Engraftment of Gene-Marked Hematopoietic Progenitors in Myeloma Patients after Transplant of Autologous Long-term Marrow Cultures

Abstract

We conducted a phase I hematopoietic stem cell (HSC) gene-marking trial in patients undergoing autologous blood or marrow stem cell transplant for the treatment of multiple myeloma. Between 500 and 1000 ml of bone marrow was harvested from each of 14 myeloma patients and 1 syngeneic donor. A mean of 3.3x10(9) cells per patient were plated in 20 to 50 long-term marrow culture (LTMC) flasks and maintained for 3 weeks. LTMCs were exposed on days 8 and 15 to clinical-grade neo(r)-containing retrovirus supernatant (G1Na). A mean of 8.23x10(8) day-21 LTMC cells containing 5.2x10(4) gene-marked granulocyte-macrophage progenitor cells (CFU-GM) were infused along with an unmanipulated peripheral blood stem cell graft into each patient after myeloablative therapy. Proviral DNA was detected in 71% of 68 tested blood and bone marrow samples and 150 of 2936 (5.1%) CFU-GM derived from patient bone marrow samples after transplant. The proportion of proviral DNA-positive CFU-GM declined from a mean of 9.8% at 3 months to a mean of 2.3% at 24 months postinfusion. Southern blots of 26 marrow and blood samples were negative. Semiquantitative PCR analysis indicated that gene transfer was achieved in 0.01-1% of total bone marrow and blood mononuclear cells (MNCs). Proviral DNA was also observed in EBV-transformed B lymphocytes, in CD34+ -enriched bone marrow cells, and in CFUs derived from the latter progenitors. Gene-modified cells were detected by PCR in peripheral blood and bone marrow for 24 months after infusion of LTMC cells. Sensitivity and specificity of the PCR assays were independently validated in four laboratories. Our data confirm that HSCs may be successfully transduced in stromal based culture systems. The major obstacle to therapeutic application of this approach remains the overall low level of genetically modified cells among the total hematopoietic cell pool in vivo.

Quantitation of HSV mass distribution in a rodent brain tumor model

“…These protocols are designed, for instance, to test novel marker proteins, 1 perform gene replacement and/or augmentation trials, 2 test prodrug activation, 3 evaluate cell-based therapies 4 and investigate oncolytic viruses. 5 During the last 5 years of vector and transgene development, it has become apparent that insufficient delivery of genetic material to tumor cells in vivo and low and/or transient gene expression are the main limitations to the effectiveness of this new treatment paradigm.…”

Long-term follow-up of gene-marked CD34+ cells after autologous stem cell transplantation for multiple myeloma

“…17 Our results corroborate with other previous, relatively shorter term, gene marking studies and furthermore help complete the overview about the nature of relapse in multiple myeloma. 20,21 In this study, no side effects were seen due to gene marking including insertional mutagenesis. However, 2% transduction rate is associated with a low number of integrations per cell; hence, the risk of insertional mutagenesis is low.…”

Hematopoietic stem cell gene therapy: progress toward therapeutic targets

“…In these genetic marking experiments, a gene with an easily detectable sequence or gene product is transferred to autologous cells of patients receiving a therapeutic autograft for an unrelated indication, typically a malignancy. Many of these studies used the neomycin resistance gene, Neo, as the marker, [29][30][31] and typically showed detectable but disappointingly low levels of transduction, of less than 5%.…”