Nuclease-mediated genome editing of primary cells and related kits

Inventors

DEVER, Daniel P. • Bak, Rasmus O. • Hendel, Ayal • Srifa, Waracharee • PORTEUS, Matthew H.

Assignees

Leland Stanford Junior University

Abstract

In certain aspects, the present invention provides methods for inducing a stable gene modification of a target nucleic acid via homologous recombination in a primary cell, such as a primary blood cell and/or a primary mesenchymal cell. In certain other aspects, the present invention provides methods for enriching a population of genetically modified primary cells having targeted integration at a target nucleic acid. The methods of the present invention rely on the introduction of a DNA nuclease such as a Cas polypeptide and a homologous donor adeno-associated viral (AAV) vector into the primary cell to mediate targeted integration of the target nucleic acid. Also provided herein are methods for preventing or treating a disease in a subject in need thereof by administering to the subject any of the genetically modified primary cells or pharmaceutical compositions described herein to prevent the disease or ameliorate one or more symptoms of the disease.

Core Innovation

The invention relates to stably integrating a polynucleotide sequence into the genome of one or more primary cells via homologous recombination. The method introduces a modified single guide RNA (sgRNA) together with a Cas polypeptide formed in a ribonucleoprotein (RNP) complex prior to introducing into the primary cells, and introduces a recombinant adeno associated viral vector serotype 6 (rAAV6) comprising the polynucleotide sequence. The modified sgRNA guides the Cas polypeptide to a target nucleic acid sequence, where Cas cleavage generates a double-strand break to enable homologous recombination.

A key feature is that the first nucleotide sequence that is complementary to the target nucleic acid sequence, the second nucleotide sequence that interacts with the CRISPR-associated Cas polypeptide, or both comprise a 2′-O-methyl 3′-phosphorothioate nucleotide. Stable integration is defined as the polynucleotide sequence being stably integrated into the target nucleic acid sequence in the genome of greater than 10% of primary cells in the population of primary cells.

The core approach is further directed to disease-relevant genome targeting, including stably integrating a polynucleotide sequence into the HBB gene of one or more primary hematopoietic stem or progenitor cells (HSPCs) isolated from a subject having sickle cell disease. In addition, a corresponding kit is provided, where one or more primary cells in the population comprises the modified sgRNA, a Cas polypeptide formed in an RNP complex, and an rAAV6 donor template having two non-overlapping homologous portions of the target nucleic acid positioned at the 5′ and 3′ ends to enable homologous recombination, with stable integration into the target nucleic acid sequence in greater than 10% of primary cells.

Claims Coverage

The document provides three independent claims covering a method for stably integrating a polynucleotide sequence into the genome of primary cells via homologous recombination, a method specifically targeting stable integration into the HBB gene of primary HSPCs from a subject having sickle cell disease, and a kit comprising primary cells configured for the same overall genome editing components and requirements. Across the independent claims, the inventive features are driven by the modified sgRNA chemistry, Cas delivered as an RNP complex, and an rAAV6 donor template enabling homologous recombination with stable integration in >10% of primary cells.

Overall, the independent claims define genome editing in primary cells and HBB-specific targeting by homologous recombination, using a modified sgRNA containing 2′-O-methyl 3′-phosphorothioate nucleotides, a Cas polypeptide delivered as an RNP complex with the modified sgRNA, and an rAAV6 donor template configured for homologous recombination, with a requirement that stable integration occurs in greater than 10% of the relevant primary cells.

Stated Advantages

Documented Applications