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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/90—Stable introduction of foreign DNA into chromosome
  • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
  • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor

Definitions

• the present disclosure relates to genome editing compositions with improved stability and activity. More particularly, the disclosure relates to nuclease variants with improved stability and/or activity, compositions, and methods of making and using the same for genome editing.
• Genome editing strategies based on programmable nucleases such as meganucleases, zinc finger nucleases, transcription activator-like effector nucleases and the clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease Cas9 hold tremendous, but as yet unrealized, potential for the treatment of diseases, disorders, and conditions with a genetic component.
• CRISPR clustered regularly interspaced short palindromic repeat
• nuclease-based genome editing tools include, but are not limited to low genome editing efficiencies, nuclease specificity, nuclease stability, and delivery challenges.
• the current state of the art for most genome editing strategies fails to meet some or all of these criteria.
• the present disclosure generally relates, in part, to compositions comprising homing endonuclease (HE) variants and megaTALs with improved stability and activity that cleave a target site in the human genome and methods of using the same.
• HE variants and megaTALs are engineered to improve or enhance the thermostability of the enzyme and/or improve the catalytic activity of the enzyme.
• the present disclosure contemplates, in part, a polypeptide comprising an engineered homing endonuclease that has been engineered to improve stability and binding and cleavage of a target site.
• an I-Onul homing endonuclease (HE) variant comprises one or more amino acid substitutions relative to a parent I-Onul HE comprising the amino acid sequence set forth in SEQ ID NO: 1, wherein the one or more amino acid substitutions increase the thermostability of the I-Onul HE variant compared to the parent I-Onul HE.
• the one or more amino acid substitutions is at an amino acid position selected from the group consisting of: 114, A19, VI 16, F168, D208, N246, and L263.
• the amino acid substitutions are at the following amino acid positions: 114, A19, FI 68, D208, and N246.
• the one or more amino acid substitutions is at an amino acid position selected from the group consisting of: K108, K156, S176, E231, V261, E277, and G300. In some embodiments, the one or more amino acid substitutions is at an amino acid position selected from the group consisting of: N31, N33, K52, Y97, K124, K147, 1153, K209, E264, and D268.
• an I-Onul HE variant comprises three or more amino acid substitutions is at an amino acid position selected from the group consisting of: 114, A19, K108, VI 16, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300.
• an I-Onul HE variant comprises three or more amino acid substitutions is at an amino acid position selected from the group consisting of: 114, A19, K108, VI 16, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300; and one or more amino acid substitutions is at an amino acid position selected from the group consisting of: N31, N33, K52, Y97, K124, K147, 1153, K209, E264, and D268.
• the I-Onul HE variant comprises five or more amino acid substitutions is at an amino acid position selected from the group consisting of: 114, A19, K108, VI 16, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300.
• an I-Onul HE variant comprises five or more amino acid substitutions is at an amino acid position selected from the group consisting of: 114, A19, K108, VI 16, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300; and one or more amino acid substitutions is at an amino acid position selected from the group consisting of: N31, N33, K52, Y97, K124, K147, 1153, K209, E264, and D268.
• an I-Onul HE variant has a TMso at least 10°C higher than the TM50 of the parent I-Onul HE.
• an I-Onul HE variant has a TM50 at least 15°C higher than the TM50 of the parent I-Onul HE.
• an I-Onul HE variant has a TM50 at least 20°C higher than the TM50 of the parent I-Onul HE.
• an I-Onul HE variant has a TM50 at least 25 °C higher than the TM50 of the parent I-Onul HE.
• an I-Onul homing endonuclease (HE) variant comprising one or more amino acid substitutions relative to a parent I-Onul HE sequence, wherein the one or more amino acid substitutions increase the thermostability of the I-Onul HE variant compared to the parent I-Onul HE.
• HE I-Onul homing endonuclease
• a parent I-Onul HE amino acid sequence set is forth in SEQ ID NO: 1.
• the amino acid substituted for 114 is selected from the group consisting of: T and V.
• amino acid substituted for A19 is selected from the group consisting of: T and V.
• the amino acid substituted for VI 16 is selected from the group consisting of: F, D, A, L and I.
• amino acid substituted for VI 16 is selected from the group consisting of: L and I.
• the amino acid substituted for F168 is selected from the group consisting of: H, Y, I, V, P, L and S.
• the amino acid substituted for FI 68 is selected from the group consisting of: L and S.
• the amino acid substituted for D208 is selected from the group consisting of: N, V, Y, and E. In particular embodiments, the amino acid substituted for D208 is E.
• the amino acid substituted for N246 is selected from the group consisting of: H, I, D, R, S, T, V, Y, and K.
• the amino acid substituted for N246 is K.
• amino acid substituted for L263 is R.
• the one or more amino acid substitutions is at an amino acid position selected from the group consisting of: K108, K156, S176, E231, V261, E277, and G300.
• the amino acid substituted for K108 is selected from the group consisting of: E, N, Q, R, T, V, and M.
• the amino acid substituted for K108 is M
• the amino acid substituted for K156 is selected from the group consisting of: N, Q, R, T, V, I, and E.
• amino acid substituted for K156 is selected from the group consisting of: I and E
• amino acid substituted for SI 76 is selected from the group consisting of: P, N and A.
• the amino acid substituted for E231 is selected from the group consisting of: D, K, V, and G.
• the amino acid substituted for E231 is selected from the group consisting of: K and G.
• the amino acid substituted for V261 is selected from the group consisting of: D, G, I, L, S, T, and A.
• the amino acid substituted for V261 is A.
• the amino acid substituted for E277 is selected from the group consisting of: A, D, G, Q, V, and K.
• the amino acid substituted for E277 is K.
• the amino acid substituted for G300 is selected from the group consisting of: S, V, D, C, and R.
• the one or more amino acid substitutions is at an amino acid position selected from the group consisting of: N31, N33, K52, Y97, K124, K147, 1153, K209, E264, and D268.
• the amino acid substituted for N31 is selected from the group consisting of: D, H, I, R, K, S, T and Y.
• the amino acid substituted for N33 is selected from the group consisting of: D, G, H, I, K, S, T and Y.
• the amino acid substituted for N33 is K.
• the amino acid substituted for K52 is selected from the group consisting of: Q, R, T, Y, N, E, and M.
• the amino acid substituted for K52 is M.
• the amino acid substituted for Y97 is selected from the group consisting of: F, N and H.
• the amino acid substituted for Y97 is F.
• the amino acid substituted for K124 is N.
• amino acid substituted for K147 is selected from the group consisting of: E, I, N, R and T.
• the amino acid substituted for K147 is I.
• the amino acid substituted for 1153 is selected from the group consisting of: D, H, K, T, Y, S, V and N.
• the amino acid substituted for K209 is selected from the group consisting of: E, M, N, Q and R.
• the amino acid substituted for K209 is R.
• the amino acid substituted for E264 is selected from the group consisting of: A, D, G, K, Q, R and V.
• the amino acid substituted for E264 is K.
• the amino acid substituted for D268 is N.
• an I-Onul HE variant comprises three or more amino acid substitutions.
• the I-Onul HE variant comprises five or more amino acid substitutions.
• an I-Onul HE variant comprises three or more amino acid substitutions is at an amino acid position selected from the group consisting of: 114, A19, K108, VI 16, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300; and one or more amino acid substitutions is at an amino acid position selected from the group consisting of: N31, N33, K52, Y97, K124, K147, 1153, K209, E264, and D268.
• an I-Onul HE variant comprises five or more amino acid substitutions is at an amino acid position selected from the group consisting of: 114, A19, K108, VI 16, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300.
• an I-Onul HE variant comprises five or more amino acid substitutions is at an amino acid position selected from the group consisting of: 114, A19, K108, VI 16, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300; and one or more amino acid substitutions is at an amino acid position selected from the group consisting of: N31, N33, K52, Y97, K124, K147, 1153, K209, E264, and D268.
• an I-Onul HE variant has a TIVEo at least 10°C higher than the TM50 of the parent I-Onul HE.
• an I-Onul HE variant has a TM50 at least 15°C higher than the TM50 of the parent I-Onul HE. In particular embodiments, an I-Onul HE variant has a TMso at least 20°C higher than the TM50 of the parent I-Onul HE.
• an I-Onul HE variant has a TM50 at least 25 °C higher than the TM50 of the parent I-Onul HE.
• an I-Onul HE variant targets a site in a gene selected from the group consisting of: HBA, HBB, HBGl, HBG2, BCL11 A, PCSK9, TCRA, TCRB, B2M, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, CUT A, AHR, PD-1, CTLA4, TIGIT, TGFBR2, LAG-3, TIM-3, BTLA, IL4R, IL6R, CXCR1, CXCR2, IL10R, IL13Ra2,
• an I-Onul homing endonuclease (HE) variant that cleaves a target site in the human BCL11 A gene, comprises one or more amino acid substitutions relative to a parent I-Onul HE sequence, wherein the one or more amino acid substitutions increase the thermostability of the I-Onul HE variant compared to the parent I-Onul HE.
• HE I-Onul homing endonuclease
• an I-Onul homing endonuclease (HE) variant that cleaves a target site in the human PCSK9 gene, comprises one or more amino acid substitutions relative to a parent I-Onul HE sequence, wherein the one or more amino acid substitutions increase the thermostability of the I-Onul HE variant compared to the parent I-Onul HE.
• HE I-Onul homing endonuclease
• an I-Onul homing endonuclease (HE) variant that cleaves a target site in the human PDCD-1 gene, comprises one or more amino acid substitutions relative to a parent I-Onul HE sequence, wherein the one or more amino acid substitutions increase the thermostability of the I-Onul HE variant compared to the parent I-Onul HE.
