High-level protein production in erythroid cells derived from in vivo transduced hematopoietic stem cells

Blood Adv. 2019 Oct 8;3(19):2883-2894. doi: 10.1182/bloodadvances.2019000706.


We developed an in vivo hematopoietic stem cell (HSC) transduction approach that involves HSC mobilization from the bone marrow into the peripheral bloodstream and the IV injection of an integrating, helper-dependent adenovirus (HDAd5/35++) vector system. HDAd5/35++ vectors target human CD46, a receptor that is abundantly expressed on primitive HSCs. Transgene integration is achieved by a hyperactive Sleeping Beauty transposase (SB100x) and transgene marking in peripheral blood cells can be increased by in vivo selection. Here we directed transgene expression to HSC-derived erythroid cells using β-globin regulatory elements. We hypothesized that the abundance and systemic distribution of erythroid cells can be harnessed for high-level production of therapeutic proteins. We first demonstrated that our approach allowed for sustained, erythroid-lineage specific GFP expression and accumulation of GFP protein in erythrocytes. Furthermore, after in vivo HSC transduction/selection in hCD46-transgenic mice, we demonstrated stable supraphysiological plasma concentrations of a bioengineered human factor VIII, termed ET3. High-level ET3 production in erythroid cells did not affect erythropoiesis. A phenotypic correction of bleeding was observed after in vivo HSC transduction of hCD46+/+/F8-/- hemophilia A mice despite high plasma anti-ET3 antibody titers. This suggests that ET3 levels were high enough to provide sufficient noninhibited ET3 systemically and/or locally (in blood clots) to control bleeding. In addition to its relevance for hemophilia A gene therapy, our approach has implications for the therapy of other inherited or acquired diseases that require high levels of therapeutic proteins in the blood circulation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Erythroid Cells / metabolism*
  • Hematopoietic Stem Cells / metabolism*
  • Humans
  • Mice