TALE base editor for gene and cell therapy

Abstract

The present invention relates to a method for efficiently genetically engineering cells, in particular primary hematopoietic stem cells (HSCs) and primary immune cells, using a base editor. In particular, the present invention relates to rules for designing a highly active and specific TALE-base editor exhibiting an improved on-target / off-target activity ratio for use in the manufacture of therapeutic levels of complex gene editing cells or in vivo gene therapy. In various gene therapy methods, the resulting TALE-base editor can be used either alone or in combination with a rare-cleavage endonuclease.

Description

Technical Field

[0001] The present invention relates to methods for efficiently genetically engineering cells using base editors, especially primary hematopoietic stem cells (HSCs) and primary immune cells. Specifically, the present invention relates to rules for designing highly active and specific TALE-base editors that exhibit an improved on-target / off-target activity ratio and can be used to manufacture therapeutic-grade complex gene-edited cells or perform in vivo gene therapy. The resulting TALE-base editors can be used alone or in combination with rare-cutting nucleases in various gene therapy methods. Background Art

[0002] Artificial transcription activator-like effectors (TALEs) form a special class of proteins that can bind to DNA originally derived from the plant pathogen Xanthomonas [Kay S. et al. (2007) A bacterial effectoracts as a plant transcription factor and induces a cell size regulator. Science 318: 648-651]. Artificial TALE proteins have become versatile and sequence-specific genetic tools that, after elucidation of the DNA recognition "code", connect the amino acid sequence of the TALE to the genomic DNA sequence to which it binds, providing flexible applications [Moscou JM et al. (2009) A Simple Cipher Governs DNA Recognition by TAL Effectors. Science. 326: 1501].

[0003] TALE binding is driven by a series of 33 to 35 amino acid long repeats that are essentially different at two positions, the so-called repeat variable dipeptides (RVDs). Each base of one strand of the DNA target contacts a separate repeat, with predictable specificity due to the linear arrangement of the RVDs. Biochemical structure-function studies have shown that the amino acid at position 13 uniquely recognizes a nucleotide in the major groove of the DNA target [Deng D. et al. (2012) Structural basis for sequence-specific recognition of DNA by TAL effectors. Science 335: 720-723; Stella S. et al. (2013) Structure of the AvrBs3-DNA complex provides new insights into the initial thymine-recognition mechanism. Acta Crystallogr Sect. D. Bio. l Crystallogr. 69 (9): 1707-1716]. This DNA-protein interaction unit is stabilized by the amino acid at position 12. In order to form TALEs with different precision and binding affinity, six conventional RVDs (NG, HD, NI, NK, NH and NN) are generally used. HD and NG are associated with cytosine (C) and thymine (T), respectively. NN is a degenerate RVD that exhibits binding affinity for both guanine (G) and adenine (A), but is reported to be more specific for guanine. RVD NI binds to A, and NK binds to G. It is worth noting that the binding affinity of TALE is affected by the methylation state of the target DNA sequence [Streubel J et al. (2012) TAL effector RVD specificities and efficiencies. Nat Biotechnol 30 (7): 593-595]. Methylated cytosine cannot be effectively bound by typical RVDs.However, as described by Valton J et al., they can be accommodated in TALEs by a certain degree of degeneracy [Overcoming transcription activator-like effector (TALE) DNA binding domain sensitivity to cytosine methylation (2012) J. Biol. Chem. 287 (46): 38427-38432]. This code is used to efficiently engineer the scaffold specificity of TALE DNA-binding through modular assembly to form different combinations of TALE proteins with various enzyme domains (such as transcription activators, repressors, base editors, or nucleases with the potential to act on genomic sequences) [Voytas et al. (2011) TAL effectors: Customizable proteins for DNA targeting. Science. 333 (6051): 1843-6].

[0004] TALE-base editors (BE) have recently appeared more as fusions of TALE with deaminases (and sometimes with other DNA repair proteins). The base editor catalytic domain can introduce single nucleotide variants at the desired site in DNA (nuclear DNA or organelle DNA) or RNA of both dividing cells and non-dividing cells. In general, DNA base editors can be divided into cytosine base editors (CBE), adenine base editors (ABE), C- to -G base editors (CGBE), dual base editors, and organelle base editors.

[0005] Mok et al. [A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing (2020) Nature. 583: 631-637] recently developed a base editing method by fusing the TALE binding domain to the bacterial cytidine deaminase toxin DddAtox to demonstrate the efficient conversion of C- to -T bases on the mitochondrial genome in vitro. In this method, DddAtox was split into two non-toxic halves and fused to the C-termini of paired (left and right) TALE binding domains to form heterodimeric TALE base editors.

[0006] In this case, when the two halves of the deaminase DddAtox recognize the predetermined target DNA sequence in the genome through the TALE binding domain and are close enough, the deaminase DddAtox becomes active by forming a functional heterodimeric cytosine deaminase that converts the C base located between the two binding sites to T. So far, this DddA-TALE fusion deaminase construct has achieved mitochondrial DNA editing in mice [Lee, H., et al. (2021) Mitochondrial DNA editing in mice with DddA-TALE fusion deaminases. Nat Commun 12: 1190].

[0007] However, the mitochondrial genome is much smaller than the nuclear genome of human cells.

[0008] The use of such base editors in human cells, especially for immunotherapy, has proven to be very challenging. In human gene therapy in particular, it becomes critical to determine the editing window to induce C-to-T base editing at the target site to avoid unwanted substitution of any C bases located elsewhere into proximal genomic regions.

[0009] Depending on the sequences to be targeted in the genome and their intrinsic variability in the human population, TALE-base editors need to be further improved to fully exert their activity and reduce the risk of potential off-target substitutions.

[0010] As shown in the experimental section of this article, the inventors have conducted extensive research to determine rules that allow the determination of optimal target genomic sequences associated with the design of efficient TALE base editors. They combined the screening of dozens of TALE base editors targeting various endogenous sites with the development of medium / high-throughput cell-based analysis, which can take full advantage of the bias caused by the confounding effects of factors such as epigenomic factors or modifications. This method relies on creating a pool of cells containing artificial targets for base editors. These cells are generated by inserting a set of (30 to 191 members) carefully designed BE target sequences into predetermined genomic sites. The cell pool is then treated with various TALE base editors to perform gene editing on different sets of target sequences. Next-generation sequencing (NGS) analysis of editing frequencies on BE targets can better characterize the activity and substrate specificity of TALE base editors within the editing window. The accumulated knowledge was then used to create a new TALE base editor scaffold, referred to herein as "TALEB", which can effectively knock out several genes in primary T cells, especially the CD52 gene (up to 87% phenotypically and 86% edited at the genomic level) and the β2m gene (a potential target gene for allogeneic CAR-T cell adoptive therapy). The knowledge gained from this study clarified the editing guidelines and rules, which helped to develop the TALE base editors of the present invention and their use in therapeutic immune cells. In addition to the new scaffold TALEB, the present invention also provides a platform for the rational design of higher therapeutic-level TALE base editors based on the selection of appropriate endogenous genomic targets. Summary of the invention

[0011] TALE recombinant DddA-derived cytosine base editors are heterodimers produced by a fusion of a transcription activator-like effector array protein (TALE), a split DddA deaminase half, and a uracil glycosylase inhibitor (UGI). It is a recent improvement on the available base editor tools that can directly edit double-stranded DNA, converting cytosine (C) to thymine (T). This TALE base editor has been used to edit in mitochondria and produce heritable modifications. However, the editing rules for this particular base editor have not been fully elucidated. In order to further dissect the editing rules of TALE base editors, the inventors used nuclease-based targeted knock-in technology and created a cell pool, each containing a unique BE target sequence at the same genomic site. These cells were then treated with TALE base editors, followed by NGS analysis of the mutation patterns on the target sequence. As shown in the experimental section of this article, this approach enables the generation of a large number of different TALE base editor target pools and in-depth access to editing rules within cells, while excluding confounding factors such as epigenetic and microenvironmental differences between different genomic sites. Using the knowledge gained from this innovative approach, the inventors designed new scaffolds, termed "TALEBs," targeting a range of endogenous genes, such as those encoding CD52, TCR, B2M, and PD1 (for knockout in therapeutic immune cells).

[0012] As one aspect, the present invention is directed to identifying target sequences in the genome that specifically allow TALE base editors to specifically focus on converting the desired cytosine (C) to thymine (T) while limiting off-target mutations. This "more precise" target sequence is defined by:

[0013] 5′-T0-N 左 -N y -RTC-N X -N 右 -A0-3′; and

[0014] 5′-T0-N 左 -N x -GAY-N y -N 右 -A0-3′

[0015] in

[0016] N can be A, T, C or G

[0017] R can be G or A, preferably G

[0018] Y can be C or T, preferably C;

[0019] N 左It may be a polynucleotide sequence comprising 9 to 20 nucleotides, wherein each individual nucleotide may be A, T, C or G;

[0020] N 右 It may be a polynucleotide sequence comprising 9 to 20 nucleotides, wherein each individual nucleotide may be A, T, C or G;

[0021] G is the complementary base of C.

[0022] x=2 to 6

[0023] y = 6 to 10

[0024] Preferably, x+y≥11, and more preferably, x+y=12.

[0025] like Figure 3 As shown, the above general formula interprets the surface on the double-stranded DNA available for deaminase to act, which has a length of about 7 nucleotides (L = 0.34nm × 6 = 2.4nm), which represents the optimal target window for targeting the desired C in the genomic sequence using the TALE base editor. This surface has an arc f = 4 × 34,3° = 136,8° = 2,38 radians (an angle spanning 5 nucleotide bases). Assuming that the radius of the double-stranded DNA helix is ​​about 1nm, the surface for targeting C is equivalent to L×R×f=2,4×1×2,38=5,71nm 2 .

[0026] Therefore, when N 左 and N 右 When the interval is 15 bases, the target surface bounded by the diagonal lines connecting the bases at positions N11, N-13 (opposite strand) and N9, N-9 (opposite strand) is about 4.87 nm. 2 .

[0027] As shown in the experiments in the Examples, more specific TALE base editors, such as the "TALEB" described herein, can be designed to more specifically target genomic sequences defined as follows:

[0028] 5′-T0-N 左 -N y -RTCC-N X -N 右 -A0-3′; and

[0029] 5′-T0-N 左 -N x -GGAY-N y -N 右 -A0-3′

[0030] in

[0031] N can be A, T, C or G

[0032] R can be G or A, preferably G

[0033] Y can be C or T, preferably C;

[0034] N 左 It may be a polynucleotide sequence comprising 9 to 20 nucleotides, wherein each individual nucleotide may be A, T, C or G;

[0035] N 右 It may be a polynucleotide sequence comprising 9 to 20 nucleotides, wherein each individual nucleotide may be A, T, C or G;

[0036] G is the complementary base of C.

[0037] x = 1 to 5

[0038] y=5 to 9

[0039] Spacer, defined as the number of binding sites N 左 and N 右 The number of base pairs between the two is preferably 13 or 15 bp.

[0040] According to experiments, the TALE base editor monomer of the present invention, which contains a C-terminus of a TALE of less than 40 amino acids, such as C40 and C11 described herein, shows higher specificity on a target sequence containing a 15 bp interval. The TALEB monomer containing a C-terminus of a TALE of less than 12 amino acids, such as C11 described herein, exhibits the highest specificity, especially when bound to a 15 bp interval, but also when bound to a 13 bp interval. Therefore, this TALE base editor monomer is particularly suitable for target sequences containing an interval of about 10 to 20 bp, more preferably 13 to 16 bp, and even more preferably 12 to 15 bp.

[0041] TALE base editor monomers at the C-terminus of TALEs containing less than 12 amino acids, particularly C11 described in this article, also show stronger discrimination ability when there is a series of Cs in the target sequence, such as at least two CCs, three CCCs, or four CCCCs. This series of Cs may or may not be preceded by T, but it is best to be preceded by T, and the first C is usually converted to T (C>T) by the TALE base editor.

[0042] One benefit of this embodiment is that the TALE base editors of the present invention are able to target genomic sequences that do not have a "T" immediately preceding the C to be edited, or a series of CCCs following such a "T". Thus, the present invention broadens the number of sequences that can be edited using TALE base editors.

[0043] In view of these findings, the present invention provides a method for designing a TALE base editor that can accurately target the C position in a gene sequence, the method comprising one of the following steps:

[0044] i) identifying a target sequence in a genome as defined above;

[0045] ii) Synthetic coding and N 左 Polynucleotide sequences and N 右 The polynucleotide sequences of the left TALE-binding polypeptide and the right TALE-binding polypeptide bound by the polynucleotide sequence.

[0046] iii) fusing the polynucleotide sequence encoding the left TALE binding polypeptide with a polynucleotide encoding the N-terminal split DddAtox;

[0047] iv) fusing the polynucleotide sequence encoding the right TALE binding polypeptide with a polynucleotide encoding the C-terminal split DddAtox;

[0048] v) fusing a polynucleotide sequence encoding a polypeptide that inhibits uracil glycosylation (such as UGI, (uracil glycosylase inhibitor)) to at least one polynucleotide sequence encoding the polynucleotide sequence produced by steps ii) and iii).

[0049] vi) optionally, co-expressing the two generated polynucleotide sequences to obtain TALE base editor heterodimers.

[0050] According to certain aspects, the left TALE-binding polypeptide and the right TALE-binding polypeptide comprise a C-terminus of 1 to 50 amino acids, preferably 8 to 40, more preferably 10 to 30, even more preferably about 11 amino acids or about 40 amino acids.

[0051] According to certain aspects, the left and right TALE-binding polypeptides comprise a C-terminus of about 13 or 40 amino acids derived from the original AvrBs3 TALE protein, which is typically at least 90%, 95% or 99% identical to SEQ ID NO:4.

[0052] According to certain aspects, the N-terminal and / or C-terminal members of the split DddAtox contain at least one mutation that reduces the affinity of the two split DddAtox members to each other to avoid TALE-independent nonspecific binding of DddAtox in the genome, thereby improving the specificity of the TALE base editor.

[0053] According to certain aspects, the present invention can be viewed as a method for introducing mutations into a cell genome, the method comprising the following steps: introducing a TALE base editor into a cell or expressing it in a cell, the TALE base editor consisting of a left TALE binding polypeptide and a right TALE binding polypeptide having a C-terminal domain of about 1 to 50 amino acids, respectively, and a heterodimeric fusion of the C-terminus and N-terminus of split DddATox, wherein the heterodimeric TALE base editor binds to a genomic sequence as defined above.

[0054] A preferred embodiment is a method of gene editing using the TALE base editor of the present invention in gene therapy, especially engineering and manufacturing primary cells in vitro, more specifically HSC and immune cells (such as T cells and NK cells for cell therapy). The manufacture of therapeutic cells more specifically includes the following steps: using a base editor to make it allogeneic and / or hidden to the patient's immune system, such as by destroying the TCR or B2M gene; and other steps using rare-cutting nucleases for gene targeted insertion or replacement, such as, for example, at immune checkpoint gene sites.

[0055] This manufacturing strategy is particularly effective when TCR inactivation by using base editors is combined with insertion / replacement of chimeric antigen receptors or recombinant TCRs at different sites, such as B2M or PD1. Another example is the opposite strategy, where B2M is inactivated and inserted at the TCR site by using base editors.

[0056] A preferred method comprises the following steps: making cells resistant to immunosuppressive drugs by inactivating genes (such as CD52) using base editors, and integrating exogenous polynucleotide sequences at another site by using rare-cutting nucleases. These steps can be performed simultaneously by co-electroporating immune cells or their precursors using base editing agents and at least one nuclease agent.

[0057] In this regard, the present invention provides specific reagents and target sequences to successfully achieve the production of such therapeutic immune cells as well as examples of various TALE base editing proteins designed according to the principles and rules of the present invention.

[0058] The TALE base editors according to the present invention can also be used for in vivo gene therapy to correct mutations or inactivate genetic defective genes, such as ApoC3 in hepatocytes.

[0059] The present invention encompasses vectors comprising polynucleotide sequences and polypeptide sequences or agents obtainable by the present invention, and their use for cell transformation and genetic modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 : Schematic diagram of the TALE base editor of the present invention. TALEB consists of the N-terminal portion of TALE (such as N152 truncation of AvrBs3), a repeat array, the C-terminal portion of TALE (preferably AvrBs3C11 or C40 truncation), split DddATox (for example, at position G1397), and UGI (uracil glycosylase inhibitor).

