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 CD34^Hi to CD34^Lo cells in Fanconi patients relative to healthy donors, with exclusive in vitro repopulating ability in only CD34^Hi 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).

Figure 1.

Diminished CD34^Hi hematopoietic cells from Fanconi Anemia A genetic defect (FA-A) patients. CD34 expression in baseline bone marrow (BM) (Patients 1, 2, 3, and healthy donor 1) or mobilized leukapheresis (mAPH) (Patient 3 and healthy donor 2) products was determined by fluorescence staining and flow cytometry analysis. Positive cell fractions are gated based on unstained and isotype stained control samples into two levels of CD34 expression: low expression, CD34^Lo, or high expression, CD34^Hi. The average mean fluorescence intensity (MFI) of CD34^Lo population = 3453; standard error of the mean (SEM) = 516 and CD34^Hi population = 19731; SEM = 4103.

Figure 2.

In vitro repopulation potential restricted to CD34^Hi hematopoietic cells. Mobilized leukapheresis from FA-A Patient 3 (Panel A) and a healthy donor (Panel B) were in parallel fluorescence stained with anti-CD34 antibody and sort-purified for CD34^Hi and CD34^Lo cells. Total nucleated cells (TNC) equivalent to 1500 CD34-expressing cells were seeded in CFC assays. Percentage of CD34⁺ cells seeded in the assay that gave rise to colonies is represented as the % of colony-forming cells.

Figure 3.

Direct CD34 enrichment versus depletion of lineage positive (+) cells. Products can include bone marrow (BM) or mobilized apheresis product (mAPH) (1). BM products were first processed through hetastarch sedimentation to deplete red blood cells (RBCs). Leukapheresis products were first subjected to several washes to deplete platelets. For direct CD34⁺ cell selection, anti-CD34 antibody-bound immunomagnetic beads (microbeads) are used, whereas for lineage depletion anti-CD3⁺, CD14⁺, CD16⁺, and CD19⁺, microbeads are used (2). In both cases, microbead-bound cells are retained on the column and subjected to wash steps. When lineage depletion is used, CD34-expressing cells undergo minimal manipulation during purification. Following purification, cells are cultured and transduced with a VSV-G pseudotyped lentiviral vector at a multiplicity of infection (MOI) of 5–10 IU/ cell (3). Following ~16 hours of incubation cells are harvested (4). ^*These processes were performed on the CliniMACS Prodigy™ device from Miltenyi Biotec GmbH.

Figure 4.

Multi-lineage engraftment of lineage depleted and transduced bone marrow (BM) and mobilized apheresis products (mAPH) in NSG mice. Recovery of total nucleated cells (TNC), CD34⁺ cells and lineage positive (+) cells (A and B). (C-E) Gene transfer efficiency. The colony-forming potential of transduced cells in standard CFC assays is defined as the plating efficiency (TNC). The colony-forming potential normalized to the number of CD34⁺ cells seeded is depicted as plating efficiency (CD34⁺). The percentage of colonies analyzed positive for the presence of lentivirus (LV) backbone by PCR analysis on DNA extracted from individual colonies is depicted as transduction efficiency. The vector copy number per cell in the bulk transduced population is depicted as VCN. The average VCN per cell in the individual CFC is depicted as single colony VCN. Data are representative of the average of 9 healthy BM products and 10 healthy mAPH products. Error bars represent the standard error of the mean. (F) Engraftment of human CD45⁺ cells and lineage development into T cells (CD3⁺), monocytes (CD14⁺) and B cells (CD20⁺) was determined by flow cytometry over 20 weeks following infusion of lineage-depleted cell products. Data are representative of 36 mice from 6 mAPH donors and 42 mice from 6 BM donors, respectively. Error bars represent the Standard Error of the Mean.

Figure 5.

Multi-lineage engraftment levels of lineage-depleted cell products in NSG (NOD/SCID/IL2rg^null) mice is comparable to CD34-enriched cell products from the same donor. (A) Graph depicts percent recovery of total nucleated cells (TNC) and CD34⁺ cells from each arm following depletion or enrichment. (B) Numbers of total and transduced colony-forming cells (CFC) normalized to 1×10⁸ cells processed to each arm, and vector copy number (VCN) in the bulk transduced cells following ten days of culture. Data are representative of 2 healthy donor bone marrow products. Error bars represent the Standard Error of the Mean. (C) Engraftment of human CD45⁺ cells and lineage development into T cells (CD3⁺), monocytes (CD14⁺) and B cells (CD20⁺) was determined by flow cytometry over 26 weeks following infusion. Data are representative of 9 mice for the lineage depleted (Lin-) arm and 6 mice for the CD34 enriched (CD34) arm, respectively.