the microfluidic chip using a NanoAssemblr® instrument (Precision Nanosystems, Vancouver, British Columbia, Canada) according to the manufacturer's instructions to form an LNP composition containing the encapsulated mRNA. The NanoAssemblr process parameters for mRNA encapsulation are shown in Table 8.

[0406] [Table 8]

[0407] The resulting mRNA LNP composition was then transferred to a Repligen Float-A-Lyzer dialysis device (Spectrum Chemical Mfg. Corp., CA, USA) with a molecular weight cutoff (MWCO) of 8-10 kDa and processed by dialysis overnight at 4 °C (or alternatively, for at least 4 h at room temperature) against phosphate-buffered saline (PBS) (dialysate:dialysis buffer volume at least 1:200 v / v), pH 7.4, to remove 25% ethanol and achieve complete buffer exchange. In some experiments, the LNPs were further concentrated using Amicon® Ultra-4 centrifugal filter units, MWCO-30 kDa (Millipore Sigma, USA), and spun at approximately 4100 × g in an ultracentrifuge. The mRNA LNPs were then stored at 4 °C until further use. The average particle size of the LNPs was approximately 70 nm.

[0408] Gene editing in primary human hepatocytes (PHH) and HepaRG cell lines

[0409] PHH or HepaRG cells were seeded at a density of 5E5 cells / ml in 12-well plates and transfected with different concentrations of gRNA targeting the human KLKB1 gene (SEQ ID NO: 72 and SEQ ID NO: 73) and either mRNA encoding 5'-CleanCap-5MeC-Cas-CLOVER (SEQ ID NO: 69) or mRNA encoding 5'-CleanCap-5MeC-Cas-CLOVER S44P+E99K (SEQ ID NO: 71), formulated into the LNP composition of Example 3A. After 48 hours, the medium was removed, cells were lysed, and genomic DNA was isolated from the cell extract using a QuickExtract Kit (Lucigen Corp.) according to the manufacturer's instructions. The isolated genomic DNA was then analyzed by droplet digital PCR (ddPCR) using a drop-off assay containing a fluorescent probe hybridizing to the Cas-CLOVER target site. The percentage of wild-type and S44P+E99K indels in PHH and HepaRG cell lines is shown in Table 9.

[0410] [Table 9]

[0411] Example 4 - S44P and E99K mutations in Cas-CLOVER result in increased in vivo gene editing activity compared to wild-type Cas-CLOVER This example shows that a Cas-CLOVER amino acid sequence with S44P and E99K mutations in the Clo051 nuclease domain exhibits increased in vivo gene editing activity at multiple genomic loci compared to wild-type Cas-CLOVER.

[0412] A. Human KLKB1 gene editing in liver-humanized mice

[0413] Fully humanized adult male NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-UL23)7-2 / ShiJic mice (TK-Nog, Taconic; n=3 / group) were intravenously co-administered with a pair of KLKB1-targeting gRNAs (SEQ ID NO:74 and SEQ ID NO:75) and 2.0 mg / kg of mRNA encoding 5'-CleanCap-5MeC-Cas-CLOVER (SEQ ID NO:69), 5'-CleanCap-5MeC-Cas-CLOVER S44P (SEQ ID NO:70), or 5'-CleanCap-5MeC-Cas-CLOVER S44P and E99K (SEQ ID NO:71), formulated into the LNP composition of Example 3A. One group of mice was treated with vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0414] Mice were euthanized 7 days after administration, and DNA was isolated from liver tissue. Briefly, liver biopsies were excised after euthanasia, flash-frozen in liquid nitrogen, mixed with lysis buffer (15 mg of tissue in 200 μL of lysis buffer + 10 μL of proteinase K), and pulverized in a TissueLyser II (Qiagen) using triple-pure zirconium beads (Fisher Scientific). The homogenized tissue was incubated at 56°C for 30 minutes and column-purified using the New England Biolabs Monarch Genomic DNA Purification Kit according to the manufacturer's instructions. The final DNA elution was performed in 50 μL of elution buffer (10 mM Tris-Cl, pH 8.5). The concentration and purity of the DNA samples were assessed by measuring absorbance at 260 and 280 nm.

[0415] The extent of gene editing observed for the Cas-CLOVER and Cas-CLOVER S44P+E99K mRNA delivered to mice was measured by droplet digital PCR (ddPCR) using a drop-off assay containing a fluorescent probe that hybridizes to the Cas-CLOVER target site. The percentage of indels for wild-type Cas-CLOVER and Cas-CLOVER S44P+E99K at the KLKB1 locus in the livers of treated mice is shown in Table 10.

[0416] [Table 10]

[0417] As shown in Table 10, Cas-CLOVER with the S44P+E99K mutation resulted in an approximately 3.2-fold increase in indel percentage compared to wild-type Cas-CLOVER, indicating improved in vivo activity of the S44P+E99K mutant.

