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

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.

Battling Neurodegenerative Diseases with Adeno-Associated Virus-Based Approaches

“…Viral gene therapy vectors specifically designed for NDD applications should be able to efficiently cross the blood-brain barrier (BBB), if delivered by iv infusion [44,49], and support an efficient transgene expression, regardless of aberrant cell signaling caused by the accumulation of toxic proteins or restoration of an autophagic state in neuronal cells [50]. For instance, vectors based on genomes of adeno-associated virus (AAV) [44,49,[51][52][53][54][55][56], lentivirus [57], type 1 herpes simplex virus [58], and Semliki Forest virus (SFV) [59] have been broadly used for neuron targeting. Extensive use of the above viruses in neuroscience is dictated by their natural ability to efficiently and selectively transduce and provide high transgene expression in neurons.…”

Adenovirus vectors in hematopoietic stem cell genome editing

“…In our approach, the HSCs residing in the bone marrow niche were mobilized into the peripheral circulation through administration of granulocyte colonystimulating factor and the CXCR4 antagonist, Plerixafor/AMD3100. After in vivo transduction and selection with three low doses of O 6 -BG/ BCNU administered intraperitoneally with interval of 2 weeks, transgene marking in PBMCs was increased to > 50% [176]. HSPCs transduced in peripheral blood homed back to bone marrow shortly after transduction.…”

“…This was likely in part due to a lack of a proliferative stimulus for the modified HSCs. To overcome this drawback, we therefore combined the in vivo transduction with an in vivo selection mechanism by incorporating a mutant O 6methylguanine-DNA methyltransferase (mgmt P140K ) gene that confers resistance to O 6 -BG/BCNU (O 6 -Benzylguanine/Carmustine) [176][177][178]. After in vivo transduction and selection with three low doses of O 6 -BG/ BCNU administered intraperitoneally with interval of 2 weeks, transgene marking in PBMCs was increased to > 50% [176].…”

“…To overcome this drawback, we therefore combined the in vivo transduction with an in vivo selection mechanism by incorporating a mutant O 6methylguanine-DNA methyltransferase (mgmt P140K ) gene that confers resistance to O 6 -BG/BCNU (O 6 -Benzylguanine/Carmustine) [176][177][178]. After in vivo transduction and selection with three low doses of O 6 -BG/ BCNU administered intraperitoneally with interval of 2 weeks, transgene marking in PBMCs was increased to > 50% [176]. Importantly, the approach has been tested by delivering several therapeutic genes, for instance, c-globin, and with optimized procedures we routinely obtained more than 80% gene marking in HSCs and peripheral blood cells [53, 103,179].…”

Non-viral therapeutic cell engineering with the Sleeping Beauty transposon system

“…A potential disadvantage is that the efficiency of gene manipulation was not as high as that typically seen in clinical trials using lentiviral vectors and ex vivo cell processing. Importantly, in a recent follow-up work it has been shown that a low-dose drug selection regimen provides gene-modified HSPCs that carry a O 6methylguanine-DNA methyltransferase (mgmt P140K ) transgene a selective survival and proliferation advantage, resulting in transgene expression in up to 80% of peripheral blood cells [82]. Taken together, we consider that Table 1 Estimated costs of GMP-grade viral versus non-viral gene transfer vectors per CAR-T cell product.…”