[0061] Figure 2 : A. The graph shows the distribution of the 37 TALE-nucleases tested in Example 2 based on their nuclease activity. B. Comparison of TALE-nuclease activity (Y-axis) and TALE base editor (X-axis) frequency for 37 TALE target sequences: There was no significant correlation between the activity of TALE-nucleases and TALE-base editing at those target sequences.

[0062] Figure 3 : A. 3D schematic diagram of a double-stranded DNA structure showing sites (black circles) that can be edited by a TALE base editor (using a TALE base editor to split a DddATox heterodimer) according to the experimental analysis provided herein. B. 3D schematic diagram of a surface that can be edited by a TALE base editor.

[0063] Figure 4 :A. Graphical display of insertion and deletion (indel) frequency (Y-axis) and the percentage of CT conversion induced by the TALE base editors of the present invention (X-axis). B. Percentage of editing purity. For each TALE base editor tested, the percentage of CT conversion detected in the interval for all conversions ("Editing Only") or all conversions and insertions and deletions ("Editing and Indels"). C. Schematic diagram showing the different events induced by each of the 37 TALE base editors in Example 2. These figures show that for all activity levels induced by TALE base editors, the final purity of the edited cell population is very high.

[0064] Figure 5:A. Design of the first TALE base editor screening described in Example 3. The oligonucleotide pool contains the left and right homology arms of the TRAC site, the left and right binding sequences of T-25, and TC / GA sequences placed at different positions within a 15bp interval. After double transfection (TRAC TALE-nuclease and ssODN pool, and T-25 base editor), genomic DNA was analyzed by NGS. B. Represents the different events (CT conversion: editing, indels, other mutations, none) obtained on 2 different donors. C. Correlation of CT conversion frequencies between donors obtained on the bottom chain (left figure) or the top chain (right figure). D. Percentage of CT conversion according to the positioning of C on the top chain (top figure) or the bottom chain (bottom figure).

[0065] Figure 6 :A. Design of the second TALE base editor screening performed in Example 3. The oligonucleotide pool contains the left and right homology arms of the TRAC site, the left and right binding sequences of T-25, and TC / GA sequences placed at different positions of intervals of different lengths. In addition, a barecode (a unique specific sequence) is inserted between the right binding site and the right homology arm of the TALE of each interval length. B. Display of different events (CT conversion: "editing", insertion and deletion, other mutations, none) obtained on 2 different donors. C. Correlation between donors in the frequency of CT conversions obtained on the bottom chain (left figure) or the top chain (right figure). D. The frequency of CT conversion of the top chain (left figure) or the bottom chain (right figure) according to the position of the CT within the interval and the length of the interval.

[0066] Figure 7 : Heat map of C to T conversion according to TC context (NTCCNN). N=2, independent T cell donors, experiments as shown in Example 3 showed that G or A before TC is conducive to efficient BE editing.

[0067] Figure 8 : A. Schematic diagram of the base editing strategy according to the present invention, inactivating the CD52 gene by mutating the splice acceptor site of CD52, thereby creating therapeutic immune cells. B. The percentage of CD52-negative cells obtained using the indicated TALE base editors in Example 4. C. The frequency of CT conversion (E) or indel (I) obtained using the indicated TALE base editors.

[0068] Fig. 9: A. Schematic diagram of the base editing strategy according to the present invention, inactivating the CD52 gene by mutating the signal peptide of CD52, thereby creating therapeutic immune cells. B. The percentage of CD52 negatives obtained with the indicated TALE base editors. C. The frequency of CT conversions obtained at the indicated positions or indels. D. The frequency of different sequences obtained after treatment with TALE base editors (amino acid substitutions, shown in gray).

[0069] Fig.10 : Flow cytometric analysis of primary T cells (TCR as X-axis and CD52 as Y-axis), untreated (upper panel), treated with TALENs targeting TRAC and CD52 (lower left panel), treated with TALENs targeting TRAC and TALEBs targeting CD52 (according to the experiment from Example 4 of the present invention).

[0070] Fig.11 : A graph comparing the translocation readout of primary T cells treated with TALEN targeting TRAC and CD52 (TALEN+TALEN), or with TALEN targeting TRAC and a TALE base editor targeting CD52 (according to Example 4 of the present invention).

[0071] Fig.12 , 13 and 14: Schematic diagram of a gene therapy method according to the present invention, which method may include the use of sequence-specific nucleases to insert a functional copy of a gene or a correction sequence thereof, combined with the use of sequence-specific base editing reagents, which are used to inactivate residual endogenous sequences that act as "proofreaders". In the exemplified case, the correct sequence is rewritten relative to the wild-type allele sequence by using alternative codons and introduced into the genome by using site-directed nuclease integration. Depending on the extent of allele replacement, different results can be expected from this integration (mainly carried out by homologous recombination) in the cell genome (schemes A to C). A: Both the dominant mutant allele and the wild-type functional allele have been replaced, resulting in a functional homozygous cell. B: Only the dominant mutant allele has been replaced, resulting in a functional heterozygous cell. C: No insertion has occurred and the heterozygous cell is still defective. D: Only the wild-type allele has been replaced, resulting in a heterozygous cell that is still defective. In Fig.14 In the present invention, sequence-specific base editors (such as the TALE base editors described in this specification) are introduced into cells to inactivate endogenous sequences (i.e., non-rewritten sequences) that are not replaced / corrected by integration of functional rewriting sequences.

[0072] Fig.15: Schematic diagram of inserting artificial exons (Artex) into endogenous genes guided by sequence-specific nucleases, so that exon expression is regulated by the endogenous gene promoter. This strategy of correcting exon insertion can be combined with the introduction of base editors to "proofread" and inactivate uncorrected exons, which is the aforementioned Figures 12 to 14 Specific implementations of the described methods.

[0073] Fig.16 and 17 : As Figures 12 to 14 Embodiments of gene therapy methods, these figures illustrate the combination of sequence-specific nucleases with base editors, where the specific nuclease can be co-electroporated with a DNA matrix encoding a therapeutic gene cassette, which contains an exogenous promoter for integration into a predetermined site between exon 1 and exon 2 of a specific gene. Schemes 1 to 4 correspond to the possible outcomes of gene cassette integration relative to defective endogenous exon 3 alleles, and the benefits of using base editors to inactivate the expression of exon 3 to address each of these situations, either sequentially (as shown) or simultaneously (e.g., co-transfection). In this example, for simplicity, it is assumed that the base editor edits two alleles. However, this editing can also distinguish alleles carrying pathogenic mutations.

[0074] Fig.18 and 19 : As Figures 12 to 14 Embodiments of gene therapy methods, these figures illustrate the integration of a promoter-less corrected copy of an exon, which is placed under the control of a gene's endogenous promoter by Artex (e.g. Fig.15 ), and subsequent inactivation of the originally defective exon by base editing.

[0075] Fig. 20 :A. An example of a nuclease / base editor-mediated gene therapy according to the present invention, in which a promoter-less therapeutic cDNA matrix (encoding a correction sequence of exons 2 to 24) is integrated by the Artex method to correct a dominant negative mutation causing ADPS1 occurring in exon 24 of PIK3CD ( Fig.15 ). When the insertion itself is not blocked, the expression of the originally defective exon 24 is inactivated by base editing, as detailed in Example 5. Using this approach, all reagents, in particular the site-specific endonuclease and the sequence-specific base editor bases, can be introduced into the cell simultaneously, such as by co-electroporation. B. Schematic diagram detailing the different elements that make up the therapeutic repair matrix.

[0076] Fig.21: Schematic diagram of the site-specific integration of a promoter-less PIK3CD corrected copy (including exon 24) into intron 2 of this gene in HSCs by Artex, and the subsequent inactivation of the originally defective exon by base editing using a TALE base editor according to the present invention as detailed in Example 5.

[0077] Fig. 22 : Schematic diagram of artificial STAT3TALEB target sequences including 5, 7, 11, 13, 15 and 17 bp interval lengths / editing windows to be inserted at the TRAC site to test C to T editing efficiency as detailed in Example 6.

[0078] Fig.23 : Detailed representation of TALEBs tested in Example 6 for optimal C to T editing efficiency, including alternative TALE C-terminal "linkers" C0, C11 and C40.

[0079] Fig.24 : Graphical analysis of sequencing data obtained by NGS analysis from Example 6 experiments evaluating C0, C11 and C40 TALEB scaffolds relative to different spacer lengths. A: Editing target with 5 bp spacer. B: Editing target with 7 bp spacer. C: Editing target with 9 bp spacer. D: Editing target with 11 bp spacer. E: Editing target with 13 bp spacer. F: Editing target with 15 bp spacer. G: Editing target with 17 bp spacer.

[0080] Fig.25 : Graphical analysis of sequencing data obtained by NGS analysis resulting from the experiment of Example 6 evaluating the combination of C11 and C40 heterodimers on targets with a 15 bp interval.

[0081] Fig.26 : Schematic diagram of the environmental changes around "TC" edited when using the STAT3TALEB scaffold involving C0, C11 and C40 linker structures to test the target sequence library to be inserted in the TCR site through the experiment of Example 6.

[0082] Fig. 27 : Fig.26 The positions of the changes in the target sequence library are described in .

[0083] Fig.28 : Bioinformatics data analysis to determine the contribution of each surrounding base pair to C editing efficiency in the context of 15 bp intervals. A: Using the C40 TALEB scaffold. B: Using the C11 TALEB scaffold.

[0084] Fig.29: Bioinformatics data analysis showing the efficacy of TCC→TTT based on each base around TCC in a 15 bp interval environment. A: Using C40TALEB scaffold. B: Using C11TALEB scaffold.

[0085] Fig.30 : Bioinformatics data analysis to determine the contribution of each base pair to C editing efficiency in the context of a 13 bp interval. A: Using the C40 TALEB scaffold. B: Using the C11 TALEB scaffold.

[0086] Fig.31 : Bioinformatics data analysis showing the efficacy of TCC→TTT based on each base around TCC in the context of 13 bp intervals. A: Using C40TALEB scaffold. B: Using C11TALEB scaffold.

[0087] Fig.32 : Experimental results detailed in Example 7, which relates to a strategy for gene editing in T cells combining TALEB (TCRKO) and TALEN (KI of HLAE using AAV matrix and homologous recombination). The results show efficient gene editing and avoidance of "AAV capture" at the TRAC site. A: A graphical representation showing the percentage of gene-edited cells. B: Flow cytometric analysis comparing TCR inactivation using TALEN and TALEB in the presence of HLAE AAV matrix.

[0088] Table 1: 37 genomic target sequences used in Example 2.

[0089] Table 2: Sequences of 2×15 individual ssODNs used in Example 3 to determine the editing window with 15 bp intervals.

[0090] Table 3: Sequences of 191 individual ssODNs used in Example 3 to evaluate the effect of spacer length on editing.

[0091] Table 4: Sequences of individual ssODNs used to evaluate TC context in TALE base editor target sequences in Example 4.

[0092] Table 5: Examples of KO CD52 TALEB polypeptides and polynucleotides of interest according to the invention.

[0093] Table 6: Predicted potential off-target locations of the four TALEBs targeting CD52 evaluated in Example 4.

[0094] Table 7: List of TALEB target sequence windows for introducing mutations in TRAC genes following the rules of the present invention.

[0095] Table 8: List of TALEB target sequence windows for introducing mutations in the CD52 gene following the rules of the present invention.

[0096] Table 9: List of TALEB target sequence windows for introducing mutations in the PD1 gene following the rules of the present invention.

[0097] Table 10: List of TALEB target sequence windows for introducing mutations in the B2m gene following the rules of the present invention.

[0098] Table 11: List of TALEB target sequence windows for introducing mutations in the ApoC3 gene following the rules of the present invention.

[0099] Table 12: Base editor target locations in exon 1, 2 or 3 of the PK13 gene according to the combined gene therapy (nuclease + base editor) method of the present invention described in Example 5 herein.

[0100] Table 13: Polypeptide sequences of the C-terminal lengths of different TALEs used in TALEBs designated C40, C11 and C0 backbones.

[0101] Table 14: TALEB heterodimers tested in Example 6.

[0102] Table 15: ssODN pool containing 5′ TC at 11 positions flanked by optimal spacer lengths (13 or 15 bp spacer length) integrated at the TCR locus targeted by the STAT3 TALEB target.

[0103] Table 16: ssODN pool used in Example 6 to evaluate the impact of TC surrounding environment in 15 bp interval length.

[0104] Table 17: ssODN pool used in Example 6 to evaluate the impact of the TC surrounding environment in 13 bp interval lengths.

[0105] Table 18: Polynucleotide and polypeptide sequences used in Example 7.

[0106] Table 19: Figures 12 to 17 A list of exemplary diseases and alleles that can be cured by a gene therapy approach exemplified in , which can include combining a site-specific nuclease (to rewrite the gene sequence for targeted insertion correction) with a sequence-specific base editor (to inactivate the remaining endogenous deleterious allele sequence). DETAILED DESCRIPTION

[0107] Unless specifically defined herein, all technical and scientific terms used have the same meanings as commonly understood by one of ordinary skill in the fields of gene therapy, biochemistry, genetics, and molecular biology.

[0108] All methods and materials similar or equivalent to those described herein can be used for the practice or testing of the present invention, and suitable methods and materials are described herein. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification including definitions will take precedence. In addition, unless otherwise stated, materials, methods and embodiments are illustrative only and are not intended to be limiting.

[0109] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art. Such techniques are fully explained in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; Oligonucleotide Synthesis (MJ Gait ed., 1984); Mullis et al. US Pat. No. 4,683,195; Nucleic Acid Hybridization (BD Harries&S.J.Higgins eds.1984); Transcription And Translation (BDHames&S.J.Higgins eds.1984); Culture OfAnimal Cells (RIFreshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols. 154 and 155 (Wu et al.eds.) andVol.185, "Gene Expression Technology" (D.Goeddel, ed.); Gene Transfer Vectors ForMammalian Cells (JHMiller and MPCalos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (DMWeir and CC Blackwell, eds., 1986); Harbor, NY, 1986].

[0110] Therefore, the purpose of the present invention is to provide a method for designing and producing TALE proteins to convert a specific C or its complementary G position in a double-stranded nucleic acid sequence into A / T. Although it is not explicitly stated throughout this document, the present teaching will directly convert the targeted C position into a G on the opposite DNA strand.

[0111] According to some embodiments, the method of the present invention comprises the step of identifying a target sequence in a polynucleotide sequence (such as a genomic sequence), the target sequence having the following characteristics:

[0112] 5′-T0-N 左 -N y -RTC-N x -N 右 -A0-3′; or

[0113] 5′-T0-N 左 -N x -GAY-N y -N 右 -A0-3′,

[0114] in:

[0115] N can be A, T, C, or G;

[0116] R can be G or A, preferably G;

[0117] Y can be C or T;

[0118] N 左 It may be a polynucleotide sequence comprising 9 to 20 nucleotides, wherein each individual nucleotide may be A, T, C or G;

[0119] N 右It may be a polynucleotide sequence comprising 9 to 20 nucleotides, wherein each individual nucleotide may be A, T, C or G;

[0120] G is the complementary base of C.

[0121] x = 2 to 6;

[0122] y = 6 to 10;

[0123] Preferably, x+y≥11, more preferably, x+y=12.

[0124] Also preferably, x is 2-5, and more preferably 3-5.

[0125] The inventors also show that TALE base editors, in particular TALEB of the present invention, are more specific to polynucleotide target sequences represented by formula i) or ii):

[0126] i) 5′-T0-N 左 -N y -RTCC-N x -N 右 -A0-3′; or

[0127] ii) 5′-T0-N 左 -N x -GGAY-N y -N 右 -A0-3′,

[0128] And even more specific for target sequences represented by formula iii) and iv):

[0129] iii) 5′-T0-N 左 -N y -GTCC-N x -N 右 -A0-3′; or

[0130] iv) 5′-T0-N 左 -N x -GGAC-N y -N 右 -A0-3′,

[0131] in:

[0132] N can be A, T, C, or G;

[0133] R can be G or A, preferably G;

[0134] Y can be C or T;

[0135] N 左It may be a polynucleotide sequence comprising 9 to 20 nucleotides, wherein each individual nucleotide may be A, T, C or G;

[0136] N 右 It may be a polynucleotide sequence comprising 9 to 20 nucleotides, wherein each individual nucleotide may be A, T, C or G;

[0137] G is the complementary base of C.

[0138] Where x and y are preferably defined as follows:

[0139] x=2 to 4

[0140] y = 6 to 8

[0141] At the same time, 11≥x+y≥9, more preferably x+y=9.

