Recombinase for accurate insertion of DNA sequences in eukaryotic cells
By using a recombinase gene editing system and combining HIV integrase with TALE, highly accurate DNA insertion in eukaryotic cells has been achieved, solving the problem of off-target mutations in existing technologies and improving the reliability and safety of gene therapy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-04-29
- Publication Date
- 2026-03-20
AI Technical Summary
Existing gene editing technologies lack accuracy when inserting DNA into the body, making them prone to off-target mutations. This limits the widespread application of gene therapy, especially in treatments involving CRISPR or its derivatives.
The recombinase gene editing system, which includes HIV integrase and transcription activator-like effector (TALE), connects the HIV IN monomer subunit to TALE through a polypeptide linker to form a gene editing construct. This construct is then delivered to target cells using a lentiviral vector, achieving efficient and accurate gene editing.
It achieves highly accurate DNA insertion in eukaryotic cells, reduces off-target mutations, improves the reliability and safety of gene therapy, and is suitable for clinical applications.
Smart Images
Figure CN121712899A_ABST
Abstract
Description
[0001] Cross Reference to Related Applications
[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 639,007, filed April 26, 2024, and U.S. Provisional Patent Application No. 63 / 462,377, filed April 27, 2023, which are incorporated by reference herein in their entirety as if disclosed herein. BACKGROUND
[0003] In the field of genomics, there are a variety of mechanisms that have evolved naturally to insert transgenes and exogenous DNA fragments into the genome of an organism to accomplish a particular goal. Many of the mechanisms of these enzymes have been elucidated, and their goal is to translocate genetic material to a new location in the genome. Unfortunately, many of these enzymes come from transposons or viruses, which do not support the life of a cell, nor the stability of its genome. Thus, many of the mechanisms in nature are not accurate enough to reliably insert DNA into a specific locus, with little off-target mutations. While there are other mechanisms, such as different recombination enzymes, their ability to insert DNA is also limited, as they require that the target sequence already exist in the genome to perform the insertion, which often requires performing an initial insertion.
[0004] People have attempted to alter certain proteins to increase their sequence specificity for DNA insertion, but in most cases, these proteins have not shown the accuracy necessary to make them useful for many clinical applications, or they are unable to effectively transfect cells in vivo. In recent years, methods have been developed to insert, edit, or remove small regions of DNA on the scale of less than a hundred nucleotides, such as guided editors, dual-guided editors, and base editors. However, almost all of these methods rely on the same Cas backbone in some way, and are susceptible to similar issues in accuracy. Even “high accuracy” variants, like Sniper-Cas9, still exhibit off-target mutation rates as high as 10% in some cases, while other variants show a clear tradeoff between increasing accuracy and maintaining modification efficiency.
[0005] Due to the difficulty in accurately editing genomic material, and the high risk of off-target changes, especially in vivo, current gene therapy treatments, especially those involving forms of CRISPR or its derivatives, are limited to the most severe cases, and require the extraction of cells after which the cells are modified, thereby minimizing the overall utility of these treatments. Thus, there is a need for a gene editing system and method that is capable of site-specific genome editing in vivo, and thereby provides a reliable gene therapy treatment to patients with genetic abnormalities. SUMMARY
[0006] Aspects of the present disclosure relate to a recombinase gene editing system. In some embodiments, the recombinase gene editing system comprises a recombinase comprising an HIV integrase (HIV IN) and two or more transcription activator-like effectors (TALEs) bound to the HIV IN. In some embodiments, the recombinase comprises one or more polynucleotides bound to the 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 a combination thereof.
[0007] In some embodiments, the HIV IN comprises four monomeric subunits comprising 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 monomeric subunits have a TALE bound to its N-terminal domain. In some embodiments, a first TALE is bound to the first external monomer of the HIV IN via a first polypeptide linker and a second TALE is bound to the second external monomer of the HIV IN via 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 repeating sequences of a GGGS domain, an EAAAK domain, or a combination thereof. In some embodiments, the polypeptide linker comprises alternating repeating sequences 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 first TALE and the second TALE 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 at a first region and the second TALE is configured to bind to the target DNA structure at a second region, wherein the first region and the second region are spaced apart on the target DNA structure between about 15 base pairs and about 25 base pairs. In some embodiments, the HIV IN comprises a wild-type HIV IN with a mutation to reduce association of the HIV IN with LEDGF / p75; an E152Q mutation for each of the first external monomer and the second external monomer; a K186Q mutation for each of the first external monomer and the second external monomer, or a combination thereof.
