The future of blood stem cell gene therapy is in vivo

From the Lieber and Kiem Labs, Hematologic Malignancies Program, Cancer Consortium.

Nestled in the pillowy, marrow-filled interior of your bones is a special group of cells called hematopoietic stem cells (HSCs) that are responsible for generating and re-generating your red and white blood cells throughout life. Unfortunately, in hereditary diseases, HSCs that carry mutations just like any other cell, pass them along to their progeny. For instance, hemoglobinopathies, mutations that affect the hemoglobin protein that is responsible for binding oxygen in red blood cells, arise from germline mutations in HSCs and include diseases such as thalassemia and sickle cell anemia. For decades, therapies for these diseases have been supportive, not curative, including either red blood cell transfusion from an unaffected donor or HSC transplant. Transfusions have limited utility as red blood cells only live for 100-120 days.  However, researchers have pivoted recently towards gene therapy approaches that reprogram a portion of a patient’s own HSCs. This therapy relies on retrieving HSCs from the bone marrow, introducing a corrected copy of the affected gene, then dosing the patient with high dose chemotherapy to make room in their bone marrow for engraftment of the reprogrammed cells. It’s a risky, high-cost, and technically complex process – the perfect target for technological innovation. A collaborative study published this month in Molecular Therapy: Methods & Clinical Development by the Lieber and Kiem lab groups describes a technique that would allow clinicians to reprogram HSCs inside patients’ bodies without the need for high-dose chemotherapy and HSC transplantation.

“We have developed an in vivo HSC transduction approach that requires only intravenous injections and could be provided as an outpatient treatment,” said Dr. Andre Lieber, MD, PhD, a professor of Medical Genetics at the University of Washington, and one of the two lab leaders driving the study. The secret to this approach? Helper-dependent adenovirus vectors (HDAds). These man-made gene-delivery tools replicate the capsid, or shell, of a virus and pack it with a payload of therapeutic genes. “HSCs are mobilized from the bone marrow into the peripheral blood stream and transduced with [HDAds] that target receptors present on primitive HSCs,” Dr. Lieber explained. “HSCs transduced in the periphery return to the bone marrow, persist there long-term, and contribute to all blood cell lineages.” Previously, the Lieber and Kiem groups had reported that this method worked well in mice. However, for this publication they wanted to test the method in a more relevant model. “The physiological similarities of the human and macaque hematopoietic systems make rhesus macaques (Macaca mulatta) a better model for assessing the safety and efficacy of our in vivo HSC gene therapy approach,” said Dr. Lieber.

A graphic shows how hematopoietic stem cells (HSCs) are mobilized from the bone marrow, treated with gene therapy, and re-homed to the bone marrow in order to treat hematologic malignancies such as thalassemia and sickle cell anemia.
In their recent study, Drs. Li and Wang show proof-of-principle for a new gene therapy technique to mobilize, transduce (“reprogram”), and re-home hematopoietic stem cells (HSC) – the cells that live in your bone marrow and give rise to blood cells. Efficiently reprogramming these cells is key to solving several human blood disorders, such as hemophilia and sickle cell anemia. Figure provided by Dr. Andre Lieber, MD, PhD

The study showed that HSCs were well-mobilized and transduced, and returned to their niche in the bone marrow  – all good news. “This together with the good safety profile suggests that in vivo HSC gene therapy could be feasible in humans upon further improvements,” said Dr. Lieber. Additionally, the study pointed out a couple factors limiting the efficacy. First, the cellular receptor that HDAd vectors originally bound to, CD46, is expressed on both HSCs and red blood cells in macaques – this meant that some of the gene-therapy delivery vehicles were sequestered by those red blood cells which “decrease[d] the vector dose that is available for transduction of mobilized HSCs,” explained Dr. Lieber. To solve this problem, they engineered the HDAds so that they would bind a receptor called desmoglein 2 (DSG2) with high-affinity instead and saw significantly better (~10-fold) HSC reprogramming in the macaque context. “[Second, we observed] the preferential return/survival of mobilized transduced HSCs to/in the spleen, a secondary, much smaller hematopoietic organ.” They tried to encourage the HSCs to return to the bone marrow by including the gene for a specific receptor, CXC motif chemokine receptor 4 (CXCR4), in the HDAd payload so that it would be expressed following reprogramming. Expressing this receptor increased the percentage of reprogrammed HSCs found in the bone marrow to 7%, a decent percentage when you consider that these HSCs will give rise to generations of red blood cells that express the corrected gene.

As members of the Cancer Consortium, the Lieber and Kiem labs have a history of working together on complex hematologic diseases. In fact, “the basis that allowed us to perform in vivo HSC transduction studies was the collaboration with the Kiem lab,” said Dr. Lieber. “This group has decades of experience in gene therapy studies in large animals.” Asked what’s next for the two labs, Dr. Lieber said: “Current collaborations with the Kiem group are focused on in vivo HSC gene therapy of HIV using secreted virus decoy receptors.” Luckily for the patients awaiting cures, Cancer Consortium collaborations such as this one are persistently pushing the front-edge of what can be achieved with gene therapy.

Li C, Wang H, Gil S, Germond A, Fountain C, Baldessari A, Kim J, Liu Z, Georgakopoulou A, Radtke S, Raskó T, Pande A, Chiang C, Chin E, Yannaki E, Izsvak Z, Papayannopoulou T, Kiem HP, and Lieber A. 2022. Safe and efficient in vivo hematopoietic stem cell transduction in nonhuman primates using HDAd5/35++ vectors. Molecular Therapy: Methods & Clinical Development. 24: 127-141. doi: 10.1016/j.omtm.2021.12.003.

This work was funded by the National Institutes of Health, Ensoma Bio, the Bill and Melinda Gates Foundation, and the Foundation of the Hellenic Society of Hematology.

Fred Hutch/UW Cancer Consortium members Dr. André Lieber (UW), Dr. Hans-Peter Kiem (Fred Hutch), and Dr. Thalia Papayannopoulou (UW) contributed to this work.