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Meganuclease variants cleaving a DNA target sequence from a xp gene and uses thereof

A coloring and variant technology, applied in the direction of stable introduction of foreign DNA into chromosomes, recombinant DNA technology, applications, etc., can solve problems such as difficulty in fully evaluating the sequence range

Inactive Publication Date: 2009-03-11
赛莱提斯
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it remains difficult to fully evaluate the range of sequences that can be obtained using this combinatorial approach

Method used

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  • Meganuclease variants cleaving a DNA target sequence from a xp gene and uses thereof
  • Meganuclease variants cleaving a DNA target sequence from a xp gene and uses thereof
  • Meganuclease variants cleaving a DNA target sequence from a xp gene and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0200] Example 1 : Transformation of I-CreI variants with specific changes at positions ±8 to ±10

[0201] Methods for generating meganuclease variants, as well as assays based on recombination-induced cleavage in mammalian or yeast cells, were used to screen for variants with altered specificity, which have been described in the literature (PCT Application WO2004 / 067736; Arnould et al., J.Mol.Biol., 2006, 355, 443-458.Epinat et al., N.A.R., 2003, 31, 2952-2962 and Chames et al., Nucleic Acids Res., 2005, 33, e178). These assays yield a functional lacZ reporter that can be monitored by standard methods.

[0202] A) Materials and methods

[0203] a) Construction of libraries Ulib4, Ulib5 and Lib4

[0204] The I-Cre Iwt and I-CreI D75N open reading frames were synthesized as previously described (Epinat et al., 2003, 31, 2952-2962). Mutation D75N was introduced by replacing codon 75 with aac. Three combinatorial libraries (Ulib4, Ulib5, and Lib4) were derived by exchang...

Embodiment 2

[0232] Example 2 : A strategy for the construction of novel meganucleases that cleave targets from the XPC gene

[0233] The first series of I-CreI variants having at least one substitution at positions 44, 68 and / or 70 of I-CreI and capable of cleaving mutant I-CreI sites with changes in positions ±3 to 5 identified (Arnould et al., J. Mol. Biol., 2006, 355, 443-458). The cleavage patterns of the variants are shown in Figure 7.

[0234] According to the method described in Example 1, it is identified that there is at least one substitution at positions 28, 30, 33 or 28, 33, 38 and 40 of I-CreI, and it is possible to cleave cells with changes in positions ±8 to 10 A second series of I-Crel variants at the mutant I-Crel site. The cleavage patterns of the variants are shown in Figure 6.

[0235] On the same DNA-binding fold, positions 28, 30, 33, 38, and 40 are on one side, and 44, 68, and 70 are on the other, and there is no structural evidence that they work independently...

Embodiment 3

[0242] Example 3 : Preparation of a meganuclease that cleaves Xx.3

[0243] This example shows that the I-Crel mutant can palindromically cleave the Xx.3 DNA target sequence derived from the left portion of the Xx.2 target ( Figure 9 and 23). The target sequence described in this example is a 22bp palindromic sequence. Therefore, only their first 11 nucleotides are described, followed by the suffix _P; for example, target Xa.3 would be annotated as ctgccttttgt_P.

[0244] Xa.3 is similar to 5TTT_P positions ±1, ±2, ±3, ±4, ±5 and ±11, and to 10TGC_P positions ±1, ±2, ±8, ±9, ±10 and ±11.

[0245] Xb.3 is similar to 5GGG_P bits ±1, ±2, ±3, ±4, ±5, and ±11, and to 10GGG_P bits ±1, ±2, ±8, ±9, ±10, and ±11.

[0246] Xc.3 is similar to C1221 bits ±1, ±2, ±3, ±4, ±5, ±7, and ±11, and to 10GAG_P bits ±1, ±2, ±7, ±8, ±9, ± 10 and ±11 bits.

[0247] It is known that wild-type I-CreI allows nucleotide substitutions at positions ±11, ±7 and ±6 (Chevalie et al., J. Mol. Biol., 20...

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PUM

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Abstract

An I-CreI variant which has at least two substitutions, one in each of the two functional subdomains of the LAGLIDADG core domain situated from positions 28 to 40 and 44 to 70 of I-CreI, said variant being able to cleave a DNA target sequence from a XP gene. Use of said variant and derived products for the prevention and the treatment of the disease associated with Xeroderma pigmentosum.

Description

technical field [0001] The present invention relates to a meganuclease variant that cleaves a DNA target sequence from the xeroderma pigmentosa gene (XP gene), to a vector encoding said variant, to a cell transformed by said vector , animals or plants, and relates to the use of said meganuclease variants and derived products thereof for in vivo and in vitro genome therapy (gene cell therapy), and genome engineering. Background technique [0002] Xeroderma pigmentosa (XP) is a rare autosomal recessive disorder characterized by hypersensitivity to ultraviolet (UV) exposure, a high predisposition to skin cancer in sun-exposed areas, and Sometimes insane (Hengge, U.R. and W. Bardenheuer, Am. J. Med. Genet. C. Semin. Med. Genet., 2004, 131: 93-100; Magnaldo, T. and A. Sarasin, Cells Tissues Organs , 2004, 177: 189-198; Cleaver, J.E., Nat. Rev. Cancer, 2005, 5: 564-573; Hengge U.R., Clin. Dermatol., 2005, 23: 107-114). Cells from XP patients are less able to eliminate UV-induced...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N15/55C12N15/90A61K48/00
CPCC12N9/22A61P17/00A61P17/02
Inventor 西尔万·阿尔努克里斯托夫·佩雷-米绍朱莉安娜·斯米特
Owner 赛莱提斯
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