[0008] Aspects of the present disclosure relate to a method of editing genomic material in a target organism (e.g., a patient). In some embodiments, the method comprises: providing a recombinase; combining one or more polynucleotides with the recombinase to form a gene editing construct; administering an effective amount of the gene editing construct to the patient; transporting a concentration of the gene editing construct to the nucleus in the patient; positioning the gene editing construct on a target DNA structure via a TALE; and inserting the one or more polynucleotides into the genomic material of the patient at the target DNA structure.
[0009] In some embodiments, the recombinase comprises an HIV IN tetramer and two or more TALEs bound to the HIV IN tetramer. In some embodiments, the HIV IN is designed to comprise four monomeric subunits comprising 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 the external monomers comprise mutations that preferably bind in a desired pattern to increase the likelihood of correct assembly of the desired tetrameric structure. In some embodiments, at least two of the monomeric subunits have a TALE bound to their N-terminal domain. In some embodiments, the first TALE is bound to the first external monomer of the HIV IN via a first polypeptide linker and the second TALE is bound to the second external monomer of the HIV IN via 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 repeating sequences of GGGS domains, EAAAK domains, or combinations thereof. In some embodiments, the polypeptide linker comprises alternating repeating sequences of GGGS domains, EAAAK domains, or combinations 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 first TALE and the second TALE are of 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 at a first region and the second TALE is configured to bind to the target DNA structure at a second region, wherein the first region and the second region are spaced apart on the target DNA structure between about 15 base pairs and about 25 base pairs. In some embodiments, the gene editing construct is provided in a lentiviral capsid, as part of a lentiviral vector, or combinations thereof. In some embodiments, the HIV IN comprises a wild-type HIV IN with a mutation to reduce HIV IN association with LEDGF / p75; an E152Q mutation for each of the first external monomer and the second external monomer; a K186Q mutation for each of the first external monomer and the second external monomer, or combinations thereof.
[0010] This disclosure relates to a recombinase gene editing system comprising a recombinase. In some embodiments, the recombinase comprises an HIV integrase (HIV IN) tetramer, the integrase 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, and the first external monomer is bound to the first internal monomer and the second external monomer is bound to the second internal monomer; and a first TALE, which binds to the N-terminal region of the first external monomer via a first polypeptide linker, and a second TALE, which binds to the N-terminal region of the second external monomer via a second polypeptide linker. In some embodiments, the one or more polynucleotides bind 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.
[0011] In some embodiments, the peptide linker is rigid. In some embodiments, the peptide linker is flexible. In some embodiments, the peptide linker is composed of glycine residues. In some embodiments, the peptide linker includes repeating sequences of GGGS domains, EAAAK domains, or combinations thereof. In some embodiments, the peptide linker includes alternating repeating sequences of GGGS domains, EAAAK domains, or combinations thereof. In some embodiments, the peptide linker includes GGGS GGGS GGGS GGGS (SEQ.ID NO.: 5). In some embodiments, the first and second peptide linkers are composed of glycine residues. In some embodiments, the first and second tags have different lengths. In some embodiments, the first tag is longer than the second tag. In some embodiments, each tag targets a DNA structural segment between about 8 and about 31 base pairs. In some embodiments, the first tag targets a DNA structural segment of about 30 base pairs. In some embodiments, the HIV IN tetramer includes wild-type HIV IN with the following mutations: for the first internal monomer, Y99D, K103E, K173E, and K186E; for the second internal monomer, E96K, Y99E, K103E, V201H, D25K, and E11K; for the first external monomer, E87K, E96K, E152Q, K186Q, and K215E; for the second external monomer, E152Q, K173E, T174K, K186Q, and I204D; and mutations to reduce the association of HIV IN with LEDGF / p75. In some embodiments, the HIV IN tetramer comprises wild-type HIV IN having the following mutations: for the first internal monomer, Y99D, K103E, K173E, and K186E; for the second internal monomer, E96K, Y99E, K103E, V201H, D25K, and E11K; for the first external monomer, E87K, E96K, E152Q, K186Q, and K215E; for the second external monomer, E152Q, K173E, T174K, K186Q, and I204D; mutations for reducing HIV IN association with LEDGF / p75; and its functional equivalents. Attached Figure Description
