Transduction of human CD34+ repopulating cells with a self-inactivating lentiviral vector for SCID-X1 produced at clinical scale by a stable cell line

Abstract

Self-inactivating (SIN)-lentiviral vectors have safety and efficacy features that are well suited for transduction of hematopoietic stem cells (HSCs), but generation of vector at clinical scale has been challenging. Approximately 280 liters of an X-Linked Severe Combined Immunodeficiency Disorder (SCID-X1) SIN-lentiviral vector in two productions from a stable cell line were concentrated to final titers of 4.5 and 7.2×10(8) tu/ml. These two clinical preparations and three additional development-scale preparations were evaluated in human CD34(+) hematopoietic cells in vitro using colony forming cell (CFU-C) assay and in vivo using the NOD/Lt-scid/IL2Rγ(null) (NSG) mouse xenotransplant model. A 40-hour transduction protocol using a single vector exposure conferred a mean NSG repopulating cell transduction of 0.23 vector genomes/human genome with a mean myeloid vector copy number of 3.2 vector genomes/human genome. No adverse effects on engraftment were observed from vector treatment. Direct comparison between our SIN-lentiviral vector using a 40-hour protocol and an MFGγ(c) γ-retroviral vector using a five-day protocol demonstrated equivalent NSG repopulating cell transduction efficiency. Clonality survey by linear amplification-mediated polymerase chain reaction (LAM-PCR) with Illumina sequencing revealed common clones in sorted myeloid and lymphoid populations from engrafted mice demonstrating multipotent cell transduction. These vector preparations will be used in two clinical trials for SCID-X1.

Figures

FIG. 1.

Vector production from stable cell line using a WAVE bioreactor. (a) Kinetics of vector particle production for three development and two clinical scale runs after induction by media exchange to doxycycline-free D10. Daily harvests from day 3 to end of run were included in final product. Vector positivity was determined by IL-2Rγc surface expression of transduced ED7R cells analyzed by flow cytometry compared to untransduced control. (b) Characteristics of the two clinical scale runs, Wave9 and Wave12. Final titers were determined using the same ED7R method.

FIG. 2.

Performance of large-scale vector products using human G-CSF mobilized peripheral blood CD34+ cells in in vitro transduction assay. Measurement of CD34+ transduction by in vitro CFU-C assay. Experiments with the same letter name performed simultaneously. Each point represents a transduction assayed by qualitative PCR of 30 individual colonies.

FIG. 3.

Transduction protocol and experimental outline. This diagram shows the timeline in boxes on the left and sequence of procedures for transplant in boxes in the center for the assays used in this manuscript. Cell, cytokine, and vector doses are indicated, as is culture media for our standard 40-hour transduction protocol. Donor selection and conditioning regimen is also noted.

FIG. 4.

Engraftment characteristics of vector and mock-transduced human G-CSF mobilized peripheral blood CD34+ cells obtained from whole bone marrow harvested from NSG recipient mice 12–16 weeks post-transplant. (a) Representative flow cytometry of single mouse bone marrow. (b) Isotype control is shown at left. Human CD45 engraftment comparison of all mock-treated to all vector-treated NSG recipients. Each point represents the result of one mouse's terminal bone marrow evaluation. (c) Representative flow cytometry of human CD45+ fraction for expression of human myeloid (CD33) and lymphoid (CD19/3) markers. (d) Human lineage-specific engraftment comparison of all mock-treated to all vector-treated NSG recipients. Each point represents the result of one mouse's terminal bone marrow evaluation. Bars represent the mean.

FIG. 5.

Transduction of human CD34+-derived NSG repopulating cells with large-scale vector preparations. (a) Transduction efficiency as measured in genomic DNA from FACS-purified human CD45+ bone marrow obtained 12–16 weeks post-transplant using quantitative real-time PCR assay. Each data point represents the result of a single mouse. Number of separate experiments performed with each vector preparation is indicated below the X-axis. Bars represent mean. (b) Myeloid vector copy number measured by quantitative real-time PCR on individual vector-positive colonies obtained from CFU-C assay performed on human CD45+ cells FACS-purified from the bone marrow of NSG mice 12–16 weeks post-transplant. There was no significant difference between arms in any intradonor comparison (not including Donor6-Wave9 because n=1 and thus not evaluable by t-test/ANOVA). The bars represent the mean of all evaluable colonies from all evaluable mice treated in one arm or one experiment. Error bars represent the mean±SEM. Number of evaluated colonies indicated as n.

FIG. 6.

Transduction of human CD34+-derived NSG repopulating cells with large-scale vector preparations at higher doses. (a) Engraftment of CD34+ transductions in NSG mice measured 12–15 weeks post-transplant. Each point represents the result from a single mouse bone marrow evaluated by flow cytometry. Bars represent the mean. (b) Transduction of NSG repopulating cells measured 12–15 weeks post-transplant. Each point represents the result of a quantitative real-time PCR assay of genomic DNA purified from human CD45+ FACS-sorted bone marrow from a single transplanted NSG mouse. Bars represent the mean. W9(30), Wave9 at 30% volume (MOI 135); W12(19), Wave12 at 19% volume (MOI 135); W12(30), Wave12 at 30% volume (MOI 225); x1, single 24-hr vector exposure; x2, two 24-hr vector exposures.

FIG. 7.

Comparison of human CD34+ cell transduction with Wave9 clinical SIN-lentivirus prep and an MFGγc gamma-retroviral prep. (a) Transduction protocol outline used for each vector. (b) Pre-transplant CFU-C graft transduction measured in individual colonies by qualitative PCR. There was a significant difference between the Wave9 and MFGγc arms in both mPB and CB. *indicates which comparisons are statistically significant (p<0.05) (c) Human CD45 engraftment of NSG recipients with all treatment arms. Each point represents the result of one mouse's terminal bone marrow evaluation. Bars represent the mean. (d) Transduction of human cord blood and mobilized peripheral blood CD34+ cells treated with either Wave9 clinical lentiviral preparation or MFGγc γ-retroviral preparation as measured by quantitative real-time PCR in genomic DNA from FACS-purified human CD45+ bone marrow obtained after 12–16 weeks post-transplant. Each data point represents the result of a single mouse. Bars represent the mean.