• HE I-Onul homing endonuclease
• an I-Onul homing endonuclease (HE) variant that cleaves a target site in the human TCRa gene, comprises one or more amino acid substitutions relative to a parent I-Onul HE sequence, wherein the one or more amino acid substitutions increase the thermostability of the I-Onul HE variant compared to the parent I-Onul HE.
• HE I-Onul homing endonuclease
• an I-Onul homing endonuclease (HE) variant that cleaves a target site in the human CBLB gene, comprises one or more amino acid substitutions relative to a parent I-Onul HE sequence, wherein the one or more amino acid substitutions increase the thermostability of the I-Onul HE variant compared to the parent I-Onul HE.
• HE I-Onul homing endonuclease
• an I-Onul homing endonuclease (HE) variant that cleaves a target site in the human CTLA-4 gene, comprises one or more amino acid substitutions relative to a parent I-Onul HE sequence, wherein the one or more amino acid substitutions increase the thermostability of the I-Onul HE variant compared to the parent I-Onul HE.
• HE I-Onul homing endonuclease
• an I-Onul homing endonuclease (HE) variant that cleaves a target site in the human TGFpRII gene, comprises one or more amino acid substitutions relative to a parent I-Onul HE sequence, wherein the one or more amino acid substitutions increase the thermostability of the I-Onul HE variant compared to the parent I-Onul HE.
• HE I-Onul homing endonuclease
• I-Onul homing endonuclease (HE) variant that cleaves a target site in the human TIM3 gene, comprises one or more amino acid substitutions relative to a parent I-Onul HE sequence, wherein the one or more amino acid substitutions increase the thermostability of the I-Onul HE variant compared to the parent I-Onul HE.
• HE homing endonuclease
• an I-Onul HE variant comprises three or more amino acid substitutions is at an amino acid position selected from the group consisting of: 114, A19, K108, VI 16, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300.
• an I-Onul HE variant comprises the amino acid
• an I-Onul HE variant comprises three or more amino acid substitutions is at an amino acid position selected from the group consisting of: 114, A19, K108, VI 16, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300; and one or more amino acid substitutions is at an amino acid position selected from the group consisting of: N31, N33, K52, Y97, K124, K147, 1153, K209, E264, and D268.
• an I-Onul HE variant comprises five or more amino acid substitutions is at an amino acid position selected from the group consisting of: 114, A19, K108, VI 16, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300.
• an I-Onul HE variant comprises five or more amino acid substitutions is at an amino acid position selected from the group consisting of: 114, A19, K108, VI 16, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300; and one or more amino acid substitutions is at an amino acid position selected from the group consisting of: N31, N33, K52, Y97, K124, K147, 1153, K209, E264, and D268.
• an I-Onul HE variant has a TIVLo at least 10°C higher than the TM50 of the parent I-Onul HE.
• an I-Onul HE variant has a TM50 at least 15°C higher than the TM50 of the parent I-Onul HE.
• an I-Onul HE variant has a TM50 at least 25 °C higher than the TM50 of the parent I-Onul HE.
• FIG 1 shows the melt curves for I-Onul LHE, and I-Onul LHE variants
• Figure 2 shows a stacked bar graph of the percent change at each position from eight I- Onul LHE variants randomly mutagenized and sorted for greater stability. The positions with a percent change greater than 2 standard deviations above the average are identified.
• Figure 3A shows the melt curves for a parent BCL11 A I-Onul LHE variant endonuclease, a BCL11 A I-Onul LHE variant endonuclease with individual stabilizing point mutations (F168S, I14T, N246I, V261 A), and a BCL11 A I-Onul LHE variant that was randomly mutagenized (stable HE variant).
• Figure 4A-4C shows that the A5 thermostability enhancing mutations can stabilize I- Onul LHE variants that target PCDC-1 (Figure 4A), CBLB ( Figure 4B), and TCRa ( Figure 4C).
• Figure 5A shows a cartoon of a reporter construct used for assessing homing endonuclease stability.
• Figure 5B shows a western blot comparing protein expression of I-Onul, a parental BCL11 A I-Onul LHE variant, and the BCL11 A I-Onul LHE A5 variant.
• Figure 5C shows a timecourse of expression of a GFP reporter compared to I-Onul, a parental BCL11 A I-Onul LHE variant, and the BCL11 A I-Onul LHE A5 variant.
• Figure 6 shows that increasing thermostability of a PDCD-1 megaTAL significantly increases editing activity compared to its parent PDCD-1 megaTAL.
• SEQ ID NO: 1 is an amino acid sequence of a wild type I-Onul LAGLIDADG homing endonuclease (LHE).
• SEQ ID NO: 2 is an amino acid sequence of a wild type I-Onul LHE.
• SEQ ID NO: 3 is an amino acid sequence of a biologically active fragment of a wild- type I-Onul LHE.
• SEQ P) NO: 5 is an amino acid sequence of a biologically active fragment of a wild- type I-Onul LHE.
• SEQ P) NO: 6 is an amino acid sequence of an I-Onul LHE variant reprogrammed to bind and cleave a target site in the human TCRa gene.
• SEQ ID NO: 7 is an amino acid sequence of an I-Onul LHE variant reprogrammed to bind and cleave a target site in the human CBLB gene.
• SEQ ID NO: 8 is an amino acid sequence of an I-Onul LHE variant reprogrammed to bind and cleave a target site in the human BCL11 A gene.
• SEQ ID NO: 15 is an amino acid sequence of an I-Onul LHE variant reprogrammed to bind and cleave a target site in the human PDCD-1 gene.
• SEQ ID NO: 16 is an amino acid sequence of an I-Onul LHE thermostable variant reprogrammed to bind and cleave a target site in the human PDCD-1 gene.
• SEQ ID NO: 17 is an amino acid sequence of an I-Onul LHE thermostable variant reprogrammed to bind and cleave a target site in the human TCRa gene.
• SEQ ID NO: 18 is an amino acid sequence of an I-Onul LHE thermostable variant reprogrammed to bind and cleave a target site in the human CBLB gene.
• the present disclosure generally relates to, in part, improved genome editing compositions and methods of use thereof.
• Genome editing enzymes hold tremendous promise for treating diseases, disorders, and conditions with a genetic component.
• genome editing enzymes engineered to bind and cleave target sites in the genome may have short half- lives in vivo and/or fail to cleave with high efficiency.
• the inventors have discovered that homing endonuclease scaffolds can be engineered to increase thermostability and catalytic activity and that homing endonuclease activity unexpectedly increased when the enzymes were engineered to have greater thermostability.
• amino acid positions of homing endonucleases altered to increase thermostability and activity and reprogrammed to bind and cleave one target site are conserved and can be used to increase thermostability of other homing endonucleases reprogrammed to bind and cleave other target sites.
• Genome editing compositions and methods contemplated in various embodiments comprise nuclease variants with enhanced stability and activity, designed to bind and cleave a target sequence present in a genome.
• the nuclease variants contemplated in particular embodiments can be used to introduce a double-strand break in a target polynucleotide sequence, which may be repaired by non-homologous end joining (NHEJ) in the absence of a polynucleotide template, e.g., a donor repair template, or by homology directed repair (HDR), i.e., homologous recombination, in the presence of a donor repair template.
• NHEJ non-homologous end joining
• HDR homology directed repair
• Nuclease variants contemplated in certain embodiments can also be designed as nickases, which generate single- stranded DNA breaks that can be repaired using the cell ' s base-excision-repair (BER) machinery or homologous recombination in the presence of a donor repair template.
• NHEJ is an error-prone process that frequently results in the formation of small insertions and deletions that disrupt gene function.
• Homologous recombination requires homologous DNA as a template for repair and can be leveraged to create a limitless variety of modifications specified by the introduction of donor DNA containing the desired sequence at the target site, flanked on either side by sequences bearing homology to regions flanking the target site.
• homing endonucleases comprise one or more amino acid substitutions that increase stability and/or activity. In particular embodiments, homing endonucleases comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions that increase stability and/or activity.
• homing endonucleases comprise one or more amino acid substitutions that increase stability and/or activity are formatted as a megaTAL.
• a megaTAL comprises a homing endonuclease that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions that increase stability and/or activity.
• a cell or population of cells comprises a homing endonuclease variant or megaTAL that has been modified to increase stability and/or activity. Accordingly, the methods and compositions contemplated herein represent a quantum improvement compared to existing adoptive cell therapies.
• the term“about” or“approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
• a range e.g., 1 to 5, about 1 to 5, or about 1 to about 5, refers to each numerical value encompassed by the range.
• ex vivo refers generally to activities that take place outside an organism, such as experimentation or measurements done in or on living tissue in an artificial
• “ex vivo" procedures involve living cells or tissues taken from an organism and cultured or modulated in a laboratory apparatus, usually under sterile conditions, and typically for a few hours or up to about 24 hours, but including up to 48 or 72 hours, depending on the circumstances. In certain embodiments, such tissues or cells can be collected and frozen, and later thawed for ex vivo treatment. Tissue culture experiments or procedures lasting longer than a few days using living cells or tissue are typically considered to be“in vitro " though in certain embodiments, this term can be used interchangeably with ex vivo.
• An“increased” or“enhanced” amount is typically a“statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g, 1.5, 1.6, 1.7. 1.8, etc.) the response produced by vehicle or control.
• By“maintain,” or“preserve,” or“maintenance,” or“no change,” or“no substantial change,” or“no substantial decrease” refers generally to the ability of a nuclease variant to produce, elicit, or cause a substantially similar or comparable physiological response (i.e., downstream effects) in as compared to the response caused by either vehicle or control.
• a comparable response is one that is not significantly different or measurable different from the reference response.
• binding affinity or“specifically binds” or“specifically bound” or “specific binding” or“specifically targets” as used herein, describe binding of one molecule to another, e.g, DNA binding domain of a polypeptide binding to DNA, at greater binding affinity than background binding.
