Assay for safety assessment of therapeutic genetic manipulations, gene therapy vectors and compounds
View Patent ↗The invention is directed to methods of assessing the safety of therapeutic compounds and therapeutic genetic manipulations, including integrating gene therapy vectors and genome editing. In particular, the invention provides a method, wherein the oncogenic potential of therapeutic compounds and therapeutic genetic manipulations, including integrating gene therapy vectors and genome editing, is determined by determining the percentage of differentiation blocked hematopoietic progenitor cells.
1. An in vitro method for assessing oncogenic potential of a recombinant viral construct that integrates into a patient's genome, said method comprising:
a) transducing CD4-CD8-CD25-CD44+ (DN1) and/or CD4-CD8-CD25+CD44 30 (DN2) early thymic progenitor (ETP) cells with the recombinant viral construct;
b) culturing the cells transduced in step (a) under conditions that allow their development into mature CD4+-CD8+, CD4-CD8+or CD4+CD8- hematopoietic cells for about 10-40 days;
c) identifying cells blocked at an early differentiation stage, wherein the early differentiation stage is selected from the group consisting of (DN1), (D2), CD4-CD8-CD44- CD25+(DN3), and CD4-CD8-CD44-CD25- (DN4):
d) performing gene expression analysis and/or insertion site analysis of the blocked cells in step c), wherein the presence of recurrent insertion sites is used as evidence that the recombinant viral construct is not safe for therapeutic use, and
e) determining the oncogenic potential of the recombinant viral construct based on the gene expression analysis and/or insertion site analysis in step (d) and/or based on the percentage of the blocked cells in step (c) relative to the total number of cells in culture.
2. The method of claim 1 , wherein the determination of the oncogenic potential in step (e) involves comparison to a predetermined standard or to cultured ETP cells which have not been transduced with the recombinant viral construct.
3. The method of claim 1 , wherein the construct is a retroviral or lentiviral gene therapy vector.
4. The method of claim 1 , wherein the patient is human.
5. The method of claim 1 , wherein the insertion site analysis in step (d) is performed using a method selected from the group consisting of sequencing, quantitative shearing linear amplification PCR (qsLAM PCR), LAM-PCR, inverse PCR, and transposase-based methods.
6. The method of claim 1 , wherein the identification of the blocked cells in step (c) is performed using flow cytometry or fluorescence activated cell sorting (FACS).
7. The method of claim 1 , wherein step (d) comprises isolating DNA or RNA from the blocked cells and performing its analysis.
8. The method of claim 1 , further comprising harvesting and/or isolating the ETP cells prior to step (a).
9. The method of claim 1 , wherein the cells in step (b) are cultured on a cell line that is adherent and promotes growth and differentiation of said cells.
10. The method of claim 1 , wherein the cells in step (b) are cultured in the presence of Notch signal.
11. The method of claim 1 , wherein the cells in step (b) are cultured in the presence of Flt3 ligand and IL-7.
12. The method of claim 1 , wherein the ETP cells are murine ETP cells.
13. The method of claim 1 , wherein the early differentiation stage in step (c) is DN2.