In vivo transduction of primitive mobilized hematopoietic stem cells after intravenous injection of integrating adenovirus vectors

Abstract

• Mobilized hematopoietic stem cells transduced with IV injected HD-Ad5/35 11 vectors home to the BM persist long term.• Our approach allows for the stable genetic modification of primitive, long-term persisting HSPCs.Current protocols for hematopoietic stem/progenitor cell (HSPC) gene therapy, involving the transplantation of ex vivo genetically modified HSPCs are complex and not without risk for the patient. We developed a new approach for in vivo HSPC transduction that does not require myeloablation and transplantation. It involves subcutaneous injections of granulocyte-colony-stimulating factor/AMD3100 to mobilize HSPCs from the bone marrow (BM) into the peripheral blood stream and the IV injection of an integrating, helper-dependent adenovirus (HD-Ad5/35 11 ) vector system. These vectors target CD46, a receptor that is uniformly expressed on HSPCs. We demonstrated in human CD46 transgenic mice and immunodeficient mice with engrafted human CD34 1 cells that HSPCs transduced in the periphery home back to the BM where they stably express the transgene. In hCD46 transgenic mice, we showed that our in vivo HSPC transduction approach allows for the stable transduction of primitive HSPCs. Twenty weeks after in vivo transduction, green fluorescent protein (GFP) marking in BM HSPCs (Lin 2 Sca1 1 Kit 2 cells) in most of the mice was in the range of 5% to 10%. The percentage of GFP-expressing primitive HSPCs capable of forming multilineage progenitor colonies (colony-forming units [CFUs]) increased from 4% of all CFUs at week 4 to 16% at week 12, indicating transduction and expansion of long-term surviving HSPCs. Our approach was well tolerated, did not result in significant transduction of nonhematopoietic tissues, and was not associated with genotoxicty. The ability to stably genetically modify HSPCs without the need of myeloablative conditioning is relevant for a broader clinical application of gene therapy. (Blood. 2016;128(18):2206-2217

Rapid immune reconstitution of SCID-X1 canines after G-CSF/AMD3100 mobilization and in vivo gene therapy

“…Similarly, G-CSF/ AMD3100 mobilization of immunodeficient humanized mice increased colony forming units isolated from peripheral blood by 2.3-and 8.2-fold, respectively. 42 Our mobilization of SCID-X1 pups with G-CSF/AMD3100 showed a comparable sevenfold increase in circulating CD34 1 cells at 6 hours posttreatment.…”

“…39,40,42 Both G-CSF and AMD3100 have mobilizing properties by acting on distinct cellular pathways, and combinatory treatment has resulted in additive effects. 51 G-CSF suppresses osteoblast lineage cells in the bone marrow niche, leading to reduced levels of signaling molecules (eg, CXCL12, VLA-4, c-Kit), which are essential for HSPC retention.…”

“…Previous studies showed that a combination of G-CSF and AMD3100 efficiently mobilizes multipotent progenitor cells in peripheral blood in animal models and in patients. [39][40][41][42] On the basis of these findings, we treated 2 SCID-X1 canine pups at ;3 weeks of age with 5 mg/kg of G-CSF twice per day from day 24 to 21 before FV injection and a single dose of G-CSF with 4 mg/kg of AMD3100 on the morning of the injection (animals H864 and H867; Table 1; Figure 2A). Treatment was well tolerated and resulted in a 6.4-to 7.2-fold increase in circulating CD34 1 cells at 6 hours post-AMD3100 administration (2.30% and 2.59% of total white blood cells in the 2 mobilized SCID-X1 animals, respectively, as compared with 0.36% in a nonmobilized littermate normal control, consistent with values for steady state hematopoiesis 9 ; Figure 2B).…”

Sleeping Beauty transposon vectors for therapeutic applications: advances and challenges

“…This gene transduction system is based on a hybrid transposon/ adenovirus vector and the hyperactive SB transposase (SB100X) [74]. This in vivo strategy is effective and safe in hematopoietic stem cells (HSCs), and performs without the requirement of ex vivo expansion and transduction [75,96]. This system may overcome some of the current difficulties associated with cell collection and manufacturing, and provide technical advances for gene therapy.…”

“…Currently, one of the most promising strategies is the in vivo gene transduction system based on a hybrid transposon/adenovirus vector [73] and hyperactive SB transposase (SB100X) [74]. This in vivo strategy is effective and safe, and performs without the requirement of ex vivo expansion and transduction of hematopoietic stem cells (HSCs) [75].…”