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Adeno-associated virus compositions for restoring hbb gene function and methods of use thereof

A genome and gene technology, applied in the field of adeno-associated virus composition for restoring HBB gene function and its application, can solve safety issues and other issues

Pending Publication Date: 2020-07-24
CITY OF HOPE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, each of these techniques raises safety concerns due to the possibility of off-target mutations at sites in the human genome that are sequence-similar to the intended target site

Method used

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  • Adeno-associated virus compositions for restoring hbb gene function and methods of use thereof
  • Adeno-associated virus compositions for restoring hbb gene function and methods of use thereof
  • Adeno-associated virus compositions for restoring hbb gene function and methods of use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0160] Example 1: Selection of AAV capsids for gene editing of HBB mutant cells

[0161] This example characterizes the integration efficiency of the gene-editing AAV vector AAVS1-FP packaged in clade F AAV capsids (e.g., AAVHSC7, AAVHSC15, and AAVHSC17), also known as AAVF7, AAVHSC17, respectively, in HBB mutant cells and AAVF17, fully described in WO2016049230A1, which is hereby incorporated by reference in its entirety.

[0162] The gene editing vector AAVS1-FP used herein is fully described in WO2016049230A1. It contains from 5' to 3': AAV2 5' inverted terminal repeat (ITR), 5' homology arm consisting of 800 nucleotides with DNA sequence upstream of the target locus, splice acceptor, 2A element, fluorescence Coding sequence for the protein (FP), a 3' homology arm consisting of 800 nucleotides with the DNA sequence downstream of the target locus, and the AAV2 3'ITR, where the target locus is in the AAVS1 locus on chromosome 19 Intron 1 of human PPP1R12C, and wherein after...

Embodiment 2

[0173] Example 2: In Vitro Correction of HBB Mutations

[0174] produced as figure 2 AAV-based HBB correction vector designated hHBB-hL-014 is shown. This correction vector was designed to correct HBB mutations, such as the A to T mutation at nucleotide 20 in the exon 1 coding region (starting from the start codon) of the HBB gene in sickle cell disease. The hHBB-hL-014 vector includes 5′ and 3′ AAV2 ITRs, flanked by HBB and a portion of its adjacent genomic sequence, in which the A to T mutation at nucleotide position 20 in exon 1 is reversed. A portion of the HBB genome sequence was obtained from wild-type HBB and its adjacent loci using the amplification primers shown in Table 2 using NCBIPrimer Blast (www.ncbi.nlm.nih.gov / tools / primer-blast / )design. The PCR product covered all exons and introns of HBB and also included 1678 nucleotides upstream of the HBB transcription start site and 234 nucleotides downstream of the HBB polyadenylation sequence. This vector contains...

Embodiment 3

[0194] Example 3: HBB Correction Vectors Comprising HBB Genomic Sequences

[0195] This example provides an AAV-based HBB correction vector comprising the genome sequence of HBB capable of correcting HBB gene mutations.

[0196] a) HBB Correction Vector hHBB-hL-001

[0197] Such as Figure 4A As shown, the HBB correction vector hHBB-hL-001 contains the HBB genome sequence including all exons, all introns and polyadenylation sequences. The vector additionally includes: a 5' region from the HBB transcription initiation site (at Figure 4A referred to as "HBB HAL") upstream contains 800bp, and a 3' region from the HBB polyadenylation sequence (in Figure 4A referred to as "HBBHAR") downstream contains 800bp. The hHBB-hL-001 vector comprises the nucleotide sequence set forth in SEQ ID NO: 31 (with TI RE linker) or SEQ ID NO: 32 (without TI RE linker), and also includes a 5'ITR (for example, with SEQ ID NO: ID NO: 18) and 3'ITR (for example, having the sequence of SEQ ID NO:...

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Abstract

Provided herein are adeno-associated virus (AAV) compositions for correcting a mutation in a beta globin gene (HBB) gene and methods of using the same to correct an HBB gene mutation in a cell. Also provided are packaging systems for making the adeno-associated virus compositions.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Application No. 62 / 574,163, filed October 18, 2017, and U.S. Provisional Application No. 62 / 621,102, filed January 24, 2018, each of which is hereby incorporated by reference in its entirety. [0003] sequence listing [0004] This application contains a Sequence Listing, which has been electronically filed in ASCII format, created on October 12, 2018, named 606107_HMT-023PC_Sequence_Listing_ST25.txt, and is 200,630 bytes in size. The Sequence Listing is hereby incorporated by reference in its entirety. [0005] Statement of Government Interest [0006] This invention was made with government support under Grant No. P30CA033572 awarded by the National Institutes of Health. The government has certain rights in this invention. Background technique [0007] Hemoglobinopathies include a family of genetic disorders in which the production, structure and / or function o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C12N7/04C12N15/864A61K35/76C12N15/90
CPCA61K48/005C07K14/805C12N15/86C12N15/907A01K2207/12A01K2227/105A01K2267/0381C12N15/62A61K35/761A61K48/0058A61K48/0016C07K14/075A61K35/12A61P7/06C12N2750/14143
Inventor S·查特瑞K·K·王M·本哈杰萨拉赫L·J·史密斯A·B·西摩J·B·莱特J·A·麦克斯维根S·N·多里维T·B·圣马丁J·普劳特
Owner CITY OF HOPE
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