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.
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:
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.
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
Proof of principle
In vitro human data
|Hong Kong||Published Application||HK1248755||10/19/2018||2015-118|
|European Patent Office||Published Application||3 250 693||12/06/2017||2015-118|
|Rep Of Korea||Published Application||2015-118|
|New Zealand||Published Application||2015-118|
Additional Patent Pending
CRISPR/Cas9, Primary human T cells, Genome engineering, Cas9 ribonucleoprotein (RNP), Gene correction/replacement, Homology-directed repair (HDR)