Efficient Ex Vivo Engineering and Expansion of Highly Purified Human Hematopoietic Stem and Progenitor Cell Populations for Gene Therapy.
Ex vivo gene therapy based on CD34+hematopoietic stem cells (HSCs) has shown promising results in clinical trials, but genetic engineering to high levels and in large scale remains challenging. We devised a sorting strategy that captures more than 90% of HSC activity in less than 10% of mobilized peripheral blood (mPB) CD34+cells, and modeled a transplantation protocol based on highly purified, genetically engineered HSCs co-infused with uncultured progenitor cells. Prostaglandin E2stimulation allowed near-complete transduction of HSCs with lentiviral vectors during a culture time of less than 38 hr, mitigating the negative impact of standard culture on progenitor cell function. Exploiting the pyrimidoindole derivative UM171, we show that transduced mPB CD34+CD38-cells with repopulating potential could be expanded ex vivo. Implementing these findings in clinical gene therapy protocols will improve the efficacy, safety, and sustainability of gene therapy and generate new opportunities in the field of gene editing.
San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Milan 20132, Italy.
Erika
E
Zonari
San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Milan 20132, Italy.
Giacomo
G
Desantis
San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Milan 20132, Italy; Vita-Salute San Raffaele University, Milan 20132, Italy.
Carolina
C
Petrillo
San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Milan 20132, Italy.
Francesco E
FE
Boccalatte
San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Milan 20132, Italy.
Maria Rosa
MR
Lidonnici
San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Milan 20132, Italy.
Anna
A
Kajaste-Rudnitski
San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Milan 20132, Italy; Vita-Salute San Raffaele University, Milan 20132, Italy; Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCSS Ospedale San Raffaele, Milan 20132, Italy.
Alessandro
A
Aiuti
San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Milan 20132, Italy; Vita-Salute San Raffaele University, Milan 20132, Italy.
Giuliana
G
Ferrari
San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Milan 20132, Italy; Vita-Salute San Raffaele University, Milan 20132, Italy.
Luigi
L
Naldini
San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Milan 20132, Italy; Hematology and Bone Marrow Transplantation Unit, IRCSS Ospedale San Raffaele, Milan 20132, Italy. Electronic address: gentner.bernhard@hsr.it.
Bernhard
B
Gentner
D051997:ADP-ribosyl Cyclase 1 / Q000032:analysis; D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D018929:Cell Culture Techniques; D060846:Cell Engineering / Q000379:methods*; D049109:Cell Proliferation; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D016688:Mice, Inbred NOD; D014161:Transduction, Genetic / Q000379:methods*
D018952:Antigens, CD34; D051997:ADP-ribosyl Cyclase 1
HSC expansion; HSC gene therapy; UM171; lentiviral vector transduction; prostaglandin E2; purified HSCs