Recombinant enzymes for precisely inserting DNA sequences into eukaryotic cells

Abstract

The construct for site-specific insertion of DNA fragments and transgenes into the genome comprises a recombinant enzyme. This enzyme contains a modified HIV-1 integrase (HIV IN) tetramer with multiple mutations favoring a heterodimer structure in which the first and second outer monomers are bound to the first and second inner monomers, respectively. A transcription activator-like effector (TALE) is bound to the N-terminal region of the outer monomer via a polypeptide linker. The two TALEs confer sequence specificity to the enzyme and prevent non-specific interactions with the target DNA structure. The modified HIV IN monomer ensures that the TALE binds to the outermost monomer and that the TALE is positioned relative to the catalytic region of HIV IN. A polynucleotide is bound to the inner monomer of HIV IN. When the TALE is transported to the nucleus, either alone or within a lentiviral capsid, it interacts with the DNA to localize HIV IN, which then incorporates the polynucleotide.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 639,007, filed on April 26, 2024, and U.S. Provisional Patent Application No. 63 / 462,377, filed on April 27, 2023, and these applications are incorporated by reference in their entirety as if fully disclosed herein.

Background Art

[0002] In the field of genomics, there are various mechanisms that have evolved naturally to insert transgenes and exogenous DNA fragments into the genome of an organism to achieve specific purposes. Many of these enzyme mechanisms have been elucidated, and their purpose is to transfer genetic material to a new location within the genome. Unfortunately, many of these enzymes are derived from transposons or viruses and do not support the lifespan of the cell nor its genomic stability. For this reason, many mechanisms in nature are not accurate enough to reliably insert DNA into a specific locus without substantially accompanying off - target mutations. There are other mechanisms such as various recombinases, but their ability to insert DNA also has limitations. This is because a target sequence needs to already exist within the genome for insertion to occur, which is usually because the first insertion needs to be made.

[0003] Attempts have been made to modify specific proteins to increase sequence specificity for DNA insertions, but in most cases, these have not demonstrated the precision required for many clinical applications or have been unable to effectively transfect cells in vivo. In recent years, methods have been developed for inserting, editing, or removing small regions of DNA on a scale of less than 100 nucleotides, such as prime editors, twin prime editors, and base editors. However, almost all of these methods rely in some way on the same Cas backbone and tend to suffer from similar problems with precision. Even "highly precise" variations like Sniper-Cas9 still have off-target mutation rates of up to 10% in some cases, and other variations show a clear trade-off between improved precision and maintaining modification efficiency.

[0004] Precisely editing genomic material is difficult, and the risk of off-target modifications is particularly high in vivo. Therefore, current gene therapies (especially those involving CRISPR or its derivatives) are limited to the most severe cases, require cell extraction before modification, and minimize the overall usefulness of these therapies. Consequently, there is a need for gene editing systems and methods that enable site-specific in vivo genome editing, thereby allowing for reliable gene therapy for patients with genetic abnormalities. [Overview of the project]

[0005] Aspects of this disclosure relate to recombinant enzyme gene editing systems. In some embodiments, the recombinant enzyme gene editing system comprises a recombinant enzyme including HIV integrase (HIV IN) and two or more transcription activator-like effectors (TALEs) bound to HIV IN. In some embodiments, the recombinant enzyme comprises one or more polynucleotides bound to HIV IN to form a gene editing construct. In some embodiments, the gene editing construct is provided in a lentiviral capsid, as part of a lentiviral vector, or in combination thereof.

[0006] In some embodiments, HIV IN comprises four monomer subunits, including a first internal monomer and a second internal monomer, and a first external monomer and a second external monomer. In some embodiments, at least two of the monomer subunits have a TALE attached to its N-terminal domain. In some embodiments, the first TALE is attached to the first external monomer of HIV IN by a first polypeptide linker, and the second TALE is attached to the second external monomer of HIV IN by a second polypeptide linker. In some embodiments, the polypeptide linker is rigid. In some embodiments, the polypeptide linker is flexible. In some embodiments, the polypeptide linker is composed of glycine residues. In some embodiments, the polypeptide linker comprises repeats of a GGGS domain, an EAAAK domain, or a combination thereof. In some embodiments, the polypeptide linker comprises alternating repeats of a GGGS domain, an EAAAK domain, or a combination thereof. In some embodiments, the polypeptide linker comprises GGGS GGGS GGGS GGGS (SEQ ID NO: 5). In some embodiments, the first polypeptide linker and the second polypeptide linker are composed of glycine residues. In some embodiments, the lengths of the first TALE and the second TALE are different. In some embodiments, the first TALE is longer than the second TALE. In some embodiments, the first TALE is configured to bind to a target DNA structure in a first region, and the second TALE is configured to bind to a target DNA structure in a second region, with the first and second regions separated by approximately 15 to 25 base pairs on the target DNA structure. In some embodiments, the HIV IN includes wild-type HIV IN having mutations that reduce association of HIV IN with LEDGF / p75, E152Q mutations for the first and second extra-monomers respectively, K186Q mutations for the first and second extra-monomers respectively, or combinations thereof.