• a binding domain“specifically binds” to a target site if it binds to or associates with a target site with an affinity or K a (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) of, for example, greater than or equal to about 10 5 M 1 .
• a binding domain binds to a target site with a K a greater than or equal to about 10 6 M 1 , 10 7 M 1 , 10 8 M 1 , 10 9 M 1 , 10 10 M 1 , 10 11 M 1 , 10 12 M or 10 13 M 1 .
• “High affinity” binding domains refers to those binding domains with a K a of at least 10 7 M 1 , at least 10 8 M 1 , at least 10 9 M 1 , at least 10 10 M 1 , at least 10 11 M 1 , at least 10 12 M 1 , at least 10 13 M 1 , or greater.
• affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g, 10 5 M to 10 13 M, or less).
• Kd equilibrium dissociation constant
• Affinities of nuclease variants comprising one or more DNA binding domains for DNA target sites contemplated in particular embodiments can be readily determined using conventional techniques, e.g., yeast cell surface display, or by binding association, or displacement assays using labeled ligands.
• the affinity of specific binding is about 2 times greater than background binding, about 5 times greater than background binding, about 10 times greater than background binding, about 20 times greater than background binding, about 50 times greater than background binding, about 100 times greater than background binding, or about 1000 times greater than background binding or more.
• the terms“selectively binds” or“selectively bound” or“selectively binding” or “selectively targets” and describe preferential binding of one molecule to a target molecule (on- target binding) in the presence of a plurality of off-target molecules.
• an HE or megaTAL selectively binds an on-target DNA binding site about 5, 10, 15, 20, 25, 50, 100, or 1000 times more frequently than the HE or megaTAL binds an off-target DNA target binding site.
• On-target refers to a target site sequence.
• Off-target refers to a sequence similar to but not identical to a target site sequence.
• A“target site” or“target sequence” is a chromosomal or extrachromosomal nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind and/or cleave, provided sufficient conditions for binding and/or cleavage exist.
• a polynucleotide sequence or SEQ ID NO. that references only one strand of a target site or target sequence
• the target site or target sequence bound and/or cleaved by a nuclease variant is double-stranded and comprises the reference sequence and its complement.
• the target site is a sequence in a human PDCD-1 gene.
• Protein stability refers to the net balance of forces, which determine whether a protein will be its native folded conformation or a denatured (unfolded or extended) state. Protein unfolding, either partial or complete, can result in loss of function along with degradation by the cellular machinery. Polypeptide stability can be measured in response to various conditions including but not limited to temperature, pressure, and osmolyte
• thermostability refers to the ability of a protein to properly fold or remain in its native folded conformation and resist denaturation or unfolding upon exposure to temperature fluctuations. At non-ideal temperatures a protein will either not be able to efficiently fold into an active confirmation or will have the propensity to unfold from its active confirmation. A protein with increased thermostability will fold properly and retain activity over an increased range of temperatures when compared to a protein that is less thermostable.
• TM50 refers to the temperature at which 50% of an amount of protein is unfolded.
• the TM50 is the temperature at which an amount of protein has 50% maximum activity.
• TM50 is a specific value determined by fitting multiple data points to a Boltzmann sigmoidal curve.
• the TM50 of a protein is measured in a yeast surface display activity assay by expressing the protein on the yeast surface at ⁇ 25°C, aliquoting the yeast into multiple wells and exposing to a range of higher temperatures, cooling the yeast, and then measuring cleavage activity of the enzyme.
• the temperature at which 50% of yeast display population is active as compared to the non-heat shocked population is the TM50.
• Recombination refers to a process of exchange of genetic information between two polynucleotides, including but not limited to, donor capture by non-homologous end joining (NHEJ) and homologous recombination.
• NHEJ non-homologous end joining
• HR homologous recombination
• HDR homology-directed repair
• This process requires nucleotide sequence homology, uses a“donor” molecule as a template to repair a“target” molecule (i.e., the one that experienced the double-strand break), and is variously known as“non-crossover gene conversion” or“short tract gene conversion,” because it leads to the transfer of genetic information from the donor to the target.
• a“donor” molecule i.e., the one that experienced the double-strand break
• “short tract gene conversion” “short tract gene conversion”
• such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and/or“synthesis- dependent strand annealing,” in which the donor is used to resynthesize genetic information that will become part of the target, and/or related processes.
• Such specialized HR often results in an alteration of the sequence of the target molecule such that part of or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.
• NHEJ non-homologous end joining
• cNHEJ The classical NHEJ pathway (cNHEJ) requires the KU/DNA-PKcs/Lig4/XRCC4 complex, ligates ends back together with minimal processing and often leads to precise repair of the break.
• Alternative NHEJ pathways cNHEJ
• exonucleases e.g., Trex2
• Trex2 may increase the likelihood of imprecise repair.
• “Cleavage” refers to the breakage of the covalent backbone of a DNA molecule.
• Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double- stranded cleavage are possible. Double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends.
• polypeptides and nuclease variants e.g, homing endonuclease variants, megaTALs, etc. contemplated herein are used for targeted double-stranded DNA cleavage. Endonuclease cleavage recognition sites may be on either DNA strand.
• exogenous molecule is a molecule that is not normally present in a cell, but that is introduced into a cell by one or more genetic, biochemical or other methods.
• exogenous molecules include, but are not limited to small organic molecules, protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules.
• lipid-mediated transfer i.e ., liposomes, including neutral and cationic lipids
• electroporation direct injection, cell fusion, particle bombardment, biopolymer nanoparticle, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer.
• An“endogenous” molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. Additional endogenous molecules can include proteins.
• A“gene,” refers to a DNA region encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences.
• a gene includes, but is not limited to, promoter sequences, enhancers, silencers, insulators, boundary elements, terminators, polyadenylation sequences, post-transcription response elements, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, replication origins, matrix attachment sites, and locus control regions.
• Gene expression refers to the conversion of the information, contained in a gene, into a gene product.
• a gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA.
• Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.
• genetically engineered or“genetically modified” refers to the chromosomal or extrachromosomal addition of extra genetic material in the form of DNA or RNA to the total genetic material in a cell. Genetic modifications may be targeted or non- targeted to a particular site in a cell ' s genome. In one embodiment, genetic modification is site specific. In one embodiment, genetic modification is not site specific.
• Genome editing refers to the substitution, deletion, and/or introduction of genetic material at a target site in the cell ' s genome, which restores, corrects, disrupts, and/or modifies expression and/or function of a gene or gene product.
• Genome editing contemplated in particular embodiments comprises introducing one or more nuclease variants into a cell to generate DNA lesions at or proximal to a target site in the cell ' s genome, optionally in the presence of a donor repair template.
• the term“gene therapy” refers to the introduction of extra genetic material into the total genetic material in a cell that restores, corrects, or modifies expression of a gene or gene product, or for the purpose of expressing a therapeutic polypeptide.
• introduction of genetic material into the cell ' s genome by genome editing that restores, corrects, disrupts, or modifies expression of a gene or gene product, or for the purpose of expressing a therapeutic polypeptide is considered gene therapy.
• nucleases may lack sufficient stability to be used in a clinical setting.
• Nuclease variants contemplated herein have been modified to increase thermostability and enzymatic activity to enable clinical use of previously unstable enzymes.
• the nuclease variants are suitable for genome editing a target site and comprise one or more DNA binding domains and one or more DNA cleavage domains (e.g., one or more endonuclease and/or exonuclease domains), and optionally, one or more linkers contemplated herein.
• the engineered nucleases comprise one or amino acid substitutions that increase thermostability and/or activity compared to a reference or parent nuclease.
• reprogrammed nuclease “engineered nuclease,” or“nuclease variant” are used interchangeably and refer to a nuclease comprising one or more DNA binding domains and one or more DNA cleavage domains, wherein the nuclease has been designed to bind and cleave a double-stranded DNA target sequence and modified to increase thermostability and/or activity of the nuclease.
• A“reference nuclease” or“parent nuclease” refers to a wild type nuclease, a nuclease found in nature, or a nuclease or variant that is modified to increase basal activity, affinity, specificity, selectivity, and/or stability to generate a subsequent nuclease variant.
• a nuclease variant binds comprises at least 1 amino acid substitution that increases the stability and/or activity of the variant relative to a parent nuclease. In particular embodiments, a nuclease variant binds comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid substitution that increases the stability and/or activity of the variant relative to a parent nuclease. Nuclease variants may be designed and/or modified from a naturally occurring nuclease or from an existing nuclease variant.
• a nuclease variant comprises increased thermostability and/or enzymatic activity compared to a parent nuclease variant.
• Nuclease variants contemplated in particular embodiments may further comprise one or more additional functional domains, e.g., an end-processing enzymatic domain of an end processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' - 5 ' exonuclease (e.g, Trex2), 5 ' flap endonuclease, helicase, template-dependent DNA polymerases or template-independent DNA polymerase activity.
• additional functional domains e.g., an end-processing enzymatic domain of an end processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' - 5 ' exonuclea
• nuclease variants reprogrammed to bind and cleave a target sequence and engineered to have increased thermostability include, but are not limited to, homing endonuclease (meganuclease) variants and megaTALs.
• the nuclease variants are reprogrammed to bind a target site or sequence in a gene selected from the group consisting of: HBA, HBB, HBG1, HBG2, BCL11 A, PCSK9, TCRA, TCRB, B2M, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, CUT A, AHR, PD-1, CTLA4, TIGIT, TGFBR2, LAG-3, TIM-3, BTLA, IL4R, IL6R, CXCR1, CXCR2, IL10R, IL13Ra2,
• Homing endonucleases are genome editing enzymes that can reprogrammed to bind and cleave selected target sites. However, some reprogrammed homing endonucleases were not sufficiently stable to allow further development or clinical use.