[0012] For the purpose of illustrating the invention, the accompanying drawings show embodiments of the disclosed subject matter. However, it should be understood that this application is not limited to the precise arrangements and means shown in the drawings, wherein:
[0013] Figure 1 This is a schematic diagram of a recombinase gene editing system according to some embodiments of the present disclosure;
[0014] Figures 2A to 2D show the protein structures of modified monomeric subunits in HIV integrase (HIV IN) according to some embodiments of the present disclosure;
[0015] Figure 2E A graph illustrating simulated contact energy data between modified monomer subunits in HIV IN according to some embodiments of the present disclosure;
[0016] Figures 3A-3B Images of HIV IN dimers according to some embodiments of this disclosure; and
[0017] Figure 4 This is a schematic diagram of a method for editing genomic material in a target organism (e.g., a human patient) according to some embodiments of the present disclosure. Detailed Implementation
[0018] Now for reference Figure 1 Some embodiments of this disclosure relate to a recombinase gene editing system 100. In some embodiments, system 100 includes a gene editing construct 102. Gene editing construct 102 is designed and configured to transport desired polynucleotides (also referred to herein as “transgenic” or “DNA fragments”) from an extracellular environment to a nuclear environment and further insert the polynucleotides into the genome with high accuracy, as discussed in more detail below. As discussed herein, embodiments of this disclosure relate to inserting DNA fragments into the genome of a target organism at a targeted genomic location, for example, in a clinical setting, in vivo, etc.; however, this disclosure is not intended to be limited thereto, as genome editing performed by gene editing construct 102 can also be performed in vitro or other ex vivo settings, for example, editing a single cell or multiple cells in vitro. In some embodiments, the target organism includes any eukaryote comprising 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 the DNA fragment results in an increased yield of one or more desired compounds to the target organism / cell, such as an increased yield of chemicals.
[0019] Still referencing Figure 1Gene editing construct 102 includes a recombinase 104. In some embodiments, recombinase 104 includes HIV integrase (HIV IN) 106. In some embodiments, HIV IN 106 is a tetramer comprising four monomeric subunits, the four monomeric subunits being 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 bind to each other. In some embodiments, the first external monomer 106B binds to the first internal monomer 106A. In some embodiments, the second external monomer 106D binds to the second internal monomer 106C. In some embodiments, an internal monomer (e.g., 106A) and an external monomer (e.g., 106B) first dimerize (bind) together to form a dimer, the interface being stronger, for example, than the interface between the first internal monomer 106A and the second internal monomer 106C. Then, in some embodiments, two dimers in the dimer aggregate together to form a dimer of the timer construct having a relatively weak tetramer interface.
[0020] HIV IN 106 facilitates the integration of DNA fragments into target DNA structures, presenting as dimerized fragments. In some embodiments, each dimer binds to at least a portion of the polynucleotide used for integration and catalyzes a half-site reaction, collectively leading to complete integration at the target site. In addition to containing nuclear localization signals (NLS), HIV IN 106 relies on cellular proteins ubiquitous in eukaryotes, allowing for broad applicability to host organisms. Among the various enzymes used to achieve transgene integration, HIV-1 (IN) is an attractive candidate due to its high insertion efficiency and robust activity. HIV-1 IN also achieves DNA integration without the unpredictable risk of deletion, a risk seen in other systems using the non-homologous end joining (NHEJ) pathway.
[0021] In HIV IN 106, the residues involved in forming the dimer interface are located in the catalytic core domain (CCD) of the monomer. The dimer-dimer arrival interface is generated by the N-terminal domain of an inner monomer (e.g., 106A) and the CCD of the opposing inner monomer (e.g., 106C). These two inner monomers perform the DNA integration reaction, with the outer monomers (e.g., 106B and 106D) playing a stabilizing role. The outer integrase monomers have their free N-terminal domains, and additional domains attached to these domains associate relatively freely. In some embodiments, the desired HIV IN monomer pair is produced as a fusion of a larger protein with a cleavable linker, which can be cleaved post-protein production, for example, by a native protease or by an HIV-1 protease to release them and allow them to associate.
[0022] In some embodiments, HIV IN 106 comprises 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, such as HIV-1 (IN). In some embodiments, HIV IN 106 maintains DNA integration function substantially equivalent to that of wild-type HIV integrase. In some embodiments, HIV IN 106 maintains DNA integration function substantially equivalent to that of HIV-1 (IN).