[0007] Aspects of this disclosure relate to methods for editing genomic material in a target organism, such as a patient. In some embodiments, the method includes providing a recombinant enzyme; attaching one or more polynucleotides to the recombinant enzyme to form a gene editing construct; administering an effective amount of the gene editing construct to a patient; transporting a certain concentration of the gene editing construct to the patient's cell nucleus; localizing the gene editing construct onto a target DNA structure via TALE; and inserting one or more polynucleotides into the patient's genomic material at the target DNA structure.

[0008] In some embodiments, the recombinant enzyme comprises an HIV IN tetramer and two or more TALEs bound to the HIV IN tetramer. In some embodiments, HIV IN is designed to contain four monomer subunits, including a first internal monomer and a second internal monomer, and a first external monomer and a second external monomer. In some embodiments, the internal monomers and / or external monomers include mutations to increase the likelihood of preferential binding in a desired pattern to correctly assemble the desired tetrameric structure. In some embodiments, at least two of the monomer subunits have TALEs bound to their N-terminal domains. In some embodiments, the first TALE is bound to the first external monomer of HIV IN by a first polypeptide linker, and the second TALE is bound to the second external monomer of HIV IN by a second polypeptide linker. In some embodiments, the polypeptide linker is rigid. In some embodiments, the polypeptide linker is flexible. In some embodiments, the polypeptide linker is composed of glycine residues. In some embodiments, the polypeptide linker includes repeats of a GGGS domain, an EAAAK domain, or a combination thereof. In some embodiments, the polypeptide linker includes alternating repeats of a GGGS domain, an EAAAK domain, or a combination thereof. In some embodiments, the polypeptide linker includes GGGS GGGS GGGS GGGS (SEQ ID NO: 5). In some embodiments, the first and second polypeptide linkers are composed of glycine residues. In some embodiments, the first and second TALEs have different lengths. In some embodiments, the first TALE is longer than the second TALE. In some embodiments, the first TALE is configured to bind to a target DNA structure in a first region, and the second TALE is configured to bind to a target DNA structure in a second region, with the first and second regions separated by approximately 15 to 25 base pairs on the target DNA structure. In some embodiments, the gene editing construct is provided in a lentiviral capsid, as part of a lentiviral vector, or in a combination thereof.In some embodiments, the HIV IN includes wild-type HIV IN having mutations that reduce the association of HIV IN with LEDGF / p75, E152Q mutations for the first and second external monomers respectively, K186Q mutations for the first and second external monomers respectively, or a combination thereof.

[0009] Aspects of this disclosure relate to recombinant gene editing systems comprising recombinant enzymes. In some embodiments, the recombinant enzyme comprises an HIV integrase (HIV IN) tetramer comprising a first internal monomer and a second internal monomer, and a first external monomer and a second external monomer, wherein the first internal monomer and the second internal monomer are bound to each other, the first external monomer is bound to the first internal monomer, and the second external monomer is bound to the second internal monomer; a first TALE bound to the N-terminal region of the first external monomer via a first polypeptide linker; and a second TALE bound to the N-terminal region of the second external monomer via a second polypeptide linker. In some embodiments, one or more polynucleotides are bound to at least one of the first internal monomer and the second internal monomer to form a gene editing construct. In some embodiments, a lentiviral capsid encapsulates the gene editing construct.

[0010] In some embodiments, the polypeptide linker is rigid. In some embodiments, the polypeptide linker is flexible. In some embodiments, the polypeptide linker is composed of glycine residues. In some embodiments, the polypeptide linker includes repeats of a GGGS domain, an EAAAK domain, or a combination thereof. In some embodiments, the polypeptide linker includes alternating repeats of a GGGS domain, an EAAAK domain, or a combination thereof. In some embodiments, the polypeptide linker includes GGGS GGGS GGGS GGGS (SEQ ID NO: 5). In some embodiments, the first and second polypeptide linkers are composed of glycine residues. In some embodiments, the first and second TALEs have different lengths. In some embodiments, the first TALE is longer than the second TALE. In some embodiments, each TALE targets a DNA structural segment of about 8 to 31 base pairs. In some embodiments, the first TALE targets a DNA structural segment of about 30 base pairs. In some embodiments, the HIV IN tetramer includes, for the first internal monomer, Y99D, K103E, K173E, and K186E mutations; for the second internal monomer, E96K, Y99E, K103E, V201H, D25K, and E11K mutations; for the first external monomer, E87K, E96K, E152Q, K186Q, and K215E mutations; for the second external monomer, E152Q, K173E, T174K, K186Q, and I204D mutations, as well as wild-type HIV IN having mutations that reduce association of HIV IN with LEDGF / p75.In some embodiments, the HIV IN tetramer includes, for the first internal monomer, Y99D, K103E, K173E, and K186E mutations; for the second internal monomer, E96K, Y99E, K103E, V201H, D25K, and E11K mutations; for the first external monomer, E87K, E96K, E152Q, K186Q, and K215E mutations; for the second external monomer, E152Q, K173E, T174K, K186Q, and I204D mutations, mutations that reduce the association of HIV IN with LEDGF / p75, and wild-type HIV IN having functional equivalents thereof.