• the present inventors have unexpectedly discovered that certain amino acid positions in homing endonucleases affect stability, e.g, thermostability, of the enzymes; and further, substitution of amino acids at these positions can enhance enzyme stability compared to the parent enzyme, without sacrificing affinity or activity of the enzyme.
• a homing endonuclease or meganuclease is reprogrammed to introduce a double-strand break (DSB) in a target site and engineered to increase its thermostability, affinity, specificity, selectivity, and/or enzymatic activity.
• a homing endonuclease is reprogrammed to bind and cleave a target site and engineered to increase the enzyme’s thermostability relative to the thermostability of the enzyme from which it was designed.
• Homing endonuclease and“meganuclease” are used interchangeably and refer to naturally-occurring homing endonucleases that recognize 12-45 base-pair cleavage sites and are commonly grouped into five families based on sequence and structure motifs:
• LAGLIDADG LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D/E)XK.
• A“reference homing endonuclease,”“reference meganuclease,”“parent homing endonuclease,” or“parent meganuclease” refers to a wild type homing endonuclease, a homing endonuclease found in nature, or a homing endonuclease that has been modified to increase basal activity, affinity, and/or stability to generate a subsequent homing endonuclease variant.
• An“engineered homing endonuclease,”“reprogrammed homing endonuclease,” “homing endonuclease variant,”“engineered meganuclease,”“reprogrammed meganuclease,” or“meganuclease variant” refers to a homing endonuclease comprising one or more DNA binding domains and one or more DNA cleavage domains, wherein the homing endonuclease has been designed and/or modified from a parental or naturally occurring homing endonuclease to bind and cleave a DNA target sequence; has optionally undergone one or more rounds of refining affinity, selectivity, specificity, and/or activity; and has further been modified to have increased thermostability.
• Homing endonuclease variants may be designed and/or modified from a naturally occurring homing endonuclease or from another homing endonuclease variant.
• Homing endonuclease variants contemplated in particular embodiments may further comprise one or more additional functional domains, e.g., an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' -5 ' exonuclease (e.g, Trex2), 5 ' flap endonuclease, helicase, template dependent DNA polymerase or template-independent DNA polymerase activity.
• additional functional domains e.g., an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' -3 ' alkaline ex
• Homing endonuclease variants do not exist in nature and can be obtained by recombinant DNA technology or by random mutagenesis. Homing endonuclease variants may be obtained by making one or more amino acid alterations, e.g, mutating, substituting, adding, or deleting one or more amino acids, in a naturally occurring homing endonuclease or homing endonuclease variant.
• a homing endonuclease variant comprises one or more amino acid alterations to the DNA recognition interface to bind and cleave a selected target sequence and one or more amino acid substitutions to increase thermostability.
• Homing endonuclease variants contemplated in particular embodiments may further comprise one or more linkers and/or additional functional domains, e.g., an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' -5 ' exonuclease (e.g, Trex2), 5 ' flap endonuclease, helicase, template- dependent DNA polymerase or template-independent DNA polymerase activity.
• linkers and/or additional functional domains e.g., an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' -5 ' exonuclease (e.g, Trex2), 5 ' flap endonucleas
• homing endonuclease variants are introduced into a cell with an end-processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' -5 ' exonuclease (e.g, Trex2), 5 ' flap endonuclease, helicase, template-dependent DNA polymerase or template- independent DNA polymerase activity.
• an end-processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' -5 ' exonuclease (e.g, Trex2), 5 ' flap endonuclease, helicase, template-dependent DNA polymerase or template- independent DNA polymerase activity.
• the homing endonuclease variant and 3 ' processing enzyme may be introduced separately, e.g, in different vectors or separate mRNAs, or together, e.g, as a fusion protein, or in a polycistronic construct separated by a viral self- cleaving peptide or an IRES element.
• A“DNA recognition interface” refers to the homing endonuclease amino acid residues that interact with nucleic acid target bases as well as those residues that are adjacent.
• the DNA recognition interface comprises an extensive network of side chain-to-side chain and side chain-to-DNA contacts, most of which is necessarily unique to recognize a particular nucleic acid target sequence.
• the amino acid sequence of the DNA recognition interface corresponding to a particular nucleic acid sequence varies significantly and is a feature of any natural or homing endonuclease variant.
• a homing endonuclease variant contemplated in particular embodiments may be derived by constructing libraries of HE variants in which one or more amino acid residues localized in the DNA recognition interface of the natural homing endonuclease (or a previously generated homing endonuclease variant) are varied.
• the libraries may be screened for target cleavage activity against each target site using cleavage assays (see e.g, Jai journey el al. , 2009. Nuc. Acids Res. 37(20): 6871-6880).
• LAGLIDADG homing endonucleases are the most well studied family of homing endonucleases, are primarily encoded in archaea and in organellar DNA in green algae and fungi, and display the highest overall DNA recognition specificity.
• a reprogrammed LHE or LHE variant that has been engineered to enhance thermostability is an I-Onul HE variant (I-Onul LHE variant). See e.g., SEQ ID NOs: 8-14 and 16-18.
• a reprogrammed I-Onul HE or I-Onul HE variant engineered to increase thermostability is generated from a natural I-Onul, I-Onul HE variant, or biologically active fragment thereof (e.g ., SEQ ID NOs: 1-8 and 15).
• a reprogrammed I-Onul HE or I-Onul HE variant engineered to increase thermostability is generated from an existing I-Onul HE variant.
• a reprogrammed I-Onul HE or I-Onul HE variant engineered to increase thermostability comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid substitutions to increase
• thermostability of the enzyme compared to the thermostability of an existing parent I-Onul HE variant.
• an I-Onul HE variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or 7 amino acid substitutions of the following amino acid positions that have been identified to individually and collectively increase homing
• thermostability 114, A19, VI 16, F168, D208, N246, and L263 of representative I-Onul amino acid sequences (SEQ ID NOs: 1-8 and 15), biologically active fragments thereof, and/or further variants thereof.
• an I-Onul HE variant comprises amino acid substitutions of the following amino acid positions that have been identified to individually and collectively increase homing endonuclease thermostability: 114, A 19, FI 68, D208, and N246 of representative I-Onul amino acid sequences (SEQ ID NOs: 1-8 and 15), biologically active fragments thereof, and/or further variants thereof.
• the amino acid substitution for 114 is selected from the group consisting of: I14S, I14N, I14M, I14K, I14F, I14D, I14T, and I14V.
• the amino acid substitution for 114 is selected from the group consisting of: I14S, I14N, I14M, I14K, I14F, I14D, I14T, and I14V.
• the amino acid substitution for 114 is I14T or I14V.
• the amino acid substitution for A19 is selected from the group consisting of: A19C, A19D, A19I, A19L, A19S, A19T, and A19V.
• the amino acid substitution for A19 is A19T or A19V.
• the amino acid substitution for VI 16 is selected from the group consisting of: VI 16F, VI 16D, VI 16A, VI 16L, and VI 161.
• the amino acid substitution for VI 16 is VI 16L or VI 161.
• the amino acid substitution for FI 68 is selected from the group consisting of: F168H, F168Y, F168I, F168V, F168P, F168L, and F168S.
• the amino acid substitution for F168 is F168L and F168S.
• the amino acid substitution for D208 is selected from the group consisting of: D208N, D208Y, D208V, and D208E.
• the amino acid substitution for D208 is D208E.
• the amino acid substitution for F168 is F168L, and F168S.
• the amino acid substitution for N246 is selected from the group consisting of: N246H, N246I, N246D, N246R, N246S, N246T, N246V, N246Y, and N246K. In preferred embodiments, the amino acid substitution for N246 is N246K.
• the amino acid substitution for L263 is selected from the group consisting of: L263H, L263F, L263P, L263T, L263V, and L263R. In preferred embodiments, the amino acid substitution for L263 is L263R.
• an I-Onul HE variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or 7 amino acid substitutions of the following amino acid positions that have been identified to individually and collectively increase homing
• thermostability K108, K156, S176, E231, V261, E277, and G300 of representative I-Onul amino acid sequences (SEQ ID NOs: 1-8 and 15), biologically active fragments thereof, and/or further variants thereof.
• the amino acid substitution for K108 is selected from the group consisting of: K108E, K108N, K108Q, K108R, K108T, K108V, and K108M. In preferred embodiments, the amino acid substitution for K108 is K108M. In particular embodiments, the amino acid substitution for K156 is selected from the group consisting of: K156N, K156Q, K156R, K156T, K156V, K156I and K156E. In preferred embodiments, the amino acid substitution for K156 is K156I or K156E. In particular embodiments, the amino acid substitution for S176 is S176P, S176N or S176A. In preferred embodiments, the amino acid substitution for S176 is S176A.
• the amino acid substitution for E231 is selected from the group consisting of: E23 ID, E23 IV, E23 IK, and E231G. In preferred embodiments, the amino acid substitution for E231 is E23 IK or E231G.
• the amino acid substitution for V261 is selected from the group consisting of: V261D, V261G, V261I, V261L, V261 S, V261T and V261A. In preferred embodiments, the amino acid substitution for V261 is V261 A.
• the amino acid substitution for E277 is selected from the group consisting of: E277A, E277D, E277G,
• the amino acid substitution for E277 is E277K.
• the amino acid substitution for G300 is selected from the group consisting of: G300S, G300V, G300D, G300C, and G300R. In preferred embodiments, the amino acid substitution for G300 is G300R.
• an I-Onul HE variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10 amino acid substitutions of the following amino acid positions that have been identified to individually, and in some cases collectively, increase homing endonuclease thermostability: N31, N33, K52, Y97, K124,
• the amino acid substitution for N31 is selected from the group consisting of: N3 ID, N31H, N3 II, N31R, N3 IK, N31 S, N31 T and N31 Y.
• the amino acid substitution for N31 is N3 IK.
