Taking the good out of viruses to cure immunodeficiencies by in vivo gene therapy

from the Kiem lab, Clinical Research Division

SCID-X1 (X-linked severe combined immunodeficiency disease) is an inherited genetic disorder affecting the interleukin receptor (IL2RG) that is manifest by an absence of functional T and B lymphocytes as well as NK cells. Unless treated by a stem cell transplant, such immunodeficiency can be fatal within the first two years of life because of opportunistic infections. Bone marrow transplant using cells from a matched donor is a standard curative therapy whose efficiency can be limited by the risk of graft-versus-host-disease. SCID-X1 is a monogenic disorder, which can also be treated by gene therapy. To this end, the hematopoietic stem and progenitor cells (HSPCs) from the patient are isolated and modified ex vivo to express the functional IL2RG gene. However, ex vivo manipulation of the cells is a labor-intensive and costly procedure.

In a recent study by Dr. Olivier Humbert, staff scientist in the group of Dr. Hans-Peter Kiem (Clinical Research Division, Stem Cell and Gene Therapy Program) and collaborators Drs. Rawlings, Scharenberg and Torgerson from Seattle Children’s hospital, direct intravenous injection of a therapeutic retrovirus called foamy virus allowed for efficient immune reconstitution in SCID-X1 subject without the need to modify cells ex vivo. Dr. Humbert explained, “While ex vivo stem cell gene therapy clinical trials for SCID-X1 are showing promising results, this approach is expensive, requires extensive manipulation of bone marrow cells and is not accessible worldwide. With this study we are offering a simpler, more accessible and affordable treatment option for SCID-X1 by using direct intravenous injection of the therapeutic vector.” The results of this study were recently published in the journal Blood Advances.

The study was performed in a canine model of SCID-X1, the closest SCID model to humans available to date to assess therapies. Foamy virus vectors are especially advantageous for such an application as they are non-pathogenic, resistant to serum inactivation in vivo and have safer integration profile than commonly used lentiviral vectors. Two SCID-X1 animals were injected competitively with two foamy virus vectors controlled by either the PGK (PhosphoGlycerate Kinase) or the EF1a  (Elongation Factor 1 alpha) promoter that could be distinguished with different fluorochromes. The PGK promoter proved significantly more efficient than the EF1a promoter, as 70% to 90% of the circulating lymphocytes were gene marked 2.5 years post-treatment as compared to 5 to 10% when using the EF1a promoter. Thus, the PGK promoter was selected for further studies.

More than one year following treatment, animals injected with foamy vectors containing a PGK promoter showed stable T cell reconstitution in the circulating blood. Prior mobilization of the hematopoietic stem and progenitor cells from the bone marrow to the blood by G-CSF and AMD3100 increased the kinetic of reconstitution.
More than one year following treatment, animals injected with foamy vectors containing a PGK promoter showed stable T cell reconstitution in the circulating blood. Prior mobilization of the hematopoietic stem and progenitor cells from the bone marrow to the blood by G-CSF and AMD3100 increased the kinetic of reconstitution. Figure provided by Dr. Olivier Humbert.

In a second set of experiments, the kinetics of reconstitution by gene-marked cells was also improved by administration of granulocyte-colony stimulating factor (G-CSF) and AMD3100, an immunostimulant chemokine receptor antagonist, prior to virus injection. These drugs decrease HSPC retention in the bone marrow and allow their mobilization to the peripheral blood, increasing their chance to be targeted by the intravenously administered foamy virus. Six hours post-administration, the percentage of circulating HSPCs increased by six- to seven-fold and the foamy virus was injected. Six weeks following the treatment, 80% of lymphocytes were gene-marked compared to 40% gene-marking in the absence of G-CSF/AMD3100 treatment at the same time points.

Mobilization of HSPCs also allowed stable persistence of modified naïve T lymphocytes in the peripheral blood up to 15 months post-treatment, while the non-mobilized animals presented a decrease in gene-marked naïve T lymphocytes. T cell recovery was assessed using TREC assay, or T-Cell Receptor Excision Circles, which gives a measure of the byproducts produced by TCR rearrangement in T cells. The mobilized animals presented a normal T cell recovery by opposition to non-mobilized animals 2.5 years post-treatment. Additionally, in both mobilized and non-mobilized dogs, circulating mature CD4 and CD8 T cells were observed and the modified cells also expressed a wide range of rearranged TCRab. Isolated T cells responded to interleukin-21 stimulation by STAT3 phosphorylation and to phytohemaglutinin by increased cell proliferation, demonstrating functionality of the modified T cells. Following immunization by bacteriophage j-X174, both mobilized and non-mobilized animals elicited immunoglobulins G, M and A antibodies, demonstrating B cells functionality. Retroviral integration site analyses showed no dominant clone that could be indicative of a transformation event and no integration in germinal cells , demonstrating the safety of the strategy.

The results of this study are promising for patients suffering from immunodeficiencies that can be corrected by gene therapy and bring therapeutic possibilities one step closer to the clinical side. “Our current study shows very good reconstitution of T-lymphocytes in the SCID-X1 subjects but other cell lineages such as B-lymphocytes or myeloid cells are not efficiently corrected even though they are important players for a fully functional immune system. We are thus looking to further engineer our viral vector to increase efficiency and specificity of targeting of the most primitive, multipotent stem cells. In addition, we would also like to explore the idea of directly delivering the vector to the bone marrow where the majority of these stem cells reside”, concluded Dr. Humbert.


Funding for this study was provided by the National Institutes of Health, National Institute of Allergy and Infectious Diseases grant and National Heart, Lung, and Blood Institute.

Fred Hutch/UW Cancer Consortium faculty members Drs. Hans-Peter Kiem, Jennifer Adair, David Rawlings and Andrew Scharenberg contributed to this research


Humbert O,Chan F,Rajawat YS,Torgerson TR,Burtner CR,Hubbard NW,Humphrys D,Norgaard ZK,O'Donnell P,Adair JE,Trobridge GD,Scharenberg AM,Felsburg PJ,Rawlings DJ,Kiem H-P. 2018. Rapid immune reconstitution of SCID-X1 canines after G-CSF/AMD3100 mobilization and in vivo gene therapy. Blood Advances. 2(9), 987-999.