A Combined In Vivo HSC Transduction/Selection Approach Results in Efficient and Stable Gene Expression in Peripheral Blood Cells in Mice

André Lieber,Anja Ehrhardt,Jiho Kim,Benjamin Cao,Nikoletta Psatha,Donna Palmer,Hongjie Wang,Philip Ng,Hans-Peter Kiem,Susan K Nilsson,Chang Li,Maximilian Richter,Zsuzsanna Izsvák,Thalia Papayannopoulou,Jing Liu,Kevin G Haworth

doi:10.1016/j.omtm.2017.11.004

Abstract

We recently reported on an in vivo hematopoietic stem cell (HSC) gene therapy approach. It involves the subcutaneous injections of G-CSF/AMD3100 to mobilize HSCs from the bone marrow into the peripheral blood stream and the intravenous injection of an integrating helper-dependent adenovirus vector system. HSCs transduced in the periphery homed back to the bone marrow, where they persisted long-term. However, high transgene marking rates found in primitive bone marrow HSCs were not reflected in peripheral blood cells. Here, we tested small-molecule drugs to achieve selective mobilization and transduction of HSCs. We found more efficient GFP marking in bone marrow HSCs but no increased marking in the peripheral blood cells. We then used an in vivo HSC chemo-selection based on a mutant of the O6-methylguanine-DNA methyltransferase (mgmtP140K) gene that confers resistance to O6-BG/BCNU and should give stably transduced HSCs a proliferation stimulus and allow for the selective survival and expansion of progeny cells. Short-term exposure of G-CSF/AMD3100-mobilized, in vivo-transduced mice to relatively low selection drug doses resulted in stable GFP expression in up to 80% of peripheral blood cells. Overall, the further improvement of our in vivo HSC transduction approach creates the basis for a simpler HSC gene therapy.

Highlights

Current hematopoietic stem cell (HSC) gene therapy protocols are based on the transplantation of ex-vivo-transduced HSCs into myelo-conditioned recipients
We demonstrated in transgenic mice expressing human CD46 in a pattern similar to humans[2] and in immunodeficient mice with engrafted human CD34+ cells that HSCs transduced with HDAd5/35++ in the periphery home back to the bone marrow, where they persist and stably express the transgene long-term.[1]
In contrast to marking rates in bone marrow, GFP expression in peripheral blood mononuclear cells (PBMCs) at 30 weeks was less than 1% in all animals (Figure 1E)

Summary

Introduction

Current hematopoietic stem cell (HSC) gene therapy protocols are based on the transplantation of ex-vivo-transduced HSCs into myelo-conditioned recipients. To simplify HSC gene therapy, we recently developed an approach for in vivo HSC transduction It involves subcutaneous injections of granulocyte colony-stimulating factor (G-CSF)/AMD3100 to mobilize HSCs from the bone marrow into the peripheral blood stream and the intravenous injection of an integrating helper-dependent adenovirus (HDAd5/35++) vector system.[1] These vectors target CD46, a receptor that is expressed at higher levels in HSCs than in more differentiated bone marrow and blood cells. The percentage of GFP-expressing peripheral blood mononuclear cells (PBMCs) was on average less than 1% at 20 weeks post-transduction This is a shortcoming of our approach because for most genetic blood disorders to be cured, the transgene product must be expressed in differentiated peripheral blood cells

Objectives

Methods

Results