[0142] The improved target sequence according to the present invention can be used to design and express corresponding appropriate specific base editing tools, in particular, by synthesizing polynucleotide sequences encoding left TALE binding polypeptides and right TALE binding polypeptides, which are respectively combined with the above-defined N 左 Polynucleotide sequences and N 右 Polynucleotide sequences. Such polynucleotide sequences encoding the left TALE binding polypeptide and the right TALE binding polypeptide can be fused with a polynucleotide sequence encoding a member of the split DddAtox to form a TALE-DddATox heterodimer, which is usually achieved by fusing the member of the split DddaTox to the C-terminus of the TALE binding polypeptide. The method of the present invention generally also includes the step of fusing a polynucleotide sequence encoding UGI (uracil glycosylase inhibitor) to a monomer of the TALE-DddATox heterodimer, such as Figure 1 shown.

[0143] According to some embodiments, the left TALE binding polypeptide and the right TALE binding polypeptide are connected to the split DddAtox via the C-terminus of a TALE of 1 to 50 amino acids, preferably 8 to 40, more preferably 10 to 30, and even more preferably about 40 amino acids. The present invention provides an optimal scaffold comprising a C-terminal linker of about 11 amino acids or, alternatively, about 40 amino acids, which is generally derived from the AvrBs3 original Xanthomonas TALE protein [Christian, M. et al. TAL effector nucleases create targeted DNA double-strand breaks (2010) Genetics 186: 757-761].

[0144] "TALE protein" as referred to herein refers to a polypeptide that generally comprises a core DNA binding domain that is at least 50%, preferably at least 60%, 70%, 80% or 90% identical to the DNA binding domain of wild-type AvrBs3 [also known as TalC Uniprot-G7TLQ9], which represents the prototype of the transcription activator-like (TAL) effector (effetor, sensor) family from phytopathogenic Xanthomonas phytopathogenicus. This DNA binding domain is characterized by a repeat sequence of about 30 and 34 amino acids, including variable double residues usually found at positions 12 and 13.

[0145] "AvrBs3-like repeats" refers to artificial arrays of about 30 to 33 amino acids, which typically contain variable double residues that interact with A, C, G or T at positions 12 and 13, similar to the consensus AvrBs3 repeats described above. In other words, AvrBs3-like repeats are similar and can be combined with AvrBs3 repeats, but are generally not completely identical to consensus or wild-type AvrBs3 repeats. It should be noted that in some cases, the double residues at positions 12 or 13 may not be present - the so-called * (asterisk) - to accommodate methylated bases in genomic DNA, as described in [Valton et al. (2012) Overcoming Transcription Activator-like Effector (TALE) DNA Binding Domain Sensitivity to Cytosine Methylation. DNA and Chromosomes. 287 (46): 38427].

[0146] The AvrBs3-like repeats of the present invention generally show at least 60%, preferably at least 70%, 75%, 80%, 90% or 95% identity with any of the AvrBs3 consensus repeats of SEQ ID NOs: 12 to 15 above. They generally include D4 and D32 substitutions, such as the following repeat sequences of the present invention SEQ ID NOs: 5 to 11:

[0147] LTP D QVVAIASX 12 X 13 GGKQALETVQRLLPVLCQ D HG (SEQ ID NO: 5),

[0148] LTP D QVVAIASX 12 X 13GGKQALETVQALLPVLCQDHG(SEQ ID NO:6),

[0149] LTP D QVVAIASX 12 X 13 GGKQALETVQQLLPVLCQDHG(SEQ ID NO:7),

[0150] LTP D QLVAIASX 12 X 13 GGKQALETVQRLLPVLCQDHG(SEQ ID NO:8),

[0151] LTP D QMVAIASX 12 X 13 GGKQALETVQRLLPVLCQDHG(SEQ ID NO:9),

[0152] LTP D QVVAIASX 12 X 13 GGKQALETVQRLLPVLCQDQG (SEQ ID NO: 10), or

[0153] LTL D QVVAIASX 12 X 13 GGKQALETVQRLLPVLCQDHG(SEQ ID NO:11),

[0154] Where X 12 X 13 is a di-residue that interacts with a given nucleotide base pair in the target sequence.

[0155] The variable di-residues (X) present in the AvrBs3-like repeats and associated with recognition of different nucleotides 12 X 13) are typically: HD for recognition of C; NG for recognition of T; NI for recognition of A; NN for recognition of G or A; NS for recognition of A, C, G or T; HG for recognition of T; IG for recognition of T; NK for recognition of G; HA for recognition of C; ND for recognition of C; HI for recognition of C; HN for recognition of G; NA for recognition of G; SN for recognition of G or A and YG for recognition of T; TL for recognition of A; VT for recognition of A or G and SW for recognition of A. More preferably, the RVDs associated with recognition of nucleotides C, T, A, G / A and G are selected from the group consisting of: NN or NK for recognition of G; HD for recognition of C; NG for recognition of T and NI for recognition of A; TL for recognition of A; VT for recognition of A or G and SW for recognition of A, respectively. More typically, the RVD associated with recognition of nucleotide C is selected from the group consisting of N*, and the RVD associated with recognition of nucleotide T is selected from the group consisting of N* and H*, wherein * may represent a vacancy in the repeat sequence, corresponding to the lack of an amino acid residue at the second position of the RVD. In some embodiments, X 12 X 13 Can represent unusual or unconventional amino acid residues to tune their specificity for the nucleotides A, T, C, and G as described by Juillerat et al. [Optimized tuning of TALEN specificity using non-conventional RVDs (2015) Sci Rep 5:8150].

[0156] AvrBs3-like repeats are usually represented by a polypeptide sequence, where X 12 and X 13 They are NI (preferably targeting A), HD (preferably targeting C), NN (preferably targeting G) and NG (preferably targeting T), as shown in SEQ ID NOs: 12, 13, 14 and 15, respectively.

[0157] In some embodiments, the present invention also provides a recombinant transcription activator-like effector (TALE) base editor, which includes one or more AvrBs3-like repeats including D (aspartic acid) residues at positions 4 and 32, such as shown in the above-mentioned polynucleotide sequences SEQ ID NO: 5 to 11. Including or in addition to the D4 and D32 positions, this AvrBs3-like repeat may further mutate at 1 to 5 amino acid positions. This recombinant transcription activator-like effector (TALE) base editor may include one or more such repeats to bind N 左 and N 右, to form a polypeptide comprising typically 9 to 20 repeat segments, preferably 10 to 18, more preferably 11 to 15 and alternatively 5 to 12 repeat segments (in the case of smaller genomes (such as the mitochondrial genome)).

[0158] Although not mandatory, the core DNA binding domain generally comprises half a RVD consisting of 20 amino acids at the C-terminus. Thus, the core DNA binding domain comprises 9.5 to 20.5 RVDs, more preferably 10.5 to 18.5 RVDs, even more preferably 11.5 to 15.5 RVDs.

[0159] According to the present invention, the core DNA binding domain as described above preferably comprises an RVD with D4 and / or D32 substitutions, flanked by N-terminal and C-terminal sequences, and the N-terminal and C-terminal sequences preferably have one of the following detailed features.

[0160] In some embodiments, the N-terminal sequence is derived from the N-terminal domain of a naturally occurring TAL effector (such as AvrBs3). In another embodiment, the additional N-terminal domain is the full-length N-terminal domain of a naturally occurring TAL effector N-terminal domain. In a further embodiment, the additional N-terminal domain is a variant that is able to overcome sequence restrictions associated with the so-called "RVD0" (i.e., the first cryptic repeat), such as, for example, the necessity of having T as the first base in the binding nucleic acid sequence.

[0161] In another embodiment, the N-terminal sequence is derived from a naturally occurring TAL effector or a variant thereof. In another embodiment, the N-terminal sequence is a truncated N-terminus of such a naturally occurring TAL effector or variant. In another embodiment, the additional domain is a truncated version of the AvrBs3 TAL effector. In another embodiment, the truncated version lacks its N-terminal fragment away from the core TALE binding domain, such as the first 152 N-terminal amino acid residues of wild-type AvrBs3, or at least 152 amino acid residues.

[0162] In some embodiments, the C-terminal sequence corresponds to the complete or preferably truncated C-terminal region of a naturally occurring TAL effector (such as AvrBs3). Typically, the C-terminal sequence is a truncated version of the AvrBs3 TAL effector near the core TALE binding domain, such as SEQ ID NO: 2 (11 amino acids), SEQ ID NO: 3 (40 amino acids) or SEQ ID NO: 4 (50 amino acids) or natural variants thereof. Therefore, the C-terminal sequence typically comprises or consists of: a polypeptide sequence having at least 85%, 90%, 95% or 99% identity with the following SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, as follows:

[0163] -SEQ ID NO: 2 (C-11AA):

[0164] SIVAQLSRPDP

[0165] -SEQ ID NO: 3 (C-40AA):

[0166] SIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVX1X2GL

[0167] -SEQ ID NO:4 (C-50AA):

[0168] SIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVX1X2GLPHAPALIX3RT

[0169] In the above sequence, X1, X2 and X3 represent K or an amino acid substitution introduced into the C-terminal polypeptide sequence of wild-type AvrBs3, which is preferably an R (arginine) or H (histidine) residue, most preferably R. X1, X2 and X3 may be the same or different.

[0170] The N-terminal sequence or C-terminal sequence may comprise a localization sequence (or signal) that allows the chimeric protein to be targeted to a given organelle, tissue or cell in an organism. Non-limiting examples of such localization signals are nuclear localization signals, chloroplast localization signals or mitochondrial localization signals. In another embodiment, the additional N-terminal domain may comprise a nuclear export signal having an opposite effect to the nuclear localization signal to help target organelles such as chloroplasts or mitochondria. Additional C-terminal or N-terminal sequences with a variety of localization signal combinations are also included within the scope of the present invention. As a non-limiting example, this combination can be a nuclear localization signal (NLS) and / or a tissue-specific signal to help localize the fusion protein of the present invention in the nucleus of tissue-specific cells. In a preferred embodiment, an NLS is typically included in the N-terminal region of the TALE protein.

[0171] Throughout this specification, "identity" refers to the sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing the position arranged for the purpose of comparison in each sequence. When the position in the compared sequence is occupied by the same base, then these molecules are identical in this position. The degree of similarity or identity between nucleic acid or amino acid sequences depends on the number of nucleotides that are identical or matched in the position shared by the nucleic acid sequence. Various alignment algorithms and / or programs can be used to calculate the identity between two sequences, including FASTA or BLAST, which are available as part of the GCG sequence analysis software package (University of Wisconsin, Madison, Wis.), and can be used with, for example, default settings. This specification generally includes polypeptides and polynucleotides with at least 70%, 85%, 90%, 95%, 98% or 99% identity to specific polypeptides and polynucleotide sequences described herein, which exhibit substantially the same function or can be considered as equivalents.

[0172] In the present invention, DddAtox refers to a wild-type cytosine deaminase derived from the microorganism Burkholderia cenocepacia as described by Mok et al. [A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing (2020) Nature. 583: 631-637], which can be split into two inactive halves called DddAtoxspNter (SEQ ID NO: 28) and DddAtoxspCter (SEQ ID NO: 29) at residues 1333 or 1397. When these halves are assembled adjacently on a target DNA driven by a TALE binding domain, these halves will reconstruct deamination activity. In a preferred embodiment, DddAtox splits at residue 1397.

[0173] According to a preferred embodiment (which can itself be regarded as an invention), the specificity of the TALE base editor can be further enhanced by introducing mutations to DddAtoxspNter (SEQ ID NO: 28) and DddAtoxCter (SEQ ID NO: 29) in order to reduce the stability of the interaction between the two split parts. In this way, only the stronger interaction caused by TALE-mediated binding between the mutant split monomers can prevail. As a result, deamination will occur more specifically at the correct target C position. Mutations can be introduced at any position of SEQ ID NO: 28 (DddAtoxNter split) and / or SEQ ID NO: 29 (DddAtoxCter split, preferably at any position of SEQ ID NO: 29DddAtoxCter split). In addition, in the method according to the present invention, the TALE base editor monomer preferably includes the Nter and / or Cter members of the split DddAtox, which preferably include at least one mutation or modification that reduces the affinity of the two split DddAtox members to each other.

[0174] As another way to enhance the specificity of TALE base editors (which can be regarded as a further invention), it is a method to reduce off-target genomic mutations, in which the polypeptide sequence of the TALE base editor heterodimer is mutated to reduce its interaction with auxiliary proteins such as CTCF (CCCTC-binding factor). CTCF is a well-known transcription factor in organizing 3D genome structure, which forms a ring domain in the process involving the cohesin complex [Merkenschlager, M. & Nora, EP (2016) CTCF and Cohesin in Genome Folding and Transcriptional Gene Regulation. Annu Rev Genomics Hum Genet 17: 17-43]. Recently, Lei, Z. et al. [Mitochondrial base editor induces substantial nuclear off-target mutations. Nature. (2022) doi.org / 10.1038 / s41586-022-04836-5] found that CTCF recognition positions bias specific TALE base editors to bind to their target positions, which can lead to serious off-target genome ranges. Therefore, it can be expected that according to the method of the present invention, the step of selecting an appropriate target sequence combined with the step of reducing the interaction between the TALE base editor and CTCF not only significantly increases the frequency of the desired mutations, but also significantly reduces off-target mutations within the nuclear genome.

[0175] The method of the present invention comprises the steps of expressing the polynucleotide constructs described herein (such as DNA or mRNA) in cells in order to obtain their transcription and / or translation to obtain polypeptides that introduce mutations into the genome of the cells.

[0176] The present invention also relates to any polypeptide or polypeptide sequence included in the methods described herein, in particular those encoding TALE base editors that act on the genomic target sequences defined herein, as well as cells transformed or engineered with these sequences or cells comprising the genomic target sequences.

[0177] In fact, the present invention can also be regarded as a method for introducing mutations into the genome of a cell, in particular by converting C to A or G to T, the method comprising the following steps: introducing or expressing into the cell a polynucleotide encoding a TALE base editor as described above, such as a polynucleotide consisting of a left and / or right TALE binding polypeptide having a C-terminal domain of about 1 to 50 amino acids and a C-terminal and / or N-terminal split DddATox fusion. This method preferably comprises targeting a genomic sequence selected from the following:

[0178] 5′-T0-N 左 -N y -RTC-N x -N 右 -A0;

[0179] 5′-T0-N 左 -N x -GAY-N y -N 右 -A0;

[0180] in:

[0181] N can be A, T, C, or G;

[0182] R can be G or A;

[0183] Y can be C or T;

[0184] N 左 It may be a polynucleotide sequence comprising 9 to 20 A, T, C or G;

[0185] N 右 It may be a polynucleotide sequence comprising 9 to 20 A, T, C or G;

[0186] G is the complementary base of C.

[0187] x = 2 to 6;

[0188] y = 6 to 10;

[0189] Preferably, x+y≥11, more preferably, x+y=12,

[0190] The heterodimeric TALE base editor is combined with N 左 Polynucleotide sequences and N 右 Polynucleotide sequence binding.

[0191] According to a preferred embodiment, the left TALE binding polypeptide and the right TALE binding polypeptide of the TALE base editor are connected to the split deaminase through the C-terminus of 1 to 50 amino acids, preferably 8 to 40, more preferably 10 to 30, and even more preferably about 11 amino acids or about 40 amino acids.

[0192] According to a preferred embodiment, x (determining the number of nucleotide bases in the interval) is comprised between 2 and 5, preferably between 3 and 5, to obtain the best specificity.

[0193] According to a preferred embodiment, the TALE base editor of the present invention has a structure comprising a TALE C-terminus comprising about 11 amino acids, such as SEQ ID NO: 2 or SEQ ID NO: 551, the latter comprising an additional GGS linker. This TALE base editor structure is specifically suitable for targeting sequences represented by formulas i), ii), iii) and iv) as defined above, more specifically formula iii) and formula iv) (11≥x+y≥9, more preferably x+y=9). The present invention can be advantageously used to introduce specific mutations in living cells, in vitro or in vivo to produce therapeutic cells, especially therapeutic immune cells.

[0194] "Immune cell" refers to a cell of hematopoietic origin, which is functionally related to the initiation and / or execution of an intrinsic and / or adaptive immune response, typically such as CD3 or CD4 positive cells. Immune cells according to the present invention can be dendritic cells, killer dendritic cells, mast cells, NK-cells, B-cells or T-cells (selected from the group consisting of: inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes or helper T-lymphocytes). Cells can be obtained from many non-restrictive sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, infection site tissue, ascites, pleural effusion, spleen tissue and from tumors (such as tumor infiltrating lymphocytes). In some embodiments, the immune cell can be derived from a healthy donor, from a patient diagnosed with cancer or from a patient diagnosed with infection. In another embodiment, the cell is a part of a mixed immune cell population presenting different phenotypic characteristics (such as positive cells including CD4, CD8 and CD56).

