Prime Assembly enabled the integration of large DNA fragments and the generation of CAR T cells that target B-cell tumors
Prime Assembly enabled the integration of large DNA fragments and the generation of CAR T cells that target B-cell tumors
On August 28, Nature Biotechnology published a study of Prime Assembly, a form of prime editing that modifies DNA according to instructions encoded by guide RNAs. The authors tested the method in laboratory cell lines, primary human T cells, and mouse liver.
Replacing a large DNA segment in a cell requires delivery of a donor sequence and its precise joining to the selected genomic site. Conventional prime editing rewrites a short region using instructions encoded by RNA. With Prime Assembly, the authors extended this principle to kilobase-scale fragments containing thousands of base pairs.
Prime editing has already been used clinically to correct a two-nucleotide deletion in the NCF1 gene in a patient’s blood stem cells. In the Prime Assembly study, the authors tested whether the same class of tools could assemble a donor fragment thousands of base pairs long at a specified genomic site.
The method creates complementary 3′ overhangs on the genomic DNA and the donor DNA. These short single-stranded ends are designed to match each other. When the ends pair, the donor sequence is completed at the junction. Two pairs of these ends allow the selected region to be removed and replaced with the donor fragment in the same procedure.
The authors first tested assembly in the HEK293T laboratory cell line. The donors ranged from 1.0 to 6.5 kilobases in length. With a 2.9-kilobase donor, the replacement frequency reached 57.8%. Long-read DNA sequencing confirmed that most integrated fragments retained the intended sequence.
The same approach was then applied to primary human T cells. The researchers inserted a CAR, an engineered receptor that helps an immune cell recognize CD19, a protein found on B cells and many B-cell tumors. After seven days, the receptor was present on the surface of 28.1% of cells when two pairs of overhangs were used. The resulting CAR T cells killed CD19-positive tumor cells in culture. In immunocompromised mice, they suppressed tumor growth and prolonged survival compared with the control group.
In the final experiment, the authors injected a large volume of solution through the tail vein to deliver circular DNA molecules called plasmids to the mouse liver. Among the liver cells that received the plasmids and therefore fluoresced green, targeted genomic integration reached 4.3% with one Prime Assembly configuration.
In this study, DNA segment replacement and CAR T-cell generation relied on the same principle: joining donor DNA to a specified genomic site. The cell-line experiments tested the assembly process itself. The T-cell experiments tested the function of the resulting cellular product. The mouse liver experiment tested whether the joining process could occur within tissue in a living animal.