[0023] In some embodiments, HIV IN 106 includes multiple mutations relative to wild-type HIV integrase. In some embodiments, specific mutations are designed to favor the binding of different monomers to each other. In some embodiments, the first internal monomer 106A includes the mutations Y99D, K103E, K173E, K186E, or combinations thereof. In some embodiments, the first internal monomer 106A includes functional equivalents of these mutations. In some embodiments, the first internal monomer 106A includes SEQ.ID NO.: 1. In some embodiments, the second internal monomer 106C includes the mutations E96K, Y99E, K103E, V201H, D25K, E11K, or combinations thereof. In some embodiments, the second internal monomer 106C includes functional equivalents of these mutations. In some embodiments, the second internal monomer 106C includes 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 exomer 106B comprises a functional equivalent of these mutations. In some embodiments, the first exomer 106B comprises SEQ.ID NO.: 3. In some embodiments, the second exomer 106D comprises the mutations E152Q, K173E, T174K, K186Q, I204D, or a combination thereof. In some embodiments, the second exomer 106D comprises a functional equivalent of these mutations. In some embodiments, the second exomer 106D comprises SEQ.ID NO.: 4. In some embodiments, each of the first exomer 106B and the second exomer 106D comprises the E152Q mutation. In some embodiments, each of the first exomer 106B and the second exomer 106D comprises the K186Q mutation. In some embodiments, HIV IN 106 comprises a mutation to reduce the association of HIV IN with LEDGF / p75, which typically imparts affinity of the tetrameric complex to target DNA structures and biases the insertion of DNA fragments toward transcriptionally active regions of chromosomes. In some embodiments, HIV IN 106 includes a mutation at V165A of one or more monomers of HIV IN 106.
[0024] Still referencing Figure 1Gene editing construct 102 includes one or more transcription activator-like effector (TALE) 108. In some embodiments, recombinase 104 includes one or more TALE 108. In some embodiments, TALE 108 binds to HIV IN 106. In some embodiments, at least two TALE 108 (e.g., a first TALE 108A and a second TALE 108B) bind to HIV IN 106. In some embodiments, TALE 108 binds to external monomers (e.g., 106B, 106D, or combinations thereof). In some embodiments, TALE 108 binds to external monomers (e.g., 106B, 106D, or combinations thereof) at the N-terminal regions of those external monomers. In some embodiments, the first TALE 108A binds to the first external monomer 106B. In some embodiments, the second TALE 108B binds to the second external monomer 106D.
[0025] TALE is a naturally found protein that recognizes and binds to specific DNA fragments to label them. TALE's DNA targeting is based on two amino acids within repeating fragments, called repeat variable residues, or RVDs. TALE possesses a highly specific, rotationally decoupled linear search mechanism along DNA, which confers excellent accuracy even in tightly packed DNA regions and especially in regions of edited heterochromatin. Therefore, TALE represents a strong candidate for conferring DNA site specificity to gene-editing construct 102. TALE can also be delivered in lentiviral vectors and can be encapsulated within the HIV-1 internal capsid (discussed in more detail below), and therefore will not affect the activity of System 100 when targeting non-dividing cells.
[0026] By creating different versions of HIV IN monomers as described in the exemplary embodiments above, multiple pairs of dimers are formed, each pair of dimers preferably binding to form two specific heterodimers, which then form a heterotetramer, thereby greatly increasing the likelihood of having the desired TALE both in the tetramer and on the external monomer. In some embodiments, TALE 108 binds to HIV IN 106 in the gene-editing construct 102 via peptide linker 110. In some embodiments, a first TALE 108A binds to a first external monomer 106B via a first peptide linker 110A. In some embodiments, the first TALE 108A binds to the N-terminal region of the first external monomer 106B via the first peptide linker 110A. In some embodiments, a second TALE 108B binds to a second external monomer 106D via a second peptide linker 110B. In some embodiments, the second TALE 106B binds to the N-terminal region of the second external monomer 106D via the second peptide linker 110B. In some embodiments, the peptide linker 110 is rigid. In some embodiments, the peptide linker 110 is flexible. In some embodiments, the peptide linker 110 is composed of glycine residues. In some embodiments, the peptide linker 110 includes repeating sequences of GGGS domains, EAAAK domains, or combinations thereof. In some embodiments, the peptide linker 110 includes alternating repeating sequences of GGGS domains, EAAAK domains, or combinations thereof. In some embodiments, the peptide linker 110 includes GGGS GGGSGGGS GGGS (SEQ. ID NO.: 5). Without being bound by theory, as the rigidity of the peptide linker 110 increases, the specificity of the gene editing construct 102 also increases.