[0195] In a preferred embodiment, the immune cells are tumor infiltrating lymphocytes (TILs): TILs include, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17CD4+ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs can generally be defined biochemically (using cell surface markers) or functionally (by their ability to infiltrate tumors and affect treatment). TILs can generally be classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. In addition and alternatively, TILs can be functionally defined based on their ability to infiltrate solid tumors when reintroduced into a patient.

[0196] In a preferred embodiment, therapeutic cells are primary cells obtained from healthy donors." primary cells " refer to cells directly obtained from living tissue (e.g., biopsy material) and established in vitro for a limited time of growth, which means that they can undergo a limited number of population doublings. Primary cells are contrary to continuous tumorigenicity or artificial immortalized cell lines. Non-limiting examples of this cell line are CHO-K1 cells, HEK293 cells, Caco2 cells, U2-OS cells, NIH 3T3 cells, NSO cells, SP2 cells, CHO-S cells, DG44 cells, K-562 cells, U-937 cells, MRC5 cells, IMR90 cells, Jurkat cells, HepG2 cells, HeLa cells, HT-1080 cells, HCT-116 cells, Hu-h7 cells, Huvec cells, Molt 4 cells. Primary cells are generally used in cell therapy because they are considered to be more functional and less tumorigenic.

[0197] Typically, primary immune cells are provided by a donor or patient by a variety of methods known in the art, such as by the leukocyte separation technique reviewed by Schwartz J. et al. (Guidelines on the use of therapeutic apheresis in clinical practice-evidence-based approach from the Writing Committee of the American Society for Apheresis: the sixth special issue (2013) J Clin Apher. 28 (3): 145-284). Primary immune cells according to the present invention can also be differentiated from stem cells, such as umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, hematopoietic stem cells (HSC) and induced pluripotent stem cells (iPS).

[0198] In a preferred embodiment, the therapeutic cell of the present method is a T-cell or NK cell that can be endowed with a chimeric antigen receptor (CAR) or a recombinant TCR, as described in the prior art (such as, for example, in WO2013176915).

[0199] By following the teachings of the present invention, prioritized safer TALE base editor target sequences in various genes have been identified for the production of engineered therapeutic immune cells.

[0200] In a preferred embodiment, the method can be used to inhibit or inactivate genes encoding TCR components (such as genes encoding TCRα or TCRβ) in T cells to produce lower alloreactive T-cells that can be used in allogeneic therapeutic environments. More specifically, the present invention provides a list of target window sequences entering the TCRα (TRAC) gene (Table 7), which are particularly easy for TALE base editors to introduce specific mutations while reducing the risk of off-target mutations throughout the human genome.

[0201] In a preferred embodiment, the method can be used to inhibit or inactivate genes such as CD52, which encodes the target of immunosuppressive drugs (such as alemtuzumab). By inactivating this gene, therapeutic cells can be resistant to drugs used in standard anticancer treatments. In other preferred embodiments, the GR and DCK genes can be inactivated by mutation, respectively, to render cells resistant to glucocorticoids and purine analogs.

[0202] In a preferred embodiment, the method of the present invention includes a step of introducing a TALE base editor into an immune cell, which binds to a genomic sequence contained in a gene encoding an immunosuppressive drug target (such as CD52). More particularly, the present invention provides a list of target window sequences entering the CD52 gene (Table 8), particularly in the splicing acceptor site and signal peptide of exon 2, which are particularly easy for TALE base editors to introduce specific mutations while reducing the risk of off-target mutations throughout the human genome.

[0203] In a further embodiment, the method of the present invention includes a step of introducing a TALE base editor into an immune cell, which binds to a genomic sequence contained in a gene encoding an immune checkpoint protein (such as PD1, CISH, CTLA4, TIM3 or LAG3). More particularly, the present invention provides a list of target window sequences entering the PD1 gene (Table 9), which are particularly easy for TALE base editors to introduce specific mutations while reducing the risk of off-target mutations throughout the human genome.

[0204] In a further embodiment, the method of the present invention includes a step of introducing a TALE base editor into an immune cell, which binds to a genomic sequence contained in a gene encoding β2-microglobulin (B2M) or human leukocyte antigen (HLA). More particularly, the present invention provides a list of target window sequences entering the B2M gene (Table 10), which are particularly easy for TALE base editors to introduce specific mutations while reducing the risk of off-target mutations throughout the human genome.

[0205] The target window sequence refers to the genomic sequence encompassed by the following general formula:

[0206] 5′-T0-N 左 -N y -RTC-N x -N 右 -A0-3′; or

[0207] 5′-T0-N 左 -N x -GAY-N y -N 右 -A0-3′,

[0208] As defined above,

[0209] According to the present invention, taking into account the changes in x and y and N 左 and N 右 The number of nucleotides contained in a sequence that can be spanned by one or more TALE base editor heterodimers.

[0210] Further examples of immune checkpoint genes and mutations of genes are provided in the literature, in particular in WO2019016360, to generate therapeutic engineered immune cells with different properties.

[0211] According to a preferred embodiment, the present method combines the use of TALE base editors with rare-cutting endonucleases (particularly TALE-nucleases) for multi-gene editing in immune cells.

[0212] In some embodiments, the TALE base editor and the rare-cutting endonuclease can be co-expressed, co-transfected, or introduced sequentially in a manner that minimizes the risk of chromosomal defects.

[0213] For example, as shown in Example 4, specific combinations have resulted in very low levels of translocations, off-targets and / or chromosomal rearrangements:

[0214] - Inactivation of the TCR using rare-cutting endonucleases and introduction of one or several point mutations into the CD52 gene by using TALE base editors;

[0215] - Inactivate the TCR using a rare-cutting endonuclease and introduce one or a few point mutations into the TGFBRII gene by using a TALE base editor;

[0216] - Inactivation of immune checkpoint genes (such as PD1, CISH, CTLA4, TIM3 or LAG3) using rare-cutting endonucleases and introduction of one or a few point mutations into TCR by using TALE base editors;

[0217] - Inactivation of TCR using rare-cutting endonucleases and introduction of one or several point mutations into immune checkpoint genes (such as PD1, CISH, CTLA4, TIM3 or LAG3) by using TALE base editors;

[0218] - Inactivation of TCR using rare-cutting endonucleases and introduction of one or a few point mutations into the genomic components of MHC (such as HLA-A, HLA-B, HLA-C or B2M) by using TALE base editors;

[0219] - Inactivation of genomic components of the MHC (such as HLA-A, HLA-B, HLA-C or B2M) using rare-cutting endonucleases and introduction of one or a few point mutations into the TCR by using TALE base editors;

[0220] This combined approach is an important part of the present invention and is particularly useful for combining multiplex gene knock-ins (e.g., targeted gene insertion) and / or gene knockouts (e.g., gene inactivation) in immune cells. Specifically, rare-cutting nucleases can be used to introduce exogenous polynucleotide sequences at the first site of the genome through site-directed gene integration, while accompanying TALE base editors can be used to introduce one or several point mutations at another site (particularly a site that needs to be inactivated).

[0221] For example:

[0222] - Inactivate B2M expression using a rare-cutting endonuclease and introduce an exogenous polynucleotide sequence encoding HLAE at this site to render the cell invisible to NK cells, while at the same time, one or several point mutations can be introduced into TCR and / or CD52 using a TALE base editor as proposed above;

[0223] - Inactivation of immune checkpoint genes (such as PD1, CISH, CTLA4, TIM3 or LAG3) using rare-cutting endonucleases and introduction of exogenous polynucleotide sequences encoding chimeric antigen receptors (CARs) at such sites, while at the same time, TALE base editors can be used to introduce one or several point mutations into TCR and / or CD52 as proposed above;

[0224] - Inactivation of immune checkpoint genes (such as PD1, CISH, CTLA4, TIM3 or LAG3) using rare-cutting endonucleases and introduction of exogenous polynucleotide sequences encoding cytokines (such as IL-2, IL-12, IL-18...) at such sites, while at the same time, one or several point mutations can be introduced into TCR and / or CD52 using TALE base editors as proposed above;

[0225] - Use rare-cutting nucleases to inactivate the expression of components of the TCR (such as TRAC) and introduce exogenous polynucleotide sequences encoding CAR or recombinant TCR at this site, while at the same time, TALE base editors can be used to introduce one or several point mutations into immune checkpoint genes and / or CD52 as proposed above.

[0226] As shown in the examples, the above-mentioned embodiments combining gene knockout and targeted gene insertion, such as by using an AAV vector containing a transgene, for example, by homologous recombination (HDR) to introduce the transgene, prevent accidental transgene capture (more particularly referred to as "AAV capture") when the genome is simultaneously knocked out at another site. In this way, nucleases can be used for gene insertion, while TALE base editors can be used simultaneously to inactivate genes located elsewhere in the genome.

[0227] "Rare-cutting endonuclease" refers to a sequence-specific endonuclease agent that does not naturally occur in mammalian cells and whose recognition sequence generally ranges from 10 to 50 consecutive base pairs, preferably 12 to 30 bp, more preferably 14 to 20 bp. Such endonuclease agents are typically nucleic acids encoding "engineered" or "programmable" rare-cutting endonucleases, such as homing endonucleases (e.g., described by Arnould S. et al. in [WO2004067736]), zinc finger nucleases (ZFNs) (e.g., described by Urnov F. et al. in [Highly efficient endogenous human gene correctionusing designed zinc-finger nucleases(2005)Nature 435:646-651]), TALE-nucleases (e.g., described by Mussolino et al. in [A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity(2011)Nucl.Acids Res.39(21):9283-9293]), or MegaTAL nucleases (e.g., described by Boissel et al. in [MegaTALs:a rare-cleaving nuclease architecture for therapeutic genome engineering(2013)Nucleic Acids Research Due to their higher specificity, TALE-nucleases have been shown to be particularly suitable sequence-specific nuclease agents for therapeutic applications, especially in the form of heterodimers - i.e., with one "right" monomer (also called "5'" or "forward") and one "left" monomer (also called "3'" or "reverse"), as reported by Mussolino et al. [TALEN facilitates targeted genome editing in human cells with high specificity and low cytotoxicity (2014) Nucl. Acids Res. 42 (10): 6762-6773].RNA-guides are used in combination with RNA-guided endonucleases such as Cas9 or Cpf1, which, among other things, are rare-cutting endonucleases contemplated by the present invention in accordance with the teachings of Doudna J. and Charpentier, E. [The new frontier of genome engineering with CRISPR-Cas9 (2014) Science 346(6213):1077].

[0228] According to a preferred aspect of the present invention, the endonuclease agent is transiently expressed in the cell, such as the following: RNA (more specifically mRNA), protein or a complex of a mixed protein and a nucleic acid conjugate (involving polynucleotides and polypeptides, such as so-called "ribonucleoprotein"). This conjugate can be more specifically formed by the agent Cas9 or Cpf1 (RNA-guided endonuclease) and their RNA guides, such as described by Zetsche, B. et al. [Cpf1Is a SingleRNA-Guided Endonuclease of a Class 2CRISPR-Cas System (2015) Cell 163 (3): 759-771].

[0229] Typically, one or both of the nuclease and the TALE base editor are transfected into the immune cells using an electroporation step, which is typically performed in a closed chamber comprising parallel plate electrodes between which a pulsed electric field greater than 100 volts / cm and less than 5000 volts / cm is generated that is substantially uniform throughout the treatment volume, as described in WO2004083379 (incorporated by reference, particularly from page 23, line 25 to page 29, line 11). One such electroporation chamber preferably has a square (cm) of the electrode gap. 2 ) divided by the chamber volume (cm 3 ) is defined as the geometric factor (cm -1 ), where the geometric factor is less than or equal to 0.1 cm -1, wherein the cell suspension and the sequence-specific reagent are in an adjusted medium so that the conductivity of the medium is in the range of 0.01 to 1.0 millisiemens. Typically, the cell suspension is subjected to one or more pulsed electric fields. In this way, the processing volume of the suspension is scalable and the processing time of the cells in the chamber is substantially uniform. Multiple treatments of rare-cutting nucleases and TALE base editors in immune cells can be performed according to the protocols previously reported for nucleases [Poirot et al. (2013) Blood. 122 (21): 1661 and Sachdeva et al. (2019) Nat Commun. 10 (1)].

[0230] "Exogenous sequence" refers to any nucleotide or nucleic acid sequence that was not originally present at the selected site. This sequence can be homologous to the genomic sequence, or a copy thereof, or an exogenous sequence introduced into the cell. The exogenous sequence preferably encodes a polypeptide, and its expression has a therapeutic advantage relative to sister cells that do not integrate this exogenous sequence at this site. Exogenous sequences are usually introduced into cells as donor templates and integrated into the genome by homologous recombination induced by rare-cutting endonucleases. This donor template can be introduced into cells by transduction in the form of a viral vector (such as AAV), or can be introduced as a polynucleotide (such as a single-stranded oligonucleotide (ssODN)), as described in, for example, WO2021224395.

[0231] The present method can generate immune cells that contain and / or co-express rare-cutting nucleases and TALE base editors as described herein as cell populations or intermediate cells for producing engineered therapeutic cells or cell compositions.

[0232] According to some aspects of the present invention, the TALE base editor of the present invention is used in gene therapy for in vivo gene correction or inactivation of defective gene expression. Specifically, the TALE base editor according to the present invention can be directly directed to hepatocytes in vivo to target viral genomes, such as cccDNA (covalently closed circular DNA) of hepadnaviruses, especially HBV (hepatitis B virus), which reside in hepatocytes in a tolerant form.

[0233] Encapsulation of mRNA or polypeptides into nanocarriers (such as liposomes, polymers, and inorganic nanoparticles) has shown great potential in delivering gene editing reagents to hepatocytes [Witzigmann, D. et al. (2020) Lipidnanoparticle technology for therapeutic gene regulation in the liver. Advanced Drug Delivery Reviews, 159: 344-363].

[0234] Depending on the structure of the biodegradable matrix involved and the monomers forming the core hydrophobic domain and polar domain, various types of biodegradable delivery capsules containing RNA reagents can be manufactured. By connecting the targeting domain to the proximal polar domain of the nanocarrier, the delivery specificity can be improved so that the delivery capsule can bind to surface antigens of different cell types. These delivery capsules are particularly suitable for intravenous injection to target endogenous genetic sequences into cells. This delivery capsule according to the present invention is used to deliver TALE base editors into cells in the form of RNA, in particular, co-delivery of messenger RNA encoding right and left heterodimeric TALE base editors.

[0235] The present application more specifically claims that a pharmaceutical composition comprising a biodegradable delivery capsule of the present invention is incorporated into a treatment involving a TALE base editor according to the present invention. Such treatment can be part of a gene therapy, in which a specific gene sequence must be knocked out or repaired, or part of an anti-infective treatment, by targeting the genome of an infectious agent or a genetically defective gene, such as ApoC3, transthyretin (TTR), ANGPTL3, and PCSK9 genes, which can be used to treat or prevent atherosclerosis, transthyretin (TTR)-mediated amyloidosis (ATTR), hyperlipidemia, and hypercholesterolemia, respectively.

[0236] In a preferred embodiment, the present invention provides a list of target window sequences entering the ApoC3 gene (Table 11), which are particularly easy for TALE base editors to introduce specific mutations while reducing the risk of off-target mutations throughout the human genome.

[0237] According to a more specific embodiment, the present invention provides a method for introducing mutations into TRAC, CD52, PD1, B2m and ApoC3 by targeting any target sequence presented in Tables 7 to 11, respectively, using a TALE base editor as described herein.

[0238] Specifically, the present invention includes the following method: wherein the TALE base editor binds to a genomic sequence contained in a gene encoding TRAC, the sequence being selected from any one of SEQ ID NO:366 to SEQ ID NO:407 shown in Table 7.

[0239] Specifically, the present invention includes the following method: wherein the TALE base editor binds to a genomic sequence contained in a gene encoding CD52, which sequence is selected from any one of SEQ ID NO:408 to SEQ ID NO:422 shown in Table 8.

[0240] Specifically, the present invention includes the following method: wherein the TALE base editor binds to a genomic sequence contained in a gene encoding PD1, which sequence is selected from any one of SEQ ID NO:423 to SEQ ID NO:466 shown in Table 9.