[0011] The drawings illustrate embodiments of the subject matter disclosed for the purpose of illustrating the present invention. However, it should be understood that this application is not limited to the exact arrangements and means shown in the drawings. [Brief explanation of the drawing]

[0012] [Figure 1] This is a schematic diagram of a recombinant enzyme gene editing system according to some embodiments of the present disclosure.

[0013] [Figure 2-1] A to D are protein structures of modified monomeric subunits in HIV integrase (HIV IN) according to some embodiments of the present disclosure.

[0014] [Figure 2-2] E is a graph showing simulated contact energy data between modified monomer subunits in HIV IN according to some embodiments of the present disclosure.

[0015] [Figure 3] Images A and B are Western blot images of HIV IN dimers according to some embodiments of the present disclosure.

[0016] [Figure 4]This is a chart of methods for editing genomic material in a target organism, such as a human patient, according to some embodiments of the present disclosure. [Modes for carrying out the invention]

[0017] Referring to Figure 1, some embodiments of this disclosure relate to a recombinant enzyme gene editing system 100. In some embodiments, the system 100 includes a gene editing construct 102. The gene editing construct 102 is designed and configured to transport a desired polynucleotide, also referred to herein as a “transgene” or “DNA fragment,” from the extracellular environment to the cellular nuclear environment, and further to insert the polynucleotide into the genome with high precision, as will be discussed in more detail below. As discussed herein, embodiments of this disclosure refer to the insertion of a DNA fragment into the genome of a target organism at a target genomic site, for example, in a clinical setting, in vivo, but genome editing by the gene editing construct 102 can also be achieved in vitro or otherwise in an ex vivo setting (e.g., editing of individual cells or multiple cells in vitro), and therefore this disclosure is not intended to limit it in this respect. In some embodiments, the target organism includes any eukaryote (including one or more of its cells). In some embodiments, the target organism is a human patient. In some embodiments, the insertion of the DNA fragment has a therapeutic effect on the target organism / cell. In some embodiments, the insertion of DNA fragments increases the production of one or more desired compounds, such as chemical substances, by the target organism / cell.

[0018] Continuing to refer to Figure 1, the gene editing construct 102 includes recombinant enzyme 104. In some embodiments, recombinant enzyme 104 includes HIV integrase (HIV IN) 106. In some embodiments, HIV IN 106 includes four monomer subunits, including a first internal monomer 106A and a second internal monomer 106C, and a first external monomer 106B and a second external monomer 106D. In some embodiments, the first internal monomer 106A and the second internal monomer 106C are bound to each other. In some embodiments, the first external monomer 106B is bound to the first internal monomer 106A. In some embodiments, the second external monomer 106D is bound to the second internal monomer 106C. In some embodiments, one internal monomer (e.g., 106A) and one external monomer (e.g., 106B) first dimerize (combine) to form a dimer, the interface of which is stronger than, for example, the interface between the first internal monomer 106A and the second internal monomer 106C. Subsequently, in some embodiments, the two dimers combine to form a dimer structure with a relatively weak tetrameric interface.

[0019] HIV IN 106 facilitates the integration of DNA fragments into target DNA structures, presenting them as dimer-dimers, and in some embodiments, each dimer binds to at least a portion of the polynucleotide for integration, jointly catalyzing a half-site reaction to lead to complete integration at the target site. In addition to containing a nuclear localization signal (NLS), HIV IN 106 also relies on cellular proteins widely present in eukaryotes, making it usable in a wide range of host organisms. Among the various enzymes that enable transgene integration, HIV-1 (IN) is an attractive candidate due to its high insertion efficiency and strong activity. HIV-1 IN also enables DNA integration without the risk of unpredictable deletions seen in other systems using the non-homologous end-joining (NHEJ) pathway.

[0020] The residues involved in the formation of the dimer interface in HIV IN 106 are located in the monomer's catalytic core domain (CCD). The dimer-dimer interface is formed by the N-terminal domain of one internal monomer (e.g., 106A) relative to the CCD of the opposite internal monomer (e.g., 106C). These two internal monomers perform the DNA integration reaction, while the external monomers (e.g., 106B and 106D) play a stabilizing role. The N-terminal domains of the external integrase monomers are free, and any further domains bound to these domains associate relatively freely. In some embodiments, the desired pair of HIV IN monomers is produced as a larger protein fusion with a cleavable linker, which can be cleaved after protein production, for example, by a natural protease or HIV-1 protease, to release and allow the monomers to associate.