[0418] B. Mouse klkb1 gene editing in wild-type BALB / c mice

[0419] Female BALB / c mice (n=3 / group) were intravenously co-administered with a pair of mouse klkb1-targeting gRNAs (SEQ ID NO:74 and SEQ ID NO:75) and 2.0 mg / kg of mRNA encoding 5'-CleanCap-5MeC-Cas-CLOVER (SEQ ID NO:69), 5'-CleanCap-5MeC-Cas-CLOVER S44P (SEQ ID NO:70), or 5'-CleanCap-5MeC-Cas-CLOVER S44P and E99K (SEQ ID NO:71), formulated into the LNP composition of Example 3A. One group of mice was treated with vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0420] Mice were euthanized 7 days after administration, and DNA was isolated from liver tissue. Briefly, liver biopsies were excised after euthanasia, flash-frozen in liquid nitrogen, mixed with lysis buffer (15 mg of tissue in 200 μL of lysis buffer + 10 μL of proteinase K), and pulverized in a TissueLyser II (Qiagen) using triple-pure zirconium beads (Fisher Scientific). The homogenized tissue was incubated at 56°C for 30 minutes and column-purified using the New England Biolabs Monarch Genomic DNA Purification Kit according to the manufacturer's instructions. The final DNA elution was performed in 50 μL of elution buffer (10 mM Tris-Cl, pH 8.5). The concentration and purity of the DNA samples were assessed by measuring absorbance at 260 and 280 nm.

[0421] The extent of gene editing observed for the Cas-CLOVER and Cas-CLOVER S44P+E99K mRNA delivered to mice was measured by droplet digital PCR (ddPCR) using a drop-off assay containing a fluorescent probe that hybridizes to the Cas-CLOVER target site. The percentage of indels for wild-type Cas-CLOVER and Cas-CLOVER S44P+E99K at the KLKB1 locus in the livers of treated mice is shown in Table 11.

[0422] [Table 11]

[0423] As shown in Table 11, Cas-CLOVER with the S44P+E99K mutation resulted in an approximately 2.2-fold increase in indel percentage compared to wild-type Cas-CLOVER, indicating improved in vivo activity of the S44P+E99K mutant.

Claims

1. A polypeptide comprising the amino acid sequence of SEQ ID NO:

76.

2. A polynucleotide encoding the polypeptide of claim 1.

3. A vector comprising the polynucleotide of claim 2.

4. A pharmaceutical composition comprising the vector of claim 3 and at least one pharmaceutically acceptable excipient or diluent.

5. 1. A method for modifying a target sequence in the genome of a plurality of cells, comprising: a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 76 or a polynucleotide encoding said polypeptide of SEQ ID NO: 76; b) introducing a composition comprising at least one guide RNA (gRNA); thereby producing an alteration in said target sequence in said genome; wherein 1.1-fold to 100-fold more of the plurality of cells comprise the modification in the target sequence in the genome compared to a plurality of modified cells into which a composition comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or a polynucleotide encoding the polypeptide of SEQ ID NO: 10 has been introduced.

6. 6. The method of claim 5, wherein 1.6-fold to 3.2-fold more of the cells comprise the modification in the target sequence in the genome compared to a plurality of modified cells into which a composition comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or a polynucleotide encoding the polypeptide of SEQ ID NO: 10 has been introduced.

7. 6. The method of claim 5, wherein 4-75 times more of the plurality of cells comprise the modification in the target sequence in the genome compared to a plurality of modified cells into which a composition comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or a polynucleotide encoding the polypeptide of SEQ ID NO: 10 has been introduced.

8. The method of any one of claims 5 to 7, wherein the modification in the target sequence in the genome is a deletion, insertion, substitution, inversion and / or rearrangement.

9. The composition comprises: about 40.75 mol % of the compound of formula (I); about 51.75 mol % cholesterol; about 5 mol % DOPC, and encapsulated in at least one lipid nanoparticle comprising about 2.5 mol% DMG-PEG2000; 8. The method of any one of claims 5 to 7, wherein the polynucleotide encoding the polypeptide of SEQ ID NO: 76 is an RNA molecule, and the ratio of lipid to RNA molecule in the at least one nanoparticle is about 120:1 (w / w).

10. The composition comprises: encapsulated in at least one lipid nanoparticle comprising about 54 mol% SS-OP, about 35 mol% cholesterol, about 5 mol% DOPC, about 5 mol% DSPC, and about 1 mol% DMG-PEG2000; 8. The method of any one of claims 5 to 7, wherein the polynucleotide encoding the polypeptide of SEQ ID NO: 76 is an RNA molecule, the ratio of lipid to RNA molecule in the at least one nanoparticle is about 100:1 (w / w), and the total lipid is 25 nM.

11. the plurality of cells (a) hepatocytes, preferably hepatocytes, hepatic parenchymal cells, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal endothelial cells; (b) T cells, preferably activated T cells, resting T cells, or stem memory T cells (T ＳＣＭ T cells, or The method of any one of claims 5 to 10, comprising (c) hematopoietic stem cells (HSCs).

12. A cell modified according to the method of any one of claims 5 to 11.

13. A composition comprising a population of cells modified according to the method of claims 5 to 11.

14. A method for treating at least one disease or disorder in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of at least one of the pharmaceutical composition of claim 4, the cell of claim 12, or the composition of claim 13.

15. The at least one disease or disorder is a liver disease or disorder, and preferably the liver disease or disorder is: (a) metabolic liver disorders, 15. The method of claim 14, wherein (b) the UCD is a urea cycle disorder (UCD), preferably wherein the UCD is N-acetylglutamate synthetase (NAGS) deficiency, carbamoylphosphate synthetase I deficiency (CPSI deficiency), ornithine transcarbamylase (OTC) deficiency, argininosuccinate synthetase deficiency (ASSD) (citrullinemia I), citrin deficiency (citrullinemia II), argininosuccinate lyase deficiency (argininosuccinic aciduria), arginase deficiency (hyperargininemia), ornithine translocase deficiency (HHH syndrome), or any combination thereof.

16. 15. The method of claim 14, wherein the at least one disease or disorder is cancer.

17. 15. The method of claim 14, wherein the at least one disease or disorder is hemophilia A.

Images