[0241] Specifically, the present invention includes the following method: wherein the TALE base editor binds to a genomic sequence contained in a gene encoding B2m, and the sequence is selected from any one of SEQ ID NO:467 and SEQ ID NO:501 shown in Table 10.

[0242] Specifically, the present invention includes the following method: wherein the TALE base editor binds to a genomic sequence contained in a gene encoding ApoC3, which sequence is selected from any one of SEQ ID NO:502 and SEQ ID NO:523 shown in Table 11.

[0243] According to another aspect of the present invention, mutations can be induced directly in RNA transcripts in cells by TALE base editors. This RNA editing method combines the introduction of single-stranded DNA (such as ssODN) into cells with the heterodimer TALE base editors described herein, wherein the target RNA transcript hybridizes with the single-stranded DNA to form a double-stranded nucleic acid, which is bound by the heterodimer TALE base editor, resulting in the introduction of mutations directly at the transcript level at the C (or G) position required for the target sequence.

[0244] According to a further embodiment of the present invention, it is a method for correcting genetic defects (particularly dysfunctional dominant alleles) by combining targeted gene integration (such as one produced by homologous recombination) with inactivation of endogenous genes by sequence-specific base editors (such as the TALE-base editors described above). As an example, provided herein is Figures 12 to 17The principle and schematic diagram are described. This gene therapy method may include the use of sequence-specific nucleases to insert a functional copy of a gene or a portion thereof, or a corrected sequence thereof, combined with the introduction of a sequence-specific base editor reagent into the cell for inactivating the residual endogenous sequence that has not been replaced or corrected. In some cases, the corrected sequence integrated at the endogenous site has been rewritten relative to the original endogenous sequence by using alternative codons. Sequence-specific base editors that recognize the remaining complete endogenous allele sequence (preferably a defect that causes a genetic disease) can be introduced into cells by different tools known to those skilled in the art (such as purified proteins, mRNA, or viral or non-viral expression vectors).

[0245] According to a preferred embodiment, gene therapy includes a site-specific endonuclease, such as a TALE-nuclease, a zinc finger nuclease, a meganuclease or an RNA-guided endonuclease, in combination with a sequence-specific base editor such as a TALE base editor as described above for targeted gene integration. The site-specific endonuclease is co-transfected with a DNA template encoding a functional allele sequence (such as an AAV vector or single-stranded DNA) in order to promote its integration by homologous recombination.

[0246] According to a preferred embodiment, the site-specific nuclease and sequence-specific base editor can be introduced into cells sequentially or simultaneously (such as by co-transfection). Co-transfection by electroporation of mRNA encoding the two agents is preferred, but other technical solutions are also possible, such as combining viral vectorization, electroporation, nanoparticles, ribonucleotides or purified protein transfection.

[0247] According to a preferred embodiment, the introduction of the site-specific nuclease and the sequence-specific base editor is performed ex vivo, such as in blood immune cells, preferably primary immune cells (such as HSCs or their progeny).

[0248] According to a preferred embodiment, in order to correct the genetic defect, the sequence integrated into the genome is "rewritten", meaning that alternative genetic codons are used, usually by replacing codon usage (different from the codons of the endogenous allele). Therefore, the rewritten sequence after integration will not be recognized by the sequence-specific base editor for the corresponding endogenous allele sequence.

[0249] According to a preferred embodiment, in order to correct a genetic defect, the functional gene sequence may be a functional gene sequence of an exon or a portion thereof, which may be introduced into the genome, for example according to Fig.15 and the “Artex” strategy described in WO2021224416, incorporated by reference.

[0250] According to a preferred embodiment, the gene therapy method of the present invention targets a dysfunctional allele causing a disease selected from the alleles listed in Table 19.

[0251] Variations of the above method may also be considered to improve its efficiency by changing various parameters, such as one of the following:

[0252] - The therapeutic integration sequence can be inserted at any preferred site in the genome, not necessarily at the site where the defective allele is located.

[0253] - The therapeutic integration sequence can be promoterless and inserted upstream of the disease-associated mutation. In this case, the base editor used is preferably designed to edit the exon downstream of the therapeutic insertion.

[0254] -Can involve multiple sequence-specific base editors targeting different exons of a defective gene.

[0255] Accordingly, the present invention is directed to methods of treatment comprising one or more of the following steps:

[0256] - introducing a transgene inserted into an endogenous locus into a cell and / or expressing it in the cell to correct a genetic defect,

[0257] - Introducing and / or expressing in said cell a sequence-specific base editor that targets the endogenous allele sequence causing said genetic defect to inactivate its expression.

[0258] The above steps can be performed simultaneously or sequentially.The introduction of the transgene can be performed by different tools known in the art, viral or non-viral, such as by introducing a DNA template encoding the transgene that binds a site-specific rare-cutting endonuclease.

[0259] The combination of site-specific nucleases and base editors in this method is an advantage because they can be introduced into cells simultaneously, such as by electroporation, without the risk of interaction. Unlike the use of multiple nucleases, which may cause chromosomal deletions or rearrangements, the combined use of site-specific nucleases and sequence-specific base editors (particularly TALE nucleases and TALE base editors) is considered safe and there are no known negative interactions.

[0260] The present gene therapy method is not limited to the combined use of TALE base editors and TALE-nucleases described in the embodiments, and can be implemented using other site-specific nuclease reagents, such as RNA-guided nucleases (e.g., Cas9, Cas12...), and other types of sequence-specific base editors, such as those composed of catalytically dead Cas9 (dCas9) or nickase Cas9 (nCas9) fused to a deaminase and guided to the site of interest by a single-stranded guide RNA (sgRNA), and any combination thereof.

[0261] Preferably, the transgenic sequence has an overwritten or different genetic sequence relative to the endogenous allele that causes the genetic defect, so that a sequence-specific base editor can easily distinguish between the endogenous defective allele and the transgene that corrects the genetic defect.

[0262] In some embodiments, one or more of the following steps may be performed sequentially or simultaneously:

[0263] - introducing or expressing a rare-cutting endonuclease targeted to an endogenous site into a cell containing a defective gene sequence causing a genetic defect,

[0264] - Correction of the genetic defect by introducing into said cell a DNA template through genetic integration of an endogenous site targeted by said rare-cutting endonuclease.

[0265] - Introducing or expressing a base editor, preferably a TALE base editor (such as one described herein), to inactivate at least one endogenous allele causing the genetic defect.

[0266] The above methods are particularly suitable for genetic defects caused by dominant alleles because they can inactivate all alleles associated with the genetic defect while providing exogenous functional copies of such alleles. Table 19 provides a non-limiting list of such genetic defects. Considering administration to patients, the methods of the present invention are shown to be particularly suitable for ex vivo engineering HSC or T-cells for the treatment of genetic defects, particularly for the treatment of ADPS1 and STAT3.

[0267] One aspect of the present invention is an engineered therapeutic cell, such as an HSC or its progeny, obtainable and / or involving the above-mentioned gene therapy method, which generally comprises a transgene for correcting a genetic defect, which transgene is generally a corrected and / or rewritten version of an endogenous allele causing the genetic defect, wherein the endogenous allele causing the genetic defect has been inactivated (mutated) by at least one base editor.

[0268] Such engineered therapeutic cells, such as HSCs or their progeny, obtained and / or involved by the above-mentioned gene therapy methods may generally contain (1) a transgene for correcting a genetic defect, which is generally a corrected and / or rewritten version of a defective endogenous allele that causes the genetic defect, and (2) a base editor or a transgenic sequence encoding the base editor for inactivating the endogenous allele that causes the genetic defect, and optionally (3) a rare-cutting endonuclease or a transgenic sequence encoding the rare-cutting endonuclease for integrating the transgene into a selected endogenous site.

[0269] Having generally described the present invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to limit the scope of the claimed invention.

[0270] Example

[0271] Example 1

[0272] Materials and methods

[0273] T cell culture

[0274] Cryopreserved human PBMCs were obtained from ALLCELLS. PBMCs were cultured in X-vivo-15 medium (Lonza Group) containing 20 ng / ml human IL-2 (Miltenyi Biotec) and 5% human AB serum (Seralab). On the day after thawing PBMCs, human T cell activator TransAct (Miltenyi Biotec) was used to activate T cells at a concentration of 100 μg / ml CD3+ / -100 μg / ml. + T cells were activated with 25 μl of TransAct. TransAct was maintained in the culture medium for 72 hours.

[0275] Preparation of TALE-nucleases and TALEBs

[0276] according to Figure 1The TALEN (fused TALE N-terminus (δ152)-repeat 15,5-C-terminus (40)-Fok1 nuclease domain) and TALE-base editor (left TALE binding domain N-terminus (δ152)-repeat 15,5-C-terminus (40)-DddAtoxsp-N-terminus-UGI and right TALE binding domain N-terminus (δ152)-repeat 15,5-C-terminus (40)-DddAtoxsp-C-terminus) heterodimers shown were assembled using standard molecular biology and / or microbiology techniques, such as enzyme restriction digestion, ligation, bacterial transformation, and plasmid DNA extraction (NEB 10-β competent E. coli for ccdB screening or NEB stable competent E. coli for blue / white screening). TALE DNA targeting arrays were assembled and cloned into the corresponding TALEN backbones (pCLS32783) and / or TALE base editor backbones (pCLS35714 and pCLS35715).

[0277] Small-scale mRNA preparation

[0278] The plasmids of 37 TALE base editors and 37 matching TALE-nucleases derived from the above backbone, containing T7 promoter and poly A sequence, were prepared in a non-clonal form after assembly (transformants were directly inoculated for culture and plasmid preparation). The plasmids were then linearized with SapI (NEB) and mRNA was prepared by in vitro transcription (NEB HiScribe ARCA, NEB).

[0279] Small-scale TALE-nuclease and TALE base editor testing (37 endogenous targets and TRAC / CD52 multiplex engineering)

[0280] 10-12 hours before transfection, T cells activated for 3 days with TransAct (Miltenyi Biotec) were transferred to fresh complete medium containing 20 ng / ml human IL-2 (Miltenyi Biotec) and 5% human AB serum (Seralab). The collected cells were washed once with warm PBS. 1×10 6The PBS-washed cells were pelleted and resuspended in 20 μl Lonza P3 primary cell buffer (Lonza). 1 μg / arm / million cells of TALE-nuclease or TALE base editor were mixed with the cells, and the cell mixture was electroporated under the EO115 program using the Lonza 4D-nucleofector to obtain stimulated human T cells. After electroporation, 80 μl of warm complete medium was added to the cuvette to dilute the electroporation buffer, and the mixture was carefully transferred to 400 ml of preheated complete medium in a 48-well plate. The TALE-nuclease-transfected cells were incubated at 30°C overnight and then transferred back to the 37°C incubator. The TALE-base editor-transfected cells were cultured at 37°C throughout the process. Cells were collected on day 6 after transfection for gDNA extraction and NGS analysis.

[0281] Large-scale TALE-nuclease and TALEB mRNA preparation (CD52-targeted base editor)

[0282] The plasmid encoding TRAC TALE-nuclease contains a T7 promoter and a poly A sequence. TALE-nuclease mRNA from the TRAC TALE-nuclease plasmid was produced by Trilink. Sequences targeted by TRACTALE-nuclease (17 bp recognition site, capital letters, separated by 15 bp spacers):

[0283] 5′-TTCCTCCTACTCACCATcagcctcctggttatGGTACAGGTAAGAGCAA-3′ (SEQ ID NO: 31)

[0284] TALE-nuclease mRNA from CD52 TALE-nuclease plasmid was prepared by Trilink. Sequence targeted by CD52 TALE-nuclease (17 bp recognition site, capital letters, separated by 15 bp spacers):

[0285] 5′-TTCCTCCTACTCACCATcagcctcctggttatGGTACAGGTAAGAGCAACGCCTGGCA-3′(SEQID NO:32)

[0286] Plasmids encoding the TALE base editors T-25 and CD52 contain a T7 promoter and poly A sequence. Sequence-verified plasmids were linearized with SapI (NEB) prior to in vitro mRNA synthesis. mRNA was expressed using NEB HiScribe TM Prepared using T7 Rapid High Yield RNA Synthesis Kit (NEB). TMThe 5′ end capping reaction was performed using the m7G capping system (Cellscript). The capped mRNA was treated with Antarctic Phosphatase (NEB) and finally cleaned using Mag-Bind TotalPure NGS beads (Omega bio-tek) and Invitrogen DynaMag-2 magnet (ThermoFisher).

[0287] ssODN repair template transfection

[0288] A pool of ssODNs targeting the TRAC site (SEQ ID NOs: 33 to 69; see Table 1) was ordered from Integrated DNA Technologies (IDT) and resuspended in ddH2O at 50 pmol / μl.

[0289] 10-12 hours before transfection, T cells activated for 3 days with TransACT were transferred to fresh complete medium containing 20 ng / ml human IL-2 (Miltenyi Biotec) and 5% human AB type serum (Seralab).

[0290] Wash the collected cells once with warm PBS. 6 The PBS-washed cells were centrifuged and resuspended in 20 μl of Lonza P3 primary cell buffer (Lonza). 200 pmol of ssODN pool and 1 μg / arm of TRACTALE-nuclease were mixed with the cells, and the cell mixture was electroporated using the Lonza 4D-nucleofector under the EO115 program to obtain stimulated human T cells. After electroporation, 80 μl of warm complete medium was added to the cuvette to dilute the electroporation buffer, and the mixture was carefully transferred to 400 ml of pre-warmed complete medium in a 48-well plate. ssODN and TALE-nuclease transfected cells were cultured at 30°C until 24 hours after TALE-nuclease transfection and then transferred back to 37°C.

[0291] After two days of culture, cells containing ssODN KI were collected for TALE base editor treatment. The collected cells were washed once with warm PBS. 1×10 6The PBS-washed cells were centrifuged and resuspended in 20 μl Lonza P3 primary cell buffer (Lonza). 1 μg / arm of TALE base editor T-25 was mixed with the cells, and the cell mixture was electroporated using the Lonza 4D-nucleofector under the EO115 program to obtain stimulated human T cells. After electroporation, 80 μl of warm complete medium was added to the cuvette to dilute the electroporation buffer, and the mixture was carefully transferred to 400 ml of preheated complete medium in a 48-well plate. The TALE base editor-transfected cells were cultured at 37°C for another two days and then collected for gDNA extraction and NGS analysis.

[0292] Large-scale CD52 TALE base editor testing

[0293] 10-12 hours before transfection, T cells activated for 3 days with TransACT were transferred to fresh complete medium containing 20 ng / ml human IL-2 (Miltenyi Biotec) and 5% human AB type serum (Seralab).

[0294] Wash the collected cells twice with Cytoporation Media T (BTXpress, 47-0002). 6 The washed cells were pelleted by centrifugation and resuspended in 180 μl Cytoporation Media T. 2 μg / arm / million cells of TALE base editor were mixed with cells to a final volume of 200 μl, and the cell / mRNA mixture was electroporated using BTX Pulse Agile in a 0.4 cm gap cuvette. After electroporation, 180 μl of warm complete medium was added to the cuvette to dilute the electroporation buffer, and the mixture was carefully transferred to 2 ml of pre-warmed complete medium in a 12-well plate. TALE-base editor-transfected cells were cultured at 37°C throughout the process. Cells were collected on day 6 after transfection for gDNA extraction and NGS analysis.

[0295] Genomic DNA extraction

[0296] Cells were collected and washed once with PBS. Genomic DNA extraction was performed using the Mag-Bind Blood & Tissue DNA HDQ Kit (OmegaBio-Tek) according to the manufacturer's instructions.

[0297] Targeted PCR and NGS

[0298] In 50 μl reactions, 100 μl of genomic DNA was used per reaction using Phusion High Fidelity PCR Master Mix (NEB). PCR conditions were set as follows: 1 cycle: 30 s at 98°C; 30 cycles: 10 s at 98°C, 30 s at 60°C, 30 s at 72°C; 1 cycle: 5 min at 72°C; hold at 4°C. PCR products were then purified using Omega NGS beads (1:1.2 ratio) and eluted into 30 ml of 10 mM Tris buffer at pH 7.4. The purified products from the first PCR were then subjected to a second PCR (combined with NGS indices). 15 ul of the first PCR product was placed in a 50 ul reaction and Phusion High Fidelity PCR Master Mix (NEB) was added. PCR conditions were set as follows: 1 cycle: 98°C for 30 s; 8 cycles: 98°C for 10 s, 62°C for 30 s, 72°C for 30 s; 1 cycle: 72°C for 5 min; hold at 4°C. Purified PCR products were sequenced on a MiSeq (Illumina) on a 2x250 nanoV2 cassette.