[0027] In some embodiments, one or more polynucleotides 112 bind to gene-editing construct 102. In some embodiments, polynucleotide 112 is any polynucleotide sequence (or transgene or DNA fragment) of genomic material to be inserted into a cell. In some embodiments, polynucleotide 112 binds 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 specific polynucleotide 112 (HIV IN 106 can be modified by preparing additional mutations, for example, to allow directed insertion into a transgene).
[0028] As discussed above, the remainder of gene-editing construct 102 (e.g., HIV IN 106 and TALE 108) binds to other lentiviral vector components, facilitating the transport of polynucleotide 112 from the extracellular environment to the nuclear environment, where it can be inserted into the genome and subsequently replicated. Gene-editing construct 102, consistent with embodiments of this disclosure, also enables such integration to occur with high site specificity and avoids off-target delivery. Previous attempts have attempted to alter certain proteins to increase their sequence specificity for DNA insertion, but these previous attempts have not shown the ability to produce the accuracy necessary for their clinical use. One example of how these weaknesses can affect the feasibility of treatments is a recent study that attempted to use CRISPR to edit genes in patients with sickle cell anemia. In this study, two of the eight participants developed acute myeloid leukemia for unknown reasons.
[0029] In some embodiments, a first TALE (e.g., 108A) is configured to bind a target DNA structure at a first region, and a second TALE (e.g., 108B) is configured to bind a target DNA structure at a second region. In some embodiments, the first and second regions are spaced apart by a predetermined number of base pairs on the target DNA structure. In some embodiments, the first and second regions are spaced apart by approximately 15 to approximately 25 base pairs on the target DNA structure. After the first and second TALEs bind to the target DNA structure, HIV IN 106 is located in a catalytic region adjacent to the desired insertion site in the genome.
[0030] In some embodiments, each TALE 108 targets a DNA structural fragment longer than about 8 base pairs. In some embodiments, each TALE 108 targets a DNA structural fragment between about 8 and about 31 base pairs. In some embodiments, the first TALE 108A and the second TALE 108B have 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 fragment of about 30 base pairs. In these embodiments, the larger TAL array will diffuse more slowly than the smaller array. Not wanting to be bound by theory, the smaller array compresses the adapter (e.g., peptide adapter 110A) to HIV IN 106 and inhibits HIV IN activity until the target DNA sequence is found. Once the smaller TAL array recognizes the target DNA sequence, the larger TAL array extends back to its own target DNA sequence, thereby opening the active site of HIV IN 106 and facilitating the integration of the target DNA.
[0031] Now refer to Figure 2A to Figure 2EBased on the examination of protein charge plaques, dimer interface and charge-exchange mutations on key residues of exemplary HIV IN 106 were selected and analyzed. In these exemplary embodiments, HIV IN 106 has complementary charge regions on the relative monomers. As can be seen from the contact energy comparisons in Figures 2A to 2D, monomers A and B have much higher affinity for each other, while having lower affinity for any other monomer, with the same trend observed for monomers C and D. The nonpolar binding region of the integrase dimerization interface was not mutated because HIV-1 IN, once dimerized, exhibits excellent resistance to dissociation, where K… d The concentration is approximately 68 pM. To facilitate proper pairing of dimer pairs, another set of mutations is introduced, for example, around K186 in the N-terminal region of the inner monomer of the second dimer pair and its corresponding binding pair. These residues are known to prevent tetramerization when mutated. Both outer monomers B and D carry inactivation mutations, such as E152Q, which prevent them from performing integration reactions on their own, and also contain mutations, such as K186Q, that prevent them from becoming inner monomers in the tetramer structure. Mutations are introduced to reduce binding to LEDGF / p75, for example at V165A, which typically gives the tetramer complex an affinity for DNA and causes DNA insertion to be biased towards transcriptionally active regions of the chromosome.