• the amino acid substitution for N33 is selected from the group consisting of: N33D, N33G, N33H, N33I, N33K, N33S, N33T and N33Y.
• the amino acid substitution for N33 is N33K.
• the amino acid substitution for K52 is selected from the group consisting of: K52Q, K52R, K52T, K52Y, K52N, K52E, and K52M. In preferred embodiments, the amino acid substitution for K52 is K52M. In particular embodiments, the amino acid substitution for Y97 is selected from the group consisting of: Y97H, Y97N, and Y97F. In particular embodiments, the amino acid substitution for Y97 is Y97F. In particular embodiments, the amino acid substitution for K124 is selected from the group consisting of: K124E, K124N, K124R and K124T. In particular embodiments, the amino acid substitution for K124 is K124N.
• the amino acid substitution for K147 is selected from the group consisting of: K147E, K147I, K147N, K147R and K147T.
• the amino acid substitution for K147 is K147I.
• the amino acid substitution for 1153 is selected from the group consisting of: I153D, I153H, I153K, I153T, I153Y, I153S, II 53 V and I153N.
• the amino acid substitution for 1153 is I153N.
• the amino acid substitution for K209 is selected from the group consisting of: K209E, K209M, K209N, K209Q and K209R.
• the amino acid substitution for K209 is K209R.
• the amino acid substitution for E264 is selected from the group consisting of: E264A, E264D, E264G, E264K, E264Q, E264R and E264V.
• the amino acid substitution for E264 is E264K.
• the amino acid substitution for D268 is selected from the group consisting of: D268A, D268E, D268G, D268H, D268N, D268V and D268Y.
• the amino acid substitution for D268 is D268N.
• an I-Onul HE variant comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 11 or more, 12 or more, 13 or more, or 14 amino acid substitutions of the following amino acid positions that have been identified to individually and collectively increase homing endonuclease thermostability: 114, A19, K108, VI 16, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300 of representative I-Onul amino acid sequences (SEQ ID NOs: 1-8 and 15), biologically active fragments thereof, and/or further variants thereof.
• an I-Onul HE variant comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 11 or more, 12 or more, 13 or more, or 14 amino acid substitutions of the following amino acid positions that have been identified to individually and collectively increase homing endonuclease thermostability: 114, A19, K108, VI 16, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300; and one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or 10 amino acid substitutions of the following amino acid positions that have been identified to individually, and in some cases collectively, increase homing endonuclease thermostability:
• an I-Onul HE variant that binds and cleaves a target sequence comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or 10 amino acid substitutions to increase thermostability and is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1-18 or a biologically active fragment thereof.
• an I-Onul HE variant has increased thermostability compared to a parent I-Onul LHE variant.
• an I-Onul HE variant has a TM50 about 5°C higher to about 35°C higher, about 10°C higher to about 35 °C higher, about 10°C higher to about 30°C higher, about 10°C higher to about 25°C higher about 15°C higher to about 35°C higher about 15°C higher to about 30°C, or about 15°C higher to about 25 °C higher than the TM50 of a parent I-Onul HE or reference I-Onul HE.
• an I-Onul HE variant has increased thermostability compared to a parent or reference I-Onul LHE variant.
• an I-Onul HE variant has a TM50 about 5°C higher, about 6°C higher, about 7°C higher, about 8°C higher, about 9°C higher, about 10°C higher, about 11°C higher, about 12°C higher, about 13°C higher, about 14°C higher, about 15°C, about 16°C higher, about 17°C higher, about 18°C higher, about 19°C higher, about 20°C higher, about 21°C higher, about 22°C higher, about 23 °C higher, about 24°C higher, about 25 °C higher, about 26°C higher, about 27°C higher, about 28°C higher, about 29°C higher, about 30°C higher, about 31°C higher, about 32°C higher, about 33°C higher, about 34°C higher, or about 35°C higher than the TM50 of a parent I-Onul
• an I-Onul HE variant comprising one or more mutations to enhance thermostability is reprogrammed to bind a target site or sequence in a gene selected from the group consisting of: HBA, HBB, HBGl, HBG2, BCL11 A, PCSK9, TCRA, TCRB, B2M, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, CIITA, AHR, PD-1, CTLA4, TIGIT, TGFBR2, LAG-3, TIM-3, BTLA, IL4R, IL6R, CXCR1, CXCR2, IL10R, IL13Ra2, TRAILRl, RCAS1R, and FAS.
• a gene selected from the group consisting of: HBA, HBB, HBGl, HBG2, BCL11 A, PCSK9, TCRA, TCRB, B2M, HLA-A, HLA-B, HLA-C, HLA-E
• MegaTALs are genome editing enzymes that combine the DNA binding properties of TAL DNA binding domains with the DNA binding and cleavage activities of homing endonucleases. Without wishing to be bound by any particular theory, it is believed that when a relatively unstable homing endonuclease is formatted as a megaTAL, the megaTAL is not inherently stabilized. Accordingly, introducing one or more stabilizing mutations into a homing endonuclease similarly stabilizes the corresponding megaTAL.
• a megaTAL comprises one or more TAL DNA binding domains and a homing endonuclease or meganuclease reprogrammed to introduce a double strand break (DSB) in a target site and engineered to increase its thermostability, affinity, specificity, selectivity, and/or enzymatic activity of the enzyme.
• the increased thermostability of a megaTAL comprising a homing endonuclease engineered to increase the enzyme’s thermostability is relative to the thermostability of a megaTAL comprising the homing endonuclease before engineering to increase its thermostability.
• A“megaTAL” refers to a polypeptide comprising a TALE DNA binding domain and a homing endonuclease variant that binds and cleaves a DNA target sequence and that has been engineered for increased thermostability, and optionally comprises one or more linkers and/or additional functional domains, e.g., an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' -5 ' exonuclease (e.g, Trex2), 5 ' flap endonuclease, helicase or template-independent DNA polymerase activity.
• an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' -5 ' exonucle
• A“reference megaTAL” or“parent megaTAL” refers to a megaTAL that comprises a TALE DNA binding domain and a wild type homing endonuclease, a homing endonuclease found in nature, or a homing endonuclease that is modified to increase basal activity, affinity, and/or stability to generate a subsequent homing endonuclease variant.
• a megaTAL can be introduced into a cell along with an end processing enzyme that exhibits 5 ' -3 ' exonuclease, 5 ' -3 ' alkaline exonuclease, 3 ' -5 ' exonuclease (e.g, Trex2), 5 ' flap endonuclease, helicase, template-dependent DNA
• the megaTAL and 3 ' processing enzyme may be introduced separately, e.g., in different vectors or separate mRNAs, or together, e.g, as a fusion protein, or in a polycistronic construct separated by a viral self cleaving peptide or an IRES element.
• A“TALE DNA binding domain” is the DNA binding portion of transcription activator-like effectors (TALE or TAL-effectors), which mimics plant transcriptional activators to manipulate the plant transcriptome ( see e.g., Kay etal. , 2007. Science 318:648-651).
• TALE DNA binding domains contemplated in particular embodiments are engineered de novo or from naturally occurring TALEs, e.g., AvrBs3 from Xanthomonas campestris pv.
• vesicatoria Xanthomonas gardneri, Xanthomonas translucens, Xanthomonas axonopodis, Xanthomonas perforans, Xanthomonas alfalfa, Xanthomonas citri, Xanthomonas euvesicatoria, and
• a megaTAL comprises a TALE DNA binding domain comprising one or more repeat units that are involved in binding of the TALE DNA binding domain to its corresponding target DNA sequence.
• a single“repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length.
• Each TALE DNA binding domain repeat unit includes 1 or 2 DNA-binding residues making up the Repeat Variable Di -Residue (RVD), typically at positions 12 and/or 13 of the repeat.
• RVD Repeat Variable Di -Residue
• a megaTAL comprises a TAL effector architecture comprising an“N-terminal domain (NTD)” polypeptide, one or more TALE repeat domains/units, a“C-terminal domain (CTD)” polypeptide, and a homing endonuclease variant.
• NTD N-terminal domain
• TALE repeats and/or CTD domains are from the same species.
• one or more of the NTD, TALE repeats, and/or CTD domains are from different species.
• a megaTAL comprises a homing endonuclease variant and a TALE DNA binding domain that binds a nucleotide sequence that is within about 2, 3, 4, 5, or 6 nucleotides upstream of the binding site of the reprogrammed homing endonuclease.
• a megaTAL contemplated herein comprises one or more TALE DNA binding repeat units and an I-Onul HE variant comprising increased
• thermostability and/or enzymatic activity compared to a parent I-Onul HE variant thermostability and/or enzymatic activity compared to a parent I-Onul HE variant.
• a megaTAL contemplated herein comprises an NTD, one or more TALE DNA binding repeat units, a CTD, and an I-Onul HE variant comprising increased thermostability and/or enzymatic activity compared to a parent I-Onul HE variant.
• a megaTAL contemplated herein comprises an NTD, about 9.5 to about 15.5 TALE DNA binding repeat units, and an I-Onul HE variant comprising increased thermostability and/or enzymatic activity compared to a parent I-Onul HE variant.
• a megaTAL contemplated herein comprises an NTD of about 122 amino acids to 137 amino acids, about 9.5, about 10.5, about 11.5, about 12.5, about 13.5, about 14.5, or about 15.5 binding repeat units, a CTD of about 20 amino acids to about 85 amino acids, and an I-Onul HE variant comprising increased thermostability and/or enzymatic activity compared to a parent I-Onul HE variant.
• any one of, two of, or all of the NTD, DNA binding domain, and CTD can be designed from the same species or different species, in any suitable combination.