[0299] Flow cytometry

[0300] TRAC KO is monitored using anti-TCRα / β antibodies (Biolegend, #306732, clone IP26, BV605).CD52KO is monitored using anti-52 ​​antibodies (BD Biosciences, #563609, clone 4C8, AlexaFlour488).Flow cytometry is performed on BD FACSCanto (BD Biosciences), and data analysis is processed using FlowJo.First, cell populations are gated for lymphocytes (SSC-A and FSC-A) and single cells (singlet) (FSC-H and FSC-A). Lymphocyte gating is further analyzed to express CD52 and-TCRα / β from the gated population.

[0301] In silico off-target prediction

[0302] To evaluate the possible off-target editing of the CD52TALE base editor, we generated a list of potential off-target targets of these base editors in silico. The list was generated as follows. The TALE base editor has two 17bp binding sequences separated by a gap. These binding sequences must start with T. Therefore, we first selected all genomic sequences that start with T, end with A, and are 27bp to 67bp in length (including both), with an allowed gap range of 10 to 40bp as potential targets. Then, the number of mismatches between the binding sequence of the potential target and the actual TALE base editor target is calculated. If the total number is greater than 8, the potential target is deleted. Finally, all potential targets that lack G in the left half of the gap or C in the right half of the gap (editing window) are discarded.

[0303] Off-target and translocation multiplex amplicon sequencing

[0304] rhAmp primers were designed based on on-target and / or off-target sites identified by in silico off-target predictions. Site-specific forward and reverse primers were obtained from Integrated DNA Technologies (IDT) either as ready-to-use primer pools or as individual plates and used according to the IDT protocol for RNase H2-dependent multiplex detection amplification (1 cycle: 95°C for 10 min; 14 cycles: 95°C for 15 s, then 65°C for 8 min; 1 cycle: 99.5°C for 15 min; hold at 4°C) followed by universal PCR to add NGS indexes (i5 or i7) (1 cycle: 95°C for 3 min; 24 cycles: 95°C for 15 s, then 60°C for 30 s and 72°C for 30 s; 1 cycle: 72°C for 1 min; hold at 4°C). Purified PCR amplicons were sequenced on NextSeq (Illumina) using the NextSeq 500 / 550 mid-throughput kit (150 cycles).

[0305] Example 2: Comparison of TALE-nuclease and TALEB efficiency

[0306] To identify key determinants of efficient TALEB editing (C-to-T conversion) using the previously described split-DddaTox strategy, we first selected a gene that exhibited high activity (median = 82%, SD = 12) in primary T cells ( Figure 2A) of 37 TALE-nuclease subsets. These 37 target sequences (SEQ ID NO: 33 to 69 in Table 1) are carefully selected to target regions with different chromatin states in T cells. The spacer sequence (sequence between two TALE binding regions) is also kept at a constant 15bp because it has previously been shown to optimize TALE-nucleases [Juillerat, A. et al. Comprehensive analysis of the specificity of transcription activator-like effector nucleases (2014) Nucleic Acids Research, 42 (8): 5390-5402]. The spacer sequence contains different numbers of evenly distributed C, G, TC or GA, as previous studies have shown that it has a strong editing preference in the environment of 5′-TC-3′ (Mok. et al. A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing (2020) Nature 583, 631-637). As described in Example 1, 37 TALE base editors with the DddAtox cleavage and uracil glycosidase inhibitor (UGI) were prepared, replacing the FokI catalytic domain. The G1397 cleavage was used because this fusion showed better editing activity. The maximum editing within a given TALE base editor was compared to the indel frequency produced by the corresponding TALE-nuclease counterpart ( Figure 2 B). The complete lack of correlation between these two datasets (TALE-nuclease vs. TALE base editing frequency) (Spearman correlation coefficient = 0.16, p-value = 0.33) suggests that the key determinant of efficient editing may be the position of the target cytosine within the spacer. Indeed, analysis of editing efficiency at positions within the spacer revealed a clear 4-5 bp editing window on both the top and bottom strands ( Figure 3 ).

[0307] Interestingly, only low frequencies (<0.5%) of indels (small insertions and deletions) were observed for 35 of the 37 base editors (indel frequency: median = 0.06%, SD = 0.17). Indels at the target site were moderately correlated with editing frequencies within the interval (Spearman correlation coefficient = 0.44, p-value = 0.007) ( Figure 4A). In addition, we detected low side product (C-to-A / G) editing within the editing window, overall indicating a very high final purity of the edited cell population ( Figure 4 B and Figure 4 C).

[0308] Table 1: Genomic target sequences used in Example 2

[0309]

[0310]

[0311] Example 3: Screening and rules for optimal base editing

[0312] To more comprehensively investigate DddA-derived cytosine base editors, a medium- to high-throughput format was designed to screen by generating primary T-cell pools containing predefined TALE base editor target sequences precisely inserted at the TRAC gene in a specific genomic context. Each TALE base editor target contains a unique TC or GA (target of the DddA deaminase) located within a spacer sequence and flanked by two fixed TALE binding sequences (RVD-L and RVD-R, Figure 5 A). This setup allows for uniform TALE binding to the artificial target site, eliminating editing differences caused by (i) different DNA binding affinities of different TALE array proteins and (ii) the influence of epigenomic factors, such as chromosomal relaxation around the artificial base editor target site.

[0313] A set of 30 ssODNs was created that contained the previous polynucleotide TALE binding sequence of T-25 (SEQ ID NO:57 in Table 1) separated by 15 bp variable spacer sequences (similar to our previous set of TALE base editors targeting endogenous sites), as follows:

[0314] 5′TCTAAGAAGTTCCTGCT (variable interval 15 nucleotides)GAATGTGGTTAGAGACA 3′ (SEQ ID NOs: 70 to 100 in Table 2).

[0315] Table 2: Individual ssODN sequences used to identify editing windows with 15 bp intervals

[0316]

[0317]

[0318]

[0319] These ssODNs were used to generate a primary T cell pool containing a collection of base editor targets. 30 ssODN oligonucleotides were mixed in equal amounts and transfected in primary T cells by electroporation (200 pmol per million cells), along with mRNA encoding the TALE-nuclease targeting TRAC (left TALEN monomer of SEQ ID NO:16 and right TALEN monomer of SEQ ID NO:17). In the second step, two days after transfection of the ssODN pool, the mRNA encoding the T-25TALE base editor was vectorized by electroporation. The genomic DNA of the transfected cells was then collected on day 2 after TALE base editor transfection for editing analysis ( Figure 5 A). NGS analysis showed that ssODN integrated efficiently and uniformly at the TRAC site (read counts: median = 1667.5, mean = 1686.2, standard deviation = 351.7). Control samples not treated with TALE base editors showed low background mutation frequencies, while samples treated with TALE base editors showed detectable and reproducible levels of C to T conversions ( Figure 5 B and Figure 5 C). This analysis further highlights editing windows comparable to those observed for 37 TALE base editors targeting endogenous sequences ( Figure 5 D), thus fully validating this combined approach.

[0320] The ssODN collection was expanded to have different number of intervals of length, from 5 to 39 bp (i.e., 5, 7, 9, 11…37, 39 bp). A TCGA quadruplex target sequence was inserted at every other position in the interval ( Figure 6 A). This design contains 191 unique ssODNs (SEQ ID NOs: 103 to 293 in Table 3), allowing the editing efficiency on both strands to be examined simultaneously using a single ssODN. In addition, to facilitate sequence analysis, a unique barcode ( Figure 6 A). After filtering the NGS data to remove reads with inconsistent barcodes and spacer sequences, a high and homogeneous representation of each ssODN was obtained (number of reads: median = 545, mean = 3522.6, standard deviation = 7122.5). As in the previous set (15 bp intervals), low mutation frequencies were observed without TALE base editors, while with TALE base editors, C-to-T conversions were robustly detected on both the positive and negative strands ( Figure 6 B and 6C). Data analysis indicated that optimal editing was achieved with interval lengths ranging from 11 to 17 bp, with 4 to 5 bp editing windows at different intervals ( Figure 6 D and Figure 6 E).

[0321] To study the effect of the sequence surrounding the TC environment on base editing efficiency, another ssODN set was designed, which contained two fixed TALE array protein binding sites (SEQ ID NO: 294 to 357 in Table 4) separated by a 16bp spacer sequence from the T-25TALE base editor (SEQ ID NO: 57 in Table 1). The spacer sequence consists of a 10bp molecular barcode followed by the NTCCNN sequence (target of the base editor). Cell treatment, transfection, and gDNA analysis were performed as previously described.

[0322] After NGS data screening and analysis, the results clearly showed that G or A before TC facilitates efficient editing ( Figure 7 ).

[0323] Table 3: Individual ssODN sequences used to evaluate the effect of spacer length on editing

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334] Table 4: Individual ssODN sequences used to evaluate the TC environment in the TALE base editor target sequence in Example 4

[0335]

[0336]

[0337]

[0338]

[0339]

[0340] Example 4: Application of TALE base editor rules to generate CD52-negative T cells

[0341] In the setting of allogeneic CAR-T therapy, CD52 is often knocked out by gene editing to generate resistance to alemtuzumab, a CD52-targeting monoclonal antibody used in lymphodepleting regimens. Since the CD52 gene has only two exons and exon 2 contains the sequence encoding the mature peptide, a splice site mutation at the intron 1 / exon 2 junction was selected to cause exon 2 skipping, resulting in the loss of CD52. Therefore, the TALE base editor rules defined above were applied to identify the best targets, resulting in 3 major TALE base editors (out of 34 potential base editors, Figure 8 A). Primary T cells were transfected with mRNA encoding these three pairs of TALE base editors (TALEB#1, SEQ ID NO:20 and SEQ ID NO:21; TALEB#2, SEQ ID NO:22 and SEQ ID NO:23; TALEB#3, SEQ ID NO:24 and SEQ ID NO:25). Seven days after transfection, phenotypic CD52 knockout was monitored by flow cytometry and splice site editing was measured by NGS. We observed high levels of phenotypic knockout for these three TALE base editors ( Figure 8 B, TALEB#1 average 81.1% + / - 4.7%, TALEB#2SA-2 average 83% + / - 3.4%, TALEB#3 average 81.9%, + / - 5.3%, which correlates with editing levels (TALEB#1 average 72.6%, + / - 1.7%, TALEB#2 average 74.5%, + / - 0.6%, TALEB#3 average 74.2%, + / - 2.3%, Figure 8 C). As expected from our previous dataset, NGS data analysis results showed very low levels of indels at these loci (TALEB#1 average 0.16%, + / -0.05%; TALEB#2 average 0.28%, + / -0.06%; TALEB#3 average 0.12%, + / -0.02%, mock transfection average 0.01%, + / -0.005%; Figure 8 C). The polypeptide and polynucleotide target sequences are reported in Table 5.

[0342] Table 5: KO CD52 TALEB polypeptides and target polynucleotides according to the present invention

[0343]

[0344]

[0345]

[0346]

[0347] Next, we attempted to use TALE base editors to create mutations within the CD52 signal peptide sequence (SEQ ID NO:365). Mutations in the signal peptide have been shown to disrupt the processing and translocation of the nascent peptide, thereby affecting the surface expression of certain genes. Therefore, we designed TALEB:TALE base editor SP (SEQ ID NO:26 and SEQ ID NO:27), which could cause (i) silent mutations at the Leu23 residue and (ii) several amino acid changes in the signal peptide (Gly22Lys, Ser24Leu, and Gly25Lys) ( Fig. 9 A). Residue changes (mutating hydrophobic glycine to highly charged lysine and polar serine to hydrophobic leucine in the signal peptide) significantly affected the ability of the signal peptide to properly guide translocation. Indeed, 6 days after TALE base editor mRNA transfection (ex2SP), flow cytometry observed an average of 84.2% (+ / -1.8%) CD52-negative cells ( Fig. 9 B). NGS sequencing analysis showed that all 6 positions were mutated, albeit at different levels (average editing frequencies: G[4]: 73.65+ / -1%, G[5]: 85.65+ / -0.7%, C[9]: 11.4+ / -0.1%, C

[11] : 56.5+ / -0.9%, G

[13] : 0.6+ / -0.1, G

[14] : 6.5+ / -0.5%) ( Fig. 9 C). Sequence analysis showed that 34 different species (including the wild type) were identified at the protein level, and they existed in different proportions ( Fig. 9 D).

[0348] Overall, the 4 CD52 TALEBs achieved very high phenotypic KO (median of CD52 negative population: 82.1%) and editing purity (median = 99.7, standard deviation = 0.6). In order to evaluate the possible off-target editing of these 4 CD52 TALEBs, a computer list of 276 potential off-target targets was generated (Table 6) and monitored using multiple amplicon sequencing analysis. Target amplicon sequencing of these sites did not show evidence of editing higher than the control experiment (N = 2, independent T cell donors).

[0349] Table 6: Predicted potential off-target locations of the four TALEBs targeting CD52

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359] Finally, since the TALEB CD52 splice site BE produced only marginal levels of indels, we hypothesized that multiple gene editing (i.e., the simultaneous use of base editors and nucleases, such as TALE-nucleases) should not produce chromosomal translocations (a phenomenon often observed in cells treated with multiple nucleases). As a proof of concept, TRAC-targeting TALE-nucleases (SEQ ID NO: 16 and SEQ ID NO: 17) were combined with CD52 TALE-nucleases (SEQ ID NO: 18 and SEQ ID NO: 19) or base editors TALE-BE SP (SEQ ID NO: 26 and SEQ ID NO: 27). Although high and similar levels of phenotypic double gene KO were detected by flow cytometry in both TALE-nuclease / TALE-nuclease and TALE-nuclease / TALE-base editor-treated cells (79% and 75%, respectively), Fig.10 ), but translocation between the two target sites (detected by multiplex amplicon sequencing) was only observed in TALE-nuclease / TALE-nuclease treated samples (479 reads out of 224,406 reads in TALE-nuclease / TALE-nuclease samples and 0 reads out of 144,323 reads in TALE-nuclease / BE samples, N = 1, 1 individual T cell donor, see Fig.11 ).

[0360] discuss

[0361] Base editing represents one of the latest gene editing technologies. Recently, it has been shown that the TALE backbone is compatible with the creation of a new class of DddA-derived cytosine base editors. In the above experimental studies, several base editors targeting various endogenous sites were screened using a simple and robust medium-throughput method developed to investigate the determinants of TALE-base editor editing. This throughput screening strategy exploits the efficient and precise TALE-nuclease-mediated ssODN knock-in in primary T cells and allows the evaluation of the editing efficiency of TALE base editors on hundreds of different targets in cells. Because the artificial target sequences of all base editors are inserted into the same predefined site in the genome, this approach can focus on how changes in the target / spacer sequence affect the TALE base editors while excluding factors such as DNA binding affinity or epigenetic variation. The experimental results show that the optimal spacer length window for editing is 13-17 bp, and the TALE base editor with the G1397C arm is placed 4-7 bp below the 3′ direction of the target TC to obtain the best editing activity.

[0362] Although extremely precise introduction of expected mutations (highly pure final products) is a prerequisite for gene correction, the generation of DSBs by base editors may cause great concern, especially because the recently discovered CRISPR / Cas base nucleases are associated with genomic instability or chromosomal abnormalities on major targets [Weisheit,, et al. (2020). Detection of Deleterious On-Target Effects after HDR-Mediated CRISPR Editing. Cell Rep. 31. Boutin, J., et al. (2022). ON-Target Adverse Events of CRISPR-Cas9 Nuclease: More Chaotic than Expected. Cris. J. 5, 19-30]. In this study, even at high editing frequencies (>80% in a large population), only very few side product mutations (C- to -A / G) and more importantly, very few insertions and deletions were detected by TALE base editors (dozens of these molecular tools were observed). However, careful design of the base editor positions can prevent or minimize bystander mutations.

[0363] Base editors have been used to edit or mutate conserved genetic elements such as enhancers [Zeng, J., et al. (2020). Therapeutic base editing of human hematopoietic stem cells. Nat. Med. 26, 535-541], start codons, splice sites [Kluesner, MG et al. (2021). CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary and immortalized cells. Nat. Commun. 12: 1-12], branch points and conserved active sites [Hanna, RE, et al. (2021). Massively parallel assessment of human variants with base editor screens. Cell 184, 1064-1080]. It is estimated that according to the present invention, approximately 46,000 (46,608) splice sites in the genome may be potentially targeted by TALE base editors, affecting 15,279 different transcripts, accounting for 76.57% of all transcripts in the human genome, and overall, indicating that splice site editing may be a feasible method for gene knockout by TALE base editors. To demonstrate the feasibility of this approach, an efficient TALE base editor targeting the conserved G at the splice site at the intron 1 / exon 2 junction of the CD52 gene has been designed. It has also been demonstrated that, as an alternative to splice site editing, targeting the signal peptide can also lead to efficient surface CD52 protein knockout.