[0032] Now for reference Figures 3A-3B After creating four exemplary monomeric mutants using site-directed mutagenesis, sfGFP was fused with INA, the His7 tag with INB, mCherry with INB, and the strep tag with INB. First, plasmids containing INB and INB were expressed in BL21. The proteins were resuspended in lysis buffer containing 1 M urea and only 0.1 M NaCl. Purification was performed using a His-Ni column with an increased concentration of imidazole to reduce contamination from potential homodimers. Due to the addition of GFP to one monomer rather than the other, there was a size difference between INB (58.9 kDa) and INB (33.1 kDa); the same was true for INB (59.1 kDa) and INB (33.5 kDa). Figure 3AThe results of the protein blot are shown in the image. In the GFP protein blot of samples AB, a well-defined dimer band and a lower monomer band were identified at approximately 90 kDa. Furthermore, to verify the presence of GFP in the observed bands, the protein blot was performed using an anti-GFP antigen. The results of SDS-PAGE and the protein blot show a single band at approximately 90 kDa in 1 M imidazole elution, which is the expected band for the desired heterodimer, where no homodimer band (approximately 66 kDa) was observed at this point or in other elutions. The presence of the dimer band in the SDS-PAGE indicates the presence of a strong binding interface, which is expected, as HIV IN does indeed maintain a dimer under the denaturing conditions used (SDS and β-mercaptoethanol). The same procedure was repeated for IN C and D, where filtration was performed using a strep-tactin XT 4flow resin column. Figure 3B The image shows an mCherry protein blot, indicating a monomeric band at approximately 60 kDa, which appears to be a secondary transcription product at <40 kDa (this is likely the complete mCherry product, as methionine was not removed during fusion), and a dimeric band is also visible at approximately 90 kDa. In this sample, the weaker binding resulted in fewer dimers observed under denaturing conditions, which is expected, as the binding energies between monomers A and B should be significantly stronger than those between C and D.
[0033] Refer again Figure 1 In some embodiments, the gene-editing construct 102 is encapsulated in a lentiviral capsid 114. In some embodiments, system 100 is incorporated into a composition for administration to a target organism (e.g., a patient). In some embodiments, the composition is formulated for any desired route of administration, such as intravenous, nasal, topical, oral, inhalation, etc., or combinations thereof. In some embodiments, system 100 is incorporated into target cells via a suitable transformation process (e.g., in vitro cytochemical transformation). In some embodiments, system 100 is incorporated into target cells via a suitable biological ballistic particle delivery system (e.g., a gene gun) for use with plant cells. In some embodiments, the composition contains an effective amount of gene-editing construct 102, for example, sufficient to achieve the desired editing of the genome of the target organism and to achieve the desired therapeutic outcome. In some embodiments, the composition contains one or more additional active ingredients, pharmaceutical adjuvants, diluents, excipients, carriers, or combinations thereof.
[0034] Now for reference Figure 4Some embodiments of this disclosure include a method 400 for editing genomic material, such as in a patient, cells, etc. As discussed above, in some embodiments, method 400 edits genomic material in an effort to provide a therapeutic effect to a cell / target organism. In some embodiments, method 400 edits genomic material in an effort to increase the yield of one or more desired compounds, such as the yield of chemicals, to a cell / target organism. At 402, a recombinase is provided. As discussed above, in some embodiments, the recombinase includes HIV IN and two or more TALEs that bind to HIV IN. In some embodiments, a first TALE binds to a first external monomer in HIV IN. In some embodiments, a second TALE binds to a second external monomer in HIV IN. In some embodiments, the TALE binds in a gene-editing construct, for example, at HIV IN, via a peptide linker. In some embodiments, the first TALE binds to the N-terminal region of the first external monomer via a first peptide linker. In some embodiments, the second TALE binds to the N-terminal region of the second external monomer via a second peptide linker. In some embodiments, the first TALE and the second TALE have different lengths. In some embodiments, the first TALE is longer than the second TALE. In some embodiments, the peptide linker is rigid. In some embodiments, the peptide linker is flexible. In some embodiments, the peptide linker is composed of glycine residues. In some embodiments, the peptide linker includes repeating sequences of GGGS domains, EAAAK domains, or combinations thereof. In some embodiments, the peptide linker includes alternating repeating sequences of GGGS domains, EAAAK domains, or combinations thereof. In some embodiments, the peptide linker includes GGGS GGGS GGGS GGGS (SEQ.ID NO.:5).
[0035] In some embodiments, HIV IN includes 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, such as HIV-1 (IN). In some embodiments, HIV IN maintains DNA integration function substantially equivalent to that of wild-type HIV integrase. In some embodiments, HIV IN maintains DNA integration function substantially equivalent to that of HIV-1 (IN).
