Robust Genome Engineering in Primary Human T Cells using CRISPR/Cas9 Ribonucleoproteins

Invention Novelty

This invention enables highly effective experimental and therapeutic genomic engineering of primary human T cells and other hematopoietic cells with CRISPR/Cas9 ribonucleoprotein (RNP) technology.  

Value Proposition

CRISPR/Cas9-mediated genome editing provides an exceptional opportunity to engineer human T cells for research and therapeutic purposes, including cell-based therapies for cancer, viral infections and autoimmune diseases. However, therapeutic applications of CRISPR/Cas9 have been limited until now by inefficient DNA editing and inability to perform targeted DNA sequence replacement in human T cells. This invention augments the efficiency of CRISPR/Cas9-mediated genome editing in human T cells and raises the prospect of the therapeutic application of gene correction in T cells for treatment of myriad human diseases. Additional advantages of this invention include: 

• First CRISPR-mediated homology directed repair (HDR) in human immune cells could allow therapeutic editing of disease mutations in patient cells and introduction of specific sequences into TCR and CAR-transduced T cells.
• Strict temporal control over genome editing with Cas9 RNPs could increase the CRISPR safety profile for therapeutic applications.     

Technology Description

UCSF researchers have developed a powerful Cas9 RNP-based technology that uses purified Cas9 ribonucleoproteins (RNP) for successful and efficient genome editing in primary human CD4⁺ T cells. Cas9 protein pre-complexed with a single guide RNA (sgRNA) is introduced as an RNP into human T cells by transient electroporation. The active complexes enabled the first successful Cas9-mediated homology directed repair (HDR) in primary human T cells. Cas9 RNPs have allowed generation of ‘knock-in’ primary human T cells with targeted genetic replacement of specific nucleotides, which was previously unattainable.

Application

1) Unprecedented flexibility to ‘knock-out’ and ‘knock-in’ specific genetic elements in engineered T cells for cancer immunotherapy

2) New opportunity for therapeutic gene correction for primary immune deficiencies, treatment of infections and autoimmune diseases

3) Diverse research applications examining the function of coding and non-coding genetic variation in human immune regulation

Stage of Development

Proof of principle

Related Materials

• Not available at this time

Data Availability

In vitro human data

Patent Status

Additional Patents Pending