• PCSK9 TCRA, TCRB, B2M, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, CIITA, AHR, PD-1, CTLA4, TIGIT, TGFBR2, LAG-3, TIM-3, BTLA, IL4R, IL6R, CXCR1,
• Genome editing compositions and methods contemplated in particular embodiments comprise editing cellular genomes using an I-Onul HE variant comprising increased thermostability and/or enzymatic activity compared to a parent I-Onul HE variant and one or more copies of an end-processing enzyme.
• a single polynucleotide encodes a homing endonuclease variant and an end-processing enzyme, separated by a linker, a self-cleaving peptide sequence, e.g., 2 A sequence, or by an IRES sequence.
• genome editing compositions comprise a polynucleotide encoding a nuclease variant and a separate polynucleotide encoding an end-processing enzyme.
• genome editing compositions comprise a polynucleotide encoding a homing endonuclease variant end-processing enzyme single polypeptide fusion in addition to a tandem copy of the end-processing enzyme separated by a self-cleaving peptide.
• An end-processing enzyme may modify ends at endonuclease cut sites or at ends generated by other chemical or mechanical means, such as shearing (for example by passing through fine-gauge needle, heating, sonicating, mini bead tumbling, and nebulizing), ionizing radiation, ultraviolet radiation, oxygen radicals, chemical hydrolysis and chemotherapy agents.
• genome editing compositions and methods contemplated in particular embodiments comprise editing cellular genomes using and an I-Onul HE variant comprising increased thermostability and/or enzymatic activity compared to a parent I-Onul HE variant or megaTAL and a DNA end-processing enzyme.
• DNA end-processing enzyme refers to an enzyme that modifies the exposed ends of DNA.
• a DNA end-processing enzyme may modify blunt ends or staggered ends (ends with 5 ' or 3 ' overhangs).
• a DNA end-processing enzyme may modify single stranded or double stranded DNA.
• a DNA end-processing enzyme may modify ends at endonuclease cut sites or at ends generated by other chemical or mechanical means, such as shearing (for example by passing through fine-gauge needle, heating, sonicating, mini bead tumbling, and nebulizing), ionizing radiation, ultraviolet radiation, oxygen radicals, chemical hydrolysis and chemotherapy agents.
• DNA end-processing enzyme may modify exposed DNA ends by adding one or more nucleotides, removing one or more nucleotides, removing or modifying a phosphate group and/or removing or modifying a hydroxyl group.
• DNA end-processing enzymes suitable for use in particular embodiments contemplated herein include but are not limited to: 5 ' -3 ' exonucleases, 5 ' -3 ' alkaline exonucleases, 3 ' -5 ' exonucleases, 5 ' flap endonucleases, helicases, phosphatases, hydrolases and template-independent DNA polymerases.
• DNA end-processing enzymes suitable for use in particular embodiments contemplated herein include, but are not limited to, Trex2, Trexl, Trexl without transmembrane domain, Apollo, Artemis, DNA2, Exol, ExoT, EcoPI, Fenl, Fanl, Mrell, Rad2, Rad9, TdT (terminal deoxynucleotidyl transferase), PNKP, RecE, RecJ, RecQ, Lambda exonuclease, Sox, Vaccinia DNA polymerase, exonuclease I, exonuclease III, exonuclease VII, NDK1, NDK5, NDK7, NDK8, WRN, T7 -exonuclease Gene 6, avian myeloblastosis virus integration protein (IN), Bloom, Antartic Phophatase, Alkaline
• Phosphatase Poly nucleotide Kinase (PNK), Apel, Mung Bean nuclease, Hexl, TTRAP (TDP2), Sgsl, Sae2, CUP, Pol mu, Pol lambda, MUS81, EME1, EME2, SLX1, SLX4 and UL- 12
• Polypeptides include“polypeptide variants.”
• Polypeptide variants may differ from a naturally occurring polypeptide in one or more amino acid substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more amino acids of the above polypeptide sequences. For example, in particular embodiments, it may be desirable to improve the biological properties of a homing endonuclease, megaTAL or the like that binds and cleaves a target site by introducing one or more substitutions, deletions, additions and/or insertions into the polypeptide that increase thermal stability.
• polypeptide variants include homing endonucleases or megaTALs that have been engineered to increase their thermostability and/or activity.
• I-Onul HE polypeptides or fragments thereof can be reprogrammed to bind and cleave a target site.
• a reprogrammed I-Onul HE variant has relatively low thermostability and/or activity compared to a parent I-Onul HE.
• an I-Onul homing endonuclease or fragment thereof is engineered to bind and cleave a target site and to increase thermostability and/or activity of the enzyme.
• the biological activity is binding affinity and/or cleavage activity for a target sequence.
• a polypeptide fragment can comprise an amino acid chain at least 5 to about 1700 amino acids long. It will be appreciated that in certain embodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
• a polypeptide comprises a biologically active fragment of a homing endonuclease variant.
• a polypeptide comprises a biologically active fragment of a homing endonuclease variant or a megaTAL.
• the polypeptides set forth herein may comprise one or more amino acids denoted as“X.”“X’ if present in an amino acid SEQ ID NO, refers to any amino acid.
• One or more“X” residues may be present at the N- and C-terminus of an amino acid sequence set forth in particular SEQ ID NOs contemplated herein. If the“X’ amino acids are not present the remaining amino acid sequence set forth in a SEQ ID NO may be considered a biologically active fragment.
• a biologically active fragment may comprise an N-terminal truncation and/or C- terminal truncation.
• a biologically active fragment lacks or comprises a deletion of the 1, 2, 3, 4, 5, 6, 7, or 8 N-terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence, more preferably a deletion of the 4 N-terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence.
• a biologically active fragment lacks or comprises a deletion of the 1, 2, 3, 4, or 5 C-terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence, more preferably a deletion of the 2 C-terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence.
• a biologically active fragment lacks or comprises a deletion of the 4 N-terminal amino acids and 2 C-terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence.
• Fusion polypeptides contemplated in particular embodiments include fusion polypeptides.
• fusion polypeptides and polynucleotides encoding fusion polypeptides are provided.
• Fusion polypeptides and fusion proteins refer to a polypeptide having at least two, three, four, five, six, seven, eight, nine, or ten polypeptide segments.
• two or more polypeptides can be expressed as a fusion protein that comprises one or more self-cleaving polypeptide sequences as disclosed elsewhere herein.
• a fusion protein contemplated herein comprises one or more DNA binding domains and one or more nucleases, and one or more linker and/or self-cleaving polypeptides.
• the polypeptides of the fusion protein can be in any order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired activity of the fusion polypeptide is preserved. Fusion polypeptides may be produced by chemical synthetic methods or by chemical linkage between the two moieties or may generally be prepared using other standard techniques.
• Fusion polypeptides may optionally comprise a linker that can be used to link the one or more polypeptides or domains within a polypeptide.
• a peptide linker sequence may be employed to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds into its appropriate secondary and tertiary structures so as to allow the polypeptide domains to exert their desired functions.
• Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques in the art.
• Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
• Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence.
• Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea el al. , Gene 40:39-46, 1985; Murphy et al. , Proc. Natl. Acad. Sci.
• flexible linkers can be rationally designed using a computer program capable of modeling both DNA-binding sites and the peptides themselves (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91 : 11099-11103 (1994) or by phage display methods.
• Suitable protease cleavages sites and self-cleaving peptides are known to the skilled person (see, e.g., in Ryan el al, 1997. J. Gener. Virol. 78, 699-722; Scymczak el al. (2004) Nature Biotech. 5, 589-594).
• Exemplary protease cleavage sites include, but are not limited to the cleavage sites of potyvirus Nla proteases (e.g, tobacco etch virus protease), potyvirus HC proteases, potyvirus PI (P35) proteases, byovirus Nla proteases, byovirus RNA-2-encoded proteases, aphthovirus L proteases, enterovirus 2A proteases, rhinovirus 2A proteases, picoma 3C proteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase.
• potyvirus Nla proteases e.g, tobacco etch virus protease
• potyvirus HC proteases e.g, tobacco etch
• TEV tobacco etch virus protease cleavage sites
• EXXYXQ(G/S) SEQ ID NO: 35
• ENLYFQG SEQ ID NO: 36
• ENLYFQS SEQ ID NO: 37
• X represents any amino acid (cleavage by TEV occurs between Q and G or Q and S).
• the polypeptide cleavage signal is a viral self-cleaving peptide or ribosomal skipping sequence.
• ribosomal skipping sequences include but are not limited to: a 2A or 2A-like site, sequence or domain (Donnelly el al., 2001. J. Gen. Virol. 82: 1027-1041).
• the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.
• the viral 2A peptide is selected from the group consisting of: a foot-and-mouth disease virus (FMDV) 2A peptide, an equine rhinitis A virus (ERAV) 2A peptide, a Thosea asigna virus (TaV) 2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.
• FMDV foot-and-mouth disease virus
• EAV equine rhinitis A virus
• TaV Thosea asigna virus
• PTV-1 porcine teschovirus-1
• Exemplary 2A sites include the following sequences:
• Polynucleotides include, but are not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, genomic RNA (gRNA), plus strand RNA (RNA(+)), minus strand RNA (RNA(-)), tracrRNA, crRNA, single guide RNA (sgRNA), synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.
• pre-mRNA pre-messenger RNA
• mRNA messenger RNA
• RNA short interfering RNA
• shRNA short hairpin RNA
• miRNA microRNA
• ribozymes genomic RNA (gRNA), plus strand RNA (RNA(+)), minus strand RNA (RNA(-)), tracrRNA, crRNA, single guide RNA (sg
• Polynucleotides refer to a polymeric form of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide, as well as all intermediate lengths.
• polynucleotides may be codon-optimized.
• codon-optimized refers to substituting codons in a polynucleotide encoding a polypeptide in order to increase the expression, stability and/or activity of the polypeptide.
• nucleotide refers to a heterocyclic nitrogenous base in N- glycosidic linkage with a phosphorylated sugar.