[0036] In some embodiments, HIV IN comprises multiple mutations relative to wild-type HIV integrase. In some embodiments, specific mutations are designed to favor the binding of different monomers to each other. In some embodiments, the first internal monomer comprises the mutations Y99D, K103E, K173E, K186E, or combinations thereof. In some embodiments, the first internal monomer comprises SEQ.ID NO.: 1. In some embodiments, the second internal monomer comprises the mutations E96K, Y99E, K103E, V201H, D25K, E11K, or combinations thereof. In some embodiments, the second internal monomer comprises SEQ.ID NO.: 2. In some embodiments, the first external monomer comprises the mutations E87K, E96K, E152Q, K186Q, K215E, or combinations thereof. In some embodiments, the first external monomer comprises SEQ.ID NO.: 3. In some embodiments, the second external monomer comprises the mutations E152Q, K173E, T174K, K186Q, I204D, or combinations thereof. In some embodiments, the second external monomer 106D comprises SEQ.ID NO.: 4. In some embodiments, each of the first and second outer monomers includes the E152Q mutation. In some embodiments, each of the first and second outer monomers includes the K186Q mutation. In some embodiments, HIV IN includes a mutation to reduce the association of HIV IN with LEDGF / p75, which typically imparts affinity of the tetrameric complex to target DNA structures and biases the insertion of DNA fragments toward transcriptionally active regions of the chromosome. In some embodiments, HIV IN includes a mutation at V165A of one or more monomers of HIV IN.
[0037] Still referencing Figure 4 In some embodiments, method 400 includes conjugating one or more polynucleotides with a recombinase to form a gene-editing construct 404, such as gene-editing construct 102 discussed above. In some embodiments, the polynucleotide is conjugated to at least one of a first internal monomer and a second internal monomer 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.
[0038] At 406, an effective amount of the gene-editing construct is administered, for example, to the target organism or patient. As discussed 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, etc., or combinations thereof). At 408, a certain concentration of the gene-editing construct is transported to the cell nucleus. At 410, the gene-editing construct is localized to the target DNA structure via a TALE. In some embodiments, a first TALE is configured to bind the target DNA structure at a first region, and a second TALE is configured to bind the target DNA structure at a second region. In some embodiments, the first and second regions are spaced apart by a predetermined number of base pairs on the target DNA structure. In some embodiments, the first and second regions are spaced apart by approximately 15 to approximately 25 base pairs on the target DNA structure. After the first and second TALEs bind to the target DNA structure, HIV IN is localized to a catalytic region adjacent to the desired insertion site. At 412, a polynucleotide is inserted into the target DNA structure via HIV IN, i.e., into the target genomic material.
[0039] The systems and methods of this disclosure advantageously utilize a recombinase comprising a modified HIV-1 integrase tetramer fused to two separate TALEs for use as a site-specific gene editor capable of editing eukaryotic cells via a standard transformation process. HIV-1 integrase functions reliably in non-dividing cells, is resistant to degradation and adverse conditions, exhibits low immunogenicity, and further does not depend on non-ubiquitous cellular coenzyme / repair mechanisms, and has been shown to function in a variety of organisms, including *Saccharomyces cerevisiae*. The modified HIV-1 integrase can be combined with polynucleotides to form pre-integrated gene-editing constructs to simplify construct construction and delivery.
[0040] The HIV IN tetramer according to embodiments of this disclosure comprises a mutated monomeric subunit and two sets of two mutant variants that specifically pair with each other using a charge-exchange method to generate a greater binding affinity (compared to a much lower binding affinity associated with mismatched pairs) to facilitate the generation of correct monomer pairing. These mutations are applied along both monomer-monomer interfaces and dimer-dimer interfaces, wherein an additional inactivating mutation is made on the outer monomer to ensure activation / inactivation of the desired monomer.
[0041] The presence of two TALEs endows the recombinase with full-sequence specificity and prevents non-specific interactions with the target DNA structure. The modified HIV-1 integrase monomer ensures proper binding of the TALE to the outermost monomer and the arrangement of the TALE relative to the catalytic region of HIV IN, enabling the entire construct to rapidly, efficiently, and site-specifically target and integrate the DNA fragment into the target DNA structure. These gene-editing constructs can be used independently and / or integrated into existing lentiviral vectors to allow for efficient and accurate insertion of transgenes into desired genomic locations, with a focus on achieving accuracy measured by virtually no off-target mutations or insertions / deletions.
[0042] Although the present invention has been described and illustrated with respect to exemplary embodiments thereof, those skilled in the art will understand that various other changes, omissions and additions may be made therein and thereon without departing from the spirit and scope of the invention.
Claims
1. A recombinase gene editing system, comprising: Recombinase, the enzyme comprising: HIV integrase (HIV IN) tetramer structure; as well as Two or more transcription activator-like effector (TALE) factors, which bind to the HIV IN tetramer. One or more polynucleotides, said polynucleotides being bound to HIV IN to form a gene-editing construct.
2. The system of claim 1, wherein the HIV IN comprises four monomer subunits, the four monomer subunits comprising 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 that binds to its N-terminal domain.
