Science Spotlight

Gene-modified cells in the brain

From the Kiem Lab, Clinical Research Division

Hematopoietic stem and progenitor cells (HSPCs) develop into all types of blood cells, including lymphoid and myeloid white blood cells as well as red blood cells. Genetically modifying HSPCs allows researchers to change the behavior of the progeny of these stem cells. Therapeutically desirable changes include generating immune cells that can become resistant to high-dose chemotherapy through modification of a gene known as methylguanine methyltransferase (MGMT). MGMT repairs DNA damage caused by alkylating agents such as temozolomide or BCNU used in the treatment of brain tumors including glioblastoma.  Transplanting chemotherapy-resistant immune cells before treating glioblastoma multiforme demonstrated improved patient outcomes in previous research published by Dr. Hans-Peter Kiem (Clinical Research Division) and members of his lab1.  

A team of researchers led by Dr. Chris Peterson, a staff scientist in the Kiem lab, conducted a study revealing more possibilities for treatments involving MGMT-modified HSPCs. The results were published in the journal Stem Cell Reports. Along with the mutant form of MGMT which confers resistance to chemotherapy, the authors inserted green fluorescent protein (GFP) DNA into HSPCs, allowing them to track which cells are modified. They then infused these cells into a large animal preclinical model of stem cell engraftment. The authors found GFP+ leukocytes, those derived from gene-modified HSPCs, were enriched in peripheral blood after two rounds of chemotherapy. These results confirmed that the mutant MGMT gene conferred resistance to chemotherapy in the infused HSPCs. They tracked the cells over nearly ten years and found GFP+ leukocytes remained at higher levels than GFP- leukocytes in the peripheral blood. Next, the authors used CD45, a marker common among hematopoietic cells, to determine gene-modified cell numbers in tissues. They found GFP+CD45+ cells in lymphoid tissue, kidney, lung, liver, and brain. 

Confocal microscope image of cerebellum stained with antibodies GFP (green) and the microglia marker Iba1 (red); double-positive cells are in yellow, and DAPI staining for nuclei are gray.
Confocal microscope image of cerebellum stained with antibodies GFP (green) and the microglia marker Iba1 (red); double-positive cells are in yellow, and DAPI staining for nuclei are gray. Image from the article CC BY-NC-ND 4.0

Finding these cells in the brain such a long time after their infusion had the authors thinking of new possibilities. Dr. Peterson said, “This work demonstrates novel, outside-the-box applications for hematopoietic stem cell gene therapy. The center is founded on the use of hematopoietic stem cells to treat leukemias, but what we’re showing here is that the same strategy can also be used to deliver distinct gene-modified cells in the brain. These subsets could then be used to treat genetic diseases of the central nervous system, and maybe even brain cancers.”

The authors visualized these cells in the brain using confocal microscopy. They used fluorescent antibodies against GFP and Iba1 to locate modified brain-resident macrophages, including subsets known as microglia. They had two ideas of how these GFP+ cells made it into the brain. They could have stemmed from monocytes that recently crossed the blood-brain barrier (BBB) or they could be derived from HSPCs which migrated soon after infusion when the BBB was temporarily broken down by the transplant conditioning. The authors used integration site (IS) analysis to track down the answer. The gene-modified cells have the mutated MGMT DNA inserted into unique positions of their genome. Therefore, any cells with the same insertion site will have arisen from a common ancestral clone. The IS analysis showed one very dominant clone and other patterns of GFP+ cells common across all tissues except brain tissue. This suggests that the GFP+ cells in the brain are derived from HSPCs that entered shortly after transplantation. Dr. Peterson noted that learning the origin of these cells was a breakthrough. He said, “There is a great deal of evidence showing that microglia in the brain are derived from an embryonic source, rather than hematopoietic stem cells per se. What our data suggest is that both sources most likely play a role.”

Dr. Peterson said that future research will focus on these hematopoietic cells found in the brain. He said, “We measured them at relatively low levels in the brain, so the two options are to devise strategies to increase their numbers, or gene therapy-based approaches to maximize these cells’ contribution. For example, they could act as delivery vehicles that supply functional enzymes or anti-cancer antibodies to the central nervous system, but we need to engineer the cells so that they very efficiently secrete these molecules. This is something that Dr. Anne-Sophie Kuhlmann in the Kiem lab is working on2.”

This work was supported by the National Institutes of Health and Fred Hutch.

Fred Hutch/UW Cancer Consortium members Hans-Peter Kiem and Jennifer Adair contributed to this research.

Peterson CW, Adair JE, Wohlfahrt ME, Deleage C, Radtke S, Rust B, Norman KK, Norgaard ZK, Schefter LE, Sghia-Hughes GM, Repetto A, Baldessari A, Murnane RD, Estes JD, Kiem HP. 2019. Autologous, Gene-Modified Hematopoietic Stem and Progenitor Cells Repopulate the Central Nervous System with Distinct Clonal Variants. Stem Cell Reports. 2019 Jul 9;13(1):91-104. doi: 10.1016/j.stemcr.2019.05.016. Epub 2019 Jun 13. 

Additional citations:

1.    Adair JE, Beard BC, Trobridge GD, Neff T, Rockhill JK, Silbergeld DL, Mrugala MM, Kiem HP. 2012. Extended survival of glioblastoma patients after chemoprotective HSC gene therapy. Sci Transl Med. 2012 May 9;4(133):133ra57. doi: 10.1126/scitranslmed.3003425. 

2.    Kuhlmann AS, Haworth KG, Barber-Axthelm IM, Ironside C, Giese MA, Peterson CW, Kiem HP. 2019. Long-Term Persistence of Anti-HIV Broadly Neutralizing Antibody-Secreting Hematopoietic Cells in Humanized Mice. Mol Ther. 2019 Jan 2;27(1):164-177. doi: 10.1016/j.ymthe.2018.09.017.