“We performed a comprehensive single-cell multi-omic analysis of this gene therapy to understand whether a functional copy of the corrected gene was in patient cells, to what extent the gene was expressed, and the chromatin organization at a single-cell level,” said first author Koon-Kiu Yan, Ph.D., St. Jude Department of Computational Biology. “Before this study, we could measure the general gene expression of a bulk group of cells. But with bone marrow samples from two patients, we saw at a single-cell level which genes were expressed in which cell types.”

Using that single-cell analysis, along with their other work, Yan showed that the safety and efficacy of treatment are also related to integration into compartments near the nuclear pore. Previous gene therapy efforts integrated into promoters near to or directly into oncogenes, ultimately causing cancer. The lentiviral vector used by St. Jude selectively avoids promoters without disrupting oncogenes, improving treatment safety. The same pattern was observed with a lentiviral gene therapy used to create chimeric antigen receptor (CAR) T cells, suggesting that the phenomenon may be a general mechanism not restricted to the SCID-X1 vector. 

“The integration pattern data could serve as a map of potentially safe integration sites,” Yu and Gottschalk explained. “The single-cell analysis is like deep cartography, a map with a near pixel-perfect resolution. The large number of integration sites could be used as a safety reference for future lentiviral gene therapies.” 

One case was especially notable: the patient who required a second dose of the gene therapy to achieve a cure was found to have a different integration pattern than the patients who had responded to the first dose. After receiving the second dose, the patient’s integration pattern resembled the others’, and the therapy was effective. This study provides a foundation for understanding differences in treatment response so that future gene therapies may be improved.

Authors and funding

The study’s other authors are Jose Condori, Zhijun Ma, Jean-Yves Metais, Bensheng Ju, Liang Ding, Yogesh Dhungana, Lance Palmer, Deanna Langfitt, Francesca Ferrara, Robert Throm, Hao Shi, Isabel Risch, Sheetal Bhatara, Bridget Shaner, Timothy Lockey, Aimee Talleur, John Easton, Michael Meagher, Sheng Zhou and Ewelina Mamcarz, all of St. Jude; and Jennifer Puck and Morton Cowan, University of California San Francisco Benioff Children’s Hospital.

The study was supported by grants from the National Institutes of Health (R01-GM13482, P01-HL053749 and U54-AI082973), National Institute of Allergy and Infectious Diseases (U54-AI082973), California Institute of Regenerative Medicine (CLIN2-09504 and CLIN2-10830), National Cancer Institute Comprehensive Cancer Center (P30CA021765), Assisi Foundation of Memphis and ALSAC, the fundraising and awareness organization of St. Jude.