3. The system of claim 2, wherein the first TALE binds to the first external monomer of the HIV IN via a first polypeptide linker, and the second TALE binds to the second external monomer of the HIV IN via a second polypeptide linker.
4. The system of claim 3, wherein the first polypeptide connector and the second polypeptide connector are flexible, rigid, or a combination thereof.
5. The system of claim 3, wherein the first tag and the second tag have different lengths.
6. The system of claim 3, wherein the first TALE is configured to bind a target DNA structure at a first region, and the second TALE is configured to bind the target DNA structure at a second region, wherein the first region and the second region are spaced apart on the target DNA structure by approximately 15 base pairs and approximately 25 base pairs.
7. The system of claim 3, wherein the HIV IN comprises wild-type HIV IN having the following: Used to reduce mutations associated with HIV IN and LEDGF / p75; For each of the first and second external monomers, the E152Q mutation; For the K186Q mutation in each of the first and second external monomers, Or a combination thereof.
8. The system of 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 method comprising: Provide a recombinase, said enzyme comprising: The structure of the HIV integrase (HIV IN) tetramer; and Two or more transcription activator-like effector (TALE) factors, which bind to the HIV IN tetramer. One or more polynucleotides are combined with the recombinase to form a gene-editing construct; Administer an effective amount of the gene-editing construct to the target organism; A certain concentration of the gene-editing construct is transported into the cell nucleus of the target organism; The gene editing construct is positioned on the target DNA structure via the TALE; and The one or more polynucleotides are inserted into the genomic material of the target organism at the target DNA structure.
10. The method according to claim 9, wherein: HIV IN comprises four monomeric subunits, including a first inner monomer and a second inner monomer, as well as a first outer monomer and a second outer monomer, wherein at least two of the monomeric subunits have a TALE that binds to its N-terminal domain, and The first TALE binds to the first external monomer of HIV IN via a first polypeptide linker, and the second TALE binds to the second external monomer of HIV IN via a second polypeptide linker.
11. The method of claim 10, wherein the first polypeptide connector and the second polypeptide connector are flexible, rigid, or a combination thereof.
12. The method of claim 10, wherein the first tag is longer than the second tag.
13. The method of claim 10, wherein the first TALE is configured to bind the target DNA structure at a first region, and the second TALE is configured to bind the target DNA structure at a second region, wherein the first region and the second region are spaced apart on the target DNA structure between approximately 15 base pairs and approximately 25 base pairs.
14. The method of claim 10, wherein the HIV IN comprises wild-type HIV IN having the following: Used to reduce mutations associated with HIV IN and LEDGF / p75; For each of the first and second external monomers, the E152Q mutation; For the K186Q mutation in each of the first and second external monomers, Or a combination thereof.
15. The method of claim 9, wherein the gene-editing construct is provided in a lentiviral capsid, as part of a lentiviral vector, or in combination thereof.
16. A recombinase gene editing system, comprising: Recombinase, the enzyme comprising: HIV integrase (HIV IN) tetramer, the HIV integrase tetramer comprising a first internal monomer and a second internal monomer, a first external monomer and a second external monomer, wherein the first internal monomer and the second internal monomer are bound to each other, and the first external monomer is bound to the first internal monomer and the second external monomer is bound to the second internal monomer. as well as A first transcription activator-like effector (TALE), which binds to the N-terminal region of the first external monomer via a first polypeptide linker, and a second TALE, which binds to the N-terminal region of the second external monomer via a second polypeptide linker. One or more polynucleotides, said polynucleotides being bound to at least one of the first internal monomer and the second internal monomer to form a gene-editing construct, and A lentiviral capsid that encapsulates the gene-editing construct.
17. The system of claim 16, wherein the first polypeptide adapter and the second polypeptide adapter comprise GGGS GGGS GGGS GGGS (SEQ.ID NO.: 5).
18. The system of claim 16, wherein the first tag is longer than the second tag.
19. The system of claim 18, wherein the first TALE targets a DNA structural fragment of about 30 base pairs.
20. The system of claim 16, wherein the HIV IN tetramer comprises wild-type HIV IN having the following mutations: For the first internal monomer, Y99D, K103E, K173E, and K186E; For the second internal monomer, E96K, Y99E, K103E, V201H, D25K, and E11K; For the first external monomer, E87K, E96K, E152Q, K186Q and K215E; For the second external monomer, E152Q, K173E, T174K, K186Q, and I204D; and Used to reduce mutations associated with HIV IN and LEDGF / p75.