Sorting Out the Best: Enriching Hematopoietic Stem Cells for Gene Therapy and Editing
Authors: Stefan Radtke, Olivier Humbert, Hans-Peter Kiem
Journal: Molecular Therapy (2018)

Hematopoietic stem cell (HSC) gene therapy has become a real treatment option for diseases such as the hemoglobinopathies, immunodeficiencies, and other genetic disorders that affect the hematopoietic system. The development and pre-clinical testing of these therapies has been greatly facilitated by increasing interest and financial support from biotech companies. While major progress has been accomplished in the field to improve the efficiency and specificity of gene-editing tools, less effort has been spent on defining a better cell target for HSC gene therapy. A significant bottleneck for the clinical translation of current gene therapy applications is the large amount of good manufacturing practices-grade viral vector or gene-editing reagents needed to modify CD34+ cells, the current clinical gold standard target cell population used by most investigators and companies. To tackle this issue, research groups have developed strategies to reduce the target cell population by focusing on long-term engrafting HSCs. This strategy would not only reduce reagent expenses, but it could also potentially increase safety by minimizing off-target events. Purification of an HSC-enriched subset from CD34+ hematopoietic stem and progenitor cells (HSPCs) has recently been reported for phenotypical subsets such as CD34+CD38low/− or CD34+CD90+CD45RA− cells.1Masiuk K.E. Brown D. Laborada J. Hollis R.P. Urbinati F. Kohn D.B. Improving Gene Therapy Efficiency through the Enrichment of Human Hematopoietic Stem Cells.Mol Ther. 2017; 25: 2163-2175Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 2Zonari E. Desantis G. Petrillo C. Boccalatte F.E. Lidonnici M.R. Kajaste-Rudnitski A. Aiuti A. Ferrari G. Naldini L. Gentner B. Efficient Ex Vivo Engineering and Expansion of Highly Purified Human Hematopoietic Stem and Progenitor Cell Populations for Gene Therapy.Stem Cell Reports. 2017; 8: 977-990Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 3Radtke S. Adair J.E. Giese M.A. Chan Y.Y. Norgaard Z.K. Enstrom M. Haworth K.G. Schefter L.E. Kiem H.P. A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates.Sci. Transl. Med. 2017; 9: 9Crossref Scopus (65) Google Scholar Masiuk et al.1Masiuk K.E. Brown D. Laborada J. Hollis R.P. Urbinati F. Kohn D.B. Improving Gene Therapy Efficiency through the Enrichment of Human Hematopoietic Stem Cells.Mol Ther. 2017; 25: 2163-2175Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar used a bead-based magnetic-assisted cell sorting strategy to reduce the number of target cells and reagents up to 7-fold without compromising the gene-editing efficiency of long-term engrafting HSCs. Enriching for the same phenotype but focusing on a fluorescence-activated cell sorting approach, Zonari et al.2Zonari E. Desantis G. Petrillo C. Boccalatte F.E. Lidonnici M.R. Kajaste-Rudnitski A. Aiuti A. Ferrari G. Naldini L. Gentner B. Efficient Ex Vivo Engineering and Expansion of Highly Purified Human Hematopoietic Stem and Progenitor Cell Populations for Gene Therapy.Stem Cell Reports. 2017; 8: 977-990Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar further reduced the target cell number up to 10-fold, increased the transduction efficiency of purified cells by exposing them to prostaglandin E2, and culture-expanded gene-modified HSCs with the small molecule UM171. Lastly, using the pre-clinical nonhuman primate (NHP) stem cell transplantation and gene therapy model, sort-purified CD34+CD90+CD45RA− cells reduced the target cell number up to 20-fold, were responsible for rapid short- and persistent long-term multilineage engraftment of gene-modified cells, and accurately predicted recovery onset.3Radtke S. Adair J.E. Giese M.A. Chan Y.Y. Norgaard Z.K. Enstrom M. Haworth K.G. Schefter L.E. Kiem H.P. A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates.Sci. Transl. Med. 2017; 9: 9Crossref Scopus (65) Google Scholar Despite obvious differences in the methodology and target cell phenotype, all strategies successfully reduced the number of target cells without compromising the efficiency of gene modification or engraftment potential. With the goal of translating these findings into a therapeutic application, we investigated the use of the CD34+CD90+CD45RA− cell fraction for the treatment of beta-hemoglobinopathies and recently presented results at the Presidential Symposium during the 2018 Annual Meeting of the American Society of Gene and Cell Therapy. Using a CRISPR/Cas9-based system to disable a binding site for a repressor of fetal hemoglobin (HbF), we demonstrated substantial long-term engraftment and HbF reactivation after editing and transplantation of the refined CD34+CD90+CD45RA− cell fraction in the NHP model, with results that were comparable to the editing and transplantation of the entire CD34+ cell population. These observations bode well for the ability to target a more HSC-enriched cell population for clinical applications. In fact, a similar selection approach is currently being studied in the allogeneic setting in severe-combined immunodeficiency patients (ClinicalTrials.gov: NCT02963064). A second aspect currently impacting the availability and portability of HSC gene therapy and editing is the requirement of ultra-sterile cell processing facilities (CPFs). Unfortunately, CPFs are very costly and can only be found in a few specialized institutions and companies worldwide. To increase availability, a promising option is the use of CPF-independent, semi- or fully-automated and portable closed-system applications, such as the CliniMACS Prodigy (Miltenyi Biotech), to perform a bead-based purification of phenotypically defined HSPCs.4Adair J.E. Waters T. Haworth K.G. Kubek S.P. Trobridge G.D. Hocum J.D. Heimfeld S. Kiem H.P. Semi-automated closed system manufacturing of lentivirus gene-modified haematopoietic stem cells for gene therapy.Nat. Commun. 2016; 7: 13173Crossref PubMed Scopus (28) Google Scholar Complementary to this system, the availability of a closed-system cell sorter, such as the MACSQuant Tyto (Miltenyi Biotech), has the potential to turn flow-based cell-sorting for HSC gene therapy into a portable and CPF-independent approach that could potentially reach more than 50 million patients worldwide. Thus, we think the future is very bright for HSC gene therapy and editing. Newer technologies are converging to ultimately make this a very efficient and portable treatment strategy not only for many genetic diseases affecting the hematopoietic system, but also for certain cancer applications and HIV. S.R. O.H., and H.P.K. wrote and reviewed the manuscript. This work was supported by grants to H.P.K. from the NIH (R01 HL135136 and R01 AI135953-01). H.P.K. is a Markey Molecular Medicine Investigator and received support as the inaugural recipient of the José Carreras/E. Donnall Thomas Endowed Chair for Cancer Research and the Fred Hutch Endowed Chair for Cell and Gene Therapy.