[0021] In some embodiments, HIV IN 106 has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with wild-type HIV integrase, e.g., HIV-1(IN). In some embodiments, HIV IN 106 maintains substantially equivalent DNA integration function to wild-type HIV integrase. In some embodiments, HIV IN 106 maintains substantially equivalent DNA integration function to HIV-1(IN).

[0022] In some embodiments, HIV IN 106 contains multiple mutations compared to wild-type HIV integrase. In some embodiments, specific mutations are designed to bias the binding of different monomers to each other. In some embodiments, the first internal monomer 106A contains mutations Y99D, K103E, K173E, K186E, or a combination thereof. In some embodiments, the first internal monomer 106A contains functional equivalents to these mutations. In some embodiments, the first internal monomer 106A contains SEQ ID NO: 1. In some embodiments, the second internal monomer 106C contains mutations E96K, Y99E, K103E, V201H, D25K, E11K, or a combination thereof. In some embodiments, the second internal monomer 106C contains functional equivalents to these mutations. In some embodiments, the second internal monomer 106C contains SEQ ID NO: 2. In some embodiments, the first external monomer 106B includes the mutations E87K, E96K, E152Q, K186Q, K215E, or combinations thereof. In some embodiments, the first external monomer 106B includes functional equivalents to these mutations. In some embodiments, the first external monomer 106B includes SEQ ID NO: 3. In some embodiments, the second external monomer 106D includes the mutations E152Q, K173E, T174K, K186Q, I204D, or combinations thereof. In some embodiments, the second external monomer 106D includes functional equivalents to these mutations. In some embodiments, the second external monomer 106D includes SEQ ID NO: 4. In some embodiments, each of the first external monomer 106B and the second external monomer 106D includes the E152Q mutation. In some embodiments, each of the first external monomer 106B and the second external monomer 106D contains the K186Q mutation. In some embodiments, HIV IN 106 contains a mutation that reduces the association of HIV IN with LEDGF / p75, which typically confers affinity to the tetrameric complex to the target DNA structure, biasing the insertion of the DNA fragment to the transcriptionally active region of the chromosome. In some embodiments, HIV IN 106 contains a mutation in V165A of one or more monomers of HIV IN 106.

[0023] Continuing to refer to FIG. 1, the gene editing construct 102 includes one or more transcription activator-like effectors (TALEs) 108. In some embodiments, the recombinant enzyme 104 includes one or more TALEs 108. In some embodiments, the TALE 108 is bound to the HIV IN 106. In some embodiments, at least two TALEs 108, for example, a first TALE 108A and a second TALE 108B, are bound to the HIV IN 106. In some embodiments, the TALE 108 is bound to an external monomer, such as 106B, 106D, or a combination thereof. In some embodiments, the TALE 108 is bound to an external monomer, such as 106B, 106D, or a combination thereof, at the N-terminal region of those external monomers. In some embodiments, the first TALE 108A is bound to the first external monomer 106B. In some embodiments, the second TALE 108B is bound to the second external monomer 106D.

[0024] TALEs are naturally occurring proteins that identify and bind to specific DNA regions and label them. TALE DNA targeting is based on two amino acids within a repetitive segment called the repetitive variable residue or RVD. TALEs have a highly specific and rotationally separated linear search mechanism along DNA, which provides excellent accuracy even in the editing of dense DNA regions, particularly heterochromatin regions. Thus, TALEs represent a strong candidate for conferring DNA site specificity to the gene editing construct 102. TALEs can also be delivered in a lentiviral vector and encapsulated in the HIV-1 inner capsid (as will be discussed in more detail below), and thus do not affect the activity of the system 100 when targeting non-dividing cells.

[0025] By creating different versions of the HIV IN monomer as described in the above exemplary embodiments, pairs of dimers are formed, each pair of dimers preferentially binds to form two specific heterodimers, and the heterodimers then form a heterotetramer, significantly increasing the likelihood that both desired TALEs are present on the inner and outer monomers of the tetramer. In some embodiments, the TALE 108 is attached to the HIV IN 106 in the gene editing construct 102, for example, by a polypeptide linker 110. In some embodiments, the first TALE 108A is attached to the first outer monomer 106B by a first polypeptide linker 110A. In some embodiments, the first TALE 108A is attached to the N-terminal region of the first outer monomer 106B by a first polypeptide linker 110A. In some embodiments, the second TALE 108B is attached to the second outer monomer 106D by a second polypeptide linker 110B. In some embodiments, the second TALE 106B is attached to the N-terminal region of the second outer monomer 106D by a second polypeptide linker 110B. In some embodiments, the polypeptide linker 110 is rigid. In some embodiments, the polypeptide linker 110 is flexible. In some embodiments, the polypeptide linker 110 is composed of glycine residues. In some embodiments, the polypeptide linker 110 includes repeats of the GGGS domain, the EAAAK domain, or a combination thereof. In some embodiments, the polypeptide linker 110 includes alternating repeats of the GGGS domain, the EAAAK domain, or a combination thereof. In some embodiments, the polypeptide linker 110 includes GGGS GGGS GGGS GGGS (SEQ ID NO: 5). Without wishing to be bound by theory, as the rigidity of the polypeptide linker 110 increases, the specificity of the gene editing construct 102 also increases.

