Novel lineage depletion preserves autologous blood stem cells for gene therapy of Fanconi anemia complementation group A

Abstract

A hallmark of Fanconi anemia is accelerated decline in hematopoietic stem and progenitor cells (CD34 +) leading to bone marrow failure. Long-term treatment requires hematopoietic cell transplantation from an unaffected donor but is associated with potentially severe side-effects. Gene therapy to correct the genetic defect in the patient’s own CD34+ cells has been limited by low CD34+ cell numbers and viability. Here we demonstrate an altered ratio of CD34Hi to CD34Lo cells in Fanconi patients relative to healthy donors, with exclusive in vitro repopulating ability in only CD34Hi cells, underscoring a need for novel strategies to preserve limited CD34+ cells. To address this need, we developed a clinical protocol to deplete lineage+(CD3+, CD14+, CD16+ and CD19+) cells from blood and marrow products. This process depletes >90% of lineage+cells while retaining ≥60% of the initial CD34+cell fraction, reduces total nucleated cells by 1–2 logs, and maintains transduction efficiency and cell viability following gene transfer. Importantly, transduced lineage− cell products engrafted equivalently to that of purified CD34+ cells from the same donor when xenotransplanted at matched CD34+ cell doses. This novel selection strategy has been approved by the regulatory agencies in a gene therapy study for Fanconi anemia patients (NCI Clinical Trial Reporting Program Registry ID NCI-2011-00202; clinicaltrials.gov identifier: 01331018).

Purification of Human CD34+CD90+ HSCs Reduces Target Cell Population and Improves Lentiviral Transduction for Gene Therapy

“…Next, sorted CD90 + as well as bulk CD34 + cells were transduced with a lentiviral vector encoding for green fluorescent protein (GFP) according to our clinically approved CD34-mediated gene therapy protocol. 39 , 40 Five days post-transduction, the gene modification efficiency in the CD90 + subset of bulk-transduced CD34 + cells reached 16.5% ± 3.4% (SEM), whereas 2.3-fold (Tyto, 38.1% ± 5.5%, SEM, p = 0.012) and 3.1-fold (Sony, 51.9% ± 5.8%, SEM, p = 0.006) higher frequencies of GFP were seen in the FACS-purified CD90 + cell fractions ( Figure 5 E). Despite significant differences in the gene modification efficiency, no significant increase in the overall mean fluorescence intensity (MFI) of GFP was observed transducing purified CD90 + cells ( Figure 5 E).…”

Envelope-Specific Adaptive Immunity following Transplantation of Hematopoietic Stem Cells Modified with VSV-G Lentivirus

“…In vitro transduction success and cell dosing in subjects 002 and 004 were previously reported. 15 However, within the first 100 days following transplantation, engraftment of gene-modified cells was not observed in either subject ( Figure 1).…”

“…This procedure has been previously described. 15 Briefly, the vector copy number (VCN) per genome equivalent was assessed by quantitative real-time PCR with an LV-specific primer/probe combination (forward, This assay was adapted from previously published experiments. 31 Serum was mixed with VSV-G-pseudotyped vector preparations with 5 Â 10 5 EGFP transducing units (TU) in triplicate, incubated at 37 C for 30 min, and then added to 1 Â 10 5 HT1080 cells (ATCC, Manassas, VA, USA).…”

Sort-purification of human CD34+CD90+cells reduces target cell population and improves lentiviral transduction for gene therapy

“…Next, sorted CD90 + as well as bulk CD34 + cells were transduced with a lentivirus encoding for GFP according to our clinically approved CD34-mediated gene therapy protocol (43,44). Five days post-transduction, the gene-modification efficiency in the CD90 + subset of bulk-transduced CD34 cells reached 16.5±3.4% (SEM), whereas 2.3-fold (Tyto: 38.1±5.5%, SEM, p=0.012) and…”