• Nucleotides are understood to include natural bases, and a wide variety of art-recognized modified bases. Such bases are generally located at the 1 ' position of a nucleotide sugar moiety.
• Nucleotides generally comprise a base, sugar and a phosphate group.
• RNA ribonucleic acid
• DNA deoxyribonucleic acid
• deoxyribose i.e., a sugar lacking a hydroxyl group that is present in ribose.
• polynucleotide variant and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, or modified, or replaced with different nucleotides.
• certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide.
• sequence identity refers to the extent that sequences are identical on a nucleotide-by- nucleotide basis or an amino acid-by -amino acid basis over a window of comparison.
• a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g A, T, C, G, I) or the identical amino acid residue (e.g, Ala,
• Pro Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met
• sequence relationships between two or more polynucleotides or polypeptides include“reference sequence,”“comparison window,”“sequence identity,” “percentage of sequence identity,” and“substantial identity”.
• A“reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length.
• A“comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
• the comparison window may comprise additions or deletions ⁇ i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• an“isolated polynucleotide,” as used herein, refers to a polynucleotide that has been purified from the sequences which flank it in a naturally -occurring state, e.g, a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment.
• an“isolated polynucleotide” refers to a complementary DNA
• mRNAs encoding a homing endonuclease variant or megaTAL comprise a 5 ' cap that is a 7-methyl guanylate (“m 7 G”) linked via a triphosphate bridge to the 5 ' -end of the first transcribed nucleotide, resulting in m 7 G(5 ' )ppp(5 ' )N, where N is any nucleoside.
• m 7 G 7-methyl guanylate
• an mRNA comprises a m 7 G(5')ppp(5')G cap.
• an mRNA encoding a homing endonuclease variant or megaTAL comprises one or more modified nucleosides.
• pseudouridine pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5- carboxymethyl -uridine, 1-carboxymethyl -pseudouridine, 5-propynyl -uridine, 1-propynyl- pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl -pseudouridine, 5-taurinomethyl-2- thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl -uridine, 1 -methyl -pseudouridine, 4-thio l-methyl-pseudouridine, 2-thio-l -methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouri dine, 2-
• an mRNA encoding a homing endonuclease variant or megaTAL comprises one or more modified nucleosides selected from the group consisting of:
• pseudouridine pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5- carboxymethyl -uridine, 1-carboxymethyl -pseudouridine, 5-propynyl -uridine, 1-propynyl- pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl -pseudouridine, 5-taurinomethyl-2- thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl -uridine, 1 -methyl -pseudouridine, 4-thio l-methyl-pseudouridine, 2-thio-l -methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouri dine, 2-
• an mRNA encoding a homing endonuclease variant or megaTAL comprises one or more modified nucleosides selected from the group consisting of: 5-aza- cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4- methylcytidine, 5-hydroxymethylcytidine, 1 -methyl -pseudoisocyti dine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio- 1 -methyl -pseudoi socytidine, 4-thio- 1 -methyl- 1 -deaza-pseudoi socytidine, 1 -methyl- 1 - deaza-pseu
• an mRNA encoding a homing endonuclease variant or megaTAL comprises one or more modified nucleosides selected from the group consisting of: inosine, 1- methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio- guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6- thio-7-m ethyl -guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2- methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1- methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine
• an mRNA comprises one or more pseudouridines, one or more 5- methyl-cytosines, and/or one or more 5-methyl-cytidines.
• an mRNA comprises one or more 5-methyl-cytosines.
• an mRNA encoding a homing endonuclease variant or megaTAL comprises a poly(A) tail to help protect the mRNA from exonuclease degradation, stabilize the mRNA, and facilitate translation.
• an mRNA comprises a 3 ' poly(A) tail structure.
• the length of the poly(A) tail is at least about 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or at least about 500 or more adenine nucleotides or any intervening number of adenine nucleotides.
• the length of the poly(A) tail is at least about 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,
• the length of the poly(A) tail is about 10 to about 500 adenine nucleotides, about 50 to about 500 adenine nucleotides, about 100 to about 500 adenine nucleotides, about 150 to about 500 adenine nucleotides, about 200 to about 500 adenine nucleotides, about 250 to about 500 adenine nucleotides, about 300 to about 500 adenine nucleotides, about 50 to about 450 adenine nucleotides, about 50 to about 400 adenine nucleotides, about 50 to about 350 adenine nucleotides, about 100 to about 500 adenine nucleotides, about 100 to about 450 adenine nucleotides, about 100 to about 400 adenine nucleotides, about 100 to about 350 adenine nucleotides, about 100 to about 300 adenine nucleotides, about 150 to about 500 adenine nucleot
• Polynucleotide sequences can be annotated in the 5 ' to 3 ' orientation or the 3 ' to 5 ' orientation.
• the 5 ' to 3 ' strand is designated the“sense,”“plus,” or“coding” strand because its sequence is identical to the sequence of the pre-messenger (pre-mRNA) [except for uracil (U) in RNA, instead of thymine (T) in DNA]
• pre-mRNA pre-messenger
• the complementary 3' to 5' strand which is the strand transcribed by the RNA polymerase is designated as“template,”“antisense,”“minus,” or“non-coding” strand.
• the term“reverse orientation” refers to a 5' to 3' sequence written in the 3 ' to 5 ' orientation or a 3 ' to 5 ' sequence written in the 5 ' to 3 ' orientation.
• “complementary” and“complementarity” refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules.
• the complementary strand of the DNA sequence 5' A G T C A T G 3' is 3' T C A G T A C 5'.
• the latter sequence is often written as the reverse complement with the 5 ' end on the left and the 3 ' end on the right, 5 C A T G A C T 3 ' .
• a sequence that is equal to its reverse complement is said to be a palindromic sequence.
• Complementarity can be“partial,” in which only some of the nucleic acids ' bases are matched according to the base pairing rules. Or, there can be“complete” or “total” complementarity between the nucleic acids.
• polynucleotides contemplated in particular embodiments may be combined with other DNA sequences, such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g LoxP, FRT, and Att sites), termination codons,
• promoters and/or enhancers untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g LoxP, FRT, and Att sites), termination codons,
• transcriptional termination signals post-transcription response elements, and polynucleotides encoding self-cleaving polypeptides, epitope tags, as disclosed elsewhere herein or as known in the art, such that their overall length may vary considerably. It is therefore contemplated in particular embodiments that a polynucleotide fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
• Polynucleotides can be prepared, manipulated, expressed and/or delivered using any of a variety of well-established techniques known and available in the art.
• a nucleotide sequence encoding the polypeptide can be inserted into appropriate vector.
• a desired polypeptide can also be expressed by delivering an mRNA encoding the polypeptide into the cell.
• vectors include, but are not limited to plasmid, autonomously replicating sequences, and transposable elements, e.g., Sleeping Beauty, PiggyBac.
• vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses.
• YAC yeast artificial chromosome
• BAC bacterial artificial chromosome
• PAC PI -derived artificial chromosome
• bacteriophages such as lambda phage or M13 phage
• animal viruses include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses.
• viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g, herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g, SV40).
• retrovirus including lentivirus
• adenovirus e.g, adeno-associated virus
• herpesvirus e.g, herpes simplex virus
• poxvirus baculovirus
• papillomavirus papillomavirus
• papovavirus e.g, SV40
• expression vectors include, but are not limited to pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DESTTM, pLenti6/V5- DESTTM, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.
• coding sequences of polypeptides disclosed herein can be ligated into such expression vectors for the expression of the polypeptides in mammalian cells.
• the vector is an episomal vector or a vector that is maintained extrachromosomally.
• the term“episomal” refers to a vector that is able to replicate without integration into host ' s chromosomal DNA and without gradual loss from a dividing host cell also meaning that said vector replicates extrachromosomally or episomally.
• “Expression control sequences,”“control elements,” or“regulatory sequences” present in an expression vector are those non-translated regions of the vector including but not limited to an origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgamo sequence or Kozak sequence) introns, post-transcriptional regulatory elements, a polyadenylation sequence, 5 ' and 3 ' untranslated regions, which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters may be used.
• operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
• the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, and/or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide- of-interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
• vectors comprise a polyadenylation sequence 3' of a polynucleotide encoding a polypeptide to be expressed.
• the term“polyA site” or“polyA sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase P.
• Polyadenylation sequences can promote mRNA stability by addition of a polyA tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency.
• Cleavage and polyadenylation is directed by a poly(A) sequence in the RNA.
• the core poly(A) sequence for mammalian pre-mRNAs has two recognition elements flanking a cleavage-polyadenylation site. Typically, an almost invariant AAUAAA hexamer lies 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5' cleavage product.
• the core poly(A) sequence is an ideal polyA sequence (e.g, AATAAA, ATTAAA, AGTAAA).
• the poly(A) sequence is an SV40 polyA sequence, a bovine growth hormone polyA sequence (BGHpA), a rabbit b-globin polyA sequence (rPgpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art.
• the poly(A) sequence is synthetic.
• polynucleotides encoding one or more nuclease variants, megaTALs, end-processing enzymes, or fusion polypeptides may be introduced into a cell by both non-viral and viral methods.
• the term“vector” is used herein to refer to a nucleic acid molecule capable transferring or transporting another nucleic acid molecule.
• the transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule.
• a vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA.
• non-viral vectors are used to deliver one or more polynucleotides contemplated herein to a T cell.
• non-viral vectors include, but are not limited to plasmids (e.g, DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.
• Illustrative methods of non-viral delivery of polynucleotides contemplated in particular embodiments include, but are not limited to: electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran-mediated transfer, gene gun, and heat-shock.
• adeno-associated virus retrovirus
• retrovirus e.g, lentivirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.
