A non-toxic cas9 enzyme and application thereof

A nuclease, exonuclease technology, used in hydrolase, recombinant DNA technology, DNA/RNA fragments, etc.

Pending Publication Date: 2021-12-17
克里斯普赫尔治疗公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The frequency of introduction of mutations (e.g., deletions and insertions) at target nucleic acids by non-homologous end-joining (NHEJ) repair mechanisms limits the utility of gene targeting and editing in therapeutic agent development

Method used

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  • A non-toxic cas9 enzyme and application thereof
  • A non-toxic cas9 enzyme and application thereof
  • A non-toxic cas9 enzyme and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0235] Example 1 - Reduced cytotoxicity in A549 cells

[0236] refer to figure 1 , generated several plasmid constructs with different polynucleotides. Each polynucleotide encodes a different fusion protein consisting of the hExo1 fragment (amino acids 1-352 of SEQ ID NO:1) linked to Cas9 (any one of SEQ ID NO:2-18) through a specific linker peptide )constitute. Some plasmid constructs encode Cas9 enzymes with one N-terminal nuclear localization sequence (NLS) or C-terminal NLS, while some plasmid constructs encode Cas9 enzymes with both C-terminal NLS and N-terminal NLS. All plasmid constructs were sequenced to ensure no mutations occurred in the polynucleotide sequence. Each plasmid construct also contains a nucleotide sequence encoding a gRNA directed to the desired chromosomal locus. The predicted chromosomal locus is in the intergenic region between VSP33A on 5′ and CLIP1 on 3′ on chromosome 12, which has no predicted genes or long noncoding RNAs. Once cells are tra...

Embodiment 2

[0239] Example 2 - Reduction of cytotoxicity in A549 cells using gRNA targeting the HBB gene

[0240]Similar to the experiment carried out in Example 1, several plasmids containing polynucleotides encoding the fusion protein hExo1-Cas9 enzyme were produced ( figure 1 ). Each plasmid construct also contained a nucleotide sequence encoding a gRNA recognizing exon 1 of the human HBB gene. Compared to the experiments performed in Example 1, an additional control was introduced in which the cells were transfected with plasmids having the nucleotide sequence encoding the wild-type Cas9 enzyme and the nucleotide sequence encoding hExo1, respectively. Three gRNA sequences listed in Table 2 for recognition of one of the exons of the HBB gene were used. A control plasmid was prepared to encode an unmodified Cas9 (any one of SEQ ID NO: 2-18) enzyme.

[0241] A similar cell culture and transfection protocol to the experiments performed in Example 1 was used. Referring to FIG. 6 , gRN...

Embodiment 3

[0242] Example 3 - Treatment of sickle cell anemia in a patient

[0243] A biological sample is obtained from a subject with sickle cell anemia. Genomic DNA was extracted from biological samples and sequenced to verify single nucleotide substitutions (A to T) in the amino acid 6 codon of the β-globin gene. This mutation converts a glutamic acid codon (GAG) to a valine codon (GTG). Hematopoietic stem cells are isolated from the patient's bone marrow cavity and cultured ex vivo. A nucleic acid vector encoding the protein fusion complex and gRNA portion of hExo1-Cas9 was delivered into cultured hematopoietic stem cells. In addition, a DNA template sequence with an integration cassette encoding the wild-type sequence of exon 1 of the β-globin gene was delivered into cultured hematopoietic stem cells. The gRNA portion comprises at least 10 nucleotides complementary to the GTG locus of exon 1 of the β-globin gene. The DNA template sequence comprises an integration cassette flank...

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Abstract

Compositions related to engineered Cas9 enzyme in reducing cellular toxicity and methods using thereof related to the selective targeting and editing endogenous nucleic acid segment in both normal cell and in cell associated with genetic diseases are disclosed. In some cases, a polypeptide comprising a human Exo1 enzyme or a first functional fragment thereof and a Cas9 enzyme or a second functional fragment thereof, which are connected by a linker peptide, is disclosed. In some cases, a polynucleotide encoding the polypeptide and a guide RNA (gRNA) is disclosed. Further, methods for treating single gene disorders utilizing either the polypeptide or the polynucleotide are disclosed.

Description

[0001] cross reference [0002] This application claims U.S. Provisional Application 62 / 789,347, filed January 7, 2019; U.S. Provisional Application 62 / 823,477, filed March 25, 2019; U.S. Provisional Application 62 / 824,164, filed March 26, 2019 and priority to U.S. Provisional Application 62 / 855,612, filed May 31, 2019, which is hereby incorporated by reference in its entirety. [0003] sequence listing [0004] This application contains a Sequence Listing, which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 3, 2020, is named 55190-701_601_SL.txt and is 236,317 bytes in size. Background technique [0005] Targeted editing of nucleic acids is a very promising approach to study genetic function as well as to treat and ameliorate the symptoms of genetic disorders and diseases. The most notable target-specific genetic modification approaches involve the engineering and use of zinc fi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/90C12N9/22
CPCC12N9/22C12N15/62C12N2320/31C12N15/113C12N2310/20C12N15/907C12N15/90C07K2319/00C12N15/111
Inventor 克里斯多佛·哈克利
Owner 克里斯普赫尔治疗公司
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