Haematopoietic stem cells (HSC) are important gene therapy targets, e.g. in delivering treatments for immunodeficiency disorders such as adenosine deaminase (ADA) severe combined immunodeficiency (SCID) and X-linked SCID (Gaspar et al., 2011; Gaspar et al., 2004; Hacein-Bey-Abina et al., 2014). Currently patients have bone marrow removed, HSC are transduced ex vivo with retroviral vectors to deliver the corrective gene therapy, and the cells are then enriched in the laboratory and transplanted back into patients who have had myeloablation to wipe out their own bone marrow HSC. The gene therapy-treated HSC then re-populate the bone marrow, allowing correction of the immunodeficiency.
There have been fantastic successes in the clinic with these gene therapy applications. There are risks for the patients during these procedures, including susceptibility of the patient to infection following the myeloablation procedure and theoretical culturing effects on the stem cells when enriched in the laboratory. Thus, the ability to transduce the stem cells directly in situ could offer advantages in safety and efficacy if it could be achieved with high enough efficiency.
Here, Richter et al., 2016 present a method for in vivo transduction of HSC via the bloodstream. Mice were injected with a growth factor called granulocyte-colony-stimulating-factor which mobilised the HSC to leave the bone marrow and enter the bloodstream. Subsequently, the mice were injected via tail vein with a modified viral gene transfer vector and the HSC were directly transduced in the bloodstream.
The authors utilised a viral vector based on adenovirus, unlike the previous clinical trials which used retroviral gene transfer. Adenoviral vectors, particularly those based on serotype 5 (Ad5), have been widely used in gene therapy applications for a variety of diseases and when Ad5 is injected into the bloodstream it normally delivers genes to the liver. However, here the authors used an alternative adenovirus based on serotype 35 (Ad35) which binds a receptor called CD46 widely expressed on HSC. Hence, Ad35 efficiently transduces HSC.
Adenoviral vectors do not integrate their genome (unlike retroviral vectors) and therefore only provide short term gene transfer, typically in the region of 3-4 weeks. Therefore, the authors also used a modified Ad35, termed helper-dependent Ad35. Helper dependent adenoviral vectors do integrate into the genome and therefore have the potential to provide sustained gene transfer.
Using the new genetically engineered helper-dependent Ad35 the authors demonstrated that mobilized HSC were successfully transduced directly in the bloodstream and that gene transfer (using the marker gene green fluorescent protein) was still measurable 20 weeks later. Gene transfer into other non-target tissues including liver was found to be not significant.
The transduced HSC homed back to the bone marrow and were able to form multi-lineage progenitor colonies demonstrating their ability to differentiate into different mature cell types, highlighting the potential for this technology.
Important future work is still required to develop this technology further, including assessment of its efficacy in modulating a disease phenotype, longevity of gene transfer to HSC and scale up into larger animal models before clinical application.
Furthermore, the risks associated with ex vivo modification of bone marrow-derived HSC with retroviral vectors will need to be balanced with those associated with in vivo intravascular administration of adenoviral vectors.
A key part of future work will be how this new technology compares to the exemplar clinical work already performed in ex vivo stem cell gene therapy for trials in inherited SCID. Overall, however, this interesting and novel study does demonstrate proof of concept for the potential to develop alternative gene delivery strategies able to modify HSC via direct n vivo gene delivery.
Gaspar, H. B., Cooray, S., Gilmour, K. C., Parsley, K. L., Zhang, F., Adams, S., . . . Thrasher, A. J. (2011). Hematopoietic stem cell gene therapy for adenosine deaminase-deficient severe combined immunodeficiency leads to long-term immunological recovery and metabolic correction. Sci Transl Med, 3(97), 97ra80. doi:10.1126/scitranslmed.3002716
Gaspar, H. B., Parsley, K. L., Howe, S., King, D., Gilmour, K. C., Sinclair, J., . . . Thrasher, A. J. (2004). Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector. Lancet, 364(9452), 2181-2187. doi:10.1016/S0140-6736(04)17590-9
Hacein-Bey-Abina, S., Pai, S. Y., Gaspar, H. B., Armant, M., Berry, C. C., Blanche, S., . . . Thrasher, A. J. (2014). A modified gamma-retrovirus vector for X-linked severe combined immunodeficiency. N Engl J Med, 371(15), 1407-1417. doi:10.1056/NEJMoa1404588
Richter, M., Saydaminova, K., Yumul, R., Krishnan, R., Liu, J., Nagy, E. E., . . . Lieber, A. (2016). In vivo transduction of primitive mobilized hematopoietic stem cells after intravenous injection of integrating adenovirus vectors. Blood, 128(18), 2206-2217. doi:10.1182/blood-2016-04-711580