[0364] Base editors therefore represent promising molecular tools for multiplex genetic engineering, although so far they have been limited to gene knockout or gene correction. Here, the feasibility of using a combination of two different molecular tools (nucleases and base editors) for effective multiplex genetic engineering has been demonstrated. This multiplex / multi-tool strategy shows several advantages. First, it prevents the occurrence of translocations observed when several (>1) nucleases are used simultaneously, and secondly, it allows the possibility of going beyond multiple knockouts while still allowing gene knock-in at the nuclease target site, generally expanding the range of possible applications while better controlling the outcomes of engineered cell populations (e.g., avoiding translocations). The precise positional rules determined here for TALE base editors allow for a reduction in the frequency of unwanted insertions and deletions and an increase in availability to other cellular compartments beyond traditional nuclear targets. Thus, they expand the potential scope of TALE-based multiplex / multi-tool strategies beyond the capabilities of most other non-TALE editing tools.

[0365] Example 5: Gene therapy to correct exon 24 of the PIK3CD gene that causes combined immunodeficiency ADPS1

[0366] The methods of the invention described herein are intended to improve the efficiency and safety of TALEM-mediated therapeutic gene insertion in long-term hematopoietic stem cells (LT-HSC) of individuals with dominant negative genetic diseases. The treatment includes the insertion of TALEN-mediated therapeutic repair matrix (cDNA of mutant gene) into the intron or exon of the defective gene, followed by TALE base editor-mediated inactivation of the same defective gene. The TALE base editor treatment proposed by this method can theoretically increase the frequency of cells with normal phenotypes without generating additional genomic adverse events due to the simultaneous generation of double-strand breaks. In general, inactivating the remaining defective genes is expected to improve the therapeutic effect of gene therapy interventions.

[0367] APDS1 is a combined immunodeficiency disorder caused by a gain-of-function mutation (E1021K) in exon 24 of the PIK3CD gene. This indication could benefit from TALEN / TALEN base editor-mediated targeted repair approaches, based on the principle of Figures 12 to 16 Description (Artex integration of the rewritten PIK3CD correction sequence + inactivation of the downstream original exon by using a base editor). This TALEN / TALEN base editor-mediated targeted repair / inactivation approach for exon 24 of PIK3CD is described below Fig. 20 and Fig.21As an example. Treatment of APDS1 cells with TALEN targeting intron 1 (between exons 1 and 2) promoted the insertion of a recoded therapeutic cDNA matrix carrying the correct version of PIK3CD cDNA (from exon 2 to exon 24). At the same time, treatment with a TALE base editor targeting exon 3 (see Table 12 for the selection of TALE base editor target sites) generated a stop codon downstream of the therapeutic cassette insertion site, thereby preventing the expression of the mutant allele.

[0368] Example 6: Effect of spacer length on C-to-T editing efficiency of C0, C11, and C40

[0369] TALE base editor heterodimers are double-stranded bacterial deaminases characterized by the following fusions: 1) The catalytic domain is divided into two inactive halves that, when reconstructed, catalyze the conversion of cytosine (C) to thymine (T); 2) A transcription activator-like effector domain (TALE) for DNA binding; 3) Uracil glycosidase inhibitor (UGI) (Mok B.Y. et all., Nature 2020). These TALE base editors have been used for several applications, including generating mutations in mitochondrial DNA mitochondria (Mok BY et all., Nature 2020) or chloroplasts (Beum-Chang Kang, et al., Nature Plants 2021). Despite these successful applications, the editing rules and target sequence specificity of TALE base editors are still limited. Therefore, more detailed and comprehensive studies are needed to create a further generation of TALE base editors. However, such progress is challenging. In vitro studies require purified recombinant TALE base editors, and cell-based approaches are tedious because many different TALEs targeting various sites are needed to rule out confounding effects such as epigenomic factors or modifications.

[0370] For the reported 15 bp interval length / editing window ( Figure 1 ) to define the key determinants of efficient TALE base editing (C-to-T conversion), the inventors established a medium- to high-throughput screen in a specific genomic context by generating a panel of cells containing a predefined TALE base editor target sequence ( Figure 5 ) of primary T-cell pools. Each TALE base editor target contains a unique TC or GA (target of DddA deaminase) in the spacer sequence, which is flanked by two fixed TALE binding sequences (RVD-L and RVD-R, Fig. 22This setup allows TALE base editors to bind uniformly to artificial target sites, eliminating editing variability caused by different DNA binding affinities of different TALE array proteins and the influence of epigenomic factors (such as chromosome relaxation around the artificial BE target site).

[0371] To investigate whether the length of the linker connecting the TALE array to the cleavage heads on both arms could affect the mobility of the DddA cleavage head and thus alter target specificity, STAT3 TALE base editors with different TALE C-terminal lengths (referred to as C40, C11, and C0 backbones) were constructed (Table 13, Fig.23 ).

[0372] Table 13: C-termini of TALEs used in the C40, C11 and C0TALEB backbones in Example 6.

[0373] TALE C-terminus Amino acid sequence SEQ ID NO# C40 SIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS 551 C11 SIVAQLSRPDPSGSGSGGGS 552 C0 GGS NA

[0374] Effects of spacer lengths (C0, C11, and C40) on C-to-T editing efficiency

[0375] like Fig.24 As shown, a collection of ssODN was constructed, which contains two fixed TALE array protein binding sites separated by intervals with different lengths from STAT3TALE base editors, and the interval spans from 5 to 17bp (i.e., 5, 7, 9, 11...17bp) to evaluate the differences in the STAT3 target sequence related to the interval length. A TCGA quadruplex target sequence was inserted every other position in the interval to generate a primary T cell pool containing a BE target set. In addition, for the convenience of sequence analysis, each construct was added with a unique barcode. The 37 unique ssODNs (Table 15) obtained were mixed in equal amounts and transfected into primary T cells by electroporation (200pmol per million cells), and mRNA (SEQ ID NO: 16 and 17) encoding the TALE-nuclease targeting TRAC was transfected at the same time. In the second step, two days after transfection of the TRAC TALEN and ssODN pools, the mRNA encoding the STAT3TALE base editor (mixed linker length) was co-electroporated.

[0376] Table 14: TALEB heterodimer structures tested in Example 6.

[0377]

[0378] Then, genomic DNA of transfected cells was collected on day 2 after TALE base editor transfection for editing analysis. Fig.24(C11 / C11 and C40 / C40 TALEB heterodimers) and Fig.25 The NGS analysis data summarized and presented in the graphs (mixed C11 / C40 and C40 / C11 TALEB heterodimers) are shown as follows:

[0379] Editing was best when the interval was between 11 bp (iv) and 15 bp (vi), reaching a maximum at 13 bp (v);

[0380] Overall, C11 / C11 has better editing, followed by C40 / C40;

[0381] The position of C / G and the size of the interval are still the parameters that have the greatest impact on editing efficiency.

[0382] Effect of TC surrounding environment: 15 bp interval length

[0383] To evaluate the effect of the surrounding environment on TALEB editing efficiency within a 15 bp interval length, a library containing 256 unique ssODNs was designed, such as Fig.26 and 27 As shown and detailed in Table 16. PBMCs from 2 donors were transfected with TRACTALEN and three different oligonucleotide pools to be inserted into the TRAC site, and then transfected with STAT3BE C40 / C40 or C11 / C11 to edit the cells with oligonucleotide KI. gDNA was prepared from cells treated with the 3 oligonucleotide pools, and samples were sequenced in MiSeq.

[0384] Bioinformatic data analysis determined the contribution of each surrounding base pair to C editing efficiency, such as Fig.28 As shown in (A and B), it is found that the situation of the two architectures is similar:

[0385] At position M2: A≤T< <G<C。

[0386] At position M1: T ≤ C <A<<G。

[0387] In position 1: T ≤ G <A<<C。

[0388] In position 2: T <C<G<<A。

[0389] We also studied multiple editing where C was immediately followed by a central TC (TCC to TTT), and editing analysis showed that the C40 architecture was more tolerated than C11 ( Fig.29 ).

[0390] These results show for the first time that for gene editing projects where a single point mutation (C->T) is desired, the C11 architecture is best suited for this situation, especially for target sequences that display a 15 bp interval. Such a target sequence can be defined by the following general formula:

[0391] 5′-T0-N 左 -N y -RTC-N x -N 右 -A0-3′; or

[0392] 5′-T0-N 左 -N x -GAY-N y -N 右 -A0-3′,

[0393] in:

[0394] N can be A, T, C, or G;

[0395] R can be G or A, preferably G;

[0396] Y can be C or T;

[0397] N 左 It may be a polynucleotide sequence comprising 9 to 20 nucleotides, wherein each individual nucleotide may be A, T, C or G;

[0398] N 右 It may be a polynucleotide sequence comprising 9 to 20 nucleotides, wherein each individual nucleotide may be A, T, C or G;

[0399] G is the complementary base of C.

[0400] And preferably the following formula:

[0401] 5′-T0-N 左 -N y -RTCC-N x -N 右 -A0-3′; or

[0402] 5′-T0-N 左 -N x -GGAY-N y -N 右 -A0-3′,

[0403] More preferably, it is the following formula:

[0404] 5′-T0-N 左 -N y -RTCC-N x -N 右-A0-3′; or

[0405] 5′-T0-N 左 -N x -GGAY-N y -N 右 -A0-3′,

[0406] where:

[0407] x = 2 to 6;

[0408] y = 6 to 10;

[0409] and x + y = 11.

[0410] Effect of the TC surrounding environment: 13 bp spacer length

[0411] For the 13 bp spacer length, other libraries containing 256 unique ssODNs were designed (detailed in Table 17). PBMCs from 2 donors were transfected with TRAC TALEN and three different oligonucleotide pools to be inserted into the TRAC locus, and then transfected with STAT3BE C40 / C40 or C11 / C11 to edit the cells with oligonucleotide KI. gDNA was prepared from the cells treated with 8 oligonucleotide pools, and the samples were sequenced on MiSeq. Fig.30 Bioinformatics data analysis as shown in A and B determined the contribution of each surrounding base pair to the C editing efficiency, and found that the situations of the two architectures were similar.

[0412] · At position M2: T < A << G = C. Although this position is not adjacent to TC, it seems to be important.

[0413] · At position M1: T < C < A < G.

[0414] · At position 1: T < G < A < C.

[0415] · At position 2: T < A = C < G. This position seems to be the least important, just like C11 on the same spacer.

[0416] We also studied multiplex editing, where C is immediately followed by the central TC (TCC to TTT), and the editing analysis showed that for both architectures, editing of two Ts at 13 bp was more tolerant ( Fig.31 ).

[0417] Surprisingly, TALEB was more tolerant when targeting sequences showing 13 bp spacing than when targeting sequences showing 15 bp spacing. These results suggest that for gene editing projects where multiple edits are desired, the design of TALE base editors should preferably be designed for genomic sequences showing 13 bp spacing. Such a target sequence can be defined by the following general formula:

[0418] 5′-T0-N 左 -N y -RTC-N x -N 右 -A0-3′; or

[0419] 5′-T0-N 左 -N x -GAY-N y -N 右 -A0-3′.

[0420] in:

[0421] N can be A, T, C, or G;

[0422] R can be G or A;

[0423] Y can be C or T;

[0424] N 左 It may be a polynucleotide sequence comprising 9 to 20 nucleotides, wherein each individual nucleotide may be A, T, C or G;

[0425] N 右 It may be a polynucleotide sequence comprising 9 to 20 nucleotides, wherein each individual nucleotide may be A, T, C or G;

[0426] G is the complementary base of C.

[0427] And preferably the following formula:

[0428] 5′-T0-N 左 -N y -RTCC-N x -N 右 -A0-3′; or

[0429] 5′-T0-N 左 -N x -GGAY-N y -N 右 -A0-3′,

[0430] and more preferably:

[0431] 5′-T0-N 左 -Ny -RTCC-N x -N 右 -A0-3′; or

[0432] 5′-T0-N 左 -N x -GGAY-N y -N 右 -A0-3′,

[0433] in:

[0434] x=2 to 4;

[0435] y = 6 to 8;

[0436] And x+y=9.

[0437] like Fig.28 As shown in (A and B), bioinformatic data analysis aimed at determining the contribution of each surrounding base to the efficiency of C editing surprisingly showed that for highly edited targets containing different spacings, the results were very similar regarding the surrounding bases of TC. However, regardless of the spacing, the C11TALEB backbone showed stronger specificity on those target sequences.

[0438] Materials and methods

[0439] T cell culture

[0440] Cryopreserved human PBMCs were obtained from ALLCELLS. PBMCs were cultured in X-vivo-15 medium (Lonza Group) containing 20 ng / ml human IL-2 (Miltenyi Biotec) and 5% human AB serum (Seralab). On the day after thawing PBMCs, human T cell activator TransAct (Miltenyi Biotec) was used to activate T cells at a concentration of 100 μg / ml CD3+ / -100 μg / ml. + T cells were activated with 25 μl of TransAct. TransAct was maintained in the culture medium for 72 hours.

[0441] Preparation of TALE-nucleases or TALE-base editors

[0442] The backbones of TALEN (pCLS32783) and TALE-base editors (pCLS35714, pCLS35715, pCLS37473, and pCLS37474, Table 13) were assembled using standard molecular biology and / or microbiology techniques such as enzyme restriction digestion, ligation, bacterial transformation, and plasmid DNA extraction. TALE DNA targeting arrays were assembled and cloned into TALEN and / or TALE base editor backbones using standard molecular biology and / or microbiology techniques such as enzyme restriction digestion, ligation, bacterial transformation (NEB 10-β competent E. coli for ccdB screening or NEB stable competent E. coli for blue / white screening), and plasmid DNA extraction.

[0443] Large-scale TALE-nuclease and TALE-base editor mRNA preparation (STAT3 target TALEB).

[0444] The plasmid encoding TRAC TALE-nuclease contains a T7 promoter and a poly A sequence. TALE-nuclease mRNA from the TRAC TALE-nuclease plasmid was produced by Trilink. The sequence targeted by TRACTALE-nuclease (17 bp recognition site, capital letters, separated by 15 bp spacers).

[0445] The plasmid encoding the STAT3 TALE base editor contains a T7 promoter and a poly A sequence. The sequence-verified plasmid was linearized with SapI (NEB) prior to in vitro mRNA synthesis. mRNA was expressed using NEB HiScribe TM Prepared using T7 Rapid High Yield RNA Synthesis Kit (NEB). TM The 5′ end capping reaction was performed using the m7G capping system (Cellscript). The capped mRNA was treated with thermosensitive phosphatase (NEB) and finally cleaned using Mag-Bind TotalPure NGS beads (Omega bio-tek) and Invitrogen DynaMag-2 magnet (ThermoFisher).

[0446] ssODN repair template transfection

[0447] Pools of ssODNs targeting the TRAC site (Table 15, Table 16, and Table 17) were ordered from Integrated DNA Technologies (IDT) and resuspended in ddH2O at 50 pmol / μl.

[0448] 10-12 hours before transfection, T cells activated for 3 days with TransACT were transferred into fresh complete medium containing 20 ng / ml human IL-2 (Miltenyi Biotec) and 5% human AB type serum (Seralab).

[0449] Wash the collected cells once with warm PBS. 6 The PBS-washed cells were centrifuged and resuspended in 20 μl of Lonza P3 primary cell buffer (Lonza). 200 pmol of ssODN pool and 1 μg / arm of TRACTALE-nuclease were mixed with the cells, and the cell mixture was electroporated under the EO115 program using the Lonza 4D-nucleofector to obtain stimulated human T cells. After electroporation, 80 μl of warm complete medium was added to the cuvette to dilute the electroporation buffer, and the mixture was carefully transferred to 400 ml of preheated complete medium in a 48-well plate. The ssODN and TALE-nuclease transfected cells were then incubated at 30°C until 24 hours after TALE-nuclease transfection and then transferred back to 37°C.