[0026] In some embodiments, one or more polynucleotides 112 are bound to the gene editing construct 102. In some embodiments, the polynucleotide 112 is any polynucleotide sequence (or transgene or DNA fragment) desirable for insertion into the cell's genomic material. In some embodiments, the polynucleotide 112 is bound to at least one of a first internal monomer 106A and a second internal monomer 106C. In some embodiments, HIV IN 106 selectively binds to the end of a particular polynucleotide 112 (HIV IN 106 can be modified, for example, by introducing further mutations to enable directional insertion into a transgene).

[0027] As described above, the remaining parts of the gene editing construct 102, such as HIV IN 106 and TALE 108, along with other lentiviral vector components, facilitate the transport of polynucleotide 112 from the extracellular environment to the nuclear environment, where it becomes available for insertion into the genome and subsequent replication. Gene editing construct 102 consistent with embodiments of this disclosure also enables such integration with high site specificity while avoiding off-target delivery. Previous attempts to modify specific proteins to increase sequence specificity for DNA insertion have not been shown to achieve the precision required for clinical use. As an example of how these weaknesses affect the feasibility of therapeutics, a recent study attempted to edit the genes of patients with sickle cell anemia using CRISPR. In this study, two of the eight participants developed acute myeloid leukemia of unknown cause.

[0028] In some embodiments, a first TALE (e.g., 108A) is configured to bind to a target DNA structure in a first region, and a second TALE (e.g., 108B) is configured to bind to a target DNA structure in a second region. In some embodiments, the first and second regions are separated by a predetermined number of base pairs on the target DNA structure. In some embodiments, the first and second regions are separated by approximately 15 to 25 base pairs on the target DNA structure. Once the first and second TALEs bind to the target DNA structure, HIV IN 106 is localized so that the catalytic region is adjacent to the desired insertion site in the genome.

[0029] In some embodiments, each TALE 108 targets a DNA structural segment greater than approximately 8 base pairs. In some embodiments, each TALE 108 targets a DNA structural segment of approximately 8 to 31 base pairs. In some embodiments, the first TALE 108A and the second TALE 108B are of different lengths. In some embodiments, the first TALE 108A is longer than the second TALE 108B. In some embodiments, the first TALE 108A targets a DNA structural segment of approximately 30 base pairs. In these embodiments, the larger TAL array diffuses more slowly than the smaller array. While we do not wish to be constrained by theory, the smaller array compresses the linker to HIV IN 106, e.g., polypeptide linker 110A, and inhibits the activity of HIV IN until the target DNA sequence is found. Once the smaller TAL array identifies the target DNA sequence, the larger TAL array returns to its own target DNA sequence, opening the active site of HIV IN 106 and facilitating target DNA integration.

[0030] Referring here to Figures 2A to 2E, charge exchange mutations at the dimer interface and on major residues in exemplary embodiments of HIV IN 106 were selected and analyzed based on protein charge patch analysis. In these exemplary embodiments, HIV IN 106 has complementary charge regions on opposing monomers. As can be seen from the contact energy comparison in Figures 2A to 2D, monomers A and B have a much higher affinity for each other and a lower affinity for any other monomer, and a similar trend is observed for monomers C and D. When HIV-1 IN forms a dimer, approximately 68 pM of K d Because it exhibits excellent resistance to dissociation, the nonpolar binding region of the integrase dimerization interface was left unmutated. To promote correct pairing of the dimer pairs, another set of mutations were introduced, for example, around its corresponding binding pair on the N-terminal region of the inner monomer of the second dimer pair, and at K186, a residue known to prevent tetramerization when mutated. Both outer monomers B and D have inactivating mutations (e.g., E152Q) that prevent them from performing the integration reaction alone, and also include mutations (e.g., K186Q) that prevent them from becoming inner monomers within the tetramer structure. To reduce binding to LEDGF / p75, a mutation was introduced at, for example, V165A, which typically confers affinity to DNA to the tetramer complex, biasing DNA insertion towards transcriptionally active regions of chromosomes.