• compositions contemplated in particular embodiments may comprise one or more homing endonuclease variants and megaTALs engineered to increase thermostability and/or enzymatic activity, polynucleotides, vectors comprising same, and genome editing
• compositions and genome edited cell compositions are useful for editing a target site in the human genome in a cell or a population of cells.
• An“isolated cell” refers to a non-naturally occurring cell, e.g, a cell that does not exist in nature, a modified cell, an engineered cell, a recombinant cell etc., that has been obtained from an in vivo tissue or organ and is substantially free of extracellular matrix.
• the term“population of cells” refers to a plurality of cells that may be made up of any number and/or combination of homogenous or heterogeneous cell types.
• a genome editing composition is used to edit a target site in an embryonic stem cell or an adult stem or progenitor cell.
• a genome editing composition is used to edit a target site in a stem or progenitor cell selected from the group consisting of: mesodermal stem or progenitor cells, endodermal stem or progenitor cells, and ectodermal stem or progenitor cells.
• mesodermal stem or progenitor cells include but are not limited to bone marrow stem or progenitor cells, umbilical cord stem or progenitor cells, adipose tissue derived stem or progenitor cells, hematopoietic stem or progenitor cells (HSPCs), mesenchymal stem or progenitor cells, muscle stem or progenitor cells, kidney stem or progenitor cells, osteoblast stem or progenitor cells, chondrocyte stem or progenitor cells, and the like.
• HSPCs hematopoietic stem or progenitor cells
• Illustrative examples of ectodermal stem or progenitor cells include but are not limited to neural stem or progenitor cells, retinal stem or progenitor cells, skin stem or progenitor cells, and the like.
• Illustrative examples of endodermal stem or progenitor cells include but are not limited to liver stem or progenitor cells, pancreatic stem or progenitor cells, epithelial stem or progenitor cells, and the like.
• a genome editing composition is used to edit a target site in a bone cell, osteocyte, osteoblast, adipose cell, chondrocyte, chondroblast, muscle cell, skeletal muscle cell, myoblast, myocyte, smooth muscle cell, bladder cell, bone marrow cell, central nervous system (CNS) cell, peripheral nervous system (PNS) cell, glial cell, astrocyte cell, neuron, pigment cell, epithelial cell, skin cell, endothelial cell, vascular endothelial cell, breast cell, colon cell, esophagus cell, gastrointestinal cell, stomach cell, colon cell, head cell, neck cell, gum cell, tongue cell, kidney cell, liver cell, lung cell, nasopharynx cell, ovary cell, follicular cell, cervical cell, vaginal cell, uterine cell, pancreatic cell, pancreatic parenchymal cell, pancreatic duct cell, pancreatic islet cell, prostate cell, penile cell, gonadal
• the I-Onul HE variant may be in the form of an mRNA that is introduced into a cell via polynucleotide delivery methods disclosed supra , e.g, electroporation, lipid nanoparticles, etc.
• a composition comprising an mRNA encoding an I-Onul HE variant or megaTAL, and optionally a 3 -5' exonuclease, is introduced in a cell via polynucleotide delivery methods disclosed supra.
• the composition may be used to generate a genome edited cell or population of genome edited cells by error prone NHEJ.
• the compositions contemplated herein comprise a donor repair template.
• the composition may be delivered to a cell that expresses or will express an I- Onul HE variant, and optionally an end-processing enzyme.
• the composition may be delivered to a cell that expresses or will express an I-Onul HE variant or megaTAL, and optionally a 3 ' -5 ' exonuclease.
• Expression of the gene editing enzymes in the presence of the donor repair template can be used to generate a genome edited cell or population of genome edited cells by HDR.
• a composition comprises a cell containing one or more homing endonuclease variants and megaTALs engineered to increase thermostability and/or enzymatic activity, polynucleotides, vectors comprising same.
• the cells may be autologous/autogeneic (“self’) or non-autologous (“non-self,” e.g, allogeneic, syngeneic or xenogeneic).“Autologous,” as used herein, refers to cells from the same subject.
• “Allogeneic,” as used herein, refers to cells of the same species that differ genetically to the cell in comparison.“Syngeneic,” as used herein, refers to cells of a different subject that are genetically identical to the cell in comparison.“Xenogeneic,” as used herein, refers to cells of a different species to the cell in comparison.
• the cells are obtained from a mammalian subject. In a more preferred embodiment, the cells are obtained from a primate subject, optionally a non-human primate. In the most preferred embodiment, the cells are obtained from a human subject.
• compositions contemplated herein comprise a population of cells, an I-Onul HE variant, and optionally, a donor repair template.
• compositions contemplated herein comprise a population of cells, an I-Onul HE variant, an end-processing enzyme, and optionally, a donor repair template.
• the I-Onul HE and/or end-processing enzyme may be in the form of an mRNA that is introduced into the cell via polynucleotide delivery methods disclosed supra.
• compositions contemplated herein comprise a population of cells, an I-Onul HE variant or megaTAL engineered to increase thermostability and/or activity of the enzyme, and optionally, a donor repair template.
• the compositions contemplated herein comprise a population of cells, an I-Onul HE variant or megaTAL, a 3 -5 ' exonuclease, and optionally, a donor repair template.
• the I- Onul HE variant, megaTAL, and/or 3 '-5 ' exonuclease may be in the form of an mRNA that is introduced into the cell via polynucleotide delivery methods disclosed supra.
• a yeast surface display assay was used to identify mutations that increase the thermostability of LAGLIDADG homing endonucleases.
• I-Onul e.g., SEQ ID NO: 1
• engineered nucleases e.g, SEQ ID NOs: 6 and 7
• each yeast population was subjected to heat shock at multiple temperatures for 15 minutes, and the percent of nuclease expressing cells still able to cleave its DNA target was measured by flow cytometry.
• This assay generates a standard protein melt curve with an associated TMso value for each endonuclease.
• I-Onul derived homing endonucleases were subjected to random mutagenesis via PCR over the entire open reading frame. These mutant libraries were expressed in yeast and sorted for active nuclease activity after heat shock at or above the TMso of the library. After two rounds of sorting, HE variants were sequenced with either PacBio or Sanger sequencing to determine the identity and frequency of mutations at each position. The cumulative mutation frequencies are shown in Figure 2 as a stacked bar graph, and top mutations at each position are shown in Table 2.
• Figure 3 A shows the thermostability on a parent I-Onul HE variant targeting BCL11 A (SEQ ID NO 8), I-Onul HE variants with single amino acid substitutions and their impact on thermostability (SEQ ID NOs: 9-12), and a representative I-Onul HE variant (SEQ ID NO 13) generated from random mutagenesis that has a slightly higher overall TMso than the individual variants.
• Figure 3 A shows the thermostability on a parent I-Onul HE variant targeting BCL11 A (SEQ ID NO 8), I-Onul HE variants with single amino acid substitutions and their impact on thermostability (SEQ ID NOs: 9-12), and a representative I-Onul HE variant (SEQ ID NO 13) generated from random mutagenesis that has a slightly higher overall TMso than the individual variants.
• Figure 3 A shows the thermostability on a parent I-Onul HE variant targeting BCL11 A (SEQ ID NO 8), I-Onul HE
• Combinatorial mutations derived from either random mutagenesis or directed mutagenesis increase I-Onu HE variant thermostability.
• the mutations from the BCL11 A A5 I-OnuI HE variant were transferred to I-OnuI HE variants that target PDCD-1 (SEQ ID NO: 6), TCRa (SEQ ID NO: 7), or CBLB (SEQ ID NO: 15).
• the TMso of an I-OnuI HE variant that targets the human PDCD-1 gene was increased by 16°C (SEQ ID NO: 16)
• the TMso of an I-OnuI HE variant that targets the human TCRa gene was increased by ⁇ 14°C (SEQ ID NO: 17)
• the TMso of an I-OnuI HE variant that targets the human CBLB gene was increased by 19°C (SEQ ID NO 18).
• I-OnuI HE variant thermostability was also assessed by measuring expression of the enzymes in 293 T cells. Briefly, each I-OnuI HE variant was formatted as an mRNA with a c- terminal HA tag followed by T2A GFP to track mRNA transfection efficiency ( Figure 5 A). mRNA was prepared by in vitro transcription and co-transcriptionally capped with Anti- Reverse Cap Analog and enzymatically polyadenylated with poly(A) polymerase. The mRNA was purified and equal amounts electroporated into 293T cells (protein SEQ ID NOs 2,8,14: mRNA SEQ ID NOs: 19-21). At each time point cells were run on a cytometer to measure GFP expression, as well as lysed and frozen for western blot analysis.
• the dynamics for GFP protein expression were similar for each polycistronic mRNA; but the amount of HA tagged HE protein varied (Figure 5B and 5C).
• the amount of stabilized BCL11 A A5 HE protein was significant higher compared to the amount of parent BCL11 A HE protein, when normalized to the actin loading control.
• the amount of parent BCL11 A HE protein was undetectable; in contrast, the BCL11 A A5 HE variant was still near its peak expression levels.
• the stabilized BCL11 A A5 HE variant was present in the cells for almost twice as long as the parent HE.
• the effects of the stabilizing mutations on PDCD-1 editing was measured by comparing editing rates of a parental megaTAL that lacks the stabilizing mutations (SEQ ID NO: 22) with a megaTAL comprising stabilizing mutations (SEQ ID NO: 23).
• megaTAL mRNA was prepared by in vitro transcription, co-transcriptionally capped with Anti-Reverse Cap Analog (ARCA) and enzymatically polyadenylated with poly(A) polymerase. Purified mRNA was used to measure PDCD-1 editing efficiency in primary human T cells.
• Primary human peripheral blood mononuclear cells (PBMCs) from two donors were activated with anti-CD3 and anti-CD28 antibodies and cultured in the presence of 250U/mL IL-2.

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