[0450] After two days of culture, cells containing ssODN KI were collected for TALEB treatment. The collected cells were washed once with warm PBS. 1×10 6 The PBS-washed cells were centrifuged and resuspended in 20 μl Lonza P3 primary cell buffer (Lonza). The cells were mixed with 1 μg / arm of STAT3 TALEB (C0, C11, or C40), and the cell mixture was electroporated under the EO115 program using the Lonza 4D-nucleofector to obtain stimulated human T cells. After electroporation, 80 μl of warm complete medium was added to the cuvette to dilute the electroporation buffer, and the mixture was carefully transferred to 400 ml of preheated complete medium in a 48-well plate. Cells transfected with TALE base editors were incubated at 37 ° C for another 2 days and then collected for gDNA extraction and NGS analysis.

[0451] Genomic DNA extraction

[0452] Cells were collected and washed once with PBS. Genomic DNA extraction was performed using the Mag-Bind Blood & Tissue DNA HDQ Kit (OmegaBio-Tek) according to the manufacturer's instructions.

[0453] Targeted PCR and NGS

[0454] In a 50 ml reaction, 100 mg of genomic DNA was used per reaction using Phusion High Fidelity PCR Master Mix (NEB). The PCR conditions were set as follows: 1 cycle: 30 s at 98°C; 30 cycles: 10 s at 98°C, 30 s at 60°C, 30 s at 72°C; 1 cycle: 5 min at 72°C; hold at 4°C. The PCR products were then purified using Omega NGS beads (1:1.2 ratio) and eluted into 30 ml of 10 mM Tris buffer, pH 7.4. The purified products from the first PCR were then subjected to a second PCR (combined with NGS indices). 15 ul of the first PCR product was placed in a 50 ml reaction and Phusion High Fidelity PCR Master Mix (NEB) was added. PCR conditions were set as follows: 1 cycle: 98°C for 30 s; 8 cycles: 98°C for 10 s, 62°C for 30 s, 72°C for 30 s; 1 cycle: 72°C for 5 min; hold at 4°C. Purified PCR products were sequenced on a MiSeq (Illumina) on a 2x250 nanoV2 cassette.

[0455] Example 7: TALEB according to the present invention prevents AAV capture

[0456] Day 0: Frozen human peripheral blood mononuclear cells (PBMCs) from AllCells (Alameda, CA 94502) were thawed, washed, counted, and resuspended in OpTmizer medium (Gibco: A1048501) supplemented with 5% AB serum (GeminiBio: 100-318) and 20 ng / mL recombinant human IL-2 (Miltenyi: 130-097-743). The cells were then transferred to an incubator set at 37°C, 5% CO2.

[0457] Day 1: PBMCs were counted, analyzed by flow cytometry to assess the percentage of CD3+ cells, centrifuged and resuspended in OpTmizer medium supplemented with 5% AB serum, 20 ng / mL human IL-2 and Transact beads CD3CD28 (Miltenyi: 130-111-160). The cells were then transferred to an incubator set at 37°C, 5% CO2.

[0458] Day 4: T cells were subcultured into fresh OpTmizer medium supplemented with 5% AB serum and 20 ng / mL IL-2. The culture dish was then transferred to an incubator set at 37°C and 5% CO2.

[0459] Day 5: Cells were co-electroporated using AgilePulse technology with 1 μg of mRNA encoding the left and right arms of TRAC TALEN (SEQ ID NO: 562 and 563) or B2M TALEN (SEQ ID NO: 564 and 565) or TRAC TALEB (SEQ ID NO: 566 and 567) targeting the TRAC genomic sequence SEQ ID NO: 561. After transfection, cells were incubated in fresh OpTmizer medium at 37°C for 15 minutes and then transferred to 30°C for another 15 minutes. Cells were then counted and concentrated to 8×10 6 Cells / mL were transduced with or without AAV6 particles encoding HLA-E (SEQ ID NO: 560) at 50,000 vg / cell for targeted integration at the B2M site (SEQ ID NO: 559) according to previous reports [Jo et al (2022) Nat Commun 13 (1) and Sachdeva et al. (2019) Nat Commun. 10 (1)]. The modified cells were cultured overnight at 30°C and subcultured the next day into fresh OpTmizer medium supplemented with 5% AB serum and 20 ng / mL IL-2. The cells were then transferred to an incubator set at 37°C and 5% CO2.

[0460] Day 8: Modified T cells were collected and analyzed by flow cytometry using anti-TCRαβ, anti-HLA-ABC, and anti-HLA-E antibodies.

[0461] The sequences of the reagents used in these experiments are reported in Table 18.

[0462] like Fig.32 As shown in A, approximately 80%, 60%, and 10% of the cells were TCRαβ negative when treated with TRAC TALEN, TRAC TALEB, and B2M TALEN, respectively.

[0463] This result shows that both TRAC TALEN and TALE base editors are very efficient. In addition, when transduced with AAV6 particles, 50% of HLA-E positive cells were detected in cells transfected with B2M TALEN, indicating its high targeting efficiency. When cells were transfected with TRAC TALEN and transduced with AAV6 particles (used as template DNA designed to insert the HLAE coding sequence into the B2M site by homologous recombination), approximately 0.5% of HLA-E positive cells were detected. Fig.32As shown in Figure B, these HLA-E positive cells are not artifacts, and these results indicate that AAV6 constructs can be captured at the TRAC site, even though the DNA template was not originally designed to insert into the TRAC site. Importantly, when cells were transfected with the TRAC TALE base editor and transduced with AAV6 particles ( Fig.32 A and 32B), no HLA-E positive cells were detected, indicating that this capture can be eliminated when TALE base editors are used. Therefore, it was found that the combination of TALEN and TALEB is highly efficient and can quickly ensure higher genome integrity when multiplexing gene editing of therapeutic immune cells, especially when combined with gene editing of transgenes knocked in using TALE nucleases and knocking out endogenous genes using TALEB according to the present invention.

[0464] Table 18: Polynucleotides and polypeptides used in Example 7

[0465]

[0466]

[0467]

[0468]

[0469] Table 7: List of TALEB target sequence windows for introducing mutations in the TRAC gene according to the rules of the present invention.

[0470]

[0471]

[0472] Table 8: List of TALEB target sequence windows for introducing mutations into the CD52 gene according to the rules of the present invention.

[0473]

[0474]

[0475] Table 9: List of TALEB target sequence windows for introducing mutations into the PD1 gene according to the rules of the present invention.

[0476]

[0477]

[0478] Table 10: List of TALEB target sequence windows for introducing mutations into the B2m gene according to the rules of the present invention.

[0479]

[0480]

[0481]

[0482] Table 11: List of TALEB target sequence windows for introducing mutations into the ApoC3 gene according to the rules of the present invention.

[0483]

[0484]

[0485] Table 12: Base editor target sites in exon 1, 2 or 3 of the PK13 gene according to the combined gene therapy approach shown in Example 5.

[0486]

[0487]

[0488] Table 15: Initial STAT3 TALE target sequence library spanning 5 to 17 bp

[0489]

[0490]

[0491]

[0492] Table 16: Library containing 256 target sequences (ssODN) with 15 bp intervals designed to test TC environment in Example 6

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507] Table 17: Library containing 256 target sequences (ssODN) with 13 bp intervals designed to test TC environment in Example 6

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522] Table 18: List of diseases caused by deleterious alleles (target genes) that can be addressed by the present invention

[0523] Disease Name Target genes Hereditary amyloid angiopathy APP Marfan syndrome FBN1 Neurofibromatosis type I NF1 Familial adenomatous polyposis APC Tuberous sclerosis complex (TSC) TSC1 or TSC2 Brugada syndrome SCN5A APDS2 and SHORT syndrome PIK3R1 Vascular Ehlers-Danlos syndrome COL3A1 APDS1 PIK3CD Congenital neutropenia SRP54 Autosomal Dominant Hyper-IgE Syndrome (AD-HIES) stat3 Liver disease with severe immunodeficiency NFKBIA Laron syndrome GHR, growth hormone receptor Pituitary hormone deficiency POU1F1, POU domain, level 1 Growth hormone deficiency GH1 Thyroid hormone receptor beta Thyroid hormone resistance THRB GnRH receptor hypogonadotropic hypogonadism GNRHR, gonadotropin-releasing hormone receptor UDP-glycosyltransferase 1 superfamily Gilbert syndrome GLI2, GLI UDP-glycosyltransferase 1 superfamily Gilbert syndrome UGT1A1 Li-Fraumeni syndrome TP53, tumor protein p53 Rubinstein-Taybi syndrome CREBBP Wilms tumor 1 Denys-Drash syndrome WT1 .

Claims

1. A method for designing and producing a TALE base editor heterodimer in a double-stranded nucleic acid sequence to convert a specific C to A and / or convert a C-complementary G position to T, the method comprising the following steps: i) recognizing a target sequence in the nucleic acid sequence selected from the group consisting of: 5′-T0-N 左 -N y -RTC-N x -N 右 -A0-3′; as well as 5′-T0-N 左 -N x -GAY-N y -N 右 -A0-3′, in: N can be A, T, C or G; R can be G or A; Y can be C or T; N 左 can be a polynucleotide sequence comprising 9 to 20 nucleotides, wherein each individual nucleotide can be A, T, C or G; N 右 can be a polynucleotide sequence comprising 9 to 20 nucleotides, wherein each individual nucleotide can be A, T, C or G; G is the complementary base of C; x = 2 to 6; y=6 to 10, wherein preferably x+y≥11, more preferably x+y=12; ii) Synthetic coding and N 左 Polynucleotide sequences and N 右 The polynucleotide sequences of the left TALE-binding polypeptide and the right TALE-binding polypeptide to which the polynucleotide sequence binds; iii) fusing the polynucleotide sequence encoding the left TALE binding polypeptide with a polynucleotide encoding the N-terminal split DddAtox; iv) fusing the polynucleotide sequence encoding the right TALE binding polypeptide with the polynucleotide encoding the C-terminal split DddAtox, and fusing the polynucleotide sequence encoding UGI (uracil glycosylase inhibitor) with at least one polynucleotide sequence encoding the polynucleotide sequence obtained by ii) and iii).

2. The method of claim 1, wherein the left TALE-binding polypeptide and the right TALE-binding polypeptide comprise a C-terminal domain of about 11 amino acids or about 40 amino acids from SEQ ID NO:

270.

3. The method according to claim 1 or 2, wherein x is 2 to 5, preferably 3 to 5.

4. The method according to any one of claims 1 to 3, wherein the sequence of the N-terminal split DddAtox and / or the C-terminal split DddAtox comprises at least one mutation which reduces the affinity of the split DddAtox for each other.

5. The method of claim 4, wherein the mutation is introduced into the C-terminal split DddAtox of SEQ ID NO:

29.

6. The method of any one of claims 1 to 5, wherein the left TALE-binding polypeptide and the right TALE-binding polypeptide comprise AvrBs3-like repeats of a canonical sequence selected from SEQ ID NOs: 12 to 15.

7. The method according to any one of claims 1 to 6, wherein the left TALE-binding polypeptide and the right TALE-binding polypeptide comprise AvrBs3-like repeats, wherein the AvrBs3-like repeats comprise D (aspartic acid) residues at positions 4 and 32 relative to any of the canonical sequences of AvrBs3.

8. The method of claim 7, wherein at least one of the AvrBs3-like repeats comprises a polypeptide sequence selected from the group consisting of: LTPDQVVAIASX 12 X 13 GGKQALETVQRLLPVLCQDHG(SEQ ID NO:5), LTPDQVVAIASX 12 X 13 GGKQALETVQALLPVLCQDHG(SEQ ID NO:6), LTPDQVVAIASX 12 X 13 GGKQALETVQQLLPVLCQDHG (SEQ ID NO: 7), or LTPDQLVAIASX 12 X 13 GGKQALETVQRLLPVLCQDHG(SEQ ID NO:8), LTPDQMVAIASX 12 X 13 GGKQALETVQRLLPVLCQDHG(SEQ ID NO:9), LTPDQVVAIASX 12 X 13 GGKQALETVQRLLPVLCQDQG(SEQ ID NO:10), LTLDQVVAIASX 12 X 13 GGKQALETVQRLLPVLCQDHG(SEQ ID NO:11), Where X 12 X 13 are amino acids that form variable di-residues.

9. The method of any one of claims 1 to 8, wherein the C-terminal domain of the TALE-binding polypeptide consists of a polypeptide sequence having 40 to 80 residues, the polypeptide sequence having at least 85% identity to: SIVAQLSRPDP (SEQ ID NO: 2); SIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVX1X 2 GL(SEQ ID NO:3); SIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVX1X 2 GL PHAPAL I X3RT (SEQ ID NO: 4); or in, X1, X2 and X3 are K (lysine), H (histidine) or R (arginine) residues.

10. The method according to any one of claims 1 to 8, further comprising the following steps: The polynucleotide encoding the TALE base editor heterodimer obtained in step iv) is expressed in a cell, preferably to introduce mutations into therapeutic immune cells.

11. A method for introducing a mutation into the genome of a cell, comprising the steps of: A TALE base editor is introduced or expressed into the cell, wherein the TALE base editor consists of a left TALE binding polypeptide and a right TALE binding polypeptide having a C-terminal domain of about 1 to 50 amino acids and a heterodimeric fusion of C-terminal and N-terminal split DddATox, respectively, wherein the heterodimeric TALE base editor binds to a genomic sequence selected from the following: 5′-T0-N 左 -N y -RTC-N x -N 右 -A0-3′; as well as 5′-T0-N 左 -N x -GAY-N y -N 右 -A0-3′; in: N can be A, T, C or G; R can be G or A; Y can be C or T; N 左 can be a polynucleotide sequence in which each individual nucleotide can be A, T, C or G; N 右 can be a polynucleotide sequence in which each individual nucleotide can be A, T, C or G; G is the complementary base of C; x = 2 to 6; y = 6 to 10; Preferably, x+y≥11, and more preferably, x+y=12.

12. The method of claim 11, wherein the left TALE-binding polypeptide and the right TALE-binding polypeptide have a C-terminal domain of 1 to 50 amino acids, preferably 8 to 40 amino acids, more preferably 10 to 30 amino acids.

13. The method of claim 11, wherein the left TALE-binding polypeptide and the right TALE-binding polypeptide have a C-terminal domain of about 11 or 40 amino acids.

14. The method according to any one of claims 11 to 13, wherein x is 2 to 5, preferably 3 to 5.

15. The method according to any one of claims 11 to 14, wherein the cells are hematopoietic stem cells.

16. The method according to any one of claims 11 to 14, wherein the cell is an immune cell.

17. The method of claim 16, wherein the immune cells are primary cells.

18. The method according to claim 16 or 17, wherein the immune cell is a T cell or a NK cell. The method according to claim 18 , wherein the expression of TCR in the T cells is inhibited or inactivated.

20. The method of any one of claims 11 to 19, wherein the immune cells are endowed with a chimeric antigen receptor (CAR) or a recombinant TCR.

21. The method of any one of claims 11 to 20, wherein the TALE base editor binds to a genomic sequence contained in a gene encoding TRAC.

22. The method of claim 21, wherein the TALE base editor binds to a genomic sequence selected from any one of SEQ ID NO:366 to SEQ ID NO:

407.

23. The method of any one of claims 11 to 20, wherein the TALE base editor binds to a genomic sequence contained in a gene encoding a target for an immunosuppressive drug.

24. The method of claim 23, wherein the TALE base editor binds to a genomic sequence contained in a gene encoding CD52.

25. The method of claim 24, wherein the TALE base editor binds to a genomic sequence selected from any one of SEQ ID NO:408 to SEQ ID NO:

422.

26. The method of any one of claims 11 to 20, wherein the TALE base editor binds to a genomic sequence contained in a gene encoding an immune checkpoint.

27. The method of claim 26, wherein the TALE base editor binds to a genomic sequence in a PD1 gene selected from any one of SEQ ID NO: 423 to SEQ ID NO:

466.

28. The method of any one of claims 11 to 20, wherein the TALE base editor binds to a genomic sequence contained in a gene encoding B2M.

29. The method of claim 28, wherein the TALE base editor binds to a genomic sequence in a B2M gene selected from any one of SEQ ID NO: 467 and SEQ ID NO:

501.

30. The method of any one of claims 11 to 29, wherein the TALE base editor introduces a mutation into a genomic sequence encoding a functional protein domain.

31. The method of claim 30, wherein the functional domain is a catalytic site, a signal peptide or a structural domain.

32. The method of any one of claims 11 to 29, wherein the TALE base editor introduces a mutation into a splice site.

33. The method of any one of claims 11 to 14, wherein the genomic sequence is targeted to the liver for gene therapy.

34. The method of claim 31, wherein the genomic sequence encodes a defective ApoC3 protein.

35. A method according to claim 34, wherein the TALE base editor binds to a genomic sequence in ApoC3 selected from any one of SEQ ID NO:502 and SEQ ID NO:523.

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