[0031] Referring to Figures 3A and 3B, four exemplary monomeric mutants were generated by site-directed mutagenesis, and then sfGFP was fused to IN A, the His7 tag to IN B, mCherry to IN C, and the strep tag to IN D. First, plasmids containing IN A and IN B were expressed at BL21. The proteins were resuspended in lysis buffer containing only 1M urea and 0.1M NaCl. Purification was performed using a His nickel column while increasing the imidazole concentration to reduce contamination by potential homodimers. Since GFP was added to one monomer but not the other, there was a size difference between IN A (58.9 kDa) and IN B (33.1 kDa), which also applies to IN C (59.1 kDa) and IN D (33.5 kDa), as shown in the Western blot in Figure 3A. GFP Western blots of samples A and B revealed a distinct dimer band at approximately 90 kDa, with a monomer band also identified below that level. Furthermore, Western blotting was performed using anti-GFP antigen to confirm the presence of GFP in the observed bands. SDS-PAGE and Western blotting results showed a single band at approximately 90 kDa in the 1 M imidazole eluate, which is the expected band for the desired heterodimer; no homodimer band (approximately 66 kDa) was observed in this or other elutes. The presence of the dimer band in SDS-PAGE indicates a strong binding interface, as expected given that HIV IN maintains dimerization under denaturing conditions (SDS and beta-mercaptoethanol). Similar procedures were repeated for IN C and D, and filtration was performed using a strep-tactin XT 4flow resin column. The results can be seen in Figure 3B. Figure 3B shows an mCherry Western blot with a monomer band at approximately 60 kDa, what appears to be a secondary transcript at less than 40 kDa (this is likely a complete mCherry product as methionine was not removed during fusion), and a dimer band at approximately 90 kDa.In this sample, the weaker the bond, the fewer dimers are observed under denaturation conditions, which is expected since the bond energy between monomers A and B must be significantly stronger than the bond energy between C and D.

[0032] Referring again to Figure 1, in some embodiments, the gene editing construct 102 is encapsulated in a lentiviral capsid 114. In some embodiments, the system 100 is incorporated into a composition for administration to a target organism, e.g., a patient. In some embodiments, the composition is formulated to suit any desired route of administration (e.g., intravenous, nasal, topical, oral, inhalation, or a combination thereof). In some embodiments, the system 100 is incorporated into target cells via a suitable transformation process, e.g., chemical transformation of cells in vitro. In some embodiments, the system 100 is incorporated into target cells via a suitable bioristic particle delivery system, e.g., a gene gun for use with plant cells. In some embodiments, the composition contains a sufficient amount of gene editing construct 102 to achieve, for example, the desired editing of the genome of a target organism and to achieve the desired therapeutic outcome. In some embodiments, the composition includes one or more additional active ingredients, pharmaceutically acceptable adjuvants, diluents, excipients, carriers, or a combination thereof.

[0033] Referring here to Figure 4, some embodiments of the present disclosure include methods 400 for editing genomic material in, for example, a patient, a cell, etc. As described above, in some embodiments, method 400 edits genomic material to provide a therapeutic effect to a cell / target organism. In some embodiments, method 400 edits genomic material to increase the production of one or more desired compounds by a cell / target organism, for example, to increase the production of a chemical substance. In 402, recombinant enzymes are provided. As described above, in some embodiments, recombinant enzymes include HIV IN and two or more TALEs bound to HIV IN. In some embodiments, a first TALE is bound to a first external monomer of HIV IN. In some embodiments, a second TALE is bound to a second external monomer of HIV IN. In some embodiments, the TALEs are linked in a gene editing construct, for example, in HIV IN, by a polypeptide linker. In some embodiments, the first TALE is linked to the N-terminal region of the first external monomer by a first polypeptide linker. In some embodiments, the second TALE is linked to the N-terminal region of the second external monomer by a second polypeptide linker. In some embodiments, the lengths of the first and second TALEs are different. In some embodiments, the first TALE is longer than the second TALE. In some embodiments, the polypeptide linker is rigid. In some embodiments, the polypeptide linker is flexible. In some embodiments, the polypeptide linker is composed of glycine residues. In some embodiments, the polypeptide linker includes repeats of a GGGS domain, an EAAAK domain, or a combination thereof. In some embodiments, the polypeptide linker includes alternating repeats of a GGGS domain, an EAAAK domain, or a combination thereof. In some embodiments, the polypeptide linker includes GGGS GGGS GGGS GGGS (SEQ ID NO: 5).

[0034] In some embodiments, HIV IN has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with wild-type HIV integrase, e.g., HIV-1(IN). In some embodiments, HIV IN maintains substantially equivalent DNA integration function to wild-type HIV integrase. In some embodiments, HIV IN maintains substantially equivalent DNA integration function to HIV-1(IN).

[0035] In some embodiments, HIV IN contains multiple mutations compared to wild-type HIV integrase. In some embodiments, specific mutations are designed to bias the binding of different monomers to each other. In some embodiments, the first internal monomer includes mutations Y99D, K103E, K173E, K186E, or a combination thereof. In some embodiments, the first internal monomer includes SEQ ID NO: 1. In some embodiments, the second internal monomer includes mutations E96K, Y99E, K103E, V201H, D25K, E11K, or a combination thereof. In some embodiments, the second internal monomer includes SEQ ID NO: 2. In some embodiments, the first external monomer includes mutations E87K, E96K, E152Q, K186Q, K215E, or a combination thereof. In some embodiments, the first external monomer includes SEQ ID NO: 3. In some embodiments, the second external monomer includes mutations E152Q, K173E, T174K, K186Q, I204D, or a combination thereof. In some embodiments, the second external monomer 106D includes SEQ ID NO: 4. In some embodiments, each of the first and second external monomers includes the E152Q mutation. In some embodiments, each of the first and second external monomers includes the K186Q mutation. In some embodiments, HIV IN includes mutations that reduce the association of HIV IN with LEDGF / p75, which typically confers affinity to the tetramer complex for the target DNA structure, biasing the insertion of DNA fragments to the transcriptionally active region of the chromosome. In some embodiments, HIV IN includes mutations in V165A of one or more monomers of HIV IN.

[0036] Continuing to refer to Figure 4, in some embodiments, method 400 includes conjugating one or more polynucleotides to a recombinant enzyme to form a gene editing construct (e.g., gene editing construct 102 described above) (404). In some embodiments, the polynucleotide is conjugated to at least one of the first and second internal monomers of HIV IN. In some embodiments, the gene editing construct is encapsulated in a lentiviral capsid. In some embodiments, the gene editing construct is part of a lentiviral vector.

[0037] In 406, an effective amount of the gene-editing construct is administered to, for example, a target organism or a patient. As described above, in some embodiments, the gene-editing construct is administered as part of a composition formulated for any desired route of administration (e.g., intravenous, nasal, topical, oral, inhalation, or a combination thereof). In 408, a certain concentration of the gene-editing construct is transported to the cell nucleus. In 410, the gene-editing construct is localized onto the target DNA structure via the TALE. In some embodiments, the first TALE is configured to bind to the target DNA structure in a first region, and the second TALE is configured to bind to the target DNA structure in a second region. In some embodiments, the first and second regions are separated by a predetermined number of base pairs on the target DNA structure. In some embodiments, the first and second regions are separated by approximately 15 to 25 base pairs on the target DNA structure. Once the first and second TALEs bind to the target DNA structure, HIV IN is localized such that the catalytic region is adjacent to the desired insertion site. In step 412, polynucleotides are inserted into the target DNA structure, i.e., into the target genomic material, via HIV IN.

[0038] The systems and methods of this disclosure advantageously utilize a recombinant enzyme comprising a modified HIV-1 integrase tetramer fused to two distinct TALEs for use as a site-directed gene editor capable of editing eukaryotic cells via a standard transformation process. HIV-1 integrase has been shown to function reliably in non-dividing cells, be resistant to degradation and adverse conditions, have low immunogenicity, and be non-ubiquitous, independent of cellular coenzyme / repair mechanisms, and function in diverse organisms such as Saccharomyces cerevisiae. Modified HIV-1 integrase can form pre-integration gene-editing constructs with polynucleotides to simplify construct construction and delivery.

[0039] The HIV IN tetramer according to embodiments of this disclosure comprises mutated monomer subunits, which are two sets of two mutated variants, which specifically pair with each other using a charge exchange approach for greater binding affinity related to the matching of correct pairing by integrase compared to a much lower binding affinity with unmatched pairs, in order to facilitate the generation of correct monomer pairing. These mutations are applied to both monomer-monomer and dimer-dimer interfaces, and further inactivation mutations are added to the outer monomer to ensure the activation / inactivation of the desired monomer.

[0040] The presence of two TALEs confers complete sequence specificity to the recombinant enzyme, preventing nonspecific interactions with the target DNA structure. The modified HIV-1 integrase monomer ensures that the TALEs properly bind to the outermost monomer and are positioned relative to the catalytic region of HIV IN, allowing the overall construct to rapidly and efficiently site-specifically target DNA fragments and integrate them into the target DNA structure. These gene editing constructs can be used alone and / or incorporated into existing lentiviral vectors to efficiently and accurately insert transgenes into desired genomic locations, with a focus on precision based on the metric of virtually no off-target mutations or indels occurring.

[0041] While the present invention has been described and illustrated with respect to exemplary embodiments thereof, it will be understood by those skilled in the art that the foregoing and various other modifications, omissions, and additions can be made in and to the present invention without departing from the spirit and scope of the invention.

Claims

1. Recombinant enzyme gene editing system, Recombinant enzyme, wherein the enzyme is HIV integrase (HIV IN) tetramer structure and Two or more transcription activator-like effectors (TALEs) bound to the HIV IN tetramer, Recombinant enzymes, The HIV IN comprises one or more polynucleotides that bind to the HIV IN to form a gene editing construct, Recombinant enzyme gene editing system.

2. The system according to claim 1, wherein the HIV IN comprises four monomer subunits including a first internal monomer and a second internal monomer, and a first external monomer and a second external monomer, and at least two of the monomer subunits have a TALE attached to their N-terminal domain.

3. The system according to claim 2, wherein a first TALE is bonded to the first external monomer of the HIV IN by a first polypeptide linker, and a second TALE is bonded to the second external monomer of the HIV IN by a second polypeptide linker.

4. The system according to claim 3, wherein the first polypeptide linker and the second polypeptide linker are flexible, rigid, or a combination thereof.

5. The system according to claim 3, wherein the lengths of the first TALE and the second TALE are different.

6. The system according to claim 3, wherein the first TALE is configured to bind to a target DNA structure in a first region, and the second TALE is configured to bind to the target DNA structure in a second region, and the first region and the second region are separated by approximately 15 to 25 base pairs on the target DNA structure.

7. The HIV-IN is wild-type HIV-IN, Mutations that reduce the association of HIV IN with LEDGF / p75, E152Q mutations in the first external monomer and the second external monomer, K186Q mutations in the first external monomer and the second external monomer, or a combination of those The system according to claim 3, comprising wild-type HIV IN having

8. The system according to claim 1, wherein the gene editing construct is provided in a lentiviral capsid as part of a lentiviral vector, or in combination thereof.

9. A method for editing genomic material in a target organism, The objective is to provide a recombinant enzyme, wherein the enzyme is HIV integrase (HIV IN) tetramer structure and Two or more transcription activator-like effectors (TALEs) bound to the HIV IN tetramer, A step of providing a recombinant enzyme containing, The steps include: forming a gene editing construct by attaching one or more polynucleotides to the recombinant enzyme; The steps include administering an effective amount of the gene editing construct to the target organism, The steps include transporting the gene editing construct at a certain concentration to the cell nucleus of the target organism, The steps include: localizing the gene editing construct onto a target DNA structure via the TALE; The steps include inserting one or more polynucleotides into the target DNA structure of the target organism's genome, Methods that include...

10. The HIV IN comprises four monomer subunits, including a first internal monomer and a second internal monomer, and a first external monomer and a second external monomer, wherein at least two of the monomer subunits have a TALE attached to their N-terminal domain. The method according to claim 9, wherein a first TALE is bonded to the first external monomer of the HIV IN by a first polypeptide linker, and a second TALE is bonded to the second external monomer of the HIV IN by a second polypeptide linker.

11. The method according to claim 10, wherein the first polypeptide linker and the second polypeptide linker are flexible, rigid, or a combination thereof.

12. The method according to claim 10, wherein the first TALE is longer than the second TALE.

13. The method according to claim 10, wherein the first TALE is configured to bind to the target DNA structure in a first region, and the second TALE is configured to bind to the target DNA structure in a second region, and the first region and the second region are separated by approximately 15 to 25 base pairs on the target DNA structure.

14. The HIV-IN is wild-type HIV-IN, Mutations that reduce the association of HIV IN with LEDGF / p75, E152Q mutations in the first external monomer and the second external monomer, K186Q mutations in the first external monomer and the second external monomer, or a combination of those The method according to claim 10, comprising wild-type HIV IN having

15. The method according to claim 9, wherein the gene editing construct is provided in a lentiviral capsid as part of a lentiviral vector, or in combination thereof.

16. Recombinant enzyme gene editing system, Recombinant enzyme, wherein the enzyme is An HIV integrase (HIV IN) tetramer comprising a first internal monomer and a second internal monomer, and a first external monomer and a second external monomer, wherein the first internal monomer and the second internal monomer are bound to each other, the first external monomer is bound to the first internal monomer, and the second external monomer is bound to the second internal monomer, and the HIV IN tetramer A first transcription activator-like effector (TALE) bound to the N-terminal region of the first external monomer via a first polypeptide linker, and a second TALE bound to the N-terminal region of the second external monomer via a second polypeptide linker, Recombinant enzymes, One or more polynucleotides that bind to at least one of the first internal monomer and the second internal monomer to form a gene editing construct, A lentiviral capsid containing the aforementioned gene editing construct, Recombinant enzyme gene editing systems, including [specific components / methods].

17. The system according to claim 16, wherein the first polypeptide linker and the second polypeptide linker include GGGS GGGS GGGS GGGS (SEQ ID NO: 5).

18. The system according to claim 16, wherein the first TALE is longer than the second TALE.

19. The system according to claim 18, wherein the first TALE targets a DNA structural segment of about 30 base pairs.

20. The HIV-IN tetramer is wild-type HIV-IN, The first internal monomer is characterized by the Y99D, K103E, K173E, and K186E mutations. The second internal monomer is one of the following mutants: E96K, Y99E, K103E, V201H, D25K, and E11K. The first external monomers include E87K, E96K, E152Q, K186Q, and K215E mutations. The second external monomers include the E152Q, K173E, T174K, K186Q, and I204D mutations, and, Mutations that reduce the association of HIV IN with LEDGF / p75 The system according to claim 16, comprising wild-type HIV IN having

Images